Genetic Basis of Virulence Attenuation Revealed by Comparative Genomic Analysis of Mycobacterium tuberculosis Strain H37Ra versus H37Rv

Abstract

Tuberculosis, caused by Mycobacterium tuberculosis, remains a leading infectious disease despite the availability of chemotherapy and BCG vaccine. The commonly used avirulent M. tuberculosis strain H37Ra was derived from virulent strain H37 in 1935 but the basis of virulence attenuation has remained obscure despite numerous studies. We determined the complete genomic sequence of H37Ra ATCC25177 and compared that with its virulent counterpart H37Rv and a clinical isolate CDC1551. The H37Ra genome is highly similar to that of H37Rv with respect to gene content and order but is 8,445 bp larger as a result of 53 insertions and 21 deletions in H37Ra relative to H37Rv. Variations in repetitive sequences such as IS6110 and PE/PPE/PE-PGRS family genes are responsible for most of the gross genetic changes. A total of 198 single nucleotide variations (SNVs) that are different between H37Ra and H37Rv were identified, yet 119 of them are identical between H37Ra and CDC1551 and 3 are due to H37Rv strain variation, leaving only 76 H37Ra-specific SNVs that affect only 32 genes. The biological impact of missense mutations in protein coding sequences was analyzed in silico while nucleotide variations in potential promoter regions of several important genes were verified by quantitative RT-PCR. Mutations affecting transcription factors and/or global metabolic regulations related to in vitro survival under aging stress, and mutations affecting cell envelope, primary metabolism, in vivo growth as well as variations in the PE/PPE/PE-PGRS family genes, may underlie the basis of virulence attenuation. These findings have implications not only for improved understanding of pathogenesis of M. tuberculosis but also for development of new vaccines and new therapeutic agents.

Glycolytic and Non-glycolytic Functions of Mycobacterium tuberculosis Fructose-1,6-bisphosphate Aldolase, an Essential Enzyme Produced by Replicating and Non-replicating Bacilli

“…The choice of using M. tuberculosis H37Ra is justified by the fact that the H37Ra strain is considered a class II organism yet requires FBA-tb for growth like its virulent counterpart, M. tuberculosis H37Rv, with which it shares the exact same glycolytic/gluconeogenetic and pentose phosphate pathway enzymes (100% identical in their primary sequence) (47). Mutants in which the wild-type chromosomal copy of the gene was inactivated were easily isolated using this merodiploid strain, confirming the requirement of fba-tb for growth of M. tuberculosis on 7H11-OADC-sucrose plates (Fig.…”

Evaluation of mycobacterial virulence using rabbit skin liquefaction model

“…Since the above findings suggest that the rabbit skin model is useful to distinguish the virulence of BCG, H37Ra and M. smegmatis, we decided to use the rabbit skin model to rapidly evaluate virulence of H37Ra complemented strains based on the mutations found in H37Ra. 9 The rabbits were injected by the intradermal route with high (5 x 10 7 CFU), intermediate (5 x 10 6 CFU), and low (5 x 10 5 CFU) doses of H37Ra complemented strains ( Table 2) and also the H37Ra strain transformed with the vector pOLYG control, 10 and also H37Ra alone as control on both the right and left flanks of rabbits, respectively. Forty-two days later, they were reinjected with the same bacilli at the same dose.…”

“…In H37Ra, compared with H37Rv, the pstA1 gene has a point mutation of C to T change causing Arg at the 913 rd nt to a stop codon. 9 Complementation of H37Ra with the pstA1 caused the biggest granuloma but not the most obvious liquefaction lesion. This suggests that the gene products involved in producing liquefaction seems to be different from those that induce granuloma.…”

“…M. tuberculosis H37Ra were harvested by centrifugation at 3,000 g for 20 min at room temperature, washed with sterile distilled H 2 O at room temperature and resuspended in 1-2 ml of 10% the nrdH gene has a 14 bp deletion in its putative promoter region compared to that of Rv3053c in H37Rv. 9 Since ribonucleotide reductase is essential for DNA biosynthesis and DNA repair which are affected in the presence or absence of oxygen, 19 it is likely that defect in nrdH expression in H37Ra due to the promoter region deletion may affect the survival of the bacilli in vivo under low oxygen conditions and thus may be involved in attenuation of virulence in H37Ra. It is interesting to note that when H37Ra is complemented with wild type nrdH containing its own promoter region from H37Rv, the complemented strain caused more severe lesions and more pronounced liquefaction ( Table 3).…”

“…H37Ra has a S219L mutation in the phoP gene, 9,22,23 and the PhoP mutation is thought to be involved in virulence attenuation in H37Ra. [22][23][24] However, it is surprising that phoP complemented H37Ra did not seem to be involved in virulence in the rabbit skin model, as it did not show more severe skin lesions than the H37Ra control ( Table 3).…”