scholarly journals CesL Regulates Type III Secretion Substrate Specificity of the Enteropathogenic E. coli Injectisome

2021 ◽  
Vol 9 (5) ◽  
pp. 1047
Author(s):  
Miguel Díaz-Guerrero ◽  
Meztlli O. Gaytán ◽  
Eduardo Soto ◽  
Norma Espinosa ◽  
Elizabeth García-Gómez ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Type Iii Secretion ◽  
Pathogenic Bacteria ◽  
Effector Proteins ◽  
Host Cells ◽  
Target Cells ◽  
Remarkable Feature ◽  
Type Iii ◽  
Pairwise Interactions ◽  
Effector Secretion

The type III secretion system (T3SS) is a complex molecular device used by several pathogenic bacteria to translocate effector proteins directly into eukaryotic host cells. One remarkable feature of the T3SS is its ability to secrete different categories of proteins in a hierarchical manner, to ensure proper assembly and timely delivery of effectors into target cells. In enteropathogenic Escherichia coli, the substrate specificity switch from translocator to effector secretion is regulated by a gatekeeper complex composed of SepL, SepD, and CesL proteins. Here, we report a characterization of the CesL protein using biochemical and genetic approaches. We investigated discrepancies in the phenotype among different cesL deletion mutants and showed that CesL is indeed essential for translocator secretion and to prevent premature effector secretion. We also demonstrated that CesL engages in pairwise interactions with both SepL and SepD. Furthermore, while association of SepL to the membrane does not depended on CesL, the absence of any of the proteins forming the heterotrimeric complex compromised the intracellular stability of each component. In addition, we found that CesL interacts with the cytoplasmic domains of the export gate components EscU and EscV. We propose a mechanism for substrate secretion regulation governed by the SepL/SepD/CesL complex.

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 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 Genetic Analysis of Assembly of theSalmonella enterica Serovar Typhimurium Type III Secretion-Associated Needle Complex

2001 ◽  
Vol 183 (4) ◽  
pp. 1159-1167 ◽  
Author(s):  
Anand Sukhan ◽  
Tomoko Kubori ◽  
James Wilson ◽  
Jorge E. Galán
Keyword(s):  
Type Iii Secretion ◽  
Pathogenic Bacteria ◽  
Effector Proteins ◽  
Host Cells ◽  
Loss Of Function ◽  
Type Iii ◽  
Serovar Typhimurium ◽  
Protein Secretion System ◽  
Independent Process ◽  
Body Complex

ABSTRACT Several pathogenic bacteria have evolved a specialized protein secretion system termed type III to secrete and deliver effector proteins into eukaryotic host cells. Salmonella entericaserovar Typhimurium uses one such system to mediate entry into nonphagocytic cells. This system is composed of more than 20 proteins which are encoded within a pathogenicity island (SPI-1) located at centisome 63 of its chromosome. A subset of these components form a supramolecular structure, termed the needle complex, that resembles the flagellar hook-basal body complex. The needle complex is composed of a multiple-ring cylindrical base that spans the bacterial envelope and a needle-like extension that protrudes from the bacterial outer surface. Although the components of this structure have been identified, little is known about its assembly. In this study we examined the effect of loss-of-function mutations in each of the type III secretion-associated genes encoded within SPI-1 on the assembly of the needle complex. This analysis indicates that the assembly of this organelle occurs in discrete, genetically separable steps. A model for the assembly pathway of this important organelle is proposed that involves asec-dependent step leading to the assembly of the base substructure followed by a sec-independent process resulting in the assembly of the needle portion.

scholarly journals Molecular Targets and Strategies for Inhibition of the Bacterial Type III Secretion System (T3SS); Inhibitors Directly Binding to T3SS Components

Biomolecules ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 316 ◽  
Author(s):  
Julia A. Hotinger ◽  
Heather A. Pendergrass ◽  
Aaron E. May
Keyword(s):  
Type Iii Secretion ◽  
Pathogenic Bacteria ◽  
Type Iii Secretion System ◽  
Systemic Infection ◽  
Inhibitory Effect ◽  
Secretion System ◽  
Effector Proteins ◽  
Host Cells ◽  
Complex Nature ◽  
Type Iii

The type III secretion system (T3SS) is a virulence apparatus used by many Gram-negative pathogenic bacteria to cause infections. Pathogens utilizing a T3SS are responsible for millions of infections yearly. Since many T3SS knockout strains are incapable of causing systemic infection, the T3SS has emerged as an attractive anti-virulence target for therapeutic design. The T3SS is a multiprotein molecular syringe that enables pathogens to inject effector proteins into host cells. These effectors modify host cell mechanisms in a variety of ways beneficial to the pathogen. Due to the T3SS’s complex nature, there are numerous ways in which it can be targeted. This review will be focused on the direct targeting of components of the T3SS, including the needle, translocon, basal body, sorting platform, and effector proteins. Inhibitors will be considered a direct inhibitor if they have a binding partner that is a T3SS component, regardless of the inhibitory effect being structural or functional.

