The Type VI Secretion System: A Dynamic System for Bacterial Communication?

1Bacteria are perpetually at war against multiple competitors and thus require weapons to conquer new territory or persist in an ecological niche. Among the mechanisms that aid in the struggle against other bacterial species are the Type VI Secretion Systems (T6SSs). The T6SSs of Gram-negative bacteria are effector translocation apparatuses, resembling an inverted bacteriophage-puncturing device, composed of at least 13 proteins called core-components (TssA-M, for Type six secretion). Auxiliary components can be associated with these conserved proteins (Tag, for Type six secretion associated genes). T6SSs participate in a broad variety of functions, including virulence and antibacterial activity. T6SSs also participate in metal ion uptake, such as that of iron, manganese, and zinc, conferring an advantage during bacteria–bacteria competition. In this review, we provide an overview of the data on T6SS assembly and emphasize connections between T6SSs and bacterial communication.

2Type VI Secretion System contractile nanomachines allow bacteria to inject toxins directly into prey cell membranes or cytoplasm. The machinery of the T6SS is assembled in an orderly manner. It starts with membrane complex formation, allowing baseplate positioning. The baseplate serves as a platform for contractile tail elongation. Contraction of the sheath propels effectors across membranes. Finally, the ATPase, TssH (ClpV), recycles the sheath and probably other T6SS components such as TssA, whereas the membrane-anchoring complex can be used to fire a new salvo (Figure 1).

3Type VI Secretion Systems are anchored to the cell envelope by a membrane core complex, which serves as a T6SS docking station and platform for baseplate assembly and prevents membrane cell damage during effector injection. The membrane core complex is a 1.7 MDa structure with a fivefold symmetry composed of 10 heterotrimeric complexes containing the three proteins TssJ, TssL, and TssM. Hierarchical biogenesis of this complex is initiated by the insertion of the lipoprotein TssJ in the outer membrane. TssJ then interacts with the large periplasmic domain of the inner membrane protein TssM. The cytoplasmic domain of TssM interacts with the inner membrane protein TssL and the cytoplasmic domain of another TssM subunit, thus enabling oligomerization. Similarly, the cytoplasmic domain of TssL mediates self-polymerization. The TssM periplasmic domain recruits a lytic transglycosylase (LTG), which is required for local peptidoglycan layer degradation, necessary for proper assembly of the 1.7 MDa TssJLM complex (Figure 1, assembly steps 1 and 2). Associated proteins with a peptidoglycan-binding domain, such as TagL, which can bind to truncated TssL, can associate with the membrane complex. TagL corresponds in this case to an “ancestral TssL” domain.

4The T6SS baseplate complex, composed of TssA, TssE, TssF, TssG, TssK, and Valine-glycine repeat protein G (VgrG or TssI) proteins, is recruited by the membrane complex. This structure serves as a platform for contractile sheath assembly and is essential for the correct assembly of the inner tube, comprised of hexameric rings of Hemolysin-coregulated protein (Hcp or TssD). TssA forms a dodecamer complex, which first binds to the membrane complex (Figure 1, assembly step 3). Positioning of the baseplate complex may be initiated by recruitment of TssE, TssK, and VgrG via TssA. The cytoplasmic domains of TssM and TssL, located at the base of the membrane complex, interact with the TssG/TssK and TssK/TssE baseplate subunits, respectively. The baseplate complex thus forms an interface between the membrane complex and the T6SS tail: both the Hcp and TssC sheath subunits interact with baseplate components (Figure 1, assembly step 4). After TssE recruitment, TssA likely properly attaches the sheath onto the baseplate and/or stabilizes the sheath structure.

5The TssE baseplate component may initiate sheath assembly and anchors the sheath to the baseplate. Hcp proteins assemble into hexameric rings, stacked head-to-tail, under the control of baseplate components. The TssBC sheath component then wraps around the inner Hcp tube. Formation of the inner tube precedes TssBC assembly and is primordial for proper stacking of the subunits. The Hcp tube has an inner diameter of ∼40 Å, forming a lumen with a neutral surface, suggesting passive effector translocation into the Hcp tube. The diameter of the internal sheath, measuring approximately 100–110 Å, coincides with the ∼80–85 Å outer diameter of the Hcp hexamer. The TssA dodecamer is located at the distal end of the tail in Escherichia coli, whereas TssA1 is a component of the baseplate/tail subcomplex in Pseudomonas aeruginosa. The TssA complex appears to possess short, flexible arm-like extensions, which may grasp TssBC or Hcp and incorporate them, one by one, at the distal end of the contractile tail. Moreover, the diameter of the central channel of the ring-shaped TssA structure measures approximately 100 Å, comparable to the dimension of the Hcp hexamers. Hcp components perhaps pass through the large central lumen of the TssA dodecamer (Figure 1, assembly step 4) and are added to the growing structure. Contrary to bacteriophages, the length of the T6SS tail does not appear to be controlled by a specialized protein. The length of the T6SS tail can exceed 1 μm. It is possible that contact with the opposite cell membrane is the physical signal to stop tail elongation (Figure 1, assembly step 5).

