Formylglycine-Generating Enzyme-Like Proteins Constitute a Novel Family of Widespread Type VI Secretion System Immunity Proteins.

Competition is a critical aspect of bacterial life, as it enables niche establishment and facilitates the acquisition of essential nutrients. Warfare between Gram-negative bacteria is largely mediated by the type VI secretion system (T6SS), a dynamic nanoweapon that delivers toxic effector proteins from an attacking cell to adjacent bacteria in a contact-dependent manner. Effector-encoding bacteria prevent self-intoxication and kin cell killing by the expression of immunity proteins, which neutralize effector toxicity by specifically binding their cognate effector and either occluding its active site or preventing the structural rearrangements necessary for effector activation. In this study, we investigate Tsi3, a previously uncharacterized T6SS immunity protein present in multiple strains of the human pathogen Acinetobacter baumannii. We show that Tsi3 is the cognate immunity protein of an antibacterial effector of unknown function, Tse3. Our bioinformatic analyses indicate that Tsi3 homologs are widespread among Gram-negative bacteria, often encoded within T6SS effector-immunity modules. Surprisingly, we found that Tsi3 homologs are predicted to possess a characteristic formylglycine-generating enzyme (FGE) domain, which is present in various enzymatic proteins. Our data show that Tsi3-mediated immunity is dependent on Tse3-Tsi3 protein-protein interactions and that Tsi3 homologs from various bacteria do not provide immunity against nonkin Tse3. Thus, we conclude that Tsi3 homologs are unlikely to be functional enzymes. Collectively, our work identifies FGE domain-containing proteins as important mediators of immunity against T6SS attacks and indicates that the FGE domain can be coopted as a scaffold in multiple proteins to carry out diverse functions. IMPORTANCE Despite the wealth of knowledge on the diversity of biochemical activities carried out by T6SS effectors, comparably little is known about the various strategies that bacteria employ to prevent susceptibility to T6SS-dependent bacterial killing. Our work establishes a novel family of T6SS immunity proteins with a characteristic FGE domain. This domain is present in enzymatic proteins with various catalytic activities. Our characterization of Tsi3 expands the known functions carried out by FGE-like proteins to include defense during T6SS-mediated bacterial warfare. Moreover, it highlights the evolution of FGE domain-containing proteins to carry out diverse biological functions.

Polymorphic Toxins and Their Immunity Proteins: Diversity, Evolution, and Mechanisms of Delivery.

All bacteria must compete for growth niches and other limited environmental resources. These existential battles are waged at several levels, but one common strategy entails the transfer of growth-inhibitory protein toxins between competing cells. These antibacterial effectors are invariably encoded with immunity proteins that protect cells from intoxication by neighboring siblings. Several effector classes have been described, each designed to breach the cell envelope of target bacteria. Although effector architectures and export pathways tend to be clade specific, phylogenetically distant species often deploy closely related toxin domains. Thus, diverse competition systems are linked through a common reservoir of toxin-immunity pairs that is shared via horizontal gene transfer. These toxin-immunity protein pairs are extraordinarily diverse in sequence, and this polymorphism underpins an important mechanism of self/nonself discrimination in bacteria. This review focuses on the structures, functions, and delivery mechanisms of polymorphic toxin effectors that mediate bacterial competition.

Identification of a new effector-immunity pair of Aeromonas hydrophila type VI secretion system.

The type VI secretion system (T6SS) is a multiprotein weapon that kills eukaryotic predators or prokaryotic competitors by delivering toxic effectors. Despite the importance of T6SS in bacterial environmental adaptation, it is still challenging to systematically identify T6SS effectors because of their high diversity and lack of conserved domains. In this report, we discovered a putative effector gene, U876-17730, in the whole genome of Aeromonas hydrophila NJ-35 based on the reported conservative domain DUF4123 (domain of unknown function), with two cognate immunity proteins encoded downstream. Phylogenetic tree analysis of amino acids indicates that AH17730 belongs to the Tle1 (type VI lipase effector) family, and therefore was named Tle1AH. The deletion of tle1AH resulted in significantly decreased biofilm formation, antibacterial competition ability and virulence in zebrafish (Danio rerio) when compared to the wild-type strain. Only when the two immunity proteins coexist can bacteria protect themselves from the toxicity of Tle1AH. Further study shows that Tle1AH is a kind of phospholipase that possesses a conserved lipase motif, Gly-X-Ser-X-Gly (X is for any amino acid). Tle1AH is secreted by T6SS, and this secretion requires its interaction with an associated VgrG (valine-glycine repeat protein G). In conclusion, we identified a T6SS effector-immunity pair and verified its function, which lays the foundation for future research on the role of T6SS in the pathogenic mechanism of A. hydrophila.

