Effector conformational plasticity enables lineage-specific secretion via Hcp heterohexamers in gut symbionts

Domain: chemistry
Published: 2026-02-21
Editorial outcome: minor_revision

Abstract
The Type VI Secretion System (T6SS) delivers effectors to mediate inter-bacterial competition. While the conserved hexameric Hcp ring is acknowledged as a key effector carrier, the mechanism by which it recognizes and accommodates structurally diverse cargos remains unclear. Notably, gut-dominant Bacteroidota species encode at least five hcp genes within their T6SS loci, yet the biological significance of this multiplicity is unknown. Here, we show that Hcp2 and Hcp3 form an obligate heterohexamer essential for effector translocation. Cryo-EM structures reveal that the effector Bte1 undergoes substantial conformational rearrangements to fit into the rigid Hcp pore. Structural, evolutionary, and functional analyses demonstrate that lineage-specific Hcp2-Hcp3 complexes selectively associate with distinct effectors. This specificity is mediated primarily by hypervariable interfaces on Hcp3, which recognize a structurally conserved N-terminal module in effectors via a “lock-and-key” co-evolutionary mechanism. Our findings provide insights into effector-specific delivery in gut symbionts through combinatorial Hcp assembly, redefine the rules of T6SS cargo selection by highlighting conformational adaptability, and suggest potential engineering applications.

Introduction
Bacterial competition in polymicrobial environments is intense, driving an evolutionary arms race between antagonistic and susceptible strains1–4. Among the molecular weapons employed, the Type VI Secretion System (T6SS)5–9 is one of the most widespread and potent nanomachines, functioning across many Gram-negative species to deliver toxic effectors into neighboring cells for interbacterial antagonism or host interaction through lytic10–12, enzymatic13–15, or immunomodulatory16–19 activities.

Effector translocation by T6SS is mechanistically diverse. The T6SS core is structurally analogous to contractile phage tails20,21, comprising a central Hcp hexameric tube22,23 surrounded by a contractile sheath formed by the TssB-TssC complex24,25. Effectors are delivered through either the central Hcp tube or the terminal VgrG-PAAR spike complex26,27. Importantly, structural components such as VgrG, PAAR, and Hcp have evolved bifunctionally, serving not only as scaffolds for the secretion machinery but also harboring effector domains28. High-molecular-weight effectors (>40 kDa) are typically loaded onto the VgrG or PAAR spikes27,29,30, with or without adapter assistance31–33, whereas low-molecular-weight effectors are often trafficked through the central channel of the Hcp hexamer34–38. Despite extensive functional studies, the molecular principles guiding Hcp-effector specificity and how Hcp accommodates structurally diverse effectors remain poorly understood.

A T6SS with potent antagonistic capabilities has recently been described in Bacteroidota, the dominant Gram-negative phylum of the human gut microbiota39. The Bacteroidales T6SS is categorized into three genetically distinct architectures (GAs 1–3) based on gene organization40. GA1 and GA2 loci, embedded within integrative conjugative elements (ICEs), can be horizontally transferred among strains40,41. While toxic effectors have been identified in GA3 T6SS loci19,42–44, neither GA1 nor GA2 has shown strong antagonistic activity in vitro, with only modest levels of interbacterial killing observed41,45. In contrast, the GA3 T6SS, which is not associated with an ICE, forms a highly conserved genetic locus uniquely present across Bacteroides fragilis species and exhibits robust antagonistic activity in vitro19,42,43. It includes a conserved core essential for T6SS function, as well as two variable regions (V1 and V2) encoding effector-immunity protein pairs40,42–44. In the B. fragilis NCTC9343 strain (hereafter referred to as NCTC9343), V1 encodes the effector Bte1 and its cognate immunity protein Bti1, while V2 encodes Bte2 with two immunity proteins Bti2a and Bti2b43 (Fig. 1a). Remarkably, unlike most Proteobacteria, which typically encode a single hcp gene per T6SS locus, the GA3 system of B. fragilis encodes five distinct hcp homologs (hcp1–hcp5) (Fig. 1a and Supplementary Fig. 1a). However, the functional relevance of this expansion and how these multiple Hcp variants mediate effector export have remained unclear.

Here, we uncover a unique effector delivery mechanism mediated by combinatorial Hcp assembly in B. fragilis. We show that Hcp2 and Hcp3 form a heterohexamer that is essential for V1 effector Bte1 export and for T6SS-mediated interbacterial competition. Neither Hcp2 nor Hcp3 alone is sufficient for Bte1 recruitment. Structural and biochemical analyses reveal that Bte1 undergoes a major conformational rearrangement, enabling selective recognition and encapsulation by the Hcp2-Hcp3 heterohexamer. This plasticity in effector structure is essential for its compatibility with the mixed Hcp pore. Phylogenetic and structural comparisons reveal that lineage-specific Hcp2-Hcp3 pairs co-evolve with distinct V1 effectors, all of which share a structurally conserved N-terminal module, supporting a lock-and-key model of effector recognition. Our findings redefine the rule of Hcp in effector selection, demonstrating that combinatorial Hcp assemblies and effector conformational plasticity jointly determine substrate specificity. This work provides a mechanistic foundation for understanding effector diversification in gut-resident symbionts and suggests strategies for engineering T6SS-based delivery systems.

Results
B. fragilis Hcp1, Hcp2, and Hcp3 are essential for Bte1-mediated bacterial competition
In the T6SS locus of NCTC9343, the hcp2-hcp3 and hcp4-hcp5 gene pairs are located adjacent to the variable region 1 (V1), prompting the hypothesis that these Hcps may be involved in the delivery of V1 effector, Bte1 (Fig. 1a). To investigate the roles of hcp variants in Bte1-mediated antagonism within the gut microbiota, we performed competitive co-colonization experiments in SPF C57BL/6J mice. After antibiotic cocktails treatment, mice were orally gavaged with a 1:1 mixture of donor strains (wild-type NCTC9343, the T6SS-deficient ΔtssC mutant, or individual hcp deletion mutants Δhcp1–hcp5) together with a Bte1-sensitive recipient strain (ΔtssCΔV1-ErmR) (Fig. 1b). Fecal samples were collected at the indicated time points post-gavage, homogenized, diluted, and plated on selective BHI agar to quantify recipient abundance (Fig. 1b). Consistent with the essential role of the T6SS in Bte1 delivery, the ΔtssC donor strain exhibited no killing activity against the sensitive recipient. The individual deletion of hcp1, hcp2, or hcp3 abolished Bte1-mediated killing, phenocopying the ΔtssC mutant. In contrast, Δhcp4 and Δhcp5 retained full antagonistic capacity (Fig. 1c).

To further validate these observations, we conducted parallel in vitro co-culture assays using the same donor and recipient strain combinations. Consistent with the in vivo results, deletion of hcp1, hcp2, or hcp3 completely abolished Bte1-dependent killing, whereas Δhcp4 and Δhcp5 mutants exhibited antagonistic activity comparable to that of the wild type (Fig. 1d). Collectively, these data demonstrate that Hcp1, Hcp2, and Hcp3—but not Hcp4 or Hcp5—are specifically required for Bte1-mediated interbacterial competition both in vivo and in vitro.

Given Hcp1’s established role in forming the T6SS inner tube42, the observed loss of killing in Δhcp1 mutants likely reflects disruption of core structural assembly. However, the requirement for Hcp2 and Hcp3 was unexpected. Immunoblot analyses showed that Δhcp2 and Δhcp3 mutants displayed partial defects in Hcp1 secretion and a complete block in Bte1 secretion, while Δhcp4 and Δhcp5 mutants exhibited normal secretion profiles (Fig. 1e). Functional restoration of Bte1 killing and secretion was achieved via plasmid-based complementation (Fig. 1f, g), confirming that Hcp2 and Hcp3 are specifically required for Bte1 delivery.

Hcp2 and Hcp3 assemble into heterohexamers that mediate Bte1 delivery
To define the interaction relationships between Hcp1, Hcp2, and Hcp3 and to identify which components directly associate with Bte1, we performed a series of in vivo co-immunoprecipitation assays. These experiments showed that Bte1 binds specifically to the assembled Hcp2-Hcp3 complex, whereas none of the individual Hcp proteins (Hcp1, Hcp2, or Hcp3 alone) displayed detectable interaction with Bte1 (Fig. 2a and Supplementary Fig. 1b). Critically, our data indicate that Hcp1 and Hcp2-Hcp3 do not form hybrid hexamers, suggesting they remain as independent structural units (Fig. 2a and Supplementary Fig. 1b).

Next, we purified individual Hcp1, Hcp2, and Hcp3 proteins in vitro to compare their assembly states (Supplementary Fig. 2a). As expected, Hcp1 formed a stable homohexamer (Supplementary Fig. 2b). In contrast, although both Hcp2 and Hcp3 were expressible, neither formed stable homomeric assemblies when isolated. Instead, they predominantly aggregated, as shown by gel filtration chromatography and negative-stain electron microscopy (Supplementary Fig. 2a–c). Notably, when co-expressed, Hcp2 and Hcp3 formed a stable complex. Gel filtration analysis showed an elution profile similar to that of the Hcp1 hexamer, and negative-stain EM revealed uniform hexameric rings, confirming the formation of a stable Hcp2-Hcp3 heterohexamer (Supplementary Fig. 2a–c).

Furthermore, in vitro pull-down assays demonstrated that Bte1 specifically interacts with the Hcp2-Hcp3 heterohexamer (Fig. 2b and Supplementary Fig. 2d), but not with Hcp1 (Fig. 2c and Supplementary Fig. 2e), consistent with our in vivo observations (Fig. 2a and Supplementary Fig. 1b). Together, these results indicate that Hcp2 and Hcp3 form a heterohexamer, which is essential for efficient Bte1 delivery.

To investigate the structural basis of Bte1 recognition and secretion mediated by the Hcp2-Hcp3 complex, we incubated purified Hcp2-Hcp3 with Bte1 in vitro and successfully obtained a stable ternary Bte1-Hcp2-Hcp3 complex, which enabled subsequent structural analysis by cryo-EM. During initial data processing, two distinct particle populations emerged: an apo Hcp2-Hcp3 hexamer and a Bte1-bound complex (Supplementary Fig. 3), consistent with previous observations35.

To determine the stoichiometry of Hcp2 and Hcp3 within the heterocomplex, we firstly predicted the structures of Hcp2 and Hcp3 using AlphaFold246 (Supplementary Data 1), and compared them to the B. fragilis Hcp1 crystal structure (PDB ID: 7YW0). Despite low sequence identity (<17%, relative to Hcp1), all three Hcp proteins exhibited a remarkable structural similarity (Cα RMSD≈0.9 Å) (Supplementary Fig. 4a–d), except for a unique feature observed in Hcp2—a short α-helix (residues 12–23) that protrudes from the periphery of the hexameric assembly (Supplementary Fig. 4d). Notably, this feature is clearly discernible in the 2D class averages (red dashed box, Fig. 2d and Supplementary Fig. 4e).

To experimentally validate this observation, we purified a stable binary complex of Hcp2Δ12–23 strep and Hcp36×His (Supplementary Fig. 4f). Cryo-EM analysis of this mutant complex revealed that the characteristic exterior helical density was absent in 2D class averages (Supplementary Fig. 4g, h). This result confirms that the helical density can be used as a reliable indicator for estimating the Hcp2-to-Hcp3 stoichiometry.

Based on initial 2D classification, the Hcp2-Hcp3 complex assembles into two predominant distinct stoichiometries in vitro: 3:3 and 4:2 (Hcp2:Hcp3) (Fig. 2d and Supplementary Fig. 4e, i). Subsequent cryo-EM data processing enabled the reconstruction of a 3.2 Å cryo-EM map of the Hcp24-Hcp32 complex (Supplementary Figs. 3, 5a–d and Supplementary Table 1). In contrast, particles corresponding to the 3:3 heterohexamer exhibited severe preferred orientation that could not be resolved despite multiple attempts (Supplementary Figs. 3, 5e, f).

To validate the 4:2 stoichiometry in the final model, we identified residue-specific markers distinguishing Hcp2 and Hcp3. In particular, Tyr55 and Ile106 in Hcp2 corresponded to Ser53 and Arg103 in Hcp3, providing unambiguous identifiers for subunit assignment (Fig. 2e and Supplementary Fig. 4a). Detailed cryo-EM density analysis confirmed a symmetric hexameric architecture containing four Hcp2 and two Hcp3 subunits (Fig. 2f). Notably, the two Hcp3 monomers were located opposite each other within the hexameric ring, generating a C2-symmetric arrangement (Fig. 2g, h). Further structural analysis revealed the molecular basis governing Hcp2-Hcp3 (Fig. 2i, j) and Hcp2-Hcp2 (Fig. 2k) interactions. These interfaces are stabilized through a combination of hydrophobic contacts and main-chain polar interactions, which together preserve the structural integrity of the heterohexameric assembly. Collectively, these data demonstrate that Hcp2 and Hcp3 form a defined heterohexamer that specifically mediates the recruitment and export of Bte1 in NCTC9343.

