Long-term flaxseed oil supplementation diet protects BALB/c mice against Streptococcus pneumoniae infection

Intense host immune response to infection contributes significantly to the pathology of pneumococcal pneumonia. Therefore, the regulation of host immune response is critical for the successful outcome of pneumonia in such patients. The aim of the present study was to investigate the effect of n-3 PUFA, i.e. flaxseed oil supplementation for short (4 weeks) as well as long (9 weeks) term, on the course of S. pneumoniae D39 serotype 2 infection in mice. The efficacy of flaxseed oil supplementation was investigated in terms of survival of animals and production of various inflammatory molecules (malondialdehyde, myeloperoxidase, nitric oxide) in the lung homogenate of animals. This was correlated with bacteriological and histopathological parameters. The immunomodulation was studied in terms of cytokines in the lungs following infection with Streptococcus pneumoniae. Results suggest that long-term flaxseed supplementation protected the mice against bacterial colonization of lungs with Streptococcus pneumoniae with reduced histopathological involvement of lung tissue. Moderate pneumonia was observed in supplemented, infected mice compared to severe pneumonia seen in control mice. This was accompanied by decreased inflammatory markers (malondialdehyde, myeloperoxidase, nitric oxide) as the disease progressed. In addition, difference in the levels of pro-inflammatory (TNF-α and IL-1β) and anti-inflammatory (IL-10) cytokines was observed in the flaxseed fed animals. On the contrary, short-term supplementation did not show such an effect on lung colonization.     

Risk factors for levofloxacin-nonsusceptible Streptococcus pneumoniae in community-acquired pneumococcal pneumonia: a nested case–control study

This study was performed to evaluate the clinical features of community-onset levofloxacin-nonsusceptible pneumococcal pneumonia and to identify risk factors for levofloxacin resistance. Using the database of a surveillance study of community-acquired pneumococcal infections in Asian countries, we conducted a nested case–control study to identify risk factors for levofloxacin-nonsusceptible S. pneumoniae in community-acquired pneumonia in adults. Of 981 patients with pneumococcal pneumonia, 46 (4.7 %) had levofloxacin-nonsusceptible S. pneumoniae, of whom 39 evaluable cases were included in the analysis. All cases were from Korea, Taiwan, and Hong Kong. Among patients with levofloxacin-susceptible S. pneumoniae, 490 controls were selected based on patient country. Of the 39 cases of levofloxacin-nonsusceptible pneumococcal pneumonia, 23 (59.0 %) were classified as healthcare-associated, while 164 (33.5 %) of the 490 controls of levofloxacin-susceptible S. pneumoniae (P?=?0.001) were classified as healthcare-associated. Multivariate analysis showed that previous treatment with fluoroquinolones, cerebrovascular disease, and healthcare-associated infection were significantly associated with levofloxacin-nonsusceptible pneumococcal pneumonia (all P?<?0.05). Levofloxacin-nonsusceptible pneumococci pose an important new public health threat in our region, and more information on the emergence and spread of these resistant strains will be necessary to prevent spread throughout the population.     

Interleukin‐10 plays a key role in the modulation of neutrophils recruitment and lung inflammation during infection by Streptococcus pneumoniae

Streptococcus pneumoniae is a major aetiological agent of pneumonia worldwide, as well as otitis media, sinusitis, meningitis and sepsis. Recent reports have suggested that inflammation of lungs due to S. pneumoniae infection promotes bacterial dissemination and severe disease. However, the contribution of anti-inflammatory molecules to the pathogenesis of S. pneumoniae remains unknown. To elucidate whether the production of the anti-inflammatory cytokine interleukin-10 (IL-10) is beneficial or detrimental for the host during pneumococcal pneumonia, we performed S. pneumoniae infections in mice lacking IL-10 (IL-10^−/− mice). The IL-10^−/− mice showed increased mortality, higher expression of pro-inflammatory cytokines, and an exacerbated recruitment of neutrophils into the lungs after S. pneumoniae infection. However, IL-10^−/− mice showed significantly lower bacterial loads in lungs, spleen, brain and blood, when compared with mice that produced this cytokine. Our results support the notion that production of IL-10 during S. pneumoniae infection modulates the expression of pro-inflammatory cytokines and the infiltration of neutrophils into the lungs. This feature of IL-10 is important to avoid excessive inflammation of tissues and to improve host survival, even though bacterial dissemination is less efficient in the absence of this cytokine.     

