Filamentous prophage Pf4 promotes genetic exchange in Pseudomonas aeruginosa.

Filamentous prophages are widespread among bacteria and play crucial functions in virulence, antibiotic resistance, and biofilm structures. The filamentous Pf4 particles, extruded by an important pathogen Pseudomonas aeruginosa, can protect producing cells from adverse conditions. Contrary to the conventional belief that the Pf4-encoding cells resist reinfection, we herein report that the Pf4 prophage is reciprocally and commonly exchanged within P. aeruginosa colonies, which can repair defective Pf4 within the community. By labeling the Pf4 locus with antibiotic resistance and fluorescence markers, we demonstrate that the Pf4 locus is frequently exchanged within colony biofilms, in artificial sputum media, and in infected mouse lungs. We further show that Pf4 trafficking is a rapid process and capable of rescuing Pf4-defective mutants. The Pf4 phage is highly adaptable and can package additional DNA doubling its genome size. We also report that two clinical P. aeruginosa isolates are susceptible to the Pf4-mediated exchange, and the Pf5 prophage can be exchanged between cells as well. These findings suggest that the genetic exchanging interactions by filamentous prophages may facilitate defect rescue and the sharing of prophage-dependent benefits and costs within the P. aeruginosa community.

VgrG Spike Dictates PAAR Requirement for the Assembly of the Type VI Secretion System.

Widely employed by Gram-negative pathogens for competition and pathogenesis, the type six protein secretion system (T6SS) can inject toxic effectors into neighboring cells through the penetration of a spear-like structure comprising a long Hcp tube and a VgrG-PAAR spike complex. The cone-shaped PAAR is believed to sharpen the T6SS spear for penetration but it remains unclear why PAAR is required for T6SS functions in some bacteria but dispensable in others. Here, we report the conditional requirement of PAAR for T6SS functions in Aeromonas dhakensis, an emerging human pathogen that may cause severe bacteremia. By deleting the two PAAR paralogs, we show that PAAR is not required for T6SS secretion, bacterial killing, or specific effector delivery in A. dhakensis. By constructing combinatorial PAAR and vgrG deletions, we demonstrate that deletion of individual PAAR moderately reduced T6SS functions but double or triple deletions of PAAR in the vgrG deletion mutants severely impaired T6SS functions. Notably, the auxiliary-cluster-encoded PAAR2 and VgrG3 are less critical than the main-cluster-encoded PAAR1 and VgrG1&2 proteins to T6SS functions. In addition, PAAR1 but not PAAR2 contributes to antieukaryotic virulence in amoeba. Our data suggest that, for a multi-PAAR T6SS, the variable role of PAAR paralogs correlates with the VgrG-spike composition that collectively dictates T6SS assembly. IMPORTANCE Gram-negative bacteria often encode multiple paralogs of the cone-shaped PAAR that sits atop the VgrG-spike and is thought to sharpen the spear-like T6SS puncturing device. However, it is unclear why PAAR is required for the assembly of some but not all T6SSs and why there are multiple PAARs if they are not required. Our data delineate a VgrG-mediated conditional requirement for PAAR and suggest a core-auxiliary relationship among different PAAR-VgrG modules that may have been acquired sequentially by the T6SS during evolution.

A Dueling-Competent Signal-Sensing Module Guides Precise Delivery of Cargo Proteins into Target Cells by Engineered Pseudomonas aeruginosa.

To recognize and manipulate a specific microbe of a crowded community is a highly challenging task in synthetic biology. Here we introduce a highly selective protein delivery platform, termed DUEC, which responds to direct contact of attacking cells by engineering the tit-for-tat/dueling response of H1-T6SS (type VI secretion system) in Pseudomonas aeruginosa. Using a Cre-recombinase-dependent reporter, we screened H1-T6SS-secreted substrates and developed Tse6N as the most effective secretion tag for Cre delivery. DUEC cells can discriminately deliver the Tse6N-Cre cargo into the cytosol of T6SS+ but not T6SS- Vibrio cholerae cells. DUEC could also deliver a nuclease cargo, Tse6N-NucSe1, to selectively kill provoking cells in a mixed community. These data demonstrate that the DUEC cell not only is a prototypical physical-contact sensor and delivery platform but also may be coupled with recombination-based circuits with the potential for complex tasks in mixed microbial communities.

Heterologous Assembly of the Type VI Secretion System Empowers Laboratory Escherichia coli with Antimicrobial and Cell Penetration Capabilities.

