Attenuated Virulence of a Burkholderia cepacia Type III Secretion Mutant in a Murine Model of Infection

ABSTRACT Type III secretion systems are utilized by a number of gram-negative bacterial pathogens to deliver virulence-associated proteins into host cells. Using a PCR-based approach, we identified homologs of type III secretion genes in the gram-negative bacterium Burkholderia cepacia, an important pulmonary pathogen in immunocompromised patients and patients with cystic fibrosis. One of the genes, designated bscN, encodes a member of a family of ATP-binding proteins believed to generate energy driving virulence protein secretion. Genetic dissection of the regions flanking the bscN gene revealed a locus consisting of at least 10 open reading frames, predicted to encode products with significant homology to known type III secretion proteins in other bacteria. A defined null mutation was generated in the bscN gene, and the null strain and wild-type parent strain were examined by use of a murine model of B. cepacia infection. Quantitative bacteriological analysis of the lungs and spleens of infected C57BL/6 mice revealed that the bscN null strain was attenuated in virulence compared to the parent strain, with significantly lower bacterial recovery from the lungs and spleens at 3 days postinfection. Moreover, histopathological changes, including an inflammatory cell infiltrate, were more pronounced in the lungs of mice infected with the wild-type parent strain than in those of mice infected with the isogenic bscN mutant. These results implicate type III secretion as an important determinant in the pathogenesis of B. cepacia.

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Roles of the Tol-Pal system in the Type III secretion system and flagella-mediated virulence in enterohemorrhagic Escherichia coli

Scientific Reports ◽

10.1038/s41598-020-72412-w ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Hidetada Hirakawa ◽

Kazutomo Suzue ◽

Ayako Takita ◽

Chikako Awazu ◽

Jun Kurushima ◽

...

Keyword(s):

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Enterohemorrhagic Escherichia Coli ◽

Host Cells ◽

Gram Negative Bacteria ◽

Wild Type ◽

E Coli ◽

Type Iii ◽

Wild Type Parent

Abstract The Tol-Pal system is a protein complex that is highly conserved in many gram-negative bacteria. We show here that the Tol-Pal system is associated with the enteric pathogenesis of enterohemorrhagic E. coli (EHEC). Deletion of tolB, which is required for the Tol-Pal system decreased motility, secretion of the Type III secretion system proteins EspA/B, and the ability of bacteria to adhere to and to form attaching and effacing (A/E) lesions in host cells, but the expression level of LEE genes, including espA/B that encode Type III secretion system proteins were not affected. The Citrobacter rodentium, tolB mutant, that is traditionally used to estimate Type III secretion system associated virulence in mice did not cause lethality in mice while it induced anti-bacterial immunity. We also found that the pal mutant, which lacks activity of the Tol-Pal system, exhibited lower motility and EspA/B secretion than the wild-type parent. These combined results indicate that the Tol-Pal system contributes to the virulence of EHEC associated with the Type III secretion system and flagellar activity for infection at enteric sites. This finding provides evidence that the Tol-Pal system may be an effective target for the treatment of infectious diseases caused by pathogenic E. coli.

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Role of Autocleavage in the Function of a Type III Secretion Specificity Switch Protein in Salmonella enterica Serovar Typhimurium

mBio ◽

10.1128/mbio.01459-15 ◽

2015 ◽

Vol 6 (5) ◽

Cited By ~ 31

Author(s):

Julia V. Monjarás Feria ◽

Matthew D. Lefebre ◽

York-Dieter Stierhof ◽

Jorge E. Galán ◽

Samuel Wagner

Keyword(s):

