The type VI secretion system of Acinetobacter: mechanisms, biology and therapeutic potential

Abstract

The Type VI secretion system (T6SS) is widely recognized as a contractile nanomachine that mediates interbacterial antagonism, yet its biological roles and evolutionary logic vary substantially across bacterial lineages. In this Review, we synthesize recent advances in the Acinetobacter T6SS field and propose a unifying perspective in which the system functions as a context-dependent fitness module rather than a constitutive virulence weapon. We highlight how Acinetobacter has rewired a single T6SS platform through non-canonical structural solutions, multilayered regulatory integration, and an unusually expansive effector repertoire. Beyond microbial competition, emerging clinical and experimental evidence links T6SS activity to host immune amplification, disease severity, and the dynamics of horizontal gene transfer and antibiotic resistance. By integrating structural biology, regulatory logic, effector function, and clinical observations, this Review reframes the Acinetobacter T6SS as an adaptable system that balances aggression, persistence, and metabolic cost in polymicrobial and host-associated environments. This perspective not only advances conceptual understanding of T6SS diversity but also highlights translational opportunities for diagnostics, vaccines, and anti-virulence strategies targeting multidrug-resistant Acinetobacter infections.

We synthesize advances in Acinetobacter T6SS biology, revealing how non-canonical structural solutions and multilayered regulation create an adaptable system for competition, persistence, and host immune modulation.

Introduction

The genus Acinetobacter comprises a diverse group of Gram-negative, strictly aerobic coccobacilli that are ubiquitously distributed in natural environments such as soil, freshwater, and marine ecosystems^1,2. While many Acinetobacter species are considered environmental or commensal organisms with limited pathogenic potential, a subset has emerged as clinically relevant opportunistic pathogens. These include Acinetobacter baumannii, A. nosocomialis, A. pittii, A. seifertii, and A. calcoaceticus, which together constitute the A. calcoaceticus–baumannii (ACB) complex frequently isolated from human infections². Among them, A. baumannii has gained particular notoriety in healthcare settings, where it is a leading cause of hospital-acquired infections such as ventilator-associated pneumonia, bloodstream infections, and wound infections³. Its extraordinary capacity to survive under harsh conditions and to acquire antibiotic resistance determinants has led to the global emergence of multidrug-resistant (MDR) and pan-drug-resistant strains^3–5. Consequently, carbapenem-resistant A. baumannii has been designated by the World Health Organization as a “critical priority” pathogen, reflecting its high mortality burden, limited therapeutic options, and major contribution to antimicrobial resistance–associated deaths worldwide (https://www.who.int/publications/i/item/9789240093461). These features underscore the urgent need to understand not only the virulence of pathogenic Acinetobacter, but also the broader ecological strategies that allow members of this genus to persist, compete, and dominate in diverse niches.

A major mechanism by which bacteria adapt to complex and competitive environments is the deployment of specialized secretion systems. Among these, the Type VI secretion system (T6SS) has emerged as one of the most versatile and widespread molecular machines in Gram-negative bacteria^6,7. First identified in Vibrio cholerae and Pseudomonas aeruginosa, the T6SS is now recognized as a phage tail–like contractile apparatus capable of injecting toxic effector proteins directly into neighboring cells^8–10. Over the past two decades, research on the T6SS has expanded rapidly, revealing its central roles in interbacterial antagonism, niche competition, community structuring, and, in certain contexts, interactions with eukaryotic hosts⁶. Mechanistically, the T6SS consists of a membrane-anchored baseplate, a contractile sheath, and an inner tube tipped with a spike complex, which together function as a spring-loaded nanoweapon^6,11. Upon sheath contraction, effector-loaded spike–tube assemblies are propelled into adjacent cells, delivering a diverse repertoire of toxic cargos^6,7,12. Studies across multiple bacterial genera have demonstrated that T6SS activity is tightly regulated and often coordinated with environmental cues, population density, and stress responses, highlighting its role as a context-dependent rather than constitutively active weapon^6,12.

Within the Acinetobacter genus, and particularly among pathogenic members of the ACB complex, the T6SS represents a key strategy for survival in hostile and polymicrobial environments. Notably, unlike organisms such as P. aeruginosa, Serratia marcescens, Neisseria spp. or Klebsiella pneumoniae, which often harbor multiple T6SS clusters with specialized functions^13–16, pathogenic Acinetobacter species typically encode a single T6SS locus¹⁷. Accumulating evidence suggests that the Acinetobacter T6SS differs from classical T6SS architectures in both composition and regulation, and is embedded within an unusually multilayered regulatory network that integrates chromosomal and plasmid-borne signals^18–20. In parallel, bioinformatic and comparative genomic analyses reveal that the Acinetobacter genus encodes a remarkably large and diverse repertoire of putative T6SS effectors, encompassing a wide range of predicted structural architectures and catalytic domains^17,21. However, despite this apparent richness, only a small fraction of these effectors has been experimentally characterized to date, and the biochemical activities, delivery mechanisms, and ecological roles of most predicted toxins remain unknown. This gap highlights a central challenge in the field: understanding how a single T6SS platform in Acinetobacter supports such functional diversity and how effector deployment contributes to ecological fitness and pathogenic success.

In this review, we summarize recent advances in the study of the T6SS in the Acinetobacter genus, with a particular focus on its unique architectural features, multilayered regulatory mechanisms, and expanding effector repertoire. We integrate insights from structural biology, genetics, microbiology, and clinical studies to highlight emerging concepts and unresolved questions. Finally, we discuss methodological advances and future directions, and consider how a deeper understanding of Acinetobacter T6SS biology may inform new strategies to combat infections caused by multidrug-resistant Acinetobacter species, at a time when therapeutic options are increasingly limited.