scholarly journals SsaV Interacts with SsaL to Control the Translocon-to-Effector Switch in theSalmonellaSPI-2 Type Three Secretion System

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Xiu-Jun Yu ◽  
Grzegorz J. Grabe ◽  
Mei Liu ◽  
Luís Jaime Mota ◽  
David W. Holden
Keyword(s):  
Amino Acids ◽  
Protein Secretion ◽  
Type Iii Secretion ◽  
Secretion System ◽  
Effector Proteins ◽  
Host Cells ◽  
Intracellular Bacteria ◽  
Type Iii ◽  
Encoded Proteins ◽  
Effector Secretion

ABSTRACTNonflagellar type III secretion systems (nf T3SSs) form a cell surface needle-like structure and an associated translocon that deliver bacterial effector proteins into eukaryotic host cells. This involves a tightly regulated hierarchy of protein secretion. A switch involving SctP and SctU stops secretion of the needle protein. The gatekeeper protein SctW is required for secretion of translocon proteins and controls a second switch to start effector secretion.Salmonella entericaserovar Typhimurium encodes two T3SSs inSalmonellapathogenicity island 1 (SPI-1) and SPI-2. The acidic vacuole containing intracellular bacteria stimulates assembly of the SPI-2 T3SS and its translocon. Sensing the nearly neutral host cytosolic pH is required for effector translocation. Here, we investigated the involvement of SPI-2-encoded proteins SsaP (SctP), SsaU (SctU), SsaV (SctV), and SsaL (SctW) in regulation of secretion. We found that SsaP and SsaU are involved in the first but not the second secretion switch. A random-mutagenesis screen identified amino acids of SsaV that regulate translocon and effector secretion. Single substitutions in subdomain 4 of SsaV or InvA (SPI-1-encoded SctV) phenocopied mutations of their corresponding gatekeepers with respect to translocon and effector protein secretion and host cell interactions. SsaL interacted with SsaV in bacteria exposed to low ambient pH but not after the pH was raised to 7.2. We propose that SsaP and SsaU enable the apparatus to become competent for a secretion switch and facilitate the SsaL-SsaV interaction. This mediates secretion of translocon proteins until neutral pH is sensed, which causes their dissociation, resulting in arrest of translocon secretion and derepression of effector translocation.IMPORTANCESalmonellaTyphimurium is an intracellular pathogen that uses the SPI-2 type III secretion system to deliver virulence proteins across the vacuole membrane surrounding intracellular bacteria. This involves a tightly regulated hierarchy of protein secretion controlled by two molecular switches. We found that SPI-2-encoded proteins SsaP and SsaU are involved in the first but not the second secretion switch. We identify key amino acids of the inner membrane protein SsaV that are required to interact with the so-called gatekeeper protein SsaL and show that the dissociation of SsaV-SsaL causes the second switch, leading to delivery of effector proteins. Our results provide insights into the molecular events controlling virulence-associated type III secretion and suggest a broader model describing how the process is regulated.

The YscU/FlhB homologue HrcU from Xanthomonas controls type III secretion and translocation of early and late substrates

Microbiology ◽  
2014 ◽  
Vol 160 (3) ◽  
pp. 576-588 ◽  
Author(s):  
Jens Hausner ◽  
Daniela Büttner
Keyword(s):  
Substrate Specificity ◽  
Type Iii Secretion ◽  
Xanthomonas Campestris ◽  
Pathogenic Bacteria ◽  
Effector Proteins ◽  
Effector Protein ◽  
Amino Acid Residues ◽  
Type Iii ◽  
Cytoplasmic Region ◽  
Essential Components

