Development of loop-mediated isothermal amplification (LAMP) assay using SYBR safe and gold-nanoparticle probe for detection of Leishmania in HIV patients.

Asymptomatic leishmaniasis cases have continuously increased, especially among patients with HIV who are at risk to develop further symptoms of cutaneous and visceral leishmaniasis. Thus, early diagnosis using a simple, sensitive and reliable diagnostic assay is important because populations at risk mostly reside in rural communities where laboratory equipment is limited. In this study, the highly sensitive and selective determination of Leishmania infection in asymptomatic HIV patients was achieved using dual indicators (SYBR safe and gold-nanoparticle probe; AuNP-probe) in one-step LAMP method based on basic instruments. The assay can be simply evaluated under the naked eye due to clear interpretation of fluorescent emission of LAMP-SYBR safe dye-complex and colorimetric precipitate of specific AuNP-probes. The sensitivities and specificities of fluorescent SYBR safe dye and AuNP-probe indicators were equal, which were as high as 94.1 and 97.1%, respectively. Additionally, detection limits were 102 parasites/mL (0.0147 ng/µL), ten times more sensitivity than other related studies. To empower leishmaniasis surveillance, this inexpensive one-step SYBR safe and AuNP-LAMP assay is reliably fast and simple for field diagnostics to point-of-care settings, which can be set up in all levels of health care facilities including resource limited areas, especially in low to middle income countries.

Acute shock caused by Clonorchis sinensis infection: a case report.

BACKGROUND: Clonorchiasis, caused by Clonorchis sinensis (C. sinensis) infection, is a serious food-borne zoonotic disease that is often asymptomatic or shows only mild symptoms, which leads to delayed treatment and chronic clonorchiasis and results in various complications, such as cholelithiasis, cholangitis, cholecystitis and cholangiocarcinoma. However, acute shock caused by C. sinensis infection has not been reported. Here, for the first time, we describe a fatal case of acute shock caused by C. sinensis infection. CASE PRESENTATION: A patient with a history of eating raw or undercooked freshwater fish was hospitalized with acute shock caused by severe abdominal pain. Physical examination suggested acute abdomen with severe abdominal pain and rigidity. Computed tomography (CT) detection indicated acute cholecystitis and cholelithiasis. After cholecystectomy, several liver flukes were found in the drainage tube. Furthermore, morphological analysis and polymerase chain reaction (PCR) identified the pathogen as C. sinensis. The liver gradually restored normal function after anthelmintic therapy with praziquantel. CONCLUSIONS: In this fatal case, C. sinensis infection was the cause of acute shock, which is rarely found in the clinic environment. This report aims to increase awareness of the hazards and complications related to clonorchiasis. The PCR diagnosis method used in this report might be helpful in reducing the misdiagnosis of clonorchiasis and unnecessary cholecystectomy.

A Cross-Sectional Study of Entamoeba histolytica/dispar/moshkovskii Complex in Salvador, Bahia, Brazil.

Epidemiological studies on species-specific Entamoeba infections are scarce due to the morphological similarity of pathogenic Entamoeba histolytica and nonpathogenic E. dispar and E. moshkovskii. The diagnosis of E. histolytica is frequently based on coproantigen (E. histolytica-Gal/GalNAc lectin specific) detection by immunoassays. However, specific E. histolytica-lectin is not expressed in cysts, which are eliminated by asymptomatic individuals leading to false-negative results and an underestimation of amebiasis prevalence. Molecular techniques based on the amplification of parasite DNA have been shown to be a highly sensitive and specific method that allows the detection of different Entamoeba species. This study aimed to assess the frequency of the species from E. histolytica/dispar/moshkovskii complex by molecular and immunological techniques in individuals attended at a public health system in Salvador-Bahia, Brazil. A cross-sectional study involving 55,218 individuals was carried out. The diagnosis was based on microscopy revealing E. histolytica/dispar/moshkovskii complex. The species differentiation was performed by E. histolytica-specific antigen, serological evaluation and by molecular technique. The overall prevalence of E. histolytica/dispar/moshkovskii complex determined by microscopy was approximately 0.49% (273/55,218). E. histolytica-specific antigen detection and molecular characterization returned 100% negativity for E. histolytica. However, serological evaluation returned an 8.9% positivity (8/90). In the stool samples analysed by PCR, it was not possible to identify E. histolytica and E. moshkovskii, although circulating IgG anti-E. histolytica has been detected.

