Identification of Mammalian Proteins That Collaborate with Type III Secretion System Function: Involvement of a Chemokine Receptor in Supporting Translocon Activity

ABSTRACTThe type III secretion system (T3SS) is a highly conserved protein delivery system found in multiple Gram-negative pathogens, includingYersinia pseudotuberculosis. Most studies of Yersinia species type III intoxication of host cells have focused on the bacterial determinants that promote assembly and function of the secretion system. In this study, we performed a pooled RNA interference (RNAi) screen to identify mammalian host proteins required for the cytotoxic effects associated with the Yersinia translocated substrate YopE, a GTPase-activating protein (GAP) that inactivates the small Rho GTPases. Cell populations were positively selected for short hairpin RNAs (shRNAs) that interfere with YopE activity using a combination of fluorescence resonance energy transfer (FRET) and flow cytometry, and the degree of enrichment was determined by deep sequencing. Analysis of the candidates identified by the enrichment process revealed that many were important for the initial step of Y. pseudotuberculosis T3SS function, YopB/D pore formation. These candidates included shRNA that depleted downstream effectors of RhoA signaling, coated pit formation, and receptors involved in cell signaling, including the chemokine receptor CCR5 (chemokine [C-C motif] receptor 5). Depletion of CCR5 in 293T cells yielded a defect in YopB/D pore formation and effector translocation, while both phenotypes could be complemented by overexpression of CCR5 protein. Yop effector translocation was also decreased in isolated primary phagocytic cells from aCcr5−/−knockout mouse. We postulate that CCR5 acts to promote translocation by modulating cytoskeletal activities necessary for proper assembly of the YopB/D translocation pore. Overall, this study presents a new approach to investigating the contribution of the host cell to T3SS in Y. pseudotuberculosis.IMPORTANCEMany Gram-negative bacteria require type III secretion systems (T3SS) for host survival, making these highly specialized secretion systems good targets for antimicrobial agents. After the bacterium binds to host cells, T3SS deposit proteins into the cytosol of host cells through a needle-like appendage and a protein translocon channel. Translocation of proteins via this system is highly regulated, and the contribution of the host cell in promoting assembly and insertion of the channel into the plasma membrane, folding of the bacterial proteins, and trafficking of these substrates are all poorly characterized events. In this study, we identified host cell proteins important for activity of YopE, aYersinia pseudotuberculosisT3SS-delivered protein. The results demonstrate that insertion and assembly of the translocon are complex processes, requiring a variety of membrane trafficking and cytoskeletal processes, as well as a surprising role for cell surface signaling molecules in supporting proper function.

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Microbiota and Pathogen Proteases Modulate Type III Secretion Activity in EnterohemorrhagicEscherichia coli

mBio ◽

10.1128/mbio.02204-18 ◽

2018 ◽

Vol 9 (6) ◽

Cited By ~ 7

Author(s):

Elizabeth A. Cameron ◽

Meredith M. Curtis ◽

Aman Kumar ◽

Gary M. Dunny ◽

Vanessa Sperandio

Keyword(s):

Gut Microbiota ◽

Host Cell ◽

Type Iii Secretion ◽

Cell Function ◽

Enteric Pathogens ◽

Host Cells ◽

Secretion Systems ◽

Gram Negative ◽

Content Type ◽

Type Iii

ABSTRACTEnteric pathogens have complex interactions with the gut microbiota. Most of what is known about them has focused on microbiota-derived metabolites or small molecules that serve as nutrients and/or signals to aid in growth or transcriptionally regulate virulence gene expression. A common virulence strategy is to express a type III secretion system (T3SS), which is a molecular syringe deployed by many Gram-negative pathogens to hijack host cell function. EnterohemorrhagicEscherichiacoli(EHEC) requires its T3SS to colonize the intestinal tract and cause disease. Here we report that a prominent member of the intestinal microbiota,Bacteroides thetaiotamicron(Bt), secretes proteases that cleave the translocon of the T3SS of EHEC to enhance effector translocation into host cells. This is in contrast from an endogenous protease from EHEC itself (namely, EspP) that cleaves the translocon protein EspB in a different site to limit effector translocation. The EspB protein forms the T3SS pore in mammalian cells, and pore proteins are conserved in the T3SSs from several pathogens. This is the first demonstration of a commensal species directly processing a pathogen’s T3SS, posing a new paradigm for how the microbiota can influence the severity of disease caused by bacterial pathogens. Because T3SSs are employed by many pathogens, this phenomenon has broad implications to commensal-pathogen relationships.IMPORTANCEThe gut microbiota is usually regarded as providing colonization resistance against enteric pathogens. However, some pathogens evolved to thrive with the aid of certain members of the microbiota. Several Gram-negative bacteria employ type three secretion systems (T3SSs), which are molecular syringes that deliver effector proteins to host cells, hijacking host cell function. Here we show that the T3SS of enterohemorrhagicE. coli(EHEC) is cleaved by self and microbiota-derived proteases. Self-cleavage limits effector translocation, while cleavage by the microbiota memberBacteroides thetaiotamicron(Bt) exacerbates effector translocation and lesion formation on epithelial cells.