Effects of Mycoplasma pneumoniae infection on sphingolipid metabolism in human lung carcinoma A549 cells

The role of sphingolipids in bacterial pathogenesis has been gradually recognized. In an effort to identify the possible involvement of sphingolipids during Mycoplasma pneumoniae (M. pneumoniae) infection, we first adopted a lipidomic approach to achieve the profiles of major sphingolipid species of M. pneumoniae as well as human lung carcinoma A549 cells, and further evaluated the effects of M. pneumoniae infection on sphingolipid metabolism in A549 cells. It was shown that M. pneumoniae and A549 cells share many common sphingolipid species, however, M. pneumoniae possesses certain specific molecular species that are not found in A549 cells. On the other hand, M. pneumoniae infection could alter sphingolipid metabolism in A549 cell, including the generation of new ceramide and sphingomyelin species, or the increase/decrease of intensities, which varies depending on the different infection doses and times. The effects of M. pneumoniae infection on two key enzymes in sphingolipid metabolism, serine palmitoyltransferase (SPT) and acid sphingomyelinase (ASM), were also examined. It was found that M. pneumoniae infection could affect the expression of SPT or the distribution of ASM at certain concentrations. These data suggest that M. pneumoniae infection could influence sphingolipid metabolism of its host, which might be related to its pathogenicity.     

Predictive value of Thomsen-Friedenreich antigen activation for Streptococcus pneumoniae infection and severity in pediatric lobar pneumonia

BackgroundMost cases of complicated pneumonia in children are caused by pneumococcal infections. Thomsen-Friedenreich antigen (TA) is present on erythrocytes, platelets and glomeruli, and it can be activated during pneumococcal infection. The aim of this study was to investigate the predictive value of TA activation for pneumococcal infection and association with the severity of complicated pneumonia.Materials and methodsPatients with lobar pneumonia were routinely tested for TA at the Department of Pediatrics, Mackay Memorial Hospital from January 2010 to December 2015. We retrospectively reviewed and analyzed their charts and data including age, sex, etiology of infection, chest tube insertion or video-assisted thoracoscopic surgery, length of hospital stay, TA activation, white blood cell count and level of C reactive protein.ResultsA total of 142 children with lobar pneumonia were enrolled, including 35 with empyema, 31 with effusion, 11 with necrotizing pneumonia and four with lung abscess. Streptococcus pneumoniae was the most commonly identified pathogen. Twenty-two patients (15.4%) had activated TA, all of whom were infected with S. pneumoniae. TA activation had 100% specificity and 100% positive predictive value for pneumococcal infection. In the multivariate analysis in lobar pneumonia, TA activation (OR, 15.8; 95% CI, 3.0–83.5; p = 0.001), duration of fever before admission (OR, 1.2; 95% CI, 1.1–1.5; p = 0.013) and initial CRP level (OR, 1.1; 95% CI, 1.0–1.1; p = 0.004) were independent predictors of empyema.ConclusionsTA activation is a specific marker for pneumococcal pneumonia and might indicate higher risk for complicated pneumonia.     

Pleiotropic Effects of Cell Wall Amidase LytA on Streptococcus pneumoniae Sensitivity to the Host Immune Response

The complement system is a key component of the host immune response for the recognition and clearance of Streptococcus pneumoniae. In this study, we demonstrate that the amidase LytA, the main pneumococcal autolysin, inhibits complement-mediated immunity independently of effects on pneumolysin by a complex process of impaired complement activation, increased binding of complement regulators, and direct degradation of complement C3. The use of human sera depleted of either C1q or factor B confirmed that LytA prevented activation of both the classical and alternative pathways, whereas pneumolysin inhibited only the classical pathway. LytA prevented binding of C1q and the acute-phase protein C-reactive protein to S. pneumoniae, thereby reducing activation of the classical pathway on the bacterial surface. In addition, LytA increased recruitment of the complement downregulators C4BP and factor H to the pneumococcal cell wall and directly cleaved C3b and iC3b to generate degradation products. As a consequence, C3b deposition and phagocytosis increased in the absence of LytA and were markedly enhanced for the lytA ply double mutant, confirming that a combination of LytA and Ply is essential for the establishment of pneumococcal pneumonia and sepsis in a murine model of infection. These data demonstrate that LytA has pleiotropic effects on complement activation, a finding which, in combination with the effects of pneumolysin on complement to assist with pneumococcal complement evasion, confirms a major role of both proteins for the full virulence of the microorganism during septicemia.     