Host Cell ◽

Type Iii Secretion ◽

Effector Proteins ◽

Switching Function ◽

Gram Negative Bacteria ◽

Wild Type ◽

Gram Negative ◽

Type Iii ◽

Autocatalytic Cleavage ◽

Serovar Typhimurium

ABSTRACTType III secretion systems (T3SSs) are multiprotein machines employed by many Gram-negative bacteria to inject bacterial effector proteins into eukaryotic host cells to promote bacterial survival and colonization. The core unit of T3SSs is the needle complex, a supramolecular structure that mediates the passage of the secreted proteins through the bacterial envelope. A distinct feature of the T3SS is that protein export occurs in a strictly hierarchical manner in which proteins destined to form the needle complex filament and associated structures are secreted first, followed by the secretion of effectors and the proteins that will facilitate their translocation through the target host cell membrane. The secretion hierarchy is established by complex mechanisms that involve several T3SS-associated components, including the “switch protein,” a highly conserved, inner membrane protease that undergoes autocatalytic cleavage. It has been proposed that the autocleavage of the switch protein is the trigger for substrate switching. We show here that autocleavage of theSalmonella entericaserovar Typhimurium switch protein SpaS is an unregulated process that occurs after its folding and before its incorporation into the needle complex. Needle complexes assembled with a precleaved form of SpaS function in a manner indistinguishable from that of the wild-type form. Furthermore, an engineered mutant of SpaS that is processed by an external protease also displays wild-type function. These results demonstrate that the cleavage eventper sedoes not provide a signal for substrate switching but support the hypothesis that cleavage allows the proper conformation of SpaS to render it competent for its switching function.IMPORTANCEBacterial interaction with eukaryotic hosts often involves complex molecular machines for targeted delivery of bacterial effector proteins. One such machine, the type III secretion system of some Gram-negative bacteria, serves to inject a multitude of structurally diverse bacterial proteins into the host cell. Critical to the function of these systems is their ability to secrete proteins in a strict hierarchical order, but it is unclear how the mechanism of switching works. Central to the switching mechanism is a highly conserved inner membrane protease that undergoes autocatalytic cleavage. Although it has been suggested previously that the autocleavage event is the trigger for substrate switching, we show here that this is not the case. Rather, our results show that cleavage allows the proper conformation of the protein to render it competent for its switching function. These findings may help develop inhibitors of type III secretion machines that offer novel therapeutic avenues to treat various infectious diseases.

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Type III Secretion Decreases Bacterial and Host Survival following Phagocytosis of Yersinia pseudotuberculosis by Macrophages

Infection and Immunity ◽

10.1128/iai.00183-08 ◽

2008 ◽

Vol 76 (9) ◽

pp. 4299-4310 ◽

Cited By ~ 24

Author(s):

Yue Zhang ◽

James Murtha ◽

Margaret A. Roberts ◽

Richard M. Siegel ◽

James B. Bliska

Keyword(s):

Cell Death ◽

Type Iii Secretion ◽

Yersinia Pseudotuberculosis ◽

De Novo ◽

Host Cells ◽

Host Responses ◽

Intracellular Bacteria ◽

Wild Type ◽

Macrophage Response ◽

Type Iii

ABSTRACT Yersinia pseudotuberculosis uses a plasmid (pYV)-encoded type III secretion system (T3SS) to translocate a set of effectors called Yops into infected host cells. YopJ functions to induce apoptosis, and YopT, YopE, and YopH act to antagonize phagocytosis in macrophages. Because Yops do not completely block phagocytosis and Y. pseudotuberculosis can replicate in macrophages, it is important to determine if the T3SS modulates host responses to intracellular bacteria. Isogenic pYV-cured, pYV+ wild-type, and yop mutant Y. pseudotuberculosis strains were allowed to infect bone marrow-derived murine macrophages at a low multiplicity of infection under conditions in which the survival of extracellular bacteria was prevented. Phagocytosis, the intracellular survival of the bacteria, and the apoptosis of the infected macrophages were analyzed. Forty percent of cell-associated wild-type bacteria were intracellular after a 20-min infection, allowing the study of the macrophage response to internalized pYV+ Y. pseudotuberculosis. Interestingly, macrophages restricted survival of pYV+ but not pYV-cured or ΔyopB Y. pseudotuberculosis within phagosomes: only a small fraction of the pYV+ bacteria internalized replicated by 24 h. In addition, ∼20% of macrophages infected with wild-type pYV+ Y. pseudotuberculosis died of apoptosis after 20 h. Analysis of yop mutants expressing catalytically inactive effectors revealed that YopJ was important for apoptosis, while a role for YopE, YopH, and YopT in modulating macrophage responses to intracellular bacteria could not be identified. Apoptosis was reduced in Toll-like receptor 4-deficient macrophages, indicating that cell death required signaling through this receptor. Treatment of macrophages harboring intracellular pYV+ Y. pseudotuberculosis with chloramphenicol reduced apoptosis, indicating that the de novo bacterial protein synthesis was necessary for cell death. Our finding that the presence of a functional T3SS impacts the survival of both bacterium and host following phagocytosis of Y. pseudotuberculosis suggests new roles for the T3SS in Yersinia pathogenesis.

