Search

Dengue virus (DENV) and Zika virus (ZIKV) belong to the same viral family, the Flaviviridae. They cause recurring threats to the public health systems of tropical countries such as Brazil. The primary Brazilian vector of both viruses is the mosquito Aedes aegypti. After the mosquito ingests a blood meal from an infected person, the viruses infect and replicate in the midgut, disseminate to secondary tissues and reach the salivary gland (SG), where they are ready to be transmitted to a vertebrate host. It is thought that the intrinsic discrepancies among mosquitoes could affect their ability to deal with viral infections. This study confirms that the DENV and ZIKV infection patterns of nine Ae. aegypti field populations found in geographically separate health districts of an endemic Brazilian city vary. We analyzed the infection rate, disseminated infection, vector competence, and viral load through quantitative PCR. Mosquitoes were challenged using the membrane-feeding assay technique and were tested seven and fourteen days post-infection (early and late infection phases, respectively). The infection responses varied among the Ae. aegypti populations for both flaviviruses in the two infection phases. There was no similarity between DENV and ZIKV vector competencies or viral loads. According to the results of our study, the risk of viral transmission overtime after infection either increases or remains unaltered in ZIKV infected vectors. However, the risk may increase, decrease, or remain unaltered in DENV-infected vectors depending on the mosquito population. For both flaviviruses, the viral load persisted in the body even until the late infection phase. In contrast to DENV, the ZIKV accumulated in the SG over time in all the mosquito populations. These findings are novel and may help direct the development of control strategies to fight dengue and Zika outbreaks in endemic regions, and provide a warning about the importance of understanding mosquito responses to arboviral infections., Author summary Dengue and Zika are neglected diseases caused by viruses transmitted to humans by mosquitoes (vector-borne diseases). The primary vector of both diseases is Aedes aegypti, a highly abundant mosquito in tropical countries and adapted to the urban habitat. The viral cycle in the vector starts when the mosquito bites an infected person and acquires the viruses through the blood meal. When the infected blood reaches the mosquito’s midgut, the viruses invade the epithelial cells and disseminate in several organs until they reach the salivary glands, enabling viral transmission to the next person. However, the mosquitoes have developed strategies to combat the viral invasion and dissemination in their body, making this journey a challenge to the viruses. Herein, we show that the mosquito responses against dengue and Zika viruses are distinct. In addition, mosquitoes from separate populations of the same city have different abilities to deal with the viruses in both cases, dengue and Zika infections. Our results show the diversity of responses that the mosquitoes may present to viral infections. These findings may better direct disease control strategies to combat dengue and Zika outbreaks in endemic regions.

Free living amoeba of the genus Acanthamoeba are opportunist protozoan involved in corneal, systemic, and encephalic infections in humans. Most of the mechanisms underlying intraspecies variations and pathogenicity are still unknown. Recently, the release of extracellular vesicles (EVs) by Acanthamoeba was reported. However, comparative characterization of EVs from distinct strains is not available. The aim of this study was to evaluate EVs produced by Acanthamoeba from different genotypes, comparing their proteases profile and immunomodulatory properties. EVs from four environmental or clinical strains (genotypes T1, T2, T4, and T11) were obtained by ultracentrifugation, quantitated by nanoparticle tracking analysis and analyzed by scanning and transmission electron microscopy. Proteases profile was determined by zymography and functional properties of EVs (measure of nitrite and cytokine production) were determined after peritoneal macrophage stimulation. Despite their genotype, all strains released EVs and no differences in size and/or concentration were detected. EVs exhibited a predominant activity of serine proteases (pH 7.4 and 3.5), with higher intensity in T4 and T1 strains. EVs from the environmental, nonpathogenic T11 strain exhibited a more proinflammatory profile, inducing higher levels of Nitrite, tumor necrosis factor alpha and interleukin-6 via TLR4/TLR2 than those strains with pathogenic traits (T4, T1, and T2). Preincubation with EVs treated with protease inhibitors or heating drastically decreased nitrite concentration production in macrophages. Those data suggest that immunomodulatory effects of EVs may reflect their pathogenic potential depending on the Acanthamoeba strains and are dependent on protease integrity.