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The Ruler Protein EscP of the Enteropathogenic Escherichia coli Type III Secretion System Is Involved in Calcium Sensing and Secretion Hierarchy Regulation by Interacting with the Gatekeeper Protein SepL

mBio ◽

10.1128/mbio.01733-16 ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 23

Author(s):

Lihi Shaulov ◽

Jenia Gershberg ◽

Wanyin Deng ◽

B. Brett Finlay ◽

Neta Sal-Man

Keyword(s):

Host Cell ◽

Type Iii Secretion ◽

Bacterial Pathogens ◽

Type Iii Secretion System ◽

Secretion System ◽

Bacterial Virulence ◽

Effector Proteins ◽

Host Cells ◽

Gram Negative ◽

Type Iii

ABSTRACT The type III secretion system (T3SS) is a multiprotein complex that plays a central role in the virulence of many Gram-negative bacterial pathogens. To ensure that effector proteins are efficiently translocated into the host cell, bacteria must be able to sense their contact with the host cell. In this study, we found that EscP, which was previously shown to function as the ruler protein of the enteropathogenic Escherichia coli T3SS, is also involved in the switch from the secretion of translocator proteins to the secretion of effector proteins. In addition, we demonstrated that EscP can interact with the gatekeeper protein SepL and that the EscP-SepL complex dissociates upon a calcium concentration drop. We suggest a model in which bacterial contact with the host cell is accompanied by a drop in the calcium concentration that causes SepL-EscP complex dissociation and triggers the secretion of effector proteins. IMPORTANCE The emergence of multidrug-resistant bacterial strains, especially those of pathogenic bacteria, has serious medical and clinical implications. At the same time, the development and approval of new antibiotics have been limited for years. Recently, antivirulence drugs have received considerable attention as a novel antibiotic strategy that specifically targets bacterial virulence rather than growth, an approach that applies milder evolutionary pressure on the bacteria to develop resistance. A highly attractive target for the development of antivirulence compounds is the type III secretion system, a specialized secretory system possessed by many Gram-negative bacterial pathogens for injecting virulence factors (effectors) into host cells. In this study, we shed light on the molecular mechanism that allows bacteria to sense their contact with the host cell and to respond with the timed secretion of effector proteins. Understanding this critical step for bacterial virulence may provide a new therapeutic strategy. IMPORTANCE The emergence of multidrug-resistant bacterial strains, especially those of pathogenic bacteria, has serious medical and clinical implications. At the same time, the development and approval of new antibiotics have been limited for years. Recently, antivirulence drugs have received considerable attention as a novel antibiotic strategy that specifically targets bacterial virulence rather than growth, an approach that applies milder evolutionary pressure on the bacteria to develop resistance. A highly attractive target for the development of antivirulence compounds is the type III secretion system, a specialized secretory system possessed by many Gram-negative bacterial pathogens for injecting virulence factors (effectors) into host cells. In this study, we shed light on the molecular mechanism that allows bacteria to sense their contact with the host cell and to respond with the timed secretion of effector proteins. Understanding this critical step for bacterial virulence may provide a new therapeutic strategy.

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Edwardsiella tarda-Induced Cytotoxicity Depends on Its Type III Secretion System and Flagellin

Infection and Immunity ◽

10.1128/iai.01065-13 ◽

2014 ◽

Vol 82 (8) ◽

pp. 3436-3445 ◽

Cited By ~ 22

Author(s):

Hai-Xia Xie ◽

Jin-Fang Lu ◽

Nathalie Rolhion ◽

David W. Holden ◽

Pin Nie ◽

...