Envelope stress responses defend against type six secretion system attacks independently of immunity proteins.

The arms race among microorganisms is a key driver in the evolution of not only the weapons but also defence mechanisms. Many Gram-negative bacteria use the type six secretion system (T6SS) to deliver toxic effectors directly into neighbouring cells. Defence against effectors requires cognate immunity proteins. However, here we show immunity-independent protection mediated by envelope stress responses in Escherichia coli and Vibrio cholerae against a V. cholerae T6SS effector, TseH. We demonstrate that TseH is a PAAR-dependent species-specific effector highly potent against Aeromonas species but not against its V. cholerae immunity mutant or E. coli. A structural analysis reveals TseH is probably a NlpC/P60-family cysteine endopeptidase. We determine that two envelope stress-response pathways, Rcs and BaeSR, protect E. coli from TseH toxicity by mechanisms including capsule synthesis. The two-component system WigKR (VxrAB) is critical for protecting V. cholerae from its own T6SS despite expressing immunity genes. WigR also regulates T6SS expression, suggesting a dual role in attack and defence. This deepens our understanding of how bacteria survive T6SS attacks and suggests that defence against the T6SS represents a major selective pressure driving the evolution of species-specific effectors and protective mechanisms mediated by envelope stress responses and capsule synthesis.

Human gut bacteria contain acquired interbacterial defence systems.

The human gastrointestinal tract consists of a dense and diverse microbial community, the composition of which is intimately linked to health. Extrinsic factors such as diet and host immunity are insufficient to explain the constituents of this community, and direct interactions between co-resident microorganisms have been implicated as important drivers of microbiome composition. The genomes of bacteria derived from the gut microbiome contain several pathways that mediate contact-dependent interbacterial antagonism1-3. Many members of the Gram-negative order Bacteroidales encode the type VI secretion system (T6SS), which facilitates the delivery of toxic effector proteins into adjacent cells4,5. Here we report the occurrence of acquired interbacterial defence (AID) gene clusters in Bacteroidales species that reside within the human gut microbiome. These clusters encode arrays of immunity genes that protect against T6SS-mediated intra- and inter-species bacterial antagonism. Moreover, the clusters reside on mobile elements, and we show that their transfer is sufficient to confer resistance to toxins in vitro and in gnotobiotic mice. Finally, we identify and validate the protective capability of a recombinase-associated AID subtype (rAID-1) that is present broadly in Bacteroidales genomes. These rAID-1 gene clusters have a structure suggestive of active gene acquisition and include predicted immunity factors of toxins derived from diverse organisms. Our data suggest that neutralization of contact-dependent interbacterial antagonism by AID systems helps to shape human gut microbiome ecology.

PAAR proteins act as the 'sorting hat' of the type VI secretion system.

Bacteria exist in polymicrobial environments and compete to prevail in a niche. The type VI secretion system (T6SS) is a nanomachine employed by Gram-negative bacteria to deliver effector proteins into target cells. Consequently, T6SS-positive bacteria produce a wealth of antibacterial effector proteins to promote their survival among a prokaryotic community. These toxins are loaded onto the VgrG-PAAR spike and Hcp tube of the T6SS apparatus and recent work has started to document the specificity of effectors for certain spike components. Pseudomonas aeruginosa encodes several PAAR proteins, whose roles have been poorly investigated. Here we describe a phospholipase family antibacterial effector immunity pair from Pseudomonas aeruginosa and demonstrate that a specific PAAR protein is necessary for the delivery of the effector and its cognate VgrG. Furthermore, the PAAR protein appears to restrict the delivery of other phospholipase effectors that utilise distinct VgrG proteins. We provide further evidence for competition for PAAR protein recruitment to the T6SS apparatus, which determines the identities of the delivered effectors.

Immunogenicity and protective efficacy of mucosal delivery of recombinant hcp of Campylobacter jejuni Type VI secretion system (T6SS) in chickens.