6demonstrated that the sheath of Francisella tularensis has a quaternary structure with handedness opposite to that of the contracted sheath of T4 phage tails. The sheath contracts within a few milliseconds, propelling the Hcp-VgrG spike and effectors, punching either indiscriminately or in a focused manner into neighboring bacteria. The sheath contracts and becomes shorter and wider than in the extended state. Once the sheath is contracted, the ClpV recognition motif of TssC, which is buried in the elongated state, becomes accessible, permitting TssBC recycling by the ATPase. Thus, TssB and TssC can be reused for a new round of sheath elongation (Figure 1, assembly steps 6 and 7). An alternative mode of sheath disassembly may involve the TagJ accessory protein. TagJ is structurally related to particular TssA C-terminal extensions. In this case, TagJ interacts with TssB and recruits ClpV to the sheath. ClpV can also interact with TssA and may be involved in recycling TssA rings.

7The puncturing device, consisting of the VgrG trimer, is located at the top of the inner tube and may be crucial for piercing the prey cell envelope. The VgrG trimer sometimes terminates with a Pro-Ala-Ala-Arg (PAAR)-repeat-containing protein, sharpening the tip.

8Effectors transported by T6SS fall into two groups: “specialized” effectors and “cargo” effectors. Specialized effectors are extension domains of a structural component, whereas cargo effectors interact directly with VgrG, PAAR, or Hcp proteins, with or without the help of accessory proteins. Four main classes of antibacterial effectors have been described, according to the target (Figure 1). Peptidoglycan targeting effectors are comprised of both Type six amidase effectors (Tae) and Type six glycoside hydrolase effectors (Tge). Type six lipase effectors (Tle) hydrolyse membrane phospholipids, whereas Type six DNase effectors (Tde) have nuclease activity. Some toxins do not belong to any of these four main classes. Pore-forming toxins, such as VasX from Vibrio cholerae, disrupt the inner membrane integrity of target cells. Whitney and collaborators identified a NAD(P)+ glycohydrolase toxin in P. aeruginosa. This toxin depletes cellular NAD(P)+ levels and induces bacteriostasis. The T6SS is not only an injection mechanism, it also enables the release of a proteinaceous metallophore into the extracellular medium and plays a role in the transport of Mn2+ under conditions of oxidative stress and iron uptake.

9Bacteria that secrete antibacterial toxins also produce immunity proteins, which interact with toxic effectors, to allow self-protection and prevent the killing of sibling cells (called Tai, Tgi, Tli, and Tdi, corresponding to their effector family). Immunity proteins and effector targets are located within the same cellular compartment. Therefore, tli genes encoding outer membrane lipoproteins or periplasmic exposed lipoproteins, the Tle, should act in the periplasm (Figure 1).

10Some other proteins secreted by the T6SS are involved in self-recognition, allowing communication between bacteria. In bacteria, secreted proteins are involved in many functions and are essential for bacterial fitness. In some strains, the T6SS is activated in response of T6SS aggression by neighboring bacteria during cell–cell contact. Thus, the T6SS can modulate the fitness of other bacteria. In addition, T6SSs can be active, even in pure culture, and the presence of Hcp and VgrG in the culture medium is often used as evidence of a functional T6SS. What purpose, however, does a functional T6SS serve in the absence of a competitor or prey?

11Pseudomonas aeruginosa is a widely-used model for T6SS studies. P. aeruginosa possesses three T6SS clusters comprised of TssA-M core component proteins. They are called H1-T6SS, H2-T6SS, and H3-T6SS. Among them, the H1-T6SS machinery is the most widely studied and is involved in antibacterial activity. The H2-T6SS and H3-T6SS are involved in virulence in eukaryotes but also display antibacterial activity by secreting trans-kingdom effectors, such as PldA and TplE via the H2-T6SS or PldB via the H3-T6SS machinery.

