Mechanistic Insights into the Impact of Air Pollution on Pneumococcal Pathogenesis and Transmission.

Streptococcus pneumoniae (the pneumococcus) is the leading cause of pneumonia and bacterial meningitis. A number of recent studies indicate an association between the incidence of pneumococcal disease and exposure to air pollution. Although the epidemiological evidence is substantial, the underlying mechanisms by which the various components of air pollution (particulate matter and gases such as NO2 and SO2) can increase susceptibility to pneumococcal infection are less well understood. In this review, we summarize the various effects air pollution components have on pneumococcal pathogenesis and transmission; exposure to air pollution can enhance host susceptibility to pneumococcal colonization by impairing the mucociliary activity of the airway mucosa, reducing the function and production of key antimicrobial peptides, and upregulating an important pneumococcal adherence factor on respiratory epithelial cells. Air pollutant exposure can also impair the phagocytic killing ability of macrophages, permitting increased replication of S. pneumoniae. In addition, particulate matter has been shown to activate various extra- and intracellular receptors of airway epithelial cells, which may lead to increased proinflammatory cytokine production. This increases recruitment of innate immune cells, including macrophages and neutrophils. The inflammatory response that ensues may result in significant tissue damage, thereby increasing susceptibility to invasive disease, because it allows S. pneumoniae access to the underlying tissues and blood. This review provides an in-depth understanding of the interaction between air pollution and the pneumococcus, which has the potential to aid the development of novel treatments or alternative strategies to prevent disease, especially in areas with high concentrations of air pollution.

Structure-Based Discovery of Lipoteichoic Acid Synthase Inhibitors.

Lipoteichoic acid synthase (LtaS) is a key enzyme for the cell wall biosynthesis of Gram-positive bacteria. Gram-positive bacteria that lack lipoteichoic acid (LTA) exhibit impaired cell division and growth defects. Thus, LtaS appears to be an attractive antimicrobial target. The pharmacology around LtaS remains largely unexplored with only two small-molecule LtaS inhibitors reported, namely "compound 1771" and the Congo red dye. Structure-based drug discovery efforts against LtaS remain unattempted due to the lack of an inhibitor-bound structure of LtaS. To address this, we combined the use of a molecular docking technique with molecular dynamics (MD) simulations to model a plausible binding mode of compound 1771 to the extracellular catalytic domain of LtaS (eLtaS). The model was validated using alanine mutagenesis studies combined with isothermal titration calorimetry. Additionally, lead optimization driven by our computational model resulted in an improved version of compound 1771, namely, compound 4 which showed greater affinity for binding to eLtaS than compound 1771 in biophysical assays. Compound 4 reduced LTA production in S. aureus dose-dependently, induced aberrant morphology as seen for LTA-deficient bacteria, and significantly reduced bacteria titers in the lung of mice infected with S. aureus. Analysis of our MD simulation trajectories revealed the possible formation of a transient cryptic pocket in eLtaS. Virtual screening (VS) against the cryptic pocket led to the identification of a new class of inhibitors that could potentiate ß-lactams against methicillin-resistant S. aureus. Our overall workflow and data should encourage further drug design campaign against LtaS. Finally, our work reinforces the importance of considering protein conformational flexibility to a successful VS endeavor.

Exposure to diesel exhaust particles increases susceptibility to invasive pneumococcal disease.

