The genomic sequences of several closely related pathogenic and non-pathogenic E

Question

The genomic sequences of several closely related pathogenic and non-pathogenic Escherichia coli strains that are associated with extra-intestinal infections of humans have been determined. Upon detailed bioinformatic analysis of their genomic sequences, you identify an interesting DNA sequence that is present in the pathogenic E. coli strains but absent in the non-pathogenic E. coli strains. Based on your analysis of this DNA sequence, you predict that it encodes an outer membrane protein that is a member of the Type V(a) autotransporter family of proteins; autotransporter proteins are known to contribute to E. coli virulence.

i) Explain how and why you have concluded that the protein is a Type V(a) autotransporter protein.

ii) Assuming that the protein is an autotransporter protein, describe a series of experiments that you would need to undertake:

to prove that the protein is located in the outer membrane of the pathogenic coli strains,

to determine the protein’s intrinsic biological activity that promotes disease,

and to show that this autotransporter protein is a virulence factor that contributes to the ability of the pathogenic coli strains to cause disease.

Explanation / Answer

i) an autotransporter domain is a structural domain found in some bacterial outer membrane proteins. The domain is always located at the C-terminal end of the protein and forms a beta-barrel structure. The barrel is oriented in the membrane such that the N-terminal portion of the protein, termed the passenger domain, is presented on the cell surface.

ii) In one of the experiments  the DNA sequence of a cloned fragment (4.6 kb) revealed a single gene coding for a 169-kDa precursor of IgA1 protease; the precursor contained three functional domains: the N-terminal leader (which is assumed to initiate the inner membrane transport of the precursor), the extracellular IgA1 protease, and a C-terminal “helper” domain (which is required for secretion across the outer membrane.

The autotransporters are present only in the Bacteria kingdom and are most prevalent in the phylum Proteobacteria, including the -, -, -, and -Proteobacteria classes.It has been proposed that autotransporter proteins could have evolved by domain shuffling .

Proteins exported by the autotransporter secretion mechanism are translated as a polyprotein possessing three domains. The three domains of the polyprotein (the leader sequence, the passenger domain, and the C-terminal -domain) are indicated. The leader sequence directs secretion via the sec apparatus and is cleaved at the inner membrane by a signal peptidase releasing the remaining portion of the molecule into the periplasm. Once in the periplasm the -domain assumes a biophysically favored state characterized by a -barrel shaped structure which inserts itself into the outer membrane to form a pore. After insertion into the outer membrane the passenger domain is translocated to the bacterial cell surface where it may remain intact or undergo processing. A processed protein may be released into the extracellular milieu or remain associated with the bacterial cell surface.

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