Pneumococcal sialidase promotes bacterial survival by fine-tuning of pneumolysin-mediated membrane disruption.

Pneumolysin (Ply) is an indispensable cholesterol-dependent cytolysin for pneumococcal infection. Although Ply-induced disruption of pneumococci-containing endosomal vesicles is a prerequisite for the evasion of endolysosomal bacterial clearance, its potent activity can be a double-edged sword, having a detrimental effect on bacterial survivability by inducing severe endosomal disruption, bactericidal autophagy, and scaffold epithelial cell death. Thus, Ply activity must be maintained at optimal levels. We develop a highly sensitive assay to monitor endosomal disruption using NanoBiT-Nanobody, which shows that the pneumococcal sialidase NanA can fine-tune Ply activity by trimming sialic acid from cell-membrane-bound glycans. In addition, oseltamivir, an influenza A virus sialidase inhibitor, promotes Ply-induced endosomal disruption and cytotoxicity by inhibiting NanA activity in vitro and greater tissue damage and bacterial clearance in vivo. Our findings provide a foundation for innovative therapeutic strategies for severe pneumococcal infections by exploiting the duality of Ply activity.

Estimates of differential toxin expression governing heterogeneous intracellular lifespans of Streptococcus pneumoniae.

Following invasion of the host cell, pore-forming toxins secreted by pathogens compromise vacuole integrity and expose the microbe to diverse intracellular defence mechanisms. However, the quantitative correlation between toxin expression levels and consequent pore dynamics, fostering the intracellular life of pathogens, remains largely unexplored. In this study, using Streptococcus pneumoniae and its secreted pore-forming toxin pneumolysin (Ply) as a model system, we explored various facets of host-pathogen interactions in the host cytosol. Using time-lapse fluorescence imaging, we monitored pore formation dynamics and lifespans of different pneumococcal subpopulations inside host cells. Based on experimental histograms of various event timescales such as pore formation time, vacuolar death or cytosolic escape time and total degradation time, we developed a mathematical model based on first-passage processes that could correlate the event timescales to intravacuolar toxin accumulation. This allowed us to estimate Ply production rate, burst size and threshold Ply quantities that trigger these outcomes. Collectively, we present a general method that illustrates a correlation between toxin expression levels and pore dynamics, dictating intracellular lifespans of pathogens.

Time-resolved RNA-seq analysis to unravel the in vivo competence induction by Streptococcus pneumoniae during pneumonia-derived sepsis.

Competence development in Streptococcus pneumoniae (pneumococcus) is tightly intertwined with virulence. In addition to genes encoding genetic transformation machinery, the competence regulon also regulates the expression of allolytic factors, bacteriocins, and cytotoxins. Pneumococcal competence system has been extensively interrogated in vitro where the short transient competent state upregulates the expression of three distinct phases of "early," "late," and "delayed" genes. Recently, we have demonstrated that the pneumococcal competent state develops naturally in mouse models of pneumonia-derived sepsis. To unravel the underlying adaptive mechanisms driving the development of the competent state, we conducted a time-resolved transcriptomic analysis guided by the spatiotemporal live in vivo imaging system of competence induction during pneumonia-derived sepsis. Mouse lungs infected by the serotype 2 strain D39 expressing a competent state-specific reporter gene (D39-ssbB-luc) were subjected to RNA sequencing guided by monitoring the competence development at 0, 12, 24, and, at the moribund state, >40 hours post-infection (hpi). Transcriptomic analysis revealed that the competence-specific gene expression patterns in vivo were distinct from those under in vitro conditions. There was significant upregulation of early, late, and some delayed phase competence-specific genes as early as 12 hpi, suggesting that the pneumococcal competence regulon is important for adaptation to the lung environment. Additionally, members of the histidine triad (pht) gene family were sharply upregulated at 12 hpi followed by a steep decline throughout the rest of the infection cycle, suggesting that Pht proteins participate in the early adaptation to the lung environment. Further analysis revealed that Pht proteins execute a metal ion-dependent regulatory role in competence induction.IMPORTANCEThe induction of pneumococcal competence for genetic transformation has been extensively studied in vitro but poorly understood during lung infection. We utilized a combination of live imaging and RNA sequencing to monitor the development of a competent state during acute pneumonia. Upregulation of competence-specific genes was observed as early as 12 hour post-infection, suggesting that the pneumococcal competence regulon plays an important role in adapting pneumococcus to the stressful lung environment. Among others, we report novel finding that the pneumococcal histidine triad (pht) family of genes participates in the adaptation to the lung environment and regulates pneumococcal competence induction.

