Faculty Bibliography

This cross-sectional study at a tertiary-care hospital in Botswana from 2000 to 2007 was performed to determine the epidemiologic characteristics of Staphylococcus aureus bacteremia. We identified a high prevalence (11.2% of bacteremia cases) of methicillin-resistant S. aureus (MRSA) bacteremia. MRSA isolates had higher proportions of resistance to commonly used antimicrobials than did methicillin-susceptible isolates, emphasizing the need to revise empiric prescribing practices in Botswana.

Currently there is pressing need to develop novel therapeutic agents for the treatment of infections by the human respiratory pathogens Pseudomonas aeruginosa and Streptococcus pneumoniae. The neuraminidases of these pathogens are important for host colonization in animal models of infection and are attractive targets for drug discovery. To aid in the development of inhibitors against these neuraminidases, we have determined the crystal structures of the P. aeruginosa enzyme NanPs and S. pneumoniae enzyme NanA at 1.6 and 1.7A resolution, respectively. In situ proteolysis with trypsin was essential for the crystallization of our recombinant NanA. The active site regions of the two enzymes are strikingly different. NanA contains a deep pocket that is similar to that in canonical neuraminidases, while the NanPs active site is much more open. The comparative studies suggest that NanPs may not be a classical neuraminidase, and may have distinct natural substrates and physiological functions. This work represents an important step in the development of drugs to prevent respiratory tract colonization by these two pathogens.

Bacterial vaginosis (BV) is the most common vaginal infection worldwide and is associated with significant adverse sequelae. We have recently characterized vaginolysin (VLY), the human-specific cytotoxin produced by Gardnerella vaginalis and believed to play a critical role in the pathogenesis of BV and its associated morbidities. We hypothesize that novel antibody-based strategies may be useful for detection of VLY and for inhibition of its toxic effects on human cells. Using purified toxin as an immunogen, we generated polyclonal rabbit immune serum (IS) against VLY. A western blot of G. vaginalis lysate was probed with IS and a single band (57 kD) identified. Immunofluorescence techniques using IS detected VLY production by G. vaginalis. In addition, we have developed a sandwich ELISA assay capable of VLY quantification at ng/ml concentrations in the supernatant of growing G. vaginalis. To investigate the potential inhibitory role of IS on VLY-mediated cell lysis, we exposed human erythrocytes to VLY or VLY pretreated with IS and determined the percent hemolysis. Pretreatment with IS resulted in a significant reduction in VLY-mediated lysis. Similarly, both human cervical carcinoma cells and vaginal epithelial cells exhibited reduced cytolysis following exposure to VLY with IS compared to VLY alone. These results confirm that antibody-based techniques are an effective means of VLY detection. Furthermore, VLY antiserum functions as an inhibitor of VLY-CD59 interaction, mitigating cell lysis. These strategies may have a potential role in the diagnosis and treatment of BV.

Pore-forming toxins are essential to the virulence of a wide variety of pathogenic bacteria. Gardnerella vaginalis is a bacterial species associated with bacterial vaginosis (BV) and its significant adverse sequelae, including preterm birth and acquisition of human immunodeficiency virus. G. vaginalis makes a protein toxin that generates host immune responses and has been hypothesized to be involved in the pathogenesis of BV. We demonstrate that G. vaginalis produces a toxin (vaginolysin [VLY]) that is a member of the cholesterol-dependent cytolysin (CDC) family, most closely related to intermedilysin from Streptococcus intermedius. Consistent with this predicted relationship, VLY lyses target cells in a species-specific manner, dependent upon the complement regulatory molecule CD59. In addition to causing erythrocyte lysis, VLY activates the conserved epithelial p38 mitogen-activated protein kinase pathway and induces interleukin-8 production by human epithelial cells. Transfection of human CD59 into nonsusceptible cells renders them sensitive to VLY-mediated lysis. In addition, a single amino acid substitution in the VLY undecapeptide [VLY(P480W)] generates a toxoid that does not form pores, and introduction of the analogous proline residue into another CDC, pneumolysin, significantly decreases its cytolytic activity. Further investigation of the mechanism of action of VLY may improve understanding of the functions of the CDC family as well as diagnosis and therapy for BV.

