Faculty Bibliography

Apicomplexans such as Toxoplasma gondii actively invade host cells using a unique parasite-dependent mechanism termed gliding motility. Calcium-mediated protein secretion by the parasite has been implicated in this process, but the precise role of calcium signaling in motility remains unclear. Here we used calmidazolium as a tool to stimulate intracellular calcium fluxes and found that this drug led to enhanced motility by T. gondii. Treatment with calmidazolium increased the duration of gliding and resulted in trails that were twice as long as those formed by control parasites. Calmidazolium also increased microneme secretion by T. gondii, and studies with a deletion mutant of the accessory protein m2AP specifically implicated that adhesin MIC2 was important for gliding. The effects of calmidazolium on gliding and secretion were due to increased release of calcium from intracellular stores and calcium influx from the extracellular milieu. In addition, we demonstrate that calmidazolium-stimulated increases in intracellular calcium were highly dynamic, and that rapid fluxes in calcium levels were associated with parasite motility. Our studies suggest that oscillations in intracellular calcium levels may regulate microneme secretion and control gliding motility in T. gondii.

During infection of B. subtilis by bacteriophage SPO1, host-takeover is accomplished primarily by the products of a 24-gene cluster in the SPO1 terminal redundancy. We are studying the roles of specific genes by observing the effect of knockout mutations on SPO1 infection, and the effect of expression of cloned genes on uninfected cells. Genes 38, 39 and 40, genes 44, 50 and 51, and genes 53, 54 and 55 appear to be involved in shutoff of bacterial DNA, RNA, and protein synthesis, respectively. This was suggested by the following observations: (1) a gene 40 knockout mutant shut off host DNA synthesis more slowly, and synthesized phage DNA more slowly, than did wild-type SPO1; (2) expression of genes 38 and 39 together in uninfected cells inhibited DNA synthesis by more than 80%; (3) expression of either gene 44 or the gene 50/51 operon in uninfected cells inhibited bacterial transcription; (4) GP44 was previously shown to target bacterial RNA polymerase; and (5) when expressed in uninfected cells, the gene 55-53 operon inhibited bacterial protein synthesis without affecting RNA synthesis. Gene 56 targets host cell division: its expression in uninfected B. subtilis specifically prevented cell division; SPO1 infection inhibited cell-division; and a gene 56 knockout mutation prevented this inhibition. Many of the genes (38/39, 41, 44, 45/46, 50/51, 52, and 56), were lethal when expressed in E. coli and/or B. subtilis, suggesting that new antibiotics might be based on their lethal mechanisms. The combination of genes 44, 50, and 51 killed more than 20 times as rapidly as any of the individual genes. Knockout mutations in genes 40, 44, 50, 51 or 56 caused no decrease in burst-size, or in shutoff of transcription, arguing that those genes are either redundant or unnecessary under laboratory conditions. A 44/51 double mutant did decrease the burst-size, but not the shutoff. The shutoffs of DNA, RNA, or protein synthesis, by individual genes or by combinations such as 44/50/51, were not as rapid or complete as the shutoffs caused by SPO1 infection, suggesting that we have yet to identify all components of the machinery for any of these shutoffs. C1 Sampath, A.; Myles, B.; Cox, K.; Chen, L.; Stewart, C.; Rice University, Houston, TX, USA