The oxidative stress response of Streptococcus pneumoniae: its contribution to both extracellular and intracellular survival.

Streptococcus pneumoniae is a gram-positive, aerotolerant bacterium that naturally colonizes the human nasopharynx, but also causes invasive infections and is a major cause of morbidity and mortality worldwide. This pathogen produces high levels of H2O2 to eliminate other microorganisms that belong to the microbiota of the respiratory tract. However, it also induces an oxidative stress response to survive under this stressful condition. Furthermore, this self-defense mechanism is advantageous in tolerating oxidative stress imposed by the host's immune response. This review provides a comprehensive overview of the strategies employed by the pneumococcus to survive oxidative stress. These strategies encompass the utilization of H2O2 scavengers and thioredoxins, the adaptive response to antimicrobial host oxidants, the regulation of manganese and iron homeostasis, and the intricate regulatory networks that control the stress response. Here, we have also summarized less explored aspects such as the involvement of reparation systems and polyamine metabolism. A particular emphasis is put on the role of the oxidative stress response during the transient intracellular life of Streptococcus pneumoniae, including coinfection with influenza A and the induction of antibiotic persistence in host cells.

Hexameric rings of the scaffolding protein CheW enhance response sensitivity and cooperativity in Escherichia coli chemoreceptor arrays.

The Escherichia coli chemoreceptor array is a supramolecular assembly that enables cells to respond to extracellular cues dynamically and with great precision and sensitivity. In the array, transmembrane receptors organized as trimers of dimers are connected at their cytoplasmic tips by hexameric rings of alternating subunits of the kinase CheA and the scaffolding protein CheW (CheA-CheW rings). Interactions of CheW molecules with the members of receptor trimers not directly bound to CheA-CheW rings may lead to the formation of hexameric CheW rings in the chemoreceptor array. Here, we detected such CheW rings with a cellular cysteine-directed cross-linking assay and explored the requirements for their formation and their participation in array assembly. We found that CheW ring formation varied with cellular CheW abundance, depended on the presence of receptors capable of a trimer-of-dimers arrangement, and did not require CheA. Cross-linking studies of a CheA~CheW fusion protein incapable of forming homomeric CheW oligomers demonstrated that CheW rings were not essential for the assembly of CheA-containing arrays. Förster resonance energy transfer (FRET)-based kinase assays of arrays containing variable amounts of CheW rings revealed that CheW rings enhanced the cooperativity and the sensitivity of the responses to attractants. We propose that six-membered CheW rings provide the additional interconnectivity required for optimal signaling and gradient tracking performance by chemosensory arrays.

The pneumococcal two-component system SirRH is linked to enhanced intracellular survival of Streptococcus pneumoniae in influenza-infected pulmonary cells.

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.

Identification of a Kinase-Active CheA Conformation in Escherichia coli Chemoreceptor Signaling Complexes.

Escherichia coli chemotaxis relies on control of the autophosphorylation activity of the histidine kinase CheA by transmembrane chemoreceptors. Core signaling units contain two receptor trimers of dimers, one CheA homodimer, and two monomeric CheW proteins that couple CheA activity to receptor control. Core signaling units appear to operate as two-state devices, with distinct kinase-on and kinase-off CheA output states whose structural nature is poorly understood. A recent all-atom molecular dynamic simulation of a receptor core unit revealed two alternative conformations, "dipped" and "undipped," for the ATP-binding CheA.P4 domain that could be related to kinase activity states. To explore possible signaling roles for the dipped CheA.P4 conformation, we created CheA mutants with amino acid replacements at residues (R265, E368, and D372) implicated in promoting the dipped conformation and examined their signaling consequences with in vivo Förster resonance energy transfer (FRET)-based kinase assays. We used cysteine-directed in vivo cross-linking reporters for the dipped and undipped conformations to assess mutant proteins for these distinct CheA.P4 domain configurations. Phenotypic suppression analyses revealed functional interactions among the conformation-controlling residues. We found that structural interactions between R265, located at the N terminus of the CheA.P3 dimerization domain, and E368/D372 in the CheA.P4 domain played a critical role in stabilizing the dipped conformation and in producing kinase-on output. Charge reversal replacements at any of these residues abrogated the dipped cross-linking signal, CheA kinase activity, and chemotactic ability. We conclude that the dipped conformation of the CheA.P4 domain is critical to the kinase-active state in core signaling units.IMPORTANCE Regulation of CheA kinase in chemoreceptor arrays is critical for Escherichia coli chemotaxis. However, to date, little is known about the CheA conformations that lead to the kinase-on or kinase-off states. Here, we explore the signaling roles of a distinct conformation of the ATP-binding CheA.P4 domain identified by all-atom molecular dynamics simulation. Amino acid replacements at residues predicted to stabilize the so-called "dipped" CheA.P4 conformation abolished the kinase activity of CheA and its ability to support chemotaxis. Our findings indicate that the dipped conformation of the CheA.P4 domain is critical for reaching the kinase-active state in chemoreceptor signaling arrays.

