RsmA and AmrZ orchestrate the assembly of all three type VI secretion systems in Pseudomonas aeruginosa

The type VI secretion system (T6SS) is a weapon of bacterial warfare and host cell subversion. The Gram-negative pathogen Pseudomonas aeruginosa has three T6SSs involved in colonization, competition, and full virulence. H1-T6SS is a molecular gun firing seven toxins, Tse1–Tse7, challenging survival of other bacteria and helping P. aeruginosa to prevail in specific niches. The H1-T6SS characterization was facilitated through studying a P. aeruginosa strain lacking the RetS sensor, which has a fully active H1-T6SS, in contrast to the parent. However, study of H2-T6SS and H3-T6SS has been neglected because of a poor understanding of the associated regulatory network. Here we performed a screen to identify H2-T6SS and H3-T6SS regulatory elements and found that the posttranscriptional regulator RsmA imposes a concerted repression on all three T6SS clusters. A higher level of complexity could be observed as we identified a transcriptional regulator, AmrZ, which acts as a negative regulator of H2-T6SS. Overall, although the level of T6SS transcripts is fine-tuned by AmrZ, all T6SS mRNAs are silenced by RsmA. We expanded this concept of global control by RsmA to VgrG spike and T6SS toxin transcripts whose genes are scattered on the chromosome. These observations triggered the characterization of a suite of H2-T6SS toxins and their implication in direct bacterial competition. Our study thus unveils a central mechanism that modulates the deployment of all T6SS weapons that may be simultaneously produced within a single cell.

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AT6SS trans-kingdom effector is required for the delivery of a novel antibacterial toxin in Pseudomonas aeruginosa

10.1101/551929 ◽

2019 ◽

Author(s):

Benjamin Berni ◽

Chantal Soscia ◽

Djermoun Sarah ◽

Ize Bérengère ◽

Sophie Bleves

Keyword(s):

Pseudomonas Aeruginosa ◽

Surrounding Medium ◽

Host Cells ◽

List Type ◽

Type Vi Secretion System ◽

Multiple Strategies ◽

Type Vi Secretion ◽

Host Infection ◽

Opportunist Pathogen

AbstractPseudomonas aeruginosa has evolved multiple strategies to disarm and take advantage of its host. For this purpose this opportunist pathogen has particularly developed protein secretion in the surrounding medium or injection into host cells. Among this, the Type VI Secretion System (T6SS) is utilized to deliver effectors into eukaryotic host as well as target bacteria. It assembles into a contractile bacteriophage tail-like structure that functions like a crossbow, injecting an arrow loaded with effectors into the target cell. The repertoire of T6SS antibacterial effectors of P. aeruginosa is remarkably broad to promote environmental adaptation and survival in various bacterial communities, and presumably in the eukaryotic host too.Here we report the discovery a novel pair of antibacterial effector and immunity of P. aeruginosa, Tle3 and Tli3. Tli3 neutralizes the toxicity of Tle3 in the periplasm to protect from fratricide intoxication. The characterization of the secretion mechanism of Tle3 indicates that it requires a cytoplasmic adaptor, Tla3, to be targeted and loaded onto the VgrG2b spike and thus delivered by the H2-T6SS machinery. Tla3 is different from the other adaptors discovered so far, and defines a novel family among T6SS.Interestingly this led us to discover that VgrG2b that we previously characterized as an anti-eukaryotic effector possesses an antibacterial activity as well, as it is toxic towards Escherichia coli. VgrG2b is thus a novel trans-kingdom effector targeting both bacteria and eukaryotes. VgrG2b represents an interesting target for fighting against P. aeruginosa in the environment and in the context of host infection.HighlightsTle3 and Tli3 are a novel pair of antibacterial toxin and immunity of P. aeruginosaTla3 recruits Tle3 in the cytoplasm, and targets it to VgrG2bVgrG2b is required for Tle3 delivery into target bacteria by the H2-T6SSTla3 defines a novel type of T6SS adaptor with a DUF2875VgrG2b is a new trans-kingdom effector targeting both bacteria and eukaryotes

