Date Published: June 8, 2018
Publisher: Public Library of Science
Author(s): 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, Timothy J. Mitchell.
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.
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. Signal transduction is mainly carried out by protein kinases, which autophosphorylate upon sensing stimuli and then catalyze the phosphorylation of a specific substrate that initiates an adaptive cellular response. In prokaryotes, signaling pathways are mainly mediated by two-component 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. Generally, 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.
Two-component systems (TCSs) represent one of the most important mechanisms of gene regulation in bacteria. Alternatively, eukaryotic-like serine-threonine kinases (STKs) constitute another signaling mechanism that bacteria utilize to regulate different cellular functions, such as stress response and pathogenesis. STKs are more promiscuous than the TCS-associated kinases and can phosphorylate different protein substrates producing pleiotropic effects [16,51,52]. STKs are also able to interact with TCSs by direct phosphorylation of RRs, as reviewed in [3,53]. STK-mediated RR activation takes place on either serine or threonine residues, instead of aspartate, which is the typical residue target for HK phosphorylation. STK-mediated phosphorylation on DNA-binding domains of RR have been reported, as described for GraR in S. aureus , YvcK in Listeria monocytogenes  and RitR in S. pneumoniae . STKs may also phosphorylate on receiver domains, as observed for CovR in S. pyogenes , WalR in B. subtillis , DosR in M. tuberculosis , or in both domains, as demonstrated for VraR in S. aureus .