Heterogeneity among Homologs of Cutinase-Like Protein Cut5 in Mycobacteria.

The study of genomic variability within various pathogenic and non-pathogenic strains of mycobacteria provides insight into their evolution and pathogenesis. The mycobacterial genome encodes seven cutinase-like proteins and each one of these exhibit distinct characteristics. We describe the presence of Cut5, a member of the cutinase family, in mycobacteria and the existence of a unique genomic arrangement in the cut5 gene of M. tuberculosis (Mtb) strains. A single nucleotide (T) insertion is observed in the cut5 gene, which is specific for Mtb strains. Using in silico analysis and RT-PCR, we demonstrate the transcription of Rv3724/cut5 as Rv3724a/cut5a and Rv3724b/cut5b in Mtb H37Rv and as full length cut5 in M. bovis. Cut5b protein of Mtb H37Rv (MtbCut5b) was found to be antigenically similar to its homologs in M. bovis and M. smegmatis, without any observed cross-reactivity with other Mtb cutinases. Also, the presence of Cut5b in Mtb and its homologs in M. bovis and M. smegmatis were confirmed by western blotting using antibodies raised against recombinant Cut5b. In Mtb H37Rv, Cut5b was found to be localized in the cell wall, cytosol and membrane fractions. We also report the vast prevalence of Cut5 homologs in pathogenic and non pathogenic species of mycobacteria. In silico analysis revealed that this protein has three possible organizations in mycobacteria. Also, a single nucleotide (T) insertion in Mtb strains and varied genomic arrangements within mycobacterial species make Rv3724/Cut5 a potential candidate that can be exploited as a biomarker in Mtb infection

Expression of Cut5b at different <i>in vitro</i> growth phases of <i>Mtb</i> H37Rv.

<p>Expression of Cut5b at different <i>in vitro</i> growth phases of <i>Mtb</i> H37Rv.</p

Percentage homology based on amino acid sequences of cutinases of <i>Mtb</i>H37Rv.

<p>*Tuberculist database (genolist.Pasteur.fr/Tuberculist),</p><p>**West <i>et al</i>., 2009</p><p>***Adapted from West <i>et al</i>., 2008</p><p>Percentage homology based on amino acid sequences of cutinases of <i>Mtb</i>H37Rv.</p

Specificity of Cut5 homologs in mycobacteria.

<p>Specificity of Cut5 homologs in mycobacteria.</p

Existence of Cut5b and its homologs in mycobacteria.

<p>Existence of Cut5b and its homologs in mycobacteria.</p

Proposed evolutionary pathway of <i>Rv3724a</i> (<i>cut5a</i>) and <i>Rv3724b</i> (<i>cut5b</i>) (Panel A) and possible organizations (Org. 1, 2 & 3) of Cut5 protein and its homologs (Panel B) in mycobacteria.

<p>Proposed evolutionary pathway of <i>Rv3724a</i> (<i>cut5a</i>) and <i>Rv3724b</i> (<i>cut5b</i>) (Panel A) and possible organizations (Org. 1, 2 & 3) of Cut5 protein and its homologs (Panel B) in mycobacteria.</p

Homologs of <i>M</i>. <i>tuberculosis</i> H37Rv Cut5a/Rv3724a and Cut5b/Rv3724b present in various mycobacterial and bacterial species.

<p>Names of the homologs in the table are mentioned as; specie; gene; % identity; amino acid overlap/total amino acids, e.g. <i>Kineococcus radiotolerance</i><b>(specie)</b>; <i>Krad_4111</i><b>(gene)</b>; (53.2%) <b>(% identity)</b>; [62/294] <b>(overlap/total amino acids)</b></p><p>Homologs of <i>M</i>. <i>tuberculosis</i> H37Rv Cut5a/Rv3724a and Cut5b/Rv3724b present in various mycobacterial and bacterial species.</p

Clustal omega alignment of <i>Rv3724b</i> in <i>Mtb</i> H37Rv and its homologs.

<p>Clustal omega alignment of <i>Rv3724b</i> in <i>Mtb</i> H37Rv and its homologs.</p