Why does bacteria produce coagulase

The inability of strict anaerobes to synthesize catalase, peroxidase, or superoxide dismutase may explain why oxygen is poisonous to these microorganisms. In the absence of these enzymes, the toxic concentration of H 2 O 2 cannot be degraded when these organisms are cultivated in the presence of oxygen. Organisms capable of producing catalase rapidly degrade hydrogen peroxide which is a tetramer containing four polypeptide chains, which are usually amino acids long.

It also contains four porphyrin heme groups ie. The enzyme catalase is present in most cytochrome-containing aerobic and facultative anaerobic bacteria. Catalase is the enzyme which has one of the highest turnover numbers compared to all other enzymes; one molecule of catalase has the ability to convert millions of molecules of hydrogen peroxide to water and oxygen in each second.

Catalase production and activity can be detected by adding the substrate H 2 O 2 to an appropriately incubated to hour tryptic soy agar slant culture.

Organisms which produce the enzyme break down the hydrogen peroxide, and the resulting O 2 production produces bubbles in the reagent drop, indicating a positive test. Organisms lacking the cytochrome system also lack the catalase enzyme and are unable to break down hydrogen peroxide, into O 2 and water and are catalase negative.

Catalase activity is very useful in differentiating between groups of bacteria. For example, the morphologically similar Enterococcus catalase negative and Staphylococcus catalase positive can be differentiated using the catalase test. Coagulases are enzymes that clot blood plasma by a mechanism that is similar to normal clotting.

Clumping factor activity has been attributed to two cell wall anchored surface proteins, ClfA and ClfB, each of which binds to fibrinogen [42] , [43].

Three additional secreted factors of S. One of these, Eap, has been demonstrated to contribute to disease pathogenesis and antibodies against Eap generate a moderate amount of protection against abscess formation [5]. Thus, S. We surmise that during bloodstream infections, Coa and vWbp promote establishment of microthrombi, thereby disrupting local bloodflow and promoting staphylococcal adhesion to the vasculature.

Endothelial cells in association with staphylococci would thus be exposed to high concentrations of toxins, precipitating local injury and inflammation. These events may ultimately promote colonization and staphylococcal invasion across blood vessels and into organ tissues. These predictions can now be tested to unravel the many unique attributes of coagulases towards the pathogenesis of staphylococcal diseases. Nucleotide sequence specifying the D1 domain of coagulase displays variation between different staphylococcal isolates, providing the basis for classification of S.

This variation also underwrites staphylocoagulase serotyping, i. It is not known whether the recently reported D1 sequence variation can provide for an escape from protective immunity afforded by coagulase-specific antibodies as is reported here. Future work will need to define the molecular basis of protective immunity by Coa and vWbp specific immune responses and address the scope of protection against the different coagulase types of S. Experiments with blood from human volunteers involved a protocol that was reviewed, approved and performed under the regulatory supervision of The University of Chicago's Institutional Review Board IRB.

Written, informed consent was provided by all volunteers. Veterinary care is under the direction of full-time resident veterinarians boarded by the American College of Laboratory Animal Medicine. The signs for judging morbidity have been specified by the IACUC to be any one of the following: rapid respiration; slow, shallow or labored respiration; rapid weight loss and ruffled fur; hunched posture; inappetence.

Signs for judging animals to be moribund included any one of the following: signs of morbidity plus; impaired ambulation; evidence for muscle atrophy or emaciation; lethargy; inability to remain upright. Signs of pain in infected animals were judged if any one of the following was observed: animal not alert, abnormal postures or abnormal movement; inappetence or dehydration; guarding reaction upon contact; vocalization when palpated or moved; self-mutilation, restlessness or lethargy; shock.

Animals that were judged to be moribund were euthanized with CO 2 , a method that is approved by the Panel on Euthanasia of the American Veterinary Medical Association. These vectors were electroporated into S. Plasmid p coa did not restore the expression of Coa to wild-type levels Fig. Plasmids constructs were verified by DNA sequencing and electroporated into staphylococcal strains.

After confirming that the data sets abide by a normal distribution, the statistical analysis of staphylococcal burden in renal tissue was analyzed using the unpaired two-tailed student's t-test. Quantification of mouse renal abscesses in histopathology slides were analyzed for statistical significance using the non-parametric Mann-Whitney test.

Statistical significance of the mouse lethal challenge data was calculated with the two-tailed log-rank test. All animal experiments were conducted at least twice to assess reproducibility.

Blood samples were then tested in the manner described above for mouse blood. All experiments were repeated in at least three independent trials to examine reproducibility. Supernatant was loaded onto a HiTrap desalting column to remove remnants of Triton-X Proteins were then loaded onto a Superdex 75 GE Healthcare column for final purification. On day 60, rabbits were bled and serum recovered for immunoblotting or passive transfer experiments.

