Regulation of inflammation and protection against invasive pneumococcal infection by the long pentraxin PTX3.

Streptococcus pneumoniae is a major pathogen in children, elderly subjects, and immunodeficient patients. Pentraxin 3 (PTX3) is a fluid-phase pattern recognition molecule (PRM) involved in resistance to selected microbial agents and in regulation of inflammation. The present study was designed to assess the role of PTX3 in invasive pneumococcal infection. In a murine model of invasive pneumococcal infection, PTX3 was strongly induced in non-hematopoietic (particularly, endothelial) cells. The IL-1ß/MyD88 axis played a major role in regulation of the Ptx3 gene expression. Ptx3-/- mice presented more severe invasive pneumococcal infection. Although high concentrations of PTX3 had opsonic activity in vitro, no evidence of PTX3-enhanced phagocytosis was obtained in vivo. In contrast, Ptx3-deficient mice showed enhanced recruitment of neutrophils and inflammation. Using P-selectin-deficient mice, we found that protection against pneumococcus was dependent upon PTX3-mediated regulation of neutrophil inflammation. In humans, PTX3 gene polymorphisms were associated with invasive pneumococcal infections. Thus, this fluid-phase PRM plays an important role in tuning inflammation and resistance against invasive pneumococcal infection.

TCS01 Two-Component System Influenced the Virulence of Streptococcus pneumoniae by Regulating PcpA.

Streptococcus pneumoniae relies on two-component systems (TCSs) to regulate the processes of pathogenicity, osmotic pressure, chemotaxis, and energy metabolism. The TCS01 system of S. pneumoniae is composed of HK01 (histidine kinase) and RR01 (response regulator). Previous studies have reported that an rr01 mutant reduced the pneumococcal virulence in rat pneumonia, bacteremia, a nasopharyngeal model, and infective endocarditis. However, the mechanism of TCS01 (HK/RR01) regulating pneumococcal virulence remains unclear. Here, pneumococcal mutant strains Δrr01, Δhk01, and Δrr01&hk01 were constructed, and bacterial adhesion and invasion to A549 cells were compared. RNA sequencing was performed in D39 wild-type and Δrr01 strains, and transcript profile changes were analyzed. Differentially expressed virulence genes in the Δrr01 strain were screened out and identified by quantitative real-time PCR (qRT-PCR). Our results showed that pneumococcal mutant strains exhibited attenuated adhesion and invasion to A549 cells and differential transcript profiles. Results of qRT-PCR identification showed that the differential virulence genes screened out were downregulated. Among those changed virulence genes in the Δrr01 strain, the downregulated expression level of choline binding protein pcpA was the most obvious. Complementation of rr01 and overexpression of pcpA in the Δrr01 strain partially restored both pneumococcal adhesion and invasion, and rr01 complementation made the expression of pcpA upregulated. These findings revealed that rr01 influenced pneumococcal virulence by regulating pcpA.

Mitochondrial ROS production by neutrophils is required for host antimicrobial function against Streptococcus pneumoniae and is controlled by A2B adenosine receptor signaling.

