Molecular mechanism of toxin neutralization in the HipBST toxin-antitoxin system of Legionella pneumophila

Zhen, Xiangkai; Wu, Yongyu; Ge, Jinli; Fu, Jiaqi; Ye, Le; Lin, Niannian; Huang, Zhijie; Liu, Zihe; Luo, Zhao-qing; Qiu, Jiazhang; Ouyang, Songying

doi:10.1038/s41467-022-32049-x

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Published: 26 July 2022

Molecular mechanism of toxin neutralization in the HipBST toxin-antitoxin system of Legionella pneumophila

Xiangkai Zhen¹^na1,
Yongyu Wu¹^na1,
Jinli Ge²^na1,
Jiaqi Fu³,
Le Ye¹,
Niannian Lin¹,
Zhijie Huang¹,
Zihe Liu¹,
Zhao-qing Luo ORCID: orcid.org/0000-0001-8890-6621³,
Jiazhang Qiu² &
…
Songying Ouyang ORCID: orcid.org/0000-0002-1120-1524¹

Nature Communications volume 13, Article number: 4333 (2022) Cite this article

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Abstract

Toxin-antitoxin (TA) systems are ubiquitous genetic modules in bacteria and archaea. Here, we perform structural and biochemical characterization of the Legionella pneumophila effector Lpg2370, demonstrating that it is a Ser/Thr kinase. Together with two upstream genes, lpg2370 constitutes the tripartite HipBST TA. Notably, the toxin Lpg2370 (HipT_Lp) and the antitoxin Lpg2369 (HipS_Lp) correspond to the C-terminus and N-terminus of HipA from HipBA TA, respectively. By determining crystal structures of autophosphorylated HipT_Lp, its complex with AMP-PNP, and the structure of HipT_Lp-HipS_Lp complex, we identify residues in HipT_Lp critical for ATP binding and those contributing to its interactions with HipS_Lp. Structural analysis reveals that HipS_Lp binding induces a loop-to-helix shift in the P-loop of HipT_Lp, leading to the blockage of ATP binding and inhibition of the kinase activity. These findings establish the L. pneumophila effector Lpg2370 as the HipBST TA toxin and elucidate the molecular basis for HipT neutralization in HipBST TA.

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Introduction

Toxin–antitoxin (TA) systems are bacterial and archaeal genetic modules enriched in mobile genetic elements and chromosomes that comprise two or more closely linked genes encoding a toxin protein and its cognate antitoxin¹. Since the discovery that the ccdB/ccdA TA system maintains stable inheritance of the mini-F plasmid in Escherichia coli², the biological roles of TA systems have been demonstrated to include maintaining stabilization and fitness of mobile genetic elements such as plasmids³ and protection against phages⁴. Toxins are stable enzymes (e.g., RNases and kinases) or other proteins (e.g., gyrase inhibitors and pore-like toxins) that, in the absence of cognate antitoxin, interfere with vital cellular processes such as DNA replication and protein translation⁵. Antitoxins are unstable proteins or RNAs that counteract toxins. Based on the antitoxin nature and toxin-neutralization mechanism, TA systems can be divided into types I–VIII^6,7.

In type II TA systems such as HipBA modules, toxin neutralization depends on direct binding of a proteinaceous antitoxin^1,8. HipA from HipBA TA module of the E. coli strain K-12 is a 440-amino acid (aa) Ser/Thr kinase that phosphorylates the tRNA^Glu-bound glutamate-tRNA ligase GltX at Ser239, thereby inhibiting protein translation^9,10. The growth arrest induced by E. coli HipA can be counteracted by HipB, a cro/C1-type helix–turn–helix (HTH) domain-containing protein¹¹. The structures of HipBA modules from E. coli (HipBA_Ec) and Shewanella onesidensis (HipBA_So) reveal that both TA modules form a HipA₂–HipB₂ heterotetramer in which HipB binds far from the kinase catalytic center of HipA¹². Such neutralization strategy differs from most of other type II TA systems, where antitoxins binding usually occludes the active site¹³ or mediate allosteric regulation of the toxin^14,15,16.

Recent studies demonstrated that TA systems containing toxins homologous to the E. coli HipA are widely distributed in bacterial genomes¹⁷, suggesting the diversity of HipBA TA systems^17,18. Among these TA systems, a tripartite system designated HipBST was recently identified and experimentally characterized in the enteropathogenic E. coli serotype O127:H6¹⁸. HipT, which serves as the toxin in the HipBST system, phosphorylates TrpS at Ser197, and its toxicity can be counteracted by the small protein encoded by the adjacent gene hipS¹⁷. Importantly, the toxin HipT and the antitoxin HipS of the HipBST system were found to correspond to the N-terminal subdomain 1 and the core kinase domain of the E. coli HipA, respectively. The third protein of the HipBST module, HipB, is analogous to HipB of the HipBA system and appears to enhance the neutralization effect of HipS by binding to an already formed HipT–HipS heterodimer¹⁸. Recently, a preprint study reported the structure of HipBST heterotrimer from E. coli serotype O127:H6 and concluded that ATP binding in HipT is prevented by comparing its structure in the heterotrimer to the available structures of E. coli HipBA complex¹⁹. However, the general mechanism for toxin neutralization in HipBST TA systems is not fully elucidated.

Legionella pneumophila, the causative agent of Legionnaires’ disease, extensively modifies host signal transduction pathway, especially the post-translational modifications such as ubiquitination and phosphorylation, by translocating hundreds of effectors into the host cell via the Dot/Icm system^20,21,22,23. One such effector is the recently identified Lpg2370²⁴, which was previously predicted to be an E3 ligase but has not been characterized²⁵.

In this work, we find that Lpg2370 in fact shares sequence identity with the C-terminus of the E. coli K-12 HipA, the toxin of type II TA system HipBA, which is then confirmed by experimental validation and determining the crystal structure of autophosphorylated Lpg2370 (pLpg2370). Furthermore, gene locus analysis indicates that lpg2370 is grouped with lpg2369 and lpg2368 into a tricistronic operon, which we proceed to characterize respectively as the toxin HipT_Lp, the antitoxin HipS_Lp, and HipB_Lp, that constitute the tripartite TA system HipBST. We also determine high-resolution structures of the toxin pHipT_Lp in complex with the ATP analog AMP–PNP and the binary complex with the antitoxin HipS_Lp and identify key HipT_Lp residues involved in ATP binding and interactions with HipS_Lp. Lastly, a comparison of the three structures determined in this study allowed us to propose the mechanism of toxin neutralization in the type II TA system HipBST.