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

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.

Noncritical Signaling Role of a Kinase-Receptor Interaction Surface in the Escherichia coli Chemosensory Core Complex.

In Escherichia coli chemosensory arrays, transmembrane receptors, a histidine autokinase CheA, and a scaffolding protein CheW interact to form an extended hexagonal lattice of signaling complexes. One interaction, previously assigned a crucial signaling role, occurs between chemoreceptors and the CheW-binding P5 domain of CheA. Structural studies showed a receptor helix fitting into a hydrophobic cleft at the boundary between P5 subdomains. Our work aimed to elucidate the in vivo roles of the receptor-P5 interface, employing as a model the interaction between E. coli CheA and Tsr, the serine chemoreceptor. Crosslinking assays confirmed P5 and Tsr contacts in vivo and their strict dependence on CheW. Moreover, the P5 domain only mediated CheA recruitment to polar receptor clusters if CheW was also present. Amino acid replacements at CheA.P5 cleft residues reduced CheA kinase activity, lowered serine response cooperativity, and partially impaired chemotaxis. Pseudoreversion studies identified suppressors of P5 cleft defects at other P5 groove residues or at surface-exposed residues in P5 subdomain 1, which interacts with CheW in signaling complexes. Our results indicate that a high-affinity P5-receptor binding interaction is not essential for core complex function. Rather, P5 groove residues are probably required for proper cleft structure and/or dynamic behavior, which likely impact conformational communication between P5 subdomains and the strong binding interaction with CheW that is necessary for kinase activation. We propose a model for signal transmission in chemotaxis signaling complexes in which the CheW-receptor interface plays the key role in conveying signaling-related conformational changes from receptors to the CheA kinase.

Networked Chemoreceptors Benefit Bacterial Chemotaxis Performance.

Motile bacteria use large receptor arrays to detect and follow chemical gradients in their environment. Extended receptor arrays, composed of networked signaling complexes, promote cooperative stimulus control of their associated signaling kinases. Here, we used structural lesions at the communication interface between core complexes to create an Escherichia coli strain with functional but dispersed signaling complexes. This strain allowed us to directly study how networking of signaling complexes affects chemotactic signaling and gradient-tracking performance. We demonstrate that networking of receptor complexes provides bacterial cells with about 10-fold-heightened detection sensitivity to attractants while maintaining a wide dynamic range over which receptor adaptational modifications can tune response sensitivity. These advantages proved especially critical for chemotaxis toward an attractant source under conditions in which bacteria are unable to alter the attractant gradient. IMPORTANCE: Chemoreceptor arrays are found in many motile bacteria. However, although our understanding of bacterial chemotaxis is quite detailed, the signaling and behavioral advantages of networked receptor arrays had not been directly studied in cells. We have recently shown that lesions in a key interface of the E. coli receptor array diminish physical connections and functional coupling between core signaling complexes while maintaining their basic signaling capacity. In this study, we exploited an interface 2 mutant to show, for the first time, that coupling between core complexes substantially enhances stimulus detection and chemotaxis performance.

The source of high signal cooperativity in bacterial chemosensory arrays.

