100 research outputs found
Separate Ion Pathways in a Clâ/H+ Exchanger
CLC-ec1 is a prokaryotic CLC-type Clâ/H+ exchange transporter. Little is known about the mechanism of H+ coupling to Clâ. A critical glutamate residue, E148, was previously shown to be required for Clâ/H+ exchange by mediating proton transfer between the protein and the extracellular solution. To test whether an analogous H+ acceptor exists near the intracellular side of the protein, we performed a mutagenesis scan of inward-facing carboxyl-bearing residues and identified E203 as the unique residue whose neutralization abolishes H+ coupling to Clâ transport. Glutamate at this position is strictly conserved in all known CLCs of the transporter subclass, while valine is always found here in CLC channels. The x-ray crystal structure of the E203Q mutant is similar to that of the wild-type protein. Clâ transport rate in E203Q is inhibited at neutral pH, and the double mutant, E148A/E203Q, shows maximal Clâ transport, independent of pH, as does the single mutant E148A. The results argue that substrate exchange by CLC-ec1 involves two separate but partially overlapping permeation pathways, one for Clâ and one for H+. These pathways are congruent from the protein's extracellular surface to E148, and they diverge beyond this point toward the intracellular side. This picture demands a transport mechanism fundamentally different from familiar alternating-access schemes
A high-throughput method to detect Plasmodium falciparum clones in limiting dilution microplates
<p>Abstract</p> <p>Background</p> <p>Molecular and cellular studies of <it>Plasmodium falciparum </it>require cloning of parasites by limiting dilution cultivation, typically performed in microplates. The parasite's slow replication rate combined with laborious methods for identification of positive wells has limited these studies. A new high-throughput method for detecting growth without compromising parasite viability is reported.</p> <p>Methods</p> <p><it>In vitro </it>parasite cultivation is associated with extracellular acidification. A survey of fluorescent pH indicators identified 5-(and-6)-carboxy SNARF-1 as a membrane-impermeant dye with a suitable <it>pK<sub>a </sub></it>value. Conditions for facile detection of viable parasites in 96-well microplates were optimized and used for limiting dilution cloning of genetic cross progeny and transfected parasites.</p> <p>Results</p> <p>5-(and-6)-carboxy SNARF-1 is a two-emission wavelength dye that accurately reported extracellular pH in parasite cultures. It readily detected parasite growth in microplate wells and yielded results comparable to labour-intensive examination of Giemsa-stained smears. The dye is non-toxic, allowing parasite detection without transfer of culture material to additional plates for separate assays. This dye was used with high-throughput limiting dilution culture to generate additional progeny clones from the HB3 Ă Dd2 genetic cross.</p> <p>Conclusions</p> <p>This fluorescence-based assay represents a low-cost, efficient method for detection of viable parasites in microplate wells; it can be easily expanded by automation.</p
Highly heterogeneous residual malaria risk in western Thailand
Over the past decades, the malaria burden in Thailand has substantially declined. Most infections now originate from the national border regions. In these areas, the prevalence of asymptomatic infections is still substantial and poses a challenge for the national malaria elimination program. To determine epidemiological parameters as well as risk factors for malaria infection in western Thailand, we carried out a cohort study in Kanchanaburi and Ratchaburi provinces on the Thailand-Myanmar border. Blood samples from 999 local participants were examined for malaria infection every 4âŻweeks between May 2013 and Jun 2014. Prevalence of Plasmodium falciparum and Plasmodium vivax was determined by quantitative PCR (qPCR) and showed a seasonal variation with values fluctuating from 1.7% to 4.2% for P. vivax and 0% to 1.3% for P. falciparum. Ninety percent of infections were asymptomatic. The annual molecular force of blood-stage infection (molFOB) was estimated by microsatellite genotyping to be 0.24 new infections per person-year for P. vivax and 0.02 new infections per person-year for P. falciparum. The distribution of infections was heterogenous, that is, the vast majority of infections (>80%) were found in a small number of individuals (<8% of the study population) who tested positive at multiple timepoints. Significant risk factors were detected for P. vivax infections, including previous clinical malaria, occupation in agriculture and travel to Myanmar. In contrast, indoor residual spraying was associated with a protection from infection. These findings provide a recent landscape of malaria epidemiology and emphasize the importance of novel strategies to target asymptomatic and imported infections
Malaria Parasite clag3 Genes Determine Channel-Mediated Nutrient Uptake by Infected Red Blood Cells
SummaryDevelopment of malaria parasites within vertebrate erythrocytes requires nutrient uptake at the host cell membrane. The plasmodial surface anion channel (PSAC) mediates this transport and is an antimalarial target, but its molecular basis is unknown. We report a parasite gene family responsible for PSAC activity. We used high-throughput screening for nutrient uptake inhibitors to identify a compound highly specific for channels from the Dd2 line of the human pathogen P. falciparum. Inheritance of this compound's affinity in a Dd2 à HB3 genetic cross maps to a single parasite locus on chromosome 3. DNA transfection and in vitro selections indicate that PSAC-inhibitor interactions are encoded by two clag3 genes previously assumed to function in cytoadherence. These genes are conserved in plasmodia, exhibit expression switching, and encode an integral protein on the host membrane, as predicted by functional studies. This protein increases host cell permeability to diverse solutes.PaperFlic
