Use of high-density tiling microarrays to identify mutations globally and elucidate mechanisms of drug resistance in Plasmodium falciparum

Background

The identification of genetic changes that confer drug resistance or other phenotypic changes in pathogens can help optimize treatment strategies, support the development of new therapeutic agents, and provide information about the likely function of genes. Elucidating mechanisms of phenotypic drug resistance can also assist in identifying the mode of action of uncharacterized but potent antimalarial compounds identified in high-throughput chemical screening campaigns against Plasmodium falciparum.

Results

Here we show that tiling microarrays can detect de novo a large proportion of the genetic changes that differentiate one genome from another. We show that we detect most single nucleotide polymorphisms or small insertion deletion events and all known copy number variations that distinguish three laboratory isolates using readily accessible methods. We used the approach to discover mutations that occur during the selection process after transfection. We also elucidated a mechanism by which parasites acquire resistance to the antimalarial fosmidomycin, which targets the parasite isoprenoid synthesis pathway. Our microarray-based approach allowed us to attribute in vitro derived fosmidomycin resistance to a copy number variation event in the pfdxr gene, which enables the parasite to overcome fosmidomycin-mediated inhibition of isoprenoid biosynthesis.

Conclusions

We show that newly emerged single nucleotide polymorphisms can readily be detected and that malaria parasites can rapidly acquire gene amplifications in response to in vitro drug pressure. The ability to define comprehensively genetic variability in P. falciparum with a single overnight hybridization creates new opportunities to study parasite evolution and improve the treatment and control of malaria.     

Effects of light on cyanide‐resistant respiration and alternative oxidase function in Arabidopsis seedlings

Mitochondrial alternative oxidase (AOX), the unique respiratory terminal oxidase in plants, catalyzes the energy wasteful cyanide (CN)‐resistant respiration and plays a role in optimizing photosynthesis. Although it has been demonstrated that leaf AOX is upregulated after illumination, the in vivo mechanism of AOX upregulation by light and its physiological significance are still unknown. In this report, red light and blue light‐induced AOX (especially AOX1a) expressions were characterized. Phytochromes, phototropins and cryptochromes, all these photoreceptors mediate the light‐response of AOX1a gene. When aox1a mutant seedlings were grown under a high‐light (HL) condition, photobleaching was more evident in the mutant than the wild‐type plants. More reactive oxygen species (ROS) accumulation and inefficient dissipation of chloroplast reducing‐equivalents in aox1a mutant may account for its worse adaptation to HL stress. When etiolated seedlings were exposed to illumination for 4 h, chlorophyll accumulation was largely delayed in aox1a plants. We first suggest that more reduction of the photosynthetic electron transport chain and more accumulation of reducing‐equivalents in the mutant during de‐etiolation might be the main reasons.     

Polymorphisms in the beta-tubulin gene of Cryptosporidium parvum differentiate between isolates based on animal host but not geographic origin

Polymerase chain reaction primers were designed to target a region of the Cryptosporidium parvum beta-tubulin gene spanning an intron. Amplification products contained 11 polymorphic positions, representing a sequence divergence of 1.8%, which discriminated between isolates of C. parvum found solely in humans (genotype 1) and those found in humans and animals (genotype 2). Seven of the polymorphic sites were located outside of the intron and the polymorphism between isolates was readily demonstrated by HaeIII restriction digestion. However, all of the sequences from genotype 1 human-derived oocysts isolated in the United States and Australia were conserved. Also, there were no sequence differences between bovine isolates obtained from both continents. Therefore, isolates could not be differentiated based on geographic source of origin.     