Conformational transitions of Bte1 upon incorporation into the Hcp2-Hcp3 heterohexamer
To investigate how Bte1 engages the Hcp2-Hcp3 heterohexamer, we reconstructed a 3.3 Å cryo-EM map of the Bte1-Hcp24-Hcp32 complex (Fig. 3a, Supplementary Figs. 3, 5g–j and Supplementary Table 1). Consistent with prior analysis of the apo Hcp2-Hcp3 complex, evaluation of side-chain densities confirmed a 4:2 stoichiometry of Hcp2 to Hcp3 in the Bte1-bound heterohexamer (Supplementary Fig. 6a). In contrast, datasets corresponding to the Bte1-Hcp23-Hcp33 complex exhibited strong preferred orientation, precluding high-resolution structural determination (Supplementary Figs. 3, 5k, l). Structural comparison of the Hcp2-Hcp3 heterohexamer between the apo and Bte1-bound Hcp2-Hcp3 complexes revealed minimal conformational change (Cα RMSD=0.708 Å), indicating that Bte1 binding does not significantly alter the architecture of the Hcp ring (Supplementary Fig. 6b, c).

Due to the inherent conformational flexibility, peripheral regions of the Hcp2-Hcp3 ring and the C-terminal domain of Bte1 exhibited reduced local resolution in the ternary complex map (Supplementary Figs. 5g, 6b). To generate a complete and visually interpretable model, we firstly aligned the apo Hcp2-Hcp3 complex into the Bte1-bound cryo-EM map, observing no major conformational changes between them (Supplementary Fig. 6c). We then fitted the cryo-EM-resolved N-terminal domain of Bte1, and finally integrated a C-terminal helical domain built with ModelAngelo. This procedure yielded a composite structural model for visualization and analysis (Fig. 3b). This composite model showed excellent agreement with the overall cryo-EM density of the Bte1-Hcp24-Hcp32 complex (Supplementary Fig. 6d).

Effector loading into Hcp rings and the associated conformational changes remain poorly understood. To gain insight, we docked the Bte1 crystal structure (PDB id: 8ZHS) into the Bte1-Hcp24-Hcp32 map by aligning their shared structures of the N-terminal domain (Fig. 3c, d). This comparison revealed extensive steric clashes involving α3, α5, and α6 helices in unbound Bte1 with the stacked Hcp rings (Fig. 3c). Structural alignment showed that Bte1 undergoes substantial conformational rearrangement upon binding to the Hcp2-Hcp3 hexamer, with α5 and α6 rotating ~70°, and α3 rotating nearly 120° to alleviate these steric conflicts (Fig. 3d). These transitions reposition the Bte1 C-terminal domain to accommodate stacking within the hexameric tube (Fig. 3c).

To test whether these conformational rearrangements are required for Bte1 loading, we designed mutations to physically restrict the movement of the α3 helix in Bte1 (Supplementary Fig. 6e). Structural analysis revealed that Ala115 on the α3 lies in close proximity to Pro61 in the N-terminal loading region (Supplementary Fig. 6f). We therefore generated a Bte1 P61C/A115C double-cysteine mutant to promote the formation of an intramolecular disulfide bond, thereby locking α3 in place and preventing conformational rearrangement (Supplementary Fig. 6g). In vitro pull-down assays demonstrated that Bte1 P61C/A115C displayed markedly reduced binding to the Hcp2-Hcp3 complex. Importantly, the addition of 1 mM TCEP, which disrupts the engineered disulfide bond, restored binding to a level comparable to that of wild-type Bte1 (Supplementary Fig. 6h). These results confirm that restricting α3 movement directly impairs the interaction between Bte1 and the Hcp2-Hcp3 heterohexamer.

We further evaluated the functional consequence of this restricted conformation using co-culture competition assays. Complementation of Bte1 P61C/A115C in the NCTC9343 Δbte1 strain resulted in reduced killing activity relative to wild-type Bte1, suggesting impaired effector loading and secretion efficiency (Supplementary Fig. 6i). Together, these findings demonstrate that conformational rearrangements in Bte1 are essential for its efficient loading and secretion onto the Hcp2-Hcp3 heterohexamer.

Critical residues in Hcp2-Hcp3 required for Bte1 binding and secretion
Although a high-resolution map of the Bte1-Hcp23-Hcp33 complex could not be obtained, top views of 2D class averages suggested that Bte1 consistently engages a neighboring pair of Hcp2 and Hcp3 subunits in both stoichiometries (Fig. 3e and Supplementary Fig. 4e, j). To dissect this interaction, we used the PISA server47 to analyze the interface between Bte1 and the Hcp24-Hcp32 complex. Bte1 primarily contacted Hcp2 Chain C, Hcp3 Chain B, and Hcp2 Chain A, with buried surface areas of 805.4 Å2, 633.2 Å2 and 210.2 Å2, respectively (Fig. 3f). Notably, the extensive interfaces observed with Hcp2 Chain C and Hcp3 Chain B indicate their critical roles in stabilizing the complex.

Electrostatic surface analysis revealed complementary charge patterns between Bte1 and Hcp2 Chain C-Hcp3 Chain B (Fig. 3g). Further examination identified specific polar interactions: Ser53, Gln114, Arg100, and Asn102 of Hcp3 Chain B formed side-chain contacts with Bte1 residues Thr38, Thr40, and Thr76 (Fig. 3h). Simultaneously, Hcp2 Chain C residues Thr57, Lys119, and Asn30, along with Hcp2 Chain A residue Lys111, interacted with Bte1 backbone residues Ala19, Gly20, and Tyr24, respectively. Additionally, a salt bridge between Arg50 of Hcp2 Chain C and Glu73 of Bte1 may further stabilize the interaction (Fig. 3h).

To evaluate the functional relevance of these contacts, we introduced point mutations in Hcp2 and Hcp3 at key interface residues and assessed their impact via in vitro pull-down assays. Substitution of Asn30 in Hcp2 or Arg100 in Hcp3 with alanine completely abolished Bte1 binding (Fig. 3i). When these mutations were integrated into a Δhcp2-hcp3 background in NCTC9343 using a pNBU2_ErmR integrative plasmid, both Bte1 T6SS-dependent killing and secretion were abolished (Fig. 3j, k). These results identify Asn30Hcp2 and Arg100Hcp3 as essential for Bte1 recognition and translocation.

Interestingly, sequence alignments revealed that these residues (Asn30Hcp2 and Arg100Hcp3) are not conserved across B. fragilis strains (Fig. 3l), suggesting a co-evolutionary mechanism wherein Hcp2-Hcp3 complexes may exhibit selective compatibility with specific V1-region effectors.

Co-evolution drives selective effector recognition by Hcp2-Hcp3 complexes
Building on the finding that the Hcp2-Hcp3 heterohexamer mediates Bte1 delivery, we explored whether this mechanism extends to other V1 effectors in B. fragilis. To this end, we performed a comparative phylogenetic analysis using concatenated full-length amino acid sequences of Hcp2-Hcp3 and Hcp4-Hcp5 from 38 B. fragilis strains. These strains encompass all four known classes of GA3 T6SS-associated V1 putative effectors. Maximum-likelihood phylogeny based on the fused Hcp sequences resolved four distinct clades (Fig. 4a and Supplementary Fig. 7a–c). Mapping the distribution of V1 effectors onto this phylogeny revealed a strong association with Hcp2-Hcp3, but not Hcp4-Hcp5 clades. In contrast, V2 effectors showed no discernible pattern of association (Fig. 4a and Supplementary Fig. 7a). These results suggest that Hcp2-Hcp3 diversification is tightly coupled to the co-evolution of their cognate V1 effectors in a lineage-specific manner.

To test whether Hcp2-Hcp3-dependent V1 effector delivery is conserved across strains, we examined B. fragilis GS08648 (hereafter referred to as GS086), which encodes a distinct V1 effector, Bfe142 (Supplementary Fig. 1a). GS086 killer strains with deletions in tssC, hcp1, hcp2-hcp3, or hcp4-hcp5 were co-cultured with a V1-deficient (Δbfe1-bfi1) prey strain. As observed with Bte1 in NCTC9343, deletion of hcp2-hcp3 in GS086 abolished Bfe1-dependent killing, whereas hcp4-hcp5 deletion had no effect (Supplementary Fig. 7d). To evaluate cross-strain functional compatibility, we complemented the hcp2-hcp3 deletion in NCTC9343 with hcp2-hcp3 gene pairs from NCTC9343 (Bte1-associated), GS086 (Bfe1-associated), and HMW616 (LysM-associated)44 (Supplementary Fig. 1a). Only the native NCTC9343 pair restored Bte1-mediated killing (Fig. 4b) and secretion (Fig. 4c). In vitro pull-down assays confirmed that the NCTC9343 Hcp2-Hcp3 complex bound specifically to Bte1, while the GS086 complex interacted exclusively with Bfe1 (Fig. 4d). These findings indicate that Hcp2-Hcp3 complexes exhibit strain-specific recognition of their cognate V1 effectors through a conserved selective mechanism.

Hcp3 functions as the primary determinant of effector-recognition specificity within the Hcp2-Hcp3 complex
Given that Hcp3 shows greater sequence variability than Hcp2 across T6SS loci (Fig. 4a and Supplementary Fig. 8a, b), we hypothesized that Hcp3 plays a key role in effector specificity. To test this, we assessed the binding of Bte1 and Bfe1 to four distinct Hcp2-Hcp3 combinations: Hcp2NCTC9343-Hcp3NCTC9343, Hcp2GS086-Hcp3GS086, Hcp2NCTC9343-Hcp3GS086, and Hcp2GS086-Hcp3NCTC9343. Gel filtration analysis confirmed that all cross-strain combinations, Hcp2NCTC9343-Hcp3GS086 and Hcp2GS086-Hcp3NCTC9343, assembled into stable heterohexamers (Supplementary Fig. 8c). Binding assays revealed that complexes containing Hcp3NCTC9343 (i.e., Hcp2NCTC9343-Hcp3NCTC9343 and Hcp2GS086-Hcp3NCTC9343) bound Bte1 but not Bfe1. In contrast, those containing Hcp3GS086 (i.e., Hcp2GS086-Hcp3GS086 and Hcp2NCTC9343-Hcp3GS086) bound Bfe1 but not Bte1 (Fig. 4e, f). These results demonstrate that, although Hcp2 also contributes to effector interaction and selection (Fig. 3f–l), Hcp3 serves as the primary determinant of effector specificity within the Hcp2-Hcp3 complex (Fig. 4e, f).

To further validate Hcp3’s critical role, we performed gain-of-function mutagenesis on the Hcp2GS086-Hcp3NCTC9343 complex—which binds Bte1 but not Bfe1—to convert its binding specificity toward Bfe1. Structural analysis of Hcp3NCTC9343 identified three non-conserved regions (variable regions 1–3) on the inner surface of the Hcp ring, predicted to mediate effector recognition (Supplementary Fig. 8b, d). Individual mutations in any single region did not confer Bfe1 binding. However, simultaneous mutation of regions 2 and 3 (VR2+3) or all three regions (VR1+2+3) to GS086-type residues enabled Bfe1 binding, with the triple mutant showing the strongest interaction (Supplementary Fig. 8e). These findings indicate that effector recognition by Hcp3 depends on a combinatorial contribution of residues from multiple variable regions, and variation in these regions across strains likely reflects co-evolution of Hcp transporters with their cognate effectors.

Conservation analysis of the Bte1-Hcp2-Hcp3 structure indicated that the interfacial residues mediating Hcp2-Hcp3 interactions are highly conserved, supporting the formation of stable heterohexamers across bacterial strains. In contrast, the effector-binding region—particularly on Hcp3—was poorly conserved, suggesting a primary role for Hcp3 in effector discrimination (Fig. 4e–g and Supplementary Fig. 8f). Together, these results identify Hcp3 as the primary determinant of effector specificity, with its hypervariable inner-surface residues driving selective effector recognition.