Streptococcus pneumoniae favors tolerance via metabolic adaptation over resistance to circumvent fluoroquinolones.

Streptococcus pneumoniae is a major human pathogen of global health concern and the rapid emergence of antibiotic resistance poses a serious public health problem worldwide. Fluoroquinolone resistance in S. pneumoniae is an intriguing case because the prevalence of fluoroquinolone resistance does not correlate with increasing usage and has remained rare. Our data indicate that deleterious fitness costs in the mammalian host constrain the emergence of fluoroquinolone resistance both by de novo mutation and recombination. S. pneumoniae was able to circumvent such deleterious fitness costs via the development of antibiotic tolerance through metabolic adaptation that reduced the production of reactive oxygen species, resulting in a fitness benefit during infection of mice treated with fluoroquinolones. These data suggest that the emergence of fluoroquinolone resistance is tightly constrained in S. pneumoniae by fitness tradeoffs and that mutational pathways involving metabolic networks to enable tolerance phenotypes are an important contributor to the evasion of antibiotic-mediated killing.IMPORTANCEThe increasing prevalence of antibiotic resistant bacteria is a major global health concern. While many species have the potential to develop antibiotic resistance, understanding the barriers to resistance emergence in the clinic remains poorly understood. A prime example of this is fluroquinolone resistance in Streptococcus pneumoniae, whereby, despite continued utilization, resistance to this class of antibiotic remains rare. In this study, we found that the predominant pathways for developing resistance to this antibiotic class severely compromised the infectious capacity of the pneumococcus, providing a key impediment for the emergence of resistance. Using in vivo models of experimental evolution, we found that S. pneumoniae responds to repeated fluoroquinolone exposure by modulating key metabolic pathways involved in the generation of redox molecules, which leads to antibiotic treatment failure in the absence of appreciable shifts in resistance levels. These data underscore the complex pathways available to pathogens to evade antibiotic mediating killing via antibiotic tolerance.

Daptomycin avoids drug resistance mediated by the BceAB transporter in Streptococcus pneumoniae.

Drug-resistant bacteria are a serious threat to human health as antibiotics are gradually losing their clinical efficacy. Comprehending the mechanism of action of antimicrobials and their resistance mechanisms plays a key role in developing new agents to fight antimicrobial resistance. The lipopeptide daptomycin is an antibiotic that selectively disrupts Gram-positive bacterial membranes, thereby showing slower resistance development than many classical drugs. Consequently, it is often used as a last resort antibiotic to preserve its use as one of the least potent antibiotics at our disposal. The mode of action of daptomycin has been debated but was recently found to involve the formation of a tripartite complex between undecaprenyl precursors of cell wall biosynthesis and the anionic phospholipid phosphatidylglycerol. BceAB-type ABC transporters are known to confer resistance to antimicrobial peptides that sequester some precursors of the peptidoglycan, such as the undecaprenyl pyrophosphate or lipid II. The expression of these transporters is upregulated by dedicated two-component regulatory systems in the presence of antimicrobial peptides that are recognized by the system. Here, we investigated whether daptomycin evades resistance mediated by the BceAB transporter from the bacterial pathogen Streptococcus pneumoniae. Although daptomycin can bind to the transporter, our data showed that the BceAB transporter does not mediate resistance to the drug and its expression is not induced in its presence. These findings show that the pioneering membrane-active daptomycin has the potential to escape the resistance mechanism mediated by BceAB-type transporters and confirm that the development of this class of compounds has promising clinical applications.IMPORTANCEAntibiotic resistance is rising in all parts of the world. New resistance mechanisms are emerging and dangerously spreading, threatening our ability to treat common infectious diseases. Daptomycin is an antimicrobial peptide that is one of the last antibiotics approved for clinical use. Understanding the resistance mechanisms toward last-resort antibiotics such as daptomycin is critical for the success of future antimicrobial therapies. BceAB-type ABC transporters confer resistance to antimicrobial peptides that target precursors of cell-wall synthesis. In this study, we showed that the BceAB transporter from the human pathogen Streptococcus pneumoniae does not confer resistance to daptomycin, suggesting that this drug and other calcium-dependent lipopeptide antibiotics have the potential to evade the action of this type of ABC transporters in other bacterial pathogens.

