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Salmonella enterica expresses two virulence-related type III secretion systems (T3SSs) encoded in Salmonella pathogenicity island 1 (SPI1) and SPI2, respectively. SseK1 is a poorly characterized substrate of the SPI2-encoded T3SS. Here, we show that this effector is essential to get full virulence both in oral and intraperitoneal mice infections, i...
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SrfJ is an effector of the Salmonella pathogenicity island 2-encoded type III secretion system. Salmonella enterica serovar Typhimurium expresses srfJ under two disparate sets of conditions: media with low Mg²⁺ and low pH, imitating intravacuolar conditions, and media with myo-inositol (MI), a carbohydrate that can be used by Salmonella as sole car...
Salmonella enterica serovar Typhimurium is a human and animal pathogen that uses type III secretion system effectors to manipulate the host cell and fulfill infection. SseK1 is a Salmonella effector with glycosyltransferase activity. We carried out a yeast two-hybrid screen and have identified tubulin-binding cofactor B (TBCB) as a new binding part...
The ability of Pseudomonas syringae pv. tomato DC3000 to be pathogenic on plants is dependent upon the type III secretion system (TTSS) and the effector proteins it translocates into plant cells. A draft sequence of DC3000 is currently being analysed for hop (Hrp outer protein) genes that encode TTSS substrates. Several different approaches were us...
Enteric pathogens employ sophisticated strategies to colonize and infect mammalian hosts. Gram-negative bacteria, such as Escherichia coli , Salmonella , and Campylobacter jejuni , are among the leading causes of gastrointestinal tract infections worldwide. The virulence strategies of many of these Gram-negative pathogens rely on type III secretion...
Dozens of Gram negative pathogens use one or more type III secretion systems (T3SS) to disarm host defenses or occupy a beneficial niche during infection of a host organism. While the T3SS represents an attractive drug target and dozens of compounds with T3SS inhibitory activity have been identified, few T3SS inhibitors have been validated and mode...
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... Salmonella harbors two Pathogenicity Islands-SPI1 and SPI2. Although encoded by a gene located outside SPI1 and SPI2, SseK1 was shown to be secreted by both T3SS1 and T3SS2 secretion systems 49 . Once translocated, SseK1 glycosylates host proteins to reduce host immune responses during infection 10 . ...
Type III secretion system (T3SS) effector proteins are primarily recognized for binding host proteins to subvert host immune response during infection. Besides their known host target proteins, several T3SS effectors also interact with endogenous bacterial proteins. Here we demonstrate that the Salmonella T3SS effector glycosyltransferase SseK1 glycosylates the bacterial two-component response regulator OmpR on two arginine residues, R15 and R122. Arg-glycosylation of OmpR results in reduced expression of ompF, a major outer membrane porin gene. Glycosylated OmpR has reduced affinity to the ompF promoter region, as compared to the unglycosylated form of OmpR. Additionally, the Salmonella ΔsseK1 mutant strain had higher bile salt resistance and increased capacity to form biofilms, as compared to WT Salmonella, thus linking OmpR glycosylation to several important aspects of bacterial physiology.
... Several in vivo studies have explored the role of the SseKs in murine infection, albeit with mixed results [123,124,139,[146][147][148][149]. Clearly more studies are required to clarify the contribution of SseK effectors to Salmonella pathogenicity in vivo, as well as continued efforts to further define the physiological substrates of each effector. ...
Salmonella injects over 40 virulence factors, termed effectors, into host cells to subvert diverse host cellular processes. Of these 40 Salmonella effectors, at least 25 have been described as mediating eukaryotic-like, biochemical post-translational modifications (PTMs) of host proteins, altering the outcome of infection. The downstream changes mediated by an effector's enzymatic activity range from highly specific to multifunctional, and altogether their combined action impacts the function of an impressive array of host cellular processes, including signal transduction, membrane trafficking, and both innate and adaptive immune responses. Salmonella and related Gram-negative pathogens have been a rich resource for the discovery of unique enzymatic activities, expanding our understanding of host signalling networks, bacterial pathogenesis as well as basic biochemistry. In this review, we provide an up-to-date assessment of host manipulation mediated by the Salmonella type III secretion system injectosome, exploring the cellular effects of diverse effector activities with a particular focus on PTMs and the implications for infection outcomes. We also highlight activities and functions of numerous effectors that remain poorly characterized.
