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Regulation of phenotypic heterogeneity permits Salmonella evasion of the host caspase-1 inflammatory response
Abstract
Sensing and adapting to the environment is one strategy by which bacteria attempt to maximize fitness in an unpredictable world; another is the stochastic generation of phenotypically distinct subgroups within a genetically clonal population. In culture, Salmonella Typhimurium populations are bistable for the expression of flagellin. We report that YdiV controls this expression pattern by preventing transcription of the sigma factor that recruits RNA polymerase to the flagellin promoter. Bistability ensues when the sigma factor is repressed in a subpopulation of cells, resulting in two phenotypes: flagellin expressors and flagellin nonexpressors. Although the ability to swim is presumably a critical survival trait, flagellin activates eukaryotic defense pathways, and Salmonella restrict the production of flagellin during systemic infection. Salmonella mutants lacking YdiV are unable to fully repress flagellin at systemic sites, rendering them vulnerable to caspase-1 mediated colonization restriction. Thus, a regulatory mechanism producing bistability also impacts Salmonella virulence.
... The cooperation between injectisome and flagellum bistable populations has been demonstrated in Salmonella enterica [117,118]. Both nanomachines are highly conserved across bacterial species and function as a pathogen-associated molecular pattern (PAMP), triggering host immune responses during infection [19,20]. ...
... The subpopulation nonexpressing injectisome thrives in the gut lumen, focusing on replication, while the expressing subpopulation infiltrates tissues, eliciting immune responses (Figure 4c). For the flagellum non-expressing subpopulation, it enables the evasion of eukaryotic defense pathways at the host level and even within macrophage intracellular infections by preventing the caspase-1 inflammatory response [118]. Conversely, flagellum-expressing bacteria trigger an inflammatory cascade, leading to the pyroptosis of macrophages [119]. ...
Bacterial nanomachines represent remarkable feats of evolutionary engineering, showcasing intricate molecular mechanisms that enable bacteria to perform a diverse array of functions essential to persist, thrive, and evolve within ecological and pathological niches. Injectosomes and bacterial flagella represent two categories of bacterial nanomachines that have been particularly well studied both at the molecular and functional levels. Among the diverse functionalities of these nanomachines, bistability emerges as a fascinating phenomenon, underscoring their dynamic and complex regulation as well as their contribution to shaping the bacterial community behavior during the infection process. In this review, we examine two closely related bacterial nanomachines, the type 3 secretion system, and the flagellum, to explore how the bistability of molecular-scale devices shapes the bacterial eco-pathological life cycle.
... Repression of flagellar genes can be beneficial to Salmonella during its pathogenesis. For example, YdiV represses flagellar genes in response to nutritional cues, such as poor nutrient conditions inside macrophages (Stewart et al., 2011;Wada et al., 2011). Additionally, YdiV represses flagellar genes in systemic tissues, which protects Salmonella from caspase-1-mediated bacterial clearance (Lara-Tejero et al., 2006;Miao et al., 2006;Stewart et al., 2011). ...
... For example, YdiV represses flagellar genes in response to nutritional cues, such as poor nutrient conditions inside macrophages (Stewart et al., 2011;Wada et al., 2011). Additionally, YdiV represses flagellar genes in systemic tissues, which protects Salmonella from caspase-1-mediated bacterial clearance (Lara-Tejero et al., 2006;Miao et al., 2006;Stewart et al., 2011). The regulation of Salmonella flagellar expression reflects the importance of reducing flagella in specific environments for survival. ...
Salmonella enterica subspecies enterica serovar Heidelberg (Salmonella Heidelberg) has caused several multistate foodborne outbreaks in the United States, largely associated with the consumption of poultry. However, a 2015–2017 multidrug-resistant (MDR) Salmonella Heidelberg outbreak was linked to contact with dairy beef calves. Traceback investigations revealed calves infected with outbreak strains of Salmonella Heidelberg exhibited symptoms of disease frequently followed by death from septicemia. To investigate virulence characteristics of Salmonella Heidelberg as a pathogen in bovine, two variants with distinct pulse-field gel electrophoresis (PFGE) patterns that differed in morbidity and mortality during the multistate outbreak were genotypically and phenotypically characterized and compared. Strain SX 245 with PFGE pattern JF6X01.0523 was identified as a dominant and highly pathogenic variant causing high morbidity and mortality in affected calves, whereas strain SX 244 with PFGE pattern JF6X01.0590 was classified as a low pathogenic variant causing less morbidity and mortality. Comparison of whole-genome sequences determined that SX 245 lacked ~200 genes present in SX 244, including genes associated with the IncI1 plasmid and phages; SX 244 lacked eight genes present in SX 245 including a second YdiV Anti-FlhC(2)FlhD(4) factor, a lysin motif domain containing protein, and a pentapeptide repeat protein. RNA-sequencing revealed fimbriae-related, flagella-related, and chemotaxis genes had increased expression in SX 245 compared to SX 244. Furthermore, SX 245 displayed higher invasion of human and bovine epithelial cells than SX 244. These data suggest that the presence and up-regulation of genes involved in type 1 fimbriae production, flagellar regulation and biogenesis, and chemotaxis may play a role in the increased pathogenicity and host range expansion of the Salmonella Heidelberg isolates involved in the bovine-related outbreak.
... Flagellar gene expression is bimodal in S. enterica (3)(4)(5)(6)(7)(8)(9). Under certain growth conditions, some cells express the flagellar genes, whereas others do not. ...
... The key finding in the present work is that class 3 bimodality results from the 28 -FlgM checkpoint. Stewart and coworkers proposed that motility is bimodal in S. enterica because it generates mixed populations of invasive and noninvasive cells due to the coupling of motility and virulence (6,34,35). This model, however, does not explain why S. enterica employs two mechanisms to induce bimodal expression of the flagellar genes when one alone would suffice. ...
Flagellar gene expression is bimodal in Salmonella enterica . Under certain growth conditions, some cells express the flagellar genes whereas others do not. This results in mixed populations of motile and non-motile cells. In the present study, we found that two independent mechanisms control bimodal expression of the flagellar genes. One was previously found to result from a double negative-feedback loop involving the flagellar regulators RflP and FliZ. This feedback loop governs bimodal expression of class 2 genes. In this work, a second mechanism was found to govern bimodal expression of class 3 genes. In particular, class 3 gene expression is still bimodal even when class 2 gene expression is not. Using a combination of experimental and modeling approaches, we found that class 3 bimodalilty results from the σ ²⁸ -FlgM developmental checkpoint.
IMPORTANCE Many bacterial use flagella to swim in liquids and swarm over surface. In Salmonella enterica, over fifty genes are required to assemble flagella. The expression of these genes is tightly regulated. Previous studies have found that flagella gene expression is bimodal in S. enterica , which means that only a fraction of cells express flagellar genes and are motile. In the present study, we found that two separate mechanisms induce this bimodal response. One mechanism, which was previously identified, tunes the fraction of motile cells in response to nutrients. The other results from a developmental checkpoint that couples flagellar gene expression to flagellar assembly. Collectively, these results further our understanding of how flagellar gene expression is regulated in S. enterica .
... However, flagella are also highly immunogenic, being rapidly recognized by the innate immune system (5). Known strategies to avoid this immune response used by such pathogens as Salmonella, Campylobacter, and Helicobacter rely on stochastic variation in the expression of flagellar genes, due either to genetic changes (6)(7)(8)(9)(10) or to changes at the transcriptional level (11)(12)(13). The latter regulation in Salmonella is mediated by RflP (formerly called YdiV) (14), which represses fliA expression by sequestering the FlhDC complex and targeting it for degradation in a subpopulation of cells (15,16). ...
... In this study, we demonstrate that the expression of flagellar genes in a number of pathogenic E. coli strains is inhibited in liquid but that their expression is induced when bacteria are grown either in porous medium or on a surface. Because this regulation was not observed in commensal or laboratory strains of E. coli, it likely represents a specific mechanism to avoid the adaptive immune response of the host, akin to genetic or transcriptional variation in the expression of flagellar genes observed in other bacterial pathogens (6)(7)(8)(9)(10)(11)(12)28). However, unlike with the stochastic antigenic variation or transcriptional switching observed in these bacteria, the control of flagellar gene expression in pathogenic E. coli apparently relies on sensing the mechanical load on the flagellar motor. ...
Flagella and motility are widespread virulence factors among pathogenic bacteria. Motility enhances the initial host colonization, but the flagellum is a major antigen targeted by the host immune system. Here, we demonstrate that pathogenic E. coli strains employ a mechanosensory function of the flagellar motor to activate flagellar expression under high loads, while repressing it in liquid culture. We hypothesize that this mechanism allows pathogenic E. coli to regulate its motility dependent on the stage of infection, activating flagellar expression upon initial contact with the host epithelium, when motility is beneficial, but reducing it within the host to delay the immune response.
... The peritrichous flagella of S. typhimurium are energetically costly both during biosynthesis and at functionality such as swimming and swarming motility, using approximately 20% of the cellular energy. Tight regulation of these multifunctional appendages on the transcriptional, post-transcriptional, and functional level is a prerequisite for the multifactorial and even opposite roles of flagella in motility, biofilm formation, and virulence including interactions with biotic and abiotic surfaces, the environment and association with animal and plant hosts [34,69,95,96]. In various Gram-positive and Gram-negative bacterial species, the cyclic di-GMP signaling network contributes to the tuning of flagellar biosynthesis and functionality from the transcriptional to the post-translational level [97,98] that reaches far beyond regulation of swimming and surface swarming motility. ...
... Instead, these proteins bind to FlhD 4 C 2 , the class 1 master regulator of the flagella regulon, which prevents promoter binding of FlhD 4 C 2 , promotes removal of FlhD 4 C 2 from a target promoter, and provides potential adaptor function for degradation by ClpXP [18,26,106]. The physiological role of STM1344 and STM1697 spans from energy-saving under nutrient deprivation, sensing of envelope stress, maintenance of bistable flagella production of planktonic cells, persistence on leaf surfaces to concerted regulation of flagella biosynthesis and resistance to phagocyte oxidase in the animal host in order to withstand and avoid innate and adaptive immune system recognition for successful systemic infection [26,34,95,96,107,108]. As such, STM1344 and STM1697 contribute to the tight regulation of flagella biosynthesis in diverse environments. ...
Cyclic di-GMP is perhaps the most abundant nucleotide-based second messenger in bacteria. In the gamma-proteobacterium Salmonella enterica serovar Typhimurium, a gastrointestinal pathogen, this signaling network regulates biofilm formation, flagella-associated physiology, and acute virulence properties. This chapter summarizes the impact of the complex cyclic di-GMP signaling network on the physiology of S. typhimurium in different environments and compares its consequences, when appropriate, with the close relative, the commensal and pathogenic Escherichia coli. The substantial diversity and variability in the cyclic di-GMP turnover protein network span from single amino acid replacements and stop codon variants in individual proteins to deletion and acquisition of novel cyclic di-GMP turnover genes by horizontal transfer. Despite differences in enzyme activities and gene combinations, cyclic di-GMP signaling modules become integrated into a common but even isolate-specific regulation of lifestyle transitions that are coordinated with cell cycle regulation. On a wider phylogenetic perspective, the observed conservation of cyclic di-GMP turnover proteins with a similar domain structure found in S. enterica throughout the phylogenetic tree poses a quest for the origin and maintenance of common principles in cyclic di-GMP signaling.
... Even under homogeneous conditions, individual bacteria are capable of executing markedly distinct gene expression programs from the rest of the population (12)(13)(14)(15)(16)(17). For example, flagellar gene expression in the closely related Salmonella appears to occur in only a subpopulation of cells (18,19). On the other hand, there are notable differences in the proteins that regulate flagellar expression in Salmonella and E. coli (20,21), including the master regulator FlhDC itself (22), which suggests that E. coli could have its own unique pattern of flagellar gene regulation. ...
