Oxidation of hemoglobin in the lung parenchyma facilitates the differentiation of pneumococci into encapsulated bacteria.

Pneumococcal pneumonia causes cytotoxicity in the lung parenchyma but the underlying mechanism involves multiple factors contributing to cell death. Here, we discovered that hydrogen peroxide produced by Streptococcus pneumoniae (Spn-H 2 O 2 ) plays a pivotal role by oxidizing hemoglobin, leading to its polymerization and subsequent release of labile heme. At physiologically relevant levels, heme selected a population of encapsulated pneumococci. In the absence of capsule and Spn-H 2 O 2 , host intracellular heme exhibited toxicity towards pneumococci, thus acting as an antibacterial mechanism. Further investigation revealed that heme-mediated toxicity required the ABC transporter GlnPQ. In vivo experiments demonstrated that pneumococci release H 2 O 2 to cause cytotoxicity in bronchi and alveoli through the non-proteolytic degradation of intracellular proteins such as actin, tubulin and GAPDH. Overall, our findings uncover a mechanism of lung toxicity mediated by oxidative stress that favor the growth of encapsulated pneumococci suggesting a therapeutic potential by targeting oxidative reactions. Highlights: Oxidation of hemoglobin by Streptococcus pneumoniae facilitates differentiation to encapsulated pneumococci in vivo Differentiated S. pneumoniae produces capsule and hydrogen peroxide (Spn-H 2 O 2 ) as defense mechanism against host heme-mediated toxicity. Spn-H 2 O 2 -induced lung toxicity causes the oxidation and non-proteolytic degradation of intracellular proteins tubulin, actin, and GAPDH. The ABC transporter GlnPQ is a heme-binding complex that makes Spn susceptible to heme toxicity.

Oligopeptide Transporters of Nonencapsulated Streptococcus pneumoniae Regulate CbpAC and PspA Expression and Reduce Complement-Mediated Clearance.

Streptococcus pneumoniae colonizes the human nasopharynx and causes several diseases. Pneumococcal vaccines target the polysaccharide capsule and prevent most serious disease, but there has been an increase in the prevalence of nonencapsulated S. pneumoniae (NESp). Previously, it was thought that a capsule was necessary to cause invasive disease. NESp strains expressing the oligopeptide transporters AliC and AliD have been isolated from patients with invasive disease. The AliC and AliD oligopeptide transporters regulate the expression of several genes, including choline binding protein AC (CbpAC) (a homolog of PspA), which aids in reducing C3b deposition. It is hypothesized that by altering CbpAC expression, AliC and AliD provide protection from classical complement-mediated clearance by reducing C-reactive protein (CRP) binding. Our study demonstrates that AliC and AliD regulate CbpAC expression in NESp and that AliD found in certain serotypes of encapsulated strains regulates PspA expression. C3b deposition was increased in the NESp ΔaliD and encapsulated mutants in comparison to the wild type. NESp strains expressing AliC and AliD have a significant decrease in C1q and CRP deposition in comparison to the ΔaliC ΔaliD mutant. The complement protein C1q is required for NESp clearance in a murine model and increases opsonophagocytosis. By regulating CbpAC expression, NESp inhibits CRP binding to the bacterial surface and blocks classical complement activation, leading to greater systemic survival and virulence. Due to the increase in the prevalence of NESp, it is important to gain a better understanding of NESp virulence mechanisms that aid in establishing disease and persistence within a host by avoiding clearance by the immune system. IMPORTANCE Streptococcus pneumoniae (pneumococcus) can cause a range of diseases. Although there is a robust pneumococcal vaccination program that reduces invasive pneumococcal disease by targeting various polysaccharide capsules, there has been an increase in the isolation of nonvaccine serotypes and nonencapsulated S. pneumoniae (NESp) strains. While most studies of pneumococcal pathogenesis have focused on encapsulated strains, there is little understanding of how NESp causes disease. NESp lacks a protective capsule but contains novel genes, such as aliC and aliD, which have been shown to regulate the expression of numerous genes and to be required for NESp virulence and immune evasion. Furthermore, NESp strains have high transformation efficiencies and harbor resistance to multiple drugs. This could be deleterious to current treatment strategies employed for pneumococcal disease as NESp can be a reservoir of drug resistance genes. Therefore, deciphering how NESp survives within a host and facilitates disease is a necessity that will allow the fabrication of improved, broad-spectrum treatments and preventatives against pneumococcal disease. Our study provides a better understanding of NESp virulence mechanisms during host-pathogen interactions through the examination of genes directly regulated by the NESp proteins AliC and AliD.

