PfClpC Is an Essential Clp Chaperone Required for Plastid Integrity and Clp Protease Stability in Plasmodium falciparum.

The deadly malaria parasite Plasmodium falciparum contains a nonphotosynthetic plastid, known as the apicoplast, that functions to produce essential metabolites, and drugs that target the apicoplast are clinically effective. Several prokaryotic caseinolytic protease (Clp) genes have been identified in the Plasmodium genome. Using phylogenetic analysis, we focused on the Clp members that may form a regulated proteolytic complex in the apicoplast. We genetically targeted members of this complex and generated conditional mutants of the apicoplast-localized PfClpC chaperone and PfClpP protease. Conditional inhibition of the PfClpC chaperone resulted in growth arrest and apicoplast loss and was rescued by addition of the essential apicoplast-derived metabolite IPP. Using a double-conditional mutant parasite line, we discovered that the chaperone activity is required to stabilize the mature protease, revealing functional interactions. These data demonstrate the essential function of PfClpC in maintaining apicoplast integrity and its role in regulating the proteolytic activity of the Clp complex.

Dose response of Listeria monocytogenes invasion, fetal morbidity, and fetal mortality after oral challenge in pregnant and nonpregnant Mongolian gerbils.

Listeria monocytogenes is a food-borne pathogen that can result in adverse pregnancy outcomes, such as stillbirth or premature delivery. The Mongolian gerbil was recently proposed as the most appropriate small-animal model of listeriosis due to its susceptibility to the same invasion pathways as humans. The objectives of this study were to investigate invasion and adverse pregnancy outcomes in gerbils orally exposed to L. monocytogenes, to compare the dose-response data to those of other animal models, and to investigate differences in the responses of pregnant versus nonpregnant gerbils. Gerbils were orally exposed to 0 (control), 10(3), 10(5), 10(7), or 10(9) CFU L. monocytogenes in whipping cream. L. monocytogenes was recovered in a dose-dependent manner from fecal samples, adult organs, and pregnancy-associated tissues. Dams exposed to 10(9) CFU had more invaded organs and higher concentrations of L. monocytogenes in almost all organs than nonpregnant animals, though no differences in fecal shedding were seen between the two groups. Adverse pregnancy outcomes occurred only in the dams treated with 10(9) CFU. A 50% infectivity dose (ID50) of 2.60 × 10(6) CFU for fetuses was calculated by fitting the data to a logistic model. Our results suggest that the 50% lethal dose (LD50) falls within the range of 5 × 10(6) to 5 × 10(8) CFU. This range includes the guinea pig and nonhuman primate LD50s, but the observation that L. monocytogenes-induced stillbirths can be seen in guinea pigs and primates exposed to lower doses than those at which stillbirths were seen in gerbils indicates that gerbils are not more sensitive to L. monocytogenes invasion.

Producing timed-pregnant Mongolian gerbils for developmental studies.

The developing fetus is differentially susceptible to chemicals and pathogens depending on the stage of its development. In developmental studies, embryos or fetuses therefore must be exposed to experimental interventions at the same gestational stage. Acquiring sufficient numbers of embryos of the same developmental stage requires the use of timed-pregnant animals. Timed-pregnant Mongolian gerbils currently are not available for purchase. The authors developed a novel method for timed mating of virgin female gerbils. Female gerbils were housed in the same cage as males, but physically separated by a partition, for 3 d in order to expose the females to the males' pheromones before they were allowed to interact. Females were monitored for lordosis to determine sexual receptivity. Lordosis was observed in 10 of 15 females (67%), and 9 of these females (90%) became pregnant. When lordosis was not observed, none of the females became pregnant. These results demonstrate that the timed mating method produces a high rate of mating success and indicate that lordosis is a reliable predictor of sexual receptivity and subsequent successful mating in the Mongolian gerbil.