12Two types of T6SS behavior can be distinguished: that of defensive (targeted firing) and offensive cells (arbitrarily firing). P. aeruginosa can discern T6SS-mediated aggression of neighboring sister cells. Similarly, P. aeruginosa can perceive T6SS attacks coming from V. cholerae or Acinetobacter baylyi cells. In both cases, P. aeruginosa is first assaulted by a nearby bacterium and then attacks, in turn, the aggressive cell. This mechanism is called “T6SS dueling” and is mediated by the H1-T6SS. Thus, P. aeruginosa only counterattacks in response to T6SS firing of V. cholerae or A. baylyi. In general, P. aeruginosa does not target T6SS-defective bacteria, suggesting that it is a defensive bacterium, in contrast to offensive V. cholerae and A. baylyi strains. However, P. aeruginosa strains affected in the hybrid sensor kinase RetS, can attack T6SS-defective bacteria in a H1-T6SS-dependent manner. The perception of a T6SS attack involves the TagQRST threonine phosphorylation pathway, following envelope perturbation after T6SS-mediated perforation (Figure 2). Indeed, Polymyxin B, which alters cell membranes of Gram-negative bacteria, mediates activation of the T6SS, confirming that envelope perturbation triggers the T6SS counterattack. The TagQRST trans-membrane signaling cascade, essential for activation of the H1-T6SS of P. aeruginosa, is composed of four proteins. The ABC transporter complex TagST, anchored to the inner membrane, has ATPase activity. In this complex, TagT is required for T6SS activation after cell membrane perturbation. TagQ, an outer membrane lipoprotein, is necessary for outer membrane localisation of TagR, which is required for protein kinase PpkA phosphorylation. PpkA phosphorylates, in turn, Fha1 (which has a forkhead-associated domain), thus activating H1-T6SS assembly (Figure 2). The phosphatase PppA counteracts the role of PpkA by dephosphorylating Fha1. T6SS dueling appears to be an indirect means of communication, in which the occurrence of T6SS attacks may correlate with cell density. As the population increases, the likelihood of targeting sister cells also intensifies. In other words, as the population grows, the incidence of T6SS aggression rises. Thus, the perception of T6SS attacks provides an overall view of bacterial density and a form of social interaction (Figure 2).

13Two-Component Signal Transduction systems (TCSTs) are involved in external signal perception via a “sensor” and translate the signal via a “response regulator.” Thus, TCSTs play a key role in adaptive responses during environmental stress. GacS/GacA is a TCST in P. aeruginosa that perceives unknown signals and regulates numerous networks. The GacS/GacA cascade activates the transcription of the small RNAs (sRNAs) RsmZ and RsmY. RsmZ has a high affinity for the RNA-binding protein RsmA and can sequester it, permitting the translation of genes encoding H1-T6SS mRNAs. A rsmZ mutation results in downregulation of the transcription of genes encoding the H1-T6SS and H3-T6SS of P. aeruginosa. The GacS/GacA system is under the control of two hybrid sensors, RetS and LadS. The hybrid sensor kinase RetS decreases RsmZ/RsmY transcription via the inhibition of GacS/GacA phospho-relay. In contrast, LadS, enhances GacA phosphorylation via GacS. In summary, H1-T6SS is upregulated by the GacS/GacA/RsmZ regulatory pathway, which depends on the balance between RetS and LadS activation from external signals, unlike the H2-T6SS and H3-T6SS (Figure 2).

14Quorum Sensing (QS) is a system that allows social synchronization, based on the perception of population density, according to signal molecule concentration. QS is crucial for collective adaptive responses (similar to a social behavior) and regulates both bacterial virulence and biofilm formation. P. aeruginosa has four QS networks consisting of three classes of diffusible auto-inducers. The first class includes two types of Homoserine Lactones (HSLs): N-(3-oxododecanoyl)-Homoserine Lactones (odDHL, 3-oxo-C12-HSL) and N-butyrylhomoserine Lactones (BHL, C4-HSL) controlled by the Las and Rhl cascades, respectively. P. aeruginosa also produces Pseudomonas Quinolone Signal (PQS), 2-heptyl-3-hydroxy-4-quinolone, of which the production is regulated by the PqsR cascade (also known as MvfR). The last QS system consists of 2-(2-hydroxyphenyl)-thiazole-4-carbaldehyde, which is involved in the Integrated Quorum Sensing system (IQS). The IQS can enhance PQS production depending on the P. aeruginosa strain. These QS networks are all interconnected and positively regulated by the Las cascade. At the same time, RsmA is a negative post-transcriptional regulator of both 3-oxo-C12-HSL and C4-HSL production, affecting the Las/Rhl quorum sensing cascades. Similarly, PqsR is post-transcriptionally repressed by RsmA. However, the PQS system and Rhl cascade are upregulated via the Las pathway and PQS positively regulates the Rhl cascade. In summary, the GacS/GacA system is a global activator of QS communication, because the action of the RNA-binding protein RsmA is jointly antagonized by RsmY and RsmZ (Figure 2). At the same time, QS positively regulates H2-T6SS and H3-T6SS, whereas it suppresses H1-T6SS associated gene expression.