BACKGROUND: The World Health Organization estimates that air pollution is responsible for 7 million deaths per annum, with 7% of these attributable to pneumonia. Many of these fatalities have been linked to exposure to high levels of airborne particulates, such as diesel exhaust particles (DEPs). OBJECTIVES: We sought to determine whether exposure to DEPs could promote the progression of asymptomatic nasopharyngeal carriage of Streptococcus pneumoniae to invasive pneumococcal disease. METHODS: We used mouse models and in vitro assays to provide a mechanistic understanding of the link between DEP exposure and pneumococcal disease risk, and we confirmed our findings by using induced sputum macrophages isolated from healthy human volunteers. RESULTS: We demonstrate that inhaled exposure to DEPs disrupts asymptomatic nasopharyngeal carriage of S pneumoniae in mice, leading to dissemination to lungs and blood. Pneumococci are transported from the nasopharynx to the lungs following exposure to DEPs, leading to increased proinflammatory cytokine production, reduced phagocytic function of alveolar macrophages, and consequently, increased pneumococcal loads within the lungs and translocation into blood. These findings were confirmed by using DEP-exposed induced sputum macrophages isolated from healthy volunteers, demonstrating that impaired innate immune mechanisms following DEP exposure are also at play in humans. CONCLUSION: Lung inhaled DEPs increase susceptibility to pneumococcal disease by leading to loss of immunological control of pneumococcal colonisation, increased inflammation, tissue damage, and systemic bacterial dissemination.

Impaired Alanine Transport or Exposure to d-Cycloserine Increases the Susceptibility of MRSA to ß-lactam Antibiotics.

Prolonging the clinical effectiveness of ß-lactams, which remain first-line antibiotics for many infections, is an important part of efforts to address antimicrobial resistance. We report here that inactivation of the predicted d-cycloserine (DCS) transporter gene cycA resensitized methicillin-resistant Staphylococcus aureus (MRSA) to ß-lactam antibiotics. The cycA mutation also resulted in hypersusceptibility to DCS, an alanine analogue antibiotic that inhibits alanine racemase and d-alanine ligase required for d-alanine incorporation into cell wall peptidoglycan. Alanine transport was impaired in the cycA mutant, and this correlated with increased susceptibility to oxacillin and DCS. The cycA mutation or exposure to DCS were both associated with the accumulation of muropeptides with tripeptide stems lacking the terminal d-ala-d-ala and reduced peptidoglycan cross-linking, prompting us to investigate synergism between ß-lactams and DCS. DCS resensitized MRSA to ß-lactams in vitro and significantly enhanced MRSA eradication by oxacillin in a mouse bacteremia model. These findings reveal alanine transport as a new therapeutic target to enhance the susceptibility of MRSA to ß-lactam antibiotics.

Early Signals of Vaccine-driven Perturbation Seen in Pneumococcal Carriage Population Genomic Data.

BACKGROUND: Pneumococcal conjugate vaccines (PCVs) have reduced pneumococcal diseases globally. Pneumococcal genomic surveys elucidate PCV effects on population structure but are rarely conducted in low-income settings despite the high disease burden. METHODS: We undertook whole-genome sequencing (WGS) of 660 pneumococcal isolates collected through surveys from healthy carriers 2 years from 13-valent PCV (PCV13) introduction and 1 year after rollout in northern Malawi. We investigated changes in population structure, within-lineage serotype dynamics, serotype diversity, and frequency of antibiotic resistance (ABR) and accessory genes. RESULTS: In children <5 years of age, frequency and diversity of vaccine serotypes (VTs) decreased significantly post-PCV, but no significant changes occurred in persons ≥5 years of age. Clearance of VT serotypes was consistent across different genetic backgrounds (lineages). There was an increase of nonvaccine serotypes (NVTs)-namely 7C, 15B/C, and 23A-in children <5 years of age, but 28F increased in both age groups. While carriage rates have been recently shown to remain stable post-PCV due to replacement serotypes, there was no change in diversity of NVTs. Additionally, frequency of intermediate-penicillin-resistant lineages decreased post-PCV. Although frequency of ABR genes remained stable, other accessory genes, especially those associated with mobile genetic element and bacteriocins, showed changes in frequency post-PCV. CONCLUSIONS: We demonstrate evidence of significant population restructuring post-PCV driven by decreasing frequency of vaccine serotypes and increasing frequency of few NVTs mainly in children under 5. Continued surveillance with WGS remains crucial to fully understand dynamics of the residual VTs and replacement NVT serotypes post-PCV.