... Several approaches involving chromosomally-encoded fusions have been developed as they are advantageous over plasmid-encoded fusions since they result in a single-copy gene fusion whose expression depends on native promoters [6,[15][16][17]. In this context, cyaA' has arisen as an appealing reporter gene for studying the expression, secretion, and translocation of effector proteins in the case of several pathogenic bacteria [5,15,[18][19][20][21][22]. The advantage of using a CyaA' enzymatic tag is that the expression and secretion of the corresponding fusion protein can be detected in bacterial cultures in vitro by immunoblotting [18,21,22], and also translocation of these fusions into host cells can be ...
... Several approaches involving chromosomally-encoded fusions have been developed as they are advantageous over plasmid-encoded fusions since they result in a single-copy gene fusion whose expression depends on native promoters [6,[15][16][17]. In this context, cyaA' has arisen as an appealing reporter gene for studying the expression, secretion, and translocation of effector proteins in the case of several pathogenic bacteria [5,15,[18][19][20][21][22]. The advantage of using a CyaA' enzymatic tag is that the expression and secretion of the corresponding fusion protein can be detected in bacterial cultures in vitro by immunoblotting [18,21,22], and also translocation of these fusions into host cells can be monitored by its calmodulin-dependent adenylate cyclase activity, measuring the cAMP levels in infected cells [5,15,[18][19][20][21][22]. ...
... In this context, cyaA' has arisen as an appealing reporter gene for studying the expression, secretion, and translocation of effector proteins in the case of several pathogenic bacteria [5,15,[18][19][20][21][22]. The advantage of using a CyaA' enzymatic tag is that the expression and secretion of the corresponding fusion protein can be detected in bacterial cultures in vitro by immunoblotting [18,21,22], and also translocation of these fusions into host cells can be monitored by its calmodulin-dependent adenylate cyclase activity, measuring the cAMP levels in infected cells [5,15,[18][19][20][21][22]. ...
The type III secretion systems (T3SS) encoded in pathogenicity islands SPI-1 and SPI-2 are key virulence factors of Salmonella. These systems translocate proteins known as effectors into eukaryotic cells during infection. To characterize the functionality of T3SS effectors, gene fusions to the CyaA’ reporter of Bordetella pertussis are often used. CyaA’ is a calmodulin-dependent adenylate cyclase that is only active within eukaryotic cells. Thus, the translocation of an effector fused to CyaA’ can be evaluated by measuring cAMP levels in infected cells. Here, we report the construction of plasmids pCyaA’-Kan and pCyaA’-Cam, which contain the ORF encoding CyaA’ adjacent to a cassette that confers resistance to kanamycin or chloramphenicol, respectively, flanked by Flp recombinase target (FRT) sites. A PCR product from pCyaA’-Kan or pCyaA’-Cam containing these genetic elements can be introduced into the bacterial chromosome to generate gene fusions by homologous recombination using the Red recombination system from bacteriophage λ. Subsequently, the resistance cassette can be removed by recombination between the FRT sites using the Flp recombinase. As a proof of concept, the plasmids pCyaA’-Kan and pCyaA’-Cam were used to generate unmarked chromosomal fusions of 10 T3SS effectors to CyaA’ in S. Typhimurium. Each fusion protein was detected by Western blot using an anti-CyaA’ monoclonal antibody when the corresponding mutant strain was grown under conditions that induce the expression of the native gene. In addition, T3SS-1-dependent secretion of fusion protein SipA-CyaA’ during in vitro growth was verified by Western blot analysis of culture supernatants. Finally, efficient translocation of SipA-CyaA’ into HeLa cells was evidenced by increased intracellular cAMP levels at different times of infection. Therefore, the plasmids pCyaA’-Kan and pCyaA’-Cam can be used to generate unmarked chromosomal cyaA’ translational fusion to study regulated expression, secretion and translocation of Salmonella T3SS effectors into eukaryotic cells.
... NleB expression is induced and secreted in conditions that permit the expression of the EspADB translocon 38 . In Salmonella, SseK1 is expressed in both SPI1-and SPI2-inducing conditions 39 , but is higher in SPI2-inducing conditions 40 . SseK2 and SseK3 are expressed in SPI2-inducing conditions and are dependent upon the SsrA/SsrB two-component regulatory system 20 . ...