A conserved N-terminal fold in V1 effectors suggests a modular mechanism for recruitment by Hcp2-Hcp3
To define the molecular basis of V1 effector recognition by the Hcp2-Hcp3 complex, we used AlphaFold2 to predict the structures of Bfe1 and LysM (Supplementary Data 1 and Supplementary Fig. 9a, b). Our cryo-EM analysis of the Bte1-Hcp2-Hcp3 complex revealed that Bte1 binds via its N-terminal Regions (NTRs) (Fig. 3f, h). Structural comparisons among the NTRs of Bte1 and the AlphaFold2-predicted NTRs of Bfe1 and LysM revealed a conserved fold despite low sequence identity (<17%, relative to Bte1) (Fig. 5a and Supplementary Fig. 9c, d). In contrast, the C-terminal domains were highly divergent (Supplementary Fig. 9c, d). Although the NTRs of Bte1, Bfe1 and LysM exhibit structural conservation, a distinct V1 effector (BteO49) in B. fragilis GS074 shows a divergent N-terminal fold and is encoded within an atypical T6SS cluster (Fig. 4a and Supplementary Figs. 1a, 9e). This configuration may indicate an alternative mechanism of Hcp-mediated effector recognition. Together, these observations support a model in which V1 effectors (Bte1, Bfe1 and LysM) utilize a structurally conserved NTRs as a modular interface for Hcp recognition.

To further elucidate the modular nature of this mechanism, we docked AlphaFold2-predicted structures of Bfe1 and LysM, along with their respective Hcp3 partners from GS086 and HMW616 (Supplementary Data 1 and Supplementary Fig. 10a, b), onto the cryo-EM map of the Bte1-Hcp24-Hcp32 complex. This enabled the generation of putative structural models for the Bfe1-Hcp3GS086 (Fig. 5b) and LysM-Hcp3HMW616 (Fig. 5c) interactions. Structural analysis revealed that an antiparallel β-sheet pair within the effector N-terminal loading region mediates binding to Hcp3 (Figs. 3f, h, 5b, c and supplementary Fig. 10c). Notably, this β-sheet pair is not conserved at the sequence level among different effectors (Supplementary Fig. 9d, blue dashed box), likely to complement variable regions in Hcp3 across different bacterial strains.

To assess whether the conserved N-terminal module found in other V1 effectors also mediates Hcp3 binding, we introduced mutations into residues within the NTRs of Bfe1 and LysM corresponding to the Bte1-Hcp2-Hcp3 interface. In vitro pull-down assays demonstrated that single-residue substitutions at Trp34, Met35, Val39, or Ile64 in Bfe1 each impaired its binding to the GS086 Hcp2-Hcp3 complex, with the I64S mutation exhibiting the most pronounced effect (Fig. 5d). Similarly, LysM mutants T36A/K37A/K39A and L65S/R67A failed to interact with the HMW616 Hcp2-Hcp3 complex (Fig. 5e). These findings support the idea that conserved structural features within the NTRs constitute a modular interface for Hcp recognition across diverse V1 effectors.

We next examined whether non-conserved residues in the Hcp3 inner surface from GS086 and HMW616 contribute to the interaction with the N-terminal module of their respective V1 effectors, Bfe1 and LysM. Structural modeling identified candidate residues on Hcp3 that likely interact with the NTRs of Bfe1 and LysM, respectively (Fig. 5b, c and Supplementary Fig. 8b). Site-directed mutagenesis of these residues, followed by in vitro pull-down assays, confirmed their importance: Arg51 in Hcp3GS086 was essential for Bfe1 binding (Fig. 5f), while Leu53/Ser55 and Lys112/Asp114 in Hcp3HMW616 were required for LysM binding (Fig. 5g). In both cases, disruption of these regions impaired effector interaction without affecting Hcp2-Hcp3 complex formation.

Based on these biochemical data, we further conducted residue-specific co-evolutionary analysis of the Bte1-Hcp3NCTC9343, Bfe1-Hcp3GS086 and LysM-Hcp3HMW616 putative interfaces (Fig. 3h and Supplementary Fig. 10d). In the resolved Bte1-Hcp2-Hcp3 structure, Hcp3 interacts with Bte1 through a polar interaction network (Fig. 3h). In contrast, in the Bfe1-Hcp3GS086 and LysM-Hcp3HMW616 putative interfaces, we observed a combination of hydrophobic and polar interactions, indicating diversified co-evolutionary adaptation (Supplementary Fig. 10d). For example, Ser53 in Hcp3NCTC9343 forms a polar interaction with Thr40 of Bte1. However, when Ser53 is substituted by leucine in Hcp3GS086 or Hcp3HMW616, the interacting residues in the cognate effector’s N-terminal β-sheet pair also shift to uncharged counterparts. This mutual change facilitates hydrophobic interactions, thereby maintaining a stable interface. Importantly, while the overall interaction networks differ markedly pattern among the Bte1-Hcp3NCTC9343, Bfe1-Hcp3GS086, and LysM-Hcp3HMW616 interfaces, they follow a regular pattern (Fig. 3h and Supplementary Fig. 10c, d). This provides direct evidence for residue-level co-evolution underlying effector specificity.

To further test whether co-evolved effector residues are sufficient to alter Hcp recognition, we replaced the β-sheet pair of Bte1 with the corresponding residues from Bfe1 and examined its interaction with the GS086 Hcp2-Hcp3 complex. The resulting mutant gained the ability to bind the GS086 Hcp2-Hcp3 complex (Supplementary Fig. 10e).

Together, our data demonstrate that effector selectivity is governed by co-evolved, residue-specific interaction networks between the hypervariable inner surface of Hcp3 and the structurally conserved but sequence-divergent N-terminal loading regions of effectors, supporting a refined lock-and-key co-evolutionary model for T6SS effector loading.

Methods
Bacterial strains and growth conditions
All strains used in this study are listed in Supplementary Table 2. E. coli strains used for genetic manipulation, recombinant protein purification, and plasmid conjugation into Bacteroides species were maintained under aerobic conditions using LB broth at 37°C for liquid cultures or agar plates (15% m/v agar). All Bacteroides strains were cultured at 37°C in brain heart infusion (BHI) broth supplemented with hemin (5 mg/L) and L-cysteine (1 g/L), and maintained in an anaerobic condition (20% CO2, 10% H2 and 70% N2). Antibiotics were added as follows: kanamycin (50 μg/mL), ampicillin (100 μg/mL), gentamicin (200 μg/mL), erythromycin (50 μg/mL), and chloramphenicol (15 μg/mL) for plasmid retention or selection. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was prepared as a stock solution (0.5 M) and used at 0.2 mM to induce gene expression.

Plasmid construction
All plasmid constructs used in this study are listed in Supplementary Table 3. To co-express Hcp2 and Hcp3 in E. coli, their coding sequences were subcloned into a modified pET21b backbone, with a C-terminal Strep tag fused to hcp2 and a 6×His tag fused to hcp3 by site-directed mutagenesis. bte1 was subcloned into pET15b with an N-terminal 6×His-MsyB-SUMO fusion tag. Sequence analysis predicted that both Bfe1 and LysM contain C-terminal transmembrane regions. As full-length constructs could not be expressed in a soluble form in E. coli, truncated constructs covering the predicted soluble domains were designed for biochemical studies. The truncated Bfe1 (residues 1–242) fragment was cloned into the pET28a vector with a C-terminal 6×His tag, while the LysM (residues 1–244) was cloned into the pET15b vector with an N-terminal 6×His-MBP tag. All constructs were verified by DNA sequencing prior to protein expression. For in situ genetic manipulation in Bacteroides strains, 1 kb homology arms flanking genomic loci were amplified and ligated into the pSIE-Bfe1 vector containing Bfe1 as a counterselection marker. Complementation or overexpression in Bacteroides utilized the pNBU2_CamR or pNBU2_ErmR integrative plasmids, where target genes were placed under transcriptional control of the constitutive tonB3 promoter54 to enhance expression levels. All constructs were sequence-verified through capillary electrophoresis (Sangon Biotech, China).

Conjugation and resistance selection
Plasmid transfer was achieved through conjugation using E. coli S17-1 λ pir as the donor strain and Bacteroides species as the recipient strain. Briefly, overnight cultures of E. coli S17-1 λ pir donor strains were diluted at a 1:200 ratio in LB broth containing ampicillin, while B. fragilis recipients were diluted at a 1:100 ratio in BHI broth. After approximately 3 h of growth (donor OD600 0.2-0.6; recipient OD600 0.1-0.2), cells were mixed at a 1:10 donor-to-recipient volume ratio and pelleted by centrifugation at 9000×g for 10 min. The pellet was resuspended in 100 μL BHI and spotted onto a non-selective BHI agar for aerobic mating (37°C, 20 h). Conjugants were harvested in 1 mL BHI, serially diluted (undiluted, 1:5, 1:10), and plated on selective BHI agar containing gentamicin (200 μg/mL) and chloramphenicol (15 μg/mL) or erythromycin (25 μg/mL). The single colonies were restreaked and verified by PCR after 2 to 3 days of anaerobic selection.

In situ genetic manipulation in Bacteroides
In situ genetic manipulation in Bacteroides species was achieved through double-crossover allelic exchange. The verified single-crossover merodiploid strains (refer to “Conjugation and Selection”) were incubated overnight in 1 mL of non-selective BHI broth, followed by serial dilution (10−3-10–4) and platted on BHI agar supplemented with anhydrotetracycline (aTC, 200 ng/mL) to select colonies that had successfully excised the counter-selection cassette. Following 36–48 h of anaerobic growth, isolated colonies were restreaked and subjected to PCR genotyping. Sanger sequencing was performed to validate wild-type and mutant strains and further confirm excision of the counter-selection cassette.

Competitive colonization experiments in mice gut
All animal experiments were supervised and approved by the Animal Research Ethical Inspection Form of Shandong University School of Life Sciences (SYDWLL-2025-034). Only female mice were used for the competitive colonization experiments, and no sex-based analysis was performed. Seven-week-old female C57BL/6J SPF-grade mice were purchased from GemPharmatech (Jiangsu, China) and housed under SPF conditions (25 ± 2°C, 45 ± 5% humidity, 12 h light/dark cycle) with unlimited access to autoclaved water and irradiated food. After 1-week acclimatization (all mice remained healthy), animals received antibiotic cocktails treatment: oral gavage (10 mg/mouse/day of vancomycin, metronidazole, neomycin, ampicillin) for 5 days, followed by antibiotic-supplemented drinking water (0.5 g/L vancomycin, 1.0 g/L each of metronidazole, neomycin, ampicillin) for 7 days, then regular drinking water for 4 days. Fecal samples were cultured on BHIS agar (200 μg/mL gentamycin + 15 μg/mL erythromycin); only mice with no bacterial growth were included. Antibiotic-treated mice (n = 5/group) received oral gavage of 2.5 × 109 c.f.u. prey strains plus 2.5 × 109 c.f.u. killer strains. Fecal samples collected at specified time points were homogenized in PBS, diluted, and plated on selective BHI agar (200 μg/mL gentamycin + 15 μg/mL erythromycin) for c.f.u. counting.

Co-culture assay in vitro
The killer and prey strains were grown anaerobically in BHI broth at 37°C until reaching an optical density at 600 nm (OD600) of 0.3. Then the killer and prey strains were mixed at a 10:1 ratio, pelleted by centrifugation, and resuspended in 200 μL of PBS. 5 μL of each mixture was spotted on BHI agar plates and incubated at 37°C anaerobically for 20–24 h. Then the bacterial spots were resuspended in 1 mL PBS (serve as 100 dilution) and serial dilutions (100 to 10−5 dilutions) were plated onto BHI agar supplemented with appropriate antibiotics to select prey strains.

Protein expression and purification
The E. coli BL21 (DE3) strains carrying plasmids for overexpression of recombinant proteins were cultured in LB broth supplemented with appropriate antibiotics at 37°C until reaching an optical density at 600 nm (OD600) of 0.8, then induced overnight at 20°C by the addition of 0.2 mM IPTG. Bacterial cells were harvested by centrifugation and lysed using a high-pressure cell disruptor (Union-Biotech) in lysis buffer (20 mM Tris 8.0, 150 mM NaCl) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and 50 μg/mL DNase I. The lysate was further centrifuged to eliminate cellular debris. The Hcp2strep-Hcp36×His and Hcp2Δ12-23 strep-Hcp36×His complex was purified through tandem affinity chromatography. The resulting supernatant was firstly loaded onto Ni-NTA agarose (Qiagen) for initial purification. The elute protein was then loaded onto Strep-affinity resin (IBA Lifesciences) for further purification. The Ni-NTA column was eluted with 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 250 mM imidazole, while the Strep-Tactin column was eluted with 100 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, and 50 mM biotin to obtain the purified Hcp2strep-Hcp36×His complex. The 6×His-MsyB-SUMO tag fusion protein was cleaved with ULP1 protease at room temperature for 3 h, followed by elution from the Ni-NTA column using lysis buffer (20 mM Tris 8.0, 150 mM NaCl). Purified Hcp2strep-Hcp36his complex and untagged Bte1 were mixed at a molar ratio of 2:1 and incubated at 4°C for 1 h to allow complex formation. Subsequently, the sample was further purified through gel filtration chromatography (Superdex 200 Increase 10/300 GL, GE Healthcare). The truncated Bfe1 (1–242) and LysM (1–244) constructs were purified using Ni-affinity chromatography, followed by gel filtration chromatography to further improve purity and homogeneity. The resulting proteins were obtained in soluble form and used for subsequent biochemical and structural analyses.

