A Novel Molecular Mechanism for Effector Protein Wrapping and Delivery by the Bacterial Type VI Secretion System

A Novel Molecular Mechanism for Effector Protein Wrapping and Delivery by the Bacterial Type VI Secretion System | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A Novel Molecular Mechanism for Effector Protein Wrapping and Delivery by the Bacterial Type VI Secretion System Patricia Paracuellos*, Ambre Bexter*, Jonasz B. Patkowski, Oleksii Omelchenko, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7023205/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Pathogenic bacteria deploy sophisticated strategies to endure hostile environments and outcompete host microbiota or immune cells. Up to 30% of Gram-negative bacteria, including Pseudomonas aeruginosa, harbor a Type VI secretion system (T6SS), a supramolecular nanomachine that operates like a crossbow, for firing effectors into prokaryotic and eukaryotic prey cells. Despite its widespread distribution in nature, the mechanism by which effectors are loaded into ca 120 Hcp ring assemblies that form the T6SS injection tube, and the diversity of effectors delivered per firing event, remain undefined. Here, we reveal this mechanism by solving the cryo-electron microscopy structure of the Tce1 cargo effector loaded into a hexameric Hcp ring. Our structure reveals that a single cargo is enclosed by multiple rings and interacts asymmetrically with individual Hcp protomers. Our data delineate a conceptually novel mode of effector recognition and a stepwise loading mechanism, whereby an initial heterodimeric Hcp-cargo complex forms prior to ring formation occurring around the effector. We showed that another effector, Tce2, which exhibits anti-fungal properties, is similarly a Hcp3 cargo. We thus propose a novel and foundational mechanism by which distinct cargos are wrapped and simultaneously loaded into a single T6SS molecular device, enabling the coordinated delivery of a broad and potent payload into target cells. *Patricia Paracuellos & Ambre Bexter contributed equally. Biological sciences/Microbiology/Bacteriology Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Microbes have been competing for billions of years, and this has led to the evolution of numerous strategies to capture scarce resources and eliminate competitors 1 . The Type VI secretion system (T6SS) is a sophisticated and dynamic bacterial supramolecular machine that injects toxic effectors into target cells 2-4 . T6SSs are versatile and can target many cell types such as eukaryotic cells, including fungi, or prokaryotes such as multi-drug-resistant Gram-negative bacteria 5 . This nanomachine is often compared to a crossbow loaded with toxic arrows and there is extensive knowledge on how its assembly is initiated, then extended and finally contracted to fire effectors into susceptible cells 6,7 . The T6SS tip is a puncturing device, which includes a spike complex comprised of PAAR and VgrG proteins 8 . The polymerisation and thus extension of a cytosolic contractile T6SS sheath is a coordinated event 9 during which hexameric rings of the Hcp 10,11 protein first interact with the flat base of the VgrG-PAAR spike complex and then stack on top of one another to form a tube structure that is simultaneously encapsulated within the sheath 12 . The rapid contraction of the sheath 13 propels the PAAR-VgrG-Hcp puncturing device through the bacterial envelope and into target cells, delivering effectors along with it. Despite many notable advances in visualizing the structure and dynamics of the T6SS apparatus, gaps remain in our understanding of the mechanisms by which toxins/effectors are recruited and loaded into the T6SS. In recent years, some of these mechanisms have begun to be defined. For example, effectors can be targeted to the T6SS through the recognition of conserved protein sequences such as the MIX, RIX, PIX, WHIX or FIX motifs 14,15 , but these are found in a limited number of effectors and how each of them functions at a molecular level has not yet been examined. Some effectors have been shown to require the assistance of chaperones or adaptors such as Eag or Tap proteins 16,17 , respectively, which may help prevent premature folding in the producing cell cytoplasm. T6SS effector loading has been proposed to occur by attachment of effectors to either one part of the PAAR-VgrG-Hcp complex 8 . Effectors can either exist as C-terminal extension of any of these T6SS spike elements, in which case they are termed specialized effectors, or they are recruited to one of these components in which case they are denoted cargo effectors 18 . In general, the association of effectors with VgrG, e.g. Escherichia coli Tle1 19 , and PAAR, e.g. Pseudomonas aeruginosa Tse6 20 , is well described at a molecular and structural level. VgrG and PAAR 21,22 , which are a trimer and a monomer, respectively, could load at maximum a total of 4 effectors on one single T6SS spike complex. By contrast, Hcp rings exists in as many as 120 copies within the elongated T6SS sheath 23-25 , dramatically increasing the ability of the system to deliver a huge cocktail of possibly functionally distinct effectors. As such, T6SS loading through Hcp may result in a highly effective system against any type of prey cells, but comparatively little is known about how toxins/effectors are recruited to Hcp. Interestingly, Hcp has been proposed to exhibit chaperone activity by stabilizing small effectors such as P. aeruginosa Tse2 and it was hypothesized that Tse2 sits within the lumen of Hcp1 hexameric rings 26 . P. aeruginosa is equipped with 3 T6SSs known as the H1-, H2- or H3-T6SS 27 , and each system is associated with the Hcp proteins, Hcp1, Hcp2 and Hcp3, respectively. In previous work, using in vivo pulldown experiments, we identified potential cargo effectors associated with these Hcp proteins 28,29 . Here, we describe two novel T ype VI Secretion System delivered c ysteine protease e ffector (Tce) proteins bound to Hcp3, Tce1 (PA0256) and Tce2 (PA2372). We present the first high resolution structure of an Hcp-cargo effector complex using cryo-electron microscopy. The 3.8 Å resolution of the Hcp3-Tce1 complex reveals the molecular details behind Hcp3 toxin recognition, the molecular events that lead to the formation of the complex, and their stoichiometric arrangements. Our data indicate that a 1:1 Hcp3-Tce1 heterocomplex forms first, followed by the sequential oligomerisation of Hcp around the toxin until the final Hcp3 hexameric ring filled with the Tce1 toxin in its lumen is formed. We also show that the loading of a T6SS toxin requires two rings for it to be fully embedded with the Hcp tube. These findings offer an entirely new concept for T6SS effector loading that is driven by the cellular concentration of Hcp. Overall, our work provides a novel mechanism and original molecular rules on how T6SS cargo complexes are recruited by and loaded into Hcp. Exploiting such unique knowledge can be tapped on to design T6SS-wielding bacteria as biocontrol agents for microbiome stewardship in human and natural ecosystems. Results Hcp3 interacts with two H3-T6SS effector candidates In a previous study, we identified two potential P. aeruginosa H3-T6SS effectors – PA0256 and PA2372 28,29 , hereafter referred to as Tce1 and Tce2 – through an in vivo pull-down approach using Hcp3, which is encoded within the H3-T6SS cluster (Supplementary Fig. 1). A distance-matrix alignment (DALI) 30 was performed using the AlphaFold3 31 predictions of both Tce1 and Tce2 to identify structural homology to proteins with known enzymatic activity (Fig. 1a). A top hit for the analysis of each protein was the cysteine protease domain for the Clostridium difficile toxin TcdB 32 (PDB:3PEE). A multiple sequence alignment of Tce1, Tce2 and TcdB, and other known cysteine proteases revealed conserved cysteine and histidine residues across these proteins (Fig. 1b) Structural alignment showed that His186 and Cys240 in Tce1, as well as His65 and Cys115 in Tce2, are spatially positioned to align with the catalytic dyad of TcdB (His110 and Cys155), supporting the hypothesis that both Tce1 and Tce2 are cysteine protease-like enzymes (Fig. 1c). It is also noteworthy that Tce1 is a larger protein (310 a.a) than Tce2 (190 a.a.) with a C terminus of unknown function (110-310) encompassing the putative cysteine protease catalytic site and an extended N terminus (1-101) (Fig. 1a). Both effectors co-purified with Hcp3 via affinity chromatography (Fig. 1d), with Tce1 showing a stronger interaction consistent with the in vivo pull-down where Tce1 also shows a stronger interaction (8.4-fold enrichment) compared to Tce2 (5.8-fold enrichment). Based on these data, we further prioritised Tce1 toobtain a high resolution cryo-EM structure of a Hcp3-effector complex. Cryo-EM reconstruction reveals that two Hcp3 rings are needed to fully enclose Tce1 The hcp3 and tce1 genes were cloned, co-expressed in E. coli , and co-purified via affinity and size-exclusion chromatography. Hcp3 and Tce1 remained stably associated throughout the purification (Fig. 2a,b), eluting as a complex at 12.6 ml. Given that the Hcp3 hexamer alone elutes at 12.8 ml 29 (Fig. 2a), this shift suggests that Hcp3 binds Tce1 in its hexameric form, with the presence of the toxin accounting for the earlier elution, indicative of a heavier particle. Upon purification, the assembly and homogeneity of the complex was assessed through negative stain-EM and following successful inspection, the complex was frozen onto thin carbon layer grids and imaged through cryo-EM (Supplementary Table 1). After subsequent beam induced motion and contrast transfer function (CTF) correction, particles were extracted from the collected micrographs and rounds of 2D classification were performed. From this, a dataset of ~1 million particles was acquired, yielding high resolution 2D class averages which display a clear ring structure with additional density observable within the central lumen only when Tce1 is present in the sample (Supplementary Fig. 2 and Fig. 2c). By looking at the side views we could also infer that a portion of the toxin density slightly protrudes out of the ring. The high resolution 2D classes were subjected to iterative rounds of Ab initio classification followed by non-uniform refinement with C1 symmetry applied. The final map achieved an overall resolution of 3.8Å, with local resolution extending to sub-3.4Å across the complex (Supplementary Fig. 3). The densities corresponding to the Hcp3 ring and Tce1 C-terminus were identified in the map and used to build an atomic model of the entire Hcp3 ring in complex with the C-terminal domain of Tce1 (Fig. 3a,b). The model of the Hcp3 ring displays six repeating units in a homohexameric conformation with each Hcp3 monomer made up of nine β-strands (β1-β9) forming two interfacing β-sheets, alongside a single α-helix (Fig. 3c,d). Hcp3 monomer-monomer interactions are stabilised through hydrogen bonds between the α1 and β6 of one monomer with β2 and β9 of its neighbour, with specific interacting residues listed in Supplementary Table 2. These interacting residues are repeated across all Hcp3 monomers in the hexameric ring. Our model also showed that only the C-terminal domain of Tce1 is structurally resolved in the Hcp3-Tce1 complex and is comprised of ten β-strands (β9-β18) forming one central β-sheet, surrounded by four α-helices (α5-α8). Because our model of Tce1 starts from Lys110, we modelled the full length Tce1 using AlphaFold3 31 to predict the structure of residues 1-109 (Fig. 3d). From this, we observe that the N-terminal domain possesses a β-sandwich domain arrangement (β1-β8) alongside four short α-helices (α1-α4) and a short linker sequence (residues 106-109) that connects the two domains. We measured the Hcp3 ring to have an outer diameter of 85 Å and an inner diameter of 45 Å. Based on these dimensions, the lumen can accommodate only the C-terminal domain of