Faculty Bibliography

Infections are a common cause of infant mortality worldwide, especially due to Streptococcus pneumoniae. Colonization is the prerequisite to invasive pneumococcal disease, and is particularly frequent and prolonged in children, though the mechanisms underlying this susceptibility are unknown. We find that infant mice exhibit prolonged pneumococcal carriage, and are delayed in recruiting macrophages, the effector cells of clearance, into the nasopharyngeal lumen. This lack of macrophage recruitment is paralleled by a failure to upregulate chemokine (C-C) motif ligand 2 (Ccl2 or Mcp-1), a macrophage chemoattractant that is required in adult mice to promote clearance. Baseline expression of Ccl2 and the related chemokine Ccl7 is higher in the infant compared to the adult upper respiratory tract, and this effect requires the infant microbiota. These results demonstrate that signals governing macrophage recruitment are altered at baseline in infant mice, which prevents the development of appropriate innate cell infiltration in response to pneumococcal colonization, delaying clearance of pneumococcal carriage.

Respiratory tract infections are an important cause of morbidity and mortality worldwide. Chief among these are infections involving the lower airways. The opportunistic bacterial pathogens responsible for most cases of pneumonia can cause a range of local and invasive infections. However, bacterial colonization (or carriage) in the upper airway is the prerequisite of all these infections. Successful colonizers must attach to the epithelial lining, grow on the nutrient-limited mucosal surface, evade the host immune response, and transmit to a susceptible host. Here, we review the molecular mechanisms underlying these conserved stages of carriage. We also examine how the demands of colonization influence progression to disease. A range of bacteria can colonize the upper airway; nevertheless, we focus on strategies shared by many respiratory tract opportunistic pathogens. Understanding colonization opens a window to the evolutionary pressures these pathogens face within their animal hosts and that have selected for attributes that contribute to virulence and pathogenesis.

Regulation of neutrophil activity is critical for immune evasion among extracellular pathogens, yet the mechanisms by which many bacteria disrupt phagocyte function remain unclear. Here, we have shown that the respiratory pathogen Streptococcus pneumoniae disables neutrophils by exploiting molecular mimicry to degrade platelet-activating factor (PAF), a host-derived inflammatory phospholipid. Using mass spectrometry and murine upper airway infection models, we demonstrated that phosphorylcholine (ChoP) moieties that are shared by PAF and the bacterial cell wall allow S. pneumoniae to leverage a ChoP-remodeling enzyme (Pce) to remove PAF from the airway. S. pneumoniae-mediated PAF deprivation impaired viability, activation, and bactericidal capacity among responding neutrophils. In the absence of Pce, neutrophils rapidly cleared S. pneumoniae from the airway and impeded invasive disease and transmission between mice. Abrogation of PAF signaling rendered Pce dispensable for S. pneumoniae persistence, reinforcing that this enzyme deprives neutrophils of essential PAF-mediated stimulation. Accordingly, exogenous activation of neutrophils overwhelmed Pce-mediated phagocyte disruption. Haemophilus influenzae also uses an enzyme, GlpQ, to hydrolyze ChoP and subvert PAF function, suggesting that mimicry-driven immune evasion is a common paradigm among respiratory pathogens. These results identify a mechanism by which shared molecular structures enable microbial enzymes to subvert host lipid signaling, suppress inflammation, and ensure bacterial persistence at the mucosa.

Streptococcus pneumoniae (the pneumococcus), a leading cause of bacterial disease, is most commonly carried in the human nasopharynx. Colonization induces inflammation that promotes the organism's growth and transmission. This inflammatory response is dependent on intracellular sensing of bacterial components that access the cytosolic compartment via the pneumococcal pore-forming toxin pneumolysin. In vitro, cytosolic access results in cell death that includes release of the proinflammatory cytokine interleukin-1beta (IL-1beta). IL-1 family cytokines, including IL-1beta, are secreted upon activation of inflammasomes, although the role of this activation in the host immune response to pneumococcal carriage is unknown. Using a murine model of pneumococcal nasopharyngeal colonization, we show that mice deficient in the interleukin-1 receptor type 1 (Il1r1(-/-)) have reduced numbers of neutrophils early after infection, fewer macrophages later in carriage, and prolonged bacterial colonization. Moreover, intranasal administration of Il-1beta promoted clearance. Macrophages are the effectors of clearance, and characterization of macrophage chemokines in colonized mice revealed that Il1r1(-/-) mice have lower expression of the C-C motif chemokine ligand 6 (CCL6), correlating with reduced macrophage recruitment to the nasopharynx. IL-1 family cytokines are known to promote adaptive immunity; however, we observed no difference in the development of humoral or cellular immunity to pneumococcal colonization between wild-type and Il1r1(-/-) mice. Our findings show that sensing of IL-1 cytokines during colonization promotes inflammation without immunity, which may ultimately benefit the pneumococcus.

