Molecular characterization of the point mutation in the Rhomboid encoding gene in Mycobacterium avium subspecies paratuberculosis

Abstract

Mycobacterium avium subspecies paratuberculosis (MAP) is the etiological agent for a paratuberculosis an incurable, chronic and contagious disease affecting ruminants characterized by progressive weight loss, intermittent diarrhoea and eventually death. MAP has been associated with Crohn’s a chronic inflammatory disease of the gastrointestinal tract raising public health concerns. Despite challenges associated with MAP detection, molecular methods allow rapid and accurate detection. The standard marker for MAP i.e., IS900 element was reported in other mycobacteria and this led to research into MAP specific targets however, these are not as sensitive as IS900 therefore, there is need to search for more MAP specific targets. The rhomboid encoding gene in MAP is one of such targets. One study reported a SNP (G>A) at position 219 which resulted into truncated proteins (MAP_2426c and MAP_2425c). Point mutations are less mutable compared to other polymorphism and hence they are stable and informative. Rhomboids are intramembrane serine proteases that are ubiquitous therefore functional characterization of these proteases has revealed important roles such as quorum sensing, antibiotic resistance, and cell division which are crucial for survival of organisms. Bioinformatics reveals that rhomboid proteases of mycobacteria are involved in DNA replication and metabolite pathways. In this investigation, plasmid DNA (n=32) cloned with rhomboid encoding gene of MAP was successfully amplified using PCR and the products (n=16) sequenced. Multiple sequence alignment using MEGA software version X confirmed presence and conservation of the SNP in all the samples. A bioinformatics analysis (BLAST-search) in the NCBI database revealed presence of SNP in MAP isolates of Type C strain (15) but absent in isolates of Type S strain (3). The primers and probe sequences targeting the SNP (G>A) in the rhomboid encoding gene to detect MAP were designed manually and synthesized by Eurofins-MWG. The real-time PCR SNP assay designed detected all the 39 samples (n=32 clones and 7 field isolates) and discriminated MAP from 13 non-tuberculosis Mycobacteria, 6 strains of Mycobacterium tuberculosis and 1 isolate of Mycobacterium bovis. The analytical specificity of the assay was 100%. In conclusion, the SNP (G>A) at nucleotide position 219 is apparently conserved in Type C isolates but not Type S isolates. The real-time SNP assay detected all the samples with no cross reactivity which allowed quick and accurate detection. There is need to investigate the role of SNP in Type C isolates but also evaluate the SNP assay with MAP isolates from different regions of the world.