Infection and Immunity

The diameter, length, and shape of bacteria are maintained with such high fidelity that these parameters are classically used as metrics in the distinction of bacterial species. Increasing evidence indicates that bacteria transiently shift their shapes into distinctive morphologies in response to environmental changes. Elongation of bacterial length into a filamentous shape provides unique survival advantages for many bacterial species. Analysis of 42 clinical isolates of uropathogenic Escherichia coli (UPEC) revealed that filamentation to host-derived antimicrobials is a conserved phenotype. Therefore, we hypothesize that filamentation represents a conserved mechanism of pathogenic bacterial persistence that can be targeted for narrow-spectrum, anti-virulence therapies. We demonstrate that cranberries prevent SulA-mediated filamentation of UPEC. Furthermore, we identify multiple fractions of cranberries that retain anti-filamentation properties. These studies provide mechanistic insight into the clinical efficacy of cranberry for patients with recurrent urinary tract infections. Inhibition of filamentation represents a novel approach to promote bacterial pathogen susceptibility to immune and antibiotic-mediated clearance to attenuate disease.

The O5-specific monoclonal IgA antibody, Sal4, mediates the conversion of Salmonella enterica serovar Typhimurium (STm) from virulent, free-swimming cells to non-motile, multicellular biofilm-like aggregates within a matter of hours. We hypothesize that the rapid transition from an invasive to a non-invasive state is an adaptation of STm to Sal4 IgA exposure. In this report, we performed a genome-wide CRISPR interference (CRISPRi) screen to identify STm genes that influence multicellular aggregate formation in response to Sal4 IgA treatment. From a customized library of >36,000 spacers, ~1% (373) were enriched at the top of the culture supernatant after two consecutive rounds of Sal4 IgA treatment. The enriched spacers mapped to a diversity of targets, including genes involved in O-antigen modification, cyclic-di-GMP metabolism, outer membrane biosynthesis/signaling, and invasion/virulence, with the most frequently targeted gene being fimW , which encodes a negative regulator of type 1 fimbriae (T1F) expression. Generation of a STm Δ fimW strain confirmed that the loss of FimW activity results in a hyperfimbriated phenotype and evasion of Sal4 IgA-mediated agglutination in solution. Closer examination of the fimW mutant revealed its propensity to form biofilms at the air–liquid interface in response to Sal4 exposure, suggesting that T1F “primes” STm to transition from a planktonic to a sessile state, possibly by facilitating bacterial attachment to abiotic surfaces. These findings shed light on the mechanism by which IgA antibodies influence STm virulence in the intestinal environment.

Haemophilus ducreyi causes cutaneous ulcers in children who live in yaws-endemic countries and the genital ulcer disease chancroid. In the human host, H. ducreyi resides in an abscess and may need to resist both heat and oxidative stress, which result in aggregation and misfolding of bacterial proteins. In Escherichia coli , the hslUV ( clpYQ ) operon encodes a proteasome-like complex that degrades misfolded proteins and is upregulated during heat shock. In previous studies, we showed that hslUV transcripts are upregulated in experimental lesions caused by H. ducreyi in human volunteers, suggesting that HslUV may help H. ducreyi adapt to the abscess environment. Here, we constructed an unmarked hslUV operon deletion mutant, 35000HPΔ hslUV , in H. ducreyi . Whole-genome sequencing showed that compared to its parent (35000HP), the mutant contained only the deletion of interest. Six volunteers were inoculated at three sites on skin overlying the deltoid on opposite arms with 35000HP and 35000HPΔ hslUV . Within 24 h, papules formed at 88.9% (95% CI [69%, 100%]) at both parent and mutant-inoculated sites ( P = 1.0). Pustules formed at 44.4% (95% CI [25.6%, 64.3%]) at parent-inoculated sites and 33.3% (95% CI [2.5%, 64.1%]) at mutant-inoculated sites ( P = 0.17). Thus, the proteosome-like complex encoded by hslUV was dispensable for H. ducreyi virulence in humans. In the absence of hslUV , H. ducreyi likely utilizes other systems such as the Lon protease, ClpXP, and ClpB/DnaK to combat protein aggregation and misfolding, underscoring the importance of the functional redundancy of such systems in gram-negative pathogens.

Cryptococcal meningitis is a common and refractory central nervous system (CNS) infection with high mortality and disability. For Cryptococcus neoformans ( C. neoformans ) to penetrate the CNS, it first adheres to and breaches the blood‒brain barrier (BBB). Here, we explored the roles of CD146, an adhesion molecule expressed on the surface of brain microvascular endothelial cells (BMECs), in cryptococcal vascular adhesion and BBB invasion. Following cryptococcal infection, we observed a reduction in CD146 expression in BMECs, which was at least partially mediated by metalloproteinase-9. Once overexpressed on BMECs, CD146 increased C. neoformans adhesion; in contrast, CD146 knockout decreased the attachment of fungi to endothelial cells in vitro . Unexpectedly, CD146 knockout failed to reduce fungal infection in the brain following intravascular instillation of C. neoformans . However, the anti-CD146 antibody AA98 significantly increased the fungemia (spleen CFU), suggesting that CD146 may be involved in the early adhesion and invasion of Cryptococcus into cerebral vessels. AA98, however, failed to extend the survival of C. neoformans infected mice. These results suggest that CD146 may play dispensable roles in the C. neoformans brain infection.

