Molecular Basis of Hcp-Dependent Toxin Delivery during Interbacterial Competition
Ontology highlight
ABSTRACT:
ORGANISM(S): Pseudomonas aeruginosa PAO1
PROVIDER: GSE334501 | GEO | 2026/06/20
REPOSITORIES: GEO
Similar Datasets
Project description:Secretion systems are used as weapons by a variety of Gram-negative bacteria. Among them the Type VI Secretion System (T6SS) gained more interest throughout the last years. The system functions as a molecular nano-weapon: it is used in inter-kingdom competition by various bacteria to deliver toxic effectors in target cells. Here we describe the role of the T6SS in Photorhabdus laumondii subsp. laumondii strain DJC, an entomopathogenic biocontrol agent able to live in different environmental niches, such as in symbiosis with nematodes and in the rhizosphere on plant roots. Using bioinformatic and protein motif analyses we identified four T6SS gene clusters (T6SS-1, T6SS-2, T6SS-3 and T6SS-4) and multiple orphan T6SS related genes in the genome of P. laumondii. Furthermore, we highlighted 11 T6SS effector-immunity pairs, including three undescribed membrane disrupting effectors, each with putatively different antibacterial activities. By label-free mass spectrometry of P. laumondii wild type cells and respective T6SS-deficient strains, we could point out a cross-link between T6SS and other Photorhabdus’ virulence related mechanisms such as PVCs, T3SS and pyocins. Furthermore, a change in motility as well as in the secondary metabolism was observed upon T6SS-deficiency. Here, we shed light on the T6SS in P. laumondii DJC and suggesting a cross-link of various virulence mechanisms, which could help to gain knowledge on T6SS and better figure out the Photorhabdus ability to live in polymicrobial environments.
2024-04-05 | PXD035720 | Pride
Project description:Bacteria have evolved effectors or toxins to outcompete other bacteria or to hijack host cell pathways. One broad family of bacterial polymorphic toxins regroups multidomain proteins with a modular organization, that comprise a C-terminal toxin domain fused to a N-terminal domain that adapt to the delivery apparatus. Polymorphic toxins include bacteriocins, contact-dependent growth inhibition systems, and specialized Hcp, VgrG, PAAR or Rhs Type VI secretion (T6SS) components. We recently described and characterized Tre23, a toxin domain fused to a T6SS-associated Rhs protein in Photorhabdus laumondii. Here, we show that the Rhs polymorphic toxin forms a complex with the T6SS spike protein VgrG and a cognate chaperone EagR. Using truncation derivatives and cross-linking mass spectrometry, we demonstrate that VgrG-EagR-Rhs complex formation requires the VgrG C-terminal β-helix and the Rhs Nterminal prePAAR and PAAR domains. We then report the cryo-electron-microscopy structure of the Rhs-EagR complex, demonstrating that the Rhs central region forms a β-barrel cage-like structure that encapsulates the C-terminal toxin domain, and provide evidence for processing of the Rhs protein through aspartyl autoproteolysis. We propose a model for Rhs loading on the T6SS, transport and delivery into the target cell.
2021-11-30 | PXD028652 | Pride
Project description:The Type VI Secretion System (T6SS) is a molecular nanomachine that injects toxic effector proteins into the environment or neighbouring cells, playing an important role in interbacterial competition and host antagonism during infection. Pseudomonas aeruginosa encodes three different T6SSs. The H1-T6SS delivers toxins into aggressive bacteria in response to attacks mediated by their own T6SS. This suggests that P. aeruginosa has the ability to survive T6SS assaults. However, the resistance mechanisms are poorly characterized. In this work, we performed a CRISPRi screen to identify pathways involved in resistance to T6SS effectors of Acinetobacter baylyi and Vibrio cholerae. We show that members of the GacA/GacS regulon, such as the mag operon, and GacA-independent factors, such as the outer membrane protein OprF, confer resistance to T6SS toxins. Specifically, MagD protects mainly against PG-targeting effectors, whereas OprF confers general protection against multiple effector types. We show that these mechanisms are crucial for resistance against T6SS attacks, as well as for resistance to certain antibiotics. Interestingly, some of these mechanisms also lead to higher antibiotic susceptibility, suggesting more complex links between general T6SS and antibiotic resistance.
2025-01-07 | MSV000096798 | MassIVE
Project description:The Type VI secretion system (T6SS) is a bacterial mechanism that delivers effectors to eliminate competitors and modulate host responses. We previously discovered that the oral symbiont Aggregatibacter aphrophilus uses its T6SS to kill the pathobiont Aggregatibacter actinomycetemcomitans. To understand how the T6SS-dependent mechanism of A. aphrophilus affects host cells and A. actinomycetemcomitans, we co-cultured our immortalised epithelial cell line with A. actinomycetemcomitans and various strains of A. aphrophilus: wild-type (HK83 + JP2, n = 6), a T6SS-deficient strain (HK83 Δhcp + JP2, n = 6), and double mutants (HK83 Δtle5 Δglh + JP2, n = 6). As a baseline, we also examined single inoculations of A. aphrophilus (HK83, n = 6), the Δhcp mutant (HK83 Δhcp, n = 6), the Δtle5 Δglh mutant (HK83 Δtle5 Δglh, n = 6), and A. actinomycetemcomitans JP2 (JP2, n = 6). Cells not challenged served as controls. This study aims to identify specific effectors and characterize host responses to A. aphrophilus T6SS.