Tce1, with a portion of it protruding from the ring. This indicates that in the full-length protein, the N-terminal domain of Tce1 would extend further outward from the ring and into an adjacent Hcp3 hexamer. (Fig. 3e). Overall, our data strongly support a model in which two stacked Hcp3 rings fully encapsulate full-length Tce1, with the C- and N-terminal domains each positioned within the lumen of a ring (Fig. 3e). Tce1 interacts with key Hcp3 residues at the inner surface of the ring With Tce1 existing as a monomer within the Hcp3 hexameric ring, it is expected that its interface with each monomer would differ. This is indeed what our structure reveals, with each Hcp3 molecule displaying a distinct degree of interaction with Tce1 (Fig. 3 and 4a,b), and key interacting residues forming hydrogen bonds or salt bridges (Fig.4b and Supplementary Table 2). We arbitrarily numbered the Hcp3 protomers A through F and found that monomers B, C, and D interact with Tce1 via residues G121 and K123. These residues exist in a loop region between β6 and β7 that extends into the lumen of the assembled Hcp3 ring. This observation suggests that G121 and K123 play a critical role in anchoring and positioning Tce1 within the ring structure. To validate this hypothesis and the assembly revealed by our cryo-EM structure, we generated mutants in these key interacting residues to sterically modify the Hcp3 inner surface. Both G121W and K123W substitutions were generated to introduce, via the bulky nature of the aromatic tryptophan side chain, the greatest steric hindrance within the Hcp ring lumen and restrict access to Tce1 (Fig. 4c). We then compared the gel filtration chromatography profile of Tce1 co-purified with Hcp3 G121W and Hcp3 G121W+K123W with our previous co-purification with the native Hcp3 WT (Fig. 4d). In support of our structural data, we observe that in the presence of Tce1, both Hcp3 G121W and Hcp3 G121W+K123W elute at the same retention volume of Hcp3 WT alone, indicating that they do not interact with Tce1 (Fig. 4d). This observation is further supported by SDS-PAGE analysis of the elution fractions (Fig. 4e), where only a band corresponding to Hcp3 is detected. Together, these results validate our 3D structure of the Hcp3-Tce1 complex and demonstrate that Tce1 is positioned within the Hcp3 ring, interacting with its inner surface. Hcp3 sequentially wraps around Tce1 revealing the molecular basis for T6SS effector loading The asymmetric interaction between Tce1 and individual Hcp3 monomers suggests an assembly mechanism in which Tce1 first interacts with a single Hcp3 monomer followed by the rest of the ring assembling around it. To test this hypothesis, we first assessed the solution state behaviour of the Hcp3 ring at different protein concentrations. We purified Hcp3 WT at 4.0, 1.0, 0.5 and 0.1 mg/ml using affinity and gel filtration chromatography. At the higher concentrations, Hcp3 WT elutes at 12.9ml (Fig. 5a), consistent with the previously observed retention volume expected for a hexamer (Fig. 2a). However, at 0.1mg/ml, the retention volume increases to 16.2ml, indicating a substantial reduction in molecular weight consistent with monomeric Hcp3 (Fig. 5a). These results demonstrate that Hcp3 exists as a monomer at low concentrations and as a hexamer at higher concentrations, suggesting that ring formation is concentration dependent. Since Hcp3 elutes as a hexamer at concentrations of 0.5 mg/ml and above, the threshold concentration for hexamer assembly appears to lie between 0.1 mg/ml and 0.5 mg/ml. Next, we examined how Tce1 interacts with Hcp3 under conditions that favor incomplete ring formation. We co-purified Tce1 with Hcp3 WT at 4.0, 1.0, 0.5 and 0.1mg/ml, following the same protocol as for Hcp3 WT alone. The chromatographs for the 4.0,1.0 and 0.5mg/ml purifications depict the characteristic peak at 12.6ml, corresponding to the fully assembled Tce1-filled hexameric ring (Fig. 5b). At 0.1 mg/ml, consistent with the behavior of Hcp3 WT alone, the retention volume increases, but in this case to 15.4 ml—less than the 16.2 ml observed for monomeric Hcp3 alone. This shift likely reflects the addition of Tce1 (~39 kDa) to a monomeric or partially assembled Hcp3 species. Western blot analysis confirmed that both Tce1 and Hcp3 are present in the 15.4 ml elution fraction, demonstrating that Tce1 can interact with non-hexameric Hcp3 (Fig. 5a,b). To further bolster these findings, we re-analyzed the 2D classifications from the original cryo-EM dataset used to solve the Hcp3–Tce1 complex structure. Remarkably, we identified distinct 2D classes corresponding to intermediate stages of ring assembly in which Tce1 is only partially enclosed (Fig. 5c). Altogether these results support a novel molecular model in which Hcp3 ring formation is a sequential process rather than a discrete transition from monomer to hexamer. Furthermore, they reveal that Tce1 initially binds to a small number of Hcp3 monomers, with the rest of the ring assembling around the toxin, even though not all subunits directly interact with it. Tce1 and Tce2 are cysteine protease with analogous structural and functional properties. Based on our experimentally derived Hcp3-Tce1 complex and using AlphaFold3 31 we could predict that as for Tce1, Tce2 nicely sits in the lumen of the Hcp3 ring with asymmetrical contact with the various Hcp3 protomers (Fig. 6a). Tce1 and Tce2 both possess central β-sheets encircled by α-helices (Fig.1a,c). Both β-sheets align, localising Tce2 to the same region of the Hcp3 lumen as where Tce1 is loaded. Tce2 sits in the lower half of the Hcp3 ring, where it partially protrudes out from the underside of the ring (Fig. 6a). We then revisited our previous suggestion that like most T6SS effectors, Tce1 and Tce2 likely possess antibacterial activity 33 . Our data showed that this is likely not the case, since heterologous expression of these enzymes in E. coli did not affect growth (Fig. 6b,c). This is also supported by the observation that no obvious immunity protein 34-37 encoding genes are found in the vicinity of the genes encoding these effector proteins (Supplementary Fig. 1). We then further investigated potential anti-eukaryotic toxicity 38,39 by expressing Tce1 and Tce2 in Saccharomyces cerevisiae . Strikingly, we observed that Tce2 significantly impairs yeast growth. Notably, this toxicity was alleviated by a mutation in the predicted active site of the cysteine protease, confirming its functional relevance (Fig. 6d). In contrast, Tce1 expression did not significantly affect yeast growth. Western blot analysis revealed detectable expression of Tce2, but not Tce1, suggesting Tce1 may be unstable in this context (Fig. 6e,f). To mitigate this instability, we co-expressed Tce1 with Hcp3, and although Tce1 could now be detected by western blot, no impact was observed on yeast growth. A possible hypothesis to the lack of toxicity is that the extended N-terminus of Tce1, as compared to Tce2, may interfere with cysteine protease activity. We thus produced a N-terminally truncated version of Tce1 and expressed in yeast in presence/absence of Hcp3, but in all cases no toxicity could be observed (Fig. 6d,f). Overall, we identified for the first time a T6SS-dependent antifungal property that may be associated with the H3-T6SS function through Tce2, while the absence of a toxic phenotype for Tce1 in these assays may suggest the requirement of auxiliary molecules for its enzymatic activity. Discussion T6SS-dependent effector delivery is a challenging conceptual question to address experimentally and there is a plethora of mechanisms that have been proposed 40 . Some effectors are called specialized with a cargo domain that is an extension of a T6SS component, namely PAAR, VgrG or Hcp. In this case the loading simply relies on these subunits being assembled into the system and the protein-protein interfaces with other elements of the T6SS machine are relatively well described 8 . There are many examples of VgrG- or PAAR-specialized effectors 18,41 , but specialized Hcp effectors have been poorly studied. However three specialized Hcp effectors have been recently described in Enterobacter cloacae , Hcp-ET1 (DNase), Hcp-ET2 (Phospholipase) and Hcp-ET3 (Pyocin) 42 , while further genomic screening identified around 350 Hcp-specialized effectors mostly in Enterobacteriaceae . It was initially proposed that large effectors associate with the T6SS tip, i.e. VgrG or PAAR, whereas effectors that are smaller in size insert into the Hcp lumen such as the P. aeruginosa Tse2 cargo effector 26 . Here our study reveals the detailed molecular mechanism on how cargo effectors are loaded into Hcp. The cryo-EM structure of the P. aeruginosa Hcp3-Tce1 complex at 3.8 Å resolution provides unprecedented insight on how Tce1 is wrapped within the lumen of a homohexameric Hcp3 ring. One of the major findings is the sequential nature of the process during which Tce1 initially interacts with monomeric Hcp3 through specific contacts, notably through the formation of hydrogen bonds and salt bridges (Supplementary Table 2). This initial 1:1 interaction appears to nucleate the subsequent oligomerisation of Hcp3 around Tce1, culminating in the formation of a fully formed effector-loaded hexamer. In this process, S29, E56, and T60 serve as key hydrogen bond-forming residues, interacting with Tce1 across multiple Hcp3 protomers. Additional hydrogen bonds form between each of the five interacting Hcp3 monomers and Tce1, facilitating stable capture of the toxin. This mechanism of Hcp wrapping around the effector is conceptually novel compared to the previously proposed mechanism of effector insertion into Hcp rings. This assembly mechanism can to some extent be compared with the Tat system 43 , which translocates folded proteins across the bacterial cytoplasmic membrane. In this case, TatA monomers once associated with a Tat substrate further wrap around the substrate through oligomerization 44 . This mechanism helps accommodate proteins of varying sizes by recruiting more TatA protomers for large substrates as compared to smaller ones. In the case of Hcp, such modulation in the number of protomers in the ring is likely not possible due to the size and structure of the Hcp rings being fixed and rigid. However, another striking discovery we made is that effectors can be enclosed by more than one Hcp ring. This creates additional room to pack larger effectors than initially thought. Another implication of our findings is that for specialized Hcp effectors 42 , the ring cannot be formed by more than one Hcp-extended effector due to the steric incompatibility of packing more than one effector in the Hcp lumen. Instead, the extended Hcp may bind to 5 other ‘regular’ Hcps that complete the hexameric ring around one Hcp-ET. This conclusion is supported by our observation that the Tce1 effector initially interacts with a single Hcp3 protomer. It is also striking that two effectors, Tce1 and Tce2, are packed within Hcp3 rings. The stronger binding of Tce1 to Hcp3, compared to Tce2, may be explained by their genomic context. While tce2 is encoded within the H3-T6SS cluster and co-transcribed with hcp3 (Supplementary Fig. 1), tce1 is located distantly. It is therefore plausible that Tce1 has evolved a higher affinity for Hcp3 to compensate for its genomic separation. Alternatively, it may suggest a mechanism that drives loading preference, where effectors with higher affinity are loaded first. This strategy is used in other biological context such as fimbrial assembly in which fimbriae subunits that need to be recruited first display higher affinity for the outer membrane usher protein compared to subunits that should be assembled subsequently 45 . In the T6SS, such mechanism would indicate that there are more Tce1 molecules loaded into the H3-T6SS apparatus than there are of Tce2, thus introducing a level of regulatory control in terms of T6SS function and impact on target