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

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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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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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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Fur-Dam Regulatory Interplay at an Internal Promoter of the Enteroaggregative Escherichia coli Type VI Secretion sci1 Gene Cluster

Journal of Bacteriology ◽

10.1128/jb.00075-20 ◽

2020 ◽

Vol 202 (10) ◽

Cited By ~ 3

Author(s):

Yannick R. Brunet ◽

Christophe S. Bernard ◽

Eric Cascales

Keyword(s):

Escherichia Coli ◽

Gene Cluster ◽

Gene Clusters ◽

Secretion System ◽

Target Cells ◽

Type Vi Secretion System ◽

Content Type ◽

Internal Promoter ◽

E Coli ◽

Type Vi Secretion

ABSTRACT The type VI secretion system (T6SS) is a weapon for delivering effectors into target cells that is widespread in Gram-negative bacteria. The T6SS is a highly versatile machine, as it can target both eukaryotic and prokaryotic cells, and it has been proposed that T6SSs are adapted to the specific needs of each bacterium. The expression of T6SS gene clusters and the activation of the secretion apparatus are therefore tightly controlled. In enteroaggregative Escherichia coli (EAEC), the sci1 T6SS gene cluster is subject to a complex regulation involving both the ferric uptake regulator (Fur) and DNA adenine methylase (Dam)-dependent DNA methylation. In this study, an additional, internal, promoter was identified within the sci1 gene cluster using +1 transcriptional mapping. Further analyses demonstrated that this internal promoter is controlled by a mechanism strictly identical to that of the main promoter. The Fur binding box overlaps the −10 transcriptional element and a Dam methylation site, GATC-32. Hence, the expression of the distal sci1 genes is repressed and the GATC-32 site is protected from methylation in iron-rich conditions. The Fur-dependent protection of GATC-32 was confirmed by an in vitro methylation assay. In addition, the methylation of GATC-32 negatively impacted Fur binding. The expression of the sci1 internal promoter is therefore controlled by iron availability through Fur regulation, whereas Dam-dependent methylation maintains a stable ON expression in iron-limited conditions. IMPORTANCE Bacteria use weapons to deliver effectors into target cells. One of these weapons, the type VI secretion system (T6SS), assembles a contractile tail acting as a spring to propel a toxin-loaded needle. Its expression and activation therefore need to be tightly regulated. Here, we identified an internal promoter within the sci1 T6SS gene cluster in enteroaggregative E. coli. We show that this internal promoter is controlled by Fur and Dam-dependent methylation. We further demonstrate that Fur and Dam compete at the −10 transcriptional element to finely tune the expression of T6SS genes. We propose that this elegant regulatory mechanism allows the optimum production of the T6SS in conditions where enteroaggregative E. coli encounters competing species.

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YbeY controls the type III and type VI secretion systems and biofilm formation through RetS in Pseudomonas aeruginosa

Applied and Environmental Microbiology ◽

10.1128/aem.02171-20 ◽

2020 ◽

Author(s):

Yushan Xia ◽

Congjuan Xu ◽

Dan Wang ◽

Yuding Weng ◽

Yongxin Jin ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Type Iii Secretion ◽

Biofilm Formation ◽

Small Rnas ◽

Type Iii Secretion System ◽

Secretion System ◽

Type Vi Secretion System ◽

Chronic Infections ◽

Type Iii ◽

Type Vi Secretion

YbeY is a highly conserved RNase in bacteria and plays essential roles in the maturation of 16S rRNA, regulation of small RNAs (sRNAs) and bacterial responses to environmental stresses. Previously, we verified the role of YbeY in rRNA processing and ribosome maturation in Pseudomonas aeruginosa and demonstrated YbeY-mediated regulation of rpoS through a sRNA ReaL. In this study, we demonstrate that mutation of the ybeY gene results in upregulation of the type III secretion system (T3SS) genes as well as downregulation of the type VI secretion system (T6SS) genes and reduction of biofilm formation. By examining the expression of the known sRNAs in P. aeruginosa, we found that mutation of the ybeY gene leads to downregulation of the small RNAs RsmY/Z that control the T3SS, the T6SS and biofilm formation. Further studies revealed that the reduced levels of RsmY/Z are due to upregulation of retS. Taken together, our results reveal the pleiotropic functions of YbeY and provide detailed mechanisms of YbeY-mediated regulation in P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa causes a variety of acute and chronic infections in humans. The type III secretion system (T3SS) plays an important role in acute infection and the type VI secretion system (T6SS) and biofilm formation are associated with chronic infections. Understanding of the mechanisms that control the virulence determinants involved in acute and chronic infections will provide clues for the development of effective treatment strategies. Our results reveal a novel RNase mediated regulation on the T3SS, T6SS and biofilm formation in P. aeruginosa.

