316 research outputs found
The influence of host genetics on erythrocytes and malaria infection: is there therapeutic potential?
As parasites, Plasmodium species depend upon their host for survival. During the blood stage of their life-cycle parasites invade and reside within erythrocytes, commandeering host proteins and resources towards their own ends, and dramatically transforming the host cell. Parasites aptly avoid immune detection by minimizing the exposure of parasite proteins and removing themselves from circulation through cytoadherence. Erythrocytic disorders brought on by host genetic mutations can interfere with one or more of these processes, thereby providing a measure of protection against malaria to the host. This review summarizes recent findings regarding the mechanistic aspects of this protection, as mediated through the parasites interaction with abnormal erythrocytes. These novel findings include the reliance of the parasite on the host enzyme ferrochelatase, and the discovery of basigin and CD55 as obligate erythrocyte receptors for parasite invasion. The elucidation of these naturally occurring malaria resistance mechanisms is increasing the understanding of the host-parasite interaction, and as discussed below, is providing new insights into the development of therapies to prevent this disease.We acknowledge funding support from the National Health and Medical
Research Council (Grant APP605524, 490037 and 1047082), the Australian
Research Council (Grant DP12010061), the National Collaborative Research
Infrastructure Strategy of Australia and the Education investment fund from
the Department of Innovation, Industry, Science and Research. PML is a recipient
of an Australian Postgraduate award
A flow cytometric assay to quantify invasion of red blood cells by rodent Plasmodium parasites in vivo
BACKGROUND Malaria treatments are becoming less effective due to the rapid spread of drug resistant parasites. Increased understanding of the host/parasite interaction is crucial in order to develop treatments that will be less prone to resistance. Parasite invasion of the red blood cell (RBC) is a critical aspect of the parasite life cycle and is, therefore, a promising target for the development of malaria treatments. Assays for analysing parasite invasion in vitro have been developed, but no equivalent assays exist for in vivo studies. This article describes a novel flow cytometric in vivo parasite invasion assay. METHODS Experiments were conducted with mice infected with erythrocytic stages of Plasmodium chabaudi adami strain DS. Exogenously labelled blood cells were transfused into infected mice at schizogony, and collected blood samples stained and analysed using flow cytometry to specifically detect and measure proportions of labelled RBC containing newly invaded parasites. A combination of antibodies (CD45 and CD71) and fluorescent dyes, Hoechst (DNA) and JC-1 (mitochondrial membrane potential), were used to differentiate parasitized RBCs from uninfected cells, RBCs containing Howell-Jolly bodies, leukocytes and RBC progenitors. Blood cells were treated ex vivo with proteases to examine the effects on in vivo parasite invasion. RESULTS The staining and flow cytometry analysis method was accurate in determining the parasitaemia down to 0.013% with the limit of detection at 0.007%. Transfused labelled blood supported normal rates of parasite invasion. Protease-treated red cells resulted in 35% decrease in the rate of parasite invasion within 30 minutes of introduction into the bloodstream of infected mice. CONCLUSIONS The invasion assay presented here is a versatile method for the study of in vivo red cell invasion efficiency of Plasmodium parasites in mice, and allows direct comparison of invasion in red cells derived from two different populations. The method also serves as an accurate alternative method of estimating blood parasitaemia.We acknowledge funding support from the National Health and Medical Research Council (grant APP605524, 490037 and 1047082), the Australian Research Council (grant DP12010061), the National Collaborative Research Infrastructure Strategy of Australia and the Education investment fund from the Department of Innovation, Industry, Science and Research. PML is a recipient of an Australian Postgraduate award
Sporadic sampling, not climatic forcing, drives observed early hominin diversity.
