RETRACTED ARTICLE: Role of inhibitors of serine peptidases in protecting Leishmania donovani against the hydrolytic peptidases of sand fly midgut

Verma, Sudha; Das, Sushmita; Mandal, Arabinda; Ansari, Yousuf; Kumari, Sujata; Mansuri, Rani; Kumar, Ajay; Singh, R. A.; Saini, Savita; Abhishek, Kumar; Kumar, Vijay; Sahoo, Ganesh Chandra; Das, Pradeep

doi:10.1186/s13071-017-2239-9

Cited by 12 publications

(18 citation statements)

References 51 publications

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“…Similarly, all but one publications on Leishmania ecotin orthologs indicated that the main function of these proteins is protection against intestinal proteases in the gut of the insect Ecotin protects bacteria against human serum-mediated lysis vector, or against contact pathway-mediated bactericidal effects of the host [47,48], or downregulation of inflammatory monocytes and monocyte-derived cells [49]. We found only a single, very recent paper reporting that Leishmania donovani ecotin ortholog LnISP2 is not only a neutrophil elastase inhibitor, but also a MASP-2 inhibitor [40].…”

Section: Discussionmentioning

confidence: 99%

Ecotin, a microbial inhibitor of serine proteases, blocks multiple complement dependent and independent microbicidal activities of human serum

et al. 2019

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Ecotin is a serine protease inhibitor produced by hundreds of microbial species, including pathogens. Here we show, that ecotin orthologs from Escherichia coli, Yersinia pestis, Pseudomonas aeruginosa and Leishmania major are potent inhibitors of MASP-1 and MASP-2, the two key activator proteases of the complement lectin pathway. Factor D is the key activator protease of another complement activation route, the alternative pathway. We show that ecotin inhibits MASP-3, which is the sole factor D activator in resting human blood. In pathway-specific ELISA tests, we found that all ecotin orthologs are potent lectin pathway inhibitors, and at high concentration, they block the alternative pathway as well. In flow cytometry experiments, we compared the extent of complement-mediated opsonization and lysis of wild-type and ecotin-knockout variants of two E. coli strains carrying different surface lipopolysaccharides. We show, that endogenous ecotin provides significant protections against these microbicidal activities for both bacteria. By using pathway specific complement inhibitors, we detected classical-, lectin-and alternative pathway-driven complement attack from normal serum, with the relative contributions of the activation routes depending on the lipopolysaccharide type. Moreover, in cell proliferation experiments we observed an additional, complement-unrelated antimicrobial activity exerted by heat-inactivated serum. While ecotin-knockout cells are highly vulnerable to these activities, endogenous ecotin of wild-type bacteria provides complete protection against the lectin pathwayrelated and the complement-unrelated attack, and partial protection against the alternative pathway-related damage. In all, ecotin emerges as a potent, versatile self-defense tool that blocks multiple antimicrobial activities of the serum. These findings suggest that ecotin might be a relevant antimicrobial drug target.Bloodstream infections are major cause of morbidity and mortality in many countries around the globe. As the number of multi-drug resistant pathogenic strains is growing, it is urgent to identify their virulence factors and unveil the corresponding mechanisms of action that enable the pathogen to avoid potent immune response. A microbial inhibitor of serine proteases, ecotin was previously implicated in protecting various pathogenic bacteria and eukaryotic Leishmania species against the host immune system by inhibiting leukocyte elastase. However, the interaction of ecotin with the complement system, which provides a first line defense against pathogens, remained unexplored. We found that ecotin blocks activation of the complement lectin pathway by inhibiting its key activator enzymes, MASP-1 and MASP-2. Furthermore, by inhibiting MASP-3, ecotin also disrupts a fundamental link between the lectin-and the alternative pathways. We provide evidence that E. coli cells devoid of ecotin are extremely vulnerable to complement-mediated lysis and they are also potently killed by some complement-independent antimicrobial factors o...

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Section: Discussionmentioning

confidence: 99%

Ecotin, a microbial inhibitor of serine proteases, blocks multiple complement dependent and independent microbicidal activities of human serum

et al. 2019

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“…L. donovani promastigotes (AG83:MHOM/IN/1983/AG83) were used in our experiments. ISP2KD and ISP2 overexpressed (ISP2OE) Leishmania parasites were prepared in our lab as described previously ( 24 ). All the cell lines promastigotes were maintained according to Mandal et al ( 25 ) at 25°C in 25 cm 2 flasks in fresh Medium-199 supplemented with 10% FBS.…”

Section: Methodsmentioning

confidence: 99%

“…All the cell lines promastigotes were maintained according to Mandal et al ( 25 ) at 25°C in 25 cm 2 flasks in fresh Medium-199 supplemented with 10% FBS. The ISP2KD and ISP2OE cells were maintained in M199 media supplemented with neomycin (G418) at 200 µg/ml concentration ( 24 ).…”

