Leishmania (Sauroleishmania) tarentolae versus pathogenic species: comparative evaluation of protease activity, glycoconjugates, resistance to complement and metabolome composition.

BACKGROUND: Leishmania tarentolae is a non-pathogenic species found in lizards representing an important model for Leishmania biology. However, several aspects of this Sauroleishmania remain unknown to explain its low level of virulence. OBJECTIVES: We reported several aspects of L. tarentolae biology including glycoconjugates, proteolytic activities and metabolome composition in comparison to pathogenic species (Leishmania amazonensis, Leishmania braziliensis, Leishmania infantum and Leishmania major). METHODS: Parasites were cultured for extraction and purification of lipophosphoglycan (LPG), immunofluorescence probing with anti-gp63 and resistance against complement. Parasite extracts were also tested for proteases activity and metabolome composition. FINDINGS: Leishmania tarentolae does not express LPG on its surface. It expresses gp63 at lower levels compared to pathogenic species and, is highly sensitive to complement-mediated lysis. This species also lacks intracellular/extracellular activities of proteolytic enzymes. It has metabolic differences with pathogenic species, exhibiting a lower abundance of metabolites including ABC transporters, biosynthesis of unsaturated fatty acids and steroids, TCA cycle, glycine/serine/threonine metabolism, glyoxylate/dicarboxylate metabolism and pentose-phosphate pathways. MAIN CONCLUSIONS: The non-pathogenic phenotype of L. tarentolae is associated with alterations in several biochemical and molecular features. This reinforces the need of comparative studies between pathogenic and non-pathogenic species to elucidate the molecular mechanisms of virulence during host-parasite interactions.

Analysis of KRAS, BRAF, and EGFR mutational status in respiratory epithelial adenomatoid hamartoma (REAH).

BACKGROUND: Respiratory epithelial adenomatoid hamartoma (REAH) is a sinonasal glandular overgrowth arising from the surface respiratory epithelium and invaginating into the stroma. Clinically, it appears as a polypoid mass that may cause nasal obstruction, anosmia, and epistaxis. The presence of cartilaginous and/or osseous areas move the lesion to a chondro-osseous respiratory epithelial (CORE) hamartoma subtype. Scattered small seromucinous glands may be observed between typical REAH glands and when it is the only feature, it represents seromucinous hamartoma (SH). The molecular pathogenesis of REAH has been poorly explored and remains unclear. Given that KRAS, BRAF, and EGFR mutations have been detected in a variety of sinonasal tumors, we aimed to assess these mutations in REAH and SH. METHODS: Ten REAH (including one CORE subtype), in addition to two SH cases, were Sanger sequenced by standard techniques. The targeted regions included KRAS exons 2-4 (encompassing hotspots codons 12, 13, 61, and 146), BRAF exons 11 and 15 (spanning the V600 codon), and EGFR exons 19 and 20. RESULTS: All REAH and SH samples showed wild-type sequences for KRAS, BRAF, and EGFR genes. CONCLUSION: Our results demonstrate a lack of KRAS, BRAF, or EGFR pathogenic variants with further evaluation of REAH and SH needed to elucidate driver genetic events.

Leptomonas pyrrhocoris: Genomic insight into Parasite's Physiology.

BACKGROUND: Leptomonas pyrrhocoris is a parasite of the firebug Pyrrhocoris apterus. This flagellate has been recently proposed as a model species for studying different aspects of the biology of monoxenous trypanosomatids, including host - parasite interactions. During its life cycle L. pyrrhocoris never tightly attaches to the epithelium of the insect gut. In contrast, its dixenous relatives (Leishmania spp.) establish a stable infection via attachment to the intestinal walls of their insect hosts. MATERIAL AND METHODS: This process is mediated by chemical modifications of the cell surface lipophosphoglycans. In our study we tested whether the inability of L. pyrrhocoris to attach to the firebug's midgut is associated with the absence of these glycoconjugates. We also analyzed evolution of the proteins involved in proper lipophosphoglycan assembly, cell attachment and establishment of a stable infection in L. pyrrhocoris, L. seymouri, and Leishmania spp. Our comparative analysis demonstrated differences in SCG/L/R repertoire between the two parasite subgenera, Leishmania and Viannia, which may be related to distinct life strategies in various Leishmania spp. The genome of L. pyrrhocoris encodes 6 SCG genes, all of which are quite divergent from their orthologs in the genus Leishmania. Using direct probing with an antibody recognizing the ß-Gal side chains of lipophosphoglycans, we confirmed that these structures are not synthesized in L. pyrrhocoris. CONCLUSION: We conclude that either the SCG enzymes are not active in this species (similarly to SCG5/7 in L. major), or they possess a different biochemical activity.

Intraspecies Polymorphisms in the Lipophosphoglycan of L. braziliensis Differentially Modulate Macrophage Activation via TLR4.

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-1ß, 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.