Keyword(s):

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Host Cells ◽

Edwardsiella Tarda ◽

Dependent Manner ◽

Gram Negative ◽

Content Type ◽

Murine Bone Marrow ◽

Type Iii

ABSTRACTMany Gram-negative bacteria utilize a type III secretion system (T3SS) to translocate virulence proteins into host cells to cause diseases. In responding to infection, macrophages detect some of the translocated proteins to activate caspase-1-mediated cell death, called pyroptosis, and secretion of proinflammatory cytokines to control the infection.Edwardsiella tardais a Gram-negative enteric pathogen that causes hemorrhagic septicemia in fish and both gastrointestinal and extraintestinal infections in humans. In this study, we report that the T3SS ofE. tardafacilitates its survival and replication in murine bone marrow-derived macrophages, andE. tardainfection triggers pyroptosis of infected macrophages from mice and fish and increased secretion of the cytokine interleukin 1β in a T3SS-dependent manner. Deletion of the flagellin genefliCofE. tardaresults in decreased cytotoxicity for infected macrophages and does not attenuate its virulence in a fish model of infection, whereas upregulated expression of FliC in thefliCmutant strain reduces its virulence. We propose that the host controlsE. tardainfection partially by detecting FliC translocated by the T3SS, whereas the bacteria downregulate the expression of FliC to evade innate immunity.

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Type III Secretion System-Dependent Translocation of Ectopically Expressed Yop Effectors into Macrophages by Intracellular Yersinia pseudotuberculosis

Infection and Immunity ◽

10.1128/iai.05396-11 ◽

2011 ◽

Vol 79 (11) ◽

pp. 4322-4331 ◽

Cited By ~ 12

Author(s):

Yue Zhang ◽

Galina Romanov ◽

James B. Bliska

Keyword(s):

Cell Death ◽

Type Iii Secretion ◽

Yersinia Pseudotuberculosis ◽

Bacterial Pathogen ◽

Type Iii Secretion System ◽

Inducible Promoter ◽

Secretion System ◽

Host Cells ◽

Content Type ◽

Type Iii

ABSTRACTYersinia pseudotuberculosisis a Gram-negative bacterial pathogen. Virulence inY. pseudotuberculosisrequires the plasmid-encoded Ysc type III secretion system (T3SS), which functions to translocate a set of effectors called Yops into infected host cells. The effectors function to antagonize phagocytosis (e.g., YopH) or to induce apoptosis (YopJ) in macrophages infected withY. pseudotuberculosis. Additionally, when antiphagocytosis is incomplete andY. pseudotuberculosisis internalized by macrophages, the bacterium can survive in phagosomes. Previous studies have shown that delivery of effectors into host cells occurs efficiently whenYersiniais extracellular. However, it is not clear whether the T3SS can be utilized by intracellularY. pseudotuberculosisto translocate Yops. This possibility was investigated here usingY. pseudotuberculosisstrains that express YopJ or YopH under the control of an inducible promoter. Bone marrow-derived murine macrophages were infected with these strains under conditions that prevented the survival of extracellular bacteria. Effector translocation was detected by measuring apoptosis or the activities of Yop-β-lactamase fusion proteins. Results showed that macrophages underwent apoptosis when YopJ expression was induced prior to phagocytosis, confirming that delivery of this effector prior to or during uptake is sufficient to cause cell death. However, macrophages also underwent apoptosis when YopJ was ectopically expressed after phagocytosis; furthermore, expression of the translocator YopB from intracellular bacteria also resulted in increased cell death. Analysis by microscopy showed that translocation of ectopically expressed YopH- or YopJ-β-lactamase fusions could be correlated with the presence of viableY. pseudotuberculosisin macrophages. Collectively, our results suggest that the Ysc T3SS ofY. pseudotuberculosiscan function within macrophage phagosomes to translocate Yops into the host cytosol.