Polymorphonuclear cells (PMNs) control Streptococcus pneumoniae (pneumococcus) infection through various antimicrobial activities. We previously found that reactive oxygen species (ROS) were required for optimal antibacterial function, however, the NADPH oxidase is known to be dispensable for the ability of PMNs to kill pneumococci. In this study, we explored the role of ROS produced by the mitochondria in PMN antimicrobial defense against pneumococci. We found that the mitochondria are an important source of overall intracellular ROS produced by murine PMNs in response to infection. We investigated the host and bacterial factors involved and found that mitochondrial ROS (MitROS) are produced independent of bacterial capsule or pneumolysin but presence of live bacteria that are in direct contact with PMNs enhanced the response. We further found that MyD88-/- PMNs produced less MitROS in response to pneumococcal infection suggesting that released bacterial products acting as TLR ligands are sufficient for inducing MitROS production in PMNs. To test the role of MitROS in PMN function, we used an opsonophagocytic killing assay and found that MitROS were required for the ability of PMNs to kill pneumococci. We then investigated the role of MitROS in host resistance and found that MitROS are produced by PMNs in response to pneumococcal infection. Importantly, treatment of mice with a MitROS scavenger prior to systemic challenge resulted in reduced survival of infected hosts. In exploring host pathways that control MitROS, we focused on extracellular adenosine, which is known to control PMN anti-pneumococcal activity, and found that signaling through the A2B adenosine receptor inhibits MitROS production by PMNs. A2BR-/- mice produced more MitROS and were significantly more resistant to infection. Finally, we verified the clinical relevance of our findings using human PMNs. In summary, we identified a novel pathway that controls MitROS production by PMNs, shaping host resistance against S. pneumoniae.

Ethanol Intoxication Impairs Respiratory Function and Bacterial Clearance and Is Associated With Neutrophil Accumulation in the Lung After Streptococcus pneumoniae Infection.

Alcohol consumption is commonplace in the United States and its prevalence has increased in recent years. Excessive alcohol use is linked to an increased risk of infections including pneumococcal pneumonia, mostly commonly caused by Streptococcus pneumoniae. In addition, pneumonia patients with prior alcohol use often require more intensive treatment and longer hospital stays due to complications of infection. The initial respiratory tract immune response to S. pneumoniae includes the production of pro-inflammatory cytokines and chemokines by resident cells in the upper and lower airways which activate and recruit leukocytes to the site of infection. However, this inflammation must be tightly regulated to avoid accumulation of toxic by-products and subsequent tissue damage. A majority of previous work on alcohol and pneumonia involve animal models utilizing high concentrations of ethanol or chronic exposure and offer conflicting results about how ethanol alters immunity to pathogens. Further, animal models often employ a high bacterial inoculum which may overwhelm the immune system and obscure results, limiting their applicability to the course of human infection. Here, we sought to determine how a more moderate ethanol exposure paradigm affects respiratory function and innate immunity in mice after intranasal infection with 104 colony forming units of S. pneumoniae. Ethanol-exposed mice displayed respiratory dysfunction and impaired bacterial clearance after infection compared to their vehicle-exposed counterparts. This altered response was associated with increased gene expression of neutrophil chemokines Cxcl1 and Cxcl2 in whole lung homogenates, elevated concentrations of circulating granulocyte-colony stimulating factor (G-CSF), and higher neutrophil numbers in the lung 24 hours after infection. Taken together, these findings suggest that even a more moderate ethanol consumption pattern can dramatically modulate the innate immune response to S. pneumoniae after only 3 days of ethanol exposure and provide insight into possible mechanisms related to the compromised respiratory immunity seen in alcohol consumers with pneumonia.

Streptococcus pneumoniae binds collagens and C1q via the SSURE repeats of the PfbB adhesin.

The binding of Streptococcus pneumoniae to collagen is likely an important step in the pathogenesis of pneumococcal infections, but little is known of the underlying molecular mechanisms. Streptococcal surface repeats (SSURE) are highly conserved protein domains present in cell wall adhesins from different Streptococcus species. We find here that SSURE repeats of the pneumococcal adhesin plasminogen and fibronectin binding protein B (PfbB) bind to various types of collagen. Moreover, deletion of the pfbB gene resulted in a significant impairment of the ability of encapsulated or unencapsulated pneumococci to bind collagen. Notably, a PfbB SSURE domain is also bound to the complement component C1q that bears a collagen-like domain and promotes adherence of pneumococci to host cells by acting as a bridge between bacteria and epithelial cells. Accordingly, deletion of PfbB or pre-treatment with anti-SSURE antibodies markedly decreased pneumococcal binding to C1q as well as C1q-dependent adherence to epithelial and endothelial cells. Further data indicated that C1q promotes pneumococcal adherence by binding to integrin α2 ß1 . In conclusion, our results indicate that the SSURE domains of the PfbB protein promote interactions of pneumococci with various types of collagen and with C1q. These repeats may be useful targets in strategies to control S. pneumoniae infections.

