Identification of an immunogenic histone-like protein (HLP_Mt) of Mycobacterium tuberculosis

Abstract

We report the identification of the first histone-like protein ofMycobacterium tuberculosis(MTB) (HLP_Mt). The T cell blot assay was used to identify antigens of MTB associated with human immune response in healthy contacts. Fraction 21 corresponding to proteins in the molecular weight range of approximately 30 kDa were found to be immunogenic in tuberculin reactors. None of the fractions were found to be immunogenic by this assay in non-reactors to tuberculin. All sera, irrespective of the source, showed reactivity with MTB antigen(s) over a wide molecular weight range (205 16 kDa). In the present study fraction 21 was processed for the generation of murine polyclonal sera and amino acid sequencing. The sequence of a 16-amino acid long peptide showed a 100% homology with an open reading frame (ORF) in the translated sequence of cosmid cY349 (Sanger Centre, Cambridge, UK). The ORF was predicted to code for a protein of 214 amino acids. Oligonucleotide primers were synthesized based on the nucleotide sequence located at the 5' and 3' regions of the gene. The gene encoding the predicted protein was PCR-amplified, cloned, sequenced and expressed in Escherichia colias a protein of 28 kDa. The expressed HLP_Mt protein was shown to react with the polyclonal murine sera originally raised against fraction 21. Human immune response to the recombinant HLP_Mtprotein was demonstrated by its ability to induce lymphoproliferation in peripheral blood derived mononuclear cells, and the presence of anti-HLP_Mtantibodies in pooled patient sera by immunoblot. The recombinant HLP_Mtprotein elicited a vigorous lymphoproliferative response especially in healthy tuberculin reactors compared to non-reactors and patients of tuberculosis, (P≪ 0.05). The protein has unique dual domains with homology to both bacterial histone-like proteins (HU) and eukaryotic histone H1. Homology to prokaryotic and eukaryotic deoxyribonucleic acid (DNA)-binding proteins suggested that HLP_Mtcould bind DNA. DNA-binding properties were confirmed by South-Western analysis strongly suggesting an interaction between HLP_Mt and the MTB chromosome.