scholarly journals Functional Characterization of the Type III Secretion ATPase HrcN from the Plant Pathogen Xanthomonas campestris pv. vesicatoria

2008 ◽  
Vol 191 (5) ◽  
pp. 1414-1428 ◽  
Author(s):  
Christian Lorenz ◽  
Daniela Büttner
Keyword(s):  
Type Iii Secretion ◽  
Xanthomonas Campestris ◽  
Pathogenic Bacteria ◽  
Functional Characterization ◽  
Effector Proteins ◽  
Host Cells ◽  
Dependent Manner ◽  
Phosphate Binding ◽  
Type Iii

ABSTRACT Many gram-negative plant and animal pathogenic bacteria employ a type III secretion (T3S) system to inject effector proteins into the cytosol of eukaryotic host cells. The membrane-spanning T3S apparatus is associated with an ATPase that presumably provides the energy for the secretion process. Here, we describe the role of the predicted ATPase HrcN from the plant pathogenic bacterium Xanthomonas campestris pathovar vesicatoria. We show that HrcN hydrolyzes ATP in vitro and is essential for T3S and bacterial pathogenicity. Stability of HrcN in X. campestris pv. vesicatoria depends on the conserved HrcL protein, which interacts with HrcN in vitro and in vivo. Both HrcN and HrcL bind to the inner membrane protein HrcU and specifically localize to the bacterial membranes under T3S-permissive conditions. Protein-protein interaction studies revealed that HrcN also interacts with the T3S substrate specificity switch protein HpaC and the global T3S chaperone HpaB, which promotes secretion of multiple effector proteins. Using an in vitro chaperone release assay, we demonstrate that HrcN dissociates a complex between HpaB and the effector protein XopF1 in an ATP-dependent manner, suggesting that HrcN is involved in the release of HpaB-bound effectors. Effector release depends on a conserved glycine residue in the HrcN phosphate-binding loop, which is crucial for enzymatic activity and protein function during T3S. There is no experimental evidence that T3S can occur in the absence of the ATPase, in contrast to recent findings reported for animal pathogenic bacteria.

scholarly journals The C-terminal domain of the type III secretion chaperone HpaB contributes to dissociation of chaperone-effector complex in Xanthomonas campestris pv. campestris

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0246033
Author(s):  
Yong-Liang Gan ◽  
Li-Yan Yang ◽  
Li-Chao Yang ◽  
Wan-Lian Li ◽  
Xue-Lian Liang ◽  
...  
Keyword(s):  
Xanthomonas Campestris ◽  
Pathogenic Bacteria ◽  
Terminal Region ◽  
Effector Proteins ◽  
Host Cells ◽  
Dependent Manner ◽  
In Planta ◽  
Terminal Domain ◽  
Self Association

Many animal and plant pathogenic bacteria employ a type three secretion system (T3SS) to deliver type three effector proteins (T3Es) into host cells. Efficient secretion of many T3Es in the plant pathogen Xanthomonas campestris pv. campestris (Xcc) relies on the global chaperone HpaB. However, how the domain of HpaB itself affects effector translocation/secretion is poorly understood. Here, we used genetic and biochemical approaches to identify a novel domain at the C-terminal end of HpaB (amino acid residues 137–160) that contributes to virulence and hypersensitive response (HR). Both in vitro secretion assay and in planta translocation assay showed that the secretion and translocation of T3E proteins depend on the C-terminal region of HpaB. Deletion of the C-terminal region of HpaB did not affect binding to T3Es, self-association or interaction with T3SS components. However, the deletion of C-terminal region sharply reduced the mounts of free T3Es liberated from the complex of HpaB with the T3Es, a reaction catalyzed in an ATP-dependent manner by the T3SS-associated ATPase HrcN. Our findings demonstrate the C-terminal domain of HpaB contributes to disassembly of chaperone-effector complex and reveal a potential molecular mechanism underpinning the involvement of HpaB in secretion of T3Es in Xcc.

scholarly journals Identification of Novel Type III Secretion Effectors in Xanthomonas oryzae pv. oryzae

2009 ◽  
Vol 22 (1) ◽  
pp. 96-106 ◽  
Author(s):  
Ayako Furutani ◽  
Minako Takaoka ◽  
Harumi Sanada ◽  
Yukari Noguchi ◽  
Takashi Oku ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Pathogenic Bacteria ◽  
Regulatory Protein ◽  
Effector Proteins ◽  
Xanthomonas Oryzae ◽  
Dependent Manner ◽  
Plant Pathogenic Bacteria ◽  
Type Iii ◽  
Genes Encoding