Helminth infections predispose mice to pneumococcal pneumonia but not to other pneumonic pathogens

Pneumonia is the leading killer of children worldwide. Here, we report that helminth-infected mice develop fatal pneumonia when challenged with Streptococcus pneumoniae. Mice were chronically infected with either the flatworm Taenia crassiceps or the roundworm Heligmosomoides polygyrus. Upon challenge with a pneumonic type 3 strain of S. pneumoniae (A66.1), the worm-infected mice developed pneumonia at a rate and to a degree higher than age-matched control mice as measured by bioluminescent imaging and lung titers. This predisposition to pneumonia appears to be specific to S. pneumoniae, as worm-infected mice did not show evidence of increased morbidity when challenged with a lethal dose of influenza virus or sublethal doses of Staphylococcus aureus or Listeria monocytogenes. The defect was also present when worm-infected mice were challenged with a type 2 sepsis-causing strain (D39); an increased rate of pneumonia, decreased survival, and increased lung and blood titers were found. Pneumococcal colonization and immunity against acute otitis media were unaffected. Anti-helminthic treatment in the H. polygyrus model reversed this susceptibility. We conclude that helminth coinfection predisposes mice to fatal pneumococcal pneumonia by promoting increased outgrowth of bacteria in the lungs and blood. These data have broad implications for the prevention and treatment for pneumonia in the developing world, where helminth infections are endemic and pneumococcal pneumonia is common.     

Enhanced vulnerability for Streptococcus pneumoniae sepsis during asplenia is determined by the bacterial capsule

Patients without a spleen are susceptible for overwhelming sepsis with Streptococcus pneumoniae. We investigated the relative contribution of the pneumococcal capsule in the reduced host defense after splenectomy.Sham-operated or splenectomized mice were inoculated with serotype 2 or 4 S. pneumoniae (D39, TIGR4) or the isogenic nonencapsulated mutants (D39Δcps, TIGR4Δcps). After splenectomy, intranasal infection with D39 resulted in increased mortality, increased bacterial dissemination and exaggerated systemic inflammation rather then altering inflammation in the lungs. Intravenous infection also resulted in enhanced mortality, bacterial growth and systemic inflammation after splenectomy. In contrast, the spleen did not contribute to host defense during infection with D39Δcps. Similar observations were made for TIGR4: increased bacterial growth and inflammation after intravenous infection with wild-type, but not nonencapsulated bacteria in splenectomized mice.These results indicate that the capsule of S. pneumoniae is indeed responsible for increased vulnerability of asplenic mice to invasive pneumococcal disease.     

Cross-protective mucosal immunity mediated by memory Th17 cells against Streptococcus pneumoniae lung infection

Pneumonia caused by Streptococcus pneumoniae (Sp) remains a leading cause of serious illness and death worldwide. Immunization with conjugated pneumococcal vaccine has lowered the colonization rate and consequently invasive diseases by inducing serotype-specific antibodies. However, many of the current pneumonia cases result from infection by serotype strains not included in the vaccine. In this study, we asked if cross-protection against lung infection by heterologous strains can be induced, and investigated the underlying immune mechanism. We found that immune mice recovered from a prior infection were protected against heterologous Sp strains in the pneumonia challenge model, as evident by accelerated bacterial clearance, reduced pathology, and apoptosis of lung epithelial cells. Sp infection in the lung induced strong T-helper type 17 (Th17) responses at the lung mucosal site. Transfer of CD4⁺ T cells from immune mice provided heterologous protection against pneumonia, and this protection was abrogated by interleukin-17A (IL-17A) blockade. Transfer of memory CD4⁺ T cells from IL-17A-knockout mice failed to provide protection. These results indicate that memory Th17 cells had a key role in providing protection against pneumonia in a serotype-independent manner and suggest the feasibility of developing a broadly protective vaccine against bacterial pneumonia by targeting mucosal Th17 T cells.     