Dairy Heifers Naturally Exposed to Fasciola hepatica Develop a Type 2 Immune Response and Concomitant Suppression of Leukocyte Proliferation.

Fasciola hepatica is a parasitic trematode of global importance in livestock. Control strategies reliant on anthelmintics are unsustainable due to the emergence of drug resistance. Vaccines are under development, but efficacies are variable. Evidence from experimental infection suggests that vaccine efficacy may be affected by parasite-induced immunomodulation. Little is known about the immune response to F. hepatica following natural exposure. Hence, we analyzed the immune responses over time in calves naturally exposed to F. hepatica infection. Cohorts of replacement dairy heifer calves (n = 42) with no prior exposure to F. hepatica, on three commercial dairy farms, were sampled over the course of a grazing season. Exposure was determined through an F. hepatica-specific serum antibody enzyme-linked immunosorbent assay (ELISA) and fluke egg counts. Concurrent changes in peripheral blood leukocyte subpopulations, lymphocyte proliferation, and cytokine responses were measured. Relationships between fluke infection and immune responses were analyzed by using multivariable linear mixed-effect models. All calves from one farm showed evidence of exposure, while cohorts from the remaining two farms remained negative over the grazing season. A type 2 immune response was associated with exposure, with increased interleukin-4 (IL-4) production, IL-5 transcription, and eosinophilia. Suppression of parasite-specific peripheral blood mononuclear cell (PBMC) proliferation was evident, while decreased mitogen-stimulated gamma interferon (IFN-γ) production suggested immunomodulation, which was not restricted to parasite-specific responses. Our findings show that the global immune response is modulated toward a nonproliferative type 2 state following natural challenge with F. hepatica This has implications in terms of the timing of the administration of vaccination programs and for host susceptibility to coinfecting pathogens.

E-cigarette vapour enhances pneumococcal adherence to airway epithelial cells.

E-cigarette vapour contains free radicals with the potential to induce oxidative stress. Since oxidative stress in airway cells increases platelet-activating factor receptor (PAFR) expression, and PAFR is co-opted by pneumococci to adhere to host cells, we hypothesised that E-cigarette vapour increases pneumococcal adhesion to airway cells.Nasal epithelial PAFR was assessed in non-vaping controls, and in adults before and after 5 min of vaping. We determined the effect of vapour on oxidative stress-induced, PAFR-dependent pneumococcal adhesion to airway epithelial cells in vitro, and on pneumococcal colonisation in the mouse nasopharynx. Elemental analysis of vapour was done by mass spectrometry, and oxidative potential of vapour assessed by antioxidant depletion in vitroThere was no difference in baseline nasal epithelial PAFR expression between vapers (n=11) and controls (n=6). Vaping increased nasal PAFR expression. Nicotine-containing and nicotine-free E-cigarette vapour increased pneumococcal adhesion to airway cells in vitro Vapour-stimulated adhesion in vitro was attenuated by the PAFR blocker CV3988. Nicotine-containing E-cigarette vapour increased mouse nasal PAFR expression, and nasopharyngeal pneumococcal colonisation. Vapour contained redox-active metals, had considerable oxidative activity, and adhesion was attenuated by the antioxidant N-acetyl cysteine.This study suggests that E-cigarette vapour has the potential to increase susceptibility to pneumococcal infection.

Airborne dust and high temperatures are risk factors for invasive bacterial disease.