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A Type III Secretion System Inhibitor Targets YopD while Revealing Differential Regulation of Secretion in Calcium-Blind Mutants of Yersinia pestis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01170-13 ◽

2013 ◽

Vol 58 (2) ◽

pp. 839-850 ◽

Cited By ~ 22

Author(s):

Danielle L. Jessen ◽

David S. Bradley ◽

Matthew L. Nilles

Keyword(s):

Yersinia Pestis ◽

Type Iii Secretion ◽

Yersinia Pseudotuberculosis ◽

Type Iii Secretion System ◽

Secretion System ◽

Secretion Systems ◽

Content Type ◽

Type Iii ◽

Regulatory Processes ◽

Secretion Inhibition

ABSTRACTNumerous Gram-negative pathogens rely upon type III secretion (T3S) systems to cause disease. Several small-molecule inhibitors of the type III secretion systems have been identified; however, few targets of these inhibitors have been elucidated. Here we report that 2,2′-thiobis-(4-methylphenol) (compound D), inhibits type III secretion inYersinia pestis,Yersinia pseudotuberculosis, andPseudomonas aeruginosa. YopD, a protein involved in the formation of the translocon and regulatory processes of the type III secretion system, appears to play a role in the inhibition of secretion by compound D. The use of compound D in T3S regulatory mutants demonstrated a difference in secretion inhibition in the presence and absence of calcium. Interestingly, compound D was effective only under conditions without calcium, indicating that a secretion-active needle structure may be necessary for compound D to inhibit secretion.

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Cloning and Transfer of the Salmonella Pathogenicity Island 2 Type III Secretion System for Studies of a Range of Gram-Negative Genera

Applied and Environmental Microbiology ◽

10.1128/aem.00952-07 ◽

2007 ◽

Vol 73 (18) ◽

pp. 5911-5918 ◽

Cited By ~ 14

Author(s):

James W. Wilson ◽

Clint Coleman ◽

Cheryl A. Nickerson

Keyword(s):

Type Iii Secretion ◽

Vaccine Development ◽

Type Iii Secretion System ◽

Pathogenicity Island ◽

Secretion System ◽

Host Cells ◽

Secretion Systems ◽

Gram Negative ◽

Type Iii ◽

Type Iii Secretion Systems

ABSTRACT The engineering of bacterial strains with specific phenotypes frequently requires the use of blocks or “cassettes” of genes that act together to perform a desired function. The potential benefits of utilizing type III secretion systems in this regard are becoming increasingly realized since these systems can be used to direct interactions with host cells for beneficial purposes such as vaccine development, anticancer therapies, and targeted protein delivery. However, convenient methods to clone and transfer type III secretion systems for studies of a range of different types of bacteria are lacking. In addition to functional applications, such methods would also reveal important information about the evolution of a given type III secretion system, such as its ability to be expressed and functional outside of the strain of origin. We describe here the cloning of the Salmonella enterica serovar Typhimurium pathogenicity island 2 (SPI-2) type III secretion system onto a vector that can be easily transferred to a range of gram-negative bacterial genera. We found that expression of the cloned SPI-2 system in different Gammaproteobacteria and Alphaproteobacteria (as monitored by SseB protein levels) is dependent on the bacterial strain and growth medium. We also demonstrate that the cloned system is functional for secretion, can direct interactions with macrophages, and can be used as a novel tool to analyze the predicted interaction of SseB with host cells. This work provides a foundation for future applications where the cloned SPI-2 region (or other cloned type III systems) can provide a desired function to an engineered gram-negative strain.

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Vibrio parahaemolyticusSenses Intracellular K+To Translocate Type III Secretion System 2 Effectors Effectively

mBio ◽

10.1128/mbio.01366-18 ◽

2018 ◽

Vol 9 (4) ◽

Cited By ~ 3

Author(s):

Sarunporn Tandhavanant ◽

Shigeaki Matsuda ◽

Hirotaka Hiyoshi ◽

Tetsuya Iida ◽

Toshio Kodama

Keyword(s):

