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Identification of a substrate-like cleavage-resistant thrombin inhibitor from the saliva of the flea Xenopsylla cheopis

ABSTRACT: The salivary glands of the flea Xenopsylla cheopis, a vector of the plague bacterium, Yersinia pestis, express proteins and peptides thought to target the hemostatic and inflammatory systems of its mammalian hosts. Past transcriptomic analyses of salivary gland tissue revealed the presence of two similar peptides (XC-42 and XC-43) having no extensive similarities to any other deposited sequences. Here we show that these peptides specifically inhibit coagulation of plasma and the amidolytic activity of α-thrombin. XC-43, the smaller of the two peptides, is a fast, tight-binding inhibitor of thrombin with a dissociation constant of less than 10 pM. XC-42 exhibits similar selectivity as well as kinetic and binding properties. The crystal structure of XC-43 in complex with thrombin shows that despite its substrate-like binding mode, XC-43 is not detectably cleaved by thrombin and that it interacts with the thrombin surface from the enzyme catalytic site through the fibrinogen-binding exosite I. The low rate of hydrolysis was verified in solution experiments with XC-43, which show the substrate to be largely intact after 2 h of incubation with thrombin at 37 °C. The low rate of XC-43 cleavage by thrombin may be attributable to specific changes in the catalytic triad observable in the crystal structure of the complex or to extensive interactions in the prime sites that may stabilize the binding of cleavage products. Based on the increased arterial occlusion time, tail bleeding time, and blood coagulation parameters in rat models of thrombosis XC-43 could be valuable as an anticoagulant.

SUBMITTER: Lu S

PROVIDER: S-EPMC8573170 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background/aimsArthropod-borne pathogens are transmitted into a unique intradermal microenvironment that includes the saliva of their vectors. Immunomodulatory factors in the saliva can enhance infectivity; however, in some cases the immune response that develops to saliva from prior uninfected bites can inhibit infectivity. Most rodent reservoirs of Yersinia pestis experience fleabites regularly, but the effect this has on the dynamics of flea-borne transmission of plague has never been investigated. We examined the innate and acquired immune response of mice to bites of Xenopsylla cheopis and its effects on Y. pestis transmission and disease progression in both naïve mice and mice chronically exposed to flea bites.Methods/principal findingsThe immune response of C57BL/6 mice to uninfected flea bites was characterized by flow cytometry, histology, and antibody detection methods. In naïve mice, flea bites induced mild inflammation with limited recruitment of neutrophils and macrophages to the bite site. Infectivity and host response in naïve mice exposed to flea bites followed immediately by intradermal injection of Y. pestis did not differ from that of mice infected with Y. pestis without prior flea feeding. With prolonged exposure, an IgG1 antibody response primarily directed to the predominant component of flea saliva, a family of 36-45 kDa phosphatase-like proteins, occurred in both laboratory mice and wild rats naturally exposed to X. cheopis, but a hypersensitivity response never developed. The incidence and progression of terminal plague following challenge by infective blocked fleas were equivalent in naïve mice and mice sensitized to flea saliva by repeated exposure to flea bites over a 10-week period.ConclusionsUnlike what is observed with many other blood-feeding arthropods, the murine immune response to X. cheopis saliva is mild and continued exposure to flea bites leads more to tolerance than to hypersensitivity. The immune response to flea saliva had no detectable effect on Y. pestis transmission or plague pathogenesis in mice.

Project description:Yersinia pestis, the causative agent of plague, is a highly lethal pathogen transmitted by the bite of infected fleas. Once ingested by a flea, Y. pestis establish a replicative niche in the gut and produce a biofilm that promotes foregut colonization and transmission. The rat flea Xenopsylla cheopis is an important vector to several zoonotic bacterial pathogens including Y. pestis. Some fleas naturally clear themselves of infection; however, the physiological and immunological mechanisms by which this occurs are largely uncharacterized. To address this, RNA was extracted, sequenced, and distinct transcript profiles were assembled de novo from X. cheopis digestive tracts isolated from fleas that were either: 1) not fed for 5 days; 2) fed sterile blood; or 3) fed blood containing ~5x108 CFU/ml Y. pestis KIM6+. Analysis and comparison of the transcript profiles resulted in identification of 23 annotated (and 11 unknown or uncharacterized) digestive tract transcripts that comprise the early transcriptional response of the rat flea gut to infection with Y. pestis. The data indicate that production of antimicrobial peptides regulated by the immune-deficiency pathway (IMD) is the primary flea immune response to infection with Y. pestis. The remaining infection-responsive transcripts, not obviously associated with the immune response, were involved in at least one of 3 physiological themes: 1) alterations to chemosensation and gut peristalsis; 2) modification of digestion and metabolism; and 3) production of chitin-binding proteins (peritrophins). Despite producing several peritrophin transcripts shortly after feeding, including a subset that were infection-responsive, no thick peritrophic membrane was detectable by histochemistry or electron microscopy of rat flea guts for the first 24 hours following blood-feeding. Here we discuss the physiological implications of rat flea infection-responsive transcripts, the function of X. cheopis peritrophins, and the mechanisms by which Y. pestis may be cleared from the flea gut.

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Identification of a substrate-like cleavage-resistant thrombin inhibitor from the saliva of the flea Xenopsylla cheopis

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