Type III secretion system effector proteins have primarily been characterized for their interactions with host cell proteins and their ability to disrupt host signaling pathways. We are testing the hypothesis that some effectors are active within the bacterium, where they modulate bacterial signal transduction and physiology. We previously determined that the Citrobacter rodentium effector NleB possesses an intra-bacterial glycosyltransferase activity that increases glutathione synthetase activity to protect the bacterium from oxidative stress. Here we investigated the potential intra-bacterial activities of NleB orthologs in Salmonella enterica and found that SseK1 and SseK3 mediate resistance to methylglyoxal. SseK1 glycosylates specific arginine residues on four proteins involved in methylglyoxal detoxification, namely GloA (R9), GloB (R190), GloC (R160), and YajL (R149). SseK1-mediated Arg-glycosylation of these four proteins significantly enhances their catalytic activity, thus providing another important example of the intra-bacterial activities of type three secretion system effector proteins. These data are also the first demonstration that a Salmonella T3SS effector is active within the bacterium.
... Intracellular pathogen Salmonella entrica strains secreted three Salmonella pathogenicity island 2 (SPI-2) effector SseK1, SseK2, and SseK3 (Kujat Choy et al., 2004;Brown et al., 2011;Baison-Olmo et al., 2015;El Qaidi et al., 2017;Gunster et al., 2017;Yang et al., 2018;Araujo-Garrido et al., 2020;Meng et al., 2020). Crystal structure studies show that NleB, SseK1, and SseK3 belong to the GT-A family glycosyltransferase (Esposito et al., 2018;Park et al., 2018;Ding et al., 2019;Araujo-Garrido et al., 2020;Pan et al., 2020). ...
Death receptor signaling is critical for cell death, inflammation, and immune homeostasis. Hijacking death receptors and their corresponding adaptors through type III secretion system (T3SS) effectors has been evolved to be a bacterial evasion strategy. NleB from enteropathogenic Escherichia coli (EPEC) and SseK1/2/3 from Salmonella enterica serovar Typhimurium (S. Typhimurium) can modify some death domain (DD) proteins through arginine-GlcNAcylation. Here, we performed a substrate screen on 12 host DD proteins with conserved arginine during EPEC and Salmonella infection. NleB from EPEC hijacked death receptor signaling through tumor necrosis factor receptor 1 (TNFR1)-associated death domain protein (TRADD), FAS-associated death domain protein (FADD), and receptor-interacting serine/threonine-protein kinase 1 (RIPK1), whereas SseK1 and SseK3 disturbed TNF signaling through the modification of TRADD Arg235/Arg245 and TNFR1 Arg376, respectively. Furthermore, mouse infection studies showed that SseK1 but not SseK3 rescued the bacterial colonization deficiency contributed by the deletion of NleBc (Citrobacter NleB), indicating that TRADD was the in vivo substrate. The result provides an insight into the mechanism by which attaching and effacing (A/E) pathogen manipulate TRADD-mediated signaling and evade host immune defense through T3SS effectors.
... Since most of the isolated clones corresponded to TBCB, we focused our study on the interaction of SseK1 with this host protein. To confirm the results obtained with the yeast two-hybrid approach, the bacterial glutathione S-transferase (GST) expression system was used to produce GST or GST-TBCB in a strain of Salmonella expressing a chromosomal SseK1-3xFLAG fusion (strain SV7071) [15]. Then, we performed affinity purification using glutathione-agarose beads to isolate GST proteins, and we further analyzed the presence of SseK1 by western blot. ...
Salmonella enterica serovar Typhimurium is a human and animal pathogen that uses type III secretion system effectors to manipulate the host cell and fulfill infection. SseK1 is a Salmonella effector with glycosyltransferase activity. We carried out a yeast two-hybrid screen and have identified tubulin-binding cofactor B (TBCB) as a new binding partner for this effector. SseK1 catalyzed the addition of N-acetylglucosamine to arginine on TBCB, and its expression promoted the stabilization of the microtubule cytoskeleton of HEK293T cells. The conserved Asp-x-Asp (DxD) motif that is essential for the activity of SseK1 was required for the binding and modification of TBCB and for the effect on the cytoskeleton. Our study has identified a novel target for SseK1 and suggests that this effector may have a role in the manipulation of the host cell microtubule network to provide a safe niche for this pathogen.
