Deciphering the structural intricacy in virulence effectors for proton-motive force mediated unfolding and type-III protein secretion

ABSTRACTMany gram-negative pathogenic bacteria use type III secretion system (T3SS) to inject virulence effectors directly into the cytosol of targeted host cells. Given that the protein unfolding requisite for secretion via nano-size pore of T3SS injectisome is an energetically unfavorable process, “How do pathogenic bacteria unfold and secrete hundreds of toxic proteins in seconds” remain largely unknown. In this study, first, from an in-depth analysis of folding and stability of T3SS effector ExoY, we show that the proton-concentration gradient (∼pH 5.8-6.0) generated by proton-motive force (PMF) can significantly amortize tertiary structural folding and stability of effectors without significant entropic cost. Strikingly, it was found that the lower energetic cost associated with the global unfolding of ExoY is mainly due to its weakly folded geometry and abundance of geometrical frustrations stemming from buried water molecules and native-like folded intermediates in the folded cores. From in-silico structural analysis of 371 T3SS effectors, it can be curtained that T3SS effectors belong to typical class (disorder globules) of IDPs and have evolved similar conserved intrinsic structural archetypes to mediate early-stage unfolding. The slower folding kinetics in effector proteins requisite for efficient T3SS-mediated secretion mostly stems from reduced hydrophobic density and enhanced polar-polar repulsive interactions in their sequence landscapes. Lastly, the positively evolved histidine-mediated stabilizing interactions and gate-keeper residues in effector proteins shed light on collaborative role of evolved structural chemistry in T3SS effectors and PMF in the spatial-temporal regulation of effector folding and stability essential for maintaining balance in secretion and function trade-off.

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

Infection and Immunity ◽

10.1128/iai.72.7.4004-4009.2004 ◽

2004 ◽

Vol 72 (7) ◽

pp. 4004-4009 ◽

Cited By ~ 75

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.

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

Antibodies ◽

10.3390/antib9030035 ◽

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.

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

mSystems ◽

10.1128/msystems.00032-15 ◽

2016 ◽

Vol 1 (4) ◽

Cited By ~ 9

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.

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

Microorganisms ◽

10.3390/microorganisms9051047 ◽

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.

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

Journal of Bacteriology ◽

10.1128/jb.183.4.1159-1167.2001 ◽

2001 ◽

Vol 183 (4) ◽

pp. 1159-1167 ◽

Cited By ~ 137

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.

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

Biomolecules ◽

10.3390/biom11020316 ◽

2021 ◽

Vol 11 (2) ◽

pp. 316 ◽

Cited By ~ 1

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.

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Deciphering the structural intricacy in virulence effectors for proton-motive force mediated unfolding in type-III protein secretion

International Journal of Biological Macromolecules ◽

10.1016/j.ijbiomac.2020.04.266 ◽

2020 ◽

Vol 159 ◽

pp. 18-33

Author(s):

Basavraj Khanppnavar ◽

Anupam Roy ◽

Kausik Chandra ◽

Vladimir N. Uversky ◽

Nakul Chandra Maiti ◽

...

Keyword(s):

Protein Secretion ◽

Proton Motive Force ◽

Motive Force ◽

Type Iii ◽

Type Iii Protein Secretion ◽

Virulence Effectors

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

Journal of Bacteriology ◽

10.1128/jb.01446-08 ◽

2008 ◽

Vol 191 (5) ◽

pp. 1414-1428 ◽

Cited By ~ 47

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.

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

Journal of Bacteriology ◽

10.1128/jb.01004-08 ◽

2008 ◽

Vol 191 (2) ◽

pp. 563-570 ◽

Cited By ~ 71

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.

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A Salmonella type III effector, PipA, works in a different manner than the PipA family effectors GogA and GtgA

PLoS ONE ◽

10.1371/journal.pone.0248975 ◽

2021 ◽

Vol 16 (3) ◽

pp. e0248975

Author(s):

Momo Takemura ◽

Takeshi Haneda ◽

Hikari Idei ◽

Tsuyoshi Miki ◽

Nobuhiko Okada

Keyword(s):

Hela Cells ◽

Critical Role ◽

Effector Proteins ◽

Host Cells ◽

Microbial Pathogens ◽

Secretion Systems ◽

Type Iii ◽

Zinc Metalloprotease ◽

Type Iii Secretion Systems ◽

Serovar Typhimurium

Nuclear factor-kappa B (NF-κB) plays a critical role in the host defense against microbial pathogens. Many pathogens modulate NF-κB signaling to establish infection in their host. Salmonella enterica serovar Typhimurium (S. Typhimurium) possesses two type III secretion systems (T3SS-1 and T3SS-2) and directly injects many effector proteins into host cells. It has been reported that some effectors block NF-κB signaling, but the molecular mechanism of the inactivation of NF-κB signaling in S. Typhimurium is poorly understood. Here, we identified seven type III effectors—GogA, GtgA, PipA, SseK1, SseK2, SseK3, and SteE—that inhibited NF-κB activation in HeLa cells stimulated with TNF-α. We also determined that only GogA and GtgA are involved in regulation of the activation of NF-κB in HeLa cells infected with S. Typhimurium. GogA, GtgA, and PipA are highly homologous to one another and have the consensus zinc metalloprotease HEXXH motif. Our experiments demonstrated that GogA, GtgA, and PipA each directly cleaved NF-κB p65, whereas GogA and GtgA, but not PipA, inhibited the NF-κB activation in HeLa cells infected with S. Typhimurium. Further, expressions of the gogA or gtgA gene were induced under the SPI-1-and SPI-2-inducing conditions, but expression of the pipA gene was induced only under the SPI-2-inducing condition. We also showed that PipA was secreted into RAW264.7 cells through T3SS-2. Finally, we indicated that PipA elicits bacterial dissemination in the systemic stage of infection of S. Typhimurium via a T3SS-1-independent mechanism. Collectively, our results suggest that PipA, GogA and GtgA contribute to S. Typhimurium pathogenesis in different ways.

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