Sensitive detection of Plasmodium vivax malaria by the rotating-crystal magneto-optical method in Thailand
The rotating-crystal magneto-optical detection (RMOD) method has been developed for the rapid and quantitative diagnosis of malaria and tested systematically on various malaria infection models. Very recently, an extended field trial in a high-transmission region of Papua New Guinea demonstrated its great potential for detecting malaria infections, in particular Plasmodium vivax. In the present small-scale field test, carried out in a low-transmission area of Thailand, RMOD confirmed malaria in all samples found to be infected with Plasmodium vivax by microscopy, our reference method. Moreover, the magneto-optical signal for this sample set was typically 1â3 orders of magnitude higher than the cut-off value of RMOD determined on uninfected samples. Based on the serial dilution of the original patient samples, we expect that the method can detect Plasmodium vivax malaria in blood samples with parasite densities as low as âŒ5â10 parasites per microliter, a limit around the pyrogenic threshold of the infection. In addition, by investigating the correlation between the magnitude of the magneto-optical signal, the parasite density and the erythrocytic stage distribution, we estimate the relative hemozoin production rates of the ring and the trophozoite stages of in vivo Plasmodium vivax infections
Asymptomatic and sub-microscopic malaria infection in Kayah State, eastern Myanmar
Myanmar has the heaviest burden of malaria in the Greater Mekong Sub-region. Asymptomatic Plasmodium spp. infections are common in this region and may represent an important reservoir of transmission that must be targeted for malaria elimination.; A mass blood survey was conducted among 485 individuals from six villages in Kayah State, an area of endemic but low transmission malaria in eastern Myanmar. Malaria infection was screened by rapid diagnostic test (RDT), light microscopy and real-time polymerase chain reaction (PCR), and its association with demographic factors was explored.; The prevalence of asymptomatic Plasmodium spp. infection was 2.3% (11/485) by real-time PCR. Plasmodium vivax accounted for 72.7% (8/11) and Plasmodium falciparum for 27.3% (3/11) of infections. Men were at greater risk of infection by Plasmodium spp. than women. Individuals who worked as farmers or wood and bamboo cutters had an increased risk of infection.; A combination of RDT, light microscopy and PCR diagnostics were used to identify asymptomatic malaria infection, providing additional information on asymptomatic cases in addition to the routine statistics on symptomatic cases, so as to determine the true burden of disease in the area. Such information and risk factors can improve malaria risk stratification and guide decision-makers towards better design and delivery of targeted interventions in small villages, representative of Kayah State
Asymptomatic Plasmodium vivax infections induce robust IgG responses to multiple blood-stage proteins in a low-transmission region of western Thailand
BACKGROUND: Thailand is aiming to eliminate malaria by the year
2024. Plasmodium vivax has now become the dominant species
causing malaria within the country, and a high proportion of
infections are asymptomatic. A better understanding of antibody
dynamics to P. vivax antigens in a low-transmission setting,
where acquired immune responses are poorly characterized, will
be pivotal for developing new strategies for elimination, such
as improved surveillance methods and vaccines. The objective of
this study was to characterize total IgG antibody levels to 11
key P. vivax proteins in a village of western Thailand. METHODS:
Plasma samples from 546 volunteers enrolled in a cross-sectional
survey conducted in 2012 in Kanchanaburi Province were utilized.
Total IgG levels to 11 different proteins known or predicted to
be involved in reticulocyte binding or invasion (ARP, GAMA, P41,
P12, PVX_081550, and five members of the PvRBP family), as well
as the leading pre-erythrocytic vaccine candidate (CSP) were
measured using a multiplexed bead-based assay. Associations
between IgG levels and infection status, age, and spatial
location were explored. RESULTS: Individuals from a
low-transmission region of western Thailand reacted to all 11 P.
vivax recombinant proteins. Significantly greater IgG levels
were observed in the presence of a current P. vivax infection,
despite all infected individuals being asymptomatic. IgG levels
were also higher in adults (18 years and older) than in
children. For most of the proteins, higher IgG levels were
observed in individuals living closer to the Myanmar border and
further away from local health services. CONCLUSIONS: Robust IgG
responses were observed to most proteins and IgG levels
correlated with surrogates of exposure, suggesting these
antigens may serve as potential biomarkers of exposure,
immunity, or both
Utility of ultra-sensitive qPCR to detect Plasmodium falciparum and Plasmodium vivax infections under different transmission intensities
Background: The use of molecular diagnostics has revealed an unexpectedly large number of asymptomatic
low-density malaria infections in many malaria endemic areas. This study compared the gains in parasite prevalence
obtained by the use of ultra-sensitive (us)-qPCR as compared to standard qPCR in cross-sectional surveys conducted
in Thailand, Brazil and Papua New Guinea (PNG). The compared assays differed in the copy number of qPCR targets in
the parasite genome.