BACKGROUND: The Sahel region of West Africa has the highest bacterial meningitis attack and case fatality rate in the world. The effect of climatic factors on patterns of invasive respiratory bacterial disease is not well documented. OBJECTIVE: We aimed to assess the link between climatic factors and occurrence of invasive respiratory bacterial disease in a Sahel region of Niger. METHODS: We conducted daily disease surveillance and climatic monitoring over an 8-year period between January 1, 2003, and December 31, 2010, in Niamey, Niger, to determine risk factors for bacterial meningitis and invasive bacterial disease. We investigated the mechanistic effects of these factors on Streptococcus pneumoniae infection in mice. RESULTS: High temperatures and low visibility (resulting from high concentrations of airborne dust) were identified as significant risk factors for bacterial meningitis. Dust inhalation or exposure to high temperatures promoted progression of stable asymptomatic pneumococcal nasopharyngeal carriage to pneumonia and invasive disease. Dust exposure significantly reduced phagocyte-mediated bacterial killing, and exposure to high temperatures increased release of the key pneumococcal toxin pneumolysin through increased bacterial autolysis. CONCLUSION: Our findings show that climatic factors can have a substantial influence on infectious disease patterns, altering density of pneumococcal nasopharyngeal carriage, reducing phagocytic killing, and resulting in increased inflammation and tissue damage and consequent invasiveness. Climatic surveillance should be used to forecast invasive bacterial disease epidemics, and simple control measures to reduce particulate inhalation might reduce the incidence of invasive bacterial disease in regions of the world exposed to high temperatures and increased airborne dust.

Comparative Genomic Analysis and In Vivo Modeling of Streptococcus pneumoniae ST3081 and ST618 Isolates Reveal Key Genetic and Phenotypic Differences Contributing to Clonal Replacement of Serotype 1 in The Gambia.

Streptococcus pneumoniae serotype 1 is one of the leading causes of invasive pneumococcal disease (IPD) in West Africa, with ST618 being the dominant cause of IPD in The Gambia. Recently however, a rare example of clonal replacement was observed, where the ST3081 clone of serotype 1 replaced the predominant ST618 clone as the main cause of IPD. In the current study, we sought to find the reasons for this unusual replacement event. Using whole-genome sequence analysis and clinically relevant models of in vivo infection, we identified distinct genetic and phenotypic characteristics of the emerging ST3081 clone. We show that ST3081 is significantly more virulent than ST618 in models of invasive pneumonia, and is carried at higher densities than ST618 during nasopharyngeal carriage. We also observe sequence type-specific accessory genes and a unique sequence type-specific fixed mutation in the pneumococcal toxin pneumolysin, which is associated with increased hemolytic activity in ST3081 and may contribute to increased virulence in this clone. Our study provides evidence that, within the same serotype 1 clonal complex, biological properties differ significantly from one clone to another in terms of virulence and host invasiveness, and that these differences may be the result of key genetic differences within the genome.

Redeploying ß-Lactam Antibiotics as a Novel Antivirulence Strategy for the Treatment of Methicillin-Resistant Staphylococcus aureus Infections.

Innovative approaches to the use of existing antibiotics is an important strategy in efforts to address the escalating antimicrobial resistance crisis. We report a new approach to the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections by demonstrating that oxacillin can be used to significantly attenuate the virulence of MRSA despite the pathogen being resistant to this drug. Using mechanistic in vitro assays and in vivo models of invasive pneumonia and sepsis, we show that oxacillin-treated MRSA strains are significantly attenuated in virulence. This effect is based primarily on the oxacillin-dependent repression of the accessory gene regulator quorum-sensing system and altered cell wall architecture, which in turn lead to increased susceptibility to host killing of MRSA. Our data indicate that ß-lactam antibiotics should be included in the treatment regimen as an adjunct antivirulence therapy for patients with MRSA infections. This would represent an important change to current clinical practice for treatment of MRSA infection, with the potential to significantly improve patient outcomes in a safe, cost-effective manner.

Phage therapy is highly effective against chronic lung infections with Pseudomonas aeruginosa.

With an increase in cases of multidrug-resistant Pseudomonas aeruginosa, alternative and adjunct treatments are needed, leading to renewed interest in bacteriophage therapy. There have been few clinically relevant studies of phage therapy against chronic lung infections. Using a novel murine model that uses a natural respiratory inhalation route of infection, we show that phage therapy is an effective treatment against chronic P. aeruginosa lung infections. We also show efficacy against P. aeruginosa in a biofilm-associated cystic fibrosis lung-like environment. These studies demonstrate the potential for phage therapy in the treatment of established and recalcitrant chronic respiratory tract infections.

