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.

Figure 1. Atlas of the Chromosome of M. tuberculosis H37Ra ATCC25177 and its Comparison with M. tuberculosis H37Rv.

Moving inside, each concentric circle represents genomic data for M. tuberculosis H37Ra and its comparison with M. tuberculosis H37Rv. The outer circle illustrates predicted coding sequences on the plus and minus strands, respectively, colored by functional categories according to COG classification. The 2^nd circle represents location of nucleotide substitution in coding regions (purple for synonymous substitutions; green for nonsynonymous substitutions) and noncoding regions (red), respectively. The 3^rd circle displays loci of insertions in H37Ra, distinguished by coding regions (red) and noncoding regions (purple). The 4^th circle shows loci of deletions in H37Ra, distinguished by coding regions (red) and noncoding regions (purple). The 5^th circle presents H37Ra specific genes compared with H37Rv and CDC151 genes, red for CDSs with variations and green for genes with promoter variations. The 6^th circle (innermost) represents GC skew (G−C)/(G+C) calculated using a 5 kb window.

Figure 2. Transcription Analysis of Selected Genes with Promoter Mutations in H37Ra Compared to H37Rv in culture and in macrophages.

The in vitro cultured M. tuberculosis H37Ra and H37Rv were grown to log phase. The bacterial cells in macrophages were harvested 24 hours after infection. Mycobacterial RNA was isolated and 16 S rRNA gene and sigA were used as internal controls for normalization of the transcriptional analysis for in vitro and macrophage derived mycobacteria, respectively. The transcription levels of the selected genes (lpdA, pabB, phoH2, sigC and nrdH) were determined by qRT-PCR as described in the Methods. Error bars were given as standard deviations (SDs) of the means. E phase: exponential (log) phase. Μφ: macrophage. The RT-PCR data were repeated at least three times. ** indicates p value <0.05.

Figure 3. Schematic Diagram of IS6110-Mediated Genetic Variations Detected among H37Ra, H37Rv And CDC1551.

A. IS6110 distributions in the chromosomes of known M. tuberculosis complex strains. Blue arrow represents IS6110 elements shared by more than one strain. Red arrow indicates IS6110 elements specifically detected only in one strain. The direction of arrow indicates the transcription orientation of the transposase genes. The numbers above the arrows indicate the location (kb) of IS6110 in the strains relative to the oriC in clockwise direction. B. Schematic diagram of a hot spot region for IS6110 insertion. ‘ATT’, ‘CGA’ are the DRs flanking each IS6110, the same DRs on both sides of one IS6110 indicate that no IS6110 recombination had occurred here. In CDC1551, no IS6110 is found in this region but instead, a gene MT3275.1 is present, which is disrupted by IS6110 insertion(s) in H37Ra and H37Rv. C. Schematic diagram of the plcD-cut1 region (RvD2) in H37Ra, H37Rv and CDC1551. The H37Ra CDSs are predicted to encode hypothetical proteins (MRA_1768A, and MRA_1768B), encoding a putative sulfite oxidase (MRA_1768C) and a putative transmembrane transport protein MmpL14 (MRA_1768D). ‘GAG’, ‘TAG’, ‘AAG’ are the DRs flanking the IS element. The coding sequences of MT1800, MT1801 and MT1802 of CDC1551 are identical to that of MRA_1768B, MRA_1768C and mmpL14 gene, respectively. D. Schematic diagram of the H37Ra MRA_1776-MRA_1780 region,with IS6110 related 247 bp insertion (MRA_1779). Corresponding regions in H37Rv and CDC1551 are shown for comparison. The MT1812, MT1813 and 3′ end of MT1814 of CDC1551 correspond to the RvD3 region of H37Rv. The 247 bp remnant and its probable original counterpart in H37Rv are shaded. Referring to CDC1551 and the DRs, it might be inferred that two IS6110 elements were once inserted in the RvD3 region in the process of evolving H37Ra and H37Rv. Subsequent recombination between these two IS elements caused deletion of RvD3 in H37Ra and H37Rv. The recombination in H37Ra underwent an incomplete exchange, leaving the remnant observed. E. Schematic diagram of the 2064 bp insertion region containing 2 esat-6 like protein genes (MRA_2374, MRA_2375) and a PPE family protein gene (MRA_2376). A neighboring IS6110 insertion is shown which might be H37-specific. H37Ra and H37Rv share the same IS6110 insertion which disrupted the MT2423, generating a new gene, MRA_2377 (Rv2353c).

Figure 4. Multiple Sequence Alignment of MazG (A) and the C-Terminal Effector Domains of PhoP (B).

The amino acid sequences used are from M. tuberculosis H37Rv (Rv), H37Ra (Ra), CDC1551(CDC), M. bovis (MB), M. bovis BCG (BCG), M. avium (MA), M. smegmatis (MS), Escherichia coli (EC) and Bacillus subtilis (BS). The numbers refer to the amino acid residues of MazG and PhoP. The key secondary structure element (α3-helix) of PhoP is indicated above the sequence. The site of substitution is indicated by an asterisk under the sequence.

Figure 5. A Proposed Phylogenetic Relationship of H37Ra, H37Rv, H37, and the Closely Related Clinical Isolate CDC1551.

The process of strain evolution as well as major phenotypes and related genotypes are illustrated in the text. The origination of H37Rv and H37Ra is described according to the literature . Multiple appearances of both H37Ra and H37Rv represent their possible repeated in vitro passage, which may cause subsequent genetic variations.

Figure 6. Schematic Diagram of Major H37Ra Genes with Functions Affected by Mutations.

The genes in blue and in green indicate variations that occurred in coding sequences and in promoter regions, respectively.