Results

The L. pneumophila effector Lpg2370 is a Ser/Thr kinase

We first verified whether Lpg2370 is translocated into host cells via the type IV secretion system (T4SS) Dot/Icm as an effector protein. To this end, we performed TEM-1 β-lactamase translocation assay by infecting RAW264.7 macrophages with the fusion protein-expressing L. pneumophila cells grown to post-exponential phase. Vectors expressing TEM-1-RaIF (positive control), TEM-1-FabI (negative control) or TEM-1-Lpg2370 fusion proteins were introduced into the T4SS-competent L. pneumophila strain Lp02 or the Dot/Icm-deficient strain Lp03, which were then assessed for the delivery of the β-lactamase fusions into the host macrophage cells by visual inspection under a fluorescence microscope. Cells infected by Lp02 cells expressing the TEM-RalF fusion protein emitted blue fluorescence, whereas infection with the TEM-FabI-expressing cells did not result in any emission of blue fluorescence by host cells (Fig. 1a). In addition, none of the fusion proteins were detectably translocated upon infection with the Dot/Icm-deficient strain Lp03 (Fig. 1a). Consistent with previous studies²⁴, the Lpg2370-overexpressing L. pneumophila strain Lp02 can be secreted into host cells, though at very low translocation efficiencies, suggesting that Lpg2370 is indeed a L. pneumophila effector protein.

**Fig. 1: *L. pneumophila* effector Lpg2370 is a Ser/Thr kinase.**

Although previous studies implied that Lpg2370 is an E3 ubiquitin ligase based on sequence similarity with the RING-type E3 ubiquitin ligase FANCL^25,26,27, we repeated primary sequence analysis and did not find any significant similarity between the two proteins. However, we found notable sequence identity (~20%) between Lpg2370 and the residues 64–440 of E. coli HipA, an atypical Ser/Thr kinase from the E. coli K-12 strain (Supplementary Fig. 1)²⁸. The results particularly indicated conservation of the sequences corresponding to the P-loop (RISVAGAQ), the signature motif responsible for ATP binding, and the catalytic loop, which contains the catalytic residue D310 required for the kinase activity of E. coli HipA^29,30,31 (Fig. 1b). Moreover, comparing the Lpg2370 sequence to the NCBI database using basic local alignment search tool (BLAST) identified Lpg2370 as a HipA-like Ser/Thr kinase. These findings led us to hypothesize that Lpg2370 could be a kinase.

Kinases frequently undergo autophosphorylation on an invariable Ser or Thr residue in the P-loop³². For instance, autophosphorylation of Ser150 on the P-loop has been observed in HipA kinases from E. coli (HipA_Ec) and the proteobacterium Shewanella oneidensis (HipA_So)^30,31. To investigate whether Lpg2370 is also autophosphorylated, we incubated purified recombinant Lpg2370 expressed in E. coli with the N6-benzyladenosine-5’-O-(3-thiotriphosphate) (N⁶-Bn-ATPγS) (Fig. 1c)³³. Immunoblotting with anti-N⁶-Bn-ATPγS antibody detected a protein band corresponding to Lpg2370 (35 kDa) (Fig. 1d), which clearly indicated that Lpg2370 can be autophosphorylated. We next performed LC-MS/MS to identify the autophosphorylation site on Lpg2370. A mass shift of 79.97 Da (m/z = 684.85, z = 2) was consistently observed in the putative P-loop (53-MSVQGVQKK-61), revealing that the residue Ser54 within the P-loop is the autophosphorylation site (Fig. 1e). Taken together, these results suggest that Lpg2370 is a Ser/Thr kinase, though its substrates are currently unknown.

Lpg2370 adopts a Ser/Thr kinase-like fold

To gain deeper insight into the molecular function of Lpg2370, we set out to determine its crystal structure. Diffraction phases for the SeMet-labeled Lpg2370 were determined using the single-wavelength anomalous diffraction method and the final structural mode was refined at 1.46 Å (Table 1).

Table 1 X-ray data collection and refinement statistics

Full size table

Like other members of the protein kinase superfamily, Lpg2370 has a globular kinase fold that can be further divided into N-lobe and C-lobe. The N-lobe, which contains the P-loop, is composed of sheets β1–5 sandwiched by helices α1 and α2, whereas the C-lobe is predominantly α-helical and consists of helix bundles α3–α6 and α8–α11 and a short β-sheet β6–8 (Fig. 2a). In line with the results of primary sequence analysis, there is no apparent structural similarity between Lpg2370 and the E3 ligase FANCL (Supplementary Fig. 2a), and residues 4–28 of Lpg2370, which are relatively well aligned with FANCL²⁵, are a part of the typical kinase N-lobe. In agreement with our LC-MS/MS results, we can observe a phosphate group covalently attached to the Ser54 residue (Fig. 2b). However, unlike HipA_Ec and HipA_So whose P-loops are disordered upon serine autophosphorylation²⁹, the electron density of the Lpg2370 P-loop is well defined in the present structure (Fig. 2b). Interestingly, the P-loop of Lpg2370 differs from the counterparts of typical protein kinases in that it contains a single glycine residue³⁴. The positioning of the phosphorylated Ser54 (pSer54) is stabilized by the positively charged side chains of K40, R131, and R134, as well as hydrogen bonds between side chain of Gln56 and side chains of Asp145 and Lys201 (Supplementary Fig. 2b).

**Fig. 2: Crystal structure of pLpg2370.**

Dali search suggested that Lpg2370 shares the highest structural homology score with HipA_Ec and HipA_So (Supplementary Table 1)³⁵. Lpg2370 and HipA_Ec superimpose with a relatively large root-mean-square deviation (RMSD) value of 3.846 Å over 221 Cα atoms (Fig. 2c, d). In addition to lacking a counterpart to the N-terminal region of HipA_Ec (i.e., helices α1–α4 and strands β1–β3 of HipA), Lpg2370 differs from HipA_Ec mainly in the configuration of the N-terminal lobe (Fig. 2c). One prominent difference is that the Lpg2370 P-loop with phosphorylated Ser54 is exposed to solvent in an orientation similar to that of unphosphorylated P-loop of HipA_Ec, which upon serine autophosphorylation rotates by ~180° and bends away from the N-terminal lobe by 17.3 Å (Fig. 2e, f). Conversely, the six C-terminal α-helices of Lpg2370 and HipA_Ec, including the catalytic residues and some ATP-binding residues, are almost perfectly aligned (Fig. 2e).

