Influence of parasite encoded inhibitors of serine peptidases in early infection of macrophages withLeishmania major

Abstract

SummaryEcotin is a potent inhibitor of family S1A serine peptidases, enzymes lacking in the protozoan parasite Leishmania major. Nevertheless, L. major has three ecotin-like genes, termed inhibitor of serine peptidase (ISP). ISP1 is expressed in vectorborne procyclic and metacyclic promastigotes, whereas ISP2 is also expressed in the mammalian amastigote stage. Recombinant ISP2 inhibited neutrophil elastase, trypsin and chymotrypsin with Kis between 7.7 and 83 nM. L. major ISP2-ISP3 double null mutants (Disp2/3) were created. These grew normally as promastigotes, but were internalized by macrophages more efficiently than wild-type parasites due to the upregulation of phagocytosis by a mechanism dependent on serine peptidase activity. Disp2/3 promastigotes transformed to amastigotes, but failed to divide for 48 h. Intracellular multiplication of Disp2/3 was similar to wild-type parasites when serine peptidase inhibitors were present, suggesting that defective intracellular growth results from the lack of serine peptidase inhibition during promastigote uptake. Disp2/3 mutants were more infective than wild-type parasites to BALB/c mice at the early stages of infection, but became equivalent as the infection progressed. These data support the hypothesis that ISPs of L. major target host serine peptidases and influence the early stages of infection of the mammalian host.

Inhibitor of serine peptidase 2 enhancesLeishmania majorsurvival in the skin through control of monocytes and monocyte‐derived cells

“…Overall, these data suggest that ISP2 is important for L. major survival at the site of infection; it contributes to the control of parasite load early in infection for enhanced survival at later stages of infection. The ISP2/3 reexpressing line, which has a higher level of ISP2 than L. major WT (18), demonstrated slower parasite expansion early in infection, with significantly increased burdens compared with Δ isp2/3 later. These observations suggest that the fine-tuning of ISP2 expression can have a major impact on skin infection in a physiologic model of infection.…”

“…Parasite lines that were deficient in ISP2 and ISP3 (Δ isp2/3 ) and that reexpressed ISP2 and ISP3 (Δ isp2/3:ISP2/3 ) were generated by gene replacement and reintroduction, as described previously (18). Promastigotes of Δ isp2/3 and Δ isp2/3:ISP2/3 grew similarly to L. major wild type (WT) in vitro (Supplemental Fig.…”

“…We have recently identified another Leishmania virulence factor, termed inhibitor of serine peptidase 2 (ISP2), that is important for the establishment of murine macrophage infections in vitro (18, 19). ISP2 is one of 3 orthologs of the bacterial protease inhibitor, ecotin, which inactivates serine peptidases from the S1A family, including neutrophil elastase (NE), cathepsin G, and proteinase 3 (20, 21).…”

“…Leishmania lacks genes that encode S1A serine peptidases, and ISP2 has been shown to be a potent inhibitor of NE (18, 28), a peptidase that is found in the azurophilic granules of neutrophils and on the surface of monocytes and macrophages. Inhibition of NE activity by ISP2 during Leishmania– macrophage interaction in vitro prevents the activation of TLR4-mediated responses, including reactive oxygen species production, favoring parasite survival and intracellular development (19).…”

Macrophages and neutrophils cooperate in immune responses to Leishmania infection

“…These results suggest that the proinflammatory and leishmanicidal activities of B6 neutrophils are due to efficient amounts of NE, and perhaps other granule proteins released into the medium. Interestingly, L. major expresses inhibitor of serine peptidase-2, a virulence factor that prevents TLR4 activation by NE, and promotes parasite survival in macrophages [36,37].…”