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Addressing the challenges of quiescence and post-treatment relapse is of utmost importance in the microbiology field. This study shows that Leishmania infantum and L. donovani parasites rapidly enter into quiescence after an estimated 2–3 divisions in both human and mouse bone marrow stem cells. Interestingly, this behavior is not observed in macrophages, which are the primary host cells of the Leishmania parasite. Transcriptional comparison of the quiescent and non-quiescent metabolic states confirmed the overall decrease of gene expression as a hallmark of quiescence. Quiescent amastigotes display a reduced size and signs of a rapid evolutionary adaptation response with genetic alterations. Our study provides further evidence that this quiescent state significantly enhances resistance to treatment. Moreover, transitioning through quiescence is highly compatible with sand fly transmission and increases the potential of parasites to infect cells. Collectively, this work identified stem cells in the bone marrow as a niche where Leishmania quiescence occurs, with important implications for antiparasitic treatment and acquisition of virulence traits. Author summary: Quiescence and post-treatment relapse are crucial aspects of treatment failure across the microbiology field. This study shows that Leishmania infantum and L. donovani parasites rapidly enter into quiescence in both human and mouse bone marrow stem cells, but not in macrophages. Besides a reduced size, quiescent amastigotes show signs of a rapid evolutionary adaptation with notable genetic alterations. Transitioning through a quiescent state allows escape from treatment, efficient transmission by sand flies and the acquisition of an increased cellular infectivity. Transcriptional profiling of quiescent and non-quiescent parasites isolated from the stem cell niche confirmed a generalized transcriptional downregulation as a hallmark of quiescence. [ABSTRACT FROM AUTHOR]

It is well-known that Leishmania parasites can alter the behavior of the sand fly vector in order to increase their transmission potential. However, little is known about the contribution of the infecting host's blood composition on subsequent sand fly infection and survival. This study focused on the host's glucose metabolism and the insulin/insulin-like growth factor 1 (IGF-1) pathway as both metabolic processes are known to impact vector-parasite interactions of other protozoa and insect species. The focus of this study was inspired by the observation that the glycemic levels in the blood of infected Syrian golden hamsters inversely correlated to splenic and hepatic parasite burdens. To evaluate the biological impact of these findings on further transmission, Lutzomyia longipalpis sand flies were infected with blood that was artificially supplemented with different physiological concentrations of several monosaccharides, insulin or IGF-1. Normoglycemic levels resulted in transiently higher parasite loads and faster appearance of metacyclics, whereas higher carbohydrate and insulin/IGF-1 levels favored sand fly survival. Although the recorded effects were modest or transient of nature, these observations support the concept that the host blood biochemistry may affect Leishmania transmission and sand fly longevity. Author summary: Past research on the interaction between the Leishmania parasite and the sand fly vector has revealed that Leishmania is capable of changing vector behavior to favor transmission of parasites in the environment. Little is known about the impact of host blood composition on parasite development inside the vector and on vector survival. Here, we showed that parasite burdens in the spleen and the liver inversely correlated to the serum blood glucose levels of infected animals, which triggered us to further investigate the effect of blood monosaccharides, insulin and insulin-like growth factor 1 (IGF-1) on sand fly infection and survival. We demonstrated that normal serum glucose levels in the initial parasitized blood meal resulted in transiently higher parasite loads and a faster appearance of infectious parasites, whereas higher sugar and insulin/IGF-1 levels favored sand fly survival, which supports the concept that the host blood biochemistry may affect Leishmania transmission and sand fly longevity. [ABSTRACT FROM AUTHOR]

Sodalis glossinidius, a secondary bacterial symbiont of the tsetse fly, is currently considered as a potential delivery system for anti-trypanosomal components interfering with African trypanosome transmission (i.e. paratransgenesis). Nanobodies (Nbs) have been proposed as potential candidates to target the parasite during development in the tsetse fly. In this study, we have generated an immune Nb-library and developed a panning strategy to select Nbs against the Trypanosoma brucei brucei procyclic developmental stage present in the tsetse fly midgut. Selected Nbs were expressed, purified, assessed for binding and tested for their impact on the survival and growth of in vitro cultured procyclic T. b. brucei parasites. Next, we engineered S. glossinidius to express the selected Nbs and validated their ability to block T. brucei development in the tsetse fly midgut. Genetically engineered S. glossinidius expressing Nb_88 significantly compromised parasite development in the tsetse fly midgut both at the level of infection rate and parasite load. Interestingly, expression of Nb_19 by S. glossinidius resulted in a significantly enhanced midgut establishment. These data are the first to show in situ delivery by S. glossinidius of effector molecules that can target the trypanosome-tsetse fly crosstalk, interfering with parasite development in the fly. These proof-of-principle data represent a major step forward in the development of a control strategy based on paratransgenic tsetse flies. Finally, S. glossinidius-based Nb delivery can also be applied as a powerful laboratory tool to unravel the molecular determinants of the parasite-vector association. Author summary: Tsetse flies are the main vectors of African trypanosomiasis, a group of diseases caused by the protozoan Trypanosoma parasites which poses a severe burden on human/animal health and agricultural development in sub-Saharan Africa. The lack of prophylactic drugs and limitations of the existing control programs urge the need for developing alternative strategies to complement the existing control programs. Paratransgenesis, the genetic manipulation of insect symbiotic microorganisms to block pathogen transmission, is a promising strategy for controlling vector-borne diseases. In this experimental study we successfully genetically modified the tsetse fly gut symbiont S. glossinidius to express trypanosome-interfering proteins (i.e. Nanobodies) thereby impairing trypanosome development in the fly. The application of the concept of using pathogen-targeting Nbs delivered by insect symbiotic bacteria could be extended to other vector-pathogen systems. Furthermore, our symbiont-based Nb delivery system can also be applied as a powerful laboratory tool to unravel the molecular determinants of the vector-pathogen association. [ABSTRACT FROM AUTHOR]

