Potential virulence factors of Streptococcus dysgalactiae associated with bovine mastitis
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Potential virulence factors of Streptococcus dysgalactiae associated with bovine mastitis
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Potential virulence factors of Streptococcus dysgalactiae associated with bovine mastitis
- 1. Potential virulence factors of Streptococcus dysgalactiae associated with bovine mastitis Prepared by – Dr. Shoaib Ahmad Shakhes
- 2. Introduction • S.dysgalactiae, the Lancefield serological group C bacterium, is one of the most common environmental pathogens capable of causing bovine mastitis. • Mastitis caused by environmental pathogens is a major problem that affects many well-managed dairy herds. • A disease responsible for large economic losses in the dairy industry. (Song et al.,2002)
- 3. Contd. • Contagious pathogens live and multiply on and in the cow's mammary gland and are spread from animal to animal primarily during milking. • Environmental pathogens are those whose primary reservoir is the environment where cows live and not infected mammary glands. (Smith and Hogan, 1993)
- 4. Among the environmental streptococci, S.uberis and S.dysgalactiae are the most prevalent, infecting mammary glands as favorable conditions arise. (Smith et al., 1985; Oliver, 1988; Todhunter et al., 1995) Contagious Mastitis • Staphylococcus aureus • CNS • Streptococcus agalactiae • Streptococcus dysgalactiae • Arcanobacterium pyogenes • Mycoplasma • Leptospira Environmental Mastitis • Escherichia coli • Klebsiella pneumoniae • Klebsiella oxytoca • Enterobacter aerogenes • S. uberis / S. parauberis • S. bovis • S.dysgalactiae
- 5. Streptococcus dysgalactiae • Taxonomically, S.dysgalactiae belongs to: • Family: Streptococcaceae • Genus: Streptococcus • Species: S.dysgalactiae subsp.dysgalactiae S.dysgalactiae subsp. equisimilis • It is Gram-positive cocci, arranging in chains. • The name S.dysgalactiae subsp. dysgalactiae is proposed for strains of animal origin. These strains belong to Lancefield groups C and L, are α, β, or nonhemolytic, and do not exhibit streptokinase activity on human plasminogen or proteolytic activity on human fibrin.
- 6. Potential virulence factors of S.dysgalactiae • The ability to initiate growth in vivo and stably infect a host requires acquisition by the invading pathogen of virulence factors capable of neutralizing nonspecific mechanisms of the host's defense. • These factors include: Structural components Toxins Enzymes (Calvinho et al.,1998)
- 7. Interaction of S.dysgalactiae with host- derived proteins S.dysgalactiae can interact with several plasma and extracellular host derived proteins. S.dysgalactiae IgA IgG Albumin Fibronectin Collagen Vitronectin Plasminogen α2-macroglobulin
- 8. IgG-binding protein S.dysgalactiae isolated from bovine IMI are capable of binding to IgG in a nonimmune fashion. (LaÈmmler and Frede, 1989; RantamaÈki and MuÈ ller, 1995) A receptor for the Fc fraction of IgG (FcR) has been isolated from 8 of 26 culture supernatants of group C streptococcal strains.
- 9. Contd. Specificity of IgG binding reaction was confirmed through inhibition by prior addition of IgG or Fc- fractions of IgG to FcR. S.dysgalactiae isolated from bovine IMI interacted both with IgG and albumin. Role of IgG-binding proteins as a microbial factor is that may interfere with host defense mechanisms at sites with low concentrations of IgG.