The quorum sensing com system regulates pneumococcal colonisation and invasive disease in a pseudo-stratified airway tissue model.

BACKGROUND: The effects of the com quorum sensing system during colonisation and invasion of Streptococcus pneumoniae (Spn) are poorly understood. METHODS: We developed an ex vivo model of differentiated human airway epithelial (HAE) cells with beating ciliae, mucus production and tight junctions to study Spn colonisation and translocation. HAE cells were inoculated with Spn wild-type TIGR4 (wtSpn) or its isogenic ΔcomC quorum sensing-deficient mutant. RESULTS: Colonisation density of ΔcomC mutant was lower after 6 h but higher at 19 h and 30 h compared to wtSpn. Translocation correlated inversely with colonisation density. Transepithelial electric resistance (TEER) decreased after pneumococcal inoculation and correlated with increased translocation. Confocal imaging illustrated prominent microcolony formation with wtSpn but disintegration of microcolony structures with ΔcomC mutant. ΔcomC mutant showed greater cytotoxicity than wtSpn, suggesting that cytotoxicity was likely not the mechanism leading to translocation. There was greater density- and time-dependent increase of inflammatory cytokines including NLRP3 inflammasome-related IL-18 after infection with ΔcomC compared with wtSpn. ComC inactivation was associated with increased pneumolysin expression. CONCLUSIONS: ComC system allows a higher organisational level of population structure resulting in microcolony formation, increased early colonisation and subsequent translocation. We propose that ComC inactivation unleashes a very different and possibly more virulent phenotype that merits further investigation.

Oxidative Reactions Catalyzed by Hydrogen Peroxide Produced by Streptococcus pneumoniae and Other Streptococci Cause the Release and Degradation of Heme from Hemoglobin.

Streptococcus pneumoniae (Spn) strains cause pneumonia that kills millions every year worldwide. Spn produces Ply, a hemolysin that lyses erythrocytes releasing hemoglobin, and also produces the pro-oxidant hydrogen peroxide (Spn-H2O2) during growth. The hallmark of the pathophysiology of hemolytic diseases is the oxidation of hemoglobin, but oxidative reactions catalyzed by Spn-H2O2 have been poorly studied. We characterized the oxidation of hemoglobin by Spn-H2O2. We prepared a series of single-mutant (ΔspxB or ΔlctO), double-mutant (ΔspxB ΔlctO), and complemented strains in TIGR4, D39, and EF3030. We then utilized an in vitro model with oxyhemoglobin to demonstrate that oxyhemoglobin was oxidized rapidly, within 30 min of incubation, by Spn-H2O2 to methemoglobin and that the main source of Spn-H2O2 was pyruvate oxidase (SpxB). Moreover, extended incubation caused the release and the degradation of heme. We then assessed oxidation of hemoglobin and heme degradation by other bacterial inhabitants of the respiratory tract. All hydrogen peroxide-producing streptococci tested caused the oxidation of hemoglobin and heme degradation, whereas bacterial species that produce <1 µM H2O2 neither oxidized hemoglobin nor degraded heme. An ex vivo bacteremia model confirmed that oxidation of hemoglobin and heme degradation occurred concurrently with hemoglobin that was released from erythrocytes by Ply. Finally, gene expression studies demonstrated that heme, but not red blood cells or hemoglobin, induced upregulated transcription of the spxB gene. Oxidation of hemoglobin may be important for pathogenesis and for the symbiosis of hydrogen peroxide-producing bacteria with other species by providing nutrients such as iron.