Background: 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. Methodology/Principal findings: 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-γ levels, finally resulting in a less efficient establishment in liver macrophages. The elevated IFN-γ 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-γ production. Conclusions/Significance: Differential induction of innate immune responses in the liver was found to underlie the attenuated phenotype of a MIL-R parasite and its peculiar feature of drug-dependency. The impact of MIL on hepatic NK and NKT activation and IFN-γ production following recognition of a MIL-R strain indicates that this mechanism may sustain infections with resistant parasites and contribute to treatment failure. Author summary: Visceral leishmaniasis is a neglected tropical disease that is fatal if left untreated. Miltefosine is currently the only oral drug available but is increasingly failing to cure patients, resulting in its discontinuation as first-line drug in some endemic areas. To understand these treatment failures, we investigated the complex interplay of the parasite with the host immune system in the presence and absence of miltefosine. Our data indicate that miltefosine-resistant Leishmania parasites become severely hampered in their in vivo infectivity, which could be attributed to the induction of a pronounced innate immune response. Interestingly, the infection deficit was partially restored in the presence of miltefosine. Our results further indicate that miltefosine can exacerbate infections with resistant parasites by reducing innate immune recognition. This study provides new insights into the complex interplay between parasite, drug and host and discloses an immune-related mechanism of treatment failure. [ABSTRACT FROM AUTHOR]

Leishmaniasis is a major infectious disease with hundreds of thousands of new cases and over 20,000 deaths each year. The current drugs to treat this life-threatening infection have several drawbacks such as toxicity and long treatment regimens. A library of 1.8 million compounds, from which the hits reported here are publicly available, was screened against Leishmania infantum as part of an optimization program; a compound was found with a 2-aminobenzimidazole functionality presenting moderate potency, low metabolic stability and high lipophilicity. Several rounds of synthesis were performed to incorporate chemical groups capable of reducing lipophilicity and clearance, leading to the identification of compounds that are active against different parasite strains and have improved in vitro properties. As a result of this optimization program, a group of compounds was further tested in anticipation of in vivo evaluation. In vivo tests were carried out with compounds 29 (L. infantum IC50: 4.1 μM) and 39 (L. infantum IC50: 0.5 μM) in an acute L. infantum VL mouse model, which showed problems of poor exposure and lack of efficacy, despite the good in vitro potency. Author summary: Leishmaniasis is a neglected tropical disease affecting millions of people worldwide and, in the case of visceral leishmaniasis (VL), is potentially fatal if untreated. Protozoan parasites of the genus Leishmania spp. are the causative agents of leishmaniasis, which has different clinical manifestations, including the visceral form and a cutaneous form that causes disfiguring skin lesions. The current treatment options are limited either by the length of treatment or toxic side effects. Starting from a promising hit in an in vitro phenotypic screen, hundreds of analogues were synthesized with the aim of finding a molecule capable of killing the parasite whilst causing little or no harm to the patient. The program led to several active compounds with good in vitro activity against L. infantum intracellular amastigotes, however, in vivo data showed low parasiticidal efficacy. [ABSTRACT FROM AUTHOR]

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 naïve 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 N-glycans. In overlay assays, these sugars were differentially recognized by the mannose receptor and DC-SIGN C-type lectins. 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. Author summary: In addition to helping the ingestion of a bloodmeal, the saliva of vector insects can modulate vertebrate immune responses. However, most research has focused on the salivary proteins, while the sugars (glycans) that modify them remain unexplored. Here we studied N-glycosylation, a common post-translational modification where sugar structures are attached to specific sites of a protein. Insect salivary N-glycans may affect how the saliva is recognized by the host, possibly playing a role during pathogen transmission. In this manuscript, we present the first detailed structural characterization of the salivary N-glycans in the tsetse fly Glossina morsitans, vector of African trypanosomiasis. We found that tsetse fly glycoproteins are mainly modified by simple N-glycans with short mannose modifications, which are recognised by mammalian C-type lectins (mannose receptor and DC-SIGN). Furthermore, we show that salivary glycoproteins bind to the surface of the trypanosomes that are transmitted to the vertebrate host; this opens up interesting questions as to the role of these glycoproteins in the successful establishment of infection by this parasite. Overall, our work represents a novel contribution towards the salivary N-glycome of an important insect vector, and towards the understanding of vector saliva and its complex effects in the vertebrate host. [ABSTRACT FROM AUTHOR]

In most low-resource settings, microscopy still is the standard method for diagnosis of cutaneous leishmaniasis, despite its limited sensitivity. In Ethiopia, the more sensitive molecular methods are not yet routinely used. This study compared five PCR methods with microscopy on two sample types collected from patients with a suspected lesion to advise on optimal diagnosis of Leishmania aethiopica. Between May and July 2018, skin scrapings (SS) and blood exudate from the lesion spotted on filter paper (dry blood spot, DBS) were collected for PCR from 111 patients of four zones in Southern Ethiopia. DNA and RNA were simultaneously extracted from both sample types. DNA was evaluated by a conventional PCR targeting ITS-1 and three probe-based real-time PCRs: one targeting the SSU 18S rRNA and two targeting the kDNA minicircle sequence (the 'Mary kDNA PCR' and a newly designed 'LC kDNA PCR' for improved L. aethiopica detection). RNAs were tested with a SYBR Green-based RT-PCR targeting spliced leader (SL) RNA. Giemsa-stained SS smears were examined by microscopy. Of the 111 SS, 100 were positive with at least two methods. Sensitivity of microscopy, ITS PCR, SSU PCR, Mary kDNA PCR, LC kDNA PCR and SL RNA PCR were respectively 52%, 22%, 64%, 99%, 100% and 94%. Microscopy-based parasite load correlated well with real-time PCR Ct-values. Despite suboptimal sample storage for RNA detection, the SL RNA PCR resulted in congruent results with low Ct-values. DBS collected from the same lesion showed lower PCR positivity rates compared to SS. The kDNA PCRs showed excellent performance for diagnosis of L. aethiopica on SS. Lower-cost SL RNA detection can be a complementary high-throughput tool. DBS can be used for PCR in case microscopy is negative, the SS sample can be sent to the referral health facility where kDNA PCR method is available. Author summary: Cutaneous leishmaniasis is a neglected tropical disease and causing a public health problem in Ethiopia. Microscopy is still the standard method for detection of the parasite in Ethiopia, and also in many other low resource settings. A more sensitive method is needed for timely diagnosis and treatment. In this study, we compared five molecular methods on samples collected from patients with a skin lesion suspected of cutaneous leishmaniasis to advice on optimal diagnosis of L. aethiopica. We collected two sample types from the same lesion (skin scrapings and lesion fluid on filter paper) and isolated both DNA and RNA of them. Majority (90.1%) of the samples from skin scrapings were positive in two or more methods and the molecular methods had a higher sensitivity than the conventional methods. Interestingly, we evaluated for the first time a new molecular method designed to improve L. aethiopica detection. Also, we showed that RNA detection performed well for samples that were collected under difficult field conditions. Samples collected on filter paper showed less positive results than skin scraped samples, but could still be the method of choice for easy sampling and transport in resource-limited settings as it performed better than microscopy. [ABSTRACT FROM AUTHOR]

