scholarly journals Identification of YsaP, the Pilotin of the Yersinia enterocolitica Ysa Type III Secretion System

2015 ◽  
Vol 197 (17) ◽  
pp. 2770-2779 ◽  
Author(s):  
Reina Rau ◽  
Andrew J. Darwin
Keyword(s):  
Outer Membrane ◽  
Yersinia Enterocolitica ◽  
Type Iii Secretion ◽  
Outer Membrane Proteins ◽  
State Level ◽  
Secretion Systems ◽  
Membrane Pore ◽  
Content Type ◽  
Type Iii ◽  
Null Strain

ABSTRACTSecretins are multimeric outer membrane pore-forming proteins found in complex export systems in Gram-negative bacteria. All type III secretion systems (T3SSs) have a secretin, and one of these is the YsaC secretin of the chromosomally encoded Ysa T3SS ofYersinia enterocolitica. In some cases, pilotin proteins, which are outer membrane lipoproteins, are required for their cognate secretins to multimerize and/or localize to the outer membrane. However, if secretin multimers mislocalize to the inner membrane, this can trigger the protective phage shock protein (Psp) stress response. During a screen for mutations that suppress YsaC toxicity to apspnull strain, we isolated several independent mutations predicted to increase expression of the YE3559 gene within the Ysa pathogenicity island. YE3559, which we have namedysaP, is predicted to encode a small outer membrane lipoprotein, and this location was confirmed by membrane fractionation. ElevatedysaPexpression increased the steady-state level of YsaC but made it less toxic to apspnull strain, and it also decreased YsaC-dependent induction ofpspgene expression. Subsequent experiments showed that YsaP was not required for YsaC multimerization but was required for the multimers to localize to the outer membrane. Consistent with this, aysaPnull mutation compromised protein export by the Ysa T3SS. All these observations suggest that YsaP is the pilotin for the YsaC secretin. This is only the second pilotin to be characterized forYersiniaand one of only a small number of pilotins described for all bacteria.IMPORTANCESecretins are essential for the virulence of many bacterial pathogens and also play roles in surface attachment, motility, and competence. This has generated considerable interest in understanding how secretins function. However, their fundamental differences from typical outer membrane proteins have raised various questions about secretins, including how they are assembled into outer membrane multimers. Pilotin proteins facilitate the assembly of some secretins, but only a small number of pilotins have been identified, slowing efforts to understand common and distinct features of secretin assembly. This study provides an important advance by identifying a novel member of the pilotin family and also demonstrating a method of pilotin discovery that could be broadly applied.

scholarly journals Identification of Potential Type III Secretion Proteins via Heterologous Expression of Vibrio parahaemolyticus DNA

2012 ◽  
Vol 78 (9) ◽  
pp. 3492-3494 ◽  
Author(s):  
Xiaohui Zhou ◽  
Seth D. Nydam ◽  
Jeffrey E. Christensen ◽  
Michael E. Konkel ◽  
Lisa Orfe ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Yersinia Enterocolitica ◽  
Type Iii Secretion ◽  
Vibrio Parahaemolyticus ◽  
Genomic Islands ◽  
Secretion Systems ◽  
Content Type ◽  
Type Iii ◽  
Type Iii Secretion Systems ◽  
Secretion Assay

ABSTRACTWe employed a heterologous secretion assay to identify proteins potentially secreted by type III secretion systems (T3SSs) inVibrio parahaemolyticus. N-terminal sequences from 32 proteins within T3SS genomic islands and seven proteins from elsewhere in the chromosome included proteins that were recognized for export by theYersinia enterocoliticaflagellar T3SS.

