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

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.

The pneumococcal two-component system VisRH is linked to enhanced intracellular survival ofStreptococcus pneumoniaein influenza-infected pneumocytes

“…Virus replication was allowed to progress for 24 h before infection with the S. pneumoniae R801 strain at a MOI of 30. Flow cytometry using Annexin-V-ACP/PI labeling to test necrosis/apoptosis levels revealed that a MOI of 10 of IAV led to ∼5% increase in the number of necrotic/apoptotic cells compared to non-infected cells (Fig S1A) and ∼15% after bacterial superinfection using a bacterial MOI of 30 (Fig S1B), as described [16]. In further studies, we used IAV at a MOI of 10 and S. pneumoniae at a MOI of 30 in the superinfection model.…”

“…We previously demonstrated that the S. pneumoniae R801 strain can survive inside pneumocytes for several hours [16]. To further define whether a concomitant influenza virus infection would affect S. pneumoniae intracellular survival, we established an in vitro IAV- S. pneumoniae superinfection model in human-derived A549 pneumocyte cells.…”

“…In further studies, we used IAV at a MOI of 10 and S. pneumoniae at a MOI of 30 in the superinfection model. Gentamicin was used to eliminate extracellular bacteria before evaluation of intracellular S. pneumoniae following the classical protection assay [16]. Prior IAV inoculation consistently increased bacterial survival by ∼2 fold in A549 pneumocytes (Fig 1A).…”

“…We hypothesized that pneumococcal two-component systems (TCSs) sense physiological changes induced by IAV-infection of pneumocytes and mediate adaptative responses that lead to increased intracellular bacterial survival. Next, we considered that the intracellular changes induced by IAV-infection generate stress conditions sensed by S. pneumoniae via TCSs other than ComE and CiaR [15, 16]. From a previous systematic screening of insertion-duplication histidine kinase ( hk ) mutants of S. pneumoniae [15] (Table S1), we focused the search on hk mutations that were null for pneumococcal intracellular survival in the absence of IAV infection.…”

“…We previously reported that the two-component systems (TCSs) ComDE and CiaRH are involved in the pneumococcal stress response to acidic conditions and in the intracellular survival of S. pneumoniae in pneumocytes [15]. In addition, we recently reported that the crosstalk signaling between the serine/threonine kinase StkP and ComE controls H 2 O 2 production in S. pneumoniae modulating its intracellular survival in pneumocytes [16].…”