- 10. MAG Protein (α2-macroglobulin/albumin/IgG-binding) Trifunctional protein receptor . (Jonsson et al., 1994) There are three binding activities reside in discrete domains of the protein: Single IgG-binding domain Stretch of 50 amino acids that mediate albumin binding α2M-binding domain is located in the N terminus of the molecule
- 11. Role of MAG • Inhibition of opsonization by binding to Fc fraction of IgG Capsule Cell wall S.dysgalactiae MAG proteinα2-M Albumin
- 12. Fibronectin-binding S.dysgalactiae binds to fibronectin (Myhre and Kuusela, 1983; Mamo et al., 1987) Fibronectin is a high - molecular-weight glycoprotein found in soluble form in plasma and body fluids and in an insoluble form in connective tissue and basement membranes. (Yamada and Olden, 1978)
- 13. S.dysgalactiae
- 14. Contd. Presence of a ligand binding site in a fibronectin- binding receptor termed Au these amino acid residues adopt a specific conformation on binding to fibronectin. Conformational changes in the Au sequence induced by binding to fibronectin may favor bacterial adherence & internalization to mammary tissue. (Speziale et al.,1996)
- 15. FnB
- 16. α2-Macroglobulin-binding Strains of S.dysgalactiae from cattle bound to α2-macroglobulin-trypsin (α2-M-T) ( Valentin-Weigand et al., 1990; RantamaÈki and MuÈller, 1995) α2-macroglobulin is a plasma glycoprotein that can inhibit nearly all endoproteases by a unique trap mechanism.
- 17. Contd. This protein also detected in mastitic milk as a result of increased vascular permeability. Binding of α2-M-T to S.dysgalactiae is specific and can inhibit by homologous α2-M-T and not by other proteins such as fibrinogen, fibronectin, IgG or albumin.
- 18. Pretreatment of S.dysgalactiae with α2-M-T led to a concentration-dependent inhibition in phagocytosis of this bacterium by bovine neutrophils. Digestion of the streptococcal binding sites for α2M and α2M-T with pronase abolished the inhibition of phagocytosis of S.dysgalactiae. Binding of α2M or its complexes appeared to play a role in streptococcal pathogenicity. (Valentin-Weigand et al., 1990) Contd.
- 19. Vitronectin-binding S.dysgalactiae from bovine IMI can bind to vitronectin (Filippsen et al.,1990; RantamaÈki and MuÈller, 1995) Vitronectin (Vn) is a multifunctional plasma and extracellular matrix glycoprotein involved in : Cell attachment Coagulation Phagocytosis Protection of bystander cells from complement- and T cell-mediated lysis (Sobel et al., 2009)
- 20. Contd. The binding of S protein to bacteria was saturable and inhibit only by unlabeled S protein but not by albumin. Trypsinization and heat treatment of bacteria can not destroy the S-protein binding capacity in case of group A & C streptococci. Facilitate the adherence of bacteria to host cells in particular to endothelial cells.
- 21. M-like proteins (Fg & IgG binding) These are similar to M proteins the major virulence factors of S.pyogenes by mediating resistance to phagocytosis. Fg and IgG binding is a common feature among these proteins.
- 23. Contd. There is a gene called demA, encoding for a protein (DemA) with a molecular mass of 58 kDa, was characterized. DemA displays both plasma protein binding properties and sequence similarities with the M and M-like proteins of other streptococcal species.
- 24. Mig protein The Mig protein of S.dysgalactiae is a type III IgG- binding protein, expressing IgG- and α2-M-binding receptors. This protein also displays binding activities to bovine immunoglobulin A (B-IgA) It is an M-like protein that considered a potential virulence factor of S.dysgalactiae. (Song et al., 2001)
- 25. Role of Mig α2-M bound to the bacterial surface via Mig Inhibiting the activity of proteases Protecting important virulence factors from proteolytic degradation Interferes with phagocytosis by bovine neutrophils Binding IgA helping the microorganism to evade the immunological surveillance of the host.