Section: Methodsmentioning

confidence: 99%

Leishmania donovani Inhibitor of Serine Peptidases 2 Mediated Inhibition of Lectin Pathway and Upregulation of C5aR Signaling Promote Parasite Survival inside Host

et al. 2018

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Leishmania donovani, the causative agent of Indian visceral leishmaniasis has to face several barriers of the immune system inside the mammalian host for its survival. The complement system is one of the first barriers and consists of a well-balanced network of proteases including S1A family serine proteases (SPs). Inhibitor of serine peptidases (ISPs) is considered as inhibitor of S1A family serine peptidases and is reported to be present in trypanosomes, including Leishmania. In our previous study, we have deciphered the role of ISPs [LdISP1 and L. donovani inhibitor of serine peptidases 2 (LdISP2)] in the survival of L. donovani inside the sandfly midgut. However, the role of theses ISPs in the survival of L. donovani inside mammalian host still remains elusive. In the present study, we have deciphered the inhibitory effect of LdISPs on the host complement S1A serine peptidases, such as C1r/C1s and MASP1/MASP2. Our study suggested that although both rLdISP1 and rLdISP2 inferred strong interaction with C1complex and MBL-associated serine proteases (MASPs) but rLdISP2 showed the stronger inhibitory effect on MASP2 than rLdISP1. Moreover, we found that rLdISP2 significantly reduces the formation of C3, C5 convertase, and membrane attacking complex (MAC) by lectin pathway (LP) resulting in significant reduction in serum mediated lysis of the parasites. The role of LdISP2 on neutrophil elastase-mediated C5aR signaling was also evaluated. Notably, our results showed that infection of macrophages with ISP2-overexpressed Leishmania parasites significantly induces the expression of C5aR both at the transcript and translational level. Simultaneously, infection with ISP2KD parasites results in downregulation of host PI3K/AKT phosphorylation and increased in IL-12 production. Taken together, our findings clearly suggest that LdISP2 promotes parasite survival inside host by inhibiting MAC formation and complement-mediated lysis via LP and by upregulation of C5aR signaling.

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“…Those observations helped to define ISP2 as a virulence factor required for successful infection by L. major (21, 22), but the potential contribution of ISP2 in modulating NE activity and infection in VL has not been investigated. mRNA to ISP2 was detected in L. donovani (23), and studies in vitro using recombinant ISP2 from this species [ L. donovani inhibitor of serine peptidase 2 (LdISP2)] showed that it can inhibit trypsin and NE (24, 25). Recombinant ISP2 also inhibited serine peptidases of the sand fly midgut in vitro , and transgenic lines knocked down to ISP2 were more susceptible to sand fly peptidases and less fit to survive in the midgut (23).…”

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confidence: 99%

“…mRNA to ISP2 was detected in L. donovani (23), and studies in vitro using recombinant ISP2 from this species [ L. donovani inhibitor of serine peptidase 2 (LdISP2)] showed that it can inhibit trypsin and NE (24, 25). Recombinant ISP2 also inhibited serine peptidases of the sand fly midgut in vitro , and transgenic lines knocked down to ISP2 were more susceptible to sand fly peptidases and less fit to survive in the midgut (23). Other studies applying treatment of infected RAW cells with recombinant LdISP2 showed slight reduction of infection and of NO production (25).…”

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confidence: 99%

Neutrophil elastase promotes Leishmania donovani infection via interferon‐β

et al. 2019

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Visceral leishmaniasis is a deadly illness caused by Leishmania donovani that provokes liver and spleen inflammation and tissue destruction. In cutaneous leishmaniasis, the protein of L. major, named inhibitor of serine peptidases (ISP) 2, inactivates neutrophil elastase (NE) present at the macrophage surface, resulting in blockade of TLR4 activation, prevention of TNF‐α* and IFN‐β production, and parasite survival. We report poor intracellular growth of L. donovani in macrophages from knockout mice for NE (ela−/−), TLR4, or TLR2. NE and TLR4 colocalized with the parasite in the parasitophorous vacuole. Parasite load in the liver and spleen of ela−/− mice were reduced and accompanied by increased NO and decreased TGF‐β production. Expression of ISP2 was not detected in L. donovani, and a transgenic line constitutively expressing ISP2, displayed poor intracellular growth in macrophages and decreased burden in mice. Infected ela−/− macrophages displayed significantly lower IFN‐β mRNA than background mice macrophages, and the intracellular growth was fully restored by exogenous IFN‐β. We propose that L. donovani utilizes the host NE‐TLR machinery to induce IFN‐β necessary for parasite survival and growth during early infection. Low or absent expression of parasite ISP2 in L. donovani is necessary to preserve the activation of the NE‐TLR pathway.—Dias, B. T., Dias‐Teixeira, K. L., Godinho, J. P., Faria, M. S., Calegari‐Silva, T., Mukhtar, M. M., Lopes, U. G., Mottram, J. C., Lima, A. P. C. A. Neutrophil elastase promotes Leishmania donovani infection via interferon‐β. FASEB J. 33,10794–10807 (2019). http://www.fasebj.org

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RETRACTED ARTICLE: Role of inhibitors of serine peptidases in protecting Leishmania donovani against the hydrolytic peptidases of sand fly midgut

Cited by 12 publications

References 51 publications

Ecotin, a microbial inhibitor of serine proteases, blocks multiple complement dependent and independent microbicidal activities of human serum

Ecotin, a microbial inhibitor of serine proteases, blocks multiple complement dependent and independent microbicidal activities of human serum

Leishmania donovani Inhibitor of Serine Peptidases 2 Mediated Inhibition of Lectin Pathway and Upregulation of C5aR Signaling Promote Parasite Survival inside Host

Neutrophil elastase promotes Leishmania donovani infection via interferon‐β

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