Multiple mutations in heterogeneous miltefosine-resistant Leishmania major population as determined by whole genome sequencing

Background

Miltefosine (MF) is the first oral compound used in the chemotherapy against leishmaniasis. Since the mechanism of action of this drug and the targets of MF in Leishmania are unclear, we generated in a step-by-step manner Leishmania major promastigote mutants highly resistant to MF. Two of the mutants were submitted to a short-read whole genome sequencing for identifying potential genes associated with MF resistance.

Methods/Principal Findings

Analysis of the genome assemblies revealed several independent point mutations in a P-type ATPase involved in phospholipid translocation. Mutations in two other proteins—pyridoxal kinase and α-adaptin like protein—were also observed in independent mutants. The role of these proteins in the MF resistance was evaluated by gene transfection and gene disruption and both the P-type ATPase and pyridoxal kinase were implicated in MF susceptibility. The study also highlighted that resistance can be highly heterogeneous at the population level with individual clones derived from this population differing both in terms of genotypes but also susceptibility phenotypes.

Conclusions/Significance

Whole genome sequencing was used to pinpoint known and new resistance markers associated with MF resistance in the protozoan parasite Leishmania. The study also demonstrated the polyclonal nature of a resistant population with individual cells with varying susceptibilities and genotypes.     

Spatial genetic structure reflects extensive clonality,low genotypic diversity and habitat fragmentation in Grevillea renwickiana (Proteaceae), a rare,sterile shrub from south-eastern Australia

Background and Aims

The association of clonality, polyploidy and reduced fecundity has been identified as an extinction risk for clonal plants. Compromised sexual reproduction limits both their ability to adapt to new conditions and their capacity to disperse to more favourable environments. Grevillea renwickiana is a prostrate, putatively sterile shrub reliant on asexual reproduction. Dispersal is most likely limited by the rate of clonal expansion via rhizomes. The nine localized populations constituting this species provide an opportunity to examine the extent of clonality and spatial genotypic diversity to evaluate its evolutionary prospects.

Methods

Ten microsatellite loci were used to compare genetic and genotypic diversity across all sites with more intensive sampling at four locations (n = 185). The spatial distribution of genotypes and chloroplast DNA haplotypes based on the trnQ–rps16 intergenic spacer region were compared. Chromosome counts provided a basis for examining genetic profiles inconsistent with diploidy.

Key Results

Microsatellite analysis identified 46 multilocus genotypes (MLGs) in eight multilocus clonal lineages (MLLs). MLLs are not shared among sites, with two exceptions. Spatial autocorrelation was significant to 1·6 km. Genotypic richness ranged from 0 to 0·33. Somatic mutation is likely to contribute to minor variation between MLGs within clonal lineages. The eight chloroplast haplotypes identified were correlated with eight MLLs defined by ordination and generally restricted to single populations. Triploidy is the most likely reason for tri-allelic patterns.

Conclusions

Grevillea renwickiana comprises few genetic individuals. Sterility has most likely been induced by triploidy. Extensive lateral suckering in long-lived sterile clones facilitates the accumulation of somatic mutations, which contribute to the measured genetic diversity. Genetic conservation value may not be a function of population size. Despite facing evolutionary stagnation, sterile clonal species can play a vital role in mitigating ecological instability as floras respond to rapid environmental change.     

Monoculture of Leafcutter Ant Gardens

Background

Leafcutter ants depend on the cultivation of symbiotic Attamyces fungi for food, which are thought to be grown by the ants in single-strain, clonal monoculture throughout the hundreds to thousands of gardens within a leafcutter nest. Monoculture eliminates cultivar-cultivar competition that would select for competitive fungal traits that are detrimental to the ants, whereas polyculture of several fungi could increase nutritional diversity and disease resistance of genetically variable gardens.

