Functional characterization of a small heat shock protein from Mycobacterium leprae

<p>Abstract</p> <p>Background</p> <p>Small heat shock proteins are ubiquitous family of stress proteins, having a role in virulence and survival of the pathogen. <it>M. leprae</it>, the causative agent of leprosy is an uncultivable organism in defined media, hence the biology and function of proteins were examined by cloning <it>M. leprae </it>genes in heterologous hosts. The study on sHsp18 was carried out as the knowledge about the functions of this major immunodominant antigen of <it>M. leprae </it>is scanty.</p> <p>Results</p> <p>The gene encoding <it>Mycobacterium leprae </it>small heat shock protein (sHsp18) was amplified from biopsy material of leprosy patients, and cloned and expressed in <it>E. coli</it>. The localization and <it>in vitro </it>characterization of the protein are detailed in this report. Data show that major portion of the protein is localized in the outer membrane of <it>E. coli</it>. The purified sHsp18 functions as an efficient chaperone as shown by their ability to prevent thermal inactivation of restriction enzymes <it>Sma</it>I and <it>Nde</it>I. Physical interaction of the chaperone with target protein is also demonstrated. Size exclusion chromatography of purified protein shows that the protein can form multimeric complexes under <it>in vitro </it>conditions as is demonstrated for several small heat shock proteins.</p> <p>Conclusion</p> <p>The small heat shock protein sHsp18 of <it>M. leprae </it>is a chaperone and shows several properties associated with other small heat shock proteins. Membrane association and <it>in vitro </it>chaperone function of sHsp18 shows that the protein may play a role in the virulence and survival of <it>M. leprae </it>in infected host.</p

Cloning of mce1 locus of Mycobacterium leprae in Mycobacterium smegmatis mc2 155 SMR5 and evaluation of expression of mce1 genes in M. smegmatis and M. leprae

Plasmid pSET152 is a broad host range mobilizable vector which integrates into streptomyces chromosome utilizing att site and int function of &#216;C31. Transformation of this plasmid into Mycobacterium smegmatis mc2 155 SMR5 gave stable transformants carrying the pSET152 as an integrated copy. Integration occurred at the cross over sequence 5'TTG disrupting the gatA gene (Glu-tRNAGln amidotransferase subunitA), which is non-essential under conditions used. Recombinant pSET152 plasmids carrying mce1 locus of Mycobacterium leprae were used to construct M. smegmatis transformants carrying the mce1 locus in their chromosome. RT-PCR analysis revealed specific transcripts of M. leprae mce in M. smegmatis. The transcribed mRNA carried intergenic regions between genes of mce1 locus indicating that mce1 locus is an operon. Examination of M. leprae specific mRNA from lepromatous leprosy patient's biopsy showed that mce locus is transcribed as an operon in the pathogen also