Developing a Novel Plague Antibiotic by Targeting Protein Secretion

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DESCRIPTION (provided by applicant): Yersinia pestis, the causative agent of plague, was the most deadly of all the bacterial pathogens in ancient and medieval times. However, in recent years there has been a steady increase in plague morbidity, in the extent and number of plague-endemic areas, and most frighteningly, for the first time strains possessing broad resistance to most antibiotics use to treat plague. Combined with the need for immediate effective treatment, the emergence of resistant strains is especially troubling and highlights the need for an improved understanding of any unique susceptibilities that this bacterium might have, and for molecules that take advantage of the susceptibility. We have recently observed that Y. pestis is hypersensitive to the arylomycin class of natural product antibiotics. In other bacteria, we and others have demonstrated that these antibiotics act via a novel mechanism, namely the inhibition of the bacterial enzyme signal peptidase I (SPase), and thus the inhibition of protein secretion, for which SPase is required. Indeed, it is possible that the obligate parasite Y. pestis is hyperdependent on protein secretion to assemble the systems required for the establishing infections and for scavenging nutrients from its environment. The distinct hypersensitivity supports a stronger reliance on protein secretion for viability in Y. pestis, suggesting that antibiotics targeting secretion might be of particular utility for treating plague. We will seek to define the origins of the hypersensitivity of Y. pestis to the arylomycins via four Aims. First, biochemical and genetic methods will be used to determine whether Y. pestis SPase is produced at unusually low levels or whether the arylomycins bind it with an unusually high affinity (Aim 1). Next, transcriptional profiling will be used to determine whether Y. pestis is deficient in initiating stress responses used by other bacteria to better tolerate the protein secretion stress mediated by the arylomycins (Aim 2). We will then use 2D-LC/MS/MS to determine if the Y. pestis hypersensitivity results from inhibiting the secretion of specific proteins, either that form toxic intermediates or that are required for nutrient acquisition (Aim 3). Lastly, the mechanism by which Y. pestis evolves resistance to the arylomycins will be determined by using whole genome re-sequencing to characterize clones evolved to be resistant in vitro (Aim 4). This approach will provide the first detailed analysis of the arylomycins as anti-plague antibiotics and should reveal the biochemical origins of their unique anti-Y. pestis activity. In addition, the results should determine whether Y. pestis is particularly susceptible to inhibitors of protein secretion, with fundamental implications for our understanding of its physiology and for how antibiotics might be developed for its treatment. PUBLIC HEALTH RELEVANCE: The emergence of drug resistance in Yersinia pestis, the causative bacterium of plague, brings with it the threat that the most devastating disease in human history will return. As an obligate parasite, Yersinia pestis relies on protein secretion to deliver toxins and scavenge nutrients, and we have found that it is hypersensitive to a novel class of natural products that are known to inhibit protein secretion in other bacteria. We propose to characterize the origins of the hypersensitivity, in an effort to validate the inhibition of protein secretion as a new and especially promising approach to developing drugs to combat this ancient but re-emergent disease.