Leishmania chagasi causes visceral leishmaniasis, a potentially fatal disease of humans. Within the sand fly vector, L. chagasi replicates as promastigotes which undergo complex changes in morphology as they progress from early stage procyclic promastigotes, to intermediate stage leptomonad and nectomonad promastigotes, and ultimately to terminal stage metacyclic promastigotes that are highly infective to vertebrates. This developmental progression is largely recapitulated in vitro using axenic promastigote cultures that have been passaged only a few times. Within a single passage (which takes about a week), axenic cultures progress from logarithmic to stationary growth phases; parasites within those growth phases progress from stages that do not have metacyclic cell properties to ones that do. Interestingly, repeated serial passage of promastigote cultures will result in cell populations that exhibit perturbations in developmental progression, in expression levels of surface macromolecules (major surface protease, MSP, and promastigote surface antigen, PSA), and in virulence properties, including resistance to serum lysis. Experiments were performed to determine whether there exists a direct relationship between promastigote developmental form and perturbations associated with repeated serial passage. Passage 2 to passage 4 L. chagasi cultures at stationary growth phase were predominately (>85%) comprised of metacyclic promastigotes and exhibited high resistance to serum lysis and high levels of MSP and PSA. Serial passaging 8, or more, times resulted in a stationary phase population that was largely (>85%) comprised of nectomonad promastigotes, almost completely devoid (<2%) of metacyclic promastigotes, and that exhibited low resistance to serum lysis and low levels of MSP and PSA. The study suggests that the loss of particular cell properties seen in cells from serially passaged cultures is principally due to a dramatic reduction in the proportion of metacyclic promastigotes. Additionally, the study suggests that serially passaged cultures may be a highly enriched source of nectomonad-stage promastigotes, a stage that has largely been characterized only in mixtures containing other promastigote forms.

Serial passage of axenically cultured Leishmania chagasi promastigotes results in a progressive diminution in resistance to complement-mediated lysis (CML), whereas high CML resistance is seen in infectious metacyclic promastigotes from the sandfly vector as well as metacyclic-like promastigotes within low-passage cultures at stationary growth phase. As we previously reported, in a screen seeking to identify novel genes involved in CML resistance: (1) a genomic cosmid library derived from DNA of CML-resistant L. chagasi promastigotes was transfected into high-passage (constitutively CML-sensitive) L. chagasi promastigotes; (2) transformants were screened for acquisition of CML-resistance; (3) multiple cosmid-transfectants exhibited partial CML resistance; and (4) the sequence for one of the cosmids (Cosmid 51) was determined. This report extends the analysis of Cosmid 51, and identifies by deletion analysis a subregion of the cosmid insert that is critical to the CML-resistance phenotype of Cosmid 51 transformants. We also report the sequence determination and initial CML-resistance activity of another cosmid that also confers partial resistance to CML.

BackgroundGene identification and sequence determination are critical requirements for many biological, genomic, and bioinformatic studies. With the advent of next generation sequencing (NGS) technologies, such determinations are predominantly accomplished in silico for organisms for which the genome is known or for which there exists substantial gene sequence information. Without detailed genomic/gene information, in silico sequence determination is not straightforward, and full coding sequence determination typically involves time- and labor-intensive PCR-based amplification and cloning methods.ResultsAn improved method was developed with which to determine full length gene coding sequences in silico using de novo assembly of RNA-Seq data. The scheme improves upon initial contigs through contig-to-gene identification by BLAST nearest–neighbor comparison, and through single-contig refinement by iterative-binning and -assembly of reads. Application of the iterative method produced the gene identification and full coding sequence for 9 of 12 genes and improved the sequence of 3 of the 12 genes targeted by benzimidazole, macrocyclic lactone, and nicotinic agonist classes of anthelminthic drugs in the swine nodular parasite Oesophagostomum dentatum. The approach improved upon the initial optimized assembly with Velvet that only identified full coding sequences for 2 genes.ConclusionsOur reiterative methodology represents a simplified pipeline with which to determine longer gene sequences in silico from next generation sequence data for any nematode for which detailed genetic/gene information is lacking. The method significantly improved upon an initial Velvet assembly of RNA-Seq data that yielded only 2 full length sequences. The identified coding sequences for the 11 target genes enables further future examinations including: (i) the use of recombinant target protein in functional assays seeking a better understanding of the mechanism of drug resistance, and (ii) seeking comparative genomic and transcriptomic assessments between parasite isolates that exhibit varied drug sensitivities.

Nematode anthelminthic resistance is widespread for the 3 major drug classes commonly used in agriculture: benzamidazoles, macrocyclic lactones, and nicotinic agonists e.g. levamisole. In parasitic nematodes the genetics of resistance are unknown other than to the benzimidazoles which primarily involve a single gene. In previous work with a levamisole resistant Oesophagostomum dentatum isolate, the nicotinic acetylcholine receptor (nAChR) exhibited decreased levamisole sensitivity. Here, using a transcriptomic approach on the same isolate, we investigate whether that decreased nAChR sensitivity is achieved via a 1-gene mechanism involving 1 of 27 nAChR pathway genes. 3 nAChR receptor subunit genes exhibited ≥ 2-fold change in transcript abundance: acr-21 and acr-25 increased, and unc-63 decreased. 4 SNPs having a ≥ 2-fold change in frequency were also identified. These data suggest that resistance is likely polygenic, involving modulated abundance of multiple subunits comprising the heteropentameric nAChR, and is not due to a simple 1-gene mechanism.

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