Airway infection by the Gram-positive pathogen Streptococcus pneumoniae (Sp) leads to recruitment of neutrophils but limited bacterial killing by these cells. Co-colonization by Sp and a Gram-negative species, Haemophilus influenzae (Hi), provides sufficient stimulus to induce neutrophil and complement-mediated clearance of Sp from the mucosal surface in a murine model. Products from Hi, but not Sp, also promote killing of Sp by ex vivo neutrophil-enriched peritoneal exudate cells. Here we identify the stimulus from Hi as its peptidoglycan. Enhancement of opsonophagocytic killing was facilitated by signaling through nucleotide-binding oligomerization domain-1 (Nod1), which is involved in recognition of gamma-D-glutamyl-meso-diaminopimelic acid (meso-DAP) contained in cell walls of Hi but not Sp. Neutrophils from mice treated with Hi or compounds containing meso-DAP, including synthetic peptidoglycan fragments, showed increased Sp killing in a Nod1-dependent manner. Moreover, Nod1(-/-) mice showed reduced Hi-induced clearance of Sp during co-colonization. These observations offer insight into mechanisms of microbial competition and demonstrate the importance of Nod1 in neutrophil-mediated clearance of bacteria in vivo.

Air-liquid interface models using murine tracheal respiratory epithelium have revolutionized the in vitro study of pulmonary diseases. This model is often impractical because of the small number of respiratory epithelial cells that can be isolated from the mouse trachea. We describe a simple technique to harvest the murine nasal septum and grow the epithelial cells in an air-liquid interface. The degree of ciliation of mouse trachea, nasal septum, and their respective cultured epithelium at an air-liquid interface were compared by scanning electron microscopy (SEM). Immunocytochemistry for type IV beta-tubulin and zona occludens-1 (Zo-1) are performed to determine differentiation and confluence, respectively. To rule out contamination with olfactory epithelium (OE), immunocytochemistry for olfactory marker protein (OMP) was performed. Transepithelial resistance and potential measurements were determined using a modified vertical Ussing chamber SEM reveals approximately 90% ciliated respiratory epithelium in the nasal septum as compared with 35% in the mouse trachea. The septal air-liquid interface culture demonstrates comparable ciliated respiratory epithelium to the nasal septum. Immunocytochemistry demonstrates an intact monolayer and diffuse differentiated ciliated epithelium. These cultures exhibit a transepithelial resistance and potential confirming a confluent monolayer with electrically active airway epitheliumn containing both a sodium-absorptive pathway and a chloride-secretory pathway. To increase the yield of respiratory epithelial cells harvested from mice, we have found the nasal septum is a superior source when compared with the trachea. The nasal septum increases the yield of respiratory epithelial cells up to 8-fold.

Siderophores are low-molecular-weight iron chelators secreted by microbes to obtain iron under deprivation. We hypothesized that the catecholate siderophore enterobactin, produced by Enterobacteriaceae, serves as a proinflammatory signal for respiratory epithelial cells. Respiratory tract responses were explored, since at this site siderocalin, an enterobactin-binding mammalian gene product, is expressed inducibly at high levels and enterobactin-secreting respiratory flora is rare, suggesting selection against a dependence on enterobactin. Addition of aferric, but not iron-saturated, enterobactin elicits a dose-dependent increase in secretion of the proinflammatory chemokine interleukin-8 by human respiratory epithelial cells in culture. This response to purified enterobactin is potentiated by recombinant siderocalin at physiologically relevant concentrations. Conditioned media from genetically modified Escherichia coli strains expressing various levels of enterobactin induce an enterobactin-mediated proinflammatory response. Siderocalin has been shown to deliver enterobactin to other mammalian cell types, exogenously supplied siderocalin can be detected within epithelial cells, and siderocalin increases delivery of enterobactin to the intracellular compartment. Although many siderophores perturb labile cellular iron pools, only enterobactin elicits interleukin-8 secretion, suggesting that iron chelation is necessary but not sufficient. Thus, aferric enterobactin may be a proinflammatory signal for respiratory epithelial cells, permitting detection of microbial communities that have disturbed local iron homeostasis, and siderocalin expression by the host amplifies this signal. This may be a novel mechanism for the mucosa to respond to metabolic signals of expanding microbial communities.