To enrich the native Bte1-Hcp2-Hcp3 complex, a 6×His tag was introduced at the C-terminus of Bte1 in B. fragilis NCTC9343, and HA tags were inserted at the C-termini of Hcp2 and Hcp3. Cells were harvested and lysed under native conditions, and the lysates were subjected to Ni-affinity chromatography. Eluted fractions were analyzed by immunoblotting using anti-Bte1 and anti-HA antibodies to confirm the presence of the ternary complex. The Ni-eluted sample was further fractionated by gel filtration chromatography, and the ~120 kDa fraction corresponding to the Bte1-Hcp2-Hcp3 complex was collected, concentrated, and directly used for cryo-EM grid preparation.

In vitro pull-down assays
To evaluate in vitro complex assembly, affinity-purified bait and prey proteins were preincubated at a molar ratio of 2:1 (4°C incubation, 1 h). The Ni-NTA resin (Qiagen) or Strep-Tactin resin (IBA Lifesciences) was then introduced (50 μL/sample) and incubated for 1 h. Unbound proteins were removed by three sequential washes with lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, optional 20 mM imidazole for Ni-NTA resin wash). Bound complexes were competitively eluted using either Ni-NTA elution buffer or Strep-Tactin elution buffer. Eluates were separated and evaluated by electrophoresis using 15% Glycine-SDS-PAGE gels combined with Coomassie staining. Uncropped and unprocessed scans of the SDS-PAGE gels are provided in the Source Data file.

Western immunoblot analysis
The strains to be tested were cultured anaerobically in fresh BHI broth for approximately 4 h until reaching OD600 of 0.6. Subsequently, the 5 mL collected culture was centrifuged. The pellet was then resuspended in 100 μL PBS buffer, followed by 100 μL 2× SDS loading buffer (WCL: Whole Cell Lysate). Meanwhile, the supernatant was filtered through a 0.22 μm filter and concentrated to ~200 μL by ultrafiltration and then complemented 200 μL 2× SDS loading buffer (SUP: Supernatant). For Western immunoblot analysis, the WCL and SUP samples were electrophoresed on 15% Glycine-SDS-PAGE gels. The contents of the gels were transferred to PVDF membranes (Millipore), blocked, and probed with primary antibodies α-Dnak (cytoplasmic control), α-Bte1 and α-Hcp1 (Rabbit α-Bte1 and α-Hcp1, this study, used at a dilution of 1:3000; Rabbit α-DnaK, Cusabio #CSB-PA633459HA01EGW, used at a dilution of 1:3000; Mouse α-HA, MBL #M180-3, used at a dilution of 1:2000), followed by horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit, MBL #458, diluted to 1:5000; goat anti-mouse, MBL #330, diluted to 1:5000). All protein bands were visualized using the M5 HiPer ECL Western HRP Substrate (Mei5 Biotechnology, MF074-05) and captured using the Tanon-5200 Chemiluminescent Imaging System (Tanon). Uncropped and unprocessed scans of the Western blots are provided in the Source Data file.

Co-immunoprecipitation and immunoblotting analyses
To validate the in vivo assembly of the Hcp2-Hcp3-Bte1 complex, NCTC9343-Hcp2HA-Hcp3HA-Bte16×His (and corresponding isogenic mutants) were cultured anaerobically overnight in BHI broth. Cultures were diluted 1:100 into fresh BHI broth (1 L) and incubated until reaching mid-log phase (OD600 ~0.8). Cell pellets were resuspended in 25 mL lysis buffer and lysed by a high-pressure cell crusher (Union-Biotech). The supernatant from the lysate was collected and subjected to at least three sequential binding cycles to Ni-NTA resin (50 μL), followed by three sequential washes cycles (10 mL lysis buffer per cycle). The Ni-NTA eluted fractions (100 μL) were collected and boiled with 100 μL of 2× SDS loading buffer to prepare output samples. Unprocessed lysates (“input”) and affinity-purified samples (“output”) were separated by SDS-PAGE (10 μL/lane) and transferred to PVDF membranes for subsequent western blot analysis according to the protocol described above “Western immunoblot analysis”.

AlphaFold2-predicted structures for initial model building and structural analysis
To facilitate initial model building and structural analysis, 3D structures of the target proteins were predicted using AlphaFold246 via ColabFold v1.5.5. The resulting models and corresponding Predicted Aligned Error (PAE) files are available in Supplementary Data 1. The predicted models of Hcp2NCTC9343 (Uniport id: Q5LDT5) and Hcp3NCTC9343 (Uniport id: Q5LDT6) served as starting templates for manual model building and refinement. Predicted structures of Hcp3GS086 (Uniport id: E1WVU6), Hcp3HMW616 (Uniport id: A0A413JT53), Bfe1GS086 (Uniport id: E1WVU5), LysMHMW616 (Uniport id: A0A413JTG1) and BteOGS074 (Uniport id: A0A015XAP0) were used for V1 effectors modular assembly analysis.

Cryo-EM sample preparation and data collection
Aliquots of 4 μL of sample at a concentration of approximately 0.5–1 mg/mL was applied onto the glow-discharged cryo-EM grid (QUANTIFOIL Cu R1.2/1.3, 300 mesh) and then plunge-frozen into liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific, Waltham, MA) (blotting force, 0; blotting time, 3 s; wait time, 5 s). Purified Bte1-Hcp2strep-Hcp36×His cryo-EM data were collected using a 300 kV Titan Krios G4 microscope equipped with a K3 direct electron detector (nominal magnification, 81,000×; pixel size, 0.526 Å; defocus range, −0.8 to −1.8 μm). The total electron dose for data collection was 60 e-/Å2. For the native Bte16×His-Hcp2HA-Hcp3HA complex, ~10,000 micrographs were collected on a 200 kV cryo-EM microscope with a total electron dose of ~40 e⁻/Å². For the purified Hcp2Δ12-23 strep-Hcp36×His complex, ~4000 micrographs were collected under the same conditions (nominal magnification, 130,000×; pixel size, 0.89 Å; defocus range, −0.6 to −1.6 μm).

Cryo-EM data processing
Cryo-EM data for the Bte1-Hcp2-Hcp3 complex were processed using cryoSPARC55 and RELION56. A flowchart illustrating the process is provided in Supplementary Fig. 3. A total of 11,619 movies were collected and subjected to Patch motion correction and Patch CTF estimation in cryoSPARC. After discarding low-quality micrographs, 9138 were selected for particle picking. After initial particles selection with blob- and template-based picking, followed by 2D classification to remove bad particles. The selected particles were subjected to Ab initio Reconstruction and Heterogeneous Refinement in cryoSPARC to remove junk classes. This resulted in four distinct classes, including Bte1-Hcp24-Hcp32, Bte1-Hcp23-Hcp33, Hcp24-Hcp32 and Hcp23-Hcp33 complexes. Particles were further refined by 3D classification and non-uniform refinement in cryoSPARC. Due to severe preferred orientation in the data, we firstly applied misalignment correction using the SPisonet57 module integrated into the RELION 3D auto-refine process. To further address anisotropic distribution, we performed anisotropy correction using SPisonet. Final Postprocess in RELION yielded a 3.2 Å map of Hcp24-Hcp32 complex and a 3.3 Å map of Bte1-Hcp24-Hcp32 complex. However, reconstruction of the Hcp23-Hcp33 complex and Bte1-Hcp23-Hcp33 complex remained heavily affected by preferred orientation, resulting in a low-quality map.

Model building into cryo-EM maps
The predicted models of Hcp2NCTC9343 and Hcp3NCTC9343 from AlphaFold2 were docked into the Hcp24-Hcp32 and Bte1-Hcp24-Hcp32 complex cryo-EM maps using UCSF ChimeraX58 and Phenix59. For Bte1, we used the previously solved crystal structure (PDB ID: 7YW0) as the starting model. These models were manually adjusted in Coot60 to better fit the density and correct geometric issues. The whole assembly was refined through several rounds of real-space refinement in Phenix. Detailed information on data processing and model refinement can be found in Supplementary Table 1.

Analysis of Hcp variants
The amino acid sequences of Hcp2, Hcp3, Hcp4, and Hcp5 from NCTC9343 were employed as query sequences (e-value ≤ 1e−10) for homology searches against the NCBI Nr/Nt database (BLAST+ v2.12.0) restricted to B. fragilis. Resulting homologs were subjected to multiple sequence alignment via MAFFT (v7.487)61 and visualized by Escript server 3.062. To investigate the linkage between Hcp variant pairs (Hcp2-Hcp3 or Hcp4-Hcp5) and V1 effectors, the two proteins adjacent V1 region-associated Hcp pairs were fused in the order of Hcp2 and Hcp3, or Hcp4 and Hcp5. Representative chimeric sequences were generated by in silico fusion of adjacent V1-associated Hcp pairs in their genomic order. The representative fused protein sequences were aligned using MAFFT while retaining the alignment blocks. Maximum-likelihood phylogenetic trees were constructed using IQTREE (v.2.1.4_beta)63 after automatic model selection, with nodal support assessed through 1000 ultrafast phylogenetic bootstraps.

Gene locus visualization
Gene locus visualization is generated using Clinker64.

