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 VgrG-dependent effectors and chaperones modulate the assembly of the type VI secretion system

2021 ◽  
Vol 17 (12) ◽  
pp. e1010116
Author(s):  
Xiaoye Liang ◽  
Tong-Tong Pei ◽  
Hao Li ◽  
Hao-Yu Zheng ◽  
Han Luo ◽  
...  
Keyword(s):  
Secretion System ◽  
Effector Proteins ◽  
Host Cells ◽  
Structural Proteins ◽  
Wild Type ◽  
Type Vi Secretion System ◽  
Double Deletion ◽  
Type Vi Secretion ◽  
Structural Determinant

The type VI secretion system (T6SS) is a spear-like nanomachine found in gram-negative pathogens for delivery of toxic effectors to neighboring bacterial and host cells. Its assembly requires a tip spike complex consisting of a VgrG-trimer, a PAAR protein, and the interacting effectors. However, how the spike controls T6SS assembly remains elusive. Here we investigated the role of three VgrG-effector pairs in Aeromonas dhakensis strain SSU, a clinical isolate with a constitutively active T6SS. By swapping VgrG tail sequences, we demonstrate that the C-terminal ~30 amino-acid tail dictates effector specificity. Double deletion of vgrG1&2 genes (VgrG3+) abolished T6SS secretion, which can be rescued by ectopically expressing chimeric VgrG3 with a VgrG1/2-tail but not the wild type VgrG3. In addition, deletion of effector-specific chaperones also severely impaired T6SS secretion, despite the presence of intact VgrG and effector proteins, in both SSU and Vibrio cholerae V52. We further show that SSU could deliver a V. cholerae effector VasX when expressing a plasmid-borne chimeric VgrG with VasX-specific VgrG tail and chaperone sequences. Pull-down analyses show that two SSU effectors, TseP and TseC, could interact with their cognate VgrGs, the baseplate protein TssK, and the key assembly chaperone TssA. Effectors TseL and VasX could interact with TssF, TssK and TssA in V. cholerae. Collectively, we demonstrate that chimeric VgrG-effector pairs could bypass the requirement of heterologous VgrG complex and propose that effector-stuffing inside the baseplate complex, facilitated by chaperones and the interaction with structural proteins, serves as a crucial structural determinant for T6SS assembly.

scholarly journals The Type VI Secretion System Modulates Flagellar Gene Expression and Secretion in Citrobacter freundii and Contributes to Adhesion and Cytotoxicity to Host Cells

2015 ◽  
Vol 83 (7) ◽  
pp. 2596-2604 ◽  
Author(s):  
Liyun Liu ◽  
Shuai Hao ◽  
Ruiting Lan ◽  
Guangxia Wang ◽  
Di Xiao ◽  
...  
Keyword(s):  
Secretion System ◽  
Host Cells ◽  
Target Cells ◽  
Deletion Mutants ◽  
Citrobacter Freundii ◽  
Wild Type ◽  
Type Vi Secretion System ◽  
Content Type ◽  
Type Vi Secretion ◽  
Mutant Strains

The type VI secretion system (T6SS) as a virulence factor-releasing system contributes to virulence development of various pathogens and is often activated upon contact with target cells.Citrobacter freundiistrain CF74 has a complete T6SS genomic island (GI) that containsclpV,hcp-2, andvgrT6SS genes. We constructedclpV,hcp-2,vgr, and T6SS GI deletion mutants in CF74 and analyzed their effects on the transcriptome overall and, specifically, on the flagellar system at the levels of transcription and translation. Deletion of the T6SS GI affected the transcription of 84 genes, with 15 and 69 genes exhibiting higher and lower levels of transcription, respectively. Members of the cell motility class of downregulated genes of the CF74ΔT6SS mutant were mainly flagellar genes, including effector proteins, chaperones, and regulators. Moreover, the production and secretion of FliC were also decreased inclpV,hcp-2,vgr, or T6SS GI deletion mutants in CF74 and were restored upon complementation. In swimming motility assays, the mutant strains were found to be less motile than the wild type, and motility was restored by complementation. The mutant strains were defective in adhesion to HEp-2 cells and were restored partially upon complementation. Further, the CF74ΔT6SS, CF74ΔclpV, and CF74Δhcp-2mutants induced lower cytotoxicity to HEp-2 cells than the wild type. These results suggested that the T6SS GI in CF74 regulates the flagellar system, enhances motility, is involved in adherence to host cells, and induces cytotoxicity to host cells. Thus, the T6SS plays a wide-ranging role inC. freundii.

