Biology subjects

Caljon, G.

Publications and source records attributed to Caljon, G..

8 recordsLinked to original sources

bioRxiv2025

Elevated Plasmodium falciparum sexual conversion in HbAC and HbAS red blood cells without altered IgG/IgM levels to trophozoite and stage I gametocyte-specific antigens

Malaria transmission relies on the differentiation of asexual parasites into gametocytes, a process initiated by sexual conversion (SC). Mutations in the host hemoglobin beta (HBB) gene are known to influence parasite growth and disease outcome, yet their impact on SC remains unclear. We investigated the effect of HBB mutant genotypes on Plasmodium falciparum SC and humoral immunity in individuals from Nanoro, Burkina Faso. To measure SC rates in natural human malaria infections, we developed a new ex vivo SC assay (evSCA). We found that in human natural P. falciparum infections, SC rates were higher in individuals with HbAS or HbAC than in individuals with wild-type HBB (HbAA). Consistently, using an in vitro SC assay (ivSCA) based on the NF54-gexp02-Tom reporter line we found that cultures grown in HbAS red blood cells (RBCs) had higher SC rates than those grown in HbAA RBCs. Furthermore, IgG and IgM responses against trophozoite- and stage I gametocyte-infected RBC antigens, quantified by flow cytometry, did not differ between plasma from individuals with different HBB genotypes. These results demonstrate that exposure to RBCs with HBB mutations enhances SC, highlighting a host-genetic factor that may influence malaria transmission potential. AUTHOR SUMMARYTransmission of Plasmodium falciparum -a malaria causing parasite- depends on the parasites ability to produce gametocytes, the stage that infects mosquitoes. In many African regions where malaria is common, hemoglobin (Hb) variants such as hemoglobin S (the sickle-cell trait) and C are highly prevalent reflecting their role in protecting against severe disease. However, it remains unclear whether these variants also influence the parasites early shift toward gametocyte development (i.e., sexual conversion [SC]). In this study, we combined ex vivo and in vitro SC assays to measure SC in red blood cells with different hemoglobin genotypes. We found that SC was higher in parasites growing in red blood cells carrying hemoglobin AC and AS genotypes compared to normal AA hemoglobin. These findings provide experimental support for earlier epidemiological observations reporting that individuals with these hemoglobin variants often carry more gametocytes and are more infectious to mosquitoes. We also measured antibody levels in individuals with mutant RBCs and found no differences in immunity that could explain the variation in SC. Our results show that host genetic background can influence gametocyte development and may shape the infectious reservoir in high-burden settings. This knowledge is important for designing effective strategies for malaria elimination.

microbiology↗

bioRxiv2024

Allosteric inhibition of trypanosomatid pyruvate kinases by a camelid single-domain antibody

African trypanosomes are the causative agents of neglected tropical diseases affecting both humans and livestock. Disease control is highly challenging due to an increasing number of drug treatment failures. African trypanosomes are extracellular, blood-borne parasites that mainly rely on glycolysis for their energy metabolism within the mammalian host. Trypanosomal glycolytic enzymes are therefore of interest for the development of trypanocidal drugs. Here, we report the serendipitous discovery of a camelid single-domain antibody (sdAb aka Nanobody) that selectively inhibits the enzymatic activity of trypanosomatid (but not host) pyruvate kinases through an allosteric mechanism. By combining enzyme kinetics, biophysics, structural biology, and transgenic parasite survival assays, we provide a proof-of-principle that the sdAb-mediated enzyme inhibition negatively impacts parasite fitness and growth.

