Biology subjects

Salomon, D.

Publications and source records attributed to Salomon, D..

27 records · Page 2Linked to original sources

bioRxiv2022

The MIX domain is required for T6SS-mediated effector secretion

ABTRACTGram-negative bacteria often employ the type VI secretion system (T6SS) to deliver diverse cocktails of antibacterial effectors into rival bacteria. In many cases, even when the identity of the delivered effectors is known, their toxic activity and mechanism of secretion are not. Here, we investigate VPA1263, a Vibrio parahaemolyticus T6SS effector that belongs to a widespread class of polymorphic effectors containing a MIX domain. We reveal a C-terminal DNase toxin domain belonging to the HNH nuclease superfamily, and we show that it mediates the antibacterial toxicity of this effector during bacterial competition. Furthermore, we demonstrate that the VPA1263 MIX domain is necessary for T6SS-mediated secretion and intoxication of recipient bacteria. These results are the first indication of a functional role for MIX domains in T6SS secretion. IMPORTANCESpecialized protein delivery systems are used during bacterial competition to deploy cocktails of toxins that target conserved cellular components. Although numerous toxins have been revealed, the activity of many remains unknown. In this study, we investigated such a toxin from the pathogen Vibrio parahaemolyticus. Our findings indicated that the toxin employs a DNase domain to intoxicate competitors. We also showed that a domain used as a marker for secreted toxins is required for secretion of the toxin via a type VI secretion system.

microbiology↗

bioRxiv2021

A dynamic antibacterial T6SS in Pantoea agglomerans pv. betae delivers a lysozyme-like effector to antagonize competitors

The type VI secretion system (T6SS) is deployed by numerous Gram-negative bacteria to deliver toxic effectors into neighboring cells. The genome of Pantoea agglomerans pv. betae (Pab) phytopathogenic bacteria contains a gene cluster (T6SS1) predicted to encode a complete T6SS. Using secretion and competition assays, we found that T6SS1 in Pab is a functional antibacterial system that allows this pathogen to outcompete rival plant-associated bacteria found in its natural environment. Computational analysis of the T6SS1 gene cluster revealed that antibacterial effector and immunity proteins are encoded within three dynamic genomic islands that harbor arrays of orphan immunity genes or toxin and immunity cassettes. Functional analysis demonstrated that the specialized antibacterial effector VgrG contains a C-terminal catalytically active glucosaminidase domain that is used to degrade prey peptidoglycan. Moreover, we confirmed that a bicistronic unit at the end of the T6SS1 cluster encodes a novel antibacterial T6SS effector and immunity pair. Together, these results demonstrate that Pab T6SS1 is an antibacterial system delivering a lysozyme-like effector to eliminate competitors, and indicate that this bacterium contains novel T6SS effectors. Significance StatementIn this work, we describe the identification of a Pantoea agglomerans T6SS as an antibacterial determinant used by this phytopathogen to outcompete bacterial rivals. Furthermore, we provide an in-depth analysis of the T6SS gene cluster and the putative effector and immunity genes that comprise it, and we propose explanations for its dynamic evolution and effector diversification in Pantoea strains. Lastly, we experimentally validate two predicted effector and immunity pairs, and we demonstrate that one is a potent lysozyme-like toxin.

microbiology↗

bioRxiv2021

A binary effector module secreted by a type VI secretion system

Gram-negative bacteria use type VI secretion systems (T6SSs) to deliver toxic effector proteins into neighboring cells. Cargo effectors are secreted by binding non-covalently to the T6SS apparatus. Occasionally, effector secretion is assisted by an adaptor protein, although the adaptor itself is not secreted. Here, we report a new T6SS secretion mechanism, in which an effector and a co-effector are secreted together. Specifically, we identified a novel periplasm-targeting effector that is secreted together with its co-effector, which contains a MIX (marker for type sIX effector) domain previously reported only in polymorphic toxins. The effector and co-effector directly interact, and they are dependent on each other for secretion. We termed this new secretion mechanism "a binary effector module", and we show that it is widely distributed in marine bacteria.