Host Cell ◽

Type Iii Secretion ◽

Cell Attachment ◽

Type Iii Secretion System ◽

Secretion System ◽

Host Cells ◽

Content Type ◽

Type Iii ◽

Key Factor ◽

Effective Delivery

ABSTRACTMany Gram-negative bacterial symbionts and pathogens employ a type III secretion system (T3SS) to live in contact with eukaryotic cells. Because T3SSs inject bacterial proteins (effectors) directly into host cells, the switching of secretory substrates between translocators and effectors in response to host cell attachment is a crucial step for the effective delivery of effectors. Here, we show that the protein secretion switch ofVibrio parahaemolyticusT3SS2, which is a main contributor to the enteropathogenicity of a food poisoning bacterium, is regulated by two gatekeeper proteins, VgpA and VgpB. In the absence of these gatekeepers, effector secretion was activated, but translocator secretion was abolished, causing the loss of virulence. We found that the K+concentration, which is high inside the host cell but low outside, is a key factor for VgpA- and VgpB-mediated secretion switching. Exposure of wild-type bacteria to K+ions provoked both gatekeeper and effector secretions but reduced the level of secretion of translocators. The secretion protein profile of wild-type bacteria cultured with 0.1 M KCl was similar to that of gatekeeper mutants. Furthermore, depletion of K+ions in host cells diminished the efficiency of T3SS2 effector translocation. Thus, T3SS2 senses the high intracellular concentration of K+of the host cell so that T3SS2 effectors can be effectively injected.IMPORTANCEThe pathogenesis of many Gram-negative bacterial pathogens arises from a type III secretion system (T3SS), whereby bacterial proteins (effectors) are directly injected into host cells. The injected effectors then modify host cell functions. For effective delivery of effector proteins, bacteria need to both recognize host cell attachment and switch the type of secreted proteins. Here, we identified gatekeeper proteins that play important roles in a T3SS2 secretion switch ofVibrio parahaemolyticus, a causative agent of food-borne gastroenteritis. We also found that K+, which is present in high concentrations inside the host cell but in low concentrations outside, is a key factor for the secretion switch. Thus,V. parahaemolyticussenses the high intracellular K+concentration, triggering the effective injection of effectors.

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An NF-κB-Based High-Throughput Screen Identifies Piericidins as Inhibitors of the Yersinia pseudotuberculosis Type III Secretion System

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02025-13 ◽

2013 ◽

Vol 58 (2) ◽

pp. 1118-1126 ◽

Cited By ~ 21

Author(s):

Miles C. Duncan ◽

Weng Ruh Wong ◽

Allison J. Dupzyk ◽

Walter M. Bray ◽

Roger G. Linington ◽

...

Keyword(s):

Natural Products ◽

Type Iii Secretion ◽

Mammalian Cells ◽

Yersinia Pseudotuberculosis ◽

Type Iii Secretion System ◽

Secretion System ◽

Host Cells ◽

Block Type ◽

Content Type ◽

Type Iii

ABSTRACTThe type III secretion system (T3SS) is a bacterial appendage used by dozens of Gram-negative pathogens to subvert host defenses and cause disease, making it an ideal target for pathogen-specific antimicrobials. Here, we report the discovery and initial characterization of two related natural products with T3SS-inhibitory activity that were derived from a marine actinobacterium. Bacterial extracts containing piericidin A1 and the piericidin derivative Mer-A 2026B inhibitedYersinia pseudotuberculosisfrom triggering T3SS-dependent activation of the host transcription factor NF-κB in HEK293T cells but were not toxic to mammalian cells. As theYersiniaT3SS must be functional in order to trigger NF-κB activation, these data indicate that piericidin A1 and Mer-A 2026B block T3SS function. Consistent with this, purified piericidin A1 and Mer-A 2026B dose-dependently inhibited translocation of theY. pseudotuberculosisT3SS effector protein YopM inside CHO cells. In contrast, neither compound perturbed bacterial growthin vitro, indicating that piericidin A1 and Mer-A 2026B do not function as general antibiotics inYersinia. In addition, whenYersiniawas incubated under T3SS-inducing culture conditions in the absence of host cells, Mer-A 2026B and piericidin A1 inhibited secretion of T3SS cargo as effectively as or better than several previously described T3SS inhibitors, such as MBX-1641 and aurodox. This suggests that Mer-A 2026B and piericidin A1 do not block type III secretion by blocking the bacterium-host cell interaction, but rather inhibit an earlier stage, such as T3SS needle assembly. In summary, the marine-derived natural products Mer-A 2026B and piericidin A1 possess previously uncharacterized activity against the bacterial T3SS.

