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. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy
| Identifer | oai:union.ndltd.org:HKU/oai:hub.hku.hk:10722/174533 |
| Date | January 2011 |
| Creators | Lam, T. H., Jason., 林梓軒. |
| Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
| Source Sets | Hong Kong University Theses |
| Language | English |
| Detected Language | English |
| Type | PG_Thesis |
| Source | http://hub.hku.hk/bib/B47849757 |
| Rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works., Creative Commons: Attribution 3.0 Hong Kong License |
| Relation | HKU Theses Online (HKUTO) |
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