Background: Cutaneous 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 studied the role of livestock in CL transmission in southwestern Ethiopia. Methods: Blood 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 livestock were screened for the presence of Leishmania parasites. Sand flies were captured indoor and outdoor with human landing catches and CDC light traps to determine at which time and where P. pedifer is mostly active. Principal findings: A total of 180 P. pedifer sand flies were found to bite hosts of 12 genera. Humans were the predominant blood meal source indoors (65.9%, p < 0.001), while no significant differences were determined outdoors and in caves. In caves, hyraxes were represented in blood meals equally as humans (45.5% and 42.4%, respectively), but the host choice experiment revealed that sand flies have a significant preference for feeding on hyraxes (p = 0.009). 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 number of sand flies was active in the early evening, which increased over time reaching its maximum around midnight. Conclusion: In contrast to earlier suggestions of exclusive zoonotic Leishmania transmission, we propose that there is also human-to-human transmission of CL in southwestern Ethiopia. Livestock does not play a role in CL transmission and combined indoor and outdoor vector control measures at night are required for efficient vector control. Author summary: Cutaneous leishmaniasis is a major public health problem in Ethiopia. It is mainly 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 reservoir 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 an important source of 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. [ABSTRACT FROM AUTHOR]

Monitoring the drug susceptibility of Leishmania isolates still largely relies on standard in vitro cell-based susceptibility assays using (patient-isolated) promastigotes for infection. Although this assay is widely used, no fully standardized/harmonized protocol is yet available hence resulting in the application of a wide variety of host cells (primary cells and cell lines), different drug exposure times, detection methods and endpoint criteria. Advocacy for standardization to decrease inter-laboratory variation and improve interpretation of results has already repeatedly been made, unfortunately still with unsatisfactory progress. As a logical next step, it would be useful to reach at least some agreement on the type of host cell and basic experimental design for routine amastigote susceptibility determination. The present laboratory study using different L. infantum strains as a model for visceral leishmaniasis species compared primary cells (mouse peritoneal exudate (PEC), mouse bone marrow derived macrophages and human peripheral blood monocyte derived macrophages) and commercially available cell lines (THP-1, J774, RAW) for either their susceptibility to infection, their role in supporting intracellular amastigote multiplication and overall feasibility/accessibility of experimental assay protocol. The major findings were that primary cells are better than cell lines in supporting infection and intracellular parasite multiplication, with PECs to be preferred for technical reasons. Cell lines require drug exposure of >96h with THP-1 to be preferred but subject to a variable response to PMA stimulation. The fast dividing J774 and RAW cells out-compete parasite-infected cells precluding proper assay read-out. Some findings could possibly also be applicable to cutaneous Leishmania strains, but this still needs cross-checking. Besides inherent limitations in a clinical setting, susceptibility testing of clinical isolates may remain problematic because of the reliance on patient-derived promastigotes which may exhibit variable degrees of metacyclogenesis and infectivity. Author summary: Leishmaniasis is a neglected tropical disease caused by parasites belonging to the genus of Leishmania and transmitted by the bite of infected female sand flies. Concerns about the effective control of the disease are rising in view of the increasing number of treatment failures that may be related to drug resistance. Monitoring of drug susceptibility in the field should become an essential asset, however, there is still insufficient harmonization in the laboratory assays. This study focused on the standard intracellular amastigote susceptibility assay and compared protocol variables, such as type of macrophage host cell (primary versus cell lines), multiplicity of infection and duration of drug exposure. Primary cells perform best with little difference between cells derived from Swiss mice or BALB/c mice. From a practical point of view, mouse peritoneal exudate cells can be recommended. If mice would not be available, THP-1 cells are the best alternative. For field strains, metacyclic promastigotes should be used at a multiplicity of infection of 10–15 parasites per cell with drug exposure starting at 24h post-infection and continued for 120h. Unfortunately, susceptibility testing of clinical isolates will remain problematic because of the reliance on promastigotes which may exhibit variable degrees of metacyclogenesis and infectivity. Opting for cell-based assays may be complicated by the fact that dedicated laboratory infrastructure may sometimes be lacking in disease-endemic countries. [ABSTRACT FROM AUTHOR]

It was recently hypothesized that Leishmania amastigotes could constitute a semi-quiescent stage characterized by low replication and reduced metabolic activity. This concept developed with Leishmania (Leishmania) mexicana and Leishmania (Leishmania) major models might explain numerous clinical and sub-clinical features of Leishmania (Viannia) braziliensis infections, like reactivation of the disease, non-response to chemotherapy or asymptomatic infections. We compared here in vitro the proliferative capability of L. (V.) braziliensis amastigotes and promastigotes, assessed the expression of key molecular parameters and performed metabolomic analysis. We found that contrary to the highly proliferative promastigotes, amastigotes (axenic and intracellular) do not show evidence of extensive proliferation. In parallel, amastigotes showed a significant decrease of (i) the kDNA mini-circle abundance, (ii) the intracellular ATP level, (iii) the ribosomal components: rRNA subunits 18S and 28S α and ribosomal proteins RPS15 and RPL19, (iv) total RNA and protein levels. An untargeted metabolomic study identified clear differences between the different life stages: in comparison to logarithmic promastigotes, axenic amastigotes showed (a) a strong decrease of 14 essential and non-essential amino acids and eight metabolites involved in polyamine synthesis, (b) extensive changes in the phospholipids composition and (c) increased levels of several endogenous and exogenous sterols. Altogether, our results show that L. (V.) braziliensis amastigotes can show a phenotype with negligible rate of proliferation, a lower capacity of biosynthesis, a reduced bio-energetic level and a strongly altered metabolism. Our results pave the way for further exploration of quiescence among amastigotes of this species. [ABSTRACT FROM AUTHOR]

