scholarly journals High throughput identification of genes conferring resistance or sensitivity to toxic effectors delivered by the type VI secretion system

2021 ◽  
Author(s):  
Steven J Hersch ◽  
Rehnuma T Sejuty ◽  
Kevin Manera ◽  
Tao G Dong
Keyword(s):  
High Throughput ◽  
Gene Knockout ◽  
Secretion System ◽  
Effector Proteins ◽  
Type Vi Secretion System ◽  
Numerous Species ◽  
E Coli ◽  
Metabolic Genes ◽  
Defence Genes ◽  
Immunity Genes

The type six secretion system (T6SS) is a prevalent bacterial weapon delivering toxic effector proteins into nearby competitors. In addition to immunity genes that protect against a particular effector, alternate yet crucial nonspecific defences have also recently been identified. To systematically identify genes influencing T6SS susceptibility in numerous species, we designed a Tn-Seq-based competition assay. Combined with follow-up analyses using E. coli and V. cholerae gene knockout collections, we demonstrate that our Tn-Seq competition technique can be used to identify both immunity and non-immunity defences against the T6SS. We also identify E. coli proteins that facilitate T6SS-mediated cell death, including metabolic genes such as cyaA and gltA, where mutant strains were resistant to attack. Our findings act as a proof-of-concept for the technique while also illuminating novel genes of interest. Since Tn-Seq can be applied in numerous species, our method has broad potential for identifying diverse T6SS defence genes across genomes in a high-throughput manner.

scholarly journals Identification of Novel Acinetobacter baumannii Type VI Secretion System Antibacterial Effector and Immunity Pairs

2018 ◽  
Vol 86 (8) ◽  
Author(s):  
Timothy C. Fitzsimons ◽  
Jessica M. Lewis ◽  
Amy Wright ◽  
Oded Kleifeld ◽  
Ralf B. Schittenhelm ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Secretion System ◽  
Effector Proteins ◽  
Host Cells ◽  
Type Vi Secretion System ◽  
Acinetobacter Baylyi ◽  
Content Type ◽  
Type Vi Secretion ◽  
Immunity Genes

ABSTRACT The type VI secretion system (T6SS) is a macromolecular machine that delivers protein effectors into host cells and/or competing bacteria. The effectors may be delivered as noncovalently bound cargo of T6SS needle proteins (VgrG/Hcp/PAAR) or as C-terminal extensions of these proteins. Many Acinetobacter baumannii strains produce a T6SS, but little is known about the specific effectors or how they are delivered. In this study, we show that A. baumannii AB307-0294 encodes three vgrG loci, each containing a vgrG gene, a T6SS toxic effector gene, and an antitoxin/immunity gene. Each of the T6SS toxic effectors could kill Escherichia coli when produced in trans unless the cognate immunity protein was coproduced. To determine the role of each VgrG in effector delivery, we performed interbacterial competitive killing assays using A. baumannii AB307-0294 vgrG mutants, together with Acinetobacter baylyi prey cells expressing pairs of immunity genes that protected against two toxic effectors but not a third. Using this approach, we showed that AB307-0294 produces only three T6SS toxic effectors capable of killing A. baylyi and that each VgrG protein is specific for the carriage of one effector. Finally, we analyzed a number of A. baumannii genomes and identified significant diversity in the range of encoded T6SS VgrG and effector proteins, with correlations between effector types and A. baumannii global clone lineages.

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 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 A New Front in Microbial Warfare—Delivery of Antifungal Effectors by the Type VI Secretion System

2019 ◽  
Vol 5 (2) ◽  
pp. 50 ◽  
Author(s):  
Katharina Trunk ◽  
Sarah J. Coulthurst ◽  
Janet Quinn
Keyword(s):  
Bacterial Species ◽  
Fungal Species ◽  
Secretion System ◽  
Effector Proteins ◽  
Primary Role ◽  
Bacterial Cells ◽  
Type Vi Secretion System ◽  
Type Vi Secretion ◽  
Bacteria And Fungi ◽  
Polymicrobial Communities