Investigation of the role of the BAM complex and SurA chaperone in outer-membrane protein biogenesis and type III secretion system expression in Salmonella

Microbiology ◽  
2009 ◽  
Vol 155 (5) ◽  
pp. 1613-1622 ◽  
Author(s):  
Yann Fardini ◽  
Jérôme Trotereau ◽  
Elisabeth Bottreau ◽  
Charlène Souchard ◽  
Philippe Velge ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Type Iii Secretion ◽  
Outer Membrane Proteins ◽  
Transcriptional Analysis ◽  
Secretion Systems ◽  
Type Iii ◽  
Bam Complex ◽  
Periplasmic Chaperones

In Escherichia coli, the assembly of outer-membrane proteins (OMP) requires the BAM complex and periplasmic chaperones, such as SurA or DegP. Previous work has suggested a potential link between OMP assembly and expression of the genes encoding type-III secretion systems. In order to test this hypothesis, we studied the role of the different lipoproteins of the BAM complex (i.e. BamB, BamC, BamD and BamE), and the periplasmic chaperones SurA and DegP, in these two phenotypes in Salmonella. Analysis of the corresponding deletion mutants showed that, as previously described with the ΔbamB mutant, BamD, SurA and, to a lesser extent, BamE play a role in outer-membrane biogenesis in Salmonella Enteritidis, while the membrane was not notably disturbed in ΔbamC and ΔdegP mutants. Interestingly, we found that BamD is not essential in Salmonella, unlike its homologues in Escherichia coli and Neisseria gonorrhoeae. In contrast, BamD was the only protein required for full expression of T3SS-1 and flagella, as demonstrated by transcriptional analysis of the genes involved in the biosynthesis of these T3SSs. In line with this finding, bamD mutants showed a reduced secretion of effector proteins by these T3SSs, and a reduced ability to invade HT-29 cells. As ΔsurA and ΔbamE mutants had lower levels of OMPs in their outer membrane, but showed no alteration in T3SS-1 and flagella expression, these results demonstrate the absence of a systematic link between an OMP assembly defect and the downregulation of T3SSs in Salmonella; therefore, this link appears to be related to a more specific mechanism that involves at least BamB and BamD.

scholarly journals An Amino-Terminal Secretion Signal Is Required for YplA Export by the Ysa, Ysc, and Flagellar Type III Secretion Systems of Yersinia enterocolitica Biovar 1B

2005 ◽  
Vol 187 (17) ◽  
pp. 6075-6083 ◽  
Author(s):  
Sasha M. Warren ◽  
Glenn M. Young
Keyword(s):  
Yersinia Enterocolitica ◽  
Type Iii Secretion ◽  
Distinct Type ◽  
Host Cells ◽  
Amino Acid Residues ◽  
Secretion Systems ◽  
Type Iii ◽  
Secretion Signal ◽  
Amino Terminal ◽  
Type Iii Secretion Systems

ABSTRACT Yersinia enterocolitica biovar 1B maintains three distinct type III secretion (TTS) systems, which independently operate to target proteins to extracellular sites. The Ysa and Ysc systems are prototypical contact-dependent TTS systems that translocate toxic effectors to the cytosols of targeted eukaryotic host cells during infection. The flagellar TTS system is utilized during the assembly of the flagellum and is required for secretion of the virulence-associated phospholipase YplA to the bacterial milieu. When ectopically produced, YplA is also a secretion substrate for the Ysa and Ysc TTS systems. In this study, we define elements that allow YplA recognition and export by the Ysa, Ysc, and flagellar TTS systems. Fusion of various amino-terminal regions of YplA to Escherichia coli alkaline phosphatase (PhoA) lacking its native secretion signal demonstrated that the first 20 amino acids or corresponding mRNA codons of YplA were sufficient for export of YplA-PhoA chimeras by each TTS system. Export of native YplA by each of the three TTS systems was also found to depend on the integrity of its amino terminus. Introduction of a frameshift mutation or deletion of yplA sequences encoding the amino-terminal 20 residues negatively impacted YplA secretion. Deletion of other yplA regions was tolerated, including that resulting in the removal of amino acid residues 30 through 40 of the polypeptide and removal of the 5′ untranslated region of the mRNA. This work supports a model in which independent and distantly related TTS systems of Y. enterocolitica recognize protein substrates by a similar mechanism.