Background: Since miltefosine monotherapy against visceral leishmaniasis (VL) caused by Leishmania donovani has been discontinued in the Indian subcontinent due to an increase in the number of treatment failures, single dose liposomal amphotericin B is now advocated as a treatment option of choice. Paromomycin-miltefosine combination therapy can be used as substitute first-line treatment in regions without cold-chain potential. Previous laboratory studies in the closely related species Leishmania infantum have demonstrated that paromomycin monotherapy fairly rapidly selects for resistance producing a phenotype with increased fitness. Given the possible clinical implications of these findings for the current field situation, the present study aimed to identify the potential hazards of paromomycin-miltefosine combination therapy. Principal findings: Drug interaction studies using the fixed-ratio isobologram method revealed an indifferent interaction between paromomycin and miltefosine. In hamsters infected with L. infantum, the combination resulted in cumulative efficacy in reducing parasite burdens in the liver, spleen and bone marrow. Selected resistant lines against the single drugs did not display cross-resistance. When the intracellular amastigote stage was repeatedly exposed to the paromomycin-miltefosine combination, either in vitro or in vivo, no significant susceptibility decrease towards either drug was noted. Conclusions: These results suggest that implementation of paromomycin-miltefosine combination therapy indeed could represent a safe and affordable treatment option for L. donovani VL as miltefosine appears to overrule the anticipated rapid development of PMM resistance. [ABSTRACT FROM AUTHOR]

Host-directed therapies (HDTs) constitute promising alternatives to traditional therapy that directly targets the pathogen but is often hampered by pathogen resistance. HDT could represent a new treatment strategy for leishmaniasis, a neglected tropical disease caused by the obligate intracellular parasite Leishmania. This protozoan develops exclusively within phagocytic cells, where infection relies on a complex molecular interplay potentially exploitable for drug targets. We previously identified naloxonazine, a compound specifically active against intracellular but not axenic Leishmania donovani. We evaluated here whether this compound could present a host cell-dependent mechanism of action. Microarray profiling of THP-1 macrophages treated with naloxonazine showed upregulation of vATPases, which was further linked to an increased volume of intracellular acidic vacuoles. Treatment of Leishmania-infected macrophages with the vATPase inhibitor concanamycin A abolished naloxonazine effects, functionally demonstrating that naloxonazine affects Leishmania amastigotes indirectly, through host cell vacuolar remodeling. These results validate amastigote-specific screening approaches as a powerful way to identify alternative host-encoded targets. Although the therapeutic value of naloxonazine itself is unproven, our results further demonstrate the importance of intracellular acidic compartments for host defense against Leishmania, highlighting the possibility of targeting this host cell compartment for anti-leishmanial therapy. [ABSTRACT FROM AUTHOR]

Animal African trypanosomosis is a major threat to the economic development and human health in sub-Saharan Africa. Trypanosoma congolense infections represent the major constraint in livestock production, with anemia as the major pathogenic lethal feature. The mechanisms underlying anemia development are ill defined, which hampers the development of an effective therapy. Here, the contribution of the erythropoietic and erythrophagocytic potential as well as of hemodilution to the development of T. congolense-induced anemia were addressed in a mouse model of low virulence relevant for bovine trypanosomosis. We show that in infected mice, splenic extramedullary erythropoiesis could compensate for the chronic low-grade type I inflammation-induced phagocytosis of senescent red blood cells (RBCs) in spleen and liver myeloid cells, as well as for the impaired maturation of RBCs occurring in the bone marrow and spleen. Rather, anemia resulted from hemodilution. Our data also suggest that the heme catabolism subsequent to sustained erythrophagocytosis resulted in iron accumulation in tissue and hyperbilirubinemia. Moreover, hypoalbuminemia, potentially resulting from hemodilution and liver injury in infected mice, impaired the elimination of toxic circulating molecules like bilirubin. Hemodilutional thrombocytopenia also coincided with impaired coagulation. Combined, these effects could elicit multiple organ failure and uncontrolled bleeding thus reduce the survival of infected mice. MIF (macrophage migrating inhibitory factor), a potential pathogenic molecule in African trypanosomosis, was found herein to promote erythrophagocytosis, to block extramedullary erythropoiesis and RBC maturation, and to trigger hemodilution. Hence, these data prompt considering MIF as a potential target for treatment of natural bovine trypanosomosis. [ABSTRACT FROM AUTHOR]

Tsetse flies are the sole vectors of Trypanosoma brucei parasites that cause sleeping sickness. Our knowledge on the early interface between the infective metacyclic forms and the mammalian host skin is currently highly limited. Glossina morsitans flies infected with fluorescently tagged T. brucei parasites were used in this study to initiate natural infections in mice. Metacyclic trypanosomes were found to be highly infectious through the intradermal route in sharp contrast with blood stream form trypanosomes. Parasite emigration from the dermal inoculation site resulted in detectable parasite levels in the draining lymph nodes within 18 hours and in the peripheral blood within 42 h. A subset of parasites remained and actively proliferated in the dermis. By initiating mixed infections with differentially labeled parasites, dermal parasites were unequivocally shown to arise from the initial inoculum and not from a re-invasion from the blood circulation. Scanning electron microscopy demonstrated intricate interactions of these skin-residing parasites with adipocytes in the connective tissue, entanglement by reticular fibers of the periadipocytic baskets and embedment between collagen bundles. Experimental transmission experiments combined with molecular parasite detection in blood fed flies provided evidence that dermal trypanosomes can be acquired from the inoculation site immediately after the initial transmission. High resolution thermographic imaging also revealed that intradermal parasite expansion induces elevated skin surface temperatures. Collectively, the dermis represents a delivery site of the highly infective metacyclic trypanosomes from which the host is systemically colonized and where a proliferative subpopulation remains that is physically constrained by intricate interactions with adipocytes and collagen fibrous structures. [ABSTRACT FROM AUTHOR]

