The dimerisable Cre recombinase allows conditional genome editing in the mosquito stages of Plasmodium berghei

ABSTRACTAsexual blood stages of the malaria parasite are readily amenable to genetic modification via homologous recombination, allowing functional studies of parasite genes that are not essential in this part of the life cycle. However, conventional reverse genetics cannot be applied for the functional analysis of genes that are essential during asexual blood-stage replication. Various strategies have been developed for conditional mutagenesis of Plasmodium, including recombinase-based gene deletion, regulatable promoters, and mRNA or protein destabilization systems. Among these, the dimerisable Cre (DiCre) recombinase system has emerged as a powerful approach for conditional gene targeting in P. falciparum. In this system, the bacteriophage Cre is expressed in the form of two separate, enzymatically inactive polypeptides, each fused to a different rapamycin-binding protein. Rapamycin-induced heterodimerization of the two components restores recombinase activity. We have implemented the DiCre system in the rodent malaria parasite P. berghei, and show that rapamycin-induced excision of floxed DNA sequences can be achieved with very high efficiency in both mammalian and mosquito parasite stages. This tool can be used to investigate the function of essential genes not only in asexual blood stages, but also in other parts of the malaria parasite life cycle.

Staphylococcus aureus TargetArray: Comprehensive Differential Essential Gene Expression as a Mechanistic Tool To Profile Antibacterials

ABSTRACT The widespread emergence of antibiotic-resistant bacteria and a lack of new pharmaceutical development have catalyzed a need for new and innovative approaches for antibiotic drug discovery. One bottleneck in antibiotic discovery is the lack of a rapid and comprehensive method to identify compound mode of action (MOA). Since a hallmark of antibiotic action is as an inhibitor of essential cellular targets and processes, we identify a set of 308 essential genes in the clinically important pathogen Staphylococcus aureus. A total of 446 strains differentially expressing these genes were constructed in a comprehensive platform of sensitized and resistant strains. A subset of strains allows either target underexpression or target overexpression by heterologous promoter replacements with a suite of tetracycline-regulatable promoters. A further subset of 236 antisense RNA-expressing clones allows knockdown expression of cognate targets. Knockdown expression confers selective antibiotic hypersensitivity, while target overexpression confers resistance. The antisense strains were configured into a TargetArray in which pools of sensitized strains were challenged in fitness tests. A rapid detection method measures strain responses toward antibiotics. The TargetArray antibiotic fitness test results show mechanistically informative biological fingerprints that allow MOA elucidation.

In Vitro and In Vivo Effects of 14α-Demethylase (ERG11) Depletion in Candida glabrata

ABSTRACT Sterol 14α-demethylase (ERG11) is the target enzyme of azole antifungals that are widely used for the treatment of fungal infections. Candida glabrata is known to be less susceptible to fluconazole than most Candida albicansstrains, and the incidence of C. glabrata infection has been increasing mostly in conjunction with the use of azole antifungals. Recently, it has been reported that C. glabrata can rescue the defect of ergosterol biosynthesis by incorporating cholesterol from serum. To explore the effect of inactivating Erg11p in C. glabrata, we generated mutant strains in which the ERG11 gene was placed under the control of tetracycline-regulatable promoters. In these mutants, expression of the ERG11 gene can be repressed by doxycycline (DOX). All mutants showed a growth defect in the presence of DOX. The numbers of CFU of the mutants were lowered by only 1/10 with DOX treatment. In these mutants, accumulation of 4,14-dimethylzymosterol, which differs from an accumulated abnormal sterol detected in C. albicans and Saccharomyces cerevisiae treated with fluconazole, was observed by DOX treatment. Although such phenotypes were also observed in serum-containing media by DOX treatment, they were alleviated. Furthermore, the mutant could grow in DOX-treated mice without a severe reduction in the number of cells. Thus, depleting the expression of theERG11 gene lowered the number of CFU by only 1/10 due to the accumulation of 4,14-demethylzymosterol in vitro, and it did not result in the defective growth of fungal cells in mice. These results suggested that Erg11p is not an ideal target molecule of antifungals for C. glabrata.