Faculty Bibliography

Host-microbe interactions often begin with colonization of mucosal surfaces. These relationships are highly specific, as certain microbial species are found only in particular microenvironments. Transcriptional microarrays were used to screen host genes whose expression in the murine nasal mucosa was affected by colonization with the Gram-positive bacterium Streptococcus pneumoniae. Siderocalin (Scn, or lipocalin 2 or neutrophil gelatinase-associated lipocalin) expression was increased up to 65-fold during colonization by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Western analysis showed that Scn was secreted into airway surface fluid in colonized animals. Immunohistochemical analysis localized Scn expression primarily to secretory Bowman's glands. Similar results were observed during colonization with the Gram-negative bacterium Haemophilus influenzae, suggesting that Scn secretion is a general response. Western analysis of human nasal secretions also demonstrated secretion of Scn at potentially bacteriostatic levels. This is a previously unrecognized response that may have a role in determining the establishment or maintenance of mucosal colonization. Scn contributes to antimicrobial defence by sequestration of a subset of microbial siderophores. As neither S. pneumoniae nor H. influenzae are known to produce or utilize siderophores, successful colonizers of the nasal passages may have evolved siderophore-independent mechanisms to acquire essential iron and to evade the inhibitory effects of Scn.

Since mucosal surfaces may be simultaneously colonized by multiple species, the success of an organism may be determined by its ability to compete with co-inhabitants of its niche. To explore the contribution of host factors to polymicrobial competition, a murine model was used to study the initiation of colonization by Haemophilus influenzae and Streptococcus pneumoniae. Both bacterial species, which occupy a similar microenvironment within the nasopharynx, persisted during colonization when given individually. Co-colonization, however, resulted in rapid clearance of S. pneumoniae from the upper respiratory tract, associated with increased recruitment of neutrophils into paranasal spaces. Systemic depletion of either neutrophil-like cells or complement was sufficient to eliminate this competitive effect, indicating that clearance was likely due to enhanced opsonophagocytic killing. The hypothesis that modulation of opsonophagocytic activity was responsible for host-mediated competition was tested using in vitro killing assays with elicited neutrophil-like cells. Components of H. influenzae (but not S. pneumoniae) stimulated complement-dependent phagocytic killing of S. pneumoniae. Thus, the recruitment and activation of neutrophils through selective microbial pattern recognition may underlie the H. influenzae-induced clearance of S. pneumoniae. This study demonstrates how innate immune responses may mediate competitive interactions between species and dictate the composition of the colonizing flora.

The epithelial surfaces of the upper respiratory tract are continuously exposed to a wide variety of commensal microorganisms. In addition to acting as a physical barrier, epithelial cells respond to specific microbial products with the generation of signals, such as cytokines, that trigger inflammation. Because they are common components of the nasopharyngeal flora that share the potential to cause disease, we investigated the effects of Haemophilus influenzae and Streptococcus pneumoniae, alone and in combination, on human respiratory epithelial cells in culture and in a murine model of nasopharyngeal colonization. Exposure of A549 or Detroit 562 epithelial cells to both S. pneumoniae and H. influenzae led to a synergistic increase in production of IL-8, the major neutrophil chemokine in the airway, through an NF-kappaB-dependent mechanism. Likewise, nasal cocolonization of mice caused a synergistic rise in local production of macrophage inflammatory protein 2 in nasal lavage fluid and subsequent recruitment of neutrophils. This synergistic effect depended on production of the pore-forming cytolytic toxin, pneumolysin, by S. pneumoniae and activation of host p38 mitogen-activated protein kinase. Although both H. influenzae and S. pneumoniae have ligands for Toll-like receptors (TLRs) TLR2 and TLR4, synergistic activation was TLR2- and TLR4-independent. Thus, epithelial surfaces are capable of amplifying proinflammatory responses during concurrent stimulation by multiple microbial species. These synergistic responses, demonstrated both in vitro and in vivo, may contribute to inflammation of heavily colonized mucosal barriers.