Bacteriophages are the dominant members of the human enteric virome and can shape bacterial communities in the gut; however, our understanding of how they directly impact health and disease is limited. Previous studies have shown that specific bacteriophage populations are expanded in patients with Crohn’s disease (CD) and ulcerative colitis (UC), suggesting that fluctuations in the enteric virome may contribute to intestinal inflammation. Based on these studies, we hypothesized that a high bacteriophage burden directly induces intestinal epithelial responses. We found that filamentous bacteriophages M13 and Fd induced dose-dependent IL-8 expression in the human intestinal epithelial cell line HT-29 to a greater degree than their lytic counterparts, T4 and ϕX174. We also found that M13, but not Fd, reduced bacterial internalization in HT-29 cells. This led us to investigate the mechanism underlying M13-mediated inhibition of bacterial internalization by examining the antiviral and antimicrobial responses in these cells. M13 upregulated type I and III IFN expressions and augmented short-chain fatty acid (SCFA)-mediated LL-37 expression in HT-29 cells. Taken together, our data establish that filamentous bacteriophages directly affect human intestinal epithelial cells. These results provide new insights into the complex interactions between bacteriophages and the intestinal mucosa, which may underlie disease pathogenesis.

Helicobacter pylori strains containing the cag pathogenicity island (PAI) deliver an effector protein (CagA) and non-protein substrates into gastric cells through a process that requires the Cag type IV secretion system (T4SS). The Cag T4SS outer membrane core complex (OMCC) contains multiple copies of five proteins, two of which are species-specific proteins. By using modifications of a previously described OMCC immunopurification method and optimized mass spectrometric methods, we have now isolated additional cag PAI-encoded proteins that are present in lower relative abundance. Four of these proteins (CagW, CagL, CagI, and CagH) do not exhibit sequence relatedness to T4SS components in other bacterial species. Size exclusion chromatography analysis of immunopurified samples revealed that CagW, CagL, CagI, and CagH co-elute with OMCC components. These four Cag proteins are copurified with the OMCC in immunopurifications from a Δ cag3 mutant strain (lacking peripheral OMCC components), but not from a Δ cagX mutant strain (defective in OMCC assembly). Negative stain electron microscopy analysis indicated that OMCC preparations isolated from Δ cagW, cagL::kan, Δ cagI, and Δ cagH mutant strains are indistinguishable from wild-type OMCCs. In summary, by using several complementary methods, we have identified multiple species-specific Cag proteins that are associated with the Cag T4SS OMCC and are required for T4SS activity.

No licensed vaccines are available for the largely antibiotic-resistant Shigella or enterotoxigenic Escherichia coli (ETEC), the two most common bacteria causing children’s diarrhea and travelers’ diarrhea. Virulence heterogeneity is a key obstacle to developing vaccines against Shigella or ETEC. By applying a multiepitope fusion antigen (MEFA) vaccinology platform, we recently constructed epitope- and structure-based polyvalent proteins to induce cross-protective antibodies against heterogeneous Shigella or ETEC strains. In this study, we combined a polyvalent Shigella protein with two polyvalent ETEC proteins, examined antigen compatibility and broad immunogenicity, and evaluated the potential of developing a combined vaccine against the two groups of bacteria. Data showed that mice intramuscularly immunized with the combined vaccine candidate (ShecVax) developed antibodies to all the following target virulence factors: Shigella IpaB, IpaD, VirG, GuaB, StxA, Stx2A, and StxB, and ETEC STa, LT, CFA/I, CS1, CS2, CS3, CS4, CS5, and CS6. ShecVax-induced antibodies significantly inhibited the invasion of all Shigella species and important serotypes, prevented the adherence of all important ETEC pathotypes, and neutralized the enterotoxicity of ETEC toxins STa and LT. Moreover, ShecVax prevented mice from lethal pulmonary infection with Shigella sonnei or S. flexneri 2a, significantly reduced ETEC bacterial colonization in rabbit small intestines, and passively protected newborn pigs against ETEC toxin-mediated clinical diarrhea. These results indicated that ShecVax is broadly immunogenic and cross-protective against Shigella and ETEC, suggesting ShecVax can be a Shigella /ETEC combined vaccine against children’s and travelers’ diarrhea, and the MEFA platform can be generally applied for vaccine development against heterogeneous pathogens or different diseases. IMPORTANCE There are no effective countermeasures against Shigella and enterotoxigenic E. coli (ETEC), two antibiotic-resistant groups of bacteria and the leading causes of diarrhea in children in developing countries (children’s diarrhea) and international travelers (travelers’ diarrhea). Vaccines are a more practical approach to protect against infectious diseases, including diarrhea caused by Shigella or ETEC. A combined vaccine cross-protective against Shigella and ETEC can save hundreds of thousands of lives and prevent hundreds of millions of diarrhea cases yearly; it can also reduce antibiotic prescription and decrease antibiotic resistance, thus significantly improving global health. In addition, we may apply the MEFA platform to develop combined vaccines against heterogeneous pathogens or different diseases to accommodate an increasingly crowded expanded program on immunization (EPI).