Endogenously produced H2O2 is intimately involved in iron metabolism in Streptococcus pneumoniae.

IMPORTANCE: Heme degradation provides pathogens with growth essential iron, leveraging on the host heme reservoir. Bacteria typically import and degrade heme enzymatically, and here, we demonstrated a significant deviation from this dogma. We found that Streptococcus pneumoniae liberates iron from met-hemoglobin extracellularly, in a hydrogen peroxide (H2O2)- and cell-dependent manner; this activity serves as a major iron acquisition mechanism for S. pneumoniae. Inhabiting oxygen-rich environments is a major part of pneumococcal biology, and hence, H2O2-mediated heme degradation likely supplies iron during infection. Moreover, H2O2 reaction with ferrous hemoglobin but not with met-hemoglobin is known to result in heme breakdown. Therefore, the ability of pneumococci to degrade heme from met-hemoglobin is a new paradigm. Lastly, this study will inform other research as it demonstrates that extracellular degradation must be considered in the interpretations of experiments in which H2O2-producing bacteria are given heme or hemoproteins as an iron source.

Peptidoglycan biosynthesis-associated enzymatic kinetic characteristics and ß-lactam antibiotic inhibitory effects of different Streptococcus pneumoniae penicillin-binding proteins.

Penicillin-binding proteins (PBPs) include transpeptidases, carboxypeptidases, and endopeptidases for biosynthesis of peptidoglycans in the cell wall to maintain bacterial morphology and survival in the environment. Streptococcus pneumoniae expresses six PBPs, but their enzymatic kinetic characteristics and inhibitory effects on different ß-lactam antibiotics remain poorly understood. In this study, all the six recombinant PBPs of S. pneumoniae displayed transpeptidase activity with different substrate affinities (Km = 1.56-9.11 mM) in a concentration-dependent manner, and rPBP3 showed a greater catalytic efficiency (Kcat = 2.38 s-1) than the other rPBPs (Kcat = 3.20-7.49 × 10-2 s-1). However, only rPBP3 was identified as a carboxypeptidase (Km = 8.57 mM and Kcat = 2.57 s-1). None of the rPBPs exhibited endopeptidase activity. Penicillin and cefotaxime inhibited the transpeptidase and carboxypeptidase activity of all the rPBPs but imipenem did not inhibited the enzymatic activities of rPBP3. Except for the lack of binding of imipenem to rPBP3, penicillin, cefotaxime, and imipenem bound to all the other rPBPs (KD = 3.71-9.35 × 10-4 M). Sublethal concentrations of penicillin, cefotaxime, and imipenem induced a decrease of pneumococcal pbps-mRNA levels (p < 0.05). These results indicated that all six PBPs of S. pneumoniae are transpeptidases, while only PBP3 is a carboxypeptidase. Imipenem has no inhibitory effect on pneumococcal PBP3. The pneumococcal genes for encoding endopeptidases remain to be determined.

Oxidation of hemoproteins by Streptococcus pneumoniae collapses the cell cytoskeleton and disrupts mitochondrial respiration leading to the cytotoxicity of human lung cells.

IMPORTANCE: Streptococcus pneumoniae (Spn) colonizes the lungs, killing millions every year. During its metabolism, Spn produces abundant amounts of hydrogen peroxide. When produced in the lung parenchyma, Spn-hydrogen peroxide (H2O2) causes the death of lung cells, and details of the mechanism are studied here. We found that Spn-H2O2 targets intracellular proteins, resulting in the contraction of the cell cytoskeleton and disruption of mitochondrial function, ultimately contributing to cell death. Intracellular proteins targeted by Spn-H2O2 included cytochrome c and, surprisingly, a protein of the cell cytoskeleton, beta-tubulin. To study the details of oxidative reactions, we used, as a surrogate model, the oxidation of another hemoprotein, hemoglobin. Using the surrogate model, we specifically identified a highly reactive radical whose creation was catalyzed by Spn-H2O2. In sum, we demonstrated that the oxidation of intracellular targets by Spn-H2O2 plays an important role in the cytotoxicity caused by Spn, thus providing new targets for interventions.