Microbes typically exist in mixed communities and display complex synergistic and antagonistic interactions. The Type VI secretion system (T6SS) is widespread in Gram-negative bacteria and represents a contractile nano-machine that can fire effector proteins directly into neighbouring cells. The primary role assigned to the T6SS is to function as a potent weapon during inter-bacterial competition, delivering antibacterial effectors into rival bacterial cells. However, it has recently emerged that the T6SS can also be used as a powerful weapon against fungal competitors, and the first fungal-specific T6SS effector proteins, Tfe1 and Tfe2, have been identified. These effectors act via distinct mechanisms against a variety of fungal species to cause cell death. Tfe1 intoxication triggers plasma membrane depolarisation, whilst Tfe2 disrupts nutrient uptake and induces autophagy. Based on the frequent coexistence of bacteria and fungi in microbial communities, we propose that T6SS-dependent antifungal activity is likely to be widespread and elicited by a suite of antifungal effectors. Supporting this hypothesis, homologues of Tfe1 and Tfe2 are found in other bacterial species, and a number of T6SS-elaborating species have been demonstrated to interact with fungi. Thus, we envisage that antifungal T6SS will shape many polymicrobial communities, including the human microbiota and disease-causing infections.

scholarly journals A Degenerate Type III Secretion System from Septicemic Escherichia coli Contributes to Pathogenesis

2005 ◽  
Vol 187 (23) ◽  
pp. 8164-8171 ◽  
Author(s):  
Diana Ideses ◽  
Uri Gophna ◽  
Yossi Paitan ◽  
Roy R. Chaudhuri ◽  
Mark J. Pallen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Gene Clusters ◽  
Secretion System ◽  
Effector Proteins ◽  
E Coli ◽  
Type Iii

ABSTRACT The type III secretion system (T3SS) is an important virulence factor used by several gram-negative bacteria to deliver effector proteins which subvert host cellular processes. Enterohemorrhagic Escherichia coli O157 has a well-defined T3SS involved in attachment and effacement (ETT1) and critical for virulence. A gene cluster potentially encoding an additional T3SS (ETT2), which resembles the SPI-1 system in Salmonella enterica, was found in its genome sequence. The ETT2 gene cluster has since been found in many E. coli strains, but its in vivo role is not known. Many of the ETT2 gene clusters carry mutations and deletions, raising the possibility that they are not functional. Here we show the existence in septicemic E. coli strains of an ETT2 gene cluster, ETT2sepsis, which, although degenerate, contributes to pathogenesis. ETT2sepsis has several premature stop codons and a large (5 kb) deletion, which is conserved in 11 E. coli strains from cases of septicemia and newborn meningitis. A null mutant constructed to remove genes coding for the putative inner membrane ring of the secretion complex exhibited significantly reduced virulence. These results are the first demonstration of the importance of ETT2 for pathogenesis.

scholarly journals DeepT3_4: A Hybrid Deep Neural Network Model for the Distinction Between Bacterial Type III and IV Secreted Effectors

2021 ◽  
Vol 12 ◽  
Author(s):  
Lezheng Yu ◽  
Fengjuan Liu ◽  
Yizhou Li ◽  
Jiesi Luo ◽  
Runyu Jing
Keyword(s):  
Neural Network ◽  
Neural Networks ◽  
Secretion System ◽  
Effector Proteins ◽  
Host Cells ◽  
Sequence Motifs ◽  
Type Vi Secretion System ◽  
Type Iii ◽  
Type Iv