The oral bacterium Tannerella forsythia is associated with periodontitis, an inflammatory disease affecting tooth-supporting tissues. The bacterium produces a dicarbonyl compound, methylglyoxal (MGO), whose levels correlate with the severity of periodontitis. MGO can induce inflammation directly or via the generation of glycation products called advanced glycation end products (AGEs). T. forsythia -produced MGO has been shown to cause tissue collagen glycation, which in turn can induce pro-inflammatory cytokine secretion in monocytes via receptor for advanced glycation end product (RAGE) receptor activation. The current study investigated the impact of T. forsythia -secreted MGO on human gingival fibroblasts and endothelial cells. For assessing the in vivo impact of T. forsythia -secreted MGO, we employed an oral gavage-induced mouse model of periodontitis utilizing the wild-type and MGO-deficient strains of T. forsythia . Our results showed that the apoptotic activity was enhanced, and cell migration was reduced in fibroblasts exposed to collagen treated with the T. forsythia wild-type culture supernatant. Moreover, monocyte binding, reactive oxygen species production, and inflammatory cytokine secretion were increased in fibroblasts, and neutrophil transendothelial migration was enhanced in response to the T. forsythia wild type-treated collagen. In vivo , increased AGE accumulation in gingival tissues with increased alveolar bone loss was observed in wild-type T. forsythia as compared to the MGO-deficient strain-infected mice. These data demonstrated that T. forsythia -secreted MGO contributes to periodontal tissue destruction by mitigating gingival fibroblast-mediated tissue healing and promoting endothelial cell dysfunction. These findings provide a basis for targeting the T. forsythia -associated AGE-RAGE axis in alleviating periodontitis.

Extracellular vesicles (EVs) are heterogeneous particles encapsulated with a phospholipid bilayer membrane. EVs have evolved diverse biological functions, serving mainly as prominent mediators and regulators of cell-cell communication. This study investigated whether candidalysin, a key virulence factor in Candida albicans infections, is present within EVs derived from C. albicans biofilms and retains activity by inducing host immune responses. We found that biofilm EVs contain candidalysin and can permeabilize planar lipid bilayer membranes in a dose-dependent manner. However, biofilm EVs were unable to damage oral epithelial cells (OECs) but were able to induce cytokine responses. Notably, EVs obtained from biofilms cultured for 24 h and 48 h exhibited differences in cargo composition and their ability to activate OECs. This study highlights the potential of biofilm EVs as a toxin delivery system during C. albicans infection and identifies temporal differences in the ability of EVs to activate epithelial cells.

Babesia microti is the primary cause of human babesiosis in North America. Despite the emergence of the disease in recent years, the pathogenesis and immune response to B. microti infection remain poorly understood. Studies in laboratory mice have shown a critical role for macrophages in the elimination of parasites and infected red blood cells (iRBCs). Importantly, the underlying mechanisms that activate macrophages are still unknown. Recent evidence identified the release of extracellular vesicles (EVs) from Babesia iRBCs. EVs are spherical particles released from cell membranes under natural or pathological conditions that have been suggested to play roles in host–pathogen interactions among diseases caused by protozoan parasites. The present study examined whether EVs released from cultured Babesia iRBCs could activate macrophages and alter cytokine secretion. An analysis of vesicle size in EV fractions from Babesia iRBCs showed diverse populations in the <100 nm size range compared to EVs from uninfected RBCs. In co-culture experiments, EVs released by B. microti iRBCs appeared to be associated with macrophage membranes and cytoplasm, indicating uptake of these vesicles in vitro . Interestingly, the incubation of macrophages with EVs isolated from Babesia iRBC culture supernatants resulted in the activation of NF-κB and modulation of pro-inflammatory cytokines. These results support a role for Babesia -derived EVs in macrophage activation and provide new insights into the mechanisms involved in the induction of the innate immune response during babesiosis.