cells. Furthermore, our observation that ring assembly is concentration-dependent suggests that intracellular levels of Hcp3 and its cargo might regulate the formation of secretion-competent complexes, adding an additional layer of control to T6SS function. The relative concentration between Hcp and cargo could be a modulator of T6SS activity. It was recently shown in Vibrio cholerae that Hcp accumulation can drive T6SS gene repression through interaction with the VasH regulator 46 . We could thus predict that accumulation of free Hcp in the absence of effectors may result in T6SS down regulation. This would also align with the onboard checking mechanism described for VgrG-PAAR in V. cholerae , which described T6SS assembly failure in the absence of effectors 47 . Tce2 is a probable cysteine protease activity that negatively impacts yeast growth in a manner that is dependent on its predicted catalytic cysteine. When compared with Tce2, Tce1 exhibits the same conserved cysteine and histidine residues that align with the catalytic diad of the well-characterised Clostridium difficile toxin TcdB 48 . However, unlike Tce2, Tce1 does not cause significant growth inhibition in yeast. This may imply that Tce1 protease activity is tightly regulated and potentially activated only upon delivery into a physiologically relevant target cell. Hcp3 is part of the P. aeruginosa H3-T6SS which was shown to be involved mainly in anti-eukaryotic activity 49 . Intriguingly, and in contrast to the H1-T6SS and H2-T6SS, for which numerous effectors have been described, effectors associated with H3-T6SS are scarce 50,51 . Two have been associated with VgrG3, TepB 52 and TseF 53 , both having elusive role in being involved in common goods acquisition 54 and/or injected in eukaryotic cells as is the case for TepB 52 . Our findings that Tce2, and possibly Tce1, are anti-eukaryotic effectors strengthen this concept. The molecular target(s) for Tce2 and Tce1 are unknown and comparison with other cysteine protease effectors underscores the evolutionary diversity of these effectors, making potential targets challenging to predict. For example, Cpe1 is a papain-like cysteine protease that targets type II DNA topoisomerases GyrB and ParE 55 , whereas OspB secreted by the Shigella flexneri T3SS cleaves the metabolic regulator TORC1 in yeast and mammalian cells 56 . Although sharing a conserved catalytic core, differences in accessory domains and activation motifs likely reflect adaptation to specific host targets and regulatory environments, providing a broader perspective on how T6SS effector function is fine-tuned. The observation that the Hcp3 ring can be loaded with either Tce1 or Tce2 also supports the concept of the injection of a cocktail of effectors and toxins by one single system, not even accounting for those loaded on the VgrG3 tip. How many Hcp3 rings contain Tce1 compared to Tce2 remains to be understood and will need to take in account the concept that some of the rings might need to remain empty to fulfil steric requirements when the ca 120 Hcp rings are contained in the T6SS sheath. Together, a fully loaded apparatus constitutes a very powerful effector/toxin injection device, and one may even consider that empty Hcp rings could still have potential toxic activity as proposed for Hcp in Acidovorax citrulli 57 . In summary, by defining the molecular basis of T6SS effector loading and establishing an unprecedent link between toxin recognition, sequential ring assembly, and concentration-dependent oligomerisation, this work provides a novel framework for understanding how bacteria deploy T6SS effectors to modulate intercellular interactions. Methods Bacterial cultures and Hcp3-Tce1 expression and purification The bacterial strains and plasmids used in this study are listed in Supplementary Table 3. Bacteria in this study were cultured in lysogeny broth (LB) (Miller) or on LB agar (Miller plates) and grown at 37°C unless stated otherwise. The sequences coding for Hcp3-His variantsand HA-Tce1were cloned into pACYCDuet-1 and pET22b vectors respectively and transformed into BL21 cells. Cells were grown in 1L LB with appropriate antibiotics and protein expression was induced at OD 600 - 0.6 by addition of 0.5mM IPTG for 20h at 20°C. The cells were harvested by centrifugation at 7000 x g, 30 min at 4°C. The pellet was resuspended in 20mM HEPES (pH 7.5), 250mM NaCl, 100mgml -1 Lysozyme, 100μlml -1 Triton X-100, 0.5mM PMSF. Following lysis by sonication on ice, cellular debris was removed by centrifugation at 18,000 x g for 45 min. The supernatant was incubated with 1ml of TALON â metal affinity resin pre-equilibrated with equilibration buffer (20mM HEPES, 250mM NaCl) for 1h. The incubated resin was loaded onto a Econo-Pac â gravity flow chromatography column and washed with 100ml of wash buffer (20mM HEPES, 250mM NaCl, 7mM Imidazole) before elution with 10ml elution buffer (20mM HEPES, 250mM NaCl, 500mM Imidazole). Elution buffer was added 2ml at a time to be collected into 5x 2ml elution fractions. Fractions containing protein were pooled into 3.5 kDa MWCO SnakeSkin TM dialysis tubing and dialysed in 20mM HEPES pH 7.5, 150mM NaCl overnight at 4°C. Following dialysis, the sample was concentrated using a centrifugal concentrator with a 3 kDa MWCO. Size exclusion chromatography (SEC) was performed on the concentrated sample using a Superdex 200 GL 10/300 column (GE healthcare) preequilibrated in SEC buffer (20mM HEPES pH7.5, 150mM NaCl). Presence of both Hcp3-His and HA-Tce1 following SEC were both analysed using Coomassie stain and Western blot analysis. Electron microscopy sample preparation and data collection For the structural determination of the Hcp3-Tce1 complex, a Hcp3-Tce1 SEC purification was incubated with 0.1% glutaraldehyde crosslinking followed by another SEC purification. A 4 µl aliquot from the peak fraction, with the same retention volume as the uncrosslinked Hcp3-Tce1 SEC purification, at a concentration of 0.25 mg/ml was applied onto a glow-discharged Quantifoil R1.2/1.3 Cu 300 mesh holey grid (Agar Scientific). After 60s wait time, the excess sample was blotted off with a blot time of 4s and blot force -2 under 95% relative humidity at 4 °C, and plunge-frozen in liquid ethane using Vitrobot Mark IV (Thermofisher). The dataset was collected on FEI Titan Krios microscope operating at 300 kV at the UK national electron bio-imaging centre (eBIC). Imaging was automated using EPU software (FEI) on a Falcon 4i detector with a pixel size of 0.921 Å (130k magnification). A total of 26626 movies were recorded, with a nominal defocus range of approximately −1.0 to −2.7 µm with a total dose of 40 e-/Å2 over 4.89 s, corresponding to a dose rate of 7.44 e-/px/s. Cryo-EM image processing and reconstruction All image processing was performed with CryoSPARC v4.4.1 58 unless stated otherwise. The movies were aligned using patch motion correction and CTF-corrected through patch CTF estimation, then curated to select for optimum ice thickness, CTF estimation and resolution. From 20,121 selected micrographs, particles were picked and 2D classified to generate a template for automated particle picking. After the particle picking parameters were refined using a small number of micrographs, a total of 1,039,601 particles were auto picked from the entire dataset and extracted using a box size of 320x320 pixels with subsequent down sampling by Fourier cropping to 160 pixels. Several rounds of 2D classification were implemented to remove low quality particles, yielding a stack of 711,248 particles from which ab-initio maps were generated. The maps from the best classes were selected and a secondary round of 2D classification followed by ab-initio modelling was performed. The suitable classes underwent non-uniform refinement, producing a map with an estimated overall resolution of 3.8Å (Supplementary Fig. 2 and 3). The local resolution of the final map was estimated using CryoSPARC 58 and visualised with ChimeraX 59 . Model building and refinement An AlphaFold model of a single Hcp3 chain was used as the starting model. This model was rigid-body fitted into the density corresponding to one of the Hcp3 chains using Chimera. This process was repeated five more times, each time placing a new AlphaFold model with a unique chain label into unoccupied density, thereby constructing the hexameric ring of the Hcp3 protein. Similarly, an AlphaFold model of the Tce1 toxin was placed into the density using Chimera 59 . As density was available for only one of the two domains, the models of the N-terminal domain (NTD) and C-terminal domain (CTD) were separated and independently rigid-body fitted into the map. These fittings were visually using Chimera 59 and assessed to determine which domain best corresponded to the observed density. The CTD model provided a better fit and was retained, while the NTD model was discarded. An initial model of the Hcp3–toxin complex was thus constructed, comprising six Hcp3 chains (chain labels A–F) and one toxin chain (label G). Due to anisotropy in the map, density at the periphery appeared fragmented or disconnected. To improve the fit of the model to the map, each chain was manually inspected in Coot, and residues not supported by density were removed. The revised Hcp3-Tce1 model underwent several iterative rounds of real-space refinement using Phenix 60 . Model quality and refinement progress were monitored using Ramachandran plots and MolProbity 61 . Structure analysis and presentation The visualisation and analysis of the cryo-EM maps and atomic models were carried out using ChimeraX, PyMol (Molecular Graphics System, Schrödiner) and PDBePISA. Strains, plasmids and growth conditions Bacterial strains and plasmids used are listed in (Supplementary Table 3) . Bacterial cultures were grown in lysogeny broth (LB) with antibiotics where applicable (Trimethoprim 200 ug/mL). Growth of yeast strains was accomplished with synthetic complete media lacking uracil (SC-Ura – BioShop). Yeast plasmids were first cloned in E. coli and subsequently introduced using the lithium acetate method 62 . Plasmid construction and genomic mutagenesis Plasmids were constructed using restriction cloning with Phusion DNA polymerase, restriction enzymes and T4 DNA ligase from New England Biolabs or with Gibson assembly (NEB). All primers were synthesized by Integrated DNA Technologies (Supplementary Table 4). Plasmids were confirmed through Plasmidsaurus sequencing. Purification of Hcp3 for antibody generation Overnight cultures of BL21 (DE3) pLysS expressing Hcp3 were subcultured 1:100 into 1L of LB and grown to mid-log phase. Cultures were induced by the addition of 0.5mM IPTG and grown for 18 hours at 18 °C. Cultures were collected and resuspended in buffer A (50mM Tris pH 7.5, 500mM NaCl) and lysed by sonication. Lysates were then cleared by centrifugation at 36,000 x g for 45 minutes. The cleared lysate was loaded onto 1mL Ni-NTA resin, washed with subsequent additions of buffer A with 10mM imidazole followed by buffer A with 30mM imidazole. Bound protein was eluted by the addition of buffer A containing 250mM imidazole. The elution was concentrated and buffer exchanged using PD-10 column (Cytiva). Spot plate assays Spot plates were prepared by normalizing overnight cultures to and OD 600 of 1 and making serial dilutions. 10 mL spots were plated and allowed to dry and bacterial plates were incubated for 24 hours at 37 °C while yeast plates were incubated for 48 hours and 30 °C after which time plates were imaged. Western blotting For overexpression of proteins in E. coli , overnight cultures were diluted 1:100 in 25 mL of LB. Cultures were grown to mid-exponential phase and induced by the addition of 0.1% L-rhamnose. Cultures were grown for 2 hours and then collected and resuspended to a final volume of 0.5 mL. For overexpression of proteins in yeast, overnight cultures grown in 2% raffinose were diluted 1:100 and grown in 2% raffinose until log phase. Cultures were induced by the addition of 2% galactose and grown for an additional 18 hours after which time they were collected. Pellets were resuspended in 10mL of lysis buffer (50mM Tris pH 7.4, 500mM NaCl and cells were lysed using an emulsiflex. Lysates were cleared by centrifugation and mixed 1:1 with loading dye and boiled for 10 minutes. 