Genome analysis identifies a spontaneous nonsense mutation in ppsD leading to attenuation of virulence in laboratory-manipulated Mycobacterium tuberculosis

Shyamasree De Majumdar,Jaya Sivaswami Tyagi,Kriti Sikri,Malobi Nandi,Sheetal Gandotra,Payel Ghosh,Shekhar Mande,Neetika Jaisinghani

doi:10.1186/s12864-019-5482-y

Abstract

BackgroundA previous laboratory study involving wild type, mutant and devR/dosR complemented strains of Mycobacterium tuberculosis reported the attenuation phenotype of complemented strain, Comp1. This phenotype was intriguing since the parental strain H37Rv, devR mutant (Mut1) and additional complemented strains, Comp9 and Comp11, were virulent in the guinea pig model.ResultsTowards deciphering the mechanism underlying the attenuation of Comp1, a whole genome sequencing approach was undertaken. Eight Single Nucleotide Polymorphisms (SNPs) unique to the Comp1 strain were identified. Of these, 5 SNPs were non-synonymous and included a G➞A mutation resulting in a W1591Stop mutation in ppsD gene of the phthiocerol dimycocerosate (PDIM) biosynthetic cluster. Targeted sequence analysis confirmed this mutation in only Comp1 strain and not in wild type (H37Rv), devR knockout (Mut1) or other complemented (Comp9 and Comp11) bacteria. Differential expression of the PDIM locus in Comp1 bacteria was observed which was associated with a partial deficiency of PDIM, an increased sensitivity to detergent and a compromised ability to infect human THP-1 cells.ConclusionsIt is proposed that a spontaneous mutation in the ppsD gene of Comp1 underlies down-modulation of the PDIM locus which is associated with defects in permeability and infectivity as well as virulence attenuation in guinea pigs. Our study demonstrates the value of whole genome sequencing for resolving unexplainable bacterial phenotypes and recommends the assessment of PDIM status while assessing virulence properties of laboratory-manipulated strains of M. tuberculosis.

Highlights

A previous laboratory study involving wild type, mutant and devR/dosR complemented strains of Mycobacterium tuberculosis reported the attenuation phenotype of complemented strain, Comp1
Phthiocerol dimycocerosates (PDIM) are produced by a limited group of mycobacterial species, most of which are pathogenic for humans such as M. tb, M. bovis, M. leprae, M. ulcerans, and M. marinum [2] and have attracted special attention due to the key role they play in the pathogenesis of tuberculosis and bacterial-host interactions [5,6,7,8,9,10,11]
M. tb Comp1 strain is attenuated in guinea pigs We previously reported that devR complemented strain Comp1, was severely attenuated in guinea pigs, whereas the parental H37Rv and the devR mutant (Mut1) strains were virulent [16]

Summary

Introduction

A previous laboratory study involving wild type, mutant and devR/dosR complemented strains of Mycobacterium tuberculosis reported the attenuation phenotype of complemented strain, Comp. A previous laboratory study involving wild type, mutant and devR/dosR complemented strains of Mycobacterium tuberculosis reported the attenuation phenotype of complemented strain, Comp1 This phenotype was intriguing since the parental strain H37Rv, devR mutant (Mut1) and additional complemented strains, Comp and Comp, were virulent in the guinea pig model. A role for PDIM in tuberculosis (TB) pathogenesis was suggested over four decades ago when a PDIM deficient H37Rv-derived strain of M. tb was found to be attenuated in guinea pigs [10]. Mutants lacking or deficient in PDIM synthesis or translocation display altered colony morphology, increased membrane permeability phenotypes and are severely attenuated in animal models [8, 9, 14].

Results

Discussion

Conclusion