... S12). By contrast, flow cytometry data in Salmonella show well-separated bimodal distributions for both class II and class III promoter activity (19). These differences suggest that flagellar genes in E. coli are considerably more dynamic and pulse at a much higher frequency than the previously reported bistable behavior in Salmonella, which is defined by more stable active or inactive states. ...
The classic picture of flagellum biosynthesis in Escherichia coli , inferred from population measurements, depicts a deterministic program where promoters are sequentially up-regulated and are maintained steadily active throughout exponential growth. However, complex regulatory dynamics at the single-cell level can be masked by bulk measurements. Here, we discover that in individual E. coli cells, flagellar promoters are stochastically activated in pulses. These pulses are coordinated within specific classes of promoters and comprise “on” and “off” states, each of which can span multiple generations. We demonstrate that in this pulsing program, the regulatory logic of flagellar assembly dictates which promoters skip pulses. Surprisingly, pulses do not require specific transcriptional or translational regulation of the flagellar master regulator, FlhDC, but instead appears to be essentially governed by an autonomous posttranslational circuit. Our results suggest that even topologically simple transcriptional networks can generate unexpectedly rich temporal dynamics and phenotypic heterogeneities.
... YdiV, a negative regulator of the flagellar main transcriptional activator complex FlhD 4 C 2 , downregulates the expression of the flagellar gene fliC through the CadC-YdiV-FlhDC pathway (82,83). fliC can induce pyroptosis in human and murine macrophages, and YdiV inhibits the expression of fliC, preventing macrophage pyroptosis and releasing inflammatory factors (Figure 4), thereby facilitating the colonization and immune escape of Salmonella in host cells (84,85). ...
The genus Salmonella contains the most common foodborne pathogens frequently isolated from food-producing animals and is responsible for zoonotic infections in humans and animals. Salmonella infection in humans and animals can cause intestinal damage, resulting in intestinal inflammation and disruption of intestinal homeostasis more severe cases can lead to bacteremia. Pyroptosis, a proinflammatory form of programmed cell death, is involved in many disease processes. Inflammasomes, pyroptosis, along with their respective signaling cascades, are instrumental in the preservation of intestinal homeostasis. In recent years, with the in-depth study of pyroptosis, our comprehension of the virulence factors and effector proteins in Salmonella has reached an extensive level, a deficit persists in our knowledge regarding the intrinsic pathogenic mechanisms about pyroptosis, necessitating a continued pursuit of understanding and investigation. In this review, we discuss the occurrence of pyroptosis induced by Salmonella effectors to provide new ideas for elucidating the regulatory mechanisms through which Salmonella virulence factors and effector proteins trigger pyroptosis could pave the way for novel concepts and strategies in the clinical prevention of Salmonella infections and the treatment of associated diseases.
... Bet-hedging in S. Typhimurium is observed through the bistable ON-OFF regulated expression of the flagellin protein gene fliC, which is expressed in a bistable manner when grown in homogenous conditions (Stewart et al. 2011). Transcription of flagellar genes in S. Typhimurium is regulated through a three-tiered transcriptional cascade (Class I-III, Fig. 2). ...
Bacteria encounter various stressful conditions within a variety of dynamic environments, which they must overcome for survival. One way they achieve this is by developing phenotypic heterogeneity to introduce diversity within their population. Such distinct subpopulations can arise through endogenous fluctuations in regulatory components, wherein bacteria can express diverse phenotypes and switch between them, sometimes in a heritable and reversible manner. This switching may also lead to antigenic variation, enabling pathogenic bacteria to evade the host immune response. Therefore, phenotypic heterogeneity plays a significant role in microbial pathogenesis, immune evasion, antibiotic resistance, host niche tissue establishment, and environmental persistence. This heterogeneity can result from stochastic and responsive switches, as well as various genetic and epigenetic mechanisms. The development of phenotypic heterogeneity may create clonal populations that differ in their level of virulence, contribute to the formation of biofilms, and allow for antibiotic persistence within select morphological variants. This review delves into the current understanding of the molecular switching mechanisms underlying phenotypic heterogeneity, highlighting their roles in establishing infections caused by select bacterial pathogens.
... Flagella are also crucial for Salmonella biofilm formation, a long-lasting form of pathogenic bacteria that can enhance the persistence of its transmission and colonization in vivo [10,11]. In addition to contributing to motility and invasion, the flagellar filaments FliC and FljB can be recognized by the TLR5 receptor, Nalp3, and Ipaf, leading to the activation of the host's immune response and inflammation [12][13][14]. Hence, flagella play a key role in Salmonella invasion and infection. ...
Flagella play a crucial role in the invasion process of Salmonella and function as a significant antigen that triggers host pyroptosis. Regulation of flagellar biogenesis is essential for both pathogenicity and immune escape of Salmonella. We identified the conserved and unknown function protein STM0435 as a new flagellar regulator. The ∆stm0435 strain exhibited higher pathogenicity in both cellular and animal infection experiments than the wild-type Salmonella. Proteomic and transcriptomic analyses demonstrated dramatic increases in almost all flagellar genes in the ∆stm0435 strain compared to wild-type Salmonella. In a surface plasmon resonance assay, purified STM0435 protein-bound c-di-GMP had an affinity of ~8.383 µM. The crystal structures of apo-STM0435 and STM0435&c-di-GMP complex were determined. Structural analysis revealed that R33, R137, and D138 of STM0435 were essential for c-di-GMP binding. A Salmonella with STM1987 (GGDEF protein) or STM4264 (EAL protein) overexpression exhibits completely different motility behaviours, indicating that the binding of c-di-GMP to STM0435 promotes its inhibitory effect on Salmonella flagellar biogenesis.
... • "These average MFIs did not differ significantly between the strains ( Fig. 3B ) , indicating that YdiV does not regulate flhDC transcription ." [77]. • "We have also shown that FadD , FliZ , and EnvZ do not regulate hilA expression by modulating hilD expression ." ...
Curation of biomedical literature has been the traditional approach to extract relevant biological knowledge; however, this is time-consuming and demanding. Recently, Large language models (LLMs) based on pre-trained transformers have addressed biomedical relation extraction tasks outperforming classical machine learning approaches. Nevertheless, LLMs have not been used for the extraction of transcriptional regulatory interactions between transcription factors and regulated elements (genes or operons) of bacteria, a first step to reconstruct a transcriptional regulatory network (TRN). These networks are incomplete or missing for many bacteria. We compared six state-of-the-art BERT architectures (BERT, BioBERT, BioLinkBERT, BioMegatron, BioRoBERTa, LUKE) for extracting this type of regulatory interactions. We fine-tuned 72 models to classify sentences in four categories: activator , repressor , regulator , and no relation . A dataset of 1562 sentences manually curated from literature of Escherichia coli was utilized. The best model of LUKE architecture obtained a relevant performance in the evaluation dataset (Precision: 0.8601, Recall: 0.8788, F1-Score Macro: 0.8685, MCC: 0.8163). An examination of model predictions revealed that the model learned different ways to express the regulatory effect. The model was applied to reconstruct a TRN of Salmonella Typhimurium using 264 complete articles. We were able to accurately reconstruct 82% of the network. A network analysis confirmed that the transcription factor PhoP regulated many genes (uppermost degree), some of them responsible for antimicrobial resistance. Our work is a starting point to address the limitations of curating regulatory interactions, especially for the reconstruction of TRNs of bacteria or diseases of biological interest.
... The products of Class II genes form the basal flagellar structure and hook-basal body complex, while Class III genes encode proteins required for filament formation, flagellar rotation (motility), and chemotaxis (Chilcott & Hughes, 2000;Ide et al., 1999;Karlinsey et al., 2000;Minamino et al., 2021;Tomoyasu et al., 2003). The presence of flagellar is also closely associated with the pathogenicity of S. Typhimurium (Lee et al., 2022;Miao et al., 2010;Stecher et al., 2004Stecher et al., , 2007Stewart et al., 2011). Upon entry into the intestinal tract, S. Typhimurium utilizes its flagellum to navigate viscous fluids, facilitating attachment to host cells. ...
Salmonella enterica serovar Typhimurium (S. Typhimurium) is a globally recognized foodborne pathogen that affects both animals and humans. Endoribonucleases mediate RNA processing and degradation in the adaptation of bacteria to environmental changes and have been linked to the pathogenicity of S. Typhimurium. Not much is known about the specific regulatory mechanisms of these enzymes in S. Typhimurium, particularly in the context of environmental adaptation. Thus, this study carried out a comparative transcriptomic analysis of wild-type S. Typhimurium SL1344 and its mutant (∆rnc), which lacks the rnc gene encoding RNase III, thereby elucidating the detailed regulatory characteristics that can be attributed to the rnc gene. Global gene expression analysis revealed that the ∆rnc strain exhibited 410 upregulated and 301 downregulated genes (fold-change > 1.5 and p < 0.05), as compared to the wild-type strain. Subsequent bioinformatics analysis indicated that these differentially expressed genes are involved in various physiological functions, in both the wild-type and ∆rnc strains. This study provides evidence for the critical role of RNase III as a general positive regulator of flagellar-associated genes and its involvement in the pathogenicity of S. Typhimurium.
... Clonal populations of pathogens were shown to functionally diversify to effectively colonize host surfaces 4 , stabilize cooperative virulence programmes 5,6 , diversify their metabolic capacities [7][8][9] and modulate stress tolerance during infections [10][11][12] . Pathogens like Salmonella species and Escherichia coli generate subpopulations that stochastically express virulence factors, including components of flagellar motility 13,14 and type 3 secretion systems 5 . Similarly, the human pathogen Pseudomonas aeruginosa generates functionally distinct subpopulations to enhance host colonization 4,15 . ...
Efficient colonization of mucosal surfaces is essential for opportunistic pathogens like Pseudomonas aeruginosa, but how bacteria collectively and individually adapt to optimize adherence, virulence and dispersal is largely unclear. Here we identified a stochastic genetic switch, hecR–hecE, which is expressed bimodally and generates functionally distinct bacterial subpopulations to balance P. aeruginosa growth and dispersal on surfaces. HecE inhibits the phosphodiesterase BifA and stimulates the diguanylate cyclase WspR to increase c-di-GMP second messenger levels and promote surface colonization in a subpopulation of cells; low-level HecE-expressing cells disperse. The fraction of HecE⁺ cells is tuned by different stress factors and determines the balance between biofilm formation and long-range cell dispersal of surface-grown communities. We also demonstrate that the HecE pathway represents a druggable target to effectively counter P. aeruginosa surface colonization. Exposing such binary states opens up new ways to control mucosal infections by a major human pathogen.
... Low-resolution structures identified that the flagellum comprises various rings [9,10], which is consistent with a role as a rotary motor. Indeed, non-flagellated variants of Salmonella and a non-chemotactic mutant of Heliobacter pylori, have a reduced rate of infection [3,11]. Both the assembly and the rotation of bacterial flagellum depend upon the MS-ring of the flagellar motor. ...
The flagellar motor supports bacterial chemotaxis, a process that allows bacteria to move in response to their environment. A central feature of this motor is the MS-ring, which is composed entirely of repeats of the FliF subunit. This MS-ring is critical for the assembly and stability of the flagellar switch and the entire flagellum. Despite multiple independent cryoEM structures of the MS-ring, there remains a debate about the stoichiometry and organization of the ring-building motifs (RBMs). Here, we report the cryoEM structure of a Salmonella MS-ring that was purified from the assembled flagellar switch complex (MSC-ring). We term this the ‘post-assembly’ state. Using 2D class averages, we show that under these conditions, the post-assembly MS-ring can contain 32, 33, or 34 FliF subunits, with 33 being the most common. RBM3 has a single location with C32, C33, or C34 symmetry. RBM2 is found in two locations with RBM2inner having C21 or C22 symmetry and an RBM2outer-RBM1 having C11 symmetry. Comparison to previously reported structures identifies several differences. Most strikingly, we find that the membrane domain forms 11 regions of discrete density at the base of the structure rather than a contiguous ring, although density could not be unambiguously interpreted. We further find density in some previously unresolved areas, and we assigned amino acids to those regions. Finally, we find differences in interdomain angles in RBM3 that affect the diameter of the ring. Together, these investigations support a model of the flagellum with structural plasticity, which may be important for flagellar assembly and function.