scholarly journals The Type VI Secretion System Encoded in Salmonella Pathogenicity Island 19 Is Required for Salmonella enterica Serotype Gallinarum Survival within Infected Macrophages

2013 ◽  
Vol 81 (4) ◽  
pp. 1207-1220 ◽  
Author(s):  
Carlos J. Blondel ◽  
Juan C. Jiménez ◽  
Lorenzo E. Leiva ◽  
Sergio A. Álvarez ◽  
Bernardo I. Pinto ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Pathogenicity Island ◽  
Secretion System ◽  
Host Cells ◽  
Economic Losses ◽  
Type Vi Secretion System ◽  
Content Type ◽  
Type Vi Secretion ◽  
Core Components

ABSTRACTSalmonella entericaserotype Gallinarum is the causative agent of fowl typhoid, a disease characterized by high morbidity and mortality that causes major economic losses in poultry production. We have reported thatS. Gallinarum harbors a type VI secretion system (T6SS) encoded inSalmonellapathogenicity island 19 (SPI-19) that is required for efficient colonization of chicks. In the present study, we aimed to characterize the SPI-19 T6SS functionality and to investigate the mechanisms behind the phenotypes previously observedin vivo. Expression analyses revealed that SPI-19 T6SS core components are expressed and produced underin vitrobacterial growth conditions. However, secretion of the structural/secreted components Hcp1, Hcp2, and VgrG to the culture medium could not be determined, suggesting that additional signals are required for T6SS-dependent secretion of these proteins.In vitrobacterial competition assays failed to demonstrate a role for SPI-19 T6SS in interbacterial killing. In contrast, cell culture experiments with murine and avian macrophages (RAW264.7 and HD11, respectively) revealed production of a green fluorescent protein-tagged version of VgrG soon afterSalmonellauptake. Furthermore, infection of RAW264.7 and HD11 macrophages with deletion mutants of SPI-19 or strains with genes encoding specific T6SS core components (clpVandvgrG) revealed that SPI-19 T6SS contributes toS. Gallinarum survival within macrophages at 20 h postuptake. SPI-19 T6SS function was not linked toSalmonella-induced cytotoxicity or cell death of infected macrophages, as has been described for other T6SS. Our data indicate that SPI-19 T6SS corresponds to a novel tool used bySalmonellato survive within host cells.

scholarly journals Light Modulates Metabolic Pathways and Other Novel Physiological Traits in the Human Pathogen Acinetobacter baumannii

2017 ◽  
Vol 199 (10) ◽  
Author(s):  
Gabriela L. Müller ◽  
Marisel Tuttobene ◽  
Matías Altilio ◽  
Maitena Martínez Amezaga ◽  
Meaghan Nguyen ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Metabolic Pathways ◽  
Secretion System ◽  
Human Pathogen ◽  
Type Vi Secretion System ◽  
Bacterial Persistence ◽  
Content Type ◽  
Light Sensing ◽  
Type Vi Secretion ◽  
Novel Concept