Sterols are essential membrane components in eukaryotes, and sterol synthesis inhibitors can have potent effects against pathogenic fungi and trypanosomatids. Understanding the roles of sterols will facilitate the development of new drugs and counter drug resistance. LSO is required for the formation of the C-5–C-6 double bond in the sterol core structure in mammals, fungi, protozoans, plants, and algae. Functions of this C-5–C-6 double bond are not well understood. In this study, we generated and characterized a lathosterol oxidase-null mutant in Leishmania major. Our data suggest that LSO is vital for the structure and membrane-stabilizing functions of leishmanial sterols. In addition, our results imply that while mutations in lathosterol oxidase can confer resistance to amphotericin B, an important antifungal and antiprotozoal agent, the alteration in sterol structure leads to significant defects in stress response that could be exploited for drug development., Lathosterol oxidase (LSO) catalyzes the formation of the C-5–C-6 double bond in the synthesis of various types of sterols in mammals, fungi, plants, and protozoa. In Leishmania parasites, mutations in LSO or other sterol biosynthetic genes are associated with amphotericin B resistance. To investigate the biological roles of sterol C-5–C-6 desaturation, we generated an LSO-null mutant line (lso−) in Leishmania major, the causative agent for cutaneous leishmaniasis. lso− parasites lacked the ergostane-based sterols commonly found in wild-type L. major and instead accumulated equivalent sterol species without the C-5–C-6 double bond. These mutant parasites were replicative in culture and displayed heightened resistance to amphotericin B. However, they survived poorly after reaching the maximal density and were highly vulnerable to the membrane-disrupting detergent Triton X-100. In addition, lso− mutants showed defects in regulating intracellular pH and were hypersensitive to acidic conditions. They also had potential alterations in the carbohydrate composition of lipophosphoglycan, a membrane-bound virulence factor in Leishmania. All these defects in lso− were corrected upon the restoration of LSO expression. Together, these findings suggest that the C-5–C-6 double bond is vital for the structure of the sterol core, and while the loss of LSO can lead to amphotericin B resistance, it also makes Leishmania parasites vulnerable to biologically relevant stress. IMPORTANCE Sterols are essential membrane components in eukaryotes, and sterol synthesis inhibitors can have potent effects against pathogenic fungi and trypanosomatids. Understanding the roles of sterols will facilitate the development of new drugs and counter drug resistance. LSO is required for the formation of the C-5–C-6 double bond in the sterol core structure in mammals, fungi, protozoans, plants, and algae. Functions of this C-5–C-6 double bond are not well understood. In this study, we generated and characterized a lathosterol oxidase-null mutant in Leishmania major. Our data suggest that LSO is vital for the structure and membrane-stabilizing functions of leishmanial sterols. In addition, our results imply that while mutations in lathosterol oxidase can confer resistance to amphotericin B, an important antifungal and antiprotozoal agent, the alteration in sterol structure leads to significant defects in stress response that could be exploited for drug development.

Brazilian Aedes aegypti from Amazonas is highly permissive to monoinfection and coinfection with dengue virus and Zika virus (ZIKV) and capable of cotransmitting both arboviruses by bite to vertebrate hosts. Coinfection strongly influences vector competence, favoring transmission of ZIKV., Background Several tropical cities are permissive to Aedes aegypti and dengue virus (DENV) endemicity and have allowed for invasion and circulation of Zika virus (ZIKV) in the same areas. People living in arbovirus-endemic regions have been simultaneously infected with ≥2 arboviruses. Methods A. aegypti mosquitoes from Manaus, the capital city of Amazonas State in Brazil, were coinfected with circulating strains of DENV and ZIKV. The coinfected vectors were allowed to bite BALB/c mice. Results A. aegypti from Manaus is highly permissive to monoinfection and coinfection with DENV and ZIKV and is capable of cotransmitting both pathogens by bite. Coinfection strongly influences vector competence, favoring transmission of ZIKV to the vertebrate host. Conclusions This finding suggests that A. aegypti is an efficient vector of ZIKV and that ZIKV would be preferentially transmitted by coinfected A. aegypti. Coinfection in the vector population should be considered a new critical epidemiological factor and may represent a major public health challenge.

