Molecular evolution of virulence genes and non-virulence genes in clinical, natural and artificial environmental Legionella pneumophila isolates

BACKGROUND: L. pneumophila is the main causative agent of Legionnaires’ disease. Free-living amoeba in natural aquatic environments is the reservoir and shelter for L. pneumophila. From natural water sources, L. pneumophila can colonize artificial environments such as cooling towers and hot-water systems, and then spread in aerosols, infecting the susceptible person. Therefore, molecular phylogeny and genetic variability of L. pneumophila from different sources (natural water, artificial water, and human lung tissue) might be distinct because of the selection pressure in different environments. Several studies researched genetic differences between L. pneumophila clinical isolates and environmental isolates at the nucleotide sequence level. These reports mainly focused on the analysis of virulence genes, and rarely distinguished artificial and natural isolates. METHODS: We have used 139 L. pneumophila isolates to study their genetic variability and molecular phylogeny. These isolates include 51 artificial isolates, 59 natural isolates, and 29 clinical isolates. The nucleotide sequences of two representative non-virulence (NV) genes (trpA, cca) and three representative virulence genes (icmK, lspE, lssD) were obtained using PCR and DNA sequencing and were analyzed. RESULTS: Levels of genetic variability including haplotypes, haplotype diversity, nucleotide diversity, nucleotide difference and the total number of mutations in the virulence loci were higher in the natural isolates. In contrast, levels of genetic variability including polymorphic sites, theta from polymorphic sites and the total number of mutations in the NV loci were higher in clinical isolates. A phylogenetic analysis of each individual gene tree showed three to six main groups, but not comprising the same L. pneumophila isolates. We detected recombination events in every virulence loci of natural isolates, but only detected them in the cca locus of clinical isolates. Neutrality tests showed that variations in the virulence genes of clinical and environmental isolates were under neutral evolution. TrpA and cca loci of clinical isolates showed significantly negative values of Tajima’s D, Fu and Li’s D* and F*, suggesting the presence of negative selection in NV genes of clinical isolates. DISCUSSION: Our findingsreinforced the point that the natural environments were the primary training place for L. pneumophila virulence, and intragenic recombination was an important strategy in the adaptive evolution of virulence gene. Our study also suggested the selection pressure had unevenly affected these genes and contributed to the different evolutionary patterns existed between NV genes and virulence genes. This work provides clues for future work on population-level and genetics-level questions about ecology and molecular evolution of L. pneumophila, as well as genetic differences of NV genes and virulence genes between this host-range pathogen with different lifestyles.