Identification and characterization of the factor(s) that regulate the expression of Mycobacterium tuberculosis H37Rv kas operon

Abstract

Mycobacterial cell wall comprises around 60% long chain fatty acids rich in mycolic acids and unique to mycobacterial species. Cell wall mycolates of pathogenic mycobacteria play a crucial role in virulence and survival in adverse conditions, as they provide a protective barrier to bacteria against harsh physiological conditions. Mycolic acids are fatty acid derivatives which are synthesized by two different pathways in mycobacteria, namely fatty acid synthase type 1 (FAS-I) and fatty acid synthase type 2 (FAS-II), unlike other bacteria where only FAS-I exists. FAS-I encodes for a single polypeptide with multiple catalytic activities that generate shorter Co-A esters from acetyl-Co-A primers and create precursors for elongation. FAS-II consists of disparate enzymes, incapable of de novo fatty acid synthesis, but elongates palmitoyl-ACP to unusually long carbon chain fatty acids whose length is defined in species-specific manner. Elongation pathway in M. tuberculosis is accomplished by the concerted action of genes residing at two loci (Cole et al., 1998). One locus, dubbed as kas operon, contains a set of five genes, malonyl CoA-ACP transacylase (fabD, Rv2243), acyl carrier protein (acpM, Rv2244), β-ketoacyl-ACP synthase (kasA, Rv2245), beta-ketoacyl-ACP synthase (kasB, Rv2246) and acetyl Co-A carboxylase (accD6, RV2247) (Fig. I, Table I). The ketoreductase (mabA) and enoyl reductase (inhA) genes are present as another operon at other locus. Mycolic acids are the target for several anti-mycobacterial drugs like INH (Iso-Nicotinic acid Hydrazide), ETH (Ethionamide), EMB (Ethambutol), and TLM (Thiolactomycin) which arrest biosynthesis of mycolic acid at different steps leading to cell death. Isoniazid (INH), a first line drug for tuberculosis, blocks the FAS-II elongation pathway leading to progressive depletion of mature mycolates (Slayden and Barry, 2002). Wilson et al., (1999) studied the effect of INH on the tuberculosis transcriptome and observed a significant transcriptional upregulation of the kas operon genes. A similar upregulation was also obtained when the culture was treated with Ethionamide. Both INH and Ethionamide are acclaimed inhibitors of mycolic acid synthesis and as recent reports have established, a single point mutation in InhA can mediate coresistance to both drugs suggesting that inhA is the primary target of both drugs (Vilcheze et al., 2006). INH forms a covalent complex with AcpM and KasA and it has been shown that inhibition of InhA induces the KasA-containing complex (Kremer et al., 2003). The transcriptional upregulation of kas operon following INH and ethionamide treatments has also been reported in other studies (Slayden et al., 2000; Betts et al., 2003). Combined together, these results suggest that the disruption of FAS-II pathway leads to a cascade of molecular reactions in bacteria that are sensed by regulatory factor(s), which in turn affects the expression of these genes either by direct or indirect interaction with upstream regulatory sequences of the kas operon. The present project is directed towards the identification of minimal promoter region that regulates the transcription of the operon during basal and under different stress conditions, and the transcriptional regulatory factors which directly/indirectly associate with signature sequences in upstream regulatory region. To enable this, several promoter deletion constructs were prepared in fusion with reporter gene so that the reporter gene activity, imparted by cloned upstream sequence, could be directly monitored. This allowed the identification of minimal promoter region flanked by positive and negative regulatory elements. The global influence of flanking regulatory sequences was revalidated by juxtaposing them to Mycobacterium bovis BCG hsp60 promoter. The cis-regulatory motifs present in the upstream region were searched by computing over-representation of the motifs in kas operon upstream regions of different mycobacterial species. In silico identified motifs with higher significance value, present in the positive and negative regions, were validated by deletion mutagenesis. Motifs were specifically deleted/punctuated while creating promoter-reporter constructs to examine their role in promoter activity. Further, motif‘ sequences were matched to E.coli databases of transcription factor footprints according to PWM (Position Weight Matrix) in order to identify E. coli factors with similar binding sites. Subsequently, Mycobacterium tuberculosis orthologs of these factors were identified in H37Rv genome by BLAST homology search. EMSA and super shift assay were performed with chosen promoter elements to confirm the binding ability of the purified recombinant factors as well as total cell lysate of M. bovis BCG and M. aurum. Objectives of the study 1. Identification of the optimal promoter elements that impart basal level and induced expression of kas operon. 2. Identification of regulatory factor(s) affecting the expression of kas operon genes under antibiotic stress condition. 3. Characterization of the regulatory factor(s).