During mucosal colonization, epithelial cells are concurrently exposed to numerous microbial species. Epithelial cytokine production is an early component of innate immunity and contributes to mucosal defence. We have previously demonstrated a synergistic response of respiratory epithelial cells to costimulation by two human pathogens, Streptococcus pneumoniae and Haemophilus influenzae. Here we define a molecular mechanism for the synergistic activation of epithelial signalling during polymicrobial colonization. H. influenzae peptidoglycan synergizes with the pore-forming toxin pneumolysin from S. pneumoniae. Radiolabelled peptidoglycan enters epithelial cells more efficiently in the presence of pneumolysin, consistent with peptidoglycan gaining access to the cytoplasm via toxin pores. Other pore-forming toxins (including anthrolysin O from Bacillus anthracis and Staphylococcus aureus alpha-toxin) can substitute for pneumolysin in the generation of synergistic responses. Consistent with a requirement for pore formation, S. pneumoniae expressing pneumolysin but not an isogenic mutant expressing a non-pore-forming toxoid prime epithelial responses. Nod1, a host cytoplasmic peptidoglycan-recognition molecule, is crucial to the epithelial response. Taken together, these findings demonstrate a role for cytosolic recognition of peptidoglycan in the setting of polymicrobial epithelial stimulation. We conclude that combinations of extracellular organisms can activate innate immune pathways previously considered to be reserved for the detection of intracellular microorganisms.

Expression of a polysaccharide capsule is required for the full pathogenicity of many mucosal pathogens such as Streptococcus pneumoniae. Although capsule allows for evasion of opsonization and subsequent phagocytosis during invasive infection, its role during mucosal colonization, the organism's commensal state, remains unknown. Using a mouse model, we demonstrate that unencapsulated mutants remain capable of nasal colonization but at a reduced density and duration compared to those of their encapsulated parent strains. This deficit in colonization was not due to increased susceptibility to opsonophagocytic clearance involving complement, antibody, or the influx of Ly-6G-positive cells, including neutrophils seen during carriage. Rather, unencapsulated mutants remain agglutinated within lumenal mucus and, thus, are less likely to transit to the epithelial surface where stable colonization occurs. Studies of in vitro binding to immobilized human airway mucus confirmed the inhibitory effect of encapsulation. Likewise, pneumococcal variants expressing larger amounts of negatively charged capsule per cell were less likely to adhere to surfaces coated with human mucus and more likely to evade initial clearance in vivo. Removal of negatively charged sialic acid residues by pretreatment of mucus with neuraminidase diminished the antiadhesive effect of encapsulation. This suggests that the inhibitory effect of encapsulation on mucus binding may be mediated by electrostatic repulsion and offers an explanation for the predominance of anionic polysaccharides among the diverse array of unique capsule types. In conclusion, our findings demonstrate that capsule confers an advantage to mucosal pathogens distinct from its role in inhibition of opsonophagocytosis--escape from entrapment in lumenal mucus.

Epithelial cells act as an interface between human mucosal surfaces and the surrounding environment. As a result, they are responsible for the initiation of local immune responses, which may be crucial for prevention of invasive infection. Here we show that epithelial cells detect the presence of bacterial pore-forming toxins (including pneumolysin from Streptococcus pneumoniae, alpha-hemolysin from Staphylococcus aureus, streptolysin O from Streptococcus pyogenes, and anthrolysin O from Bacillus anthracis) at nanomolar concentrations, far below those required to cause cytolysis. Phosphorylation of p38 MAPK appears to be a conserved response of epithelial cells to subcytolytic concentrations of bacterial poreforming toxins, and this activity is inhibited by the addition of high molecular weight osmolytes to the extracellular medium. By sensing osmotic stress caused by the insertion of a sublethal number of pores into their membranes, epithelial cells may act as an early warning system to commence an immune response, while the local density of toxin-producing bacteria remains low. Osmosensing may thus represent a novel innate immune response to a common bacterial virulence strategy.