Sub-inhibitory fosmidomycin exposures elicits oxidative stress in Salmonella enterica Serovar typhimurium LT2

Fosmidomycin is a time-dependent nanomolar inhibitor of methylerythritol phosphate (MEP) synthase, which is the enzyme that catalyzes the first committed step in the MEP pathway to isoprenoids. Importantly, fosmidomycin is one of only a few MEP pathway-specific inhibitors that exhibits antimicrobial activity. Most inhibitors identified to date only exhibit activity against isolated pathway enzymes. The MEP pathway is the sole route to isoprenoids in many bacteria, yet has no human homologs. The development of inhibitors of this pathway holds promise as novel antimicrobial agents. Similarly, analyses of the bacterial response toward MEP pathway inhibitors provides valuable information toward the understanding of how emergent resistance may ultimately develop to this class of antibiotics. We have examined the transcriptional response of Salmonella enterica serovar typhimurium LT2 to sub-inhibitory concentrations of fosmidomycin via cDNA microarray and RTPCR. Within the regulated genes identified by microarray were a number of genes encoding enzymes associated with the mediation of reactive oxygen species (ROS). Regulation of a panel of genes implicated in the response of cells to oxidative stress (including genes for catalases, superoxide dismutases, and alkylhydrogen peroxide reductases) was investigated and mild upregulation in some members was observed as a function of fosmidomycin exposure over time. The extent of regulation of these genes was similar to that observed for comparable exposures to kanamycin, but differed significantly from tetracycline. Furthermore, S. typhimurium exposed to sub-inhibitory concentrations of fosmidomycin displayed an increased sensitivity to exogenous H2O2 relative to either untreated controls or kanamycin-treated cells. Our results suggest that endogenous oxidative stress is one consequence of exposures to fosmidomycin, likely through the temporal depletion of intracellular isoprenoids themselves, rather than other mechanisms that have been proposed to facilitate ROS accumulation in bacteria (e.g. cell death processes or the ability of the antibiotic to redox cycle)

Rapid Thermostabilization of <i>Bacillus thuringiensis</i> Serovar Konkukian 97–27 Dehydroshikimate Dehydratase through a Structure-Based Enzyme Design and Whole Cell Activity Assay

Thermostabilization of an enzyme with complete retention of catalytic efficiency was demonstrated on recombinant 3-dehydroshikimate dehydratase (DHSase or wtAsbF) from <i>Bacillus thuringiensis</i> serovar konkukian 97–27 (hereafter, <i>B. thuringiensis</i> 97–27). The wtAsbF is relatively unstable at 37 °C, <i>in vitro</i> (<i>t</i>_1/2³⁷ = 15 min), in the absence of divalent metal. We adopted a structure-based design to identify stabilizing mutations and created a combinatorial library based upon predicted mutations at specific locations on the enzyme surface. A diversified <i>asbF</i> library (∼2000 variants) was expressed in <i>E. coli</i> harboring a green fluorescent protein (GFP) reporter system linked to the product of wtAsbF activity (3,4-dihydroxybenzoate, DHB). Mutations detrimental to DHSase function were rapidly eliminated using a high throughput fluorescence activated cell sorting (FACS) approach. After a single sorting round and heat screen at 50 °C, a triple AsbF mutant (Mut1), T61N, H135Y, and H257P, was isolated and characterized. The half-life of Mut1 at 37 °C was >10-fold higher than the wtAsbF (<i>t</i>_1/2³⁷ = 169 min). Further, the second-order rate constants for both wtAsbF and Mut1 were approximately equal (9.9 × 10⁵ M^–1 s^–1, 7.8 × 10⁵ M^–1 s^–1, respectively), thus demonstrating protein thermostability did not come at the expense of enzyme thermophilicity. In addition, <i>in vivo</i> overexpression of Mut1 in <i>E. coli</i> resulted in a ∼60-fold increase in functional enzyme when compared to the wild-type enzyme under the identical expression conditions. Finally, overexpression of the thermostable AsbF resulted in an approximate 80–120% increase in DHB accumulation in the media relative to the wild-type enzyme

Biosynthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP).

<p>IPP and DMAPP are formed through the mevalonate pathway (left) in mammals, fungi and plants and through the methylerythritol phosphate (MEP) pathway (right) in many bacteria, green algae, and plant chloroplasts. Fosmidomycin inhibits formation of IPP and DMAPP (and thus late stage isoprenoid compounds) through disruption of the MEP pathway.</p

Potential Effects of Isoprenoid Deprivation on Cellular Respiration.

<p>Disruption of the MEP pathway reduces the cell’s ability to form late stage isoprenoid products such as ubiquinone and menaquinone. Impairment of bacterial respiration via inhibition of ubiquinone/menaquinone synthesis may decrease the cell’s ability to reduce oxygen into water, and ultimately may foster the production of reactive oxygen species through direct reduction of molecular oxygen.</p

Cell viability of antibiotic challenged cells upon hydrogen peroxide exposure.

<p>Viable colony forming units/mL upon H₂O₂ (10 mM) treatment for untreated control cells, or cells previously challenged with fosmidomycin, kanamycin or tetracycline. The data represents the average of three independent tests and the error reflects the standard error of the mean.</p

Regulation of selected genes of relevance to oxidative stress identified in microarray analysis of short (20 min) sub-inhibitory exposures to fosmidomycin or kanamycin.

<p>Regulation of selected genes of relevance to oxidative stress identified in microarray analysis of short (20 min) sub-inhibitory exposures to fosmidomycin or kanamycin.</p