This document discusses the virulence factors of Staphylococcus aureus associated with bovine mastitis. It begins by introducing S. aureus as a common cause of bovine mastitis. It then describes several virulence factors that allow S. aureus to colonize and infect the mammary gland, including adhesins that help it adhere to epithelial cells, such as fibronectin binding proteins; toxins that damage tissue and immune cells, like alpha-toxin; and antiphagocytic factors that help it evade the immune system, like capsular polysaccharides. The document stresses that the combined action of these various surface proteins, toxins, and enzymes enable the pathogenicity of S. aureus in bovine mastitis.

2. Prepared by – Dr. Shoaib Ahmad Shakhes
Virulence factors of Staphylococcus aureus
associated in Bovine mastitis

3. Introduction
 S. aureus is one of the most frequently isolated contagious
mastitis pathogens that cause either clinical or subclinical
mammary gland infection.
(Fox et al.,1989)
 Usually of sub clinical in nature.
 Accounts for 25-30% of total mastitis in cattle.
 The prevalence of S. aureus mastitis could increase from < 5%
to more than 30% in a year, causing significant increases in
milk somatic cell count.
(Smith et al.,1998)
 Loss accounts for nearly 25% of total yield.

4. Staphylococcus aureus
 Taxonomically, S. aureus belongs to:
 Family: Staphylococcaceae
 Genus: Staphylococcus
 It is Gram-positive cocci, arranging in bunch of grapes.
 It is catalase and coagulase positive.
 More than 95% of all coagulase positive staphylococci from
bovine mastitis belong to S. aureus.
(Fox et al.,1989)

5. S.aureus Virulence Factors
 S. aureus colonizes the teat end and it moves to the
intramammary area either by progressive colonization or
by the changes in intramammary pressure, especially at
the end of milking.
(Anderson,1983)
 In the intramammary area it can adhere to epithelial cells,
multiply and colonize the tissue.
(Sandholm et al.,1989)
 The S. aureus virulence factors comprise cell surface
structural components and extracellular components such
as proteins, lipids, carbohydrates, proteoglycans,
glycolipids and secretory products.

6. Contd..
Based on their biological activities, S. aureus
virulence factors can be divided into three general
functional categories:
 Those that mediate adhesion of bacteria to host cells or
tissue. (adhesins)
 Those that promote tissue damage and spread. (invasins)
 Those that protect the bacteria from the host immune
system. Thus, the pathogenicity of S. aureus depends on
the combined action of cell surface structural
components, different extracellular toxins and enzymes.
(Arvidson and Tegmark ,2001)

7. Molecular Kotch’s postulates
 Suspected virulence factor should be associated with
pathogen under study.
 Inactivation of genes responsible for the virulent
factors should significantly decrease its virulence.
 Reintroduction of these genes should restore the
virulence.

8. Entry of the Pathogen
 Main sources are
 Infected quarter
 Udder skin
 Teat skin
 Spreads by hands, Milking machines, clothes used to clean
 Teat orifice is the main source for entry, later the bacteria
colonize by adhering to the keratinized mammary epithelial
cells
 Finally enter the MG while milking, walking or standing up.

9. Colonisation of the mammary gland
 Bacterial surface properties play a major role in the
host-bacterium relationship during S. aureus IMI.
 Surface components of S.aureus participate in
adhesion to host mammary tissues and to resistance
to phagocytosis by milk cells.
(Sutra et al.,1994)

10. Role of Adhesins
 Staphylococcal adhesions is by two mechanisms:
I. Non-specific physiochemical interaction
II. Specific interactions between bacterial and cell
S.aureus have high surface hydrophobicity favoring
fixation of bacteria to host cell through hydrophobic
interactions with the cell membrane.

11. Specific interactions
 Usually attaches to collagen and fibronectin on cell
 There are two distinct types of Fnbp on bacteria which
attach to receptor on the cell
 D1-D4 is the specific domain of Fnbp that attaches to
Fn and collagen expressed on the mammary
epithelium and micro lesions on the mammary
epithelium
 Specific and nonspecific mediators are proteins,
treatment of S.aureus with proteases reduces
virulence of bacteria
 S.aureus can spread by binding to fat globules

13. Clumping factor (ClfA/ClfB)
 Fibrinogen/fibrin binding protein (the clumping
factor) which promotes attachment to blood clots
and traumatized tissue
 ClfB is an important determinant of adhesion to
desquamated epithelium and promotes adhesion
to squames
 Binds cytokeratin 10 (as well as fibrinogen)
 Colonization of the bovine udder teat