5uL of sample was separated on a 12% Tris-glycine SDS-PAGE gel and subsequently transferred to nitrocellulose at 100 V for 30 minutes. Blots were probed with either anti-FLAG (1:5,000 Sigma), anti-VSV-G (1:5,000 Sigma), anti-His 6 (1:5,000 GenScript) or anti-Hcp3 (1:1000 GenScript). Anti-Hcp3 antibody was generated by GenScipt using purified Hcp3 as an antigen. The produced antiserum was validated for Hcp3 detection by probing purified Hcp3. Secondary antibodies were 1:5,000 goat anti-mouse horseradish peroxidase (New England Biolabs) for anti-His 6 blots or 1:10,000 goat anti-rabbit (New England Biolabs) for anti-VSV-G, anti-Hcp3 or anti-FLAG blots. Declarations Data Availability The atomic coordinates have been deposited at the Protein Data Bank with accession code 9QPE. The density map has been deposited at the Electron Microscopy Data Bank with accession code EMD-53274. Acknowledgements Imaging was done at the eBIC (Diamond, UK). We thank Diamond for access and support of the cryo-EM facilities at the UK national electron bio-imaging centre (eBIC), proposal BI25127, funded by the Wellcome Trust, Medical Research Council and BBSRC. We thank Paul Simpson (Electron Microscopy Facility, Centre for Structural Biology, Imperial College London) for in-house EM support and Tom Wood for helpful discussion on the role of cysteine proteases. S.D.K is jointly supported by postdoctoral fellowship awards from the Natural Sciences and Engineering Research Council of Canada (NSERC) and Cystic Fibrosis Canada. This work was supported by a Canadian Institutes of Health Research project grant to J.C.W. 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Costa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYBADOTYwZcAgA+EfwK2UB0wmMBjDtPAQrSWxAUUAnxZ7BuaHD3/+sEvvEzt78HFFgR0PA/vhB8w8Z/DZwmZszJOQnNsmnZdseMYgmYeBJ82AmecGPi08bNIMCcxALTlmkg1AxQwMOQzMPB/wa5H8kVCfziadY/6zwaCeh4H/DWEtEjwJhxOAWswYGwwO8zBIgGzB57DDIL+kHTcE+QXosONAE54ZHJyDx/vs7c0PH/6wqZaXn5178GPDn2o5fv7khw/eHMOthYEZyY1gAIrTA3g0oHprFIyCUTAKRgFWAADIez95wfJu+AAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-2716-9669","institution":"Centre for Bacterial Resistance Biology, Imperial College, London, SW7 2AZ, UK; Department of Life Sciences, Imperial College, London, SW7 2AZ, UK","correspondingAuthor":true,"prefix":"","firstName":"Tiago","middleName":"R.D.","lastName":"Costa","suffix":""}],"badges":[],"createdAt":"2025-07-01 19:45:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7023205/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7023205/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88401828,"identity":"1ef1deae-3354-4f30-b2a4-b2780628ac02","added_by":"auto","created_at":"2025-08-06 07:06:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":8693251,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional evaluation of the two putative Hcp3 effectors, Tce1 (PA0256) and Tce2 (PA2372). a \u003c/strong\u003eAlphaFold3 models of the Tce1 and Tce2 structures coloured by pIDDT (sequences accessed from PAO1 genome database)\u003cstrong\u003e b \u003c/strong\u003eSequence alignment of Tce1 with Tce2 and other cysteine proteases TcdB (\u003cem\u003eClostridium difficile \u003c/em\u003ecysteine protease PDB:3PEE), a top hit from each of the distance matrix alignment search performed against Tce1 and Tce2, TcdA (\u003cem\u003eClostridium difficile \u003c/em\u003ecysteine protease PDB: 3HO6), OspB (\u003cem\u003eShigella \u003c/em\u003esp. T3SS cysteine protease effector), RtxA (\u003cem\u003eVibrio cholerae \u003c/em\u003ecysteine protease virulence factor). The conserved histidine and cysteine residues across all three proteins are indicated with a star. \u003cstrong\u003ec \u003c/strong\u003eStructural comparison of Tce1, Tce2 (AF3 models) and TcdB (PDB:3PEE) with 90º rotations displaying the shared localisation of the conserved catalytic diad \u003cstrong\u003ec (i)\u003c/strong\u003e Overlap of all three secondary structures to highlight positioning of the catalytic diad. \u003cstrong\u003ed \u003c/strong\u003eSDS-PAGE gels of Tce1-Hcp3 and Tce2-Hcp3 following nickel affinity chromatography where the stronger intensity band for Tce1 than Tce2 suggests that more Tce1 is being pulled down with Hcp3 and thus has a stronger interaction.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7023205/v1/5bcfdb2db4fcb5ab3ff92767.png"},{"id":88401827,"identity":"48aeb187-1643-467f-bce6-00e27d0a2def","added_by":"auto","created_at":"2025-08-06 07:06:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1913309,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePurification of the Hcp3-Tce1 complex. a\u003c/strong\u003e SEC chromatograph of purified Hcp3\u003csub\u003eWT\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003ealone (blue) and copurified Hcp3\u003csub\u003eWT\u003c/sub\u003e and Tce1 (purple). Schematics of the Tce1 filled and empty Hcp3 rings display the assembly of the complex present in each peak. \u003cstrong\u003eb\u003c/strong\u003e SDS-PAGE gel of the peak fractions from chromatograph ‘\u003cstrong\u003ea\u003c/strong\u003e’ stained with Coomassie blue. \u003cstrong\u003ec\u003c/strong\u003e End and side view 2D classes of Tce1 filled and empty Hcp3 hexameric rings.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7023205/v1/ba9059de3c98761b35a63df2.png"},{"id":88401831,"identity":"d40a7d0e-d3f1-41e5-aade-ebc0a4e6d962","added_by":"auto","created_at":"2025-08-06 07:06:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":13414303,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTce1 binding to the Hcp3 hexameric ring. a\u003c/strong\u003e Overview of the tilted, end and side view of the electron density map for the complete Hcp3-Tce1 complex. Each chain is labelled and coloured to represent the individual proteins. \u003cstrong\u003eb\u003c/strong\u003e Cartoon representation of the tilted, end and side view of the model of the Hcp3-Tse1 complex. \u003cstrong\u003ec\u003c/strong\u003e Topology secondary structure diagrams of Hcp3 and the full length Tce1 with individual β-sheets highlighted. The AlphaFold3 predicted N-terminus of Tce1 is outlined to differentiate it from the experimentally derived model. \u003cstrong\u003ed\u003c/strong\u003e Cartoon representation of a Hcp3 monomer and Tce1 with a 180° rotated view. The AlphaFold3 predicted N-terminus is outlined corresponding to ‘\u003cstrong\u003ec\u003c/strong\u003e’ and the linker has been added to visualise the relative positions of the two domains.\u003cstrong\u003e e\u003c/strong\u003e AlphaFold3 prediction demonstrating how two Hcp3 rings can accommodate the full length Tce1. The fitting of the C-terminus within the end ring is from the experimentally derived model.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7023205/v1/cfbeac62ca9fc8205253bb90.png"},{"id":88401835,"identity":"0b885331-4af2-49f4-ad17-d1c52143a689","added_by":"auto","created_at":"2025-08-06 07:06:29","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":58476918,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructural features involved in the interaction between the Hcp3 hexamer and Tce1. a\u003c/strong\u003e Zoom in of the interfaces between Tce1 with each Hcp3 monomer. All interacting residues forming hydrogen/disulfide bonds, salt bridges or covalent links have their side chains displayed and are labelled. \u003cstrong\u003eb\u003c/strong\u003e \u0026nbsp;A table listing all key interacting and interfacing (underlined) residues between Tce1 and each Hcp3 monomer. All key interacting residues with Tce1 are depicted in ‘a’ \u003cstrong\u003ec\u003c/strong\u003e Cartoon representation of the Hcp3 ring with a surface representation of Tce1. The side chain of the G121W point mutation in each Hcp3 monomer is circled to highlight the steric clash introduced between these Hcp3 residues and Tce1. \u003cstrong\u003ed\u003c/strong\u003e Chromatograph of the Hcp3\u003csub\u003eG121W\u003c/sub\u003e + Tce1 and Hcp3\u003csub\u003eG121W+K123W \u003c/sub\u003e+ Tce1 co-purifications presented alongside the Hcp3\u003csub\u003eWT\u003c/sub\u003e, Hcp3\u003csub\u003eWT\u003c/sub\u003e + Tce1 from ‘\u003cstrong\u003e1a\u003c/strong\u003e’ to visualise the shift in elution volumes between the WT and mutant complexes. \u003cstrong\u003ee\u003c/strong\u003e SDS-PAGE gel of the peak fractions from chromatograph ‘\u003cstrong\u003ed\u003c/strong\u003e’ stained with Coomassie blue.\u003c/p\u003e","description":"","filename":"Figure4New.png","url":"https://assets-eu.researchsquare.com/files/rs-7023205/v1/b0e9dbc50bb8e4b5f0018f38.png"},{"id":88401829,"identity":"899add5a-7f21-440e-8632-050ac3b02d77","added_by":"auto","created_at":"2025-08-06 07:06:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3912365,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssembly mechanism of the Hcp3-Tce1 complex. a\u003c/strong\u003e Left: chromatograph of Hcp3\u003csub\u003eWT\u003c/sub\u003e purifications at 4.0, 1.0, 0.5 and 0.1mg/ml with schematics representing the oligomeric state of Hcp3 at both retention volumes. Right: the anti-his Western Blot from the peak fractions of the 4.0mg/ml and 0.1mg/ml purifications. \u003cstrong\u003eb\u003c/strong\u003e Left: chromatograph of Hcp3\u003csub\u003eWT\u003c/sub\u003e + Tce1 co-purifications at 4.0, 1.0, 0.5 and 0.1mg/ml with schematics representing the oligomeric states of Hcp3 interacting with Tce1 at both retention volumes. Right: The anti-his and anti-HA Western Blots from the peak fractions of the 4.0mg/ml and 0.1mg/ml purifications. \u003cstrong\u003ec\u003c/strong\u003e 2D classes capturing the sequential assembly states of the Hcp3 ring in complex with Tce1 alongside a colour coded representation of the proteins present in the 2D classes.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7023205/v1/a26799e7d9e7f34c6e2017ff.png"},{"id":88402562,"identity":"dc177603-8eb1-41d7-823e-77a8ac772714","added_by":"auto","created_at":"2025-08-06 07:14:29","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3336377,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTce2 is a cysteine protease with antifungal activity which is also loaded into the Hcp3 lumen for toxin delivery. a \u003c/strong\u003eThe predicted localisation of the Tce2 AlphaFold3 structure within the experimentally derived Hcp3 ring, aligned to the experimentally derived Tce1 within the ring. \u003cstrong\u003eb \u003c/strong\u003eViability of \u003cem\u003eE. coli \u003c/em\u003eharbouring plasmids encoding Tce1 or Tce2. The positive control is a known antibacterial toxin Tse6. Growth was performed in the absence of inducer, or in the presence of inducer (0.1% L-rhamnose). \u003cstrong\u003ec\u003c/strong\u003e Western blot analysis of liquid cultures of \u003cem\u003eE. coli \u003c/em\u003eexpressing Tce1 or Tce2. \u003cstrong\u003ed \u003c/strong\u003eViability of \u003cem\u003eS. cerevisiae \u003c/em\u003eBY4742 harbouring plasmids encoding Tce1, Tce2, or a Tce2 catalytic variant Tce2\u003csup\u003eC115S\u003c/sup\u003e. Viability of \u003cem\u003eS. cerevisiae \u003c/em\u003ecoexpressing Tce1 and Hcp3 or the Tce1 C-terminal domain (CTD) with Hcp3 was also evaluated. Plates either contained 2% glucose for repression of expression or 2% galactose for induction.\u003cstrong\u003e \u003c/strong\u003eWestern blot analysis of \u003cstrong\u003ee\u003c/strong\u003e Tce2 and catalytic mutant Tce2\u003csup\u003eC115S\u003c/sup\u003e and \u003cstrong\u003ef\u003c/strong\u003e Tce1 or Tce1\u003csup\u003eCTD\u003c/sup\u003e with and without Hcp3 coexpression in \u003cem\u003eS. cerevisiae \u003c/em\u003eBY4742.