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Identification of Novel Acinetobacter baumannii Type VI Secretion System Antibacterial Effector and Immunity Pairs.

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.

SUBMITTER: Fitzsimons TC

PROVIDER: S-EPMC6056853 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Identification of Novel Acinetobacter baumannii Type VI Secretion System Antibacterial Effector and Immunity Pairs.

Fitzsimons Timothy C TC Lewis Jessica M JM Wright Amy A Kleifeld Oded O Schittenhelm Ralf B RB Powell David D Harper Marina M Boyce John D JD

Infection and immunity 20180723 8

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 vg ...[more]

PMID: 29735524

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Project description:Many Gram-negative bacteria use a type VI secretion system (T6SS) for microbial warfare and/or host manipulation. Acinetobacter baumannii is an important nosocomial pathogen and many A. baumannii strains utilize a T6SS to deliver toxic effector proteins to surrounding bacterial cells. These toxic effectors are usually delivered together with VgrG proteins, which form part of the T6SS tip complex. All previously identified A. baumannii T6SS effectors are encoded within a three- or four-gene locus that also encodes a cognate VgrG and immunity protein, and sometimes a chaperone. In order to characterize the diversity and distribution of T6SS effectors and immunity proteins in this species, we first identified all vgrG genes in 97 A. baumannii strains via the presence of the highly conserved VgrG domain. Most strains encoded between two and four different VgrG proteins. We then analyzed the regions downstream of the identified vgrG genes and identified more than 240 putative effectors. The presence of conserved domains in these effectors suggested a range of functions, including peptidoglycan hydrolases, lipases, nucleases, and nucleic acid deaminases. However, 10 of the effector groups had no functionally characterized domains. Phylogenetic analysis of these putative effectors revealed that they clustered into 32 distinct groups that appear to have been acquired from a diverse set of ancestors. Corresponding immunity proteins were identified for all but two of the effector groups. Effectors from eight of the 32 groups contained N-terminal rearrangement hotspot (RHS) domains. The C-terminal regions of these RHS proteins, which are predicted to confer the toxic effector function, were very diverse, but the N-terminal RHS domains clustered into just two groups. While the majority of A. baumannii strains contained an RHS type effector, no strains encoded two RHS effectors with similar N-terminal sequences, suggesting that the presence of similar N-terminal RHS domains leads to competitive exclusion. Together, these analyses define the extreme diversity of T6SS effectors within A. baumannii and, as many have unknown functions, future detailed characterization of these effectors may lead to the identification of proteins with novel antibacterial properties.

Project description:UNLABELLED:The type VI secretion system (T6SS) is a dynamic macromolecular organelle that many Gram-negative bacteria use to inhibit or kill other prokaryotic or eukaryotic cells. The toxic effectors of T6SS are delivered to the prey cells in a contact-dependent manner. In Vibrio cholerae, the etiologic agent of cholera, T6SS is active during intestinal infection. Here, we describe the use of comparative proteomics coupled with bioinformatics to identify a new T6SS effector-immunity pair. This analysis was able to identify all previously identified secreted substrates of T6SS except PAAR (proline, alanine, alanine, arginine) motif-containing proteins. Additionally, this approach led to the identification of a new secreted protein encoded by VCA0285 (TseH) that carries a predicted hydrolase domain. We confirmed that TseH is toxic when expressed in the periplasm of Escherichia coli and V. cholerae cells. The toxicity observed in V. cholerae was suppressed by coexpression of the protein encoded by VCA0286 (TsiH), indicating that this protein is the cognate immunity protein of TseH. Furthermore, exogenous addition of purified recombinant TseH to permeabilized E. coli cells caused cell lysis. Bioinformatics analysis of the TseH protein sequence suggest that it is a member of a new family of cell wall-degrading enzymes that include proteins belonging to the YD repeat and Rhs superfamilies and that orthologs of TseH are likely expressed by species belonging to phyla as diverse as Bacteroidetes and Proteobacteria. IMPORTANCE:The Gram-negative bacterium Vibrio cholerae causes cholera, a severe and often lethal diarrheal disease. The 2010-2012 epidemic in Haiti and new explosive epidemics in Africa show that cholera remains a significant global public health problem. The type VI secretion system (T6SS) is a dynamic organelle expressed by many Gram-negative bacteria, which use it to inject toxic effector proteins into eukaryotic and bacterial prey cells. In this study, we applied a comparative proteomics approach to the V. cholerae T6SS secretome to identify new substrates of this secretion apparatus. We show that the product of the gene VCA0285 is likely a new peptidoglycan hydrolase that is secreted by T6SS and that its cognate immunity protein is encoded by the gene that is immediately downstream (VCA0286). Bioinformatics analysis shows that VCA0285 carries four conserved motifs that likely define a large family of hydrolases with antibacterial activity. The identification of new antibacterial T6SS effectors provides useful information for the development of novel antibiotics and therapeutic agents.

Project description:The type VI secretion system (T6SS) is a critical weapon in bacterial warfare between Gram-negative bacteria. Although invaluable for niche establishment, this machine represents an energetic burden to its host bacterium. Acinetobacter baumannii is an opportunistic pathogen that poses a serious threat to public health due to its high rates of multidrug resistance. In some A. baumannii strains, the T6SS is transcriptionally downregulated by large multidrug resistance plasmids. Other strains, such as the clinical isolate AbCAN2, express T6SS-related genes but lack T6SS activity under laboratory conditions, despite not harboring these plasmids. This suggests that alternative mechanisms exist to repress the T6SS. Here, we used a transposon mutagenesis approach in AbCAN2 to identify novel T6SS repressors. Our screen revealed that the T6SS of this strain is inhibited by a homolog of VgrG, an essential structural component of all T6SSs reported to date. We named this protein inhibitory VgrG (VgrGi). Biochemical and in silico analyses demonstrated that the unprecedented inhibitory capability of VgrGi is due to a single amino acid mutation in a widely conserved C-terminal domain of unknown function, DUF2345. We also show that unlike in other bacteria, the C terminus of VgrG is essential for functional T6SS assembly in A. baumannii Our study provides insight into the architectural requirements underlying functional assembly of the T6SS of A. baumannii We propose that T6SS-inactivating point mutations are beneficial to the host bacterium, since they eliminate the energy cost associated with maintaining a functional T6SS, which appears to be unnecessary for A. baumannii virulence.IMPORTANCE Despite the clinical relevance of A. baumannii, little is known about its fundamental biology. Here, we show that a single amino acid mutation in VgrG, a critical T6SS structural protein, abrogates T6SS function. Given that this mutation was found in a clinical isolate, we propose that the T6SS of A. baumannii is probably not involved in virulence; this idea is supported by multiple genomic analyses showing that the majority of clinical A. baumannii strains lack proteins essential to the T6SS. We also show that, unlike in other species, the C terminus of VgrG is a unique architectural requirement for functional T6SS assembly in A. baumannii, suggesting that over evolutionary time, bacteria have developed changes to their T6SS architecture, leading to specialized systems.

Dataset Information

Identification of Novel Acinetobacter baumannii Type VI Secretion System Antibacterial Effector and Immunity Pairs.

Publications

Identification of Novel Acinetobacter baumannii Type VI Secretion System Antibacterial Effector and Immunity Pairs.

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