Burkholderia thailandensis uses a type VI secretion system to lyse protrusions without triggering host cell responses.

To spread within a host, intracellular Burkholderia form actin tails to generate membrane protrusions into neighboring host cells and use type VI secretion system-5 (T6SS-5) to induce cell-cell fusions. Here, we show that B. thailandensis also uses T6SS-5 to lyse protrusions to directly spread from cell to cell. Dynamin-2 recruitment to the membrane near a bacterium was followed by a short burst of T6SS-5 activity. This resulted in the polymerization of the actin of the newly invaded host cell and disruption of the protrusion membrane. Most protrusion lysis events were dependent on dynamin activity, caused no cell-cell fusion, and failed to be recognized by galectin-3. T6SS-5 inactivation decreased protrusion lysis but increased galectin-3, LC3, and LAMP1 accumulation in host cells. Our results indicate that B. thailandensis specifically activates T6SS-5 assembly in membrane protrusions to disrupt host cell membranes and spread without alerting cellular responses, such as autophagy.

Lipopolysaccharide transport regulates bacterial sensitivity to a cell wall-degrading intermicrobial toxin.

Gram-negative bacteria can antagonize neighboring microbes using a type VI secretion system (T6SS) to deliver toxins that target different essential cellular features. Despite the conserved nature of these targets, T6SS potency can vary across recipient species. To understand the functional basis of intrinsic T6SS susceptibility, we screened for essential Escherichia coli (Eco) genes that affect its survival when antagonized by a cell wall-degrading T6SS toxin from Pseudomonas aeruginosa, Tae1. We revealed genes associated with both the cell wall and a separate layer of the cell envelope, lipopolysaccharide, that modulate Tae1 toxicity in vivo. Disruption of genes in early lipopolysaccharide biosynthesis provided Eco with novel resistance to Tae1, despite significant cell wall degradation. These data suggest that Tae1 toxicity is determined not only by direct substrate damage, but also by indirect cell envelope homeostasis activities. We also found that Tae1-resistant Eco exhibited reduced cell wall synthesis and overall slowed growth, suggesting that reactive cell envelope maintenance pathways could promote, not prevent, self-lysis. Together, our study reveals the complex functional underpinnings of susceptibility to Tae1 and T6SS which regulate the impact of toxin-substrate interactions in vivo.

Lipopolysaccharide integrity primes bacterial sensitivity to a cell wall-degrading intermicrobial toxin.

Gram-negative bacteria can antagonize neighboring microbes using a type VI secretion system (T6SS) to deliver toxins that target different essential cellular features. Despite the conserved nature of these targets, T6SS potency can vary across recipient species. To understand the molecular basis of intrinsic T6SS susceptibility, we screened for essential Escherichia coli genes that affect its survival when antagonized by a cell wall-degrading T6SS toxin from Pseudomonas aeruginosa , Tae1. We revealed genes associated with both the cell wall and a separate layer of the cell envelope, surface lipopolysaccharide, that modulate Tae1 toxicity in vivo . Disruption of lipopolysaccharide synthesis provided Escherichia coli (Eco) with novel resistance to Tae1, despite significant cell wall degradation. These data suggest that Tae1 toxicity is determined not only by direct substrate damage, but also by indirect cell envelope homeostasis activities. We also found that Tae1-resistant Eco exhibited reduced cell wall synthesis and overall slowed growth, suggesting that reactive cell envelope maintenance pathways could promote, not prevent, self-lysis. Together, our study highlights the consequences of co-regulating essential pathways on recipient fitness during interbacterial competition, and how antibacterial toxins leverage cellular vulnerabilities that are both direct and indirect to their specific targets in vivo .

Subcellular localization of Type VI secretion system assembly in response to cell-cell contact.