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Host Adaptation Predisposes Pseudomonas aeruginosa to Type VI Secretion System-Mediated Predation by the Burkholderia cepacia Complex

10.1101/2020.04.10.036012 ◽

2020 ◽

Author(s):

Andrew I Perault ◽

Courtney E Chandler ◽

David A Rasko ◽

Robert K Ernst ◽

Matthew C Wolfgang ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Burkholderia Cepacia ◽

Secretion System ◽

Burkholderia Cenocepacia ◽

Burkholderia Cepacia Complex ◽

Dependent Manner ◽

Type Vi Secretion System ◽

Secretion Systems ◽

Type Vi Secretion

SUMMARYPseudomonas aeruginosa (Pa) and Burkholderia cepacia complex (Bcc) species are opportunistic lung pathogens of individuals with cystic fibrosis (CF). While Pa can initiate long-term infections in younger CF patients, Bcc infections only arise in teenagers and adults. Both Pa and Bcc use type VI secretion systems (T6SS) to mediate interbacterial competition. Here, we show that Pa isolates from teenage/adult CF patients, but not those from young CF patients, are outcompeted by the epidemic Bcc isolate Burkholderia cenocepacia strain AU1054 (BcAU1054) in a T6SS-dependent manner. The genomes of susceptible Pa isolates harbor T6SS-abrogating mutations, the repair of which, in some cases, rendered the isolates resistant. Moreover, seven of eight Bcc strains outcompeted Pa strains isolated from the same patients. Our findings suggest that certain mutations that arise as Pa adapts to the CF lung abrogate T6SS activity, making Pa and its human host susceptible to potentially fatal Bcc superinfection.

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Type VI Secretion Systems of Erwinia amylovora Contribute to Bacterial Competition, Virulence, and Exopolysaccharide Production

Phytopathology ◽

10.1094/phyto-11-16-0393-r ◽

2017 ◽

Vol 107 (6) ◽

pp. 654-661 ◽

Cited By ~ 8

Author(s):

Yanli Tian ◽

Yuqiang Zhao ◽

Linye Shi ◽

Zhongli Cui ◽

Baishi Hu ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Erwinia Amylovora ◽

Antibacterial Activities ◽

Type Vi Secretion System ◽

Secretion Systems ◽

Pear Fruit ◽

Exopolysaccharide Production ◽

Bacterial Competition ◽

Type Vi Secretion

The type VI secretion system (T6SS) plays a major role in mediating interbacterial competition and might contribute to virulence in plant pathogenic bacteria. However, the role of T6SS in Erwinia amylovora remains unknown. In this study, 33 deletion mutants within three T6SS clusters were generated in E. amylovora strain NCPPB1665. Our results showed that all 33 mutants displayed reduced antibacterial activities against Escherichia coli as compared with that of the wild-type (WT) strain, indicating that Erwinia amylovora T6SS are functional. Of the 33 mutants, 19 exhibited reduced virulence on immature pear fruit as compared with that of the WT strain. Among them, 6, 1, and 12 genes belonged to T6SS-1, T6SS-2, and T6SS-3 clusters, respectively. Interestingly, these 19 mutants also produced less amylovoran or levan or both. These findings suggest that E. amylovora T6SS play a role in bacterial competition and virulence possibly by influencing exopolysaccharide production.

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Pantoea ananatis Utilizes a Type VI Secretion System for Pathogenesis and Bacterial Competition

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-07-14-0219-r ◽

2015 ◽

Vol 28 (4) ◽

pp. 420-431 ◽

Cited By ~ 28

Author(s):

Divine Y. Shyntum ◽

Jacques Theron ◽

Stephanus N. Venter ◽

Lucy N. Moleleki ◽

Ian K. Toth ◽

...

