Alissa Myrick

Mapping of the Plasmodium falciparum multidrug resistance gene 5′‐upstream region, and evidence of induction of transcript levels by antimalarial drugs in chloroquine sensitive parasites

The Plasmodium falciparum multidrug resistance gene, pfmdr1, has been shown to be involved in the mediation of the parasites response to various antimalarial drugs. Previous studies of pfmdr1 expression have shown that transcript levels are increased in drug‐resistant isolates. However, a detailed examination of the transcriptional regulation of this gene has not been completed. The aim of this study was to map the 5′ UTR of pfmdr1, and to examine the transcriptional profile of the gene in sensitive parasites treated with four different antimalarial drugs. RT‐PCR and 5′‐RACE mapping showed that the 5′ UTR has a length of 1.94 kb. A putative promoter has been identified via transient transfection. Northern analysis revealed a 2.1‐ to 2.7‐fold increase in pfmdr1 expression in 3D7 parasites treated with 50 nM chloroquine for 6 h, confirming results from Serial Analysis of Gene Expression. 3D7 parasites were subsequently treated with experimentally derived IC50 concentrations of mefloquine, quinine and pyrimethamine. pfmdr1 transcript levels specifically increased 2.5‐fold at 6 h in mefloquine‐treated parasites and threefold in parasites treated with quinine for 30 min. There was no evidence of transcript induction in pyrimethamine‐treated parasites. This is the first evidence of induction of pfmdr1 expression in sensitive cells; and suggests a novel method of transcriptional control for this gene.

Experimental malaria infection triggers early expansion of natural killer cells.

ABSTRACT In order to gain a better understanding of gene expression during early malaria infection, we conducted microarray analysis of early blood responses in mice infected with erythrocytic-stage Plasmodium chabaudi. Immediately following infection, we observed coordinated and sequential waves of immune responses, with interferon-associated gene transcripts dominating by 16 h postinfection, followed by strong increases in natural killer (NK) cell-associated and major histocompatibility complex class I-related transcripts by 32 h postinfection. We showed by flow cytometry that the observed elevation in NK cell-associated transcripts was the result of a dramatic increase in the proportion of NK cells in the blood during infection. Subsequent microarray analysis of NK cells isolated from the peripheral blood of infected mice revealed a cell proliferation expression signature consistent with the observation that NK cells replicate in response to infection. Early proliferation of NK cells was directly observed in studies with adoptively transferred cells in infected mice. These data indicate that the early response to P. chabaudi infection of the blood is marked by a primary wave of interferon with a subsequent response by NK cells.

Compounds targeting disulfide bond forming enzyme DsbB of Gram-negative bacteria

In bacteria, disulfide bonds confer stability on many proteins exported to the cell envelope or beyond. These proteins include numerous bacterial virulence factors. Thus, bacterial enzymes that promote disulfide bond formation represent targets for compounds inhibiting bacterial virulence. Here, we describe a novel target- and cell-based screening methodology for identifying compounds that inhibit the disulfide bond-forming enzymes E. coli DsbB (EcDsbB) or M. tuberculosis VKOR (MtbVKOR). MtbVKOR can replace EcDsbB although the two are not homologues. Initial screening of 51,487 compounds yielded six specifically inhibiting EcDsbB. These compounds share a structural motif and do not inhibit MtbVKOR. A medicinal chemistry approach led us to select related compounds some of which are much more effective DsbB inhibitors than those found in the screen. These compounds inhibit purified DsbB and prevent anaerobic E. coli growth. Furthermore, these compounds inhibit all but one of the DsbBs of nine other gram-negative pathogenic bacteria tested.

Binding of MgtR, a Salmonella Transmembrane Regulatory Peptide, to MgtC, a Mycobacterium tuberculosis Virulence Factor: A Structural Study

MgtR, a highly hydrophobic peptide expressed in Salmonella enterica serovar Typhimurium, inhibits growth in macrophages through binding to the membrane protein MgtC that has been identified as essential for replication in macrophages. While the Mycobacterium tuberculosis MgtC is highly homologous to its S. Typhi analogue, there does not appear to be an Mtb homologue for MgtR, raising significant pharmacological interest in this system. Here, solid-state NMR and EPR spectroscopy in lipid bilayer preparations were used to demonstrate the formation of a heterodimer between S. Typhi MgtR and the transmembrane helix 4 of Mtb MgtC. Based on the experimental restraints, a structural model of this heterodimer was developed using computational techniques. The result is that MgtR appears to be ideally situated in the membrane to influence the functionality of MgtC.