Methodology/Principal Findings

Using three experimental approaches, we assessed cultivar diversity within nests of Atta leafcutter ants, which are most likely among all fungus-growing ants to cultivate distinct cultivar genotypes per nest because of the nests'' enormous sizes (up to 5000 gardens) and extended lifespans (10–20 years). In Atta texana and in A. cephalotes, we resampled nests over a 5-year period to test for persistence of resident cultivar genotypes within each nest, and we tested for genetic differences between fungi from different nest sectors accessed through excavation. In A. texana, we also determined the number of Attamyces cells carried as a starter inoculum by a dispersing queens (minimally several thousand Attamyces cells), and we tested for genetic differences between Attamyces carried by sister queens dispersing from the same nest. Except for mutational variation arising during clonal Attamyces propagation, DNA fingerprinting revealed no evidence for fungal polyculture and no genotype turnover during the 5-year surveys.

Conclusions/Significance

Atta leafcutter ants can achieve stable, fungal monoculture over many years. Mutational variation emerging within an Attamyces monoculture could provide genetic diversity for symbiont choice (gardening biases of the ants favoring specific mutational variants), an analog of artificial selection.     

Female and male fitness consequences of clonal growth in a dwarf bamboo population with a high degree of clonal intermingling

Background and Aims

Although many studies have reported that clonal growth interferes with sexual reproduction as a result of geitonogamous self-pollination and inbreeding depression, the mating costs of clonal growth are expected to be reduced when genets are spatially intermingled with others. This study examined how clonal growth affects both female and male reproductive success by studying a population of a mass-flowering plant, Sasa veitchii var. hirsuta, with a high degree of clonal intermingling.

Methods

In a 10 × 10 m plot, genets were discriminated based on the multilocus genotypes of 11 nuclear microsatellite loci. The relationships between genet size and the components of reproductive success were then investigated. Male siring success and female and male selfing rates were assessed using paternity analysis.

Key Results

A total of 111 genets were spatially well intermingled with others. In contrast to previous studies with species forming distinct monoclonal patches, seed production linearly increased with genet size. While male siring success was a decelerating function of genet size, selfing rates were relatively low and not related to genet size.

Conclusions

The results, in conjunction with previous studies, emphasize the role of the spatial arrangement of genets on both the quantity and quality of offpsring, and suggest that an intermingled distribution of genets can reduce the mating costs of clonal growth and enhance overall fitness, particularly female fitness.     

Motif depletion in bacteriophages infecting hosts with CRISPR systems

Background

CRISPR is a microbial immune system likely to be involved in host-parasite coevolution. It functions using target sequences encoded by the bacterial genome, which interfere with invading nucleic acids using a homology-dependent system. The system also requires protospacer associated motifs (PAMs), short motifs close to the target sequence that are required for interference in CRISPR types I and II. Here, we investigate whether PAMs are depleted in phage genomes due to selection pressure to escape recognition.

Results

To this end, we analyzed two data sets. Phages infecting all bacterial hosts were analyzed first, followed by a detailed analysis of phages infecting the genus Streptococcus, where PAMs are best understood. We use two different measures of motif underrepresentation that control for codon bias and the frequency of submotifs. We compare phages infecting species with a particular CRISPR type to those infecting species without that type. Since only known PAMs were investigated, the analysis is restricted to CRISPR types I-C and I-E and in Streptococcus to types I-C and II. We found evidence for PAM depletion in Streptococcus phages infecting hosts with CRISPR type I-C, in Vibrio phages infecting hosts with CRISPR type I-E and in Streptococcus thermopilus phages infecting hosts with type II-A, known as CRISPR3.

Conclusions

The observed motif depletion in phages with hosts having CRISPR can be attributed to selection rather than to mutational bias, as mutational bias should affect the phages of all hosts. This observation implies that the CRISPR system has been efficient in the groups discussed here.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-663) contains supplementary material, which is available to authorized users.     

Genotypes and Mouse Virulence of Toxoplasma gondii Isolates from Animals and Humans in China

Background

Recent population structure studies of T. gondii revealed that a few major clonal lineages predominated in different geographical regions. T. gondii in South America is genetically and biologically divergent, whereas this parasite is remarkably clonal in North America and Europe with a few major lineages including Types I, II and III. Information on genotypes and mouse virulence of T. gondii isolates from China is scarce and insufficient to investigate its population structure, evolution, and transmission.

