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

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.

In-vitro effects of the FS50 protein from salivary glands of Xenopsylla cheopis on voltage-gated sodium channel activity and motility of MDA-MB-231 human breast cancer cells.

Voltage-gated sodium channel activity enhances the motility and oncogene expression of metastasic cancer cells that express a neonatal alternatively spliced form of the NaV1.5 isoform. We reported previously that FS50, a salivary protein from Xenopsylla cheopis, showed inhibitory activity against the NaV1.5 channel when assayed in HEK 293T cells and antiarrhythmia effects on rats and monkeys after induction of arrhythmia by BaCl2. This study aims to identify the effect of FS50 on voltage-gated sodium channel activity and the motility of MDA-MB-231 human breast cancer cells in vitro. NaV1.5 was abnormally expressed in the highly metastatic breast cancer cell line MDA-MB-231, but not in the MCF-7 cell line. FS50 significantly inhibited sodium current, migration, and invasion in a dose-dependent manner, but had no effect on the proliferation of MDA-MB-231 cells at the working concentrations (1.5-12 µmol/l) after a long-term treatment for 48 h. Meanwhile, FS50 decreased NaV1.5 mRNA expression without altering the total protein level in MDA-MB-231 cells. Correspondingly, the results also showed that MMP-9 activity and the ratio of MMP-9 mRNA to TIMP-1 mRNA were markedly decreased by FS50. Taken together, our findings highlighted for the first time an inhibitory effect of a salivary protein from a blood-feeding arthropod on breast cancer cells through the NaV1.5 channel. Furthermore, this study provided a new candidate leading molecule against antitumor cells expressing NaV1.5.

The in vitro anticancer effects of FS48 from salivary glands of Xenopsylla cheopis on NCI-H460 cells via its blockage of voltage-gated K+ channels.

Voltage-gated K+ (Kv) channels play a role in the cellular processes of various cancer cells, including lung cancer cells. We previously identified and reported a salivary protein from the Xenopsylla cheopis, FS48, which exhibited inhibitory activity against Kv1.1-1.3 channels when assayed in HEK 293T cells. However, whether FS48 has an inhibitory effect on cancer cells expressing Kv channels is unclear. The present study aims to reveal the effects of FS48 on the Kv channels and the NCI-H460 human lung cancer cells through patch clamp, MTT, wound healing, transwell, gelatinase zymography, qRT-PCR and WB assays. The results demonstrated that FS48 can be effective in suppressing the Kv currents, migration, and invasion of NCI-H460 cells in a dose-dependent manner, despite the failure to inhibit the proliferation. Moreover, the expression of Kv1.1 and Kv1.3 mRNA and protein were found to be significantly reduced. Finally, FS48 decreases the mRNA level of MMP-9 while increasing TIMP-1 mRNA level. The present study highlights for the first time that blood-sucking arthropod saliva-derived protein can inhibit the physiological activities of tumour cells via the Kv channels. Furthermore, FS48 can be taken as a hit compound against the tumour cells expressing Kv channels.

Effects of host diet and thermal state on feeding performance of the flea Xenopsylla ramesis.

We examined feeding performance of the flea Xenopsylla ramesis on three different hosts: its natural, granivorous, rodent host, Sundevall's jird (Meriones crassus); the frugivorous Egyptian fruit bat (Rousettus aegyptiacus); and an insectivorous bat, Kuhl's pipistrelle (Pipistrellus kuhlii). Because these fleas are not known to occur on bats, we hypothesized that the fleas' feeding performance (i.e. feeding and digestion rates) would be higher when feeding on their natural host than on either of the bats that they do not naturally parasitize. We found that mass-specific blood-meal size of both male and female fleas was significantly lower when feeding on Kuhl's pipistrelles than on the other two species, but was not different in female fleas feeding on fruit bats or on jirds at all stages of digestion. However, more male fleas achieved higher levels of engorgement if they fed on Sundevall's jirds than if they fed on Egyptian fruit bats. The fleas digested blood of fruit bats and jirds significantly faster than blood of Kuhl's pipistrelle. In addition, after a single blood meal, the survival time of fleas fed on normothermic Kuhl's pipistrelles was significantly shorter than that of fleas fed on Sundevall's jirds and even lower when male fleas fed on Egyptian fruit bats. Thus, our prediction was partially supported: normothermic Kuhl's pipistrelles were inferior hosts for fleas compared with Sandevall's jirds and Egyptian fruit bats. Interestingly, the proportion of engorged fleas that fed on torpid Kuhl's pipistrelles was significantly higher than the proportion of the fleas that fed on normothermic individuals, indicating that becoming torpid might be a liability, rather than an effective defense against parasites.

Structure and Function of FS50, a salivary protein from the flea Xenopsylla cheopis that blocks the sodium channel NaV1.5.

Naturally occurring toxins have been invaluable tools for the study of structural and functional relationships of voltage-gated sodium channels (VGSC). Few studies have been made of potential channel-modulating substances from blood-feeding arthropods. He we describe the characterization FS50, a salivary protein from the flea, Xenopsylla cheopis, that exhibits an inhibitory activity against the NaV1.5 channel with an IC50 of 1.58 µM. The pore-blocking mechanism of this toxin is evident from the kinetics of activation and inactivation suggesting that FS50 does not interfere with the voltage sensor of NaV1.5. FS50 exhibits high specificity for NaV1.5, since 10 µM FS50 had no discernable effect on voltage-gated Na+, K+ and Ca2+ channels in rat dorsal root ganglia or VGSC forms individually expressed in HEK 293T cells. Furthermore, intravenous injection of FS50 into rats and monkeys elicited recovery from arrhythmia induced by BaCl2, as would be expected from a blockade of NaV1.5. The crystal structure of FS50 revealed a ßαßß domain similar to that of scorpion ß toxin and a small N-terminal ßαß domain. Site-directed mutagenesis experiments have implicated a basic surface including the side chains of Arg 6, His 11 and Lys 32 as potentially important in the FS50 NaV1.5 interaction.