Charged matrix was washed with 50 column volumes of PBS, antibodies eluted with elution buffer 1 M glycine pH 2. Affinity and rates of association and dissociation were measured on a BIAcore Buffers were sterile filtered and degassed. A CM5 chip was prepared for amine linkage by injection of human prothrombin nM, pH 4.

For the coagulase-prothrombin interaction, coagulase was injected at 10 pM, 0. For the coagulase-fibrinogen interaction, coagulase was injected at pM, pM, 1 nM, 5 nM, 10 nM, 25 nM, 50 nM, nM, and nM for 60 seconds of association followed by 60 seconds of dissociation and the chip was regenerated as described above.

All experiments were repeated in triplicate on at least three occasions. Two-tailed t- tests were used to determine statistically significant differences between the means of binding observations in the presence or absence of antibodies. The change in absorbance was measured at nm for 10 minutes in a spectrophotometer, plotted as a function of time, and fit to a linear curve.

On the fifth or fifteenth day post infection, mice were killed by CO 2 asphyxiation and their kidneys excised. All organs were examined for surface lesions and 8—10 right kidneys were sent for histopathology sectioning and hematoxylin-eosin staining. These slides were examined by light microscopy for internal abscesses. Sectioned kidneys were deparaffinized and rehydrated through xylen and serial dilutions of ethanol to distilled water.

Antigen-antibody binding was detected with the DAB substrate chromogen system. The slides were briefly immersed in hematoxylin for counterstaining and evaluated by light microscopy.

Cohorts of 10 mice were used, with 3 mice reserved for bleeding and antibody titers. Cohorts of 10 mice were used. Immunohistochemical staining with secondary antibody. Kidneys were removed, embedded in paraffin, and, as a control to Figure 1 , thin-sections stained by immunochemistry using HRP-conjugated secondary antibody alone. No specific staining of tissues is observed. B The same strains as above were incubated with lepirudin-anticoagulated human blood for 30 minutes, upon which an aliquot was removed and plated.

Staphylococcal survival was assessed by colony formation on agar and the counts were normalized against the initial CFU inoculum. Culture supernatants from S. The survival of infected mice was recorded over 10 days. Data are representative of two independent experimental trials.

Antibodies against Coa and vWbp block S. B Lepirudin-treated human blood was treated with antibodies as above. Surface plasmon resonance measurements of the association between staphylococcal coagulases and human coagulation proteins.

Prothrombin A, C and fibrinogen B,D were injected over the chip at increasing concentrationso shown in inset and the response increase was measured over time. The affinity was calculated by the kinetic data using BiaEvaluation software E and the residual difference from the fit are shown below the sensorgram. Immunization with coagulases protects mice against S. Bacterial load and abscess formation were determined following necropsy in the kidneys of animals that had been killed five days A—H or fifteen days I—P following infection.

Renal tissues were thin-sectioned, stained with hematoxylin-eosin and histopathology images acquired by light microscopy. Animals were monitored for survival up to hrs after challenge. Statistical significance was calculated via the log-rank test. Abstract The bacterial pathogen Staphylococcus aureus seeds abscesses in host tissues to replicate at the center of these lesions, protected from host immune cells via a pseudocapsule.

Author Summary Clinical isolates of the human pathogen Staphylococcus aureus secrete coagulase Coa , a polypeptide that binds to and activates prothrombin, thereby converting fibrinogen to fibrin and promoting clotting of plasma or blood. Introduction Staphylococcus aureus is a frequent cause of bacteremia, pneumonia, skin and soft tissue infection as well as osteomyelitis and septic arthritis [1].

Download: PPT. Figure 1. Localization of prothrombin, fibrinogen, coagulase Coa , and von Willebrand factor binding protein vWbp in staphylococcal abscesses.

Figure 2. Staphylococcus aureus coa and vWbp mutants display defects in blood clotting. Coa and vWbp are required for the pathogenesis of S. Figure 3. Contributions of coa and vWbp to bacterial survival in blood and S. Table 1. Figure 4. Tube Test to detect free coagulase Dilute the plasma 1 in 10 in physiological saline mix 0. Test is hour broth culture, Positive control is hr S. Pipette 0. Add 5 drops 0. After mixing, incubate the three tubes at Degree Celsius. Examine for clotting after 1 hours.

If no clotting has occurred, examine at 30 minutes intervals for up to 6 hours. All the negative slide test must be confirmed using the tube test. MSA also differentiates on the basis of mannitol fermentation. Microorganisms that can ferment mannitol create acidic byproducts, which decrease the pH of the medium and cause the pH indicator, phenol red, to turn yellow.

Gram-positive cocci are often isolated from clinical samples. Catalase is an enzyme that converts hydrogen peroxide to water and oxygen gas. The test is easy to perform; bacteria are simply mixed with H 2O 2.