The insect microbiome is comprised of extracellular microbial communities that colonize the host surfaces and endosymbionts that reside inside host cells and tissues. Both of these communities participate in essential aspects of host biology, including the immune response and interactions with pathogens. In recent years, our knowledge about the role of the insect microbiome in infection has increased tremendously. While many studies have highlighted the microbiome’s protective effect against various natural enemies of insects, unexpected discoveries have shown that some members of the microbiota can facilitate pathogenic infections. Here, we summarize studies in the fruit fly, Drosophila melanogaster , that have substantially progressed our understanding of host-pathogen-microbiome interactions during infection. We summarize studies on the protective mechanisms of Drosophila gut microbiota, highlight examples of microbiome exploitation by pathogens, and detail the mechanisms of endosymbiont-mediated host protection. In addition, we delve into a previously neglected topic in Drosophila microbiome research—the crosstalk between endosymbionts and gut microbiota. Finally, we address how endosymbionts and gut microbiota remain resilient to host immune responses and stably colonize the host during infection. By examining how the microbiome is influenced by and reciprocally affects infection outcomes, this review provides timely and cohesive coverage of the roles of Drosophila endosymbionts and gut microbiota during infections.

Salmonella, a leading cause of foodborne illnesses, is primarily transmitted to humans through the consumption of contaminated poultry products. The increasing resistance of Salmonella to antibiotics and lack of cross-protection by vaccines necessitate new control strategies in poultry production systems. This study assessed the efficacy of probiotics against Salmonella Typhimurium (ST) and Salmonella Enteritidis (SE). Lactobacillus acidophilus (LA), Lacticaseibacillus rhamnosus GG (LGG), and Bifidobacterium animalis subsp. lactis (Bb12) showed inhibition of ST and SE in agar well diffusion assay, with stable inhibitory properties. In co-culture assay, both LGG and Bb12 completely suppressed ST and SE growth. Liquid chromatography-with tandem mass spectrometry (LC-MS/MS) analysis of the LGG and Bb12 cell-free culture supernatant identified novel bioactive peptides with anti- Salmonella properties. Administering LGG in drinking water of chickens raised on built-up litter floor in experimental conditions significantly reduced the ST load (5.95 logs and 3.74 on 7 days post-infection [dpi] and 14 dpi, respectively). Gut microbiota analysis revealed increased abundance of several beneficial genera such as Butyricicoccus , Erysipelatoclostridium , Flavonifractor, and Bacillus in LGG-treated groups. Histomorphometry analysis demonstrated increased villus height (VH) and VH by crypt depth ratio in the ileum of the LGG-treated group on 14 dpi. These results highlight LGG as a promising probiotic for controlling Salmonella in chickens and reducing transmission to humans. The beneficial properties of LGG are attributed to the production of antimicrobial peptides, microbiota modulation, and enhanced intestinal integrity. IMPORTANCE Salmonella is the leading cause of foodborne illnesses in the United States and worldwide. It is primarily transmitted through contaminated poultry and poultry products (eggs and poultry meat). Increasing resistance of Salmonella to antibiotics and lack of cross-protection by vaccines necessitate new control strategies to reduce Salmonella in poultry production system and minimize human infections. Probiotics, which are live beneficial microorganisms when administered in an optimum amount, have been increasingly used in recent years as alternatives to antibiotics to promote health. Our study showed that LGG exhibited superior probiotics properties and significantly reduced Salmonella load in chickens. Thus, LGG supplementation is a promising approach to prevent Salmonella infection and enhance performance of poultry thereby enhance food safety, proper antibiotic stewardship and public health.

Treponema denticola is an obligate colonizer of the human gingival crevice and, along with other pathobionts, is highly associated with the development of periodontal disease. As periodontal disease develops, significant environmental changes occur in the subgingival crevice and oral microbiome. The ability to sense and respond to changing environmental conditions is essential to the ability of T. denticola to thrive and cause disease. Yet, our understanding of T. denticola sensory transduction and gene regulatory mechanisms is nearly absent. The AtcSR two-component system has been predicted to regulate several cellular processes, but its role in T. denticola adaptive responses has not been investigated. To address this knowledge gap, we constructed a deletion of the atcS gene, encoding the histidine kinase. We performed RNA sequencing, demonstrating that the deletion of atcS results in significant changes in the transcriptome of T. denticola . Most notably, the transcription of genes encoding proteins involved in motility and the dentilisin protease complex was reduced. Consistent with this, the deletion mutant displayed reduced dentilisin activity and motility. These phenotypes are critical to interactions with host cells and the pathogenicity of T. denticola . This aligns with our observation that the atcS -deficient strain had attenuated attachment and invasion of gingival epithelial cells and failed to induce alveolar bone loss in a murine periodontitis model, processes that are central to T. denticola virulence. This study is a significant step toward defining the role of the AtcSR two-component system in T. denticola pathogenicity.

