Identification and Functional Characterization of Gene Components of Type VI Secretion System in Bacterial Genomes

ABSTRACTPseudomonas aeruginosais a Gram-negative opportunistic pathogen of humans, particularly those with cystic fibrosis. As a global regulator, RpoN controls a group of virulence-related factors and quorum-sensing (QS) genes inP. aeruginosa. To gain further insights into the direct targets of RpoNin vivo, the present study focused on identifying the direct targets of RpoN regulation in QS and the type VI secretion system (T6SS). We performed chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) that identified 1,068 binding sites of RpoN, mostly including metabolic genes, a group of genes in QS (lasI,rhlI, andpqsR) and the T6SS (hcpAandhcpB). The direct targets of RpoN have been verified by electrophoretic mobility shifts assays (EMSA),luxreporter assay, reverse transcription-quantitative PCR, and phenotypic detection. TheΔrpoN::Tc mutant resulted in the reduced production of pyocyanin, motility, and proteolytic activity. However, the production of rhamnolipids and biofilm formation were higher in theΔrpoN::Tc mutant than in the wild type. In summary, the results indicated that RpoN had direct and profound effects on QS and the T6SS.IMPORTANCEAs a global regulator, RpoN controls a wide range of biological pathways, including virulence inP. aeruginosaPAO1. This work shows that RpoN plays critical and global roles in the regulation of bacterial pathogenicity and fitness. ChIP-seq provided a useful database to characterize additional functions and targets of RpoN in the future. The functional characterization of RpoN-mediated regulation will improve the current understanding of the regulatory network of quorum sensing and virulence inP. aeruginosaand other bacteria.

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Characterization of the Pseudomonas aeruginosa T6SS PldB immunity proteins PA5086, PA5087 and PA5088 explains a novel stockpiling mechanism

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x2000566x ◽

2020 ◽

Vol 76 (5) ◽

pp. 222-227

Author(s):

Haiying Wen ◽

Zhi Geng ◽

Zengqiang Gao ◽

Zhun She ◽

Yuhui Dong

Keyword(s):

Pseudomonas Aeruginosa ◽

Secretion System ◽

Type Vi Secretion System ◽

Structural Comparison ◽

Interesting Phenomenon ◽

Potential Surfaces ◽

Type Vi Secretion ◽

Host Infection ◽

Bacterial Type

The bacterial type VI secretion system (T6SS) secretes many toxic effectors to gain advantage in interbacterial competition and for eukaryotic host infection. The cognate immunity proteins of these effectors protect bacteria from their own effectors. PldB is a T6SS trans-kingdom effector in Pseudomonas aeruginosa that can infect both prokaryotic and eukaryotic cells. Three proteins, PA5086, PA5087 and PA5088, are employed to suppress the toxicity of PldB-family proteins. The structures of PA5087 and PA5088 have previously been reported, but the identification of further distinctions between these immunity proteins is needed. Here, the crystal structure of PA5086 is reported at 1.90 Å resolution. A structural comparison of the three PldB immunity proteins showed vast divergences in their electrostatic potential surfaces. This interesting phenomenon provides an explanation of the stockpiling mechanism of T6SS immunity proteins.

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Identification of divergent type VI secretion effectors using a conserved chaperone domain

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1505317112 ◽

2015 ◽

Vol 112 (29) ◽

pp. 9106-9111 ◽

Cited By ~ 78

Author(s):

Xiaoye Liang ◽

Richard Moore ◽

Mike Wilton ◽

Megan J. Q. Wong ◽

Linh Lam ◽

...

Keyword(s):

Functional Characterization ◽

Large Family ◽

Effector Proteins ◽

Gram Negative Bacteria ◽

Type Vi Secretion System ◽

Gram Negative ◽

Effector Genes ◽

Type Vi Secretion ◽

Bacterial Fitness

The type VI secretion system (T6SS) is a lethal weapon used by many bacteria to kill eukaryotic predators or prokaryotic competitors. Killing by the T6SS results from repetitive delivery of toxic effectors. Despite their importance in dictating bacterial fitness, systematic prediction of T6SS effectors remains challenging due to high effector diversity and the absence of a conserved signature sequence. Here, we report a class of T6SS effector chaperone (TEC) proteins that are required for effector delivery through binding to VgrG and effector proteins. The TEC proteins share a highly conserved domain (DUF4123) and are genetically encoded upstream of their cognate effector genes. Using the conserved TEC domain sequence, we identified a large family of TEC genes coupled to putative T6SS effectors in Gram-negative bacteria. We validated this approach by verifying a predicted effector TseC in Aeromonas hydrophila. We show that TseC is a T6SS-secreted antibacterial effector and that the downstream gene tsiC encodes the cognate immunity protein. Further, we demonstrate that TseC secretion requires its cognate TEC protein and an associated VgrG protein. Distinct from previous effector-dependent bioinformatic analyses, our approach using the conserved TEC domain will facilitate the discovery and functional characterization of new T6SS effectors in Gram-negative bacteria.

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Characterization of Type VI Secretion System in Xanthomonas oryzae pv. oryzae and Its Role in Virulence to Rice

The Plant Pathology Journal ◽

10.5423/ppj.nt.02.2020.0026 ◽

2020 ◽

Vol 36 (3) ◽

pp. 289-296 ◽

Cited By ~ 2

Author(s):

Yeounju Choi ◽

Namgyu Kim ◽

Mohamed Mannaa ◽

Hongsup Kim ◽

Jungwook Park ◽

...

Keyword(s):

Secretion System ◽

Xanthomonas Oryzae ◽

Type Vi Secretion System ◽

Type Vi Secretion

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Characterization of a Type VI Secretion System vgrG2 Gene in the Pathogenicity of Burkholderia thailandensis BPM

Frontiers in Microbiology ◽

10.3389/fmicb.2021.811343 ◽

2022 ◽

Vol 12 ◽

Author(s):

Jin Li ◽

Wei-wei Hu ◽

Guo-xin Qu ◽

Xiao-rong Li ◽

Yi Xiang ◽

...

Keyword(s):

Secretion System ◽

Host Cells ◽

Type Vi Secretion System ◽

Burkholderia Thailandensis ◽

Gram Negative ◽

Type Vi Secretion ◽

New Perspective ◽

Protein Secreting

Burkholderia thailandensis is a clinically underestimated conditional pathogen in the genus Burkholderia, the pathogenicity of the infection caused by B. thailandensis remains poorly understood. According to previous studies, Type-VI secretion system (T6SS) is a protein secreting device widely existing in Gram-negative bacilli. Valine-glycine repeat protein G (VgrG) is not only an important component of T6SS, but also a virulence factor of many Gram-negative bacilli. In one of our previous studies, a unique T6SS vgrG gene (vgrG2 gene) was present in a virulent B. thailandensis strain BPM (BPM), but not in the relatively avirulent B. thailandensis strain E264 (E264). Meanwhile, transcriptome analysis of BPM and E264 showed that the vgrG2 gene was strongly expressed in BPM, but not in E264. Therefore, we identified the function of the vgrG2 gene by constructing the mutant and complemented strains in this study. In vitro, the vgrG2 gene was observed to be involved in the interactions with host cells. The animal model experiment showed that the deletion of vgrG2 gene significantly led to the decrease in the lethality of BPM and impaired its ability to trigger host immune response. In conclusion, our study provides a new perspective for studying the pathogenicity of B. thailandensis and lays the foundation for discovering the potential T6SS effectors.

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