3′-Nucleotidase/Nuclease Activity Allows Leishmania Parasites To Escape Killing by Neutrophil Extracellular Traps

Abstract

Leishmaniasis is a widespread neglected tropical disease caused by parasites of the Leishmania genus. These parasites express the enzyme 3=-nucleotidase/nuclease (3=NT/NU), which has been described to be involved in parasite nutrition and infection. Bacteria that express nucleases escape the toxic effects of neutrophil extracellular traps (NETs). Hence, we investigated the role of 3=NT/NU in Leishmania survival of NET-mediated killing. Promastigotes of Leishmania infantum were cultured in high-phosphate (HP) or low-phosphate (LP) medium to modulate nuclease activity. We compared the survival of the two different groups of Leishmania during interaction with human neutrophils, assessing the role of neutrophil extracellular traps. As previously reported, we detected higher nuclease activity in parasites cultured in LP medium. Both LP and HP promastigotes were capable of inducing the release of neutrophil extracellular traps from human neutrophils in a dose-and time-dependent manner. LP parasites had 2.4 times more survival than HP promastigotes. NET disruption was prevented by the treatment of the parasites with ammonium tetrathiomolybdate (TTM), a 3=NT/NU inhibitor. Inhibition of 3=NT/NU by 3=-AMP, 5=-GMP, or TTM decreased promastigote survival upon interaction with neutrophils. Our results show that Leishmania infantum induces NET release and that promastigotes can escape NET-mediated killing by 3=-nucleotidase/nuclease activity, thus ascribing a new function to this enzyme. N eutrophils are short-lived cells and the most abundant leukocytes in the blood circulation; they constitute one of the first lines of defense against invading microorganisms (1). These granulocytes can kill microorganisms by phagocytosis, degranulation, and neutrophil extracellular traps (NETs). NETs are weblike structures composed of chromatin, granules, and cytoplasmic proteins that are extruded when neutrophils undergo NETosis, a unique cell death mechanism (2-5). However, recent work challenges NETosis as a cell death mechanism because live neutrophils were detected after NET extrusion in in vivo studies (6). NETs function by killing and containing pathogens, thereby preventing the pathogen's dissemination through the organism. In addition, some studies have indicated that NETs play a role in autoimmune diseases (7-10).A diverse group of stimuli has been described as activating NETosis (5, 11). Among the parasites, Leishmania promastigotes were demonstrated to activate release of NETs (12, 13). Leishmania amazonensis promastigotes interact intimately with NETs and are killed by web-associated histones (12). However, although promastigotes of Leishmania donovani trigger NET release, these parasites escape the toxicity of NETs (13). Groups of microorganisms have evolved different mechanisms of escaping the toxic effects of NETs. Streptococcus pneumoniae, group A Streptococcus, Streptococcus agalactiae, Staphylococcus aureus, and Vibrio cholerae express endonucleases that efficiently degrade DNA filaments from NETs, allowing these bacteria...

Evasion of Neutrophil Extracellular Traps by Respiratory Pathogens

“…The parasite Leishmania infantum was shown to express high levels of 39-nucleotidase/nuclease when cultured in low-phosphate media, a phenotype that showed higher survival in NETs (40). A highly active nuclease is also present in the saliva of the parasite's sandfly carrier, Lutzomyia longipalpis (41), and a recombinant form of the nuclease, Lutzomyia NET destroying protein, was shown to degrade NETs and increase parasite survival when coincubated with neutrophils.…”

A Thermonuclease ofNeisseria gonorrhoeaeEnhances Bacterial Escape From Killing by Neutrophil Extracellular Traps

“…Species of Streptococcus, Staphylococcus, Vibrio, and Leishmania express nucleases that aid in escape from NETs, to promote pathogen survival and spread [24][25][26][27][28]. We now add N. gonorrhoeae to this list, with 3 pieces of evidence to show that N. gonorrhoeae Nuc can degrade NETs.…”

Leishmania, the phagosome, and host responses: The journey of a parasite

“…The ability to survive NETs is conferred by either LPG or the 3′-Nucleotidase/Nuclease, as parasites defective in LPG expression and parasites with diminished 3'-Nucleotidase/Nuclease activity were more susceptible to NETs killing [34,35]. The 3'-Nucleotidase/Nuclease is a membrane anchored enzyme that was previously associated with nutrition since its activity is able to generate nucleotides and phosphate from nucleic acids [36][37][38].…”