Keyword(s):

Wild Type Strain ◽

Gene Clusters ◽

Wild Type ◽

Type Vi Secretion System ◽

Secretion Systems ◽

Pantoea Ananatis ◽

Bacterial Competition ◽

Type Vi Secretion ◽

Pineapple Fruit

Type VI secretion systems (T6SSs) are a class of macromolecular machines that are recognized as an important virulence mechanism in several gram-negative bacteria. The genome of Pantoea ananatis LMG 2665T, a pathogen of pineapple fruit and onion plants, carries two gene clusters whose predicted products have homology with T6SS-associated gene products from other bacteria. Nothing is known regarding the role of these T6SS-1 and T6SS-3 gene clusters in the biology of P. ananatis. Here, we present evidence that T6SS-1 plays an important role in the pathogenicity of P. ananatis LMG 2665T in onion plants, while a strain lacking T6SS-3 remains as pathogenic as the wild-type strain. We also investigated the role of the T6SS-1 system in bacterial competition, the results of which indicated that several bacteria compete less efficiently against wild-type LMG 2665T than a strain lacking T6SS-1. Additionally, we demonstrated that these phenotypes of strain LMG 2665T were reliant on the core T6SS products TssA and TssD (Hcp), thus indicating that the T6SS-1 gene cluster encodes a functioning T6SS. Collectively, our data provide the first evidence demonstrating that the T6SS-1 system is a virulence determinant of P. ananatis LMG 2665T and plays a role in bacterial competition.

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Characterization of the Pseudomonas aeruginosa T6SS PldB immunity proteins PA5086, PA5087 and PA5088 explains a novel stockpiling mechanism

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x2000566x ◽

2020 ◽

Vol 76 (5) ◽

pp. 222-227

Author(s):

Haiying Wen ◽

Zhi Geng ◽

Zengqiang Gao ◽

Zhun She ◽

Yuhui Dong

Keyword(s):

Pseudomonas Aeruginosa ◽

Secretion System ◽

Type Vi Secretion System ◽

Structural Comparison ◽

Interesting Phenomenon ◽

Potential Surfaces ◽

Type Vi Secretion ◽

Host Infection ◽

Bacterial Type

The bacterial type VI secretion system (T6SS) secretes many toxic effectors to gain advantage in interbacterial competition and for eukaryotic host infection. The cognate immunity proteins of these effectors protect bacteria from their own effectors. PldB is a T6SS trans-kingdom effector in Pseudomonas aeruginosa that can infect both prokaryotic and eukaryotic cells. Three proteins, PA5086, PA5087 and PA5088, are employed to suppress the toxicity of PldB-family proteins. The structures of PA5087 and PA5088 have previously been reported, but the identification of further distinctions between these immunity proteins is needed. Here, the crystal structure of PA5086 is reported at 1.90 Å resolution. A structural comparison of the three PldB immunity proteins showed vast divergences in their electrostatic potential surfaces. This interesting phenomenon provides an explanation of the stockpiling mechanism of T6SS immunity proteins.

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The Pseudomonas aeruginosa T6SS-VgrG1b spike is topped by a PAAR protein eliciting DNA damage to bacterial competitors

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1814181115 ◽

2018 ◽

Vol 115 (49) ◽

pp. 12519-12524 ◽

Cited By ~ 31

Author(s):

Panayiota Pissaridou ◽

Luke P. Allsopp ◽

Sarah Wettstadt ◽

Sophie A. Howard ◽

Despoina A. I. Mavridou ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Gene Clusters ◽