scholarly journals Evidence for Targeting of Yop Effectors by the Chromosomally Encoded Ysa Type III Secretion System of Yersinia enterocolitica

2002 ◽  
Vol 184 (20) ◽  
pp. 5563-5571 ◽  
Author(s):  
Briana M. Young ◽  
Glenn M. Young
Keyword(s):  
Yersinia Enterocolitica ◽  
Type Iii Secretion ◽  
Immunoblot Analysis ◽  
Effector Proteins ◽  
Secretion Systems ◽  
Necrosis Factor Alpha ◽  
Type Iii ◽  
Type Iii Secretion Systems ◽  
Molecular Masses ◽  
Factor Alpha

ABSTRACT Yersinia enterocolitica O:8 has two contact-dependent type III secretion systems (TTSSs). The Ysa TTSS is encoded by a set of genes located on the chromosome and exports Ysp proteins. The Ysc TTSS and the Yop effector proteins it exports are encoded by genes located on plasmid pYVe8081. In this study, secretion of YspG, YspH, and YspJ by the Ysa TTSS was shown to require pYVe8081. Furthermore, mutations that blocked the function of the Ysc TTSS did not affect YspG, YspH, and YspJ production. This indicated that YspG, YspH, and YspJ are encoded by genes located on pYVe8081 and that they may correspond to Yops. A comparison of Ysps with Yop effectors secreted by Y. enterocolitica indicated that YspG, YspH, and YspJ have apparent molecular masses similar to those of YopN, YopP, and YopE, respectively. Immunoblot analysis demonstrated that antibodies directed against YopN, YopP, and YopE recognized YspG, YspH, and YspJ. Furthermore, mutations in yopN, yopP, and yopE specifically blocked YopN, YopP, and YopE secretion by the Ysc TTSS and YspG, YspH, and YspJ secretion by the Ysa TTSS. These results indicate YspG, YspH, and YspJ are actually YopN, YopP, and YopE. Additional analysis demonstrated that YopP and YspH secretion was restored to yopP mutants by complementation in trans with a wild-type copy of the yopP gene. Examination of Y. enterocolitica-infected J774A.1 macrophages revealed that both the Ysc and Ysa TTSSs contribute to YopP-dependent suppression of tumor necrosis factor alpha production. This indicates that both the Ysa and Ysc TTSSs are capable of targeting YopP and that they influence Y. enterocolitica interactions with macrophages. Taken together, these results suggest that the Ysa and Ysc TTSSs contribute to Y. enterocolitica virulence by exporting both unique and common subsets of effectors.

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

2004 ◽  
Vol 72 (7) ◽  
pp. 4004-4009 ◽  
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.

scholarly journals The Bacterium Pantoea stewartii Uses Two Different Type III Secretion Systems To Colonize Its Plant Host and Insect Vector

2012 ◽  
Vol 78 (17) ◽  
pp. 6327-6336 ◽  
Author(s):  
Valdir R. Correa ◽  
Doris R. Majerczak ◽  
El-Desouky Ammar ◽  
Massimo Merighi ◽  
Richard C. Pratt ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Flea Beetle ◽  
Insect Vector ◽  
Secretion Systems ◽  
Plant Host ◽  
Content Type ◽  
Type Iii ◽  
Pantoea Stewartii ◽  
Type Iii Secretion Systems ◽  
Animal Pathogens