Structural of the Acinetobacter T6SS

In most Gram-negative bacteria, the T6SS is built from a conserved set of ~13 core components that assemble into three functional modules: the membrane complex (TssJ–TssL–TssM), the baseplate complex (TssE–TssF–TssG–TssK), and the contractile tail complex comprising an inner Hcp tube and an outer TssB/TssC sheath, capped by a VgrG/PAAR spike and recycled by the ClpV ATPase (Fig. 1A). Rather than expanding T6SS copy number or functional specialization, as observed in several other pathogens^13–15, Acinetobacter species appear to have modified key architectural nodes of a single T6SS to support broad functionality and tight regulation—an arrangement that is increasingly recognized as structurally and operationally non-canonical in this genus.

Fig. 1. Structural comparison of assembly mechanisms for canonical and non-canonical Type VI secretion systems.

A Assembly model of the canonical, TssJ-dependent T6SS. The process initiates with the formation of a trans-envelope membrane complex (MC). The outer membrane (OM, grey bilayer) lipoprotein TssJ (red ovals) anchors the transmembrane protein TssM (green pillars) to the OM, while TssL (light purple units) anchors the complex at the inner membrane (IM, grey bilayer). Following MC completion, the cytoplasmic baseplate complex (orange and brown subunits) docks to TssM/TssL, and the VgrG/PAAR spike (purple spike) is loaded. Finally, the cytoplasmic tail, comprising the inner Hcp tube (yellow) and the outer contractile TssB/C sheath (blue and yellow rings), polymerizes downwards from the baseplate, extending into the cytoplasm to reach a primed, high-tension state. B Proposed model for the dynamic assembly of the non-canonical, TssJ-independent T6SS in Acinetobacter baumannii. The assembly proceeds in three stages. In the Sensing & Foundation stage, the process is triggered by OmpA-mediated contact sensing (blue barrel in OM), establishing a specific inner membrane foundation via the interaction between the scaffold protein TsmK (purple cylinders) and the transmembrane anchor TssL (light purple units). During the Tunnel Extension & PG Remodeling stage, TssM (green) polymerizes from the IM foundation. A specific flexible GS-linker (dashed lines) within TssM is crucial for the oligomerization of the TssM periplasmic domain. This growing structure is stabilized by TslA (light purple ovals) in the periplasm, while the glycoside hydrolase TagX (red shapes) locally degrades the peptidoglycan (PG) layer (purple mesh) to facilitate passage. Note the absence of TssJ in the OM. In the Assembly Complete & Priming stage, the fully assembled TssM channel forms a rigid trans-envelope tunnel. The GS-linker functions as a molecular spring or orientation guide, allowing the complex to cross the OM in the absence of TssJ. The VgrG1 spike punctures outwards through the OM, and the Hcp tube/TssB/C sheath dock onto the baseplate in the cytoplasm, completing the super-complex.

A central departure from the canonical blueprint is the absence of the outer-membrane lipoprotein TssJ in Acinetobacter²², which typically anchors and stabilizes the TssM–TssL trans-envelope channel in other bacteria²³. To compensate, Acinetobacter employs a specialized pentameric super-complex comprising lineage-adapted factors that remodel both the structural stability and spatiotemporal positioning of the apparatus. Early work identified the periplasmic protein AsaA (later renamed TslA) as a key accessory component that directly interacts with the TssM periplasmic domain and is indispensable for assembly¹⁹. Subsequent super-resolution live-cell imaging revealed that TslA is not merely structural but functions as a spatial regulator: it directs sheath initiation events specifically to sites of cell–cell contact by responding to the outer membrane porin OmpA, thereby linking membrane complex positioning to a contact-dependent firing mechanism²⁴. Most recently, structural analysis confirmed this non-canonical architecture, identifying the membrane-associated factor TsmK (formerly AsaB)—a repurposed polyketide synthase-like protein—as the inner-membrane foundation that stabilizes TssL and recruits TssM to the assembly site¹⁸. Together, these studies support a working model in which TsmK secures the complex at the inner membrane, while TslA stabilizes the extended TssM channel, specifically protecting and guiding its unique, lineage-specific Glycine-Serine-rich linker (GS-linker); this flexible linker is critical for enabling TssM to oligomerize and span the peptidoglycan layer to reach the outer membrane without the aid of TssJ. Through this intricate evolutionary remodeling, Acinetobacter has successfully engineered a functional alternative to the canonical membrane complex, preserving robust secretion capabilities despite significant structural divergence.

Beyond stabilizing the membrane core, TslA is also responsible for recruiting TagX, a recently identified peptidoglycan hydrolase required for T6SS assembly. TagX is an L, D-endopeptidase that cleaves the peptide linkage between L-alanine and D-glutamate in the peptidoglycan stem. Deleting TagX in A. baumannii (strain ATCC 17978) or in A. baylyi completely abolishes Hcp secretion and T6SS activity, indicating that TagX performs an essential step of local cell wall remodeling, creating a passage through the rigid peptidoglycan for the assembling T6SS puncturing device to traverse²⁵. While the recruitment of housekeeping peptidoglycan hydrolases for secretion system assembly has been documented (e.g., MltE in EAEC T6SS²⁶), and such dedicated peptidoglycanases are known in other secretion systems^27,28, TagX represents the first specialized peptidoglycan hydrolase encoded within a T6SS gene cluster essential for the system to breach the cell envelope. Consistent with this specialization, TagX is conserved across T6SS clusters of Acinetobacter species, and homologs have been identified in a limited number of other taxa, including Burkholderia thailandensis and Ralstonia solanacearum²⁵. The use of TagX highlights how critical structural adaptation (beyond the core Tss proteins) is for T6SS function in these bacteria²⁹.

The spike apparatus itself exhibits an unusually stringent structural requirement in Acinetobacter. Although many strains encode multiple VgrG paralogs, genetic analyses consistently identify a dominant VgrG—often referred to as VgrG1—as indispensable for basal T6SS activity, while additional VgrGs primarily modulate effector delivery specificity^25,29. Strikingly, single amino-acid substitutions within the conserved C-terminal region of VgrG, including the DUF2345-associated segment, can abolish T6SS activity altogether, and repairing these mutations restores function³⁰. This behavior contrasts with several other bacteria in which the VgrG C-terminus is largely dispensable for core assembly and instead serves mainly as an effector carrier or PAAR docking platform^30–32. In Acinetobacter, by contrast, spike integrity appears to be a prerequisite for productive assembly or firing, suggesting that the spike plays a more architectural role within the system.