- 26. Lipoteichoic acid (LTA) LTA is present in strains of S.dysgalactiae isolated from bovine IMI. (Calvinho et al., (1997) All S.dysgalactiae isolates cross-reacted with monoclonal antibody directed against the polyglycerophosphate backbone of LTA from S.pyogenes LTA play a role during early host-pathogen interactions that lead to: Cytotoxic effects on mammalian cells Adherence of S.dysgalactiae to mammalian cells Establishment of IMI (Simpson et al., 1982)
- 27. Capsule • Presence of capsule was detected in only some fresh isolates of S.dysgalactiae from IMI. HYDROPHILIC MORE RESISTANT TO PHAGOCYTIC ACTION OF MΦ HYDROPHOBIC SUSCEPTIBLE TO PHAGOCYTIC ACTION OF MΦ
- 28. Role of Capsule • Presence of capsule resist phagocytosis by: Masking opsonin binding Shielding of bacterial surface proteins
- 30. Fibrinolysin • S.dysgalactiae produces a fibrinolysin specific for bovine but not for human fibrin. • Responsible for cleavage of fibrin and fibrinogen. • Breaks the clots and fibrin deposit, there by extending areas of infection. Segura and Gottschalk(2004)
- 31. Hyaluronidase Extracellular hyaluronidase is an enzyme that degrades hyaluronic acid. Hyaluronan is the main polysaccharide component of the host connective tissues. Hyaluronidase enzyme also called the spreading factor, is considered an important pathogenic factor for several hyaluronidase-producing streptococci.
- 32. Contd. It has been isolated and purified from S.dysgalactiae isolates from bovine IMI as a protein of approximately 55 kDa (Sting et al., 1990) Hyaluronidase contribute to streptococci tissue-invasive properties
- 33. Streptokinase Plasminogen activator, derived from the bovine mastitis-inducing S.dysgalactiae show specificity to bovine plasminogen. (McCoy et al., 1991) It forms a strong complex with plasminogen causing its activation to plasmin which hydrolyses fibrin as well as connective tissue proteins.
- 34. Contd. Plasmin has proteolytic activity may enhance the ability of streptococci to: Spread in host tissues Facilitates bacterial growth and colonization during the very early stages of infection in the lactating gland by promoting the release of nutrients. (Leigh, 1993, 1994)
- 36. Superantigen (SDM) Streptococcus dysgalactiae-derived mitogen, a 25 kDa protein, is a recently discovered superantigen isolated from S.dysgalactiae culture supernatant. (Anastassios et al., 2006) SDM shows about 30% homology with other superantigens at the amino-acid sequence level.
- 37. SDM belongs to a family distinct from other known bacterial superantigens with a zinc- binding site. Crystal structure of SDM SDM -Systemic shock -Disseminated intravascular coagulation(DIC) -Immunosuppression
- 38. Adherence Adherence of bacteria to host cells has been suggested as prerequisite for the colonization and establishment of infection. Adhesion of S.dysgalactiae to the epithelia of the mammary gland may help to: Prevent removal of the pathogen from the gland during milking Play a role in bacteria to colonize Cause bovine mastitis (Frost et al. 1977; Harper et al. 1977)
- 39. Contd. Adherence of strains of S.dysgalactiae seems to be mediated by collagen and fibronectin. S.dysgalactiae adheres to epithelial cells and to extracellular matrix proteins and invades the immune system. (Calvinho et al., 1997)
- 40. Uptake of S.dysgalactiae by mammary epithelial cells Internalization of bacteria by mammary epithelial cells may result in protection of bacteria from : Host defense mechanisms Action of antimicrobial agents This feature aids in development of persistent infection and may provide a route for bacterial colonization of sub-epithelial tissues.
- 41. Contd. Microfilaments, but not microtubules, were found to be necessary for bacterial entry into eukaryotic cells (Almeida et al., 1995). Involvement of cell kinases & de novo eukaryotic protein synthesis are required for internalization of S.dysgalactiae into bovine mammary gland epithelial cells through receptor–mediated endocytosis mechanisms.
- 42. Contd. S.dysgalactiae did not appear to cause cell injury at any bacterial density or time point evaluated. S.dysgalactiae can survive within mammary epithelial cells for an extended time without losing viability.
- 43. Conclusion Streptococcal species have developed a wide array of strategies to evade the host defense mechanisms thus they may also exhibit persistency in the host / udder tissue. A detailed knowledge about these strategies may be very essential in development of newer ways to counteract the streptococcal infections. Knowledge of these evasion strategies may also help in designing the new drugs and vaccines that may even target the evading streptococcal species.