During the last decade miltefosine (MIL) has been used as first-line treatment for visceral leishmaniasis in endemic areas with antimonial resistance, but a decline in clinical effectiveness is now being reported. While only two MIL-resistant Leishmania infantum strains from HIV co-infected patients have been documented, phenotypic MIL-resistance for L. donovani has not yet been identified in the laboratory. Hence, a better understanding of the factors contributing to increased MIL-treatment failure is necessary. Given the paucity of defined MIL-resistant L. donovani clinical isolates, this study used an experimental amastigote-selected MIL-resistant L. infantum isolate (LEM3323). In-depth exploration of the MIL-resistant phenotype was performed by coupling genomic with phenotypic data to gain insight into gene function and the mutant phenotype. A naturally MIL-resistant L. infantum clinical isolate (LEM5159) was included to compare both datasets. Phenotypically, resistance was evaluated by determining intracellular amastigote susceptibility in vitro and actual MIL-uptake. Genomic analysis provided supportive evidence that the resistance selection model on intracellular amastigotes can be a good proxy for the in vivo field situation since both resistant strains showed mutations in the same inward transporter system responsible for the acquired MIL-resistant phenotype. In line with previous literature findings in promastigotes, our data confirm a defective import machinery through inactivation of the LiMT/LiRos3 protein complex as the main mechanism for MIL-resistance also in intracellular amastigotes. Whole genome sequencing analysis of LEM3323 revealed a 2 base pair deletion in the LiMT gene that led to the formation an early stop codon and a truncation of the LiMT protein. Interestingly, LEM5159 revealed mutations in both the LiMT and LiRos3 genes, resulting in an aberrant expression of the LiMT protein. To verify that these mutations were indeed accountable for the acquired resistance, transfection experiments were performed to re-establish MIL-susceptibility. In LEM3323, susceptibility was restored upon expression of a LiMT wild-type gene, whereas the MIL-susceptibility of LEM5159 could be reversed after expression of the LiRos3 wild-type gene. The aberrant expression profile of the LiMT protein could be restored upon rescue of the LiRos3 gene both in the LEM5159 clinical isolate and a ΔLiRos3 strain, showing that expression of LdMT is dependent on LdRos3 expression. The present findings clearly corroborate the pivotal role of the LiMT/LiRos3 complex in resistance towards MIL. [ABSTRACT FROM AUTHOR]

Collagen-induced arthritis is a B cell-mediated autoimmune disease. Recently published studies have demonstrated that in some rare cases pathogens can confer protection from autoimmunity. Trypanosoma brucei parasites are tsetse fly transmitted extracellular protozoans causing sleeping sickness disease in humans and Nagana in livestock in sub-Saharan endemic areas. In the past, we demonstrated that trypanosome infections impair B cell homeostasis and abolish vaccine-induced protection against unrelated antigens. Hence, here we hypothesized that trypanosome infection can affect the onset of CIA by specifically dampening specific B-cell responses and type II collagen antibody titers in DBA/1 prone mice. We observed a substantial delay in the onset of collagen-induced arthritis in T. brucei-infected DBA/1 mice that correlates with a drastic decrease of type II collagen titers of the different IgG isotypes in the serum. Treatment of infected mice with Berenil, a trypanocidal drug, restored the development of CIA-associated clinical symptoms. Interestingly, these data were confirmed by the challenge of immunized DBA/1 prone mice with T. brucei-infected tsetse flies. Together, these results demonstrate that T. brucei infection is impairing the maintenance of the antigen specific plasma B cell pool driving the development of CIA in DBA/1 prone mice. [ABSTRACT FROM AUTHOR]

Background: Tsetse flies are the main vectors of human and animal African trypanosomes. The Tsal proteins in tsetse fly saliva were previously identified as suitable biomarkers of bite exposure. A new competitive assay was conceived based on nanobody (Nb) technology to ameliorate the detection of anti-Tsal antibodies in mammalian hosts. Methodology/Principal Findings: A camelid-derived Nb library was generated against the Glossina morsitans morsitans sialome and exploited to select Tsal specific Nbs. One of the three identified Nb families (family III, TsalNb-05 and TsalNb-11) was found suitable for anti-Tsal antibody detection in a competitive ELISA format. The competitive ELISA was able to detect exposure to a broad range of tsetse species (G. morsitans morsitans, G. pallidipes, G. palpalis gambiensis and G. fuscipes) and did not cross-react with the other hematophagous insects (Stomoxys calcitrans and Tabanus yao). Using a collection of plasmas from tsetse-exposed pigs, the new test characteristics were compared with those of the previously described G. m. moristans and rTsal1 indirect ELISAs, revealing equally good specificities (> 95%) and positive predictive values (> 98%) but higher negative predictive values and hence increased sensitivity (> 95%) and accuracy (> 95%). Conclusion/Significance: We have developed a highly accurate Nb-based competitive immunoassay to detect specific anti-Tsal antibodies induced by various tsetse fly species in a range of hosts. We propose that this competitive assay provides a simple serological indicator of tsetse fly presence without the requirement of test adaptation to the vertebrate host species. In addition, the use of monoclonal Nbs for antibody detection is innovative and could be applied to other tsetse fly salivary biomarkers in order to achieve a multi-target immunoprofiling of hosts. In addition, this approach could be broadened to other pathogenic organisms for which accurate serological diagnosis remains a bottleneck. Author Summary: Our previous studies have revealed that the saliva of the savannah tsetse fly (Glossina morsitans morsitans) and the main constituting Tsal proteins are sensitive immunological probes to detect contact with tsetse flies. A nanobody (Nb) library was generated against tsetse salivary gland proteins and used to select Nbs against the highly immunogenic Tsal proteins by a procedure of phage display and selection for binding onto the recombinant Tsal proteins. One Nb family was identified with the appropriate characteristics for the development of a competitive assay to detect Tsal-specific antibodies raised by the mammalian host when exposed to tsetse fly bites. In this immunoassay, exposure was detected by the inhibition of Nb binding by tsetse fly saliva induced antibodies in plasma. Evaluation of the competitive ELISA test using a set of porcine plasmas revealed an improved accuracy as compared to previously described tests. Moreover, the advantage of this assay is that it does not require adaptation to the sampled host species. We propose the Nb-based competitive ELISA as an additional tool to the indirect ELISA to serologically detect tsetse bite exposure and to monitor the impact of vector control programs and to detect re-invasion of cleared areas by tsetse flies on the African continent. In addition, the concept of using Nbs for the development of competitive antibody detection tests is innovative and broadens the scope of medical diagnostic applications of Nbs. [ABSTRACT FROM AUTHOR]

