Crosstalk between the serine/threonine kinase StkP and the response regulator ComE controls the stress response and intracellular survival of Streptococcus pneumoniae

by Germán E. Piñas, Nicolás M. Reinoso-Vizcaino, Nubia Y. Yandar Barahona, Paulo R. Cortes, Rosario Duran, Chandan Badapanda, Ankita Rathore, Dario R. Bichara, Melina B. Cian, Nadia B. Olivero, Daniel R. Perez, José Echenique

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 (ComE^T128A) and phosphomimetic (ComE^T128E) 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 comE^T128A 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 comE^T128A 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 comE^T128A 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 comE^T128A 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.