Statistical analysis
Data are presented as the arithmetic mean ± SEM. All statistical analyses were performed using GraphPad Prism version 9.3.0. Unpaired two-tailed Student’s t-tests were employed for mean comparisons between two groups, unless otherwise indicated. The significance of mean differences was denoted as follows: NS (not significant); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. A significance threshold of P < 0.05 was defined as statistically significant. Sample sizes were not pre-determined using specific statistical methods; however, they are comparable to those reported in prior studies. Where applicable, samples and experimental animals were randomly assigned to different groups. Data collection and analysis were not performed in a blinded manner with respect to experimental conditions. No data points were excluded from analysis.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In the manuscript \"Effector Conformational Plasticity Enables Lineage-Specific Secretion via Hcp Heterohexamers in Gut Symbionts,\" Zheng et al. investigate the role of heterohexameric Hcp rings in Type VI Secretion System (T6SS) effector delivery. The authors identify the crucial roles of Hcp1, Hcp2, and Hcp3 in Bte1 secretion and the T6SS-mediated bacterial killing effect. They co-expressed Hcp2-3 and solved the cryo-EM structures of both the Hcp2-3 complex and its interaction with Bte1. Notably, the Hcp2-3 complex exists in two different chemical stoichiometric forms (4:2 and 3:3). The authors propose a potential conformational change in the toxic Bte1 effector upon loading. Additionally, they demonstrate that Hcp3 specifically recognizes the V1 effectors. All key residues discussed in the manuscript have been validated through mutation studies. Through evolutionary analysis of the Hcp2-3 pair and V1 effectors, the authors extend their findings to the genus Bacteroides, further supporting their conclusions with AlphaFold models and biochemical methods. Overall, this manuscript is well-prepared and presents an excellent study that provides robust evidence for the loading and delivery of effector proteins by Hcp rings. The discovery of the heterohexameric Hcp rings is particularly exciting. Moreover, the authors have experimentally verified all their key conclusions. The innovation and significance of this research are undeniable and meet the standards for publication. However, several issues should be addressed to improve the manuscript: 1. While the manuscript emphasizes the importance of the Hcp2-3 heterohexamer, it remains unclear whether Hcp2 and Hcp3 can function independently. Specifically, can Hcp2 and Hcp3 form homohexamers, either in vivo or in vitro, similar to Hcp1? This could provide valuable insights into their other potential roles. 2. The gene names for hcp1, hcp2, and hcp3 should be displayed in Figure 1a or the figure legend. The order of hcp1-5 can vary across studies, and this would aid in clarifying the gene naming conventions used in the manuscript. 3. Lines 128-129 lack a citation to support the statements made. This should be corrected. 4. The authors note that Hcp2/3 exhibit high sequence identity within the Bte1 class (Figure 4a). To enhance this section, it would be beneficial to include typical sequences from this class in the sequence alignment shown in Extended Data Figure 6. 5. It would be helpful to add a panel at the bottom right of Figure 4f to display the conservatism of Hcp2/3 monomers separately. 6. A scale bar should be added to the electrostatic surface representation in Figure 3g to provide a reference for the measurements. 7. Some citations are formatted incorrectly, with missing issue or page numbers. These should be corrected to comply with the journal's citation guidelines.", "reviewer_2": "In this manuscript, Zheng et al. investigate the molecular basis of effector delivery by the Type VI Secretion System (T6SS) in Bacteroides fragilis. The authors demonstrate that Hcp2 and Hcp3 form an obligate heterohexameric ring that is indispensable for secretion of the effector Bte1 and for T6SS-mediated bacterial competition. Using cryo-EM, they reveal that Bte1 undergoes conformational rearrangements to fit into the Hcp pore, thereby establishing effector conformational plasticity as a key determinant of substrate compatibility. Comparative structural and phylogenetic analyses further show that lineage-specific Hcp2–Hcp3 complexes co-evolve with cognate effectors through hypervariable interfaces, supporting a “lock-and-key” model of recognition. These findings redefine the role of Hcp proteins in cargo selection and expand our understanding of how combinatorial Hcp assembly contributes to effector diversification in B. fragilis, with potential implications for engineering T6SS-based delivery systems. Overall, this study is clear and valuable, with only a few points that would benefit from further clarification or discussion. Major: 1. In lines 123–126 (Fig. 1d), the prey strain used in the in vitro competition assay carries pNBU2_ErmR, whereas in lines 133–135 (Fig. 1f), the prey strain used in the complementation assay carries pNBU2_CamR. Although this difference reflects only a change in antibiotic resistance, the rationale for using distinct prey strains should be clarified, and it should be explicitly demonstrated that this variation does not affect the comparability of the results. Furthermore, in Fig. 1g, Bte1 expression in the hcp2 complementation strain appears markedly weaker than in other complementation strains. Is this trend consistent across replicates? Please interpret this observation in the context of the distinct functional roles of hcp2 and hcp3. 2. The authors report that Hcp2 and Hcp3 form a stable heterohexameric complex, whereas Hcp1 forms a homohexamer. Have the authors attempted to express and purify Hcp2 or Hcp3 individually in vitro? If so, were either of them able to stably assemble into homohexamers on their own? Such information would be valuable for understanding the assembly mechanism of the Hcp2–Hcp3 heterohexamer. If Hcp2 or Hcp3 cannot form stable hexamers independently, this would further support a model in which hetero-oligomerization is required for their structural stability. I suggest that the authors provide experimental data if available. 3. The pull-down assay data could benefit from reorganization for consistency and clarity, as the presentation in Fig. 2a–c is not fully standardized. Although the assays were conducted with in vitro purified proteins, inclusion of confirmatory Western blot data would strengthen the identification of target proteins and enhance the overall robustness of the conclusions. 4. Lines 284–286: Does the statement “interface residues were highly conserved” refer to the interface between Hcp2 and Hcp3? If so, a magnified view of the contact surface should be provided. In addition, the specific thresholds used to define “Conserved” and “Variable” in Fig. 4f should be clearly stated. If instead this refers to conservation at the interface between Hcp hexameric rings, please consider whether this interface may interact with Hcp1 and VgrG. Based on the schematic in Fig. 6, Hcp1 and Hcp2/3 hexamers appear to co-assemble. Such mixed assembly could in principle compromise tube stability, yet the manuscript does not present direct evidence on this point. One possible hypothesis is that cargo-loaded Hcp2/3 hexamers are positioned exclusively at the distal tip for delivery, with the remainder of the tube composed of Hcp1. This model could account for the observation in Fig. 1e that the absence of any of the three Hcps abolishes Bte1 secretion. Do the authors have experimental evidence or informed speculation supporting this division-of-labor assembly model? Minor: 5. Line 149: The phrase “consistent with previous observations” requires supporting references. 6. In line 161, the description “Hcp2–Hcp3 complex assembles into two distinct stoichiometries” would better align with the figure–text flow if the stoichiometric order matched that in Fig. 2d. 7. Lines 199–203: Regarding the modeling of the Bte1–Hcp24–Hcp32 complex, could the use of the apo Hcp24–Hcp32 structure as a template potentially obscure subtle conformational changes in the Hcp ring upon Bte1 binding? Please provide a reasonable explanation to address this concern. 8. Lines 307–311: Only multiple-residue mutagenesis results are presented. Inclusion of single-residue mutagenesis data would help identify the critical residues mediating effector–Hcp interactions. Additionally, please verify the description in line 320: based on Fig. 5e, Arg51 in GS086 of Hcp3 appears to be essential for Bfe1 binding. 9. In Fig. 4a and Extended Data Fig. 7a, light purple circles are used to indicate branch support values in the phylogenetic trees, but the legend does not explain their scale, making it difficult to assess branch support quantitatively. 10. Line 553: The word “from” should be removed. 11. How are the V1 effectors Bfe1 and the putative LysM effector expressed and purified as predicted membrane proteins? I did not find relevant information in the Methods section. Please provide clarification.", "reviewer_3": "Overall The study by Shuaining Zheng et al. focuses on a unique T6SS effector delivery mechanism mediated by combinatorial Hcp assembly in Bacteroides fragilis. The authors have used an amazing combination of in vivo (up to co-colonization in mice) and in vitro approaches culminating with the cryo-EM structure of the complex Hcp2-Hcp3-Bte1 to illustrate the specific recruitment of the toxin into the T6SS needle. Especially, the back and forth flow between in vitro results and in vivo validation is particularly convincing in demonstrating the relevance of their findings. The authors clearly demonstrated that Hcp1 and Hcp2 are crucial for Bte1 transport and mediated killing, both using co-colonization in mice and in vitro. They also nicely demonstrated that Hcp2 and Hcp3 form a defined heterohexamer that specifically mediates the recruitment and export of Bte1 in NCTC9343. They evidenced for the first time the conformational transitions of the Bte1 effector upon incorporation into the Hcp2-Hcp3 heterohexamer. They identified critical domains (down to amino acid residues resolution) in Bte1 and Hcp3 that mediate the specificity of the transport. Ultimately, they broadened their study towards the notion of co-evolution of effector and Hcp transporters. The structural novelty is clear for the T6SS field and the originally of the data in this study meets the Nature Communications’ standards. Consequently, I’m mentioning here some comments that should however be addressed to increase the quality of the manuscript. Major Comments 1) The authors identified two types of Hcp2-Hcp3 heterohexamer. They encountered difficulties to obtain the cryo-EM structure of the Hcp2(3)-Hcp3(3) heterohexamer. They should try to assess the in vivo relevance of the two types of heterohexamer stoichiometries (4:2 or 3:3). 2) Cryo-EM and 2D-classes: the authors located the Hcp2 through an additional helical domain at the exterior of the Hcp ring (Figure 2d). It would be more convincing to confirm this location by deleting this region and perform 2D classification again to see if this feature has disappeared. 3) Gain of function mutation: the authors demonstrated that replacing Hcp3NCTC9343 with its GS086 ortholog abolished Bte1 binding. They should incorporate registering residues in Hcp3 to see if they can change the binding specificity from Bte1 to Bfe1… showing that these residues are the only ones governing the specific binding. 4) Since the authors map precisely the effector domain required for loading onto the Hcp2-Hcp3 heterohexamers, they should have used this information to construct chimeric effector (between Bte1, Bfe1 and LysM), to evaluate their transport and to validate that this is indeed the minimal sequence for transport. 5) The authors performed a deep analysis of sequence conservation across different bacterial species. They should use these data to run co-evolution analysis to predict residue-specific binding and change specificity for effector loading. 6) The authors revealed that the Bte1 effector undergoes massive conformational changes upon binding to the Hcp2-Hcp3 heterohexamer. They should investigate if the proposed conformational changes prevent the enzymatic activity of Bte1. Where are located the catalytic residues in Bte1 free or bound? 7) Additionally, they should confirm the proposed conformational change by designing mutations preventing it. These mutations should block the interaction with Hcp2-Hcp3 and consequently effector loading into Hcp tube. 8) Writing style: In some places, the description of the results is very brief. It jumps straight to the main findings without describing the methodology. For example, lines 119-126, the in vivo competition is very briefly described. Another example, lines 138-140, the result paragraph starts right away with a key result without contextualizing it. Lines 125-126: “These findings were confirmed by in vitro competition assays (Fig. 1d)”. A whole panel is not described and summarized in one single sentence. Line 145: there is no description on the way the Bte1-Hcp2-Hcp3 complex has been purified for cryo-EM. This should be briefly described in the result section. Slightly awkward. Please, add some description when necessary. Minor Comments 1) Please provide a more explicit text for the legends of the figures. The style is very brief.. 2) Figure 2b Test in vitro pulldown with only Hcp2, to parallel the in vivo experiment. Western blot to better differentiate Hcp2 from Hcp3 (anti-strep, anti-his) 3) Figure 4e Binding of Hcp2 (GS086)-Hcp3 (NCTC9343) to Bte1 is weaker as compare to Hcp2 (NCTC9343)-Hcp3 (NCTC9343): the authors should check the stability of Hcp hexamers by cryo-EM. 4) Discussion: Overall very short, the authors should improve and broaden their discussion. The authors should have cited earlier works on the same topic preprinted in bioRxiv recently (doi: https://doi.org/10.1101/2025.01.09.632221) and compare their results. Conclusion The structural novelty is clear for the T6SS field and the originally of the data in this study meets the Nature Communications’ standards. Consequently, I’m mentioning here some comments that should however be addressed to increase the quality of the manuscript. The present work (after including the requested change) will support the conclusions and claims. The findings and quality of the data reach the Nature Communications standards."