scholarly journals Show me your secret(ed) weapons: a multifaceted approach reveals novel type III-secreted effectors of a plant pathogenic bacterium

2019 ◽  
Author(s):  
Irene Jiménez Guerrero ◽  
Francisco Pérez-Montaño ◽  
Gustavo Mateus da Silva ◽  
Naama Wagner ◽  
Dafna Shkedy ◽  
...  
Keyword(s):  
Machine Learning ◽  
Endoplasmic Reticulum ◽  
Cell Nucleus ◽  
Type Iii Secretion ◽  
Pathogenic Bacteria ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Gram Negative ◽  
Type Iii ◽  
Acidovorax Citrulli

AbstractMany Gram-negative plant and animal pathogenic bacteria employ a type III secretion system (T3SS) to secrete protein effectors into the cells of their hosts and promote disease. The plant pathogen Acidovorax citrulli requires a functional T3SS for pathogenicity. As with Xanthomonas and Ralstonia spp., an AraC-type transcriptional regulator, HrpX, regulates expression of genes encoding T3SS components and type III-secreted effectors (T3Es) in A. citrulli. A previous study reported eleven T3E genes in this pathogen, based on the annotation of a sequenced strain. We hypothesized that this was an underestimation. Guided by this hypothesis, we aimed at uncovering the T3E arsenal of the A. citrulli model strain, M6. We carried out a thorough sequence analysis searching for similarity to known T3Es from other bacteria. This analysis revealed 51 A. citrulli genes whose products are similar to known T3Es. Further, we combined machine learning and transcriptomics to identify novel T3Es. The machine learning approach ranked all A. citrulli M6 genes according to their propensity to encode T3Es. RNA-Seq revealed differential gene expression between wild-type M6 and a mutant defective in HrpX. Data combined from these approaches led to the identification of seven novel T3E candidates, that were further validated using a T3SS-dependent translocation assay. These T3E genes encode hypothetical proteins, do not show any similarity to known effectors from other bacteria, and seem to be restricted to plant pathogenic Acidovorax species. Transient expression in Nicotiana benthamiana revealed that two of these T3Es localize to the cell nucleus and one interacts with the endoplasmic reticulum. This study not only uncovered the arsenal of T3Es of an important pathogen, but it also places A. citrulli among the “richest” bacterial pathogens in terms of T3E cargo. It also revealed novel T3Es that appear to be involved in the pathoadaptive evolution of plant pathogenic Acidovorax species.Author summaryAcidovorax citrulli is a Gram-negative bacterium that causes bacterial fruit blotch (BFB) disease of cucurbits. This disease represents a serious threat to cucurbit crop production worldwide. Despite the agricultural importance of BFB, the knowledge about basic aspects of A. citrulli-plant interactions is rather limited. As many Gram-negative plant and animal pathogenic bacteria, A. citrulli employs a complex secretion system, named type III secretion system, to deliver protein virulence effectors into the host cells. In this work we aimed at uncovering the arsenal of type III-secreted effectors (T3Es) of this pathogen by combination of bioinformatics and experimental approaches. We found that this bacterium possesses at least 51 genes that are similar to T3E genes from other pathogenic bacteria. In addition, our study revealed seven novel T3Es that seem to occur only in A. citrulli strains and in other plant pathogenic Acidovorax species. We found that two of these T3Es localize to the plant cell nucleus while one partially interacts with the endoplasmic reticulum. Further characterization of the novel T3Es identified in this study may uncover new host targets of pathogen effectors and new mechanisms by which pathogenic bacteria manipulate their hosts.

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 Developing Cyclic Peptomers as Broad-Spectrum Gram-negative Bacterial Type III Secretion System Inhibitors

2021 ◽  
Author(s):  
Hanh N. Lam ◽  
Tannia Lau ◽  
Adam Lentz ◽  
Jessica Sherry ◽  
Alejandro Cabrera-Cortez ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Pathogenic Bacteria ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Resistant Bacteria ◽  
Gram Negative Bacteria ◽  
Antibiotic Resistant ◽  
Gram Negative ◽  
Type Iii ◽  
Twin Arginine Translocation