The majority of Gram-negative plant- and animal-pathogenic bacteria employ a type III secretion (T3S) system to deliver effector proteins to eukaryotic cells. Members of the YscU protein family are essential components of the T3S system and consist of a transmembrane and a cytoplasmic region that is autocatalytically cleaved at a conserved NPTH motif. YscU homologues interact with T3S substrate specificity switch (T3S4) proteins that alter the substrate specificity of the T3S system after assembly of the secretion apparatus. We previously showed that the YscU homologue HrcU from the plant pathogen Xanthomonas campestris pv. vesicatoria interacts with the T3S4 protein HpaC and is required for the secretion of translocon and effector proteins. In the present study, analysis of HrcU deletion, insertion and point mutant derivatives led to the identification of amino acid residues in the cytoplasmic region of HrcU (HrcUC) that control T3S and translocation of the predicted inner rod protein HrpB2, the translocon protein HrpF and the effector protein AvrBs3. Mutations in the vicinity of the NPTH motif interfered with HrcU cleavage and/or the interaction of HrcUC with HrpB2 and the T3S4 protein HpaC. However, HrcU function was not completely abolished, suggesting that HrcU cleavage is not crucial for pathogenicity and T3S. Given that mutations in HrcU differentially affected T3S and translocation of HrpB2 and effector proteins, we propose that HrcU controls the secretion of different T3S substrate classes by independent mechanisms.

scholarly journals Molecular Pathogenesis of Shigella spp.: Controlling Host Cell Signaling, Invasion, and Death by Type III Secretion

2008 ◽  
Vol 21 (1) ◽  
pp. 134-156 ◽  
Author(s):  
Gunnar N. Schroeder ◽  
Hubert Hilbi
Keyword(s):  
Host Cell ◽  
Type Iii Secretion ◽  
Pathogenic Bacteria ◽  
Molecular Mechanisms ◽  
Inflammatory Responses ◽  
Shigella Flexneri ◽  
Effector Proteins ◽  
Target Cells ◽  
Type Iii ◽  
Shigella Spp

SUMMARY Shigella spp. are gram-negative pathogenic bacteria that evolved from harmless enterobacterial relatives and may cause devastating diarrhea upon ingestion. Research performed over the last 25 years revealed that a type III secretion system (T3SS) encoded on a large plasmid is a key virulence factor of Shigella flexneri. The T3SS determines the interactions of S. flexneri with intestinal cells by consecutively translocating two sets of effector proteins into the target cells. Thus, S. flexneri controls invasion into EC, intra- and intercellular spread, macrophage cell death, as well as host inflammatory responses. Some of the translocated effector proteins show novel biochemical activities by which they intercept host cell signal transduction pathways. An understanding of the molecular mechanisms underlying Shigella pathogenesis will foster the development of a safe and efficient vaccine, which, in parallel with improved hygiene, should curb infections by this widespread pathogen.

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 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 A NanoLuc luciferase-based assay enabling the real-time analysis of protein secretion and injection by bacterial type III secretion systems

2019 ◽  
Author(s):  
Sibel Westerhausen ◽  
Melanie Nowak ◽  
Claudia Torres-Vargas ◽  
Ursula Bilitewski ◽  
Erwin Bohn ◽  
...  
Keyword(s):  
Protein Secretion ◽  
High Throughput ◽  
Type Iii Secretion ◽  
Molecular Mechanisms ◽  
Host Cells ◽  
Target Cells ◽  
Secretion Systems ◽  
Type Iii ◽  
Nanoluc Luciferase ◽  
Type Iii Secretion Systems

AbstractThe elucidation of the molecular mechanisms of secretion through bacterial protein secretion systems is impeded by a lack of assays to quantitatively assess secretion kinetics. Also the analysis of the biological role of these secretion systems as well as the identification of inhibitors targeting these systems would greatly benefit from the availability of a simple, quick and quantitative assay to monitor principle secretion and injection into host cells. Here we present a versatile solution to this need, utilizing the small and very bright NanoLuc luciferase to assess secretion and injection through the type III secretion system encoded by Salmonella pathogenicity island 1. The NanoLuc-based secretion assay features a very high signal-to-noise ratio and sensitivity down to the nanoliter scale. The assay enables monitoring of secretion kinetics and is adaptable to a high throughput screening format in 384-well microplates. We further developed NanoLuc and split-NanoLuc-based assays that enable the monitoring of type III secretion-dependent injection of effector proteins into host cells.ImportanceThe ability to secrete proteins to the bacterial cell surface, to the extracellular environment, or even into target cells is one of the foundations of interbacterial as well as pathogen-host interaction. While great progress has been made in elucidating assembly and structure of secretion systems, our understanding of their secretion mechanism often lags behind, not last because of the challenge to quantitatively assess secretion function. Here, we developed a luciferase-based assay to enable the simple, quick, quantitative, and high throughput-compatible assessment of secretion and injection through virulence-associated type III secretion systems. The assay allows detection of minute amounts of secreted substrate proteins either in the supernatant of the bacterial culture or within eukaryotic host cells. It thus provides an enabling technology to elucidate the mechanisms of secretion and injection of type III secretion systems and is likely adaptable to assay secretion through other bacterial secretion systems.

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