Lpg2368–Lpg2369–Lpg2370 constitute the tripartite HipBST TA system of L. pneumophila

Further analysis of Lpg2370 showed that proteins containing the C-terminal domain of HipA are widespread in bacteria (Fig. 3a). Moreover, the structural similarity between Lpg2370 and E. coli HipA and the fact that E. coli HipA along with HipB from the same genomic locus composes a type II TA system prompted us to examine the locus of lpg2370. Indeed, we found that lpg2370 is preceded by open reading frames (ORFs) of lpg2368 and lpg2369. Analogously to TA systems such as HipBA²⁸, lpg2368, and lpg2369 as well as lpg2369 and lpg2370 overlap by 4 bp. Further analysis suggested that lpg2369 encodes a 102-aa protein similar to the N-terminal region of E. coli HipA and that lpg2368 encodes a 72-aa protein homologous to the helix–turn–helix (HTH) domain of HipB (Fig. 3b and Supplementary Fig. 3a, b). Such locus organization is reminiscent of the HipBST TA module in E. coli O127:H6^17,18, suggesting that the lpg2368–lpg2369-lpg2370 locus is a potential tricistronic operon encoding component of a HipBST TA system.

**Fig. 3: L. *pneumophila* genes *lpg2370*, *lpg2369*, and *lpg2368* belong to the same operon and constitute a tripartite HipBST family TA system.**

In the HipBST TA system of E. coli O127:H6, the toxin HipT (denoted HipT_O127) can form a heterotrimeric complex with the antitoxin HipS_O127 and the HTH domain protein HipB_O127¹⁸. We therefore performed size-exclusion chromatography, pull-down assays and isothermal titration calorimetry (ITC) to analyze interactions between Lpg2370, Lpg2369, and Lpg2368. The co-expressed 6×His-tag Lpg2369 and untagged Lpg2370 were co-eluted using Ni affinity chromatography, and the size-exclusion chromatography analysis revealed that the peak is shifted forward by 0.4 mL compared to the peak of Lpg2370 alone, suggesting Lpg2369 can interact with Lpg2370 (Fig. 3c). Moreover, size-exclusion chromatography indicated that Lpg2368 co-elutes with the co-expressed 6×His-tagged Lpg2369–Lpg2370 complex and binds to the Lpg2369–Lpg2370 complex assembled in vitro (Fig. 3c), which was then further confirmed by the pull-down assays (Supplementary Fig. 4). These results suggest that Lpg2370 directly interacts with Lpg2369, whereas Lpg2368 binds to a stable Lpg2369–Lpg2370 complex. Moreover, the results of ITC assays demonstrated that the dissociation constants between Lpg2370 and Lpg2369 and Lpg2370-Lpg2369 complex and Lpg2368 are 42 nM and 1.5 µM, respectively (Supplementary Fig. 5a, b), which is in agreement with the previously published data on HipBST_O127¹⁸.

Given the established analogy between the L. pneumophila Lpg2368–Lpg2369–Lpg2370 operon and the HipBST TA system, we next aimed to functionally characterize Lpg2370 by investigating its potential toxicity to host bacteria. Heterogeneous expression of the recombinant Lpg2370 in E. coli had no observable effect on cell growth (Supplementary Fig. 6). The toxicity assays were performed in the L. pneumophila strain Lp02. To avoid undesirable effects from endogenous expression, we prepared deletion strain ∆lpg2368-∆lpg2369-∆lpg2370 (∆3) and examined bacterial growth upon overexpression of recombinant Lpg2370. Overexpression of Lpg2370 significantly inhibited the growth of L. pneumophila, both on plates and in liquid medium (Fig. 3d). Moreover, the catalytically inactive H199A (H/A) mutant failed to inhibit bacterial growth (Fig. 3d), indicating that the kinase activity of Lpg2370 is strictly required for its toxicity (Fig. 3d).

To assess the impacts of Lpg2368 and Lpg2369 on bacterial growth, we inserted lpg2370 into the low-copy-number IPTG-inducible vector pZL507, and lpg2368, lpg2369, or lpg2368–lpg2369 were separately inserted into the plasmid pJL03 with the arabinose-inducible pBAD promoter. Growth and viability of the L. pneumophila Δ3 strain carrying combinations of these plasmids was then monitored. Growth inhibition caused by the expression of Lpg2370 was counteracted by co-expression of Lpg2369, suggesting that Lpg2369 functions as the antitoxin (Fig. 3e). Co-expression of Lpg2368 and Lpg2369 was also found to counteract Lpg2370-dependent growth inhibition, whereas the expression of Lpg2368 without Lpg2369 could not prevent the growth inhibition (Fig. 3e). Taken together, these results are consistent with the findings on the E. coli O127:H6 HipBST module^17,18 and demonstrate that Lpg2368, Lpg2369, and Lpg2370 from L. pneumophila constitute the tripartite HipBST TA system¹⁸ and will thus hereafter be referred to as HipT_Lp, HipS_Lp, and HipB_Lp, respectively^17,18.

The kinase activity of HipT_Lp is likely independent of P-loop serine autophosphorylation

A comparison of the crystal structure of pHipT_Lp and the structures deposited in the PDB revealed that the autophosphorylated P-loop in HipT_Lp adopts an orientation similar to that of the P-loop in the crystal structure of E. coli HipA S150A mutant (Fig. 2f)³⁰. This observation led us to speculate that pHipT_Lp can bind ATP. Thermal shift assays performed with the purified wild-type HipT_Lp revealed a 2.5 °C-increase in the melting temperature (Tm) in the presence of non-hydrolysable ATP analogue adenylyl-imidodiphosphate (AMP–PNP), suggesting that pHipT_Lp indeed binds ATP (Supplementary Fig. 7). Likewise, isothermal calorimetry determined that the dissociation constant between pHipT_Lp and AMP–PNP was about 70 µM (Fig. 4a), which is within the range of ATP-binding affinity expected for other kinases²⁹.

**Fig. 4: Crystal structure of the pHipT_Lp–AMP–PNP complex.**

To elucidate how pHipT_Lp binds ATP, we determined the crystal structure of pHipT_Lp in complex with AMP–PNP at 1.36 Å resolution (Table 1). The structure of pHipT_Lp–ATP reveals that AMP–PNP is bound to the P-loop like in other representative kinases (Fig. 4b). The backbone of pHipT_Lp in the complex is virtually identical to the apo structure (RMSD = 0.35 Å), with the exception of P-loop that bends towards helix α2 to accommodate the AMP–PNP. ATP (AMP–PNP)-interacting residues appear to be conserved among the bacterial HipT toxins, implying a shared mechanism for ATP binding (Fig. 4c). In pHipT_Lp, the γ-phosphate of AMP–PNP is stabilized by V58, H199, and D219, the β-phosphate forms hydrogen bonds with Q59, K61, and K85, whereas the α-phosphate interacts with K85 and N202. The adenosine moiety interacts with the main chain of K130 and forms π-stacking interactions with the side chain of F132 (Fig. 4b).