Methodology/Principal Findings

Genotyping of 23 T. gondii isolates from different hosts using 10 markers for PCR-restriction fragment length polymorphism analyses (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) revealed five genotypes; among them three genotypes were atypical and two were archetypal. Fifteen strains belong to the Chinese 1 lineage, which has been previously reported as a widespread lineage from swine, cats, and humans in China. Two human isolates fall into the type I and II lineages and the remaining isolates belong to two new atypical genotypes (ToxoDB#204 and #205) which has never been reported in China. Our results show that these genotypes of T. gondii isolates are intermediately or highly virulent in mice except for the strain TgCtwh6, which maintained parasitemia in mice for 35 days post infection although it possesses the uniform genotype of Chinese 1. Additionally, phylogenetic network analyses of all isolates of genotype Chinese 1 are identical, and there is no variation based on the sequence data generated for four introns (EF1, HP2, UPRT1 and UPRT7) and two dense granule proteins (GRA6 and GRA7).

Conclusion/Significance

A limited genetic diversity was found and genotype Chinese 1 (ToxoDB#9) is dominantly circulating in mainland China. The results will provide a useful profile for deep insight to the population structure, epidemiology and biological characteristics of T. gondii in China.     

Lentiviral and targeted cellular barcoding reveals ongoing clonal dynamics of cell lines in vitro and in vivo

Background

Cell lines are often regarded as clonal, even though this simplifies what is known about mutagenesis, transformation and other processes that destabilize them over time. Monitoring these clonal dynamics is important for multiple areas of biomedical research, including stem cell and cancer biology. Tracking the contributions of individual cells to large populations, however, has been constrained by limitations in sensitivity and complexity.

Results

We utilize cellular barcoding methods to simultaneously track the clonal contributions of tens of thousands of cells. We demonstrate that even with optimal culturing conditions, common cell lines including HeLa, K562 and HEK-293 T exhibit ongoing clonal dynamics. Starting a population with a single clone diminishes but does not eradicate this phenomenon. Next, we compare lentiviral and zinc-finger nuclease barcode insertion approaches, finding that the zinc-finger nuclease protocol surprisingly results in reduced clonal diversity. We also document the expected reduction in clonal complexity when cells are challenged with genotoxic stress. Finally, we demonstrate that xenografts maintain clonal diversity to a greater extent than in vitro culturing of the human non-small-cell lung cancer cell line HCC827.

Conclusions

We demonstrate the feasibility of tracking and quantifying the clonal dynamics of entire cell populations within multiple cultured cell lines. Our results suggest that cell heterogeneity should be considered in the design and interpretation of in vitro culture experiments. Aside from clonal cell lines, we propose that cellular barcoding could prove valuable in modeling the clonal behavior of heterogeneous cell populations over time, including tumor populations treated with chemotherapeutic agents.     

The genotype specific competitive ability does not correlate with infection in natural Daphnia magna populations

Background

Different evolutionary hypotheses predict a correlation between the fitness of a genotype in the absence of infection and the likelihood to become infected. The cost of resistance hypothesis predicts that resistant genotypes pay a cost of being resistant and are less fit in the absence of parasites. The inbreeding-infection hypothesis predicts that the susceptible individuals are less fit due to inbreeding depression.

Methods and Results

Here we tested if a host''s natural infection status was associated with its fitness. First, we experimentally confirmed that cured but formerly infected Daphnia magna are genetically more susceptible to reinfections with Octosporea bayeri than naturally uninfected D. magna. We then collected from each of 22 populations both uninfected and infected D. magna genotypes. All were treated against parasites and kept in their asexual phase. We estimated their relative fitness in an experiment against a tester genotype and in another experiment in direct competition. Consistently, we found no difference in competitive abilities between uninfected and cured but formerly infected genotypes. This was the case both in the presence as well as in the absence of sympatric parasites during the competition trials.

Conclusions

Our data do not support the inbreeding-infection hypothesis. They also do not support a cost of resistance, however ignoring other parasite strains or parasite species. We suggest as a possible explanation for our results that resistance genes might segregate largely independently of other fitness associated genes in this system.     

Investigating the effects of topography and clonality on genetic structuring within a large Norwegian population of Arabidopsis lyrata

Background and Aims

The gene flow through pollen or seeds governs the extent of spatial genetic structure in plant populations. Another factor that can contribute to this pattern is clonal growth. The perennial species Arabidopsis lyrata ssp. petraea (Brassicaceae) is a self-incompatible, clonal species found in disjunctive populations in central and northern Europe.

