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A host-directed adjuvant sensitizes intracellular bacterial persisters to antibiotics

ABSTRACT: Intracellular bacterial reservoirs contribute to antibiotic treatment failure by fostering metabolically dormant persister cells that are highly tolerant to killing. However, strategies to effectively target intracellular persister cells remain limited. Here, we developed a high-throughput screen to identify compounds that modulate the metabolic activity of intracellular Staphylococcus aureus. The identified compound, KL1, increases intracellular bacterial metabolic activity and sensitizes persister populations of S. aureus to antibiotics, without causing cytotoxicity or bacterial outgrowth. KL1 also exhibits adjuvant activity against intramacrophage Salmonella enterica Typhimurium and Mycobacterium tuberculosis, as well as in murine infection models of S. aureus and S. Typhimurium infection. Transcriptomic analysis and further mechanistic studies reveal that KL1 modulates host immune response genes and suppresses the production of reactive species in host macrophages, alleviating a key inducer of antibiotic tolerance. Our findings highlight the potential to target intracellular persisters by stimulating their metabolism.

ORGANISM(S): Mus musculus

PROVIDER: GSE280093 | GEO | 2025/08/01

REPOSITORIES: GEO

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Project description:Staphylococcus aureus causes invasive infections and easily acquires antibiotic resistances. Even antibiotic susceptible S. aureus can survive antibiotic therapy and persist, requiring prolonged treatment and surgical interventions. These so-called persisters display an arrested-growth phenotype, tolerate high antibiotic concentrations and are associated with chronic and recurrent infections. To characterize these persisters, we assessed S. aureus recovered directly from a patient suffering from a persistent infection. We show that host-mediated stress, including acidic-pH, abscesses-environment, and antibiotic exposure promoted persister formation in-vitro and in-vivo. Multi-omics analysis identified molecular changes in S. aureus in response to acid-stress leading to an overall virulent population. However, further analysis of a persister-enriched population revealed major molecular reprogramming in persisters including downregulation of virulence and cell division, and upregulation of ribosomal proteins, nucleotide-, and amino acid- metabolic pathways, suggesting their requirement to fuel and maintain the persister phenotype and highlighting that persisters are not completely metabolically inactive. Additionally, decreased aconitase activity and ATP-levels and accumulation of insoluble proteins involved in transcription, translation and energy-production correlated with persistence in S. aureus, underpinning the molecular mechanisms that drive the persister phenotype. Upon regrowth, these persisters regained their virulence potential and metabolically active phenotype including reduction of insoluble proteins, exhibiting a reversible state, crucial for recurrent infections. We further show that a targeted anti-persister combination therapy using retinoid derivatives and antibiotics significantly reduced lag-phase heterogeneity and persisters in a murine infection model. Our results provide molecular insights into persisters and help explain why persistent S. aureus infections are so difficult-to-treat.

Project description:The ability to eliminate bacterial persister cells is still a medical challenge that has yet to be overcome. These cells represent a unique subpopulation within bacterial communities and are characterized by a reduced susceptibility to antibiotics with growth retardation. In this study, we investigated the molecular basis of persister formation in Salmonella Typhimurium 14028s under aminoglycoside stress.We analyzed the crystal structure of the STM14_5441–STM14_5442 complex, which belongs to the type II toxin-antitoxin system, and identified key ribosome binding residues in STM14_5441. Changes in the antibiotic susceptibility of Salmonella caused by the loss of the ribosome binding property of STM14_5441 were assessed. We conducted intracellular ATP assays under aminoglycoside stress and RNA-seq analysis following STM14_5441 induction.Our studies demonstrated the critical role of STM14_5441 in the formation of persister cells in Salmonella, particularly those under aminoglycoside stress. We observed that a loss of ribosome binding in STM14_5441 resulted in increased antibiotic susceptibility. Additionally, intracellular ATP assays revealed increased ATP levels in bacterial persister cells, and RNA-seq analysis identified several genes that play a role in the formation of these persister cells.The present data suggest that persister forms under aminoglycoside stress through the following mechanisms: i) inhibition of membrane hyperpolarization by impeding F1Fo ATP synthase activity and ii) enhanced poststress recovery by ATP storage and increased protein synthesis capacity. Based on this suggestion, we reannotated the STM14_5441-STM14_5442 TA pair as the ResTA (RNA cleavage-induced energy storage toxin-antitoxin) system. Furthermore, new insights into the function of TA systems may lay the groundwork for developing novel strategies to target bacterial persister cells, thereby preventing the accelerated emergence of antibiotic resistance in bacterial populations.

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A host-directed adjuvant sensitizes intracellular bacterial persisters to antibiotics

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