The answer to question 7
Salmonella and Y. pestis have shown difference during their expression and residence. Salmonella is a facultative intracellular bacterium residing mainly intolerant macrophages, expressing a shortened O-antigen and active PgtE. Yersinia pestis has Pla active on its surface
naturally with rough lipopolysaccharides type due to frame-shift mutations within the O-antigen biosynthesis gene cluster. Y. pestis modifies its LPS structure upon transfer from the vector to the human temperature to favor enzymatic activity of Pla suppresses innate immune responses to LPS. S. enteric spreads inside the host as an intracellular pathogen that expresses rough LPS during its growth within or instantly after release from phagocytes. PgtE cleaves factor B and H whereas Pla of Y. pestis cleaves factor H but is inactive to B. O-antigen is a major defense structure of Salmonella against complement killing spreading in the host by repeated cycles of phagocytes infections, and in the phagocytes, the activity of PgtE is high due to decreased expression of the O-antigen that inhibits substrate binding by PgtE. The pgte expression is beneficial for Salmonella during degradation of serum complement pl9rotein in host leading to decreased opsonization and phagocytosis (Riva et al. 2015).
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The answer to question 8
OmpT and ompP were expressed by Salmonella and Y. pestis were expressed in E. coli because are required in cleaving factors B and H. B was cleaved by E . coli expressing their PgtE generating four immune-reactive fragments within a certain period. H was expressed incompletely by Pla expressed by E. coli. The virulence factor of pgtE is on the outer surface of membrane protease with a short chain Many pathogens recruit H on their surfaces to inhibit complement attacks by limiting assembly and delay association of the AP c3 convertase c3bBb. Cleavage of H abolishes its complement regulatory activity to increase the formation of c3b and cleavage of B results to lesser active c3 convertase and formation of c3b reduced.
The answer to question 9
A pathogen that produces a substance that would dade both factor B and H has an advantage since it gets sheltered from the host immune system by ensuring less of C3 accumulates on its surfaces and alliance with neutrophils is reduced. These fragments so make.
certain of the pathogenesis of the disease by aiming both the activating and inhibitory regions complement system. Factor B and H compete with c3/c3b which substrates for cleavage by are PgtE where c3-convertase contains Bb which cleaves c3 while H is a cofactor for c3b cleavage by factor I.
The answer to question 10
There were major steps followed in the paper were: For cleavage factors for B and H; E. coli strains were grown and omptin expression was induced by 5Um IPTG and other strains grown with shaking at 370 C overnight. For cleavage assay the plasmid –complemented Salmonella strains were diluted 1:50 with fresh broth in the morning and grown for two hours more, harvested by centrifugation and washed three times with PBS with Ph 7.4-7.5. The results obtained showed that both factor B and H were cleaved indicating that PgtE is required by Salmonella for B and H cleavage. For opsonophagocytosis assay; Salmonella strains were grown overnight in pgtE-inducing medium and labeled with FITC-Celite by incubating for fifteen minutes with shaking at 370 c in 0.5ug FITC-Celite in the dark and washed three times. Later it was incubated in 50% NHS for five minutes with agitation at 370 showing that expression of pgtE impaired complement-mediated phagocytosis of 14028R.
References
Riva, R., Korhonen. T. k., and Meri. S.(2015). The outer membrane protease PgtE of Salmonella enterica interferes with the alternative complement pathway by cleaving factors B and H. Frontiers in microbiology, 6, 63.
