Genetic crosses within and between species of Cryptosporidium.

Parasites and their hosts are engaged in reciprocal coevolution that balances competing mechanisms of virulence, resistance, and evasion. This often leads to host specificity, but genomic reassortment between different strains can enable parasites to jump host barriers and conquer new niches. In the apicomplexan parasite Cryptosporidium, genetic exchange has been hypothesized to play a prominent role in adaptation to humans. The sexual lifecycle of the parasite provides a potential mechanism for such exchange; however, the boundaries of Cryptosporidium sex are currently undefined. To explore this experimentally, we established a model for genetic crosses. Drug resistance was engineered using a mutated phenylalanyl tRNA synthetase gene and marking strains with this and the previously used Neo transgene enabled selection of recombinant progeny. This is highly efficient, and genomic recombination is evident and can be continuously monitored in real time by drug resistance, flow cytometry, and PCR mapping. Using this approach, multiple loci can now be modified with ease. We demonstrate that essential genes can be ablated by crossing a Cre recombinase driver strain with floxed strains. We further find that genetic crosses are also feasible between species. Crossing Cryptosporidium parvum, a parasite of cattle and humans, and Cryptosporidium tyzzeri a mouse parasite resulted in progeny with a recombinant genome derived from both species that continues to vigorously replicate sexually. These experiments have important fundamental and translational implications for the evolution of Cryptosporidium and open the door to reverse- and forward-genetic analysis of parasite biology and host specificity.

Genomic Approaches to Drug Resistance in Malaria.

Although the last two decades have seen a substantial decline in malaria incidence and mortality due to the use of insecticide-treated bed nets and artemisinin combination therapy, the threat of drug resistance is a constant obstacle to sustainable malaria control. Given that patients can die quickly from this disease, public health officials and doctors need to understand whether drug resistance exists in the parasite population, as well as how prevalent it is so they can make informed decisions about treatment. As testing for drug efficacy before providing treatment to malaria patients is impractical, researchers need molecular markers of resistance that can be more readily tracked in parasite populations. To this end, much work has been done to unravel the genetic underpinnings of drug resistance in Plasmodium falciparum. The aim of this review is to provide a broad overview of common genomic approaches that have been used to discover the alleles that drive drug response phenotypes in the most lethal human malaria parasite.

Enabling Medicago truncatula forward genetics: identification of genetic crossing partner for R108 and development of mapping resources for Tnt1 mutants.

Though Medicago truncatula Tnt1 mutants are widely used by researchers in the legume community, they are mainly used for reverse genetics because of the availability of the BLAST-searchable large-scale flanking sequence tags database. However, these mutants should have also been used extensively for forward genetic screens, an effort that has been hindered due to the lack of a compatible genetic crossing partner for the M. truncatula genotype R108, from which Tnt1 mutants were generated. In this study, we selected three Medicago HapMap lines (HM017, HM018 and HM022) and performed reciprocal genetic crosses with R108. After phenotypic analyses in F1 and F2 progenies, HM017 was identified as a compatible crossing partner with R108. By comparing the assembled genomic sequences of HM017 and R108, we developed and confirmed 318 Indel markers evenly distributed across the eight chromosomes of the M. truncatula genome. To validate the effectiveness of these markers, by employing the map-based cloning approach, we cloned the causative gene in the dwarf mutant crs isolated from the Tnt1 mutant population, identifying it as gibberellin 3-ß-dioxygenase 1, using some of the confirmed Indel markers. The primer sequences and the size difference of each marker were made available for users in the web-based database. The identification of the crossing partner for R108 and the generation of Indel markers will enhance the forward genetics and the overall usage of the Tnt1 mutants.

Detecting selection with a genetic cross.

Distinguishing which traits have evolved under natural selection, as opposed to neutral evolution, is a major goal of evolutionary biology. Several tests have been proposed to accomplish this, but these either rely on false assumptions or suffer from low power. Here, I introduce an approach to detecting selection that makes minimal assumptions and only requires phenotypic data from ∼10 individuals. The test compares the phenotypic difference between two populations to what would be expected by chance under neutral evolution, which can be estimated from the phenotypic distribution of an F2 cross between those populations. Simulations show that the test is robust to variation in the number of loci affecting the trait, the distribution of locus effect sizes, heritability, dominance, and epistasis. Comparing its performance to the QTL sign test-an existing test of selection that requires both genotype and phenotype data-the new test achieves comparable power with 50- to 100-fold fewer individuals (and no genotype data). Applying the test to empirical data spanning over a century shows strong directional selection in many crops, as well as on naturally selected traits such as head shape in Hawaiian Drosophila and skin color in humans. Applied to gene expression data, the test reveals that the strength of stabilizing selection acting on mRNA levels in a species is strongly associated with that species' effective population size. In sum, this test is applicable to phenotypic data from almost any genetic cross, allowing selection to be detected more easily and powerfully than previously possible.

