Faculty Bibliography

Cationic antimicrobial peptides are believed to exert their primary activities on anionic bacterial cell membranes; however, this model does not adequately account for several important structure-activity relationships. These relationships are likely to be influenced by the bacterial response to peptide challenge. In order to characterize the genomic aspect of this response, transcription profiles were examined for Escherichia coli isolates treated with sublethal and lethal concentrations of the cationic antimicrobial peptide cecropin A. Transcript levels for 26 genes changed significantly following treatment with sublethal peptide concentrations, and half of the transcripts corresponded to protein products with unknown function. The pattern of response is distinct from that following treatment with lethal concentrations and is also distinct from the bacterial response to nutritional, thermal, osmotic, or oxidative stress. These results demonstrate that cecropin A induces a genomic response in E. coli apart from any lethal effects on the membrane and suggest that a complete understanding of its mechanism of action may require a detailed examination of this response.

Both Neisseria meningitidis and Haemophilus influenzae are capable of mimicking host structures by decorating their lipopolysaccharides with sialic acid. We show that a neuraminidase expressed by Streptococcus pneumoniae (NanA) is able to desialylate the cell surfaces of both these species, which reside in and possibly compete for the same host niche.

Numerous major bacterial pathogens in the human respiratory tract, including Streptococcus pneumoniae and Haemophilus influenzae, express cell-surface phosphorylcholine (ChoP), a ligand for the receptor for platelet-activating factor (rPAF). ChoP is also bound by C-reactive protein (CRP), which, in the presence of complement, may be bactericidal. This study found that CRP can block the attachment of bacteria expressing cell-surface ChoP to host cells. Concentrations of CRP equivalent to those on the mucosal surface of the human airway blocked most adherence of both S. pneumoniae and H. influenzae to human pharyngeal epithelial cells. ChoP-mediated adherence was also reduced in the presence of an rPAF antagonist. The antiadhesive effects of the rPAF antagonist and CRP were not additive, suggesting that CRP activity is specific to the area of adherence mediated by the receptor. The binding of CRP to ChoP and the effect of CRP on adherence were inhibited by human surfactant (primarily ChoP). The antiadhesive effect of CRP may be diminished in the terminal airway, where surfactant is abundant.

Loss-of-function mutations in the following seven pneumococcal genes were detected and analyzed: pspA, spxB, xba, licD2, lytA, nanA, and atpC. Factors associated with these mutations included (i) frameshifts caused by reversible gain and loss of single bases within homopolymeric repeats as short as 6 bases, (ii) deletions caused by recombinational events between nontandem direct repeats as short as 8 bases, and (iii) substitutions of guanine residues caused at an increased frequency by the high levels of hydrogen peroxide (>2 mM) typically generated by this species under aerobic growth conditions. The latter accounted for a frequency as high as 2.8 x 10(-6) for spontaneous mutation to resistance to optochin and was 10- to 200-fold lower in the absence of detectable levels of H2O2. Some of these mutations appear to have been selected for in vivo during pneumococcal infection, perhaps as a consequence of immune pressure or oxidative stress.

Innate immunity serves as a first line defense in vertebrate organisms by providing an initial barrier to microorganisms and triggering antigen-specific responses. Antimicrobial peptides are thought to be effectors of innate immunity through their antibiotic activity and direct killing of microorganisms. Evidence to support this hypothesis in vertebrates is indirect, based on expression profiles and in vitro assays using purified peptides. Here we investigated the function of antimicrobial peptides in vivo using mice deficient in an antimicrobial peptide, mouse beta-defensin-1 (mBD-1). We find that loss of mBD-1 results in delayed clearance of Haemophilus influenzae from lung. These data demonstrate directly that antimicrobial peptides of vertebrates provide an initial block to bacteria at epithelial surfaces.

Colonization of the nasopharynx is the initial step in all infections caused by Streptococcus pneumoniae. The antibody response to carriage was examined in an experimental model of human colonization in healthy adults. Asymptomatic colonization was detected in 6/14 subjects and continued for up to 122 d. Susceptibility to carriage did not correlate with total serum immunoglobulin (Ig)G to the homotypic capsular polysaccharide. All of the colonized subjects, in contrast, developed a serum IgG and secretory IgA response to a 22 kD protein, whereas 7 of 8 subjects who did not become colonized had preexisting antibody to this protein. Analysis of the 22 kD protein identified it as the NH(2)-terminal region of pneumococcal surface protein A (PspA). Our findings provide evidence for the role of antibody to this protein fragment in preventing pneumococcal carriage by humans.

Toll-like receptors (TLRs) have been implicated in the regulation of host responses to microbial Ags. This study characterizes the role of TLR4 in the innate immune response to intrapulmonary administration of Haemophilus influenzae in the mouse. Two different strains of mice efficiently cleared aerosolized H. influenzae concurrent with a brisk elaboration of IL-1beta, IL-6, TNF-alpha, macrophage-inflammatory protein (MIP)-1alpha, and MIP-2 in bronchoalveolar lavage and a corresponding mobilization of intrapulmonary neutrophils. Congenic strains of mice deficient in TLR4 demonstrated a substantial delay in clearance of H. influenzae with diminished IL-1beta, IL-6, TNF-alpha, MIP-1alpha, and MIP-2 in bronchoalveolar lavage and a notable absence of intrapulmonary neutrophils. In TLR4-expressing animals, but not TLR4-deficient animals, TNF-alpha and MIP-1alpha expression was up-regulated in epithelial cells of the conducting airway in response to H. influenzae which was preceded by an apparent activation of the NF-kappaB pathway in these cells based on the findings of decreased overall IkappaB and an increase in its phosphorylated form. This study demonstrates a critical role of TLR4 in mediating an effective innate immune response to H. influenzae in the lung. This suggests that the airway epithelia might contribute to sensing of H. influenzae infection and signaling the innate immune response.

