Baseplate assembly of phage Mu: Defining the conserved core components of contractile-tailed phages and related bacterial systems

Contractile phage tails are powerful cell puncturing nanomachines that have been co-opted by bacteria for self-defense against both bacteria and eukaryotic cells. The tail of phage T4 has long served as the paradigm for understanding contractile tail-like systems despite its greater complexity compared with other contractile-tailed phages. Here, we present a detailed investigation of the assembly of a “simple” contractile-tailed phage baseplate, that of Escherichia coli phage Mu. By coexpressing various combinations of putative Mu baseplate proteins, we defined the required components of this baseplate and delineated its assembly pathway. We show that the Mu baseplate is constructed through the independent assembly of wedges that are organized around a central hub complex. The Mu wedges are comprised of only three protein subunits rather than the seven found in the equivalent structure in T4. Through extensive bioinformatic analyses, we found that homologs of the essential components of the Mu baseplate can be identified in the majority of contractile-tailed phages and prophages. No T4-like prophages were identified. The conserved simple baseplate components were also found in contractile tail-derived bacterial apparatuses, such as type VI secretion systems, Photorhabdus virulence cassettes, and R-type tailocins. Our work highlights the evolutionary connections and similarities in the biochemical behavior of phage Mu wedge components and the TssF and TssG proteins of the type VI secretion system. In addition, we demonstrate the importance of the Mu baseplate as a model system for understanding bacterial phage tail-derived systems.

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

Infection and Immunity ◽

10.1128/iai.01165-12 ◽

2013 ◽

Vol 81 (4) ◽

pp. 1207-1220 ◽

Cited By ~ 36

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.

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A new family of Type VI secretion system-delivered effector proteins displays ion-selective pore-forming activity

10.1101/676247 ◽

2019 ◽

Author(s):

Giuseppina Mariano ◽

Katharina Trunk ◽

David J. Williams ◽

Laura Monlezun ◽

Henrik Strahl ◽

...

Keyword(s):

Effector Proteins ◽

Type Vi Secretion System ◽

Secretion Systems ◽

New Family ◽

Type Vi Secretion ◽

Outer Membrane Permeability ◽

Polymicrobial Communities ◽

Bacterial Effectors

AbstractType VI secretion systems (T6SSs) are nanomachines widely used by bacteria to compete with rivals. T6SSs deliver multiple toxic effector proteins directly into neighbouring cells and play key roles in shaping diverse polymicrobial communities. A number of families of T6SS-dependent anti-bacterial effectors have been characterised, however the mode of action of others remains unknown. Here we report that Ssp6, an anti-bacterial effector delivered by theSerratia marcescensT6SS, is an ion-selective pore-forming toxin.In vivo, Ssp6 inhibits growth by causing depolarisation of the inner membrane of intoxicated cells and also leads to increased outer membrane permeability, whilst reconstruction of Ssp6 activityin vitrodemonstrated that it forms cation-selective pores. A survey of bacterial genomes revealed that Ssp6-like effectors are widespread in Enterobacteriaceae and often linked with T6SS genes. We conclude that Ssp6 represents a new family of T6SS-delivered anti-bacterial effectors, further diversifying the portfolio of weapons available for deployment during inter-bacterial conflict.

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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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A family of Type VI secretion system effector proteins that form ion-selective pores

Nature Communications ◽

10.1038/s41467-019-13439-0 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 18

Author(s):

Giuseppina Mariano ◽

Katharina Trunk ◽

David J. Williams ◽

Laura Monlezun ◽

Henrik Strahl ◽

...

Keyword(s):

Opportunistic Pathogen ◽

Effector Proteins ◽

Type Vi Secretion System ◽

Secretion Systems ◽

New Family ◽

Type Vi Secretion ◽

Genes Encoding ◽

Outer Membrane Permeability ◽

Bacterial Effectors

AbstractType VI secretion systems (T6SSs) are nanomachines widely used by bacteria to deliver toxic effector proteins directly into neighbouring cells. However, the modes of action of many effectors remain unknown. Here we report that Ssp6, an anti-bacterial effector delivered by a T6SS of the opportunistic pathogen Serratia marcescens, is a toxin that forms ion-selective pores. Ssp6 inhibits bacterial growth by causing depolarisation of the inner membrane in intoxicated cells, together with increased outer membrane permeability. Reconstruction of Ssp6 activity in vitro demonstrates that it forms cation-selective pores. A survey of bacterial genomes reveals that genes encoding Ssp6-like effectors are widespread in Enterobacteriaceae and often linked with T6SS genes. We conclude that Ssp6 and similar proteins represent a new family of T6SS-delivered anti-bacterial effectors.