biochemistry↗

bioRxiv2023

Long-term hematopoietic stem cells trigger quiescence in Leishmania parasites

Quiescence and posttreatment relapse constitute an important therapeutic constraint across the microbiological spectrum. This study unveils that Leishmania infantum and L. donovani parasites rapidly enter quiescence after an estimated 4-6 divisions in both mouse and human stem cells of the bone marrow but not in macrophages as primary host cells. Quiescent amastigotes display a reduced size and evidence for a rapid evolutionary adaptation response with genetic alterations. We formally demonstrate that acquisition of a quiescent phenotype endows parasites with a capacity to survive antileishmanial treatment. Transition through quiescence also results in an increased cellular infectivity and high transmission capacity through the sand fly vector. Transcriptional profiling of quiescent and non-quiescent metabolic states identified a limited set of 26 upregulated genes that are of particular interest given their predicted involvement as regulators of cell cycle progression and of gene expression at various levels. The differential gene set constitutes a reliable source for the identification of novel markers and potential drivers of quiescence, a metabolic state bestowing parasites the capacity to escape drug treatment.

microbiology↗

bioRxiv2021

Miltefosine enhances infectivity of miltefosine-resistant Leishmania infantum by attenuating innate immune recognition

Miltefosine (MIL) is currently the only oral drug available to treat visceral leishmaniasis but its use as first-line monotherapy has been compromised by an increasing treatment failure. Despite the scarce number of resistant clinical isolates, MIL-resistance by mutations in a single aminophospholipid transporter gene can easily be selected in a laboratory environment. These mutations result in a reduced survival in the mammalian host, which can partially be restored by exposure to MIL, suggesting a kind of drug-dependency. To enable a combined study of the infection dynamics and underlying immunological events for differential in vivo survival, firefly luciferase (PpyRE9) / red fluorescent protein (DsRed) double-reporter strains were generated of MIL-resistant (MIL-R) and syngeneic MIL-sensitive (MIL-S) Leishmania infantum. Results in C57Bl/6 and BALB/c mice show that MIL-R parasites induce an increased innate immune response that is characterized by enhanced influx and infection of neutrophils, monocytes and dendritic cells in the liver and elevated serum IFN-{gamma} levels, finally resulting in a less efficient establishment in liver macrophages. The elevated IFN-{gamma} levels were shown to originate from an increased response of hepatic NK and NKT cells to the MIL-R parasites. In addition, we demonstrated that MIL could increase the in vivo fitness of MIL-R parasites by lowering NK and NKT cell activation, leading to a reduced IFN-{gamma} production. These data provide an immunological basis for the MIL-R-associated attenuated phenotype and for the peculiar drug-dependency that may constitute one of the mechanisms of treatment failure. ImportanceRecently, our laboratory demonstrated an in vivo fitness loss of experimentally selected MIL-R parasites in both the sand fly vector and vertebrate host. These findings could explain the scarce number of MIL-R clinical isolates. Surprisingly, MIL-R parasites developed a MIL-dependency which could partially rescue their fitness loss and which may constitute a mechanism of treatment failure. This research aimed to better understand the immunological basis of the attenuated phenotype and the effect of MIL on infectivity traits. Together, this study provides new insights into the complex interplay between the parasite, drug and host and discloses an immune-related mechanism of treatment failure.

microbiology↗

bioRxiv2020

Tsetse salivary glycoproteins are modified with paucimannosidic N-glycans, are recognised by C-type lectins and bind to trypanosomes

African sleeping sickness is caused by Trypanosoma brucei, a parasite transmitted by the bite of a tsetse fly. Trypanosome infection induces a severe transcriptional downregulation of tsetse genes encoding for salivary proteins, which reduces its anti-hemostatic and anti-clotting properties. To better understand trypanosome transmission and the possible role of glycans in insect bloodfeeding, we characterized the N-glycome of tsetse saliva glycoproteins. Tsetse salivary N-glycans were enzymatically released, tagged with either 2-aminobenzamide (2-AB) or procainamide, and analyzed by HILIC-UHPLC-FLR coupled online with positive-ion ESI-LC-MS/MS. We found that the N-glycan profiles of T. brucei-infected and naive tsetse salivary glycoproteins are almost identical, consisting mainly (>50%) of highly processed Man3GlcNAc2 in addition to several other paucimannose, high mannose, and few hybrid-type glycans. In overlay assays, these sugars were differentially recognized by the C-type lectins mannose receptor and DC-SIGN. We also show that salivary glycoproteins bind strongly to the surface of transmissible metacyclic trypanosomes. We suggest that although the repertoire of tsetse salivary N-glycans does not change during a trypanosome infection, the interactions with mannosylated glycoproteins may influence parasite transmission into the vertebrate host.