... the flagella mediate the colonization, adhesion, and invasion process of Salmonella (4, 5); after invasion, flagellin molecules are recognized by the host Toll-like receptor 5 (TLR5), triggering the host immune response (6,7). Wild Salmonella can rapidly shut down flagellar synthesis after entering host cells to achieve immune escape, while Salmonella that continuously expresses flagella will be rapidly recognized and killed by the host (8,9). ...
When Salmonella enters host cells, the synthesis of flagella is quickly turned off to escape the host immune system. In this study, we investigated the cooperative regulatory mechanism of flagellar synthesis by two EAL-like proteins, STM1344 and STM1697, in Salmonella. We found that Salmonella upregulated the expression of both STM1344 and STM1697 to various degrees upon invading host cells. Importantly, deletion of STM1697 or STM1344 led to failure of Salmonella flagellar control within host cells, suggesting that the two factors are not redundant but indispensable. STM1697 was shown to modulate Salmonella flagellar biogenesis by preventing the flagellar master protein FlhDC from recruiting RNA polymerase. However, STM1344 was identified as a bifunctional factor that inhibits RNA polymerase recruitment of FlhDC at low molar concentrations and the DNA binding activity of FlhDC at high molar concentrations. Structural analysis demonstrated that STM1344-FlhD binds more tightly than STM1697-FlhD, and size exclusion chromatography (SEC) experiments showed that STM1344 could replace STM1697 in a STM1697-FlhDC complex. Our data suggest that STM1697 might be a temporary flagellar control factor upon Salmonella entry into the host cell, while STM1344 plays a more critical role in persistent flagellar control when Salmonella organisms survive and colonize host cells for a long period of time. Our study provides a more comprehensive understanding of the complex flagellar regulatory mechanism of Salmonella based on regulation at the protein level of FlhDC. IMPORTANCE Salmonella infection kills more than 300,000 people every year. After infection, Salmonella mainly parasitizes host cells, as it prevents host cell pyroptosis by turning off the synthesis of flagellar antigen. Previous studies have determined that there are two EAL-like proteins, STM1344 and STM1697, encoded in the Salmonella genome, both of which inhibit flagellar synthesis by interacting with the flagellar master protein FlhDC. However, the expression order and simultaneous mechanism of STM1344 and STM1697 are not clear. In this study, we determined the expression profiles of the two proteins after Salmonella infection and demonstrated the cooperative mechanism of STM1344 and STM1697 interaction with FlhDC. We found that STM1344 might play a more lasting regulatory role than STM1697. Our results reveal a comprehensive flagellar control process after Salmonella entry into host cells.
... Importantly, S. typhimurium lacking the flagellin genes fliC and fljB exhibit a weakened ability to cause inflammation in vivo, probably due to its lack of motility and/or impaired activation of flagellin-sensing NLRC4-caspases-1 inflammasome and TLR5 (147)(148)(149)(150). In contrast, S. typhimurium strains that overexpress flagellin because of lack of YdiV, a transcriptional suppressor of flagellin-encoding gene fliC, cause macrophage pyroptosis and induce elevated levels of IL-1b and TNF, ultimately fail in mouse tissue colonization (133). Caspase-1 is also involved in the cytoplasmic recognition of L. pneumophila flagellin by mouse macrophages, thereby limiting their infection (131). ...
In the early 2000s, caspase-1, an important molecule that has been shown to be involved in the regulation of inflammation, cell survival and diseases, was given a new function: regulating a new mode of cell death that was later defined as pyroptosis. Since then, the inflammasome, the inflammatory caspases (caspase-4/5/11) and their substrate gasdermins (gasdermin A, B, C, D, E and DFNB59) has also been reported to be involved in the pyroptotic pathway, and this pathway is closely related to the development of various diseases. In addition, important apoptotic effectors caspase-3/8 and granzymes have also been reported to b involved in the induction of pyroptosis. In our article, we summarize findings that help define the roles of inflammasomes, inflammatory caspases, gasdermins, and other mediators of pyroptosis, and how they determine cell fate and regulate disease progression.
... The expression of the major flagellar antigen FliC quickly decreases more than 10-fold upon wild-type Salmonella entry into host cells (8,9). Remarkably, Salmonella organisms continuously expressing FliC exhibit weaker pathogenicity and elicit stronger host immune responses (10)(11)(12), suggesting that precise regulation of the flagellar pathway is critical for successful infection by Salmonella. ...
Salmonella is an important facultative pathogen of foodborne illness and typhoid fever in humans. Flagella allow bacterial motility and are required for Salmonella to successfully invade the host cells.
... The assembly and rotation of flagella both require proton motive force (PMF) and are energetically costly (19,20). It has been shown that shutting off flagellar expression helps Salmonella cells evade the host immune response (21) and improve growth in culture (22). Interestingly, flagellar and SPI-1 expression is heterogeneous in Salmonella (23)(24)(25)(26)(27). ...
Antibiotic resistance and tolerance pose a severe threat to human health. How bacterial pathogens acquire antibiotic tolerance is not clear.
... Salmonella could rely on mobility of a part of the population, to migrate from one niche to another and to established inside the (new) host. The phenotypic heterogeneity in Salmonella agellar gene expression has been previously shown as a mechanism to maximize fitness within the mammalian host (Stewart et al., 2011). This strategy is illustrated in Fig. 2B, showing that Salmonella is able to internalize the host plant without inducing strong defenses via changing the expression of its agellin. ...
Fruits and vegetables consumed fresh or as minimally-processed produce, have multiple benefits for our diet. Unfortunately, they bring a risk of food-borne diseases, for example salmonellosis. Interactions between Salmonella and crop plants are indeed a raising concern for the global health. Salmonella uses multiple strategies to manipulate the host defense system, including plant's defense responses. The main focus of this review are strategies used by this bacterium during the interaction with crop plants. Emphasis was put on how Salmonella avoids the plant defense responses and successfully colonizes plants. In addition, several factors were reviewed assessing their impact on Salmonella persistence and physiological adaptation to plants and plant-related environment. The understanding of those mechanisms, their regulation and use by the pathogen, while in contact with plants, has significant implication on the growth, harvest and processing steps in plant production system. Consequently, it requires both the authorities and science to advance and definite methods aiming at prevention of crop plants contamination. Thus, minimizing and/or eliminating the potential of human diseases.
... At the stage of epithelial cell invasion in the intestine, expression of pathogenicity island 1 (SPI-1) and of the flagellar gene network is regulated by interacting transcription factors [4,5,18,21,22]. It is also well known that both the flagellar regulon and SPI-1 exhibit bistable expression [1][2][3]7,8,[15][16][17][18][23][24][25]. None of these previous studies analyzed simultaneous expression of both systems in single cells, which was the main goal of this study. ...
Bistable expression of the Salmonella enterica pathogenicity island 1 (SPI-1) and the flagellar network (Flag) has been described previously. In this study, simultaneous monitoring of OFF and ON states in SPI-1 and in the flagellar regulon reveals independent switching, with concomitant formation of four subpopulations: SPI-1OFF FlagOFF, SPI-1OFF FlagON, SPI-1ON FlagOFF, and SPI-1ON FlagON. Invasion assays upon cell sorting show that none of the four subpopulations is highly invasive, thus raising the possibility that FlagOFF cells might contribute to optimal invasion as previously proposed for SPI-1OFF cells. Time lapse microscopy observation indicates that expression of the flagellar regulon contributes to the growth impairment previously described in SPI-1ON cells. As a consequence, growth resumption in SPI-1ON FlagON cells requires switching to both SPI-1OFF and FlagOFF states.
... It was observed under each growth condition that some bacteria did not produce any flagella, while the number of flagella on bacteria that produced them roughly follows a normal distribution, where the mean depended on the growth condition. This phenotypic heterogeneity enables the evasion of the host immune system during acute infection [4]. It is not yet clear what mechanisms determine flagella heterogeneity and how the bacteria regulate the number of flagella produced. ...
Millions of people worldwide develop foodborne illnesses caused by Salmonella enterica (S. enterica) every year. The pathogenesis of S. enterica depends on flagella, which are appendages that the bacteria use to move through the environment. Interestingly, populations of genetically identical bacteria exhibit heterogeneity in the number of flagella. To understand this heterogeneity and the regulation of flagella quantity, we propose a mathematical model that connects the flagellar gene regulatory network to flagellar construction. A regulatory network involving more than 60 genes controls flagellar assembly. The most important member of the network is the master operon, flhDC, which encodes the FlhD4C2 protein. FlhD4C2 controls the construction of flagella by initiating the production of hook basal bodies (HBBs), protein structures that anchor the flagella to the bacterium. By connecting a model of FlhD4C2 regulation to a model of HBB construction, we investigate the roles of various feedback mechanisms. Analysis of our model suggests that a combination of regulatory mechanisms at the protein and transcriptional levels induce bistable FlhD4C2 levels and heterogeneous numbers of flagella. Also, the balance of regulatory mechanisms that become active following HBB construction is sufficient to provide a counting mechanism for controlling the total number of flagella produced.
... In addition to commensals, pathogenic microorganisms likewise show a high degree of within-population heterogeneity in composition and function (Balaban et al., 2004;Stewart et al., 2011;Claudi et al., 2014). This raises the question of how cell-to-cell variability predicts or alters the host relationship with microbial organisms in a pathological context. ...
Microbes are the most prevalent form of life yet also the least well-understood in terms of their diversity. Due to a greater appreciation of their role in modulating host physiology, microbes have come to the forefront of biological investigation of human health and disease. Despite this, capturing the heterogeneity of microbes, and that of the host responses they induce, has been challenging due to the bulk methods of nucleic acid and cellular analysis. One of the greatest recent advancements in our understanding of complex organisms has happened in the field of single-cell analysis through genomics, transcriptomics, and spatial resolution. While significantly advancing our understanding of host biology, these techniques have only recently been applied to microbial systems to shed light on their diversity as well as interactions with host cells in both commensal and pathogenic contexts. In this review, we highlight emerging technologies that are poised to provide key insights into understanding how microbe heterogeneity can be studied. We then take a detailed look into how host single-cell analysis has uncovered the impact of microbes on host heterogeneity and the effect of host biology on microorganisms. Most of these insights would have been challenging, and in some cases impossible, without the advent of single-cell analysis, suggesting the importance of the single-cell paradigm for progressing the microbiology field forward through a host-microbiome perspective and applying these insights to better understand and treat human disease.
... Such heterogeneous expression has been already observed, and even associated to higher virulence in animal hosts. The phenotypic heterogeneity in Salmonella flagellar gene expression has been shown to provide a mechanism by which the pathogen maximizes fitness within the mammalian host [60]. Whether such behavior is part of Salmonella's adaptation to colonize plant tissues or plays a role beyond the plant host as part of an adaptation to the eventual progress to an herbivorous host, is a very intriguing question. ...
Minimally processed or fresh fruits and vegetables are unfortunately linked to an increasing number of food-borne diseases, such as salmonellosis. One of the relevant virulence factors during the initial phases of the infection process is the bacterial flagellum. Although its function is well studied in animal systems, contradictory results have been published regarding its role during plant colonization. In this study, we tested the hypothesis that Salmonella's flagellin plays a versatile function during the colonization of tomato plants. We have assessed the persistence in plant tissues of a Salmonella enterica wild type strain, and of a strain lacking the two flagellins, FljB and FliC. We detected no differences between these strains concerning their respective abilities to reach distal, non-inoculated parts of the plant. Analysis of flagellin expression inside the plant, at both the population and single cell levels, shows that the majority of bacteria down-regulate flagellin production, however, a small fraction of the population continues to express flagellin at a very high level inside the plant. This heterogeneous expression of flagellin might be an adaptive strategy to the plant environment. In summary, our study provides new insights on Salmonella adaption to the plant environment through the regulation of flagellin expression.
