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.

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The prevalence of antibiotic resistance genes in pathogenic bacteria is a major challenge to treating many infectious diseases. The spread of these genes is driven by the strong selection imposed by the use of antibacterial drugs. However, in the absence of drug selection, antibiotic resistance genes impose a fitness cost, which can be ameliorated by compensatory mutations. In Streptococcus pneumoniae, β-lactam resistance is caused by mutations in three penicillin-binding proteins, PBP1a, PBP2x, and PBP2b, all of which are implicated in cell wall synthesis and the cell division cycle. We found that the fitness cost and cell division defects conferred by pbp2b mutations (as determined by fitness competitive assays in vitro and in vivo and fluorescence microscopy) were fully compensated by the acquisition of pbp2x and pbp1a mutations, apparently by means of an increased stability and a consequent mislocalization of these protein mutants. Thus, these compensatory combinations of pbp mutant alleles resulted in an increase in the level and spectrum of β-lactam resistance. This report describes a direct correlation between antibiotic resistance increase and fitness cost compensation, both caused by the same gene mutations acquired by horizontal transfer. The clinical origin of the pbp mutations suggests that this intergenic compensatory process is involved in the persistence of β-lactam resistance among circulating strains. We propose that this compensatory mechanism is relevant for β-lactam resistance evolution in Streptococcus pneumoniae.

In Streptococcus pneumoniae, autolysis is considered a programmed cell-death process executed principally by the major autolysin (LytA), and the underlying mechanism causing its activation is not completely understood. It is known that autolysis is triggered by competence development at alkaline pH and regulated by a two-component system, ComDE, which senses a competence-stimulating peptide (CSP) and behaves as a quorum-sensing mechanism. In this work, we found that acidic stress triggered a LytA-mediated autolysis and, curiously, this phenomenon was regulated by a CSP-independent ComE pathway. A further analysis of a hyperactive ComD mutant revealed that ComE needs to be phosphorylated to activate acidic stress-induced lysis (ASIL). The comE transcripts were induced by acidic culture conditions, suggesting that ComE could be sensing acidic stress. We also investigated CiaRH, a twocomponent system whose null mutants show a comE derepression and a CSP-dependent autolysis induction at alkaline pH. By analysis of cia comE double mutants, we demonstrated that CiaRH protected cells from ASIL by a ComE-independent pathway. Here, we propose that ComE is the principal route of the signalling pathway that determines a global stress response, and clearly regulates the induction of the LytA-mediated programmed cell death in S. pneumoniae. Acidic stress may represent for S. pneumoniae an alternative condition, in addition to competence and antibiotics, to assure the release of virulence factors, DNA and cell-wall compounds by autolysis, favouring genetic exchange and contributing to its pathogenesis.

Optochin susceptibility is a key test used for pneumococcal diagnosis, but optochin-resistant (Opt r ) pneumococci have been reported in the last 2 decades. In this work, we characterized eight Opt r clinical strains which presented a new mutation, G47V, a predominant A49S mutation (recently reported in Brazil) and A49T. These mutations were found in the c subunit of the F 0 F 1 ATPase encoded by the atpC gene, and W206C was found in the a subunit encoded by the atpA gene. The Opt r clinical isolates were analyzed by BOX PCR, multilocus sequence typing, and serotype and antimicrobial resistance profiles, and they showed no epidemiological relationship. To characterize the Opt r mutations that could emerge among clinical strains, we studied a pool of spontaneous Opt r colonies obtained in vitro from the virulent D39 strain. We compared the atpAC mutations of these Opt r pneumococci (with or without passage through C57BL/6 mice) with those described in the clinical isolates. This analysis revealed three new mutations, G47V and L26M in the c subunit and L184S in the a subunit. Most of the mutations identified in the laboratory-generated Opt r strains were also found in clinical strains, with the exception of the L26M and L184S mutations, and we suppose that both mutations could emerge among invasive strains in the future. Considering that atpAC are essential genes, we propose that all spontaneous mutations that confer in vitro optochin resistance would not present severe physiological alterations in S. pneumoniae and may be carried by circulating pneumococcal strains.Streptococcus pneumoniae is one of the most important pathogens in children and in elderly populations, being the most common cause of invasive bacterial infections such as pneumonia, bacteremia, and meningitis. The laboratory characterization of S. pneumoniae is based on phenotypic tests such as optochin susceptibility, bile solubility, the Quellung reaction, and genotypic tests performed in specialized centers. The optochin susceptibility test is critical in the identification of S. pneumoniae, and it has been used for decades in bacteriological laboratories. However, optochin-resistant (Opt r ) strains have been reported since 1987 in the United States, Spain, and Israel and more recently in Portugal and Brazil (1,2,7,9,16,17,[23][24][25]28), thus complicating the pneumococcal diagnosis. When additional tests are not applied, these Opt r strains are probably misidentified and overlooked, resulting in inappropriate antimicrobial therapies for patients. Despite several clinical reports describing Opt r isolates, there are only a few mutants characterized at the molecular level. It was reported that point mutations in the atpAC genes, which encode subunits of F 0 F 1 ATPase, conferred optochin resistance on S. pneumoniae (5,10,29).In this work, our objectives were to characterize the Opt r strains isolated in our country, to compare the atpAC mutations with those identified in Opt r strains isolated in other geographical regions, and to investigate a p...