The synthetic biology toolbox has amassed a vast number of diverse functional modules, but protein translocation modules for cell penetration and cytosol-to-cytosol delivery remain relatively scarce. The type VI secretion system (T6SS), commonly found in many Gram-negative pathogens, functions as a contractile device to translocate protein toxins to prokaryotic and eukaryotic cells. Here, we have assembled the T6SS of Aeromonas dhakensis, an opportunistic waterborne pathogen, in the common laboratory strain Escherichia coli BL21(DE3). We constructed a series of plasmids (pT6S) carrying the T6SS structural and effector genes under native or tetracycline-inducible promoters, the latter for controlled expression. Using fluorescence microscopy and biochemical analyses, we demonstrate a functional T6SS in E. coli capable of secreting proteins directly into the cytosol of neighboring bacteria and outcompeting a number of drug-resistant pathogens. The heterologous assembly of T6SS not only confers the lab workhorse E. coli with the cytosol-to-cytosol protein delivery capability but also demonstrates the potential for harnessing the T6SS of various pathogens for general protein delivery and antibacterial applications. IMPORTANCE The T6SS is a powerful and versatile protein delivery system. However, the complexity of its macromolecular structure and gene regulation makes it not a trivial task to reconstitute the T6SSs of pathogens in a nonpathogenic host. In this study, we have assembled an inducible T6SS in E. coli BL21(DE3) and demonstrated its functions in protein delivery and antimicrobial activities. The engineered T6SS empowers E. coli to deliver protein cargos into a wide range of prokaryotic and eukaryotic cells.

Abiotic factors modulate interspecies competition mediated by the type VI secretion system effectors in Vibrio cholerae.

Vibrio cholerae, the etiological pathogen of cholera, employs its type VI secretion system (T6SS) as an effective weapon to survive in highly competitive communities. Antibacterial and anti-eukaryotic functions of the T6SS depend on its secreted effectors that target multiple cellular processes. However, the mechanisms that account for effector diversity and different effectiveness during interspecies competition remain elusive. Here we report that environmental cations and temperature play a key role in dictating cellular response and effector effectiveness during interspecies competition mediated by the T6SS of V. cholerae. We found that V. cholerae could employ its cell-wall-targeting effector TseH to outcompete the otherwise resistant Escherichia coli and the V. cholerae immunity deletion mutant ∆tsiH when Mg2+ or Ca2+ was supplemented. Transcriptome and genetic analyses demonstrate that the metal-sensing PhoPQ two-component system is important for Mg2+-dependent sensitivity. Competition analysis in infant mice shows that TseH was active under in vivo conditions. Using a panel of V. cholerae single-effector active mutants, we further show that E. coli also exhibited variable susceptibilities to other T6SS effectors depending on cations and temperatures, respectively. Lastly, V. cholerae effector VasX could sensitize Pseudomonas aeruginosa to its intrinsically resistant antibiotic irgasan in a temperature-dependent manner. Collectively, these findings suggest that abiotic factors, that V. cholerae frequently encounters in natural and host environments, could modulate cellular responses and dictate the competitive fitness conferred by the T6SS effectors in complex multispecies communities.

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

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.

Sensing of intracellular Hcp levels controls T6SS expression in Vibrio cholerae.

The type 6 secretion system (T6SS) is a bacterial weapon broadly distributed in gram-negative bacteria and used to kill competitors and predators. Featuring a long and double-tubular structure, this molecular machine is energetically costly to produce and thus is likely subject to diverse regulation strategies that are largely ill defined. In this study, we report a quantity-sensing control of the T6SS that down-regulates the expression of secreted components when they accumulate in the cytosol due to T6SS inactivation. Using Vibrio cholerae strains that constitutively express an active T6SS, we demonstrate that mRNA levels of secreted components, including the inner-tube protein component Hcp, were down-regulated in T6SS structural gene mutants while expression of the main structural genes remained unchanged. Deletion of both hcp gene copies restored expression from their promoters, while Hcp overexpression negatively impacted expression. We show that Hcp directly interacts with the RpoN-dependent T6SS regulator VasH, and deleting the N-terminal regulator domain of VasH abolishes this interaction as well as the expression difference of hcp operons between T6SS-active and inactive strains. We find that negative regulation of hcp also occurs in other V. cholerae strains and the pathogens Aeromonas dhakensis and Pseudomonas aeruginosa This Hcp-dependent sensing control is likely an important energy-conserving mechanism that enables T6SS-encoding organisms to quickly adjust T6SS expression and prevent wasteful build-up of its major secreted components in the absence of their efficient export out of the bacterial cell.

Characterization of Lysozyme-Like Effector TseP Reveals the Dependence of Type VI Secretion System (T6SS) Secretion on Effectors in Aeromonas dhakensis Strain SSU.