Emerging Roles of Extracellular Vesicles in Pneumococcal Infections: Immunomodulators to Potential Novel Vaccine Candidates.

The Gram-positive bacterial pathogen, Streptococcus pneumoniae is a major global health threat that kills over one million people worldwide. The pneumococcus commonly colonizes the nasopharynx asymptomatically as a commensal, but is also capable of causing a wide range of life-threatening diseases such as pneumonia, meningitis and septicemia upon migration into the lower respiratory tract and spread to internal organs. Emergence of antibiotic resistant strains and non-vaccine serotypes has led to the classification of pneumococcal bacteria as a priority pathogen by the World Health Organization that needs urgent research into bacterial pathogenesis and development of novel vaccine strategies. Extracellular vesicles are spherical membrane bound structures that are released by both pathogen and host cells, and influence bacterial pathogenesis as well as the immune response. Recent studies have found that while bacterial vesicles shuttle virulence factors and toxins into host cells and regulate inflammatory responses, vesicles released from the infected host cells contain both bacterial and host proteins that are antigenic and immunomodulatory. Bacterial membrane vesicles have great potential to be developed as cell-free vaccine candidates in the future due to their immunogenicity and biostability. Host-derived vesicles isolated from patient biofluids such as blood and bronchoalveolar lavage could be used to identify potential diagnostic biomarkers as well as engineered to deliver desired payloads to specific target cells for immunotherapy. In this review, we summarize the recent developments on the role of bacterial and host vesicles in pneumococcal infections and future prospects in developing novel therapeutics and diagnostics for control of invasive pneumococcal diseases.

Acetate Improves the Killing of Streptococcus pneumoniae by Alveolar Macrophages via NLRP3 Inflammasome and Glycolysis-HIF-1α Axis.

Short-chain fatty acids (SCFAs) are metabolites produced mainly by the gut microbiota with a known role in immune regulation. Acetate, the major SCFA, is described to disseminate to distal organs such as lungs where it can arm sentinel cells, including alveolar macrophages, to fight against bacterial intruders. In the current study, we explored mechanisms through which acetate boosts macrophages to enhance their bactericidal activity. RNA sequencing analyses show that acetate triggers a transcriptomic program in macrophages evoking changes in metabolic process and immune effector outputs, including nitric oxide (NO) production. In addition, acetate enhances the killing activity of macrophages towards Streptococcus pneumoniae in an NO-dependent manner. Mechanistically, acetate improves IL-1ß production by bacteria-conditioned macrophages and the latter acts in an autocrine manner to promote NO production. Strikingly, acetate-triggered IL-1ß production was neither dependent of its cell surface receptor free-fatty acid receptor 2, nor of the enzymes responsible for its metabolism, namely acetyl-CoA synthetases 1 and 2. We found that IL-1ß production by acetate relies on NLRP3 inflammasome and activation of HIF-1α, the latter being triggered by enhanced glycolysis. In conclusion, we unravel a new mechanism through which acetate reinforces the bactericidal activity of alveolar macrophages.

Recombinant thrombomodulin attenuates hyper-inflammation and glycocalyx damage in a murine model of Streptococcus pneumoniae-induced sepsis.