Gram-negative bacteria can deliver secreted proteins (also known as secreted effectors) directly into host cells through type III secretion system (T3SS), type IV secretion system (T4SS), and type VI secretion system (T6SS) and cause various diseases. These secreted effectors are heavily involved in the interactions between bacteria and host cells, so their identification is crucial for the discovery and development of novel anti-bacterial drugs. It is currently challenging to accurately distinguish type III secreted effectors (T3SEs) and type IV secreted effectors (T4SEs) because neither T3SEs nor T4SEs contain N-terminal signal peptides, and some of these effectors have similar evolutionary conserved profiles and sequence motifs. To address this challenge, we develop a deep learning (DL) approach called DeepT3_4 to correctly classify T3SEs and T4SEs. We generate amino-acid character dictionary and sequence-based features extracted from effector proteins and subsequently implement these features into a hybrid model that integrates recurrent neural networks (RNNs) and deep neural networks (DNNs). After training the model, the hybrid neural network classifies secreted effectors into two different classes with an accuracy, F-value, and recall of over 80.0%. Our approach stands for the first DL approach for the classification of T3SEs and T4SEs, providing a promising supplementary tool for further secretome studies.

scholarly journals Novel structural components generate distinct type VI secretion system anchoring modes

2020 ◽  
Author(s):  
Patricia Bernal ◽  
R. Christopher D. Furniss ◽  
Selina Fecht ◽  
Rhoda C.Y. Leung ◽  
Livia Spiga ◽  
...  
Keyword(s):  
Functional Diversity ◽  
Distinct Type ◽  
Secretion System ◽  
Effector Proteins ◽  
Structural Components ◽  
Type Vi Secretion System ◽  
Type Vi Secretion ◽  
Sheath Structure

ABSTRACTThe type VI secretion system (T6SS) is a phage-derived contractile nanomachine primarily involved in interbacterial competition. Its pivotal component, TssA, is indispensable for the assembly of the T6SS sheath structure, the contraction of which propels a payload of effector proteins into neighboring cells. Despite their key function, TssA proteins exhibit unexpected diversity and exist in two major forms, a short (TssAS) and a long (TssAL) TssA. Whilst TssAL proteins interact with a partner, called TagA, to anchor the distal end of the extended sheath, the mechanism for the stabilization of TssAS-containing T6SSs remains unknown. Here we discover a novel class of structural components that interact with short TssA proteins and contribute to T6SS assembly by stabilizing the polymerizing sheath from the baseplate. We demonstrate that the presence of these components is important for full sheath extension and optimal firing. Moreover, we show that the pairing of each form of TssA with a different class of sheath stabilization proteins results in T6SS apparatuses that either reside in the cell for a while or fire immediately after sheath extension, thus giving rise to different aggression behaviors. We propose that this functional diversity could contribute to the specialization of the T6SS to suit bacterial lifestyles in diverse environmental niches.

scholarly journals Structure of a bacterial Rhs effector exported by the type VI secretion system

2021 ◽  
Author(s):  
Patrick Guenther ◽  
Dennis Quentin ◽  
Shehryar Ahmad ◽  
Kartik Sachar ◽  
Christos Gatsogiannis ◽  
...  
Keyword(s):  
Bacterial Cell ◽  
Secretion System ◽  
Effector Proteins ◽  
Protein Export ◽  
Spike Protein ◽  
Gram Negative Bacteria ◽  
Structural Elements ◽  
Type Vi Secretion System ◽  
Gram Negative ◽  
Type Vi Secretion

The type VI secretion system (T6SS) is a widespread protein export apparatus found in Gram-negative bacteria. The majority of T6SSs deliver toxic effector proteins into competitor bacteria. Yet, the structure, function, and activation of many of these effectors remains poorly understood. Here, we present the structures of the T6SS effector RhsA from Pseudomonas protegens and its cognate T6SS spike protein, VgrG1, at 3.3 Å resolution. The structures reveal that the rearrangement hotspot (Rhs) repeats of RhsA assemble into a closed anticlockwise β-barrel spiral similar to that found in bacterial insecticidal Tc toxins and in metazoan teneurin proteins. We find that the C-terminal toxin domain of RhsA is autoproteolytically cleaved but remains inside the Rhs ′cocoon′ where, with the exception of three ordered structural elements, most of the toxin is disordered. The N-terminal ′plug′ domain is unique to T6SS Rhs proteins and resembles a champagne cork that seals the Rhs cocoon at one end while also mediating interactions with VgrG1. Interestingly, this domain is also autoproteolytically cleaved inside the cocoon but remains associated with it. We propose that mechanical force is required to remove the cleaved part of the plug, resulting in the release of the toxin domain as it is delivered into a susceptible bacterial cell by the T6SS.