Although Leishmania infantum is well-known as the aethiological agent of visceral leishmaniasis (VL), in some Central American countries it may cause atypical non-ulcerated cutaneous leishmaniasis (NUCL). However, the mechanisms favoring its establishment in the skin are still unknown. Lipophosphoglycan (LPG) is the major Leishmania multivirulence factor involved in parasite-host interaction. In the case of viscerotropic L. infantum, it causes an immunosuppression during the interaction with macrophages. Here, we investigated the biochemical and functional roles of LPGs from four dermotropic L. infantum strains from Honduras during in vitro interaction with murine macrophages. LPGs were extracted, purified and their repeat units analysed. They did not have side chains consisting of Gal(β1,4)Man(α1)-PO4 common to all LPGs. Peritoneal macrophages from BALB/c and C57BL/6 were exposed to LPG for nitric oxide (NO) and cytokine (TNF-α and, IL-6) production. LPGs from dermotropic strains from Honduras triggered higher NO and cytokine levels compared to those from viscerotropic strains. In conclusion, LPGs from dermotropic strains are devoid of side-chains and exhibit high pro-inflammatory activity.

International audience; Lipophosphoglycan (LPG) is the major Leishmania surface glycoconjugate having importance during the host-parasite interface. Leishmania (Viannia) braziliensis displays a spectrum of clinical forms including: typical cutaneous leishmaniasis (TL), mucocutaneous (ML), and atypical lesions (AL). Those variations in the immunopathology may be a result of intraspecies polymorphisms in the parasite's virulence factors. In this context, we evaluated the role of LPG of strains originated from patients with different clinical manifestations and the sandfly vector. Six isolates of L. braziliensis were used: M2903, RR051 and RR418 (TL), RR410 (AL), M15991 (ML), and M8401 (vector). LPGs were extracted and purified by hydrophobic interaction. Peritoneal macrophages from C57BL/6 and respective knock-outs (TLR2-/- and TLR-4-/-) were primed with IFN-γ and exposed to different LPGs for nitric oxide (NO) and cytokine production (IL-1β, IL-6, IL-12, and TNF-α). LPGs differentially activated the production of NO and cytokines via TLR4. In order to ascertain if such functional variations were related to intraspecies polymorphisms in the LPG, the purified glycoconjugates were subjected to western blot with specific LPG antibodies (CA7AE and LT22). Based on antibody reactivity preliminary variations in the repeat units were detected. To confirm these findings, LPGs were depolymerized for purification of repeat units. After thin layer chromatography, intraspecies polymorphisms were confirmed especially in the type and/size of sugars branching-off the repeat units motif. In conclusion, different isolates of L. braziliensis from different clinical forms and hosts possess polymorphisms in their LPGs that functionally affected macrophage responses.

SUMMARY Activation of the NLRP3 inflammasome by Leishmania parasites is critical for the outcome of leishmaniasis, a disease that affects millions of people worldwide. We investigate the mechanisms involved in NLRP3 activation and demonstrate that caspase-11 (CASP11) is activated in response to infection by Leishmania species and triggers the non-canonical activation of NLRP3. This process accounts for host resistance to infection in macrophages and in vivo. We identify the parasite membrane glycoconjugate lipophosphoglycan (LPG) as the molecule involved in CASP11 activation. Cytosolic delivery of LPG in macrophages triggers CASP11 activation, and infections performed with Lpg1−/− parasites reduce CASP11/NLRP3 activation. Unlike bacterial LPS, purified LPG does not activate mouse CASP11 (or human Casp4) in vitro, suggesting the participation of additional molecules for LPG-mediated CASP11 activation. Our data identify a parasite molecule involved in CASP11 activation, thereby establishing the mechanisms underlying inflammasome activation in response to Leishmania species., Graphical Abstract, In Brief Activation of the NLRP3 inflammasome is critical for the outcome of leishmaniasis. De Carvalho et al. show that Leishmania lipophosphoglycan (LPG) triggers caspase-11 activation in macrophage cytoplasm and the non-canonical activation of NLRP3, thereby establishing the mechanisms underlying NLRP3 activation in response to Leishmania.