ABSTRACT Light sensing in chemotrophic bacteria has been relatively recently ascertained. In the human pathogen Acinetobacter baumannii, light modulates motility, biofilm formation, and virulence through the blue-light-sensing-using flavin (BLUF) photoreceptor BlsA. In addition, light can induce a reduction in susceptibility to certain antibiotics, such as minocycline and tigecycline, in a photoreceptor-independent manner. In this work, we identified new traits whose expression levels are modulated by light in this pathogen, which comprise not only important determinants related to pathogenicity and antibiotic resistance but also metabolic pathways, which represents a novel concept for chemotrophic bacteria. Indeed, the phenylacetic acid catabolic pathway and trehalose biosynthesis were modulated by light, responses that completely depend on BlsA. We further show that tolerance to some antibiotics and modulation of antioxidant enzyme levels are also influenced by light, likely contributing to bacterial persistence in adverse environments. Also, we present evidence indicating that surfactant production is modulated by light. Finally, the expression of whole pathways and gene clusters, such as genes involved in lipid metabolism and genes encoding components of the type VI secretion system, as well as efflux pumps related to antibiotic resistance, was differentially induced by light. Overall, our results indicate that light modulates global features of the A. baumannii lifestyle. IMPORTANCE The discovery that nonphototrophic bacteria respond to light constituted a novel concept in microbiology. In this context, we demonstrated that light could modulate aspects related to bacterial virulence, persistence, and resistance to antibiotics in the human pathogen Acinetobacter baumannii. In this work, we present the novel finding that light directly regulates metabolism in this chemotrophic bacterium. Insights into the mechanism show the involvement of the photoreceptor BlsA. In addition, tolerance to antibiotics and catalase levels are also influenced by light, likely contributing to bacterial persistence in adverse environments, as is the expression of the type VI secretion system and efflux pumps. Overall, a profound influence of light on the lifestyle of A. baumannii is suggested to occur.

scholarly journals Structural Characterization of TssL from Acinetobacter baumannii: a Key Component of the Type VI Secretion System

2020 ◽  
Vol 202 (17) ◽  
Author(s):  
Federico M. Ruiz ◽  
Juvenal Lopez ◽  
C. Gastón Ferrara ◽  
Elena Santillana ◽  
Yanis R. Espinosa ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Secretion System ◽  
Single Locus ◽  
Gram Negative Bacteria ◽  
Cytoplasmic Protein ◽  
Type Vi Secretion System ◽  
Inner Membrane ◽  
Gram Negative ◽  
Content Type ◽  
Type Vi Secretion

ABSTRACT The type VI secretion system (T6SS) is a complex molecular nanomachine used by Gram-negative bacteria to deliver diverse effectors into adjacent cells. A membrane complex (MC) anchors this transport system to the bacterial cell wall. One of the proteins forming the MC is TssL, a cytoplasmic protein bound to the inner membrane through a single transmembrane helix. Here, we report the structure of the cytoplasmic N-terminal region of TssL from Acinetobacter baumannii, a bacterium encoding in a single locus a secretion system that is a special case among other T6SSs. The protein structure, consisting of two antiparallel alpha-helical bundles connected by a short loop, reveals several interesting particularities compared with homologous proteins from other organisms. In addition, we demonstrate the structural significance of residues Asp98 and Glu99, which are strongly conserved among T6SS-encoding Gram-negative bacteria. Mutations in these two residues strongly impact protein dynamics, expression, and functionality. Our results improve our understanding of the T6SS of A. baumannii, which remains largely understudied compared with that of other pathogens. IMPORTANCE Several Acinetobacter species carry one functional type VI secretion system (T6SS). The T6SS is encoded in a single locus containing 16 conserved genes, most of which code for proteins essential to T6SS activity. One of these key components is TssL, a cytoplasmic protein bound to the inner membrane. Despite its importance and its particular characteristics, the structure of T6SS in A. baumannii remains understudied. Here, we present structural, in silico, and in vivo studies of TssL, highlighting the importance of two well-conserved residues and improving our understanding of this secretion system in this bacterium.

scholarly journals Type VI Secretion System-Associated Gene Clusters Contribute to Pathogenesis of Salmonella enterica Serovar Typhimurium

2012 ◽  
Vol 80 (6) ◽  
pp. 1996-2007 ◽  
Author(s):  
David T. Mulder ◽  
Colin A. Cooper ◽  
Brian K. Coombes
Keyword(s):  
Salmonella Enterica ◽  
Gene Clusters ◽  
Secretion System ◽  
Host Cells ◽  
Type Vi Secretion System ◽  
Macrophage Infection ◽  
Content Type ◽  
Type Vi Secretion ◽  
Systemic Dissemination ◽  
Serovar Typhimurium

