scholarly journals Identification and analysis of bacterial virulence genes in vivo

2000 ◽  
Vol 355 (1397) ◽  
pp. 613-622 ◽  
Author(s):  
Kate E. Unsworth ◽  
David W. Holden
Keyword(s):  
Type Iii Secretion ◽  
Virulence Genes ◽  
Screening Method ◽  
Secretion System ◽  
Bacterial Virulence ◽  
Microbial Pathogens ◽  
Temperature Sensitive ◽  
Approaches To Study

Signature–tagged mutagenesis is a mutation–based screening method for the identification of virulence genes of microbial pathogens. Genes isolated by this approach fall into three classes: those with known biochemical function, those of suspected function and some whose functions cannot be predicted from database searches. A variety of in vitro and in vivo methods are available to elucidate the function of genes of the second and third classes. We describe the use of some of these approaches to study the function of the Salmonella pathogenicity island 2 type III secretion system of Salmonella typhimurium . This virulence determinant is required for intracellular survival. Secretion by this system is induced by an acidic pH, and its function may be to alter trafficking of the Salmonella –containing vacuole. Use of a temperature–sensitive non–replicating plasmid and competitive index tests with other genes show that in vivo phenotypes do not always correspond to those predicted from in vitro studies.

scholarly journals Salmonella-Induced Caspase-2 Activation in Macrophages

2000 ◽  
Vol 192 (7) ◽  
pp. 1035-1046 ◽  
Author(s):  
Veronika Jesenberger ◽  
Katarzyna J. Procyk ◽  
Junying Yuan ◽  
Siegfried Reipert ◽  
Manuela Baccarini
Keyword(s):  
Type Iii Secretion ◽  
Caspase 3 ◽  
Secretion System ◽  
Caspase 1 ◽  
Chemical Inhibition ◽  
Induction Of Apoptosis ◽  
Caspase 2 ◽  
Induced Apoptosis

The enterobacterial pathogen Salmonella induces phagocyte apoptosis in vitro and in vivo. These bacteria use a specialized type III secretion system to export a virulence factor, SipB, which directly activates the host's apoptotic machinery by targeting caspase-1. Caspase-1 is not involved in most apoptotic processes but plays a major role in cytokine maturation. We show that caspase-1–deficient macrophages undergo apoptosis within 4–6 h of infection with invasive bacteria. This process requires SipB, implying that this protein can initiate the apoptotic machinery by regulating components distinct from caspase-1. Invasive Salmonella typhimurium targets caspase-2 simultaneously with, but independently of, caspase-1. Besides caspase-2, the caspase-1–independent pathway involves the activation of caspase-3, -6, and -8 and the release of cytochrome c from mitochondria, none of which occurs during caspase-1–dependent apoptosis. By using caspase-2 knockout macrophages and chemical inhibition, we establish a role for caspase-2 in both caspase-1–dependent and –independent apoptosis. Particularly, activation of caspase-1 during fast Salmonella-induced apoptosis partially relies on caspase-2. The ability of Salmonella to induce caspase-1–independent macrophage apoptosis may play a role in situations in which activation of this protease is either prevented or uncoupled from the induction of apoptosis.

scholarly journals Pseudomonas aeruginosa Cytotoxin ExoU Is Injected into Phagocytic Cells during Acute Pneumonia

2010 ◽  
Vol 78 (4) ◽  
pp. 1447-1456 ◽  
Author(s):  
Maureen H. Diaz ◽  
Alan R. Hauser
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Immune Cells ◽  
Type Iii Secretion ◽  
Cell Types ◽  
Secretion System ◽  
Host Cells ◽  
Phagocytic Cells ◽  
Acute Pneumonia

ABSTRACT ExoU, a cytotoxin translocated into host cells via the type III secretion system of Pseudomonas aeruginosa, is associated with increased mortality and disease severity. We previously showed that impairment of recruited phagocytic cells allowed survival of ExoU-secreting P. aeruginosa in the lung. Here we analyzed types of cells injected with ExoU in vivo using translational fusions of ExoU with a β-lactamase reporter (ExoU-Bla). Cells injected with ExoU-Bla were detectable in vitro but not in vivo, presumably due to the rapid cytotoxicity induced by the toxin. Therefore, we used a noncytotoxic ExoU variant, designated ExoU(S142A)-Bla, to analyze injection in vivo. We determined that phagocytic cells in the lung were frequently injected with ExoU(S142A). Early during infection, resident macrophages constituted the majority of cells into which ExoU was injected, but neutrophils and monocytes became the predominant types of cells into which ExoU was injected upon recruitment into the lung. We observed a modest preference for injection into neutrophils over injection into other cell types, but in general the repertoire of injected immune cells reflected the relative abundance of these cells in the lung. Our results indicate that phagocytic cells in the lung are injected with ExoU and support the hypothesis that ExoU-mediated impairment of phagocytes has a role in the pathogenesis of pneumonia caused by P. aeruginosa.