Viewed together, these observations raise a focused and unresolved mechanistic question: how does the Acinetobacter T6SS engage and traverse the outer membrane in the absence of a canonical TssJ-based anchor? While the molecular dynamics of outer-membrane anchoring and penetration remain unknown, the convergence of three independent features—the lack of TssJ, the reliance on TslA/TsmK to stabilize the membrane complex, and the strict requirement for an intact VgrG1 C-terminus—suggests that spike architecture and membrane complex function are unusually tightly coupled in this lineage. One parsimonious interpretation is that, in Acinetobacter, successful envelope traversal during firing depends not only on the membrane complex itself but also on a structurally reinforced spike. In the absence of a rigid TssJ-mediated pore, the system likely operates with reduced structural tolerance; consequently, VgrG1 geometry and mechanical competence become critical to ensure the spike can traverse the non-canonical TssM channel (and its flexible GS-linker) without deflection or buckling. This does not imply that VgrG1 directly replaces TssJ, but rather that the spike C-terminus has likely co-evolved with the TslA/TagX-remodeled periplasmic tunnel to guarantee precise alignment and productive membrane engagement in this high-tension architectural context.

In this light, the Acinetobacter T6SS represents a paradigm of structural interdependence, where alternative membrane anchoring, coordinated cell wall remodeling, and enhanced spike integrity functionally compensate for the loss of canonical components (Fig. 1B). This structural variation illustrates the adaptability of the T6SS membrane complex in Acinetobacter. It demonstrates that the T6SS can bypass conserved biophysical constraints through lineage-specific architectural innovations, provided that critical mechanical requirements, such as spike rigidity, are strictly maintained. Moving forward, dissecting how the Acinetobacter membrane complex, fortified spike, and envelope barriers dynamically interact at the molecular level remains a key challenge. Solving this puzzle will not only define the mechanical limits of T6SS adaptability but is also essential for understanding how such non-canonical systems sustain competitive dominance in complex polymicrobial environments.

Regulation of T6SS in Acinetobacter

Assembly of the T6SS apparatus is an energy-intensive process, which warrants tight regulation for its deployment. Acinetobacter species are no exception: these bacteria regulate T6SS gene expression and firing in response to various environmental cues by various regulatory proteins to ensure the system is active only when it is necessary. Multiple layers of T6SS regulation have been elucidated in A. baumannii, including global transcriptional regulators, cell-density-dependent signals, metal availability, and even genetic elements like plasmids.

One important regulator is the histone-like nucleoid structuring protein H-NS, a global transcriptional silencer known to repress horizontally acquired genes in Gram-negative bacteria such as Vibrio parahaemolyticus and Salmonella enterica serovar Typhimurium. In V. parahaemolyticus, H-NS silences T6SS1 expression and activity under non-inducing conditions, such as low salinity and in the absence of surface contact. This repression is alleviated by specific environmental cues, including high osmolarity and surface sensing³³. In S. Typhimurium, H-NS binds directly to the promoter region of the SPI-6-encoded T6SS gene cluster, leading to transcriptional silencing. Genetic ablation of hns derepresses the system, resulting in the assembly of a functional T6SS apparatus and conferring potent antibacterial activity³⁴. Thus, H-NS-mediated transcriptional repression may be a conserved and fundamental negative regulatory mechanism for controlling T6SS expression. A. baumannii strains often carry H-NS homologs on both chromosome and plasmids, which might modulate T6SS expression. Specifically, Zhang et al. reported that hns deletion led to increased expression of multiple virulence-related loci including the T6SS cluster³⁵. Furthermore, they confirmed that H-NS directly binds to the upstream regulatory region of hcp, a core structural gene of the T6SS. Thus, H-NS directly binds to the T6SS operon to repress its activity in Acinetobacter under non-inducing conditions³⁵. Notably, the effect of H-NS may be context-dependent: the discovery of plasmid-encoded T6SS repressors (discussed below) indicates that H-NS-based silencing may coordinate with other means of regulation.

Another major regulatory circuit is quorum sensing (QS)³⁶. V. cholerae O1 El Tor has one cluster that is tightly regulated by QS and environment cues (only firing in nutrient-poor conditions or within host intestines) – interestingly, Acinetobacter similarly connects T6SS with quorum sensing QS, though the environmental cues differ. V. cholerae’s T6SS is repressed in high cell density in vitro, activated in biofilms whereas A. baumannii’s is activated by its N-acyl-homoserine lactone (AHL) QS signal in laboratory growth conditions^37,38. An RNA-seq study showed that deleting abaI significantly downregulated T6SS genes³⁷. The ΔabaI strain also showed diminished biofilm formation and virulence, phenotypes often associated with T6SS activity³⁷. These data indicate that the A. baumannii QS system activates T6SS at high cell density, likely preparing the bacterium for competition when the number of bacterial cells reach a critical threshold. The molecular mechanism is still under exploration, but it may involve QS-controlled regulators that directly or indirectly de-repress the T6SS operon. Interestingly, the QS mutant did not show changes in antibiotic resistance profiles, suggesting that QS mainly affects virulence/community interaction traits (like biofilm and T6SS) rather than drug efflux or cell permeability³⁷.

