scholarly journals Identification of Escherichia coli ClpAP in regulating susceptibility to type VI secretion system-mediated attack by Agrobacterium tumefaciens

2019 ◽  
Author(s):  
Hsiao-Han Lin (林筱涵) ◽  
Manda Yu (余文廸) ◽  
Manoj Kumar Sriramoju ◽  
Shang-Te Danny Hsu (徐尚德) ◽  
Chi-Te Liu (劉啟德) ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Agrobacterium Tumefaciens ◽  
Secretion System ◽  
Gram Negative Bacteria ◽  
Wild Type ◽  
Type Vi Secretion System ◽  
Competition Assay ◽  
E Coli ◽  
Type Vi Secretion ◽  
In Trans

AbstractType VI secretion system (T6SS) is an effector delivery system used by gram-negative bacteria to kill other bacteria or eukaryotic host to gain fitness. In Agrobacterium tumefaciens, T6SS has been shown to kill other bacteria such as Escherichia coli. Interestingly, the A. tumefaciens T6SS killing efficiency differs when using different E. coli strains as recipient cells. Thus, we hypothesize that a successful T6SS killing not only relies on attacker T6SS activity but also depends on recipient factors. A high-throughput interbacterial competition assay was employed to test the hypothesis by screening for mutants with reduced killing outcomes caused by A. tumefaciens strain C58. From the 3909 E. coli Keio mutants screened, 16 candidate mutants were filtered out. One strain, ΔclpP::Kan, showed ten times more resistant to T6SS-mediating killing but restored its susceptibility when complemented with clpP in trans. ClpP is a universal and highly conserved protease that exists in both prokaryotes and eukaryotic organelles. In E. coli, ClpP uses either ClpA or ClpX as an adaptor for substrate specificity. Therefore, the susceptibility of the ΔclpA::Kan and ΔclpX::Kan was also tested. The T6SS attack susceptibility of ΔclpA::Kan is at the same level as ΔclpP::Kan, while ΔclpX::Kan showed no difference as compared to that of wild-type E. coli BW25113. The data also suggest that ClpA-ClpP interaction, rather than its protease activity, is responsible for enhancing susceptibility to T6SS killing. This study highlights the importance of recipient factors in determining the outcome of T6SS killing.

scholarly journals Fur-Dam Regulatory Interplay at an Internal Promoter of the Enteroaggregative Escherichia coli Type VI Secretion sci1 Gene Cluster

2020 ◽  
Vol 202 (10) ◽  
Author(s):  
Yannick R. Brunet ◽  
Christophe S. Bernard ◽  
Eric Cascales
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Gene Clusters ◽  
Secretion System ◽  
Target Cells ◽  
Type Vi Secretion System ◽  
Content Type ◽  
Internal Promoter ◽  
E Coli ◽  
Type Vi Secretion

ABSTRACT The type VI secretion system (T6SS) is a weapon for delivering effectors into target cells that is widespread in Gram-negative bacteria. The T6SS is a highly versatile machine, as it can target both eukaryotic and prokaryotic cells, and it has been proposed that T6SSs are adapted to the specific needs of each bacterium. The expression of T6SS gene clusters and the activation of the secretion apparatus are therefore tightly controlled. In enteroaggregative Escherichia coli (EAEC), the sci1 T6SS gene cluster is subject to a complex regulation involving both the ferric uptake regulator (Fur) and DNA adenine methylase (Dam)-dependent DNA methylation. In this study, an additional, internal, promoter was identified within the sci1 gene cluster using +1 transcriptional mapping. Further analyses demonstrated that this internal promoter is controlled by a mechanism strictly identical to that of the main promoter. The Fur binding box overlaps the −10 transcriptional element and a Dam methylation site, GATC-32. Hence, the expression of the distal sci1 genes is repressed and the GATC-32 site is protected from methylation in iron-rich conditions. The Fur-dependent protection of GATC-32 was confirmed by an in vitro methylation assay. In addition, the methylation of GATC-32 negatively impacted Fur binding. The expression of the sci1 internal promoter is therefore controlled by iron availability through Fur regulation, whereas Dam-dependent methylation maintains a stable ON expression in iron-limited conditions. IMPORTANCE Bacteria use weapons to deliver effectors into target cells. One of these weapons, the type VI secretion system (T6SS), assembles a contractile tail acting as a spring to propel a toxin-loaded needle. Its expression and activation therefore need to be tightly regulated. Here, we identified an internal promoter within the sci1 T6SS gene cluster in enteroaggregative E. coli. We show that this internal promoter is controlled by Fur and Dam-dependent methylation. We further demonstrate that Fur and Dam compete at the −10 transcriptional element to finely tune the expression of T6SS genes. We propose that this elegant regulatory mechanism allows the optimum production of the T6SS in conditions where enteroaggregative E. coli encounters competing species.

