Halictine-2 antimicrobial peptide shows promising anti-parasitic activity against Leishmania spp.

The protozoan parasite Leishmania spp. causes leishmaniases, a group of diseases creating serious health problems in many parts of the world with significant resistance to existing drugs. Insect derived antimicrobial peptides are promising alternatives to conventional drugs against several human disease-causing pathogens because they do not generate resistance. Halictine-2, a novel antimicrobial peptide from the venom of eusocial honeybee, Halictus sexcinctus showed significant anti-leishmanial activity in vitro, towards two life forms of the dimorphic parasite, the free-swimming infective metacyclic promastigotes and the intracellular amastigotes responsible for the systemic infection. The anti-leishmanial activity of the native peptide (P5S) was significantly enhanced by serine to threonine substitution at position 5 (P5T). The peptide showed a propensity to form α-helices after substitution at position-5, conferring amphipathicity. Distinct pores observed on the promastigote membrane after P5T exposure suggested a mechanism of disruption of cellular integrity. Biochemical alterations in the promastigotes after P5T exposure included generation of increased oxygen radicals with mitochondrial Ca2+ release, loss of mitochondrial membrane potential, reduction in total ATP content and increased mitochondrial mass, resulting in quick bioenergetic and chemiosmotic collapse leading to cell death characterized by DNA fragmentation. P5T was able to reduce intracellular amastigote burden in an in vitro model of Leishmania infection but did not alter the proinflammatory cytokines like TNF-α and IL-6. The ability of the P5T peptide to kill the Leishmania parasite with negligible haemolytic activity towards mouse macrophages and human erythrocytes respectively, demonstrates its potential to be considered as a future antileishmanial drug candidate.

Leishmania donovani Induces Autophagy in Human Blood-Derived Neutrophils.

Neutrophils, the essential components of the innate immune system, are recruited in large numbers to the pathogen site of entry. Several pathogens induce neutrophil autophagy; however, function of autophagic events during Leishmania parasite infection remain unknown. In this article, we report a finding that is new, to our knowledge, of how Leishmania-induced human polymorphonuclear neutrophil (hPMN) autophagy regulates the silent mode of parasite transfer to macrophages by influencing the engulfment of infected cells. Leishmania infection induced a time-dependent autophagy increase responsive to block by 3-methyladenine but sensitive to ULK1/2 inhibition only after 3 h. This suggested the prevalence of canonical autophagy during later hours, ULK1/2 inhibition being able to block only canonical autophagy. Interaction of Rubicon and Beclin-1 at 1 h postinfection affirmed the prevalence of noncanonical autophagy during early infection. There was a reduction in macrophage uptake of parasite-exposed hPMNs treated with 3-methyladenine or ULK1/2 inhibitor, suggesting the involvement of both noncanonical and canonical autophagy in neutrophil engulfment. Autophagy inducer rapamycin augmented neutrophil engulfment by macrophages. Redistribution of hPMN surface CD47 encouraged neutrophil uptake. Activation of ERK, phosphoinositide 3-kinase, and NADPH oxidase-mediated reactive oxygen species generation were induced after parasite binding. The lpg1-knockout parasites expressing defective lipophosphoglycan did not induce autophagy, indicating that lipophosphoglycan is necessary for interaction with the neutrophils. Autophagy induction was TLR2/4 independent because the receptor blockade did not interfere with infection-induced autophagy. In summary, the engulfment of neutrophils by the macrophages was influenced by the escalation of hPMN autophagy, which is an important event during Leishmania infection.