Use of a whole genome approach to identify vaccine molecules affording protection against Streptococcus pneumoniae infection.

Microbial targets for protective humoral immunity are typically surface-localized proteins and contain common sequence motifs related to their secretion or surface binding. Exploiting the whole genome sequence of the human bacterial pathogen Streptococcus pneumoniae, we identified 130 open reading frames encoding proteins with secretion motifs or similarity to predicted virulence factors. Mice were immunized with 108 of these proteins, and 6 conferred protection against disseminated S. pneumoniae infection. Flow cytometry confirmed the surface localization of several of these targets. Each of the six protective antigens showed broad strain distribution and immunogenicity during human infection. Our results validate the use of a genomic approach for the identification of novel microbial targets that elicit a protective immune response. These new antigens may play a role in the development of improved vaccines against S. pneumoniae.

Adhesins as targets for vaccine development.

Blocking the primary stages of infection, namely bacterial attachment to host cell receptors and colonization of the mucosal surface, may be the most effective strategy to prevent bacterial infections. Bacterial attachment usually involves an interaction between a bacterial surface protein called an adhesin and the host cell receptor. Recent preclinical vaccine studies with the FimH adhesin (derived from uropathogenic Escherichia coli) have confirmed that antibodies elicited against an adhesin can impede colonization, block infection, and prevent disease. The studies indicate that prophylactic vaccination with adhesins can block bacterial infections. With recent advances in the identification, characterization, and isolation of other adhesins, similar approaches are being explored to prevent infections, from otitis media and dental caries to pneumonia and sepsis.

Adherence of Streptococcus pneumoniae to human bronchial epithelial cells (BEAS-2B).

Pneumococcal adherence to alveolar epithelial cells and nasopharyngeal epithelial cells has been well characterized. However, the interaction of Streptococcus pneumoniae with bronchial epithelial cells has not been studied. We have now shown that pneumococci bind specifically to a human bronchial epithelial cell line (BEAS-2B cells). Pneumococci adhered to BEAS-2B cells in a time- and dose-dependent manner. These results suggest that the bronchial epithelium may serve as an additional site of attachment for pneumococci and demonstrate the utility of the BEAS-2B cell line for studying mechanisms of pneumococcal infection.

Characterization of a recombinant fragment that contains a carbohydrate recognition domain of the filamentous hemagglutinin.

The filamentous hemagglutinin (FHA) of Bordetella pertussis plays an important role in establishing infection by attaching the bacteria to the ciliated respiratory epithelial cells. Expression of DNA encoding residues 1141 to 1279 of FHA in Escherichia coli yields a protein of 18,000 Da that exhibits some of the carbohydrate recognition properties of FHA (S. M. Prasad, Y. Yin, E. Rodzinski, E. I. Tuomanen, and H. R. Masure, Infect. Immun. 61:2780-2785, 1993). We have constructed an E. coli strain that expresses this protein, designated fragment A, in a soluble form at markedly elevated levels. Fragment A could be purified with high purity and yields and was immunogenic in mice. Both fragment A and anti-fragment A sera inhibited the binding of B. pertussis to asialo-GM2 and to rabbit ciliated cells. These observations demonstrate that this fragment of FHA contains a cellular binding domain capable of eliciting functional antibodies.

Peptide methionine sulfoxide reductase contributes to the maintenance of adhesins in three major pathogens.

Pathogenic bacteria rely on adhesins to bind to host tissues. Therefore, the maintenance of the functional properties of these extracellular macromolecules is essential for the pathogenicity of these microorganisms. We report that peptide methionine sulfoxide reductase (MsrA), a repair enzyme, contributes to the maintenance of adhesins in Streptococcus pneumoniae, Neisseria gonorrhoeae, and Escherichia coli. A screen of a library of pneumococcal mutants for loss of adherence uncovered a MsrA mutant with 75% reduced binding to GalNAcbeta1-4Gal containing eukaryotic cell receptors that are present on type II lung cells and vascular endothelial cells. Subsequently, it was shown that an E. coli msrA mutant displayed decreased type I fimbriae-mediated, mannose-dependent, agglutination of erythrocytes. Previous work [Taha, M. K., So, M., Seifert, H. S., Billyard, E. & Marchal, C. (1988) EMBO J. 7, 4367-4378] has shown that mutants with defects in the pilA-pilB locus from N. gonorrhoeae were altered in their production of type IV pili. We show that pneumococcal MsrA and gonococcal PilB expressed in E. coli have MsrA activity. Together these data suggest that MsrA is required for the proper expression or maintenance of functional adhesins on the surfaces of these three major pathogenic bacteria.