Artemisinin resistance phenotypes and K13 inheritance in a Plasmodium falciparum cross and Aotus model.

Concerns about malaria parasite resistance to treatment with artemisinin drugs (ARTs) have grown with findings of prolonged parasite clearance t1/2s (>5 h) and their association with mutations in Plasmodium falciparum Kelch-propeller protein K13. Here, we describe a P. falciparum laboratory cross of K13 C580Y mutant with C580 wild-type parasites to investigate ART response phenotypes in vitro and in vivo. After genotyping >400 isolated progeny, we evaluated 20 recombinants in vitro: IC50 measurements of dihydroartemisinin were at similar low nanomolar levels for C580Y- and C580-type progeny (mean ratio, 1.00; 95% CI, 0.62-1.61), whereas, in a ring-stage survival assay, the C580Y-type progeny had 19.6-fold (95% CI, 9.76-39.2) higher average counts. In splenectomized Aotus monkeys treated with three daily doses of i.v. artesunate, t1/2 calculations by three different methods yielded mean differences of 0.01 h (95% CI, -3.66 to 3.67), 0.80 h (95% CI, -0.92 to 2.53), and 2.07 h (95% CI, 0.77-3.36) between C580Y and C580 infections. Incidences of recrudescence were 57% in C580Y (4 of 7) versus 70% in C580 (7 of 10) infections (-13% difference; 95% CI, -58% to 35%). Allelic substitution of C580 in a C580Y-containing progeny clone (76H10) yielded a transformant (76H10C580Rev) that, in an infected monkey, recrudesced regularly 13 times over 500 d. Frequent recrudescences of ART-treated P. falciparum infections occur with or without K13 mutations and emphasize the need for improved partner drugs to effectively eliminate the parasites that persist through the ART component of combination therapy.

Genotypic and Phenotypic Analyses of Two "Isogenic" Strains of the Human Fungal Pathogen Cryptococcus neoformans var. neoformans.

The Cryptococcus neoformans species complex is a model organism for fungal studies. Many studies have used two strains, JEC20 and JEC21, and their derivatives. These two strains were obtained through 10 rounds of backcrosses and have been assumed near identical except at the mating-type locus. Here we obtained and compared the JEC20 genome sequence with the published "JEC21" genome. Our comparison revealed 5322 single nucleotide polymorphisms (SNPs) with the majority (N = 3816, 71.7%) located in three genomic regions, including the previously noted mating-type region. The remaining 1506 SNPs (28.3%) were distributed throughout all 14 chromosomes, predominantly at chromosomal ends. To study the potential effects of these three SNP-rich regions on phenotypes, 24 progenies from the JEC20 × JEC21 cross representing eight recombinant genotypes were analyzed for their mating ability, melanin production, capsule formation, and growths at 30 °C and 40 °C. Significant phenotypic variations were found among the progeny. However, the observed phenotypic variations could not be explained by the three SNP-rich regions. Further genome sequencing of our JEC21 and the 24 progenies revealed only six segregating SNPs outside of the three SNP-rich regions between JEC20 and JEC21, a result indicating that the 1500 SNPs identified in the published "JEC21" genome might be caused by sequencing errors and/or strain mixing. However, the six SNPs and the three SNP-rich regions could not explain the observed phenotypic variations. Our analyses suggest that spontaneous mutations accumulated under laboratory conditions could have significant effects on phenotypes and on our interpretations of experimental results.

Different unequal cross-over events between NCF1 and its pseudogenes in autosomal p47(phox)-deficient chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a rare congenital disorder in which phagocytes cannot generate superoxide (O2(-)) and other microbicidal oxidants due to mutations in one of the five components of the O2(-)-generating NADPH oxidase complex. The most common autosomal subtype of CGD is caused by mutations in NCF1, encoding the NADPH subunit p47(phox). Usually, these mutations are the result of unequal exchange of chromatid between NCF1 and one of its two pseudogenes. We have now investigated in detail the breakpoints within or between these (pseudo) NCF1 genes in 43 families with p47(phox)-deficient CGD by means of multiplex ligase-dependent probe amplification (MLPA). In 24 families the patients totally lacked NCF1 sequences, indicating that in these families the cross-over points are located between NCF1 and its pseudogenes. Six other families were compound heterozygous for a total NCF1 deletion and another mutation in NCF1 on the other allele. In 8 families, the patients lacked NCF1 exons 1-4 but had retained NCF1 exons 6-10, indicating that a cross-over point is located within NCF1 between exons 4 and 6. Similarly, in 4 families a cross-over point was located within NCF1 between exons 2 and 4. Similar cross-overs, in heterozygous form, were observed in family members of the patients. Several patients were compound heterozygous for total and partial NCF1 deletions. Thus, at least three different cross-over points exist within the NCF1 gene cluster, indicating that autosomal p47(phox)-deficient CGD is genetically heterogeneous but can be dissected in detail by MLPA.