Identification of a miniature Sae2/Ctp1/CtIP ortholog from Paramecium tetraurelia required for sexual reproduction and DNA double-strand break repair.

DNA double-strand breaks (DSBs) induced by genotoxic agents can cause cell death or contribute to chromosomal instability, a major driving force of cancer. By contrast, Spo11-dependent DSBs formed during meiosis are aimed at generating genetic diversity. In eukaryotes, CtIP and the Mre11 nuclease complex are essential for accurate processing and repair of both unscheduled and programmed DSBs by homologous recombination (HR). Here, we applied bioinformatics and genetic analysis to identify Paramecium tetraurelia CtIP (PtCtIP), the smallest known Sae2/Ctp1/CtIP ortholog, as a key factor for the completion of meiosis and the recovery of viable sexual progeny. Using in vitro assays, we find that purified recombinant PtCtIP preferentially binds to double-stranded DNA substrates but does not contain intrinsic nuclease activity. Moreover, mutation of the evolutionarily conserved C-terminal 'RHR' motif abrogates DNA binding of PtCtIP but not its ability to functionally interact with Mre11. Translating our findings into mammalian cells, we provide evidence that disruption of the 'RHR' motif abrogates accumulation of human CtIP at sites of DSBs. Consequently, cells expressing the DNA binding mutant CtIPR837A/R839A are defective in DSB resection and HR. Collectively, our work highlights minimal structural requirements for CtIP protein family members to facilitate the processing of DSBs, thereby maintaining genome stability as well as enabling sexual reproduction.

The epidemiology of Taenia spp. infection and Taenia solium cysticerci exposure in humans in the Central Highlands of Vietnam.

BACKGROUND: Vietnam is endemic for taeniasis and T. solium cysticercosis. Despite this, information on the epidemiological characteristics of the diseases in the Central Highlands of Vietnam are poorly described. The aims of this study were to determine the epidemiological characteristics of taeniasis (Taenia spp.) and T. solium cysticerci exposure in humans in Dak Lak province in the Central Highlands, Vietnam. METHODS: This cross-sectional study was carried out in six villages in three districts of Dak Lak. A total of 190 households were visited. From each household, between one and five individuals were asked to donate a single faecal and blood sample and respond to a questionnaire. Serum samples were subjected to lentil lectin purified glycoprotein enzyme-linked immunoelectrotransfer blot assay to detect antibodies against T. solium cysticerci. Multiplex real-time PCR was used to detect Taenia spp. infection in faecal samples. A fixed-effects logistic regression model was developed to identify factors associated with the probability of Taenia spp. infection or T. solium cysticerci exposure risk. The contribution of each of identified factor was quantified using population attributable fractions. RESULTS: The prevalence of seroexposure to T. solium in Dak Lak was 5% (95% CI 3% to 8%). Consumption of raw vegetables, sourcing drinking water from lakes, streams or ponds and the practice of outdoor defaecation were identified as primary risk factors for the prevalence of T. solium cysticerci exposure, while consuming undercooked pork and beef, pork tongue and observing Taenia proglottids in stool were associated with Taenia spp. infection. Consumption of raw vegetables attributed to 74% of T. solium cysticerci exposure-positive cases and consumption of undercooked beef attributed to 77% of taeniasis cases in these communities. CONCLUSIONS: The prevalence of T. solium seroexposure in Dak Lak is consistent with those reported in other regions of Vietnam. The identified risk factors associated with the prevalence of T. solium seroexposure and taeniasis infection in Dak Lak are modifiable and thus advocate for targeted community intervention programs to mitigating these risks.

Ecoepidemiological aspects of visceral leishmaniasis in an endemic area in the Steel Valley in Brazil: An ecological approach with spatial analysis.