Bacteria require a number of systems, including the type VI secretion system (T6SS), for interbacterial competition and pathogenesis. The T6SS is a large nanomachine that can deliver toxins directly across membranes of proximal target cells. Since major reassembly of T6SS is necessary after each secretion event, accurate timing and localization of T6SS assembly can lower the cost of protein translocation. Although critically important, mechanisms underlying spatiotemporal regulation of T6SS assembly remain poorly understood. Here, we used super-resolution live-cell imaging to show that while Acinetobacter and Burkholderia thailandensis can assemble T6SS at any site, a significant subset of T6SS assemblies localizes precisely to the site of contact between neighboring bacteria. We identified a class of diverse, previously uncharacterized, periplasmic proteins required for this dynamic localization of T6SS to cell-cell contact (TslA). This precise localization is also dependent on the outer membrane porin OmpA. Our analysis links transmembrane communication to accurate timing and localization of T6SS assembly as well as uncovers a pathway allowing bacterial cells to respond to cell-cell contact during interbacterial competition.

reComBat: batch-effect removal in large-scale multi-source gene-expression data integration.

Motivation: With the steadily increasing abundance of omics data produced all over the world under vastly different experimental conditions residing in public databases, a crucial step in many data-driven bioinformatics applications is that of data integration. The challenge of batch-effect removal for entire databases lies in the large number of batches and biological variation, which can result in design matrix singularity. This problem can currently not be solved satisfactorily by any common batch-correction algorithm. Results: We present reComBat, a regularized version of the empirical Bayes method to overcome this limitation and benchmark it against popular approaches for the harmonization of public gene-expression data (both microarray and bulkRNAsq) of the human opportunistic pathogen Pseudomonas aeruginosa. Batch-effects are successfully mitigated while biologically meaningful gene-expression variation is retained. reComBat fills the gap in batch-correction approaches applicable to large-scale, public omics databases and opens up new avenues for data-driven analysis of complex biological processes beyond the scope of a single study. Availability and implementation: The code is available at https://github.com/BorgwardtLab/reComBat, all data and evaluation code can be found at https://github.com/BorgwardtLab/batchCorrectionPublicData. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

Type VI Secretion System and Its Effectors PdpC, PdpD, and OpiA Contribute to Francisella Virulence in Galleria mellonella Larvae.

Francisella tularensis causes the deadly zoonotic disease tularemia in humans and is able to infect a broad range of organisms including arthropods, which are thought to play a major role in Francisella transmission. However, while mammalian in vitro and in vivo infection models are widely used to investigate Francisella pathogenicity, a detailed characterization of the major Francisella virulence factor, a noncanonical type VI secretion system (T6SS), in an arthropod in vivo infection model is missing. Here, we use Galleria mellonella larvae to analyze the role of the Francisella T6SS and its corresponding effectors in F. tularensis subsp. novicida virulence. We report that G. mellonella larvae killing depends on the functional T6SS and infectious dose. In contrast to other mammalian in vivo infection models, even one of the T6SS effectors PdpC, PdpD, or OpiA is sufficient to kill G. mellonella larvae, while sheath recycling by ClpB is dispensable. We further demonstrate that treatment by polyethylene glycol (PEG) activates Francisella T6SS in liquid culture and that this is independent of the response regulator PmrA. PEG-activated IglC secretion is dependent on T6SS structural component PdpB but independent of putative effectors PdpC, PdpD, AnmK, OpiB1, OpiB2, and OpiB3. The results of larvae infection and secretion assay suggest that AnmK, a putative T6SS component with unknown function, interferes with OpiA-mediated toxicity but not with general T6SS activity. We establish that the easy-to-use G. mellonella larvae infection model provides new insights into the function of T6SS and pathogenesis of Francisella.

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

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'.

Nanaerobic growth enables direct visualization of dynamic cellular processes in human gut symbionts.

