Abstract
Streptococcus pneumoniae is an opportunistic human bacterial pathogen that usually colonizes the upper respiratory tract, but the invasion and survival mechanism in respiratory epithelial cells remains elusive. Previously, we described that acidic stress-induced lysis (ASIL) and intracellular survival are controlled by ComE through a yet unknown activation mechanism under acidic conditions, which is independent of the ComD histidine kinase that activates this response regulator for competence development at pH 7.8. Here, we demonstrate that the serine/threonine kinase StkP is essential for ASIL, and show that StkP phosphorylates ComE at Thr128. Molecular dynamic simulations predicted that Thr128-phosphorylation induces conformational changes on ComE’s DNA-binding domain. Using nonphosphorylatable (ComET128A) and phosphomimetic (ComET128E) proteins, we confirmed that Thr128-phosphorylation increased the DNA-binding affinity of ComE. The non-phosphorylated form of ComE interacted more strongly with StkP than the phosphomimetic form at acidic pH, suggesting that pH facilitated crosstalk. To identify the ComE-regulated genes under acidic conditions, a comparative transcriptomic analysis was performed between the comET128A and wt strains, and differential expression of 104 genes involved in different cellular processes was detected, suggesting that the StkP/ComE pathway induced global changes in response to acidic stress. In the comET128A mutant, the repression of spxB and sodA correlated with decreased H2O2 production, whereas the reduced expression of murN correlated with an increased resistance to cell wall antibiotic-induced lysis, compatible with cell wall alterations. In the comET128A mutant, ASIL was blocked and acid tolerance response was higher compared to the wt strain. These phenotypes, accompanied with low H2O2 production, are likely responsible for the increased survival in pneumocytes of the comET128A mutant. We propose that the StkP/ComE pathway controls the stress response, thus affecting the intracellular survival of S. pneumoniae in pneumocytes, one of the first barriers that this pathogen must cross to establish an infection.
Highlights
Sensing and transducing external signals into an appropriate physiological response is part of a microorganism strategy to survive in a constantly changing environment
We report that the serine-threonine kinase StkP was able to phosphorylate the response regulator ComE to control different cellular processes in response to environmental stress
We provide evidence for the regulatory control exerted by the StkP/ComE pathway on acidinduced autolysis, the acid tolerance response, and H2O2 production to modulate tissue damage and intracellular survival, which are linked to pneumococcal pathogenesis
Summary
Sensing and transducing external (or internal) signals into an appropriate physiological response is part of a microorganism strategy to survive in a constantly changing environment. In prokaryotes, signaling pathways are mainly mediated by twocomponent systems (TCS) consisting of sensor histidine kinases (HK) that phosphorylate response regulators (RR) on a receiver domain, thereby activating the effector domains of these regulators to induce a physiological event in bacterial cells. The RR effector domains bind regions of DNA that control gene expression in response to environmental changes [1,2]. Each particular HK presents a remarkable specificity for its cognate RR and is capable of identifying particular RRs. Eukaryotic-like Ser/Thr protein kinases (STKs) are present in prokaryotes, where they play key roles in several cellular processes, including the central or secondary metabolism, developmental processes, cell division and virulence [3]. Comparison of the global expression profile of the wild-type and ΔstkP strains has revealed that the transcription of genes involved in the cell wall metabolism, pyrimidine biosynthesis, DNA repair, iron uptake, and oxidative stress response are controlled by StkP, which explain why stkP mutations have
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