15.  In S. aureus, fibronectin binding is a very important step
not only for attachment but also for the internalization
process in epithelial cells.
(Michael ,2004)
 Internalization of S. aureus suggests the presence of
specific elements associated with S. aureus that elicit the
uptake of this particular pathogen by the host cell.
(Raul et al.,1996)

16. Biofilm-associated protein & PIA
 Biofilm-forming capacity is widely considered as a
major virulence factor of S. aureus, recent evidences
suggest that a group of surface proteins play a leading
role during the development of the microbial
communities.
 The first member of this group of proteins was
described in a S.aureus bovine mastitis isolate and was
named Bap, for biofilm-associated protein.
(Cristina et al.,2006)
 Polysaccharide intracellular adhesin (PIA ) has an
important role in biofilm formation in S.aureus &
S.epidermidis
(Yazdani et al., 2006)

17. Role of Bap in pathogenesis
 Surface-associated Bap of S. aureus has been
demonstrated to promote attachment to abiotic
surfaces and intercellular adhesion in vitro
But hinder several other aspects such as:
 Colonization and infectivity, like adherence to
immobilized fibronectin and fibrinogen
 Adherence to mammary gland tissue ex vivo,
internalization by epithelial cells
 Infection of mammary glands

18.  Altogether, these results suggest that Bap hinders
interaction between bacterial MSCRAMMs receptors
and the host proteins, favouring the establishment of
chronic infections.
(Cristina et al., 2006)

19. Expression of antiphagocytic factors
 Main defence mechanism in mammary gland is
phagocytosis by PMNLs.
 If SCC increases ---induces influx of PMNLs
 PMNLs bind non specifically to bacteria and kill it
 Killing is more efficient if there is opsonization
through Fc receptor on PMNLs
 Level of IgG2 low initially –increases later due to
exudation of plasma

20. Complements and interleukins
 Activation of complements and generation of C5a
and formylated methionine group that attract the
PMNs
 Following the attachment of the bacterium, IL-8 is
released at the basal side of the epithelia
 Also IL-1B and IL-8 trigger the inflammation

21. Chemotaxis Inhibitory Protein of
Staphylococcus aureus (CHIPS)
 Leukocyte migration is a key event both in host
defense against invading pathogens as well as in
inflammation.
 Bacteria generate chemoattractants primarily by
excretion
 Activation of leukocytes

22. What is CHIPS ?
 CHIPS is a protein secreted by S.aureus that specifically
impairs the response of neutrophils and monocytes to
formylated peptides and C5a.
 This chemotaxis inhibitory protein of S. aureus (CHIPS) is
a 14.1-kD protein encoded on a bacteriophage and is
found in 60% of clinical isolates.
 Suggest a new immune escape mechanism of S. aureus and
put forward CHIPS as a potential new anti-inflammatory
therapeutic compound.
(Carla et al., 2004)

24. Staphylococcal Protein A (SpA)
 Staphylococcal protein A is a membrane-bound
exoprotein characterized and well known for its
ability to bind to the Fc region of immunoglobulins
of most mammalian species
(Alonso & Dagget 2000)
 Detected in 50-60% of IMI isolates.
 Not important in initial stages of infection because
of low levels of IgG2
 In principle this will disrupt opsonization and
phagocytosis

26.  S. aureus becomes intracellular following contact with
the cell surfaces of bovine mammary epithelial cells,
escapes the endosome to reside and possibly multiply in
the host cell cytoplasm, and induces the host cell to
become apoptotic.
(Kenneth et al.,1998)
Survival within neutrophils

27. Capsule
 About 85-95% of IMI S.aureus produce diffuse
colonies like capsulated strains.
 They produce capsule polysaccharides (CP) called
microcapsules or slime
 Do not stain by India ink and not visible in LMS
 Also called as pseudocapsules- immunogenic
 Resist phagocytosis by
 masking opsonin binding and
 shielding of bacterial surface proteins

28.  In truly encapsulated S.aureus strains the bacterial cell
wall remains hidden by the capsule. This fact does not
limit the binding of opsonins to the cell wall ,but may
inhibit the interaction b/w the bound opsonins and the
corresponding receptors located on phagocytes. Thus
encapsulated bacteria may require anti-capsular
antibodies for opsonization.
(Baselga et al.,1993)
 Because bacterial adherence is thought to be a
prerequisite for infection, the CP-mediated inhibition of
adherence suggests that encapsulated strains are less
virulent. However, CP exhibits antiphagocytic activity.
(Petra et al,2000)