Epidemiological and Molecular Characterization of an Invasive Group A Streptococcus emm32.2 Outbreak.

An emm32.2 invasive group A streptococcus (iGAS) outbreak occurred in Liverpool from January 2010 to September 2012. This genotype had not previously been identified in Liverpool, but was responsible for 32% (14/44) of all iGAS cases reported during this time period. We performed a case-case comparison of emm32.2 iGAS cases with non-emm32.2 control iGAS cases identified in the Liverpool population over the same time period to assess patient risk factors for emm32.2 iGAS infection. The emm32.2 iGAS cases were confined to the adult population. We show that homelessness, intravenous drug use, and alcohol abuse predisposed patients to emm32.2 iGAS disease; however, no obvious epidemiological linkage between the patients with emm32.2 iGAS could be identified. Comparative whole-genome sequencing analysis of emm32.2 iGAS and non-emm32.2 control isolates was also performed to identify pathogen factors which might have driven the outbreak. We identified 19 genes, five of which had previously been implicated in virulence, which were present in all of the emm32.2 iGAS isolates but not present in any of the non-emm32.2 control isolates. We report that a novel emm32.2 genotype emerged in Liverpool in 2010 and identified a specific subset of genes, which could have allowed this novel emm32.2 genotype to persist in a disadvantaged population in the region over a 3-year period.

Circulating Pneumolysin Is a Potent Inducer of Cardiac Injury during Pneumococcal Infection.

Streptococcus pneumoniae accounts for more deaths worldwide than any other single pathogen through diverse disease manifestations including pneumonia, sepsis and meningitis. Life-threatening acute cardiac complications are more common in pneumococcal infection compared to other bacterial infections. Distinctively, these arise despite effective antibiotic therapy. Here, we describe a novel mechanism of myocardial injury, which is triggered and sustained by circulating pneumolysin (PLY). Using a mouse model of invasive pneumococcal disease (IPD), we demonstrate that wild type PLY-expressing pneumococci but not PLY-deficient mutants induced elevation of circulating cardiac troponins (cTns), well-recognized biomarkers of cardiac injury. Furthermore, elevated cTn levels linearly correlated with pneumococcal blood counts (r=0.688, p=0.001) and levels were significantly higher in non-surviving than in surviving mice. These cTn levels were significantly reduced by administration of PLY-sequestering liposomes. Intravenous injection of purified PLY, but not a non-pore forming mutant (PdB), induced substantial increase in cardiac troponins to suggest that the pore-forming activity of circulating PLY is essential for myocardial injury in vivo. Purified PLY and PLY-expressing pneumococci also caused myocardial inflammatory changes but apoptosis was not detected. Exposure of cultured cardiomyocytes to PLY-expressing pneumococci caused dose-dependent cardiomyocyte contractile dysfunction and death, which was exacerbated by further PLY release following antibiotic treatment. We found that high PLY doses induced extensive cardiomyocyte lysis, but more interestingly, sub-lytic PLY concentrations triggered profound calcium influx and overload with subsequent membrane depolarization and progressive reduction in intracellular calcium transient amplitude, a key determinant of contractile force. This was coupled to activation of signalling pathways commonly associated with cardiac dysfunction in clinical and experimental sepsis and ultimately resulted in depressed cardiomyocyte contractile performance along with rhythm disturbance. Our study proposes a detailed molecular mechanism of pneumococcal toxin-induced cardiac injury and highlights the major translational potential of targeting circulating PLY to protect against cardiac complications during pneumococcal infections.

Neutrophil IL-1ß processing induced by pneumolysin is mediated by the NLRP3/ASC inflammasome and caspase-1 activation and is dependent on K+ efflux.