biochemistry↗

bioRxiv2020

Evaluation of a pan-Leishmania SL-RNA qPCR assay for parasite detection in laboratory-reared and field-collected sand flies and reservoir hosts.

BackgroundIn eco-epidemiological studies, Leishmania detection in vectors and reservoirs is frequently accomplished by high-throughput and sensitive molecular methods that target minicircle kinetoplast DNA (kDNA). A pan-Leishmania SYBR Green quantitative PCR (qPCR) assay which specifically detects the conserved spliced-leader RNA (SL-RNA) sequence has recently been developed. This study comparatively assessed the SL-RNA assay performance for the detection of Leishmania in field and laboratory infected sand flies and in tissue samples from hyraxes as reservoir hosts. Principal findingsThe qPCRs targeting SL-RNA and kDNA performed equally well on infected sand fly samples, despite preservation and extraction under presumed unfavorable conditions for downstream RNA detection. Nucleic acid extraction by a crude extraction buffer combined with a precipitation step was highly compatible with downstream SL-RNA and kDNA detection. Copy numbers of kDNA were found to be identical in culture-derived parasites and promastigotes isolated from sand fly midguts. SL-RNA levels were approximately 3-fold lower in sand fly promastigotes ({Delta}Ct 1.7). The theoretical limit of detection and quantification of the SL-RNA qPCR respectively reached down to 10-3 and 10 parasite equivalents. SL-RNA detection in stored hyrax samples was less efficient with some false negative assay results, most likely due to the long-term tissue storage in absence of RNA stabilizing reagents. ConclusionThis study shows that a crude extraction method in combination with the SL-RNA qPCR assay is suitable for the detection and quantification of Leishmania in sand flies. The assay provides complementary information to the standard kDNA assays, since it is pan-Leishmania specific and detects viable parasites, a prerequisite for identification of vectors and reservoirs. Author summaryIn order to identify vectors and reservoirs of Leishmania, a large number of sand fly and animal tissue samples needs to be screened, because the infection prevalence is generally low. Hence, sensitive low-cost methods are required for nucleic acid isolation and Leishmania detection. Most approaches amplify DNA targets, in particular minicircle kinetoplast DNA (kDNA). Recently, a qPCR was developed that detects the spliced-leader RNA (SL-RNA) sequence, which is conserved among various Leishmania species and allows detection of viable parasites. We show that the SL-RNA qPCR is highly compatible with a low-cost, crude extraction approach and performs equally well on laboratory and field infected sand fly samples as kDNA qPCR assays. The assay can detect 10-3 parasite equivalent in sand flies and enables Leishmania quantification down to 10 parasites. We found that the copy number of SL-RNA is 3-fold lower in sand fly derived promastigotes compared to cultured promastigotes. SL-RNA detection in hyrax tissue samples appeared less efficient, which is presumably due to long-term storage without RNA stabilizing reagents. Overall, our assay is complementary to kDNA assays as it can identify viable Leishmania stages, which provides pivotal information for identification of reservoirs and vectors and their transmission capacity.

microbiology↗

bioRxiv2019

Feeding behavior and activity of Phlebotomus pedifer and potential reservoir hosts of Leishmania aethiopica in southwestern Ethiopia.