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7023205/v1/e83a67efa3b7616b844e72f7.png"},{"id":88402563,"identity":"abf8e7ac-52da-4cec-94e7-6dd6ca50b83c","added_by":"auto","created_at":"2025-08-06 07:14:29","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2926521,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposed mechanism of concentration driven Hcp ring assembly and disassembly\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e in vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e The mechanism is instigated in the attacking cell where Hcp3 captures the toxin in a 1:1 interaction before sequential concentration driven assembly into a hexameric ring. In this mechanism, Hcp3 also oligomerises into hexameric rings in the absence of a bound toxin. The rings then polymerise to form the tube of the T6SS tail complex which is injected into the target cell upon T6SS contraction. In the target cell, the low Hcp3 concentration promotes ring disassembly, releasing the toxin and freeing its active site.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7023205/v1/53c92b7403379ed4776fe3c7.png"},{"id":88823555,"identity":"44fdbb7c-c98d-4743-9cc8-e2bcff18eb7a","added_by":"auto","created_at":"2025-08-11 18:11:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":76267473,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7023205/v1/afd897c1-8556-4bef-a8d5-6084ce12237e.pdf"},{"id":88401826,"identity":"a090256d-bcec-40c4-99f3-8701631e4e10","added_by":"auto","created_at":"2025-08-06 07:06:29","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":40830,"visible":true,"origin":"","legend":"Supplementary Tables","description":"","filename":"SupTables090625.docx","url":"https://assets-eu.researchsquare.com/files/rs-7023205/v1/0468fae6674b59ad5ce9a85c.docx"},{"id":88401836,"identity":"3cb6bb80-207d-4d4b-b627-ccffd4845b03","added_by":"auto","created_at":"2025-08-06 07:06:29","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2797858,"visible":true,"origin":"","legend":"Tce1 loadiing and wrapping within Hcp3 ring","description":"","filename":"hcp3tce1movie200625.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7023205/v1/1231f6bf1b9b7a118aa47d76.mp4"},{"id":88401833,"identity":"9dc4ad49-5c5d-4d98-afb3-c1b3efdee9a6","added_by":"auto","created_at":"2025-08-06 07:06:29","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2399645,"visible":true,"origin":"","legend":"Supplementary figures","description":"","filename":"SupFigures090625.docx","url":"https://assets-eu.researchsquare.com/files/rs-7023205/v1/b29706abb68c308c4a34948e.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"A Novel Molecular Mechanism for Effector Protein Wrapping and Delivery by the Bacterial Type VI Secretion System","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMicrobes have been competing for billions of years, and this has led to the evolution of numerous strategies to capture scarce resources and eliminate competitors\u003csup\u003e1\u003c/sup\u003e. The Type VI secretion system (T6SS) is a sophisticated and dynamic bacterial supramolecular machine that injects toxic effectors into target cells\u003csup\u003e2-4\u003c/sup\u003e. T6SSs are versatile and can target many cell types such as eukaryotic cells, including fungi, or prokaryotes such as multi-drug-resistant Gram-negative bacteria\u003csup\u003e5\u003c/sup\u003e. This nanomachine is often compared to a crossbow loaded with toxic arrows and there is extensive knowledge on how its assembly is initiated, then extended and finally contracted to fire effectors into susceptible cells\u003csup\u003e6,7\u003c/sup\u003e. The T6SS tip is a puncturing device, which includes a spike complex comprised of PAAR and VgrG proteins\u003csup\u003e8\u003c/sup\u003e. The polymerisation and thus extension of a cytosolic contractile T6SS sheath is a coordinated event\u003csup\u003e9\u003c/sup\u003e during which hexameric rings of the Hcp\u003csup\u003e10,11\u003c/sup\u003e protein first interact with the flat base of the VgrG-PAAR spike complex and then stack on top of one another to form a tube structure that is simultaneously encapsulated within the sheath\u003csup\u003e12\u003c/sup\u003e. The rapid contraction of the sheath\u003csup\u003e13\u003c/sup\u003e propels the PAAR-VgrG-Hcp puncturing device through the bacterial envelope and into target cells, delivering effectors along with it.\u003c/p\u003e\n\u003cp\u003eDespite many notable advances in visualizing the structure and dynamics of the T6SS apparatus, gaps remain in our understanding of the mechanisms by which toxins/effectors are recruited and loaded into the T6SS. In recent years, some of these mechanisms have begun to be defined. For example, effectors can be targeted to the T6SS through the recognition of conserved protein sequences such as the MIX, RIX, PIX, WHIX or FIX motifs\u003csup\u003e14,15\u003c/sup\u003e, but these are found in a limited number of effectors and how each of them functions at a molecular level has not yet been examined. Some effectors have been shown to require the assistance of chaperones or adaptors such as Eag or Tap proteins\u003csup\u003e16,17\u003c/sup\u003e, respectively, which may help prevent premature folding in the producing cell cytoplasm. T6SS effector loading has been proposed to occur by attachment of effectors to either one part of the PAAR-VgrG-Hcp complex\u003csup\u003e8\u003c/sup\u003e. Effectors can either exist as C-terminal extension of any of these T6SS spike elements, in which case they are termed specialized effectors, or they are recruited to one of these components in which case they are denoted cargo effectors\u003csup\u003e18\u003c/sup\u003e. In general, the association of effectors with VgrG, e.g. \u003cem\u003eEscherichia coli\u003c/em\u003e Tle1\u003csup\u003e19\u003c/sup\u003e, and PAAR, e.g. \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e Tse6\u003csup\u003e20\u003c/sup\u003e, is well described at a molecular and structural level. VgrG and PAAR\u003csup\u003e21,22\u003c/sup\u003e, which are a trimer and a monomer, respectively, could load at maximum a total of 4 effectors on one single T6SS spike complex. By contrast, Hcp rings exists in as many as 120 copies within the elongated T6SS sheath\u003csup\u003e23-25\u003c/sup\u003e, dramatically increasing the ability of the system to deliver a huge cocktail of possibly functionally distinct effectors. As such, T6SS loading through Hcp may result in a highly effective system against any type of prey cells, but comparatively little is known about how toxins/effectors are recruited to Hcp. Interestingly, Hcp has been proposed to exhibit chaperone activity by stabilizing small effectors such as \u003cem\u003eP. aeruginosa\u003c/em\u003e Tse2 and it was hypothesized that Tse2 sits within the lumen of Hcp1 hexameric rings\u003csup\u003e26\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e is equipped with 3 T6SSs known as the H1-, H2- or H3-T6SS\u003csup\u003e27\u003c/sup\u003e, and each system is associated with the Hcp proteins, Hcp1, Hcp2 and Hcp3, respectively. In previous work, using \u003cem\u003ein vivo\u003c/em\u003e pulldown experiments, we identified potential cargo effectors associated with these Hcp proteins\u003csup\u003e28,29\u003c/sup\u003e. Here, we describe two novel \u003cu\u003eT\u003c/u\u003eype VI Secretion System delivered \u003cu\u003ec\u003c/u\u003eysteine protease \u003cu\u003ee\u003c/u\u003effector (Tce) proteins bound to Hcp3, Tce1 (PA0256) and Tce2 (PA2372). We present the first high resolution structure of an Hcp-cargo effector complex using cryo-electron microscopy. The 3.8 Å resolution of the Hcp3-Tce1 complex reveals the molecular details behind Hcp3 toxin recognition, the molecular events that lead to the formation of the complex, and their stoichiometric arrangements. Our data indicate that a 1:1 Hcp3-Tce1 heterocomplex forms first, followed by the sequential oligomerisation of Hcp around the toxin until the final Hcp3 hexameric ring filled with the Tce1 toxin in its lumen is formed. We also show that the loading of a T6SS toxin requires two rings for it to be fully embedded with the Hcp tube. These findings offer an entirely new concept for T6SS effector loading that is driven by the cellular concentration of Hcp. Overall, our work provides a novel mechanism and original molecular rules on how T6SS cargo complexes are recruited by and loaded into Hcp. Exploiting such unique knowledge can be tapped on to design T6SS-wielding bacteria as biocontrol agents for microbiome stewardship in human and natural ecosystems.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eHcp3\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003einteracts with two H3-T6SS effector candidates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn\u0026nbsp;a previous study, we identified two potential\u0026nbsp;\u003cem\u003eP. aeruginosa\u003c/em\u003e H3-T6SS effectors – PA0256 and PA2372\u003csup\u003e28,29\u003c/sup\u003e, hereafter referred to as Tce1 and Tce2 – through an \u003cem\u003ein vivo\u003c/em\u003e pull-down approach using Hcp3, which is encoded within the H3-T6SS cluster (Supplementary Fig. 1). A distance-matrix alignment (DALI)\u003csup\u003e30\u003c/sup\u003e was performed using the AlphaFold3\u003csup\u003e31\u003c/sup\u003e predictions of both Tce1 and Tce2 to identify structural homology to proteins with known enzymatic activity (Fig. 1a). A top hit for the analysis of each protein was the cysteine protease domain for the \u003cem\u003eClostridium difficile\u0026nbsp;\u003c/em\u003etoxin TcdB\u003csup\u003e32\u003c/sup\u003e (PDB:3PEE). A multiple sequence alignment of Tce1, Tce2 and TcdB, and other known cysteine proteases revealed conserved cysteine and histidine residues across these proteins (Fig. 1b) Structural alignment showed that His186 and Cys240 in Tce1, as well as His65 and Cys115 in Tce2, are spatially positioned to align with the catalytic dyad of TcdB (His110 and Cys155), supporting the hypothesis that both Tce1 and Tce2 are cysteine protease-like enzymes (Fig. 1c). It is also noteworthy that Tce1 is a larger protein (310 a.a) than Tce2 (190 a.a.) with a C terminus of unknown function (110-310) encompassing the putative cysteine protease catalytic site and an extended N terminus (1-101) (Fig. 1a). Both effectors co-purified with Hcp3 via affinity chromatography (Fig. 1d), with Tce1 showing a stronger interaction consistent with the in vivo pull-down where Tce1 also shows a stronger interaction (8.4-fold enrichment) compared to Tce2 (5.8-fold enrichment). Based on these data, we further prioritised Tce1 toobtain a high resolution cryo-EM structure of a Hcp3-effector complex.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCryo-EM reconstruction reveals that two Hcp3 rings are needed to fully enclose Tce1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003ehcp3\u003c/em\u003e and \u003cem\u003etce1\u003c/em\u003e genes were cloned, co-expressed in \u003cem\u003eE. coli\u003c/em\u003e, and co-purified via affinity and size-exclusion chromatography. Hcp3 and Tce1 remained stably associated throughout the purification\u0026nbsp;(Fig. 2a,b), eluting as a complex at 12.6 ml. Given that the Hcp3 hexamer alone elutes at 12.8 ml\u003csup\u003e29\u003c/sup\u003e (Fig. 2a), this shift suggests that Hcp3 binds Tce1 in its hexameric form, with the presence of the toxin accounting for the earlier elution, indicative of a heavier particle.\u003c/p\u003e\n\u003cp\u003eUpon purification, the assembly and homogeneity of the complex was assessed through negative stain-EM and following successful inspection, the complex was frozen onto thin carbon layer grids and imaged through cryo-EM (Supplementary Table 1).