The virus-bacterial synergism implicated in secondary bacterial infections caused by Streptococcus pneumoniae following infection with epidemic or pandemic influenza A virus (IAV) is well documented. However, the molecular mechanisms behind such synergism remain largely ill-defined. In pneumocytes infected with influenza A virus, subsequent infection with S. pneumoniae leads to enhanced pneumococcal intracellular survival. The pneumococcal two-component system SirRH appears essential for such enhanced survival. Through comparative transcriptomic analysis between the ΔsirR and wt strains, a list of 179 differentially expressed genes was defined. Among those, the clpL protein chaperone gene and the psaB Mn +2 transporter gene, which are involved in the stress response, are important in enhancing S. pneumoniae survival in influenza-infected cells. The ΔsirR, ΔclpL and ΔpsaB deletion mutants display increased susceptibility to acidic and oxidative stress and no enhancement of intracellular survival in IAV-infected pneumocyte cells. These results suggest that the SirRH two-component system senses IAV-induced stress conditions and controls adaptive responses that allow survival of S. pneumoniae in IAV-infected pneumocytes.

The penicillin-resistant Spain 9V -3 clone of Streptococcus pneumoniae is widespread and presents different serotype variants originating from recombination of the capsular genes. In this work, the genetic relatedness of 29 invasive pneumococci isolated from the central region of Argentina (Cordoba, Buenos Aires, Santa Fe and La Pampa provinces) was assessed by multilocus sequence typing (MLST). All of the penicillin-non-susceptible isolates studied (21/29) belonged to a serotype 14 variant of the Spain 9V -3 clone. This clone was predominant, suggesting that it was responsible for the penicillin resistance spread in this region. Interestingly, this serotype 14 variant (named Cordoba S14V) could be differentiated from the European one by its pbp1a gene, suggesting a different recombinational replacement of the capsular genes. The putative recombination sites were analysed, resulting in the proximal crossover point being clearly localized in the spr0309 gene, with the distal site restricted to the recU gene, confirming a different recombination event. Analysis of the dexB, cpsB, aliA and pbp1a genes from these strains showed a high similarity with the corresponding genes of the Spain 14 -5 clone, suggesting that the capsular genes were provided by this international clone. Analysis of the genetic polymorphisms of the pbp1a (nt 1473-1922) and spr0309 (nt 1-790) genes is proposed as an epidemiological tool to help recognize the Cordoba S14V of the Spain 9V -3 clone. On the other hand, BOX-repeat-based PCR and MLST analyses of serotype 14 strains revealed a divergent epidemiology of the Cordoba S14V, suggesting a non-recent dissemination in the paediatric population. It is suggested that this molecular epidemiology work will be a reference for monitoring the evolution of S14Vs of Spain 9V -3, the emergence of new clones and the impact of pneumococcal vaccination programmes in Argentina.