dRecurrent Clostridium difficile infection (CDI) is of particular concern among health care-associated infections. The role of the microbiota in disease recovery is apparent given the success of fecal microbiota transplantation (FMT) for recurrent CDI. Here, we present a murine model of CDI relapse to further define the microbiota recovery following FMT. Cefoperazone-treated mice were infected with C. difficile 630 spores and treated with vancomycin after development of clinical disease. Vancomycin treatment suppressed both C. difficile colonization and cytotoxin titers. However, C. difficile counts increased within 7 days of completing treatment, accompanied by relapse of clinical signs. The administration of FMT immediately after vancomycin cleared C. difficile and decreased cytotoxicity within 1 week. The effects of FMT on the gut microbiota community were detectable in recipients 1-day posttransplant. Conversely, mice not treated with FMT remained persistently colonized with high levels of C. difficile, and the gut microbiota in these mice persisted at low diversity. These results suggest that full recovery of colonization resistance against C. difficile requires the restoration of a specific community structure. R ecently, the Centers for Disease Control identified Clostridium difficile as an urgent nosocomial threat. In particular, recurrent C. difficile infection (CDI) is a major concern (1). Up to 30% of patients experience recurrent symptoms following cessation of antibiotic therapy, and subsequent recurrences are more likely after a primary recurrence incidence (2, 3). A healthy, diverse gastrointestinal microbial community, termed the gut microbiota, is important in resistance against the development of CDI. The normal gut microbiota provides colonization resistance (the ability to resist pathogen colonization) against C. difficile (4). In both human studies and animal models, perturbation of the indigenous gut microbiota by antibiotic administration has been correlated to a susceptible community structure, ultimately leading to CDI (5-8). Although it is clear that antibiotic use is correlated with the majority of CDI cases (9), the standard therapy for CDI is the administration of an antibiotic regimen with activity against C. difficile (typically vancomycin or metronidazole). Paradoxically, this may further perturb the intestinal microbial community (10, 11). Therefore, it is important to understand the impact of antibiotic treatment for CDI on recovery of the gut microbiota and colonization resistance.In a murine model of persistent CDI, we have previously shown that the use of sequential antibiotics can delay recovery of bacterial diversity (11). Previous studies have suggested that patients who develop recurrence have a less diverse community than patients who have stable recovery (12). The idea that restoration of the gut microbiota is necessary to prevent or mitigate recurrence is supported by an ϳ90% success rate of fecal microbiota transplantation (FMT) (13), and significant recovery of diver...
In externally fertilizing animals, such as sea urchins and frogs, prolonged depolarization of the egg immediately after fertilization inhibits the entry of additional sperm-a phenomenon known as the fast block to polyspermy. In the African clawed frog , this depolarization is driven by Ca-activated Cl efflux. Although the prominent Ca-activated Cl currents generated in immature oocytes are mediated by transmembrane protein 16a (xTMEM16A) channels, little is known about the channels that contribute to the fast block in mature eggs. Moreover, the gamete undergoes a gross transformation as it develops from an immature oocyte into a fertilization-competent egg. Here, we report the results of our approach to identify the Ca-activated Cl channel that triggers the fast block. By querying published proteomic and RNA-sequencing data, we identify two Ca-activated Cl channels expressed in fertilization-competent eggs: xTMEM16A and bestrophin 2A (xBEST2A). By exogenously expressing xTMEM16A and xBEST2A in axolotl cells lacking endogenous Ca-activated currents, we characterize the effect of inhibitors on currents mediated by these channels. None of the inhibitors tested block xBEST2A currents specifically. However, 2-(4-chloro-2-methylphenoxy)--[(2-methoxyphenyl)methylideneamino]-acetamide (Ani9) and -((4-methoxy)-2-naphthyl)-5-nitroanthranilic acid (MONNA) each reduce xTMEM16A currents by more than 70% while only nominally inhibiting those generated by xBEST2A. Using whole-cell recordings during fertilization, we find that Ani9 and MONNA effectively diminish fertilization-evoked depolarizations. Additionally, these inhibitors lead to increased polyspermy in embryos. These results indicate that fertilization activates TMEM16A channels in eggs and induces the earliest known event triggered by fertilization: the fast block to polyspermy.
BackgroundThe necessity of extracellular Ca2+ for fertilization and early embryonic development in the African clawed frog, Xenopus laevis, is controversial. Ca2+ entry into X. laevis sperm is reportedly required for the acrosome reaction, yet fertilization and embryonic development have been documented to occur in high concentrations of the Ca2+ chelator BAPTA. Here we sought to resolve this controversy.Methodology/principal findingUsing the appearance of cleavage furrows as an indicator of embryonic development, we found that X. laevis eggs inseminated in a solution lacking added divalent cations developed normally. By contrast, eggs inseminated in millimolar concentrations of BAPTA or EGTA failed to develop. Transferring embryos to varying solutions after sperm addition, we found that extracellular Ca2+ is specifically required for events occurring within the first 30 minutes after sperm addition, but not after. We found that the fluorescently stained sperm were not able to penetrate the envelope of eggs inseminated in high BAPTA, whereas several had penetrated the vitelline envelope of eggs inseminated without a Ca2+ chelator, or with BAPTA and saturating CaCl2. Together these results indicate that fertilization does not occur in high concentrations of Ca2+ chelators. Finally, we found that the jelly coat includes >5 mM of readily diffusible Ca2+.Conclusions/SignificanceTaken together, these data are consistent with requirement of extracellular Ca2+ for fertilization. Based on our findings, we hypothesize that the jelly coat surrounding the egg acts as a reserve of readily available Ca2+ ions to foster fertilization in changing extracellular milieu.
The prevention of polyspermy is essential for the successful progression of normal embryonic development in most sexually reproducing species. In external fertilizers, the process of fertilization induces a depolarization of the egg's membrane within seconds, which inhibits supernumerary sperm from entering an already-fertilized egg. This fast block requires an increase of intracellular Ca in the African clawed frog, , which in turn activates an efflux of Cl that depolarizes the cell. Here we seek to identify the source of this intracellular Ca Using electrophysiology, pharmacology, bioinformatics, and developmental biology, we explore the requirement for both Ca entry into the egg from the extracellular milieu and Ca release from an internal store, to mediate fertilization-induced depolarization. We report that although eggs express Ca-permeant ion channels, blockade of these channels does not alter the fast block. In contrast, insemination of eggs in the presence of Xestospongin C-a potent inhibitor of inositol 1,4,5-trisphosphate (IP)-induced Ca release from the endoplasmic reticulum (ER)-completely inhibits fertilization-evoked depolarization and increases the incidence of polyspermy. Inhibition of the IP-generating enzyme phospholipase C (PLC) with U73122 similarly prevents fertilization-induced depolarization and increases polyspermy. Together, these results demonstrate that fast polyspermy block after fertilization in eggs is mediated by activation of PLC, which increases IP and evokes Ca release from the ER. This ER-derived Ca then activates a Cl channel to induce the fast polyspermy block. The PLC-induced cascade of events represents one of the earliest known signaling pathways initiated by fertilization.
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