Methods

Fourteen microsatellite markers were employed to study the level of kinship and clonality in a high-altitude mountain valley at Spiterstulen, Norway. The population has a continuous distribution along the banks of the River Visa for about 1·5 km. A total of 17 (10 m × 10 m) squares were laid out in a north–south transect following the river on both sides.

Key Results

It is shown that clonal growth is far more common than previously shown in this species, although the overall size of the genets is small (mean diameter = 6·4 cm). Across the whole population there is no indication of isolation by distance, and spatial genetic structure is only visible on fine spatial scales. In addition, no effect of the river on the spatial distribution of genotypes was found.

Conclusions

Unexpectedly, the data show that populations of small perennials like A. lyrata can behave like panmictic units across relatively large areas at local sites, as opposed to earlier findings in central Europe.     

The potential of plant viruses to promote genotypic diversity via genotype x environment interactions

Background and Aims

Genotype by environment (G × E) interactions are important for the long-term persistence of plant species in heterogeneous environments. It has often been suggested that disease is a key factor for the maintenance of genotypic diversity in plant populations. However, empirical evidence for this contention is scarce. Here virus infection is proposed as a possible candidate for maintaining genotypic diversity in their host plants.

Methods

The effects of White clover mosaic virus (WClMV) on the performance and development of different Trifolium repens genotypes were analysed and the G × E interactions were examined with respect to genotype-specific plant responses to WClMV infection. Thus, the environment is defined as the presence or absence of the virus.

Key Results

WClMV had a negative effect on plant performance as shown by a decrease in biomass and number of ramets. These effects of virus infection differ greatly among host genotypes, representing a strong G × E interaction. Moreover, the relative fitness and associated ranking of genotypes changed significantly between control and virus treatments. This shift in relative fitness among genotypes suggests the potential for WClMV to provoke differential selection on T. repens genotypes, which may lead to negative frequency-dependent selection in host populations.

Conclusions

The apparent G × E interaction and evident repercussions for relative fitness reported in this study stress the importance of viruses for ecological and evolutionary processes and suggest an important role for viruses in shaping population dynamics and micro-evolutionary processes.     

Clonal growth is enhanced in the absence of a mating morph: a comparative study of fertile stylar polymorphic and sterile monomorphic populations of Nymphoides montana (Menyanthaceae)

Background and Aims

Many aquatic species with stylar polymorphisms have the capacity for clonal and sexual reproduction and are sensitive to the balance of the two reproductive modes when there are a limited number of mating morphs within a population. This study asked how the clonal and sexual reproductive modes perform in populations that contain only a single morph and where fitness gain through sexual reproduction is rare. In clonal aquatic Nymphoides montana, polymorphic populations normally contain two mating morphs in equal frequencies. Populations are sexually fertile and appear to be maintained by pollen transfer between the two partners. However, in a monomorphic population of N. montana where mating opportunities are unavailable, female and male function is impaired and clonality maintains the population. Here, the consequences of intraspecific variation in sexuality were explored between monomorphic and polymorphic N. montana populations in eastern Australia.

Methods

Comparative measurements of male and female fertility, total dry mass and genotypic diversity using ISSR markers were made between populations with variable sexuality.

Key Results and Conclusions

Very few seeds were produced in the monomorphic population under natural and glasshouse conditions due to dysfunctional pollen and ovules. Stigma–anther separation was minimal in the monomorphic population, which may be a consequence of the relaxed selective pressures that regulate the maintenance of sexual function. However, clonal reproduction was favoured at the expense of sexual reproduction in the monomorphic population; this may facilitate the establishment of sterility throughout the population via resource reallocation or pleiotropic effects. The ISSR results showed that the monomorphic population was one large, single genotype, unlike the multi-genotypic fertile polymorphic populations. Evolutionary loss of sex in a clonal population in which a mating morph is absent was evident; under these conditions clonal growth may assure reproduction and expand the population via spreading stolons.     