Secretion System ◽

Supramolecular Complex ◽

Type Vi Secretion System ◽

Protein Protein Interaction ◽

Bacterial Competition ◽

Type Vi Secretion ◽

C Terminus ◽

Interstrain Competition

The type VI secretion system (T6SS) is a supramolecular complex involved in the delivery of potent toxins during bacterial competition. Pseudomonas aeruginosa possesses three T6SS gene clusters and several hcp and vgrG gene islands, the latter encoding the spike at the T6SS tip. The vgrG1b cluster encompasses seven genes whose organization and sequences are highly conserved in P. aeruginosa genomes, except for two genes that we called tse7 and tsi7. We show that Tse7 is a Tox-GHH2 domain nuclease which is distinct from other T6SS nucleases identified thus far. Expression of this toxin induces the SOS response, causes growth arrest and ultimately results in DNA degradation. The cytotoxic domain of Tse7 lies at its C terminus, while the N terminus is a predicted PAAR domain. We find that Tse7 sits on the tip of the VgrG1b spike and that specific residues at the PAAR–VgrG1b interface are essential for VgrG1b-dependent delivery of Tse7 into bacterial prey. We also show that the delivery of Tse7 is dependent on the H1-T6SS cluster, and injection of the nuclease into bacterial competitors is deployed for interbacterial competition. Tsi7, the cognate immunity protein, protects the producer from the deleterious effect of Tse7 through a direct protein–protein interaction so specific that toxin/immunity pairs are effective only if they originate from the same P. aeruginosa isolate. Overall, our study highlights the diversity of T6SS effectors, the exquisite fitting of toxins on the tip of the T6SS, and the specificity in Tsi7-dependent protection, suggesting a role in interstrain competition.

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Structural basis for effector transmembrane domain recognition by type VI secretion system chaperones

eLife ◽

10.7554/elife.62816 ◽

2020 ◽

Vol 9 ◽

Author(s):

Shehryar Ahmad ◽

Kara K Tsang ◽

Kartik Sachar ◽

Dennis Quentin ◽

Tahmid M Tashin ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Transmembrane Domain ◽

Secretion System ◽

Effector Proteins ◽

Structural Basis ◽

Type Vi Secretion System ◽

Secretion Systems ◽

Conserved Motif ◽

Type Vi Secretion ◽

Helical Packing

Type VI secretion systems (T6SSs) deliver antibacterial effector proteins between neighboring bacteria. Many effectors harbor N-terminal transmembrane domains (TMDs) implicated in effector translocation across target cell membranes. However, the distribution of these TMD-containing effectors remains unknown. Here, we discover prePAAR, a conserved motif found in over 6000 putative TMD-containing effectors encoded predominantly by 15 genera of Proteobacteria. Based on differing numbers of TMDs, effectors group into two distinct classes that both require a member of the Eag family of T6SS chaperones for export. Co-crystal structures of class I and class II effector TMD-chaperone complexes from Salmonella Typhimurium and Pseudomonas aeruginosa, respectively, reveals that Eag chaperones mimic transmembrane helical packing to stabilize effector TMDs. In addition to participating in the chaperone-TMD interface, we find that prePAAR residues mediate effector-VgrG spike interactions. Taken together, our findings reveal mechanisms of chaperone-mediated stabilization and secretion of two distinct families of T6SS membrane protein effectors.

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Structure–function analysis of HsiF, a gp25-like component of the type VI secretion system, in Pseudomonas aeruginosa

Microbiology ◽

10.1099/mic.0.051987-0 ◽

2011 ◽

Vol 157 (12) ◽

pp. 3292-3305 ◽

Cited By ~ 41

Author(s):

Nadine S. Lossi ◽

Rana Dajani ◽

Paul Freemont ◽

Alain Filloux

Keyword(s):