Paleoanthropologists have long been intrigued by the observed patterns of human evolution, including species diversity, and often invoked climatic change as the principal driver of evolutionary change. Here, we investigate whether the early hominin fossil record is of suitable quality to test these climate-forcing hypotheses. Specifically, we compare early hominin diversity to sampling metrics that quantify changes in fossil preservation and sampling intensity between 7 and 1 million years ago. We find that observed diversity patterns are governed by sporadic sampling and do not yield a genuine evolutionary signal. Many more fossil discoveries are required before existing hypotheses linking climate and evolution can be meaningfully tested.The role of climate change in the origin and diversification of early hominins is hotly debated. Most accounts of early hominin evolution link observed fluctuations in species diversity to directional shifts in climate or periods of intense climatic instability. None of these hypotheses, however, have tested whether observed diversity patterns are distorted by variation in the quality of the hominin fossil record. Here, we present a detailed examination of early hominin diversity dynamics, including both taxic and phylogenetically corrected diversity estimates. Unlike past studies, we compare these estimates to sampling metrics for rock availability (hominin-, primate-, and mammal-bearing formations) and collection effort, to assess the geological and anthropogenic controls on the sampling of the early hominin fossil record. Taxic diversity, primate-bearing formations, and collection effort show strong positive correlations, demonstrating that observed patterns of early hominin taxic diversity can be explained by temporal heterogeneity in fossil sampling rather than genuine evolutionary processes. Peak taxic diversity at 1.9 million years ago (Ma) is a sampling artifact, reflecting merely maximal rock availability and collection effort. In contrast, phylogenetic diversity estimates imply peak diversity at 2.4 Ma and show little relation to sampling metrics. We find that apparent relationships between early hominin diversity and indicators of climatic instability are, in fact, driven largely by variation in suitable rock exposure and collection effort. Our results suggest that significant improvements in the quality of the fossil record are required before the role of climate in hominin evolution can be reliably determined
Comparing genotyping algorithms for Illumina's Infinium whole-genome SNP BeadChips
Background: Illumina's Infinium SNP BeadChips are extensively used in both small and large-scale genetic studies. A fundamental step in any analysis is the processing of raw allele A and allele B intensities from each SNP into genotype calls (AA, AB, BB). Various algorithms which make use of different statistical models are available for this task. We compare four methods (GenCall, Illuminus, GenoSNP and CRLMM) on data where the true genotypes are known in advance and data from a recently published genome-wide association study.Results: In general, differences in accuracy are relatively small between the methods evaluated, although CRLMM and GenoSNP were found to consistently outperform GenCall. The performance of Illuminus is heavily dependent on sample size, with lower no call rates and improved accuracy as the number of samples available increases. For X chromosome SNPs, methods with sex-dependent models (Illuminus, CRLMM) perform better than methods which ignore gender information (GenCall, GenoSNP). We observe that CRLMM and GenoSNP are more accurate at calling SNPs with low minor allele frequency than GenCall or Illuminus. The sample quality metrics from each of the four methods were found to have a high level of agreement at flagging samples with unusual signal characteristics.Conclusions: CRLMM, GenoSNP and GenCall can be applied with confidence in studies of any size, as their performance was shown to be invariant to the number of samples available. Illuminus on the other hand requires a larger number of samples to achieve comparable levels of accuracy and its use in smaller studies (50 or fewer individuals) is not recommended
Microarray-based comparative genomic profiling of reference strains and selected Canadian field isolates of Actinobacillus pleuropneumoniae
<p>Abstract</p> <p>Background</p> <p><it>Actinobacillus pleuropneumoniae</it>, the causative agent of porcine pleuropneumonia, is a highly contagious respiratory pathogen that causes severe losses to the swine industry worldwide. Current commercially-available vaccines are of limited value because they do not induce cross-serovar immunity and do not prevent development of the carrier state. Microarray-based comparative genomic hybridizations (M-CGH) were used to estimate whole genomic diversity of representative <it>Actinobacillus pleuropneumoniae </it>strains. Our goal was to identify conserved genes, especially those predicted to encode outer membrane proteins and lipoproteins because of their potential for the development of more effective vaccines.</p> <p>Results</p> <p>Using hierarchical clustering, our M-CGH results showed that the majority of the genes in the genome of the serovar 5 <it>A. pleuropneumoniae </it>L20 strain were conserved in the reference strains of all 15 serovars and in representative field isolates. Fifty-eight conserved genes predicted to encode for outer membrane proteins or lipoproteins were identified. As well, there were several clusters of diverged or absent genes including those associated with capsule biosynthesis, toxin production as well as genes typically associated with mobile elements.</p> <p>Conclusion</p> <p>Although <it>A. pleuropneumoniae </it>strains are essentially clonal, M-CGH analysis of the reference strains of the fifteen serovars and representative field isolates revealed several classes of genes that were divergent or absent. Not surprisingly, these included genes associated with capsule biosynthesis as the capsule is associated with sero-specificity. Several of the conserved genes were identified as candidates for vaccine development, and we conclude that M-CGH is a valuable tool for reverse vaccinology.</p
Recurrent miscalling of missense variation from short-read genome sequence data
Background: Short-read resequencing of genomes produces abundant information of the genetic variation of individuals. Due to their numerous nature, these variants are rarely exhaustively validated. Furthermore, low levels of undetected variant miscalling will have a systematic and disproportionate impact on the interpretation of individual genome sequence information, especially should these also be carried through into in reference databases ofgenomic variation.
Results: We find that sequence variation from short-read sequence data is subject to recurrent-yet-intermittent miscalling that occurs in a sequence intrinsic manner and is very sensitive to sequence read length. The miscalls arise from difficulties aligning short reads to redundant genomic regions, where the rate of sequencing error
approaches the sequence diversity between redundant regions. We find the resultant miscalled variants to be sensitive to small sequence variations between genomes, and thereby are often intrinsic to an individual, pedigree, strain or human ethnic group. In human exome sequences, we identify 2–300 recurrent false positive variants per individual, almost all of which are present in public databases of human genomic variation. From the exomes of non-reference strains of inbred mice, we identify 3–5000 recurrent false positive variants per mouse – the number of which increasing with greater distance between an individual mouse strain and the reference C57BL6 mouse genome. We show that recurrently miscalled variants may be reproduced for a given genome from repeated simulation rounds of read resampling, realignment and recalling. As such, it is possible to identify more than two-thirds of false positive variation from only ten rounds of simulation.