A defining feature of T6SS regulation in A. baumannii is repression mediated by multidrug-resistant (MDR) plasmids, most notably the conjugative plasmid pAB3 and its derivatives²⁰. These plasmids encode TetR-family transcriptional repressors that directly bind to and silence the chromosomal T6SS locus, rendering plasmid-bearing cells T6SS-inactive^20,39. Loss of pAB3 rapidly derepresses the system, leading to Hcp production and acquisition of potent antibacterial killing capacity²⁰. Thus, plasmid carriage functions as a reversible molecular switch that toggles T6SS activity at the population level. Beyond serving as a simple on–off switch, plasmid-mediated repression of T6SS reveals a deeper evolutionary trade-off between antibiotic resistance and interbacterial antagonism. pAB3-like plasmids not only carry resistance determinants but also encode a functional type IV secretion system (T4SS)^40,41, enabling efficient conjugative transfer among Acinetobacter populations⁴². From this perspective, repression of T6SS likely facilitates plasmid dissemination by preventing the fratricidal killing of recipient cells, which would otherwise impede horizontal transfer.

This logic further invites a broader inference. Because pAB3-encoded TetR repressors can suppress T6SS activity across some Acinetobacter strains and species⁴², conjugative plasmid transfer may disseminate not only antibiotic resistance genes but also a shared regulatory module that enforces transient suppression of T6SS within recipient populations. In this way, plasmid transfer could function as a mechanism for coordinating social behavior across the genus, promoting a state of “intraspecies détente” in which closely related cells avoid mutual killing during periods of high plasmid flux. Such coordination would be particularly advantageous in hospital or host-associated environments, where dense populations, frequent plasmid exchange, and strong antibiotic selection coexist.

While plasmid-borne virulence or fitness factors are common in bacterial pathogens—exemplified by Shigella flexneri⁴³, Yersinia pestis⁴⁴, and Agrobacterium tumefaciens⁴⁵—the use of plasmid-encoded repressors to actively silence a specialized protein secretion system encoded on the chromosome appears, to date, unique to Acinetobacter. This distinctive arrangement positions MDR plasmids not merely as vehicles of resistance, but as active regulators of interbacterial conflict, capable of reshaping competitive interactions and stabilizing cooperative coexistence within Acinetobacter populations.

Although metabolic cues frequently regulate T6SS in other bacteria—iron limitation being a prominent example⁴⁶, direct iron-dependent control of T6SS activity has not been demonstrated in Acinetobacter. Instead, recent work has uncovered a manganese-dependent post-transcriptional regulatory circuit that actively represses T6SS under oxidative stress⁴⁷. In A. baumannii ATCC 17978, Mn²⁺ imported via MumT activates the sRNA AbsR28, which base-pairs with tssM mRNA and promotes RNase E-dependent decay, thereby repressing T6SS assembly⁴⁷. This mechanism establishes a direct link between metal homeostasis, oxidative stress resistance, and T6SS repression. Notably, T6SS-active cells exhibit impaired Mn²⁺ uptake and heightened sensitivity to oxidative killing by phagocytes, providing a strong selective pressure to suppress T6SS during infection⁴⁷. Together, these findings suggest that Acinetobacter has evolved a metal-responsive regulatory logic that prioritizes stress tolerance over interbacterial aggression, underscoring the context-dependent nature of T6SS deployment during infection.

Additional regulatory layers likely operate at the level of physical contact and cell surface architecture. In several bacteria, T6SS firing is triggered by direct cell–cell contact (“tit-for-tat” responses)^48,49, It is not yet fully clear if A. baumannii has a contact-dependence trigger. This hints that additional unknown repressors or activation signals exist. For instance, the GacA/GacS two-component system known to control T6SS in Pseudomonas may do the same in Acinetobacter⁵⁰. Instead, recent work highlights the role of capsular polysaccharide (CPS) as a critical modulator of T6SS function. CPS shields A. baumannii from incoming T6SS attacks by competitors but simultaneously restrains its own T6SS firing, effectively dampening offensive capability⁵¹. The capsule thus acts as a structural checkpoint, prioritizing defense and persistence over aggression, particularly in host-associated environments. Whether additional contact-sensing mechanisms or surface-dependent triggers exist in Acinetobacter remains an open question.

Taken together, T6SS regulation in Acinetobacter is a “well-designed” regulatory network that integrates environmental factors, metabolic state, mobile genetic elements, and surface architecture. Importantly, mechanistic evidence for individual regulatory inputs is currently derived from specific model strains—H-NS– and Mn²⁺-dependent repression in ATCC 17978^35,47 and quorum sensing–mediated control in ATCC 19606³⁷—leaving their broader conservation across Acinetobacter unresolved. By comparison, plasmid-encoded TetR-family repressors carried by MDR plasmids such as pAB3 have been shown to broadly suppress chromosomal T6SS across some clinical A. baumannii isolates and extend to A. nosocomialis and A. bayly^20,42. In addition, capsule-mediated shielding enforces a defensive bias at the cell surface. Together, this multilayered control underscores a central theme: in Acinetobacter, the T6SS is a powerful but conditional weapon, deployed only when its ecological benefits outweigh its physiological costs, and tuned by regulatory strategies that require further investigation regarding their conservation and context-dependence (Fig. 2).

Fig. 2. The integrated regulatory network of Acinetobacter T6SS.

The schematic summarizes the multi-layered control of Type VI secretion system (T6SS) machinery. (1) Transcriptional Regulation: The T6SS gene cluster (blue bar on chromosomal DNA) is repressed by the global regulator histone-like nucleoid structuring protein (H-NS, red clusters) and plasmid-encoded TetR-family repressors (red squares), while being activated by Quorum Sensing signals, N-acyl-homoserine lactones (AHLs, purple diamonds; green arrow). (2) Post-Transcriptional Regulation: High intracellular Manganese (Mn²⁺) levels (purple circles), imported via the MumT transporter (purple channel), trigger the AbsR28 sRNA (hairpin structure) to base-pair with and degrade tssM mRNA, silencing the system under oxidative stress. (3) Direct and Physical Regulation: The T6SS machinery is directly inhibited by the VgrG inhibitor VgrGi (red T-bar). Furthermore, Capsular Polysaccharide (CPS, grey outer layer) overproduction acts as a physical barrier that hinders the outward firing of the VgrG spike (red crossed-out circle). Red lines indicate inhibition or blocking; green arrows indicate activation.