ABSTRACTThe enteropathogenSalmonella entericaserovar Typhimurium employs a suite of tightly regulated virulence factors within the intracellular compartment of phagocytic host cells resulting in systemic dissemination in mice. A type VI secretion system (T6SS) withinSalmonellapathogenicity island 6 (SPI-6) has been implicated in this process; however, the regulatory inputs and the roles of noncore genes in this system are not well understood. Here we describe four clusters of noncore T6SS genes in SPI-6 based on a comparative relationship with the T6SS-3 ofBurkholderia malleiand report that the disruption of these genes results in defects in intracellular replication and systemic dissemination in mice. In addition, we show that the expression of the SPI-6-encoded Hcp and VgrG orthologs is enhanced during late stages of macrophage infection. We identify six regions that are transcriptionally active during cell infections and that have regulatory contributions from the regulators of virulence SsrB, PhoP, and SlyA. We show that levels of protein expression are very weak underin vitroconditions and that expression is not enhanced upon the deletion ofssrB,phoP,slyA,qseC,ompR, orhfq, suggesting an unknown activating factor. These data suggest that the SPI-6 T6SS has been integrated into theSalmonellaTyphimurium virulence network and customized for host-pathogen interactions through the action of noncore genes.

scholarly journals Intraspecies Competition in Serratia marcescens Is Mediated by Type VI-Secreted Rhs Effectors and a Conserved Effector-Associated Accessory Protein

2015 ◽  
Vol 197 (14) ◽  
pp. 2350-2360 ◽  
Author(s):  
Juliana Alcoforado Diniz ◽  
Sarah J. Coulthurst
Keyword(s):  
Serratia Marcescens ◽  
Secretion System ◽  
Effector Proteins ◽  
Target Cells ◽  
Primary Role ◽  
Bacterial Cells ◽  
Type Vi Secretion System ◽  
Content Type ◽  
Type Vi Secretion ◽  
Intraspecies Competition

ABSTRACTThe type VI secretion system (T6SS) is widespread in Gram-negative bacteria and can deliver toxic effector proteins into eukaryotic cells or competitor bacteria. Antibacterial T6SSs are increasingly recognized as key mediators of interbacterial competition and may contribute to the outcome of many polymicrobial infections. Multiple antibacterial effectors can be delivered by these systems, with diverse activities against target cells and distinct modes of secretion. Polymorphic toxins containing Rhs repeat domains represent a recently identified and as-yet poorly characterized class of T6SS-dependent effectors. Previous work had revealed that the potent antibacterial T6SS of the opportunistic pathogenSerratia marcescenspromotes intraspecies as well as interspecies competition (S. L. Murdoch, K. Trunk, G. English, M. J. Fritsch, E. Pourkarimi, and S. J. Coulthurst, J Bacteriol 193:6057–6069, 2011,http://dx.doi.org/10.1128/JB.05671-11). In this study, two new Rhs family antibacterial effectors delivered by this T6SS have been identified. One of these was shown to act as a DNase toxin, while the other contains a novel, cytoplasmic-acting toxin domain. Importantly, usingS. marcescens, it has been demonstrated for the first time that Rhs proteins, rather than other T6SS-secreted effectors, can be the primary determinant of intraspecies competition. Furthermore, a new family of accessory proteins associated with T6SS effectors has been identified, exemplified byS. marcescensEagR1, which is specifically required for deployment of its associated Rhs effector. Together, these findings provide new insight into how bacteria can use the T6SS to deploy Rhs-family effectors and mediate different types of interbacterial interactions.IMPORTANCEInfectious diseases caused by bacterial pathogens represent a continuing threat to health and economic prosperity. To counter this threat, we must understand how such organisms survive and prosper. The type VI secretion system is a weapon that many pathogens deploy to compete against rival bacterial cells by injecting multiple antibacterial toxins into them. The ability to compete is vital considering that bacteria generally live in mixed communities. We aimed to identify new toxins and understand their deployment and role in interbacterial competition. We describe two new type VI secretion system-delivered toxins of the Rhs class, demonstrate that this class can play a primary role in competition between closely related bacteria, and identify a new accessory factor needed for their delivery.