Animal Model of Mycoplasma pneumoniae Infection Using Germfree Mice

We have attempted to establish a gnotobiotic mouse model monoassociated with Mycoplasma pneumoniae following single or repeated infection to examine the mechanism of pathogenesis following M. pneumoniae infection. M. pneumoniae inoculated into germfree mice colonized equally well at 10⁵ CFU/lung in both single infection and repeated infection. In histopathological observation, repeatedly infected mice showed pneumonia with mild infiltration of mononuclear cells and macrophages. Antibody titers against M. pneumoniae rose in the repeatedly infected mice but not in the singly infected mice. The percentage of CD4-positive, CD8-positive, and CD25-positive lymphocytes infiltrated in the lung was increased in the repeatedly infected mice. In contrast, the lymphocyte subset in the spleen was not significantly different among mock-, singly, and repeatedly infected mice. In the study of cytokine productivity of spleen cells, production of interleukin (IL)-4 and IL-10 was significantly increased and that of gamma interferon was remarkably increased in the mice following repeated infection. These results indicate that a gnotobiotic mouse model monoassociated with M. pneumoniae was established and that immune mechanisms might be involved in the pathogenesis in pneumonia following M. pneumoniae infection.     

Contributions to Protection from Streptococcus pneumoniae Infection Using the Monovalent Recombinant Protein Vaccine Candidates PcpA,PhtD, and PlyD1 in an Infant Murine Model during Challenge

A vaccine consisting of several conserved proteins with different functions directing the pathogenesis of pneumonia and sepsis would be preferred for protection against infection by Streptococcus pneumoniae. Infants will be the major population targeted for next-generation pneumococcal vaccines. Here, we investigated the potential efficacy provided by three recombinant pneumococcal vaccine candidate proteins—pneumococcal histidine triad D (PhtD), detoxified pneumolysin derivative (PlyD1), and pneumococcal choline-binding protein A (PcpA)—for reducing pneumonia and sepsis in an infant mouse vaccine model. We found vaccination with PhtD and PcpA provided high IgG antibody titers after vaccination in infant mice, similar to adult mice comparators. PlyD1-specific total IgG was significantly lower in infant mice, with minimal boosting with the second and third vaccinations. Similar isotypes of IgG for PhtD and PlyD1 were generated in infant compared to adult mice. Although lower total specific IgG to all three proteins was elicited in infant than in adult mice, the infant mice were protected from bacteremic pneumonia and sepsis mortality (PlyD1) and had lower lung bacterial burdens (PcpA and PhtD) after challenge. The observed immune responses coupled with bacterial reductions elicited by each of the monovalent proteins support further testing in human infant clinical trials.     

Platelet Endothelial Cell Adhesion Molecule-1, a Putative Receptor for the Adhesion of Streptococcus pneumoniae to the Vascular Endothelium of the Blood-Brain Barrier

The Gram-positive bacterium Streptococcus pneumoniae is the main causative agent of bacterial meningitis. S. pneumoniae is thought to invade the central nervous system via the bloodstream by crossing the vascular endothelium of the blood-brain barrier. The exact mechanism by which pneumococci cross endothelial cell barriers before meningitis develops is unknown. Here, we investigated the role of PECAM-1/CD31, one of the major endothelial cell adhesion molecules, in S. pneumoniae adhesion to vascular endothelium of the blood-brain barrier. Mice were intravenously infected with pneumococci and sacrificed at various time points to represent stages preceding meningitis. Immunofluorescent analysis of brain tissue of infected mice showed that pneumococci colocalized with PECAM-1. In human brain microvascular endothelial cells (HBMEC) incubated with S. pneumoniae, we observed a clear colocalization between PECAM-1 and pneumococci. Blocking of PECAM-1 reduced the adhesion of S. pneumoniae to endothelial cells in vitro, implying that PECAM-1 is involved in pneumococcal adhesion to the cells. Furthermore, using endothelial cell protein lysates, we demonstrated that S. pneumoniae physically binds to PECAM-1. Moreover, both in vitro and in vivo PECAM-1 colocalizes with the S. pneumoniae adhesion receptor pIgR. Lastly, immunoprecipitation experiments revealed that PECAM-1 can physically interact with pIgR. In summary, we show for the first time that blood-borne S. pneumoniae colocalizes with PECAM-1 expressed by brain microvascular endothelium and that, in addition, they colocalize with pIgR. We hypothesize that this interaction plays a role in pneumococcal binding to the blood-brain barrier vasculature prior to invasion into the brain.     