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Salmonella enterica subspecies enterica serovar Enteritidis Salmonella pathogenicity island 2 type III secretion system: role in intestinal colonization of chickens and systemic spread

Microbiology ◽

10.1099/mic.0.038018-0 ◽

2010 ◽

Vol 156 (9) ◽

pp. 2770-2781 ◽

Cited By ~ 18

Author(s):

Amanda L. S. Wisner ◽

Taseen S. Desin ◽

Birgit Koch ◽

Po-King S. Lam ◽

Emil M. Berberov ◽

...

Keyword(s):

Type Iii Secretion ◽

Salmonella Enterica ◽

Type Strain ◽

Wild Type Strain ◽

Type Iii Secretion System ◽

Secretion System ◽

Host Cells ◽

Wild Type ◽

Type Iii ◽

Systemic Spread

Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis) has been identified as a significant cause of salmonellosis in humans. Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) each encode a specialized type III secretion system (T3SS) that enables Salmonella to manipulate host cells at various stages of the invasion/infection process. For the purposes of our studies we used a chicken isolate of S. Enteritidis (Sal18). In one study, we orally co-challenged 35-day-old specific pathogen-free (SPF) chickens with two bacterial strains per group. The control group received two versions of the wild-type strain Sal18: Sal18 attTn7 : : tet and Sal18 attTn7 : : cat, while the other two groups received the wild-type strain (Sal18 attTn7 : : tet) and one of two mutant strains. From this study, we concluded that S. Enteritidis strains deficient in the SPI-1 and SPI-2 systems were outcompeted by the wild-type strain. In a second study, groups of SPF chickens were challenged at 1 week of age with four different strains: the wild-type strain, and three other strains lacking either one or both of the SPI-1 and SPI-2 regions. On days 1 and 2 post-challenge, we observed a reduced systemic spread of the SPI-2 mutants, but by day 3, the systemic distribution levels of the mutants matched that of the wild-type strain. Based on these two studies, we conclude that the S. Enteritidis SPI-2 T3SS facilitates invasion and systemic spread in chickens, although alternative mechanisms for these processes appear to exist.

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Yersinia enterocolitica Type III Secretion Depends on the Proton Motive Force but Not on the Flagellar Motor Components MotA and MotB

Infection and Immunity ◽

10.1128/iai.72.7.4004-4009.2004 ◽

2004 ◽

Vol 72 (7) ◽

pp. 4004-4009 ◽

Cited By ~ 75

Author(s):

Gottfried Wilharm ◽

Verena Lehmann ◽

Kristina Krauss ◽

Beatrix Lehnert ◽

Susanna Richter ◽

...

Keyword(s):

Yersinia Enterocolitica ◽

Type Iii Secretion ◽

Proton Motive Force ◽

Motive Force ◽

Host Cells ◽

Infection Model ◽

Flagellar Motor ◽

Wild Type ◽

Secretion Systems ◽

Type Iii

ABSTRACT The flagellum is believed to be the common ancestor of all type III secretion systems (TTSSs). In Yersinia enterocolitica, expression of the flagellar TTSS and the Ysc (Yop secretion) TTSS are inversely regulated. We therefore hypothesized that the Ysc TTSS may adopt flagellar motor components in order to use the pathogenicity-related translocon in a drill-like manner. As a prerequisite for this hypothesis, we first tested a requirement for the proton motive force by both systems using the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Motility as well as type III-dependent secretion of Yop proteins was inhibited by CCCP. We deleted motAB, which resulted in an immotile phenotype. This mutant, however, secreted amounts of Yops to the supernatant comparable to those of the wild type. Translocation of Yops into host cells was also not affected by the motAB deletion. Virulence of the mutant was comparable to that of the wild type in the mouse oral infection model. Thus, the hypothesis that the Ysc TTSS might adopt flagellar motor components was not confirmed. The finding that, in addition to consumption of ATP, Ysc TTSS requires the proton motive force is discussed.