Leishmania major is an Old World species causing cutaneous leishmaniasis and is transmitted by Phlebotomus papatasi and Phlebotomus duboscqi. In Brazil, two isolates from patients who never left the country were characterised as L. major-like (BH49 and BH121). Using molecular techniques, these isolates were indistinguishable from the L. major reference strain (FV1). OBJECTIVES; We evaluated the lipophosphoglycans (LPGs) of the strains and their behaviour in Old and New World sand fly vectors. METHODS: LPGs were purified, and repeat units were qualitatively evaluated by immunoblotting. Experimental in vivo infection with L. major-like strains was performed in Lutzomyia longipalpis (New World, permissive vector) and Ph. papatasi(Old World, restrictive or specific vector). FINDINGS: The LPGs of both strains were devoid of arabinosylated side chains, whereas the LPG of strain BH49 was more galactosylated than that of strain BH121. All strains with different levels of galactosylation in their LPGs were able to infect both vectors, exhibiting colonisation of the stomodeal valve and metacyclogenesis. The BH121 strain (less galactosylated) exhibited lower infection intensity compared to BH49 and FV1 in both vectors. MAIN CONCLUSIONS: Intraspecific variation in the LPG of L. major-like strains occur, and the different galactosylation levels affected interactions with the invertebrate host. Leishmania major é uma espécie do Velho Mundo causadora da leishmaniose cutânea e é transmitida por Phlebotomus papatasi e Phlebotomus duboscqi. No Brasil, dois isolados de pacientes que nunca saíram do país foram caracterizados como L. major-like (BH49 e BH121). Usando técnicas moleculares, esses isolados foram indistinguíveis da cepa de referência de L. major (FV1). OBJETIVOS; Nós avaliamos os lipofosfoglicanos (LPGs) das cepas e seu comportamento em vetores de flebotomíneos do Velho e do Novo Mundo. MÉTODOS: Os GLPs foram purificados e as unidades repetidas foram avaliadas qualitativamente por immunoblotting. A infecção experimental in vivo com cepas semelhantes a L. major foi realizada em Lutzomyia longipalpis (Novo Mundo, vetor permissivo) e Ph. papatasi (Velho Mundo, vetor restritivo ou específico). DADOS: Os GLPs de ambas as cepas eram desprovidos de cadeias laterais arabinosiladas, enquanto o GLP da cepa BH49 era mais galactosilado que o da cepa BH121. Todas as cepas com diferentes níveis de galactosilação em seus GLPs foram capazes de infectar ambos os vetores, apresentando colonização da válvula estomodal e metaciclogênese. A cepa BH121 (menos galactosilada) apresentou menor intensidade de infecção em relação a BH49 e FV1 em ambos os vetores. PRINCIPAIS CONCLUSÕES: Ocorre variação intraespecífica no GLP de cepas semelhantes a L. major, e os diferentes níveis de galactosilação afetaram as interações com o hospedeiro invertebrado.

