Induction of surface-associated proteins of Streptococcus uberis by cultivation with extracellular matrix components and bovine mammary epithelial cells

Surface-associated protein expression by Streptococcus uberis was influenced by the presence of collagen, laminin and bovine mammary epithelial cells in the culture medium. After electrophoresis and silver staining, four proteins stained more intensively in samples from S. uberis cultivated with epithelial cells and extracellular matrix components than in samples from S. uberis cultivated alone. Induction of these proteins was more obvious after multiple bacterial passages. The correlation between the phenotype of S. uberis and its potential virulence status as illustrated by an immunoblotting study with sera obtained from infected cows revealed that these proteins are probably expressed in vivo during infection.     

Adhesion of Streptococcus uberis to monolayers of cultured epithelial cells derived from the bovine mammary gland

Abstract Monolayers of epithelial cells obtained by culture of isolated secretory alveoli from the bovine mammary gland were used as target cells in bacterial adhesion assays. The ability of two strains of Streptococcus uberis (EF20 and 0140J) to adhere to these cells was examined using scanning electron microscopy (SEM). The cultured monolayers consisted of two types of epithelial cell one of which possessed many microvilli and another which exhibited only sparse or no microvilli. Strain EF20 adhered more readily and in greater numbers to the cells without microvilli (MV⁻) than to cells possessing microvilli (MV⁺). Strain 0140J also interacted with a greater proportion of MV⁻ cells but adhered to both MV⁻ and MV⁺ cell types in similar numbers.     

Identification of lactoferrin-binding proteins in bovine mastitis-causing Streptococcus uberis

All strains of Streptococcus uberis evaluated bound to lactoferrin (Lf) in milk as detected by polyacrylamide gel electrophoresis and Western blotting. A biotin-avidin-based microplate binding assay and ELISA also revealed that these bacterial strains bound to purified Lf. Binding of bacteria of Lf was not inhibited by mannose and galactose, indicating that glycosidic domains of the Lf molecule were not involved in binding. Lf binding was also unaffected by bovine transferrin. Western blot analysis demonstrated that there were at least two bacterial proteins involved in Lf-binding. Lf binding by S. uberis could enable this bacterium to acquire iron necessary for its growth.     

Monoclonal antibodies directed against extracellular matrix proteins reduce the adherence of Candida albicans to HEp-2 cells

The presence of the extracellular matrix (ECM) proteins collagen types I and IV, laminin and fibronectin on the surface of HEp-2 cells was confirmed by flow cytometry using monoclonal antibodies. Monoclonal antibodies directed against these ECM proteins reduced the adherence of C. albicans ATCC 44990 to HEp-2 cells, the greatest reductions being evident in assays which incorporated anti-collagen type IV monoclonal antibody. The ability of sugaramines to inhibit the adherence of C. albicans to a variety of cell types has been demonstrated previously and the most significant reduction in C. albicans – HEp-2 adherence was in assays which incorporated 0.2M galactosamine. The combination of anti-collagen IV monoclonal antibody and galactosamine reduced the adherence of C. albicans to HEp-2 cells by approximately 70% (p < 0.05). This revised version was published online in June 2006 with corrections to the Cover Date.     

The adherence of Candida yeasts to human and bovine vascular endothelium and subendothelial extracellular matrix

Abstract The adherence of Candida albicans yeasts and other Candida species to human umbilical vein endothelial cells (HUVEC), bovine aortic endothelial cells (BAEC) and their respective subendothelial extracellular matrices (ECM) was studied. Yeast adherence to confluent HUVEC and BAEC appeared to occur at intercellular junctions and edges of endothelial cells in preference to the contoured surface of the endothelial cell. Both endothelial cell lines characteristically resisted yeast adherence. The resistance of endothelium to yeast adherence was especially pronounced in the presence of serum. On the other hand, there was avid yeast binding to the subendothelial ECMs, on the order of 200–500% greater than to monolayers of syngeneic endothelial cells.     