Haemophilus influenzae expresses heterogeneous populations of short-chain lipopolysaccharide (LPS) which exhibit extensive antigenic diversity among multiple oligosaccharide epitopes. These LPS oligosaccharide epitopes can carry phosphocholine (PCho) substituents, the expression of which is subject to high frequency phase variation mediated by genes in the lic1 genetic locus. The location and site of attachment of PCho substituents were determined by structural analysis of LPS from two type b H. influenzae strains, Eagan and RM7004. The lic2 locus is involved in phase variation of oligosaccharide expression. LPS obtained from the parent strains, from mutants generated by insertion of antibiotic resistance cassettes in the lic2 genetic locus, and from phase-variants showing high levels of PCho expression was characterized by electrospray ionization-mass spectrometry (ESI-MS) and 1H NMR spectroscopy of derived O-deacylated samples. ESI-MS of O-deacylated LPS from wild-type strains revealed mixtures of related glycoform structures differing in the number of hexose residues. Analysis of LPS from PCho-expressing phase-variants revealed similar mixtures of glycoforms, each containing a single PCho substituent. O-Deacylated LPS preparations from the lic2 mutants were much less complex than their respective parent strains, consisting only of Hex3 and/or Hex2 glycoforms, were examined in detail by high-field NMR techniques. It was found that the LPS samples contain the phosphoethanolamine (PEtn) substituted inner-core element, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1--> 3)-L-alpha-D-He pp-(1-->5)-alpha-Kdo in which the major glycoforms carry a beta-D-Glcp or beta-D-Glcp-(1-->4)-beta-D-Glcp at the O-4 position of the 3-substituted heptose (HepI) and a beta-D-Galp at the O-2 position of the terminal heptose (HepIII). LPS from the lic2 mutants of both type b strains were found to carry PCho groups at the O-6 position of the terminal beta-D-Galp residue attached to HepIII. In the parent strains, the central heptose (HepII) of the LPS inner-core element is also substituted by hexose containing oligosaccharides. The expression of the galabiose epitope in LPS of H. influenzae type b strains has previously been linked to genes comprising the lic2 locus. The present study provides definitive evidence for the role of lic2 genes in initiating chain extension from HepII. From the analysis of core oligosaccharide samples, LPS from the lic2 mutant strain of RM7004 was also found to carry O-acetyl substituents. Mono-, di-, and tri-O-acetylated LPS oligosaccharides were identified. The major O-acetylated glycoforms were found to be substituted at the O-3 position of HepIII. A di-O-acetylated species was characterized which was also substituted at the O-6 postion of the terminal beta-D-Glc in the Hex3 glycoform. This is the first report pointing to the occurrence of O-acetyl groups in the inner-core region of H. influenzae LPS. We have previously shown that in H. influenzae strain Rd, a capsule-deficient type d strain, PCho groups are expressed in a different molecular environment, being attached at the O-6 position of a beta-D-Glcp, which is in turn attached to HepI.

The structure of the saccharide part of the lipopolysaccharide from Haemophilus influenzae strain AH1-3 (lic3+) has been investigated. The saccharide was obtained from the lipopolysaccharide by mild acid hydrolysis followed by high-performance anion-exchange chromatography, and isolated fractions were studied by methylation analysis, NMR spectroscopy, and FAB mass spectrometry. The major saccharide is a heptasaccharide with the following structure, [formula: see text] in which Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid and PEA is 2-aminoethyl phosphate. Hep is identified as L-glycero-D-manno-heptose. The absolute configuration of the phosphorylated heptose is tentative only.

Monoclonal antibodies raised against Haemophilus influenzae and Neisseria gonorrhoeae were used to investigate similarities or differences in the lipopolysaccharide antigens of pathogenic and commensal strains of several Gram-negative bacteria indigenous to mucosal surfaces of humans. In immunoblotting experiments, 20 of 36 monoclonal antibodies showed cross-reactions between species of Neisseria and Haemophilus. The common epitopes were present on N. gonorrhoeae, N. meningitidis, N. lactamica, H. influenzae including biogroup aegyptius, and occasionally H. parainfluenzae. No other commensal Neisseria or Gram-negative organisms tested reacted with the monoclonal antibodies with one exception; a single strain of pathogenic Escherichia coli was recognised by a N. gonorrhoeae-specific monoclonal antibody. One monoclonal antibody, raised against N. gonorrhoeae lipopolysaccharide, reacted with N. gonorrhoeae (32 of 59 strains), N. meningitidis (9 of 26 strains), H. influenzae (6 of 16 strains). An epitope expressed by H. influenzae and implicated in its virulence was also present on 14 of 59 strains of N. gonorrhoeae and was shown to comprise a digalactoside structure, alpha-galactosyl-1,4-beta-galactose (Gal alpha 1,4Gal beta), also found on human cells.