\u0026nbsp;After subsequent beam induced motion and contrast transfer function (CTF) correction, particles were extracted from the collected micrographs and rounds of 2D classification were performed. From this, a dataset of ~1 million particles was acquired, yielding high resolution 2D class averages which display a clear ring structure with additional density observable within the central lumen only when Tce1 is present in the sample (Supplementary Fig. 2\u0026nbsp;and\u0026nbsp;Fig. 2c). By looking at the side views we could also infer that a portion of the toxin density slightly protrudes out of the ring. The high resolution 2D classes were subjected to iterative rounds of Ab initio classification followed by non-uniform refinement with C1 symmetry applied. The final map achieved an overall resolution of 3.8Å, with local resolution extending to sub-3.4Å across the complex (Supplementary Fig. 3).\u003c/p\u003e\n\u003cp\u003eThe densities corresponding to the Hcp3 ring and Tce1 C-terminus were identified in the map and used to build an atomic model of the entire Hcp3 ring in complex with the C-terminal domain of Tce1 (Fig. 3a,b). The model of the Hcp3 ring displays six repeating units in a homohexameric conformation with each Hcp3 monomer made up of nine\u0026nbsp;β-strands (β1-β9) forming two interfacing\u0026nbsp;β-sheets, alongside a single\u0026nbsp;α-helix (Fig. 3c,d). Hcp3 monomer-monomer interactions are stabilised through hydrogen bonds between the\u0026nbsp;α1 and\u0026nbsp;β6 of one monomer with\u0026nbsp;β2 and\u0026nbsp;β9 of its neighbour, with specific interacting residues listed in\u0026nbsp;Supplementary Table 2. These interacting residues are repeated across all Hcp3 monomers in the hexameric ring. Our model also showed that only the C-terminal domain of Tce1 is structurally resolved in the Hcp3-Tce1 complex and is comprised of ten\u0026nbsp;β-strands (β9-β18) forming one central\u0026nbsp;β-sheet, surrounded by four\u0026nbsp;α-helices (α5-α8).\u003c/p\u003e\n\u003cp\u003eBecause our model of Tce1 starts from Lys110, we modelled the full length Tce1 using AlphaFold3\u003csup\u003e31\u003c/sup\u003e to predict the structure of residues 1-109 (Fig. 3d). From this, we observe that the N-terminal domain possesses a\u0026nbsp;β-sandwich domain arrangement (β1-β8)\u0026nbsp;alongside four short α-helices (α1-α4) and a short linker sequence (residues 106-109) that connects the two domains.\u0026nbsp;We measured the Hcp3 ring to have an outer diameter of 85 Å and an inner diameter of 45 Å. Based on these dimensions, the lumen can accommodate only the C-terminal domain of Tce1, with a portion of it protruding from the ring. This indicates that in the full-length protein, the N-terminal domain of Tce1 would extend further outward from the ring and into an adjacent Hcp3 hexamer.\u0026nbsp;(Fig. 3e). Overall, our data strongly support a model in which two stacked Hcp3 rings fully encapsulate full-length Tce1, with the C- and N-terminal domains each positioned within the lumen of a ring\u0026nbsp;(Fig. 3e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTce1 interacts with key Hcp3 residues at the inner surface of the ring\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWith Tce1 existing as a monomer within the Hcp3 hexameric ring, it is expected that its interface with each monomer would differ. This is indeed what our structure reveals, with each Hcp3 molecule displaying a distinct degree of interaction with Tce1 (Fig. 3 and 4a,b), and key interacting residues forming hydrogen bonds or salt bridges (Fig.4b and Supplementary Table 2). We arbitrarily numbered the Hcp3 protomers A through F and found that monomers B, C, and D interact with Tce1 via residues G121 and K123. These residues exist in a loop region between β6 and β7 that extends into the lumen of the assembled Hcp3 ring. This observation suggests that G121 and K123 play a critical role in anchoring and positioning Tce1 within the ring structure.\u003c/p\u003e\n\u003cp\u003eTo validate this hypothesis and the assembly revealed by our cryo-EM structure, we generated mutants in these key interacting residues to sterically modify the Hcp3 inner surface. Both G121W and K123W substitutions were generated to introduce, via the bulky nature of the aromatic tryptophan side chain, the greatest steric hindrance within the Hcp ring lumen and restrict access to Tce1 (Fig. 4c). We then compared the gel filtration chromatography profile of Tce1 co-purified with Hcp3\u003csub\u003eG121W\u003c/sub\u003e and Hcp3\u003csub\u003eG121W+K123W\u003c/sub\u003e with our previous co-purification with the native Hcp3\u003csub\u003eWT\u003c/sub\u003e (Fig. 4d). In support of our structural data, we observe that in the presence of Tce1, both Hcp3\u003csub\u003eG121W\u003c/sub\u003e and Hcp3\u003csub\u003eG121W+K123W\u003c/sub\u003e elute at the same retention volume of Hcp3\u003csub\u003eWT\u0026nbsp;\u003c/sub\u003ealone, indicating that they do not interact with Tce1 (Fig. 4d). This observation is further supported by SDS-PAGE analysis of the elution fractions (Fig. 4e), where only a band corresponding to Hcp3 is detected. Together, these results validate our 3D structure of the Hcp3-Tce1 complex and demonstrate that Tce1 is positioned within the Hcp3 ring, interacting with its inner surface.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHcp3 sequentially wraps around Tce1 revealing the molecular basis for T6SS effector loading\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe asymmetric interaction between Tce1 and individual Hcp3 monomers suggests an assembly mechanism in which Tce1 first interacts with a single Hcp3 monomer followed by the rest of the ring assembling around it. To test this hypothesis, we first assessed the solution state behaviour of the Hcp3 ring at different protein concentrations. We purified Hcp3\u003csub\u003eWT\u0026nbsp;\u003c/sub\u003eat 4.0, 1.0, 0.5 and 0.1 mg/ml using affinity and gel filtration chromatography. At the higher concentrations, Hcp3\u003csub\u003eWT\u0026nbsp;\u003c/sub\u003eelutes at 12.9ml (Fig. 5a), consistent with the\u0026nbsp;previously observed retention volume expected for a hexamer\u0026nbsp;(Fig. 2a). However, at 0.1mg/ml, the retention volume increases to 16.2ml,\u0026nbsp;indicating a substantial reduction in molecular weight consistent with monomeric Hcp3\u0026nbsp;(Fig. 5a).\u0026nbsp;These results demonstrate that Hcp3 exists as a monomer at low concentrations and as a hexamer at higher concentrations, suggesting that ring formation is concentration dependent. Since Hcp3 elutes as a hexamer at concentrations of 0.5 mg/ml and above, the threshold concentration for hexamer assembly appears to lie between 0.1 mg/ml and 0.5 mg/ml.\u003c/p\u003e\n\u003cp\u003eNext, we examined how Tce1 interacts with Hcp3 under conditions that favor incomplete ring formation. We co-purified Tce1 with Hcp3\u003csub\u003eWT\u003c/sub\u003e at 4.0, 1.0, 0.5 and 0.1mg/ml, following the same protocol as for Hcp3\u003csub\u003eWT\u003c/sub\u003e alone. The chromatographs for the 4.0,1.0 and 0.5mg/ml purifications depict the characteristic peak at 12.6ml, corresponding to the fully assembled Tce1-filled hexameric ring (Fig. 5b).\u0026nbsp;At 0.1 mg/ml, consistent with the behavior of\u0026nbsp;Hcp3\u003csub\u003eWT\u003c/sub\u003e alone,\u0026nbsp;the retention volume increases, but in this case to 15.4 ml—less than the 16.2 ml observed for monomeric Hcp3 alone. This shift likely reflects the addition of Tce1 (~39 kDa) to a monomeric or partially assembled Hcp3 species. Western blot analysis confirmed that both Tce1 and Hcp3 are present in the 15.4 ml elution fraction, demonstrating that Tce1 can interact with non-hexameric Hcp3\u0026nbsp;(Fig. 5a,b).\u003c/p\u003e\n\u003cp\u003eTo further bolster these findings, we re-analyzed the 2D classifications from the original cryo-EM dataset used to solve the Hcp3–Tce1 complex structure. Remarkably, we identified distinct 2D classes corresponding to intermediate stages of ring assembly in which Tce1 is only partially enclosed (Fig. 5c). Altogether these results support a novel molecular model in which Hcp3 ring formation is a sequential process rather than a discrete transition from monomer to hexamer. Furthermore, they reveal that Tce1 initially binds to a small number of Hcp3 monomers, with the rest of the ring assembling around the toxin, even though not all subunits directly interact with it.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTce1 and Tce2 are cysteine protease with analogous structural and functional properties.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on our experimentally derived Hcp3-Tce1 complex and using AlphaFold3\u003csup\u003e31\u003c/sup\u003e we could predict that as for Tce1, Tce2 nicely sits in the lumen of the Hcp3 ring with asymmetrical contact with the various Hcp3 protomers (Fig. 6a).\u0026nbsp;Tce1 and Tce2 both possess central β-sheets encircled by α-helices (Fig.1a,c). Both β-sheets align, localising Tce2 to the same region of the Hcp3 lumen as where Tce1 is loaded. Tce2 sits in the lower half of the Hcp3 ring, where it partially protrudes out from the underside of the ring (Fig. 6a).\u003c/p\u003e\n\u003cp\u003eWe then revisited our previous suggestion that like most T6SS effectors, Tce1 and Tce2 likely possess antibacterial activity\u003csup\u003e33\u003c/sup\u003e. Our data showed that this is likely not the case, since heterologous expression of these enzymes in \u003cem\u003eE. coli\u003c/em\u003e did not affect growth (Fig. 6b,c). This is also supported by the observation that no obvious immunity protein\u003csup\u003e34-37\u003c/sup\u003e encoding genes are found in the vicinity of the genes encoding these effector proteins (Supplementary Fig. 1). We then further investigated potential anti-eukaryotic toxicity\u003csup\u003e38,39\u003c/sup\u003e by expressing Tce1 and Tce2 in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. Strikingly, we observed that Tce2 significantly impairs yeast growth. Notably, this toxicity was alleviated by a mutation in the predicted active site of the cysteine protease, confirming its functional relevance (Fig. 6d). In contrast, Tce1 expression did not significantly affect yeast growth. Western blot analysis revealed detectable expression of Tce2, but not Tce1, suggesting Tce1 may be unstable in this context (Fig. 6e,f). To mitigate this instability, we co-expressed Tce1 with Hcp3, and although Tce1 could now be detected by western blot, no impact was observed on yeast growth. A possible hypothesis to the lack of toxicity is that the extended N-terminus of Tce1, as compared to Tce2, may interfere with cysteine protease activity. We thus produced a N-terminally truncated version of Tce1 and expressed in yeast in presence/absence of Hcp3, but in all cases no toxicity could be observed (Fig. 6d,f). Overall, we identified for the first time a T6SS-dependent antifungal property that may be associated with the H3-T6SS function through Tce2, while the absence of a toxic phenotype for Tce1 in these assays may suggest the requirement of auxiliary molecules for its enzymatic activity.