Os parasitos tripanosomatídeos são responsáveis por graves doenças no homem. Durante seu ciclo de vida são submetidos a condições extremamente adversas. Moléculas ancoradas por glicosilfosfatidilinositol (GPI) são os principais antígenosexpressados na membrana plasmática dos tripanosomatideos e auxiliam no processo de infecção. Dentre essas, as GPI-mucinas são glicoconjugados importantes em Trypanosoma cruzi e desempenham um papel crucial na sobrevivência dos parasitos. Este parasito é capaz de liberar vesículas extracelulares (EVs) enriquecidas por GPImucinascontendo resíduos terminais de a-galactosil (a-Gal) e TS/gp85. Contudo, o comportamento de células do sistema imune no curso da infecção da doença de Chagas após a estimulação com EVs de T. cruzi de cepas de diferentes DTUs ainda é desconhecido. Assim, o este trabalho avaliou o impacto destas vesículas das cepas Colombiana (Tc I), YuYu (Tc I), Y (Tc II) e CL-14 (Tc VI) no perfil de ativação da resposta imune inata e crônica. As EVs foram purificadas e exibiram diferenças intraespecíficas na quantidade de proteína e de epítopo a-Gal. Durante a resposta imuneinata, vesículas das cepas YuYu e CL-14 induziram altos níveis de citocinas próinflamatórias (TNF-a e IL-6) e óxido nítrico via TLR2. Não foi observada diferença na ativação de MAPKs (ERK1/2, p38 e JNK) após estimulação com as vesículas. A produção de citocinas no sobrenadante de cultura de células esplênicas derivadas decamundongos crônicos infectados com os quatro tipos de cepas mostrou perfil proinflamatório após estimulação com vesículas de Colombiana e Y, independente do tipo de cepa usada na infecção in vivo. Para testar a importância funcional desta modulação, foi avaliada a expressão de citocinas intracelulares após exposição in vitro apenas nas cepas Colombiana e YuYu. As vesículas foram capazes de induzir produção de citocinas. Foi observada alta frequência de IL-10 em linfócitos T CD4+ e CD8+, além de um perfil misto de produção de TNF-a e IL-10 em células B. Finalmente, células dendríticas produziram TNF-a após estimulação com as vesículas. Em conclusão, variações intra-específicas nas EVs de T. cruzi são importantes para a ativação dosistema imune inato e modular os eventos imunopatológicos nas fases aguda e crônica da Doença de Chagas. Tripanosomatid parasites are the causative agents of a wide spectrum of clinical manifestations. The parasites have an outstanding ability to avoid destruction in the hostile environments during their life cycles. Their main strategies include the expression of major glycoconjugates attached to the membrane by glycosylphosphatidylinositol (GPI) anchors. The GPI-mucins are important glycoconjugate of Trypanosoma cruzi. Those molecules play an important role on the protozoan infection and survival. Trypomastigote forms of T. cruzi shed extracellular vesicles (EVs) enriched with trans-sialidase (TS)/gp85 glycoproteins and other a-galactosyl (a-Gal)-containing glycoconjugates, like mucins. However, the role of EVs from different T. cruzi strains and some aspects of the immune system stimulation remain unknown. Here, vesicles from pathogenic (Colombiana (Tc I), YuYu (Tc I), Y (Tc II)) and non-pathogenic (CL-14 (Tc IV)) T. cruzi strains were purified and exhibited differences in their protein and a-galactosyl contents. Later, those polymorphisms wereevaluated in the modulation of immune responses (innate and in the chronic phase) in C57BL/6 mice. EVs from YuYu and CL-14 strains induced higher levels of proinflammatory cytokines (TNF-a and IL-6) and nitric oxide by macrophages via TLR2. In general, no differences were observed in MAPKs activation (p38, JNK and ERK 1/2) after EV stimulation. In splenic cells derived from chronically infected mice,a different modulation pattern was observed, where Colombiana and Y strain EVs were more pro-inflammatory. This modulation was independent of the strain used in the mice infection. To test the functional importance of this modulation, the expression of intracellular cytokines after in vitro exposure was evaluated using EVs from YuYu andColombiana strains. Both EVs induced cytokine production with the appearance of IL- 10 in the chronically infected mice. A high frequency of IL-10 in CD4+ and CD8+ T lymphocytes was observed. A mixed profile of cytokine induction was observed in B cells with the production of TNF-a and IL-10. Finally, dendritic cells produced TNF-a after stimulation with EVs. Intraspecific variation in T. cruzi EVs are very important to immune system activation and it is responsible to modulate the immunopatologycal events in acute and cronic Chagas disease.