Antibiotic resistant bacteria are an emerging global health threat. New antimicrobials are urgently needed. The injectisome type III secretion system (T3SS), required by dozens of Gram-negative bacteria for virulence but largely absent from non-pathogenic bacteria, is an attractive antimicrobial target. We previously identified synthetic cyclic peptomers, inspired by the natural product phepropeptin D, that inhibit protein secretion through the Yersinia Ysc and Pseudomonas aeruginosa Psc T3SSs, but do not inhibit bacterial growth. Here we describe identification of an isomer, 4EpDN, that is two-fold more potent (IC50 of 4 μM) than its parental compound. Furthermore, 4EpDN inhibited the Yersinia Ysa and the Salmonella SPI-1 T3SSs, suggesting that this cyclic peptomer has broad efficacy against evolutionarily distant injectisome T3SSs. Indeed, 4EpDN strongly inhibited intracellular growth of Chlamydia trachomatis in HeLa cells, which requires the T3SS. 4EpDN did not inhibit the unrelated Twin arginine translocation (Tat) system, nor did it impact T3SS gene transcription. Moreover, although the injectisome and flagellar T3SSs are evolutionarily and structurally related, the 4EpDN cyclic peptomer did not inhibit secretion of substrates through the Salmonella flagellar T3SS, indicating that cyclic peptomers broadly but specifically target the injestisome T3SS. 4EpDN reduced the number of T3SS needles detected on the surface of Y. pseudotuberculosis as detected by microscopy. Collectively, these data suggest that cyclic peptomers specifically inhibit the injectisome T3SS from a variety of Gram-negative bacteria, possibly by preventing complete T3SS assembly.

scholarly journals Developing Cyclic Peptomers as Broad-Spectrum Gram negative Bacterial Type III Secretion System Inhibitors

2020 ◽  
Author(s):  
Hanh N. Lam ◽  
Tannia Lau ◽  
Adam Lentz ◽  
Jessica Sherry ◽  
Alejandro Cabrera-Cortez ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Chlamydia Trachomatis ◽  
Broad Spectrum ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Type Iii ◽  
Bacterial Type

ABSTRACTAntibiotic resistant bacteria are an emerging global health threat. New antimicrobials are urgently needed. The injectisome type III secretion system (T3SS), required by dozens of Gram-negative bacteria for virulence but largely absent from non-pathogenic bacteria, is an attractive antimicrobial target. We previously identified synthetic cyclic peptomers, inspired by the natural product phepropeptin D, that inhibit protein secretion through the Yersinia Ysc and Pseudomonas aeruginosa Psc T3SSs, but do not inhibit bacterial growth. Here we describe identification of an isomer, 4EpDN, that is two-fold more potent (IC50 4 μM) than its parental compound. Furthermore, 4EpDN inhibited the Yersinia Ysa and the Salmonella SPI-1 T3SSs, suggesting that this cyclic peptomer has broad efficacy against evolutionarily distant injectisome T3SSs. Indeed, 4EpDN strongly inhibited intracellular growth of Chlamydia trachomatis in HeLa cells, which requires the T3SS. 4EpDN did not inhibit the unrelated Twin arginine translocation (Tat) system, nor did it impact T3SS gene transcription. Moreover, although the injectisome and flagellar T3SSs are evolutionarily and structurally related, the 4EpDN cyclic peptomer did not inhibit secretion of substrates through the Salmonella flagellar T3SS, indicating that cyclic peptomers broadly but specifically target the injestisome T3SS. 4EpDN reduced the number of T3SS basal bodies detected on the surface of Y. enterocolitica, as visualized using a fluorescent derivative of YscD, an inner membrane ring with low homology to flagellar protein FliG. Collectively, these data suggest that cyclic peptomers specifically inhibit the injectisome T3SS from a variety of Gram-negative bacteria, possibly by preventing complete T3SS assembly.IMPORTANCETraditional antibiotics target both pathogenic and commensal bacteria, resulting in a disruption of the microbiota, which in turn is tied to a number of acute and chronic diseases. The bacterial type III secretion system (T3SS) is an appendage used by many bacterial pathogens to establish infection, but is largely absent from commensal members of the microbiota. In this study, we identify a new derivative of the cyclic peptomer class of T3SS inhibitors. These compounds inhibit the T3SS of the nosocomial ESKAPE pathogen Pseudomonas aeruginosa and enteropathogenic Yersinia and Salmonella. The impact of cyclic peptomers is specific to the T3SS, as other bacterial secretory systems are unaffected. Importantly, cyclic peptomers completely block replication of Chlamydia trachomatis, the causative agent of genital, eye, and lung infections, in human cells, a process that requires the T3SS. Therefore, cyclic peptomers represent promising virulence blockers that can specifically disarm a broad spectrum of Gram-negative pathogens.

scholarly journals Novel Type III Effectors in Pseudomonas aeruginosa

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
David Burstein ◽  
Shirley Satanower ◽  
Michal Simovitch ◽  
Yana Belnik ◽  
Meital Zehavi ◽  
...  
Keyword(s):  
Machine Learning ◽  
Pseudomonas Aeruginosa ◽  
Host Cell ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Content Type ◽  
Machine Learning Classification ◽  
Type Iii ◽  
Wide Range

