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

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 (HipTLp) and the antitoxin Lpg2369 (HipSLp) correspond to the C-terminus and N-terminus of HipA from HipBA TA, respectively. By determining crystal structures of autophosphorylated HipTLp, its complex with AMP-PNP, and the structure of HipTLp-HipSLp complex, we identify residues in HipTLp critical for ATP binding and those contributing to its interactions with HipSLp. Structural analysis reveals that HipSLp binding induces a loop-to-helix shift in the P-loop of HipTLp, 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.

Functional diversification despite structural congruence in the HipBST toxin-antitoxin system ofLegionella pneumophila

“…However, no translocation signal above control levels was detected for these mutants (Figure 2A) despite their increased expression levels (Figure S2). These findings, along with the low level of translocation reported previously (21), do not support a role for HipT Lp within the eukaryotic host. Further arguing against its role as an effector, we observe that overexpression of HipT Lp does not display any growth inhibitory effects in yeast (Figure 2B), which is a common phenotype of effector expression (56).…”

“…This was unexpected, given that both pHipA Ec and HipA Ec (S150A) cannot bind ATP and are not toxic (59). In support of this unusual behaviour, a structure of pHipT Lp bound to an ATP analogue was recently reported (21), further demonstrating the retention of HipT Lp activity despite its phosphorylation state. Why HipT Lp autoregulation differs in such a critical aspect from HipA Ec is an outstanding question, as this would seemingly prohibit cell detoxification via trans autophosphorylation, leading to a kinase with less restricted activity in the cell.…”

“…Translocation is determined by monitoring emission from a fluorescent substrate within the host cells, which shifts from emitting green fluorescence to blue fluorescence upon cleavage of an internal beta-lactam ring by the TEM-1 fusion. Given the low level of translocation previously reported for HipT Lp (21), we sought to confirm this observation. Using identical methodology, we observed that the translocation signal of HipT Lp is equivalent to that of the negative control FabI-a cytosolic protein with no function in the host (Figure 2A).…”

“…In this work, we demonstrate that HipBST Lp is a TA system-the core functionality of which is conserved with HipBST Ec -and provide a detailed comparison of the same TA family across different bacterial species. A recent report on HipBST Lp (21) included the puzzling claim that HipT Lp does not inhibit growth in E. coli. We routinely observed that overexpression of HipT Lp consistently inhibits E. coli growth, using multiple strains, vectors, and induction methods (Figure 3A, S3F-G, S6B).…”

“…Notably, the toxin HipT Lp was previously reported to be a bacterial effector that is translocated into the eukaryotic host cell (20). Recently, a bi-functional role for HipT Lp was also proposed, with activity in both the host and the bacterial cell (21). Contrary to this report, we demonstrate that HipT Lp is not translocated at a level greater than what is observed for a negative (non-effector) control and has no obvious role within the eukaryotic cell.…”