Mechanistic studies of anaerobic gut bacteria have been hindered by the lack of a fluorescent protein system to track and visualize proteins and dynamic cellular processes in actively growing bacteria. Although underappreciated, many gut "anaerobes" are able to respire using oxygen as the terminal electron acceptor. The oxygen continually released from gut epithelial cells creates an oxygen gradient from the mucus layer to the anaerobic lumen [L. Albenberg et al., Gastroenterology 147, 1055-1063.e8 (2014)], with oxygen available to bacteria growing at the mucus layer. Here, we show that Bacteroides species are metabolically and energetically robust and do not mount stress responses in the presence of 0.10 to 0.14% oxygen, defined as nanaerobic conditions [A. D. Baughn, M. H. Malamy, Nature 427, 441-444 (2004)]. Taking advantage of this metabolic capability, we show that nanaerobic growth provides sufficient oxygen for the maturation of oxygen-requiring fluorescent proteins in Bacteroides species. Type strains of four different Bacteroides species show bright GFP fluorescence when grown nanaerobically versus anaerobically. We compared four different red fluorescent proteins and found that mKate2 yields the highest red fluorescence intensity in our assay. We show that GFP-tagged proteins can be localized in nanaerobically growing bacteria. In addition, we used time-lapse fluorescence microscopy to image dynamic type VI secretion system processes in metabolically active Bacteroides fragilis The ability to visualize fluorescently labeled Bacteroides and fluorescently linked proteins in actively growing nanaerobic gut symbionts ushers in an age of imaging analyses not previously possible in these bacteria.

The evolution of the type VI secretion system as a disintegration weapon.

The type VI secretion system (T6SS) is a nanomachine used by many bacteria to drive a toxin-laden needle into other bacterial cells. Although the potential to influence bacterial competition is clear, the fitness impacts of wielding a T6SS are not well understood. Here we present a new agent-based model that enables detailed study of the evolutionary costs and benefits of T6SS weaponry during competition with other bacteria. Our model identifies a key problem with the T6SS. Because of its short range, T6SS activity becomes self-limiting, as dead cells accumulate in its way, forming "corpse barriers" that block further attacks. However, further exploration with the model presented a solution to this problem: if injected toxins can quickly lyse target cells in addition to killing them, the T6SS becomes a much more effective weapon. We tested this prediction with single-cell analysis of combat between T6SS-wielding Acinetobacter baylyi and T6SS-sensitive Escherichia coli. As predicted, delivery of lytic toxins is highly effective, whereas nonlytic toxins leave large patches of E. coli alive. We then analyzed hundreds of bacterial species using published genomic data, which suggest that the great majority of T6SS-wielding species do indeed use lytic toxins, indicative of a general principle underlying weapon evolution. Our work suggests that, in the T6SS, bacteria have evolved a disintegration weapon whose effectiveness often rests upon the ability to break up competitors. Understanding the evolutionary function of bacterial weapons can help in the design of probiotics that can both establish well and eliminate problem species.

DNA Uptake upon T6SS-Dependent Prey Cell Lysis Induces SOS Response and Reduces Fitness of Acinetobacter baylyi.

Certain Gram-negative bacteria use the type VI secretion system (T6SS) to kill and lyse competing bacteria. Here, we show that the T6SS-dependent lysis of prey cells by the naturally competent Acinetobacter baylyi results in the extensive filamentation of a subpopulation of A. baylyi cells. Filamentation is dependent on the release of DNA from the prey and its uptake by the competence system. The analysis of A. baylyi transcriptome and the response of transcriptional reporters suggest that the uptake of DNA results in the upregulation of the SOS response, which often leads to cell-division arrest. Long-term competition between competent and non-competent strains shows that the strain lacking the DNA uptake machinery outcompetes the parental strain only in the presence of the T6SS-dependent lysis of prey cells. Our data suggest that the cost of the induced SOS response may drive the selection of tight regulation or the loss of DNA uptake in bacteria capable of lysing their competitors.

Diverse roles of TssA-like proteins in the assembly of bacterial type VI secretion systems.