PURPOSE: The anticoagulant agent recombinant thrombomodulin (rTM) activates protein C to prevent excessive coagulation and also possibly regulates hyper-inflammation via neutralization of high-mobility-group B1 (HMG-B1). The glycocalyx layer in endothelial cells also plays a pivotal role in preventing septic shock-associated hyperpermeability. The present study examined the effect of rTM in a murine model of Streptococcus pneumoniae-induced sepsis. METHODS: Male C57BL/6N mice were injected intratracheally via midline cervical incision with 2 × 107 CFU of S. pneumoniae (capsular subtype 19A). Control mice were sham-treated identically but injected with saline. rTM (10 mg/kg) was injected intraperitoneally 3 h after septic insult. Blood concentrations of soluble inflammatory mediators (interleukin [IL]-1ß, IL-6, IL-10, and tumor necrosis factor [TNF]-α) were determined using a microarray immunoassay. Serum concentrations of HMG-B1 and syndecan-1, as a parameter of glycocalyx damage, were determined by enzyme-linked immunosorbent assay. The glycocalyx was also evaluated with electron microscopy. The lungs were removed, and digested to cells, which were then stained with a mixture of fluorophore-conjugated antibodies. Anti-mouse primary antibodies included PE-Cy7-conjugated anti-CD31, AlexaFluor 700-conjugated anti-CD45, PerCP-Cy5.5-conjugated anti-CD326, APC-conjugated anti-TNF-α, PE-conjugated anti-IL-6, and PE-conjugated anti-IL-10. A total of 1 × 106 cells per sample were analyzed, and 2 × 105 events were recorded by flow cytometry, and parameters were compared with/without rTM treatment. RESULTS: The blood concentration of TNF-α was significantly reduced 24 h after intratracheal injection in S. pneumoniae-challenged mice treated with rTM (P = 0.016). Levels of IL-10 in the lung endothelium of rTM-treated S. pneumoniae-challenged mice increased significantly 12 h after intratracheal injection (P = 0.03). Intriguingly, serum HMGB-1 and syndecan-1 levels decreased significantly (P = 0.010 and 0.015, respectively) in rTM-treated mice 24 h after intratracheal injection of S. pneumoniae. Electron microscopy indicated that rTM treatment preserved the morphology of the glycocalyx layer in septic mice. CONCLUSIONS: These data suggest that rTM modulates local inflammation in the lung endothelium, thus diminishing systemic inflammation, i.e., hypercytokinemia. Furthermore, rTM treatment reduced serum syndecan-1 levels, thus preventing glycocalyx damage. The use of rTM to treat sepsis caused by bacterial pneumonia could therefore help prevent both excessive inflammation and glycocalyx injury in the lung endothelium.

The Roles of Transient Receptor Potential Vanilloid 1 and 4 in Pneumococcal Nasal Colonization and Subsequent Development of Invasive Disease.

Transient receptor potential (TRP) channels, neuronal stimulations widely known to be associated with thermal responses, pain induction, and osmoregulation, have been shown in recent studies to have underlying mechanisms associated with inflammatory responses. The role of TRP channels on inflammatory milieu during bacterial infections has been widely demonstrated. It may vary among types of channels/pathogens, however, and it is not known how TRP channels function during pneumococcal infections. Streptococcus pneumoniae can cause severe infections such as pneumonia, bacteremia, and meningitis, with systemic inflammatory responses. This study examines the role of TRP channels (TRPV1 and TRPV4) for pneumococcal nasal colonization and subsequent development of invasive pneumococcal disease in a mouse model. Both TRPV1 and TRPV4 channels were shown to be related to regulation of the development of pneumococcal diseases. In particular, the influx of neutrophils (polymorphonuclear cells) in the nasal cavity and the bactericidal activity were significantly suppressed among TRPV4 knockout mice. This may lead to severe pneumococcal pneumonia, resulting in dissemination of the bacteria to various organs and causing high mortality during influenza virus coinfection. Regulating host immune responses by TRP channels could be a novel strategy against pathogenic microorganisms causing strong local/systemic inflammation.

cGAS-STING and MyD88 Pathways Synergize in Ly6Chi Monocyte to Promote Streptococcus pneumoniae-Induced Late-Stage Lung IFNγ Production.