T6SS Effectors and Their Functions

Central to the Type VI secretion system’s role in microbial competition is its arsenal of effectors. These toxic proteins are delivered into target cells by the T6SS “spring-loaded” mechanism, where they incapacitate or kill competing organisms. Acinetobacter T6SS effectors exhibit a wide range of activities, reflecting the diverse ways one bacterium can attack another^17,21. Here we categorize Acinetobacter T6SS effectors by their targets, including those that attack peptidoglycan, membrane and nucleic acid. We also highlight a unique example of antifungal T6SS activity. For each category, a cognate immunity protein is typically co-produced to prevent self-destruction²¹. An overview of known or putative Acinetobacter T6SS effectors and their activities are summarized in Fig. 3.

Fig. 3. Diverse mechanisms of Acinetobacter T6SS effector-mediated antagonism.

The Acinetobacter T6SS delivers a versatile arsenal of toxic effector proteins into target cells to establish a competitive advantage. These effectors are categorized by their cellular targets. A Peptidoglycan Breach: Peptidoglycan-degrading effectors (orange Pac-Man shapes), such as amidases and lytic transglycosylases, cleave the peptide crosslinks or glycan strands of the peptidoglycan sacculus (grey mesh). This breach of the mechanical barrier leads to cell wall degradation and rapid osmotic lysis of the prey. B Membrane Destabilization: Membrane-targeting effectors (yellow Pac-Man shapes), including members of the Tle (Type VI lipase effector) family, target the phospholipid bilayer (red lipid membrane). By catalyzing lipid deacylation, these effectors compromise membrane integrity and disrupt energetic homeostasis, resulting in cell death. C Nucleic Acid Destruction: Potent nucleases (purple Pac-Man shapes), including Tde (Type VI DNase effector) and Rhs-family toxins, are delivered to fragment chromosomal DNA (purple double helix). Notably, the effector TafE (formerly Tde) exhibits cross-kingdom activity, effectively targeting fungal cells. To prevent self-intoxication, the Acinetobacter predator (blue cell) co-produces specific immunity proteins (shield icons) that bind to and neutralize the cognate toxins (small colored circles) within the producing cell before secretion.

Peptidoglycan-degrading effectors: breaching the bacterial armor

Because the peptidoglycan sacculus constitutes the primary mechanical barrier protecting bacterial cells, it represents a highly frequently exploited target of T6SS-mediated attack. Accordingly, antibacterial activity of the Acinetobacter T6SS is commonly associated with effectors harboring amidase or muramidase/lytic transglycosylase–like activities that cleave peptide crosslinks and/or glycan strands within the cell wall.

A paradigmatic framework for this strategy was established in A. baylyi strain ADP1, a naturally competent bacterium with robust T6SS activity. Systematic effector identification revealed multiple lytic cargos, including Tae1, a canonical Type VI amidase effector predicted to hydrolyze peptidoglycan peptide stems, causing rapid osmotic lysis of prey cells⁵². The toxicity of Tae1 is put under check by a periplasmic immunity protein, which neutralizes its toxicity by direct protein-protein interaction⁵². Subsequent work in A. baumannii revealed that peptidoglycan damage can be achieved not solely by hardcore enzyme activity of the effector, but also can be strongly influenced by the local physicochemical context at the cell–cell contact sites⁵³. In A. baumannii ATCC 17978, the dominant T6SS effector Tse4 functions as a bifunctional peptidoglycan hydrolase, possessing both lytic transglycosylase–like activity and endopeptidase activity, which together enable efficient contact-dependent killing of diverse prey cells⁵³. Interestingly, its catalytic potency is enhanced by alkaline conditions, and the predator actively promotes its activity by secreting basic metabolites such as D-lysine to elevate the pH at the predator–prey interface⁵³. These findings highlight a general principle: the outcome of T6SS-mediated cell wall attack reflects not only effector catalytic potential, but also microenvironmental conditions in microbial interactions. Consistent with this view, not all T6SS effectors act as constitutive toxins. For example, A. baylyi encodes Tpe1, a PAAR-linked effector predicted to function as a Zn-dependent metallopeptidase; however, it displays no detectable toxicity toward Escherichia coli under standard competition conditions, suggesting a context-dependent role or the need of an activity modulator⁵². Such observations reinforce the notion that effector function is conditional and tightly integrated with environmental and regulatory cues.

Work in A. baumannii strain AB307-0294 has also revealed an additional layer of control over effectors that target peptidoglycan⁵⁴. By experiments designed to dissect the genetic determinants required for efficient secretion and VgrG-dependent delivery of a distinct putative bifunctional peptidoglycan hydrolase, these authors found how spike–cargo pairing constrains delivery specificity⁵⁴. Thus, it appears that the toxicity of peptidoglycan-directed T6SS effectors in A. baumannii is modulated by environmental cues and precise cargo delivery.

In parallel to the many nuances of offensive strategies, A. baumannii has evolved intrinsic defenses against incoming cell wall–targeting attacks. One such strategy is peptidoglycan editing, which involves the substitution of L-lysine with D-lysine, leading to a reduction of susceptibility to amidase-mediated cleavage, thus providing a form of immunity during T6SS-mediated interbacterial conflict⁵⁵. Collectively, these studies depict a dynamic arms race centered on the bacterial cell wall, in which enzymes of multiple functions, microenvironmental modulation, delivery constraints, and target remodeling coordinate to determine the outcome of competition.

Membrane-targeting effectors: destabilizing cellular integrity

The second major class of T6SS effectors is those that target bacterial membranes, compromising their integrity and energetic homeostasis. Members of the Tle (Type VI lipase effector) effector family catalyze deacylation of phospholipids, resulting in increased permeability and eventual cell death⁶.

In A. baylyi strain ADP1, Tle1 induces slower but sustained killing compared to Tae1, which is consistent with gradual membrane destabilization instead of immediate lysis⁵². Simultaneous use of fast-acting peptidoglycan hydrolases and slower membrane-disrupting enzymes suggests a layered killing strategy; whereby multiple effectors act synergistically to overcome potential heterogeneous defenses in prey cells.

