Analysis of genetic polymorphisms affecting the four phospholipase C (plc) genes in Mycobacterium tuberculosis complex clinical isolates

Abstract

The Mycobacterium tuberculosis genome contains four highly related genes which present significant similarity to Pseudomonas aeruginosa genes encoding phospholipase C enzymes. Three of these genes, plcA, plcB and plcC, are organized in tandem (locus plcABC). The fourth gene, plcD, is located in a different region. This study investigates variations in plcABC and plcD genes in clinical isolates of M. tuberculosis, Mycobacterium africanum and 'Mycobacterium canettii '. Genetic polymorphisms were examined by PCR, Southern blot hybridization, sequence analysis and RT-PCR. Seven M. tuberculosis isolates contain insertions of IS6110 elements within plcA, plcC or plcD. In 19 of 25 M. tuberculosis isolates examined, genomic deletions were identified, resulting in loss of parts of genes or complete genes from the plcABC and/or plcD loci. Partial plcD deletion was observed in one M. africanum isolate. In each case, deletions were associated with the presence of a copy of the IS6110 element and in all occurrences IS6110 was transposed in the same orientation. A mechanism of deletion resulting from homologous recombination of two copies of IS6110 was recognized in a group of genetically related M. tuberculosis isolates. Five M. tuberculosis isolates presented major polymorphisms in the plcABC and plcD regions, along with loss of expression competence that affected all four plc genes. Phospholipase C is a well-known bacterial virulence factor. The precise role of phospholipase C in the pathogenicity of M. tuberculosis is unknown, but considering the potential importance that the plc genes may have in the virulence of the tubercle bacillus, the study of isolates cultured from patients with active tuberculosis bearing genetic variations affecting these genes may provide insights into the significance of phospholipase C enzymes for tuberculosis pathogenicity.

Distribution of Insertion- and Deletion-Associated Genetic Polymorphisms among Four Mycobacterium tuberculosis Phospholipase C Genes and Associations with Extrathoracic Tuberculosis: a Population-Based Study

“…Previous investigations of the genetic diversity of M. tuberculosis plc genes used either convenience samples or selected samples (7,18,21,25). In our population-based sample, we observed that the plcD mutation accounted for 88% (233/266) of the mutations found in the plc genes; this is consistent with the previous findings of Talarico and colleagues, who used a convenience sample collected from Turkey (18).…”

“…tuberculosis clinical strains with major polymorphisms in all four plc genes and impaired plc gene expression have been reported previously by others (25). Raynaud et al recently demonstrated that plcABC triple mutants and plcABCD quadruple mutants were attenuated for growth in the lungs and spleens of mice, and the expression of the different plc genes was important for virulence at different time points of the infection (13).…”

“…A recent functional study of M. tuberculosis demonstrated that all four genes encode functional PLC in M. tuberculosis, and each gene contributes to overall PLC activity (13). Furthermore, in mice, when the virulence of a plcABCD quadruple mutant was compared to a plcABC triple mutant, both showed the same level of attenuation, suggesting that either plcD does not contribute to virulence or it acts in association with other phospholipase-encoding genes.Several previous studies using small, selected samples of M. tuberculosis clinical isolates have found insertions or deletions in the plcA, plcB, plcC, and plcD genes (9,18,21,(23)(24)(25). Recently, we reported the association between insertion-and deletion-associated mutations in the plcD genotype and extrathoracic TB (31); however, the potential association of mutations in the other three plc genes remains to be investigated.…”

“…Several previous studies using small, selected samples of M. tuberculosis clinical isolates have found insertions or deletions in the plcA, plcB, plcC, and plcD genes (9,18,21,(23)(24)(25). Recently, we reported the association between insertion-and deletion-associated mutations in the plcD genotype and extrathoracic TB (31); however, the potential association of mutations in the other three plc genes remains to be investigated.…”

Defining mycobacteria: Shared and specific genome features for different lifestyles

“…One of these LSP groups (LSP A) comprising four loci was judged to be suitable for phylogenetic interpreations, while other LSPs occurred in regions subject to selection (rich in PE and PPE genes). Association of genotype to phenotype was indicated for deletion in plcD [68]; extrapulmonary TB was two fold more likely with plcD mutant strains [69].…”