2025-11-10 | PXD057672 | Pride
Project description:Bacteria coexist in polymicrobial communities where they engage in complex interactions, including interbacterial antagonism. We recently described that the environmental bacterial pathogen Chromobacterium violaceum has an active type VI secretion system (T6SS), which plays a crucial role in interbacterial competition. However, the arsenal of effectors delivered by this T6SS remains unknown. Here, we identified globally the repertoire of C. violaceum T6SS effectors and characterized a novel effector of Rhs family, which we name RhsF (Rhs with a FIX domain) and its immunity protein, RhsFi. Using mass spectrometry analyses of proteins from culture supernatants and those co-immunoprecipitated with VgrG3, we identified six novel toxin candidates, namely four phospholipases (CV_0012, CV_1234, CV_3971, and CV_3990), a hypothetical protein (CV_2125), and an uncharacterized Rhs protein (CV_1431). Among these T6SS-delivered effectors, we demonstrate that RhsF (CV_1431) was toxic to bacteria in a T6SS dependent manner. The toxicity of the C-terminal domain (RhsF-CT) of RhsF was neutralized upon co-expression with RhsFi (CV_1430), indicating that RhsF-RhsFi composes a toxin-immunity pair. Structure of the RhsF-CT/RhsFi complex solved by X-ray crystallography (1.85 Å resolution) revealed that RhsF exhibits structural features of ADP-ribosyltransferases and provided insights of toxin inhibition via direct occlusion of its catalytic site by RhsFi. Indeed, functional assays confirmed that RhsF toxicity requires a catalytic triad composed by R1403, Y1456, and E1497 residues. Together, our findings uncover the profile of effectors secreted by the T6SS of C. violaceum and establish RhsF as a potent antibacterial toxin, expanding the known repertoire of bacterial arms involved in microbial competition.
2026-06-01 | PXD071363 | Pride
Project description:The Type VI Secretion System (T6SS) in bacteria is a versatile mechanism that facilitates protein transport into neighboring cells and can act as an antibacterial weapon by eliminating competing organisms in the vicinity. The objective of this study was to characterize the T6SS in Aggregatibacter aphrophilus and assess its antimicrobial capabilities through competition with Aggregatibacter actinomycetemcomitans in a multispecies biofilm. The proteomic analysis consisted of two parts, referred to as monospecies biofilm and multispecies biofilms, respectively. Initially, we examined the protein profiles of monospecies biofilms formed by two strains of Aggregatibacter aphrophilus, namely HK83 and CCUG 11575, along with their Hcp mutant derivatives (Hcp being a core protein for T6SS). Each strain was analyzed with six replicates (n=4 for HK83, HK83 hcp, CCUG 11575, and CCUG 11575 hcp). Subsequently, the HK83 and CCUG 11575 strains, as well as their Hcp mutant derivatives, were individually introduced into a multispecies biofilm. This multispecies biofilm consisted of seven species, namely A. actinomycetemcomitans JP2 strain (OMZ 295), Actinomyces oris (OMZ 745), Candida albicans (OMZ 110), Fusobacterium nucleatum subsp. nucleatum KP-F2 (OMZ 598), Streptococcus oralis SK248 (OMZ 607), Streptococcus mutans UA159 (OMZ 918), and Veillonella dispar ATCC 17748T (OMZ 493). These species were selected to mimic the natural co-habitat of A. aphrophilus and A. actinomycetemcomitans. Furthermore, control 7-species biofilms with A. aphrophilus strains HK83, HK83 hcp, CCUG 11575, and CCUG 11575 hcp (n=4 each) underwent proteomic analysis to gain insights into the protein expression and potential interactions within the biofilm community.
2024-05-24 | PXD042723 | Pride
Project description:The ability to compete with diverse competitors is essential for bacteria to succeed in microbial communities. A widespread strategy for inter-bacterial competition is the delivery of antibacterial toxins, or effector proteins, directly into rival cells using the Type VI secretion system (T6SS). Whilst a large number of broad-spectrum enzymatic T6SS effectors have been described, relatively few which form pores in target cell membranes have been reported. Here, we describe a widely-occurring new family of T6SS-dependent pore-forming effectors, exemplified by Ssp4 of Serratia marcescens Db10. We show that Ssp4 forms cation-selective pores in vitro and use molecular dynamics simulations to support a high resolution structural model of a tetrameric membrane pore formed by Ssp4. Notably, the ion selectivity of Ssp4, its distribution and its impact on intoxicated cells is distinct from that of Ssp6, the other cation-selective pore-forming toxin delivered by the same T6SS. We further discovered that Ssp4 is active against a wider range of target species than Ssp6, highlighting that T6SS effectors are not always broad-spectrum. Finally, use of Tn-seq to identify Ssp4-resistant mutants revealed that a mucA mutant of Pseudomonas fluorescens, which overproduces extracellular polysaccharide, provides resistance to T6SS attacks. We conclude that possession of two distinct T6SS-dependent pore-forming toxins may be a common strategy to ensure effective de-energisation of closely- and distantly-related competitors.