Non-typeable Haemophilus influenzae is an opportunistic pathogen of the human upper respiratory tract and is often found to cause inflammatory diseases that include sinusitis, otitis media and exacerbations of chronic obstructive pulmonary disease. To persist in the inflammatory milieu during infection, non-typeable H. influenzae must resist the antimicrobial activity of the human complement system. Here, we used Tn-seq to identify genes important for resistance to complement-mediated killing. This screen identified outer membrane protein P5 in evasion of the alternative pathway of complement activation. Outer membrane protein P5 was shown to bind human complement regulatory protein factor H directly, thereby, preventing complement factor C3 deposition on the surface of the bacterium. Furthermore, we show that amino acid variation within surface-exposed regions within outer membrane P5 affected the level of factor H binding between individual strains.

The pathogenesis of the disease caused by Streptococcus pneumoniae begins with colonization of the upper respiratory tract. Temperate phages have been identified in the genomes of up to 70% of clinical isolates. How these phages affect the bacterial host during colonization is unknown. Here, we examined a clinical isolate that carries a novel prophage element, designated Spn1, which was detected in both integrated and episomal forms. Surprisingly, both lytic and lysogenic Spn1 genes were expressed under routine growth conditions. Using a mouse model of asymptomatic colonization, we demonstrate that the Spn1(-) strain outcompeted the Spn1(+) strain >70-fold. To determine if Spn1 causes a fitness defect through a trans-acting factor, we constructed an Spn1(+) mutant that does not become an episome or express phage genes. This mutant competed equally with the Spn1(-) strain, indicating that expression of phage genes or phage lytic activity is required to confer this fitness defect. In vitro, we demonstrate that the presence of Spn1 correlated with a defect in LytA-mediated autolysis. Furthermore, the Spn1(+) strain displayed increased chain length and resistance to lysis by penicillin compared to the Spn(-) strain, indicating that Spn1 alters the cell wall physiology of its host strain. We posit that these changes in cell wall physiology allow for tolerance of phage gene products and are responsible for the relative defect of the Spn1(+) strain during colonization. This study provides new insight into how bacteria and prophages interact and affect bacterial fitness in vivo.

Human genetic polymorphisms associated with decreased expression of macrophage migration inhibitory factor (MIF) have been linked to the risk of community-acquired pneumonia. Because Streptococcus pneumoniae is the leading cause of community-acquired pneumonia and nasal carriage is a precursor to invasive disease, we explored the role of MIF in the clearance of pneumococcal colonization in a mouse model. MIF-deficient mice (Mif(-/-)) showed prolonged colonization with both avirulent (23F) and virulent (6A) pneumococcal serotypes compared with wild-type animals. Pneumococcal carriage led to both local upregulation of MIF expression and systemic increase of the cytokine. Delayed clearance in the Mif(-/-) mice was correlated with reduced numbers of macrophages in upper respiratory tract lavages as well as impaired upregulation of MCP-1/CCL2. We found that primary human monocyte-derived macrophages as well as THP-1 macrophages produced MIF upon pneumococcal infection in a pneumolysin-dependent manner. Pneumolysin-induced MIF production required its pore-forming activity and phosphorylation of p38-MAPK in macrophages, with sustained p38-MAPK phosphorylation abrogated in the setting of MIF deficiency. Challenge with pneumolysin-deficient bacteria demonstrated reduced MIF upregulation, decreased numbers of macrophages in the nasopharynx, and less effective clearance. Mif(-/-) mice also showed reduced Ab response to pneumococcal colonization and impaired ability to clear secondary carriage. Finally, local administration of MIF was able to restore bacterial clearance and macrophage accumulation in Mif(-/-) mice. Our work suggests that MIF is important for innate and adaptive immunity to pneumococcal colonization and could be a contributing factor in genetic differences in pneumococcal disease susceptibility.