scholarly journals Differential expression of Salmonella type III secretion system factors InvJ, PrgJ, SipC, SipD, SopA and SopB in cultures and in mice

Microbiology ◽  
2010 ◽  
Vol 156 (1) ◽  
pp. 116-127 ◽  
Author(s):  
Hao Gong ◽  
Gia-Phong Vu ◽  
Yong Bai ◽  
Edward Yang ◽  
Fenyong Liu ◽  
...  
Keyword(s):  
Differential Expression ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Systemic Infection ◽  
Secretion System ◽  
Flag Epitope ◽  
Type Iii ◽  
Late Stages

The type III secretion system (T3SS) encoded by Salmonella pathogenicity island 1 (SPI-1) is important for the invasion of epithelial cells during development of Salmonella-associated enterocolitis. It has been suggested that the level and timing of the expression of the SPI-1 T3SS proteins and effectors dictate the consequences of bacterial infection and pathogenesis. However, the expression of these proteins has not been extensively studied in vivo, especially during the later stages of salmonellosis when the infection is established. We have constructed recombinant Salmonella strains that contain a FLAG epitope inserted in-frame to genes invJ, prgJ, sipC, sipD, sopA and sopB, and investigated the expression of the tagged proteins both in vitro and in vivo during murine salmonellosis. Mice were inoculated intraperitoneally or intragastrically with the tagged Salmonella strains. At different time points post-infection, bacteria were recovered from various organs, and the expression of the tagged proteins was determined. Our results provide direct evidence that PrgJ and SipD are expressed in Salmonella colonizing the liver and ileum of infected animals at both the early and late stages of infection. Furthermore, our study has shown that the InvJ protein is expressed preferentially in Salmonella colonizing the ileum but not the liver, while SipC is expressed preferentially in Salmonella colonizing the liver but not the ileum. Thus, Salmonella appears to express different SPI-1 proteins and effectors when colonizing specific tissues. Our results suggest that differential expression of these proteins may be important for tissue-specific aspects of bacterial pathogenesis such as gastroenterititis in the ileum and systemic infection in the liver.

scholarly journals Chaperone mediated coupling of subunit availability to activation of flagellar Type III Secretion

2020 ◽  
Author(s):  
Owain J. Bryant ◽  
Betty Y-W. Chung ◽  
Gillian M. Fraser
Keyword(s):  
Cell Membrane ◽  
Electric Potential ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Proton Motive Force ◽  
Secretion System ◽  
Motive Force ◽  
Type Iii

AbstractBacterial flagellar subunits are exported across the cell membrane by the flagellar Type III Secretion System (fT3SS), powered by the proton motive force (pmf) and a specialized ATPase that enables the flagellar export gate to utilise the pmf electric potential (ΔΨ). Export gate activation is mediated by the ATPase stalk, FliJ, but how this process is regulated to prevent wasteful dissipation of pmf in the absence of subunit cargo is not known. Here, we show that FliJ activation of the export gate is regulated by flagellar export chaperones. FliJ binds unladen chaperones and, using novel chaperone variants specifically defective for FliJ binding, we show that disruption of this interaction attenuates motility and cognate subunit export. We demonstrate in vitro that chaperones and the FlhA export gate component compete for binding to FliJ, and show in vivo that unladen chaperones, which would be present in the cell when subunit levels are low, sequester FliJ to prevent activation of the export gate and attenuate subunit export. Our data indicate a mechanism whereby chaperones couple availability of subunit cargo to pmf-driven export by the fT3SS.

scholarly journals CesD2 of Enteropathogenic Escherichia coli Is a Second Chaperone for the Type III Secretion Translocator Protein EspD

2003 ◽  
Vol 71 (4) ◽  
pp. 2130-2141 ◽  
Author(s):  
Bianca C. Neves ◽  
Rosanna Mundy ◽  
Liljana Petrovska ◽  
Gordon Dougan ◽  
Stuart Knutton ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Effector Proteins ◽  
Translocator Protein ◽  
Type Iii ◽  
Genes Encoding

