Faculty Bibliography

Capsule-switch mutants were compared to analyze how serotype affects the success of Streptococcus pneumoniae (Spn) during colonization and transmission. Strains of multiple serotypes were tested in highly susceptible infant mice, both singly and in competitive assays. Our findings demonstrated a role of serotype, apart from genetic background, in competitive success of strains, but this depended on timing postinoculation. As is the case for natural carriage, there was a hierarchy of success among serotypes using capsule-switch strains. The long-term dominance of a serotype was established within the first 4 h after acquisition, suggesting an effect independent of Spn-induced host responses. The hierarchy of serotype dominance correlated with decreased clearance rather than increased growth in vivo. Competitive assays staggering the timing of challenge showed that the first strain to dominate the niche sustained its competitive advantage, potentially explaining how increased density from delayed early clearance could result in serotype-dependent success. Effector molecules of intrastrain competition (fratricide), regulated by the competence operon in a quorum-sensing mechanism, were required for early niche dominance. This suggested a winner-takes-all scenario in which serotype is a major factor in achieving early niche dominance, such that once a strain reaches a threshold density it is able to exclude competitors through fratricide. Serotype was also an important determinant of transmission dynamics, although transit to a recipient host depended on effects of serotype different from its contribution to the dominance of colonization in the donor host. IMPORTANCE Capsule is the major virulence factor and surface antigen of the opportunistic respiratory pathogen Streptococcus pneumoniae (Spn). Strains of Spn express at least 100 structurally and immunologically distinct types (serotypes) of capsule, but for unknown reasons only a few are common. The effect of serotypes during the commensal interactions of Spn and its host, colonization and transmission, was tested. This was carried out by comparing genetically modified strains differing only in serotype in infant mouse models. Results show that serotype is an important factor in a strain's success during colonization. This was attributed to the effect of serotype on early clearance of the organism in the host. Competitive factors expressed by Spn (in a mechanism referred to as fratricide) allow the strain gaining this initial advantage to then dominate the upper respiratory tract niche. Serotype also plays an important role in a strain's success during transmission from one host to another.

Vaccines targeting Streptococcus pneumoniae (Spn) are limited by dependence on capsular polysaccharide and its serotype diversity. More broadly-based approaches using common protein antigens have not resulted in a licensed vaccine. Herein, we used an unbiased, genome-wide approach to find novel vaccine antigens to disrupt carriage modeled in mice. A Tn-Seq screen identified 198 genes required for colonization of which 16 are known to express conserved, immunogenic surface proteins. After testing defined mutants for impaired colonization of infant and adult mice, 5 validated candidates (StkP, PenA/Pbp2a, PgdA, HtrA, and LytD/Pce/CbpE) were used as immunogens. Despite induction of antibody recognizing the Spn cell surface, there was no protection against Spn colonization. There was, however, protection against an unencapsulated Spn mutant. This result correlated with increased antibody binding to the bacterial surface in the absence of capsule. Our findings demonstrate how the pneumococcal capsule interferes with mucosal protection by antibody to common protein targets.

Young age is a risk factor for respiratory and gastrointestinal infections. Here, we compared infant and adult mice to identify age-dependent mechanisms that drive susceptibility to mucosal infections during early life. Transcriptional profiling of the upper respiratory tract (URT) epithelium revealed significant dampening of early life innate mucosal defenses. Epithelial-mediated production of the most abundant antimicrobial molecules, lysozyme, and lactoferrin, and the polymeric immunoglobulin receptor (pIgR), responsible for IgA transcytosis, was expressed in an age-dependent manner. This was attributed to delayed functional development of serous cells. Absence of epithelial-derived lysozyme and the pIgR was also observed in the small intestine during early life. Infection of infant mice with lysozyme-susceptible strains of Streptococcus pneumoniae or Staphylococcus aureus in the URT or gastrointestinal tract, respectively, demonstrated an age-dependent regulation of lysozyme enzymatic activity. Lysozyme derived from maternal milk partially compensated for the reduction in URT lysozyme activity of infant mice. Similar to our observations in mice, expression of lysozyme and the pIgR in nasopharyngeal samples collected from healthy human infants during the first year of life followed an age-dependent regulation. Thus, a global pattern of reduced antimicrobial and IgA-mediated defenses may contribute to increased susceptibility of young children to mucosal infections.