Adherence of Streptococcus pneumoniae to immobilized fibronectin.

Adherence to extracellular matrix proteins, such as fibronectin, affords pathogens with a mechanism to invade injured epithelia. Streptococcus pneumoniae was found to adhere to immobilized fibronectin more avidly than other streptococci and staphylococci do. Binding was dose, time, and temperature dependent. Trypsin treatment of the bacteria resulted in decreased binding, suggesting that the bacterial adhesive component was a protein. Fragments of fibronectin generated by proteolysis or by expression of recombinant gene segments were compared for the ability to bind pneumococci and to compete against bacterial binding to immobilized fibronectin. Fragments from the carboxy-terminal heparin binding domain were consistently active, suggesting that this region contains the pneumococcal binding site, a region distinct from that supporting the attachment of most other bacteria.

Production of nitric oxide and peroxynitrite in the lung during acute endotoxemia.

Nitric oxide is a short-lived cytotoxic mediator that has been implicated in the pathogenesis of endotoxin-induced tissue injury and septic shock. In the present studies we determined whether this mediator is produced in the lung during acute endotoxemia. We found that intravenous injection of rats with bacterially derived lipopolysaccharide (LPS), a condition that induces acute endotoxemia, caused a time-dependent increase in inducible nitric oxide synthase (iNOS) mRNA expression in the lung, which reached a maximum after 24 h. This was correlated with nitric oxide production in the lung as measured by electron paramagnetic spin trapping, which was detectable within 6 h. Alveolar macrophages (AMs) and interstitial macrophages (IMs) isolated from rats 6-12 h after induction of acute endotoxemia were also found to exhibit increased nitric oxide production in response to in vitro stimulation with interferon-gamma (IFN-gamma) and LPS measured by nitrite accumulation in the culture medium. The effects of acute endotoxemia on nitric oxide production by these cells were, however, transient and returned to control levels by 24 h in AMs and 36 h in IMs. Interestingly, although nitrite accumulation in the culture medium of IMs isolated 48 h after induction of acute endotoxemia and stimulated with low concentrations of IFN-gamma and LPS was reduced, when compared with cells from control animals, these cells, as well as AMs, continued to express high levels of iNOS protein and mRNA. This was correlated with increased peroxynitrite production by the cells. Peroxynitrite has been shown to act as a nitrating agent and can generate nitrotyrosine residues in proteins. Using a specific antibody and immunohistochemistry, we found evidence of nitrotyrosine residues in sections of lungs 48 h after treatment of rats with endotoxin. These data suggest that nitric oxide produced by IMs and AMs can react with superoxide anion to form peroxynitrite. Taken together, the present studies demonstrate that AMs and IMs are activated following acute endotoxemia to produce reactive nitrogen intermediates and that both cell types contribute to inflammatory responses in the lung.

Enhanced phagocytosis, chemotaxis, and production of reactive oxygen intermediates by interstitial lung macrophages following acute endotoxemia.

Endotoxemia is associated with enhanced release of a variety of cytotoxic and/or proinflammatory mediators from locally activated tissue macrophages. The lung is highly sensitive to damage induced by endotoxin, suggesting that pulmonary macrophages are activated by this bacterially derived product to release mediators that contribute to the pathogenesis of tissue injury. In the present studies, we used a rat model of acute endotoxemia induced by a single intravenous injection of animals with lipopolysaccharide (LPS) to determine the extent to which different lung macrophage subpopulations are activated. Alveolar macrophages (AM) and interstitial macrophages (IM) were isolated sequentially from the lung by lavage, followed by digestion with collagenase and selective adherence to tissue culture dishes. Both AM and IM were found to produce superoxide anion, as well as hydrogen peroxide in response to inflammatory stimuli. AM produced greater quantities of these reactive oxygen intermediates than did IM. Treatment of rats with LPS resulted in a significant increase in production of reactive oxygen intermediates by IM, but not by AM. Similarly, while AM from untreated rats phagocytized more opsonized sheep red blood cells than did IM, LPS treatment of rats significantly enhanced phagocytosis only in IM. In addition, this treatment caused a significant increase in chemotaxis of IM towards C5a. In contrast, although LPS treatment of rats had no effect on tumor necrosis factor-alpha release by AM, a significant reduction was observed in IM. Taken together, these data demonstrate that IM play a role in the inflammatory response of the lung to acute endotoxemia.