Probiotic therapies have been suggested for amelioration efforts of wildlife disease such as chytridiomycosis caused by Batrachochytrium spp. in amphibians. However, there is a lack of information on how probiotic application affects resident microbial communities and immune responses. To better understand these interactions, we hypothesized that probiotic application would alter microbial community composition and host immune expression in Xenopus laevis . Accordingly, we applied three amphibian-derived and anti- Batrachochytrium bacteria strains (two Pseudomonas spp. and one Stenotrophomonas sp.) to X. laevis in monoculture and also as a cocktail. We quantified microbial community structure using 16S rRNA gene sequencing. We also quantified genes involved in X. laevis immune responses using quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) and skin transcriptomics over 1 and 3-week periods. All probiotic treatments successfully colonized X. laevis skin for 3 weeks, but with differential amplicon sequence variant (ASV) sequence counts over time. Bacterial community and immune gene effects were most pronounced at week 1 post-probiotic exposure and decreased thereafter. All probiotic treatments caused initial changes to bacterial community alpha and beta diversity, including reduction in diversity from pre-exposure anti- Batrachochytrium bacterial ASV relative abundance. Probiotic colonization by Pseudomonas probiotic strain RSB5.4 reduced expression of regulatory T cell marker ( FOXP3, measured with RT-qPCR) and caused the greatest gene expression changes detected by transcriptomics. Single bacterial strains and mixed cultures, therefore, altered amphibian microbiome-immune interactions. This work will help to improve our understanding of the role of the microbiome-immune interface underlying both disease dynamics and emergent eco-evolutionary processes. IMPORTANCE Amphibian skin microbial communities have an important role in determining disease outcomes, in part through complex yet poorly understood interactions with host immune systems. Here we report that probiotic-induced changes to the Xenopus laevis frog skin microbial communities also result in significant alterations to these animals’ immune gene expression. These findings underscore the interdependence of amphibian skin immune-microbiome interactions.

Group B Streptococcus (GBS; Streptococcus agalactiae) is an important pathobiont capable of colonizing various host environments, contributing to severe perinatal infections. Surface proteins play critical roles in GBS-host interactions; however, comprehensive studies of these proteins’ functions have been limited by genetic manipulation challenges. This study leveraged a CRISPR interference (CRISPRi) library to target genes encoding surface-trafficked proteins in GBS, identifying their roles in modulating macrophage cytokine responses. Bioinformatic analysis of 654 GBS genomes revealed 66 conserved surface protein genes. Using a GBS strain expressing chromosomally integrated dCas9, we generated and validated CRISPRi strains targeting these genes. THP-1 macrophage-like cells were exposed to ethanol-killed GBS variants, and pro-inflammatory cytokines TNF-⍺ and IL-1β were measured. Notably, knockdown of the sip gene, encoding the Surface Immunogenic Protein (Sip), significantly increased IL-1β secretion, implicating Sip in caspase-1-dependent regulation. Furthermore, Δsip mutants demonstrated impaired biofilm formation, reduced adherence to human fetal membranes, and diminished uterine persistence in a mouse colonization model. These findings suggest that Sip modulates GBS-host interactions critical for pathogenesis, underscoring its potential as a therapeutic target or vaccine component.

Staphylococcus aureus and its antibiotic-resistant derivative, methicillin-resistant S. aureus (MRSA), are the leading causative agents of skin and soft tissue infections globally. S. aureus transiently colonizes the skin of healthy adults, and this transient colonization likely precedes an active infection. In recent years, there have been efforts to elucidate specific factors that help MRSA transition to an active infection, but the specific genetic determinants required for this transition following skin colonization are largely unknown. To address this question, we developed a model of asymptomatic colonization of mouse skin by MRSA. From this model, we could determine the MRSA and mouse transcriptional profiles by RNA sequencing (RNAseq) at 5- and 24-hour post-colonization. The fadXDEBA locus, required for fatty acid metabolism, was highly upregulated in our data, as were numerous virulence factors. RNAseq data were confirmed via functional in vitro and in vivo promoter-fusion assays using live bioluminescent imaging of the fadXDEBA locus promoter driving fadB transcription. We analyzed the functional capacity of members of the fadXDEBA locus, which encode crucial enzymatic components of the S. aureus β-oxidation pathway. The genes fadD and fadA modulate MRSA resistance to fosfomycin and other oxidative stressors during growth in the presence of the common skin fatty acid, palmitic acid. Overall, our data demonstrate that there are global changes to the MRSA transcriptome, priming the bacteria for survival by upregulation of known virulence factors and metabolic genes implicated in host skin-nutrient utilization. IMPORTANCE Staphylococcus aureus is a major global agent of skin and soft tissue infections. S. aureus colonizes the skin transiently, an important precursor to infection. However, little is known about how S. aureus adapts to the skin at the transcriptional level. This study provides an overview of the S. aureus transcriptome during mouse skin colonization via RNA sequencing. We identified that the most highly upregulated genes during colonization are related to fatty acid metabolism. The disruption of certain genes in the fatty acid degradation pathway altered resistance of S. aureus to the antibiotic fosfomycin. This study provides an important step in understanding the transcriptional changes that occur during S. aureus skin colonization and may reveal novel targets of therapeutic interest for preventing skin infections.