Leishmaniases are a group of infectious diseases transmitted by phlebotomine sand flies, and their distribution depends on the presence of vectors, parasites, reservoirs and susceptible hosts in the same environment. In the last decades, visceral leishmaniasis (VL) has become urbanized and reached economically important cities in countries within the transmission zone. Our study was conducted in one of those cities-Ipatinga-in the state of Minas Gerais, Brazil, where the first autochthonous case of VL dates back to 2011. Since no data regarding the epidemiological triad of VL (etiological agent/vector/domestic reservoir) were available for this city, we characterized the local entomological fauna, identified the presence of specific Leishmania DNA in the captured phlebotomine sand flies, and assessed the incidence of canine and human VL. For the entomological survey, we set twenty light traps in ten districts of the city with reports of human and canine VL. The insect captures were performed monthly, during one year, starting in March 2015. A total of 1501 specimens of phlebotomine sand flies belonging to 16 distinct species were captured, with predominance (61.9%) of Lutzomyia longipalpis. Leishmania infantum DNA was detected in L. longipalpis and in Evandromyia cortelezzii test samples. A total of 9,136 dogs were examined, 1,355 of which (14.8%) were serologically positive for VL. The cases were georeferenced and the data were plotted in thematic maps, along with human cases of VL registered by the local Department of Health, during the study period. Our results confirm that the VL transmission cycle is active in Ipatinga, with the presence of vectors carrying Leishmania DNA, canine and human cases of the disease. Spatial analysis allowed for the observation of a positive relationship between canine and human cases of VL and the identification of areas with high priority for control actions in the city. The mapping of high-risk areas, together with an epidemiological study in urban areas, is fundamental to improve the efficacy of the Program for Surveillance and Control of VL (PSCVL) in Brazil.

The transcription factor Spores Absent A is a PKA dependent inducer of Dictyostelium sporulation.

Sporulation in Dictyostelium fruiting bodies evolved from amoebozoan encystation with both being induced by cAMP acting on PKA, but with downstream components still being unknown. Using tagged mutagenesis to find missing pathway components, we identified a sporeless mutant defective in a nuclear protein, SpaA. Expression of prespore genes was strongly reduced in spaA- cells, while expression of many spore stage genes was absent. Chromatin immunoprecipitation (ChIP) of a SpaA-YFP gene fusion showed that (pre)spore gene promoters bind directly to SpaA, identifying SpaA as a transcriptional regulator. SpaA dependent spore gene expression required PKA in vivo and was stimulated in vitro by the membrane-permeant PKA agonist 8Br-cAMP. The PKA agonist also promoted SpaA binding to (pre)spore promoters, placing SpaA downstream of PKA. Sequencing of SpaA-YFP ChIPed DNA fragments revealed that SpaA binds at least 117 (pre)spore promoters, including those of other transcription factors that activate some spore genes. These factors are not in turn required for spaA expression, identifying SpaA as the major trancriptional inducer of sporulation.

Cyclic GMP-AMP Synthase Is the Cytosolic Sensor of Plasmodium falciparum Genomic DNA and Activates Type I IFN in Malaria.

Innate immune receptors have a key role in the sensing of malaria and initiating immune responses. As a consequence of infection, systemic inflammation emerges and is directly related to signs and symptoms during acute disease. We have previously reported that plasmodial DNA is the primary driver of systemic inflammation in malaria, both within the phagolysosome and in the cytosol of effector cells. In this article, we demonstrate that Plasmodium falciparum genomic DNA delivered to the cytosol of human monocytes binds and activates cyclic GMP-AMP synthase (cGAS). Activated cGAS synthesizes 2'3'-cGAMP, which we subsequently can detect using liquid chromatography-tandem mass spectrometry. 2'3'-cGAMP acts as a second messenger for STING activation and triggers TBK1/IRF3 activation, resulting in type I IFN production in human cells. This induction of type I IFN was independent of IFI16. Access of DNA to the cytosolic compartment is mediated by hemozoin, because incubation of purified malaria pigment with DNase abrogated IFN-ß induction. Collectively, these observations implicate cGAS as an important cytosolic sensor of P. falciparum genomic DNA and reveal the role of the cGAS/STING pathway in the induction of type I IFN in response to malaria parasites.

Nuclear Features of the Heterotrich Ciliate Blepharisma americanum: Genomic Amplification, Life Cycle, and Nuclear Inclusion.

