Leishmania infantum Defective in Lipophosphoglycan Biosynthesis Interferes With Activation of Human Neutrophils

Abstract

Visceral leishmaniasis (VL) is often associated with hematologic manifestations that may interfere with neutrophil response. Lipophosphoglycan (LPG) is a major molecule on the surface of Leishmania promastigotes, which has been associated with several aspects of the parasite–vector–host interplay. Here, we investigated how LPG from Leishmania (L.) infantum, the principal etiological agent of VL in the New World, influences the initial establishment of infection during interaction with human neutrophils in an experimental setting in vitro. Human neutrophils obtained from peripheral blood samples were infected with either the wild-type L. infantum (WT) strain or LPG-deficient mutant (∆lpg1). In this setting, ∆lpg1 parasites displayed reduced viability compared to WT L. infantum; such finding was reverted in the complemented ∆lpg1+LPG1 parasites at 3- and 6-h post-infection. Confocal microscopy experiments indicated that this decreased survival was related to enhanced lysosomal fusion. In fact, LPG-deficient L. infantum parasites more frequently died inside neutrophil acidic compartments, a phenomenon that was reverted when host cells were treated with Wortmannin. We also observed an increase in the secretion of the neutrophil collagenase matrix metalloproteinase-8 (MMP-8) by cells infected with ∆lpg1 L. infantum compared to those that were infected with WT parasites. Furthermore, collagen I matrix degradation was found to be significantly increased in ∆lpg1 parasite-infected cells but not in WT-infected controls. Flow cytometry analysis revealed a substantial boost in production of reactive oxygen species (ROS) during infection with either WT or ∆lpg1 L. infantum. In addition, killing of ∆lpg1 parasites was shown to be more dependent on the ROS production than that of WT L. infantum. Notably, inhibition of the oxidative stress with Apocynin potentially fueled ∆lpg1 L. infantum fitness as it increased the intracellular parasite viability. Thus, our observations demonstrate that LPG may be a critical molecule fostering parasite survival in human neutrophils through a mechanism that involves cellular activation and generation of free radicals.

Quantitative single-cell analysis of Leishmania major amastigote differentiation demonstrates variably extended expression of the lipophosphoglycan (LPG) virulence factor in different host cell types

“…As this is well beyond the estimated transit time of Leishmania through “first contact” cells to macrophages (~24 hours), these data establish that the destination macrophages likely encounter a significant number of Leishmania retaining biologically relevant levels of LPG capable of mediating virulence functions. For neutrophils, persistence of LPG expression is a necessary step for survival and likely transit [ 23 , 49 ].…”

Humoral response in Leishmaniasis

“…More recent studies with L. infantum investigated how the LPG molecule influences the initial establishment of infection during interaction with human neutrophils in an in vitro experimental environment. They observed that mutant parasites that did not express LPG had a reduced viability and that this was related to an increased lysosomal fusion in the neutrophils evaluated by confocal microscopy ( Quintela-Carvalho et al., 2022 ). Other remarkable adaptive mechanisms include inhibition of phagosome-endosome fusion ( Desjardins and Descoteaux, 1997 ), expression of hydrolytic enzymes, modulation of cell signaling pathways ( Eilam et al., 1985 ), nitric oxide production ( Wei et al., 1995 ), and cytokine induction ( Barral-Netto et al., 1992 ).…”