African trypanosomiasis is a chronic debilitating disease affecting the health and economic well-being of many people in developing countries. The pathogenicity associated with this disease involves a persistent inflammatory response, whereby M1-type myeloid cells, including Ly6Chigh inflammatory monocytes, are centrally implicated. A comparative gene analysis between trypanosusceptible and trypanotolerant animals identified MIF (macrophage migrating inhibitory factor) as an important pathogenic candidate molecule. Using MIF-deficient mice and anti-MIF antibody treated mice, we show that MIF mediates the pathogenic inflammatory immune response and increases the recruitment of inflammatory monocytes and neutrophils to contribute to liver injury in Trypanosoma brucei infected mice. Moreover, neutrophil-derived MIF contributed more significantly than monocyte-derived MIF to increased pathogenic liver TNF production and liver injury during trypanosome infection. MIF deficient animals also featured limited anemia, coinciding with increased iron bio-availability, improved erythropoiesis and reduced RBC clearance during the chronic phase of infection. Our data suggest that MIF promotes the most prominent pathological features of experimental trypanosome infections (i.e. anemia and liver injury), and prompt considering MIF as a novel target for treatment of trypanosomiasis-associated immunopathogenicity. [ABSTRACT FROM AUTHOR]

Background: Tsetse flies are obligate blood-feeding insects that transmit African trypanosomes responsible for human sleeping sickness and nagana in livestock. The tsetse salivary proteome contains a highly immunogenic family of the endonuclease-like Tsal proteins. In this study, a recombinant version of Tsal1 (rTsal1) was evaluated in an indirect ELISA to quantify the contact with total Glossina morsitans morsitans saliva, and thus the tsetse fly bite exposure. Methodology/Principal Findings: Mice and pigs were experimentally exposed to different G. m. morsitans exposure regimens, followed by a long-term follow-up of the specific antibody responses against total tsetse fly saliva and rTsal1. In mice, a single tsetse fly bite was sufficient to induce detectable IgG antibody responses with an estimated half-life of 36–40 days. Specific antibody responses could be detected for more than a year after initial exposure, and a single bite was sufficient to boost anti-saliva immunity. Also, plasmas collected from tsetse-exposed pigs displayed increased anti-rTsal1 and anti-saliva IgG levels that correlated with the exposure intensity. A strong correlation between the detection of anti-rTsal1 and anti-saliva responses was recorded. The ELISA test performance and intra-laboratory repeatability was adequate in the two tested animal models. Cross-reactivity of the mouse IgGs induced by exposure to different Glossina species (G. m. morsitans, G. pallidipes, G. palpalis gambiensis and G. fuscipes) and other hematophagous insects (Stomoxys calcitrans and Tabanus yao) was evaluated. Conclusion: This study illustrates the potential use of rTsal1 from G. m. morsitans as a sensitive biomarker of exposure to a broad range of Glossina species. We propose that the detection of anti-rTsal1 IgGs could be a promising serological indicator of tsetse fly presence that will be a valuable tool to monitor the impact of tsetse control efforts on the African continent. Author Summary: Salivary proteins of hematophagous disease vectors represent potential biomarkers of exposure and could be used in serological assays that are complementary to entomological surveys. We illustrate that a recombinant version of the highly immunogenic Tsal1 protein of the savannah tsetse fly (Glossina morsitans morsitans) is a sensitive immunological probe to detect contact with tsetse flies. Experimental exposure of mice and pigs to different regimens of tsetse fly bites combined with serological testing revealed that rTsal1 is a sensitive indicator that can differentiate the various degrees of exposure of animals. Tsetse-induced antibodies persisted relatively long, and an efficient boosting of immunity was observed upon re-exposure. Recombinant Tsal1 is a promising candidate to detect contact with various tsetse species, which would enable screening of populations or herds for exposure to tsetse flies in various areas on the African continent. This exposure indicator could be a valuable tool to monitor the impact of vector control programs and to detect re-invasion of cleared areas by tsetse flies. [ABSTRACT FROM AUTHOR]

Background: In order to promote infection, the blood-borne parasite Trypanosoma brucei releases factors that upregulate arginase expression and activity in myeloid cells. Methodology/Principal findings: By screening a cDNA library of T. brucei with an antibody neutralizing the arginase-inducing activity of parasite released factors, we identified a Kinesin Heavy Chain isoform, termed TbKHC1, as responsible for this effect. Following interaction with mouse myeloid cells, natural or recombinant TbKHC1 triggered SIGN-R1 receptor-dependent induction of IL-10 production, resulting in arginase-1 activation concomitant with reduction of nitric oxide (NO) synthase activity. This TbKHC1 activity was IL-4Rα-independent and did not mirror M2 activation of myeloid cells. As compared to wild-type T. brucei, infection by TbKHC1 KO parasites was characterized by strongly reduced parasitaemia and prolonged host survival time. By treating infected mice with ornithine or with NO synthase inhibitor, we observed that during the first wave of parasitaemia the parasite growth-promoting effect of TbKHC1-mediated arginase activation resulted more from increased polyamine production than from reduction of NO synthesis. In late stage infection, TbKHC1-mediated reduction of NO synthesis appeared to contribute to liver damage linked to shortening of host survival time. Conclusion: A kinesin heavy chain released by T. brucei induces IL-10 and arginase-1 through SIGN-R1 signaling in myeloid cells, which promotes early trypanosome growth and favors parasite settlement in the host. Moreover, in the late stage of infection, the inhibition of NO synthesis by TbKHC1 contributes to liver pathogenicity. [ABSTRACT FROM AUTHOR]