Blepharisma americanum, a member of the understudied ciliate class Heterotrichea, has a moniliform somatic macronucleus that resembles beads on a string. Blepharisma americanum is distinguishable by its pink coloration derived from the autofluorescent pigment blepharismin and tends to have a single somatic macronucleus with 3-6 nodes and multiple germline micronuclei. We used fluorescence confocal microscopy to explore the DNA content and amplification between the somatic and germline nuclei of B. americanum through its life cycle. We estimate that the DNA content of the macronucleus and micronucleus are 43 ± 8 Gbp and 83 ± 16 Mbp respectively. This correlates with an approximate DNA content difference of 500-fold from micronucleus to macronucleus and a macronuclear ploidy of ~1,100 N as compared to the presumably diploid micronucleus. We also investigate a previously reported macronuclear inclusion, which is present sporadically across all life cycle stages; this inclusion looks as if it contains blepharismin based on its fluorescent properties, but its function remains unknown. We also provide additional detail to our understanding of life cycles changes in B. americanum by analyses of fluorescent images. Overall, the data analyzed here contribute to our understanding of the diversity of nuclear architecture in ciliates by providing details on the highly polyploid somatic macronucleus of B. americanum.

Insights into the role of endonuclease V in RNA metabolism in Trypanosoma brucei.

Inosine may arise in DNA as a result of oxidative deamination of adenine or misincorporation of deoxyinosine triphosphate during replication. On the other hand, the occurrence of inosine in RNA is considered a normal and essential modification induced by specific adenosine deaminases acting on mRNA and tRNA. In prokaryotes, endonuclease V (EndoV) can recognize and cleave inosine-containing DNA. In contrast, mammalian EndoVs preferentially cleave inosine-containing RNA, suggesting a role in RNA metabolism for the eukaryotic members of this protein family. We have performed a biochemical characterization of EndoV from the protozoan parasite Trypanosoma brucei. In vitro, TbEndoV efficiently processes single-stranded RNA oligonucleotides with inosine, including A to I-edited tRNA-like substrates but exhibits weak activity over DNA, except when a ribonucleotide is placed 3' to the inosine. Immunolocalization studies performed in procyclic forms indicate that TbEndoV is mainly cytosolic yet upon nutritional stress it redistributes and accumulates in stress granules colocalizing with the DEAD-box helicase TbDhh1. RNAi-mediated depletion of TbEndoV results in moderate growth defects in procyclic cells while the two EndoV alleles could be readily knocked out in bloodstream forms. Taken together, these observations suggest an important role of TbEndoV in RNA metabolism in procyclic forms of the parasite.

Chronic amastigote-negative cutaneous leishmaniasis: A clinical, histopathologic and molecular study of 27 cases with emphasis on atypical and pseudolymphomatous presentations.

BACKGROUND: Chronic amastigote-negative cutaneous leishmaniasis (CL) is a diagnostic challenge, as the parasite load may be low, or absent in biopsy tissue sections. METHODS: A series of consecutive biopsy specimens, taken from 130 patients with a diagnosis of granulomatous dermatitis of unknown etiology, were reviewed. Polymerase chain reaction (PCR) was carried out for Leishmania-specific DNA. RESULTS: A total of 27 of 130 samples were positive for Leishmania-specific DNA. In only 3 patients was a clinical diagnosis CL made. The lesions were, single or multiple nodules or plaques of many months duration. Histopathologically, a tuberculoid granulomatous dermatitis was the least common denominator in every case, whilst in 5 cases a heavy lymphoid component was predominant. One patient had a concurrent cutaneous marginal zone lymphoma (MZL), the additional PCR study showed the presence of Leishmania DNA in tissue. CONCLUSIONS: The results of this study expand on previous observations as to the deceptive clinicopathologic manifestations of chronic CL, confirming the diagnostic value of PCR analysis for Leishmania DNA in unspecified granulomatous dermatitides. We also suggest that, in countries where Leishmaniasis is endemic, PCR for Leishmania-specific DNA be performed in any idiopathic pseudolymphomatous. More compelling evidence as to whether chronic Leishmania infection is implicated in the pathogenesis of some cutaneous MZL is warranted by further studies.

How do the alkaloids emetine and homoharringtonine kill trypanosomes? An insight into their molecular modes of action.