Protein translocation by the bacterial type VI secretion system (T6SS) is driven by a rapid contraction of a sheath assembled around a tube with associated effectors. Here, we show that TssA-like or TagA-like proteins with a conserved N-terminal domain and varying C-terminal domains can be grouped into at least three distinct classes based on their role in sheath assembly. The proteins of the first class increase speed and frequency of sheath assembly and form a stable dodecamer at the distal end of a polymerizing sheath. The proteins of the second class localize to the cell membrane and block sheath polymerization upon extension across the cell. This prevents excessive sheath polymerization and bending, which may result in sheath destabilization and detachment from its membrane anchor and thus result in failed secretion. The third class of these proteins localizes to the baseplate and is required for initiation of sheath assembly. Our work shows that while various proteins share a conserved N-terminal domain, their roles in T6SS biogenesis are fundamentally different.

Assembly and Subcellular Localization of Bacterial Type VI Secretion Systems.

Bacteria need to deliver large molecules out of the cytosol to the extracellular space or even across membranes of neighboring cells to influence their environment, prevent predation, defeat competitors, or communicate. A variety of protein-secretion systems have evolved to make this process highly regulated and efficient. The type VI secretion system (T6SS) is one of the largest dynamic assemblies in gram-negative bacteria and allows for delivery of toxins into both bacterial and eukaryotic cells. The recent progress in structural biology and live-cell imaging shows the T6SS as a long contractile sheath assembled around a rigid tube with associated toxins anchored to a cell envelope by a baseplate and membrane complex. Rapid sheath contraction releases a large amount of energy used to push the tube and toxins through the membranes of neighboring target cells. Because reach of the T6SS is limited, some bacteria dynamically regulate its subcellular localization to precisely aim at their targets and thus increase efficiency of toxin translocation.

Abundance of bacterial Type VI secretion system components measured by targeted proteomics.

The Type VI secretion system (T6SS) is important for bacterial competition as well as virulence in many Gram-negative bacteria and its dynamics and regulation varies significantly between species. To gain insights into the mechanisms regulating T6SS assembly, we apply targeted proteomics to determine the abundance of the key T6SS components in Vibrio cholerae, Pseudomonas aeruginosa and Acinetobacter baylyi. We show that while there are species specific exceptions, the abundance of most components is similar in all three bacteria and ranges from less than hundred to tens of thousands of copies per cell. The comparison of T6SS dynamics and protein abundance in V. cholerae grown under various conditions suggests that the critical component TssE and the secreted protein VasX are unstable and this diminishes T6SS assembly when protein synthesis is limited. Our quantitative analysis opens possibilities to build realistic models of T6SS assembly and to identify principles of T6SS regulation in various species.

Clinical impact of the type VI secretion system on virulence of Campylobacter species during infection.

BACKGROUND: The clinical course of Campylobacter infection varies in symptoms and severity depending on host factors, virulence of the pathogen and initiated therapy. The type VI secretion system (T6SS) has been identified as a novel virulence factor, which mediates contact-dependent injection of enzymes and toxins into competing bacteria or host cells and facilitates the colonisation of a host organism. We aimed to compare the clinical course of Campylobacter infection caused by strains with and without the T6SS and identify possible associations between this putative virulence factor and the clinical manifestations of disease. METHODS: From April 2015 to January 2017, patients with detection of Campylobacter spp. were identified at the University Hospital of Basel and the University Children's Hospital of Basel and included in this case-control study. Presence of the T6SS gene cluster was assayed by PCR targeting the hcp gene, confirmed with whole genome sequencing. Pertinent clinical data was collected by medical record review. Differences in disease- and host-characteristics between T6SS-positive (case) and -negative (control) were compared in a uni- and multi-variable analysis. Hospital admission, antibiotic therapy, admission to intensive care unit, development of bacteraemia and in-hospital mortality were considered as clinical endpoints. RESULTS: We identified 138 cases of Campylobacter jejuni infections and 18 cases of Campylobacter coli infections from a paediatric and adult population. Analyses were focused on adult patients with C. jejuni (n = 119) of which 16.8% were T6SS-positive. Comparisons between T6SS-positive and -negative C. jejuni isolates did not reveal significant differences regarding clinical manifestations or course of disease. All clinical endpoints showed a similar distribution in both groups. A higher score in the Charlson Comorbidity Index was associated with T6SS-positive C. jejuni isolates (p < 0.001) and patients were more likely to have a solid organ transplant and to be under immunosuppressive therapy. CONCLUSIONS: Our study does not provide evidence that T6SS is associated with a more severe clinical course. Interestingly, T6SS-positive isolates are more commonly found in immunocompromised patients: an observation which merits further investigation.

