Sulfated polysaccharide-directed recruitment of mammalian host proteins: a novel strategy in microbial pathogenesis

Abstract

Fundamental to the virulence of microbial pathogens is their capacity for adaptation and survival within variable, and often hostile, environments encountered in the host. We describe a novel, extragenomic mechanism of surface modulation which may amplify the adaptive and pathogenic potential of numerous bacterial species, including Staphylococcus, Yersinia, and pathogenic Neisseria species, as well as Helicobacter pylori and Streptococcus pyogenes. The mechanism involves specific bacterial recruitment of heparin, glycosaminoglycans, or related sulfated polysaccharides, which in turn serve as universal binding sites for a diverse array of mammalian heparin binding proteins, including adhesive glycoproteins (vitronectin and fibronectin), inflammatory (MCP-3, PF-4, and MIP-1alpha) and immunomodulatory (gamma interferon) intermediates, and fibroblast growth factor. This strategy impacts key aspects of microbial pathogenicity as exemplified by increased bacterial invasion of epithelial cells and inhibition of chemokine-induced chemotaxis. Our findings illustrate a previously unrecognized form of parasitism that complements classical virulence strategies encoded within the microbial genome.

FIG. 1

Sulfated-polysaccharide-mediated binding of mammalian proteins to N. gonorrhoeae producing the heparin binding adhesin OpaA. Gonococci were preincubated with buffer alone (−), heparin (Hp), or dextran sulfate (DS), washed, and subsequently incubated in buffer containing the purified mammalian protein as indicated next to each gel. Bacterium-associated proteins were detected by SDS-PAGE and immunoblotting with polyclonal antisera or monoclonal antibodies specific for each protein, as described in Materials and Methods. Each gel shows the major band(s) recognized by the corresponding antibody for each protein as determined by companion Western blots for each purified protein: vitronectin (Vn), 78 and 68 kDa; fibronectin (Fn), 220 kDa; FGF, 15.8 kDa; EGF, 6.2 kDa; IFN-γ, 16.7 kDa; PF-4, 8 kDa; MIP-1α, 8 kDa; and MCP-3, 9 kDa. αFn, Fn-specific monoclonal antibody; αVn, Vn-specific monoclonal antibody.

FIG. 2

Sulfated-polysaccharide-dependent recruitment of heparin binding proteins by various bacterial pathogens. Each of the bacterial strains was incubated in buffer containing either vitronectin (Vn), IFN-γ, or MCP-3 after preincubation in the absence (−) or presence (+) of dextran sulfate (DS), as described in Materials and Methods. Shown are immunoblots probed with antisera specific for each protein as indicated. N.g.⁻, N. gonorrhoeae producing no Opa protein; N.g.^A, N. gonorrhoeae producing OpaA; N.m., N. meningitidis; H.p., H. pylori; S.e., Staphylococcus epidermidis; S.a., Staphylococcus aureus; Y.e., Y. enterocolitica; Y.p., Y. pestis; S.p., S. pyogenes.

FIG. 3

Heparan sulfate-mediated recruitment of heparin binding proteins from human serum. Gonococci were preincubated with buffer alone (−) or with heparan sulfate (HS), washed, and subsequently incubated in buffer containing normal human serum. Bacterium-associated proteins were detected by SDS-PAGE and immunoblotting with vitronectin (Vn)- and fibronectin (Fn)-specific antisera as described in Materials and Methods. The migrations and the sizes of molecular mass standards in kilodaltons are indicated between the gels. The band migrating at >200 kDa may represent cross-reactivity of the anti-vitronectin (αVn) serum with fibronectin. The apparently exclusive binding of the 68-kDa vitronectin band represents the predominance of that band in human serum (26). αFn, anti-fibronectin antibody.

FIG. 4

Biological impact of glycosaminoglycan-mediated interactions between mammalian proteins and pathogenic bacteria. (A) Adherence (open bars) and internalization (solid bars) of S. pyogenes by CHO-pgs745 cells was assayed after preincubation of bacteria in buffer without (−) or with (+) dextran sulfate and subsequent incubation in buffer without (−Vn) or with (+Vn) vitronectin. The experiments were performed in triplicate and repeated four times; the data are presented as the mean number of bacteria per cell ± standard errors. (B) Migration of HEK293 cells transfected with the CCR1 receptor toward MCP-3 in the presence and absence of dextran sulfate (DS)-coated bacteria. The data represent the mean number of migrating cells in 20 high-power fields (final magnification, ×1,000) ± standard errors. The experiments were performed in duplicate and repeated at least three times. Bact, bacteria.

FIG. 5

Proposed model for heparin-mediated recruitment of mammalian proteins. Microbes (pictured in the center) with heparin binding activities bind heparin and functionally related glycosaminoglycans (GAG) from the environment through species-specific bacterial glycosaminoglycan receptors. The glycosaminoglycans bound at the bacterial cell surface subsequently serve as universal binding sites for theoretically any mammalian heparin binding protein present in the environment, including adhesive glycoproteins, chemokines, growth factors, and cytokines. The repertoire of surface-bound proteins may affect various aspects of microbial virulence and host defense systems, such as chemotaxis, tissue invasion, tissue integrity, and immunological responses.