IL-17RA promotes pathologic epithelial inflammation in a mouse model of upper respiratory influenza infection.

The upper respiratory tract (nasopharynx or NP) is the first site of influenza replication, allowing the virus to disseminate to the lower respiratory tract or promoting community transmission. The host response in the NP regulates an intricate balance between viral control and tissue pathology. The hyper-inflammatory responses promote epithelial injury, allowing for increased viral dissemination and susceptibility to secondary bacterial infections. However, the pathologic contributors to influenza upper respiratory tissue pathology are incompletely understood. In this study, we investigated the role of interleukin IL-17 recetor A (IL-17RA) as a modulator of influenza host response and inflammation in the upper respiratory tract. We used a combined experimental approach involving IL-17RA-/- mice and an air-liquid interface (ALI) epithelial culture model to investigate the role of IL-17 response in epithelial inflammation, barrier function, and tissue pathology. Our data show that IL-17RA-/- mice exhibited significantly reduced neutrophilia, epithelial injury, and viral load. The reduced NP inflammation and epithelial injury in IL-17RA-/- mice correlated with increased resistance against co-infection by Streptococcus pneumoniae (Spn). IL-17A treatment, while potentiating the apoptosis of IAV-infected epithelial cells, caused bystander cell death and disrupted the barrier function in ALI epithelial model, supporting the in vivo findings.

Carbon source-dependent capsule thickness regulation in Streptococcus pneumoniae.

Background: The polysaccharide capsule of Streptococcus pneumoniae plays a major role in virulence, adherence to epithelial cells, and overall survival of the bacterium in the human host. Galactose, mannose, and N-acetylglucosamine (GlcNAc) are likely to be relevant for metabolization in the nasopharynx, while glucose is the primary carbon source in the blood. In this study, we aim to further the understanding of the influence of carbon sources on pneumococcal growth, capsule biosynthesis, and subsequent adherence potential. Methods: We tested the growth behavior of clinical wild-type and capsule knockout S. pneumoniae strains, using galactose, GlcNAc, mannose, and glucose as carbon source for growth. We measured capsule thickness and quantified capsule precursors by fluorescein isothiocyanate (FITC)-dextran exclusion assays and 31P-nuclear magnetic resonance measurements, respectively. We also performed epithelial adherence assays using Detroit 562 cells and performed a transcriptome analysis (RNA sequencing). Results: We observed a reduced growth in galactose, mannose, and GlcNAc compared to growth in glucose and found capsular size reductions in mannose and GlcNAc compared to galactose and glucose. Additionally, capsular precursor measurements of uridine diphosphate-(UDP)-glucose and UDP-galactose showed less accumulation of precursors in GlcNAc or mannose than in glucose and galactose, indicating a possible link with the received capsular thickness measurements. Epithelial adherence assays showed an increase in adherence potential for a pneumococcal strain, when grown in mannose compared to glucose. Finally, transcriptome analysis of four clinical isolates revealed not only strain specific but also common carbon source-specific gene expression. Conclusion: Our findings may indicate a careful adaption of the lifestyle of S. pneumoniae according to the monosaccharides encountered in the respective human niche.

The preprogrammed anti-inflammatory phenotypes of CD11chigh macrophages by Streptococcus pneumoniae aminopeptidase N safeguard from allergic asthma.

BACKGROUND: Early microbial exposure is associate with protective allergic asthma. We have previously demonstrated that Streptococcus pneumoniae aminopeptidase N (PepN), one of the pneumococcal components, inhibits ovalbumin (OVA) -induced airway inflammation in murine models of allergic asthma, but the underlying mechanism was incompletely determined. METHODS: BALB/c mice were pretreated with the PepN protein and exposed intranasally to HDM allergen. The anti-inflammatory mechanisms were investigated using depletion and adoptive transfer experiments as well as transcriptome analysis and isolated lung CD11chigh macrophages. RESULTS: We found pretreatment of mice with PepN promoted the proliferation of lung-resident F4/80+CD11chigh macrophages in situ but also mobilized bone marrow monocytes to infiltrate lung tissue that were then transformed into CD11high macrophages. PepN pre-programmed the macrophages during maturation to an anti-inflammatory phenotype by shaping the metabolic preference for oxidative phosphorylation (OXPHOS) and also inhibited the inflammatory response of macrophages by activating AMP-activated protein kinase. Furthermore, PepN treated macrophages also exhibited high-level costimulatory signaling molecules which directed the differentiation into Treg. CONCLUSION: Our results demonstrated that the expansion of CD11chigh macrophages in lungs and the OXPHOS metabolic bias of macrophages are associated with reduced allergic airway inflammation after PepN exposure, which paves the way for its application in preventing allergic asthma.