The type VI secretion system (T6SS) has recently emerged as a new pattern of protein secretions in Campylobacter jejuni (C. jejuni). Within the T6SS cluster, hemolysin co-regulated protein (hcp) is considered as a hallmark of functional T6SS and holds key role in bacterial virulence. As poultry is the primary reservoir of C. jejuni and the major sources for human infection, we evaluated the capacity of recombinant hcp (rhcp) immunization in blocking C. jejuni colonization in chickens with an aim to control bacterial transmission to humans via poultry food chain. Considering the mucosal route is the primary portal for C. jejuni entry and gut mucosa offers the apposite site for C. jejuni adherence, we investigated the immune-protective potential of intra-gastric administration of rhcp using chitosan-based nanoparticles. To achieve this goal, full length coding sequence of hcp gene from C. jejuni was cloned and expressed in E. coli. Purified rhcp was entrapped in chitosan-Sodium tripolyphosphate nanoparticles (CS-TPP NPs) and orally gavaged in chickens. Our results suggest that intra-gastric immunization of CS-TPP-rhcp induces consistent and steady increase in intestinal (sIgA) and systemic antibody (IgY) response against rhcp with significant reduction in cecal load of C. jejuni. The protection afforded by rhcp associated cellular responses with Th1 and Th17 profile in terms of increased expression of NFkB, IL-1ß, IL-8, IL-6, IFN-γ and IL-17 A genes. Though systemic immunization of rhcp with IFA resulting in a robust systemic (IgY) and local (sIgA) antibody response, mucosal administration of rhcp loaded CS-TPP NPs was found to be superior in terms of bacterial clearance. Altogether, present study suggests that chitosan based intra-gastric delivery of rhcp have several advantages over the injectable composition and could be a promising vaccine approach to effectively control C. jejuni colonization in chickens.

Type VI Secretion Systems and the Gut Microbiota.

The human colonic microbiota is a dense ecosystem comprised of numerous microbes, including bacteria, phage, fungi, archaea, and protozoa, that compete for nutrients and space. Studies are beginning to reveal the antagonistic mechanisms that gut bacteria use to compete with other members of this ecosystem. In the healthy human colon, the majority of the Gram-negative bacteria are of the order Bacteroidales. Proteobacteria, such as Escherichia coli, are numerically fewer but confer important properties to the host, such as colonization resistance. Several enteric pathogens use type VI secretion systems (T6SSs) to antagonize symbiotic gut E. coli, facilitating colonization and disease progression. T6SS loci are also widely distributed in human gut Bacteroidales, which includes three predominant genera: Bacteroides, Parabacteroides, and Prevotella. There are three distinct genetic architectures of T6SS loci among the gut Bacteroidales, termed GA1, GA2, and GA3. GA1 and GA2 T6SS loci are contained on integrative and conjugative elements and are the first T6SS loci shown to be readily transferred in the human gut between numerous species and families of Bacteroidales. In contrast, the GA3 T6SSs are present exclusively in Bacteroides fragilis. There are divergent regions in all three T6SS GAs that contain genes encoding effector and immunity proteins, many of which function by unknown mechanisms. To date, only the GA3 T6SSs have been shown to antagonize bacteria, and they target nearly all gut Bacteroidales species analyzed. This review delves more deeply into properties of the T6SSs of these human gut bacteria and the ecological outcomes of their synthesis in vivo.

Characterization of multiple type-VI secretion system (T6SS) VgrG proteins in the pathogenicity and antibacterial activity of porcine extra-intestinal pathogenic Escherichia coli.

Porcine extra-intestinal pathogenic Escherichia coli (ExPEC) causes great economic losses to the pig industry and poses a serious threat to public health worldwide. Some secreted virulence factors have been reported to be involved in the pathogenicity of the infection caused by ExPEC. Type-VI secretion system (T6SS) is discovered in many Gram-negative bacteria and contributes to the virulence of pathogenic bacteria. Valine-glycine repeat protein G (VgrG) has been reported as an important component of the functional T6SS. In our previous studies, a functional T6SS was identified in porcine ExPEC strain PCN033. Further analysis of the PCN033 genome identified two putative vgrGs genes (vgrG1 and 0248) located inside T6SS cluster and another two (vgrG2 and 1588) outside it. This study determined the function of the four putative VgrG proteins by constructing a series of mutants and complemented strains. In vitro, the VgrG1 protein was observed to be involved in the antibacterial ability and the interactions with cells. The animal model experiment showed that the deletion of vgrG1 significantly led to the decrease in the multiplication capacity of PCN033. However, the deletion of 0248 and/or the deletion of vgrG2 and 1588 had no effect on the pathogenicity of PCN033. The study of four putative VgrGs in PCN033 indicated that only VgrG1 plays an important role in the interaction between PCN033 and other bacteria or host cells. This study can provide a novel perspective to the pathogenesis of PCN033 and lay the foundation for discovering potential T6SS effectors.