scholarly journals The Type VI Secretion System of Xanthomonas phaseoli pv. manihotis is involved in virulence and in vitro motility

2020 ◽  
Author(s):  
Nathaly Andrea Montenegro Benavides ◽  
Alejandro Alvarez Borrero ◽  
Mario Luis Arrieta Ortiz ◽  
Luis Miguel Rodriguez-R. ◽  
David Octavio Botero Rozo ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Phylogenetic Analyses ◽  
Gene Clusters ◽  
Secretion System ◽  
Type Vi Secretion System ◽  
Cellular Processes ◽  
Protein Secretion System ◽  
Different Strains

Abstract Background: The type VI protein secretion system (T6SS) is important in diverse cellular processes in Gram-negative bacteria, including interactions with other bacteria and with eukaryotic hosts. In this study we analyze the evolution of the T6SS in the genus Xanthomonas and evaluate its importance of the T6SS for virulence and in vitro motility in Xanthomonas phaseoli pv. manihotis (Xpm), the causal agent of bacterial blight in cassava (Manihot esculenta). We delineate the organization of the T6SS gene clusters in Xanthomonas and then characterize proteins of this secretion system in Xpm strain CIO151. Results: We describe the presence of three different clusters in the genus Xanthomonas that vary in their organization and degree of synteny between species. Using a gene knockout mutagenesis, we also found that vgrG and hcp are required for maximal aggressiveness of Xpm on cassava plants while clpV is important for both motility and maximal aggressiveness. Conclusion: We characterized the T6SS in 15 different strains in Xanthomonas and our phylogenetic analyses suggest that the T6SS might have been acquired by a very ancient event of horizontal gene transfer and maintained through evolution, hinting at their importance for the adaptation of Xanthomonas to their hosts. Finally, we demonstrated that the T6SS of Xpm is functional, and significantly contributes to virulence and motility. This is the first experimental study that demonstrates the role of the T6SS in the Xpm-cassava interaction and the T6SS organization in the genus Xanthomonas.

scholarly journals When the pandemic opts for the lockdown: Secretion system evolution in the cholera bacterium

2021 ◽  
Vol 8 (3) ◽  
pp. 69-72
Author(s):  
Francis J. Santoriello ◽  
Stefan Pukatzki
Keyword(s):  
Common Ancestor ◽  
Diarrheal Disease ◽  
Secretion System ◽  
Effector Proteins ◽  
Type Vi Secretion System ◽  
Human Gastrointestinal Tract ◽  
Type Vi Secretion ◽  
T4 Bacteriophage ◽  
The Common ◽  
Pandemic Strains

Vibrio cholerae, the causative agent of the diarrheal disease cholera, is a microbe capable of inhabiting two different ecosystems: chitinous surfaces in brackish, estuarine waters and the epithelial lining of the human gastrointestinal tract. V. cholerae defends against competitive microorganisms with a contact-dependent, contractile killing machine called the type VI secretion system (T6SS) in each of these niches. The T6SS resembles an inverted T4 bacteriophage tail and is used to deliver toxic effector proteins into neighboring cells. Pandemic strains of V. cholerae encode a unique set of T6SS effector proteins, which may play a role in pathogenesis or pandemic spread. In our recent study (Santoriello et al. (2020), Nat Commun, doi: 10.1038/s41467-020-20012-7), using genomic and molecular biology tools, we demonstrated that the T6SS island Auxiliary Cluster 3 (Aux3) is unique to pandemic strains of V. cholerae. We went on to show that Aux3 is related to a phage-like element circulating in environmental V. cholerae strains and that two genetic domestication events formed the pandemic Aux3 cluster during the evolution of the pandemic clone. Our findings support two main conclusions: (1) Aux3 evolution from phage-like element to T6SS cluster offers a snapshot of phage domestication in early T6SS evolution and (2) chromosomal maintenance of Aux3 was advantageous to the common ancestor of V. cholerae pandemic strains.

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