Structural basis of peptide secretion for Quorum sensing by ComA.

Quorum sensing (QS) is a crucial regulatory mechanism controlling bacterial signalling and holds promise for novel therapies against antimicrobial resistance. In Gram-positive bacteria, such as Streptococcus pneumoniae, ComA is a conserved efflux pump responsible for the maturation and secretion of peptide signals, including the competence-stimulating peptide (CSP), yet its structure and function remain unclear. Here, we functionally characterize ComA as an ABC transporter with high ATP affinity and determined its cryo-EM structures in the presence or absence of CSP or nucleotides. Our findings reveal a network of strong electrostatic interactions unique to ComA at the intracellular gate, a putative binding pocket for two CSP molecules, and negatively charged residues facilitating CSP translocation. Mutations of these residues affect ComA's peptidase activity in-vitro and prevent CSP export in-vivo. We demonstrate that ATP-Mg2+ triggers the outward-facing conformation of ComA for CSP release, rather than ATP alone. Our study provides molecular insights into the QS signal peptide secretion, highlighting potential targets for QS-targeting drugs.

NEDD4 Regulated Pyroptosis Occurred from Co-infection between Influenza A Virus and Streptococcus pneumoniae.

Co-infection of respiratory tract viruses and bacteria often result in excess mortality, especially pneumonia caused by influenza viruses and Streptococcus pneumoniae. However, the synergistic mechanisms remain poorly understood. Therefore, it is necessary to develop a clearer understanding of the molecular basis of the interaction between influenza virus and Streptococcus pneumonia. Here, we developed the BALB/c mouse model and the A549 cell model to investigate inflammation and pyroptotic cell death during co-infection. Co-infection significantly activated the NLRP3 inflammasome and induced pyroptotic cell death, correlated with excess mortality. The E3 ubiquitin ligase NEDD4 interacted with both NLRP3 and GSDMD, the executor of pyroptosis. NEDD4 negatively regulated NLRP3 while positively regulating GSDMD, thereby modulating inflammation and pyroptotic cell death. Our findings suggest that NEDD4 may play a crucial role in regulating the GSDMD-mediated pyroptosis signaling pathway. Targeting NEDD4 represents a promising approach to mitigate excess mortality during influenza pandemics by suppressing synergistic inflammation during co-infection of influenza A virus and Streptococcus pneumoniae.

Impact of Endogenous Pneumococcal Hydrogen Peroxide on the Activity and Release of Pneumolysin.

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia. The pore-forming cholesterol-dependent cytolysin (CDC) pneumolysin (PLY) and the physiological metabolite hydrogen peroxide (H2O2) can greatly increase the virulence of pneumococci. Although most studies have focused on the contribution of both virulence factors to the course of pneumococcal infection, it is unknown whether or how H2O2 can affect PLY activity. Of note, S. pneumoniae exploits endogenous H2O2 as an intracellular signalling molecule to modulate the activity of several proteins. Here, we demonstrate that H2O2 negatively affects the haemolytic activity of PLY in a concentration-dependent manner. Prevention of cysteine-dependent sulfenylation upon substitution of the unique and highly conserved cysteine residue to serine in PLY significantly reduces the toxin's susceptibility to H2O2 treatment and completely abolishes the ability of DTT to activate PLY. We also detect a clear gradual correlation between endogenous H2O2 generation and PLY release, with decreased H2O2 production causing a decline in the release of PLY. Comparative transcriptome sequencing analysis of the wild-type S. pneumoniae strain and three mutants impaired in H2O2 production indicates enhanced expression of several genes involved in peptidoglycan (PG) synthesis and in the production of choline-binding proteins (CPBs). One explanation for the impact of H2O2 on PLY release is the observed upregulation of the PG bridge formation alanyltransferases MurM and MurN, which evidentially negatively affect the PLY release. Our findings shed light on the significance of endogenous pneumococcal H2O2 in controlling PLY activity and release.