Diversity of the cell-wall associated genomic island of the archaeon Haloquadratum walsbyi

Background

Haloquadratum walsbyi represents up to 80 % of cells in NaCl-saturated brines worldwide, but is notoriously difficult to maintain under laboratory conditions. In order to establish the extent of genetic diversity in a natural population of this microbe, we screened a H. walsbyi enriched metagenomic fosmid library and recovered seven novel version of its cell-wall associated genomic island. The fosmid inserts were sequenced and analysed.

Results

The novel cell-wall associated islands delineated two major clades within H. walsbyi. The islands predominantly contained genes putatively involved in biosynthesis of surface layer, genes encoding cell surface glycoproteins and genes involved in envelope formation. We further found that these genes are maintained in the population and that the diversity of this region arises through homologous recombination but also through the action of mobile genetic elements, including viruses.

Conclusions

The population of H. walsbyi in the studied saltern brine is composed of numerous clonal lineages that differ in surface structures including the cell wall. This type of variation probably reflects a number of mechanisms that minimize the infection rate of predating viruses.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1794-8) contains supplementary material, which is available to authorized users.     

The infection processes of Sclerotinia sclerotiorum in cotyledon tissue of a resistant and a susceptible genotype of Brassica napus

Background and Aims

Sclerotinia sclerotiorum can attack >400 plant species worldwide. Very few studies have investigated host–pathogen interactions at the plant surface and cellular level in resistant genotypes of oilseed rape/canola (Brassica napus).

Methods

Infection processes of S. sclerotiorum were examined on two B. napus genotypes, one resistant cultivar ‘Charlton’ and one susceptible ‘RQ001-02M2’ by light and scanning electron microscopy from 2 h to 8 d post-inoculation (dpi).

Key Results

The resistant ‘Charlton’ impeded fungal growth at 1, 2 and 3 dpi, suppressed formation of appresoria and infection cushions, caused extrusion of protoplast from hyphal cells and produced a hypersensitive reaction. At 8 dpi, whilst in ‘Charlton’ pathogen invasion was mainly confined to the upper epidermis, in the susceptible ‘RQ001-02M2’, colonization up to the spongy mesophyll cells was evident. Calcium oxalate crystals were found in the upper epidermis and in palisade cells in susceptible ‘RQ001-02M2’ at 6 dpi, and throughout leaf tissues at 8 dpi. In resistant ‘Charlton’, crystals were not observed at 6 dpi, whereas at 8 dpi they were mainly confined to the upper epidermis. Starch deposits were also more prevalent in ‘RQ001-02M2’.

Conclusions

This study demonstrates for the first time at the cellular level that resistance to S. sclerotiorum in B. napus is a result of retardation of pathogen development, both on the plant surface and within host tissues. The resistance mechanisms identified in this study will be useful for engineering disease-resistant genotypes and for developing markers for screening for resistance against this pathogen.     

High variation in clonal vs. sexual reproduction in populations of the wild strawberry, Fragaria virginiana (Rosaceae)

Background and Aims

Many plants reproduce both clonally and sexually, and the balance between the two modes of reproduction will vary among populations. Clonal reproduction was characterized in three populations of the wild strawberry, Fragaria virginiana, to determine the extent that reproductive mode varied locally between sites. The study sites were fragmented woodlands in Cook County, Illinois, USA.

Methods

A total of 95 strawberry ramets were sampled from the three sites via transects. Ramets were mapped and genotyped at five variable microsatellite loci. The variability at these five loci was sufficient to assign plants to clones with high confidence, and the spatial pattern of genets was mapped at each site.

Key Results

A total of 27 distinct multilocus genotypes were identified. Of these, 18 genotypes were detected only once, with the remaining nine detected in multiple ramets. The largest clone was identified in 16 ramets. No genets were shared between sites, and each site exhibited markedly different clonal and sexual recruitment patterns, ranging from two non-overlapping and widespread genets to 19 distinct genets. Only one flowering genet was female; the remainder were hermaphrodites.