Biofilms are a cause of chronic, non-healing infections. Staphylococcus aureus is a proficient biofilm-forming pathogen commonly isolated from prosthetic joint infections that develop following primary arthroplasty. Extracellular adherence protein (Eap), previously characterized in planktonic or non-biofilm populations as being an adhesin and immune evasion factor, was recently identified in the exoproteome of S. aureus biofilms. This work demonstrates that Eap and its two functionally orphaned homologs EapH1 and EapH2 contribute to biofilm structure and prevent macrophage invasion and phagocytosis in these communities. Biofilms unable to express Eap proteins demonstrated increased porosity and reduced biomass. We describe the role of Eap proteins in vivo using a mouse model of S. aureus prosthetic joint infection. The Results suggest that the protection conferred to biofilms by Eap proteins is a function of biofilm structural stability that interferes with the leukocyte response to biofilm-associated bacteria.

The protozoan Entamoeba gingivalis commonly colonizes anaerobic periodontal pockets, induces a severe innate immune response, invades gingival mucosa, and kills epithelial cells. E. gingivalis infection is associated with the common oral inflammatory disease periodontitis. DNA variants in vesicle-associated membrane proteins (VAMP) -3 and -8 genes are linked to increased periodontitis risk. These genes mediate host–pathogen interactions, including mucin exocytosis to form protective barriers and matrix metalloproteinase (MMP) secretion in intestinal amoebiasis caused by Entamoeba histolytica. This study aimed to investigate the roles of VAMP3/8 in gingival defense and E. gingivalis infection mechanisms. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing was used to create VAMP3/8-deficient gingival epithelial cells and fibroblasts. Functional analyses included immunofluorescence, enzyme-linked immunosorbent assay (ELISA), cytotoxicity, and collagenase assays. VAMP8 co-localized with mucins in gingival epithelial cells (gECs), and VAMP3 with MMPs in gingival fibroblasts. In gECs, E. gingivalis infection increased mucin (MUC1: 3.6×, MUC21: 14.4×) and interleukin secretion (IL-8, IL-1B: >6×, P = 0.019). VAMP8 deficiency in gECs caused higher cell death (35% vs 4% in controls) with reduced exocytosis of mucins and interleukins. Likewise, E. gingivalis-induced VAMP8 translocation into lipid rafts was lost in VAMP8 knockout cells, validating the participation of VAMP8 in exocytosis. In wild-type but not VAMP3-deficient gingival fibroblasts, E. gingivalis strongly activated collagenases. E. gingivalis effects were more pathogenic than those of the oral anaerobic bacterium Porphyromonas gingivalis. E. gingivalis exploits VAMP8/3-driven exocytosis pathways, driving inflammation and tissue destruction, underscoring its role as a significant periodontal pathogen.

Brucella abortus infects the placenta of its natural bovine host, which results in abortion and transmission of infection to other cattle and to humans. While the metabolism of B. abortus during chronic infection of the mononuclear phagocyte system has been studied, the nutrients fueling growth of B. abortus in the placenta are unknown. We found that in mice, glucose is an important carbon source for B. abortus in the placenta. A gluP mutant lacking a major facilitator superfamily protein required for glucose uptake had diminished growth in the placenta of pregnant mice and caused reduced inflammatory pathology and fetal demise. The gluP mutant was able to replicate intracellularly in a trophoblast cellular model and to cause trophoblast cell death in infected placentas. Attenuated growth of the gluP mutant was maintained in mice conditionally deficient for peroxisome proliferator-activated receptor γ in macrophages, suggesting that M2-like macrophages were not the major site for glucose-dependent growth of B. abortus in the placenta. Our results show that the infected placenta contains multiple distinct nutrient niches and that glucose utilization within the interstitial space of the placenta is an important process contributing to bacterial growth and fetal demise during placental B. abortus infection.

