Potential virulence factors of

Streptococcus dysgalactiae associated with bovine mastitis

Prepared by – Dr. Shoaib Ahmad Shakhes

Introduction

• S.dysgalactiae, the Lancefield serological group Cbacterium, is one of the most common environmentalpathogens capable of causing bovine mastitis.

• Mastitis caused by environmental pathogens is a majorproblem that affects many well-managed dairy herds.

• A disease responsible for large

economic losses in the dairy

industry. (Song et al.,2002)

Contd.

• Contagious pathogens live and multiply onand in the cow's mammary gland and arespread from animal to animal primarily duringmilking.

• Environmental pathogens are those whoseprimary reservoir is the environment wherecows live and not infected mammary glands.

(Smith and Hogan, 1993)

Among the environmental streptococci, S.uberis andS.dysgalactiae are the most prevalent, infectingmammary 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

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 forstrains of animal origin. These strains belong to Lancefieldgroups C and L, are α, β, or nonhemolytic, and do not exhibitstreptokinase activity on human plasminogen or proteolyticactivity on human fibrin.

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)

Interaction of S.dysgalactiae with host-derived proteins

S.dysgalactiae can interact with several plasma and extracellular host derived proteins.

S.dysgalactiae

IgAIgG

Albumin

FibronectinCollagen

Vitronectin

Plasminogenα2-macroglobulin

IgG-binding protein

S.dysgalactiae isolated from bovine IMI arecapable of binding to IgG in a nonimmunefashion.

(LaÈmmler and Frede, 1989; RantamaÈki and MuÈ ller, 1995)

A receptor for the Fc fraction of IgG (FcR) hasbeen isolated from 8 of 26 culturesupernatants of group C streptococcal strains.

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.

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

Role of MAG• Inhibition of opsonization by

binding to Fc fraction of IgGCapsule

Cell wall

S.dysgalactiae

MAG proteinα2-M

Albumin

Fibronectin-binding

S.dysgalactiae binds to fibronectin

(Myhre and Kuusela, 1983; Mamo et al., 1987)

Fibronectin is a high - molecular-weightglycoprotein found in soluble form in plasmaand body fluids and in an insoluble form inconnective tissue and basement membranes.

(Yamada and Olden, 1978)

S.dysgalactiae

Contd.

Presence of a ligand binding site in a fibronectin-binding receptor termed Au these amino acidresidues adopt a specific conformation on bindingto fibronectin.

Conformational changes in the Au sequenceinduced by binding to fibronectin may favorbacterial adherence & internalization to mammarytissue.

(Speziale et al.,1996)

FnB

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.

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.

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.

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 attachmentCoagulationPhagocytosis Protection of bystander cells from complement- and T cell-mediated lysis

(Sobel et al., 2009)

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.

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.

Inhibition of C3b-Fixation

S.dysgalactiae

C3b

IgG

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.

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)

Role of Mig

α2-M bound to the bacterial surface via MigInhibiting 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.

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 cellsAdherence of S.dysgalactiae to mammalian cells Establishment of IMI

(Simpson et al., 1982)

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

Role of Capsule

• Presence of capsule resist phagocytosis by:

Masking opsonin binding

Shielding of bacterial surface proteins

Extracellular factors

Fibrinolysin

Hyaluronidase

Streptokinase

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)

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.

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

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.

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)

Inhibition of Opsonization

Streptokinase

Superantigen (SDM)

Streptococcus dysgalactiae-derived mitogen, a 25 kDa protein, is a recently discovered superantigen isolated from S.dysgalactiaeculture supernatant.

(Anastassios et al., 2006)

SDM shows about 30% homology with other superantigens at the amino-acid sequence level.

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

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)

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)

Uptake of S.dysgalactiae by mammary epithelial cells

Internalization of bacteria by mammaryepithelial cells may result in protection ofbacteria from :

Host defense mechanisms

Action of antimicrobial agents

This feature aids in development of persistentinfection and may provide a route for bacterialcolonization of sub-epithelial tissues.

Contd.

Microfilaments, but not microtubules, were found tobe necessary for bacterial entry into eukaryotic cells(Almeida et al., 1995).

Involvement of cell kinases & de novo eukaryoticprotein synthesis are required for internalization ofS.dysgalactiae into bovine mammary gland epithelialcells through receptor–mediated endocytosismechanisms.

Contd.

S.dysgalactiae did not appear to cause cellinjury at any bacterial density or time pointevaluated.

S.dysgalactiae can survive within mammaryepithelial cells for an extended time withoutlosing viability.

Conclusion

Streptococcal species have developed a wide array ofstrategies to evade the host defense mechanisms thusthey may also exhibit persistency in the host / uddertissue.

A detailed knowledge about these strategies may bevery essential in development of newer ways tocounteract the streptococcal infections.

Knowledge of these evasion strategies may also help indesigning the new drugs and vaccines that may eventarget the evading streptococcal species.