2025-10-31 | GSE301252 | GEO
Project description:Paraburkholderia phymatum is a beta-proteobacterium, which lives in the soil and is able to enter nitrogen-fixing symbiosis with different legumes. The biological nitrogen fixation (BNF) process is of great ecological and agronomic importance. We previously showed that the expression of the key P. phymatum BNF enzyme – the nitrogenase –is regulated by the sigma factor σ54 (or RpoN) inside root nodules. This study focused on identifying the σ54 regulon of P. phymatum grown in nitrogen limited conditions using RNA-Sequencing. Among the genes significantly down-regulated in absence of σ54 we found those coding for a C4-dicarboxylate transport system (Bphy_0225-27), a flagellar biosynthesis cluster (Bphy_2926-64) and one of the two type 6 secretion system (T6SS-b) present in P. phymatum genome (Bphy_5978-97). Indeed, the σ54 mutant was unable to grow on C4 dicarboxylates (fumarate, malate and succinate) as the sole carbon source and was less motile compared to the wild-type strain. Both defects were complemented by adding rpoN in trans. Additionally, using reporter fusions we confirmed that T6SS-b expression is regulated by σ54. Finally, a σ54 mutant was less competitive than its parental strain against P. diazotrophica, suggesting a role of σ54 in controlling interbacterial competition.
2020-08-12 | GSE156048 | GEO
Project description:Bacteroides fragilis (Bf)'s T6SS locus has been characterized and shown to have functional activity in competition experiments. It has been demonstrated that symbiont nontoxigenic Bf strains have a more effective “weapon” effect on pathogenic Bf, which is realized through the activity of effector-immune (E-I) protein pairs. Intensive study of the T6SS structure has led to an understanding of certain issues related to its functional activity, but the exact regulatory mechanisms of E-I protein pair activity remain unclear. Proteomic annotation of T6SS components and detailed descriptions of all immune-effector pairs are currently available. In this research, we performed detailed proteogenomic analysis and subsequent proteomic annotation of the T6SS components of the toxigenic Bf BOB25. Fractionated cells, cultivated media and vesicles were prepared for proteome analysis by HPLC-MS/MS. Proteogenomic annotation and comparative genomic study of the T6SS loci of the toxigenic Bf BOB25 were carried out by comparison with the reference genomes of the following Bf strains: JIM10, NCTC 9343 and 638R. According to the data obtained, T6SS components were represented in all types of the analysed samples. The following components of the T6SS were identified in culture media and cells: ClpV (TssH), TssK, TssC, TssB, Hcp (TssD), and TetR. The predicted effector protein AKA51715.1 (VU15_08315) was also detected in media. The greatest amount of T6SS proteins, including the Hcp protein, was detected in the vesicle samples, which was also observed by TEM. Potential effectors, including AKA51715.1 (VU15_08315), AKA51716.1 (VU15_08320), AKA51728.1 (VU15_08385) and the immune protein AKA51727.1 (VU15_08380), were detected in vesicles. The presence of the immune and effector proteins in the Bf secretome indicates the high activity of the T6SS without bacterial competition. It is possible that the T6SS is also used by bacteria to regulate population size by altering the activity of different repertoires of E-I pairs.
2025-01-31 | PXD059123 | Pride
Project description:Pseudomonas aeruginosa uses three type six secretion systems (H1-, H2- and H3-T6SS) to manipulate its environment, subvert host cells and compete with microbial competitors. These T6SS machines can be loaded with a variety of effectors/toxins, many being associated with a specific VgrG. How P. aeruginosa transcriptionally coordinates the three main T6SS clusters and the multiple vgrG islands spread through the genome is unknown. Here we show an unprecedented level of control of the regulator RsmA in repressing most known T6SS-related genes. Moreover, it is noticeable that a sigma factor activator (SFA) protein is encoded in each of H2- and H3-T6SS clusters, Sfa2 and Sfa3, respectively. SFA proteins are enhancer binding proteins that usually work in concert with the sigma factor RpoN. Using a combination of RNA-seq, ChIP-seq and molecular biology approaches, we demonstrate that RpoN coordinates the T6SSs of P. aeruginosa by activating the H2-T6SS but repressing the H1- and H3-T6SS. Furthermore, RpoN and Sfa2 control the expression of all H2-T6SS-linked VgrG and effector arsenal to enable very effective interbacterial killing. Such RpoN-dependent control is exerted both directly and indirectly. The function of the SFA protein is specific since the H3-T6SS associated Sfa3 cannot complement loss of Sfa2. Our study further delineates the multiple regulatory mechanisms that modulate the deployment of an arsenal of T6SS effectors likely to respond and adapt to a range of environmental conditions that a versatile organism like P. aeruginosa would encounter.
2022-02-23 | GSE185398 | GEO