A previous study demonstrated that the kinase activity of E. coli HipA is essential for the growth arrest of host cells³⁶, and cell growth was inhibited when HipT_O127 was expressed in E. coli BL21 (DE3) cells¹⁸. To confirm the role of the residues involved into the ATP binding in the HipT toxins in vivo, HipT_O127 TA is used to perform the growth inhibition assays due to the easy manipulation of E. coli compared to L. pneumophila. To investigate whether the above-mentioned residues responsible for ATP binding are essential for the kinase activity of HipT, we performed in vivo toxicity assays with HipT_O127 variants in which residues corresponding to the S54 and the highly conserved ATP-binding residues of HipT_Lp were substituted with alanine. Intriguingly, mutation on the residues corresponding to S54 of HipT_Lp (S57A and S57D HipT_O127) remain toxic to E. coli cells, whereas substitutions of K64 (K61), K86 (K85), H212 (H199), N215 (N202), and D233 (D219) of HipT_O127 (corresponding residues in HipT_Lp are indicated in parentheses) eliminated the toxic phenotype (Fig. 4d). Taken together, these results suggest that unlike in E. coli HipA, HipT retains the ATP-binding ability independent of the autophosphorylation on the conserved S54 in the P-loop and that HipT uses a universal mode for ATP recognition.

Structural basis for the toxin HipT_Lp recognition by the antitoxin HipS_Lp

Although the toxic activity of HipT in the HipBST TA system has been demonstrated to be counteracted by the antitoxin HipS^17,18, the underlying molecular mechanism remains unknown. We therefore sought to determine the structure of the HipT_Lp–HipS_Lp complex. To express the HipT_Lp–HipS_Lp complex, a ribosomal-binding site (RBS, AGGAGA)³⁷ was introduced between the stop codon of HipS_Lp and the start codon of HipT_Lp. The resultant HipS_Lp–RBS-HipT_Lp was cloned into pET21a (+) vector. The crystal structure of the SeMet-labeled HipT_Lp–HipS_Lp complex was determined and refined at 1.89 Å resolution (Table 1).

In the structure of HipT_Lp–HipS_Lp complex, a copy of HipT_Lp and HipS_Lp each were observed per crystal asymmetric unit. Residues belonging to helices α1 and α2 of HipT_Lp were not visible in the electron density map, whereas the density of the remaining residues was unambiguous (Fig. 5a). All 102 residues of HipS_Lp were successfully built into the model, showing that HipS_Lp is a small single-domain protein composed of five β-strands and three α-helices. The overall structure of the HipT_Lp-HipS_Lp complex is highly similar to E. coli HipA, with HipS_Lp and HipT_Lp aligning with the N- and C-terminal portions of E. coli HipA, respectively (Supplementary Fig. 8a). HipS_Lp superimposed with the N-terminus of E. coli HipA with an overall RMSD of 0.932 Å across 64 Cα. However, a notable difference can be observed on the β4–α2 loop of HipS_Lp, which is twisted and rotated by ~45° with respect to its counterpart in the N-terminus of E. coli HipA (Supplementary Fig. 8b).

**Fig. 5: Structural basis for HipT_Lp recognition by HipS_Lp.**

In the structure of HipT_Lp–HipS_Lp complex, three α-helices of HipS_Lp form a helix bundle that sits above the cleft formed by the β-sheet in the N-terminal lobe of HipT_Lp, whereas the β-strands form a flank region in HipS_Lp (Fig. 5b). HipS_Lp binds the toxin HipT_Lp via hydrogen bonding in three main interacting regions, which constitute more than 1100 Å² of total buried surface area (Fig. 5c–e). The intermolecular interactions are mainly formed between helices α1, α2 and α1–α2 loop of HipS_Lp and helix α3 and strand β5 of HipT_Lp. In the first interacting region, side chains of HipS_Lp E63 and HipT_Lp K157 form a salt bridge, side chain of HipS_Lp E58 engages in polar interactions with the main chain amide and side chain of HipT_Lp R154, and hydrogen bonds are additionally formed between main chain of HipS_Lp G59 and side chain of HipT_Lp K201 and main chains of HipS_Lp I65 and HipT_Lp G57 (Fig. 5c). The second interacting region includes a salt bridge between HipT_Lp D77 and HipS_Lp K73 and hydrogen bonds between (i) side chain of HipT_Lp D133 and HipT_Lp G94, (ii) side chain of HipT_Lp R154 and side chain of HipS_Lp N91 as well as main chain of HipS_Lp V92, (iii) side chain of HipS_Lp N91 and main chain of HipT_Lp Y79, and (iv) side chains of HipT_Lp Q78 and HipS_Lp Q90 (Fig. 5d). In the third interacting region, the side chain of HipT_Lp Q148 hydrogen bonds with the main chain of HipS_Lp F56, whereas the side chain of HipT_Lp E144 forms hydrogen bonds with the side chain of HipS_Lp S38 and the main chain of HipS_Lp L39 (Fig. 5e).

To verify the importance of these interactions for stable binding of HipS_Lp to HipT_Lp, we performed pull-down assays with untagged wild-type HipS_Lp and wild-type or mutant HipT_Lp carrying a N-terminal 6×His-tag. The HipT_Lp mutants D133A, R134A, and E144A completely lost their ability to bind HipS_Lp and the mutants R154A, K157A, K201A exhibited severely reduced HipS_Lp binding, suggesting that these residues form key interactions with HipS_Lp (Fig. 5f).

Molecular mechanism for toxin neutralization in the HipBST TA systems

One of the most striking features of the HipBST TA systems is that the role of antitoxin is taken by HipS which corresponds to the N-terminal portion of HipA toxin from the E. coli HipBA system. To better understand how the toxic activity of HipT is neutralized by HipS, we reinspected and compared the structures of apo pHipT_Lp, pHipT_Lp–AMP–PNP complex, and HipT_Lp–HipS_Lp complex. Apo pHipT_Lp and HipT_Lp from the HipT_Lp–HipS_Lp complex superimpose with RMSD of 0.464 Å over 215 Cα atoms. Notably, Ser54 of HipT_Lp is phosphorylated in the structure of apo pHipT_Lp but not in the HipT_Lp–HipS_Lp complex (please note that HipT_Lp–HipS_Lp was co-expressed in E. coli BL21) (Fig. 2a, b and Supplementary Fig. 8c). Since the residue S54 is phosphorylated when HipT_Lp is expressed alone, we wondered whether the phosphorylation on S54 influences the interaction between HipT_Lp-HipS_Lp and HipB_Lp. Size-exclusion chromatography revealed that the phosphorylation state of S54 does not appear to have a noticeable effect on interactions between HipT_Lp-HipS_Lp and HipB_Lp (Supplementary Fig. 9). Moreover, structural comparison suggests that the P-loop of HipT_Lp, which encircles ATP and is critical for catalytic activities in typical Ser/Thr kinases, underwent a conformational change from loop to helix upon HipS_Lp binding (Fig. 6a, b). Such allosteric regulation induced by the antitoxin binding has not been observed in E. coli HipBA TA system²⁹. A conformational change similar to the loop-to-helix change of the HipT_Lp P-loop in HipT_Lp-HipS_Lp can also be observed in the recently released structure of HipBST_O127 trimer¹⁹ (Supplementary Fig. 10), suggesting a common mechanism of toxin neutralization.