Background Protozoan parasites of the genus Leishmania cause a number of important diseases in humans and undergo a complex life cycle, alternating between a sand fly vector and vertebrate hosts. The parasites have a remarkable capacity to avoid destruction in which surface molecules are determinant for survival. Amongst the many surface molecules of Leishmania, the glycoconjugates are known to play a central role in host–parasite interactions and are the focus of this review. Scope of the review The most abundant and best studied glycoconjugates are the Lipophosphoglycans (LPGs) and glycoinositolphospholipids (GIPLs). This review summarizes the main studies on structure and biological functions of these molecules in New World Leishmania species. Major conclusions LPG and GIPLs are complex molecules that display inter- and intraspecies polymorphisms. They are key elements for survival inside the vector and to modulate the vertebrate immune response during infection. General significance Most of the studies on glycoconjugates focused on Old World Leishmania species. Here, it is reported some of the studies involving New World species and their biological significance on host–parasite interaction. This article is part of a Special Issue entitled Glycoproteomics.

Leishmania amazonensis is the etiologic agent of the cutaneous and diffuse leishmaniasis. This species is often associated with drug resistance, and the conventional treatments exhibit high toxicity for patients. Therefore, the search for new antileishmanial compounds is urgently needed since there is no vaccine available. In this study, using the in vitro traditional drug screening test, we have analyzed the effects of a series of diaminoalkanes monoprotected with t -butyloxycarbonyl (BOC) against L. amazonensis . Among the 18 tested compounds, 6 exhibited antileishmanial activity ( 2 , 7 – 9 , 17 , and 18 ). Best IC 50 values (10.39 ± 0.27 and 3.8 ± 0.42 μg/mL) were observed for compounds 17 and 18 (H 2 N(CH 2 ) n NHBoc, n = 10 and 12), respectively. Although those compounds had higher lipophilicity as indicated by their cLog P values, compound 17 was very toxic. Determination of the selective indexes indicated that 50% of the active compounds were very toxic for HepG2 cells. However, compounds 2 , 8 , and 18 had good lipophilicity and were less toxic among all polyamine derivatives tested. The chemical properties of antileishmanial diamine derivatives, such as lipophilicity and cytotoxicity, are relevant factors for the design of new drugs. A higher lipophilicity is likely to improve the chances of reaching this intracellular parasite.

Vector-born diseases cause millions of deaths every year globally. Alternatives for the control of diseases such as malaria and dengue fever are urgently needed and the use of transgenic mosquitoes that block parasite/virus is a sound strategy to be used within control programs. However, prior to use transgenic mosquitoes as control tools, it is important to study their fitness since different biological aspects might influence their ability to disseminate and compete with wild populations. We previously reported the construction of four transgenic Aedes fluviatilis mosquito lines expressing a Plasmodium- blocking molecule (mutated bee venom phospholipase A(2)-mPLA(2)). Presently we studied two aspects of their fitness: body size, that has been used as a fitness-related status, and the expression of major enzymes classes involved in the metabolism of xenobiotics, including insecticides. Body size analysis (recorded by geometric wing morphometrics) indicated that both male and female mosquitoes were larger than the non-transgenic counterparts, suggesting that this characteristic might have an impact on their overall fitness. By contrast, no significant difference in the activity of enzymes related to metabolic insecticide resistance was detected in transgenic mosquitoes. The implication on fitness advantage of these features, towards the implementation of this strategy, is further discussed.

