The Type III Secretion System and Cytotoxic Enterotoxin Alter the Virulence of Aeromonas hydrophila

ABSTRACT Many gram-negative bacteria use a type III secretion system (TTSS) to deliver effector proteins into host cells. Here we report the characterization of a TTSS chromosomal operon from the diarrheal isolate SSU of Aeromonas hydrophila. We deleted the gene encoding Aeromonas outer membrane protein B (AopB), which is predicted to be involved in the formation of the TTSS translocon, from wild-type (WT) A. hydrophila as well as from a previously characterized cytotoxic enterotoxin gene (act)-minus strain of A. hydrophila, thus generating aopB and act/aopB isogenic mutants. The act gene encodes a type II-secreted cytotoxic enterotoxin (Act) that has hemolytic, cytotoxic, and enterotoxic activities and induces lethality in a mouse model. These isogenic mutants (aopB, act, and act/aopB) were highly attenuated in their ability to induce cytotoxicity in RAW 264.7 murine macrophages and HT-29 human colonic epithelial cells. The act/aopB mutant demonstrated the greatest reduction in cytotoxicity to cultured cells after 4 h of infection, as measured by the release of lactate dehydrogenase enzyme, and was avirulent in mice, with a 90% survival rate compared to that of animals infected with Act and AopB mutants, which caused 50 to 60% of the animals to die at a dose of three 50% lethal doses. In contrast, WT A. hydrophila killed 100% of the mice within 48 h. The effects of these mutations on cytotoxicity could be complemented with the native genes. Our studies further revealed that the production of lactones, which are involved in quorum sensing (QS), was decreased in the act (32%) and aopB (64%) mutants and was minimal (only 8%) in the act/aopB mutant, compared to that of WT A. hydrophila SSU. The effects of act and aopB gene deletions on lactone production could also be complemented with the native genes, indicating specific effects of Act and the TTSS on lactone production. Although recent studies with other bacteria have indicated TTSS regulation by QS, this is the first report describing a correlation between the TTSS and Act of A. hydrophila and the production of lactones.

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Essential Role of the Type III Secretion System Effector NleB in Colonization of Mice by Citrobacter rodentium

Infection and Immunity ◽

10.1128/iai.74.4.2328-2337.2006 ◽

2006 ◽

Vol 74 (4) ◽

pp. 2328-2337 ◽

Cited By ~ 101

Author(s):

Michelle Kelly ◽

Emily Hart ◽

Rosanna Mundy ◽

Olivier Marchès ◽

Siouxsie Wiles ◽

...

Keyword(s):

Type Iii Secretion ◽

Type Iii Secretion System ◽

Pathogenicity Island ◽

Secretion System ◽

Effector Proteins ◽

Host Cells ◽

Virulence Determinants ◽

Citrobacter Rodentium ◽

Gene Encoding ◽

Type Iii

ABSTRACT Attaching and effacing (A/E) pathogens are a significant cause of gastrointestinal illness in humans and animals. All A/E pathogens carry a large pathogenicity island, termed the locus for enterocyte effacement (LEE), which encodes a type III secretion system that translocates several effector proteins into host cells. To identify novel virulence determinants in A/E pathogens, we performed a signature-tagged mutagenesis screen in C57BL/6 mice by using the mouse A/E pathogen Citrobacter rodentium. Five hundred seventy-six derivatives of C. rodentium were tested in pools of 12 mutants. One attenuated mutant carried a transposon insertion in nleB, which encodes a putative effector of the LEE-encoded type III secretion system (T3SS). nleB is present in a genomic pathogenicity island that also encodes another putative effector, NleE, immediately downstream. Using translational fusions with β-lactamase (TEM-1), we showed that both NleB and NleE were translocated into host cells by the LEE-encoded T3SS of enteropathogenic Escherichia coli. In addition, deletion of the gene encoding NleB in C. rodentium resulted in reduced colonization of mice in single infections and reduced colonic hyperplasia. In contrast, the deletion of other non-LEE-encoded effector genes in C. rodentium, nleC, nleD, or nleE, had no effect on host colonization or disease. These results suggest that nleB encodes an important virulence determinant of A/E pathogens.

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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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Cytosporone B, an Inhibitor of the Type III Secretion System of Salmonella enterica Serovar Typhimurium

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02421-12 ◽

2013 ◽

Vol 57 (5) ◽

pp. 2191-2198 ◽

Cited By ~ 40

Author(s):

Jianfang Li ◽

Chao Lv ◽

Weiyang Sun ◽

Zhenyu Li ◽

Xiaowei Han ◽

...

