The role of small proteins in Burkholderia cenocepacia J2315 biofilm formation, persistence and intracellular growth

Burkholderia cenocepacia infections are difficult to treat due to resistance, biofilm formation and persistence. B. cenocepacia strain J2315 has a large multi-replicon genome (8.06 Mb) and the function of a large fraction of (conserved) hypothetical genes remains elusive. The goal of the present study is to elucidate the role of small proteins in B. cenocepacia, focusing on genes smaller than 300 base pairs of which the function is unknown. Almost 10% (572) of the B. cenocepacia J2315 genes are smaller than 300 base pairs and more than half of these are annotated as coding for hypothetical proteins. For 234 of them no similarity could be found with non-hypothetical genes in other bacteria using BLAST. Using available RNA sequencing data obtained from biofilms, a list of 27 highly expressed B. cenocepacia J2315 genes coding for small proteins was compiled. For nine of them expression in biofilms was also confirmed using LC-MS based proteomics and/or expression was confirmed using eGFP translational fusions. Overexpression of two of these genes negatively impacted growth, whereas for four others overexpression led to an increase in biofilm biomass. Overexpression did not have an influence on the MIC for tobramycin, ciprofloxacin or meropenem but for five small protein encoding genes, overexpression had an effect on the number of persister cells in biofilms. While there were no significant differences in adherence to and invasion of A549 epithelial cells between the overexpression mutants and the WT, significant differences were observed in intracellular growth/survival. Finally, the small protein BCAM0271 was identified as an antitoxin belonging to a toxin-antitoxin module. The toxin was found to encode a tRNA acetylase that inhibits translation. In conclusion, our results confirm that small proteins are present in the genome of B. cenocepacia J2315 and indicate that they are involved in various biological processes, including biofilm formation, persistence and intracellular growth.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

The higBA toxin-antitoxin module from the opportunistic pathogen Acinetobacter baumannii - Regulation, activity, and evolution

Acinetobacter baumannii is one of the major causes of hard to treat multidrug-resistant hospital infections. A. baumannii features contributing to its spread and persistence in clinical environment are only beginning to be explored. Bacterial toxin-antitoxin (TA) systems are genetic loci shown to be involved in plasmid maintenance and proposed to function as components of stress response networks. Here we present a thorough characterization of type II system of A. baumannii, which is the most ubiquitous TA module present in A. baumannii plasmids. higBA of A. baumannii is a reverse TA (the toxin gene is the first in the operon) and shows little homology to other TA systems of RelE superfamily. It is represented by two variants, which both are functional albeit exhibit strong difference in sequence conservation. The higBA2 operon is found on ubiquitous 11 Kb pAB120 plasmid, conferring carbapenem resistance to clinical A. baumannii isolates and represents a higBA variant that can be found with multiple sequence variations. We show here that higBA2 is capable to confer maintenance of unstable plasmid in Acinetobacter species. HigB2 toxin functions as a ribonuclease and its activity is neutralized by HigA2 antitoxin through formation of an unusually large heterooligomeric complex. Based on the in vivo expression analysis of gfp reporter gene we propose that HigA2 antitoxin and HigBA2 protein complex bind the higBA2 promoter region to downregulate its transcription. We also demonstrate that higBA2 is a stress responsive locus, whose transcription changes in conditions encountered by A. baumannii in clinical environment and within the host. We show elevated expression of higBA2 during stationary phase, under iron deficiency and downregulated expression after antibiotic (rifampicin) treatment.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Activity, delivery and diversity of Type VI secretion effectors

International audienceThe bacterial type VI secretion system (T6SS) system is a contractile secretion apparatus that delivers proteins to neighbouring bacterial or eukaryotic cells. Antibacterial effectors are mostly toxins that inhibit growth of other species and help to dominate the niche. A broad variety of these toxins cause cell lysis of the prey cell by disrupting the cell envelope. Other effectors are delivered into the cytoplasm where they affect DNA integrity, cell division or exhaust energy resources. The modular nature of T6SS machinery allows different means of recruitment of toxic effectors to secreted inner tube and spike components that act as carriers. Toxic effectors can be translationally fused to the secreted components or interact with them through specialized structural domains. These interactions can also be assisted by dedicated chaperone proteins. Moreover, conserved sequence motifs in effector-associated domains are subject to genetic rearrangements and therefore engage in diversification of the arsenal of toxic effectors. This review discusses the diversity of T6SS secreted toxins and presents current knowledge about their loading on the T6SS machinery

T6SS: killing two bugs with one stone

International audienceBacteria deploy the type VI secretion system (T6SS) to inject effectors into bacterial rivals. Contrary to the prevailing model, a recent study expands the target range of the T6SS by demonstrating that it delivers and potentializes a peptidoglycan-targeting bifunctional toxin into Gram-positive bacteria

The Variety in the Common Theme of Translation Inhibition by Type II Toxin–Antitoxin Systems