Trypomastigote forms of Trypanosoma cruzi, the causative agent of Chagas Disease, shed extracellular vesicles (EVs) enriched with glycoproteins of the gp85/trans-sialidase (TS) superfamily and other α-galactosyl (α-Gal)-containing glycoconjugates, such as mucins. Here, purified vesicles from T. cruzi strains (Y, Colombiana, CL-14 and YuYu) were quantified according to size, intensity and concentration. Qualitative analysis revealed differences in their protein and α-galactosyl contents. Later, those polymorphisms were evaluated in the modulation of immune responses (innate and in the chronic phase) in C57BL/6 mice. EVs isolated from YuYu and CL-14 strains induced in macrophages higher levels of proinflammatory cytokines (TNF-α and IL-6) and nitric oxide via TLR2. In general, no differences were observed in MAPKs activation (p38, JNK and ERK 1/2) after EVs stimulation. In splenic cells derived from chronically infected mice, a different modulation pattern was observed, where Colombiana (followed by Y strain) EVs were more proinflammatory. This modulation was independent of the T. cruzi strain used in the mice infection. To test the functional importance of this modulation, the expression of intracellular cytokines after in vitro exposure was evaluated using EVs from YuYu and Colombiana strains. Both EVs induced cytokine production with the appearance of IL-10 in the chronically infected mice. A high frequency of IL-10 in CD4+ and CD8+ T lymphocytes was observed. A mixed profile of cytokine induction was observed in B cells with the production of TNF-α and IL-10. Finally, dendritic cells produced TNF-α after stimulation with EVs. Polymorphisms in the vesicles surface may be determinant in the immunopathologic events not only in the early steps of infection but also in the chronic phase.Keywords: Trypanosoma cruzi; extracellular vesicles; innate and chronic immunity; TLR2; α-galactosyl(Published: 26 November 2015)Citation: Journal of Extracellular Vesicles 2015, 4: 28734 - http://dx.doi.org/10.3402/jev.v4.28734

In this work, some aspects of the glycobiology of Leishmania shawi were examined, as it is a causative agent of cutaneous leishmaniasis in the New World. Additionally, the interaction of L. shawi's main glycoconjugates [lipophosphoglycan (LPG) and glycoinositolphospholipids (GIPLs)] with macrophages was evaluated in vitro. L. shawi LPG was devoid of side-chains in its repeat units, whereas monosaccharide analysis showed that GIPLs were suggestive of mannose-rich (type I or hybrid). In order to evaluate the biological roles of those molecules, BALB/c resident peritoneal macrophages were incubated with these glycoconjugates for 24h, and the levels of nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-12p70 and IL-10, were determined. In general, the GIPLs exhibited a greater proinflammatory role than the LPGs did. However, for the first time, the GIPLs from this species were able to trigger the production of IL-10, an anti-inflammatory cytokine. In conclusion, L. shawi glycoconjugates were able to interact with the innate immune compartment. These data reinforce the role of parasite glycoconjugates during parasite and host cell interactions.

Neutrophils are rapidly recruited to the site of Leishmania infection and play an active role in capturing and killing parasites. They are the main source of leukotriene B4 (LTB4), a potent proinflammatory lipid mediator. However, the role of LTB4 in neutrophil infection by Leishmania amazonensis is not clear. In this study, we show that L. amazonensis or its lipophosphoglycan can induce neutrophil activation, degranulation, and LTB4 production. Using pharmacological inhibitors of leukotriene synthesis, our findings reveal an LTB4-driven autocrine/paracrine regulatory effect. In particular, neutrophil-derived LTB4 controls L. amazonensis killing, degranulation, and reactive oxygen species production. In addition, L. amazonensis infection induces an early increase in Toll-like receptor 2 expression, which facilitates parasite internalization. Nuclear factor kappa B (NFkB) pathway activation represents a required upstream event for L. amazonensis–induced LTB4 synthesis. These leishmanicidal mechanisms mediated by neutrophil-derived LTB4 act through activation of its receptor, B leukotriene receptor 1 (BLT1).

Leishmania amazonensisis the etiologic agent of the cutaneous and diffuse leishmaniasis often associated with drug resistance. Lapachol [2-hydroxy-3-(3′-methyl-2-butenyl)-1,4-naphthoquinone] displays a wide range of antimicrobial properties against many pathogens. In this study, using the classic microscopicin vitromodel, we have analyzed the effects of a series of lapachol and chlorides complexes with antimony (V), bismuth (V), and tin (IV) againstL. amazonensis. All seven compounds exhibited antileishmanial activity, but most of the antimony (V) and bismuth (V) complexes were toxic against human HepG2 cells and murine macrophages. The best IC50values (0.17±0.03 and 0.10±0.11 μg/mL) were observed for Tin (IV) complexes (3) [(Lp)(Ph3Sn)] and (6) (Ph3SnCl2), respectively. Their selective indexes (SIs) were 70.65 and 120.35 for HepG2 cells, respectively. However, while analyzing murine macrophages, the SI decreased. Those compounds were moderately toxic for HepG2 cells and toxic for murine macrophages, still underlying the need of chemical modification in this class of compounds.