Although neutrophils are the most abundant cells in acute infection and inflammation, relatively little attention has been paid to their role in inflammasome formation and IL-1ß processing. In the present study, we investigated the mechanism by which neutrophils process IL-1ß in response to Streptococcus pneumoniae. Using a murine model of S. pneumoniae corneal infection, we demonstrated a requirement for IL-1ß in bacterial clearance, and we showed that Nod-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), and caspase-1 are essential for IL-1ß production and bacterial killing in the cornea. Neutrophils in infected corneas had multiple specks with enzymatically active caspase-1 (YVAD-FLICA 660), and bone marrow neutrophils stimulated with heat-killed S. pneumoniae (signal 1) and pneumolysin (signal 2) exhibited multiple specks when stained for NLRP3, ASC, or Caspase-1. High-molecular mass ASC complexes were also detected, consistent with oligomer formation. Pneumolysin induced K(+) efflux in neutrophils, and blocking K(+) efflux inhibited caspase-1 activation and IL-1ß processing; however, neutrophils did not undergo pyroptosis, indicating that K(+) efflux and IL-1ß processing is not a consequence of cell death. There was also no role for lysosomal destabilization or neutrophil elastase in pneumolysin-mediated IL-1ß processing in neutrophils. Taken together, these findings demonstrate an essential role for neutrophil-derived IL-1ß in S. pneumoniae infection, and they elucidate the role of the NLRP3 inflammasome in cleavage and secretion of IL-1ß in neutrophils. Given the ubiquitous presence of neutrophils in acute bacterial and fungal infections, these findings will have implications for other microbial diseases.

Ca²âº-dependent repair of pneumolysin pores: A new paradigm for host cellular defense against bacterial pore-forming toxins.

Pneumolysin (PLY), a key virulence factor of Streptococcus pneumoniae, permeabilizes eukaryotic cells by forming large trans-membrane pores. PLY imposes a puzzling multitude of diverse, often mutually excluding actions on eukaryotic cells. Whereas cytotoxicity of PLY can be directly attributed to the pore-mediated effects, mechanisms that are responsible for the PLY-induced activation of host cells are poorly understood. We show that PLY pores can be repaired and thereby PLY-induced cell death can be prevented. Pore-induced Ca²âº entry from the extracellular milieu is of paramount importance for the initiation of plasmalemmal repair. Nevertheless, active Ca²âº sequestration that prevents excessive Ca²âº elevation during the execution phase of plasmalemmal repair is of no less importance. The efficacy of plasmalemmal repair does not only define the fate of targeted cells but also intensity, duration and repetitiveness of PLY-induced Ca²âº signals in cells that were able to survive after PLY attack. Intracellular Ca²âº dynamics evoked by the combined action of pore formation and their elimination mimic the pattern of receptor-mediated Ca²âº signaling, which is responsible for the activation of host immune responses. Therefore, we postulate that plasmalemmal repair of PLY pores might provoke cellular responses that are similar to those currently ascribed to the receptor-mediated PLY effects. Our data provide new insights into the understanding of the complexity of cellular non-immune defense responses to a major pneumococcal toxin that plays a critical role in the establishment and the progression of life-threatening diseases. Therapies boosting plasmalemmal repair of host cells and their metabolic fitness might prove beneficial for the treatment of pneumococcal infections. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

Understanding pneumococcal serotype 1 biology through population genomic analysis.