Leishmania (Sauroleishmania) tarentolae versus pathogenic species: comparative evaluation of protease activity, glycoconjugates, resistance to complement and metabolome composition

BACKGROUND Leishmania tarentolae is a non-pathogenic species found in lizards representing an important model for Leishmania biology. However, several aspects of this Sauroleishmania remain unknown to explain its low level of virulence. OBJECTIVES We reported several aspects of L. tarentolae biology including glycoconjugates, proteolytic activities and metabolome composition in comparison to pathogenic species (Leishmania amazonensis, Leishmania braziliensis, Leishmania infantum and Leishmania major). METHODS Parasites were cultured for extraction and purification of lipophosphoglycan (LPG), immunofluorescence probing with anti-gp63 and resistance against complement. Parasite extracts were also tested for proteases activity and metabolome composition. FINDINGS Leishmania tarentolae does not express LPG on its surface. It expresses gp63 at lower levels compared to pathogenic species and, is highly sensitive to complement-mediated lysis. This species also lacks intracellular/extracellular activities of proteolytic enzymes. It has metabolic differences with pathogenic species, exhibiting a lower abundance of metabolites including ABC transporters, biosynthesis of unsaturated fatty acids and steroids, TCA cycle, glycine/serine/threonine metabolism, glyoxylate/dicarboxylate metabolism and pentose-phosphate pathways. MAIN CONCLUSIONS The non-pathogenic phenotype of L. tarentolae is associated with alterations in several biochemical and molecular features. This reinforces the need of comparative studies between pathogenic and non-pathogenic species to elucidate the molecular mechanisms of virulence during host-parasite interactions.

O impacto de cepas de Leishmania braziliensis isoladas de hospedeiros com diferentes formas clínicas na interação com macrófagos murinos

Leishmania (Viannia) braziliensis é o agente etiológico da leishmaniose tegumentar americana (LTA) podendo causar diferentes formas clínicas: leishmaniose cutânea (CL), muco-cutânea (LMC) e lesões atípicas (LAT). Estas últimas podem apresentar-se como pápulas, placas, nódulos verrucosos, lesões vegetativas e/ou lupóides. Nossa hipótese de trabalho seria quais os fatores responsáveis por estas diferentes manifestações clínicas dentro desta espécie. Foram avaliados diferentes parâmetros relacionados à infectividade do parasito: 1) papel do lipofosfoglicano (LPG) na interação com macrófagos peritoneais; 2) avaliação dos parasitos na biogênese de fagossomos em macrófagos medulares (BMMs); 3) avaliação e quantificação da expressão de LPG e GP63 na superfície dos parasitos e, 4) caracterização preliminar de LPGs purificados destes parasitos. Utilizamos seis cepas/isolados de L. braziliensis: M2903, RR051 e RR418 (lesões típicas), RR410 (atípica), M15991 (muco-cutânea) e M8401 (cepa isolada de vetor). LPGs das diferentes cepas/isolados de L. braziliensis foram extraídos e purificados. Macrofágos peritoneais de camundongos 57BL/6 e respectivos knock-outs (TLR2 -/- e TLR4 -/-) foram estimulados por estes LPGs para a produção de óxido nítrico (NO) e citocinas. Os LPGs, dependendo da forma clínica da cepa de que foram purificados, ativaram diferencialmente a produção de NO e citocinas (TNF-α, IL-6, IL-12) via LR4. Não houve produção de IL-1ß. Νο próximo passo, incubamos os mesmos LPGs em presença de zimozan. Os LPGs aderidos ao zimozam foram incubados com BMMs para a avaliação do marcador de biogênese LAMP1 (recrutamento e fagocitose). Não foi observada diferenças na biogênese de fagossomos de BMMs incubados com zimozan e LPGs das diferentes cepas/isolados. Posteriormente, os parasitos foram opsonizados e incubados em presença dos BMMs para a avaliação infecção. A cepa RR410 (atípica) apresentou um índice de infectividade menor quando comparada às outras cepas. Estes mesmos experimentos foram realizados na presença de LAMP1 e Lysotracker (fagocitose e acidificação). Os parasitos induziram diferenças na biogênese de fagossomos dependendo da cepa/isolado. Por exemplo, a cepa atípica (RR410) foi menos fagocitada que as outras, enquanto a cepa M15991 (muco-cutânea) acidificou menos o fagolissomo. Para a avaliação da expressão de LPG e GP63, foi observado em comparação aos controles (Leishmania donovani e Leishmania major) que ambos glicoconjugados estavam menos expressos na superfície de L. braziliensis. Nossos resultados demostraram que diferentes isolados de L. braziliensis de diferentes formas clínicas e hospedeiros podem induzir diferentes respostas em células do hospedeiro vertebrado. Este efeito se deu principalmente, na fagocitose, produção de NO e citocinas, desfechos na biogênese dos fagossomos e expressão de glicoconjugados (LPG e GP63). A caracterização preliminar das unidades repetitivas do LPG detectou polimorfismos que podem justificar os diferentes padrões de ativação em macrófagos peritoneais e de infecção com os parasitos inteiros.