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Shigella Spa32 Is an Essential Secretory Protein for Functional Type III Secretion Machinery and Uniformity of Its Needle Length

Journal of Bacteriology ◽

10.1128/jb.184.5.1244-1252.2002 ◽

2002 ◽

Vol 184 (5) ◽

pp. 1244-1252 ◽

Cited By ~ 71

Author(s):

Koichi Tamano ◽

Eisaku Katayama ◽

Takahito Toyotome ◽

Chihiro Sasakawa

Keyword(s):

Hela Cells ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretory Protein ◽

Secretion System ◽

Host Cells ◽

Functional Type ◽

Wild Type ◽

Needle Length ◽

Type Iii

ABSTRACT The Shigella type III secretion machinery is responsible for delivering to host cells the set of effectors required for invasion. The type III secretion complex comprises a needle composed of MxiH and MxiI and a basal body made up of MxiD, MxiG, and MxiJ. In S. flexneri, the needle length has a narrow range, with a mean of approximately 45 nm, suggesting that it is strictly regulated. Here we show that Spa32, encoded by one of the spa genes, is an essential protein translocated via the type III secretion system and is involved in the control of needle length as well as type III secretion activity. When the spa32 gene was mutated, the type III secretion complexes possessed needles of various lengths, ranging from 40 to 1,150 nm. Upon introduction of a cloned spa32 into the spa32 mutant, the bacteria produced needles of wild-type length. The spa32 mutant overexpressing MxiH produced extremely long (>5 μm) needles. Spa32 was secreted into the medium via the type III secretion system, but secretion did not depend on activation of the system. The spa32 mutant and the mutant overexpressing MxiH did not secrete effectors such as Ipa proteins into the medium or invade HeLa cells. Upon introduction of Salmonella invJ, encoding InvJ, which has 15.4% amino acid identity with Spa32, into the spa32 mutant, the bacteria produced type III needles of wild-type length and efficiently entered HeLa cells. These findings suggest that Spa32 is an essential secreted protein for a functional type III secretion system in Shigella spp. and is involved in the control of needle length. Furthermore, its function is interchangeable with that of Salmonella InvJ.

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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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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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OspF and OspC1 Are Shigella flexneri Type III Secretion System Effectors That Are Required for Postinvasion Aspects of Virulence

Infection and Immunity ◽

10.1128/iai.00594-06 ◽

2006 ◽

Vol 74 (10) ◽

pp. 5964-5976 ◽

Cited By ~ 50

Author(s):

Daniel V. Zurawski ◽

Chieko Mitsuhata ◽

Karen L. Mumy ◽

Beth A. McCormick ◽

Anthony T. Maurelli

Keyword(s):

Type Iii Secretion ◽

Shigella Flexneri ◽

Type Iii Secretion System ◽

Secretion System ◽

Host Cells ◽

Dependent Manner ◽

Wild Type ◽

Extracellular Signal Regulated Kinase ◽

Type Iii ◽

Extracellular Signal

ABSTRACT Shigella flexneri is the causative agent of dysentery, and its pathogenesis is mediated by a type III secretion system (T3SS). S. flexneri secretes effector proteins into the eukaryotic cell via the T3SS, and these proteins usurp host cellular functions to the benefit of the bacteria. OspF and OspC1 are known to be secreted by S. flexneri, but their functions are unknown. We transformed S. flexneri with a plasmid that expresses a two-hemagglutinin tag (2HA) in frame with OspF or OspC1 and verified that these proteins are secreted in a T3SS-dependent manner. Immunofluorescence of HeLa cells infected with S. flexneri expressing OspF-2HA or OspC1-2HA revealed that both proteins localize in the nucleus and cytoplasm of host cells. To elucidate the function of these T3SS effectors, we constructed ΔospF and ΔospC1 deletion mutants by allelic exchange. We found that ΔospF and ΔospC1 mutants invade host cells and form plaques in confluent monolayers similar to wild-type S. flexneri. However, in the polymorphonuclear (PMN) cell migration assay, a decrease in neutrophil migration was observed for both mutants in comparison to the migration of wild-type bacteria. Moreover, infection of polarized T84 intestinal cells infected with ΔospF and ΔospC1 mutants resulted in decreased phosphorylation of extracellular signal-regulated kinase 1/2 in comparison to that of T84 cells infected with wild-type S. flexneri. To date, these are the first examples of T3SS effectors implicated in mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway activation. Ultimately, OspF and OspC1 are essential for PMN transepithelial migration, a phenotype associated with increased inflammation and bacterial access to the submucosa, which are fundamental aspects of S. flexneri pathogenesis.

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