ABSTRACTPlant- and animal-pathogenic bacteria utilize phylogenetically distinct type III secretion systems (T3SS) that produce needle-like injectisomes or pili for the delivery of effector proteins into host cells.Pantoea stewartiisubsp.stewartii(herein referred to asP. stewartii), the causative agent of Stewart's bacterial wilt and leaf blight of maize, carries phylogenetically distinct T3SSs. In addition to an Hrc-Hrp T3SS, known to be essential for maize pathogenesis,P. stewartiihas a second T3SS (Pantoeasecretion island 2 [PSI-2]) that is required for persistence in its flea beetle vector,Chaetocnema pulicaria(Melsh). PSI-2 belongs to the Inv-Mxi-Spa T3SS family, typically found in animal pathogens. Mutagenesis of the PSI-2psaNgene, which encodes an ATPase essential for secretion of T3SS effectors by the injectisome, greatly reduces both the persistence ofP. stewartiiin flea beetle guts and the beetle's ability to transmitP. stewartiito maize. Ectopic expression of thepsaNgene complements these phenotypes. In addition, the PSI-2psaNgene is not required forP. stewartiipathogenesis of maize and is transcriptionally upregulated in insects compared to maize tissues. Thus, the Hrp and PSI-2 T3SSs play different roles in the life cycle ofP. stewartiias it alternates between its insect vector and plant host.

scholarly journals Type III Secretion Is Essential for the Rapidly Fatal Diarrheal Disease Caused by Non-O1, Non-O139 Vibrio cholerae

mBio ◽  
2011 ◽  
Vol 2 (3) ◽  
Author(s):  
Ok S. Shin ◽  
Vincent C. Tam ◽  
Masato Suzuki ◽  
Jennifer M. Ritchie ◽  
Roderick T. Bronson ◽  
...  
Keyword(s):  
Cholera Toxin ◽  
Vibrio Cholerae ◽  
Intestinal Epithelium ◽  
Type Iii Secretion ◽  
Diarrheal Disease ◽  
Secretion Systems ◽  
Content Type ◽  
Type Iii ◽  
Severe Diarrhea ◽  
Toxin Coregulated Pilus

ABSTRACTCholera is a severe diarrheal disease typically caused by O1 serogroup strains ofVibrio cholerae. The pathogenicity of all pandemicV. choleraeO1 strains relies on two critical virulence factors: cholera toxin, a potent enterotoxin, and toxin coregulated pilus (TCP), an intestinal colonization factor. However, certain non-O1, non-O139V. choleraestrains, such as AM-19226, do not produce cholera toxin or TCP, yet they still cause severe diarrhea. The molecular basis for the pathogenicity of non-O1, non-O139V. choleraehas not been extensively characterized, but many of these strains encode related type III secretion systems (TTSSs). Here, we used infant rabbits to assess the contribution of the TTSS to non-O1, non-O139V. choleraepathogenicity. We found that all animals infected with wild-type AM-19226 developed severe diarrhea even more rapidly than rabbits infected withV. choleraeO1. UnlikeV. choleraeO1 strains, which do not damage the intestinal epithelium in rabbits or humans, AM-19226 caused marked disruptions of the epithelial surface in the rabbit small intestine. TTSS proved to be essential for AM-19226 virulence in infant rabbits; an AM-19226 derivative deficient for TTSS did not elicit diarrhea, colonize the intestine, or induce pathological changes in the intestine. Deletion of either one of the two previously identified or two newly identified AM-19226 TTSS effectors reduced but did not eliminate AM-19226 pathogenicity, suggesting that at least four effectors contribute to this strain’s virulence. In aggregate, our results suggest that the TTSS-dependent virulence in non-O1, non-O139V. choleraerepresents a new type of diarrheagenic mechanism.IMPORTANCECholera, which is caused byVibrio cholerae, is an important cause of diarrheal disease in many developing countries. The mechanisms of virulence of nonpandemic strains that can cause a diarrheal illness are poorly understood. AM-19226, like several other pathogenic, nonpandemicV. choleraestrains, carries genes that encode a type III secretion system (TTSS), but not cholera toxin (CT) or toxin coregulated pilus (TCP). In this study, we used infant rabbits to study AM-19226 virulence. Infant rabbits orally inoculated with this strain rapidly developed a fatal diarrheal disease, which was accompanied by marked disruptions of the intestinal epithelium. This strain’s TTSS proved essential for its pathogenicity, and there was no diarrhea, intestinal pathology, or colonization in rabbits infected with a TTSS mutant. The effector proteins translocated by the TTSS all appear to contribute to AM-19226 virulence. Thus, our study provides insight intoin vivomechanisms by which a novel TTSS contributes to diarrheal disease caused by nonpandemic strains ofV. cholerae.