Phosphorylcholine (ChoP) is an antigenic component on the cell surface of many commensal and pathogenic bacteria that reside in the upper airway. In the present study, human ChoP-specific antibody was affinity-purified from pooled serum gamma globulin. This naturally acquired antibody, which is primarily of the immunoglobulin (Ig) G2 subtype, recognized ChoP on the lipoteichoic acid of Streptococcus pneumoniae and on the lipopolysaccharide of Haemophilus influenzae, 2 of the leading etiologic agents of infection involving the human respiratory tract. In in vitro killing assays, anti-ChoP IgG2 was effective against some clinical isolates of nontypeable H. influenzae and against isolates of several common serotypes of S. pneumoniae. Moreover, passively administered human anti-ChoP antibody protected mice against lethal challenge with a transparent isolate of S. pneumoniae type 6A. The effectiveness of human antibody to this conserved bacterial structure suggests that, if it can be manipulated to broaden its activity, it could function as a single vaccine antigen that targets multiple pathogens.

Colonization is the first step in the interaction between Streptococcus pneumoniae and its human host. To better understand the mechanisms contributing to natural carriage, a mouse model of pneumococcal colonization was developed with a clinical isolate of S. pneumoniae previously characterized in experimental colonization of humans. Similar to carriage events in humans, colonization of mice was self-limited and there was no evidence of lower respiratory tract or invasive disease. Carriage induced a serum antibody response to whole pneumococci that was associated temporally with clearance of colonization in three inbred strains of mice. Individual mice, however, did not demonstrate a correlation between the density of colonization and amounts of serum or of mucosal antibodies, including antibodies of different isotypes and antigenic specificities. The role of antibody in the clearance of carriage was then examined in mice with genetic defects in humoral immunity. xid mice, which have deficient responses to polysaccharide antigens, cleared colonization at the same rate as the parent strain. Finally, we showed that microMT mice, which lack mature B cells and fail to produce antibody, were unaffected in the density or duration of colonization. These results demonstrate that antibody is not required for clearance of pneumococcal colonization in mice.

Most clinical isolates of Streptococcus pneumoniae consist of heterogeneous populations of at least two colony phenotypes, opaque and transparent, selected for in the bloodstream and nasopharynx, respectively. Microarray analysis revealed 24 orfs that demonstrated differences in expression greater than twofold between variants of independent strains. Twenty-one of these showed increased expression in the transparent variants, including 11 predicted to be involved in sugar metabolism. A single genomic region contains seven of these loci including the gene that encodes the neuraminidase, NanA. In contrast to previous studies, there was no contribution of NanA to adherence of S. pneumoniae to epithelial cells or colonization in an animal model. However, we observed NanA-dependent desialylation of human airway components that bind to the organism and may mediate bacterial clearance. Targets of desialylation included human lactoferrin, secretory component, and IgA2 that were shown to be present on the surface of the pneumococcus in vivo during pneumococcal pneumonia. The efficiency of desialylation was increased in the transparent variants and enhanced for host proteins binding to the surface of S. pneumoniae. Because deglycosylation affects the function of many host proteins, NanA may contribute to a protease-independent mechanism to modify bound targets and facilitate enhanced survival of the bacterium.

Aerobic growth of Streptococcus pneumoniae results in production of amounts of hydrogen peroxide (H(2)O(2)) that may exceed 1 mM in the surrounding media. H(2)O(2) production by S. pneumoniae has been shown to kill or inhibit the growth of other respiratory tract flora, as well as to have cytotoxic effects on host cells and tissue. The mechanisms allowing S. pneumoniae, a catalase-deficient species, to survive endogenously generated concentrations of H(2)O(2) that are sufficient to kill other bacterial species is unknown. In the present study, pyruvate oxidase (SpxB), the enzyme responsible for endogenous H(2)O(2) production, was required for survival during exposure to high levels (20 mM) of exogenously added H(2)O(2). Pretreatment with H(2)O(2) did not increase H(2)O(2) resistance in the mutant, suggesting that SpxB activity itself is required, rather than an H(2)O(2)-inducible pathway. SpxB mutants synthesized 85% less acetyl-phosphate, a potential source of ATP. During H(2)O(2) exposure, ATP levels decreased more rapidly in spxB mutants than in wild-type cells, suggesting that the increased killing of spxB mutants was due to more rapid ATP depletion. Together, these data support the hypothesis that S. pneumoniae SpxB contributes to an H(2)O(2)-resistant energy source that maintains viability during oxidative stress. Thus, SpxB is required for resistance to the toxic by-product of its own activity. Although H(2)O(2)-dependent hydroxyl radical production and the intracellular concentration of free iron were similar to that of Escherichia coli, killing by H(2)O(2) was unaffected by iron chelators, suggesting that S. pneumoniae has a novel mechanism to avoid the toxic effects of the Fenton reaction.