A Plasmodium falciparum genetic cross reveals the contributions of pfcrt and plasmepsin II/III to piperaquine drug resistance.

Piperaquine (PPQ) is widely used in combination with dihydroartemisinin as a first-line treatment against malaria. Multiple genetic drivers of PPQ resistance have been reported, including mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and increased copies of plasmepsin II/III (pm2/3). We generated a cross between a Cambodia-derived multidrug-resistant KEL1/PLA1 lineage isolate (KH004) and a drug-susceptible Malawian parasite (Mal31). Mal31 harbors a wild-type (3D7-like) pfcrt allele and a single copy of pm2/3, while KH004 has a chloroquine-resistant (Dd2-like) pfcrt allele with an additional G367C substitution and multiple copies of pm2/3. We recovered 104 unique recombinant parasites and examined a targeted set of progeny representing all possible combinations of variants at pfcrt and pm2/3. We performed a detailed analysis of competitive fitness and a range of PPQ susceptibility phenotypes with these progenies, including PPQ survival assay, area under the dose response curve, and a limited point IC50. We find that inheritance of the KH004 pfcrt allele is required for reduced PPQ sensitivity, whereas copy number variation in pm2/3 further decreases susceptibility but does not confer resistance in the absence of additional mutations in pfcrt. A deep investigation of genotype-phenotype relationships demonstrates that progeny clones from experimental crosses can be used to understand the relative contributions of pfcrt, pm2/3, and parasite genetic background to a range of PPQ-related traits. Additionally, we find that the resistance phenotype associated with parasites inheriting the G367C substitution in pfcrt is consistent with previously validated PPQ resistance mutations in this transporter.IMPORTANCEResistance to piperaquine, used in combination with dihydroartemisinin, has emerged in Cambodia and threatens to spread to other malaria-endemic regions. Understanding the causal mutations of drug resistance and their impact on parasite fitness is critical for surveillance and intervention and can also reveal new avenues to limiting the evolution and spread of drug resistance. An experimental genetic cross is a powerful tool for pinpointing the genetic determinants of key drug resistance and fitness phenotypes and has the distinct advantage of quantifying the effects of naturally evolved genetic variation. Our study was strengthened since the full range of copies of KH004 pm2/3 was inherited among the progeny clones, allowing us to directly test the role of the pm2/3 copy number on resistance-related phenotypes in the context of a unique pfcrt allele. Our multigene model suggests an important role for both loci in the evolution of this multidrug-resistant parasite lineage.

Evidence of high-efficiency cross fertilization in Eimeria acervulina revealed using two lines of transgenic parasites.

Eimeria species are apicomplexan parasites with a direct life cycle consisting of a replicative phase involving multiple rounds of asexual replication in the intestine or other organs including kidneys, liver, and gallbladder, depending on the species, followed by a sexual phase or gamogony involving the development and fertilization of gametes, an essential process for Eimeria transmission. Recent advances in the genetic manipulation of these parasites made it possible to conduct genetic crosses combined with genomic approaches to elucidate the genetic determinants of Eimeria development, virulence, drug resistance, and immune evasion. Here, we employed genetic techniques to generate two transgenic Eimeria acervulina lines, EaGAM56 and EaHAP2, each expressing two unique fluorescent proteins, with one controlled by a constitutive promotor for cross-efficiency analysis and the other by a male or female gametocyte stage-specific promoter to observe sexual development. The expression of fluorescent proteins in the transgenic lines was analyzed in different developmental stages of the E. acervulina life cycle by immunoblotting and by examination of frozen sections using fluorescence microscopy. The effect of infective doses on cross-fertilization was further investigated by conducting several genetic crosses between the two transgenic lines at different doses and ratios. Two transgenic lines expressing constitutive and gametocyte-specific fluorescence proteins were generated and characterized. These transgenic parasites display synchronous development in chickens, comparable with that of the wild type. Genetic crosses between the two transgenic parasites showed that a high rate of oocysts co-expressing the two reporters could be achieved following inoculation with high doses of infective oocysts. We further showed that the proportion of co-transfected oocysts can be modulated by altering the ratio of the transgenic parental lines. Higher infective doses and similar numbers of functional gametocytes from the parents increase the rate of cross-fertilization. Our data highlight the usefulness of genetic manipulation and fluorescently-labeled transgenic gametocytes as tools to study Eimeria development and to elucidate the factors that modulate sexual development. This work sets the stage for the implementation of novel approaches to investigate other aspects of Eimeria pathogenesis, virulence, and drug susceptibility and resistance.

