scholarly journals Roles of LcrG and LcrV during Type III Targeting of Effector Yops by Yersinia enterocolitica

2001 ◽  
Vol 183 (15) ◽  
pp. 4588-4598 ◽  
Author(s):  
Kristin L. DeBord ◽  
Vincent T. Lee ◽  
Olaf Schneewind
Keyword(s):  
Plasma Membrane ◽  
Yersinia Enterocolitica ◽  
Negative Regulator ◽  
Host Cells ◽  
Eukaryotic Cells ◽  
Extracellular Medium ◽  
Bacterial Surface ◽  
Type Iii ◽  
Surface Receptor ◽  
Bacterial Cytoplasm

ABSTRACT Yersinia enterocolitica target effector Yop proteins into the cytosol of eukaryotic cells by a mechanism requiring the type III machinery. LcrG and LcrV have been suggested to fulfill essential functions during the type III targeting of effector Yops. It is reported here that knockout mutations of lcrG caused mutant yersiniae to prematurely secrete Yops into the extracellular medium without abolishing the type III targeting mechanism (Los phenotype [loss of type III targeting specificity]). Knockout mutations inlcrV reduced type III targeting of mutant yersiniae but did not promote secretion into the extracellular medium (Not [no type III targeting]). However, knockout mutations in both genes caused ΔlcrGV yersiniae to display a Los phenotype similar to that of strains carrying knockout mutations in lcrG alone. LcrG binding to LcrV resulted in the formation of soluble LcrGV complexes in the bacterial cytoplasm. Membrane-associated, bacterial-surface-displayed or -secreted LcrG could not be detected. Most of LcrV was located in the bacterial cytoplasm; however, small amounts were secreted into the extracellular medium. These data support a model whereby LcrG may act as a negative regulator of type III targeting in the bacterial cytoplasm, an activity that is modulated by LcrG binding to LcrV. No support could be gathered for the hypothesis whereby LcrG and LcrV may act as a bacterial surface receptor for host cells, allowing effector Yop translocation across the eukaryotic plasma membrane.

scholarly journals Yersinia enterocolitica TyeA, an Intracellular Regulator of the Type III Machinery, Is Required for Specific Targeting of YopE, YopH, YopM, and YopN into the Cytosol of Eukaryotic Cells

2000 ◽  
Vol 182 (11) ◽  
pp. 3183-3190 ◽  
Author(s):  
Luisa W. Cheng ◽  
Olaf Schneewind
Keyword(s):  
Plasma Membrane ◽  
Yersinia Enterocolitica ◽  
Affinity Purification ◽  
Negative Regulator ◽  
Eukaryotic Cells ◽  
Cell Fractionation ◽  
Extracellular Medium ◽  
Bacterial Surface ◽  
Type Iii ◽  
Specific Targeting

ABSTRACT Pathogenic Yersinia species employ type III machines to target effector Yops into the cytosol of eukaryotic cells.Yersinia tyeA mutants are thought to be defective in the targeting of YopE and YopH without affecting the injection of YopM, YopN, YopO, YopP, and YopT into the cytosol of eukaryotic cells. One model suggests that TyeA may form a tether between YopN (LcrE) and YopD on the bacterial surface, a structure that may translocate YopE and YopH across the plasma membrane of eukaryotic cells (M. Iriarte, M. P. Sory, A. Boland, A. P. Boyd, S. D. Mills, I. Lambermont, and G. R. Cornelis, EMBO J. 17:1907–1918, 1998). We have examined the injection of Yop proteins by tyeA mutant yersiniae with the digitonin fractionation technique. We find thattyeA mutant yersiniae not only secreted YopE, YopH, YopM, and YopN into the extracellular medium but also targeted these polypeptides into the cytosol of HeLa cells. Protease protection, cell fractionation, and affinity purification experiments suggest that TyeA is located intracellularly and binds to YopN or YopD. We propose a model whereby TyeA functions as a negative regulator of the type III targeting pathway in the cytoplasm of yersiniae, presumably by preventing the export of YopN.

