Inside out: Revealing strategies for intracellular S. aureus persister eradication

Project Summary Antibiotic treatment failure imposes a significant global health and economic burden. Even in the absence of resistance mutations, the recurrence of infections can be attributed to a resilient subset of genetically identical bacteria. This phenomenon, recognized as antibiotic-tolerant persister formation, also facilitates the development of resistance, underscoring a critical challenge that demands attention. Mounting evidence suggests that the intracellular environment plays a pivotal role in the formation of persister cells, serving as a crucial reservoir for these bacterial populations. Among the most difficult-to-treat bacterial pathogens, Staphylococcus aureus can survive antibiotic exposure within host cells, including macrophages, and subsequently resume growth when conditions become favorable again. However, the intracellular niche of S. aureus in vivo and the underlying mechanisms driving persister formation remain largely enigmatic. This knowledge gap can be attributed to the challenge of distinguishing viable persisters from non-viable bacteria and the low frequency of cells harboring live bacteria (less than 0.01% of overall cells extracted from mouse organs). To address this hurdle, I have developed inducible reporter strains that will enable the identification of intracellular reservoirs in vivo. Using single-cell approaches, I will identify the host cells containing S. aureus and the antibiotic-tolerant persisters (Aim 1.1). Both the host and intracellular bacterial transcriptomes will be probed using single-cell RNA-sequencing (scRNA-seq) and a novel single-cell bacterial RNA-seq technology called proBac-seq (Aim 1.2 & 1.3). I have also established a high-throughput screening platform aimed at identifying energy modulators of intracellular S. aureus, as the low energy state is the central characteristic underlying persister cells’ insensitivity to antibiotics. My preliminary screen of >4,700 drug-like compounds has identified a compound (KL1) that increased the energy level of intracellular S. aureus as well as its susceptibility to antibiotics. Further understanding of its mechanism of action using the chemoproteomic method thermal proteome profiling (TPP) will unravel novel therapeutic target(s) for devising antibiotic adjuvants (Aim 2.1). Lastly, I intend to explore potential host-directed energy modulators from a select library of established mammalian kinase inhibitors targeting intracellular immune responses (Aim 2.2). These studies will provide a comprehensive understanding of the interplay between host responses and antibiotic tolerance within the intracellular milieu.