Clams (Corbicula fluminea) as bioindicators of fecal contamination with Cryptosporidium and Giardia spp. in freshwater ecosystems in California

This study evaluated clams as bioindicators of fecal protozoan contamination using three approaches: (i) clam tissue spiking experiments to compare several detection techniques; (ii) clam tank exposure experiments to evaluate clams that had filtered Cryptosporidium oocysts from inoculated water under a range of simulated environmental conditions; (iii) sentinel clam outplanting to assess the distribution and magnitude of fecal contamination in three riverine systems in California. Our spiking and tank experiments showed that direct fluorescent antibody (DFA), immunomagnetic separation (IMS) in combination with DFA, and PCR techniques could be used to detect Cryptosporidium in clam tissues. The most analytically sensitive technique was IMS concentration with DFA detection of oocysts in clam digestive gland tissues, which detected 10 oocysts spiked into a clam digestive gland 83% of the time. In the tank experiment, oocyst dose and clam collection time were significant predictors for detecting Cryptosporidium parvum oocysts in clams. In the wild clam study, Cryptosporidium and Giardia were detected in clams from all three study regions by IMS-DFA analysis of clam digestive glands, with significant variation by sampling year and season. The presence of C. parvum DNA in clams from riverine ecosystems was confirmed with PCR and DNA sequence analysis.     

Intra-isolate Heterogeneity and Reproducibility of PCR-based Genotyping of Cryptosporidium parvum Using the β-tubulin Gene

Cryptosporidium parvum is a common contaminant in surface waters and presents significant problems for the water industry, public health and agriculture. Consequently, ascertaining the contaminating source of waterborne oocysts is of paramount importance. Based on currently available information, isolates of C. parvum can be differentiated into at least two genotypes using polymorphic genetic markers: genotype 1, to date isolated almost exclusively from humans, and genotype 2 isolates from humans and many other animals. Differentiation into these two genotypes has been based on either restriction fragment length polymorphisms or sequencing of PCR amplified gene fragments. The objective of this study was to evaluate the reproducibility of genotyping methods using a single isolate of C. parvum. A 620 bp fragment of the C. parvum -tubulin gene, generated by PCR from multiple aliquots of a single preparation of oocysts of the Iowa isolate, was sequenced. Significant sequence heterogeneity was detected within this single isolate; there was more sequence variation between clones originating from the Iowa isolate (up to 0.9 %) than between individual clones originating from different isolates of C. parvum. Over 6 % of the -tubulin gene sequence positions (38 out of 620 bp) were variable when comparing multiple clones from the one isolate. The results indicated that while the various procedures used for genotyping isolates may introduce some sequence errors, the Iowa isolate used for this investigation appeared to be composed of multiple sub-genotypes. While none of the sequence variations resulted in clones of the Iowa isolate (genotype 2) being mis-identified as genotype 1, the results have important implications if minor sequence variations are to be used for subtyping isolates and drawing conclusions regarding the origin of, or relationships between, C. parvum oocysts in water and the community.     

New species of the genus Plesiolacerta (Squamata: Lacertidae) from the upper Oligocene (MP28) of Southern Germany and a revision of the type species Plesiolacerta lydekkeri

Abstract: The lacertid material from the locality of Herrlingen 8 (upper Oligocene, MP28) is described as a new species of the genus Plesiolacerta. The material is disarticulated and comprises isolated elements including parietal, frontal, maxilla and dentary. It can be assigned to a single species on the basis of the external surface ornamentation. This morphology is typical for the genus Plesiolacerta, but the material differs in detail from the type species P. lydekkeri. The most significant feature of the new species is that the occipital scute of the parietal bone is narrow, rectangular in shape and anteroposteriorly short. Hitherto, the last occurrence of this genus was in the lower Oligocene. This material represents the first evidence of the existence of this genus in the upper Oligocene. Therefore, our knowledge of its evolution is expanded by providing new data on its spatial and temporal ranges and morphology. This taxon has a much longer history than we thought. In addition, the Eocene species, P. lydekkeri, is reviewed here. P. lydekkeri shares the most lacertid synapomorphies and, given our present knowledge, Plesiolacerta is a taxon very close to or possibly within crown Lacertidae. The frontal and postorbitofrontal of Plesiolacerta are described for the first time. In view of the primitive morphology and early occurrence of Plesiolacerta, it seems that the feature of a longer anterior region of the frontal could be considered as a plesiomorphic feature within lacertid lizards, and the condition in Timon (approximately the same length) as derived.     