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Crosstalk between the Type VI Secretion System and the Expression of Class IV Flagellar Genes in the Pseudomonas fluorescens MFE01 Strain

Microorganisms ◽

10.3390/microorganisms8050622 ◽

2020 ◽

Vol 8 (5) ◽

pp. 622 ◽

Cited By ~ 1

Author(s):

Mathilde Bouteiller ◽

Mathias Gallique ◽

Yvann Bourigault ◽

Artemis Kosta ◽

Julie Hardouin ◽

...

Keyword(s):

Pseudomonas Fluorescens ◽

Sigma Factor ◽

Type Vi Secretion System ◽

Secretion Systems ◽

Gene Encoding ◽

Prey Cell ◽

Over Expression ◽

Type Vi Secretion ◽

Flagellar Genes ◽

Class Iv

Type VI secretion systems (T6SSs) are contractile bacterial multiprotein nanomachines that enable the injection of toxic effectors into prey cells. The Pseudomonas fluorescens MFE01 strain has T6SS antibacterial activity and can immobilise competitive bacteria through the T6SS. Hcp1 (hemolysin co-regulated protein 1), a constituent of the T6SS inner tube, is involved in such prey cell inhibition of motility. Paradoxically, disruption of the hcp1 or T6SS contractile tail tssC genes results in the loss of the mucoid and motile phenotypes in MFE01. Here, we focused on the relationship between T6SS and flagella-associated motility. Electron microscopy revealed the absence of flagellar filaments for MFE01Δhcp1 and MFE01ΔtssC mutants. Transcriptomic analysis showed a reduction in the transcription of class IV flagellar genes in these T6SS mutants. However, transcription of fliA, the gene encoding the class IV flagellar sigma factor, was unaffected. Over-expression of fliA restored the motile and mucoid phenotypes in both MFE01Δhcp1+fliA, and MFE01ΔtssC+fliA and a fliA mutant displayed the same phenotypes as MFE01Δhcp1 and MFE01ΔtssC. Moreover, the FliA anti-sigma factor FlgM was not secreted in the T6SS mutants, and flgM over-expression reduced both motility and mucoidy. This study provides arguments to unravel the crosstalk between T6SS and motility.

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The Francisella Pathogenicity Island Protein PdpD Is Required for Full Virulence and Associates with Homologues of the Type VI Secretion System

Journal of Bacteriology ◽

10.1128/jb.00198-08 ◽

2008 ◽

Vol 190 (13) ◽

pp. 4584-4595 ◽

Cited By ~ 85

Author(s):

Jagjit S. Ludu ◽

Olle M. de Bruin ◽

Barry N. Duplantis ◽

Crystal L. Schmerk ◽

Alicia Y. Chou ◽

...

Keyword(s):

North America ◽

Outer Membrane ◽

Bacterial Pathogen ◽

Pathogenicity Island ◽

Secretion System ◽

Type Vi Secretion System ◽

Secretion Systems ◽

Intracellular Growth ◽

Type Vi Secretion ◽

Genes Encoding

ABSTRACT Francisella tularensis is a highly infectious, facultative intracellular bacterial pathogen that is the causative agent of tularemia. Nearly a century ago, researchers observed that tularemia was often fatal in North America but almost never fatal in Europe and Asia. The chromosomes of F. tularensis strains carry two identical copies of the Francisella pathogenicity island (FPI), and the FPIs of North America-specific biotypes contain two genes, anmK and pdpD, that are not found in biotypes that are distributed over the entire Northern Hemisphere. In this work, we studied the contribution of anmK and pdpD to virulence by using F. novicida, which is very closely related to F. tularensis but which carries only one copy of the FPI. We showed that anmK and pdpD are necessary for full virulence but not for intracellular growth. This is in sharp contrast to most other FPI genes that have been studied to date, which are required for intracellular growth. We also showed that PdpD is localized to the outer membrane. Further, overexpression of PdpD affects the cellular distribution of FPI-encoded proteins IglA, IglB, and IglC. Finally, deletions of FPI genes encoding proteins that are homologues of known components of type VI secretion systems abolished the altered distribution of IglC and the outer membrane localization of PdpD.

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Type VI secretion system killing by commensal Neisseria is influenced by the spatial dynamics of bacteria

10.1101/2020.11.26.400259 ◽

2020 ◽

Author(s):

Rafael Custodio ◽

Rhian M. Ford ◽

Cara J. Ellison ◽

Guangyu Liu ◽

Gerda Mickute ◽

...