The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway senses DNA and induces type I interferon (IFN) production. Whether and how the STING pathway crosstalk to other innate immune pathways during pathogen infection, however, remains unclear. Here, we showed that STING was needed for Streptococcus pneumoniae-induced late, not early, stage of lung IFNγ production. Using knockout mice, IFNγ reporter mice, intracellular cytokine staining, and adoptive cell transfer, we showed that cGAS-STING-dependent lung IFNγ production was independent of type I IFNs. Furthermore, STING expression in monocyte/monocyte-derived cells governed IFNγ production in the lung via the production of IL-12p70. Surprisingly, DNA stimulation alone could not induce IL-12p70 or IFNγ in Ly6Chi monocyte. The production of IFNγ required the activation by both DNA and heat-killed S. pneumococcus. Accordingly, MyD88-/- monocyte did not generate IL-12p70 or IFNγ. In summary, the cGAS-STING pathway synergizes with the MyD88 pathway in monocyte to promote late-stage lung IFNγ production during pulmonary pneumococcal infection.

Prophylactic Inhibition of Colonization by Streptococcus pneumoniae with the Secondary Bile Acid Metabolite Deoxycholic Acid.

Streptococcus pneumoniae colonizes the nasopharynx of children and the elderly but also kills millions worldwide yearly. The secondary bile acid metabolite deoxycholic acid (DoC) affects the viability of human pathogens but also plays multiple roles in host physiology. We assessed in vitro the antimicrobial activity of DoC and investigated its potential to eradicate S. pneumoniae colonization using a model of human nasopharyngeal colonization and an in vivo mouse model of colonization. At a physiological concentration, DoC (0.5 mg/ml; 1.27 mM) killed all tested S. pneumoniae strains (n = 48) 2 h postinoculation. The model of nasopharyngeal colonization showed that DoC eradicated colonization by S. pneumoniae strains as soon as 10 min postexposure. The mechanism of action did not involve activation of autolysis, since the autolysis-defective double mutants ΔlytAΔlytC and ΔspxBΔlctO were as susceptible to DoC as was the wild type (WT). Oral streptococcal species (n = 20), however, were not susceptible to DoC (0.5 mg/ml). Unlike trimethoprim, whose spontaneous resistance frequency (srF) for TIGR4 or EF3030 was ≥1 × 10-9, no spontaneous resistance was observed with DoC (srF, ≥1 × 10-12). Finally, the efficacy of DoC to eradicate S. pneumoniae colonization was assessed in vivo using a topical route via intranasal (i.n.) administration and as a prophylactic treatment. Mice challenged with S. pneumoniae EF3030 carried a median of 4.05 × 105 CFU/ml 4 days postinoculation compared to 6.67 × 104 CFU/ml for mice treated with DoC. Mice in the prophylactic group had an ∼99% reduction of the pneumococcal density (median, 2.61 × 103 CFU/ml). Thus, DoC, an endogenous human bile salt, has therapeutic potential against S. pneumoniae.

Metabolomic profiling of microbial disease etiology in community-acquired pneumonia.

Diagnosis of microbial disease etiology in community-acquired pneumonia (CAP) remains challenging. We undertook a large-scale metabolomics study of serum samples in hospitalized CAP patients to determine if host-response associated metabolites can enable diagnosis of microbial etiology, with a specific focus on discrimination between the major CAP pathogen groups S. pneumoniae, atypical bacteria, and respiratory viruses. Targeted metabolomic profiling of serum samples was performed for three groups of hospitalized CAP patients with confirmed microbial etiologies: S. pneumoniae (n = 48), atypical bacteria (n = 47), or viral infections (n = 30). A wide range of 347 metabolites was targeted, including amines, acylcarnitines, organic acids, and lipids. Single discriminating metabolites were selected using Student's T-test and their predictive performance was analyzed using logistic regression. Elastic net regression models were employed to discover metabolite signatures with predictive value for discrimination between pathogen groups. Metabolites to discriminate S. pneumoniae or viral pathogens from the other groups showed poor predictive capability, whereas discrimination of atypical pathogens from the other groups was found to be possible. Classification of atypical pathogens using elastic net regression models was associated with a predictive performance of 61% sensitivity, 86% specificity, and an AUC of 0.81. Targeted profiling of the host metabolic response revealed metabolites that can support diagnosis of microbial etiology in CAP patients with atypical bacterial pathogens compared to patients with S. pneumoniae or viral infections.