... Degradation rate of RflP 0.1 γ 4 Degradation rate of RflP:FlhD 4 C 2 complex 0.01 γ 5 Degradation rate of RflM 0.1 δ 1 ClpXP degradation rate of FlhD 4 C 2 0.1 ...
Millions of people worldwide develop foodborne illnesses caused by \textit{Salmonella enterica} (\textit{S. enterica}) every year. The pathogenesis of \textit{S. enterica} depends on flagella, which are appendages that the bacteria use to move through the environment. Interestingly, populations of genetically identical bacteria exhibit heterogeneity in the number of flagella. To understand this heterogeneity and the regulation of flagella quantity, we propose a mathematical model that connects the flagellar gene regulatory network to flagellar construction.
A regulatory network involving more than 60 genes controls flagellar assembly. The most important member of the network is the master operon, \textit{flhDC}, which encodes the FlhD\textsubscript{4}C\textsubscript{2} protein. FlhD\textsubscript{4}C\textsubscript{2} controls the construction of flagella by initiating the production of hook basal bodies (HBBs), protein structures that anchor the flagella to the bacterium. By connecting a model of FlhD\textsubscript{4}C\textsubscript{2} regulation to a model of HBB construction, we investigate the roles of various feedback mechanisms. Analysis of our model suggests that a combination of regulatory mechanisms at the protein and transcriptional levels induce bistable FlhD\textsubscript{4}C\textsubscript{2} levels and heterogeneous numbers of flagella. Also, the balance of regulatory mechanisms that become active following HBB construction is sufficient to provide a counting mechanism for controlling the total number of flagella produced.
... Узнавание сальмонелл иммунной системой и индукция иммунного ответа прямо коррелируют с наличием в бактериальной клетке определенных патогенассоциированных молекулярных структур. Сальмонеллы могут выжить во враждебном окружении лишь при условии модификации указанных структур либо в случае снижения экспрессии определенных иммуногенов, например флагеллинов [46,47]. Таким образом, стратегия конструирования рекомбинантного штамма должна заключаться в модификации иммуногенных мишеней при сохранении иммуногенности сальмонелл. ...
Bacterial drugs for the treatment of malignant tumors have been discovered more than a hundred years ago, but their use in clinical practice has been very limited. In the past decade, there has been a revival of interest in the development of bacterial-based cancer biotherapies, which is associated with advances in genetic engineering and in depth knowledge of the mechanisms of the infectious process and immunity. The purpose of this review is to examine the current state and prospects for the development and use of drugs based on live bacterium, intended for the treatment of malignant tumors. The review presents evaluation data on experimental models of the antitumor potential of various species and strains of bacteria; the most significant results of clinical trials of bacterial antitumor agents; current trends in the design of bacterial strains for targeted drug delivery to the tumor. It is concluded that development of bacterial drugs for cancer therapy is a perspective branch of experimental oncology.
... Downregulation of flagellin expression often occurs after pathogens reach suitable host tissues (34)(35)(36)(37)(38), and there is some evidence that this is an adaptive immune avoidance strategy in animal infection models. For example, downregulation of Salmonella flagella prevents host proinflammatory cell death response and enhances colonization of systemic sites in mice (39). Additionally, a greater proportion of gut microbiomes are flagellated in TLR5 Ϫ/Ϫ mice (40), consistent with the idea that immune activation selects for or induces deflagellation in a wide range of host-adapted bacteria. ...
Foliar plant pathogens, like Pseudomonas syringae , adjust their physiology and behavior to facilitate host colonization and disease, but the full extent of these adaptations is not known. Plant immune systems are triggered by bacterial molecules, such as the proteins that make up flagellar filaments. In this study, we found that during plant infection, AlgU, a gene expression regulator that is responsive to external stimuli, downregulates expression of fliC , which encodes the flagellin protein, a strong elicitor of plant immune systems. This change in gene expression and resultant change in behavior correlate with reduced plant immune activation and improved P. syringae plant colonization. The results of this study demonstrate the proximate and ultimate causes of flagellar regulation in a plant-pathogen interaction.
... Phenotypic heterogeneity is well-documented for laboratory cultures. For example, it has been studied in bacterial subpopulations that could tolerate antibiotics (known as 'persisters') (Dhar and McKinney, 2007), in the production of cytotoxin K in Bacillus cereus (Ceuppens et al., 2013) or in the differential expression of flagellin in Salmonella Typhimurium (Stewart et al., 2011). The challenge remains to find tools to measure and quantify this heterogeneity, in order to be able to link heterogeneity with bacterial functionality. ...
... Both the flagellar and SPI-1 genes exhibit bistable expression patterns (cf. [6,[22][23][24][25][26][27][28][29][30][31][32][33]): within a population of cells, some cells will express these genes and others will not. In other words, only a subpopulation of cells will be motile or invasive. ...
Background:
Salmonella enterica serovar Typhimurium is a common food-borne pathogen. S. enterica uses a type III secretion system encoded within Salmonella pathogenicity island 1 (SPI-1) to invade intestinal epithelial cells. A complex network of interacting transcription factors regulates SPI-1 gene expression. In addition, SPI-1 gene expression is coupled to flagellar gene expression. Both SPI-1 and flagellar gene expression are bistable, with co-existing populations of cells expressing and not expressing these genes. Previous work demonstrated that nutrients could be used to tune the fraction of cells expressing the flagellar genes. In the present study, we tested whether nutrients could also tune the fraction of cells expressing the SPI-1 genes through transcriptional crosstalk with the flagellar genes.
Results:
Nutrients alone were not found to induce SPI-1 gene expression. However, when the cells were also grown in the presence of acetate, the concentration of nutrients in the growth medium was able to tune the fraction of cells expressing the SPI-1 genes. During growth in nutrient-poor medium, acetate alone was unable to induce SPI-1 gene expression. These results demonstrate that acetate and nutrients synergistically activate SPI-1 gene expression. The response to acetate was governed by the BarA/SirA two-component system and the response to nutrients was governed by transcriptional crosstalk with the flagella system, specifically through the action of the flagellar regulator FliZ.
Conclusions:
Acetate and nutrients are capable of synergistically activating SPI-1 gene expression. In addition, these signals were found to tune the fraction of cells expressing the SPI-1 genes. The governing mechanism involves transcriptional crosstalk with the flagellar gene network. Collectively, these results further our understanding of SPI-1 gene regulation and provide the basis for future studies investigating this complex regulatory mechanism.
... This can be the expression of certain structures, physiological processes, properties, or the behavior by a subpopulation of cells that are highly suited to the new, recurrent host habitat. Up to date, several examples of phenotypic heterogeneity in populations of microbial pathogens have been discovered and were further characterized [8][9][10][11][12][13][14][15][16][17]. Based on these examples, we present the different regulatory strategies and underlying mechanisms promoting cooperative behaviors and discuss their impact on the biological fitness and pathogenicity of the pathogen. ...
Recent studies revealed an amazing phenotypic heterogeneity between genetically identical individual cells within populations of microbial pathogens. During the course of an infection, subpopulations occur, which differ in certain virulence-relevant factors, stress adaptation functions or physiological and metabolic abilities. The mechanisms driving this heterogeneity are divergent reactions of the pathogens to differences in host tissue microenvironments. In addition, certain genetic regulatory circuits with positive feedback loops and stochastic differences in gene expression can generate endogenous fluctuations in regulatory components leading to bistable expression of virulence-associated functions. Here, we focus on the occurrence of phenotypic heterogeneity in populations of well-studied examples of pathogens, which enables cooperative, social behavior where a subpopulation of producers shares fitness- and/or virulence-relevant goods and traits with non-producers. We further highlight that this strategy allows preadaptation of a subgroup of cells to recurrent and thus predictable changes of the environment that they encounter during the different stages of the infection. The diversity within bacterial communities has a significant influence on the survival of the pathogens within their hosts and the progression of the disease.
... First, phenotypic heterogeneity can allow a genotype to survive in fluctuating environments (Rainey et al., 2011;Schreiber et al., 2016). For example, in Salmonella, heterogeneous expression of virulence factors allows a subset of the population to escape host inflammatory responses (Stewart et al., 2011;Tuchscherr et al., 2011). Another illustration of such bacterial bet-hedging strategy is the formation of 'persister' cells such as those resistant to antibiotics (Balaban et al., 2004). ...
An evolution experiment with the bacterial plant pathogen Ralstonia solanacearum revealed that several adaptive mutations conferring enhanced fitness in plants arose in the efpR gene encoding a regulator of virulence and metabolic functions. In this study, we found that an efpR mutant systematically displays colonies with two morphotypes: the type S (‘smooth’, similar to the wild‐type) and the type EV (‘efpR variant’). We demonstrated that the efpH gene, a homolog of efpR, plays a key role in the control of phenotypic heterogeneity, the ΔefpR‐ΔefpH double mutant being stably locked into the EV type. Using mixed infection assays, we demonstrated that the type EV is metabolically more proficient than the type S and displays fitness gain in specific environments whereas the type S has a better fitness into the plant environment. We provide evidence that this efpR‐dependent phenotypic heterogeneity is a general feature of strains of the R. solanacearum species complex and could occur in natural conditions. This study highlights the potential role of phenotypic heterogeneity in this plant pathogen as an adaptive trait to changing environments. This article is protected by copyright. All rights reserved.
NLR family, apoptosis inhibitory proteins (NAIPs) detect bacterial flagellin and structurally related components of bacterial type III secretion systems (T3SS), and recruit NLR family CARD domain containing protein 4 (NLRC4) and caspase-1 into an inflammasome complex that induces pyroptosis. NAIP/NLRC4 inflammasome assembly is initiated by the binding of a single NAIP to its cognate ligand, but a subset of bacterial flagellins or T3SS structural proteins are thought to evade NAIP/NLRC4 inflammasome sensing by not binding to their cognate NAIPs. Unlike other inflammasome components such as NLRP3, AIM2, or some NAIPs, NLRC4 is constitutively present in resting macrophages and not known to be induced by inflammatory signals. Here, we demonstrate that Toll-like receptor (TLR)-dependent p38 mitogen-activated protein kinase signaling up-regulates NLRC4 transcription and protein expression in murine macrophages, which licenses NAIP detection of evasive ligands. In contrast, TLR priming in human macrophages did not up-regulate NLRC4 expression, and human macrophages remained unable to detect NAIP-evasive ligands even following priming. Critically, ectopic expression of either murine or human NLRC4 was sufficient to induce pyroptosis in response to immunoevasive NAIP ligands, indicating that increased levels of NLRC4 enable the NAIP/NLRC4 inflammasome to detect these normally evasive ligands. Altogether, our data reveal that TLR priming tunes the threshold for the murine NAIP/NLRC4 inflammasome to enable inflammasome responses against immunoevasive or suboptimal NAIP ligands. These findings provide insight into species-specific TLR regulation of NAIP/NLRC4 inflammasome activation.
Transcriptional regulatory networks (TRNs) give a global view of the regulatory mechanisms of bacteria to respond to environmental signals. These networks are published in biological databases as a valuable resource for experimental and bioinformatics researchers. Despite the efforts to publish TRNs of diverse bacteria, many of them still lack one and many of the existing TRNs are incomplete. In addition, the manual extraction of information from biomedical literature (“literature curation”) has been the traditional way to extract these networks, despite this being demanding and time-consuming. Recently, language models based on pretrained transformers have been used to extract relevant knowledge from biomedical literature. Moreover, the benefit of fine-tuning a large pretrained model with new limited data for a specific task (“transfer learning”) opens roads to address new problems of biomedical information extraction. Here, to alleviate this lack of knowledge and assist literature curation, we present a new approach based on the Bidirectional Transformer for Language Understanding (BERT) architecture to classify transcriptional regulatory interactions of bacteria as a first step to extract TRNs from literature. The approach achieved a significant performance in a test dataset of sentences of Escherichia coli (F1-Score: 0.8685, Matthew’s correlation coefficient: 0.8163). The examination of model predictions revealed that the model learned different ways to express the regulatory interaction. The approach was evaluated to extract a TRN of Salmonella using 264 complete articles. The evaluation showed that the approach was able to accurately extract 82% of the network and that it was able to extract interactions absent in curation data. To the best of our knowledge, the present study is the first effort to obtain a BERT-based approach to extract this specific kind of interaction. This approach is a starting point to address the limitations of reconstructing TRNs of bacteria and diseases of biological interest.