BACKGROUND: Although Trypanosoma brucei causes deadly sleeping sickness, the number of the registered medications is rather limited. Some plant alkaloids are potent trypanocidal agents. PURPOSE: In this study, we wanted to elucidate the molecular modes of trypanocidal activity of the alkaloids emetine and homoharringtonine against Trypanosoma brucei brucei. METHODS: We investigated the activity of both alkaloids regarding growth recovery from alkaloid-induced stress. We measured the inhibition of protein biosynthesis using the Click-iT® AHA Alexa Fluor® 488 Protein Synthesis HCS Assay kit. Reduction of mitochondrial membrane potential and cell cycle arrest were measured by means of flow cytometry. Additionally, we determined spectrophotometrically the inhibition of the trypanosome specific enzyme trypanothione reductase activity and DNA intercalation. RESULTS: Both alkaloids prevented that parasites could resume normal growth after pretreatment with the alkaloids. They inhibited protein biosynthesis in a time- and concentration-dependent manner. In contrast to homoharringtonine, emetine is also a DNA intercalator. Homoharringtonine decreased the mitochondrial membrane potential. Both alkaloids caused cell cycle arrest. Both alkaloids failed to affect trypanothione reductase, a crucial component of the redox system of trypanosomes. CONCLUSION: We assume that both alkaloids are primarily inhibitors of protein biosynthesis in trypanosomes, with DNA intercalation as an additional mechanism for emetine. This is the first study that elucidates the molecular mode of trypanocidal action of emetine and homoharringtonine.

Presence of Toxoplasma gondii in Pork Intended for Human Consumption in Tropical Southern Mexico.

BACKGROUND: Toxoplasmosis is caused by the protozoon Toxoplasma gondii, which is one of the most widespread parasites that infect animals and humans worldwide. One of the main routes of infection for humans is through the consumption of infected meat containing bradyzoites in tissue cysts. Pork is one of the foremost meat types associated with outbreaks of acute toxoplasmosis in humans. MATERIALS AND METHODS: Sixty blood samples were collected from finished pigs at slaughter and their sera was evaluated by an indirect-IgG ELISA. Matched muscle samples were obtained from the tongue and loin. Whole blood and tissue samples were evaluated to search for T. gondii DNA using a nested-polymerase chain reaction. RESULTS: Seroprevalence of T. gondii was 96.6% (58/60) of sampled pigs. Meanwhile, T. gondii DNA was present in 23.21% of tongue tissue samples (13/56), 7% of loin tissues (4/57), and 0% in blood samples (0/44), respectively. Two pigs were serologically indeterminate. CONCLUSION: This is the first report of the presence of T. gondii DNA in tissue samples obtained from finalized pigs. Results from the present study suggest a high exposure to T. gondii in pigs intended for human consumption from the tropical region of Mexico. Thus, the consumption of some undercooked pork meat meals typical from the southern region of Mexico could represent a significant risk for acquiring infection for the human population.

First Molecular Characterization of Leishmania Species Causing Visceral Leishmaniasis among Children in Yemen.

Visceral leishmaniasis (VL) is a debilitating, often fatal disease caused by Leishmania donovani complex; however, it is a neglected tropical disease. L. donovani complex comprises two closely related species, L. donovani that is mostly anthroponotic and L. infantum that is zoonotic. Differentiation between these two species is critical due to the differences in their epidemiology and pathology. However, they cannot be differentiated morphologically, and their speciation using isoenzyme-based methods poses a difficult task and may be unreliable. Molecular characterization is now the most reliable method to differentiate between them and to determine their phylogenetic relationships. The present study aims to characterize Leishmania species isolated from bone marrows of Yemeni pediatric patients using sequence analysis of the ribosomal internal transcribed spacer-1 (ITS1) gene. Out of 41 isolates from Giemsa-stained bone marrow smears, 25 isolates were successfully amplified by nested polymerase chain reaction and sequenced in both directions. Phylogenetic analysis using neighbor joining method placed all study isolates in one cluster with L. donovani complex (99% bootstrap). The analysis of ITS1 for microsatellite repeat numbers identified L. infantum in 11 isolates and L. donovani in 14 isolates. These data suggest the possibility of both anthroponotic and zoonotic transmission of VL-causing Leishmania species in Yemen. Exploring the possible animal reservoir hosts is therefore needed for effective control to be achieved.