Genomic analyses indicate that A. baumannii encodes putative membrane-targeting effectors, often located adjacent to T6SS structural genes or vgrG loci. The actual activity of many of these predicted membrane-targeting effectors awaits experimental validation^17,21,25. In addition to effectors of lipase activity, T6SSs in other bacteria deliver small pore-forming proteins that oligomerize within target membranes¹⁹. While such pore-forming effectors have not yet been directly demonstrated in Acinetobacter, the structural and functional flexibility of its T6SS suggests that membrane perforation may represent an underexplored component of its effector repertoire.

Nucleic acid–targeting effectors and cross-kingdom antagonism

Among the most potent cargos delivered by the T6SS are nucleases that directly attack nucleic acids within target cells. In A. baumannii ATCC 17978, Weber et al. identified a VgrG-dependent DNase effector, designated Tde (Type VI DNase effector), which is delivered by a specific VgrG variant into prey cells, causing extensive chromosomal DNA fragmentation²⁵. A later study by Luo et al. demonstrated that Tde also exhibits potent activity against fungi such as Candida albicans. The name of the effector was therefore changed to TafE (Type VI antifungal effector)⁵⁶. Again, the predator cells are protected by a specific immunity protein. Together, these studies established Tde/TafE as a T6SS effector capable of exerting antagonism in both interbacterial and cross-kingdom scenarios.

Consistent with the view that nuclease payloads (effectors)⁵⁷ are widespread and modular across the Acinetobacter genus, members of the Rhs-family proteins have been identified as T6SS effectors in many different bacteria. These proteins often are of large in size and are characterized by an extended architecture with a versatile C-terminal domain (CTD) harboring the toxic activity, frequently as a nuclease. In A. baumannii AB307-0294, proteomics-based analyses revealed two Rhs-family T6SS effectors that contribute to interbacterial antagonism⁵⁸. Similarly, in A. nosocomialis strain Ab25, a dominant Rhs effector was shown to possess intrinsic DNase activity, again with a nuclease domain mapped to its C-terminal region⁵⁹. The use of Rhs toxins as a recurrent class of DNA-degrading T6SS effectors by pathogens, including Acinetobacter raised an important mechanistic question: how are such large, multidomain nucleases efficiently packaged, protected, and delivered by the T6SS apparatus?

Recent work on an A. baumannii Rhs-family T6SS cargo (Tse15) provides a compelling mechanistic framework. Rhs toxins typically comprise a conserved N-terminal “clade”/scaffold region, a central Rhs repeat “cage,” and a diverse C-terminal toxic domain (CTD) that commonly encodes catalytic activity—frequently nucleases [effector fold 2]⁶⁰. Structural and functional analyses support a model in which the N-terminal clade domain mediates productive interaction with the cognate VgrG (via defined binding interfaces), while the Rhs β-cocoon can shield an unfolded toxin payload; importantly, the toxic CTD is released/activated through cleavage events and delivery-associated remodeling⁶⁰. This modular architecture explains how DNases can be loaded onto VgrG- or PAAR-containing assemblies and translocated without premature self-intoxication, and why spike–cargo pairing is often highly specific.

Although RNase-type T6SS effectors have not yet been experimentally characterized in Acinetobacter, other bacteria have been shown to code for T6SS effectors of RNase activity that degrade tRNA or rRNA, leading to rapid shutdown of protein synthesis in target cells⁶¹. Importantly, genome-wide analyses of the Acinetobacter genus reveal a large number of T6SS-associated cargos—particularly Rhs-family proteins—whose C-terminal toxin domains remain poorly characterized. Given that the CTDs Rhs in diverse bacterial systems frequently encode nucleic acid–targeting activities, including RNases^62,63, it is possible that some Acinetobacter T6SS effectors have RNA-degrading activity.

In sum, the effector repertoire of Acinetobacter T6SS is strikingly expansive in structural architectures^17,21, yet it converges on a relatively small set of essential cellular substrates, most prominently peptidoglycan, membrane lipids, and nucleic acids. This apparent “diversity-with-convergence” likely results from a long-term arms race: prey populations can evolve resistance to individual toxins, but broad coverage of conserved essential substrates ensures the effectiveness of T6SS weapon. A key operational consequence is that like other bacteria, Acinetobacter rarely relies on a single toxin. Instead, multi-effector payloads with distinct catalytic activities are used to attack different aspects of cellular components. Findings in A. baylyi ADP1 clearly illustrate this principle: its T6SS employs a set of effectors with distinct and non-redundant activities that target different cellular substrates, enabling layered and synergistic damage that maximizes prey killing efficiency⁵².

Killing by Acinetobacter T6SS is also shaped by context-dependent gating at the effector level. Effector potency can be tuned by microenvironmental conditions, which explains the difference in killing targets and efficiencies in accordance with the state of the niches and community⁵³. Moreover, secretion and productive delivery of certain cargos require chaperones or adaptors, and effector–delivery coupling can feed back on the assembly or firing of the T6SS itself (as suggested by VgrG-dependent effector–chaperone modules)⁶⁴. These observations are in line with emerging paradigms derived from other taxa, where conserved adaptors orchestrate co-secretion of synergistic effectors⁶⁵. These findings invite a broader question for Acinetobacter: do subsets of effectors operate as functionally coordinated “teams,” wherein one effector promotes the effectiveness of the second effectors by creating an environment more conducive for its activity or the secretion of one effector actively modulates the deployment or activity of the second one?

Although canonical T6SS effectors are typically framed as weapons for interbacterial conflict, the discovery that the DNase TafE expands the conceptual substrate range of Acinetobacter intoxication and underscores that cross-kingdom targeting is biologically plausible⁵⁶. In other bacterial systems, T6SS effectors also contribute to nutrient acquisition and community shaping, suggesting that Acinetobacter may likewise encode cargos that extend beyond direct killing—potentially influencing biofilm ecology, micronutrient access, and even host interactions^66–68.

