Type VI secretion: a beginner's guide

Type VI secretion is a newly described mechanism for protein transport across the cell envelope of Gram-negative bacteria. Components that have been partially characterised include an IcmF homologue, the ATPase ClpV, a regulatory FHA domain protein and the secreted VgrG and Hcp proteins. Type VI secretion is clearly a key virulence factor for some important pathogenic bacteria and has been implicated in the translocation of a potential effector protein into eukaryotic cells by at least one organism (Vibrio cholerae). However, type VI secretion systems (T6SSs) are widespread in nature and not confined to known pathogens. In accordance with the general rule that the expression of protein secretion systems is tightly regulated, expression of type VI secretion is controlled at both transcriptional and post-transcriptional levels

Molecular weaponry:diverse effectors delivered by the Type VI secretion system

The Type VI secretion system is a widespread bacterial nanomachine, used to deliver toxins directly into eukaryotic or prokaryotic target cells. These secreted toxins, or effectors, act on diverse cellular targets, and their action provides the attacking bacterial cell with a significant fitness advantage, either against rival bacteria or eukaryotic host organisms. In this review, we discuss the delivery of diverse effectors by the Type VI secretion system, the modes of action of the so‐called ‘anti‐bacterial’ and ‘anti‐eukaryotic’ effectors, the mechanism of self‐resistance against anti‐bacterial effectors and the evolutionary implications of horizontal transfer of Type VI secretion system‐associated toxins. Whilst it is likely that many more effectors remain to be identified, it is already clear that toxins delivered by this secretion system represent efficient weapons against both bacteria and eukaryotes

Microfluidics-based liquid chromatography/mass spectrometry multiple reaction monitoring approach for the relative quantification of Burkholderia cenocepacia secreted virulence factors

Rationale: Burkholderia cenocepacia is an opportunistic pathogen that is commonly isolated from patients with cystic fibrosis (CF). Quorum sensing has been suggested to play a role in the activity of type II and type VI secretion systems and the release of virulence factors. Apart from the classical acyl homoserine lactone quorum sensing, B. cenocepacia also uses the diffusible signal factor system (DSF). Quantitative information on the true impact of DSF molecules on the release of ZmpA and other virulence factors is lacking. Methods: Based on results of a label-free proteomics analysis addressing changes in the secretome in response to DSFs, a panel of peptides was selected to develop a microfluidics liquid chromatography/mass spectrometry (LC/MS) method implementing single reaction monitoring (SRM) to quantify B. cenocepacia virulence factors. Results: Increase in secretion of virulence factors upon treatment with BDSF was observed for ZmpA and Aida, but not for ZmpB. Type VI secretion system dependent Hcp1 and TecA were decreased. However, non-physiological amounts of BDSF were needed to provoke the effect. DSFs from P. aeruginosa and S. maltophilia were also affecting virulence factor secretion, but the effect was smaller than for the endogenous BDSF. Conclusions: Microfluidics-based SRM is a useful tool to quantitatively assess the impact of quorum sensing on the release of virulence factors by (opportunistic) pathogens

TagF-mediated repression of bacterial type VI secretion systems involves a direct interaction with the cytoplasmic protein Fha

The bacterial type VI secretion system (T6SS) delivers effectors into eukaryotic host cells or toxins into bacterial competitor for survival and fitness. The T6SS is positively regulated by the threonine phosphorylation pathway (TPP) and negatively by the T6SS-accessory protein TagF. Here, we studied the mechanisms underlying TagF-mediated T6SS repression in two distinct bacterial pathogens, Agrobacterium tumefaciens and Pseudomonas aeruginosa. We found that in A. tumefaciens, T6SS toxin secretion and T6SS-dependent antibacterial activity are suppressed by a two-domain chimeric protein consisting of TagF and PppA, a putative phosphatase. Remarkably, this TagF domain is sufficient to post-translationally repress the T6SS, and this inhibition is independent of TPP. This repression requires interaction with a cytoplasmic protein, Fha, critical for activating T6SS assembly. In P. aeruginosa, PppA and TagF are two distinct proteins that repress T6SS in a TPP-dependent and -independent pathways, respectively. P. aeruginosa TagF interacts with Fha1, suggesting that formation of this complex represents a conserved TagF-mediated regulatory mechanism. Using TagF variants with substitutions of conserved amino acid residues at predicted protein-protein interaction interfaces, we uncovered evidence that the TagF-Fha interaction is critical for TagF-mediated T6SS repression in both bacteria. TagF inhibits T6SS without affecting T6SS protein abundance in A. tumefaciens, but TagF overexpression reduces the protein levels of all analyzed T6SS components in P. aeruginosa. Our results indicate that TagF interacts with Fha, which in turn could impact different stages of T6SS assembly in different bacteria, possibly reflecting an evolutionary divergence in T6SS control

Two Independent Pathways for Self-Recognition in Proteus Mirabilis Are Linked by Type VI-Dependent Export

Swarming colonies of the bacterium Proteus mirabilis are capable of self-recognition and territorial behavior. Swarms of independent P. mirabilis isolates can recognize each other as foreign and establish a visible boundary where they meet; in contrast, genetically identical swarms merge. The ids genes, which encode self-identity proteins, are necessary but not sufficient for this territorial behavior. Here we have identified two new gene clusters: one (idr) encodes rhs-related products, and another (tss) encodes a putative type VI secretion (T6S) apparatus. The Ids and Idr proteins function independently of each other in extracellular transport and in territorial behaviors; however, these self-recognition systems are linked via this type VI secretion system. The T6S system is required for export of select Ids and Idr proteins. Our results provide a mechanistic and physiological basis for the fundamental behaviors of self-recognition and territoriality in a bacterial model system.Molecular and Cellular Biolog