Conclusion: Identification and removal of recurrent false positive variants from specific individual variant sets will improve overall data quality. Variant miscalls arising are highly sequence intrinsic and are often specific to an individual, pedigree or ethnicity. Further, read length is a strong determinant of whether given false variants will be called for any given genome – which has profound significance for cohort studies that pool datasets collected and sequenced at different points in time
Erythrocyte β spectrin can be genetically targeted to protect mice from malaria
The malaria parasite hijacks host erythrocytes to shield itself from the immune system and proliferate. Red blood cell abnormalities can provide protection from malaria by impeding parasite invasion and growth within the cell or by compromising the ability of parasites to avoid host clearance. Here, we describe 2 N-ethyl-N-nitrosourea–induced mouse lines, SptbMRI26194 and SptbMRI53426, containing single-point mutations in the erythrocyte
membrane skeleton gene, b spectrin (Sptb), which exhibit microcytosis but retain a relatively normal ratio of erythrocyte surface area to volume and are highly resistant to rodent malaria. We propose the major factor responsible for malaria protection is the specific clearance of mutant erythrocytes, although an enhanced clearance of ninfected
mutant erythrocytes was also observed (ie, the bystander effect). Using an in vivo erythrocyte tracking assay, we established that this phenomenon occurs irrespective of host environment, precluding the involvement of nonerythrocytic cells in the resistance mechanism. Furthermore, we recapitulated this phenotype by disrupting the interaction between ankyrin-1 and b spectrin in vivo using CRISPR/Cas9 genome editing technology, thereby genetically validating a potential antimalarial target. This study sheds new light on
the role of b spectrin during Plasmodium infection and highlights how changes in the erythrocyte cytoskeleton can substantially influence malaria susceptibility with minimal
adverse consequences for the host.This work was supported by the National Health and Medical
Research Council (grants APP605524 , 4 90037 and 104 7082),
the Australian Research Council (grants DP12010061 and
FL150100106), the National Collaborative Research Infrastructure
Strategy of Australia and the education investment fund from the
Department of Innovation, Industry, Science and Research via
the Australian Phenomics Network, and the Japan Society for
the Promotion of Science Fellowship Program (grant S16706)
Analysis of the F2LR3 (PAR4) single nucleotide polymorphism (rs773902) in an Indigenous Australian population
The F2RL3 gene encoding protease activated receptor 4 (PAR4) contains a single nucleotide variant, rs773902, that is functional. The resulting PAR4 variants, Thr120, and Ala120, are known to differently affect platelet reactivity to thrombin. Significant population differences in the frequency of the allele indicate it may be an important determinant in the ethnic differences that exist in thrombosis and hemostasis, and for patient outcomes to PAR antagonist anti-platelet therapies. Here we determined the frequency of rs773902 in an Indigenous Australian group comprising 467 individuals from the Tiwi Islands. These people experience high rates of renal disease that may be related to platelet and PAR4 function and are potential recipients of PAR-antagonist treatments. The rs773902 minor allele frequency (Thr120) in the Tiwi Islanders was 0.32, which is similar to European and Asian groups and substantially lower than Melanesians and some African groups. Logistic regression and allele distortion testing revealed no significant associations between the variant and several markers of renal function, as well as blood glucose and blood pressure. These findings suggest that rs773902 is not an important determinant for renal disease in this Indigenous Australian group. However, the relationships between rs773902 genotype and platelet and drug responsiveness in the Tiwi, and the allele frequency in other Indigenous Australian groups should be evaluated
Transcriptomic and Exometabolomic Profiling Reveals Antagonistic and Defensive Modes of Clonostachys rosea Action Against Fusarium graminearum
The mycoparasite Clonostachys rosea ACM941 is under development as a biocontrol organism against Fusarium graminearum, the causative agent of Fusarium head blight in cereals. To identify molecular factors associated with this interaction, the transcriptomic and exometabolomic profiles of C. rosea and F. graminearum GZ3639 were compared during coculture. Prior to physical contact, the antagonistic activity of C. rosea correlated with a response heavily dominated by upregulation of polyketide synthase gene clusters, consistent with the detected accumulation of corresponding secondary metabolite products. Similarly, prior to contact, trichothecene gene clusters were upregulated in F. graminearum, while those responsible for fusarielin and fusarin biosynthesis were downregulated, correlating with an accumulation of trichothecene products in the interaction zone over time. A concomitant increase in 15-acetyl deoxynivalenol-3-glucoside in the interaction zone was also detected, with C. rosea established as the source of this detoxified mycotoxin. After hyphal contact, C. rosea was found to predominantly transcribe genes encoding cell wall–degradation enzymes, major facilitator superfamily sugar transporters, anion:cation symporters, as well as alternative carbon source utilization pathways, together indicative of a transition to necrotropism at this stage. F. graminearum notably activated the transcription of phosphate starvation pathway signature genes at this time. Overall, a number of signature molecular mechanisms likely contributing to antagonistic activity by C. rosea against F. graminearum, as well as its mycotoxin tolerance, are identified in this report, yielding several new testable hypotheses toward understanding the basis of C. rosea as a biocontrol agent for continued agronomic development and application
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