15A study published by revealed a link between the H3-T6SS and cell–cell signaling in P. aeruginosa. The cell–cell signaling compound PQS contributes to the formation of Outer Membrane Vesicles (OMVs) and associates with vesicle membranes. The PQS in OMVs can capture iron from the extracellular medium. In parallel, the protein TseF, secreted by the H3-T6SS, associates with OMVs containing PQS. The TseF from the OMVs then interacts with the Fe(III)-pyochelin receptor FptA and the porin OprF. This enables the delivery of PQS, associated with iron, into bacterial cells. Thus, the effector TseF delivered by the H3-T6SS is involved in the PQS pathway (Figure 2).

16Proteus mirabilis strains are able to recognize isogenic cells, coordinate multicellular swarming motility, and form macroscopic boundaries with non-sister cell swarms. Macroscopic demarcation, called Dienes lines, can be observed between swarming P. mirabilis strains. Functional T6SSs are involved in this recognition phenomenon in the region of inter-strain contact. This visible boundary requires physical cell–cell interactions and is the result of T6SS recognition. Indeed, cells at the intersection between two swarming populations of P. mirabilis appear to kill each other using their T6SS effectors. The T6SSs appear to assemble and fire deeply beyond the inter-strain boundary into the opposing swarming cells, thus enhancing T6SS effector injection. Some Identification of self (Ids) proteins, involved in self-recognition and territorial behavior, are exported by the T6SS. For example, IdsD mediates identity recognition between neighboring cells in a T6SS-dependant manner. IdsD interacts specifically with the cognate IdsE protein on the surface of recipient cells. The specific interaction between the two membrane-bound self-recognition proteins IdsD and IdsE drives social behavior. These binding interactions contribute to the definition of strain identity and discrimination between self and neighboring non-self cells. The lack of binding between IdsD and IdsE correlates with the formation of the visible boundary. The authors speculate that IdsE itself contributes to the repression of swarm colony expansion. Interaction between the two cognate proteins reduces swarm restriction. IdsD and IdsE proteins may constitute a lethal effector-immunity (toxin–antitoxin) system. Contrary to QS, which is based on contact-independent recognition, T6SS-associated recognition generally requires cell-to-cell contact. P. mirabilis uses the T6SS to discriminate between strains, coordinate multicellular swarming behavior, and direct its collective movement. Thus, the T6SS is essential for boundary formation and mediates cell–cell communication of swarming P. mirabilis via specific self-identity determinants.

17The P. fluorescens MFE01 strain, like numerous other P. fluorescens strains, does not produce the QS signals of P. aeruginosa (no HSL or PQS). MFE01 is an aggressive T6SS strain which contains a unique T6SS core component cluster and three orphan hcp genes. The MFE01 T6SS is involved in biofilm formation and maturation, as shown for other T6SSs. Indeed, P. fluorescens MFE01 is unable to form biofilms once the T6SS machinery is inactive (in a tssC mutant), whereas individual mutations of the three hcp genes affect biofilm maturation, but not formation. Intra-bacterial cooperation in conditions of biofilm formation via T6SS dueling could occur. Indeed, alterations of membrane phospholipid composition increase the ppGpp stress-response signal, which in turn causes the premature production of HSL-QS signals, including in P. aeruginosa (Figure 2). Similarly, communicating pathways could be activated following membrane perturbation due to T6SS perforation in MFE01 strain during “tit-for-tat” interactions.

18A recent study showed that bacteria can reuse T6SS components from attacking cells for new T6SS assembly (Figure 1). This suggests that an increase in cell density increases the concentration of T6SS components in bacteria and the ability of the cell to fire again, forming a positive feedback loop. We postulate that T6SS could be a cell-to-cell signal between sibling cells, depending on cell density, similar to the QS pathway, especially in bacteria devoid of QS signals.

19Writing, review, and editing: MG, AM, MB.

20The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.