Rationale Cross-sectional human data suggest that enrichment of oral anaerobic bacteria in the lung is associated with increased Th17 inflammatory phenotype. In this study we evaluated the microbial and host immune response dynamics after aspiration with a oral commensals using a preclinical mouse model. Methods Aspiration with a mixture of human oral commensals (MOC; Prevotella melaninogenica, Veillonella parvula, and Streptococcus mitis) was modeled in mice followed by variable time of sacrifice. Genetic background of mice included WT, MyD88 knock out and STAT3C. Measurements 16S rRNA gene sequencing characterized changes in microbiota. Flow cytometry, cytokine measurement via Luminex and RNA host transcriptome sequencing was used to characterize host immune phenotype. Main Results While MOC aspiration correlated with lower airway dysbiosis that resolved within five days, it induced an extended inflammatory response associated with IL17-producing T-cells lasting at least 14 days. MyD88 expression was required for the IL-17 response to MOC aspiration, but not for T-cell activation or IFN-γ expression. MOC aspiration prior to a respiratory challenge with S. pneumoniae led to a decreased in host's susceptibility to this pathogen. Conclusions Thus, in otherwise healthy mice, a single aspiration event with oral commensals are rapidly cleared from the lower airways, but induce a prolonged Th17 response that secondarily decreased susceptibility to respiratory pathogens. Translationally, these data implicate an immuno-protective role of episodic microaspiration of oral microbes in the regulation of the lung immune phenotype and mitigation of host susceptibility to infection with lower airway pathogens.

Binding of Streptococcus pneumoniae (Spn) to nasal mucus leads to entrapment and clearance via mucociliary activity during colonization. To identify Spn factors allowing for evasion of mucus binding, we used a solid-phase adherence assay with immobilized mucus of human and murine origin. Spn bound large mucus particles through interactions with carbohydrate moieties. Mutants lacking neuraminidase A (nanA) or neuraminidase B (nanB) showed increased mucus binding that correlated with diminished removal of terminal sialic acid residues on bound mucus. The non-additive activity of the two enzymes raised the question why Spn expresses two neuraminidases and suggested they function in the same pathway. Transcriptional analysis demonstrated expression of nanA depends on the enzymatic function of NanB. As transcription of nanA is increased in the presence of sialic acid, our findings suggest that sialic acid liberated from host glycoconjugates by the secreted enzyme NanB induces the expression of the cell-associated enzyme NanA. The absence of detectable mucus desialylation in the nanA mutant, in which NanB is still expressed, suggests that NanA is responsible for the bulk of the modification of host glycoconjugates. Thus, our studies describe a functional role for NanB in sialic acid sensing in the host. The contribution of the neuraminidases in vivo was then assessed in a murine model of colonization. Although mucus-binding mutants showed an early advantage, this was only observed in a competitive infection, suggesting a complex role of neuraminidases. Histologic examination of the upper respiratory tract demonstrated that Spn stimulates mucus production in a neuraminidase-dependent manner. Thus, an increase production of mucus containing secretions appears to be balanced, in vivo, by decreased mucus binding. We postulate that through the combined activity of its neuraminidases, Spn evades mucus binding and mucociliary clearance, which is needed to counter neuraminidase-mediated stimulation of mucus secretions.

Understanding antibody responses to SARS-CoV-2 is indispensable for the development of containment measures to overcome the current COVID-19 pandemic. Recent studies showed that serum from convalescent patients can display variable neutralization capacities. Still, it remains unclear whether there are specific signatures that can be used to predict neutralization. Here, we performed a detailed analysis of sera from a cohort of 101 recovered healthcare workers and we addressed their SARS-CoV-2 antibody response by ELISA against SARS-CoV-2 Spike receptor binding domain and nucleoprotein. Both ELISA methods detected sustained levels of serum IgG against both antigens. Yet, the majority of individuals from our cohort generated antibodies with low neutralization capacity and only 6% showed high neutralizing titers against both authentic SARS-CoV-2 virus and the Spike pseudotyped virus. Interestingly, higher neutralizing sera correlate with detection of -IgG, IgM and IgA antibodies against both antigens, while individuals with positive IgG alone showed poor neutralization response. These results suggest that having a broader repertoire of antibodies may contribute to more potent SARS-CoV-2 neutralization. Altogether, our work provides a cross sectional snapshot of the SARS-CoV-2 neutralizing antibody response in recovered healthcare workers and provides preliminary evidence that possessing multiple antibody isotypes can play an important role in predicting SARS-CoV-2 neutralization.

The dynamics underlying respiratory contagion (the transmission of infectious agents from the airways) are poorly understood. We investigated host factors involved in the transmission of the leading respiratory pathogen Streptococcus pneumoniae Using an infant mouse model, we examined whether S. pneumoniae triggers inflammatory pathways shared by influenza A virus (IAV) to promote nasal secretions and shedding from the upper respiratory tract to facilitate transit to new hosts. Here, we show that amplification of the type I interferon (IFN-I) response is a critical host factor in this process, as shedding and transmission by both IAV and S. pneumoniae were decreased in pups lacking the common IFN-I receptor (Ifnar1^-/- mice). Additionally, providing exogenous recombinant IFN-I to S. pneumoniae-infected pups was sufficient to increase bacterial shedding. The expression of IFN-stimulated genes (ISGs) was upregulated in S. pneumoniae-infected wild-type (WT) but not Ifnar1^-/- mice, including genes involved in mucin type O-glycan biosynthesis; this correlated with an increase in secretions in S. pneumoniae- and IAV-infected WT compared to Ifnar1^-/- pups. S. pneumoniae stimulation of ISGs was largely dependent on its pore-forming toxin, pneumolysin, and coinfection with IAV and S. pneumoniae resulted in synergistic increases in ISG expression. We conclude that the induction of IFN-I signaling appears to be a common factor driving viral and bacterial respiratory contagion.IMPORTANCE Respiratory tract infections are a leading cause of childhood mortality and, globally, Streptococcus pneumoniae is the leading cause of mortality due to pneumonia. Transmission of S. pneumoniae primarily occurs through direct contact with respiratory secretions, although the host and bacterial factors underlying transmission are poorly understood. We examined transmission dynamics of S. pneumoniae in an infant mouse model and here show that S. pneumoniae colonization of the upper respiratory tract stimulates host inflammatory pathways commonly associated with viral infections. Amplification of this response was shown to be a critical host factor driving shedding and transmission of both S. pneumoniae and influenza A virus, with infection stimulating expression of a wide variety of genes, including those involved in the biosynthesis of mucin, a major component of respiratory secretions. Our findings suggest a mechanism facilitating S. pneumoniae contagion that is shared by viral infection.