Comparative genomics of drug resistance in Trypanosoma brucei rhodesiense.

Trypanosoma brucei rhodesiense is one of the causative agents of human sleeping sickness, a fatal disease that is transmitted by tsetse flies and restricted to Sub-Saharan Africa. Here we investigate two independent lines of T. b. rhodesiense that have been selected with the drugs melarsoprol and pentamidine over the course of 2 years, until they exhibited stable cross-resistance to an unprecedented degree. We apply comparative genomics and transcriptomics to identify the underlying mutations. Only few mutations have become fixed during selection. Three genes were affected by mutations in both lines: the aminopurine transporter AT1, the aquaporin AQP2, and the RNA-binding protein UBP1. The melarsoprol-selected line carried a large deletion including the adenosine transporter gene AT1, whereas the pentamidine-selected line carried a heterozygous point mutation in AT1, G430R, which rendered the transporter non-functional. Both resistant lines had lost AQP2, and both lines carried the same point mutation, R131L, in the RNA-binding motif of UBP1. The finding that concomitant deletion of the known resistance genes AT1 and AQP2 in T. b. brucei failed to phenocopy the high levels of resistance of the T. b. rhodesiense mutants indicated a possible role of UBP1 in melarsoprol-pentamidine cross-resistance. However, homozygous in situ expression of UBP1-Leu(131) in T. b. brucei did not affect the sensitivity to melarsoprol or pentamidine.

First Molecular Identification and Genetic Characterization of Theileria lestoquardi in Sheep of the Maghreb Region.

Theileria lestoquardi is the most prominent Theileria species in small ruminants that causes malignant theileriosis of sheep in Africa and Asia. In the present survey, blood samples and ticks were collected in Kebili (southern Tunisia) from 166 Queue Fine de l'Ouest sheep. Giemsa-stained blood smears, immunofluorescent antibody test (IFAT) and PCR were performed. The DNA was extracted from blood and analysed by PCR targeting 18S rRNA gene of Theileria spp. and then sequenced. A total number of 140 ticks were collected from a total number of 166 sheep during the four seasons. The ticks belonged to two genera and 4 species; the most frequent tick was Hyalomma excavatum 84.3% (118/140) and then Rhipicephalus spp. 15.7% (22/140). Only two animals had positive Giemsa-stained blood smears, and they were also positive by IFAT. The amplicons had 99.3 and 99.6% homology with the BLAST published T. lestoquardi amplicons. To our knowledge, this is the first report of T. lestoquardi in small ruminants within the Maghreb region.

Cis-acting signals modulate the efficiency of programmed DNA elimination in Paramecium tetraurelia.

In Paramecium, the regeneration of a functional somatic genome at each sexual event relies on the elimination of thousands of germline DNA sequences, known as Internal Eliminated Sequences (IESs), from the zygotic nuclear DNA. Here, we provide evidence that IESs' length and sub-terminal bases jointly modulate IES excision by affecting DNA conformation in P. tetraurelia. Our study reveals an excess of complementary base pairing between IESs' sub-terminal and contiguous sites, suggesting that IESs may form DNA loops prior to cleavage. The degree of complementary base pairing between IESs' sub-terminal sites (termed Cin-score) is positively associated with IES length and is shaped by natural selection. Moreover, it escalates abruptly when IES length exceeds 45 nucleotides (nt), indicating that only sufficiently large IESs may form loops. Finally, we find that IESs smaller than 46 nt are favored targets of the cellular surveillance systems, presumably because of their relatively inefficient excision. Our findings extend the repertoire of cis-acting determinants for IES recognition/excision and provide unprecedented insights into the distinct selective pressures that operate on IESs and somatic DNA regions. This information potentially moves current models of IES evolution and of mechanisms of IES recognition/excision forward.

Are Neutrophil Extracellular Traps Playing a Role in the Parasite Control in Active American Tegumentary Leishmaniasis Lesions?