microbiology↗

bioRxiv2021

Formylglycine-generating enzyme-like proteins constitute a novel family of widespread type VI secretion system immunity proteins

Competition is a critical aspect of bacterial life, as it enables niche establishment and facilitates the acquisition of essential nutrients. Warfare between Gram-negative bacteria is largely mediated by the type VI secretion system (T6SS), a dynamic nanoweapon that delivers toxic effector proteins from an attacking cell to adjacent bacteria in a contact-dependent manner. Effector-encoding bacteria prevent self-intoxication and kin cell killing by the expression of immunity proteins, which prevent effector toxicity by specifically binding their cognate effector and occluding its active site. In this study, we investigate Tsi3, a previously uncharacterized T6SS immunity protein present in multiple strains of the human pathogen Acinetobacter baumannii. We show that Tsi3 is the cognate immunity protein of the antibacterial effector of unknown function Tse3. Our bioinformatic analyses indicate that Tsi3 homologs are widespread among Gram-negative bacteria, often encoded within T6SS effector-immunity modules. Surprisingly, we found that Tsi3 homologs possess a characteristic formylglycine-generating enzyme (FGE) domain, which is present in various enzymatic proteins. Our data shows that Tsi3-mediated immunity is dependent on Tse3-Tsi3 protein-protein interactions and that Tsi3 homologs from various bacteria do not protect against Tse3-dependent bacterial killing. Thus, we conclude that Tsi3 homologs are unlikely to be functional enzymes. Collectively, our work identifies FGE domain-containing proteins as important mediators of immunity against T6SS attacks and indicates that the FGE domain can be co-opted as a scaffold in multiple proteins to carry out diverse functions. ImportanceDespite the wealth of knowledge on the diversity of biochemical activities carried out by T6SS effectors, comparably little is known about the various strategies bacteria employ to prevent susceptibility to T6SS-dependent bacterial killing. Our work establishes a novel family of T6SS immunity proteins with a characteristic FGE domain. This domain is present in enzymatic proteins with various catalytic activities. Our characterization of Tsi3 expands the known functions carried out by FGE-like proteins to include defense during T6SS-mediated bacterial warfare. Moreover, it highlights the evolution of FGE domain-containing proteins to carry out diverse biological functions.

microbiology↗

bioRxiv2021

Engineering a customizable antibacterial T6SS-based platform in Vibrio natriegens

Bacterial pathogens are a major risk to human, animal, and plant health. To counteract the spread of antibiotic resistance, alternative antibacterial strategies are urgently needed. Here, we constructed a proof-of-concept customizable, modular, and inducible antibacterial toxin delivery platform. By engineering a type VI secretion system (T6SS) that is controlled by an externally induced on/off switch, we transformed the safe bacterium, Vibrio natriegens, into an effective antibacterial weapon. Furthermore, we demonstrated that the delivered effector repertoire, and thus the toxicity range of this platform, can be easily manipulated and tested. We believe that this platform can serve as a foundation for novel antibacterial bio-treatments, as well as a unique tool to study antibacterial toxins.

microbiology↗

bioRxiv2019

Vibrio pore-forming leukocidin activates pyroptotic cell death via the NLRP3 inflammasome