}, "author_response": "Response to the Reviewer Comments: We thank the reviewers for their time and insightful comments, which have helped us significantly improve the quality of our manuscript. We have provided our response below in blue. Reviewer #1: In the manuscript \"Effector Conformational Plasticity Enables Lineage-Specific Secretion via Hcp Heterohexamers in Gut Symbionts,\" Zheng et al. investigate the role of heterohexameric Hcp rings in Type VI Secretion System (T6SS) effector delivery. The authors identify the crucial roles of Hcp1, Hcp2, and Hcp3 in Bte1 secretion and the T6SS-mediated bacterial killing effect. They co-expressed Hcp2-3 and solved the cryo-EM structures of both the Hcp2-3 complex and its interaction with Bte1. Notably, the Hcp2-3 complex exists in two different chemical stoichiometric forms (4:2 and 3:3). The authors propose a potential conformational change in the toxic Bte1 effector upon loading. Additionally, they demonstrate that Hcp3 specifically recognizes the V1 effectors. All key residues discussed in the manuscript have been validated through mutation studies. Through evolutionary analysis of the Hcp2-3 pair and V1 effectors, the authors extend their findings to the genus Bacteroides, further supporting their conclusions with AlphaFold models and biochemical methods. Overall, this manuscript is well-prepared and presents an excellent study that provides robust evidence for the loading and delivery of effector proteins by Hcp rings. The discovery of the heterohexameric Hcp rings is particularly exciting. Moreover, the authors have experimentally verified all their key conclusions. The innovation and significance of this research are undeniable and meet the standards for publication. We are deeply grateful for your positive feedback and encouraging assessment. However, several issues should be addressed to improve the manuscript: 1. While the manuscript emphasizes the importance of the Hcp2-3 heterohexamer, it remains unclear whether Hcp2 and Hcp3 can function independently. Specifically, can Hcp2 and Hcp3 form homohexamers, either in vivo or in vitro, similar to Hcp1? This could provide valuable insights into their other potential roles. We thank the reviewer for raising this important question regarding the potential of Hcp2 and Hcp3 to form independent homohexamers. To address this, we expressed and purified Hcp2 and Hcp3 individually in vitro (Fig. R1a). While both proteins were successfully expressed, gel filtration chromatography (Fig. R1b) and negative-stain electron microscopy (Fig. R1c) revealed that neither Hcp2 nor Hcp3 formed stable homohexameric structures when isolated. Instead, both proteins showed a tendency to aggregate under these conditions, indicating an inability to form stable homomeric assemblies comparable to Hcp1. We further examined the oligomeric states of Hcp2 and Hcp3 individually in vivo. To achieve this, we constructed strains in the Δhcp2 or Δhcp3 background expressing N-terminally 6×His-tagged and C-terminally HA-tagged versions of Hcp3 or Hcp2, respectively, for affinity enrichment and detection. It was found that Hcp2 was unstable, with most of it precipitating in the pellet and failing to be efficiently enriched. In contrast, Hcp3 was successfully enriched to a detectable level, as confirmed by both Coomassie blue-stained SDS-PAGE and western blotting (Fig. R1d). Subsequent gel filtration chromatography analysis showed that Hcp2 was below the detection limit in the soluble fraction (Fig. R1e), whereas Hcp3 eluted primarily as a monomer (Fig. R1f), with no evidence of stable hexamer formation or the aberrant aggregation observed in vitro. These findings, combined with our in vivo functional assays (Fig. 1b–g, 2a), suggest that Hcp2 and Hcp3 require heterocomplex formation to achieve structural stability and functional competence. We have added these results (Fig. R1a–c) to the revised manuscript (see lines 157–173) and Supplementary Fig. 2a–c to clarify that Hcp2 or Hcp3 alone cannot form stable homohexamers. 2. The gene names for hcp1, hcp2, and hcp3 should be displayed in Figure 1a or the figure legend. The order of hcp1-5 can vary across studies, and this would aid in clarifying the gene naming conventions used in the manuscript. The gene labels have been added to Figure 1a as suggested. 3. Lines 128-129 lack a citation to support the statements made. This should be corrected. A relevant citation (Reference [42]) has been incorporated to support our statement. 4. The authors note that Hcp2/3 exhibit high sequence identity within the Bte1 class (Figure 4a). To enhance this section, it would be beneficial to include typical sequences from this class in the sequence alignment shown in Extended Data Figure 6. As suggested, we have now updated the sequence alignment in Supplementary Fig. 7b,c to include several representative sequences from the Bte1 class. 5. It would be helpful to add a panel at the bottom right of Figure 4f to display the conservatism of Hcp2/3 monomers separately. As suggested, a new panel showing the conservation of individual monomers has been added to Figure 4g as suggested. 6. A scale bar should be added to the electrostatic surface representation in Figure 3g to provide a reference for the measurements. As suggested, a scale bar has now been added to Figure 3g to provide a clear reference for the electrostatic surface representation, and the corresponding figure legend has been updated accordingly. 7. Some citations are formatted incorrectly, with missing issue or page numbers. These should be corrected to comply with the journal's citation guidelines. We thank the reviewer for this careful observation. We have thoroughly reviewed the reference list and corrected all citation formats to ensure they are complete and fully comply with the journal's guidelines. Reviewer #2: In this manuscript, Zheng et al. investigate the molecular basis of effector delivery by the Type VI Secretion System (T6SS) in Bacteroides fragilis. The authors demonstrate that Hcp2 and Hcp3 form an obligate heterohexameric ring that is indispensable for secretion of the effector Bte1 and for T6SS-mediated bacterial competition. Using cryo-EM, they reveal that Bte1 undergoes conformational rearrangements to fit into the Hcp pore, thereby establishing effector conformational plasticity as a key determinant of substrate compatibility. Comparative structural and phylogenetic analyses further show that lineage-specific Hcp2–Hcp3 complexes co-evolve with cognate effectors through hypervariable interfaces, supporting a “lock-and-key” model of recognition. These findings redefine the role of Hcp proteins in cargo selection and expand our understanding of how combinatorial Hcp assembly contributes to effector diversification in B. fragilis, with potential implications for engineering T6SS-based delivery systems. Overall, this study is clear and valuable, with only a few points that would benefit from further clarification or discussion. We are deeply grateful for your positive feedback and encouraging assessment. Major: 1. In lines 123–126 (Fig. 1d), the prey strain used in the in vitro competition assay carries pNBU2_ErmR, whereas in lines 133–135 (Fig. 1f), the prey strain used in the complementation assay carries pNBU2_CamR. Although this difference reflects only a change in antibiotic resistance, the rationale for using distinct prey strains should be clarified, and it should be explicitly demonstrated that this variation does not affect the comparability of the results. Furthermore, in Fig. 1g, Bte1 expression in the hcp2 complementation strain appears markedly weaker than in other complementation strains. Is this trend consistent across replicates? Please interpret this observation in the context of the distinct functional roles of hcp2 and hcp3. We thank the reviewer for raising this important point regarding the use of prey strains with different antibiotic resistance markers in Figs. 1d and 1f. In the complementation assays (Fig. 1f), we used a chloramphenicol-resistant prey strain (pNBU2_CamR) to avoid potential antibiotic conflict, as the complemented killer strains themselves carried an erythromycin resistance marker. This allowed clear differentiation and reliable quantification of prey survival on chloramphenicol-containing agar. To directly assess whether the antibiotic marker influences killing efficiency, we repeated the competition assay from Fig. 1d using the CamR-marked prey strain. The results consistently showed a strong killing phenotype (new Fig. R2a), confirming that the choice of resistance marker does not affect the outcome. Accordingly, we have updated Fig. 1d in the revised manuscript with these new data to maintain experimental consistency. We also appreciate the reviewer's observation on Fig. 1g. Upon re-examining our earlier replicate data and performing additional independent experiments, we have confirmed that both the intracellular expression and secretion levels of Bte1 are similar in the hcp2, hcp3, and hcp2-hcp3 complementation strains (Fig. R2b). The variation in Bte1 secretion previously noted in the hcp2 complementation strain appears to have resulted from physiological variability between bacterial cultures, which can influence expression of the complemented gene. After revival and subculture of the strains, followed by repeated assays, the phenotype has proven consistent and reproducible. The data in Fig. 1g have been updated accordingly. 2. The authors report that Hcp2 and Hcp3 form a stable heterohexameric complex, whereas Hcp1 forms a homohexamer. Have the authors attempted to express and purify Hcp2 or Hcp3 individually in vitro? If so, were either of them able to stably assemble into homohexamers on their own? Such information would be valuable for understanding the assembly mechanism of the Hcp2–Hcp3 heterohexamer. If Hcp2 or Hcp3 cannot form stable hexamers independently, this would further support a model in which hetero-oligomerization is required for their structural stability. I suggest that the authors provide experimental data if available. We thank the reviewer for raising this important question regarding the potential of Hcp2 and Hcp3 to form independent homohexamers. You and reviewer #1 raised similar questions. Please see our response to reviewer #1, comment #1. 3. The pull-down assay data could benefit from reorganization for consistency and clarity, as the presentation in Fig. 2a–c is not fully standardized. Although the assays were conducted with in vitro purified proteins, inclusion of confirmatory Western blot data would strengthen the identification of target proteins and enhance the overall robustness of the conclusions. We agree that standardization generally enhances clarity. However, considering the overall layout of the figure, we have preserved the original presentation for Fig. 2a, which we believe remains clear and effectively conveys the experimental findings. Accordingly, we have now performed western blot analysis for the in vitro pull-down assays originally shown in Fig. 2b and 2c. The new data (Fig. R3) clearly confirm the specific formation of the Hcp2-Hcp3 complex and its interaction with Bte1, while no binding was detected between Bte1 and Hcp1. These results fully support our original Coomassie-stained findings and reinforce the specificity of the Bte1-Hcp2-Hcp3 interaction. The new data now have been included in revised manuscript and Supplementary Fig. 2d,e. 4. Lines 284–286: Does the statement “interface residues were highly conserved” refer to the interface between Hcp2 and Hcp3? If so, a magnified view of the contact surface should be provided. In addition, the specific thresholds used to define “Conserved” and “Variable” in Fig. 4f should be clearly stated. If instead this refers to conservation at the interface between Hcp hexameric rings, please consider whether this interface may interact with Hcp1 and VgrG. Based on the schematic in Fig. 6, Hcp1 and Hcp2/3 hexamers appear to co-assemble. Such mixed assembly could in principle compromise tube stability, yet the manuscript does not present direct evidence on this point. One possible hypothesis is that cargo-loaded Hcp2/3 hexamers are positioned exclusively at the distal tip for delivery, with the remainder of the tube composed of Hcp1. Do the authors have experimental evidence or informed speculation supporting this division-of-labor assembly model? We sincerely thank the reviewer for these insightful comments and the constructive model proposed. To address potential ambiguity in the original wording, we have revised the statement in lines 284–286 to: \"Conservation analysis of the Bte1-Hcp2-Hcp3 structure indicated that the interfacial residues mediating Hcp2-Hcp3 interactions are highly conserved, supporting the formation of stable heterohexamers across bacterial strains.” Furthermore, as suggested, we have provided a magnified view of the Hcp2-Hcp3 contact surface with conservation mapping in a new supplementary figure (Fig. R4 and Supplementary Fig. 8f). This addition offers a clear visualization of the evolutionary conservation at the heterohexamer interface. Regarding the classification in Fig. 4f, we defined \"Conserved\" residues as those exhibiting 100% identity across all analyzed sequences (colored pink), and \"Variable\" residues as those with 0% identity (colored cyan). This specific threshold has now been clearly stated in the revised manuscript. Regarding potential co-assembly between Hcp1 and the Hcp2-Hcp3 heterohexamer, we performed a series of in vitro and in vivo interaction assays (Fig. 2a and R5a,b). In vitro, purified Hcp1 and Hcp2-Hcp3 complexes each formed single-layer hexamers under our experimental conditions and did not assemble into stacked tubes (Fig. R1a,b). No interaction between Hcp1 and the Hcp2-Hcp3 heterohexamer was detected (Fig. R5a), which may be attributed to the absence of the stabilizing TssB-TssC sheath. In vivo, although the TssB-TssC sheath naturally stabilizes the Hcp tube, we still observed no detectable interaction between Hcp1 and the Hcp2-Hcp3 heterohexamer (Fig. 2a and R5b). This is likely due to the inherent fragility of the tubular structure, which is easily disrupted during cell lysis and sample preparation. Most importantly, our data show that Hcp1 and Hcp2-Hcp3 do not form hybrid hexamers, and only Hcp2-Hcp3 is capable of binding Bte1 (Fig. 2a–c, R3a,b and R5a,b). This indicates that these complexes act as independent structural units with specialized roles. Furthermore, unlike the well-established Hcp1-VgrG interaction (Fig. R5c, lane 5; Cell Rep, 2023, PMID: 37421630), we observed no direct binding between the (Bte1)-Hcp2-Hcp3 complex and VgrG (Fig. R5c, lane 6 and 7), indicating that cargo-loaded Hcp2-Hcp3 hexamers are unlikely to be positioned at the distal tip (tube-spike interface) for delivery. Based on previous transcriptomic data showing that Hcp1 expression is ~40-fold and ~150-fold higher than that of Hcp2 and Hcp3, respectively, in B. fragilis NCTC9343 (PNAS, 2016, PMID: 26951680), we propose a modified division-of-labor assembly model in which Hcp1 primarily constitutes the core scaffold of the inner tube, whereas the Hcp2-Hcp3 heterohexamer serves as a specific effector-loading module that may be integrated into the Hcp1-based tube either at internal positions or toward the distal end, through a random or regulated assembly mechanism. This model is consistent with the observation that all three Hcps are essential for Bte1 secretion (Fig. 1c–g), implying that Bte1 loaded Hcp2-Hcp3 complex must be physically integrated into the tube to enable effector delivery. We acknowledge the reviewer’s pertinent concern that introducing Hcp2-Hcp3 heterohexamers might compromise tube stability. We speculate that the stabilizing interaction between the TssB-TssC sheath and the Hcp inner tube may help accommodate such heteromeric modules without disrupting structural integrity (Fig. R6). In summary, although our current data do not fully validate the “division-of-labor” model, we find it highly compelling and have included a discussion of this possibility in the revised manuscript (see lines 509–530) to inspire future investigations. The new data (Fig. R5b,c, R6) and related interpretations are now included as Supplementary Fig. 1b,11, Fig. 6 and in the Discussion section. Minor: 5. Line 149: The phrase “consistent with previous observations” requires supporting references. A relevant citation (Reference [35]) has been incorporated to support our statement. 