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eT6SS-dependent effector delivery is a challenging conceptual question to address experimentally and there is a plethora of mechanisms that have been proposed\u003csup\u003e40\u003c/sup\u003e. Some effectors are called specialized with a cargo domain that is an extension of a T6SS component, namely PAAR, VgrG or Hcp. In this case the loading simply relies on these subunits being assembled into the system and the protein-protein interfaces with other elements of the T6SS machine are relatively well described\u003csup\u003e8\u003c/sup\u003e. There are many examples of VgrG- or PAAR-specialized effectors\u003csup\u003e18,41\u003c/sup\u003e, but specialized Hcp effectors have been poorly studied. However three specialized Hcp effectors have been recently described in \u003cem\u003eEnterobacter cloacae\u003c/em\u003e, Hcp-ET1 (DNase), Hcp-ET2 (Phospholipase) and Hcp-ET3 (Pyocin)\u003csup\u003e42\u003c/sup\u003e, while further genomic screening identified around 350 Hcp-specialized effectors mostly in \u003cem\u003eEnterobacteriaceae\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eIt was initially proposed that large effectors associate with the T6SS tip, i.e. VgrG or PAAR, whereas effectors that are smaller in size insert into the Hcp lumen such as the \u003cem\u003eP. aeruginosa\u003c/em\u003e Tse2 cargo effector\u003csup\u003e26\u003c/sup\u003e. Here our study reveals the detailed molecular mechanism on how cargo effectors are loaded into Hcp. The cryo-EM structure of the \u003cem\u003eP. aeruginosa\u003c/em\u003e Hcp3-Tce1 complex at 3.8 Å resolution provides unprecedented insight on how Tce1 is wrapped within the lumen of a homohexameric Hcp3 ring. One of the major findings is the sequential nature of the process during which Tce1 initially interacts with monomeric Hcp3 through specific contacts, notably through the formation of hydrogen bonds and salt bridges\u0026nbsp;(Supplementary Table 2). This initial 1:1 interaction appears to nucleate the subsequent oligomerisation of Hcp3 around Tce1, culminating in the formation of a fully formed effector-loaded hexamer. In this process,\u0026nbsp;S29, E56, and T60 serve as key hydrogen bond-forming residues, interacting with Tce1 across multiple Hcp3 protomers. Additional hydrogen bonds form between each of the five interacting Hcp3 monomers and Tce1, facilitating stable capture of the toxin.\u003c/p\u003e\n\u003cp\u003eThis mechanism of Hcp wrapping around the effector is conceptually novel compared to the previously proposed mechanism of effector insertion into Hcp rings. This assembly mechanism can to some extent be compared with the Tat system\u003csup\u003e43\u003c/sup\u003e, which translocates folded proteins across the bacterial cytoplasmic membrane. In this case, TatA monomers once associated with a Tat substrate further wrap around the substrate through oligomerization\u003csup\u003e44\u003c/sup\u003e. This mechanism helps accommodate proteins of varying sizes by recruiting more TatA protomers for large substrates as compared to smaller ones. In the case of Hcp, such modulation in the number of protomers in the ring is likely not possible due to the size and structure of the Hcp rings being fixed and rigid. However, another striking discovery we made is that effectors can be enclosed by more than one Hcp ring. This creates additional room to pack larger effectors than initially thought.\u003c/p\u003e\n\u003cp\u003eAnother implication of our findings is that for specialized Hcp effectors\u003csup\u003e42\u003c/sup\u003e, the ring cannot be formed by more than one Hcp-extended effector due to the steric incompatibility of packing more than one effector in the Hcp lumen. Instead, the extended Hcp may bind to 5 other ‘regular’ Hcps that complete the hexameric ring around one Hcp-ET. This conclusion is supported by our observation that the Tce1 effector initially interacts with a single Hcp3 protomer. It is also striking that two effectors, Tce1 and Tce2, are packed within Hcp3 rings. The stronger binding of Tce1 to Hcp3, compared to Tce2, may be explained by their genomic context. While \u003cem\u003etce2\u003c/em\u003e is encoded within the H3-T6SS cluster and co-transcribed with \u003cem\u003ehcp3\u003c/em\u003e (Supplementary Fig. 1), \u003cem\u003etce1\u003c/em\u003e is located distantly. It is therefore plausible that Tce1 has evolved a higher affinity for Hcp3 to compensate for its genomic separation.\u0026nbsp;Alternatively, it may suggest a mechanism that drives loading preference, where effectors with higher affinity are loaded first. This strategy is used in other biological context such as fimbrial assembly in which fimbriae subunits that need to be recruited first display higher affinity for the outer membrane usher protein compared to subunits that should be assembled subsequently\u003csup\u003e45\u003c/sup\u003e. In the T6SS, such mechanism would indicate that there are more Tce1 molecules loaded into the H3-T6SS apparatus than there are of Tce2, thus introducing a level of regulatory control in terms of T6SS function and impact on target cells. Furthermore, our observation that ring assembly is concentration-dependent suggests that intracellular levels of Hcp3 and its cargo might regulate the formation of secretion-competent complexes, adding an additional layer of control to T6SS function. The relative concentration between Hcp and cargo could be a modulator of T6SS activity. It was recently shown in \u003cem\u003eVibrio\u003c/em\u003e \u003cem\u003echolerae\u003c/em\u003e that Hcp accumulation can drive T6SS gene repression through interaction with the VasH regulator\u003csup\u003e46\u003c/sup\u003e. We could thus predict that accumulation of free Hcp in the absence of effectors may result in T6SS down regulation. This would also align with the onboard checking mechanism described for VgrG-PAAR in \u003cem\u003eV. cholerae\u003c/em\u003e, which described T6SS assembly failure in the absence of effectors\u003csup\u003e47\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTce2 is a probable cysteine protease activity that negatively impacts yeast growth in a manner that is dependent on its predicted catalytic cysteine. When compared with Tce2, Tce1 exhibits the same conserved cysteine and histidine residues that align with the catalytic diad of the well-characterised \u003cem\u003eClostridium difficile\u003c/em\u003e toxin TcdB\u003csup\u003e48\u003c/sup\u003e. However, unlike Tce2, Tce1 does not cause significant growth inhibition in yeast. This may imply that Tce1 protease activity is tightly regulated and potentially activated only upon delivery into a physiologically relevant target cell. Hcp3 is part of the \u003cem\u003eP. aeruginosa\u003c/em\u003e H3-T6SS which was shown to be involved mainly in anti-eukaryotic activity\u003csup\u003e49\u003c/sup\u003e. Intriguingly, and in contrast to the H1-T6SS and H2-T6SS, for which numerous effectors have been described, effectors associated with H3-T6SS are scarce\u003csup\u003e50,51\u003c/sup\u003e. Two have been associated with VgrG3, TepB\u003csup\u003e52\u003c/sup\u003e and TseF\u003csup\u003e53\u003c/sup\u003e, both having elusive role in being involved in common goods acquisition\u003csup\u003e54\u003c/sup\u003e and/or injected in eukaryotic cells as is the case for TepB\u003csup\u003e52\u003c/sup\u003e. Our findings that Tce2, and possibly Tce1, are anti-eukaryotic effectors strengthen this concept. The molecular target(s) for Tce2 and Tce1 are unknown and comparison with other cysteine protease effectors underscores the evolutionary diversity of these effectors, making potential targets challenging to predict. For example, Cpe1 is a papain-like cysteine protease that targets type II DNA topoisomerases GyrB and ParE\u003csup\u003e55\u003c/sup\u003e, whereas OspB secreted by the \u003cem\u003eShigella flexneri\u003c/em\u003e T3SS cleaves the metabolic regulator TORC1 in yeast and mammalian cells\u003csup\u003e56\u003c/sup\u003e. Although sharing a conserved catalytic core, differences in accessory domains and activation motifs likely reflect adaptation to specific host targets and regulatory environments, providing a broader perspective on how T6SS effector function is fine-tuned.\u003c/p\u003e\n\u003cp\u003eThe observation that the Hcp3 ring can be loaded with either Tce1 or Tce2 also supports the concept of the injection of a cocktail of effectors and toxins by one single system, not even accounting for those loaded on the VgrG3 tip. How many Hcp3 rings contain Tce1 compared to Tce2 remains to be understood and will need to take in account the concept that some of the rings might need to remain empty to fulfil steric requirements when the \u003cem\u003eca\u003c/em\u003e 120 Hcp rings are contained in the T6SS sheath. Together, a fully loaded apparatus constitutes a very powerful effector/toxin injection device, and one may even consider that empty Hcp rings could still have potential toxic activity as proposed for Hcp in \u003cem\u003eAcidovorax citrulli\u003c/em\u003e\u003cem\u003e\u003csup\u003e57\u003c/sup\u003e\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eIn summary, by defining the molecular basis of T6SS effector loading and establishing an unprecedent link between toxin recognition, sequential ring assembly, and concentration-dependent oligomerisation, this work provides a novel framework for understanding how bacteria deploy T6SS effectors to modulate intercellular interactions.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eBacterial cultures and Hcp3-Tce1 expression and purification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe bacterial strains and plasmids used in this study are listed in\u0026nbsp;Supplementary Table 3. Bacteria in this study were cultured in lysogeny broth (LB) (Miller) or on LB agar (Miller plates) and grown at 37°C unless stated otherwise. The sequences coding for Hcp3-His variantsand HA-Tce1were cloned into pACYCDuet-1 and pET22b vectors respectively and transformed into BL21 cells. Cells were grown in 1L LB with appropriate antibiotics and protein expression was induced at OD\u003csub\u003e600\u0026nbsp;\u003c/sub\u003e- 0.6 by addition of 0.5mM IPTG for 20h at 20°C. The cells were harvested by centrifugation at 7000 x g, 30 min at 4°C. The pellet was resuspended in 20mM HEPES (pH 7.5), 250mM NaCl, 100mgml\u003csup\u003e-1\u003c/sup\u003e Lysozyme, 100μlml\u003csup\u003e-1\u003c/sup\u003e Triton X-100, 0.5mM PMSF. Following lysis by sonication on ice, cellular debris was removed by centrifugation at 18,000 x g for 45 min. The supernatant was incubated with 1ml \u0026nbsp;of TALON\u003csup\u003eâ\u003c/sup\u003e metal affinity resin pre-equilibrated with equilibration buffer (20mM HEPES, 250mM NaCl) for 1h. The incubated resin was loaded onto a Econo-Pac\u003csup\u003eâ\u003c/sup\u003e gravity flow chromatography column and washed with 100ml of wash buffer (20mM HEPES, 250mM NaCl, 7mM Imidazole) before elution with 10ml elution buffer (20mM HEPES, 250mM NaCl, 500mM Imidazole). Elution buffer was added 2ml at a time to be collected into 5x 2ml elution fractions. Fractions containing protein were pooled into 3.5 kDa MWCO SnakeSkin\u003csup\u003eTM\u003c/sup\u003e dialysis tubing and dialysed in 20mM HEPES pH 7.5, 150mM NaCl overnight at 4°C. Following dialysis, the sample was concentrated using a centrifugal concentrator with a 3 kDa MWCO. Size exclusion chromatography (SEC) was performed on the concentrated sample using a Superdex 200 GL 10/300 column (GE healthcare) preequilibrated in SEC buffer (20mM HEPES pH7.5, 150mM NaCl). Presence of both Hcp3-His and HA-Tce1 following SEC were both analysed using Coomassie stain and Western blot analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eElectron microscopy sample preparation and data collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the structural determination of the Hcp3-Tce1 complex, a Hcp3-Tce1 SEC purification was incubated with 0.1% glutaraldehyde crosslinking followed by another SEC purification. A 4 µl aliquot from the peak fraction, with the same retention volume as the uncrosslinked Hcp3-Tce1 SEC purification, at a concentration of 0.25 mg/ml was applied onto a glow-discharged Quantifoil R1.2/1.3 Cu 300 mesh holey grid (Agar Scientific). After 60s wait time, the excess sample was blotted off with a blot time of 4s and blot force -2 under 95% relative humidity at 4 °C, and plunge-frozen in liquid ethane using Vitrobot Mark IV (Thermofisher). The dataset was collected on FEI Titan Krios microscope operating at 300 kV at the UK national electron bio-imaging centre (eBIC). Imaging was automated using EPU software (FEI) on a Falcon 4i detector with a pixel size of 0.921 Å (130k magnification). A total of 26626 movies were recorded, with a nominal defocus range of approximately −1.0 to −2.7 µm with a total dose of 40 e-/Å2 over 4.89 s, corresponding to a dose rate of 7.44 e-/px/s.