scholarly journals The P. aeruginosa Type VI Secretion System Effector Tse5 Forms Ion-Selective Membrane Pores that Disrupt the Membrane Potential of Intoxicated Cells

2021 ◽  
Author(s):  
Amaia González-Magaña ◽  
Jon Altuna ◽  
María Queralt-Martín ◽  
Eneko Largo ◽  
Itxaso Montánchez ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Secretion System ◽  
Effector Proteins ◽  
Host Cells ◽  
Type Vi Secretion System ◽  
Cellular Functions ◽  
Fitness Advantage ◽  
Type Vi Secretion ◽  
Wide Range ◽  
Membrane Depolarisation

Abstract The Type VI Secretion System (T6SS) of Pseudomonas aeruginosa injects effector proteins into neighbouring competitors and host cells, providing a fitness advantage that allows this opportunistic nosocomial pathogen to persist and prevail during the onset of infections. However, despite the high clinical relevance of P. aeruginosa, the identity and mode of action of most P. aeruginosa T6SS-dependent effectors remain to be discovered. Here, we report the molecular mechanism of Tse5-CT, which is the toxic auto-proteolytic product of the P. aeruginosa T6SS exported effector Tse5. Our results demonstrate Tse5-CT is a pore-forming toxin that can transport ions across the membrane, causing membrane depolarisation and bacterial death. The membrane potential regulates a wide range of essential cellular functions, and therefore membrane depolarisation is an efficient strategy to compete with other microorganisms in polymicrobial environments.

scholarly journals VgrG-5 Is a Burkholderia Type VI Secretion System-Exported Protein Required for Multinucleated Giant Cell Formation and Virulence

2014 ◽  
Vol 82 (4) ◽  
pp. 1445-1452 ◽  
Author(s):  
Sandra Schwarz ◽  
Pragya Singh ◽  
Johanna D. Robertson ◽  
Michele LeRoux ◽  
Shawn J. Skerrett ◽  
...  
Keyword(s):  
Bacterial Cell ◽  
Cell Formation ◽  
Secretion System ◽  
Host Cells ◽  
Type Vi Secretion System ◽  
Content Type ◽  
Type Vi Secretion ◽  
Giant Cell Formation ◽  
Infection Models ◽  
Mammalian Infection

ABSTRACTThe type VI secretion system (T6SS) has emerged as a critical virulence factor for the group of closely relatedBurkholderiaspp. that includesBurkholderia pseudomallei,B. mallei, andB. thailandensis. While the genomes of these bacteria, referred to as the Bptm group, appear to encode several T6SSs, we and others have shown that one of these, type VI secretion system 5 (T6SS-5), is required for virulence in mammalian infection models. Despite its pivotal role in the pathogenesis of the Bptm group, the effector repertoire of T6SS-5 has remained elusive. Here we used quantitative mass spectrometry to compare the secretome of wild-typeB. thailandensisto that of a mutant harboring a nonfunctional T6SS-5. This analysis identified VgrG-5 as a novel secreted protein whose export depends on T6SS-5 function. Bioinformatics analysis revealed that VgrG-5 is a specialized VgrG protein that harbors a C-terminal domain (CTD) conserved among Bptm group species. We found that avgrG-5ΔCTD mutant is avirulent in mice and is unable to stimulate the fusion of host cells, a hallmark of the Bptm group previously shown to require T6SS-5 function. The singularity of VgrG-5 as a detected T6SS-5 substrate, taken together with the essentiality of its CTD for virulence, suggests that the protein is critical for the effector activity of T6SS-5. Intriguingly, we show that unlike the bacterial-cell-targeting T6SSs characterized so far, T6SS-5 localizes to the bacterial cell pole. We propose a model whereby the CTD of VgrG-5—, propelled by T6SS-5—, plays a key role in inducing membrane fusion, either by the recruitment of other factors or by direct participation.

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