Mobilizable Plasmids for Tunable Gene Expression in Francisella novicida.

Francisella tularensis is the causative agent of the life-threatening disease tularemia. However, the molecular tools to study Francisella are limited. Especially, expression plasmids are sparse and difficult to use, as they are unstable and prone to spontaneous loss. Most Francisella expression plasmids lack inducible promoters making it difficult to control gene expression levels. In addition, available expression plasmids are mainly designed for F. tularensis, however, genetic differences including restriction-modification systems impede the use of these plasmids in F. novicida, which is often used as a model organism to study Francisella pathogenesis. Here we report construction and characterization of two mobilizable plasmids (pFNMB1 and pFNMB2) designed for regulated gene expression in F. novicida. pFNMB plasmids contain a tetracycline inducible promoter to control gene expression levels and oriT for RP4 mediated mobilization. We show that both plasmids are stably maintained in bacteria for more than 40 generations over 4 days of culturing in the absence of selection against plasmid loss. Expression levels are dependent on anhydrotetracycline concentration and homogeneous in a bacterial population. pFNMB1 and pFNMB2 plasmids differ in the sequence between promoter and translation start site and thus allow to reach different maximum levels of protein expression. We used pFNMB1 and pFNMB2 for complementation of Francisella Pathogenicity Island mutants ΔiglF, ΔiglI, and ΔiglC in-vitro and pFNMB1 to complement ΔiglI mutant in bone marrow derived macrophages.

Type VI secretion system sheath inter-subunit interactions modulate its contraction.

Secretion 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. The sheath is a six-start helical assembly of interconnected VipA/VipB subunits. The mechanism of T6SS sheath contraction is unknown. Here, we show that elongating the N-terminal VipA linker or eliminating charge of a specific VipB residue abolishes sheath contraction and delivery of effectors into target cells. Mass spectrometry analysis identified the inner tube protein Hcp, spike protein VgrG, and other components of the T6SS baseplate significantly enriched in samples of the stable non-contractile sheaths. The ability to lock the T6SS in the pre-firing state opens new possibilities for understanding its mode of action.

Cryo-EM reconstruction of Type VI secretion system baseplate and sheath distal end.

The bacterial Type VI secretion system (T6SS) assembles from three major parts: a membrane complex that spans inner and outer membranes, a baseplate, and a sheath-tube polymer. The baseplate assembles around a tip complex with associated effectors and connects to the membrane complex by TssK. The baseplate assembly initiates sheath-tube polymerization, which in some organisms requires TssA. Here, we analyzed both ends of isolated non-contractile Vibrio cholerae sheaths by cryo-electron microscopy. Our analysis suggests that the baseplate, solved to an average 8.0 Å resolution, is composed of six subunits of TssE/F2/G and the baseplate periphery is decorated by six TssK trimers. The VgrG/PAAR tip complex in the center of the baseplate is surrounded by a cavity, which may accommodate up to ~450 kDa of effector proteins. The distal end of the sheath, resolved to an average 7.5 Å resolution, shows sixfold symmetry; however, its protein composition is unclear. Our structures provide an important step toward an atomic model of the complete T6SS assembly.