BACKGROUND: Pneumococcus kills over one million children annually and over 90 % of these deaths occur in low-income countries especially in Sub-Saharan Africa (SSA) where HIV exacerbates the disease burden. In SSA, serotype 1 pneumococci particularly the endemic ST217 clone, causes majority of the pneumococcal disease burden. To understand the evolution of the virulent ST217 clone, we analysed ST217 whole genomes from isolates sampled from African and Asian countries. METHODS: We analysed 226 whole genome sequences from the ST217 lineage sampled from 9 African and 4 Asian countries. We constructed a whole genome alignment and used it for phylogenetic and coalescent analyses. We also screened the genomes to determine presence of antibiotic resistance conferring genes. RESULTS: Population structure analysis grouped the ST217 isolates into five sequence clusters (SCs), which were highly associated with different geographical regions and showed limited intracontinental and intercontinental spread. The SCs showed lower than expected genomic sequence, which suggested strong purifying selection and small population sizes caused by bottlenecks. Recombination rates varied between the SCs but were lower than in other successful clones such as PMEN1. African isolates showed higher prevalence of antibiotic resistance genes than Asian isolates. Interestingly, certain West African isolates harbored a defective chloramphenicol and tetracycline resistance-conferring element (Tn5253) with a deletion in the loci encoding the chloramphenicol resistance gene (cat pC194), which caused lower chloramphenicol than tetracycline resistance. Furthermore, certain genes that promote colonisation were absent in the isolates, which may contribute to serotype 1's rarity in carriage and consequently its lower recombination rates. CONCLUSIONS: The high phylogeographic diversity of the ST217 clone shows that this clone has been in circulation globally for a long time, which allowed its diversification and adaptation in different geographical regions. Such geographic adaptation reflects local variations in selection pressures in different locales. Further studies will be required to fully understand the biological mechanisms which makes the ST217 clone highly invasive but unable to successfully colonise the human nasopharynx for long durations which results in lower recombination rates.

A new protective role for S100A9 in regulation of neutrophil recruitment during invasive pneumococcal pneumonia.

The S100A8/A9 heterodimer is abundantly expressed by myeloid cells, especially neutrophils, but its mechanism of action is only partially determined. In this study we investigated S100A8/A9 involvement in the host response to Streptococcus pneumoniae infection making use of S100a9(-/-) mice that lack heterodimer expression in myeloid cells. S100a9(-/-) mice that were infected intranasally with pneumococci rapidly succumbed, with 80% mortality after 48 h, whereas the majority of wild-type mice recovered. Over this time period, S100a9(-/-) mice displayed an average 6-fold reduction in circulating and lung-recruited neutrophils. Taqman analysis of S100a9(-/-) lungs revealed decreased production of a dominant subset of 5 cytokines and chemokines associated with neutrophil recruitment. The greatest differential was with the cytokine granulocyte colony-stimulating factor (G-CSF) that causes bone marrow release of neutrophils into the circulation (1900-fold difference at 48 h). Treating S100a9(-/-) mice with G-CSF reversed their increased susceptibility to infection by enhancing both circulating neutrophils and neutrophil recruitment into infected lungs, by reducing pneumococcal colony forming units, and by elevation of chemokine CXCL1, cytokine IL-6, and endogenous G-CSF proteins. Thus S100A9, potentially with its partner S100A8, makes a major contribution in the host response to pneumococcal infection by increasing circulating neutrophils principally regulation of G-CSF production.

Density and duration of pneumococcal carriage is maintained by transforming growth factor ß1 and T regulatory cells.

RATIONALE: Nasopharyngeal carriage of Streptococcus pneumoniae is a prerequisite for invasive disease, but the majority of carriage episodes are asymptomatic and self-resolving. Interactions determining the development of carriage versus invasive disease are poorly understood but will influence the effectiveness of vaccines or therapeutics that disrupt nasal colonization. OBJECTIVES: We sought to elucidate immunological mechanisms underlying noninvasive pneumococcal nasopharyngeal carriage. METHODS: Pneumococcal interactions with human nasopharyngeal and bronchial fibroblasts and epithelial cells were investigated in vitro. A murine model of nasopharyngeal carriage and an experimental human pneumococcal challenge model were used to characterize immune responses in the airways during carriage. MEASUREMENTS AND MAIN RESULTS: We describe the previously unknown immunological basis of noninvasive carriage and highlight mechanisms whose perturbation may lead to invasive disease. We identify the induction of active transforming growth factor (TGF)-ß1 by S. pneumoniae in human host cells and highlight the key role for TGF-ß1 and T regulatory cells in the establishment and maintenance of nasopharyngeal carriage in mice and humans. We identify the ability of pneumococci to drive TGF-ß1 production from nasopharyngeal cells in vivo and show that an immune tolerance profile, characterized by elevated TGF-ß1 and high nasopharyngeal T regulatory cell numbers, is crucial for prolonged carriage of pneumococci. Blockade of TGF-ß1 signaling prevents prolonged carriage and leads to clearance of pneumococci from the nasopharynx. CONCLUSIONS: These data explain the mechanisms by which S. pneumoniae colonize the human nasopharynx without inducing damaging host inflammation and provide insight into the role of bacterial and host constituents that allow and maintain carriage.

