The Nonmevalonate Pathway of Isoprenoid Biosynthesis Supports Anaerobic Growth of Listeria monocytogenes

ABSTRACT Isoprenoids are an essential and diverse class of molecules, present in all forms of life, that are synthesized from an essential common precursor derived from either the mevalonate pathway or the nonmevalonate pathway. Most bacteria have one pathway or the other, but the Gram-positive, facultative intracellular pathogen Listeria monocytogenes is unusual because it carries all the genes for both pathways. While the mevalonate pathway has previously been reported to be essential, here we demonstrate that the nonmevalonate pathway can support growth of strains 10403S and EGD-e, but only anaerobically. L. monocytogenes lacking the gene hmgR, encoding the rate-limiting enzyme of the mevalonate pathway, had a doubling time of 4 h under anaerobic conditions, in contrast to the 45 min doubling time of the wild type. In contrast, deleting hmgR in two clinical isolates resulted in mutants that grew significantly faster, doubling in approximately 2 h anaerobically, although they still failed to grow under aerobic conditions without mevalonate. The difference in anaerobic growth rate was traced to three amino acid changes in the nonmevalonate pathway enzyme GcpE, and these changes were sufficient to increase the growth rate of 10403S to the rate observed in the clinical isolates. Despite an increased growth rate, virulence was still dependent on the mevalonate pathway in 10403S strains expressing the more active GcpE allele.

Targeting the Nonmevalonate Pathway inBurkholderia cenocepaciaIncreases Susceptibility to Certain β-Lactam Antibiotics

ABSTRACTThe nonmevalonate pathway is the sole pathway for isoprenoid biosynthesis inBurkholderia cenocepaciaand is possibly a novel target for the development of antibacterial chemotherapy. The goals of the present study were to evaluate the essentiality ofdxr, the second gene of the nonmevalonate pathway, inB. cenocepaciaand to determine whether interfering with the nonmevalonate pathway increases susceptibility toward antibiotics. To this end, a rhamnose-inducible conditionaldxrknockdown mutant ofB. cenocepaciastrain K56-2 (B. cenocepaciaK56-2dxr) was constructed, using a plasmid which enables the delivery of a rhamnose-inducible promoter in the chromosome. Expression ofdxris essential for bacterial growth; the growth defect observed in thedxrmutant could be complemented by expressingdxr in transunder the control of a constitutive promoter, but not by providing 2-C-methyl-d-erythritol-4-phosphate, the reaction product of DXR (1-deoxy-d-xylulose 5-phosphate reductoisomerase).B. cenocepaciaK56-2dxrshowed markedly increased susceptibility to the β-lactam antibiotics aztreonam, ceftazidime, and cefotaxime, while susceptibility to other antibiotics was not (or was much less) affected; this increased susceptibility could also be complemented byin transexpression ofdxr. A similarly increased susceptibility was observed when antibiotics were combined with FR900098, a known DXR inhibitor. Our data confirm that the nonmevalonate pathway is essential inB. cenocepaciaand suggest that combining potent DXR inhibitors with selected β-lactam antibiotics is a useful strategy to combatB. cenocepaciainfections.

Divergent Isoprenoid Biosynthesis Pathways in Staphylococcus Species Constitute a Drug Target for Treating Infections in Companion Animals

ABSTRACT Drug-resistant Staphylococcus species are a major concern in human and veterinary medicine. There is a need for new antibiotics that exhibit a selective effect in treating infections in companion and livestock animals and that would not be used to treat human bacterial infections. We have identified fosmidomycin as an antibiotic that selectively targets certain Staphylococcus species that are often encountered in skin infections in cats and dogs. These findings expand our understanding of Staphylococcus evolution and may have direct implications for treating staphylococcal infections in veterinary medicine. Staphylococcus species are a leading cause of skin and soft tissue infections in humans and animals, and the antibiotics used to treat these infections are often the same. Methicillin- and multidrug-resistant staphylococcal infections are becoming more common in human and veterinary medicine. From a “One Health” perspective, this overlap in antibiotic use and resistance raises concerns over the potential spread of antibiotic resistance genes. Whole-genome sequencing and comparative genomics analysis revealed that Staphylococcus species use divergent pathways to synthesize isoprenoids. Species frequently associated with skin and soft tissue infections in companion animals, including S. schleiferi and S. pseudintermedius, use the nonmevalonate pathway. In contrast, S. aureus, S. epidermidis, and S. lugdunensis use the mevalonate pathway. The antibiotic fosmidomycin, an inhibitor of the nonmevalonate pathway, was effective in killing canine clinical staphylococcal isolates but had no effect on the growth or survival of S. aureus and S. epidermidis. These data identify an essential metabolic pathway in Staphylococcus that differs among members of this genus and suggest that drugs such as fosmidomycin, which targets enzymes in the nonmevalonate pathway, may be an effective treatment for certain staphylococcal infections. IMPORTANCE Drug-resistant Staphylococcus species are a major concern in human and veterinary medicine. There is a need for new antibiotics that exhibit a selective effect in treating infections in companion and livestock animals and that would not be used to treat human bacterial infections. We have identified fosmidomycin as an antibiotic that selectively targets certain Staphylococcus species that are often encountered in skin infections in cats and dogs. These findings expand our understanding of Staphylococcus evolution and may have direct implications for treating staphylococcal infections in veterinary medicine.