**Fig. 6: Toxin-neutralization mechanism in the HipBST TA systems.**

These observations led us to hypothesize that the loop-to-helix conformational transition induced upon HipS_Lp binding may obstruct the access of ATP to the kinase active site, resulting in inhibition of the HipT_Lp kinase activity. Superimposition of the structures of pHipT_Lp, pHipT_Lp–AMP–PNP, and the HipT_Lp–HipS_Lp further revealed that P-loop in the HipT_Lp–HipS_Lp complex overlaps with AMP–PNP in the pHipT_Lp–AMP–PNP complex (Fig. 6c, d and Supplementary Fig. 11). More specifically, the γ-phosphate and β-phosphate groups of AMP–PNP would clash with the side chains of Q59 and D219, respectively, whereas the α-phosphate would clash with the side chains of K61 and K85 (Fig. 6d). This may account for the unphosphorylated state of the P-loop in the HipT_Lp–HipS_Lp complex when they were co-expression (Supplementary Fig. 8c). Such conformational change also occurs in the P-loop of HipBST_O127, suggesting that a similar mechanism is utilized by HipBST_O127¹⁹ (Fig. 6e, f). To further verify whether ATP binding is abolished, we measured the binding affinity between the HipT_Lp–HipS_Lp complex and AMP–PNP with ITC and found that HipT_Lp completely lost the AMP–PNP binding affinity when binding with HipS_Lp (Fig. 6g). Consistent with these results, the thermal stability of the HipT_Lp-HipS_Lp complex did not change upon the addition of 4 mM AMP–PNP (Supplementary Fig. 11a). Considering that HipA_Ec in autophosphorylated form can bind ADP and AMP but not ATP³⁰, we also investigated whether the HipT_Lp–HipS_Lp complex binds ADP and AMP. Again, the results of ITC experiments suggested that the HipT_Lp–HipS_Lp complex has no detectable affinity for ADP or AMP, even at concentrations of 1 mM (Supplementary Fig. 12). The allosteric regulation of the P-loop induced by the antitoxin binding was also observed in the HipBST_O127¹⁹. Together, these findings suggest that HipS_Lp binding induces conformational changes in the P-loop of HipT_Lp, which blocks ATP binding and consequently inhibits the HipT_Lp kinase activity (Fig. 6h).

Discussion

Although the biological functions of TA systems are often ambiguous and debatable, recent advances in the field and the discovery of numerous novel TA modules increasingly support their roles in viral defense or plasmid stability^4,7 and interactions between hosts and their mobile genetic elements. Among known TA modules, the type II TA systems are most well understood. Type II TA systems typically consist of two components, though several tripartite type II TA modules have been identified, such as the Rv1955-Rv1956-Rv1957 TA-chaperone (TAC) system of Mycobacterium tuberculosis^38,39, and the recently discovered E. coli HipA-like TA system HipBST of E. coli O127: H6¹⁸.

Most type II antitoxins are composed of an N-terminal DNA-binding domain that regulates transcription from the TA locus through direct interaction with the promoter, and a C-terminal region responsible for the toxin binding and inhibition⁴⁰. HipBA modules are representative type II TA systems and are ubiquitous in bacteria²⁸. Based on the similarity searching using E. coli HipA against the sequenced microbial genomes, a three-component widespread HipBST TA was found and experimentally verified¹⁸. Later bioinformatic analysis has suggested the presence of several other HipBA-like TA in numerous bacterial species¹⁷. While the toxin HipT_O127 of HipBST_O127 was found to exert its toxic function by phosphorylating TrpS, and in spite of the similarity between HipB_O127 and E. coli HipB, HipB_O127 however cannot neutralize the HipT_O127 kinase. This task was taken over by HipS, a small protein with homology to the N-terminal part of E. coli HipA^18,19. Our current study also experimentally identifies a tripartite HipBST_Lp TA in which the antitoxin HipS_Lp instead of HipB_Lp restores the growth inhibition induced by HipTLp in human pathogen L. pneumophila, which is in agreement with the HipBST_O127 TA¹⁸.

Together with the preprint study in ref. 19, our study identifies and elucidates the toxin-neutralization mechanism in the tripartite HipBST TA systems of L. pneumophila. It is clear that the toxin neutralization mechanism in HipBST systems is notably different from the corresponding mechanism in HipBA TA system of E. coli¹⁸. The structural study by Bærentsen et al. on the E. coli O127:H6 HipBST found that HipT adopts an inactive conformation in the HipBST complex that prevents ATP binding¹⁹. However, it was unclear whether the blockage of ATP binding arises from the binding of the antitoxin HipS or autophosphorylation of the conserved P-loop serine¹⁹. Our structure of the HipT_Lp–HipS_Lp complex clearly shows the toxicity of HipT_Lp is neutralized directly upon HipS_Lp binding, leading to blocking of the ATP-binding site through steric hindrance. Although this study demonstrates that HipB_Lp forms a heterotrimer with the HipT_Lp–HipS_Lp complex, we failed to obtain crystals of the heterotrimer structure after extensive crystal screening. However, the available structure of E. coli HipBST shows that HipB binds to HipT but does not interact with the kinase active site¹⁹. In addition, overall architecture of HipBST_O127 system is reminiscent of the structure of HipBA_So system in the HipBA_So-DNA complex, implying that HipBST can also bind DNA¹⁹. Induction of HipB_O127 was found to be required and sufficient for the transcriptional repression of the HipBST TA¹⁹. Thus, it seems that the toxin neutralization and the autoregulation of the HipBST TA are carried out by the antitoxin HipS and HipB, respectively¹⁹. Nevertheless, the exact role of HipB in the HipBST systems remains to be determined.