Editor's Notes
- Some of these bacterial factors can exert a direct effect on stromal cells while others can thwart one or more host defense mechanisms to allow for survival and persistence of the pathogen in the invaded tissue (Woolcock, 1988)
- These interactions are mediated by bacterial surface proteins, which due to the high specificity and affinity of binding are also termed receptors, although a signaling event following the binding has not been demonstrated (RantamaÈki and MuÈ ller, 1995). IgG Albumin Fibronectin Fibrinogen Collagen Vitronectin Plasminogen α2-macroglobulin
- Specificity of IgG binding reaction was confirmed through inhibition by prior addition of IgG or Fc-fractions of IgG to FcR. LaÈmmler and Frede (1989) showed that S. dysgalactiae isolated from bovine IMI interacted both with IgG and albumin. None of the strains evaluated bound albumin without simultaneously binding IgG.
- This observation supports previous speculations on the role of IgG-binding proteins as a microbial factor that may interfere with host defense mechanisms at sites with low concentrations of IgG. (Boyle, 1990) Binding activities for IgG and albumin were associated with a S. dysgalactiae protein similar to protein G of group G streptococci while a second group of binding proteins with similar molecular weight interacted with IgG but not with albumin (LaÈmmler and Frede, 1989).
- Work with another strain of S.dysgalactiae of bovine origin led to cloning and sequencing of a gene (mag,gene for a type III Fc receptor) encoding for a trifunctional protein receptor that can bind α2-macroglobulin, albumin and IgG called as protein MAG. ( Jonsson et al., 1994) By subcloning and expressing various parts of the gene as fusion proteins, we found that the three binding activities reside in discrete domains of the protein. The single IgG-binding domain, localized in the C-terminal part of the molecule, shows high homology to streptococcal type-Ill Fc receptors. In the middle of the molecule, there is a stretch of 50 amino acids (aa) mediating albumin binding. This region has partial homology with the albumin-binding domains of streptococcal protein G. The α2M-binding domain is located in the N terminus of the molecule and is composed of a unique aa sequence.
- Only one of the proteins coded by these genes was expressed under standard culture conditions. However, Southern-hybridization showed that both genes were present in 20 clinical isolates of S. dysgalactiae (Lindgren et al., 1993)
- phosphorylation of integrins has been associated with transduction of signals from the cell membrane to the actin network via intermediate proteins such as talin, vinculinWe recently cloned from S. dysgalactiae two different genes encoding proteins with Fn binding activity (10). The nucleotide sequences and corresponding amino acid sequences of the Fn receptors FnBA and FnBB have been submitted to the GenBank
- Binding of α2-macroglobulin (α2M) to streptococci and its effects on phagocytosis were investigated. Two types of streptococcal binding sites for α2M were observed: Streptococcus pyogenes from human infections interacted only with native α2M whereas S. dysgalactiae from bovine and S. equi from equine infections bound only a complex of α2M with trypsin (α2M-T). Preincubation of S. pyogenes with native α2M substantially enhanced their phagocytosis by human polymorphonuclear neutrophils (PMN) whereas preincubation with α2M-T was without any effect. On the other hand, incubation of S. dysgalactiae and S. equi with α2M-T markedly reduced their phagocytosis by PMN from the respective host species. Native α2M did not affect the phagocytosis of these streptococci. Digestion of the streptococcal binding sites for α2M and α2M-T with pronase abolished the enhancement of phagocytosis of S. pyogenes by native α2M as well as the inhibition of phagocytosis of S. dysgalactiae and S. equi by α2M-T. Thus, binding of α2M or its complexes appeared to play a role in streptococcal pathogenicity.
- Binding of α2-M-T to S.dysgalactiae might block structures on the streptococcal surface required for adherence and phagocytosis by PMN.
- Vitronectin (Vn) is a multifunctional plasma and extracellular matrix glycoprotein involved in cell attachment, coagulation, phagocytosis, and the protection of bystander cells from complement- and T cell-mediated lysis
- Trypsinization and heat treatment of bacteria destroyed the S-protein binding capacity for group G streptococci, S. aureus, and E. coli but not for group A and C streptococci. S protein plays a mediatory role in adherence of streptococci to endothelial cells and that S-protein-specific binding sites on streptococci are involved in this interaction.