The immune response to pneumococcal surface structures during colonization was examined in a model of experimental human pneumococcal carriage. Healthy uncolonized adults were given a type 23F or 6B pneumococcus, and a portion of these subjects became colonized (6 of 14 with type 23F and 6 of 8 with type 6B). Sera from colonized and uncolonized subjects were used to determine the titer of antibody specific to pneumococcal surface components under consideration in development of noncapsular polysaccharide-based vaccines. These vaccine candidates included pneumococcal surface protein A (PspA), choline binding protein A (CbpA), lipoteichoic acid, immunoglobulin A1 (IgA1) protease, pneumolysin, proteinase maturation protein A, and pneumococcal surface adhesin A. Only the two related choline binding proteins, PspA and CbpA, were immunogenic in colonized subjects as determined by a statistically significant rise in the serum IgG titer. The serum IgG response to PspA was shown previously to correlate inversely with susceptibility to carriage and was localized to a region within the N-terminal portion of PspA. This region is highly variable in amino acid sequence between pneumococcal strains. Despite the sequence diversity in the immunodominant regions of both PspA and CbpA, a significant strain-to-strain cross-reactivity in the serum IgG response following experimental human carriage was observed. These findings support the need for further investigation of the human antibody response to PspA and CbpA and the potential use of one or both of these proteins as novel vaccine antigens for the prevention of pneumococcal colonization.

Haemophilus influenzae obtains choline from either its growth medium or host cell membrane lipids and expresses it on its lipopolysaccharide (LPS) in the form of phosphorylcholine (ChoP), which contributes to its pathogenesis by mimicry of host cell molecules. Two genes (licB and betT) revealed by whole genomic analysis as encoding potential choline transporters were tested for their role in LPS-ChoP synthesis. The betT gene in H. influenzae is similar to betT in Escherichia coli, which functions in choline transport for the generation of betaine in osmoprotection. The licB gene has homology to bacterial permeases including betT and is encoded in the lic1 locus, which is essential for the expression of LPS-ChoP. In the presence of high concentrations of choline, neither licB nor betT were necessary for expression of LPS-ChoP raising the possibility that other unidentified choline uptake mechanisms may exist in this species. However, under choline limiting conditions, including growth in human nasal airway surface fluid, the licB, but not betT, gene was required for choline transport and synthesis of LPS-ChoP suggesting that LicB functions as a high affinity choline permease. The betT, but not licB, gene was shown to function in osmoprotection in H. influenzae, similar to the role of betT in E. coli. Further analysis demonstrated growth condition dependent differences in the regulation of transcription of the licB and betT genes. We conclude that H. influenzae may have multiple mechanisms for choline uptake and distinct pathways for choline utilization in LPS-ChoP biosynthesis and osmoregulation.

IgA, the major class of Ig in secretions, classically functions by interfering with microbial attachment to host tissues. Many mucosal pathogens, including Streptococcus pneumoniae, express an IgA1 protease that may circumvent the protective effects of this Ig subclass. Because these proteases are specific for human IgA1, we generated human mAbs to the major surface antigen of the pneumococcus, its capsular polysaccharide, and tested their effect in a colonization model of bacterial adherence to respiratory epithelial cells in culture. Rather than inhibiting adherence, type-specific IgA1 markedly enhanced bacterial attachment to host cells, but only when cleaved by IgA1 protease. Neither antibodies of protease-insensitive subclasses (IgA2 and IgG) nor those directed against heterologous capsules had such activity. The adherence-promoting properties of cleaved antibodies correlated with the cationic characteristics of their variable segments, suggesting that bound Fab fragments may neutralize the inhibitory effect of negatively charged capsules on adhesive interaction with host cells. Coating of pneumococci with anticapsular polysaccharide antibody unmasked the bacterial phosphorylcholine ligand, allowing for increased adherence mediated by binding to the platelet activating factor receptor on epithelial cells. In addition, our findings provide evidence for a novel function of bacterial IgA1 proteases. These enzymes may enable pathogens to subvert the antigen specificity of the humoral immune response to facilitate adhesive interactions and persistence on the mucosal surface.