Streptococcus pneumoniae, one of the most common commensal pathogens among children, is spread by close contact in daycare centers or within a family. Host innate immune responses and bacterial virulence factors promote pneumococcal transmission. However, investigations into the effects of environmental factors on transmission have been limited. Passive smoking, a great concern for children's health, has been reported to exacerbate pneumococcal diseases. Here, we describe the effect of cigarette smoke exposure on an infant mouse model of pneumococcal transmission. Our findings reveal that the effect of cigarette smoke exposure significantly promotes pneumococcal transmission by enhancing bacterial shedding from the colonized host and by increasing susceptibility to pneumococcal colonization in the new host, both of which are critical steps of transmission. Local inflammation, followed by mucosal changes (such as mucus hypersecretion and disruption of the mucosal barrier), are important underlying mechanisms for promotion of transmission by smoke exposure. These effects were attributable to the constituents of cigarette smoke rather than smoke itself. These findings provide the first experimental evidence of the impact of environmental factors on pneumococcal transmission and the mechanism of pathogenesis.

Successful infection by mucosal pathogens requires overcoming the mucus barrier. To better understand this key step, we performed a survey of the interactions between human respiratory mucus and the human pathogen Streptococcus pneumoniae. Pneumococcal adherence to adult human nasal fluid was seen only by isolates expressing pilus-1. Robust binding was independent of pilus-1 adhesive properties but required Fab-dependent recognition of RrgB, the pilus shaft protein, by naturally acquired secretory IgA (sIgA). Pilus-1 binding by specific sIgA led to bacterial agglutination, but adherence required interaction of agglutinated pneumococci and entrapment in mucus particles. To test the effect of these interactions in vivo, pneumococci were preincubated with human sIgA before intranasal challenge in a mouse model of colonization. sIgA treatment resulted in rapid immune exclusion of pilus-expressing pneumococci. Our findings predict that immune exclusion would select for nonpiliated isolates in individuals who acquired RrgB-specific sIgA from prior episodes of colonization with piliated strains. Accordingly, genomic data comparing isolates carried by mothers and their children showed that mothers are less likely to be colonized with pilus-expressing strains. Our study provides a specific example of immune exclusion involving naturally acquired antibody in the human host, a major factor driving pneumococcal adaptation.

Streptococcus pneumoniae is a major cause of pneumonia, wherein infection of respiratory mucosa drives a robust influx of neutrophils. We have previously shown that S. pneumoniae infection of the respiratory epithelium induces the production of the 12-lipoxygenase (12-LOX)-dependent lipid inflammatory mediator hepoxilin A3, which promotes recruitment of neutrophils into the airways, tissue damage, and lethal septicemia. Pneumolysin (PLY), a member of the cholesterol-dependent cytolysin (CDC) family, is a major S. pneumoniae virulence factor that generates ∼25-nm diameter pores in eukaryotic membranes and promotes acute inflammation, tissue damage, and bacteremia. We show that a PLY-deficient S. pneumoniae mutant was impaired in triggering human neutrophil transepithelial migration in vitro. Ectopic production of PLY endowed the nonpathogenic Bacillus subtilis with the ability to trigger neutrophil recruitment across human-cultured monolayers. Purified PLY, several other CDC family members, and the α-toxin of Clostridium septicum, which generates pores with cross-sectional areas nearly 300 times smaller than CDCs, reproduced this robust neutrophil transmigration. PLY non-pore-forming point mutants that are trapped at various stages of pore assembly did not recruit neutrophils. PLY triggered neutrophil recruitment in a 12-LOX-dependent manner in vitro. Instillation of wild-type PLY but not inactive derivatives into the lungs of mice induced robust 12-LOX-dependent neutrophil migration into the airways, although residual inflammation induced by PLY in 12-LOX-deficient mice indicates that 12-LOX-independent pathways also contribute to PLY-triggered pulmonary inflammation. These data indicate that PLY is an important factor in promoting hepoxilin A3-dependent neutrophil recruitment across pulmonary epithelium in a pore-dependent fashion.