scholarly journals Binding of SycH Chaperone to YscM1 and YscM2 Activates Effector yop Expression in Yersinia enterocolitica

2004 ◽  
Vol 186 (3) ◽  
pp. 829-841 ◽  
Author(s):  
Eric D. Cambronne ◽  
Joseph A. Sorg ◽  
Olaf Schneewind
Keyword(s):  
Host Cell ◽  
Yersinia Enterocolitica ◽  
Posttranscriptional Regulation ◽  
Bacterial Infections ◽  
Gel Filtration ◽  
Host Cells ◽  
Cell Contact ◽  
Extracellular Medium ◽  
Type Iii ◽  
Bacterial Cytoplasm

ABSTRACT Yersinia enterocolitica transports YscM1 and YscM2 via the type III pathway, a mechanism that is required for the establishment of bacterial infections. Prior to host cell contact, YscM1 and YscM2 exert posttranscriptional regulation to inhibit expression of effector yop genes, which encode virulence factors that travel the type III pathway into the cytoplasm of macrophages. Relief from repression has been predicted to occur via the type III secretion of YscM1 and YscM2 into the extracellular medium, resulting in the depletion of regulatory molecules from the bacterial cytoplasm. Using digitonin fractionation and fluorescence microscopy of FlAsH-labeled polypeptides in Yersinia-infected cells, we have localized YscM1 and YscM2 within the host cell cytoplasm. Type III injection of YscM1 and YscM2 required the SycH chaperone. Expression of C-terminal fusions of YscM1 and YscM2 to the neomycin phosphotransferase reporter revealed sequences required for regulatory activity and for secretion in the absence of SycH. Coexpression of SycH and glutathione S-transferase (GST)-YscM1 or GST-YscM2, hybrid GST variants that cannot be transported by the type III apparatus, also relieved repression of Yop synthesis. GST-SycH bound to YscM1 and YscM2 and activated effector yop expression without initiation of the bound regulatory molecules into the type III pathway. Further, regulation of yop expression by YscM1, YscM2, and SycH is shown to act independently of factors that regulate secretion, and gel filtration chromotography revealed populations of YscM1 and YscM2 that are not bound to SycH under conditions where Yop synthesis is repressed. Taken together, these results suggest that YscM1- and YscM2-mediated repression may be relieved through binding to the cytoplasmic chaperone SycH prior to their type III injection into host cells.

scholarly journals Small-Molecule Type III Secretion System Inhibitors Block Assembly of the Shigella Type III Secreton

2008 ◽  
Vol 191 (2) ◽  
pp. 563-570 ◽  
Author(s):  
Andreas K. J. Veenendaal ◽  
Charlotta Sundin ◽  
Ariel J. Blocker
Keyword(s):  
Type Iii Secretion ◽  
Bacterial Infections ◽  
Shigella Flexneri ◽  
Effector Proteins ◽  
Host Cells ◽  
Incomplete Penetrance ◽  
Secretion Systems ◽  
Bacterial Surface ◽  
Type Iii ◽  
Type Iii Secretion Systems

ABSTRACT Type III secretion systems (T3SSs) are essential virulence devices for many gram-negative bacteria that are pathogenic for plants, animals, and humans. They serve to translocate virulence effector proteins directly into eukaryotic host cells. T3SSs are composed of a large cytoplasmic bulb and a transmembrane region into which a needle is embedded, protruding above the bacterial surface. The emerging antibiotic resistance of bacterial pathogens urges the development of novel strategies to fight bacterial infections. Therapeutics that rather than kill bacteria only attenuate their virulence may reduce the frequency or progress of resistance emergence. Recently, a group of salicylidene acylhydrazides were identified as inhibitors of T3SSs in Yersinia, Chlamydia, and Salmonella species. Here we show that these are also effective on the T3SS of Shigella flexneri, where they block all related forms of protein secretion so far known, as well as the epithelial cell invasion and induction of macrophage apoptosis usually demonstrated by this bacterium. Furthermore, we show the first evidence for the detrimental effect of these compounds on T3SS needle assembly, as demonstrated by increased numbers of T3S apparatuses without needles or with shorter needles. Therefore, the compounds generate a phenocopy of T3SS export apparatus mutants but with incomplete penetrance. We discuss why this would be sufficient to almost completely block the later secretion of effector proteins and how this begins to narrow the search for the molecular target of these compounds.