BackgroundCutaneous leishmaniasis (CL) is a major public health concern in Ethiopia. However, knowledge about the complex zoonotic transmission cycle is limited, hampering implementation of control strategies. We explored the feeding behavior and activity of the vector (Phlebotomus pedifer) and established the role of livestock in CL transmission. MethodsBlood meal origins of engorged sand flies were determined by sequencing host DNA. A host choice experiment was performed to assess the feeding preference of P. pedifer when humans and hyraxes are equally accessible. Ear and nose biopsies from goats and bovines were screened for the presence of Leishmania parasites. Sand flies were captured indoor and outdoor with human landing catches (HLC) and CDC light traps to determine at which time and where P. pedifer is mostly active. Principal findingsA total of 180 sand flies were found to bite hosts of 12 genera. Humans were the predominant blood meal source (59.4%, p < 0.001) in all habitats, even in caves, where hyraxes are abundant. Only 10.6% of the sand flies fed on hyraxes based on the blood meal analysis, but the host choice experiment revealed that sand flies have a significant preference for feeding on hyraxes over humans (p = 0.009) when hosts are equally accessible. Only a single goat nose biopsy from 412 animal samples was found with Leishmania RNA. We found that P. pedifer is predominantly endophagic (p = 0.003), but occurs both indoors and outdoors. A substantial amount of sand flies was active in the early evening, which increased over time reaching its maximum around midnight. ConclusionWe indicate that Leishmania transmission in southwestern Ethiopia is, in contrast to earlier suggestions, likely mainly anthroponotic and that livestock does not play a role in transmission. Combined indoor and outdoor vector control measures at night are required for efficient vector control. Author summaryCutaneous leishmaniasis is a major public health problem in Ethiopia. It is caused by Leishmania aethiopica protozoa that are transmitted when female sand flies take a blood meal. Hyraxes are assigned as the reservoirs of the infection, because many were found infected with Leishmania. There is very limited knowledge about the behavior of sand flies and other potential hosts of the infection. However, this information is a prerequisite for disease control, which is currently hampered. In this study, we found that humans are likely the main source of the infection and that the role of hyraxes in disease transmission needs further investigation to decide whether they should be included in control programs. Livestock appears not play a role in transmission, even though sand flies like to feed on them. We also show that sand flies are active indoors and outdoors, but have a preference for feeding inside human dwellings and that they are mostly active around midnight. Overall, we conclude that disease prevention and control should emphasize on human protection by applying vector control indoors, at night.

ecology↗

bioRxiv2019

The Glossina Genome Cluster: Comparative Genomic Analysis of the Vectors of African Trypanosomes

BackgroundTsetse flies (Glossina sp.) are the sole vectors of human and animal trypanosomiasis throughout sub-Saharan Africa. Tsetse are distinguished from other Diptera by unique adaptations, including lactation and the birthing of live young (obligate viviparity), a vertebrate blood specific diet by both sexes and obligate bacterial symbiosis. This work describes comparative analysis of six Glossina genomes representing three sub-genera: Morsitans (G. morsitans morsitans (G.m. morsitans), G. pallidipes, G. austeni), Palpalis (G. palpalis, G. fuscipes) and Fusca (G. brevipalpis) which represent different habitats, host preferences and vectorial capacity. ResultsGenomic analyses validate established evolutionary relationships and sub-genera. Syntenic analysis of Glossina relative to Drosophila melanogaster shows reduced structural conservation across the sex-linked X chromosome. Sex linked scaffolds show increased rates of female specific gene expression and lower evolutionary rates relative to autosome associated genes. Tsetse specific genes are enriched in protease, odorant binding and helicase activities. Lactation associated genes are conserved across all Glossina species while male seminal proteins are rapidly evolving. Olfactory and gustatory genes are reduced across the genus relative to other characterized insects. Vision associated Rhodopsin genes show conservation of motion detection/tracking functions and significant variance in the Rhodopsin detecting colors in the blue wavelength ranges. ConclusionsExpanded genomic discoveries reveal the genetics underlying Glossina biology and provide a rich body of knowledge for basic science and disease control. They also provide insight into the evolutionary biology underlying novel adaptations and are relevant to applied aspects of vector control such as trap design and discovery of novel pest and disease control strategies.

genomics↗