Database URL: https://github.com/laigen-unam/BERT-trn-extraction.
Pathogen diversity within an infected organism has traditionally been explored through the lens of genetic heterogeneity. Hallmark studies have characterized how genetic diversity within pathogen subpopulations contributes to treatment escape and infectious disease progression. However, recent studies have begun to reveal the mechanisms by which phenotypic heterogeneity is established within genetically identical populations of invading pathogens. Furthermore, exciting new work highlights how these phenotypically heterogeneous subpopulations contribute to a pathogen population better equipped to handle the complex and fluctuating environment of a host organism. In this review, we focus on how bacterial pathogens, including Staphylococcus aureus , Salmonella typhimurium , Pseudomonas aeruginosa , and Mycobacterium tuberculosis , establish and maintain phenotypic heterogeneity, and we explore recent work demonstrating causative links between this heterogeneity and infection outcome.
The intestinal pathogen Salmonella enterica rapidly enters the bloodstream after the invasion of intestinal epithelial cells, but how Salmonella breaks through the gut-vascular barrier is largely unknown. Here, we report that Salmonella enters the bloodstream through intestinal CX3CR1 ⁺ macrophages during early infection. Mechanistically, Salmonella induces the migration/invasion properties of macrophages in a manner dependent on host cell actin and on the pathogen effector SteC. SteC recruits host myosin light chain protein Myl12a and phosphorylates its Ser19 and Thr20 residues. Myl12a phosphorylation results in actin rearrangement, and enhanced migration and invasion of macrophages. SteC is able to utilize a wide range of NTPs other than ATP to phosphorylate Myl12a. We further solved the crystal structure of SteC, which suggests an atypical dimerization-mediated catalytic mechanism. Finally, in vivo data show that SteC-mediated cytoskeleton manipulation is crucial for Salmonella breaching the gut vascular barrier and spreading to target organs.
Understanding the phenotypic heterogeneity of antibiotic‐resistant bacteria following treatment and the transitions between different phenotypes is crucial for developing effective infection control strategies. The study expands upon previous work by explicating chloramphenicol‐induced phenotypic heterogeneities in growth rate, gene expression, and morphology of resistant Escherichia coli using time‐lapse microscopy. Correlating the bacterial growth rate and cspC expression, four interchangeable phenotypic subpopulations across varying antibiotic concentrations are identified, surpassing the previously described growth rate bistability. Notably, bacterial cells exhibiting either fast or slow growth rates can concurrently harbor subpopulations characterized by high and low gene expression levels, respectively. To elucidate the mechanisms behind this enhanced heterogeneity, a concise gene expression network model is proposed and the biological significance of the four phenotypes is further explored. Additionally, by employing Hidden Markov Model fitting and integrating the non‐equilibrium landscape and flux theory, the real‐time data encompassing diverse bacterial traits are analyzed. This approach reveals dynamic changes and switching kinetics in different cell fates, facilitating the quantification of observable behaviors and the non‐equilibrium dynamics and thermodynamics at play. The results highlight the multi‐dimensional heterogeneous behaviors of antibiotic‐resistant bacteria under antibiotic stress, providing new insights into the compromised antibiotic efficacy, microbial response, and associated evolution processes.
The immune system of multicellular organisms protects them from harmful microbes. To establish an infection in the face of host immune responses, pathogens must evolve specific strategies to target immune defense mechanisms. One such defense is the formation of intracellular protein complexes, termed inflammasomes, that are triggered by the detection of microbial components and the disruption of homeostatic processes that occur during bacterial infection. Formation of active inflammasomes initiates programmed cell death pathways via activation of inflammatory caspases and cleavage of target proteins. Inflammasome-activated cell death pathways such as pyroptosis lead to proinflammatory responses that protect the host. Bacterial infection has the capacity to influence inflammasomes in two distinct ways: activation and perturbation. In this review, we discuss how bacterial activities influence inflammasomes, and we discuss the consequences of inflammasome activation or evasion for both the host and pathogen.
NLR family, apoptosis inhibitory proteins (NAIPs) detect bacterial flagellin and structurally related components of bacterial type III secretion systems (T3SS), and recruit NLR family, CARD domain containing protein 4 (NLRC4) and caspase-1 into an inflammasome complex that induces pyroptosis. NAIP/NLRC4 inflammasome assembly is initiated by the binding of a single NAIP to its cognate ligand, but a subset of bacterial flagellins or T3SS structural proteins are thought to evade NAIP/NLRC4 inflammasome sensing by not binding to their cognate NAIPs. Unlike other inflammasome components such as NLRP3, AIM2, or some NAIPs, NLRC4 is constitutively present in resting macrophages, and not thought to be regulated by inflammatory signals. Here, we demonstrate that Toll-like receptor (TLR) stimulation upregulates NLRC4 transcription and protein expression in murine macrophages, which licenses NAIP detection of evasive ligands. TLR-induced NLRC4 upregulation and NAIP detection of evasive ligands required p38 MAPK signaling. In contrast, TLR priming in human macrophages did not upregulate NLRC4 expression, and human macrophages remained unable to detect NAIP-evasive ligands even following priming. Critically, ectopic expression of either murine or human NLRC4 was sufficient to induce pyroptosis in response to immunoevasive NAIP ligands, indicating that increased levels of NLRC4 enable the NAIP/NLRC4 inflammasome to detect these normally evasive ligands. Altogether, our data reveal that TLR priming tunes the threshold for NAIP/NLRC4 inflammasome activation and enables inflammasome responses against immunoevasive or suboptimal NAIP ligands.
Significance Statement
Cytosolic receptors in the neuronal apoptosis inhibitor protein (NAIP) family detect bacterial flagellin and components of the type III secretion system (T3SS). NAIP binding to its cognate ligand engages the adaptor molecule NLRC4 to form NAIP/NLRC4 inflammasomes culminating in inflammatory cell death. However, some bacterial pathogens evade NAIP/NLRC4 inflammasome detection, thus bypassing a crucial barrier of the immune system. Here, we find that, in murine macrophages, TLR-dependent p38 MAPK signaling increases NLRC4 expression, thereby lowering the threshold for NAIP/NLRC4 inflammasome activation in response to immunoevasive NAIP ligands. Human macrophages were unable to undergo priming-induced upregulation of NLRC4 and could not detect immunoevasive NAIP ligands. These findings provide a new insight into species-specific regulation of the NAIP/NLRC4 inflammasome.
Microbial cell individuality is receiving increasing interest in the scientific community. Individual cells within clonal populations exhibit noticeable phenotypic heterogeneity. The advent of fluorescent protein technology and advances in single-cell analysis has revealed phenotypic cell variant in bacterial populations. This heterogeneity is evident in a wide range of phenotypes, for example, individual cells display variable degrees of gene expression and survival under selective conditions and stresses, and can exhibit differing propensities to host interactions. Last few years, numerous cell sorting approaches have been employed for resolving the properties of bacterial subpopulations. This review provides an overview of applications of cell sorting to analyze Salmonella lineage-specific traits, including bacterial evolution studies, gene expression analysis, response to diverse cellular stresses and characterization of diverse bacterial phenotypic variants.
Type III secretion system 1 (T3SS1) encoded by Salmonella pathogenicity island 1 (SPI1) is associated with invasion of host cells by Salmonella, PrgH encoded by prgH gene is an important component of T3SS1. This study aimed to explore the contribution of prgH gene for Salmonella Pullorum to virulence and the expression of NLRP3, Caspase-1 and IL-1β in chickens. A prgH gene deletion mutant (C79-13ΔprgH) was firstly generated, and the result of LD50 showed that deletion of prgH significantly decreased the virulence of Salmonella Pullorum in one-day-old HY-line white chickens, and the colonization also decreased in chickens after loss of prgH. Next, the expressions of NLRP3, Caspase-1, and IL-1β were detected in acute infection model of chickens by qRT-PCR and/or ELISA, respectively, and the results showed that the mutant strain C79-13ΔprgH reduced the expression levels of NLRP3, Caspase-1, and IL-1β in chickens compared to the group infected with the wild type strain C79-13. Taken together, all of these findings indicated that prgH promotes the virulence and the expression of NLRP3, Caspase-1, and IL-1β for Salmonella Pullorum in chickens.
Advances in technologies that permit high-resolution analysis of events in single cells have revealed that phenotypic heterogeneity is a widespread phenomenon in bacteria. Flow cytometry has the potential to describe the distribution of cellular properties within a population of bacterial cells and has yielded invaluable information about the ability of isogenic cells to diversify into phenotypic subpopulations. This review will discuss several single-cell approaches that have recently been applied to define phenotypic heterogeneity in populations of Salmonella enterica.
In fluctuating nutrient environments, isogenic microbial cells transition into “multicellular” communities composed of phenotypically heterogeneous cells, showing functional specialization. In fungi (such as budding yeast), phenotypic heterogeneity is often described in the context of cells switching between different morphotypes (e.g., yeast to hyphae/pseudohyphae or white/opaque transitions in Candida albicans). However, more fundamental forms of metabolic heterogeneity are seen in clonal Saccharomyces cerevisiae communities growing in nutrient-limited conditions. Cells within such communities exhibit contrasting, specialized metabolic states, and are arranged in distinct, spatially organized groups. In this study, we explain how such an organization can stem from self-organizing biochemical reactions that depend on special metabolites. These metabolites exhibit plasticity in function, wherein the same metabolites are metabolized and utilized for distinct purposes by different cells. This in turn allows cell groups to function as specialized, interdependent cross-feeding systems which support distinct metabolic processes. Exemplifying a system where cells exhibit either gluconeogenic or glycolytic states, we highlight how available metabolites can drive favored biochemical pathways to produce new, limiting resources. These new resources can themselves be consumed or utilized distinctly by cells in different metabolic states. This thereby enables cell groups to sustain contrasting, even apparently impossible metabolic states with stable transcriptional and metabolic signatures for a given environment, and divide labor in order to increase community fitness or survival. We speculate on possible evolutionary implications of such metabolic specialization and division of labor in isogenic microbial communities.
Bacteria communicate with each other through a plethora of small, diffusible organic molecules called autoinducers. This cell-density-dependent regulatory principle is termed quorum sensing, and in many cases the process indeed coordinates group behavior of bacterial populations. Yet, even clonal bacterial populations are not uniform entities; rather, they adopt phenotypic heterogeneity to cope with consecutive, rapid, and frequent environmental fluctuations (bet-hedging) or to concurrently interact with each other by exerting different, often complementary, functions (division of labor). Quorum sensing is mainly regarded as a coordinator of bacterial collective behavior. However, it can also be a driver or a target of individual phenotypic heterogeneity. Hence, quorum sensing increases the overall fitness of a bacterial community by orchestrating group behavior as well as individual traits.