Glycolytic Metabolism Is Critical for the Innate Antibacterial Defense in Acute Streptococcus pneumoniae Otitis Media.

Objective: Streptococcus pneumoniae (S.pn) is a common respiratory pathogen and a frequent cause of acute otitis media (AOM) in children. However, little is known about the immunometabolism during AOM. This study was to assess the presence of glucose metabolic reprogramming during AOM and its underlying mechanism affecting inflammatory response and middle ear injury. Methods: The levels of glycolytic metabolism were evaluated by measuring the expression of glycolysis-related genes and the production of metabolites. HE stain, immunofluorescence, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and Western blot were performed to measure the effect of glucose metabolic reprogramming on inflammatory response, pneumococcal clearance, hypoxia-inducible factor 1 alpha (HIF-1α) expression and cytokine secretion during AOM, respectively. Results: The analysis of microarray revealed an increase of the expression of glycolysis-related genes during S.pn-induced AOM, which was verified by real-time PCR. Increased glycolysis promoted the production of IL-1ß and TNF-α and facilitated the clearance of S.pn by enhancing phagocytosis and killing capability of neutrophils, but also aggravated the middle ear injury. Furthermore, these pathogenic effects could be reversed after glycolytic inhibitor 2DG treatment. Additionally, HIF-1α was observed to involve in glycolytic metabolism during AOM. Conclusion: S.pn infection induced increased glycolysis conversion during AOM, which promoted inflammatory responses and bacterial clearance, but also aggravated tissue damage.

Gab2 (Grb2-Associated Binder2) Plays a Crucial Role in Inflammatory Signaling and Endothelial Dysfunction.

[Figure: see text].

The Critical Role of NLRP6 Inflammasome in Streptococcus pneumoniae Infection In Vitro and In Vivo.

Streptococcus pneumoniae (S. pneumoniae) causes severe pulmonary diseases, leading to high morbidity and mortality. It has been reported that inflammasomes such as NLR family pyrin domain containing 3 (NLRP3) and absent in melanoma 2 (AIM2) play an important role in the host defense against S. pneumoniae infection. However, the role of NLRP6 in vivo and in vitro against S. pneumoniae remains unclear. Therefore, we investigated the role of NLRP6 in regulating the S. pneumoniae-induced inflammatory signaling pathway in vitro and the role of NLRP6 in the host defense against S. pneumoniae in vivo by using NLRP6-/- mice. The results showed that the NLRP6 inflammasome regulated the maturation and secretion of IL-1ß, but it did not affect the induction of IL-1ß transcription in S. pneumoniae-infected macrophages. Furthermore, the activation of caspase-1, caspase-11, and gasdermin D (GSDMD) as well as the oligomerization of apoptosis-associated speck-like protein (ASC) were also mediated by NLRP6 in S. pneumoniae-infected macrophages. However, the activation of NLRP6 reduced the expression of NF-κB and ERK signaling pathways in S. pneumoniae-infected macrophages. In vivo study showed that NLRP6-/- mice had a higher survival rate, lower number of bacteria, and milder inflammatory response in the lung compared with wild-type (WT) mice during S. pneumoniae infection, indicating that NLRP6 plays a negative role in the host defense against S. pneumoniae. Furthermore, increased bacterial clearance in NLRP6 deficient mice was modulated by the recruitment of macrophages and neutrophils. Our study provides a new insight on S. pneumoniae-induced activation of NLRP6 and suggests that blocking NLRP6 could be considered as a potential therapeutic strategy to treat S. pneumoniae infection.