... Optimal expression of T3SS1 and T3SS2 can be achieved in vitro using appropriate media and culture conditions: LB medium with high osmolarity and microaerophilic conditions for the T3SS1, and LPM minimal medium with low pH, low Mg ++ concentration, and aeration conditions for the T3SS2. Significant amounts of SseK1 are produced under both sets of conditions [112], although transcription is higher under SPI2-inducing conditions [136]. This may be a consequence of the fact that transcription of the gene sseK1 is not regulated by the main regulators of SPI1 or SPI2 (HilA, HilD, and SsrB), although it is directly activated by PhoP in a SsrB-independent manner. ...
... This may be a consequence of the fact that transcription of the gene sseK1 is not regulated by the main regulators of SPI1 or SPI2 (HilA, HilD, and SsrB), although it is directly activated by PhoP in a SsrB-independent manner. This pattern of expression is consistent with SseK1 being translocated through T3SS1 and T3SS2, with different patterns and kinetics depending on the specific host cell type (epithelial, macrophages, or fibroblasts) [136]. In contrast to SseK1, SseK2 and SseK3 were detected specifically under SPI2-inducing conditions and their expression was dependent on the SsrA/SsrB system, suggesting coordination with the expression of the T3SS2 [111,112]. ...
... No difference was detected between the sseK1 sseK2 sseK3 triple mutant and the sseK1 sseK2 double mutant, suggesting that SseK3 has no role in virulence in this system [111]. Mild attenuation was also reported for an sseK1 single mutant of S. enterica serovar Typhimurium after intraperitoneal and oral infections of BALB/c mice using the CI analysis [136]. In addition, an sseK1 deletion mutant of S. enterica serovar Enteritidis showed decreased formation of biofilm, reduced intracellular survival in activated mouse peritoneal macrophages, and reduced pathogenicity in BALB/c mice [167]. ...
Type III secretion systems are used by many Gram-negative bacterial pathogens to inject proteins, known as effectors, into the cytosol of host cells. These virulence factors interfere with a diverse array of host signal transduction pathways and cellular processes. Many effectors have catalytic activities to promote post-translational modifications of host proteins. This review focuses on a family of effectors with glycosyltransferase activity that catalyze addition of N-acetyl-d-glucosamine to specific arginine residues in target proteins, leading to reduced NF-κB pathway activation and impaired host cell death. This family includes NleB from Citrobacter rodentium, NleB1 and NleB2 from enteropathogenic and enterohemorrhagic Escherichia coli, and SseK1, SseK2, and SseK3 from Salmonella enterica. First, we place these effectors in the general framework of the glycosyltransferase superfamily and in the particular context of the role of glycosylation in bacterial pathogenesis. Then, we provide detailed information about currently known members of this family, their role in virulence, and their targets.
... The standard culture medium for all bacteria was Luria-Bertani (LB) broth. For SPI2-inducing conditions, bacteria were inoculated in LPM at pH 5.8 (61) and incubated overnight at 37°C with shaking. The following supplements were added to LPM to allow growth of aro strains: Phe, 40 g/ml; Trp, 40 g/ml; Tyr, 40 g/ml; p-aminobenzoate, 10 g/ml; 2,3-dihydroxybenzoate, 10 g/ml. ...
Pseudomonas aeruginosa is a common Gram-negative opportunistic pathogen that is intrinsically resistant to a wide range of antibiotics. The development of a broadly protective vaccine against P. aeruginosa remains a major challenge. Here, we used an attenuated strain of Salmonella enterica serovar Typhimurium as a vehicle to express P. aeruginosa antigens. A fusion between the S. enterica type III secretion effector protein SseJ and the P. aeruginosa antigen PcrV expressed under the control of the sseA promoter was translocated by Salmonella into host cells in vitro and elicited the generation of specific antibodies in mice. Mice immunized with attenuated Salmonella expressing this fusion had reduced bacterial loads in the spleens and lungs and lower serum levels of proinflammatory cytokines than control mice after P. aeruginosa infection. Importantly, immunized mice also showed significantly enhanced survival in this model. These results suggest that type III secretion effectors of S. enterica are appropriate carriers in the design of a live vaccine to prevent infections caused by P. aeruginosa .
IMPORTANCE The Gram-negative bacterium Pseudomonas aeruginosa is an important opportunistic pathogen that causes infections in cystic fibrosis and hospitalized patients. Therapeutic treatments are limited due to the emergence and spread of new antibiotic-resistant strains. In this context, the development of a vaccine is a priority. Here, we used an attenuated strain of Salmonella enterica serovar Typhimurium as a vehicle to express and deliver the Pseudomonas antigen PcrV. This vaccine induced the generation of specific antibodies in mice and protected them from lethal infections with P. aeruginosa . This is an important step toward the development of an effective vaccine for the prevention of infections caused by P. aeruginosa in humans.