scholarly journals Crystal structure of the type VI immunity protein Tdi1 (Atu4351) from Agrobacterium tumefaciens

2019 ◽  
Vol 75 (3) ◽  
pp. 153-158
Author(s):  
Lingling Shi ◽  
Zengqiang Gao ◽  
Tianyi Zhang ◽  
Heng Zhang ◽  
Yuhui Dong
Keyword(s):  
Crystal Structure ◽  
Agrobacterium Tumefaciens ◽  
Active Site ◽  
Secretion System ◽  
Gram Negative Bacteria ◽  
Type Vi Secretion System ◽  
Dispersion Method ◽  
Interspecies Competition ◽  
Immunity Protein ◽  
Type Vi Secretion

The type VI secretion system (T6SS) is a novel multiprotein needle-like apparatus that is distributed widely in Gram-negative bacteria. Bacteria harboring T6SSs inject various effectors into both eukaryotic and prokaryotic cells for interspecies competition or virulence-related processes. The toxicities of the effectors can be neutralized by their cognate immunity proteins. Tde1 (Atu4350)–Tdi1 (Atu4351) has recently been characterized as a T6SS effector–immunity pair in the soil bacterium Agrobacterium tumefaciens and the neutralization mechanism remains unknown. Here, the crystal structure of the immunity protein Tdi1 was determined at 2.40 Å resolution by the single-wavelength anomalous dispersion method. Structural analysis suggested that it is composed of a GAD-like domain and an inserted DUF1851 domain, and both domains show low structural similarities to known structures. There is a positive groove mainly located in the GAD-like domain that may be associated with nucleotide binding. The structure provides a basis for further study of the positive groove as a potential active site.

scholarly journals Characterization of IcmF of the type VI secretion system in an avian pathogenic Escherichia coli (APEC) strain

Microbiology ◽  
2011 ◽  
Vol 157 (10) ◽  
pp. 2954-2962 ◽  
Author(s):  
Fernanda de Pace ◽  
Jacqueline Boldrin de Paiva ◽  
Gerson Nakazato ◽  
Marcelo Lancellotti ◽  
Marcelo Palma Sircili ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Legionella Pneumophila ◽  
Biofilm Formation ◽  
Secretion System ◽  
Type Vi Secretion System ◽  
E Coli ◽  
Type Vi Secretion ◽  
Abiotic Surfaces ◽  
Pathogenic Escherichia Coli ◽  
Zoonotic Risk

The intracellular multiplication factor (IcmF) protein is a component of the recently described type VI secretion system (T6SS). IcmF has been shown to be required for intra-macrophage replication and inhibition of phagosome–lysosome fusion in Legionella pneumophila. In Vibrio cholerae it is involved in motility, adherence and conjugation. Given that we previously reported that two T6SS genes (hcp and clpV) contribute to the pathogenesis of a septicaemic strain (SEPT362) of avian pathogenic Escherichia coli (APEC), we investigated the function of IcmF in this strain. Further elucidation of the virulence mechanisms of APEC is important because this pathogen is responsible for financial losses in the poultry industry, and is closely related to human extraintestinal pathogenic E. coli (ExPEC) strains, representing a potential zoonotic risk, as well as serving as a reservoir of virulence genes. Here we show that an APEC icmF mutant has decreased adherence to and invasion of epithelial cells, as well as decreased intra-macrophage survival. The icmF mutant is also defective for biofilm formation on abiotic surfaces. Additionally, expression of the flagella operon is decreased in the icmF mutant, leading to decreased motility. The combination of these phenotypes culminates in this mutant being altered for infection in chicks. These results suggest that IcmF in APEC may play a role in disease, and potentially also in the epidemiological spread of this pathogen through enhancement of biofilm formation.

scholarly journals Diversification of the Type VI Secretion System in Agrobacteria

mBio ◽  
2021 ◽  
Author(s):  
Chih-Feng Wu ◽  
Alexandra J. Weisberg ◽  
Edward W. Davis ◽  
Lin Chou ◽  
Surtaz Khan ◽  
...  
Keyword(s):  
Secretion System ◽  
Effector Proteins ◽  
Gram Negative Bacteria ◽  
Type Vi Secretion System ◽  
Gram Negative ◽  
Bacterial Interactions ◽  
Type Vi Secretion

The T6SS is used by several taxa of Gram-negative bacteria to secrete toxic effector proteins to attack others. Diversification of effector collections shapes bacterial interactions and impacts the health of hosts and ecosystems in which bacteria reside.

scholarly journals The Type VI Secretion System Modulates Flagellar Gene Expression and Secretion in Citrobacter freundii and Contributes to Adhesion and Cytotoxicity to Host Cells