Mammary gland-derived Escherichia coli (E. coli) is an important pathogen causing dairy cow mastitis. YdiV, with EAL-like domains, inhibits flagellum biogenesis and motility and affects c-di-GMP (eubacterial signaling molecule) concentration changes in bacteria. However, the pathophysiological role of ydiV in host-pathogen cross-talk still needs to be elucidated. In this study, firstly constructed the ydiV mutant (NJ17ΔydiV) and ydiV complementary (cNJ17ΔydiV) E. coli strains to infect mouse mammary epithelial cells (EpH4-Ev) and macrophages (RAW264.7), as well as mouse mammary glands, respectively. Then biological characteristics, adaptor molecules in related signaling pathways, proinflammatory cytokines and the extent of host cell damage was evaluated. Compared with E. coli NJ17 infected mice, the bacterial load in the mammary gland of NJ17ΔydiV was significantly lower and the extent of the damage was alleviated. Notably, the deletion of ydiV significantly aggravated cell damage in RAW264.7 cells and compared with the wild-type strain, NJ17ΔydiV significantly activated the STING/TBK1/IRF3 pathway in macrophages. In EpH4-Ev cells, although STING did not sense E. coli NJ17 invasion, IRF3 was activated by the NJ17ΔydiV strain. Taken together, ydiV deletion significantly affects a variety of biological characteristics and induces severe cell damage, while the STING/TBK1/IRF3 pathway actively participated in pathogen elimination in the host. This study highlights a new role for ydiV in E. coli infection and provides a foundation for further studies to better understand host-bacteria interactions and potential prophylactic strategies for infectious diseases.
In the framework of the modern microbiological paradigm, colonies of genetically identical microorganisms are considered as biosocial systems consisting of several heterogeneous clonal cell clusters (bacterial phenotypes) that respond differently to changes in the environment. Phenotypic heterogeneity was found in recent decades in all isogenic populations of pathogenic bacteria. Such heterogeneity provides a selective advantage of cellular phenotypes with changes in the physicochemical parameters of the environment and competitive interaction with other microorganisms. Heterogeneity in bacterial communities is of great importance for the survival of pathogenic bacteria in the host organism, the progression and persistence of infections, as well as the decrease in the effectiveness of antibiotic therapy. The modern spectrum of analytical tools for studying cellular phenotyping is presented both by optical imaging methods and qualitative structural characteristics of single cells, and by omix technologies of quantitative analysis and monitoring of molecular intracellular processes. These diverse tools make it possible not only to identify and modulate phenotypic heterogeneity in isogenic bacterial populations, but also to evaluate the functional significance of cellular phenotypes in the development of the infectious process. The aim of the review is the integration of modern concepts of heterogeneity in isogenic bacterial populations, with an emphasis on the presentation of modern analytical technologies for assessing and monitoring phenotypic typing of single cells.
From the homeostasis of human health to the cycling of Earth's elements, microbial activities underlie environmental, medical and industrial processes. These activities occur in chemical and physical landscapes that are highly dynamic and experienced by bacteria as fluctuations. In this review, we first discuss how bacteria can experience both spatial and temporal heterogeneity in their environments as temporal fluctuations of various timescale (seconds to seasons) and type (nutrient, sunlight, fluid flow, etc.). We then focus primarily on nutrient fluctuations to discuss how bacterial communities, populations and single cells respond to environmental fluctuations. Overall, we find that environmental fluctuations are ubiquitous, diverse, and strongly shape microbial behavior, ecology and evolution when compared to environments in which conditions remain constant over time. We hope this review may serve as a guide towards understanding the significance of environmental fluctuations in microbial life, such that their contributions and implications can be better assessed and exploited.
Significance
While the effect of bacterial molecules on the host immune system is well studied, how host factors affect the expression of bacterial molecules is less appreciated. Here we uncover the impact of inflammasome activation and type 1 interferon on the expression of bacterial flagellin. Flagellin induces NLRC4 inflammasome-mediated pyroptosis causing clearance of Salmonella . We show that inflammasome activation also produces lysophospholipids which increase flagellin expression by Salmonella early in infection. We further demonstrate that as infection progresses, type 1 IFN inhibits NLRC4 and lysophospholipid synthesis, resulting in down-regulation of flagellin expression, a phenotype that the pathogen switches to during establishment of in vivo infection. These findings unravel pathways for biphasic regulation of expression of flagellin, a key Salmonella effector.
While programmed cell death was once thought to be exclusive to eukaryotic cells, there are now abundant examples of well regulated cell death mechanisms in bacteria. The mechanisms by which bacteria undergo programmed cell death are diverse, and range from the use of toxin-antitoxin systems, to prophage-driven cell lysis. Moreover, some bacteria have learned how to coopt programmed cell death systems in competing bacteria. Interestingly, many of the potential reasons as to why bacteria undergo programmed cell death may parallel those observed in eukaryotic cells, and may be altruistic in nature. These include protection against infection, recycling of nutrients, to ensure correct morphological development, and in response to stressors. In the following chapter, we discuss the molecular and signaling mechanisms by which bacteria undergo programmed cell death. We conclude by discussing the current open questions in this expanding field.
Investigating phenotypic heterogeneity can help to better understand and manage microbial communities. However, characterizing phenotypic heterogeneity remains a challenge, as there is no standardized analysis framework. Several optical tools are available, such as flow cytometry and Raman spectroscopy, which describe optical properties of the individual cell. In this work, we compare Raman spectroscopy and flow cytometry to study phenotypic heterogeneity in bacterial populations. The growth stages of three replicate Escherichia coli populations were characterized using both technologies. Our findings show that flow cytometry detects and quantifies shifts in phenotypic heterogeneity at the population level due to its high‐throughput nature. Raman spectroscopy, on the other hand, offers a much higher resolution at the single‐cell level (i.e., more biochemical information is recorded). Therefore, it can identify distinct phenotypic populations when coupled with analyses tailored toward single‐cell data. In addition, it provides information about biomolecules that are present, which can be linked to cell functionality. We propose a computational workflow to distinguish between bacterial phenotypic populations using Raman spectroscopy and validated this approach with an external data set. We recommend using flow cytometry to quantify phenotypic heterogeneity at the population level, and Raman spectroscopy to perform a more in‐depth analysis of heterogeneity at the single‐cell level. © 2019 International Society for Advancement of Cytometry
Salmonellae are important enteric pathogens that cause gastroenteritis and systemic illnesses. Macrophages are important components of both the innate and acquired immune system, acting as phagocytes with significant antimicrobial killing activities that present antigen to the adaptive immune system. Macrophages can also be cultured from a variety of sites as primary cells, and the study of the survival and interactions of Salmonellae with these cells is a very early model of infection and cellular microbiology. This review traces the history of discoveries made using Salmonellae infection of macrophages and addresses the possibility of future research in this area, in particular with regards to understanding the complexity of individual bacteria and macrophage cell variability and how such heterogeneity may alter the outcome of infection.
Salmonella enterica comprises many pathogenic serovars that are able to colonize a variety of animal hosts and therefore constitute an important source of zoonotic food-borne illness. Their pathogenicity can range from gastroenteritis to typhoid fever, and depends on a series of virulence factors that are regularly located on laterally acquired genetic elements. The regulation of these virulence factors often also includes their differential expression within clonal populations. Moreover, exploitation of the resulting population heterogeneity appears to be an integral aspect of Salmonella virulence that could also affect its survival outside the host. This review therefore addresses how the regulation and heterogeneous expression of various virulence factors supports Salmonella's success as a food-borne pathogen.
Flagellar operons are divided into three classes with respect to their transcriptional hierarchy in Salmonella enterica serovar Typhimurium. The class 1 gene products FlhD and FlhC act together in an FlhD4C2 heterohexamer, which binds upstream of the class 2 promoters to facilitate binding of RNA polymerase. In this study, we showed
that flagellar expression was much reduced in the cells grown in poor medium compared to those grown in rich medium. This
nutritional control was shown to be executed at a step after class 1 transcription. We isolated five Tn5 insertion mutants in which the class 2 expression was derepressed in poor medium. These insertions were located in the ydiV (cdgR) gene or a gene just upstream of ydiV. The ydiV gene is known to encode an EAL domain protein and to act as a negative regulator of flagellar expression. Gene disruption
and complementation analyses revealed that the ydiV gene is responsible for nutritional control. Expression analysis of the ydiV gene showed that its translation, but not transcription, was enhanced by growth in poor medium. The ydiV mutation did not have a significant effect on either the steady-state level of flhDC mRNA or that of FlhC protein. Purified YdiV protein was shown in vitro to bind to FlhD4C2 through interaction with FlhD subunit and to inhibit its binding to the class 2 promoter, resulting in inhibition of FlhD4C2-dependent transcription. Taking these data together, we conclude that YdiV is a novel anti-FlhD4C2 factor responsible for nutritional control of the flagellar regulon.
Macrophages mediate crucial innate immune responses via caspase-1-dependent processing and secretion of interleukin 1β (IL-1β) and IL-18. Although infection with wild-type Salmonella typhimurium is lethal to mice, we show here that a strain that persistently expresses flagellin was cleared by the cytosolic flagellin-detection pathway through the activation of caspase-1 by the NLRC4 inflammasome; however, this clearance was independent of IL-1β and IL-18. Instead, caspase-1-induced pyroptotic cell death released bacteria from macrophages and exposed the bacteria to uptake and killing by reactive oxygen species in neutrophils. Similarly, activation of caspase-1 cleared unmanipulated Legionella pneumophila and Burkholderia thailandensis by cytokine-independent mechanisms. This demonstrates that activation of caspase-1 clears intracellular bacteria in vivo independently of IL-1β and IL-18 and establishes pyroptosis as an efficient mechanism of bacterial clearance by the innate immune system.
FliZ is an activator of class 2 flagellar gene expression in Salmonella enterica. To understand its role in flagellar assembly, we investigated how FliZ affects gene expression dynamics. We demonstrate
that FliZ participates in a positive-feedback loop that induces a kinetic switch in class 2 gene expression.
The bacterial second messenger cyclic di-GMP (c-di-GMP) regulates the transition between sessility and motility. In Salmonella enterica serovar Typhimurium, the expression of CsgD, the regulator of multicellular rdar morphotype behavior, is a major target of
c-di-GMP signaling. CsgD expression is positively regulated by at least two diguanylate cyclases, GGDEF domain proteins, and
negatively regulated by at least four phosphodiesterases, EAL domain proteins. Here, we show that in contrast to EAL domain
proteins acting as phosphodiesterases, the EAL-like protein STM1344 regulated CsgD expression positively and motility negatively.
STM1344, however, did not have a role in c-di-GMP turnover and also did not bind the nucleotide. STM1344 acted upstream of
the phosphodiesterases STM1703 and STM3611, previously identified to participate in CsgD downregulation, where it repressed
their expression. Consequently, although STM1344 has not retained a direct role in c-di-GMP metabolism, it still participates
in the regulation of c-di-GMP turnover and has a role in the transition between sessility and motility.
To survive in rapidly changing environmental conditions, bacteria have evolved a diverse set of regulatory pathways that govern various adaptive responses. Recent research has reinforced the notion that bacteria use feedback-based circuitry to generate population heterogeneity in natural situations. Using artificial gene networks, it has been shown that a relatively simple 'wiring' of a bacterial genetic system can generate two or more stable subpopulations within an overall genetically homogeneous population. This review discusses the ubiquity of these processes throughout nature, as well as the presumed molecular mechanisms responsible for the heterogeneity observed in a selection of bacterial species.
Caspase-1 is activated by a variety of stimuli after the assembly of the "inflammasome," an activating platform made up of a complex of the NOD-LRR family of proteins. Caspase-1 is required for the secretion of proinflammatory cytokines, such as interleukin (IL)-1beta and IL-18, and is involved in the control of many bacterial infections. Paradoxically, however, its absence has been reported to confer resistance to oral infection by Salmonella typhimurium. We show here that absence of caspase-1 or components of the inflammasome does not result in resistance to oral infection by S. typhimurium, but rather, leads to increased susceptibility to infection.
Caspase-1 (Casp-1) mediates the processing of the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18 to their mature
forms. Casp-1-deficient mice succumb more rapidly to Salmonella challenge than do wild-type animals. Both Casp-1 substrates, IL-18 and IL-1β, are relevant for control of Salmonella enterica serovar Typhimurium. We used IL-18−/− and IL-1β−/− mice in addition to administration of recombinant IL-18 to Casp-1−/− mice to demonstrate that IL-18 is important for resistance to the systemic infection but not for resistance to the intestinal
phase of the infection. This suggests that IL-1β is critical for the intestinal phase of the disease. Thus, we show that Casp-1
is essential for host innate immune defense against S. enterica serovar Typhimurium and that Casp-1 substrates are required at distinct times and anatomical sites.