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 Pseudomonas aeruginosa injects NDK into host cells through a type III secretion system

Microbiology ◽  
2014 ◽  
Vol 160 (7) ◽  
pp. 1417-1426 ◽  
Author(s):  
Dennis Neeld ◽  
Yongxin Jin ◽  
Candace Bichsel ◽  
Jinghua Jia ◽  
Jianhui Guo ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Host Cells ◽  
Eukaryotic Cells ◽  
Mammalian Host ◽  
Type I ◽  
Dependent Manner ◽  
Type Iii

Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen possessing a type III secretion system (T3SS) which injects toxic effector proteins into mammalian host cells. In previous studies, P. aeruginosa strains lacking all of the known type III effectors were shown to cause cytotoxicity upon prolonged infection time. In this study, we report the identification of a new cytotoxin, nucleoside diphosphate kinase (NDK), which is injected into eukaryotic cells in a T3SS-dependent manner. Injection of NDK is inhibited by the presence of previously known effectors of the T3SS, with an effectorless strain injecting the highest amount, suggesting active competition with the known T3SS effectors. NDK is shown to cause a cytotoxic response when expressed in eukaryotic cells, and P. aeruginosa strains harbouring NDK also show a greater toxicity than strains lacking it. Interestingly, the cytotoxic effect of intracellular NDK is independent of its kinase activity. In previous studies, NDK was shown to be secreted into culture supernatants via a type I secretion system and cause cytotoxicity in a kinase-dependent manner. Therefore, the current study highlights an alternative route of NDK secretion as well as two different cytotoxic mechanisms of NDK, depending on the extra- or intra-cellular location of the protein.

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 A C-Terminal Domain Targets the Pseudomonas aeruginosa Cytotoxin ExoU to the Plasma Membrane of Host Cells

2006 ◽  
Vol 74 (5) ◽  
pp. 2552-2561 ◽  
Author(s):  
Shira D. P. Rabin ◽  
Jeffrey L. Veesenmeyer ◽  
Kathryn T. Bieging ◽  
Alan R. Hauser
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Death ◽  
Plasma Membrane ◽  
Hela Cells ◽  
Type Iii Secretion ◽  
Fluorescent Protein ◽  
Host Cells ◽  
Type Iii ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT ExoU, a phospholipase injected into host cells by the type III secretion system of Pseudomonas aeruginosa, leads to rapid cytolytic cell death. Although the importance of ExoU in infection is well established, the mechanism by which this toxin kills host cells is less clear. To gain insight into how ExoU causes cell death, we examined its subcellular localization following transfection or type III secretion/translocation into HeLa cells. Although rapid cell lysis precluded visualization of wild-type ExoU by fluorescence microscopy, catalytically inactive toxin was readily detected at the periphery of HeLa cells. Biochemical analysis confirmed that ExoU was targeted to the membrane fraction of transfected cells. Visualization of ExoU peptides fused with green fluorescent protein indicated that the domain responsible for this targeting was in the C terminus of ExoU, between residues 550 and 687. Localization to the plasma membrane occurred within 1 h of expression, which is consistent with the kinetics of cytotoxicity. Together, these results indicate that a domain between residues 550 and 687 of ExoU targets this toxin to the plasma membrane, a process that may be important in cytotoxicity.

scholarly journals Characterization of the Yersinia enterocolitica Type III Secretion ATPase YscN and Its Regulator, YscL

