Computational cloning of drug target genes of a parasitic nematode, Oesophagostomum dentatum

Background: Gene 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. Results: An 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. Conclusions: Our 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

Genetic Complementation to Identify DNA Elements That Influence Complement Resistance in Leishmania chagasi

Past studies showed that Leishmania spp. promastigotes exhibit differential sensitivity to complement mediated lysis (CML) during development in vitro and in vivo. Leishmania chagasi promastigotes in cultures during logarithmic and stationary growth phases are CML-sensitive or CML-resistant when exposed to human serum, respectively, but only in cultures recently initiated with parasites from infected animals; serially passaged cultures become constitutively CML-sensitive regardless of growth phase. Building on these observations, a genetic screen was conducted to identify novel complement resistance factors of L. chagasi. A cosmid library containing genomic DNA was transfected into a promastigote line previously subjected to \u3e50 serial passages. Selection with human serum for CML resistance yielded 12 transfectant clones. Cosmids isolated from 7 of these clones conferred CML resistance when transfected into an independent, high-passage promastigote culture; at 12% human serum, the mean survival of transfectants was 37% (±11.6%), and that of control transfectants was about 1%. Inserts within the 7 cosmids were unique. Determination of the complete DNA sequence for 1 cosmid indicated that its 32-kilobase insert was 89% identical (overall) to a 31-kilobase region of Leishmania major chromosome 36, which is predicted to encode 6 genes, all of which encode hypothetical proteins

Characterization of DNA Sequences that Confer Complement Resistance in \u3ci\u3eLeishmania chagasi\u3c/i\u3e

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 highpassage (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

Characterization of DNA Sequences that Confer Complement Resistance in \u3ci\u3eLeishmania chagasi\u3c/i\u3e

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 highpassage (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

Population changes in Leishmania chagasi promastigote developmental stages due to serial passage

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 (\u3e85%) 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 (\u3e85%) comprised of nectomonad promastigotes, almost completely devoid (\u3c2%) 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

DIFFERENTIAL SURFACE DEPOSITION OF COMPLEMENT PROTEINS ON LOGARITHMIC AND STATIONARY PHASE \u3ci\u3eLEISHMANIA CHAGASI\u3c/i\u3e PROMASTIGOTES

Previous works demonstrated that various species of Leishmania promastigotes exhibit differential sensitivity to complement-mediated lysis (CML) during development. Upon exposure to normal human serum (NHS), cultures of Leishmania chagasi promastigotes recently isolated from infected hamsters (fewer than 5 in vitro passages) are CML-sensitive when in the logarithmic growth phase but become CML-resistant upon transition to the stationary culture phase. Visualization by light and electron microscopy revealed dramatic morphological differences between promastigotes from the 2 culture phases following exposure to NHS. Flow cytometric analysis demonstrated that surface deposition of the complement components C3, C5, and C9 correlated inversely with promastigote CML-resistance. The highest levels of complement protein surface accumulation were observed for logarithmic phase promastigotes, while stationary phase promastigotes adsorbed the least amount of complement proteins. Additionally, fluorescence microscopy revealed that C3 and C5 localized in a fairly uniform pattern to the plasma membrane of promastigotes from logarithmic phase cultures, while the staining of promastigotes from stationary phase cultures was indistinguishable from background. By Western blot analysis, high levels of the complement proteins C3, C5, and C9 were detected in the total lysates of NHSexposed logarithmic phase L. chagasi promastigotes, relative to NHS-exposed stationary phase promastigotes; this finding indicates that the low levels of C3 and C5 seen on the surface of stationary phase promastigotes were not due to protein uptake/internalization. Together, these data demonstrate the differential deposition of complement proteins on the surfaces of logarithmic and stationary phase L. chagasi promastigotes. The data support a model wherein stationary phase L. chagasi promastigotes resist CML by limiting the deposition of C3 and its derivatives, which, in turn, limit surface levels of complement proteins (including C5 and C9) that form the lytic membrane attack complex

Reduced Hamster Usage and Stress in Propagating \u3ci\u3eLeishmania chagasi\u3c/i\u3e Promastigotes Using Cryopreservation and Saphenous Vein Inoculation

Leishmania chagasi, a causal agent of visceral leishmaniasis, requires passage through lab animals such as hamsters to maintain its virulence. Hamster infection is typically accomplished via cardiac puncture or intraperitoneal injection, procedures accompanied by risks of increased animal stress and death. The use of the hamster model also necessitates a regular supply of infected animals, because L. chagasi parasites newly isolated from an infected hamster can be grown in culture for only several weeks before loss of function/phenotype occurs. In an effort to decrease animal usage and animal stress, experiments were performed to assess a more gentle inoculation procedure (saphenous vein inoculation) and the use of cryopreserved parasite cells for research experiments. Of 81 hamsters inoculated by the saphenous vein, 80 became infected as determined ante mortem, by display of clinical symptoms of leishmaniasis (onset of symptoms at 105 ± 22 days post-inoculation), and postmortem by the presence of parasites within the spleen. Splenic parasite load calculated for a subset (n 5 34) of infected hamsters was 124 to 26,177 Leishmania donovani infection units. Cryopreserved, and never-stored, cells were equivalent in all properties evaluated, including developmental changes in morphology during culture, culture growth rates, parasite resistance to serum-mediated lysis, and expression of developmentally regulated surface proteins major surface protease and promastigote surface antigen

Characterization of DNA Sequences that Confer Complement Resistance in \u3ci\u3eLeishmania chagasi\u3c/i\u3e

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 highpassage (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