Streptococcus suis serotype 2 binding to extracellular matrix proteins

Streptococcus suis serotype 2 is a major swine and human pathogen that causes septicemia and meningitis. The ability of S. suis serotype 2 to bind to different extracellular matrix (ECM) proteins was evaluated by ELISA. All 23 strains tested bound to plasma and cellular fibronectin and collagen types I, III, and V, some to fibrin, vitronectin, and laminin, and none to the other ECM proteins tested. An unencapsulated isogenic mutant bound to ECM proteins better than its parental encapsulated strain, suggesting that the polysaccharide capsule interfered with binding. Cross-inhibition was observed between soluble plasma fibronectin and collagens in the ECM adherence assay, indicating that binding domains for both proteins exist on the same or nearby bacterial surface molecules. On the other hand, pre-incubation with plasma fibronectin increased binding to collagen IV, suggesting that S. suis might use fibronectin as a bridging molecule. The results of heat treatment and proteolytic digestion suggest that adhesins for these ECM proteins are proteinaceous in nature.     

Role of M3 protein in the adherence and internalization of an invasive Streptococcus pyogenes strain by epithelial cells

Streptococcus pyogenes utilizes multiple mechanisms for adherence to and internalization by epithelial cells. One of the molecules suggested of being involved in adherence and internalization is the M protein. Although strains of the M3 serotype form the second largest group isolated from patients with severe invasive diseases and fatal infections, not much information is known regarding the interactions of M3 protein with mammalian cells. In this study we have constructed an emm3 mutant of an invasive M3 serotype (SP268), and demonstrated that the M3 protein is involved in both adherence to and internalization by HEp-2 cells. Fibronectin promoted both adherence and internalization of SP268 in an M3-independent pathway. Utilizing speB and speB/emm3 double mutants, it was found that M3 protein is not essential for the maturation of SpeB, as was reported for the M1 protein. Increased internalization efficiency observed in both the speB and emm3/speB mutants suggested that inhibition of S. pyogenes internalization by SpeB is not related to the presence of an intact M3 protein. Thus, other proteins in SP268, which serve as targets for SpeB activity, have a prominent role in the internalization process.     

Glycosaminoglycans inhibit Candida albicans adherence to extracellular matrix proteins

The ability of Candida albicans to adhere to subendothelial extracellular matrix (ECM) may be important in the pathogenesis of disseminated candidiasis. ECM proteins, such as fibronectin, laminin, and types I and IV collagen bind C. albicans avidly. These proteins all possess heparin-binding domains. The influence of the glycosaminoglycans (GAGS) including heparin, heparan sulfate and dextran sulfate on C. albicans adherence to subendothelial ECM and ECM proteins was studied. It was demonstrated that the GAGS inhibited C. albicans adherence to ECM and ECM proteins. This possibly occurred by the GAGS binding to the ECM proteins and, in so doing, masking a preferred ligand for C. albicans adherence.     

Protein expression by Streptococcus uberis in co-culture with bovine mammary epithelial cells

Three strains of Streptococcus uberis isolated from dairy cows with mastitis were co-cultured with a bovine mammary epithelial cell line (MAC-T) in Dulbecco's modified Eagle's medium without fetal bovine serum. Protein profiles from culture supernatants and bacterial pellets among different treatments were compared by electrophoresis. There were proteins induced or having increased expression in both supernatant and surface-associated samples from S. uberis co-cultured with MAC-T cells. Some of these proteins were recognized by antibodies in serum obtained from a cow infected by S. uberis . In supernatant samples, there were two distinct protein bands at 35 and 36.8 kDa for all three strains of S. uberis co-cultured with MAC-T cells. These two bands were absent when bacterial protein synthesis was inhibited by chloramphenicol. This study clearly indicates that bacterial protein expression was regulated in response to co-culture with mammary epithelial cells.     

