Copper, Zinc-Superoxide Dismutase from Clinically Isolated Escherichia coli: Cloning, Analysis of sodC and Its Possible Role in Pathogenicity

Abstract

Superoxide dismutase has been discovered within the periplasm of several Gram-negative pathogens. We studied the Cu,Zn-SOD enzyme in Escherichia coli isolated from clinical samples (stool samples) collected from patients suffering from diarrhea. Antibiogram studies of the isolates were carried out to determine the sensitive and resistant strains. The metal co-factor present in the enzyme was confirmed by running samples in native gels and inhibiting with 2 mM potassium cyanide. A 519 bp sodC gene was amplified from resistant and sensitive strains of Escherichia coli. Cloning and sequencing of the sodC gene indicated variation in the protein and amino acid sequences of sensitive and resistant isolates. The presence of sodC in highly resistant Escherichia coli isolates from diarrheal patients indicates that sodC may play role in enhancing the pathogenicity by protecting cells from exogenous sources of superoxide, such as the oxidative burst of phagocytes. The presence of SodC could be one of the factors for bacterial virulence.

Keywords: Drug resistance; Escherichia coli; Gram-negative bacteria; Pathogenicity; Superoxide dismutase; sodC.

Fig. 1

Achromatic bands against the dark background indicate the presence of SOD enzymes in periplasmic extracts from isolates: Lane 1E. coli 130, Lane 2E. coli 131, Lane 3E. coli 140, Lane 4E. coli 2622, Lanes 5,6,7,8 enzyme extracts of above strains treated with 2 mM KCN indicating the inhibition of Cu, Zn-SOD activity

Fig. 2

Ethidium bromide stained 1% agarose gel showing ~520 bp PCR product of sodC gene. Lane 1 Negative control: without template, Lane 2sodC amplicon of E. coli 2622, Lane 3sodC amplicon of E. coli 130, Lane 4sodC amplicon of E. coli 131, Lane 5sodC amplicon of E. coli 140, Lane 6 DNA ladder

Fig. 3

Multiple alignment of sodC nucleotide sequences among E. coli isolates and E. coli reference strain

Fig. 4

Multiple alignment of amino acid sequences of SodC (translated) among E. coli isolates and E. coli reference strain