Much of the mortality attributed to influenza virus is due to secondary bacterial pneumonia, particularly from Streptococcus pneumoniae. However, mechanisms underlying this coinfection are incompletely understood. We find that prior influenza infection enhances pneumococcal colonization of the murine nasopharynx, which in turn promotes bacterial spread to the lungs. Influenza accelerates bacterial replication in vivo, and sialic acid, a major component of airway glycoconjugates, is identified as the host-derived metabolite that stimulates pneumococcal proliferation. Influenza infection increases sialic acid and sialylated mucin availability and enhances desialylation of host glycoconjugates. Pneumococcal genes for sialic acid catabolism are required for influenza to promote bacterial growth. Decreasing sialic acid availability in vivo by genetic deletion of the major airway mucin Muc5ac or mucolytic treatment limits influenza-induced pneumococcal replication. Our findings suggest that higher rates of disease during coinfection could stem from influenza-provided sialic acid, which increases pneumococcal proliferation, colonization, and aspiration.

Pathogen-specific antibodies (Abs) protect against respiratory infection with influenza A virus (IAV) and Streptococcus pneumoniae and are the basis of effective vaccines. Sequential or overlapping coinfections with both pathogens are common, yet the impact of coinfection on the generation and maintenance of Ab responses is largely unknown. We report here that the B cell response to IAV is altered in mice coinfected with IAV and S. pneumoniae and that this response differs, depending on the order of pathogen exposure. In mice exposed to S. pneumoniae prior to IAV, the initial virus-specific germinal center (GC) B cell response is significantly enhanced in the lung-draining mediastinal lymph node and spleen, and there is an increase in CD4(+) T follicular helper (TFH) cell numbers. In contrast, secondary S. pneumoniae infection exaggerates early antiviral antibody-secreting cell formation, and at later times, levels of GCs, TFH cells, and antiviral serum IgG are elevated. Mice exposed to S. pneumoniae prior to IAV do not maintain the initially robust GC response in secondary lymphoid organs and exhibit reduced antiviral serum IgG with diminished virus neutralization activity a month after infection. Our data suggest that the history of pathogen exposures can critically affect the generation of protective antiviral Abs and may partially explain the differential susceptibility to and disease outcomes from IAV infection in humans. IMPORTANCE: Respiratory tract coinfections, specifically those involving influenza A viruses and Streptococcus pneumoniae, remain a top global health burden. We sought to determine how S. pneumoniae coinfection modulates the B cell immune response to influenza virus since antibodies are key mediators of protection.

Neonatal colonization by microbes, which begins immediately after birth, is influenced by gestational age and the mother's microbiota and is modified by exposure to antibiotics. In neonates, prolonged duration of antibiotic therapy is associated with increased risk of late-onset sepsis (LOS), a disorder controlled by neutrophils. A role for the microbiota in regulating neutrophil development and susceptibility to sepsis in the neonate remains unclear. We exposed pregnant mouse dams to antibiotics in drinking water to limit transfer of maternal microbes to the neonates. Antibiotic exposure of dams decreased the total number and composition of microbes in the intestine of the neonates. This was associated with decreased numbers of circulating and bone marrow neutrophils and granulocyte/macrophage-restricted progenitor cells in the bone marrow of antibiotic-treated and germ-free neonates. Antibiotic exposure of dams reduced the number of interleukin-17 (IL-17)-producing cells in the intestine and production of granulocyte colony-stimulating factor (G-CSF). Granulocytopenia was associated with impaired host defense and increased susceptibility to Escherichia coli K1 and Klebsiella pneumoniae sepsis in antibiotic-treated neonates, which could be partially reversed by administration of G-CSF. Transfer of a normal microbiota into antibiotic-treated neonates induced IL-17 production by group 3 innate lymphoid cells (ILCs) in the intestine, increasing plasma G-CSF levels and neutrophil numbers in a Toll-like receptor 4 (TLR4)- and myeloid differentiation factor 88 (MyD88)-dependent manner and restored IL-17-dependent resistance to sepsis. Specific depletion of ILCs prevented IL-17- and G-CSF-dependent granulocytosis and resistance to sepsis. These data support a role for the intestinal microbiota in regulation of granulocytosis, neutrophil homeostasis and host resistance to sepsis in neonates.