The Escherichia coli chemosensory system consists of large arrays of transmembrane chemoreceptors associated with a dedicated histidine kinase, CheA, and a linker protein, CheW, that couples CheA activity to receptor control. The kinase activity responses to receptor ligand occupancy changes can be highly cooperative, reflecting allosteric coupling of multiple CheA and receptor molecules. Recent structural and functional studies have led to a working model in which receptor core complexes, the minimal units of signaling, are linked into hexagonal arrays through a unique interface 2 interaction between CheW and the P5 domain of CheA. To test this array model, we constructed and characterized CheA and CheW mutants with amino acid replacements at key interface 2 residues. The mutant proteins proved defective in interface 2-specific in vivo cross-linking assays, and formed signaling complexes that were dispersed around the cell membrane rather than clustered at the cell poles as in wild type chemosensory arrays. Interface 2 mutants down-regulated CheA activity in response to attractant stimuli in vivo, but with much less cooperativity than the wild type. Moreover, mutant cells containing fluorophore-tagged receptors exhibited greater basal anisotropy that changed rapidly in response to attractant stimuli, consistent with facile changes in loosely packed receptors. We conclude that interface 2 lesions disrupt important network connections between core complexes, preventing receptors from operating in large, allosteric teams. This work confirms the critical role of interface 2 in organizing the chemosensory array, in directing the clustered array to the cell poles, and in producing its highly cooperative signaling properties.

Stress-triggered signaling affecting survival or suicide of Streptococcus pneumoniae.

Streptococcus pneumoniae is a major human pathogen that can survive to stress conditions, such as the acidic environment of inflammatory foci, and tolerates lethal pH through a mechanism known as the acid tolerance response. We previously described that S. pneumoniae activates acidic-stress induced lysis in response to acidified environments, favoring the release of cell wall compounds, DNA and virulence factors. Here, we demonstrate that F(0)F(1)-ATPase is involved in the response to acidic stress. Chemical inhibitors (DCCD, optochin) of this proton pump repressed the ATR induction, but caused an increased ASIL. Confirming these findings, mutants of the subunit c of this enzyme showed the same phenotypes as inhibitors. Importantly, we demonstrated that F(0)F(1)-ATPase and ATR are necessary for the intracellular survival of the pneumococcus in macrophages. Alternatively, a screening of two-component system (TCS) mutants showed that ATR and survival in pneumocytes were controlled in contrasting ways by ComDE and CiaRH, which had been involved in the ASIL mechanism. Briefly, CiaRH was essential for ATR (ComE represses activation) whereas ComE was necessary for ASIL (CiaRH protects against induction). They did not regulate F0F1-ATPase expression, but control LytA expression on the pneumococcal surface. These results suggest that both TCSs and F(0)F(1)-ATPase control a stress response and decide between a survival or a suicide mechanism by independent pathways, either in vitro or in pneumocyte cultures. This biological model contributes to the current knowledge about bacterial response under stress conditions in host tissues, where pathogens need to survive in order to establish infections.

Compensatory evolution of pbp mutations restores the fitness cost imposed by ß-lactam resistance in Streptococcus pneumoniae.

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, ß-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 ß-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 ß-lactam resistance among circulating strains. We propose that this compensatory mechanism is relevant for ß-lactam resistance evolution in Streptococcus pneumoniae.

Subinhibitory concentrations of penicillin increase the mutation rate to optochin resistance in Streptococcus pneumoniae.

OBJECTIVES: The aim of this work was to study the effect of subinhibitory concentrations of penicillin, chloramphenicol and erythromycin on the mutation rate of Streptococcus pneumoniae. METHODS: The mutation rate to rifampicin and optochin resistance was estimated using fluctuation analysis in three capsulated S. pneumoniae strains, cultured both with and without different subinhibitory antibiotic concentrations. The atpAC and rpoB mutations that conferred optochin and rifampicin resistance, respectively, were identified by DNA sequencing. RESULTS: The exposure to subinhibitory concentrations of penicillin increased the mutation rate (expressed as mutation per cell division) to optochin resistance between 2.1- and 3.1-fold for all three strains studied. In contrast, the rifampicin resistance assay showed no significant variations. To analyse the putative cause of the different responses between the optochin and rifampicin tests, mutations that conferred resistance in both cases were analysed. The difference may be explained by the genetic nature of the atpAC mutations, mostly transversions, which are not efficiently repaired by the HexAB mismatch repair system. CONCLUSIONS: We demonstrated that subinhibitory concentrations of penicillin significantly increased the mutation rate of S. pneumoniae, suggesting that exposure to this antibiotic could help this pathogen to acquire mutations that confer resistance to other antibiotics. The optochin test was useful to detect this phenomenon and it should be considered for further mutability analysis in S. pneumoniae.

A new serotype 14 variant of the pneumococcal Spain9V-3 international clone detected in the central region of Argentina.

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.

Acidic stress induces autolysis by a CSP-independent ComE pathway 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 two-component 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.

Characterization of in vitro-generated and clinical optochin-resistant strains of Streptococcus pneumoniae isolated from Argentina.

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.