Subtle variation within conserved effector operon gene products contributes to T6SS-mediated killing and immunity.

Type VI secretion systems (T6SS) function to deliver lethal payloads into target cells. Many studies have shown that protection against a single, lethal T6SS effector protein requires a cognate antidote immunity protein, both of which are often encoded together in a two-gene operon. The T6SS and an effector-immunity pair is sufficient for both killing and immunity. HereIn this paper we describe a T6SS effector operon that differs from conventional effector-immunity pairs in that eight genes are necessary for lethal effector function, yet can be countered by a single immunity protein. In this study, we investigated the role that the PefE T6SS immunity protein plays in recognition between two strains harboring nearly identical effector operons. Interestingly, despite containing seven of eight identical effector proteins, the less conserved immunity proteins only provided protection against their native effectors, suggesting that specificity and recognition could be dependent on variation within an immunity protein and one effector gene product. The variable effector gene product, PefD, is encoded upstream from pefE, and displays toxic activity that can be countered by PefE independent of T6SS-activity. Interestingly, while the entire pef operon was necessary to exert toxic activity via the T6SS in P. mirabilis, production of PefD and PefE alone was unable to exert this effector activity. Chimeric PefE proteins constructed from two P. mirabilis strains were used to localize immunity function to three amino acids. A promiscuous immunity protein was created using site-directed mutagenesis to change these residues from one variant to another. These findings support the notion that subtle differences between conserved effectors are sufficient for T6SS-mediated kin discrimination and that PefD requires additional factors to function as a T6SS-dependent effector.

Francisella requires dynamic type VI secretion system and ClpB to deliver effectors for phagosomal escape.

Francisella tularensis is an intracellular pathogen that causes the fatal zoonotic disease tularaemia. Critical for its pathogenesis is the ability of the phagocytosed bacteria to escape into the cell cytosol. For this, the bacteria use a non-canonical type VI secretion system (T6SS) encoded on the Francisella pathogenicity island (FPI). Here we show that in F. novicida T6SS assembly initiates at the bacterial poles both in vitro and within infected macrophages. T6SS dynamics and function depends on the general purpose ClpB unfoldase, which specifically colocalizes with contracted sheaths and is required for their disassembly. T6SS assembly depends on iglF, iglG, iglI and iglJ, whereas pdpC, pdpD, pdpE and anmK are dispensable. Importantly, strains lacking pdpC and pdpD are unable to escape from phagosome, activate AIM2 inflammasome or cause disease in mice. This suggests that PdpC and PdpD are T6SS effectors involved in phagosome rupture.

Sequential displacement of Type VI Secretion System effector genes leads to evolution of diverse immunity gene arrays in Vibrio cholerae.

Type VI secretion systems (T6SS) enable bacteria to engage neighboring cells in contact-dependent competition. In Vibrio cholerae, three chromosomal clusters each encode a pair of effector and immunity genes downstream of those encoding the T6SS structural machinery for effector delivery. Different combinations of effector-immunity proteins lead to competition between strains of V. cholerae, which are thought to be protected only from the toxicity of their own effectors. Screening of all publically available V. cholerae genomes showed that numerous strains possess long arrays of orphan immunity genes encoded in the 3' region of their T6SS clusters. Phylogenetic analysis reveals that these genes are highly similar to those found in the effector-immunity pairs of other strains, indicating acquisition by horizontal gene transfer. Extensive genomic comparisons also suggest that successive addition of effector-immunity gene pairs replaces ancestral effectors, yet retains the cognate immunity genes. The retention of old immunity genes perhaps provides protection against nearby kin bacteria in which the old effector was not replaced. This mechanism, combined with frequent homologous recombination, is likely responsible for the high diversity of T6SS effector-immunity gene profiles observed for V. cholerae and closely related species.