Compared to the large predicted effector reservoir across the Acinetobacter genus, only a limited number of them have been assigned an activity^17,21,69; a major challenge is to prove that these proteins are indeed T6SS effectors and to elucidate their mechanism of action. The development of effective multi-omics and function annotation methods, including secretomics, comparative genomics, next-generation structural biology, and AI-enabled structure prediction should greatly accelerate our understanding of the role of these proteins in the biology of Acinetobacter.

Clinical relevance and non-canonical roles of the Acinetobacter T6SS

Although the T6SS is classically viewed as a contact-dependent weapon for interbacterial antagonism, accumulating clinical and experimental evidence suggests that the Acinetobacter T6SS exerts broader impacts that extend beyond microbial competition. In pathogenic contexts, T6SS activity appears to intersect with host immune responses, clinical disease phenotypes, and the evolutionary dynamics of antibiotic resistance, positioning this system as a potential amplifier—rather than a sole determinant—of pathogenic fitness.

T6SS activity correlates with clinical infection and disease severity

Across clinical cohorts, the relationship between the Acinetobacter type VI secretion system (T6SS) and disease severity has been repeatedly interrogated, yet with seemingly conflicting conclusions. Several studies focusing on bloodstream infections report that isolates classified as T6SS-positive are associated with higher mortality, elevated inflammatory markers, and enhanced virulence in experimental models, often coinciding with multidrug resistance and increased competitive fitness^70–72. These observations suggest that T6SS activity frequently accompanies a broader high-risk pathogenic phenotype rather than acting as an isolated virulence determinant. In contrast, other studies—particularly those based on genomic epidemiology or microbiological surveys of bacteremia isolates—failed to detect a clear correlation between T6SS status and clinical severity, or even reported no association at all^4,73.

A closer inspection indicates that much of this discrepancy likely reflects methodological rather than biological divergence. Most clinical studies define “T6SS-positive” status based on the presence of one or a few core structural genes, most commonly hcp or selected tss components. However, gene presence alone does not reliably predict functional output in Acinetobacter. As discussed earlier, a competent T6SS requires not only the canonical core apparatus but also lineage-specific accessory factors such as TslA, TsmK, and the peptidoglycan-remodeling enzyme TagX^18,19,25. Partial loci, regulatory silencing, or mutations in spike-associated components (e.g., vgrG1) can render the system inactive despite retention of hallmark genes³⁰. Even approaches that move beyond genotyping and use hcp transcription as a surrogate marker face similar limitations: although elevated hcp expression correlates with inflammatory responses and has been proposed to distinguish infection from colonization in respiratory isolates⁷⁴, secretion rather than transcription defines functional T6SS activity. Consequently, cohorts relying on limited genetic or expression markers inevitably aggregate isolates with fundamentally distinct T6SS states—fully active, conditionally repressed, or structurally defective—thereby diluting genuine associations with disease severity.

Notably, the clinical success of several epidemic A. baumannii lineages despite low or undetectable T6SS activity suggests that maintaining an active system is not universally advantageous within the host⁷⁵. This pattern is best interpreted through an evolutionary cost–benefit framework. Assembly and firing of the T6SS constitute a substantial energetic investment and may be selectively down-tuned when alternative fitness programs dominate, including antibiotic tolerance, oxidative stress resistance, biofilm-associated persistence, or functional redundancy with other virulence-adaptation modules. Moreover, sustained T6SS activity may also influence immune engagement. Beyond potential immune stimulation from contact-dependent bacterial lysis, T6SS firing in some pathogens can directly shape inflammatory responses through effector-mediated activation of inflammasome pathways^76,77. In certain infection contexts, silencing the system may therefore favor immune evasion and long-term persistence.

Consistent with this conditional deployment model, clinical and genomic surveys reveal a dynamic interplay between T6SS activity, resistance evolution, and clonal expansion. While some MDR and XDR isolates retain T6SS gene content and exhibit enhanced competitive fitness^70–72, highly resistant epidemic clones frequently show repression or erosion of T6SS loci^4,73,78. This trade-off is exemplified by large conjugative resistance plasmids that simultaneously encode antibiotic resistance determinants and transcriptionally suppress chromosomal T6SS expression, effectively toggling Acinetobacter between a “competitive mode” (T6SS active, plasmid absent) and a “drug-resistant mode” (T6SS repressed, plasmid maintained)^20,79. Such regulatory coupling highlights how T6SS participates in early ecological dominance and horizontal gene acquisition, yet becomes dispensable once resistance traits are secured.

Taken together, these observations argue that the clinical relevance of the Acinetobacter T6SS cannot be captured by binary genotypic classification. Instead, T6SS should be viewed as a dynamic, context-dependent fitness module whose contribution to pathogenic success depends on regulatory state, system integrity, and ecological conditions within the host. Future clinical studies integrating locus completeness, regulatory markers, and functional readouts—such as Hcp secretion or interbacterial competition assays—will be essential to define when and how T6SS activity meaningfully shapes disease progression in Acinetobacter infections.

T6SS as a modulator of host immune responses

In light of growing clinical observations that functional T6SS activity correlates with disease severity and patient outcomes in A. baumannii infections, it is reasonable to propose that T6SS activity may intersect with host immune regulation during infection. At the cellular level, exposure to T6SS-active A. baumannii or purified T6SS components such as Hcp results in enhanced NF-κB activation and increased production of pro-inflammatory cytokines, supporting the idea that T6SS activity correlates with heightened innate immune signaling⁷⁴. Importantly, recent work demonstrating the synergistic disruption of the T6SS and iron acquisition systems revealed that T6SS deficiency profoundly alters host–pathogen interactions at the immune interface⁸⁰. In this model, loss of hcp markedly increased bacterial susceptibility to phagocytosis and clearance, impaired intracellular persistence, and significantly reduced the induction of pro-inflammatory cytokines in macrophages. These findings suggest that an intact T6SS indirectly promotes a pro-inflammatory environment by enhancing bacterial fitness within immune cells rather than by directly delivering immunomodulatory effectors into host cytosol.