Many gram-negative bacteria secrete so-called effector proteins via a type III secretion (T3S) system. Through genome screening for genes encoding potential T3S effectors, 60 candidates were selected from rice pathogen Xanthomonas oryzae pv. oryzae MAFF311018 using these criteria: i) homologs of known T3S effectors in plant-pathogenic bacteria, ii) genes with expression regulated by hrp regulatory protein HrpX, or iii) proteins with N-terminal amino acid patterns associated with T3S substrates of Pseudomonas syringae. Of effector candidates tested with the Bordetella pertussis calmodulin-dependent adenylate cyclase reporter for translocation into plant cells, 16 proteins were translocated in a T3S system-dependent manner. Of these 16 proteins, nine were homologs of known effectors in other plant-pathogenic bacteria and seven were not. Most of the effectors were widely conserved in Xanthomonas spp.; however, some were specific to X. oryzae. Interestingly, all these effectors were expressed in an HrpX-dependent manner, suggesting coregulation of effectors and the T3S system. In X. campestris pv. vesicatoria, HpaB and HpaC (HpaP in X. oryzae pv. oryzae) have a central role in recruiting T3S substrates to the secretion apparatus. Secretion of all but one effector was reduced in both HpaB– and HpaP– mutant strains, indicating that HpaB and HpaP are widely involved in efficient secretion of the effectors.

scholarly journals Interactions between effector proteins of the Pseudomonas aeruginosa type III secretion system do not significantly affect several measures of disease severity in mammals

Microbiology ◽  
2006 ◽  
Vol 152 (1) ◽  
pp. 143-152 ◽  
Author(s):  
Ciara M. Shaver ◽  
Alan R. Hauser
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Disease Severity ◽  
Type Iii Secretion ◽  
Disease Process ◽  
Effector Proteins ◽  
Host Cells ◽  
Secreted Proteins ◽  
Secretion Systems ◽  
Type Iii

The effector proteins of the type III secretion systems of many bacterial pathogens act in a coordinated manner to subvert host cells and facilitate the development and progression of disease. It is unclear whether interactions between the type-III-secreted proteins of Pseudomonas aeruginosa result in similar effects on the disease process. We have previously characterized the contributions to pathogenesis of the type-III-secreted proteins ExoS, ExoT and ExoU when secreted individually. In this study, we extend our prior work to determine whether these proteins have greater than expected effects on virulence when secreted in combination. In vitro cytotoxicity and anti-internalization activities were not enhanced when effector proteins were secreted in combinations rather than alone. Likewise in a mouse model of pneumonia, bacterial burden in the lungs, dissemination and mortality attributable to ExoS, ExoT and ExoU were not synergistically increased when combinations of these effector proteins were secreted. Because of the absence of an appreciable synergistic increase in virulence when multiple effector proteins were secreted in combination, we conclude that any cooperation between ExoS, ExoT and ExoU does not translate into a synergistically significant enhancement of disease severity as measured by these assays.

scholarly journals Pseudolipasin A Is a Specific Inhibitor for Phospholipase A2 Activity of Pseudomonas aeruginosa Cytotoxin ExoU

2006 ◽  
Vol 75 (3) ◽  
pp. 1089-1098 ◽  
Author(s):  
Vincent T. Lee ◽  
Stefan Pukatzki ◽  
Hiromi Sato ◽  
Eriya Kikawada ◽  
Anastasia A. Kazimirova ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Specific Inhibitor ◽  
Effector Proteins ◽  
Host Cells ◽  
Cytotoxic Effects ◽  
Type Iii ◽  
Secretion Pathway ◽  
Phospholipase A2 Activity

ABSTRACT A number of bacterial pathogens utilize the type III secretion pathway to deliver effector proteins directly into the host cell cytoplasm. Certain strains of Pseudomonas aeruginosa associated with acute infections express a potent cytotoxin, exoenzyme U (ExoU), that is delivered via the type III secretion pathway directly into contacting host cells. Once inside the mammalian cell, ExoU rapidly lyses the intoxicated cells via its phospholipase A2 (PLA2) activity. A high-throughput cell-based assay was developed to screen libraries of compounds for those capable of protecting cells against the cytotoxic effects of ExoU. A number of compounds were identified in this screen, including one group that blocks the intracellular activity of ExoU. In addition, these compounds specifically inhibited the PLA2 activity of ExoU in vitro, whereas eukaryotic secreted PLA2 and cytosolic PLA2 were not inhibited. This novel inhibitor of ExoU-specific PLA2 activity, named pseudolipasin A, may provide a new lead for virulence factor-based therapeutic design.