Conclusions

Local population history or fine-scale ecological differences can result in dramatically different reproductive patterns at small spatial scales. This finding may be fairly widespread among clonal plant species, and studies that aim to characterize reproductive modes in species capable of asexual reproduction need to evaluate reproductive modes in multiple populations and sites.Key words: Clonal structure, gynodioecy, Fragaria virginiana, microsatellites, population genetic structure     

High-resolution genotyping of the endemic Salmonella Typhi population during a Vi (typhoid) vaccination trial in Kolkata

Background

Typhoid fever, caused by Salmonella enterica serovar Typhi (S. Typhi), is a major health problem especially in developing countries. Vaccines against typhoid are commonly used by travelers but less so by residents of endemic areas.

Methodology

We used single nucleotide polymorphism (SNP) typing to investigate the population structure of 372 S. Typhi isolated during a typhoid disease burden study and Vi vaccine trial in Kolkata, India. Approximately sixty thousand people were enrolled for fever surveillance for 19 months prior to, and 24 months following, Vi vaccination of one third of the study population (May 2003–December 2006, vaccinations given December 2004).

Principal Findings

A diverse S. Typhi population was detected, including 21 haplotypes. The most common were of the H58 haplogroup (69%), which included all multidrug resistant isolates (defined as resistance to chloramphenicol, ampicillin and co-trimoxazole). Quinolone resistance was particularly high among H58-G isolates (97% Nalidixic acid resistant, 30% with reduced susceptibility to ciprofloxacin). Multiple typhoid fever episodes were detected in 22 households, however household clustering was not associated with specific S. Typhi haplotypes.

Conclusions

Typhoid fever in Kolkata is caused by a diverse population of S. Typhi, however H58 haplotypes dominate and are associated with multidrug and quinolone resistance. Vi vaccination did not obviously impact on the haplotype population structure of the S. Typhi circulating during the study period.     

Resistance of Asian Cryptococcus neoformans serotype A is confined to few microsatellite genotypes

Background

Cryptococcus neoformans is a pathogenic yeast that causes cryptococcosis, a life threatening disease. The prevalence of cryptococcosis in Asia has been rising after the onset of the AIDS epidemic and estimates indicate more than 120 cases per 1,000 HIV-infected individuals per year. Almost all cryptococcal disease cases in both immunocompromised and immunocompetent patients in Asia are caused by C. neoformans var. grubii. Epidemiological studies on C. neoformans in pan-Asia have not been reported. The present work studies the genetic diversity of the fungus by microsatellite typing and susceptibility analysis of approximately 500 isolates from seven Asian countries.

Methodology/Principal Findings

Genetic diversity of Asian isolates of C. neoformans was determined using microsatellite analysis with nine microsatellite markers. The analysis revealed eight microsatellite complexes (MCs) which showed different distributions among geographically defined populations. A correlation between MCs and HIV-status was observed. Microsatellite complex 2 was mainly associated with isolates from HIV-negative patients, whereas MC8 was associated with those from HIV-positive patients. Most isolates were susceptible to amphotericin B, itraconazole, voriconazole, posaconazole, and isavuconazole, but 17 (3.4%) and 10 (2%) were found to be resistant to 5-flucytosine and fluconazole, respectively. Importantly, five Indonesian isolates (approximately 12.5% from all Indonesian isolates investigated and 1% from the total studied isolates) were resistant to both antifungals. The majority of 5-flucytosine resistant isolates belonged to MC17.

Conclusions

The findings showed a different distribution of genotypes of C. neoformans var. grubii isolates from various countries in Asia, as well as a correlation of the microsatellite genotypes with the original source of the strains and resistance to 5-flucytosine.     

Microbial-resistant Salmonella enteritidis isolated from poultry samples

Background:

Multidrug resistance in Salmonella enteritidis isolates is a public health problem worldwide; the present study, therefore, was designed for antimicrobial-resistance determination in this strain.

Methods:

Salmonella strains isolated from poultry samples by biochemical positive and negative tests were subjected to PCR and identified as Salmonella enteritidis. For detection and identification of Salmonella enteritidis isolates, sdfI gene-specific primers were used.

Results:

We found that 100% of isolates were resistant to ampicillin, 90% were resistant to cephalothin and streptomycin, 70% were resistant to cefotaxime, and 60% were resistant to kanamycin and gentamicin.

Conclusion:

Salmonella enteritidis isolates had antimicrobial resistance to mentioned antibiotics. Key Words: Antibiotic Resistance, PCR, Poultry, Salmonella enteritidis