Bacterial flagellins are potent inducers of innate immunity. Three signaling pathways have been implicated in the sensing
of flagellins; these involve toll-like receptor 5 (TLR5) and the cytosolic proteins Birc1e/Naip5 and Ipaf. Although the structural
basis of TLR5-flagellin interaction is known, little is known about how flagellin enters the host cell cytosol to induce signaling
via Birc1e/Naip5 and Ipaf. Here we demonstrate for the first time the translocation of bacterial flagellin into the cytosol
of host macrophages by the vacuolar pathogen, Salmonella enterica serotype Typhimurium. Translocation of flagellin into the host cell cytosol was directly demonstrated using β-lactamase reporter
constructs. Flagellin translocation required the Salmonella Pathogenicity Island 1 Type III secretion system (SPI-1 T3SS) but not the flagellar T3SS.
Pfam is a comprehensive collection of protein domains and families, represented as multiple sequence alignments and as profile
hidden Markov models. The current release of Pfam (22.0) contains 9318 protein families. Pfam is now based not only on the
UniProtKB sequence database, but also on NCBI GenPept and on sequences from selected metagenomics projects. Pfam is available
on the web from the consortium members using a new, consistent and improved website design in the UK (http://pfam.sanger.ac.uk/), the USA (http://pfam.janelia.org/) and Sweden (http://pfam.sbc.su.se/), as well as from mirror sites in France (http://pfam.jouy.inra.fr/) and South Korea (http://pfam.ccbb.re.kr/).
Flagellar assembly proceeds in a sequential manner, beginning at the base and concluding with the filament. A critical aspect
of assembly is that gene expression is coupled to assembly. When cells transition from a nonflagellated to a flagellated state,
gene expression is sequential, reflecting the manner in which the flagellum is made. A key mechanism for establishing this
temporal hierarchy is the σ28-FlgM checkpoint, which couples the expression of late flagellar (Pclass3) genes to the completion of the hook-basal body. In this work, we investigated the role of FliZ in coupling middle flagellar
(Pclass2) gene expression to assembly in Salmonella enterica serovar Typhimurium. We demonstrate that FliZ is an FlhD4C2-dependent activator of Pclass2/middle gene expression. Our results suggest that FliZ regulates the concentration of FlhD4C2 posttranslationally. We also demonstrate that FliZ functions independently of the flagellum-specific sigma factor σ28 and the filament-cap chaperone/FlhD4C2 inhibitor FliT. Furthermore, we show that the previously described ability of σ28 to activate Pclass2/middle gene expression is, in fact, due to FliZ, as both are expressed from the same overlapping Pclass2 and Pclass3 promoters at the fliAZY locus. We conclude by discussing the role of FliZ regulation with respect to flagellar biosynthesis based on our characterization
of gene expression and FliZ's role in swimming and swarming motility.
The assembly of large and complex organelles, such as the bacterial flagellum, poses the formidable problem of coupling temporal gene expression to specific stages of the organelle-assembly process. The discovery that levels of the bacterial flagellar regulatory protein FlgM are controlled by its secretion from the cell in response to the completion of an intermediate flagellar structure (the hook-basal body) was only the first of several discoveries of unique mechanisms that coordinate flagellar gene expression with assembly. In this Review, we discuss this mechanism, together with others that also coordinate gene regulation and flagellar assembly in Gram-negative bacteria.
Flagellin is the structural component of flagella produced by many pathogenic bacteria and is a potent proinflammatory molecule that mediates these effects through Toll-like receptor (TLR) 5. In Listeria monocytogenes (LM), flagellin expression is regulated by temperature and has been described as being shut off at 37degreesC. In this study, we demonstrate that TLR5-mediated cell activation and flagellin expression is maintained at 37degreesC in some laboratory-adapted strains and in approximate to 20% of LM clinical isolates. To determine the role of flagellin in LM infection, a targeted mutation in the structural gene for flagellin (flaA) was generated in a parental LM strain that expressed flagellin under all conditions examined. In vitro studies demonstrated that this DeltaflaA mutant was (i) non-motile; (ii) not able to activate TLR5-transfected HeLa cells; and (iii) induced tumour necrosis factor (TNF)-alpha production in approximate to50% fewer CD11b(+) cells in splenocytes from normal mice compared with the parental strain. However, there was no significant alteration in virulence of the DeltaflaA mutant after either intravenous or oral murine infection. Similarly, there was no difference in the generation of LM-specific CD8 or CD4 T cells after intravenous or oral infection. These data indicate that flagellin is not essential for LM pathogenesis or for the induction of LM-specific adaptive immune responses in normal mice.
OmpR and EnvZ differentially control the transcription of the major outer membrane porin genes, ompF and ompC, in Escherichia coli in response to the osmolarity of the medium. We have previously provided evidence that OmpR works both positively and negatively at the ompF promoter to give the characteristic switch from OmpF to OmpC production with increasing osmolarity. Here, we describe the isolation of cis-acting ompF mutations that affect negative regulation by OmpR by affecting the three-dimensional structure of the promoter region as measured by agarose gel mobility. These results further clarify the mechanism by which OmpR negatively regulates ompF expression, suggesting a model in which OmpR forms a repressive loop in the ompF promoter region.
Yersinia enterocolitica cells, when cultured at 30 degrees C or below, are flagellated and motile. Cells cultured at 37 degrees C or above lack flagella and are non-motile. To identify flagellin genes that are a target of this temperature-dependent regulation, a library of Y. enterocolitica genomic inserts in a phage lambda vector was probed with the Salmonella typhimurium fliC (flagellin) gene. A DNA fragment subcloned from a recombinant phage which hybridizes with the probe complements a non-motile S. typhimurium fliC-fljB- (flagellin-minus) mutant. DNA sequence analysis shows that Y. enterocolitica contains three tandem flagellin genes, designated fleA, fleB and fleC. All three genes are co-ordinately transcribed at low, but not high, temperature from fliA-dependent (sigma F) promoters. Flagellin transcription arrests rapidly after upshift to 37 degrees C (host temperature). In contrast, flagellin transcription resumes only after several generations when cells cultured at 37 degrees C are downshifted to 28 degrees C.
Flagellin genes (fliC) were detected in two species of the genus Shigella. The fliCSF gene cloned from Shigella flexneri produced normal-type flagella in an Escherichia coli delta fliC strain while the fliCSS genes from two Shigella sonnei strains produced curly-type flagella and their expression is repressible by Salmonella FljA repressor. The fliCSF gene (1650 bp) shared high similarity with the E. coli fliCE gene not only in the 5' and 3' constant sequences but also in the upstream and downstream sequences. The fliCSS genes (1572 bp) shared high similarity with the Salmonella typhimurium fliCS gene in the operator and 3' constant sequences and also shared high similarity with the fliCE gene in the downstream sequence, suggesting that the fliCSS gene has undergone horizontal transfer and recombination. Differences in nucleotide sequences of the central variable regions among the four fliC genes, including fliCE and fliCS, suggest that they started differentiation in each lineage approximately 80 million years ago. Loss of motility in Shigella seems to be evolutionarily a recent event.
We have purified and characterized a modified peptide pheromone that accumulates in culture medium as B. subtilis grows to high density. This pheromone is required for the development of genetic competence. When added to cells at low density, the pheromone induces the premature development of competence. The peptide moiety of the pheromone matches nine of the last ten amino acids predicted from a 55 codon open reading frame, comX. comX and comQ, the gene immediately upstream of comX, are required for production of the pheromone. Response to the pheromone requires the comP-comA two-component regulatory system and the oligopeptide permease encoded by spo0K. Spo0K could transport the pheromone into the cell, or function as a receptor, binding the pheromone and sending a transmembrane signal, leading to activation of the ComA transcription factor and induction of competence development.
The fliA gene encodes the flagellum-specific sigma factor sigma28 In Salmonella typhimurium. The transcription in vivo and in vitro of this gene was analysed and it was found that there are two promoters for the expression of this gene. One is a class 2 promoter which is recognized by sigma70-RNA polymerase in the presence of the FlhD and FlhC activator proteins. The other is a class 3 promoter which is recognized by sigma28-RNA polymerase. Therefore, the fliA operon is under dual positive control from FlhD/FlhC and from FliA itself. The nucleotide sequence downstream of the fliA gene was determined. The sequence contains two ORFs following the fliA gene. On the basis of their sequence homology, it is concluded that these two correspond to the fliZ and fliY genes of Escherichia coil. Northern blot analysis revealed that the fliZ gene is transcribed from the fliA promoters, whereas the fliY gene is transcribed from both the fliA promoters and its own FlhD/FlhC-independent promoter. A fliZ-disruption mutant was constructed by inserting a kanamycin-resistance gene cassette into the fliZ gene on the chromosome. The mutant showed poor motility, and introduction of a fliZ+ plasmid into this mutant restored the wildtype level of motility. These results suggest that the fliZ gene may be required for expression of maximal motility.
Specialized epithelia known as M cells overlying the lymphoid follicles of Peyer's patches are important in the mucosal immune system, but also provide a portal of entry for pathogens such as Salmonella typhimurium, Mycobacterium bovis, Shigella flexneri, Yersinia enterocolitica and reoviruses. Penetration of intestinal M cells and epithelial cells by Salmonella typhimurium requires the invasion genes of Salmonella Pathogenicity Island 1 (SPI1). SPI1-deficient S. typhimurium strains gain access to the spleen following oral administration and cause lethal infection in mice without invading M cells or localizing in Peyer's patches, which indicates that Salmonella uses an alternative strategy to disseminate from the gastrointestinal tract. Here we report that Salmonella is transported from the gastrointestinal tract to the bloodstream by CD18-expressing phagocytes, and that CD18-deficient mice are resistant to dissemination of Salmonella to the liver and spleen after oral administration. This CD18-dependent pathway of extraintestinal dissemination may be important for the development of systemic immunity to gastrointestinal pathogens, because oral challenge with SPI1-deficient S. typhimurium elicits a specific systemic IgG humoral immune response, despite an inability to stimulate production of specific mucosal IgA.
We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage lambda Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells.
We used flow cytometry and confocal immunofluorescence microscopy to study the localization of Salmonella typhimurium in spleens of infected mice. Animals were inoculated intragastrically or intraperitoneally with S. typhimurium strains, constitutively expressing green fluorescent protein. Independently of the route of inoculation, most bacteria were found in intracellular locations 3 days after inoculation. Using a panel of antibodies that bound to cells of different lineages, including mononuclear phagocyte subsets, we have shown that the vast majority of S. typhimurium bacteria reside within macrophages. Bacteria were located in red pulp and marginal zone macrophages, but very few were found in the marginal metallophilic macrophage population. We have demonstrated that the Salmonella SPI-2 type III secretion system is required for replication within splenic macrophages, and that sifA(-) mutant bacteria are found within the cytosol of these cells. These results confirm that SifA and SPI-2 are involved in maintenance of the vacuolar membrane and intracellular replication in vivo.
In the Salmonella-mouse model of systemic infection, high dose inoculation results in the multiplication of many of the cells present in the inoculum, rather than the clonal amplification of a small number. This characteristic has allowed the development of methods to screen multiple strains for either virulence attenuation or gene expression within the same animal. Mixed infections with mutant and wild-type strains are used to provide a sensitive measure of virulence attenuation referred to as the competitive index. We have recently used a variation of this method, involving mixed infections of single and double mutant strains, to study virulence gene interaction in vivo.
Intestinal M cells, the specialised antigen-sampling cells of the mucosal immune system, are exploited by Salmonella and other pathogens as a route of invasion. Salmonella entry into M cells and colonisation of Peyer's patches involve mechanisms critical for infection of cultured cells as well as factors not accurately modelled in vitro.