scholarly journals Dynamics of the Type III Secretion System Activity of Enteropathogenic Escherichia coli

mBio ◽  
2013 ◽  
Vol 4 (4) ◽  
Author(s):  
Erez Mills ◽  
Kobi Baruch ◽  
Gili Aviv ◽  
Mor Nitzan ◽  
Ilan Rosenshine
Keyword(s):  
Escherichia Coli ◽  
Type Iii Secretion ◽  
Public Health Problem ◽  
Effector Proteins ◽  
Host Cells ◽  
Global Public Health ◽  
Secretion Systems ◽  
Comprehensive Description ◽  
Content Type ◽  
Type Iii

ABSTRACT Type III secretion systems (TTSSs) are employed by pathogens to translocate host cells with effector proteins, which are crucial for virulence. The dynamics of effector translocation, behavior of the translocating bacteria, translocation temporal order, and relative amounts of each of the translocated effectors are all poorly characterized. To address these issues, we developed a microscopy-based assay that tracks effector translocation. We used this assay alongside a previously described real-time population-based translocation assay, focusing mainly on enteropathogenic Escherichia coli (EPEC) and partly comparing it to Salmonella. We found that the two pathogens exhibit different translocation behaviors: in EPEC, a subpopulation that formed microcolonies carried out most of the translocation activity, while Salmonella executed protein translocation as planktonic bacteria. We also noted variability in host cell susceptibility, with some cells highly resistant to translocation. We next extended the study to determine the translocation dynamics of twenty EPEC effectors and found that all exhibited distinct levels of translocation efficiency. Further, we mapped the global effects of key TTSS-related components on TTSS activity. Our results provide a comprehensive description of the dynamics of the TTSS activity of EPEC and new insights into the mechanisms that control the dynamics. IMPORTANCE EPEC and the closely related enterohemorrhagic Escherichia coli (EHEC) represent a global public health problem. New strategies to combat EPEC and EHEC infections are needed, and development of such strategies requires better understanding of their virulence machinery. The TTSS is a critical virulence mechanism employed by these pathogens, and by others, including Salmonella. In this study, we aimed at elucidating new aspects of TTSS function. The results obtained provide a comprehensive description of the dynamics of TTSS activity of EPEC and new insights into the mechanisms that control these changes. This knowledge sets the stage for further analysis of the system and may accelerate the development of new ways to treat EPEC and EHEC infections. Further, the newly described microscopy-based assay can be readily adapted to study the dynamics of TTSS activity in other pathogens.

scholarly journals Persistence of Vibrio parahaemolyticus in the Pacific Oyster, Crassostrea gigas, Is a Multifactorial Process Involving Pili and Flagella but Not Type III Secretion Systems or Phase Variation

2013 ◽  
Vol 79 (10) ◽  
pp. 3303-3305 ◽  
Author(s):  
Alisha M. Aagesen ◽  
Sureerat Phuvasate ◽  
Yi-Cheng Su ◽  
Claudia C. Häse
Keyword(s):  
Type Iii Secretion ◽  
Vibrio Parahaemolyticus ◽  
Phase Variation ◽  
Type I ◽  
Secretion Systems ◽  
Content Type ◽  
Type Iii ◽  
Type Iv ◽  
Pacific Oysters ◽  
Type Iii Secretion Systems

ABSTRACTVibrio parahaemolyticuscan resist oyster depuration, suggesting that it possesses specific factors for persistence. We show that type I pili, type IV pili, and both flagellar systems contribute toV. parahaemolyticuspersistence in Pacific oysters whereas type III secretion systems and phase variation do not.

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