Origins of a 350-Kilobase Genomic Duplication in Mycobacterium tuberculosis and Its Impact on Virulence

Abstract

bIn the present study, we have investigated the evolution and impact on virulence of a 350-kb genomic duplication present in the most recently evolved members of the Mycobacterium tuberculosis East Asian lineage. In a mouse model of infection, comparing HN878 subclones HN878-27 (no duplication) and HN878-45 (with the 350-kb duplication) revealed that the latter is impaired for in vivo growth during the initial 3 weeks of infection. Furthermore, the median survival time of mice infected with isolate HN878-45 is significantly longer (77 days) than that of mice infected with HN878-27. Whole-genome sequencing of both isolates failed to reveal any mutational events other than the duplication that could account for such a substantial difference in virulence. Although we and others had previously speculated that the 350-kb duplication arose in response to some form of hostapplied selective pressure ( , here we show that this large chromosomal amplification event is very rapidly selected within standard in vitro broth cultures in a range of isolates. Indeed, subclones harboring the duplication were detectable after just five rounds of in vitro passage. In contrast, the duplication appears to be highly unstable in vivo and is negatively selected during the later stages of infection in mice. We believe that the rapid in vitro evolution of M. tuberculosis is an underappreciated aspect of its biology that is often ignored, despite the fact that it has the potential to confound the data and conclusions arising from comparative studies of isolates at both the genotypic and phenotypic levels.

Respiratory Flexibility in Response to Inhibition of Cytochrome c Oxidase in Mycobacterium tuberculosis

“…Our results suggest that the pathways involved in respiration and energy metabolism show significant metabolic plasticity and can be altered during long-term laboratory adaption of clinical isolates. Laboratory passage of M. tuberculosis strains has likely resulted in many adaptations to in vitro growth over time (17). In this instance, the tight control of the cydAB genes in strain H37Rv seems to have been lost, allowing a buffered response to the reduced functioning of the bc 1 complex.…”

Unique Regulation of the DosR Regulon in the Beijing Lineage of Mycobacterium tuberculosis

“…The gene arrangement of each KO strain around the site of homologous recombination was confirmed by PCR, and their gene expression phenotypes were confirmed by qRT-PCR. The absence of the 350-kb duplication that can occur in strains belonging to groups 3 to 5 of the East Asian lineage was confirmed by PCR, as previously described (14,32).…”

“…Strain HN878 was originally obtained from J. Musser (Methodist Hospital Research Institute, Houston, TX) (75). The HN878-27 subclone derived in our laboratory (14) lacks the 350-kb genomic duplication generated in vitro by HN878 and certain other Beijing isolates, and it is referred to here as HN878 (32).…”

“…Next, we investigated a novel 350-kb gene duplication that was identified in members of the most recently evolved Beijing strains (14,31). The presence of this duplication, which includes a second copy of the Rv3134c-dosR-dosS operon, does increase the extent of DosR regulon expression; however, gene disruption studies coupled with the finding that it is not uniformly present among all Beijing strains highlight the fact that the duplication is not the underlying cause of the constitutive DosR phenotype (14,32). Furthermore, recent work by our laboratory has shown that this large chromosomal duplication event is rapidly selected for as a consequence of in vitro growth (32).…”

Diversity and disease pathogenesis in Mycobacterium tuberculosis

“…Some strains might not grow in laboratory media, while others might grow so well as to dominate cultured bacillary populations [26]. Moreover, important new evidence suggests that propagation in laboratory media induces genomic changes in cultured isolates: a recent study reported a strong selective advantage for a large genomic duplication (approximately 350 kb) that arose in the bacillary population after only five rounds of passage in broth media, and was associated with attenuated virulence in mice [31]. In addition to the implications for genotype-phenotype analyses, this result reinforces the potential to select inadvertently for laboratory-adapted variants, a possibility that is generally not considered in genomic studies and might influence epidemiological inferences of strain prevalence and fitness [32].…”