6. In line 161, the description “Hcp2–Hcp3 complex assembles into two distinct stoichiometries” would better align with the figure–text flow if the stoichiometric order matched that in Fig. 2d. We revised the text to state: “Based on initial 2D classification, the Hcp2-Hcp3 complex assembles into two distinct stoichiometries: 3:3 and 4:2 (Hcp2:Hcp3).” to be completely consistent with the representation in Figure 2d. 7. Lines 199–203: Regarding the modeling of the Bte1–Hcp24–Hcp32 complex, could the use of the apo Hcp24–Hcp32 structure as a template potentially obscure subtle conformational changes in the Hcp ring upon Bte1 binding? Please provide a reasonable explanation to address this concern. We thank the reviewer for raising this point and apologize for the possible misunderstanding. In our study, we did not use the apo Hcp24-Hcp32 structure as a template to model the Bte1-Hcp24-Hcp32 complex. The atomic model of the ternary complex was built based on the experimental cryo-EM density map. However, due to the relatively low local resolution of the peripheral regions of the Hcp ring in the ternary map, the corresponding Hcp densities appeared fragmented. To produce a more complete and visually interpretable representation, we aligned the apo Hcp24-Hcp32 structure to the Bte1-bound map and used it for illustration in the figure. This replacement does not affect the model refinement, interface analysis, or any structural interpretation presented in the manuscript. Accordingly, we have revised the relevant section (lines 237–243 in the revised manuscript) to prevent any potential misunderstanding. 8. Lines 307–311: Only multiple-residue mutagenesis results are presented. Inclusion of single-residue mutagenesis data would help identify the critical residues mediating effector–Hcp interactions. Additionally, please verify the description in line 320: based on Fig. 5e, Arg51 in GS086 of Hcp3 appears to be essential for Bfe1 binding. We appreciate the reviewer’s valuable suggestion. To identify critical residues in Bfe1 involved in its interaction with the GS086 Hcp2-Hcp3 complex, we conducted additional single-residue mutagenesis assays. Our results demonstrate that mutations at Trp34, Met35, Val39, and Ile64 individually impair binding affinity, with the I64S substitution exhibiting the most pronounced reduction (Fig. R7). The data in Fig. 5d and the corresponding description in the revised manuscript (lines 407–410) have been updated accordingly. In addition, we have carefully re-examined the description concerning Arg51 of Hcp3 and confirmed that this residue indeed contributes critically to Bfe1 binding, as indicated in Fig. 5f. The manuscript has been revised accordingly for clarity and accuracy. 9. In Fig. 4a and Extended Data Fig. 7a, light purple circles are used to indicate branch support values in the phylogenetic trees, but the legend does not explain their scale, making it difficult to assess branch support quantitatively. We are sorry for the confusion caused by the ambiguous representation of bootstrap values using light purple circles in the original Fig. 4a and supplementary Fig. 7a. To improve clarity and enable quantitative assessment of branch support, we have revised the phylogenetic trees by removing the light purple circles and directly labeling the exact bootstrap values at the corresponding nodes (Fig. 4a and supplementary Fig. 7a). Additionally, we have updated the figure legends to include clear descriptions of the bootstrap values. 10. Line 553: The word “from” should be removed. The word \"from\" has been removed in the revised manuscript. 11. How are the V1 effectors Bfe1 and the putative LysM effector expressed and purified as predicted membrane proteins? I did not find relevant information in the Methods section. Please provide clarification. We have now added the detailed protein expression and purification protocols for Bfe1 and LysM to the Methods section (see lines 569–576 and 653–656 in the revised manuscript). Reviewer #3: Overall The study by Shuaining Zheng et al. focuses on a unique T6SS effector delivery mechanism mediated by combinatorial Hcp assembly in Bacteroides fragilis. The authors have used an amazing combination of in vivo (up to co-colonization in mice) and in vitro approaches culminating with the cryo-EM structure of the complex Hcp2-Hcp3-Bte1 to illustrate the specific recruitment of the toxin into the T6SS needle. Especially, the back and forth flow between in vitro results and in vivo validation is particularly convincing in demonstrating the relevance of their findings. The authors clearly demonstrated that Hcp1 and Hcp2 are crucial for Bte1 transport and mediated killing, both using co-colonization in mice and in vitro. They also nicely demonstrated that Hcp2 and Hcp3 form a defined heterohexamer that specifically mediates the recruitment and export of Bte1 in NCTC9343. They evidenced for the first time the conformational transitions of the Bte1 effector upon incorporation into the Hcp2-Hcp3 heterohexamer. They identified critical domains (down to amino acid residues resolution) in Bte1 and Hcp3 that mediate the specificity of the transport. Ultimately, they broadened their study towards the notion of co-evolution of effector and Hcp transporters. The structural novelty is clear for the T6SS field and the originally of the data in this study meets the Nature Communications’ standards. Consequently, I’m mentioning here some comments that should however be addressed to increase the quality of the manuscript. We are deeply grateful for your positive feedback and encouraging assessment. Major Comments 1) The authors identified two types of Hcp2-Hcp3 heterohexamer. They encountered difficulties to obtain the cryo-EM structure of the Hcp2(3)-Hcp3(3) heterohexamer. They should try to assess the in vivo relevance of the two types of heterohexamer stoichiometries (4:2 or 3:3). We thank the reviewer for this valuable suggestion and fully agree that assessing the in vivo relevance of the two Hcp2-Hcp3 heterohexamer stoichiometries (4:2 and 3:3) is important for understanding their physiological significance. Although both stoichiometries were identified during in vitro cryo-EM analysis, severe preferred orientation prevented high-resolution reconstruction of the 3:3 form. To evaluate their physiological relevance, we performed native complex enrichment from 18L cultures of B. fragilis NCTC9343. A 6×His tag was inserted at the C-terminus of Bte1 to enable enrichment of the native Bte1-Hcp2-Hcp3 complex, and HA tags were inserted at the C-terminus of Hcp2 and Hcp3 for detection. Due to the extremely low endogenous abundance of the complex, Coomassie staining after Ni-affinity purification showed no visible target band. However, Western blot analysis confirmed the co-purification of Bte1, Hcp2, and Hcp3 (Fig. R8a). Gel filtration of the eluate revealed a ~120 kDa fraction containing all three components, consistent with the ternary complex (Fig. R8b,c). Despite extensive cryo-EM data collection (~10,000 micrographs) and particle picking, only ~20,000 particles of the native Bte1-Hcp2-Hcp3 complex were identified, again reflecting its low abundance (Fig. R8d). Strikingly, 2D classification of these particles revealed only the 3:3 stoichiometry; the 4:2 form was not observed (Fig. R8e). This suggests that the 3:3 assembly is the predominant form under the tested physiological conditions. Nevertheless, we cannot exclude that the 4:2 form may exist at very low abundance, be transient, or be condition-specific and thus not captured here. As with the in vitro sample, the native 3:3 particles exhibited strong preferred orientation, which prevented high-resolution reconstruction (Fig. R8f). Importantly, although a high-resolution structure of the Bte1-Hcp23-Hcp33 complex was not obtained, structural analysis confirms that in both stoichiometries, Bte1 engages an adjacent Hcp2-Hcp3 subunit pair (Fig. 3e, R8e, and Supplementary Fig. 4c,h). All key mechanistic conclusions regarding Bte1 conformational changes and interface interactions—derived from the Bte1-Hcp24-Hcp32 structure—have been consistently supported by in vitro and in vivo biochemical and functional assays, underscoring the robustness of our findings. Together, these results suggest that the 3:3 heterohexamer likely serves as the major functional form in vivo, whereas the 4:2 assembly may constitute a low-abundance or regulated state. Future work aimed at increasing complex yield, along with techniques such as single-molecule imaging in secretion assays and high-resolution cryo-ET or in situ cryo-EM, will be essential to further elucidate the functional relevance of distinct Hcp2-Hcp3 stoichiometries under native conditions. We have added these data to the Discussion of the revised manuscript (see lines 464–483) and to Supplementary Fig. 10. 2) Cryo-EM and 2D-classes: the authors located the Hcp2 through an additional helical domain at the exterior of the Hcp ring (Figure 2d). It would be more convincing to confirm this location by deleting this region and perform 2D classification again to see if this feature has disappeared. We thank the reviewer for this insightful suggestion. As suggested, we deleted the additional helical domain (residues 12–23) in Hcp2 that distinguishes it from Hcp3. The truncated Hcp2 (Δ12–23) and full-length Hcp3 were co-expressed and co-purified using tandem Ni-Strep affinity chromatography, yielding a stable binary complex (Fig. R9a). Gel filtration chromatography analysis further confirmed that the Hcp2 Δ12–23 strep-Hcp36×His complex forms a stable heterohexamer in solution (Fig. R9b). Cryo-EM data were collected for this mutant complex, and 2D classification revealed that the characteristic helical density previously observed at the exterior of Hcp2 in the Hcp2-Hcp3 complex was no longer visible (Fig. R9c,d). These findings support our conclusion that this structural feature corresponds to the Hcp2-specific helical region and can serve as a marker for estimating the relative proportions of Hcp2 and Hcp3 within the heterohexameric complex. We have added these data to the revised manuscript (see lines 192–197) and Supplementary Fig. 4d–f. 3) Gain of function mutation: the authors demonstrated that replacing Hcp3NCTC9343 with its GS086 ortholog abolished Bte1 binding. They should incorporate registering residues in Hcp3 to see if they can change the binding specificity from Bte1 to Bfe1… showing that these residues are the only ones governing the specific binding. To validate the critical role of Hcp3 in effector recognition and to assess whether specific residues in Hcp3 govern binding selectivity, we conducted a series of gain-of-function mutagenesis experiments as suggested. We first examined the binding of Bfe1 to four different Hcp2-Hcp3 combinations: Hcp2NCTC9343-Hcp3NCTC9343, Hcp2GS086-Hcp3GS086, Hcp2NCTC9343-Hcp3GS086, and Hcp2GS086-Hcp3NCTC9343 (Fig. R10a). The results showed that Hcp2GS086-Hcp3GS086 and Hcp2NCTC9343-Hcp3GS086 were able to bind Bfe1 (Fig. R10a) but not Bte1 (Fig. 4e), reinforcing the conclusion that Hcp3 plays a dominant role in effector recognition and binding within the Hcp2-Hcp3 complex. Based on this, we used the Hcp2GS086-Hcp3NCTC9343 complex, which binds Bte1 but not Bfe1, as the background for targeted mutagenesis. Structural analysis of Hcp3NCTC9343 revealed three non-conserved regions (variable regions 1,2 and 3) along the inner surface of the Hcp ring, predicted to mediate effector recognition (Fig. R10b,c). Individual mutations in any single region failed to confer Bfe1-binding ability. However, when variable regions 2 and 3 (VR2+3) or all three regions (VR1+2+3) were simultaneously mutated to match the GS086-type residues, the complex acquired Bfe1-binding ability, with the triple mutant showing the strongest binding (Fig. R10d). Together, these data establish Hcp3 as the primary determinant of effector specificity in the Hcp2-Hcp3 complex, while Hcp2 also contributes to effector interaction and selection (Fig. 3f–l). The non-conserved residues on the inner surface of Hcp3 directly shape effector-binding selectivity, and variation in these regions across strains likely reflects co-evolution of Hcp transporters with their cognate effectors. We have added these data to the revised manuscript (see lines 339–367), Fig. 4f and Supplementary Fig. 8b–e. 4) Since the authors map precisely the effector domain required for loading onto the Hcp2-Hcp3 heterohexamers, they should have used this information to construct chimeric effector (between Bte1, Bfe1 and LysM), to evaluate their transport and to validate that this is indeed the minimal sequence for transport. We thank the reviewer for raising this point. Based on structural analysis of the Bte1 crystal structure (PDB: 8ZHS) and the Bte1-Hcp2-Hcp3 cryo-EM complex, we defined residues 1–83 of Bte1 as the minimal sequence required for Hcp2-Hcp3-mediated loading. The corresponding regions in Bfe1 and LysM comprise residues 1–71 and 1–72, respectively (Fig. R11a). Structurally, this segment corresponds to a conserved N-terminal motif rather than a structurally independent domain. Therefore, in the revised manuscript, for accuracy, we have replaced the term “N-terminal loading domain” with “N-terminal loading region” for accuracy. Following the reviewer's suggestion, we constructed two chimeric effectors—Bfe1 1–71-Bte1 84–end and LysM 1–72-Bte1 84–end—by swapping their N-terminal loading regions. When expressed with an N-terminal 6×His tag, both chimeras were insoluble and detected only in the pellet fraction after cell lysis (Fig. R11b). To improve solubility, we introduced an N-terminal 6×His-MsyB-SUMO fusion tag. While this strategy rendered the chimeras soluble, gel filtration analysis revealed severe aggregation (Fig. R11c). These results indicate that the N-terminal loading region is structurally interdependent with the rest of the effector, and its substitution disrupts proper folding and stability (Fig. R11a). Although the instability of chimeric effectors prevented us from performing reliable binding assays, we believe that the current data—together with the mutational analysis, cross-strain binding results (Figs. 3i, 4d–f, 5d–g, R10a–d) and following co-evolution analysis (Fig. R12)—already provide strong evidence supporting the role of the identified region in effector loading. Harnessing the Hcp-cargo-mediated T6SS for targeted delivery of engineered effectors will require future systemic investigation. 