The characterization of a putative DNA repair protein in Mycobacterium Tuberculosis, encoded rv2414c.
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Date
2023
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Abstract
Mycobacterium tuberculosis (MTB) is a causative agent of the communicable disease
tuberculosis (TB), which is regarded as one of the top ten causes of death worldwide. Globally,
TB accounts for over 10 million infections and over 1.8 million deaths annually. These
statistics are subject to a constant increase due to the emergence of drug resistant strains.
Although, the recent use of next generation sequencing technology has generated complete
genome sequences and functional genomic data for various organisms (MTB included), to this
point, the biological functions of several proteins encoded for in the MTB genome are not
known or characterized hence they are called hypothetical proteins. Characterization of these
hypothetical proteins is essential, as they could be involved in key regulatory processes of
MTB, which is essential for the pathogen to retain a successful life cycle and disease
progression. For successful invasion of the host and disease progression, it is important for
MTB to retain genomic stability. Therefore, the degree of survival of MTB in the host
environment is largely dependent on the bacterium’s ability to retain genomic stability. DNA
repair mechanisms protect bacterial DNA from damage that can be induced by numerous stress
factors. The hypothetical protein rv2414c encodes a gene amongst the MTB immunogenic
protein identified in a study by Chiliza et al., (2019) which is closely associated with genes
involved in MTB DNA repair, suggesting a possible role in DNA repair pathways. Therefore,
the present study is aimed at characterizing a conserved hypothetical
protein encoded rv2414c in Mycobacterium tuberculosis to elucidate the proteins biological
function. For in-silico characterization, three bioinformatics tools were used, namely;
Mycobroswer, I-TASSER and STRING online tools. Thereafter, a CRISPR-cas9 gene
silencing mechanism was developed to elucidate the biological role of rv2414c. CRISPR
system entails the co-expression of the silenced form of RNA-guided DNA endonuclease from
the type II CRISPR system (dCas9) and a small guide RNA specific to a target sequence,
leading to the DNA recognition complex resultant in transcription interference of
corresponding DNA sequence. This CRIPSR mechanism was achieved by generating
knockdown mutants. Phenotypic characterization of the mutants was accomplished by
monitoring the mutants’ growth kinetics and biological assays were done to assign a possible
biological function to rv2414c. Bioinformatics analysis suggests rv2414c is involved in DNA
repair based on the structural networks it forms with 3 genes (dprA, recA and cinA) involved
in MTB DNA repair and 3 proteins (rv3242c, rv3737 and rv2897c) that are involved in mainly
aiding in the mechanism of DNA repair.
However, the growth kinetics showed that rv2414c has no impact on the MTB growth rate, as
all strains grew in a similar growth pattern with no statistical significance (p > 0.05) observed
at the different time points. Additionally, UV biological assay showed that rv2414c is not a
major role player in DNA repair, as UV exposure did not have an effect on bacterial survival
rate even in the knockdown strain. A slight decrease in cell survival rate was noticed after
addition of Mitomycin C (MMC) between Δrv2414c (100 ng/ml) + MMC and Δrv2414c +
MMC, however, the difference was not significant. This implies that rv2414c is not involved
in MTB DNA repair.
Description
Masters Degree. University of KwaZulu-Natal, Durban.