Coxiella burnetii is a gram-negative, obligate intracellular pathogen that causes human Q fever. Within host cells, C. burnetii proliferates in a spacious, acidic, lysosome-derived Coxiella-containing vacuole (CCV) by a process that requires the Dot/Icm type IVB secretion system to deliver effectors that manipulate host cell functions. A previous transposon mutagenesis screen identified the gene cbu0937 as being important for intracellular replication of C. burnetii. Here, the function of Cbu0937 was investigated. The cbu0937::Tn mutant had no detectable defect replicating in the axenic acidified citrate cysteine medium 2. Additionally, intracellular replication of the cbu0937::Tn mutant was not restored by co-infection of host cells with an isogenic wild-type strain of C. burnetii. Thus, the cbu0937::Tn mutant has a cell-intrinsic intracellular replication defect. Intracellular replication of the cbu0937::Tn mutant was restored by complementing the gene in trans with a plasmid encoding either untagged or an epitope-tagged version of Cbu0937. Analysis of the predicted structure of the Cbu0937 protein using AlphaFold revealed high similarity between Cbu0937 and several bacterial porins. Fractionation studies and surface labeling of C. burnetii producing a functional epitope-tagged protein indicated the localization of Cbu0937 to the bacterial outer membrane. From these data, we conclude that cbu0937 encodes a porin that plays an essential role in supporting C. burnetii intracellular replication, which likely involves the acquisition of an important metabolite in the CCV lumen.

Post-weaning diarrhea (PWD) is associated predominantly with enterotoxigenic Escherichia coli (ETEC) and continuously causes significant economic losses to swine producers worldwide. Currently, there are no effective countermeasures against this significant swine disease. Challenges persist in developing vaccines against PWD since ETEC strains produce heterogeneous virulence factors, including F4 (K88) and F18 fimbria and heat-labile toxin (LT), heat-stable toxin type I (STa), heat-stable toxin II (STb), and Shiga toxin type 2e (Stx2e, also causes edema disease). An effective PWD vaccine would induce broadly protective immunity, ideally against two fimbriae and four toxins. In this study, by applying a novel epitope- and structure-based multiepitope-fusion-antigen (MEFA) vaccinology platform, we created a monomeric polyvalent fimbria-toxin protein (fimbria-toxin MEFA) and a holotoxin-structured protein to target PWD virulence determinants (F4 and F18 fimbriae and LT, STa, STb, and Stx2e toxins) and developed a two-component multivalent PWD vaccine candidate, PWDVax. We further applied a heterologous prime-boost immunization strategy and assessed vaccine protection against F18 ETEC-associated PWD. Piglets, after being primed intramuscularly with a fimbria-toxin MEFA monomer protein and boosted orally with live Escherichia coli bacteria producing GM1-binding holotoxin-structured fimbria-toxin MEFA, developed IgG and secretory IgA responses to the target fimbriae and toxins. Challenged with an F18ac ETEC strain, the immunized piglets were protected against watery diarrhea (87.5%) or any diarrhea (66.7%). These data indicated that PWDVax protects against F18 ETEC-associated PWD and can become an effective PWD vaccine. The two-component vaccine and heterologous prime-boost immunization strategy may be instructive for developing neonatal vaccines in general. IMPORTANCE Enterotoxigenic Escherichia coli (ETEC)-associated post-weaning diarrhea (PWD) is a global swine disease, remains a major threat to pig health and well-being, and causes significant economic losses. Currently, there are no effective vaccines available against this disease because of challenges including heterogeneity among ETEC strains (or virulence factors) and difficulties in inducing protective immunity against some key virulence determinants. PWDVax, a two-component PWD vaccine candidate, unprecedentedly targeted two ETEC fimbriae (F4 and F18) and four toxins (LT, STa, STb, and Stx2e), the virulence factors associated with nearly all PWD clinical cases. Under a heterologous prime-boost immunization schedule, it induced broad systemic and mucosal antigen-specific antibodies but also protected weaned piglets against F18 ETEC diarrhea. This makes PWDVax potentially an effective vaccine to protect against PWD, particularly the current F18 ETEC-associated severe PWD outbreaks in the United States. Additionally, the two-component vaccine and heterologous prime-boost immunization strategy may also facilitate the development of effective neonatal vaccines for humans.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that if left untreated can cause reproductive harm. Failure of natural adaptive immunity results in chronic and repeat infections. In efforts to understand the failure of adaptive immunity, we have previously discovered that CD8⁺ T cells, normally integral for controlling intracellular pathogen infections, are misprogrammed by PD-1/PD-L1 signaling during in vivo C. trachomatis infection and fail to mount a protective response. Seeking to uncover the pathways and host factors involved in PD-L1 upregulation that may lead to CD8⁺ T-cell inhibition, we discovered that C. trachomatis triggers the secretion of host type I interferons (IFNs) that are necessary and sufficient to upregulate PD-L1 in vitro. Additionally, secretion of type I IFNs is dependent on C. trachomatis development and its type III secretion system. We have also validated that type I IFNs contribute to upregulation of PD-L1 during C. trachomatis infection in vivo using a mouse model of infection. Overall, these findings reveal that C. trachomatis induction of this host pathway may contribute to adaptive immune evasion.