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

2015 ◽  
Vol 81 (17) ◽  
pp. 6078-6087 ◽  
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.

scholarly journals XopC and XopJ, Two Novel Type III Effector Proteinsfrom Xanthomonas campestris pv.vesicatoria

2003 ◽  
Vol 185 (24) ◽  
pp. 7092-7102 ◽  
Author(s):  
Laurent Noël ◽  
Frank Thieme ◽  
Jana Gäbler ◽  
Daniela Büttner ◽  
Ulla Bonas
Keyword(s):  
Pseudomonas Syringae ◽  
Plant Cell ◽  
Type Iii Secretion ◽  
Xanthomonas Campestris ◽  
Pathogenic Bacteria ◽  
Effector Proteins ◽  
Type Iii ◽  
Genome Wide ◽  
A Genome ◽  
Xanthomonas Campestris Pv.Vesicatoria

ABSTRACT Pathogenicity of the gram-negative plant pathogen Xanthomonas campestris pv. vesicatoria depends on a type III secretion (TTS) system which translocates bacterial effector proteins into the plant cell. Previous transcriptome analysis identified a genome-wide regulon of putative virulence genes that are coexpressed with the TTS system. In this study, we characterized two of these genes, xopC and xopJ. Both genes encode Xanthomonas outer proteins (Xops) that were shown to be secreted by the TTS system. In addition, type III-dependent translocation of both proteins into the plant cell was demonstrated using the AvrBs3 effector domain as a reporter. XopJ belongs to the AvrRxv/YopJ family of effector proteins from plant and animal pathogenic bacteria. By contrast, XopC does not share significant homology to proteins in the database. Sequence analysis revealed that the xopC locus contains several features that are reminiscent of pathogenicity islands. Interestingly, the xopC region is flanked by 62-bp inverted repeats that are also associated with members of the Xanthomonas avrBs3 effector family. Besides xopC, a second gene of the locus, designated hpaJ, was shown to be coexpressed with the TTS system. hpaJ encodes a protein with similarity to transglycosylases and to the Pseudomonas syringae pv. maculicola protein HopPmaG. HpaJ secretion and translocation by the X. campestris pv. vesicatoria TTS system was not detectable, which is consistent with its predicted Sec signal and a putative function as transglycosylase in the bacterial periplasm.

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

Antibodies ◽  
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.

scholarly journals Role of the Salmonella Pathogenicity Island 1 (SPI-1) Protein InvB in Type III Secretion of SopE and SopE2, Two Salmonella Effector Proteins Encoded Outside of SPI-1

2003 ◽  
Vol 185 (23) ◽  
pp. 6950-6967 ◽  
Author(s):  
Kristin Ehrbar ◽  
Andrea Friebel ◽  
Samuel I. Miller ◽  
Wolf-Dietrich Hardt
Keyword(s):  
Type Iii Secretion ◽  
Inflammatory Responses ◽  
Pathogenicity Island ◽  
Effector Proteins ◽  
Host Cells ◽  
Effector Protein ◽  
Dependent Manner ◽  
Type Iii ◽  
Serovar Typhimurium

ABSTRACT Salmonella enterica subspecies 1 serovar Typhimurium encodes a type III secretion system (TTSS) within Salmonella pathogenicity island 1 (SPI-1). This TTSS injects effector proteins into host cells to trigger invasion and inflammatory responses. Effector proteins are recognized by the TTSS via signals encoded in their N termini. Specific chaperones can be involved in this process. The chaperones InvB, SicA, and SicP are encoded in SPI-1 and are required for transport of SPI-1-encoded effectors. Several key effector proteins, like SopE and SopE2, are located outside of SPI-1 but are secreted in an SPI-1-dependent manner. It has not been clear how these effector proteins are recognized by the SPI-1 TTSS. Using pull-down and coimmunoprecipitation assays, we found that SopE is copurified with InvB, the known chaperone for the SPI-1-encoded effector protein Sip/SspA. We also found that InvB is required for secretion and translocation of SopE and SopE2 and for stabilization of SopE2 in the bacterial cytosol. Our data demonstrate that effector proteins encoded within and outside of SPI-1 use the same chaperone for secretion via the SPI-1 TTSS.