Identification of Streptococcus pneumoniae genes associated with hypothiocyanous acid tolerance through genome-wide screening.

Streptococcus pneumoniae is a commensal bacterium and invasive pathogen that causes millions of deaths worldwide. The pneumococcal vaccine offers limited protection, and the rise of antimicrobial resistance will make treatment increasingly challenging, emphasizing the need for new antipneumococcal strategies. One possibility is to target antioxidant defenses to render S. pneumoniae more susceptible to oxidants produced by the immune system. Human peroxidase enzymes will convert bacterial-derived hydrogen peroxide to hypothiocyanous acid (HOSCN) at sites of colonization and infection. Here, we used saturation transposon mutagenesis and deep sequencing to identify genes that enable S. pneumoniae to tolerate HOSCN. We identified 37 genes associated with S. pneumoniae HOSCN tolerance, including genes involved in metabolism, membrane transport, DNA repair, and oxidant detoxification. Single-gene deletion mutants of the identified antioxidant defense genes sodA, spxB, trxA, and ahpD were generated and their ability to survive HOSCN was assessed. With the exception of ΔahpD, all deletion mutants showed significantly greater sensitivity to HOSCN, validating the result of the genome-wide screen. The activity of hypothiocyanous acid reductase or glutathione reductase, known to be important for S. pneumoniae tolerance of HOSCN, was increased in three of the mutants, highlighting the compensatory potential of antioxidant systems. Double deletion of the gene encoding glutathione reductase and sodA sensitized the bacteria significantly more than single deletion. The HOSCN defense systems identified in this study may be viable targets for novel therapeutics against this deadly pathogen. IMPORTANCE Streptococcus pneumoniae is a human pathogen that causes pneumonia, bacteremia, and meningitis. Vaccination provides protection only against a quarter of the known S. pneumoniae serotypes, and the bacterium is rapidly becoming resistant to antibiotics. As such, new treatments are required. One strategy is to sensitize the bacteria to killing by the immune system. In this study, we performed a genome-wide screen to identify genes that help this bacterium resist oxidative stress exerted by the host at sites of colonization and infection. By identifying a number of critical pneumococcal defense mechanisms, our work provides novel targets for antimicrobial therapy.

Isorhamnetin as a novel inhibitor of pneumolysin against Streptococcus pneumoniae infection in vivo/in vitro.

The increasing incidence of Streptococcus pneumoniae (S. pneumoniae) infection severely threatened the global public heath, causing a significant fatality in immunocompromised hosts. Notably, pneumolysin (PLY) as a pore-forming cytolysin plays a crucial role in the pathogenesis of pneumococcal pneumonia and lung injury. In this study, a natural flavonoid isorhamnetin was identified as a PLY inhibition to suppress PLY-induced hemolysis by engaging the predicted residues and attenuate cytolysin PLY-mediated A549 cells injury. Underlying mechanisms revealed that PLY inhibitor isorhamnetin further contributed to decrease the formation of bacterial biofilms without affecting the expression of PLY. In vivo S. pneumoniae infection confirmed that the pathological injury of lung tissue evoked by S. pneumoniae was ameliorated by isorhamnetin treatment. Collectively, these results presented that isorhamnetin could inhibit the biological activity of PLY, thus reducing the pathogenicity of S. pneumoniae. In summary, our study laid a foundation for the feasible anti-virulence strategy targeting PLY, and provided a promising PLY inhibitor for the treatment of S. pneumoniae infection.

Diverse regulatory pathways modulate bet hedging of competence induction in epigenetically-differentiated phase variants of Streptococcus pneumoniae.

Despite enabling Streptococcus pneumoniae to acquire antibiotic resistance and evade vaccine-induced immunity, transformation occurs at variable rates across pneumococci. Phase variants of isolate RMV7, distinguished by altered methylation patterns driven by the translocating variable restriction-modification (tvr) locus, differed significantly in their transformation efficiencies and biofilm thicknesses. These differences were replicated when the corresponding tvr alleles were introduced into an RMV7 derivative lacking the locus. RNA-seq identified differential expression of the type 1 pilus, causing the variation in biofilm formation, and inhibition of competence induction in the less transformable variant, RMV7domi. This was partly attributable to RMV7domi's lower expression of ManLMN, which promoted competence induction through importing N-acetylglucosamine. This effect was potentiated by analogues of some proteobacterial competence regulatory machinery. Additionally, one of RMV7domi's phage-related chromosomal island was relatively active, which inhibited transformation by increasing expression of the stress response proteins ClpP and HrcA. However, HrcA increased competence induction in the other variant, with its effects depending on Ca2+ supplementation and heat shock. Hence the heterogeneity in transformation efficiency likely reflects the diverse signalling pathways by which it is affected. This regulatory complexity will modulate population-wide responses to synchronising quorum sensing signals to produce co-ordinated yet stochastic bet hedging behaviour.