Clarithromycin impairs tissue-resident memory and Th17 responses to macrolide-resistant Streptococcus pneumoniae infections.

The increasing prevalence of antimicrobial resistance in pathogens is a growing public health concern, with the potential to compromise the success of infectious disease treatments in the future. Particularly, the number of infections by macrolide antibiotics-resistant Streptococcus pneumoniae is increasing. We show here that Clarithromycin impairs both the frequencies and number of interleukin (IL)-17 producing T helper (Th) 17 cells within the lungs of mice infected with a macrolide-resistant S. pneumoniae serotype 15A strain. Subsequently, the tissue-resident memory CD4+ T cell (Trm) response to a consecutive S. pneumoniae infection was impaired. The number of lung resident IL-17+ CD69+ Trm was diminished upon Clarithromycin treatment during reinfection. Mechanistically, Clarithromycin attenuated phosphorylation of the p90-S6-kinase as part of the ERK pathway in Th17 cells. Moreover, a strong increase in the mitochondrial-mediated maximal respiratory capacity was observed, while mitochondrial protein translation and mTOR sisgnaling were unimpaired. Therefore, treatment with macrolide antibiotics may favor the spread of antimicrobial-resistant pathogens not only by applying a selection pressure but also by decreasing the natural T cell immune response. Clinical administration of macrolide antibiotics as standard therapy procedure during initial hospitalization should be reconsidered accordingly and possibly be withheld until microbial resistance is determined. KEY MESSAGES: • Macrolide-resistant S. pneumoniae infection undergoes immunomodulation by Clarithromycin • Clarithromycin treatment hinders Th17 and tissue-resident memory responses • Macrolide antibiotics impair Th17 differentiation in vitro by ERK-pathway inhibition.

Hemoglobin Induces Early and Robust Biofilm Development in Streptococcus pneumoniae by a Pathway That Involves comC but Not the Cognate comDE Two-Component System.

Streptococcus pneumoniae grows in biofilms during both asymptomatic colonization and infection. Pneumococcal biofilms on abiotic surfaces exhibit delayed growth and lower biomass and lack the structures seen on epithelial cells or during nasopharyngeal carriage. We show here that adding hemoglobin to the medium activated unusually early and vigorous biofilm growth in multiple S. pneumoniae serotypes grown in batch cultures on abiotic surfaces. Human blood (but not serum, heme, or iron) also stimulated biofilms, and the pore-forming pneumolysin, ply, was required for this induction. S. pneumoniae transitioning from planktonic into sessile growth in the presence of hemoglobin displayed an extensive transcriptome remodeling within 1 and 2 h. Differentially expressed genes included those involved in the metabolism of carbohydrates, nucleotides, amino acid, and lipids. The switch into adherent states also influenced the expression of several regulatory systems, including the comCDE genes. Inactivation of comC resulted in 67% reduction in biofilm formation, while the deletion of comD or comE had limited or no effect, respectively. These observations suggest a novel route for CSP-1 signaling independent of the cognate ComDE two-component system. Biofilm induction and the associated transcriptome remodeling suggest hemoglobin serves as a signal for host colonization in pneumococcus.

Testing Anti-Pneumococcal Antibody Function Using Bacteria and Primary Neutrophils.

Antibodies against Streptococcus pneumoniae (pneumococcus) following vaccination are crucial for host protection against invasive pneumococcal infections. The antibodies induced by pneumococcal vaccines act as opsonins to mediate bacterial uptake and killing by host phagocytic cells, especially polymorphonuclear leukocytes (PMNs) also called neutrophils. Therefore, it is important to measure not only the levels of antibodies induced by a pneumococcal vaccine candidate but their actual functional capacity in mediating bacterial opsonization and killing by PMNs. Here, we describe a protocol to demonstrate effective deposition of vaccine-induced antibodies on the surface of S. pneumoniae by flow cytometry and subsequent opsonophagocytic killing (OPH) by murine bone-marrow derived PMNs.