Pseudomonas Aeruginosa ◽

Tertiary Structure ◽

Function Analysis ◽

Gene Clusters ◽

Secretion System ◽

Lysozyme Activity ◽

Type Vi Secretion System ◽

Secretion Systems ◽

Type Vi Secretion ◽

T4 Phage

Bacterial pathogens use a range of protein secretion systems to colonize their host. One recent addition to this arsenal is the type VI secretion system (T6SS), which is found in many Gram-negative bacteria. The T6SS involves 12–15 components, including a ClpV-like AAA+ ATPase. Moreover, the VgrG and Hcp components have been proposed to form a puncturing device, based on structural similarity to the tail spike components gp5/gp27 and the tail tube component gp19 of the T4 bacteriophage, respectively. Another T6SS component shows similarity to a T4 phage protein, namely gp25. The gp25 protein has been proposed to have lysozyme activity. Other T6SS components do not exhibit obvious similarity to characterized T4 phage components. The genome of Pseudomonas aeruginosa contains three T6SS gene clusters. In each cluster a gene encoding a putative member of the gp25-like protein family was identified, which we called HsiF. We confirmed this similarity by analysing the structure of the P. aeruginosa HsiF proteins using secondary and tertiary structure prediction tools. We demonstrated that HsiF1 is crucial for the T6SS-dependent secretion of Hcp and VgrG. Importantly, lysozyme activity of HsiF proteins was not detectable, and we related this observation to the demonstration that HsiF1 localizes to the cytoplasm of P. aeruginosa. Finally, our data showed that a conserved glutamate, predicted to be required for proper HsiF folding, is essential for its function. In conclusion, our data confirm the central role of HsiF in the T6SS mechanism, provide information on the predicted HsiF structure, and call for reconsideration of the function of gp25-like proteins.

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Evolution of a cis-acting SNP that controls Type VI Secretion in Vibrio cholerae

10.1101/2022.01.11.475911 ◽

2022 ◽

Author(s):

Siu Lung Ng ◽

Sophia A. Kammann ◽

Gabi Steinbach ◽

Tobias Hoffmann ◽

Peter J. Yunker ◽

...

Keyword(s):

Vibrio Cholerae ◽

Phenotypic Diversity ◽

Constitutive Expression ◽

Regulatory Elements ◽

Regulatory Control ◽

Type Vi Secretion System ◽

Type Vi Secretion ◽

Intergenic Regions

Mutations in regulatory mechanisms that control gene expression contribute to phenotypic diversity and thus facilitate the adaptation of microbes to new niches. Regulatory architecture is often inferred from transcription factor identification and genome analysis using purely computational approaches. However, there are few examples of phenotypic divergence that arise from the rewiring of bacterial regulatory circuity by mutations in intergenic regions, because locating regulatory elements within regions of DNA that do not code for protein requires genomic and experimental data. We identify a single cis-acting single nucleotide polymorphism (SNP) dramatically alters control of the type VI secretion system (T6), a common weapon for inter-bacterial competition. Tight T6 regulatory control is necessary for adaptation of the waterborne pathogen Vibrio cholerae to in vivo conditions within the human gut, which we show can be altered by this single non-coding SNP that results in constitutive expression in vitro. Our results support a model of pathogen evolution through cis-regulatory mutation and preexisting, active transcription factors, thus conferring different fitness advantages to tightly regulated strains inside a human host and unfettered strains adapted to environmental niches.

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The evolution of tit-for-tat in bacteria via the type VI secretion system

Nature Communications ◽

10.1038/s41467-020-19017-z ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

William P. J. Smith ◽

Maj Brodmann ◽

Daniel Unterweger ◽

Yohan Davit ◽

Laurie E. Comstock ◽

...

Keyword(s):

Animal Behavior ◽

Evolutionary Game ◽

Secretion System ◽

Evolutionary Strategy ◽

Type Vi Secretion System ◽

Agent Based ◽

Agent Based Modelling ◽

Bacterial Competition ◽

Type Vi Secretion ◽

Tit For Tat

Abstract Tit-for-tat is a familiar principle from animal behavior: individuals respond in kind to being helped or harmed by others. Remarkably some bacteria appear to display tit-for-tat behavior, but how this evolved is not understood. Here we combine evolutionary game theory with agent-based modelling of bacterial tit-for-tat, whereby cells stab rivals with poisoned needles (the type VI secretion system) after being stabbed themselves. Our modelling shows tit-for-tat retaliation is a surprisingly poor evolutionary strategy, because tit-for-tat cells lack the first-strike advantage of preemptive attackers. However, if cells retaliate strongly and fire back multiple times, we find that reciprocation is highly effective. We test our predictions by competing Pseudomonas aeruginosa (a tit-for-tat species) with Vibrio cholerae (random-firing), revealing that P. aeruginosa does indeed fire multiple times per incoming attack. Our work suggests bacterial competition has led to a particular form of reciprocation, where the principle is that of strong retaliation, or ‘tits-for-tat’.

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