Binding of Clostridium difficile to Caco-2 epithelial cell line and to extracellular matrix proteins

Adhesion of Clostridium difficile to Caco-2 was examined as a function of monolayers polarization and differentiation. The number of adherent C. difficile C253 bacteria per cell strongly decreased when postconfluent 15-day-old monolayers were used (1.7 bacteria per cell versus 17.3 with 3-day-old monolayers). Following disruption of intercellular junctions by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid, a significant rise in the level of bacterial adhesion was observed, above all in postconfluent monolayers. Immunofluorescence studies of bacteria and transferrin receptor, a marker of basolateral pole of polarized monolayers, showed that C. difficile C253 adheres mainly to the basolateral surface of differentiated and undifferentiated polarized Caco-2 cells. Furthermore, binding of C. difficile C253 to several extracellular matrix proteins in vitro was demonstrated by an ELISA-based assay.     

Binding of Streptococcus mutans to extracellular matrix molecules and fibrinogen

We have determined the ability of Streptococcus mutans cells to bind to extracellular matrix (ECM) molecules and fibrinogen. S. mutans cells were found to bind fibronectin, laminin, collagen type I, and fibrinogen. An isogenic S. mutans strain with a defect in the expression of the major surface protein of S. mutans, antigen I/II, possessed a reduced ability to bind fibronectin, collagen, and fibrinogen but not laminin, suggesting that antigen I/II contributes during pathological processes to the interaction of S. mutans cells with fibronectin, collagen type I, and fibrinogen.     

Partial characterization of the cohemolytic factor produced by Streptococcus uberis and comparison with the CAMP-factor

Abstract Exosubstances (cohemolysins) produced by Streptococcus agalactiae (CAMP-factor) and Streptococcus uberis (Uberis-factor) showing hemolytic synergism with β-lysin produced by Staphylococcus aureus were compared. Cohemolytic activity was evaluated in the supernatants of bacterial cultures, before and after ammonium sulfate precipitation. Sheep erythrocytes sensitized with β-lysin were used as substrate. The assays were performed in microtiter plates and results were expressed as cohemolytic units/ml. Maximum cohemolytic activity was detected, respectively, after 8 h and 14 h of growth in Columbia broth in S. uberis and S. agalactiae cultures. Cohemolytic activities of both microorganisms showed similarities when submitted to various physical and chemical treatments. They were significantly decreased by heating at 60°C and 100°C, or in presence of trypsin, and were abolished in the presence of Tween 20. Activities were found to be stable in crude supernatants and concentrated preparations maintained at −20°C for 3 months. Differences were related to levels of activity and kinetics of detection during the growth cycle. The results indicate the proteic nature, at least in part, of the Uberis factor. Analysis by PAGE in the presence or absence of SDS allowed us to correlate Uberis activity with a protein band with apparent molecular mass of 42 kDa, while CAMP activity was associated with a protein band of 27 kDa.     

The Mig protein of Streptococcus dysgalactiae inhibits bacterial internalization into bovine mammary gland epithelial cells

The role of the Mig protein of Streptococcus dysgalactiae in bacterial adhesion and internalization of bovine mammary gland epithelial cells (MAC-T) was investigated with the wild-type and isogenic mig mutant strains. While there was no difference in adhesion between the strains, the wild-type strain exhibited a significantly lower level of invasion than the mutants. The lower level of internalization of the Mig(+) strain is likely due to Mig-mediated interference with uptake of the microorganisms rather than the host protein binding properties of Mig. Avoidance of intimate interactions with the host cells might be an alternative strategy for S. dysgalactiae to survive and persist in the bovine mammary glands.     

