Heterogeneity among Homologs of Cutinase-Like Protein Cut5 in Mycobacteria

Abstract

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.

Fig 1. Complete nucleotide sequences of the genes encoding Cut5a and Cut5b (in M. tuberculosis H37Rv) and Cut5 (in M. bovis).

Fig 2. Clustal omega alignment of Rv3724b in Mtb H37Rv and its homologs.

Fig 3. Expression of Rv3724 and its homolog Mb_3751 in Mtb H37Rv and M. bovis BCG respectively.

Fig 4. Specificity of Cut5 homologs in mycobacteria.

Fig 5. Existence of Cut5b and its homologs in mycobacteria.

Fig 6. Immunoelectron microscopy showing localization of Cut5b in Mtb H37Rv.

Fig 7. Expression of Cut5b at different in vitro growth phases of Mtb H37Rv.

Fig 8. Proposed evolutionary pathway of Rv3724a (cut5a) and Rv3724b (cut5b) (Panel A) and possible organizations (Org. 1, 2 & 3) of Cut5 protein and its homologs (Panel B) in mycobacteria.