Cell death mechanisms are central to combat infectious microbes and to drive pathological inflammation. One such mechanism, the inflammasome, controls infection through either activation of caspase-1 and the subsequent secretion of the mature pro-inflammatory cytokine, interleukin 1{beta} (IL-1{beta}), or by stopping the dissemination of intracellular pathogens by inducing pyroptotic cell death in infected cells. Hemolysins, which are pore-forming toxins (PFTs), target the host cell plasma membrane by producing pores with different diameters. These pores alter the permeability of the target membrane, often leading to cell death. We previously discovered a functional and potent pore-forming, leukocidin domain-containing hemolysin produced by the Gram-negative marine bacterium Vibrio proteolyticus (V. proteolyticus), termed VPRH. Although leukocidin domains are found in other known PFTs, VPRH constitutes a distinct, understudied class within the leukocidin superfamily. Since PTFs of other pathogens were shown to induce cell death by activating the inflammasome pathway, we hypothesized that VPRH-induced cell death is mediated by direct activation of the inflammasome in mammalian immune host cells. Indeed, we found that VPRH induced a two-step cell death in primary macrophages. The first, a rapid step, was mediated by activating the NLRP3 inflammasome, leading to caspase-1 activation and GSDMD cleavage that resulted in IL-1{beta} secretion and pyroptotic cell death. The second step was independent of the inflammasome; however, its mechanism remains unknown. This study sets the foundation for better understanding the immunological consequences of inflammasome activation by a new leukocidin class of toxins.

immunology↗

bioRxiv2018

New players in the regulatory network of Vibrio parahaemolyticus type VI secretion system 1

Type VI secretion systems (T6SSs) are widespread, tightly regulated, protein delivery apparatuses used by Gram-negative bacteria to outcompete their neighbors. The pathogen, Vibrio parahaemolyticus, encodes two T6SSs. These T6SSs are differentially regulated by external conditions. T6SS1, an antibacterial system predominantly found in pathogenic isolates, requires warm marine-like conditions and surface sensing for activation. The regulatory network that governs this activation is not well understood. In this work, we devised a screening methodology that allows us to easily monitor the outcome of bacterial competitions and thus, to identify mutants that are defective in T6SS1-mediated bacterial killing. The methodology, termed Bacterial Competition Fluorescence (BaCoF), relies on detection of a fluorescent signal as an indicator of the survival and growth of a T6SS-sensitive, GFP-expressing prey that has been co-cultured with mutants derived from a T6SS+ attacker of interest. Using BaCoF, we screened a random transposon insertion mutant library and identified genes required for V. parahaemolyticus T6SS1 activation, among them TfoY and Tmk. We used epistasis experiments to determine the relationships between the newly identified components and other regulators that were previously described. Thus, we present here a detailed biological understanding of the T6SS1 regulatory network.

microbiology↗

bioRxiv2018

Association study of long-term kidney transplant rejection using whole-exome sequencing

Long-term renal allograft rejection is the most common outcome in kidney transplantation. Continuing the crusade to extend allograft function after the first year post-transplantation, we attempted to associate genetic factors that might contribute to long-term allograft outcomes by sequencing the exomes of patients diagnosed with chronic allograft nephropathy/interstitial fibrosis and tubular atrophy. A variety of association analyses were employed, but these analyses failed to identify statistically significant associations. The study was underpowered to detect the association of rare genomic variants with small effect sizes. However, it confirmed previous reports of the absence of large effects from common variants. We have made both the study data and analysis workflow available for public use, and we hope that these resources will help to power future meta-analyses that may detect smaller effects.

genetics↗

bioRxiv2017

T3SS effector VopL inhibits the host ROS response, promoting the intracellular survival of Vibrio parahaemolyticus

The production of antimicrobial reactive oxygen species by the nicotinamide dinucleotide phosphate (NADPH) oxidase complex is an important mechanism for control of invading pathogens. Herein, we show that the gastrointestinal pathogen Vibrio parahaemolyticus counteracts reactive oxygen species (ROS) production using the Type III Secretion System 2 (T3SS2) effector VopL. In the absence of VopL, intracellular V. parahaemolyticus undergo ROS-dependent filamentation, with concurrent limited growth. During infection, VopL assembles actin into non-functional filaments resulting in a dysfunctional actin cytoskeleton that can no longer mediate the assembly of the NADPH oxidase at the cell membrane, thereby limiting ROS production. This is the first example of how a T3SS2 effector contributes to the intracellular survival of V. parahaemolyticus to support the establishment of a protective intracellular replicative niche.

microbiology↗