Specific interactions between Porphyromonas gingivalis fimbriae and human extracellular matrix proteins

The interactions of the extracellular matrix (ECM) proteins (laminin, elastin, fibronectin, type I collagen, thrombospondin and vitronectin) with the fimbriae of Porphyromonas gingivalis were analyzed based on surface plasmon resonance (SPR) spectroscopy using a biomolecular interaction analyzing system (BIAcore). The BIAcore profiles demonstrated that fimbriae specifically bound to all of the ECM proteins with significant association constants (Ka). Vitronectin showed the highest affinity to fimbriae (Ka = 3.79 x 10(6) M-1), while the affinity of laminin was lowest (Ka = 2.15 x 10(6) M-1). A synthetic peptide which is a potent inhibitor of fimbrial binding to salivary proteins was not significantly effective on the fimbrial interactions with the ECM proteins. Using polystyrene microtiter plates revealed that P. gingivalis fimbriae bound markedly to immobilized fibronectin and type I collagen, while the interaction of fimbriae with the other ECM proteins was not clearly demonstrated. These results suggest that interactions between fimbriae and the ECM proteins occur with specific affinities which are not mediated by mechanisms identical to those of salivary proteins. It was also shown that SPR spectroscopy is a useful method to analyze these specific interactions.     

Effect of lipoteichoic acid on the uptake of Streptococcus pyogenes by HEp-2 cells

Lipoteichoic acid (LTA) is thought to play a role in the interactions between Streptococcus pyogenes and host cells. We have examined the effect of exogenous LTA on the adherence and entry of S. pyogenes JRS4 strain into HEp-2 epithelial cells. LTA markedly inhibited bacterial entry in a concentration-dependent manner, up to 250 microg ml(-1). In contrast, LTA had only a slight inhibitory effect on adherence. LTA also inhibited the entry but not adherence of Salmonella typhimurium strain into HEp-2 cells. Binding experiments showed a dose-dependent binding of LTA to cells up to 10 microg ml(-1). Confocal laser microscopy imaging and analysis revealed that LTA was internalized by the epithelial cells and colocalized with F-actin. These results might imply that, following binding, exogenous LTA enters HEp-2 cells and exerts a cytotoxic effect that interferes with bacterial internalization. A possible target for LTA activity might be the actin cytoskeleton, which is known to be essential for bacterial uptake.     

Evaluation of the relative expression of genes associated with adherence after different hours of co-culture between Streptococcus uberis and MAC-T cells

Streptococcus uberis is an environmental pathogen associated with subclinical and clinical IMI in both lactating and non-lactating cows. RC19 strain was isolated from a cow with subclinical mastitis, qualitatively classified as moderate biofilm producer in Todd Hewitt medium (THB), and it showed a high value of the adhered bacteria (CFU/ml). Hence, the aims of this study were (a) to determine ability to adhere to and internalize into epithelial cells MAC-T for 1, 2 and 3 h, (b) to evaluate the relative expression of adherence-associated genes from co-cultures of S. uberis with MAC-T cells at 1, 2 and 3 h. We hypothesized that upon contact with bovine mammary epithelial cells, S. uberis upregulates adherence-associated genes encoding adhesins, which enable it a higher adherence to and/or internalization into host cells. Four to six genes increased their R with regard to the control after initial contact with MAC-T cells (group 1) at 1, 2 and 3 h. The highest value of R was observed at 2 h after co-culture between RC19 and MAC-T cells.     

Immunoproteomics of extracellular proteins of Chinese virulent strains of Streptococcus suis type 2

Streptococcus suis type 2 (SS2) is a porcine zoonotic pathogen with worldwide distribution, and lacking suitable vaccine and virulent maker were bottleneck to control this infection. An immunoproteomic assay was used to identify antigenic proteins from the total extracellular proteins of the virulent Chinese SS2 strain ZY05719. The convalescent serum of a specific pathogen free (SPF) mini-pig recognized nine protein spots on PVDF membrane. Antigenic proteins on a duplicate gel, as well as those with a similar placement of extracellular proteins from another virulent strain (HA9801) and an avirulent strain (T15) on 2-D gels, were excised and identified by MALDI-TOF-MS. PMF of the protein spots were performed using the MASCOT server. Two proteins were found in all three strains. Comparative proteomic analysis between the two virulent strains and the avirulent strain revealed nine differential proteins, eight of which were successfully identified. Genes for six of the differentially expressed proteins were found in both virulent strains, and of those were present in the avirulent stain.