Novel Strategy To Protect against Influenza Virus-Induced Pneumococcal Disease without Interfering with Commensal Colonization

Streptococcus pneumoniae commonly inhabits the nasopharynx as a member of the commensal biofilm. Infection with respiratory viruses, such as influenza A virus, induces commensal S. pneumoniae to disseminate beyond the nasopharynx and to elicit severe infections of the middle ears, lungs, and blood that are associated with high rates of morbidity and mortality. Current preventive strategies, including the polysaccharide conjugate vaccines, aim to eliminate asymptomatic carriage with vaccine-type pneumococci. However, this has resulted in serotype replacement with, so far, less fit pneumococcal strains, which has changed the nasopharyngeal flora, opening the niche for entry of other virulent pathogens (e.g., Streptococcus pyogenes, Staphylococcus aureus, and potentially Haemophilus influenzae). The long-term effects of these changes are unknown. Here, we present an attractive, alternative preventive approach where we subvert virus-induced pneumococcal disease without interfering with commensal colonization, thus specifically targeting disease-causing organisms. In that regard, pneumococcal surface protein A (PspA), a major surface protein of pneumococci, is a promising vaccine target. Intradermal (i.d.) immunization of mice with recombinant PspA in combination with LT-IIb(T13I), a novel i.d. adjuvant of the type II heat-labile enterotoxin family, elicited strong systemic PspA-specific IgG responses without inducing mucosal anti-PspA IgA responses. This response protected mice from otitis media, pneumonia, and septicemia and averted the cytokine storm associated with septic infection but had no effect on asymptomatic colonization. Our results firmly demonstrated that this immunization strategy against virally induced pneumococcal disease can be conferred without disturbing the desirable preexisting commensal colonization of the nasopharynx.     

The interaction between bacterial enolase and plasminogen promotes adherence of Streptococcus pneumoniae to epithelial and endothelial cells

Binding and conversion of the plasma protein plasminogen is an important pathogenesis mechanism of the human pathogen Streptococcus pneumoniae. Once converted into plasmin, the proteolytic activity of this major fibrinolysis component promotes degradation of extracellular matrix and the dissolution of fibrin clots. Here, we present the exploitation of plasminogen-binding as a further pivotal strategy of pneumococci facilitating adherence to eukaryotic cells. Flow cytometric measurements demonstrated the immobilization of plasminogen on host cell surfaces of human alveolar type II pneumocytes (A549), nasopharyngeal epithelium (Detroit 562) and brain-derived endothelial cells (HBMEC). These host-derived cells were employed in cell culture infection analyses followed by confocal microscopy to monitor the plasminogen-mediated adherence. Results of these studies revealed that host cell-bound plasminogen promotes pneumococcal adherence to human epithelial and endothelial cells in dose-dependent manner, whereas pneumococcal internalization was not enhanced. As an opposed effect pneumococcal-bound plasminogen reduced attachment to the epithelial and endothelial cells, and increased the interaction with neutrophil granulocytes. Moreover, the surface-displayed enolase, which serves as major pneumococcal plasminogen receptor, was identified as a key factor for plasminogen-mediated bacterial attachment in infection analyses with S. pneumoniae enolase mutants.     

