Comparative Genomics ofesxGenes from Clinical Isolates of Mycobacterium tuberculosis Provides Evidence for Gene Conversion and Epitope Variation

Abstract

The 23-membered Esx protein family is involved in the host-pathogen interactions of Mycobacterium tuberculosis. These secreted proteins are among the most immunodominant antigens recognized by the human immune system and have thus been used to develop vaccines and immunodiagnostic tests for tuberculosis (TB). Gene pairs for 10 Esx proteins are contained in the ESX-1 to ESX-5 loci, encoding type VII secretion systems. A subset of Esx proteins can be further classified into the Mtb9.9, QILSS, and TB10.4 subfamilies. To survey genetic diversity in the Esx family and its potential for antigenic variation, we sequenced all esx genes from 108 clinical isolates of M. tuberculosis from different clades by using a targeted approach. A total of 109 unique single nucleotide polymorphisms (SNPs) were observed, and 59 of these were nonsynonymous. Some of the resultant amino acid substitutions affect known Esx epitopes, including two in the EsxB (CFP-10) and EsxH (TB10.4) antigens. Assessment of the SNP distribution across the Esx proteins revealed high genetic variability, especially in the Mtb9.9 and QILSS subfamilies, and more conservation in the ESX-1 to ESX-4 loci. Comparison of the DNA sequences of variable esx genes provided clear evidence for recombination events between different genes in the same strain, some of which are predicted to truncate the corresponding protein.Many of these polymorphisms escape detection by ultrahigh-throughput sequencing using short sequence reads, as such approaches cannot distinguish between closely related genes. The esx gene family is dynamic, and sequence changes likely lead to immune variation.

Quantification of circulating Mycobacterium tuberculosis antigen peptides allows rapid diagnosis of active disease and treatment monitoring

“…Serum CFP-10 was detectable at lower concentrations than ESAT-6, perhaps owing to variable expression levels of ESAT-6 and CFP-10 in different strains of Mtb (40,41). However, host β2 microglobulin also has been reported to bind ESAT-6 to mask a tryptic cleavage site (amino acids 90-95) required for the 1,900.95 m/z ESAT-6 peptide (16,42), and thus may inhibit ESAT-6 cleavage to decrease the apparent serum ESAT-6 levels in our assay.…”

Multivalent TB vaccines targeting the esx gene family generate potent and broad cell-mediated immune responses superior to BCG

“…24,25,45 Similarly, esxS, esxR, and esxH were the only dominant esx antigens that induced strong Th1 responses in BCG vaccinated mice. Interestingly, these 5 immunogenic esx antigens are categorized as part of the same esx Mtb locus family cluster (Esx3), 43 therefore, likely underlying the importance of this unique esx subfamily members as essential immunogenic esx antigens elicited by BCG and perhaps emphasizes their vital role in the efficacy of BCG.…”

“…43 Therefore to target a range of T cell epitopes and increase the breadth of immune responses we co-administrated all 5 esx constructs in a cocktail (RSQ-15 vaccine). Immunization with RSQ-15 plasmids induced a broad pattern of T cell reactivity to all 15 encoded esx members 1 mo after the last immunization (Fig.…”

After the bottleneck: Genome-wide diversification of the Mycobacterium tuberculosis complex by mutation, recombination, and natural selection

“…This includes the highly GC-rich and polymorphic PE/PPE gene family, esx genes, and Insertion Sequences. Gene conversion resulting from frequent intrachromosomal homologous recombination has been reported between members of each of these families (Ho et al 2000;McEvoy et al 2007;Karboul et al 2008;Uplekar et al 2011). Hence, exclusion of these regions prevents spurious inclusion of intrachromosomal recombination events in the analyses of recombination presented below.…”