Transmembrane Molecules for Phylogenetic Analyses of Pathogenic Protists: Leishmania -Specific Informative Sites in Hydrophilic Loops of Trans- Endoplasmic Reticulum N -Acetylglucosamine-1-Phosphate Transferase

Abstract

A sequence database was created for the Leishmania N-acetylglucosamine-1-phosphate transferase (nagt) gene from 193 independent isolates. PCR products of this single-copy gene were analyzed for restriction fragment length polymorphism based on seven nagt sequences initially available. We subsequently sequenced 77 samples and found 19 new variants (genotypes). Alignment of all 26 nagt sequences is gap free, except for a single codon addition or deletion. Phylogenetic analyses of the sequences allow grouping the isolates into three subgenera, each consisting of recognized species complexes, i.e., subgenus Leishmania (L. amazonensis-L. mexicana, L. donovani-L. infantum, L. tropica, L. major, and L. turanica-L. gerbilli), subgenus Viannia (L. braziliensis, L. panamensis), and one unclassified (L. enriettii) species. This hierarchy of grouping is also supported by sequence analyses of selected samples for additional single-copy genes present on different chromosomes. Intraspecies divergence of nagt varies considerably with different species complexes. Interestingly, species complexes with less subspecies divergence are more widely distributed than those that are more divergent. The relevance of this to Leishmania evolutionary adaptation is discussed. Heterozygosity of subspecies variants contributes to intraspecies diversity, which is prominent in L. tropica but not in L. donovani-L. infantum. This disparity is thought to result from the genetic recombination of the respective species at different times as a rare event during their predominantly clonal evolution. Phylogenetically useful sites of nagt are restricted largely to several extended hydrophilic loops predicted from hypothetical models of Leishmania NAGT as an endoplasmic reticulum transmembrane protein. In silico analyses of nagt from fungi and other protozoa further illustrate the potential value of this and, perhaps, other similar transmembrane molecules for phylogenetic analyses of single-cell eukaryotes.Many microorganisms speciate via clonal evolution. They replicate asexually, with genetic recombination as a rare event.A typical example among single-cell eukaryotes is the trypanosomatid protozoa (58, 59), which are mostly parasites, e.g., Leishmania spp. and Trypanosoma spp. Leishmania spp. live extracellularly in the digestive tracts of blood-sucking female sand flies of various species as their vectors and intracellularly in the macrophages of different mammalian hosts, i.e., human, canine, rodent, and other reservoir animals. The complexities of such unusual ecological niches undoubtedly contribute to Leishmania speciation.A large body of biological, biochemical, immunological, and molecular data (7,10,23,55) exists in the literature suggesting that the genus Leishmania consists of three groups (55) as follows: (i) subgenus Leishmania, which includes species complexes distributed in both the New World and the Old World, e.g., L. majorturanica-L. gerbilli; (ii) subgenus Viannia, whose members are restricted to the Neotropics, e.g., L. braziliensis [...

Visceral Leishmaniasis in China: an Endemic Disease under Control

“…The sequences of nuclear protein-coding genes were similarly analyzed in two additional studies. Dozens of isolates from China together with several hundred from elsewhere were examined in one study for five different single-copy genes (42). All canine and human VL isolates fell into the same clade of the L. infantum/L.…”

Leishmania spp.: Delta-aminolevulinate-inducible neogenesis of porphyria by genetic complementation of incomplete heme biosynthesis pathway

“…used for the present study include L. amazonensis (LV 78 clone 12-1), L. major (RM), L. tropica (K27) and L. infantum (Adana #7) (see http://66.99.255.20/cms/micro/sample%20list.pdf and Waki et al, 2007 for the origin of the strains used).…”

“…Of these eight enzymes, only the last one, i. e. ferrochelatase (Fe-C) has been shown to exist functionally by nutritional studies (Chang et al, 1975) and enzyme assays (Salzman et al, 1982) in different trypanosomatid protozoa. Most recently, we have found the putative Fe-C sequences of high homology in all Leishmania isolates grouped phylogenetically into some 20 genotypes, including most Old World species, i. e. L. infantum /donovani, L. major, L. tropica, L. turanica and L. gerbilli (Waki et al, 2007). Sequence conservation of Fe-C suggests that it may function beyond heme biosynthesis, since protoporphyrin (PROTO) is cytotoxic and thus would not be widely available to serve as a substrate of this enzyme to produce the heme needed by Leishmania.…”

Molecular characterization of Leishamania isolates from China by inter-simple sequence repeat polymerase chain reaction

“…So, there are also many DNA-based technique applied to phylogenetic study of Leishmania. The most common method is sequencing or RFLP analysis of the specific gene or gene fragment, such as mini-exon gene (Fernandes et al 1994), ribosomal internal transcribed spacer (El Tai et al 2001;Kuhls et al 2005), kinetoplast DNA maxicircle divergent region (Flegontov et al 2006), gp63 (Mauricio et al 2007;Waki et al 2007), genes encoding DNA polymerase catalytic subunit, RNA polymerase II largest subunit (Croan et al 1997) etc. However, phylogenetic analysis focusing on only particular sequences in the genome could be misleading.…”