2015 ◽  
Vol 83 (7) ◽  
pp. 2596-2604 ◽  
Author(s):  
Liyun Liu ◽  
Shuai Hao ◽  
Ruiting Lan ◽  
Guangxia Wang ◽  
Di Xiao ◽  
...  
Keyword(s):  
Secretion System ◽  
Host Cells ◽  
Target Cells ◽  
Deletion Mutants ◽  
Citrobacter Freundii ◽  
Wild Type ◽  
Type Vi Secretion System ◽  
Content Type ◽  
Type Vi Secretion ◽  
Mutant Strains

The type VI secretion system (T6SS) as a virulence factor-releasing system contributes to virulence development of various pathogens and is often activated upon contact with target cells.Citrobacter freundiistrain CF74 has a complete T6SS genomic island (GI) that containsclpV,hcp-2, andvgrT6SS genes. We constructedclpV,hcp-2,vgr, and T6SS GI deletion mutants in CF74 and analyzed their effects on the transcriptome overall and, specifically, on the flagellar system at the levels of transcription and translation. Deletion of the T6SS GI affected the transcription of 84 genes, with 15 and 69 genes exhibiting higher and lower levels of transcription, respectively. Members of the cell motility class of downregulated genes of the CF74ΔT6SS mutant were mainly flagellar genes, including effector proteins, chaperones, and regulators. Moreover, the production and secretion of FliC were also decreased inclpV,hcp-2,vgr, or T6SS GI deletion mutants in CF74 and were restored upon complementation. In swimming motility assays, the mutant strains were found to be less motile than the wild type, and motility was restored by complementation. The mutant strains were defective in adhesion to HEp-2 cells and were restored partially upon complementation. Further, the CF74ΔT6SS, CF74ΔclpV, and CF74Δhcp-2mutants induced lower cytotoxicity to HEp-2 cells than the wild type. These results suggested that the T6SS GI in CF74 regulates the flagellar system, enhances motility, is involved in adherence to host cells, and induces cytotoxicity to host cells. Thus, the T6SS plays a wide-ranging role inC. freundii.

scholarly journals Structure of a Peptidoglycan Amidase Effector Targeted to Gram-Negative Bacteria by the Type VI Secretion System

Cell Reports ◽  
2012 ◽  
Vol 1 (6) ◽  
pp. 656-664 ◽  
Author(s):  
Seemay Chou ◽  
Nhat Khai Bui ◽  
Alistair B. Russell ◽  
Katrina W. Lexa ◽  
Taylor E. Gardiner ◽  
...  
Keyword(s):  
Secretion System ◽  
Gram Negative Bacteria ◽  
Type Vi Secretion System ◽  
Gram Negative ◽  
Type Vi Secretion

scholarly journals Metabolic phospholipid labeling of intact bacteria enables a fluorescence assay that detects compromised outer membranes

2020 ◽  
Vol 61 (6) ◽  
pp. 870-883 ◽  
Author(s):  
Inga Nilsson ◽  
Sheng Y. Lee ◽  
William S. Sawyer ◽  
Christopher M. Baxter Rath ◽  
Guillaume Lapointe ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gram Negative Bacteria ◽  
Metabolic Labeling ◽  
Wild Type ◽  
Transport Pathways ◽  
Gram Negative ◽  
E Coli ◽  
Outer Membranes ◽  
Outer Leaflet ◽  
Promising Tool

Gram-negative bacteria possess an asymmetric outer membrane (OM) composed primarily of lipopolysaccharides (LPSs) on the outer leaflet and phospholipids (PLs) on the inner leaflet. The loss of this asymmetry due to mutations in the LPS biosynthesis or transport pathways causes the externalization of PLs to the outer leaflet of the OM and leads to OM permeability defects. Here, we used metabolic labeling to detect a compromised OM in intact bacteria. Phosphatidylcholine synthase expression in Escherichia coli allowed for the incorporation of exogenous propargylcholine into phosphatidyl(propargyl)choline and exogenous 1-azidoethyl-choline (AECho) into phosphatidyl(azidoethyl)choline (AEPC), as confirmed by LC/MS analyses. A fluorescent copper-free click reagent poorly labeled AEPC in intact wild-type cells but readily labeled AEPC from lysed cells. Fluorescence microscopy and flow cytometry analyses confirmed the absence of significant AEPC labeling from intact wild-type E. coli strains and revealed significant AEPC labeling in an E. coli LPS transport mutant (lptD4213) and an LPS biosynthesis mutant (E. coli lpxC101). Our results suggest that metabolic PL labeling with AECho is a promising tool for detecting a compromised bacterial OM, revealing aberrant PL externalization, and identifying or characterizing novel cell-active inhibitors of LPS biosynthesis or transport.­

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