Invasion status of Florida bass Micropterus floridanus (Lesueur, 1822) in South Africa

Largemouth bass Micropterus salmoides are a popular North American angling species that was introduced into South Africa in 1928. To enhance the largemouth bass fisheries, Florida bass Micropterus floridanus were introduced into KwaZulu Natal, South Africa, in 1980. Knowledge on the status of M. floridanus in South Africa is required, because it lives longer and reaches larger sizes than M. salmoides, which may result in heightened impacts on native biota. Because M. floridanus are morphologically similar, but genetically distinct from M. salmoides, the distribution of this species was assessed by genetically screening 185 Micropterus sp. individuals sampled from 20 localities across South Africa using the mitochondrial ND2 gene. Individuals with mitochondrial DNA matching M. salmoides were recovered from 16 localities, whereas M. floridanus mitochondrial DNA was recovered from 13 localities. At nine localities (45%), the mitochondrial DNA of both species was detected. These results demonstrate M. floridanus dispersal to multiple sites across South Africa.     

Estimating Maximum Possible Environmental Loading Amounts of Cryptosporidium parvum Attributable to Adult Beef Cattle

Populations of beef cattle represent a potential non-point source of environmental contamination for Cryptosporidium parvum if on-farm management practices fail to minimize transport from bovine manure to adjacent water sources. Characterizing this risk of contamination requires several parameters to be estimated, the most important being a valid and precise estimate of the oocyst loading rate per animal unit. The oocyst loading rate is defined in this study as the total number of oocysts excreted by a cohort of adult beef cows during a 24[emsp4 ]h period. We propose a methodology for estimating this parameter for low prevalent populations whereby the majority of individuals are test negative. Under specific degrees of confidence and at the population scale, this methodology generates estimates for maximal oocyst loading based on the sensitivity of the diagnostic test and the point prevalence and intensity of fecal shedding from a cross-sectional survey of the target population.Our cross-sectional survey on California beef cows generated a prevalence of infection of 1.1 % (6/557) and an intensity of oocyst shedding ranging from 219 to 5,491 oocysts/g, with a geometric mean of 835 oocysts/g from six positive cows. Negative binomial estimate of the percent recovery of the diagnostic assay was 0.235. Based on this percent recovery and using approximately 19.4[emsp4 ]mg of feces per assay, the DT₉₀ of our assay, defined as the concentration of oocysts at which our diagnostic assay had a 90 % probability of detecting one or more oocysts in a sample, was 755 oocyst/g feces. At a 95 % confidence level, the estimated maximum number of oocysts being excreted in the feces of California beef cows ranged from 4.8 to 14.4 oocysts/g feces/cow, or 7.7×10⁴ to 2.3×10⁵ oocysts/beef cow/day.     

Association of Cryptosporidium parvum with suspended particles: impact on oocyst sedimentation

The association of Cryptosporidium parvum oocysts with suspended particles can alter the oocysts' effective physical properties and influence their transport in aquatic systems. To assess this behavior, C. parvum oocysts were mixed with various suspended sediments under a variety of water chemical conditions, and the resulting settling of the oocysts was observed. Direct microscopic observations showed that oocysts attached to suspended sediments. Settling column and batch experiments demonstrated that oocysts are removed from suspension at a much higher rate when associated with sediments. The rate of oocyst sedimentation depended primarily on the type of sediment with which the oocysts were mixed. Changes in background water conditions had a relatively small impact on the extent of oocyst-particle association and the resulting oocyst deposition. We believe that the ubiquitous association of C. parvum oocysts with suspended particles enhances the sedimentation of oocysts in natural waters and that this interaction should generally be considered when predicting the migration of pathogens in the environment.