Background: The discovery of Nanobodies (Nbs) with a direct toxic activity against African trypanosomes is a recent advancement towards a new strategy against these extracellular parasites. The anti-trypanosomal activity relies on perturbing the highly active recycling of the Variant-specific Surface Glycoprotein (VSG) that occurs in the parasite's flagellar pocket. Methodology/Principal Findings: Here we expand the existing panel of Nbs with anti-Trypanosoma brucei potential and identify four categories based on their epitope specificity. We modified the binding properties of previously identified Nanobodies Nb_An05 and Nb_An33 by site-directed mutagenesis in the paratope and found this to strongly affect trypanotoxicity despite retention of antigen-targeting properties. Affinity measurements for all identified anti-trypanosomal Nbs reveal a strong correlation between trypanotoxicity and affinity (KD), suggesting that it is a crucial determinant for this activity. Half maximal effective (50%) affinity of 57 nM was calculated from the non-linear dose-response curves. In line with these observations, Nb humanizing mutations only preserved the trypanotoxic activity if the KD remained unaffected. Conclusions/Significance: This study reveals that the binding properties of Nanobodies need to be compatible with achieving an occupancy of >95% saturation of the parasite surface VSG in order to exert an anti-trypanosomal activity. As such, Nb-based approaches directed against the VSG target would require binding to an accessible, conserved epitope with high affinity. Author Summary: Nanobodies, antigen binding fragments derived from a non-conventional class of antibodies in camelids, were previously shown to exert a direct activity against African trypanosomes without the need of a toxin. Their mode-of-action relies on interference with the highly active recycling of the Variant-specific Surface Glycoprotein (VSG) that occurs in the flagellar pocket of the parasite. By expanding the panel of anti-trypanosomal Nanobodies and by modification of their binding properties through site-directed mutagenesis, we have been able to show a strong correlation between their trypanotoxic activity and affinity for the cognate antigen. From these studies it was calculated that the parasite surface saturation needs to exceed 95% in order to achieve this anti-trypanosomal effect of Nanobodies, which can be considered as a critical cut-off value for future Nanobody-based or other small molecule drug approaches against the VSG target. [ABSTRACT FROM AUTHOR]

Analysis of the tsetse fly salivary gland EST database revealed the presence of a highly enriched cluster of putative endonuclease genes, including tsal1 and tsal2. Tsal proteins are the major components of tsetse fly (G. morsitans morsitans) saliva where they are present as monomers as well as high molecular weight complexes with other saliva proteins. We demonstrate that the recombinant tsetse salivary gland proteins 1&2 (Tsal1&2) display DNA/RNA non-specific, high affinity nucleic acid binding with KD values in the low nanomolar range and a non-exclusive preference for duplex. These Tsal proteins exert only a residual nuclease activity with a preference for dsDNA in a broad pH range. Knockdown of Tsal expression by in vivo RNA interference in the tsetse fly revealed a partially impaired blood digestion phenotype as evidenced by higher gut nucleic acid, hematin and protein contents. [ABSTRACT FROM AUTHOR]

The African trypanosome Trypanosoma brucei, which persists within the bloodstream of the mammalian host, has evolved potent mechanisms for immune evasion. Specifically, antigenic variation of the variant-specific surface glycoprotein (VSG) and a highly active endocytosis and recycling of the surface coat efficiently delay killing mediated by anti-VSG antibodies. Consequently, conventional VSG-specific intact immunoglobulins are non-trypanocidal in the absence of complement. In sharp contrast, monovalent antigen-binding fragments, including 15 kDa nanobodies (Nb) derived from camelid heavy-chain antibodies (HCAbs) recognizing variant-specific VSG epitopes, efficiently lyse trypanosomes both in vitro and in vivo. This Nb-mediated lysis is preceded by very rapid immobilisation of the parasites, massive enlargement of the flagellar pocket and major blockade of endocytosis. This is accompanied by severe metabolic perturbations reflected by reduced intracellular ATP-levels and loss of mitochondrial membrane potential, culminating in cell death. Modification of anti-VSG Nbs through site-directed mutagenesis and by reconstitution into HCAbs, combined with unveiling of trypanolytic activity from intact immunoglobulins by papain proteolysis, demonstrates that the trypanolytic activity of Nbs and Fabs requires low molecular weight, monovalency and high affinity. We propose that the generation of low molecular weight VSG-specific trypanolytic nanobodies that impede endocytosis offers a new opportunity for developing novel trypanosomiasis therapeutics. In addition, these data suggest that the antigen-binding domain of an anti-microbial antibody harbours biological functionality that is latent in the intact immunoglobulin and is revealed only upon release of the antigen-binding fragment. [ABSTRACT FROM AUTHOR]

African trypanosomes of the Trypanosoma brucei species are extracellular protozoan parasites that cause the deadly disease African trypanosomiasis in humans and contribute to the animal counterpart, Nagana. Trypanosome clearance from the bloodstream is mediated by antibodies specific for their Variant Surface Glycoprotein (VSG) coat antigens. However, T. brucei infection induces polyclonal B cell activation, B cell clonal exhaustion, sustained depletion of mature splenic Marginal Zone B (MZB) and Follicular B (FoB) cells, and destruction of the B-cell memory compartment. To determine how trypanosome infection compromises the humoral immune defense system we used a C57BL/6 T. brucei AnTat 1.1 mouse model and multicolor flow cytometry to document B cell development and maturation during infection. Our results show a more than 95% reduction in B cell precursor numbers from the CLP, pre-pro-B, pro-B, pre-B and immature B cell stages in the bone marrow. In the spleen, T. brucei induces extramedullary B lymphopoiesis as evidenced by significant increases in HSC-LMPP, CLP, pre-pro-B, pro-B and pre-B cell populations. However, final B cell maturation is abrogated by infection-induced apoptosis of transitional B cells of both the T1 and T2 populations which is not uniquely dependent on TNF-, Fas-, or prostaglandin-dependent death pathways. Results obtained from ex vivo co-cultures of living bloodstream form trypanosomes and splenocytes demonstrate that trypanosome surface coat-dependent contact with T1/2 B cells triggers their deletion. We conclude that infection-induced and possibly parasite-contact dependent deletion of transitional B cells prevents replenishment of mature B cell compartments during infection thus contributing to a loss of the host's capacity to sustain antibody responses against recurring parasitemic waves. [ABSTRACT FROM AUTHOR]