Cryptococcus neoformans, an invasive basidiomycete fungal pathogen, causes one of the most prevalent, life-threatening diseases in immunocompromised individuals and accounts for ~19% of AIDS-associated deaths. Therefore, understanding the pathogenesis of C. neoformans and its interactions with the host immune system is critical for developing therapeutics against cryptococcosis. Previous studies demonstrated that C. neoformans cells lacking polyphosphate (polyP), an immunomodulatory polyanionic storage molecule, display altered cell surface architecture but unimpaired virulence in a murine model of cryptococcosis. However, the relevance of cell surface changes and the role of hyperaccumulation of polyP in the virulence of C. neoformans remain unclear. Here we show that mutants with abundant polyP due to loss of the polyphosphatases Xpp1 and Epp1 are attenuated for virulence. The double mutant differed from the wild type during disease by demonstrating a higher fungal burden in disseminated organs at the experimental endpoint and by provoking an altered immune response. An analysis of triple mutants lacking the polyphosphatases and the Vtc4 protein for polyP synthesis also caused attenuated virulence in mice, thus suggesting an influence of Xpp1 and/or Epp1 independent of polyP levels. A more detailed characterization revealed that Xpp1 and Epp1 play multiple roles by contributing to the organization of the cell surface, virulence factor production, the response to stress, and mitochondrial function. Overall, we conclude that polyphosphatases have additional functions in the pathobiology of C. neoformans beyond an influence on polyP levels. IMPORTANCE Cryptococcus neoformans causes one of the most prevalent fungal diseases in people with compromised immune systems and accounts for ~19% of AIDS-associated deaths worldwide. The continual increase in the incidence of fungal infections and limited treatment options necessitate the development of new antifungal drugs and improved diagnostics. Polyphosphate (polyP), an under-explored biopolymer, functions as a storage molecule, modulates the host immune response, and contributes to the ability of some fungal and bacterial pathogens to cause disease. However, the role of polyP in cryptococcal disease remains unclear. In this study, we report that the polyphosphatase enzymes that regulate polyP synthesis and turnover contribute to the virulence of C. neoformans in a mouse model of cryptococcosis. The polyphosphatases influenced the survival of C. neoformans in macrophages and altered the host immune response. In addition, the mutants lacking the enzymes have changes in cell surface architecture and size, as well as defects in both mitochondrial function and the stress response. By using mutants defective in the polyphosphatases and polyP synthesis, we demonstrate that many of the phenotypic contributions of the polyphosphatases are independent of polyP.

Ixodes scapularis ticks are an important vector for at least seven tick-borne human pathogens, including a North American Lyme disease spirochete, Borrelia burgdorferi. The ability for these ticks to survive in nature is credited, in part, to their ability to feed on a variety of hosts without triggering an immune response capable of preventing tick feeding. While the ability of nymphal ticks to feed on a variety of hosts has been well documented, the host-parasite interactions between larval I. scapularis and different vertebrate hosts are relatively unexplored. Here we report on the changes in the vertebrate host transcriptome present at the larval tick bite site using the natural I. scapularis host Peromyscus leucopus, a non-natural rodent host, Mus musculus (BALB/c), and humans. We note substantially less evidence of activation of canonical proinflammatory pathways in P. leucopus compared to BALB/c mice and pronounced evidence of inflammation in humans. Pathway enrichment analyses revealed a particularly strong signature of interferon gamma, tumor necrosis factor, and interleukin 1 signaling at the BALB/c and human tick bite sites. We also note that bite sites on BALB/c mice and humans, but not deer mice, show activation of wound-healing pathways. These data provide molecular evidence of the coevolution between larval I. scapularis and P. leucopus and, in addition, expand our overall understanding of I. scapularis feeding.

Respiratory infections with multiresistant Pseudomonas aeruginosa are a major clinical problem, affecting mainly patients with pre-existing lung diseases such as cystic fibrosis (CF) or chronic obstructive pulmonary disease but also immunocompromised or elderly patients. We have previously shown that sphingosine, which is abundantly present on epithelial cells of the respiratory tract in healthy humans and wild-type mice, but almost undetectable on the surface of epithelial cells of the respiratory tract from CF patients and CF mice, efficiently kills many bacterial species in vitro and in vivo. Here, we show that sphingosine very rapidly induces marked changes in the membrane of P. aeruginosa with a rolling of the membrane followed by destruction of the bacteria. Sphingosine induced a degradation of cardiolipin via the maintenance of lipid asymmetry (Mla) system in P. aeruginosa. Degradation of cardiolipin induced by sphingosine is prevented in P. aeruginosa mutants of MlaY and reduced in mutants of MlaZ and MlaA. Mutants of MlaY and MlaZ were resistant to sphingosine-induced death of P. aeruginosa. In summary, our data indicate that sphingosine induces the death of P. aeruginosa by a persisting degradation of cardiolipin by the Mla system leading to severe membrane changes in bacteria, while leaving mammalian cells, devoid of cardiolipin in their plasma membrane, alive.