5) The authors performed a deep analysis of sequence conservation across different bacterial species. They should use these data to run co-evolution analysis to predict residue-specific binding and change specificity for effector loading. We thank the reviewer for this insightful suggestion regarding residue-level co-evolution analysis. First, we performed ConSurf analysis using multiple sequence alignments of Hcp2/Hcp3 homologues from 38 B. fragilis strains (same strain set as Fig. 4a and Supplementary Fig. 7a) together with the Hcp2-Hcp3-Bte1 structure. This analysis allowed us to estimate the evolutionary conservation of individual residues in each protein. We found that the inner surfaces of Hcp2 and Hcp3 that engage effectors are highly variable, whereas the remaining regions are strongly conserved (Fig. R12a). Our experimental data further indicate that Hcp3 serves as the primary determinant of effector-recognition specificity within the Hcp2-Hcp3 complex (Fig. 4e, R12a). Consistent with this, replacing the variable regions (VR1/2/3) in Hcp3 enabled the complex to bind alternative effectors (Fig. R10d), demonstrating a direct link between the variability of interface residues and the specificity of effector loading. Although Bte1, Bfe1, and LysM share low overall sequence similarity (Fig. R12b), their N-terminal loading regions display high structural similarity (Fig. 5a, R11a). By fitting Bfe1 together with its cognate Hcp3GS086, and LysM together with its cognate Hcp3HMW616, into the Bte1-Hcp2-Hcp3 cryo-EM map, we identified the putative effector-Hcp3 interaction interfaces (Fig. 5b,c). Structural analysis revealed that an antiparallel β-sheet pair within the effector N-terminal loading region mediates binding to Hcp3 (Figs. 3f, 3h, 5b, 5c and R12c). Notably, this β-sheet pair is not conserved at the sequence level among different effectors (Fig. R12b, blue dashed box), likely to complement variable regions in Hcp3 across different bacterial strains. To validate these predicted interfaces, we performed in vitro loss-of-function mutagenesis targeting both residues within the effector N-terminal β-sheet pair and residues in the variable region of Hcp3 (Fig. 5d–g). Mutations on either side significantly impaired the interaction between effectors and the Hcp2-Hcp3 complex, confirming the reliability of the predicted residue-specific interfaces. Based on these biochemical data, we further conducted residue-specific co-evolutionary analysis of the Bte1-Hcp3NCTC9343, Bfe1-Hcp3GS086 and LysM-Hcp3HMW616 putative interfaces (Fig. R12d,e). In the resolved Bte1-Hcp2-Hcp3 structure, Hcp3 interacts with Bte1 through a polar interaction network (Fig. 3h, R12d). In contrast, in the Bfe1-Hcp3GS086 and LysM-Hcp3HMW616 putative interfaces, we observed a combination of hydrophobic and polar interactions, indicating diversified co-evolutionary adaptation (Fig. R12e). For example, Ser53 in Hcp3NCTC9343 forms a polar interaction with Thr40 of Bte1. However, when Ser53 is substituted by leucine in Hcp3GS086 or Hcp3HMW616, the interacting residues in the cognate effector's N-terminal β-sheet pair also shift to uncharged counterparts. This mutual change facilitates hydrophobic interactions, thereby maintaining a stable interface. Importantly, while the overall interaction networks differ markedly pattern among the Bte1-Hcp3NCTC9343, Bfe1-Hcp3GS086, and LysM-Hcp3HMW616 interfaces, they follow a regular pattern. This provides direct evidence for residue-level co-evolution underlying effector specificity (Fig. R12d,e). Although the attempt to generate fully functional effector chimeras to achieve complete gain-of-function specificity switching at the effector level failed (Fig. R11), substituting the β-sheet pair of Bte1 with the corresponding residues from Bfe1 enabled it to gain the ability to bind the GS086 Hcp2-Hcp3 complex (Fig. R12f). Together, these results demonstrate that effector selectivity is encoded by co-evolved, residue-specific interaction networks between the hypervariable inner surface of Hcp3 and the structurally conserved but sequence-divergent N-terminal loading regions of effectors, supporting a refined lock-and-key co-evolutionary model for T6SS effector loading. We have added these data (Fig. R12b,c,e,f) to the revised manuscript (see lines 398-403 and 427–454) and Supplementary Fig. 9. 6) The authors revealed that the Bte1 effector undergoes massive conformational changes upon binding to the Hcp2-Hcp3 heterohexamer. They should investigate if the proposed conformational changes prevent the enzymatic activity of Bte1. Where are located the catalytic residues in Bte1 free or bound? We apologize for any confusion that may have arisen. It is important to clarify that Bte1 is not an enzymatic effector; rather, its toxicity stems from its C-terminal domain, which binds and inhibits the periplasmic chaperone complex PpiD-YfgM, leading to substrate aggregation and consequent cellular toxicity (Cell Host Microbe, 2025, PMID: 39909037). Thus, the function of Bte1 depends on specific protein-protein interactions, not catalytic activity. To directly address the concern, we performed in vitro pull-down assays with Bte1-Hcp2-Hcp3 and YfgM-PpiD. The results show that Bte1 bound to Hcp2-Hcp3 complex still interacts stably with YfgM-PpiD complex, indicating that its functional binding domain remains free despite the large conformational rearrangements upon Hcp loading (Fig. R13a). Furthermore, we fitted our Bte1-Hcp2-Hcp3 model into the published cryo-EM map of the Bte1-PpiD-YfgM complex (EMD-60115). This analysis shows that Hcp2-Hcp3 binds to the N-terminal region of Bte1, while PpiD-YfgM binds to the C-terminal region, with no steric clashes, supporting the possibility that Bte1 can simultaneously engage both complexes (Fig. R13b). Although our data show that Bte1 can interact with its target while loaded onto Hcp2-Hcp3, we speculate that upon delivery into the periplasm of recipient cells, Bte1 may dissociate from the Hcp complex and adopt a more flexible conformation to fully exert its toxic function. The precise molecular details of this proposed mechanism will be an important subject for future investigation. 7) Additionally, they should confirm the proposed conformational change by designing mutations preventing it. These mutations should block the interaction with Hcp2-Hcp3 and consequently effector loading into Hcp tube. We thank the reviewer for this constructive suggestion. As described in the manuscript, structural analysis indicates that Bte1 undergoes substantial conformational rearrangements upon loading onto the Hcp2-Hcp3 heterohexamer, with helices α5 and α6 rotating ~70°, and α3 rotating nearly 120° to alleviate these steric conflicts (Fig. 3c,d). To directly test whether these conformational changes are required for effector loading, we designed targeted mutations to physically restrict the movement of α3 (Fig. R14a). Structural analysis revealed that Ala115 in α3 is positioned in close proximity to Pro61 in the N-terminal loading domain (Fig. R14b). We therefore generated a Bte1A115C/P61C double-cysteine mutant to promote the formation of an intramolecular disulfide bond, thereby locking α3 and preventing conformational rearrangement (Fig. R14c). In vitro pull-down assays demonstrated that Bte1P61C/A115C exhibited markedly reduced binding to the Hcp2-Hcp3 complex. Importantly, when 1 mM TCEP was added to disrupt the engineered disulfide bond, binding was restored to a level comparable to that of wild-type Bte1 (Fig. R14d). These results indicate that restricting α3 movement directly impairs the interaction between Bte1 and the Hcp2-Hcp3 heterohexamer. To further investigate how this restricted conformation influences Bte1 secretion, we performed co-culture competition assays. Complementation of Bte1P61C/A115C in NCTC9343Δbte1 strain resulted in markedly reduced killing activity compared to wild-type Bte1, suggesting impaired effector secretion (Fig. R14e). Together, these findings demonstrate that conformational rearrangements in Bte1 are essential for its efficient loading onto the Hcp2-Hcp3 heterohexamer. We have added these data to the revised manuscript (see lines 257–276) and Supplementary Fig. 6e–i. 8) Writing style: In some places, the description of the results is very brief. It jumps straight to the main findings without describing the methodology. For example, lines 119-126, the in vivo competition is very briefly described. Another example, lines 138-140, the result paragraph starts right away with a key result without contextualizing it. Lines 125-126: “These findings were confirmed by in vitro competition assays (Fig. 1d)”. A whole panel is not described and summarized in one single sentence. Line 145: there is no description on the way the Bte1-Hcp2-Hcp3 complex has been purified for cryo-EM. This should be briefly described in the result section. Slightly awkward. Please, add some description when necessary. We thank the reviewer for this valuable suggestion. Following the comment, we have thoroughly revised the relevant parts of the manuscript to incorporate additional methodological context prior to presenting the key findings, thereby enhancing the clarity and logical flow of the research narrative (see lines 116–128, 130–136, 148–155 and 175–178 in the revised manuscript). Minor Comments 1) Please provide a more explicit text for the legends of the figures. The style is very brief. We have checked and revised the figure legends to provide more detailed and explicit descriptions. 2) Figure 2b Test in vitro pulldown with only Hcp2, to parallel the in vivo experiment. Western blot to better differentiate Hcp2 from Hcp3 (anti-strep, anti-his) Regarding the request for a pull-down assay with Hcp2 alone, our experimental data provided in the responses to Reviewer #1 and #2 already demonstrate that neither Hcp2 nor Hcp3 can form stable homohexamers independently in vitro (Fig. R1a–c). Nevertheless, we performed the requested in vitro pull-down assay using purified Bte1 and Hcp2strep (Fig. R15). The results confirmed no direct interaction between Bte1 and Hcp2strep alone, supporting the conclusion that Hcp2 and Hcp3 require heterocomplex formation to achieve structural stability and enable Bte1 loading. Regarding the Western blot analysis, as also requested by Reviewer #2, we have now performed Western blot analysis for the pull-down assays in Fig. 2b,c using specific antibodies. Please see our response to reviewer #2, comment #3. 3) Figure 4e Binding of Hcp2 (GS086)-Hcp3 (NCTC9343) to Bte1 is weaker as compare to Hcp2 (NCTC9343)-Hcp3 (NCTC9343): the authors should check the stability of Hcp hexamers by cryo-EM. In our analyses of the interaction interface between Bte1 and the Hcp2-Hcp3 complex, we found that although Hcp3 contributes the majority of the binding interface, Hcp2 also provides several specific residues that participate in the interaction (Fig. 3f–h). Therefore, substituting Hcp2NCTC9343 with the GS086 variant, which contains substitutions at several contact residues, reasonably leads to weaker binding with Bte1, as observed in Fig. 4e. To evaluate whether the reduced binding arises from impaired hexamer stability, we assessed the structural stability of the cross-strain Hcp2-Hcp3 complexes. Gel filtration chromatography revealed a single, symmetric peak corresponding to a hexameric assembly, with no signs of dissociation or aggregation (Fig. R16). This indicates that the heterohexamers remain stable in solution. Moreover, conservation analysis shows that the residues mediating the Hcp2-Hcp3 interface are highly conserved across bacterial strains (Fig. 4g and R4), further supporting the structural robustness of cross-strain heterohexamers. Together, these results confirm that the cross-strain complex forms a stable heterohexamer comparable to the native counterpart, and that the attenuated binding to Bte1 is more likely due to the loss of specific interfacial contacts introduced by the GS086 variant. This result has already been updated in Supplementary Fig. 8c. 4) Discussion: Overall very short, the authors should improve and broaden their discussion. The authors should have cited earlier works on the same topic preprinted in bioRxiv recently (doi: https://doi.org/10.1101/2025.01.09.632221) and compare their results. In response to the reviewer’s suggestion, we have expanded the Discussion in the revised manuscript (see lines 455-550) to better situate our findings within the broader context of T6SS mechanisms. We have also cited and compared our results with a recent bioRxiv preprint (doi:10.1101/2025.01.09.632221), which reported a co-evolutionary relationship between Hcp2-Hcp3 and V1 effectors in B. fragilis NCTC9343 and provided in vitro biochemical evidence that Hcp2NCTC9343 and Hcp3NCTC9343 can form hexamers with variable stoichiometry and interact with Bte1, which further supports our findings. Beyond these corroborating observations, our study integrates in vivo mouse gut co-colonization assays, in vitro bacterial competition experiments, in vivo and in vitro biochemical interaction analysis, and high-resolution cryo-EM structures of both the apo Hcp2-Hcp3 complex and the Bte1-loaded assembly. Furthermore, we reveal that Bte1 undergoes substantial conformational rearrangements to adapt to the rigid Hcp pore, and that effector specificity is determined primarily by hypervariable interfaces on Hcp3. These interfaces recognize a structurally conserved N-terminal loading module via a “lock-and-key” co-evolutionary mechanism. Conclusion The structural novelty is clear for the T6SS field and the originally of the data in this study meets the Nature Communications’ standards. Consequently, I’m mentioning here some comments that should however be addressed to increase the quality of the manuscript. The present work (after including the requested change) will support the conclusions and claims. The findings and quality of the data reach the Nature Communications standards."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have done a thorough job of addressing the reviewers' critiques and their new data add strength to the conclusions in the paper. I recommend acceptance.", "reviewer_2": "This is a very careful and comprehensive revision of a paper that I earlier reviewed. I have no further comments.I think the manuscript is now suitable for publication.", "reviewer_3": "I would like to commend the authors for their exceptionally rigorous and skillful revision. They have addressed all the reviewers’ inquiries thoroughly, updating the figures and text to bring the manuscript to an outstanding level of quality. The structural novelty is clear for the T6SS field and the originally of the data in this study meets the Nature Communications’ standards. I personally accept this revised version."}, "author_response": ""}]

Decision letter