Improved Efficacy of Fosmidomycin against Plasmodium and Mycobacterium Species by Combination with the Cell-Penetrating Peptide Octaarginine

ABSTRACTCellular drug delivery can improve efficacy and render intracellular pathogens susceptible to compounds that cannot permeate cells. The transport of physiologically active compounds across membranes into target cells can be facilitated by cell-penetrating peptides (CPPs), such as oligoarginines. Here, we investigated whether intracellular delivery of the drug fosmidomycin can be improved by combination with the CPP octaarginine. Fosmidomycin is an antibiotic that inhibits the second reaction in the nonmevalonate pathway of isoprenoid biosynthesis, an essential pathway for many obligate intracellular pathogens, including mycobacteria and apicomplexan parasites. We observed a strict correlation between octaarginine host cell permeability and its ability to improve the efficacy of fosmidomycin.Plasmodium bergheiliver-stage parasites were only partially susceptible to an octaarginine-fosmidomycin complex. Similarly,Toxoplasma gondiiwas only susceptible during the brief extracellular stages. In marked contrast, a salt complex of octaarginine and fosmidomycin greatly enhanced efficacy against blood-stagePlasmodium falciparum. This complex and a covalently linked conjugate of octaarginine and fosmidomycin also reverted resistance ofMycobacteriato fosmidomycin. These findings provide chemical genetic evidence for vital roles of the nonmevalonate pathway of isoprenoid biosynthesis in a number of medically relevant pathogens. Our results warrant further investigation of octaarginine as a delivery vehicle and alternative fosmidomycin formulations for malaria and tuberculosis drug development.

Herbicides as Probes in Plant Biology

Herbicides are small molecules that inhibit specific molecular target sites within plant biochemical pathways and/or physiological processes. Inhibition of these sites often has catastrophic consequences that are lethal to plants. The affinity of these compounds for their respective target sites makes them useful tools to study and dissect the intricacies of plant biochemical and physiological processes. For instance, elucidation of the photosynthetic electron transport chain was achieved in part by the use of herbicides, such as terbutryn and paraquat, which act on photosystem II and I, respectively, as physiological probes. Work stemming from the discovery of the binding site of PS II–inhibiting herbicides was ultimately awarded the Nobel Prize in 1988. Although not as prestigious as the seminal work on photosynthesis, our knowledge of many other plant processes expanded significantly through the ingenious use of inhibitors as molecular probes. Examples highlight the critical role played by herbicides in expanding our understanding of the fundamental aspects of the synthesis of porphyrins and the nonmevalonate pathway, the evolution of acetyl-coenzyme A carboxylase, cell wall physiology, the functions of microtubules and the cell cycle, the role of auxin and cyanide, the importance of subcellular protein targeting, and the development of selectable markers.

The Nonmevalonate Pathway of Isoprenoid Biosynthesis in Mycobacterium tuberculosis Is Essential and Transcriptionally Regulated by Dxs

ABSTRACT Mycobacterium tuberculosis synthesizes isoprenoids via the nonmevalonate or DOXP pathway. Previous work demonstrated that three enzymes in the pathway (Dxr, IspD, and IspF) are all required for growth in vitro. We demonstrate the essentiality of the key genes dxs1 and gcpE, confirming that the pathway is of central importance and that the second homolog of the synthase (dxs2) cannot compensate for the loss of dxs1. We looked at the effect of overexpression of Dxr, Dxs1, Dxs2, and GcpE on viability and on growth in M. tuberculosis. Overexpression of dxs1 or dxs2 was inhibitory to growth, whereas overexpression of dxr or gcpE was not. Toxicity is likely to be, at least partially, due to depletion of pyruvate from the cells. Overexpression of dxs1 or gcpE resulted in increased flux through the pathway, as measured by accumulation of the metabolite 4-hydroxy-3-methyl-but-2-enyl pyrophosphate. We identified the functional translational start site and promoter region for dxr and demonstrated that it is expressed as part of a polycistronic mRNA with gcpE and two other genes. Increased expression of this operon was seen in cells overexpressing Dxs1, indicating that transcriptional control is effected by the first enzyme of the pathway via an unknown regulator.