- Binding of M proteins of group A streptococci to fibrinogen or complement factor H have been considered as mechanisms to evade phagocytosis (Kehoe, 1994).
- Resisting phagocytosis by bovine neutrophils in the presence of bovine serum by: In bovine mastitis, the mucosal defence system in the mammary gland plays a major role in the prevention of infection. Since immunoglobulin A (IgA) is the predominant class of immunoglobulin present on mucosal surfaces, and constitutes an important defence mechanism against microbial infections, IgA-binding receptors expressed by bacteria might be involved in the development of mastitis by binding IgA and helping the micro-organism evade the immunological surveillance of the host.
- Bacterial surface hydrophobicity (SH) plays a role in adhesion of bacteria to host surfaces and ingestion by phagocytic cells.
- Products released by pathogenic streptococci could favor establishment and persistence of IMI.
- The ability of bacteria to grow in mastitic milk is enhanced by the presence of the caseinolytic enzyme plasmin (Mills and Thomas 1981). The transformation of plasminogen to plasmin requires plasminogen activators, which are known to occur in blood plasma and animal tissues (Collen 1980). Plasmin is a potent serine proteinase that has an important function in physiological processes in mammals, such as degradation of extracellular matrix proteins, blood clot dissolution (fibrinolysis), cellular migration, and for cancer metastasis Lottenberg et al. (1994).
- Streptokinase and staphylokinase have unique but slightly different mechanisms of plasminogen activation. Streptokinase and staphylokinase form 1:1 stoichiometric plasminogen activator complexes with plasminogen and plasmin, respectively. Streptokinase induces a conformation of the serine proteinase domain of plasminogen, which exposes the active site of proteinase without prior proteolytic cleavage, thereby providing the streptokinase-plasminogen complex with what has been called “virgin” enzyme activity (Reddy and Markus 1972). In contrast, the staphylokinase-plasminogen complex is proteolytically inactive but it can be transformed into the active staphylokinase-plasmin complex by activation with plasmin (Collen et al. 1993).
- Superantigens are protein toxins of bacterial or viral origin able to cross-link major histocompatibility complex class II (MHCII) molecules with T-cell receptors (TcR)
- Some of the enterotoxins are known to bind to MHC molecules at different site from the peptide binding groove which results in T cell activation and cytokine production [109]. This over-stimulation by superantigens leads to systemic shock and disseminated intravascular coagulation (DIC) similar to lipopolysaccharides (LPS) of Gram-negative bacteria. Increased IFN and IL-1 cell destruction
- S. dysgulactiue interacted, and engulfment of bacteria by cellular microvilli. Collectively, these results suggested that cytoskeleton microfilaments of the mammary epithelial cell play a role in the entry of S. dysgulactiue that seems to occur by an endocytosis mechanism, as described for other invasive pathogens
- Bovine mammary epithelial cells were pretreated with inhibitors of protein kinase activity, actin polymerization and receptor-mediated endocytosis. In addition, mammary epithelial cells and Streptococcus dysgalactiae were pretreated with inhibitors of protein synthesis. Results showed that activity of tyrosine protein kinases, intact microfilaments and de novo eukaryotic protein synthesis was required for uptake of S. dysgalactiae by bovine mammary epithelial cells; a process that appeared to occur via receptor-mediated endocytosis. In contrast, de novo bacterial protein synthesis was not required for uptake of S. dysgalactiae by MAC–T cells.
- Uptake or internalization of bacteria by mammary epithelial cells is a potentially important feature in the pathogenesis of some mastitis pathogens. Bacterial uptake may result in protection from host defense mechanisms and action of antimicrobial agents and may affect secretory cell function. In addition, an infection route from the lumen into subepithelial tissues may be provided (Chandler et al., 1980; Falkow et al., 1992; GalaÂn, 1994).