Keyword(s):

Type Iii Secretion ◽

Salmonella Enterica ◽

Bacterial Resistance ◽

Type Iii Secretion System ◽

Secretion System ◽

Effector Proteins ◽

Host Cells ◽

Content Type ◽

Type Iii ◽

Serovar Typhimurium

ABSTRACTBacterial virulence factors have been increasingly regarded as attractive targets for development of novel antibacterial agents. Virulence inhibitors are less likely to generate bacterial resistance, which makes them superior to traditional antibiotics that target bacterial viability.Salmonella entericaserovar Typhimurium, an important food-borne human pathogen, has type III secretion system (T3SS) as its major virulence factor. T3SS secretes effector proteins to facilitate invasion into host cells. In this study, we identified several analogs of cytosporone B (Csn-B) that strongly block the secretion ofSalmonellapathogenicity island 1 (SPI-1)-associated effector proteins, without affecting the secretion of flagellar protein FliCin vitro. Csn-B and two other derivatives exhibited a strong inhibitory effect on SPI-1-mediated invasion to HeLa cells, while no significant toxicity to bacteria was observed. Nucleoid proteins Hha and H-NS bind to the promoters of SPI-1 regulator geneshilD,hilC, andrtsAto repress their expression and consequently regulate the expression of SPI-1 apparatus and effector genes. We found that Csn-B upregulated the transcription ofhhaandhns, implying that Csn-B probably affected the secretion of effectors through the Hha–H-NS regulatory pathway. In summary, this study presented an effective SPI-1 inhibitor, Csn-B, which may have potential in drug development against antibiotic-resistantSalmonella.

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The Salmonella enterica Serotype Typhimurium Effector Proteins SipA, SopA, SopB, SopD, and SopE2 Act in Concert To Induce Diarrhea in Calves

Infection and Immunity ◽

10.1128/iai.70.7.3843-3855.2002 ◽

2002 ◽

Vol 70 (7) ◽

pp. 3843-3855 ◽

Cited By ~ 189

Author(s):

Shuping Zhang ◽

Renato L. Santos ◽

Renee M. Tsolis ◽

Silke Stender ◽

Wolf-Dietrich Hardt ◽

...

Keyword(s):

Type Iii Secretion ◽

Salmonella Enterica ◽

Type Iii Secretion System ◽

Secretion System ◽

Effector Proteins ◽

Host Cells ◽

Loop Model ◽

Fluid Accumulation ◽

Ileal Loop ◽

Type Iii

ABSTRACT Salmonella enterica serotype Typhimurium requires a functional type III secretion system encoded by Salmonella pathogenicity island 1 (SPI1) to cause diarrhea. We investigated the role of genes encoding secreted target proteins of the SPI1-associated type III secretion system for enteropathogenicity in calves. Salmonella serotype Typhimurium strains having mutations in sptP, avrA, sspH1, or slrP induced fluid secretion in the bovine ligated ileal loop model at levels similar to that of the wild type. In contrast, mutations in sipA, sopA, sopB, sopD, or sopE2 significantly reduced fluid accumulation in bovine ligated ileal loops at 8 h postinfection. A strain carrying mutations in sipA, sopA, sopB, sopD, and sopE2 (sipA sopABDE2 mutant) caused the same level of fluid accumulation in bovine ligated ileal loops as a strain carrying a mutation in sipB, a SPI1 gene required for the translocation of effector proteins into host cells. A positive correlation was observed between the severity of histopathological lesions detected in the ileal mucosa and the levels of fluid accumulation induced by the different mutants. After oral infection of calves, the Salmonella serotype Typhimurium sipAsopABDE2 mutant caused only mild diarrhea and was more strongly attenuated than strains having only single mutations. These data demonstrate that SipA, SopA, SopB, SopD, and SopE2 are major virulence factors responsible for diarrhea during Salmonella serotype Typhimurium infection of calves.

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Type III Secretion System Translocon Component EseB Forms Filaments on and Mediates Autoaggregation of and Biofilm Formation by Edwardsiella tarda

Applied and Environmental Microbiology ◽

10.1128/aem.01254-15 ◽

2015 ◽

Vol 81 (17) ◽

pp. 6078-6087 ◽

Cited By ~ 23

Author(s):

Zhi Peng Gao ◽

Pin Nie ◽

Jin Fang Lu ◽

Lu Yi Liu ◽

Tiao Yi Xiao ◽

...

Keyword(s):

Type Iii Secretion ◽

Biofilm Formation ◽

Type Iii Secretion System ◽

Secretion System ◽

Effector Proteins ◽

Host Cells ◽

Edwardsiella Tarda ◽

Dependent Manner ◽

Content Type ◽

Type Iii

ABSTRACTThe type III secretion system (T3SS) ofEdwardsiella tardaplays an important role in infection by translocating effector proteins into host cells. EseB, a component required for effector translocation, is reported to mediate autoaggregation ofE. tarda. In this study, we demonstrate that EseB forms filamentous appendages on the surface ofE. tardaand is required for biofilm formation byE. tardain Dulbecco's modified Eagle's medium (DMEM). Biofilm formation byE. tardain DMEM does not require FlhB, an essential component for assembling flagella. Dynamic analysis of EseB filament formation, autoaggregation, and biofilm formation shows that the formation of EseB filaments occurs prior to autoaggregation and biofilm formation. The addition of an EseB antibody toE. tardacultures before bacterial autoaggregation prevents autoaggregation and biofilm formation in a dose-dependent manner, whereas the addition of the EseB antibody toE. tardacultures in which biofilm is already formed does not destroy the biofilm. Therefore, EseB filament-mediated bacterial cell-cell interaction is a prerequisite for autoaggregation and biofilm formation.