The immunomodulatory properties of lipophosphoglycans (LPG) from New World species of Leishmania have been assessed in Leishmania infantum and Leishmania braziliensis, the causative agents of visceral and cutaneous leishmaniasis, respectively. This glycoconjugate is highly polymorphic among species with variation in sugars that branch off the conserved Gal(β1,4)Man(α1)-PO4 backbone of repeat units. Here, the immunomodulatory activity of LPGs from Leishmania amazonensis, the causative agent of diffuse cutaneous leishmaniasis, was evaluated in two strains from Brazil. One strain (PH8) was originally isolated from the sand fly and the other (Josefa) was isolated from a human case. The ability of purified LPGs from both strains was investigated during in vitro interaction with peritoneal murine macrophages and CHO cells and in vivo infection with Lutzomyia migonei. In peritoneal murine macrophages, the LPGs from both strains activated TLR4. Both LPGs equally activate MAPKs and the NF-κB inhibitor p-IκBα, but were not able to translocate NF-κB. In vivo experiments with sand flies showed that both stains were able to sustain infection in L. migonei. A preliminary biochemical analysis indicates intraspecies variation in the LPG sugar moieties. However, they did not result in different activation profiles of the innate immune system. Also those polymorphisms did not affect infectivity to the sand fly., Author Summary Leishmania amazonensis, a member of the Leishmania mexicana complex, is the causative agent of localized cutaneous leishmaniasis (LCL) and anergic diffuse cutaneous leishmaniasis (ADCL) [1,2]. It is widely distributed throughout the Amazon basin, where it infects a wide range of terrestrial rodents and, less frequently, marsupials. Its main vector is Lutzomyia flaviscutellata (Diptera: Psychodidae) widely distributed in South America and a recent study has predicted its expansion towards South of Brazil [3]. Moreover, Lutzomyia migonei (França, 1920) can also harbor the infection of this species [4,5]. Although its transmission to man is very uncommon, L. amazonensis triggers an incurable and disseminated form of cutaneous leishmaniasis [2,6]. However, most of the mechanisms involved in L. amazonensis pathogenesis are still unknown, especially those related to surface molecules. Glycoconjugates have been extensively characterized as important for the establishment of infection as they protect the parasite from the early action of the host immune system and therefore acting as invasive/evasive strategies. Consequently, we here present the role of lipophosphoglycan (LPG) of L. amazonensis in the interaction with vertebrate and invertebrate hosts.

BACKGROUND Lutzomyia umbratilis, the vector for Leishmania guyanensis in northern South America, has been found naturally infected with L. guyanensis only in areas north of the Negro and Amazon rivers. While populations of this sand fly species are also found in areas south of these rivers, these populations have never been reported to be infected and/or transmitting L. guyanensis. However, no studies on the corresponding host-parasite interactions are available. OBJECTIVES This study evaluated the interaction between Lu. guyanensis promastigotes and field-collected Lu. umbratilis sand flies from Rio Preto da Eva and Manacapuru, which are located to the north and south, respectively, of the Negro River. METHODS Procyclic and metacyclic attachment was quantified using an in vitro system. FINDINGS Low attachment of parasites to the midguts of insects collected from Manacapuru was detected. Conversely, greater binding of metacyclic parasites was observed in the midguts of insects collected from Rio Preto da Eva, and this attachment was more pronounced than that observed for procyclics (p < 0.03). MAIN CONCLUSIONS The Lu. umbratilis population from an area south of the Negro River has lower in vitro interaction with L. guyanensis. The higher attachment of L. guyanensis to midguts of insects from Rio Preto da Eva may suggest better vector competence. These findings are in accordance with previously reported epidemiological information of American cutaneous leishmaniasis (ACL) transmission in the Amazon.