European Society of Mycobacteriology - Instituto Nacional de Saúde ...

PP-87
Mycobacterium tuberculosis
Arley Gómez López
Infectious Diseases and Tropical Medicine Unit; Universidad del Rosario
TlyA protein has a controversial function as a virulence factor on Mycobacterium tuberculosis (Mtb). Updated studies
have not demonstrated a possible hemolytic activity conferred by TlyA and contrary to recent evidence have suggested
a function enzymatically similar to RNA methyltransferase at ribosomal level which confers antibiotic susceptibility to
capreomycin and viomycin.
Our aim was to determine the In vitro hemolytic activity of Mtb TlyA overexpressed and purified from E. coli BL21-AI and
to carry out an in silico phylogenetic and structural analysis.
Based on tlyA gene sequence (Rv1694) from Mtb H37Rv specific primers were designed. The amplification product was
ligated on pEXP5-CT/TOPO vector (Invitrogen), transformed and overexpressed on E. coli BL-21-AI. After purification by
affinity chromatography, TlyA-His6 recombinant protein was analyzed by SDS-PAGE under denaturing conditions which
was detected as 28 KDa single band. Recombinant protein as recognized by anti-His monoclonal antibody.
Protein structural characterization by circular dichroism was carried out. On the other hand, hemolytic activity assays
using TlyA-His6 purified were negative as well as on bacterial lysates.
Hemolytic assays with TlyA-His6 supplemented with calcium and magnesium were negative suggesting lack of specific
requirements for this activity.
By using bioinformatics tools, a ribosomal binding called S4 located between 5 and 68 residues and FtsJ among 62 and
247 residues were identified on TlyA. These domains have been described on proteins associated with ribosomal translational
machinery. The Hidrophobicity profile was in disagreement with a possible transmembrane helix although non
polar amino acid composition suggesting that TlyA might not be membrane attachment. Nevertheless, three-dimensional
model (Structural homology with 1L9K model) reveals a consensus structure with a common core, comprising a parallel
β-sheet of six strands, sandwiched between two layers of α-helices corresponding to a RNA methyltransferase structure.
Phylogenetic analyses showed that TlyA is highly conserved among mycobacteria species and it does not exhibit changes
among Mtb complex strains. tlyA gene evolution might operate under purifying selection model. Additionally differences
were observed among TlyA and bacterial pore forming proteins.
This evidence supports a link between ribosomal modifications to posttranslational level and suggests a functional annotation
error of this family protein at GENBANK as well as missannotation on several genomes as Mtb genome. Studies
on resistance mechanisms of Mtb mediated by ribosomal proteins might be useful for understanding new alternative
therapeutic approaches for tuberculosis control applied to knowledge of second line antibiotics such as capreomycin.
Key words
TlyA, Mycobacterium tuberculosis, hemolysin, Structural modeling, RNA methyltransferase.
European Society of Mycobacteriology | 30 th Annual Congress | July 2009 | Porto - Portugal
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