Causalities of war: The connection between type VI secretion system and microbiota

Cellular Microbiology published by John Wiley & Sons Ltd Microbiota niches have space and/or nutrient restrictions, which has led to the coevolution of cooperation, specialisation, and competition within the population. Different animal and environmental niches contain defined resident microbiota that tend to be stable over time and offer protection against undesired intruders. Yet fluxes can occur, which alter the composition of a bacterial population. In humans, the microbiota are now considered a key contributor to maintenance of health and homeostasis, and its alteration leads to dysbiosis. The bacterial type VI secretion system (T6SS) transports proteins into the environment, directly into host cells or can function as an antibacterial weapon by killing surrounding competitors. Upon contact with neighbouring cells, the T6SS fires, delivering a payload of effector proteins. In the absence of an immunity protein, this results in growth inhibition or death of prey leading to a competitive advantage for the attacker. It is becoming apparent that the T6SS has a role in modulating and shaping the microbiota at multiple levels, which is the focus of this review. Discussed here is the T6SS, its role in competition, key examples of its effect upon the microbiota, and future avenues of researc

Biochemical analysis of TssK, a core component of the bacterial Type VI secretion system, reveals distinct oligomeric states of TssK and identifies a TssK–TssFG subcomplex

Gram-negative bacteria use the Type VI secretion system (T6SS) to inject toxic proteins into rival bacteria or eukaryotic cells. However, the mechanism of the T6SS is incompletely understood. In the present study, we investigated a conserved component of the T6SS, TssK, using the antibacterial T6SS of Serratia marcescens as a model system. TssK was confirmed to be essential for effector secretion by the T6SS. The native protein, although not an integral membrane protein, appeared to localize to the inner membrane, consistent with its presence within a membrane-anchored assembly. Recombinant TssK purified from S. marcescens was found to exist in several stable oligomeric forms, namely trimer, hexamer and higher-order species. Native-level purification of TssK identified TssF and TssG as interacting proteins. TssF and TssG, conserved T6SS components of unknown function, were required for T6SS activity, but not for correct localization of TssK. A complex containing TssK, TssF and TssG was subsequently purified in vitro, confirming that these three proteins form a new subcomplex within the T6SS. Our findings provide new insight into the T6SS assembly, allowing us to propose a model whereby TssK recruits TssFG into the membrane-associated T6SS complex and different oligomeric states of TssK may contribute to the dynamic mechanism of the system

A transcriptional regulatory mechanism finely tunes the firing of type VI secretion system in response to bacterial enemies

The ability to detect and measure danger from an environmental signal is paramount for bacteria to respond accordingly, deploying strategies that halt or counteract potential cellular injury and maximize survival chances. Type VI secretion systems (T6SSs) are complex bacterial contractile nanomachines able to target toxic effectors into neighboring bacteria competing for the same colonization niche. Previous studies support the concept that either T6SSs are constitutively active or they fire effectors in response to various stimuli, such as high bacterial density, cell-cell contact, nutrient depletion, or components from dead sibling cells. For Serratia marcescens, it has been proposed that its T6SS is stochastically expressed, with no distinction between harmless or aggressive competitors. In contrast, we demonstrate that the Rcs regulatory system is responsible for finely tuning Serratia T6SS expression levels, behaving as a transcriptional rheostat. When confronted with harmless bacteria, basal T6SS expression levels suffice for Serratia to eliminate the competitor. A moderate T6SS upregulation is triggered when, according to the aggressor-prey ratio, an unbalanced interplay between homologous and heterologous effectors and immunity proteins takes place. Higher T6SS expression levels are achieved when Serratia is challenged by a contender like Acinetobacter, which indiscriminately fires heterologous effectors able to exert lethal cellular harm, threatening the survival of the Serratia population. We also demonstrate that Serratia’s RcsB-dependent T6SS regulatory mechanism responds not to general stress signals but to the action of specific effectors from competitors, displaying an exquisite strategy to weigh risks and keep the balance between energy expenditure and fitness costs. IMPORTANCE Serratia marcescens is among the health-threatening pathogens categorized by the WHO as research priorities to develop alternative antimicrobial strategies, and it was also recently identified as one major component of the gut microbiome in familial Crohn disease dysbiosis. Type VI secretion systems (T6SSs) stand among the array of survival strategies that Serratia displays. They are contractile multiprotein complexes able to deliver toxic effectors directed to kill bacterial species sharing the same niche and, thus, competing for vital resources. Here, we show that Serratia is able to detect and measure the extent of damage generated through T6SS-delivered toxins from neighboring bacteria and responds by transcriptionally adjusting the expression level of its own T6SS machinery to counterattack the rival. This strategy allows Serratia to finely tune the production of costly T6SS devices to maximize the chances of successfully fighting against enemies and minimize energy investment. The knowledge of this novel mechanism provides insight to better understand bacterial interactions and design alternative treatments for polymicrobial infections.Fil: Lazzaro, Martina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Feldman, Mario F.. Washington University in St. Louis; Estados UnidosFil: Garcia Vescovi, Eleonora. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

Genome Sequence of Pantoea ananatis Strain AMG 501, a Plant Growth-Promoting Bacterium Isolated from Rice Leaves Grown in Paddies of Southern Spain

Pantoea ananatis AMG 501 is a plant growth-promoting bacterium isolated from rice leaves. Its genome was estimated at 5,102,640 bp with 4,994 coding sequences, encompassing genes related to the metabolism of carbohydrates, to the synthesis of auxins, siderophores, and homoserine lactones, and to the type I, II, III, IV, and VI secretion systems.España, MINECO AGL2016-77163-