Streptococcus pneumoniae Colonization Disrupts the Microbial Community within the Upper Respiratory Tract of Aging Mice

Nasopharyngeal colonization by the Gram-positive bacterium Streptococcus pneumoniae is a prerequisite for pneumonia and invasive pneumococcal diseases. Colonization is asymptomatic, involving dynamic and complex interplay between commensals, the host immune system, and environmental factors. The elderly are at an increased risk of developing pneumonia, which might be due to changes in the respiratory microbiota that would impact bacterial colonization and persistence within this niche. We hypothesized that the composition of the upper respiratory tract (URT) microbiota changes with age and subsequently can contribute to sustained colonization and inefficient clearance of S. pneumoniae. To test this, we used a mouse model of pneumococcal colonization to compare the composition of the URT microbiota in young, middle-aged, and old mice in the naive state and during the course of colonization using nasal pharyngeal washes. Sequencing of variable region 3 (V3) of the 16S rRNA gene was used to identify changes occurring with age and throughout the course of S. pneumoniae colonization. We discovered that age affects the composition of the URT microbiota and that colonization with S. pneumoniae is more disruptive of preexisting communities in older mice. We have further shown that host-pathogen interactions following S. pneumoniae colonization can impact the populations of resident microbes, including Staphylococcus and Haemophilus. Together, our findings indicate alterations to the URT microbiota could be detrimental to the elderly, resulting in increased colonization of S. pneumoniae and decreased efficiency in its clearance.     

Allergic Lung Inflammation Reduces Tissue Invasion and Enhances Survival from Pulmonary Pneumococcal Infection in Mice,Which Correlates with Increased Expression of Transforming Growth Factor β1 and SiglecFlow Alveolar Macrophages

Asthma is generally thought to confer an increased risk for invasive pneumococcal disease (IPD) in humans. However, recent reports suggest that mortality rates from IPD are unaffected in patients with asthma and that chronic obstructive pulmonary disease (COPD), a condition similar to asthma, protects against the development of complicated pneumonia. To clarify the effects of asthma on the subsequent susceptibility to pneumococcal infection, ovalbumin (OVA)-induced allergic lung inflammation (ALI) was induced in mice followed by intranasal infection with A66.1 serotype 3 Streptococcus pneumoniae. Surprisingly, mice with ALI were significantly more resistant to lethal infection than non-ALI mice. The heightened resistance observed following ALI correlated with enhanced early clearance of pneumococci from the lung, decreased bacterial invasion from the airway into the lung tissue, a blunted inflammatory cytokine and neutrophil response to infection, and enhanced expression of transforming growth factor β1 (TGF-β1). Neutrophil depletion prior to infection had no effect on enhanced early bacterial clearance or resistance to IPD in mice with ALI. Although eosinophils recruited into the lung during ALI appeared to be capable of phagocytizing bacteria, neutralization of interleukin-5 (IL-5) to inhibit eosinophil recruitment likewise had no effect on early clearance or survival following infection. However, enhanced resistance was associated with an increase in levels of clodronate-sensitive, phagocytic SiglecF^low alveolar macrophages within the airways following ALI. These findings suggest that, while the risk of developing IPD may actually be decreased in patients with acute asthma, additional clinical data are needed to better understand the risk of IPD in patients with different asthma phenotypes.     

Type I Interferon Signaling Regulates Activation of the Absent in Melanoma 2 Inflammasome during Streptococcus pneumoniae Infection

Streptococcus pneumoniae, a Gram-positive bacterial pathogen, causes pneumonia, meningitis, and septicemia. Innate immune responses are critical for the control and pathology of pneumococcal infections. It has been demonstrated that S. pneumoniae induces the production of type I interferons (IFNs) by host cells and that type I IFNs regulate resistance and chemokine responses to S. pneumoniae infection in an autocrine/paracrine manner. In this study, we examined the effects of type I IFNs on macrophage proinflammatory cytokine production in response to S. pneumoniae. The production of interleukin-18 (IL-18), but not other cytokines tested, was significantly decreased by the absence or blockade of the IFN-α/β receptor, suggesting that type I IFN signaling is necessary for IL-18 production. Type I IFN signaling was also required for S. pneumoniae-induced activation of caspase-1, a cysteine protease that plays a central role in maturation and secretion of IL-18. Earlier studies proposed that the AIM2 and NLRP3 inflammasomes mediate caspase-1 activation in response to S. pneumoniae. From our results, the AIM2 inflammasome rather than the NLRP3 inflammasome seemed to require type I IFN signaling for its optimal activation. Consistently, AIM2, but not NLRP3, was upregulated in S. pneumoniae-infected macrophages in a manner dependent on the IFN-α/β receptor. Furthermore, type I IFN signaling was found to contribute to IL-18 production in pneumococcal pneumonia in vivo. Taken together, these results suggest that type I IFNs regulate S. pneumoniae-induced activation of the AIM2 inflammasome by upregulating AIM2 expression. This study revealed a novel role for type I IFNs in innate responses to S. pneumoniae.     

