No abstract

Studies of Leishmania donovani have shown that both ornithine decarboxylase and spermidine synthase, two enzymes of the polyamine biosynthetic pathway, are critical for promastigote proliferation and required for maximum infection in mice. However, the importance of arginase (ARG), the first enzyme of the polyamine pathway in Leishmania, has not been analyzed in L. donovani. To test ARG function in intact parasites, we generated Δarg null mutants in L. donovani and evaluated their ability to proliferate in vitro and trigger infections in mice. The Δarg knockout was incapable of growth in the absence of polyamine supplementation, but the auxotrophic phenotype could be bypassed by addition of either millimolar concentrations of ornithine or micromolar concentrations of putrescine or by complementation with either glycosomal or cytosolic versions of ARG. Spermidine supplementation of the medium did not circumvent the polyamine auxotrophy of the Δarg line. Although ARG was found to be essential for ornithine and polyamine synthesis, ornithine decarboxylase appeared to be the rate-limiting enzyme for polyamine production. Mouse infectivity studies revealed that the Δarg lesion reduced parasite burdens in livers by an order of magnitude but had little impact on the numbers of parasites recovered from spleens. Thus, ARG is essential for proliferation of promastigotes but not intracellular amastigotes. Coupled with previous studies, these data support a model in which L. donovani amastigotes readily salvage ornithine and have some access to host spermidine pools, while host putrescine appears to be unavailable for salvage by the parasite.

In vitro models for hepatotoxicity can be useful tools to predict in vivo responses. In this review, we discuss the use of the transforming growth factor-α transgenic mouse hepatocyte (TAMH) cell line, which is an attractive model to study drug-induced liver injury due to its ability to retain a stable phenotype and express drug-metabolizing enzymes. Hepatotoxicity involves damage to the liver and is often associated with chemical exposure. Since the liver is a major site for drug metabolism, drug-induced liver injury is a serious health concern for certain agents. At the molecular level, various mechanisms may protect or harm the liver during drug-induced hepatocellular injury including signaling pathways and endogenous factors (e.g., Bcl-2, GSH, Nrf2, or MAPK). The interplay between these and other pathways in the hepatocyte can change upon drug or drug metabolite exposure leading to intracellular stress and eventually cell death and liver injury. This review focuses on mechanistic studies investigating drug-induced toxicity in the TAMH line and how alterations to hepatotoxic mechanisms in this model relate to the in vivo situation. The agents discussed herein include acetaminophen (APAP), tetrafluoroethylcysteine (TFEC), flutamide, PD0325901, lapatinib, and flupirtine.

Bacillus subtilis Spx is a global transcriptional regulator that is conserved among Gram-positive bacteria, in which Spx is required for preventing oxidatively induced proteotoxicity. Upon stress induction, Spx engages RNA polymerase (RNAP) through interaction with the C-terminal domain of the rpoA-encoded RNAP ␣ subunit (␣CTD). Previous mutational analysis of rpoA revealed that substitutions of Y263 in ␣CTD severely impaired Spx-activated transcription. Attempts to substitute alanine for ␣CTD R261, R268, R289, E255, E298, and K294 were unsuccessful, suggesting that these residues are essential. To determine whether these RpoA residues were required for productive Spx-RNAP interaction, we ectopically expressed the putatively lethal rpoA mutant alleles in the rpoA Y263C mutant, where "Y263C" indicates the amino acid change that results from mutation of the allele. By complementation analysis, we show that Spx-bound ␣CTD amino acid residues are not essential for Spx-activated transcription in vivo but that R261A, E298A, and E255A mutants confer a partial defect in NaCl-stress induction of Spx-controlled genes. In addition, strains expressing rpoA E255A are defective in disulfide stress resistance and produce RNAP having a reduced affinity for Spx. The E255 residue corresponds to Escherichia coli ␣D259, which has been implicated in ␣CTD-70 interaction ( 70 R603, corresponding to R362 of B. subtilis A ). However, the combined rpoA E255A and sigA R362A mutations have an additive negative effect on Spx-dependent expression, suggesting the residues' differing roles in Spx-activated transcription. Our findings suggest that, while ␣CTD is essential for Spx-activated transcription, Spx is the primary DNA-binding determinant of the Spx-␣CTD complex.IMPORTANCE Though extensively studied in Escherichia coli, the role of ␣CTD in activator-stimulated transcription is largely uncharacterized in Bacillus subtilis. Here, we conduct phenotypic analyses of putatively lethal ␣CTD alanine codon substitution mutants to determine whether these residues function in specific DNA binding at the Spx-␣CTD-DNA interface. Our findings suggest that multisubunit RNAP contact to Spx is optimal for activation while Spx fulfills the most stringent requirement of upstream promoter binding. Furthermore, several ␣CTD residues targeted for mutagenesis in this study are conserved among many bacterial species and thus insights on their function in other regulatory systems may be suggested herein.KEYWORDS alpha subunit, RNA polymerase, Spx, transcription, sporulation R egulation of gene expression at the level of transcription initiation is required for bacteria to maintain fitness and survival in a dynamic environment (e.g., perturbations in temperature, nutrient availability, exposure to toxic agents, etc.). Global control

Since the development of high-density microarray technology in the late 1990s, global host gene expression changes in response to various stimuli have been extensively studied. More than a dozen peer-reviewed publications have investigated the effect of Leishmania infection in various models since 2001. This review covers the transcriptional changes in macrophage models induced by various Leishmania species and summarizes the resulting impact these studies have on our understanding of the host response to leishmaniasis in vitro. Characterization of the similarities and differences between various model systems will not only further our understanding of Leishmania-induced changes to macrophage gene expression but also identify potential therapeutic targets in the future.