The morphogenic protein CopD controls the spatio-temporal dynamics of PBP1a and PBP2b in Streptococcus pneumoniae.

IMPORTANCE: Penicillin-binding proteins (PBPs) are essential for proper bacterial cell division and morphogenesis. The genome of Streptococcus pneumoniae encodes for two class B PBPs (PBP2x and 2b), which are required for the assembly of the peptidoglycan framework and three class A PBPs (PBP1a, 1b and 2a), which remodel the peptidoglycan mesh during cell division. Therefore, their activities should be finely regulated in space and time to generate the pneumococcal ovoid cell shape. To date, two proteins, CozE and MacP, are known to regulate the function of PBP1a and PBP2a, respectively. In this study, we describe a novel regulator (CopD) that acts on both PBP1a and PBP2b. These findings provide valuable information for understanding bacterial cell division. Furthermore, knowing that ß-lactam antibiotic resistance often arises from PBP mutations, the characterization of such a regulator represents a promising opportunity to develop new strategies to resensitize resistant strains.

Effects of Capsular Polysaccharide amount on Pneumococcal-Host interactions.

Among the many oral streptococci, Streptococcus pneumoniae (Spn) stands out for the capacity of encapsulated strains to cause invasive infection. Spread beyond upper airways, however, is a biological dead end for the organism, raising the question of the benefits of expending energy to coat its surface in a thick layer of capsular polysaccharide (CPS). In this study, we compare mutants of two serotypes expressing different amounts of CPS and test these in murine models of colonization, invasion infection and transmission. Our analysis of the effect of CPS amount shows that Spn expresses a capsule of sufficient thickness to shield its surface from the deposition of complement and binding of antibody to underlying epitopes. While effective shielding is permissive for invasive infection, its primary contribution to the organism appears to be in the dynamics of colonization. A thicker capsule increases bacterial retention in the nasopharynx, the first event in colonization, and also impedes IL-17-dependent clearance during late colonization. Enhanced colonization is associated with increased opportunity for host-to-host transmission. Additionally, we document substantial differences in CPS amount among clinical isolates of three common serotypes. Together, our findings show that CPS amount is highly variable among Spn and could be an independent determinant affecting host interactions.

Negative regulation of MurZ and MurA underlies the essentiality of GpsB- and StkP-mediated protein phosphorylation in Streptococcus pneumoniae D39.

GpsB links peptidoglycan synthases to other proteins that determine the shape of the respiratory pathogen Streptococcus pneumoniae (pneumococcus; Spn) and other low-GC Gram-positive bacteria. GpsB is also required for phosphorylation of proteins by the essential StkP(Spn) Ser/Thr protein kinase. Here we report three classes of frequently arising chromosomal duplications (≈21-176 genes) containing murZ (MurZ-family homolog of MurA) or murA that suppress ΔgpsB or ΔstkP. These duplications arose from three different repeated sequences and demonstrate the facility of pneumococcus to modulate gene dosage of numerous genes. Overproduction of MurZ or MurA alone or overproduction of MurZ caused by ΔkhpAB mutations suppressed ΔgpsB or ΔstkP phenotypes to varying extents. ΔgpsB and ΔstkP were also suppressed by MurZ amino-acid changes distant from the active site, including one in commonly studied laboratory strains, and by truncation or deletion of the homolog of IreB(ReoM). Unlike in other Gram-positive bacteria, MurZ is predominant to MurA in pneumococcal cells. However, ΔgpsB and ΔstkP were not suppressed by ΔclpCP, which did not alter MurZ or MurA amounts. These results support a model in which regulation of MurZ and MurA activity, likely by IreB(Spn), is the only essential requirement for StkP-mediated protein phosphorylation in exponentially growing D39 pneumococcal cells.