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ClpV3 of the H3-Type VI Secretion System (H3-T6SS) Affects Multiple Virulence Factors in Pseudomonas aeruginosa

Frontiers in Microbiology ◽

10.3389/fmicb.2020.01096 ◽

2020 ◽

Vol 11 ◽

Cited By ~ 1

Author(s):

Yanqi Li ◽

Lin Chen ◽

Pansong Zhang ◽

Anjali Y. Bhagirath ◽

Kangmin Duan

Keyword(s):

Pseudomonas Aeruginosa ◽

Virulence Factors ◽

Secretion System ◽

Type Vi Secretion System ◽

Type Vi Secretion

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Type VI Secretion System Dynamics Reveals a Novel Secretion Mechanism in Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.00744-17 ◽

2018 ◽

Vol 200 (11) ◽

Cited By ~ 8

Author(s):

Jacqueline Corbitt ◽

Jun Seok Yeo ◽

C. Ian Davis ◽

Michele LeRoux ◽

Paul A. Wiggins

Keyword(s):

Pseudomonas Aeruginosa ◽

Vibrio Cholerae ◽

Conformational Change ◽

Secretion System ◽

Force Generation ◽

Type Vi Secretion System ◽

Content Type ◽

Evolutionary Diversification ◽

Type Vi Secretion ◽

Generation Mechanisms

ABSTRACT The type VI secretion system (T6SS) inhibits the growth of neighboring bacterial cells through a contact-mediated mechanism. Here, we describe a detailed characterization of the protein localization dynamics in the Pseudomonas aeruginosa T6SS. It has been proposed that the type VI secretion process is driven by a conformational-change-induced contraction of the T6SS sheath. However, although the contraction of an optically resolvable TssBC sheath and the subsequent localization of ClpV are observed in Vibrio cholerae , coordinated assembly and disassembly of TssB and ClpV are observed without TssB contraction in P. aeruginosa . These dynamics are inconsistent with the proposed contraction sheath model. Motivated by the phenomenon of dynamic instability, we propose a new model in which ATP hydrolysis, rather than conformational change, generates the force for secretion. IMPORTANCE The type VI secretion system (T6SS) is widely conserved among Gram-negative bacteria and is a central determinant of bacterial fitness in polymicrobial communities. The secretion system targets bacteria and secretes effectors that inhibit the growth of neighboring cells, using a contact-mediated-delivery system. Despite significant homology to the previously characterized Vibrio cholerae T6SS, our analysis reveals that effector secretion is driven by a distinct force generation mechanism in Pseudomonas aeruginosa . The presence of two distinct force generation mechanisms in T6SS represents an example of the evolutionary diversification of force generation mechanisms.

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Vibrio cholerae Requires the Type VI Secretion System Virulence Factor VasX To Kill Dictyostelium discoideum

Infection and Immunity ◽

10.1128/iai.01266-10 ◽

2011 ◽

Vol 79 (7) ◽

pp. 2941-2949 ◽

Cited By ~ 118

Author(s):

Sarah T. Miyata ◽

Maya Kitaoka ◽

Teresa M. Brooks ◽

Steven B. McAuley ◽

Stefan Pukatzki

Keyword(s):

Vibrio Cholerae ◽

Dictyostelium Discoideum ◽

Virulence Factor ◽

Membrane Lipids ◽

Gene Clusters ◽

Secretion System ◽

Type Vi Secretion System ◽

Content Type ◽

Enzymatic Function ◽

Type Vi Secretion

ABSTRACTThe type VI secretion system (T6SS) is recognized as an important virulence mechanism in several Gram-negative pathogens. InVibrio cholerae, the causative agent of the diarrheal disease cholera, a minimum of three gene clusters—one main cluster and two auxiliary clusters—are required to form a functional T6SS apparatus capable of conferring virulence toward eukaryotic and prokaryotic hosts. Despite an increasing understanding of the components that make up the T6SS apparatus, little is known about the regulation of these genes and the gene products delivered by this nanomachine. VasH is an important regulator of theV. choleraeT6SS. Here, we present evidence that VasH regulates the production of a newly identified protein, VasX, which in turn requires a functional T6SS for secretion. Deletion ofvasXdoes not affect export or enzymatic function of the structural T6SS proteins Hcp and VgrG-1, suggesting that VasX is dispensable for the assembly of the physical translocon complex. VasX localizes to the bacterial membrane and interacts with membrane lipids. We present VasX as a novel virulence factor of the T6SS, as aV. choleraemutant lackingvasXexhibits a phenotype of attenuated virulence towardDictyostelium discoideum.

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VipA N-terminal linker and VipB-VipB interaction modulate the contraction of Type VI secretion system sheath

10.1101/152785 ◽

2017 ◽

Cited By ~ 1

Author(s):

Maximilian Brackmann ◽

Jing Wang ◽

Marek Basler

Keyword(s):

Protein Translocation ◽

Secretion System ◽

Mass Spectrometry Analysis ◽

Target Cells ◽

Extended State ◽

Type Vi Secretion System ◽

Secretion Systems ◽

Spectrometry Analysis ◽

Type Vi Secretion ◽

Bacterial Type

AbstractSecretion systems are essential for bacteria to survive and manipulate their environment. The bacterial Type VI Secretion System (T6SS) generates the force needed for protein translocation by the contraction of a long polymer called sheath, which is composed of interconnected VipA/VipB subunits forming a six-start helix. The mechanism of T6SS sheath contraction and the structure of its extended state are unknown. Here we show that elongating the N-terminal VipA linker or eliminating charge of a specific VipB residue abolished sheath contraction and delivery of effectors into target cells. The assembly of the non-contractile sheaths was dependent on the baseplate component TssE and mass-spectrometry analysis identified Hcp, VgrG and other components of the T6SS baseplate specifically associated with stable non-contractile sheaths. The ability to lock T6SS in the pre-firing state opens new possibilities for understanding its mode of action.

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