ABSTRACT Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that causes chronic and acute infections in immunocompromised patients. Most P. aeruginosa strains encode an active type III secretion system (T3SS), utilized by the bacteria to deliver effector proteins from the bacterial cell directly into the cytoplasm of the host cell. Four T3SS effectors have been discovered and extensively studied in P. aeruginosa: ExoT, ExoS, ExoU, and ExoY. This is especially intriguing in light of P. aeruginosa's ability to infect a wide range of hosts. We therefore hypothesized that additional T3SS effectors that have not yet been discovered are encoded in the genome of P. aeruginosa. Here, we applied a machine learning classification algorithm to identify novel P. aeruginosa effectors. In this approach, various types of data are integrated to differentiate effectors from the rest of the open reading frames of the bacterial genome. Due to the lack of a sufficient learning set of positive effectors, our machine learning algorithm integrated genomic information from another Pseudomonas species and utilized dozens of features accounting for various aspects of the effector coding genes and their products. Twelve top-ranking predictions were experimentally tested for T3SS-specific translocation, leading to the discovery of two novel T3SS effectors. We demonstrate that these effectors are not part of the injection structural complex and report initial efforts toward their characterization. IMPORTANCE Pseudomonas aeruginosa uses a type III secretion system (T3SS) to secrete toxic proteins, termed effectors, directly into the cytoplasm of the host cell. The activation of this secretion system is correlated with disease severity and patient death. Compared with many other T3SS-utilizing pathogenic bacteria, P. aeruginosa has a fairly limited arsenal of effectors that have been identified. This is in sharp contrast with the wide range of hosts that this bacterium can infect. The discovery of two novel effectors described here is an important step toward better understanding of the virulence and host evasion mechanisms adopted by this versatile pathogen and may provide novel approaches to treat P. aeruginosa infections.

scholarly journals Cell Individuality: The Bistable Gene Expression of the Type III Secretion System in Dickeya dadantii 3937

2012 ◽  
Vol 25 (1) ◽  
pp. 37-47 ◽  
Author(s):  
Quan Zeng ◽  
Michael D. Laiosa ◽  
Douglas A. Steeber ◽  
Eulandria M. Biddle ◽  
Quan Peng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Type Iii Secretion ◽  
Expression Patterns ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Expression Level ◽  
Dickeya Dadantii ◽  
Phytopathogenic Bacterium ◽  
Gram Negative ◽  
Type Iii

Dickeya dadantii 3937 is a gram-negative phytopathogenic bacterium that expresses genes encoding a type III secretion system (T3SS) in a bistable pattern when cultured in a homogeneous minimal media. In this work, we further characterized the bistable gene expression of T3SS at the single-cell level. We demonstrated that bistable expression of the HrpL-regulon genes, such as hrpA and hrpN, is controlled by the same regulatory mechanism. We also showed that the expression level of the T3SS master regulatory gene hrpL plays an important role in the development of the bistable expression of hrpA. A high expression level of hrpL is required but unable to guarantee the high-state expression of hrpA in a cell. In addition, bistable expression patterns of T3SS genes in other gram-negative pathogens of the Enterobacteriaceae and Pseudomonadaceae families were also described in this study. This suggests that the T3SS bistability might be a conserved population behavior in several gram-negative bacterial pathogens.

scholarly journals TRAPPC13 is a Novel Target of Mesorhizobium amorphae Type III Secretion System Effector NopP

2021 ◽  
Author(s):  
Dongying Liu ◽  
Yantao Luo ◽  
Xiaofeng Zheng ◽  
Xinye Wang ◽  
Minxia Chou ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Pathogenic Bacteria ◽  
Nitrogenase Activity ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Host Cells ◽  
Bimolecular Fluorescence Complementation ◽  
Physical Interaction ◽  
Type Iii ◽  
Early Stages

Similar to pathogenic bacteria, rhizobia can inject effector proteins into host cells directly to promote infection via the type III secretion system. Nodulation outer protein P (NopP), a specific type III secretion system effector of rhizobia, plays different roles in the establishment of multiple rhizobia-legume symbiotic systems. Mesorhizobium amorphae CCNWGS0123 (GS0123), which infects Robinia pseudoacacia specifically, secretes several type III secretion system (T3SS) effectors, including NopP. Here, we demonstrate that NopP is secreted through T3SS-Ⅰof GS0123 during the early stages of infection, and its deficiency decreases nodule nitrogenase activity of R. pseudoacacia nodules. A trafficking protein particle complex subunit 13-like protein (TRAPPC13) ishas been identified as a NopP target protein in R. pseudoacacia roots by screening a yeast two-hybrid library. The physical interaction between NopP and TRAPPC13 is verified by bimolecular fluorescence complementation and co-immunoprecipitation assays. In addition, subcellular localization analysis reveals that both NopP and its target, TRAPPC13, are co-localized on the plasma membrane. Compared with GS0123-inoculated R. pseudoacacia roots, some genes associated with cell wall remodeling and plant innate immunity down-regulated in ΔnopP-inoculated roots at 36 hpi. The results suggest that NopP in M. amorphae CCNWGS0123 acts in multiple process in R. pseudoacacia during the early stages of infection, and TRAPPC13 could participate in the process as a NopP target.

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