Tsetse flies are the notorious transmitters of African trypanosomiasis, a disease caused by the Trypanosoma parasite that affects humans and livestock on the African continent. Metacyclic infection rates in natural tsetse populations with Trypanosoma brucei, including the two human-pathogenic subspecies, are very low, even in epidemic situations. Therefore, the infected fly/host contact frequency is a key determinant of the transmission dynamics. As an obligate blood feeder, tsetse flies rely on their complex salivary potion to inhibit host haemostatic reactions ensuring an efficient feeding. The results of this experimental study suggest that the parasite might promote its transmission through manipulation of the tsetse feeding behavior by modifying the saliva composition. Indeed, salivary gland Trypanosoma brucei-infected flies display a significantly prolonged feeding time, thereby enhancing the likelihood of infecting multiple hosts during the process of a single blood meal cycle. Comparison of the two major anti-haemostatic activities i.e. anti-platelet aggregation and anti-coagulation activity in these flies versus non-infected tsetse flies demonstrates a significant suppression of these activities as a result of the trypanosome-infection status. This effect was mainly related to the parasite-induced reduction in salivary gland gene transcription, resulting in a strong decrease in protein content and related biological activities. Additionally, the anti-thrombin activity and inhibition of thrombin-induced coagulation was even more severely hampered as a result of the trypanosome infection. Indeed, while naive tsetse saliva strongly inhibited human thrombin activity and thrombin-induced blood coagulation, saliva from T. brucei-infected flies showed a significantly enhanced thrombinase activity resulting in a far less potent anti-coagulation activity. These data clearly provide evidence for a trypanosomemediated modification of the tsetse salivary composition that results in a drastically reduced anti-haemostatic potential and a hampered feeding performance which could lead to an increase of the vector/host contact and parasite transmission in field conditions. [ABSTRACT FROM AUTHOR]

Background: Tsetse flies (Glossina sp.), the African trypanosome vectors, rely on anti-hemostatic compounds for efficient blood feeding. Despite their medical importance, very few salivary proteins have been characterized and functionally annotated. Methodology/Principal Findings: Here we report on the functional characterisation of a 5' nucleotidase-related (5' Nuc) saliva protein of the tsetse fly Glossina morsitans morsitans. This protein is encoded by a 1668 bp cDNA corresponding at the genomic level with a single-copy 4 kb gene that is exclusively transcribed in the tsetse salivary gland tissue. The encoded 5' Nuc protein is a soluble 65 kDa glycosylated compound of tsetse saliva with a dual anti-hemostatic action that relies on its combined apyrase activity and fibrinogen receptor (GPIIb/IIIa) antagonistic properties. Experimental evidence is based on the biochemical and functional characterization of recombinant protein and on the successful silencing of the 5' nuc translation in the salivary gland by RNA interference (RNAi). Refolding of a 5' Nuc/SUMO-fusion protein yielded an active apyrase enzyme with Km and Vmax values of 43 ± 4 μM and 684 ± 49 nmol Pi/min × mg for ATPase and 49 ± 11 μM and 177 ± 37 nmol Pi/min×mg for the ADP-ase activity. In addition, recombinant 5' Nuc was found to bind to GPIIb/IIIa with an apparent KD of 92 ± 25 nM. Consistent with these features, 5' Nuc potently inhibited ADP-induced thrombocyte aggregation and even caused disaggregation of ADP-triggered human platelets. The importance of 5' Nuc for the tsetse fly hematophagy was further illustrated by specific RNAi that reduced the anti-thrombotic activities in saliva by approximately 50% resulting in a disturbed blood feeding process. Conclusions/Significance: These data show that this 59nucleotidase-related apyrase exhibits GPIIb/IIIa antagonistic properties and represents a key thromboregulatory compound of tsetse fly saliva. [ABSTRACT FROM AUTHOR]

Background: In order to promote infection, the blood-borne parasite Trypanosoma brucei releases factors that upregulate arginase expression and activity in myeloid cells., Methodology/principal Findings: By screening a cDNA library of T. brucei with an antibody neutralizing the arginase-inducing activity of parasite released factors, we identified a Kinesin Heavy Chain isoform, termed TbKHC1, as responsible for this effect. Following interaction with mouse myeloid cells, natural or recombinant TbKHC1 triggered SIGN-R1 receptor-dependent induction of IL-10 production, resulting in arginase-1 activation concomitant with reduction of nitric oxide (NO) synthase activity. This TbKHC1 activity was IL-4Rα-independent and did not mirror M2 activation of myeloid cells. As compared to wild-type T. brucei, infection by TbKHC1 KO parasites was characterized by strongly reduced parasitaemia and prolonged host survival time. By treating infected mice with ornithine or with NO synthase inhibitor, we observed that during the first wave of parasitaemia the parasite growth-promoting effect of TbKHC1-mediated arginase activation resulted more from increased polyamine production than from reduction of NO synthesis. In late stage infection, TbKHC1-mediated reduction of NO synthesis appeared to contribute to liver damage linked to shortening of host survival time., Conclusion: A kinesin heavy chain released by T. brucei induces IL-10 and arginase-1 through SIGN-R1 signaling in myeloid cells, which promotes early trypanosome growth and favors parasite settlement in the host. Moreover, in the late stage of infection, the inhibition of NO synthesis by TbKHC1 contributes to liver pathogenicity.