International audienceType II Toxin-antitoxin (TA) modules are bacterial operons that encode a toxic protein and its antidote, which form a self-regulating genetic system. Antitoxins put a halter on toxins in many ways that distinguish different types of TA modules. In type II TA modules, toxin and antitoxin are proteins that form a complex which physically sequesters the toxin, thereby preventing its toxic activity. Type II toxins inhibit various cellular processes, however, the translation process appears to be their favorite target and nearly every step of this complex process is inhibited by type II toxins. The structural features, enzymatic activities and target specificities of the different toxin families are discussed. Finally, this review emphasizes that the structural folds presented by these toxins are not restricted to type II TA toxins or to one particular cellular target, and discusses why so many of them evolved to target translation as well as the recent developments regarding the role(s) of these systems in bacterial physiology and evolution

Salmonella antibacterial Rhs polymorphic toxin inhibits translation through ADP-ribosylation of EF-Tu P-loop

International audienceAbstract Rearrangement hot spot (Rhs) proteins are members of the broad family of polymorphic toxins. Polymorphic toxins are modular proteins composed of an N-terminal region that specifies their mode of secretion into the medium or into the target cell, a central delivery module, and a C-terminal domain that has toxic activity. Here, we structurally and functionally characterize the C-terminal toxic domain of the antibacterial Rhsmain protein, TreTu, which is delivered by the type VI secretion system of Salmonella enterica Typhimurium. We show that this domain adopts an ADP-ribosyltransferase fold and inhibits protein synthesis by transferring an ADP-ribose group from NAD+ to the elongation factor Tu (EF-Tu). This modification is specifically placed on the side chain of the conserved D21 residue located on the P-loop of the EF-Tu G-domain. Finally, we demonstrate that the TriTu immunity protein neutralizes TreTu activity by acting like a lid that closes the catalytic site and traps the NAD+

Salmonella antibacterial Rhs polymorphic toxin inhibits translation through ADP-ribosylation of EF-Tu P-loop

International audienceAbstract Rearrangement hot spot (Rhs) proteins are members of the broad family of polymorphic toxins. Polymorphic toxins are modular proteins composed of an N-terminal region that specifies their mode of secretion into the medium or into the target cell, a central delivery module, and a C-terminal domain that has toxic activity. Here, we structurally and functionally characterize the C-terminal toxic domain of the antibacterial Rhsmain protein, TreTu, which is delivered by the type VI secretion system of Salmonella enterica Typhimurium. We show that this domain adopts an ADP-ribosyltransferase fold and inhibits protein synthesis by transferring an ADP-ribose group from NAD+ to the elongation factor Tu (EF-Tu). This modification is specifically placed on the side chain of the conserved D21 residue located on the P-loop of the EF-Tu G-domain. Finally, we demonstrate that the TriTu immunity protein neutralizes TreTu activity by acting like a lid that closes the catalytic site and traps the NAD+

Mounting, structure and autocleavage of a type VI secretion-associated Rhs polymorphic toxin

International audienceBacteria have evolved toxins to outcompete other bacteria or to hijack host cell pathways. One broad family of bacterial polymorphic toxins gathers multidomain proteins with a modular organization, comprising a C-terminal toxin domain fused to a N-terminal domain that adapts 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 , Rhs1. Here, we show that Rhs1 forms a complex with the T6SS spike protein VgrG and the EagR chaperone. Using truncation derivatives and cross-linking mass spectrometry, we demonstrate that VgrG-EagR-Rhs1 complex formation requires the VgrG C-terminal β-helix and the Rhs1 N-terminal region. We then report the cryo-electron-microscopy structure of the Rhs1-EagR complex, demonstrating that the Rhs1 central region forms a β-barrel cage-like structure that encapsulates the C-terminal toxin domain, and provide evidence for processing of the Rhs1 protein through aspartyl autoproteolysis. We propose a model for Rhs1 loading on the T6SS, transport and delivery into the target cell

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<p>Acinetobacter baumannii is one of the major causes of hard to treat multidrug-resistant hospital infections. A. baumannii features contributing to its spread and persistence in clinical environment are only beginning to be explored. Bacterial toxin-antitoxin (TA) systems are genetic loci shown to be involved in plasmid maintenance and proposed to function as components of stress response networks. Here we present a thorough characterization of type II system of A. baumannii, which is the most ubiquitous TA module present in A. baumannii plasmids. higBA of A. baumannii is a reverse TA (the toxin gene is the first in the operon) and shows little homology to other TA systems of RelE superfamily. It is represented by two variants, which both are functional albeit exhibit strong difference in sequence conservation. The higBA2 operon is found on ubiquitous 11 Kb pAB120 plasmid, conferring carbapenem resistance to clinical A. baumannii isolates and represents a higBA variant that can be found with multiple sequence variations. We show here that higBA2 is capable to confer maintenance of unstable plasmid in Acinetobacter species. HigB2 toxin functions as a ribonuclease and its activity is neutralized by HigA2 antitoxin through formation of an unusually large heterooligomeric complex. Based on the in vivo expression analysis of gfp reporter gene we propose that HigA2 antitoxin and HigBA2 protein complex bind the higBA2 promoter region to downregulate its transcription. We also demonstrate that higBA2 is a stress responsive locus, whose transcription changes in conditions encountered by A. baumannii in clinical environment and within the host. We show elevated expression of higBA2 during stationary phase, under iron deficiency and downregulated expression after antibiotic (rifampicin) treatment.</p