Ecotin‐like serine peptidase inhibitor ISP1 of<i>Leishmania major</i>plays a role in flagellar pocket dynamics and promastigote differentiation

BackgroundIn vector-borne diseases such as leishmaniasis, the sand fly midgut is considered to be an important site for vector-parasite interaction. Digestive enzymes including serine peptidases such as trypsin and chymotrypsin, which are secreted in the midgut are one of the obstacles for Leishmania in establishing a successful infection. The presence of some natural inhibitors of serine peptidases (ISPs) has recently been reported in Leishmania. In the present study, we deciphered the role of these ISPs in the survival of Leishmania donovani in the hostile sand fly midgut environment.Methods In silico and co-immunoprecipitation studies were performed to observe the interaction of L. donovani ISPs with trypsin and chymotrypsin. Zymography and in vitro enzyme assays were carried out to observe the inhibitory effect of purified recombinant ISPs of L. donovani (rLdISPs) on trypsin, chymotrypsin and the sand fly midgut peptidases. The expression of ISPs in the amastigote to promastigote transition stages were studied by semi-quantitative RT-PCR and Western blot. The role of LdISP on the survival of ISP overexpressed (OE) and ISP knocked down (KD) Leishmania parasites inside the sand fly gut was investigated by in vitro and in vivo cell viability assays.ResultsWe identified two ecotin-like genes in L. donovani, LdISP1 and LdISP2. In silico and co-immunoprecipitation results clearly suggest a strong interaction of LdISP molecules with trypsin and chymotrypsin. Zymography and in vitro enzyme assay confirmed the inhibitory effect of rLdISP on trypsin, chymotrypsin and the sand fly midgut peptidases. The expression of LdISP2 was found to be strongly associated with the amastigote to promastigote phase transition. The activities of the digestive enzymes were found to be significantly reduced in the infected sand flies when compared to uninfected. To our knowledge, our study is the first report showing the possible reduction of chymotrypsin activity in L. donovani infected sand flies compared to uninfected. Interestingly, during the early transition stage, substantial killing was observed in ISP2 knocked down (ISP2KD) parasites compared to wild type (WT), whereas ISP1 knocked down (ISP1KD) parasites remained viable. Therefore, our study clearly indicates that LdISP2 is a more effective inhibitor of serine peptidases than LdISP1.ConclusionOur results suggest that the lack of ISP2 is detrimental to the parasites during the early transition from amastigotes to promastigotes. Moreover, the results of the present study demonstrated for the first time that LdISP2 has an important role in the inhibition of peptidases and promoting L. donovani survival inside the Phlebotomus argentipes midgut.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-017-2239-9) contains supplementary material, which is available to authorized users.

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“…The morphology of the parasites was determined according to previously published methods [32–35] (detail in Additional file 1). …”

Section: Methodsmentioning

confidence: 99%

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

et al. 2017

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“…The ISPs have inhibitory effects against vertebrate macrophage serine proteases, such as neutrophil elastase and one of them (ISP2, [GeneDB: LmjF.15.0510]) has been shown to enhance parasite survival in murine macrophages [49]. The ISPs also inhibit trypsin-like activity of sand fly midguts in-vitro [50]. The possibility of ISPs having an effect on insect midgut proteases in-viv o is currently under investigation in our laboratory.…”

Section: Reviewmentioning

confidence: 99%

Leishmania development in sand flies: parasite-vector interactions overview

2012

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Leishmaniases are vector-borne parasitic diseases with 0.9 – 1.4 million new human cases each year worldwide. In the vectorial part of the life-cycle, Leishmania development is confined to the digestive tract. During the first few days after blood feeding, natural barriers to Leishmania development include secreted proteolytic enzymes, the peritrophic matrix surrounding the ingested blood meal and sand fly immune reactions. As the blood digestion proceeds, parasites need to bind to the midgut epithelium to avoid being excreted with the blood remnant. This binding is strictly stage-dependent as it is a property of nectomonad and leptomonad forms only. While the attachment in specific vectors (P. papatasi, P. duboscqi and P. sergenti) involves lipophosphoglycan (LPG), this Leishmania molecule is not required for parasite attachment in other sand fly species experimentally permissive for various Leishmania. During late-stage infections, large numbers of parasites accumulate in the anterior midgut and produce filamentous proteophosphoglycan creating a gel-like plug physically obstructing the gut. The parasites attached to the stomodeal valve cause damage to the chitin lining and epithelial cells of the valve, interfering with its function and facilitating reflux of parasites from the midgut. Transformation to metacyclic stages highly infective for the vertebrate host is the other prerequisite for effective transmission. Here, we review the current state of knowledge of molecular interactions occurring in all these distinct phases of parasite colonization of the sand fly gut, highlighting recent discoveries in the field.

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“…2 C ). In addition, infection with a cell line that overexpressed ISP2, WT ( pXG-ISP2 ) (28), demonstrated that an excess of ISP2 delayed MPO-specific bioluminescence, which increased only from 4 wk compared with 2 wk during WT and Δ isp2/3 infection (Supplemental Fig. 2 D ).…”

Section: Resultsmentioning

confidence: 99%

“…1 A ). Cell lines that overexpressed ISP2 [WT ( pXG-ISP2 )] were generated via the introduction of an episomal copy of ISP2 into L. major WT (28). Metacyclic promastigotes were isolated from a stationary phase culture by agglutination of other promastigote forms with peanut lectin, as previously described (31).…”

Section: Methodsmentioning

confidence: 99%

“…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).…”

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

et al. 2018

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Leishmania major is the causative agent of the neglected tropical disease, cutaneous leishmaniasis. In the mouse, protective immunity to Leishmania is associated with inflammatory responses. Here, we assess the dynamics of the inflammatory responses at the lesion site during experimental long-term, low-dose intradermal infection of the ear, employing noninvasive imaging and genetically modified L. major. Significant infiltrates of neutrophils and monocytes occurred at 1–4 d and 2–4 wk, whereas dermal macrophage and dendritic cell (DC) numbers were only slightly elevated in the first days. Quantitative whole-body bioluminescence imaging of myeloperoxidase activity and the quantification of parasite loads indicated that the Leishmania virulence factor, inhibitor of serine peptidase 2 (ISP2), is required to modulate phagocyte activation and is important for parasite survival at the infection site. ISP2 played a role in the control of monocyte, monocyte-derived macrophage, and monocyte-derived DC (moDC) influx, and was required to reduce iNOS expression in monocytes, monocyte-derived cells, and dermal DCs; the expression of CD80 in moDCs; and levels of IFN-γ in situ. Our findings indicate that the increased survival of L. major in the dermis during acute infection is associated with the down-regulation of inflammatory monocytes and monocyte-derived cells via ISP2.—Goundry, A., Romano, A., Lima, A. P. C. A., Mottram, J. C., Myburgh, E. Inhibitor of serine peptidase 2 enhances Leishmania major survival in the skin through control of monocytes and monocyte-derived cells.

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Ecotin‐like serine peptidase inhibitor ISP1 ofLeishmania majorplays a role in flagellar pocket dynamics and promastigote differentiation

Cited by 21 publications

References 48 publications

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

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

Leishmania development in sand flies: parasite-vector interactions overview

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

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