ABSTRACT Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli are extracellular pathogens that employ a type III secretion system to export translocator and effector proteins, proteins which facilitates colonization of the mucosal surface of the intestine via formation of attaching and effacing (A/E) lesions. The genes encoding the proteins for A/E lesion formation are located on a pathogenicity island, termed the locus of enterocyte effacement (LEE), which contains eae encoding intimin as well as the type III secretion system and effector genes. Many type III secreted proteins are stabilized and maintained in a secretion-competent conformation in the bacterial cytosol by specific chaperone proteins. Three type III chaperones have been described thus far within the EPEC LEE region: CesD, for the translocator proteins EspB and EspD; CesT, for the effector proteins Tir and Map; and CesF, for EspF. In this study we report the characterization of CesD2 (previously Orf27), a second LEE-encoded chaperone for EspD. We show specific CesD2-EspD protein interaction which appears to be necessary for proper EspD secretion in vitro and pathogenesis in vivo as demonstrated in the A/E-lesion-forming mouse pathogen Citrobacter rodentium.

scholarly journals In Vivo Genetic Analysis Indicates That PhoP-PhoQ and the Salmonella Pathogenicity Island 2 Type III Secretion System Contribute Independently to Salmonella enterica Serovar Typhimurium Virulence

2001 ◽  
Vol 69 (12) ◽  
pp. 7254-7261 ◽  
Author(s):  
Carmen R. Beuzón ◽  
Kate E. Unsworth ◽  
David W. Holden
Keyword(s):  
Virulence Factors ◽  
Type Iii Secretion ◽  
Salmonella Enterica ◽  
Type Iii Secretion System ◽  
Pathogenicity Island ◽  
Secretion System ◽  
Type Iii ◽  
Serovar Typhimurium

ABSTRACT Many virulence factors are required for Salmonella enterica serovar Typhimurium to replicate intracellularly and proliferate systemically within mice. In this work, we have carried out genetic analyses in vivo to determine the functional relationship between two major virulence factors necessary for systemic infection byS. enterica serovar Typhimurium: theSalmonella pathogenicity island 2 (SPI-2) type III secretion system (TTSS) and the PhoP-PhoQ two-component regulatory system. Although previous work suggested that PhoP-PhoQ regulates SPI-2 TTSS gene expression in vitro, in vivo competitive analysis of mutant strains indicates that these systems contribute independently toS. typhimurium virulence. Our results also suggest that mutation of phoP may compensate partially for defects in the SPI-2 TTSS by deregulating SPI-1 TTSS expression. These results provide an explanation for previous reports showing an apparent functional overlap between these two systems in vitro.

scholarly journals Influence of the Salmonella typhimuriumPathogenicity Island 2 Type III Secretion System on Bacterial Growth in the Mouse

1999 ◽  
Vol 67 (1) ◽  
pp. 213-219 ◽  
Author(s):  
Jacqueline E. Shea ◽  
Carmen R. Beuzon ◽  
Colin Gleeson ◽  
Rosanna Mundy ◽  
David W. Holden
Keyword(s):  
Bacterial Growth ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Virulence Plasmid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Type Iii ◽  
Virulence Attenuation

ABSTRACT We have investigated the in vivo growth kinetics of aSalmonella typhimurium strain (P11D10) carrying a mutation in ssaJ, a Salmonella pathogenicity island 2 (SPI2) gene encoding a component of a type III secretion system required for systemic growth in mice. Similar numbers of mutant and wild-type cells were recovered from the spleens and livers of BALB/c mice up to 8 h after inoculation by the intraperitoneal route. Thereafter, the numbers of wild-type cells continued to increase logarithmically in these organs, whereas those of P11D10 remained relatively static for several days before being cleared. Gentamicin protection experiments on spleen cell suspensions recovered from infected mice showed that viable intracellular wild-type bacteria accumulated over time but that intracellular P11D10 cells did not. Infection experiments were also performed with wild-type and P11D10 cells carrying the temperature-sensitive plasmid pHSG422 to distinguish between bacterial growth rates and killing in vivo. At 16 h postinoculation there were 10-fold more wild-type cells than mutant cells in the spleens of infected mice, but the numbers of cells of both strains carrying the nonreplicating plasmid were very similar, showing that there was little difference in the degree of killing sustained by the two strains and that the SPI2 secretion system must be required for bacterial replication, rather than survival, in vivo. The SPI2 mutant phenotype in mice is similar to that of strains carrying mutations in the Salmonella virulence plasmid spv genes. To determine if these two sets of genes interact together, a double mutant strain carrying SPI2 and spv mutations was constructed and compared with strains carrying single mutations in terms of virulence attenuation. These experiments failed to provide any evidence showing that the SPI2 and spv gene products interact together as part of the same virulence mechanism.

scholarly journals TheYersinia enterocoliticaYsa type III secretion system is expressed during infections both in vitro and in vivo