Mode of action studies confirm on-target engagement of lysyl-tRNA synthetase inhibitor and lead to new selection marker for Cryptosporidium.

Introduction: Cryptosporidiosis is a leading cause of diarrheal-associated morbidity and mortality, predominantly affecting children under 5 years old in low-and-middle-income countries. There is no effective treatment and no vaccine. New therapeutics are emerging from drug discovery efforts. It is critical that mode of action studies are performed alongside drug discovery to ensure the best clinical outcomes. Unfortunately, technology to identify and validate drug targets for Cryptosporidium is severely lacking. Methods: We used C. parvum lysyl-tRNA synthetase (CpKRS) and DDD01510706 as a target-compound pair to develop both chemical and genetic tools for mode of action studies for Cryptosporidium. We adapted thermal proteome profiling (TPP) for Cryptosporidium, an unbiased approach for target identification. Results: Using TPP we identified the molecular target of DDD01510706 and confirm that it is CpKRS. Genetic tools confirm that CpKRS is expressed throughout the life cycle and that this target is essential for parasite survival. Parasites genetically modified to over-express CpKRS or parasites with a mutation at the compound-binding site are resistant to treatment with DDD01510706. We leveraged these mutations to generate a second drug selection marker for genetic modification of Cryptosporidium, KRSR. This second selection marker is interchangeable with the original selection marker, NeoR, and expands the range of reverse genetic approaches available to study parasite biology. Due to the sexual nature of the Cryptosporidium life cycle, parental strains containing different drug selection markers can be crossed in vivo. Discussion: Selection with both drug markers produces highly efficient genetic crosses (>99% hybrid progeny), paving the way for forward genetics approaches in Cryptosporidium.

A Malaria Parasite Cross Reveals Genetic Determinants of Plasmodium falciparum Growth in Different Culture Media.

What genes determine in vitro growth and nutrient utilization in asexual blood-stage malaria parasites? Competition experiments between NF54, clone 3D7, a lab-adapted African parasite, and a recently isolated Asian parasite (NHP4026) reveal contrasting outcomes in different media: 3D7 outcompetes NHP4026 in media containing human serum, while NHP4026 outcompetes 3D7 in media containing AlbuMAX, a commercial lipid-rich bovine serum formulation. To determine the basis for this polymorphism, we conducted parasite genetic crosses using humanized mice and compared genome-wide allele frequency changes in three independent progeny populations cultured in media containing human serum or AlbuMAX. This bulk segregant analysis detected three quantitative trait loci (QTL) regions [on chromosome (chr) 2 containing aspartate transaminase AST; chr 13 containing EBA-140; and chr 14 containing cysteine protease ATG4] linked with differential growth in serum or AlbuMAX in each of the three independent progeny pools. Selection driving differential growth was strong (s = 0.10 - 0.23 per 48-hour lifecycle). We conducted validation experiments for the strongest QTL on chr 13: competition experiments between ΔEBA-140 and 3D7 wildtype parasites showed fitness reversals in the two medium types as seen in the parental parasites, validating this locus as the causative gene. These results (i) demonstrate the effectiveness of bulk segregant analysis for dissecting fitness traits in P. falciparum genetic crosses, and (ii) reveal intimate links between red blood cell invasion and nutrient composition of growth media. Use of parasite crosses combined with bulk segregant analysis will allow systematic dissection of key nutrient acquisition/metabolism and red blood cell invasion pathways in P. falciparum.

Phenotypic and genotypic analysis of benzimidazole resistance in reciprocal genetic crosses of Haemonchus contortus.