2006 ◽  
Vol 188 (10) ◽  
pp. 3525-3534 ◽  
Author(s):  
Bill Blaylock ◽  
Kelly E. Riordan ◽  
Dominique M. Missiakas ◽  
Olaf Schneewind
Keyword(s):  
Yersinia Enterocolitica ◽  
Type Iii Secretion ◽  
Atp Hydrolysis ◽  
Biochemical Properties ◽  
Host Cells ◽  
Gram Negative Bacteria ◽  
Type Iii ◽  
Bacterial Proteins

ABSTRACT Type III secretion is a mechanism used by a broad range of gram-negative bacteria to neutralize eukaryotic defenses by enabling translocation of bacterial proteins directly into the cytoplasm of host cells. The bacterial energy source for secretion is ATP, which is consumed by an ATPase that couples ATP hydrolysis to the unfolding of secreted proteins and the dissociation of their chaperones just prior to secretion. By studying the biochemical properties of YscN and YscL of Yersinia enterocolitica, we have characterized them as the ATPase and ATPase regulator, respectively, of the type III secretion system of this organism. In vivo, YscL and YscN interact with each other, and the overexpression of glutathione S-transferase-YscL abolishes secretion and down-regulates the expression of secretion apparatus components.

scholarly journals CYRI-A regulates macropinocytic cup maturation and mediates integrin uptake, limiting invasive migration

2020 ◽  
Author(s):  
Anh Hoang Le ◽  
Tamas Yelland ◽  
Nikki Paul ◽  
Loic Fort ◽  
Savvas Nikolaou ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Ewing Sarcoma ◽  
Negative Regulator ◽  
Cell Surface Receptor ◽  
Actin Dynamics ◽  
Surface Receptor ◽  
Sarcoma Cells ◽  
Wave Complex

The Scar/WAVE complex is the major driver of actin nucleation at the plasma membrane, resulting in lamellipodia and membrane ruffles. While lamellipodia aid migration, membrane ruffles can generate macropinosomes - cup-like structures - important for nutrient uptake and regulation of cell surface receptor levels. How macropinosomes are formed and the role of the actin machinery in their formation and resolution is still not well understood. Mammalian CYRI-B is a recently described negative regulator of the Scar/WAVE complex by RAC1 sequestration, but its other paralogue, CYRI-A has not been characterised. Here we implicate CYRI-A as a key regulator of macropinocytosis maturation and integrin internalisation from the cell surface. We find that CYRI-A is recruited to nascent macropinosomes in a transient but distinct burst, downstream of PIP3-mediated RAC1 activation to regulate actin polymerisation. CYRI-A precedes RAB5A recruitment to engulfed macropinocytic cups and departs as RAB5A is recruited, consistent with a role for CYRI-A as a local suppressor of actin dynamics, enabling the resolution of the macropinocytic cup. The suppression of integrin a5b1 uptake caused by the co-depletion of CYRI-A and B in Ewing sarcoma cells, leads to an enhancement of surface integrin levels and enhanced invasion and anchorage-independent growth in 3D. Thus CYRI-A is a dynamic regulator of integrin uptake via macropinocytosis, functioning together with CYRI-B to regulate integrin homeostasis on the cell surface.

scholarly journals LcrG is Required for Efficient Translocation ofYersinia Yop Effector Proteins into Eukaryotic Cells

1998 ◽  
Vol 66 (6) ◽  
pp. 2976-2979 ◽  
Author(s):  
Mahfuzur R. Sarker ◽  
Marie-Paule Sory ◽  
Aoife P. Boyd ◽  
Maite Iriarte ◽  
Guy R. Cornelis
Keyword(s):  
Immune System ◽  
Yersinia Enterocolitica ◽  
Effector Proteins ◽  
Host Cells ◽  
Eukaryotic Cells

ABSTRACT Extracellular Yersinia disables the immune system of its host by injecting effector Yop proteins into host cells. We show that a Yersinia enterocolitica nonpolar lcrGmutant is severely impaired in the translocation of YopE, YopH, YopM, YpkA/YopO, and YopP into eukaryotic cells. LcrG is thus required for efficient internalization of all the known Yop effectors.

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