A simple method for the construction of targeted transcriptional and translational fusions to the lac operon using FLP mediated site-specific recombination is described. Conditional plasmids containing promoterless lacZY genes and the FLP recognition target (FRT) site in both orientations were constructed for generating transcriptional fusions. Similarly, a plasmid used to create translational fusions was constructed in which the endogenous translational start of lacZ has been removed. These plasmids can be transformed into strains containing a single FRT site, which was previously integrated downstream of the promoter of interest using the lambda Red recombination method. The FLP protein produced from a helper plasmid that contains a conditional origin of replication promotes site-specific recombination between the FRT sites, resulting in an integrated lac fusion to the gene of interest. Transcriptional fusions to the Salmonella typhimurium genes sodCII and sitA were constructed using this method and shown to respond appropriately to mutations in the respective regulatory genes, rpoS and fur. Translational fusions were also constructed using this method. In this case, expression of beta-galactosidase was dependent on translation of the target protein. Given that the FLP recombinase does not require host factors for function and that this method requires no molecular cloning, this method should be applicable for the analysis of gene expression in a variety of organisms.
In enterobacteria such as Salmonella, flagellar biogenesis is dependent upon the master operon flhDC at the apex of the flagellar gene hierarchy, which is divided into three classes 1, 2 and 3. Previously we reported that depletion of the ClpXP ATP-dependent protease results in dramatic enhancement of class 2 and class 3 gene transcription, whereas the transcription level of the flhDC operon remains normal in Salmonella enterica serovar Typhimurium. This suggests that the ClpXP protease may be responsible for the turnover of the FlhD and FlhC master regulators (Tomoyasu, T., Ohkishi, T., Ukyo, Y., Tokumitsu, A., Takaya, A., Suzuki, M. et al., 2002, J Bacteriol 184:645-653). In this study, to establish the role of the ClpXP protease in the turnover of FlhD and FlhC proteins, we analysed levels of the FlhD and FlhC proteins in wild-type and ClpXP mutant cells using anti-FlhD and anti-FlhC antibodies. The results show that both FlhD and FlhC proteins are markedly accumulated in ClpXP mutant cells and the half-life of FlhC is approximately fivefold longer in the ClpXP mutant, suggesting that the ClpXP protease is responsible for the degradation of FlhD and FlhC. The results also show that the ClpXP protease degrades both proteins in FlhD2FlhC2 complex but does not seem to recognize the respective subunits synthesized individually. Taken together, it is suggested that the cellular concentration of the FlhD2FlhC2 master regulator is tightly controlled at the post-translational level by the ClpXP protease. We also examined the role of other members of the ATP-dependent protease family in the regulation of flagellar biogenesis and concluded that only ClpXP in this family functions as a negative regulator for flagellar biogenesis in Salmonella.
Flagellin is the structural component of flagella produced by many pathogenic bacteria and is a potent proinflammatory molecule that mediates these effects through Toll-like receptor (TLR) 5. In Listeria monocytogenes (LM), flagellin expression is regulated by temperature and has been described as being shut off at 37 degrees C. In this study, we demonstrate that TLR5-mediated cell activation and flagellin expression is maintained at 37 degrees C in some laboratory-adapted strains and in approximately 20% of LM clinical isolates. To determine the role of flagellin in LM infection, a targeted mutation in the structural gene for flagellin (flaA) was generated in a parental LM strain that expressed flagellin under all conditions examined. In vitro studies demonstrated that this deltaflaA mutant was (i). non-motile; (ii). not able to activate TLR5-transfected HeLa cells; and (iii). induced tumour necrosis factor (TNF)-alpha production in approximately 50% fewer CD11b+ cells in splenocytes from normal mice compared with the parental strain. However, there was no significant alteration in virulence of the deltaflaA mutant after either intravenous or oral murine infection. Similarly, there was no difference in the generation of LM-specific CD8 or CD4 T cells after intravenous or oral infection. These data indicate that flagellin is not essential for LM pathogenesis or for the induction of LM-specific adaptive immune responses in normal mice.
Signature-tagged transposon mutagenesis of Salmonella with differential recovery from wild-type and immunodeficient mice revealed that the gene here named cdgR[for c-diguanylate (c-diGMP) regulator] is required for the bacterium to resist host phagocyte oxidase in vivo. CdgR consists solely of a glutamate-alanine-leucine (EAL) domain, a predicted cyclic diGMP (c-diGMP) phosphodiesterase. Disruption of cdgR decreased bacterial resistance to hydrogen peroxide and accelerated bacterial killing of macrophages. An ultrasensitive assay revealed c-diGMP in wild-type Salmonella with increased levels in the CdgR-deficient mutant. Thus, besides its known role in regulating cellulose synthesis and biofilm formation, bacterial c-diGMP also regulates host-pathogen interactions involving antioxidant defence and cytotoxicity.
Macrophages respond to Salmonella typhimurium infection via Ipaf, a NACHT-leucine-rich repeat family member that activates caspase-1 and secretion of interleukin 1beta. However, the specific microbial salmonella-derived agonist responsible for activating Ipaf is unknown. We show here that cytosolic bacterial flagellin activated caspase-1 through Ipaf but was independent of Toll-like receptor 5, a known flagellin sensor. Stimulation of the Ipaf pathway in macrophages after infection required a functional salmonella pathogenicity island 1 type III secretion system but not the flagellar type III secretion system; furthermore, Ipaf activation could be recapitulated by the introduction of purified flagellin directly into the cytoplasm. These observations raise the possibility that the salmonella pathogenicity island 1 type III secretion system cannot completely exclude 'promiscuous' secretion of flagellin and that the host capitalizes on this 'error' by activating a potent host-defense pathway.
CsrA is a regulator of invasion genes in Salmonella enterica serovar Typhimurium. To investigate the wider role of CsrA in gene regulation, we compared the expression of Salmonella genes in a csrA mutant with those in the wild type using a DNA microarray. As expected, we found that expression of Salmonella pathogenicity island 1 (SPI-1) invasion genes was greatly reduced in the csrA mutant, as were genes outside the island that encode proteins translocated into eukaryotic cells by the SPI-1 type III secretion apparatus. The flagellar synthesis operons, flg and fli, were also poorly expressed, and the csrA mutant was aflagellate and non-motile. The genes of two metabolic pathways likely to be used by Salmonella in the intestinal milieu also showed reduced expression: the pdu operon for utilization of 1,2-propanediol and the eut operon for ethanolamine catabolism. Reduced expression of reporter fusions in these two operons confirmed the microarray data. Moreover, csrA was found to regulate co-ordinately the cob operon for synthesis of vitamin B12, required for the metabolism of either 1,2-propanediol or ethanolamine. Additionally, the csrA mutant poorly expressed the genes of the mal operon, required for transport and use of maltose and maltodextrins, and had reduced amounts of maltoporin, normally a dominant protein of the outer membrane. These results show that csrA controls a number of gene classes in addition to those required for invasion, some of them unique to Salmonella, and suggests a co-ordinated bacterial response to conditions that exist at the site of bacterial invasion, the intestinal tract of a host animal.
FliC is a natural antigen recognized by the innate and adaptive immune systems during Salmonella infection in mice and humans; however, the regulatory mechanisms governing its expression in vivo are incompletely understood. Here, we use flow cytometry to quantify fliC gene expression in single bacteria. In vitro, fliC transcription was not uniformly positive; a viable fliC-negative subpopulation was also identified. Intracellular Salmonella repressed transcription of fliC and its positive regulator fliA, but constitutively transcribed the master regulator flhD; fliC repression required ClpXP protease, known to degrade FlhD. In orally infected mice, fliC transcription was anatomically restricted: Salmonella transcribed fliC in the Peyer's Patches (PP) but not in the mesenteric lymph nodes and spleen. The intracellularly transcribed pagC promoter was upregulated by Salmonella in all tissues, defining the infected PP as a unique environment that initiates expression of intracellularly induced genes and yet permits transcription of fliC. Because a single bacterium can escape the GI tract to colonize deeper tissues, heterogeneous gene expression may have important implications for Salmonella pathogenesis: FliC-positive bacteria in the PP could stimulate inflammation and facilitate the priming of FliC-specific immune responses, while FliC-negative bacteria escape host detection in the gut and spread to systemic sites of replication.
Salmonella enterica serovar Typhimurium invades host macrophages and induces a unique caspase-1-dependent pathway of cell death termed pyroptosis, which is activated during bacterial infection in vivo. We demonstrate DNA cleavage during pyroptosis results from caspase-1-stimulated nuclease activity. Although poly(ADP-ribose) polymerase (PARP) activation by fragmented DNA depletes cellular ATP to cause lysis during oncosis, the rapid lysis characteristic of Salmonella-infected macrophages does not require PARP activity or DNA fragmentation. Membrane pores between 1.1 and 2.4 nm in diameter form during pyroptosis of host cells and cause swelling and osmotic lysis. Pore formation requires host cell actin cytoskeleton rearrangements and caspase-1 activity, as well as the bacterial type III secretion system (TTSS); however, insertion of functional TTSS translocons into the host membrane is not sufficient to directly evoke pore formation. Concurrent with pore formation, inflammatory cytokines are released from infected macrophages. This mechanism of caspase-1-mediated cell death provides additional experimental evidence supporting pyroptosis as a novel pathway of inflammatory programmed cell death.
Gene expression in bacteria is traditionally studied from the average behaviour of cells in a population, which has led to the assumption that under a particular set of conditions all cells express genes in an approximately uniform manner. The advent of methods for visualizing gene expression in individual cells reveals, however, that populations of genetically identical bacteria are sometimes heterogeneous, with certain genes being expressed in a non-uniform manner across the population. In some cases, heterogeneity is manifested by the bifurcation into distinct subpopulations, and we adopt the common usage, referring to this phenomenon as bistability. Here we consider four cases of bistability, three from Bacillus subtilis and one from Escherichia coli, with an emphasis on random switching mechanisms that generate alternative cell states and the biological significance of phenotypic heterogeneity. A review describing additional examples of bistability in bacteria has been published recently.
The mammalian intestine is colonized by a dense bacterial community, called microbiota. The microbiota shields from intestinal infection (colonization resistance). Recently, we have shown that enteropathogenic Salmonella spp. can exploit inflammation to compete with the intestinal microbiota. The mechanisms explaining the enhanced pathogen growth in the inflamed intestine are elusive. Here, we analysed the function of bacterial flagella in the inflamed intestine using a mouse model for acute Salmonella Typhimurium enterocolitis. Mutations affecting flagellar assembly (Fla(-)) and chemotaxis (Che(-)) impaired the pathogen's fitness in the inflamed intestine, but not in the normal gut. This was attributable to a localized source of high-energy nutrients (e.g. galactose-containing glyco-conjugates, mucin) released as an element of the mucosal defence. Motility allows Salmonella Typhimurium to benefit from these nutrients and utilize them for enhanced growth. Thus, nutrient availability contributes to enhanced pathogen growth in the inflamed intestine. Strategies interfering with bacterial motility or nutrient availability might offer starting points for therapeutic approaches.
Cytoplasmic flagellin activates caspase-1 and secretion of in-terleukin-1β via Ipaf
- Miao
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Miao EA, et al. (2006) Cytoplasmic flagellin activates caspase-1 and secretion of in-terleukin-1β via Ipaf. Nat Immunol 7:569–575.
Harwood for helpful discus-sions. This work was supported by a University of Washington Cellular and Molecular Biology Training Grant, National Institute of General Medical Sciences Public Health Service National Research Service Award Grant T32 GM07270, and National Institutes of Health Grants P50
- Acknowledgments We
ACKNOWLEDGMENTS. We thank Caroline S. Harwood for helpful discus-sions. This work was supported by a University of Washington Cellular and Molecular Biology Training Grant, National Institute of General Medical Sciences Public Health Service National Research Service Award Grant T32 GM07270, and National Institutes of Health Grants P50 HG 02360 and U19 AI 090882A.