Using a label-free proteomics method to identify differentially abundant proteins in closely related hypo- and hypervirulent clinical Mycobacterium tuberculosis Beijing isolates

Abstract

Although the genome of the Mycobacterium tuberculosis H37Rv laboratory strain has been available for over 10 years, it is only recently that genomic information from clinical isolates has been used to generate the hypothesis of virulence differences between different strains. In addition, the relationship between strains displaying differing virulence in an epidemiological setting and their behavior in animal models has received little attention. The potential causes for variation in virulence between strains, as determined by differential protein expression, have similarly been a neglected area of investigation. In this study, we used a label-free quantitative proteomics approach to estimate differences in protein abundance between two closely related Beijing genotypes that have been shown to be hyper- and hypovirulent on the basis of both epidemiological and mouse model studies. We were able to identify a total of 1668 proteins from both samples, and protein abundance calculations revealed that 48 proteins were over-represented in the hypovirulent isolate, whereas 53 were over-represented in the hypervirulent. Functional classification of these results shows that molecules of cell wall organization and DNA transcription regulatory proteins may have a critical influence in defining the level of virulence. The reduction in the presence of ESAT-6, other Esx-like proteins, and FbpD (MPT51) in the hypervirulent strain indicates that changes in the repertoire of highly immunogenic proteins can be a defensive process undertaken by the virulent cell. In addition, most of the previously well characterized gene targets related to virulence were found to be similarly expressed in our model. Our data support the use of proteomics as a complementary tool for genomic comparisons to understand the biology of M. tuberculosis virulence.

Fig. 1.

IS6110 DNA fingerprint of selected Beijing genotype strains. A, strain representative of the largest cluster (hypervirulent). B, strain that did not transmit during the study period (hypovirulent).

Fig. 2.

Virulence of M. tuberculosis clinical isolates in murine infection model. BALB/c mice were infected by intratracheal injection (2.5 × 10⁵ bacilli) with selected Beijing strains. The virulence of the strain representative of the largest cluster (white symbols) was compared with the virulence of the non-transmitting strain (black symbols). A, Kaplan-Meier survival curves. B, bacillary loads. C, morphometry (percentage of lung surface area affected by pneumonia). Asterisks represent statistical significance (p < 0.005). Error bars indicate 95% confidence intervals.

Fig. 3.

MS/MS profile of ion m/z 601.41. A tandem mass spectrum of a prevalent ion on a particular time point in the LC gradient and ionized on the LTQ-Orbitrap is shown. The peptide fragments randomly on each amide bond, resulting in carboxyl-terminal y ions or amino-terminal b ions. After the fragment masses were submitted to Mascot, the peptide was identified as FGDQVVAVLTR (inset with detected y and b ions represented) from protein Rv3220c, a probable two-component sensor kinase. * Asterisk represent the parent ion.

Fig. 4.

Protein abundance comparison of hypo- and hypervirulent cells. Protein abundance was derived from emPAI values. Solid lines delimit differentially abundant proteins (external area) from similarly abundant (internal area). HV, high virulent; LV, low virulent.

Fig. 5.

Functional classification of differentially abundant proteins. Proteins were classified according to the Tuberculist functional category (FC) groups. Gray bars represent proteins that are more abundant in the hypovirulent strain, and black bars represent proteins that are more abundant in the hypervirulent strain. FC10 represents the group of conserved hypothetical proteins.