This immune-amplifying effect becomes more evident in whole-organism infection models. In Galleria mellonella and murine infection systems, T6SS-deficient strains exhibit substantially reduced virulence and fail to induce lethal inflammatory responses, despite comparable inoculum sizes^71,72,81,82. In a mouse peritonitis model, combined inactivation of T6SS and siderophore-mediated iron uptake completely abolished mortality, accompanied by striking reductions in systemic cytokine levels, macrophage M1 polarization, and transcriptional activation of inflammatory pathways in infected tissues⁸⁰. These data indicate that T6SS activity contributes to the development of cytokine storm–like pathology, not by acting as a classical immune effector system, but by enabling sustained bacterial survival, iron acquisition, and repeated immune stimulation within the host. Complementary insights arise from zebrafish infection models, which uniquely capture host responses to microbial community dynamics. When T6SS-mediated interbacterial killing occurs in vivo, hosts mount stronger inflammatory responses and experience increased mortality compared with infections dominated by diffusible toxins⁸³.

Together, current evidence supports a model in which the Acinetobacter T6SS contributes to host immune modulation primarily through ecological and metabolic mechanisms rather than direct immune targeting. Nevertheless, whether Acinetobacter encodes bona fide T6SS effectors that directly target host immune signaling remains unresolved. Unlike trans-kingdom effectors described in other pathogens, no dedicated eukaryotic immune-modulating T6SS effectors have yet been conclusively identified in A. baumannii. An alternative, and perhaps more parsimonious, model is that T6SS influences host immunity indirectly—by reshaping microbial competition, enhancing iron scavenging and metabolic fitness, and prolonging bacterial persistence within immune niches. These processes collectively intensify host inflammatory responses and may help explain the observed association between T6SS activity, clinical disease severity, and adverse outcomes.

Horizontal gene transfer (HGT)

Beyond its role in shaping infection outcomes, T6SS activity also influences Acinetobacter evolution by promoting horizontal gene transfer in competitive microbial environments. Members of this genus—particularly A. baylyi and A. baumannii—are naturally competent^84,85, and T6SS-mediated killing generates localized pools of extracellular DNA through contact-dependent lysis of neighboring cells. Experimental studies demonstrate that efficient prey lysis by T6SS markedly enhances DNA uptake and transformation efficiency, directly coupling interbacterial antagonism to genetic acquisition^52,86.

In polymicrobial settings such as hospital surfaces, medical devices, or infected tissues, this mechanism provides a direct ecological route for Acinetobacter to sample genetic material from surrounding communities. From this perspective, the T6SS operates not only as a competitive weapon but also as an evolutionary catalyst, linking niche clearance with opportunistic genome remodeling and adaptive diversification.

Conclusion and Future Perspectives

The Acinetobacter T6SS is best viewed not simply as a conserved contractile nanomachine, but as a lineage-adapted platform that has been selectively rewired at key architectural and regulatory nodes. While it retains the canonical tube–sheath–spike core, Acinetobacter departs from textbook T6SS paradigms in several conceptually important ways. These include a non-canonical membrane complex that lacks TssJ yet remains fully functional through genus-specific components such as TslA, TsmK, and TagX; a stringent dependence on VgrG1 integrity that links spike architecture to system competence; and a multilayered regulatory logic that integrates plasmid carriage, metal homeostasis, quorum sensing, and surface architecture. Together, these features position the Acinetobacter T6SS as a conditional fitness module that flexibly balances aggression, persistence, and clinical adaptation.

Beyond mechanistic insight, this system offers emerging translational opportunities. Structural components such as Hcp are being explored as vaccine antigens, supported by in silico and experimental studies proposing multicomponent immunization strategies^87,88. In parallel, T6SS sheaths have been repurposed as antigen-display nanoparticles, establishing a modular platform for vaccine development⁸⁹. Diagnostic avenues are also beginning to take shape, with hcp expression proposed as a marker to distinguish infection from colonization—although these efforts further underscore the need for activity-proximal readouts, as gene presence or transcription alone does not capture functional secretion.

At the same time, several key questions now define the next phase of Acinetobacter T6SS research. First, the genus encodes a large and largely unexplored repertoire of predicted effectors, yet only a small fraction have assigned biochemical activities. Systematic discovery pipelines that integrate comparative genomics^17,21, structure-guided annotation^54,60, and scalable functional assays⁹⁰ will be essential to resolve this “effector dark matter”. Second, although T6SS activity correlates with disease severity and host inflammatory responses, it remains unclear whether Acinetobacter deploys dedicated effectors that directly modulate mammalian signaling, or whether these effects arise indirectly through altered microbial competition, nutrient acquisition, and bacterial persistence. Addressing this will require effector-focused genetics combined with host-pathway readouts in polymicrobial infection models. Third, because Acinetobacter commonly inhabits mixed communities and closely related strains often co-colonize, fundamental questions remain regarding how T6SS-mediated antagonism is balanced with kin tolerance—raising experimentally tractable hypotheses involving capsule-based shielding, immunity repertoires, spatial organization, and plasmid-mediated coordination.

Taken together, future progress will depend on treating the Acinetobacter T6SS not merely as a weapon, but as a systems-level determinant of ecological fitness—one that integrates envelope architecture, spike mechanics, regulatory circuitry, effector diversity, and community context. By revealing how a conserved nanomachine can be selectively remodeled to meet distinct ecological and clinical pressures, Acinetobacter provides a powerful model for understanding the evolutionary plasticity of bacterial secretion systems and for developing new diagnostic and anti-virulence strategies.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Author contributions

L.S., J.J., S.G., and D.L. conceived the topic and wrote the original draft. M.Z. and L.S. prepared the figures. Z.-Q.L. and L.S. reviewed and edited the manuscript.

Peer review

Peer review information

Communications Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editors: Ranjana Pathania and Tobias Goris. A peer review file is available.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s42003-026-09782-w.