Capsule type of Streptococcus pneumoniae determines growth phenotype.

The polysaccharide capsule of Streptococcus pneumoniae defines over ninety serotypes, which differ in their carriage prevalence and invasiveness for poorly understood reasons. Recently, an inverse correlation between carriage prevalence and oligosaccharide structure of a given capsule has been described. Our previous work suggested a link between serotype and growth in vitro. Here we investigate whether capsule production interferes with growth in vitro and whether this predicts carriage prevalence in vivo. Eighty-one capsule switch mutants were constructed representing nine different serotypes, five of low (4, 7F, 14, 15, 18C) and four of high carriage prevalence (6B, 9V, 19F, 23F). Growth (length of lag phase, maximum optical density) of wildtype strains, nontypeable mutants and capsule switch mutants was studied in nutrient-restricted Lacks medium (MLM) and in rich undefined brain heart infusion broth supplemented with 5% foetal calf serum (BHI+FCS). In MLM growth phenotype depended on, and was transferred with, capsule operon type. Colonization efficiency of mouse nasopharynx also depended on, and was transferred with, capsule operon type. Capsule production interfered with growth, which correlated inversely with serotype-specific carriage prevalence. Serotypes with better growth and higher carriage prevalence produced thicker capsules (by electron microscopy, FITC-dextran exclusion assays and HPLC) than serotypes with delayed growth and low carriage prevalence. However, expression of cpsA, the first capsule gene, (by quantitative RT-PCR) correlated inversely with capsule thickness. Energy spent for capsule production (incorporation of H3-glucose) relative to amount of capsule produced was higher for serotypes with low carriage prevalence. Experiments in BHI+FCS showed overall better bacterial growth and more capsule production than growth in MLM and differences between serotypes were no longer apparent. Production of polysaccharide capsule in S. pneumoniae interferes with growth in nutrient-limiting conditions probably by competition for energy against the central metabolism. Serotype-specific nasopharyngeal carriage prevalence in vivo is predicted by the growth phenotype.

T regulatory cells control susceptibility to invasive pneumococcal pneumonia in mice.

Streptococcus pneumoniae is an important human pathogen responsible for a spectrum of diseases including pneumonia. Immunological and pro-inflammatory processes induced in the lung during pneumococcal infection are well documented, but little is known about the role played by immunoregulatory cells and cytokines in the control of such responses. We demonstrate considerable differences in the immunomodulatory cytokine transforming growth factor (TGF)-ß between the pneumococcal pneumonia resistant BALB/c and susceptible CBA/Ca mouse strains. Immunohistochemistry and flow cytometry reveal higher levels of TGF-ß protein in BALB/c lungs during pneumococcal pneumonia that correlates with a rapid rise in lung Foxp3(+)Helios(+) T regulatory cells. These cells have protective functions during pneumococcal pneumonia, because blocking their induction with an inhibitor of TGF-ß impairs BALB/c resistance to infection and aids bacterial dissemination from lungs. Conversely, adoptive transfer of T regulatory cells to CBA/Ca mice, prior to infection, prolongs survival and decreases bacterial dissemination from lungs to blood. Importantly, strong T regulatory cell responses also correlate with disease-resistance in outbred MF1 mice, confirming the importance of immunoregulatory cells in controlling protective responses to the pneumococcus. This study provides exciting new evidence for the importance of immunomodulation during pulmonary pneumococcal infection and suggests that TGF-ß signalling is a potential target for immunotherapy or drug design.