Neutrophil extracellular traps (NETs) have been described as a network of extracellular fibers composed by DNA, histones and various proteins/enzymes. Studies have demonstrated that NETs could be responsible for the trapping and elimination of a variety of infectious agents. In order to verify the presence of NETs in American tegumentary leishmaniasis (ATL) and their relationship with the presence of amastigotes we evaluated active cutaneous lesions of 35 patients before treatment by the detection of parasites, neutrophils (neutrophil elastase) and histones through immunohistochemistry and confocal immunofluorescence. Intact neutrophils could be detected in all ATL lesions. NETs were present in 27 patients (median 1.1; range from 0.1 to 23.5/mm2) with lesion duration ranging from one to seven months. NETs were in close proximity with neutrophils (r = 0.586; p = 0.0001) and amastigotes (r = 0.710; p = 0.0001). Two patterns of NET formation were detected: small homogeneously distributed networks observed in all lesions; and large structures that could be visualized at a lower magnification in lesions presenting at least 20% of neutrophils. Lesions presenting the larger NET formation showed high parasite detection. A correlation between NET size and the number of intact amastigotes was observed (p=0.02). As we detected an association between NET and amastigotes, our results suggest that neutrophil migration and NET formation could be stimulated and maintained by stimuli derived from the parasite burden/parasite antigen in the extracellular environment. The observation of areas containing only antigens not intermingled with NETs (elastase and histone) suggests that the involvement of these structures in the control of parasite burden is a dynamic process in which the formation of NETs is exhausted with the destruction of the parasites. Since NETs were also associated with granulomas, this trapping would favor the activity of macrophages in order to control the parasite burden.

TLR9-deficiency reduces TLR1, TLR2 and TLR3 expressions in Leishmania major-infected macrophages.

The parasite Leishmania major counteractively modulates TLR2 and TLR9 expression and their functions. Although TLR1, TLR3, TLR4, and TLR7 are also implicated in Leishmania infection, whether their expression was altered in TLR2 or TLR9 deficiency remained unknown. Therefore, we examined TLR1, TLR3, TLR4 and TLR7 expression in L. major infection in TLR2-deficient or TLR9-deficient macrophages. We observed that TLR9-deficiency reduced TLR1, TLR2 and TLR3 but not TLR7 expression in the macrophages treated with live or killed L. major promastigotes. TLR2-deficiency had little effects by comparison. TLR9-deficient macrophages had reduced CD40 expression and less IL-12 and TNF-α expression. Thus, we report that TLR9 modulates TLR1, TLR2 and TLR3, but not TLR7, expression in L. major-infected macrophages.

TLR9 activation is triggered by the excess of stimulatory versus inhibitory motifs present in Trypanosomatidae DNA.

DNA sequences purified from distinct organisms, e.g. non vertebrate versus vertebrate ones, were shown to differ in their TLR9 signalling properties especially when either mouse bone marrow-derived- or human dendritic cells (DCs) are probed as target cells. Here we found that the DC-targeting immunostimulatory property of Leishmania major DNA is shared by other Trypanosomatidae DNA, suggesting that this is a general trait of these eukaryotic single-celled parasites. We first documented, in vitro, that the low level of immunostimulatory activity by vertebrate DNA is not due to its limited access to DCs' TLR9. In addition, vertebrate DNA inhibits the activation induced by the parasite DNA. This inhibition could result from the presence of competing elements for TLR9 activation and suggests that DNA from different species can be discriminated by mouse and human DCs. Second, using computational analysis of genomic DNA sequences, it was possible to detect the presence of over-represented inhibitory and under-represented stimulatory sequences in the vertebrate genomes, whereas L. major genome displays the opposite trend. Interestingly, this contrasting features between L. major and vertebrate genomes in the frequency of these motifs are shared by other Trypanosomatidae genomes (Trypanosoma cruzi, brucei and vivax). We also addressed the possibility that proteins expressed in DCs could interact with DNA and promote TLR9 activation. We found that TLR9 is specifically activated with L. major HMGB1-bound DNA and that HMGB1 preferentially binds to L. major compared to mouse DNA. Our results highlight that both DNA sequence and vertebrate DNA-binding proteins, such as the mouse HMGB1, allow the TLR9-signaling to be initiated and achieved by Trypanosomatidae DNA.