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RNase E Promotes Expression of Type III Secretion System Genes in Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.00336-19 ◽

2019 ◽

Vol 201 (22) ◽

Cited By ~ 3

Author(s):

Josh S. Sharp ◽

Arne Rietsch ◽

Simon L. Dove

Keyword(s):

Pseudomonas Aeruginosa ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Effector Proteins ◽

Host Cells ◽

Rnase E ◽

Content Type ◽

Type Iii ◽

Small Regulatory Rnas

ABSTRACT Pseudomonas aeruginosa is an important opportunistic pathogen that employs a type III secretion system (T3SS) to inject effector proteins into host cells. Using a protein depletion system, we show that the endoribonuclease RNase E positively regulates expression of the T3SS genes. We also present evidence that RNase E antagonizes the expression of genes of the type VI secretion system and limits biofilm production in P. aeruginosa. Thus, RNase E, which is thought to be the principal endoribonuclease involved in the initiation of RNA degradation in P. aeruginosa, plays a key role in controlling the production of factors involved in both acute and chronic stages of infection. Although the posttranscriptional regulator RsmA is also known to positively regulate expression of the T3SS genes, we find that RNase E does not appreciably influence the abundance of RsmA in P. aeruginosa. Moreover, we show that RNase E still exerts its effects on T3SS gene expression in cells lacking all four of the key small regulatory RNAs that function by sequestering RsmA. IMPORTANCE The type III secretion system (T3SS) is a protein complex produced by many Gram-negative pathogens. It is capable of injecting effector proteins into host cells that can manipulate cell metabolism and have toxic effects. Understanding how the T3SS is regulated is important in understanding the pathogenesis of bacteria with such systems. Here, we show that RNase E, which is typically thought of as a global regulator of RNA stability, plays a role in regulating the T3SS in Pseudomonas aeruginosa. Depleting RNase E results in the loss of T3SS gene expression as well as a concomitant increase in biofilm formation. These observations are reminiscent of the phenotypes associated with the loss of activity of the posttranscriptional regulator RsmA. However, RNase E-mediated regulation of these systems does not involve changes in the abundance of RsmA and is independent of the known small regulatory RNAs that modulate RsmA activity.

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Control of Type III Secretion System Effector/Chaperone Ratio Fosters Pathogen Adaptation to Host-Adherent Lifestyle

mBio ◽

10.1128/mbio.02074-19 ◽

2019 ◽

Vol 10 (5) ◽

Cited By ~ 3

Author(s):

Netanel Elbaz ◽

Yaakov Socol ◽

Naama Katsowich ◽

Ilan Rosenshine

Keyword(s):

Gene Expression ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Effector Proteins ◽

Host Cells ◽

Host Colonization ◽

Content Type ◽

Type Iii ◽

Attached State

ABSTRACT The transition from a planktonic lifestyle to a host-attached state is often critical for bacterial virulence. Upon attachment to host cells, enteropathogenic Escherichia coli (EPEC) employs a type III secretion system (T3SS) to inject into the host cells ∼20 effector proteins, including Tir. CesT, which is encoded from the same operon downstream of tir, is a Tir-bound chaperone that facilitates Tir translocation. Upon Tir translocation, the liberated CesT remains in the bacterial cytoplasm and antagonizes the posttranscriptional regulator CsrA, thus eliciting global regulation in the infecting pathogen. Importantly, tight control of the Tir/CesT ratio is vital, since an uncontrolled surge in free CesT levels may repress CsrA in an untimely manner, thus abrogating colonization. We investigated how fluctuations in Tir translation affect the regulation of this ratio. By creating mutations that cause the premature termination of Tir translation, we revealed that the untranslated tir mRNA becomes highly unstable, resulting in a rapid drop in cesT mRNA levels and, thus, CesT levels. This mechanism couples Tir and CesT levels to ensure a stable Tir/CesT ratio. Our results expose an additional level of regulation that enhances the efficacy of the initial interaction of EPEC with the host cell, providing a better understanding of the bacterial switch from the planktonic to the cell-adherent lifestyle. IMPORTANCE Host colonization by extracellular pathogens often entails the transition from a planktonic lifestyle to a host-attached state. Enteropathogenic E. coli (EPEC), a Gram-negative pathogen, attaches to the intestinal epithelium of the host and employs a type III secretion system (T3SS) to inject effector proteins into the cytoplasm of infected cells. The most abundant effector protein injected is Tir, whose translocation is dependent on the Tir-bound chaperon CesT. Upon Tir injection, the liberated CesT binds to and inhibits the posttranscriptional regulator CsrA, resulting in reprogramming of gene expression in the host-attached bacteria. Thus, adaptation to the host-attached state involves dynamic remodeling of EPEC gene expression, which is mediated by the relative levels of Tir and CesT. Fluctuating from the optimal Tir/CesT ratio results in a decrease in EPEC virulence. Here we elucidate a posttranscriptional circuit that prevents sharp variations from this ratio, thus improving host colonization.

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