Induction of the macrolide-resistance efflux pump Mega inhibits intoxication of Staphylococcus aureus strains by Streptococcus pneumoniae.

Streptococcus pneumoniae (Spn) kills Staphylococcus aureus (Sau) through a contact-dependent mechanism that is catalyzed by cations, including iron, to convert hydrogen peroxide (H2O2) to highly toxic hydroxyl radicals (•OH). There are two well-characterized ABC transporters that contribute to the pool of iron in Spn, named Pia and Piu. Some Spn strains have acquired genes mef(E)/mel encoding another ABC trasporter (Mega) that produces an inducible efflux pump for resistance to macrolides. In macrolide-resistant Spn clinical isolates the insertion of Mega class 1. IV and 2. IVc deleted the locus piaABCD and these strains were attenuated for intoxicating Sau. The goal of this study was to investigate if the disruption of iron acquisition, or the antimicrobial-resistance activity of Mega, contributed to inhibiting the killing mechanism. Neither depletion of iron with 2,2'-dipyridyl-d8 (DP) nor incubating with a double knockout mutant SpnΔpiaAΔpiuA, inhibited killing of Sau. Clinical Spn strains carrying Mega1. IV or Mega2. IVc showed a significant delay for killing Sau. An ex vivo recombination system was used to transfer Mega1. IV or Mega2. IVc to reference Spn strains, which was confirmed by whole genome sequencing, and recombinants TIGR4Mega2. IVc, D39Mega2. IVc, and D39Mega1. IV were delayed for killing Sau. We then compared Sau killing of selected Mega-carrying Spn strains when incubated with sub-inhibitory erythromycin (Mega-induced) or sub-inhibitory cefuroxime. Remarkably, killing of Sau was completely inhibited under the Mega-induced condition whereas incubation with cefuroxime did not interfere with killing. Both mef(E) and mel were upregulated > 400-fold, and spxB (encoding an enzyme responsible for production of most H2O2) was upregulated 14.2-fold, whereas transcription of the autolysin (lytA) gene was downregulated when incubated with erythromycin. We demonstrated that erythromycin induction of Mega inhibits the •OH-mediated intoxication of Sau and that the inhibition occurred at the post-translational level suggesting that an imbalance of ions in the membrane inhibits these reactions.

MoWa: A Disinfectant for Hospital Surfaces Contaminated With Methicillin-Resistant Staphylococcus aureus (MRSA) and Other Nosocomial Pathogens.

Introduction: Staphylococcus aureus strains, including methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA), are a main cause of nosocomial infection in the world. The majority of nosocomial S. aureus-infection are traced back to a source of contaminated surfaces including surgery tables. We assessed the efficacy of a mixture of levulinic acid (LA) and sodium dodecyl sulfate (SDS), hereafter called MoWa, to eradicate nosocomial pathogens from contaminated surfaces. Methods and Results: A dose response study demonstrated that MoWa killed 24 h planktonic cultures of S. aureus strains starting at a concentration of (LA) 8.2/(SDS) 0.3 mM while 24 h preformed biofilms were eradicated with 32/1.3 mM. A time course study further showed that attached MRSA bacteria were eradicated within 4 h of incubation with 65/2 mM MoWa. Staphylococci were killed as confirmed by bacterial counts, and fluorescence micrographs that were stained with the live/dead bacterial assay. We then simulated contamination of hospital surfaces by inoculating bacteria on a surface prone to contamination. Once dried, contaminated surfaces were sprayed with MoWa or mock-treated, and treated contaminated surfaces were swabbed and bacteria counted. While bacteria in the mock-treated samples grew at a density of ~104 cfu/cm2, those treated for ~1 min with MoWa (1.0/0.04 M) had been eradicated below limit of detection. A similar eradication efficacy was obtained when surfaces were contaminated with other nosocomial pathogens, such as Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, or Staphylococcus epidermidis. Conclusions: MoWa kills planktonic and biofilms made by MRSA and MSSA strains and showed great efficacy to disinfect MRSA-, and MSSA-contaminated, surfaces and surfaces contaminated with other important nosocomial pathogens.