Prepared by – Dr. Shoaib Ahmad Shakhes

Virulence factors of Staphylococcus aureus associated in Bovine mastitis

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.

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)

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.

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)

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.

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.

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)

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.

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

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

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)

Biofilm-associated protein & PIA

Biofilm-forming capacity is widely considered as amajor virulence factor of S. aureus, recent evidencessuggest that a group of surface proteins play a leadingrole during the development of the microbialcommunities.

The first member of this group of proteins wasdescribed in a S.aureus bovine mastitis isolate and wasnamed 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)

Role of Bap in pathogenesis

Surface-associated Bap of S. aureus has beendemonstrated to promote attachment to abioticsurfaces 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

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)

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

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 isreleased at the basal side of the epithelia

Also IL-1B and IL-8 trigger the inflammation

Chemotaxis Inhibitory Protein ofStaphylococcus 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

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)

Staphylococcal Protein A (SpA) Staphylococcal protein A is a membrane-bound

exoprotein characterized and well known for itsability to bind to the Fc region of immunoglobulinsof 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

S. aureus becomes intracellular following contact withthe cell surfaces of bovine mammary epithelial cells,escapes the endosome to reside and possibly multiply inthe host cell cytoplasm, and induces the host cell tobecome apoptotic.

(Kenneth et al.,1998)

Survival within neutrophils

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

In truly encapsulated S.aureus strains the bacterial cellwall remains hidden by the capsule. This fact does notlimit the binding of opsonins to the cell wall ,but mayinhibit the interaction b/w the bound opsonins and thecorresponding receptors located on phagocytes. Thusencapsulated bacteria may require anti-capsularantibodies for opsonization.

(Baselga et al.,1993)

Because bacterial adherence is thought to be aprerequisite for infection, the CP-mediated inhibition ofadherence suggests that encapsulated strains are lessvirulent. However, CP exhibits antiphagocytic activity.

(Petra et al,2000)

Kinetics of SR Expression Two stages

Hydrophobic surface proteins

Express CP and proteins preventing

phagocytosis

“agr” gene

Toxins S.aureus can produce four different haemolytic

toxins

α and β hemolysins are the most important inpathogenesis of the intramammary infections

(Park et al., 2004)

Leucocidin

TSST-1

Role of toxins

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)

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

β-toxin

β-toxin is produced by 75-100 % of these strains.

It is a Mg2+-dependent sphingomyelinase C,which degrades sphingomyelin in the outerphospholipid layer of the membrane

(Linehan et al., 2003)

It induces inflammation of the gland, i.e.,oedema and a flow of PMNL into mammaryducts and glandular alveoli.

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)

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

Role of extracellular enzymes & coagulase

S. aureus produces numerous extracellular enzymesincluding hyaluronidase, phosphatase, nuclease,lipase, catalase, staphylokinase and proteases, thathave been implicated in the pathogenesis of bovineIMI.

(Sutra et al.,1994)

Enzymes could allow S. aureus to use milk substratesfor their metabolism and, consequently, to adapt tomilk and grow.

(Anderson et al., 1976)

Staphylokinase

Many strains of S.aureusexpress a plasminogen activator called staphylokinase

It has the immune evasion activity

Avoidance of complement fixation as well as phagocytosis

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 thehyaluronic acid (in CT)helps in the invasion into thehost tissue.

(Farrell et al., 1995)

Coagulase Coagulase is not an enzyme.

It is an extracellular protein has the ability to turnfibrinogen into fibrin threads.

(Palma et al.,1999)

Fibrin deposition may shield staphylococci fromphagocytic cells.

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

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.