2013 ◽  
Vol 2 (6) ◽  
pp. 962-975 ◽  
Author(s):  
Zachary W. Bent ◽  
Steven S. Branda ◽  
Glenn M. Young
Keyword(s):  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Type Iii

scholarly journals In VitroReconstitution of Functional Type III Protein Export and Insights into Flagellar Assembly

mBio ◽  
2018 ◽  
Vol 9 (3) ◽  
Author(s):  
Hiroyuki Terashima ◽  
Akihiro Kawamoto ◽  
Chinatsu Tatsumi ◽  
Keiichi Namba ◽  
Tohru Minamino ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Protein Transport ◽  
Atp Hydrolysis ◽  
Secretion System ◽  
Protein Export ◽  
Type Iii ◽  
Transport Assay ◽  
Flagellar Protein

ABSTRACTThe type III secretion system (T3SS) forms the functional core of injectisomes, protein transporters that allow bacteria to deliver virulence factors into their hosts for infection, and flagella, which are critical for many pathogens to reach the site of infection. In spite of intensive genetic and biochemical studies, the T3SS protein export mechanism remains unclear due to the difficulty of accurate measurement of protein exportin vivo. Here, we developed anin vitroflagellar T3S protein transport assay system using an inverted cytoplasmic membrane vesicle (IMV) for accurate and controlled measurements of flagellar protein export. We show that the flagellar T3SS in the IMV fully retains export activity. The flagellar hook was constructed inside the lumen of the IMV by adding purified component proteins externally to the IMV solution. We reproduced the hook length control and export specificity switch in the IMV consistent with that seen in the native cell. Previousin vivoanalyses showed that flagellar protein export is driven by proton motive force (PMF) and facilitated by ATP hydrolysis by FliI, a T3SS-specific ATPase. Ourin vitroassay recapitulated these previousin vivoobservations but furthermore clearly demonstrated that even ATP hydrolysis by FliI alone can drive flagellar protein export. Moreover, this assay showed that addition of the FliH2/FliI complex to the assay solution at a concentration similar to that in the cell dramatically enhanced protein export, confirming that the FliH2/FliI complex in the cytoplasm is important for effective protein transport.IMPORTANCEThe type III secretion system (T3SS) is the functional core of the injectisome, a bacterial protein transporter used to deliver virulence proteins into host cells, and bacterial flagella, critical for many pathogens. The molecular mechanism of protein transport is still unclear due to difficulties in accurate measurements of protein transport under well-controlled conditionsin vivo. We succeeded in developing anin vitrotransport assay system of the flagellar T3SS using inverted membrane vesicles (IMVs). Flagellar hook formation was reproduced in the IMV, suggesting that the export apparatus in the IMV retains a protein transport activity similar to that in the cell. Using this system, we revealed that ATP hydrolysis by the T3SS ATPase can drive protein export without PMF.

scholarly journals The transcription factor IscR promotes Yersinia type III secretion system activity by antagonizing the repressive H-NS-YmoA histone-like protein complex

2021 ◽  
Author(s):  
David Balderas ◽  
Pablo Alvarez ◽  
Mané Ohanyan ◽  
Erin Mettert ◽  
Natasha Tanner ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Protein Complex ◽  
Type Iii Secretion ◽  
Defense Mechanisms ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Type Iii ◽  
Transcription In Vitro

The type III secretion system (T3SS) is a appendage used by many bacterial pathogens, such as pathogenic Yersinia, to subvert host defenses. However, because the T3SS is energetically costly and immunogenic, it must be tightly regulated in response to environmental cues to enable survival in the host. Here we show that expression of the Yersinia Ysc T3SS master regulator, LcrF, is orchestrated by the opposing activities of the repressive YmoA/H-NS histone-like protein complex and induction by the iron and oxygen-regulated IscR transcription factor. Although IscR has been shown to bind the lcrF promoter and is required for in vivo expression of lcrF, in this study we show IscR alone fails to enhance lcrF transcription in vitro. Rather, we find that in a ymoA mutant, IscR is no longer required for LcrF expression or T3SS activity. Additionally, a mutation in YmoA that prevents H-NS binding (ymoAD43N) rescues the T3SS defect of a ∆iscR mutant, suggesting that a YmoA/H-NS complex is needed for this repressive activity. Furthermore, chromatin immunoprecipitation analysis revealed that H-NS is enriched at the lcrF promoter at environmental temperatures, while IscR is enriched at this promoter at mammalian body temperature under aerobic conditions. Importantly, CRISPRi knockdown of H-NS leads to increased lcrF transcription. Collectively, our data suggest that as IscR levels rise with iron limitation and oxidative stress, conditions Yersinia experiences during extraintestinal infection, IscR antagonizes YmoA/H-NS-mediated repression of lcrF transcription to drive T3SS activity and manipulate host defense mechanisms.

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