To the best of our knowledge, HipT_Lp is the only identified toxin of TA systems secreted into host cells via the type IV secretion system Dot/Icm. Nevertheless, the employment of TA toxins by the pathogenic bacteria during host cell infection is not unprecedented. For instance, the type III secretion system effector AvrRxo1-ORF1 from Xanthomonas oryzae pv. oryzicola constitutes a type II TA system with adjacent AvrRxo1-ORF2⁴¹. AvrRxo1 can phosphorylate NAD in planta, leading to the suppression of the flg22-triggered ROS burst⁴². Moreover, previous investigation of fourteen effectors from Burkholderia gladioli revealed that the restriction endonuclease Tox-Rease-5 domain-containing effector TaseT_Bg can be employed to inhibit the growth of co-habiting bacterial species, and its activity is counteracted by the associated immunity protein TsiTBg⁴³. Apart from the effectors LegK1–K5 and LegK7, HipT_Lp is the sixth effector known to function as a Ser/Thr kinase in L. pneumophila. LegK1 phosphorylates NF-κB to activate the genes expression involved in inflammation during infection⁴⁴. Phosphorylation of Hsp70 at T495 by LegK4⁴⁵, leading to the inhibition of protein synthesis. LegK7 phosphorylates a conserved scaffold protein MoB1, hijacking the hippo pathway to promote its survival^33,46. The exact substrate of HipT_Lp in its host remains to be identified in future studies. In summary, these results suggest that TA systems could serve as a reservoir for additional secreted effectors, which sheds light on the evolutionary links between the TA system and the effectors secreted by the pathogenic microorganisms.

Methods

Bacterial strains and growth conditions

The L. pneumophila strain Philadelphia-1 derivative Lp02⁴⁷ was used as the progenitor of all derivative strains used in this study. To construct ∆lpg2368-∆lpg2369-∆lpg2370 triple-deletion mutant strain of L. pneumophila (termed ∆3), we first constructed deletion plasmid by cloning the upstream and downstream flanking regions into pSR47S. Briefly, the 1.2-kb fragment located upstream of lpg2368 and the 1.2-kb fragment located downstream of lpg2370 were obtained by PCR using ∆3 A1/∆3 A2 and ∆3 B1/∆3 B2 primer pairs and high-fidelity FastPfu DNA polymerase (TransGen) (Supplementary Table 2), respectively. The amplified PCR products were used as templates to produce a DNA fragment containing the flanking regions by fusion PCR with primers ∆3 A1/∆3 B2 and high-fidelity FastPfu DNA polymerase (TransGen). After digestion with BamHI and SalI, the DNA fragment was inserted into BamHI/SalI-digested pSR47S⁴⁸. The deletion plasmid was then introduced into Lp02 by triparental mating, and conjugants were selected on CYET plates containing kanamycin (20 μg/mL) and streptomycin (50 μg/mL). Deletion mutants were verified by standard colony PCR techniques using primers ∆3 A1/∆3 B2 and 2×Taq master mix (Novoprotein) from colonies grown on CYET plates containing 5% sucrose⁴⁹. Moreover, the lpg2370, lpg2368, lpg2369, and lpg2368–lpg2369 genes were inserted into pZL507 or pJL03 as BamHI/SalI fragments to construct pZL507-Lpg2370, pJL03-Lpg2368, pJL03-Lpg2369, and pJL03-Lpg2368-Lpg2369 plasmids, which were then introduced into the ∆lpg2368∆lpg2369∆lpg2370 strain. For effector translocation, fresh single colonies of Lp02 or Lp03 harboring expression plasmid for TEM-1-Lpg2370, TEM-1-RalF, or TEM-1-FabI fusion proteins were streaked onto BCYE plates 2 days before infection.

TEM-1 β-lactamase translocation assays

To test Dot/Icm-dependent transfer of the fusion proteins into host cells, L. pneumophila cells expressing the fusion proteins were grown in the presence of 0.5 mM IPTG to post-exponential phase and used to infect monolayers of RAW264.7 cells that were seeded in 96-well plates at an MOI of 20. The CCF4-AM substrates (Invitrogen, Carlsbad, CA) were mixed with medium in the wells two hours after infection. After further incubation for 1 hour at room temperature, infected cells were inspected under a Nikon IX-80 fluorescence microscope equipped with a β-lactamase FL-Cube (U-N41031, Chroma Technology Corp, Bellows Falls, VT). Images of infected cells were obtained using a DP-72 color fluorescence camera (Olympus). Translocation of the β-lactamase chimeras was assessed by the presence of cells emitting blue fluorescence signals. The percentage of infected cells was determined by counting the number of cells emitting blue fluorescence in specified areas of the wells. TEM-1-RalF and TEM-1-FabI fusion proteins were used as positive and negative controls, respectively. Experiments were performed in triplicate, and at least 300 cells were counted in each sample.

Protein expression

DNA fragments encoding full-length HipT_Lp, HipS_Lp, and HipB_Lp or their variants were inserted into pET21a (+) containing a N-terminal 6×His-tag, respectively. To obtain HipT_Lp–HipS_Lp complex, HipS_Lp–HipT_Lp was cloned in the pET21a (+) following previously described methods^37,50 to express the 6×His HipT_Lp–HipS_Lp. The recombinant plasmids were transformed into E. coli BL21 (DE3) cells for protein expression. Overexpression was induced in log phase cultures (OD₆₀₀ ≈ 0.6) by cooling cultures on ice for 20 min and adding IPTG to a final concentration of 0.4 mM, followed by overnight incubation at 16 °C and 220 rpm. The overexpression of the wild-type HipT_Lp did not lead to the growth arrest in E. coli. After target protein expression, cells were pelleted by centrifugation and resuspended in buffer A (50 mM Tris-HCl, pH 8.0, 100 mM NaCl). The cells were then lysed by ultrasonication and the lysate was centrifugated at 17,000×g and 4 °C for 30 min. The supernatant was loaded onto Ni²⁺-NTA column (Qiagen) for purification of target recombinant proteins. After washing with 100 mL of buffer A supplemented with 50 mM imidazole, the target proteins were eluted with buffer A supplemented with 250 mM imidazole. Fractions containing the target protein were pooled, concentrated to 0.5 mL and then purified with Superdex 75 increase column (GE Healthcare) equilibrated with buffer B (20 mM Tris-HCl, pH 8.0, 150 mM NaCl).

Selenomethionine (SeMet)-labeled HipT_Lp and HipT_Lp–HipS_Lp were expressed in M9 medium supplemented with 2 mM MgSO₄, 0.1 mM CaCl₂, 0.5% w/v glucose, 2 mg/L biotin, 2 mg/L thiamine, and 0.03 mg/L FeSO₄. When bacterial cultures reached OD₆₀₀ of 1.0, final concentrations of 100 mg/mL of phenylalanine, lysine, and threonine, and 50 mg/mL of isoleucine, leucine, valine, and of SeMet (Chemie Brunschwig) were added in form of solid powder, after which the cultures were incubated for 30 min. Protein expression was then induced with 0.2 mM IPTG, and the cultures were further incubated on a shaker at 16 °C for 20 h. Cells were collected at 5000×g for 15 min and 4 °C and resuspended in the lysis buffer (50 mM Tris pH 8.0, 100 mM NaCl, 5 mM β-mercaptoethanol). Protein purification was performed as described above.