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCryo-EM image processing and reconstruction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll image processing was performed with CryoSPARC v4.4.1\u003csup\u003e58\u003c/sup\u003e unless stated otherwise. The movies were aligned using patch motion correction and CTF-corrected through patch CTF estimation, then curated to select for optimum ice thickness, CTF estimation and resolution. From 20,121 selected micrographs, particles were picked and 2D classified to generate a template for automated particle picking. After the particle picking parameters were refined using a small number of micrographs, a total of 1,039,601 particles were auto picked from the entire dataset and extracted using a box size of 320x320 pixels with subsequent down sampling by Fourier cropping to 160 pixels. Several rounds of 2D classification were implemented to remove low quality particles, yielding a stack of 711,248 particles from which ab-initio maps were generated. The maps from the best classes were selected and a secondary round of 2D classification followed by ab-initio modelling was performed. The suitable classes underwent non-uniform refinement, producing a map with an estimated overall resolution of 3.8Å (Supplementary Fig. 2 and 3). The local resolution of the final map was estimated using CryoSPARC\u003csup\u003e58\u003c/sup\u003e and visualised with ChimeraX\u003csup\u003e59\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eModel building and refinement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn AlphaFold model of a single Hcp3 chain was used as the starting model. This model was rigid-body fitted into the density corresponding to one of the Hcp3 chains using Chimera. This process was repeated five more times, each time placing a new AlphaFold model with a unique chain label into unoccupied density, thereby constructing the hexameric ring of the Hcp3 protein. Similarly, an AlphaFold model of the Tce1 toxin was placed into the density using Chimera\u003csup\u003e59\u003c/sup\u003e. As density was available for only one of the two domains, the models of the N-terminal domain (NTD) and C-terminal domain (CTD) were separated and independently rigid-body fitted into the map. These fittings were visually using Chimera\u003csup\u003e59\u003c/sup\u003e and assessed to determine which domain best corresponded to the observed density. The CTD model provided a better fit and was retained, while the NTD model was discarded. An initial model of the Hcp3–toxin complex was thus constructed, comprising six Hcp3 chains (chain labels A–F) and one toxin chain (label G). Due to anisotropy in the map, density at the periphery appeared fragmented or disconnected. To improve the fit of the model to the map, each chain was manually inspected in Coot, and residues not supported by density were removed. The revised Hcp3-Tce1 model underwent several iterative rounds of real-space refinement using Phenix\u003csup\u003e60\u003c/sup\u003e. Model quality and refinement progress were monitored using Ramachandran plots and MolProbity\u003csup\u003e61\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStructure analysis and presentation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe visualisation and analysis of the cryo-EM maps and atomic models were carried out using ChimeraX, PyMol (Molecular Graphics System, Schrödiner) and PDBePISA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStrains, plasmids and growth conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBacterial strains and plasmids used are listed in (Supplementary Table 3)\u003cem\u003e.\u0026nbsp;\u003c/em\u003eBacterial cultures were grown in lysogeny broth (LB) with antibiotics where applicable (Trimethoprim 200 ug/mL). Growth of yeast strains was accomplished with synthetic complete media lacking uracil (SC-Ura – BioShop). Yeast plasmids were first cloned in \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eand subsequently introduced using the lithium acetate method\u003csup\u003e62\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmid construction and genomic mutagenesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlasmids were constructed using restriction cloning with Phusion DNA polymerase, restriction enzymes and T4 DNA ligase from New England Biolabs or with Gibson assembly (NEB). All primers were synthesized by Integrated DNA Technologies (Supplementary Table 4). Plasmids were confirmed through Plasmidsaurus sequencing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePurification of Hcp3 for antibody generation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOvernight cultures of BL21 (DE3) pLysS expressing Hcp3 were subcultured 1:100 into 1L of LB and grown to mid-log phase. Cultures were induced by the addition of 0.5mM IPTG and grown for 18 hours at 18\u0026nbsp;°C. Cultures were collected and resuspended in buffer A (50mM Tris pH 7.5, 500mM NaCl) and lysed by sonication. Lysates were then cleared by centrifugation at 36,000 x g for 45 minutes. The cleared lysate was loaded onto 1mL Ni-NTA resin, washed with subsequent additions of buffer A with 10mM imidazole followed by buffer A with 30mM imidazole. Bound protein was eluted by the addition of buffer A containing 250mM imidazole. The elution was concentrated and buffer exchanged using PD-10 column (Cytiva).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpot plate assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpot plates were prepared by normalizing overnight cultures to and OD\u003csub\u003e600\u0026nbsp;\u003c/sub\u003eof 1 and making serial dilutions. 10\u0026nbsp;mL spots were plated and allowed to dry and bacterial plates were incubated for 24 hours at 37\u0026nbsp;°C while yeast plates were incubated for 48 hours and 30 °C after which time plates were imaged.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor overexpression of proteins in \u003cem\u003eE. coli\u003c/em\u003e, overnight cultures were diluted 1:100 in 25 mL of LB. Cultures were grown to mid-exponential phase and induced by the addition of 0.1% L-rhamnose. Cultures were grown for 2 hours and then collected and resuspended to a final volume of 0.5 mL. For overexpression of proteins in yeast, overnight cultures grown in 2% raffinose were diluted 1:100 and grown in 2% raffinose until log phase. Cultures were induced by the addition of 2% galactose and grown for an additional 18 hours after which time they were collected. Pellets were resuspended in 10mL of lysis buffer (50mM Tris pH 7.4, 500mM NaCl and cells were lysed using an emulsiflex. Lysates were cleared by centrifugation and mixed 1:1 with loading dye and boiled for 10 minutes. 5uL of sample was separated on a 12% Tris-glycine SDS-PAGE gel and subsequently transferred to nitrocellulose at 100 V for 30 minutes. Blots were probed with either anti-FLAG (1:5,000 Sigma), anti-VSV-G (1:5,000 Sigma), anti-His\u003csub\u003e6\u003c/sub\u003e (1:5,000 GenScript) or anti-Hcp3 (1:1000 GenScript). Anti-Hcp3 antibody was generated by GenScipt using purified Hcp3 as an antigen. The produced antiserum was validated for Hcp3 detection by probing purified Hcp3. Secondary antibodies were 1:5,000 goat anti-mouse horseradish peroxidase (New England Biolabs) for anti-His\u003csub\u003e6\u0026nbsp;\u003c/sub\u003eblots or 1:10,000 goat anti-rabbit (New England Biolabs) for anti-VSV-G, anti-Hcp3 or anti-FLAG blots.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe atomic coordinates have been deposited at the Protein Data Bank with accession code 9QPE. The density map has been deposited at the Electron Microscopy Data Bank with accession code EMD-53274.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImaging was done at the eBIC (Diamond, UK). We thank Diamond for access and support of the cryo-EM facilities at the UK national electron bio-imaging centre (eBIC), proposal BI25127, funded by the Wellcome Trust, Medical Research Council and BBSRC. We thank Paul Simpson (Electron Microscopy Facility, Centre for Structural Biology, Imperial College London) for in-house EM support and Tom Wood for helpful discussion on the role of cysteine proteases. S.D.K is jointly supported by postdoctoral fellowship awards from the Natural Sciences and Engineering Research Council of Canada (NSERC) and Cystic Fibrosis Canada. This work was supported by a Canadian Institutes of Health Research project grant to J.C.W. (PJT-175011), UKRI-MRC award\u0026nbsp;MR/S02316X/1 and MR/N023250/1 to A.F.\u0026nbsp;and a\u0026nbsp;Welcome Trust Award 215164/Z/18/Z\u0026nbsp;to T.R.D.C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eP.P., A.B, A.F. and T.R.D.C. conceived the study; P.P., A.B., J.B.P., O.O., K.M., A.I., and S.D.K.; conducted the experiments; P.P., A.B., A.I., S.D.K., A.F. and T.R.D.C. analysed the data; P.P and A.B. made figures; A.F. and T.R.D.C. wrote the manuscript, with inputs from P.P., A.B., S.D.K., and J.C.W.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGonzalez, D., Sabnis, A., Foster, K. R. \u0026amp; Mavridou, D. A. I. 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Yeast transformation by the LiAc/SS carrier DNA/PEG method. \u003cem\u003eMethods Mol Biol\u003c/em\u003e\u003cstrong\u003e1205\u003c/strong\u003e, 1-12 (2014). https://doi.org:10.1007/978-1-4939-1363-3_1\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7023205/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7023205/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePathogenic bacteria deploy sophisticated strategies to endure hostile environments and outcompete host microbiota or immune cells. Up to 30% of Gram-negative bacteria, including Pseudomonas aeruginosa, harbor a Type VI secretion system (T6SS), a supramolecular nanomachine that operates like a crossbow, for firing effectors into prokaryotic and eukaryotic prey cells. Despite its widespread distribution in nature, the mechanism by which effectors are loaded into ca 120 Hcp ring assemblies that form the T6SS injection tube, and the diversity of effectors delivered per firing event, remain undefined. Here, we reveal this mechanism by solving the cryo-electron microscopy structure of the Tce1 cargo effector loaded into a hexameric Hcp ring. Our structure reveals that a single cargo is enclosed by multiple rings and interacts asymmetrically with individual Hcp protomers. Our data delineate a conceptually novel mode of effector recognition and a stepwise loading mechanism, whereby an initial heterodimeric Hcp-cargo complex forms prior to ring formation occurring around the effector. We showed that another effector, Tce2, which exhibits anti-fungal properties, is similarly a Hcp3 cargo. We thus propose a novel and foundational mechanism by which distinct cargos are wrapped and simultaneously loaded into a single T6SS molecular device, enabling the coordinated delivery of a broad and potent payload into target cells.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e*Patricia Paracuellos \u0026amp; Ambre Bexter contributed equally.\u003c/strong\u003e\u003c/p\u003e","manuscriptTitle":"A Novel Molecular Mechanism for Effector Protein Wrapping and Delivery by the Bacterial Type VI Secretion System","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-06 07:06:24","doi":"10.21203/rs.3.rs-7023205/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-microbiology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nmicrobiol","sideBox":"Learn more about [Nature Microbiology](http://www.nature.com/nmicrobiol/)","snPcode":"","submissionUrl":"","title":"Nature Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"94a9dd65-ca9f-4486-8661-4db5034e5c31","owner":[],"postedDate":"August 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":51680982,"name":"Biological sciences/Microbiology/Bacteriology"},{"id":51680983,"name":"Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy"}],"tags":[],"updatedAt":"2025-08-06T07:06:24+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-06 07:06:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7023205","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7023205","identity":"rs-7023205","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}