The type VI secretion system (T6SS) is a widespread weapon employed by Gram-negative bacteria for interspecies interaction in complex communities. Analogous to a contractile phage tail, the double-tubular T6SS injects toxic effectors into prokaryotic and eukaryotic neighboring cells. Although effectors dictate T6SS functions, their identities remain elusive in many pathogens. Here, we report the lysozyme-like effector TseP in Aeromonas dhakensis, a waterborne pathogen that can cause severe gastroenteritis and systemic infection. Using secretion, competition, and enzymatic assays, we demonstrate that TseP is a T6SS-dependent effector with cell wall-lysing activities, and TsiP is its cognate immunity protein. Triple deletion of tseP and two known effector genes, tseI and tseC, abolished T6SS-mediated secretion, while complementation with any single effector gene partially restored bacterial killing and Hcp secretion. In contrast to whole-gene deletions, the triple-effector inactivation in the 3effc mutant abolished antibacterial killing but not T6SS secretion. We further demonstrate that the 3effc mutation abolished T6SS-mediated toxicity of SSU to Dictyostelium discoideum amoebae, suggesting that the T6SS physical puncture is nontoxic to eukaryotic cells. These data highlight not only the necessity of possessing functionally diverse effectors for survival in multispecies communities but also that effector inactivation would be an efficient strategy to detoxify the T6SS while preserving its delivery efficiency, converting the T6SS to a platform for protein delivery to a variety of recipient cells. IMPORTANCE Delivery of cargo proteins via protein secretion systems has been shown to be a promising tool in various applications. However, secretion systems are often used by pathogens to cause disease. Thus, strategies are needed to detoxify secretion systems while preserving their efficiency. The T6SS can translocate proteins through physical puncture of target cells without specific surface receptors and can target a broad range of recipients. In this study, we identified a cell wall-lysing effector, and by inactivating it and the other two known effectors, we have built a detoxified T6SS-active strain that may be used for protein delivery to prokaryotic and eukaryotic recipient cells.

Differential Cellular Response to Translocated Toxic Effectors and Physical Penetration by the Type VI Secretion System.

The type VI secretion system (T6SS) is a lethal microbial weapon that injects a large needle-like structure carrying toxic effectors into recipient cells through physical penetration. How recipients respond to physical force and effectors remains elusive. Here, we use a series of effector mutants of Vibrio cholerae to determine how T6SS elicits response in Pseudomonas aeruginosa and Escherichia coli. We show that TseL, but no other effectors or physical puncture, triggers the tit-for-tat response of P. aeruginosa H1-T6SS. Although E. coli is sensitive to all periplasmically expressed effectors, P. aeruginosa is most sensitive to TseL alone. We identify a number of stress response pathways that confer protection against TseL. Physical puncture of T6SS has a moderate inhibitory effect only on envelope-impaired tolB and rseA mutants. Our data reveal that recipient cells primarily respond to effector toxicity but not to physical contact, and they rely on the stress response for immunity-independent protection.

Publisher Correction: Intramolecular chaperone-mediated secretion of an Rhs effector toxin by a type VI secretion system.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Intramolecular chaperone-mediated secretion of an Rhs effector toxin by a type VI secretion system.

Bacterial Rhs proteins containing toxic domains are often secreted by type VI secretion systems (T6SSs) through unclear mechanisms. Here, we show that the T6SS Rhs-family effector TseI of Aeromonas dhakensis is subject to self-cleavage at both the N- and the C-terminus, releasing the middle Rhs core and two VgrG-interacting domains (which we name VIRN and VIRC). VIRC is an endonuclease, and the immunity protein TsiI protects against VIRC toxicity through direct interaction. Proteolytic release of VIRC and VIRN is mediated, respectively, by an internal aspartic protease activity and by two conserved glutamic residues in the Rhs core. Mutations abolishing self-cleavage do not block secretion, but reduce TseI toxicity. Deletion of VIRN or the Rhs core abolishes secretion. TseI homologs from Pseudomonas syringae, P. aeruginosa, and Vibrio parahaemolyticus are also self-cleaved. VIRN and VIRC interact with protein VgrG1, while the Rhs core interacts with protein TecI. We propose that VIRN and the Rhs core act as T6SS intramolecular chaperones to facilitate toxin secretion and function.

An onboard checking mechanism ensures effector delivery of the type VI secretion system in Vibrio cholerae.

The type VI secretion system (T6SS) is a lethal yet energetically costly weapon in gram-negative bacteria. Through contraction of a long sheath, the T6SS ejects a few copies of effectors accompanied by hundreds of structural carrier proteins per delivery. The few ejected effectors, however, dictate T6SS functions. It remains elusive how the T6SS ensures effector loading and avoids futile ejection. Here, by systemically mutating the active sites of 3 Vibrio cholerae effectors, TseL, VasX, and VgrG3, we show that the physical presence but not their activities is crucial for T6SS assembly. We constructed catalytic mutants of TseL and VgrG3 and truncated VasX mutants. These mutations abolished the killing of the effector-cognate immunity mutants. We determined that the VasX-mediated antimicrobial activity is solely dependent on the C-terminal colicin domain. Removal of the colicin domain abolished VasX secretion and reduced T6SS assembly, while deletion of the colicin internal loop abolished its toxicity but had little effect on secretion and assembly. The triple effector-inactive mutant maintains an active T6SS that is capable of delivering chimeric VgrG, PAAR, and TseL proteins fused with a cargo nuclease, indicating effector activities are not required for T6SS assembly or penetration into the cytosol of recipient cells. Therefore, by recruiting effectors as critical components for T6SS assembly, it represents an effective onboard checking mechanism that ensures effectors are loaded in place to prevent futile secretion. Our study also demonstrates a detoxified secretion platform by inactivating native effector activities that could translocate engineered cargo proteins via multiple routes.