scholarly journals Identification of Novel Host Interactors of Effectors Secreted by Salmonella and Citrobacter

mSystems ◽  
2016 ◽  
Vol 1 (4) ◽  
Author(s):  
Ryan L. Sontag ◽  
Ernesto S. Nakayasu ◽  
Roslyn N. Brown ◽  
George S. Niemann ◽  
Michael A. Sydor ◽  
...  
Keyword(s):  
Host Cell ◽  
Type Iii Secretion ◽  
Defense Mechanisms ◽  
Pathogenic Bacteria ◽  
Effector Proteins ◽  
Host Cells ◽  
Secretion Systems ◽  
Type Iii ◽  
Cell Cytosol ◽  
Host Cell Cytosol

ABSTRACT During infection, pathogenic bacteria face an adverse environment of factors driven by both cellular and humoral defense mechanisms. To help evade the immune response and ultimately proliferate inside the host, many bacteria evolved specialized secretion systems to deliver effector proteins directly into host cells. Translocated effector proteins function to subvert host defense mechanisms. Numerous pathogenic bacteria use a specialized secretion system called type III secretion to deliver effectors into the host cell cytosol. Here, we identified 75 new host targets of Salmonella and Citrobacter effectors, which will help elucidate their mechanisms of action. Many pathogenic bacteria of the family Enterobacteriaceae use type III secretion systems to inject virulence proteins, termed “effectors,” into the host cell cytosol. Although host-cellular activities of several effectors have been demonstrated, the function and host-targeted pathways of most of the effectors identified to date are largely undetermined. To gain insight into host proteins targeted by bacterial effectors, we performed coaffinity purification of host proteins from cell lysates using recombinant effectors from the Enterobacteriaceae intracellular pathogens Salmonella enterica serovar Typhimurium and Citrobacter rodentium. We identified 54 high-confidence host interactors for the Salmonella effectors GogA, GtgA, GtgE, SpvC, SrfH, SseL, SspH1, and SssB collectively and 21 interactors for the Citrobacter effectors EspT, NleA, NleG1, and NleK. We biochemically validated the interaction between the SrfH Salmonella protein and the extracellular signal-regulated kinase 2 (ERK2) host protein kinase, which revealed a role for this effector in regulating phosphorylation levels of this enzyme, which plays a central role in signal transduction. IMPORTANCE During infection, pathogenic bacteria face an adverse environment of factors driven by both cellular and humoral defense mechanisms. To help evade the immune response and ultimately proliferate inside the host, many bacteria evolved specialized secretion systems to deliver effector proteins directly into host cells. Translocated effector proteins function to subvert host defense mechanisms. Numerous pathogenic bacteria use a specialized secretion system called type III secretion to deliver effectors into the host cell cytosol. Here, we identified 75 new host targets of Salmonella and Citrobacter effectors, which will help elucidate their mechanisms of action.

scholarly journals Functional Characterization of the Type III Secretion Substrate Specificity Switch Protein HpaC from Xanthomonas campestris pv. vesicatoria

2011 ◽  
Vol 79 (8) ◽  
pp. 2998-3011 ◽  
Author(s):  
Steve Schulz ◽  
Daniela Büttner
Keyword(s):  
Amino Acids ◽  
Substrate Specificity ◽  
Type Iii Secretion ◽  
Protein Function ◽  
Xanthomonas Campestris ◽  
Functional Characterization ◽  
Effector Proteins ◽  
Content Type ◽  
Type Iii ◽  
In The Wild

ABSTRACTPathogenicity ofXanthomonas campestrispv.vesicatoriadepends on a type III secretion (T3S) system which translocates effector proteins into eukaryotic cells and is associated with an extracellular pilus and a translocon in the host plasma membrane. T3S substrate specificity is controlled by the cytoplasmic switch protein HpaC, which interacts with the C-terminal domain of the inner membrane protein HrcU (HrcUC). HpaC promotes the secretion of translocon and effector proteins but prevents the efficient secretion of the early T3S substrate HrpB2, which is required for pilus assembly. In this study, complementation assays with serial 10-amino-acid HpaC deletion derivatives revealed that the T3S substrate specificity switch depends on N- and C-terminal regions of HpaC, whereas amino acids 42 to 101 appear to be dispensable for the contribution of HpaC to the secretion of late substrates. However, deletions in the central region of HpaC affect the secretion of HrpB2, suggesting that the mechanisms underlying HpaC-dependent control of early and late substrates can be uncoupled. The results of interaction and expression studies with HpaC deletion derivatives showed that amino acids 112 to 212 of HpaC provide the binding site for HrcUCand severely reduce T3S when expressed ectopically in the wild-type strain. We identified a conserved phenylalanine residue at position 175 of HpaC that is required for both protein function and the binding of HpaC to HrcUC. Taking these findings together, we concluded that the interaction between HpaC and HrcUCis essential but not sufficient for T3S substrate specificity switching.

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