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The study of virulence determinants of mycobacterium tuberculosis

Persistence in human macrophages is central to the virulence of Mycobacterium

tuberculosis, which is the causative agent of tuberculosis. Although the

intracellular parasitism is apparent, molecular determinants of mycobacterial

virulence are not well understood.

The current investigation identified virulent genes of M. tuberculosis by

measuring survivability of Mycobacterium smegmatis recombinants inside a

human monocytic cell line THP-1 after acquiring various virulent gene candidates

of M. tuberculosis. These gene candidates included nine virulent gene

candidates suggested by other studies, five genomic polymorphisms identified in

hypervirulent strains of M. tuberculosis using microarray-based comparative

genomic hybridization, and ten single nucleotide polymorphisms identified in the

hypervirulent strains using full genome sequencing. Interestingly, only

recombinants harboring a truncated Rv2820c and a known virulent gene mce1A

survived significantly better than vector control after six hours of ex vivo

infection.

As nucleotide sequencing indicated that the truncated Rv2820c loses around 60%

of gene at 3’ end, ex vivo survivability of M. smegmatis recombinants harboring

the last 60% of Rv2820c as well as the intact Rv2820c was measured, but was

similar to that of vector control. The 3’ truncated portion itself did not alter

mycobacterial survivability ex vivo, but its presence did compromise the survival

advantage gained due to the truncated Rv2820c.

To determine whether the truncated and the intact Rv2820c could enhance

mycobacterial virulence in vivo, these two alleles were transformed into

Mycobacterium marinum and their recombinants were used to infect zebrafish.

In vivo infection showed that zebrafish infected with the recombinant harboring

truncated Rv2820c died significantly faster than vector control, whereas the

recombinant harboring intact Rv2820c behaved similarly to vector control.

Results indicated that the truncated Rv2820c, but not the intact Rv2820c, could

enhance mycobacterial virulence both ex vivo and in vivo.

Additional nucleotide sequencing revealed that the 3’ truncation in Rv2820c is

caused by a Beijing/W-defining deletion RD207 and is commonly found in

Beijing/W strains of M. tuberculosis. Non-Beijing/W strains possess the intact

Rv2820c conversely. Since Beijing/W strains have proven to be more virulent

than non-Beijing/W strains both ex vivo and in vivo, the truncated Rv2820c may

be one of the Beijing/W-specific virulence determinants.

To confirm that Rv2820c of Beijing/W strains really enhances M. tuberculosis

survival in human macrophages, the truncated Rv2820c was transformed into

non-Beijing/W M. tuberculosis strains and their recombinants were used to infect

THP-1 cells. Ex vivo infection confirmed that the truncated Rv2820c could

enhance M. tuberculosis survival inside human macrophages, but is unlikely to

induce a different profile of cytokine secretion from infected macrophages.

In conclusion, the current study demonstrated that the truncated Rv2820c of

Beijing/W strains could enhance mycobacterial virulence both ex vivo and in vivo.

Enhanced phenotypic virulence, however, was not observed for the intact

Rv2820c of non-Beijing/W strains. The truncated Rv2820c may be one of the

Beijing/W-specific virulence determinants and collaboratively contribute to the

high phenotypic virulence of this family.