In vitro kinase assays

Purified Lpg2370 (1 µg) was resolved by SDS-PAGE and transferred to the PVDF membrane using a Bio-Rad wet transfer system. The membrane was blocked with 5% milk for 1 h at room temperature. The membrane was incubated overnight at 4 °C with a primary antibody against thiophosphate ester (rabbit anti-thiophosphate ester (ab92570), Abcam, dilution 1:4000 v/v). The membrane was then washed three times with TBST before being incubated with a secondary antibody (HRP-conjugated AffiniPure goat anti-rabbit (Cat No. SA00001-2), Proteintech, dilution 1:5000 v/v) for 1 h at room temperature. The membrane was washed three times with TBST, and proteins were detected using an ECL detection reagent.

Liquid chromatography-mass spectrometry (LC-MS) analysis

LC-MS was used to analyze autophosphorylation of purified recombinant Lpg2370. After staining of gels with Coomassie blue, excised gel segments were subjected to in-gel trypsin digestion and dried. Electrospray ionization mass spectrometry (ESI-MS) was performed using an integrated HPLC/ESI-MS system (1260 Infinity, Agilent Technologies/amaZon SL, Bruker Corporation) equipped with a Luna 5 µm C18 column (100 Å, 250 × 4.60 mm, 5 µm). Peptides were dissolved in 10 µl 0.1% formic acid and were auto-sampled directly onto a homemade C18 column (35 cm × 75 μm i.d., 1.9 μm 100 Å). Samples were then eluted for 60 mins with linear gradients of 3–35% acetonitrile in 0.1% formic acid at a flow rate of 300 nl/min. The mass spectrometer was equipped with a CaptiveSpray source. Survey scans were recorded over 100–1700 m/z range and the mass spectra data were acquired with a timsTOF Pro mass spectrometer (Brucker) operated in PASEF mode. PASEF setting: 10 MS/MS scans (total cycle time 1.27 s), charge range 0–5, active exclusion for 0.4 min, Scheduling Target intensity 10000, Intensity threshold 2500, CID collision energy 42 eV.

The raw files generated from LC-MS/MS were analyzed by Peaks Studio X software (Bioinformatics Solutions Inc., Waterloo, ON, Canada) against the input of the Lpg2370 amino acid sequence. The following database search criteria were set to: enzyme, trypsin; variable modification, phosphorylation, precursor ion mass tolerance, 10 ppm; MS/MS fragment mass tolerance, 0.02 Da; tryptic enzyme specificity with two missed cleavages allowed. Identifications were filtered according to mass accuracy and 1% false discovery rate.

Protein crystallization and collection of crystallographic data

The purified target protein was concentrated to 0.5 mL and loaded onto a Superdex 75 increase column (GE Healthcare) Fractions containing purified proteins were then concentrated at 4000×g, 4 °C to ~15 mg/mL using an Amicon Ultra 30 K centrifugal filter. To obtain HipT_Lp–AMP–PNP complex, the purified HipT_Lp was incubated with AMP–PNP at a 1:1.2 molar ratio at 4 °C for 30 min and concentrated to about 12 mg/mL.

For crystallization of SeMet-labeled HipT_Lp, HipT_Lp–AMP–PNP complex, and SeMet-labeled HipT_Lp–HipS_Lp complex, the purified and concentrated protein samples were mixed with the reservoir solution at equal volumes and crystallized using the sitting drop vapor diffusion method at 16 °C. Initial crystals of SeMet-labeled HipT_Lp and HipT_Lp–AMP–PNP complex were obtained within three days in condition containing 8% Tacsimate (pH 6.0) and 20% w/v PEG 3350. Initial crystals of the SeMet-labeled HipT_Lp-HipS_Lp complex were obtained in the condition containing 0.1 M sodium acetate (pH 7.0) and 12% v/w PEG 3350. After extensive optimization, diffraction-quality crystals of SeMet-labeled HipT_Lp and the HipT_Lp–AMP–PNP complex were grown in the presence of 10% Tacsimate (pH 6.2) and 20% v/w PEG 3350. Diffraction-quality crystals of the HipT_Lp–HipS_Lp complex were grown in the presence of 0.1 M sodium acetate (pH 7.2) and 15% w/v PEG 3350. Harvested crystals were preserved in the respective reservoir solutions supplemented with cryoprotectant and flash-frozen in liquid nitrogen.

Structure determination and refinement

All X-ray diffraction data were collected at the BL-02U1 station of the Shanghai Synchrotron Radiation Facility (SSRF). Single-wavelength anomalous diffraction (SAD) datasets of SeMet-labeled HipT_Lp and SeMet-labeled HipT_Lp–HipS_Lp complex were obtained at high resolution and the data were processed with the HKL-2000 package⁵¹. Autosol program of PHENIX package was used for SAD phasing and initial model building. Residues 72–312 of HipT_Lp were auto-built, whereas the residues 1–71 residues were built by iterative manual building in Coot⁵². Structure refinement was carried out with PHENIX⁵³. The HipT_Lp–AMP–PNP binary complex was determined using the molecular replacement method with the structure of HipT_Lp as the search model. Structure quality was analyzed during PHENIX refinements and later validated in the PDB validation server. Detailed crystallographic and structure refinement data are listed in Table 1. Structural images were generated using PyMol (Schrödinger, LLC).

Mutagenesis

Base substitutions in this study were introduced using two pairs of complementary primers (sense and antisense strand primers) containing the desired mutation. The primers used in this study were listed in Supplementary Table 2. All constructs were verified by DNA sequencing.

Isothermal titration calorimetry (ITC)

ITC experiments were performed in Nano ITC Low Volume (TA instruments). All samples were prepared in the buffer containing 20 mM HEPES (pH 8.0) and 150 mM NaCl. Typically, the titrant concentration in the syringe was 200–500 μM, and the titrand concentration in the reaction cell was 10–20 μM. Titration was conducted at 25 °C using multiple injection method with 150 s intervals. Obtained data were integrated, corrected, and analyzed using the NanoAnalyze software (TA Instruments) with a single-site binding model.

