Faculty Bibliography

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.

Host-to-host transmission is a necessary but poorly understood aspect of microbial pathogenesis. Herein, we screened a genomic library of mutants of the leading respiratory pathogen Streptococcus pneumoniae generated by mariner transposon mutagenesis (Tn-Seq) to identify genes contributing to its exit or shedding from the upper respiratory tract (URT), the limiting step in the organism's transmission in an infant mouse model. Our analysis focused on genes affecting the bacterial surface that directly impact interactions with the host. Among the multiple factors identified was the dlt locus, which adds d-alanine onto lipoteichoic acids (LTA) and thereby increases Toll-like receptor 2-mediated inflammation and resistance to antimicrobial peptides. The more robust proinflammatory response in the presence of d-alanylation promotes secretions that facilitate pneumococcal shedding and allows for transmission. Expression of the dlt locus is controlled by the CiaRH system, which senses cell wall stress in response to antimicrobial activity, including in response to lysozyme, the most abundant antimicrobial along the URT mucosa. Accordingly, in a lysM^-/- host, there was no longer an effect of the dlt locus on pneumococcal shedding. Thus, our findings demonstrate how a pathogen senses the URT milieu and then modifies its surface characteristics to take advantage of the host response for transit to another host.IMPORTANCEStreptococcus pneumoniae (the pneumococcus) is a common cause of respiratory tract and invasive infection. The overall effectiveness of immunization with the organism's capsular polysaccharide depends on its ability to block colonization of the upper respiratory tract and thereby prevent host-to-host transmission. Because of the limited coverage of current pneumococcal vaccines, we carried out an unbiased in vivo transposon mutagenesis screen to identify pneumococcal factors other than its capsular polysaccharide that affect transmission. One such candidate was expressed by the dlt locus, previously shown to add d-alanine onto the pneumococcal lipoteichoic acid present on the bacterial cell surface. This modification protects against host antimicrobials and augments host inflammatory responses. The latter increases secretions and bacterial shedding from the upper respiratory tract to allow for transmission. Thus, this study provides insight into a mechanism employed by the pneumococcus to successfully transit from one host to another.

Streptococcus pneumoniae is a common nasopharyngeal colonizer, but can also cause life-threatening invasive diseases such as empyema, bacteremia and meningitis. Genetic variation of host and pathogen is known to play a role in invasive pneumococcal disease, though to what extent is unknown. In a genome-wide association study of human and pathogen we show that human variation explains almost half of variation in susceptibility to pneumococcal meningitis and one-third of variation in severity, identifying variants in CCDC33 associated with susceptibility. Pneumococcal genetic variation explains a large amount of invasive potential (70%), but has no effect on severity. Serotype alone is insufficient to explain invasiveness, suggesting other pneumococcal factors are involved in progression to invasive disease. We identify pneumococcal genes involved in invasiveness including pspC and zmpD, and perform a human-bacteria interaction analysis. These genes are potential candidates for the development of more broadly-acting pneumococcal vaccines.