Methods: Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) parasites were quantified by qPCR amplifying the
low-copy Pf_ and Pv_18S rRNA genes or the multi-copy targets Pf_varATS and Pv_mtCOX1. Cross-sectional surveys at
the three study sites included 2252 participants of all ages and represented different transmission intensities.
Results: In the two low-transmission areas, P. falciparum positivity was 1.3% (10/773) (Thailand) and 0.8% (5/651) (Bra-
zil) using standard Pf_18S rRNA qPCR. In these two countries, P. falciparum positivity by Pf_varATS us-qPCR increased
to 1.9% (15/773) and 1.7% (11/651). In PNG, an area with moderate transmission intensity, P. falciparum positivity
significantly increased from 8.6% (71/828) by standard qPCR to 12.2% (101/828) by us-qPCR. The proportions of P.
falciparum infections not detected by standard qPCR were 33%, 55% and 30% in Thailand, Brazil and PNG. Plasmodium
vivax was the predominating species in Thailand and Brazil, with 3.9% (30/773) and 4.9% (32/651) positivity by Pv_18S
rRNA qPCR. In PNG, P. vivax positivity was similar to P. falciparum, at 8.0% (66/828). Use of Pv_mtCOX1 us-qPCR led to
a significant increase in positivity to 5.1% (39/773), 6.4% (42/651) and 11.5% (95/828) in Thailand, Brazil, and PNG. The
proportions of P. vivax infections missed by standard qPCR were similar at all three sites, with 23%, 24% and 31% in
Thailand, Brazil and PNG.
Conclusion: The proportional gains in the detection of P. falciparum and P. vivax infections by ultra-sensitive diag-
nostic assays were substantial at all three study sites. Thus, us-qPCR yields more precise prevalence estimates for
both P. falciparum and P. vivax at all studied levels of endemicity and represents a significant diagnostic improvement
Anopheles bionomics in a malaria endemic area of southern Thailand
Background
Ivermectin mass drug administration (MDA) could accelerate malaria elimination in the Greater Mekong Subregion. This study was performed to characterize the bionomics of Anopheles in Surat Thani province, Thailand.
Methods
Mosquitoes were collected via human landing collections between February and October 2019. Anopheles mosquitoes were morphologically identified to species. Primary Anopheles malaria vectors were dissected to assess parity status, and a subset were evaluated for molecular identification and Plasmodium detection.
Results
A total of 17,348 mosquitoes were collected during the study period; of these, 5777 were Anopheles mosquitoes. Morphological studies identified 15 Anopheles species, of which the most abundant were Anopheles minimus (s.l.) (87.16%, nâ=â5035), An. dirus s.l. (7.05%, nâ=â407) and An. barbirostris s.l. (2.86%, nâ=â165). Molecular identification confirmed that of the An. minimus s.l. mosquitoes collected, 99.80% were An. minimus (s.s.) (nâ=â484) and 0.2% were An. aconitus (nâ=â1), of the An. dirus (s.l.) collected, 100% were An. baimaii (nâ=â348), and of the An. maculatus (s.l.) collected, 93.62% were An. maculatus (s.s.) (nâ=â44) and 6.38% were An. sawadwongporni (nâ=â3). No Anopheles mosquito tested was Plasmodium positive (0/879). An average of 11.46 Anopheles were captured per collector per night. There were differences between species in hour of collection (KruskalâWallis H-test: Ï2â=ââ80.89, PâConclusions
The study area in Surat Thani province is an ideal location to evaluate the impact of ivermectin MDA on An. minimus parity
Proton block of the CLC-5 Clâ/H+ exchanger
CLC-5 is a H+/Clâ exchanger that is expressed primarily in endosomes but can traffic to the plasma membrane in overexpression systems. Mutations altering the expression or function of CLC-5 lead to Dentâs disease. Currents mediated by this transporter show extreme outward rectification and are inhibited by acidic extracellular pH. The mechanistic origins of both phenomena are currently not well understood. It has been proposed that rectification arises from the voltage dependence of a H+ transport step, and that inhibition of CLC-5 currents by low extracellular pH is a result of a reduction in the driving force for exchange caused by a pH gradient. We show here that the pH dependence of CLC-5 currents arises from H+ binding to a single site located halfway through the transmembrane electric field and driving the transport cycle in a less permissive direction, rather than a reduction in the driving force. We propose that protons bind to the extracellular gating glutamate E211 in CLC-5. It has been shown that CLC-5 becomes severely uncoupled when SCNâ is the main charge carrier: H+ transport is drastically reduced while the rate of anion movement is increased. We found that in these conditions, rectification and pH dependence are unaltered. This implies that H+ translocation is not the main cause of rectification. We propose a simple transport cycle model that qualitatively accounts for these findings
- âŠ