Human ␤ defensin 2 (h␤D-2) is thought to play an important role in cutaneous immune defense. We hypothesized that (i) keratinocyte expression of h␤D-2, measured by reverse transcription-PCR, would be upregulated in response to challenge with pathogenic bacteria, particularly highly adherent strains of Streptococcus pyogenes and Staphylococcus aureus, and (ii) h␤D-2 would have potent antimicrobial activity against pathogenic but not commensal organisms. Expression of h␤D-2 was induced consistently by S. aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa, whereas strains of S. pyogenes were poor and variable inducers of h␤D-2. No correlation was found between levels of bacterial adherence and keratinocyte expression of h␤D-2. S. pyogenes was significantly more sensitive to killing by h␤D-2 than S. epidermidis. We conclude that the ability to induce h␤D-2 expression in combination with sensitivity to its antimicrobial effects may contribute to the rarity of skin infections with the gram-negative bacterial organisms, whereas lack of stimulation of h␤D-2 expression by S. pyogenes may be important in its ability to evade innate defenses and cause skin disease. Induction of expression of h␤D-2 but relative tolerance to it may enable S. epidermidis to survive on the skin surface and modulate h␤D-2 expression when the stratum corneum barrier is disrupted.

Several bacterial pathogens have evolved the means to escape immune detection by mimicking host cell surface carbohydrates that are crucial for self/non-self recognition. Sialic acid, a terminal residue on these carbohydrates, inhibits activation of the alternate pathway of complement by recruiting the immune modulating molecule factors H, I, and iC3b. Sialylation of capsular polysaccharide (CPS) is important for virulence of group B streptococci (GBS), a significant human pathogen. We previously reported that cpsK, a gene within the cps locus of type III GBS, could complement a sialyltransferase deficient lst mutant of Haemophilus ducreyi, implicating its role in sialylation of the GBS capsule. To explore the function of cpsK in GBS capsule production, we created a mutant in cpsK. Immunoblot analysis and enzyme-linked immunosorbent assay using anti-type III CPS antisera demonstrated that the mutant CPS did not contain sialic acid. This was confirmed by highperformance liquid chromatography after mild acid hydrolysis of the CPS. Although increased CPS chain length was seen for this strain, CPS production was <20% of the parental isolate. An episomal cpsK copy restored synthesis of sialo-CPS to wild-type levels. These data support our hypothesis that cpsK encodes the GBS CPS sialyltransferase and provide further evidence that lack of CPS oligosaccharide sialylation reduces the amount of CPS expressed on the cell surface. These observations also imply that one or more of the components involved in synthesis or transport of oligosaccharide repeating units requires a sialo-oligosaccharide for complete activity.Streptococcus agalactiae (group B streptococci or GBS) remains a major cause of serious neonatal bacterial infection in the developed world despite a Ͼ65% reduction in early-onset GBS disease (infection within the first week of life) due to the advent of chemoprophylactic prevention measures (37). The GBS capsular polysaccharide (CPS) is well established as a primary virulence determinant in GBS pathogenesis (16). Nine serotypes of GBS have been identified based on their unique CPS antigens (serotypes Ia and Ib and types II through VIII). The individual serotypes arise from the synthesis of distinct CPS precursor oligosaccharide repeating units (RPUs) and/or differences in the way the CPS RPUs are polymerized. The cps loci in each serotype are organized similarly with genes in the 5Ј region involved in regulation and chain length, a 3Ј region encoding sialic acid synthesis, and a central region containing the oligosaccharide RPU structural and polymerization genes (Fig. 1). Despite significant heterogeneity in the central structural region of their cps loci, all GBS serotypes produce RPUs with side chains terminated by N-acetylneuraminic acid (sialic acid; Neu5Ac) ␣2,3 linked to galactose (Gal).Several bacterial species produce sialylated glycoconjugates on their surfaces. A number of gram-negative species, including Salmonella enterica, Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae, Ha...

To evaluate the role of putative group A streptococcal virulence factors in the initiation of skin infections, we compared the adherence of a wild-type M49-protein skin-associated strain to that of a series of 16 isogenic mutants created by insertional inactivation of virulence genes. None of the mutants, including the M-proteindeficient (emm mutant) strain, displayed reduced adherence to early-passage cultured human keratinocytes, but adherence of the mutant lacking hyaluronic acid capsule expression (has mutant) was increased 13-fold. In contrast, elimination of capsule expression in M2-, M3-, and M18-protein has mutants increased adherence only slightly (1.3-to 2.3-fold) compared to their respective wild-type strains. A mutant with inactivation of both emm and has displayed high-level adherence (34.9 ؎ 4.1%) equal to that of the has mutant strain (40.7 ؉ 8.0%), confirming the lack of involvement of M49 protein in attachment. Moreover, adherence of the M49-proteindeficient (emm mutant) and wild-type strains was increased to the same level (57 and 55%, respectively) following enzymatic digestion of their hyaluronic acid capsule. Adherence of mutants lacking oligopeptide permease (Opp) expression was increased 3.8-to 5.5-fold, in association with decreased cell-associated hyaluronic acid capsule. Finally, soluble CD46 failed to inhibit adherence of M49-and M52-serotype skin strains. We conclude that (i) bacterial M protein and keratinocyte CD46 do not mediate adherence of M49 skin-associated Streptococcus pyogenes to epidermal keratinocytes, (ii) hyaluronic acid capsule impedes the interaction of bacterial adhesins with keratinocyte receptors, (iii) modulation of capsule expression may be important in the pathogenesis of skin infections, and (iv) the molecular interactions in attachment of skin strains of S. pyogenes to keratinocytes are unique and remain unidentified.Streptococcus pyogenes (group A streptococcus) is unsurpassed among bacterial pathogens in its ability to cause a variety of skin infections ranging from self-limited superficial impetigo to fulminant life-threatening, soft-tissue destruction and necrotizing fasciitis (26-28). Increased global incidence of severe, invasive disease due to S. pyogenes over the past decade, with the skin serving as the portal of entry in more than half of the cases, has highlighted our need to understand the molecular pathogenesis of skin infections (66). Pathogenic mechanisms of streptococcal skin infections are almost entirely unknown, however, since efforts have focused on the interaction of streptococci with mucosal epithelium, and in vitro models which emulate human skin disease (23) have not been available.An initial step in group A streptococcal skin infection appears to involve adherence of the bacteria via its adhesin(s) to host cell receptor(s) (25). The type of adhesin utilized for specific binding may vary depending on the streptococcal strain and type of host cell and tissue involved. M protein is the most well-documented virulence factor of S. pyogenes. ...