Francisella tularensis Live Vaccine Strain deficient in capB and overexpressing the fusion protein of IglA, IglB, and IglC from the bfr promoter induces improved protection against F. tularensis respiratory challenge.

A safer and more effective vaccine than the unlicensed Francisella tularensis Live Vaccine Strain (LVS) is needed to protect against the biowarfare agent F. tularensis. Previously, we developed an LVS ΔcapB mutant that is significantly safer than LVS and provides potent protective immunity against F. tularensis respiratory challenge when administered intranasally but limited protection when administered intradermally unless as part of a prime-boost vaccination strategy. To improve the immunogenicity and efficacy of LVS ΔcapB, we developed recombinant LVS ΔcapB (rLVS ΔcapB) strains overexpressing various F. tularensis Francisella Pathogenicity Island (FPI) proteins - IglA, IglB and IglC, and a fusion protein (IglABC) comprising immunodominant epitopes of IglA, IglB, and IglC downstream of different Francisella promoters, including the bacterioferritin (bfr) promoter. We show that rLVS ΔcapB/bfr-iglA, iglB, iglC, and iglABC express more IglA, IglB, IglC or IglABC than parental LVS ΔcapB in broth and in human macrophages, and stably express FPI proteins in macrophages and mice absent antibiotic selection. In response to IglC and heat-inactivated LVS, spleen cells from mice immunized intradermally with rLVS ΔcapB/bfr-iglC or bfr-iglABC secrete greater amounts of interferon-gamma and/or interleukin-17 than those from mice immunized with LVS ΔcapB, comparable to those from LVS-immunized mice. Mice immunized with rLVS ΔcapB/bfr-iglA, iglB, iglC or iglABC produce serum antibodies at levels similar to LVS-immunized mice. Mice immunized intradermally with rLVS ΔcapB/bfr-iglABC and challenged intranasally with virulent F. tularensis Schu S4 survive longer than sham- and LVS ΔcapB-immunized mice. Mice immunized intranasally with rLVS ΔcapB/bfr-iglABC - but not with LVS - just before or after respiratory challenge with F. tularensis Schu S4 are partially protected; protection is correlated with induction of a strong innate immune response. Thus, rLVS ΔcapB/bfr-iglABC shows improved immunogenicity and protective efficacy compared with parental LVS ΔcapB and, in contrast to LVS, has partial efficacy as immediate pre- and post-exposure prophylaxis.

Human symbionts inject and neutralize antibacterial toxins to persist in the gut.

The human gut microbiome is a dynamic and densely populated microbial community that can provide important benefits to its host. Cooperation and competition for nutrients among its constituents only partially explain community composition and interpersonal variation. Notably, certain human-associated Bacteroidetes--one of two major phyla in the gut--also encode machinery for contact-dependent interbacterial antagonism, but its impact within gut microbial communities remains unknown. Here we report that prominent human gut symbionts persist in the gut through continuous attack on their immediate neighbors. Our analysis of just one of the hundreds of species in these communities reveals 12 candidate antibacterial effector loci that can exist in 32 combinations. Through the use of secretome studies, in vitro bacterial interaction assays and multiple mouse models, we uncover strain-specific effector/immunity repertoires that can predict interbacterial interactions in vitro and in vivo, and find that some of these strains avoid contact-dependent killing by accumulating immunity genes to effectors that they do not encode. Effector transmission rates in live animals can exceed 1 billion events per minute per gram of colonic contents, and multiphylum communities of human gut commensals can partially protect sensitive strains from these attacks. Together, these results suggest that gut microbes can determine their interactions through direct contact. An understanding of the strategies human gut symbionts have evolved to target other members of this community may provide new approaches for microbiome manipulation.

Bacteroides fragilis type VI secretion systems use novel effector and immunity proteins to antagonize human gut Bacteroidales species.