29. Kinetics of SR Expression
 Two stages
 Hydrophobic surface proteins
 Express CP and proteins preventing
phagocytosis
 “agr” gene

30. Toxins
 S.aureus can produce four different haemolytic
toxins
 α and β hemolysins are the most important in
pathogenesis of the intramammary infections
(Park et al., 2004)
 Leucocidin
 TSST-1

31. Role of toxins

32. α -toxin
 α -toxin, which is produced by about 20- 50% of strains from
bovine IMI is a extracellular protein of S. aureus
 This membrane damaging toxin is a basic polypeptide
possesses lethal, cytotoxic, dermonecrotic and hemolytic
properties
(Kenny et al,.1992)
 It binds to cell membranes and forms hexameric pores leading
to cell death as a consequence of rapid egress of cytoplasmic
components.
 Injection of α-toxin in to rabbit mammary gland causes
haemorrhagic necrosis of the gland
(Sutra et al.,1994)

33. S.aureus α-toxin is a paradigm for pore-
forming toxins that target neutrophils

34. β-toxin
 β-toxin is produced by 75-100 % of these
strains.
 It is a Mg2+-dependent sphingomyelinase C,
which degrades sphingomyelin in the outer
phospholipid layer of the membrane
(Linehan et al., 2003)
 It induces inflammation of the gland, i.e.,
oedema and a flow of PMNL into mammary
ducts and glandular alveoli.

35. Leucocidin
 It is produced by most S.aureus in IMI
 Two parts S & F—synergistically act by
cytolysis and is not hemolytic
 Leucocidin damage membranes of
phagocytosing host defence cells by inducing
Ca2+ influx and subsequent pore formation.
(Kamio et al., 1993)

36. TSST-1
 Above 20% S.aureus in IMI produce TSST-1
 which causes toxic shock due to its superantigenic activity.
(Kenny et al.,1993)
 Increased IFN and IL-1 cell destruction
-Systemic shock
-Disseminated
intravascular
coagulation(DIC)
-Immunosuppression

37. Role of extracellular enzymes &
coagulase
 S. aureus produces numerous extracellular enzymes
including hyaluronidase, phosphatase, nuclease,
lipase, catalase, staphylokinase and proteases, that
have been implicated in the pathogenesis of bovine
IMI.
(Sutra et al.,1994)
 Enzymes could allow S. aureus to use milk substrates
for their metabolism and, consequently, to adapt to
milk and grow.
(Anderson et al., 1976)

38. Staphylokinase
 Many strains of S.aureus
express a plasminogen
activator called
staphylokinase
 It has the immune
evasion activity
 Avoidance of complement
fixation as well as
phagocytosis

41. Other enzymes
 Staphylococcal nuclease (thermonuclease) it
hydrolyzes RNA and DNA to 3´-phosphomono-
nucleotides phosphodiesterase requiring Ca2+ and it
is an important diagnostic test for identification of
S. aureus . (Shortle et al., 1983)
 Hyaluronate lyase (hyaluronidase) lyses the
hyaluronic acid (in CT)helps in the invasion into the
host tissue.
(Farrell et al., 1995)

42. Coagulase
 Coagulase is not an enzyme.
 It is an extracellular protein has the ability to turn
fibrinogen into fibrin threads.
(Palma et al.,1999)
 Fibrin deposition may shield staphylococci from
phagocytic cells.

43. Conclusion
 Staphylococcus aureus has several virulence factors
mainly categorized as cell surface structural components,
secreted toxins, enzymes and proteases. Some of these S.
aureus cell surface structural components are considered
adhesins which allow the bacteria to colonize the host.
 On the other hand, secreted virulence factors comprise
toxins and enzymes, which enable bacteria to invade and
spread through the tissue of the host and they are
collectively named invasins

44.  In addition to these two broad categories of virulence
factors S. aureus has many other mechanisms to adopt
to the host environment such as immune evasion,
ability to attach and internalize into host cells and
formation of small colony variants.
 Staphylococcus aureus being a most resistant bacterium,
A detailed knowledge about these virulence factors may
help in future to develop suitable and effective control
strategies.
 Further, these virulence factors and their genes may be
used as candidates in developing the effective vaccines.