Faculty Bibliography

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.

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.

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.

Bacterial cell-surface proteins play integral roles in host-pathogen interactions. These proteins are often architecturally and functionally sophisticated and yet few studies of such proteins involved in host-pathogen interactions have defined the domains or modules required for specific functions. Streptococcus pneumoniae (pneumococcus), an opportunistic pathogen that is a leading cause of community acquired pneumonia, otitis media and bacteremia, is decorated with many complex surface proteins. These include beta-galactosidase BgaA, which is specific for terminal galactose residues beta-1-4 linked to glucose or N-acetylglucosamine and known to play a role in pneumococcal growth, resistance to opsonophagocytic killing, and adherence. This study defines the domains and modules of BgaA that are required for these distinct contributions to pneumococcal pathogenesis. Inhibitors of beta-galactosidase activity reduced pneumococcal growth and increased opsonophagocytic killing in a BgaA dependent manner, indicating these functions require BgaA enzymatic activity. In contrast, inhibitors increased pneumococcal adherence suggesting that BgaA bound a substrate of the enzyme through a distinct module or domain. Extensive biochemical, structural and cell based studies revealed two newly identified non-enzymatic carbohydrate-binding modules (CBMs) mediate adherence to the host cell surface displayed lactose or N-acetyllactosamine. This finding is important to pneumococcal biology as it is the first adhesin-carbohydrate receptor pair identified, supporting the widely held belief that initial pneumococcal attachment is to a glycoconjugate. Perhaps more importantly, this is the first demonstration that a CBM within a carbohydrate-active enzyme can mediate adherence to host cells and thus this study identifies a new class of carbohydrate-binding adhesins and extends the paradigm of CBM function. As other bacterial species express surface-associated carbohydrate-active enzymes containing CBMs these findings have broad implications for bacterial adherence. Together, these data illustrate that comprehending the architectural sophistication of surface-attached proteins can increase our understanding of the different mechanisms by which these proteins can contribute to bacterial pathogenesis.

While the importance of transmission of pathogens is widely accepted, there is currently little mechanistic understanding of this process. Nasal carriage of Streptococcus pneumoniae (the pneumococcus) is common in humans, especially in early childhood, and is a prerequisite for the development of disease and transmission among hosts. In this study, we adapted an infant mouse model to elucidate host determinants of transmission of S. pneumoniae from inoculated index mice to uninfected contact mice. In the context of co-infection with influenza A virus, the pneumococcus was transmitted among wildtype littermates, with approximately half of the contact mice acquiring colonization. Mice deficient for TLR2 were colonized to a similar density but transmitted S. pneumoniae more efficiently (100% transmission) than wildtype animals and showed decreased expression of interferon alpha and higher viral titers. The greater viral burden in tlr2-/- mice correlated with heightened inflammation, and was responsible for an increase in bacterial shedding from the mouse nose. The role of TLR2 signaling was confirmed by intranasal treatment of wildtype mice with the agonist Pam3Cys, which decreased inflammation and reduced bacterial shedding and transmission. Taken together, these results suggest that the innate immune response to influenza virus promotes bacterial shedding, allowing the bacteria to transit from host to host. These findings provide insight into the role of host factors in the increased pneumococcal carriage rates seen during flu season and contribute to our overall understanding of pathogen transmission.

The response to influenza virus (IAV) infection and severity of disease is highly variable in humans. We hypothesized that one factor contributing to this variability is the presence of specific respiratory tract (RT) microbes. One such microbe is Streptococcus pneumoniae (Sp) that is carried asymptomatically in the RT of many humans. In a mouse co-infection model we found that in contrast to secondary bacterial infection that exacerbates disease, Sp colonization 10 days prior to IAV protects from virus-induced morbidity and lung pathology. Using mutant Sp strains, we identified a critical role for the bacterial virulence factor pneumolysin (PLY) in mediating this protection. Colonization with the PLY-sufficient Sp strain induces expression of the immune-suppressive enzyme arginase 1 in alveolar macrophages (aMo) and correlates with attenuated recruitment and function of pulmonary inflammatory cells. Our study demonstrates a novel role for PLY in Sp-mediated protection by maintaining aMo as "gatekeepers" against virus-induced immunopathology.

Nontypeable Haemophilus influenzae is a frequent cause of noninvasive mucosal inflammatory diseases but may also cause invasive diseases, such as sepsis and meningitis, especially in children and the elderly. Infection by nontypeable Haemophilus influenzae is characterized by recruitment of neutrophilic granulocytes. Despite the presence of a large number of neutrophils, infections with nontypeable Haemophilus influenzae are often not cleared effectively by the antimicrobial activity of these immune cells. Herein, we examined how nontypeable Haemophilus influenzae evades neutrophil-mediated killing. Transposon sequencing (Tn-seq) was used on an isolate resistant to neutrophil-mediated killing to identify genes required for its survival in the presence of human neutrophils and serum, which provided a source of complement and antibodies. Results show that nontypeable Haemophilus influenzae prevents complement-dependent neutrophil-mediated killing by expression of surface galactose-containing oligosaccharide structures. These outer-core structures block recognition of an inner-core lipooligosaccharide epitope containing glucose attached to heptose HepIII-beta1,2-Glc by replacement with galactose attached to HepIII or through shielding HepIII-beta1,2-Glc by phase-variable attachment of oligosaccharide chain extensions. When the HepIII-beta1,2-Glc-containing epitope is expressed and exposed, nontypeable Haemophilus influenzae is opsonized by naturally acquired IgM generally present in human serum and subsequently phagocytosed and killed by human neutrophils. Clinical nontypeable Haemophilus influenzae isolates containing galactose attached to HepIII that are not recognized by this IgM are more often found to cause invasive infections. Importance: Neutrophils are white blood cells that specialize in killing pathogens and are recruited to sites of inflammation. However, despite the presence of large numbers of neutrophils in the middle ear cavity and lungs of patients with otitis media or chronic obstructive pulmonary disease, respectively, the bacterium nontypeable Haemophilus influenzae is often not effectively cleared from these locations by these immune cells. In order to understand how nontypeable Haemophilus influenzae is able to cause inflammatory diseases in the presence of neutrophils, we determined the mechanism that underlies resistance to neutrophil-mediated killing. We have shown that nontypeable Haemophilus influenzae prevents binding of antibodies of the IgM subtype through changes in their surface lipooligosaccharide structure, thereby preventing complement activation and clearance by human neutrophils.

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.

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.