Type VI secretion systems (T6SSs) are multiprotein complexes best studied in Gram-negative pathogens where they have been shown to inhibit or kill prokaryotic or eukaryotic cells and are often important for virulence. We recently showed that T6SS loci are also widespread in symbiotic human gut bacteria of the order Bacteroidales, and that these T6SS loci segregate into three distinct genetic architectures (GA). GA1 and GA2 loci are present on conserved integrative conjugative elements (ICE) and are transferred and shared among diverse human gut Bacteroidales species. GA3 loci are not contained on conserved ICE and are confined to Bacteroides fragilis Unlike GA1 and GA2 T6SS loci, most GA3 loci do not encode identifiable effector and immunity proteins. Here, we studied GA3 T6SSs and show that they antagonize most human gut Bacteroidales strains analyzed, except for B. fragilis strains with the same T6SS locus. A combination of mutation analyses,trans-protection analyses, and in vitro competition assays, allowed us to identify novel effector and immunity proteins of GA3 loci. These proteins are not orthologous to known proteins, do not contain identified motifs, and most have numerous predicted transmembrane domains. Because the genes encoding effector and immunity proteins are contained in two variable regions of GA3 loci, GA3 T6SSs of the species B. fragilis are likely the source of numerous novel effector and immunity proteins. Importantly, we show that the GA3 T6SS of strain 638R is functional in the mammalian gut and provides a competitive advantage to this organism.

Type VI Secretion System Transports Zn2+ to Combat Multiple Stresses and Host Immunity.

Type VI secretion systems (T6SSs) are widespread multi-component machineries that translocate effectors into either eukaryotic or prokaryotic cells, for virulence or for interbacterial competition. Herein, we report that the T6SS-4 from Yersinia pseudotuberculosis displays an unexpected function in the transportation of Zn2+ to combat diverse stresses and host immunity. Environmental insults such as oxidative stress induce the expression of T6SS-4 via OxyR, the transcriptional factor that also regulates many oxidative response genes. Zinc transportation is achieved by T6SS-4-mediated translocation of a novel Zn2+-binding protein substrate YezP (YPK_3549), which has the capacity to rescue the sensitivity to oxidative stress exhibited by T6SS-4 mutants when added to extracellular milieu. Disruption of the classic zinc transporter ZnuABC together with T6SS-4 or yezP results in mutants that almost completely lost virulence against mice, further highlighting the importance of T6SS-4 in resistance to host immunity. These results assigned an unconventional role to T6SSs, which will lay the foundation for studying novel mechanisms of metal ion uptake by bacteria and the role of this process in their resistance to host immunity and survival in harmful environments.

Role of the Bacterial Type VI Secretion System in the Modulation of Mammalian Host Cell Immunity.

The type VI Secretion System (T6SS) is a tool for Gram-negative pathogens to interact with other bacteria as well as with the eukaryotic host cell. While the role of T6SS in interbacterial interactions has drawn much attention in recent years, research into the T6SS as a human virulence factor continues at a slower pace. Nevertheless, T6SS has been shown to interfere with eukaryotic host cell immunity at several levels, ranging from direct attack of the host cell to attenuation of disease, allowing the pathogen to survive longer in the host environment. In this review, we aim to give a comprehensive overview focused on the ways bacteria use their T6SS in the modulation of mammalian host cell immunity. While doing so, we attempt to describe potential new avenues of research, as well as outline the ways in which T6SS could become a therapeutic target allowing to circumvent existing antibiotic resistance. Although much work remains to be done, a better comprehension of the T6SS mechanisms of action will undoubtedly lead to new strategies to counteract T6SS-bearing pathogens.

Inhibition of type VI secretion by an anti-TssM llama nanobody.

The type VI secretion system (T6SS) is a secretion pathway widespread in Gram-negative bacteria that targets toxins in both prokaryotic and eukaryotic cells. Although most T6SSs identified so far are involved in inter-bacterial competition, a few are directly required for full virulence of pathogens. The T6SS comprises 13 core proteins that assemble a large complex structurally and functionally similar to a phage contractile tail structure anchored to the cell envelope by a trans-membrane spanning stator. The central part of this stator, TssM, is a 1129-amino-acid protein anchored in the inner membrane that binds to the TssJ outer membrane lipoprotein. In this study, we have raised camelid antibodies against the purified TssM periplasmic domain. We report the crystal structure of two specific nanobodies that bind to TssM in the nanomolar range. Interestingly, the most potent nanobody, nb25, competes with the TssJ lipoprotein for TssM binding in vitro suggesting that TssJ and the nb25 CDR3 loop share the same TssM binding site or causes a steric hindrance preventing TssM-TssJ complex formation. Indeed, periplasmic production of the nanobodies displacing the TssM-TssJ interaction inhibits the T6SS function in vivo. This study illustrates the power of nanobodies to specifically target and inhibit bacterial secretion systems.