Pulmonary Immunostimulation with MALP-2 in Influenza Virus-Infected Mice Increases Survival after Pneumococcal Superinfection

Pulmonary infection with influenza virus is frequently complicated by bacterial superinfection, with Streptococcus pneumoniae being the most prevalent causal pathogen and hence often associated with high morbidity and mortality rates. Local immunosuppression due to pulmonary influenza virus infection has been identified as a major cause of the pathogenesis of secondary bacterial lung infection. Thus, specific local stimulation of the pulmonary innate immune system in subjects with influenza virus infection might improve the host defense against secondary bacterial pathogens. In the present study, we examined the effect of pulmonary immunostimulation with Toll-like receptor 2 (TLR-2)-stimulating macrophage-activating lipopeptide 2 (MALP-2) in influenza A virus (IAV)-infected mice on the course of subsequent pneumococcal superinfection. Female C57BL/6N mice infected with IAV were treated with MALP-2 on day 5 and challenged with S. pneumoniae on day 6. Intratracheal MALP-2 application increased proinflammatory cytokine and chemokine release and enhanced the recruitment of leukocytes, mainly neutrophils, into the alveolar space of IAV-infected mice, without detectable systemic side effects. Local pulmonary instillation of MALP-2 in IAV-infected mice 24 h before transnasal pneumococcal infection considerably reduced the bacterial number in the lung tissue without inducing exaggerated inflammation. The pulmonary viral load was not altered by MALP-2. Clinically, MALP-2 treatment of IAV-infected mice increased survival rates and reduced hypothermia and body weight loss after pneumococcal superinfection compared to those of untreated coinfected mice. In conclusion, local immunostimulation with MALP-2 in influenza virus-infected mice improved pulmonary bacterial elimination and increased survival after subsequent pneumococcal superinfection.     

Impact of the glpQ2 Gene on Virulence in a Streptococcus pneumoniae Serotype 19A Sequence Type 320 Strain

Glycerophosphodiester phosphodiesterase (GlpQ) metabolizes glycerophosphorylcholine from the lung epithelium to produce free choline, which is transformed into phosphorylcholine and presented on the surfaces of many respiratory pathogens. Two orthologs of glpQ genes are found in Streptococcus pneumoniae: glpQ, with a membrane motif, is widespread in pneumococci, whereas glpQ2, which shares high similarity with glpQ in Haemophilus influenzae and Mycoplasma pneumoniae, is present only in S. pneumoniae serotype 3, 6B, 19A, and 19F strains. Recently, serotype 19A has emerged as an epidemiological etiology associated with invasive pneumococcal diseases. Thus, we investigated the pathophysiological role of glpQ2 in a serotype 19A sequence type 320 (19AST320) strain, which was the prevalent sequence type in 19A associated with severe pneumonia and invasive pneumococcal disease in pediatric patients. Mutations in glpQ2 reduced phosphorylcholine expression and the anchorage of choline-binding proteins to the pneumococcal surface during the exponential phase, where the mutants exhibited reduced autolysis and lower natural transformation abilities than the parent strain. The deletion of glpQ2 also decreased the adherence and cytotoxicity to human lung epithelial cell lines, whereas these functions were indistinguishable from those of the wild type in complementation strains. In a murine respiratory tract infection model, glpQ2 was important for nasopharynx and lung colonization. Furthermore, infection with a glpQ2 mutant decreased the severity of pneumonia compared with the parent strain, and glpQ2 gene complementation restored the inflammation level. Therefore, glpQ2 enhances surface phosphorylcholine expression in S. pneumoniae 19AST320 during the exponential phase, which contributes to the severity of pneumonia by promoting adherence and host cell cytotoxicity.