Lower Density and Shorter Duration of Nasopharyngeal Carriage by Pneumococcal Serotype 1 (ST217) May Explain Its Increased Invasiveness over Other Serotypes.

Streptococcus pneumoniae is a frequent colonizer of the human nasopharynx and a major cause of life-threating invasive infections such as pneumonia, meningitis and sepsis. Over 1 million people die every year due to invasive pneumococcal disease (IPD), mainly in developing countries. Serotype 1 is a common cause of IPD; however, unlike other serotypes, it is rarely found in the carrier state in the nasopharynx, which is often considered a prerequisite for disease. The aim of this study was to understand this dichotomy. We used murine models of carriage and IPD to characterize the pathogenesis of African serotype 1 (sequence type 217) pneumococcal strains obtained from the Queen Elizabeth Central Hospital in Blantyre, Malawi. We found that ST217 pneumococcal strains were highly virulent in a mouse model of invasive pneumonia, but in contrast to the generally accepted assumption, can also successfully establish nasopharyngeal carriage. Interestingly, we found that cocolonizing serotypes may proliferate in the presence of serotype 1, suggesting that acquisition of serotype 1 carriage could increase the risk of developing IPD by other serotypes. RNA sequencing analysis confirmed that key virulence genes associated with inflammation and tissue invasiveness were upregulated in serotype 1. These data reveal important new insights into serotype 1 pathogenesis, with implications for carriage potential and risk of invasive disease through interactions with other cocolonizing serotypes, an often-overlooked factor in transmission and disease progression.IMPORTANCE The pneumococcus causes serious diseases such as pneumonia, sepsis, and meningitis and is a major cause of morbidity and mortality worldwide. Serotype 1 accounts for the majority of invasive pneumococcal disease cases in sub-Saharan Africa but is rarely found during nasopharyngeal carriage. Understanding the mechanisms leading to nasopharyngeal carriage and invasive disease by this serotype can help reduce its burden on health care systems worldwide. In this study, we also uncovered the potential impact of serotype 1 on disease progression of other coinfecting serotypes, which can have important implications for vaccine efficacy. Understanding the interactions between different serotypes during nasopharyngeal carriage may lead to improved intervention methods and therapies to reduce pneumococcal invasive disease levels.

Influenza virus-mediated suppression of bronchial Chitinase-3-like 1 secretion promotes secondary pneumococcal infection.

Infections of the lung are among the leading causes of death worldwide. Despite the preactivation of innate defense programs during viral infection, secondary bacterial infection substantially elevates morbidity and mortality rates. Particularly problematic are co-infections with influenza A virus (IAV) and the major bacterial pathogen Streptococcus pneumoniae. However, the molecular processes underlying the severe course of such co-infections are not fully understood. Previously, the absence of secreted glycoprotein Chitinase-3-like 1 (CHI3L1) was shown to increase pneumococcal replication in mice. We therefore hypothesized that an IAV preinfection decreases CHI3L1 levels to promote pneumococcal infection. Indeed, in an air-liquid interface model of primary human bronchial epithelial cells (hBECs), IAV preinfection interfered with apical but not basolateral CHI3L1 release. Confocal time-lapse microscopy revealed that the gradual loss of apical CHI3L1 localization during co-infection with influenza and S. pneumoniae coincided with the disappearance of goblet as well as ciliated cells and increased S. pneumoniae replication. Importantly, extracellular restoration of CHI3L1 levels using recombinant protein significantly reduced bacterial load in influenza preinfected bronchial models. Thus, recombinant CHI3L1 may provide a novel therapeutic means to lower morbidity and mortality associated with post-influenza pneumococcal infections.