... To determine the binding of PhoP to DNA probe of hlyF promoter, EMSAs were conducted using the commercialized EMSA kit (Invitrogen, California) according to the manufacturer's protocol (15). To obtain phosphorylated PhoP, purified PhoP was phosphorylated with acetylphosphate (Sigma) according to the previous study (37). PhoP (10 µM) was incubated in 100 µl of phosphorylation buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, and 5 mM MgCl2) containing 20 mM acetylphosphate for 2 h at 37°C. ...
The extraintestinal pathogenic Escherichia coli (ExPEC) is a typical facultative intracellular bacterial pathogen. Sensing the environmental stimuli and undertaking adaptive change are crucial for ExPEC to successfully colonize in specific extraintestinal niches. The previous studies show that pathogens exploit two-component systems (TCSs) in response to the host environments during its infection. The PhoP/PhoQ is a typical TCS which is ubiquitous in Gram-negative bacteria. However, there is an incompletely understanding about critical regulatory roles of PhoP/PhoQ in ExPEC pathogenesis. Conjugative ColV-related plasmids are responsible for ExPEC virulence, which is associated with ExPEC zoonotic risk. In this study, the molecular characteristics of HlyF, Mig-14 ortholog (Mig-14p), and OmpT variant (OmpTp) encoded by ColV plasmids were identified. Mig-14p and OmpTp played important roles in conferring ExPEC resistance to cationic antimicrobial peptides (CAMPs) during the infection. Moreover, HlyF and Mig-14p acted as intracellular survival factors to promote ExPEC resistance to macrophages killing. The hlyF and Mig-14p formed an operon in ExPEC ColV plasmid, and PhoP acted as a transcriptional activator of hlyF operon by directly binding to the PhlyF promoter. The acidic pH and CAMPs could additively stimulate ExPEC PhoQ/PhoP activities to upregulate the expression of HlyF and Mig-14p. Our studies revealed that the novel PhoP/PhoQ-HlyF signaling pathway directly upregulates the production of ExPEC outer membrane vesicles. Furthermore, our study first clarified that this PhoP/PhoQ-HlyF pathway was essential for ExPEC intracellular survival in macrophages. It was required to prevent the fusion of ExPEC-containing phagosomes with lysosomes. Moreover, PhoP/PhoQ-HlyF pathway facilitated the inhibition of the phagolysosomal acidification and disruption of the phagolysosomal membranes. In addition, this pathway might promote the formation of ExPEC-containing autophagosome during ExPEC replication in macrophages. Collectively, our studies suggested that PhoP/PhoQ system and CloV plasmids could facilitate ExPEC survival and replication within macrophages.
... Specificity of secretion is achieved, at least for some effectors, simply by coexpression between the particular T3SS and its effectors. An example is SseK1: when expressed from a constitutive promoter it can be secreted through T3SS1 at earlier time points p.i. than when expressed from its own promoter (Baisón-Olmo et al., 2015). Coexpression of T3SS1 and srfJ from PiolE may take place in plants due to the presence of MI, making it possible the delivery of the effector through this way. ...
SrfJ is an effector of the Salmonella pathogenicity island 2-encoded type III secretion system. Salmonella enterica serovar Typhimurium expresses srfJ under two disparate sets of conditions: media with low Mg²⁺ and low pH, imitating intravacuolar conditions, and media with myo-inositol (MI), a carbohydrate that can be used by Salmonella as sole carbon source. We investigated the molecular basis for this dual regulation. Here, we provide evidence for the existence of two distinct promoters that control the expression of srfJ. A proximal promoter, PsrfJ, responds to intravacuolar signals and is positively regulated by SsrB and PhoP and negatively regulated by RcsB. A second distant promoter, PiolE, is negatively regulated by the MI island repressor IolR. We also explored the in vivo activity of these promoters in different hosts. Interestingly, our results indicate that the proximal promoter is specifically active inside mammalian cells whereas the distant one is expressed upon Salmonella colonization of plants. Importantly, we also found that inappropriate expression of srfJ leads to reduced proliferation inside macrophages whereas lack of srfJ expression increases survival and decreases activation of defense responses in plants. These observations suggest that SrfJ is a relevant factor in the interplay between Salmonella and hosts of different kingdoms.
