Dados do Trabalho

Título

Exploring the role of phosphoglycans on the virulence of L. amazonensis clinical isolates: insights from in vitro and in vivo assays

Introdução

The lpg2 gene encodes a GDP-mannose transporter involved in synthesizing phosphoglycan-containing molecules (PGs), including (LPG, PPGs, PGs and sAP), which play critical roles in Leishmania infection of the mammalian host. We conducted in vitro and in vivo assays using lpg2 knockout (KO) parasites for two strains of L. amazonensis.

Objetivo (s)

In this study, we investigated the role of these glycoconjugates in two strains of L. amazonensis (La) previously isolated from patients with localized (LCL BA125) or diffuse (DCL BA336) forms of the disease.

Material e Métodos

La lpg2KO strains were generated using CRISPR/CAS9, incorporating an antibiotic resistance gene to disrupt the coding sequence. Clonal lpg2KO parasites were isolated through Zeocin selection and limiting dilution assay. Neutrophils and BMDM were infected with wild-type (WT) or lpg2KO parasites, and parasite burden was determined at 4h or 48h post-infection. Cellular migration assays were performed with human dendritic cells (hDCs) infected with WT or lpg2KO parasites in a Boyden chamber. In vivo evaluation involved the intradermal inoculation of parasites in the ear of BALB/c mice, and lesion development was monitored using an analog caliper for up to 12 weeks.

Resultados e Conclusão

A total of 6 clonal resistant parasites were characterized using PCR, Sanger sequencing and NGS, confirming the disruption of the lpg2 gene. Western blot analysis demonstrated a complete absence of LPG and PG expression in these clonal parasites. In vitro assays revealed no differences in infection and replication rates between lpg2KO and WT parasites in BMDM or human neutrophils. However, both La lpg2KO strains exhibited reduced hDC migration compared to WT parasites. Surprisingly, in vivo infection of mice yielded contrasting outcomes between the two lpg2KO strains. The BA336 lpg2KO strain displayed minimal differences in lesion development, while the BA125 lpg2KO strain failed to induce any lesion development.
Our findings demonstrate successful disruption of the lpg2 gene in BA125 and BA336 strains of La. The absence of PGs does not appear to be essential for parasite virulence in vitro assays, although it does impact hDC migration. In contrast, PGs seems to play an essential role in lesion development of BA125. Further research is needed to fully understand the factors responsible for the divergent in vivo virulence seen in BA125 and BA336 lpg2KO strains.

Palavras-chave

CRISPR, Leishmania, Lipophosphoglycans

Agradecimentos

FAPESB, CAPES, IGM, FIOCRUZ-BA