Keyword(s):

Spatial Dynamics ◽

Secretion System ◽

Type Vi Secretion System ◽

Secretion Systems ◽

Spatial Constraints ◽

Human Respiratory Tract ◽

Type Vi Secretion ◽

Close Proximity ◽

The Impact ◽

Commensal Neisseria

ABSTRACTType VI Secretion Systems (T6SS) are widespread in bacteria and can dictate the development and organisation of polymicrobial ecosystems by mediating contact dependent killing. In Neisseria species, including Neisseria cinerea a commensal of the human respiratory tract, interbacterial contacts are mediated by Type four pili (Tfp) which promote formation of aggregates and govern the spatial dynamics of growing Neisseria microcolonies. Here we show that N. cinerea expresses a plasmid-encoded T6SS that is active and can limit growth of related pathogens. We explored the impact of Tfp expression on N. cinerea T6SS-dependent killing and show that expression of Tfp by prey strains enhances their susceptibility to T6SS, by keeping them in close proximity of T6SS-wielding attacker strains. Our findings have important implications for understanding how spatial constraints during contact-dependent antagonism can shape the evolution of microbial communities.

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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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Beyond dueling: roles of the type VI secretion system in microbiome modulation, pathogenesis and stress resistance

Stress Biology ◽

10.1007/s44154-021-00008-z ◽

2021 ◽

Vol 1 (1) ◽

Author(s):

Jinshui Lin ◽

Lei Xu ◽

Jianshe Yang ◽

Zhuo Wang ◽

Xihui Shen

Keyword(s):

Stress Resistance ◽

Secretion System ◽

Bacterial Cells ◽

Type Vi Secretion System ◽

Secretion Systems ◽

Competitive Fitness ◽

Type Vi Secretion ◽

Protein Secretion Systems ◽

Extracellular Environment

AbstractBacteria inhabit diverse and dynamic environments, where nutrients may be limited and toxic chemicals can be prevalent. To adapt to these stressful conditions, bacteria have evolved specialized protein secretion systems, such as the type VI secretion system (T6SS) to facilitate their survival. As a molecular syringe, the T6SS expels various effectors into neighboring bacterial cells, eukaryotic cells, or the extracellular environment. These effectors improve the competitive fitness and environmental adaption of bacterial cells. Although primarily recognized as antibacterial weapons, recent studies have demonstrated that T6SSs have functions beyond interspecies competition. Here, we summarize recent research on the role of T6SSs in microbiome modulation, pathogenesis, and stress resistance.

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Killing by Type VI secretion drives clonal phase separation and the evolution of cooperation

10.1101/063487 ◽

2016 ◽

Author(s):

Luke McNally ◽

Eryn Bernardy ◽

Jacob Thomas ◽

Arben Kalziqi ◽

Jennifer Pentz ◽

...

Keyword(s):

Phase Separation ◽

Public Goods ◽

Universality Class ◽

Evolution Of Cooperation ◽

Type Vi Secretion System ◽

Secretion Systems ◽

Self Organized ◽

Type Vi Secretion ◽

Solid Media ◽

The Relationship

SummaryBy nature of their small size, dense growth and frequent need for extracellular metabolism, microbes face persistent public goods dilemmas1–5. Spatial assortment can act as a general solution to social conflict by allowing extracellular goods to be utilized preferentially by productive genotypes1,6,7. Established mechanisms that generate microbial assortment depend on the availability of free space8–14; however, microbes often live in densely-packed environments, wherein these mechanisms are ineffective. Here, we describe a novel class of self-organized pattern formation that facilitates the development of spatial structure within densely-packed bacterial colonies. Contact-mediated killing through the Type VI secretion system (T6SS) drives high levels of assortment by precipitating phase separation, even in initially well-mixed populations that do not necessarily exhibit net growth. We examine these dynamics using three different classes of mathematical models and experiments with mutually antagonistic strains of Vibrio cholerae growing on solid media, and find that all appear to de-mix via the same ‘Model A’ universality class of order-disorder transition. We mathematically demonstrate that contact killing should favour the evolution of public goods cooperation, and empirically examine the relationship between T6SSs and potential cooperation through phylogenetic analysis. Across 26 genera of Proteobacteria and Bacteroidetes, the proportion of a strain’s genome that codes for potentially-exploitable secreted proteins increases significantly with boththe number of Type 6 secretion systems and the number of T6SS effectors that it possesses. This work demonstrates how antagonistic traits—likely evolved for the purpose of killing competitors—can indirectlylead to the evolution of cooperation by driving genetic phase separation.

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