Haemonchus contortus is arguably one of the most economically important and ubiquitous parasites of livestock globally and commonly involved in cases of anthelmintic resistance. Here, we performed reciprocal genetic crosses using susceptible (MHco3(ISE)) and multiple anthelmintic resistant (MHco18(UGA2004)) H. contortus isolates. Resultant admixed populations were designated MHco3/18 or MHco18/3, where the lead isolate reflects the origin of the females. Three independent filial generations were generated for each cross, which were subjected to bioassays, molecular approaches and population genetic analyses to investigate the phenotypic and genotypic inheritance of benzimidazole (BZ) resistance at each stage. A panel of microsatellite markers confirmed the success of the genetic cross as markers from both parents were seen in the F1 crosses. Egg hatch tests revealed a stark difference between the two F1 crosses with ED50 estimates for MHco18/3 being 9 times greater than those for MHco3/18. Resistance factors based on ED50 estimates ranged from 6 to 57 fold in the filial progeny compared to MHco3(ISE) parents. Molecular analysis of the F167Y and F200Y SNP markers associated with BZ resistance were analysed by pyrosequencing and MiSeq deep amplicon sequencing, which showed that MHco3/18.F1 and MHco18/3.F1 both had similar frequencies of the F200Y resistant allele (45.3% and 44.3%, respectively), whereas for F167Y, MHco18/3.F1 had a two-fold greater frequency of the resistant-allele compared to MHco3/18.F1 (18.2% and 8.8%, respectively). Comparison between pyrosequencing and MiSeq amplicon sequencing revealed that the allele frequencies derived from both methods were concordant at codon 200 (rc = 0.97), but were less comparable for codon 167 (rc = 0.55). The use of controlled reciprocal genetic crosses have revealed a potential difference in BZ resistance phenotype dependent on whether the resistant allele is paternally or maternally inherited. These findings provide new insight and prompt further investigation into the inheritance of BZ resistance in H. contortus.

Genomic landscape of drug response reveals mediators of anthelmintic resistance.

Like other pathogens, parasitic helminths can rapidly evolve resistance to drug treatment. Understanding the genetic basis of anthelmintic drug resistance in parasitic nematodes is key to tracking its spread and improving the efficacy and sustainability of parasite control. Here, we use an in vivo genetic cross between drug-susceptible and multi-drug-resistant strains of Haemonchus contortus in a natural host-parasite system to simultaneously map resistance loci for the three major classes of anthelmintics. This approach identifies new alleles for resistance to benzimidazoles and levamisole and implicates the transcription factor cky-1 in ivermectin resistance. This gene is within a locus under selection in ivermectin-resistant populations worldwide; expression analyses and functional validation using knockdown experiments support that cky-1 is associated with ivermectin survival. Our work demonstrates the feasibility of high-resolution forward genetics in a parasitic nematode and identifies variants for the development of molecular diagnostics to combat drug resistance in the field.

Genetic Analyses of Amphotericin B Susceptibility in Aspergillus fumigatus.

Aspergillus fumigatus is a ubiquitous saprophytic mold that can cause a range of clinical syndromes, from allergic reactions to invasive infections. Amphotericin B (AMB) is a polyene antifungal drug that has been used to treat a broad range of systemic mycoses since 1958, including as a primary treatment option against invasive aspergillosis in regions with high rates (≥10%) of environmental triazole resistance. However, cases of AMB-resistant A. fumigatus strains have been increasingly documented over the years, and high resistance rates were recently reported in Brazil and Canada. The objective of this study is to identify candidate mutations associated with AMB susceptibility using a genome-wide association analysis of natural strains, and to further investigate a subset of the mutations in their putative associations with differences in AMB minimum inhibitory concentration (MIC) and in growths at different AMB concentrations through the analysis of progeny from a laboratory genetic cross. Together, our results identified a total of 34 candidate single-nucleotide polymorphisms (SNPs) associated with AMB MIC differences-comprising 18 intergenic variants, 14 missense variants, one synonymous variant, and one non-coding transcript variant. Importantly, progeny from the genetic cross allowed us to identify putative SNP-SNP interactions impacting progeny growth at different AMB concentrations.

Cryptococcus neoformans Mating and Genetic Crosses.

The Cryptococcus pathogenic species complex is a group of opportunistic human fungal pathogens that cause cryptococcal meningoencephalitis, an infection associated with unacceptably high mortality rates. The public health relevance of these pathogens has galvanized extensive research over the past several decades and led to characterization of their sexual cycles. This research has allowed several Cryptococcus species to develop into model fungal organisms for both pathogenesis and basic science studies. Many of these studies require observation of the meiotic process and its associated mating structures as well as generation of meiotic progeny with novel phenotypes and genotypes. Herein, we describe how to set up genetic crosses between Cryptococcus strains and observe their mating phenotypes as well as how to recover progeny from these crosses for further analysis. © 2019 by John Wiley & Sons, Inc.