High Resolution Discrimination of Clinical Mycobacterium tuberculosis Complex Strains Based on Single Nucleotide Polymorphisms

Recently, the diversity of the Mycobacterium tuberculosis complex (MTBC) population structure has been described in detail. Based on geographical separation and specific host pathogen co-evolution shaping MTBC virulence traits, at least 20 major lineages/genotypes have evolved finally leading to a clear influence of strain genetic background on transmissibility, clinical presentation/outcome, and resistance development. Therefore, high resolution genotyping for characterization of strains in larger studies is mandatory for understanding mechanisms of host-pathogen-interaction and to improve tuberculosis (TB) control. Single nucleotide polymorphisms (SNPs) represent the most reliable markers for lineage classification of clinical isolates due to the low levels of homoplasy, however their use is hampered either by low discriminatory power or by the need to analyze a large number of genes to achieve higher resolution. Therefore, we carried out de novo sequencing of 26 genes (approx. 20000 bp per strain) in a reference collection of MTBC strains including all major genotypes to define a highly discriminatory gene set. Overall, 161 polymorphisms were detected of which 59 are genotype-specific, while 13 define deeper branches such as the Euro-American lineage. Unbiased investigation of the most variable set of 11 genes in a population based strain collection (one year, city of Hamburg, Germany) confirmed the validity of SNP analysis as all strains were classified with high accuracy. Taken together, we defined a diagnostic algorithm which allows the identification of 17 MTBC phylogenetic lineages with high confidence for the first time by sequencing analysis of just five genes. In conclusion, the diagnostic algorithm developed in our study is likely to open the door for a low cost high resolution sequence/SNP based differentiation of the MTBC with a very high specificity. High throughput assays can be established which will be needed for large association studies that are mandatory for detailed investigation of host-pathogen-interaction during TB infection

High Sequence Variability of the ppE18 Gene of Clinical Mycobacterium tuberculosis Complex Strains Potentially Impacts Effectivity of Vaccine Candidate M72/AS01E

The development of an effective vaccine is urgently needed to fight tuberculosis (TB) which is still the leading cause of death from a single infectious agent worldwide. One of the promising vaccine candidates M72/AS01E consists of two proteins subunits PepA and PPE18 coded by Rv0125 and Rv1196. However, preliminary data indicate a high level of sequence variability among clinical Mycobacterium tuberculosis complex (MTBC) strains that might have an impact on the vaccine efficacy. To further investigate this finding, we determined ppE18 sequence variability in a well-characterized reference collection of 71 MTBC strains from 23 phylogenetic lineages representing the global MTBC diversity. In total, 100 sequence variations consisting of 96 single nucleotide polymorphisms (SNPs), three insertions and one deletion were detected resulting in 141 variable positions distributed over the entire gene. The majority of SNPs detected were non-synonymous (n = 68 vs. n = 28 synonymous). Strains from animal adapted lineages, e.g., M. bovis, showed a significant higher diversity than the human pathogens such as M. tuberculosis Haarlem. SNP patterns specific for different lineages as well as for deeper branches in the phylogeny could be identified. The results of our study demonstrate a high variability of the ppE18 gene even in the N-terminal domains that is normally highly conserved in ppe genes. As the N-terminal region interacts with TLR2 receptor inducing a protective anti-inflammatory immune response, genetic heterogeneity has a potential impact on the vaccine efficiency, however, this has to be investigated in future studies

Strains of the reference collection.

<p>Strains of the reference collection.</p

Distribution of 100 sequence variation among different MTBC lineages.

<p>ATCC: American Type Culture Collection; CAS: Central Asien; EAI: East African Indian; LAM: Latin American Mediterranean; Tur: Turkish.</p

Strains of the reference collection.

<p>CAS: Central Asien; EAI: East African Indian; LAM: Latin American Mediterranean; TUR: Turkish.</p

Sequence based algorithm for the classification of clinical isolates in 17 different genotypes and phylogenetic main groups.

<p><i>M. africanum</i> 1a/1b, West African 1a and West African 1b; <i>M. africanum</i> 2, West African 2; CAS, <i>M. tuberculosis</i> Central Asien; EAI, <i>M. tuberculosis</i> East African Indian; LAM, <i>M. tuberculosis</i> Latin American Mediterranean; TUR, <i>M. tuberculosis</i> Turkish; wt: wildtype.</p

26 Genes investigated.

<p>PCR: polymerase chain reaction.</p

Phylogenetic informative sequence variations.

<p>CAS: Central Asien; EAI: East African Indian; LAM: Latin American Mediterranean; TUR: Turkish.</p

Maximum Parsimomy Tree based on sequencing analyses data of 11 genes in a population based strain collection from Hamburg, Germany.

<p>WA1, <i>M. africanum</i> West African 1; CAS, <i>M. tuberculosis</i> Central Asien; EAI, <i>M. tuberculosis</i> East African Indian; LAM, <i>M. tuberculosis</i> Latin American Mediterranean; TUR, <i>M. tuberculosis</i> Turkish.</p

Maximum Likelihood Tree based on sequence data from a reference collection.

<p>Bootstrap support >90% is indicated. CAS: Central Asien; LAM: Latin American Mediterranean; TUR: Turkish.</p