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Des-, syn- and anti-oxyimino-delta 3-cephalosporins. Intrinsic reactivity and reaction with RTEM-2 serine beta-lactamase and D-alanyl-D-alanine-cleaving serine and Zn2+-containing peptidases.

ABSTRACT: The presence and configuration (syn or anti) of an oxyimino group in the 7 (beta)-acyl side chain of 3-cephems do not modify the intrinsic reactivity of the beta-lactam ring, but have highly enzyme-specific effects. When compared with the corresponding desoxyimino beta-lactam compound: (i) with the plasmid-mediated Escherichia coli RTEM-2 serine beta-lactamase, the substrate activity of the anti isomer is increased and that of the syn isomer is decreased; (ii) with the Streptomyces R61 serine D-alanyl-D-alanine cleaving peptidase (a highly penicillin-sensitive enzyme), the rate of enzyme acylation is not or only little affected when the oxyimino group is in the syn configuration, but is decreased when the oxyimino group is in the anti configuration; (iii) with the Actinomadura R39 serine D-alanyl-D-alanine-cleaving peptidase (an exceedingly highly penicillin-sensitive enzyme), the rate of enzyme acylation is unaffected whatever the configuration of the substituent. The oxidation of the sulphur atom of the dihydrothiazine ring on the beta-face of the molecule makes it both a poorer inactivator of the DD-peptidases and a poorer substrate of the beta-lactamase. The Streptomyces albus G Zn2+-containing D-alanyl-D-alanine-cleaving peptidase (a highly penicillin-resistant enzyme) remains highly resistant to all compounds tested.

SUBMITTER: Laurent G

PROVIDER: S-EPMC1153425 | biostudies-other | 1984 Mar

REPOSITORIES: biostudies-other

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Project description: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.MethodsIn 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.

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Des-, syn- and anti-oxyimino-delta 3-cephalosporins. Intrinsic reactivity and reaction with RTEM-2 serine beta-lactamase and D-alanyl-D-alanine-cleaving serine and Zn2+-containing peptidases.

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