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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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Antibodies Inhibiting the Type III Secretion System of Gram-Negative Pathogenic Bacteria

Antibodies ◽

10.3390/antib9030035 ◽

2020 ◽

Vol 9 (3) ◽

pp. 35

Author(s):

Julia A. Hotinger ◽

Aaron E. May

Keyword(s):

Type Iii Secretion ◽

Pathogenic Bacteria ◽

Type Iii Secretion System ◽

The United States ◽

Secretion System ◽

Effector Proteins ◽

Host Cells ◽

Gram Negative ◽

Type Iii ◽

Shigella Spp

Pathogenic bacteria are a global health threat, with over 2 million infections caused by Gram-negative bacteria every year in the United States. This problem is exacerbated by the increase in resistance to common antibiotics that are routinely used to treat these infections, creating an urgent need for innovative ways to treat and prevent virulence caused by these pathogens. Many Gram-negative pathogenic bacteria use a type III secretion system (T3SS) to inject toxins and other effector proteins directly into host cells. The T3SS has become a popular anti-virulence target because it is required for pathogenesis and knockouts have attenuated virulence. It is also not required for survival, which should result in less selective pressure for resistance formation against T3SS inhibitors. In this review, we will highlight selected examples of direct antibody immunizations and the use of antibodies in immunotherapy treatments that target the bacterial T3SS. These examples include antibodies targeting the T3SS of Pseudomonas aeruginosa, Yersinia pestis, Escherichia coli, Salmonella enterica, Shigella spp., and Chlamydia trachomatis.

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Bordetella pertussis Expresses a Functional Type III Secretion System That Subverts Protective Innate and Adaptive Immune Responses

Infection and Immunity ◽

10.1128/iai.00836-07 ◽

2008 ◽

Vol 76 (3) ◽

pp. 1257-1266 ◽

Cited By ~ 62

Author(s):

Neil K. Fennelly ◽

Federico Sisti ◽

Sarah C. Higgins ◽

Pádraig J. Ross ◽

Han van der Heide ◽

...

Keyword(s):

Immune Responses ◽

Bordetella Pertussis ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Effector Proteins ◽

Host Cells ◽

Adaptive Immune Responses ◽

Type Iii ◽

Adaptive Immune

ABSTRACT Certain bacteria use a type III secretion system (TTSS) to deliver effector proteins that interfere with cell function into host cells. While transcription of genes encoding TTSS components has been demonstrated, studies to date have failed to identify TTSS effector proteins in Bordetella pertussis. Here we present the first evidence of a functionally active TTSS in B. pertussis. Three known TTSS effectors, Bsp22, BopN, and BopD, were identified as TTSS substrates in B. pertussis 12743. We found expression of Bsp22 in a significant proportion of clinical isolates but not in common laboratory-adapted strains of B. pertussis. We generated a TTSS mutant of B. pertussis 12743 and showed that it induced significantly lower respiratory tract colonization in mice than the wild-type bacteria. Respiratory infection of mice with the mutant bacteria induced significantly greater innate proinflammatory cytokine production in the lungs soon after challenge, and this correlated with significantly higher antigen-specific interleukin-17, gamma interferon, and immunoglobulin G responses later in infection. Our findings suggest that the TTSS subverts innate and adaptive immune responses during infection of the lungs and may be a functionally important virulence factor for B. pertussis infection of humans.

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Identification of translocation inhibitors targeting the type III secretion system of enteropathogenic E. coli

10.1101/2021.05.11.443714 ◽

2021 ◽

Author(s):

Sabrina Mühlen ◽

Viktor Zapolskii ◽

Ursula Bilitewski ◽

Petra Dersch

Keyword(s):

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Effector Proteins ◽

Host Cells ◽

Immune Recognition ◽

E Coli ◽

Type Iii ◽

Compound Libraries ◽

High Throughput Screening Assay

Infections with enteropathogenic E. coli (EPEC) cause severe diarrhea in children. The non-invasive bacteria adhere to enterocytes of the small intestine and use a type III secretion system (T3SS) to inject effector proteins into host cells to modify and exploit cellular processes in favor of bacterial survival and replication. Several studies have shown that the T3SSs of bacterial pathogens are essential for virulence. Furthermore, the loss of T3SS-mediated effector translocation results in increased immune recognition and clearance of the bacteria. The T3SS is, therefore, considered a promising target for antivirulence strategies and novel therapeutics development. Here, we report the results of a high-throughput screening assay based on the translocation of the EPEC effector protein Tir. Using this assay, we screened more than 13,000 small molecular compounds of six different compound libraries and identified three substances which showed a significant dose-dependent effect on translocation without adverse effects on bacterial or eukaryotic cell viability. Additionally, these substances reduced bacterial binding to host cells, effector-dependent cell detachment and abolished A/E lesion formation without affecting the expression of components of the T3SS or associated effector proteins. Moreover, no effects of the inhibitors on bacterial motility or Shiga-toxin expression were observed. In summary, we have identified three new compounds that strongly inhibit T3SS-mediated translocation of effectors into mammalian cells, which could be valuable as lead substances for treating EPEC and EHEC infections.

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