Pull-down assays

To perform pull-down assays, wild-type or mutant HipT_Lp carrying C-terminal 6×His-tag were incubated with Ni-agarose beads for 30 min and then washed twice with buffer containing 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl. The beads were then incubated with untagged wild-type HipS_Lp (6×His-tag was previously cleaved with TEV protease) for 1 h and then washed twice. The proteins were eluted from beads using a buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 250 mM imidazole. Eluted samples were analyzed using SDS-PAGE analysis.

In vivo toxicity assays

For in vivo toxicity assays of HipT from E. coli serotype O127:H6 (denoted HipT_O127) to E. coli, which was performed by expressing the hipT_O127 gene or its mutant variants in E. coli BL21(DE3). The gene encoding HipT_O127 was synthesized and cloned into pET21a (+) vector. Plasmids encoding HipT_O127 single-point mutants S57A, S57D, K64A, K86A, H212A, N215A, and D233A were prepared using site-specific mutagenesis, and the primers are listed in Supplementary Table 2. Transformed E. coli BL21 (DE3) cells were plated on agar and a single bacterial colony was transferred to 10 mL LB medium for culturing. Expression of wild-type HipT_O127 and its mutant variants was induced with IPTG at 0.2 mM concertation when the bacterial cultures reached OD₆₀₀ = 0.5, after which the bacterial growth curve was measured every 30 min for 6 h.

For the Lpg2370 in vivo toxicity assays in L. pneumophila, Lpg2370, Lpg2370-Lpg2369, Lpg2370-Lpg2368_, Lpg2370-Lpg2369-Lpg2368 were overexpressed in L. pneumophila Lp02 or the ∆lpg2368∆lpg2369∆lpg2370 deletion strain. Overnight cultures of the tested bacterial strains were diluted in fresh AYE broth to OD₆₀₀ = 0.1 and split into 2-mL subcultures into which different concentrations of arabinose or IPTG were added. The subcultures were then grown at 37 °C with constant rotation at 180 rpm. Cell viability was assessed by readout of OD₆₀₀ value every 3 h and plotting the values on a log scale.

Thermal shift assays

Thermal shift assays were performed using 1 mg/mL phosphorylated HipT_Lp or its mutants incubated with ATP/AMP–PNP and varied concentrations of nucleotides (0–4 mM) in 50 mM Tris-HCl (pH 8.0) and 150 mM NaCl. Then the mixture was loaded in 96-well PCR plates, the fluorescence signals were recorded as a function of temperature using Prometheus NT.48 (NanoTemper Technologies) in FRET mode. Fluorescence intensity was measured at Ex/Em of 350/330 nm. The temperature gradient range was set as 20–95 °C with a 0.5 °C ramp over the course of 30 s. Control assays were conducted in the same buffer without ATP/AMP–PNP. The thermal unfolding value (Tm) for pHipT_Lp was calculated using the curve fitting software PR.ThermControl (NanoTemper Technologies).

Phylogenetic analysis

The sequence of Lpg2370 was blasted in the Uniprot (https://www.uniprot.org/) and all the hit sequences were used for sequence alignment in ClustalW, which was used to build the phylogenetic tree using MEGA software⁵⁴. The phylogenetic tree was visualized using iTOL⁵⁵.

Statistics and reproducibility

The TEM-1 β-lactamase translocation assays experiments in Fig. 1a, the western blotting experiment in Fig. 1d, the pull-down assays in Figs. 3c, 5f, Supplementary Fig. 4, and Supplementary Fig. 9 were performed at least triplicate at two independent times.

Data availability

The atomic coordinates and structure factors of the autophosphorylated toxin HipT_Lp, the complex of HipT_Lp with AMP–PNP, and HipT_Lp-HipS_Lp binary complex have been deposited in the Protein Data Bank under the accession codes 7VKC, 7WCF, and 7VKB. Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Key Research and Development Program of China (2021YFC2301403 to S.O), the National Nature Science Foundation of China grants (32170045 to X.K.Z., 82172287 to S.O., 31970134 and 32170182 to J.Z.Q.), the Fujian Provincial Department of Science and Technology (2020Y4007 to S.O.), and the High-level personnel introduction grant of Fujian Normal University (Z0210509 to S.O.). The diffraction data were collected at the beamline BL-02U1 of the Shanghai Synchrotron Radiation Facility (SSRF). We thank the support of the scientific research innovation program “Xiyuanjiang River Scholarship” of the College of Life Sciences, Fujian Normal University (22FSSK003 to X.K.Z.). We also thank our colleague Vanja Perčulija for the scientific and language editing of the manuscript.

Author information

These authors contributed equally: Xiangkai Zhen, Yongyu Wu, Jinli Ge.

Authors and Affiliations

Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation, the Key Laboratory of Innate Immune Biology of Fujian Province, Biomedical Research Center of South China, Key Laboratory of OptoElectronic Science and Technology for Medicine of the Ministry of Education, College of Life Sciences, Fujian Normal University, 350117, Fuzhou, China
Xiangkai Zhen, Yongyu Wu, Le Ye, Niannian Lin, Zhijie Huang, Zihe Liu & Songying Ouyang
State Key Laboratory for Zoonotic Diseases, College of Veterinary Medicine, Jilin University, Changchun, China
Jinli Ge & Jiazhang Qiu
Purdue Institute for Inflammation, Immunology and Infectious Disease and Department of Biological Sciences, Purdue University, West Lafayette, IN, USA
Jiaqi Fu & Zhao-qing Luo

Authors

Xiangkai Zhen
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Yongyu Wu
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Jinli Ge
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Jiaqi Fu
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Le Ye
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Niannian Lin
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Zhijie Huang
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Zihe Liu
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Zhao-qing Luo
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Jiazhang Qiu
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Songying Ouyang
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Contributions

X.K.Z., S.O., and J.Z.Q. conceived the project and designed the experiments, X.K.Z. and Y.Y.W. performed crystallization and resolved the structures, J.L.G. performed the in vivo toxicity assays, J.Q.F., L.Y., N.N.L., Z.J.H., and Z.H.L. contributed to the protein expression and purification. X.K.Z. analyzed the structures, X.K.Z., S.O., Z.Q.L., and J.Z.Q. designed the biochemical experiments and wrote the manuscript.

Corresponding authors

Correspondence to Jiazhang Qiu or Songying Ouyang.

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Zhen, X., Wu, Y., Ge, J. et al. Molecular mechanism of toxin neutralization in the HipBST toxin-antitoxin system of Legionella pneumophila. Nat Commun 13, 4333 (2022). https://doi.org/10.1038/s41467-022-32049-x

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Received: 28 March 2022
Accepted: 11 July 2022
Published: 26 July 2022
DOI: https://doi.org/10.1038/s41467-022-32049-x

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