Mechanistic insights into acid-induced viable but non-culturable state formation regulated by asr in Escherichia coli O157:H7: roles of intracellular protein aggregation.

Food processing exposes bacteria to multiple stresses that trigger the formation of viable but non-culturable (VBNC) state, posing food safety risks. High-pressure carbon dioxide (HPCD), a non-thermal technology, induces VBNC formation in Escherichia coli O157:H7 mainly via low-pH stress. The acid-stress gene asr is speculated to mediate intracellular protein aggregation and facilitate VBNC development, though the underlying mechanism remains unclear. Here in this study, acid stress (pH 3) was applied to explore the regulatory role of asr in VBNC formation. Following 30 h of exposure to acid stress, E. coli O157:H7 entered the VBNC state, accompanied by marked upregulation of asr and suppressed expression of acid resistance genes. This repression impeded intracellular proton consumption, reduced cytoplasmic pH (pHin), and ultimately triggered intracellular protein aggregation. The aggregated proteins were identified to be mainly enriched in energy production and conversion, amino acid/carbohydrate transport and metabolism, cell envelope biogenesis, transcription and translation, as well as replication, recombination and repair. This aggregation reduced intracellular ATP levels, while aggregate disassembly restored ATP during resuscitation. Consistently, the Δasr strain failed to form VBNC cells and protein aggregates under acid stress, and the phenotypes were restored in its complemented strain. Transcriptomic analysis revealed that the Δasr strain exhibited impaired stress perception, accompanied by upregulation of genes associated with protein refolding and virulence, enhanced utilization of efficient carbon sources for energy provision, increased acid responses and repression of toxin-antitoxin systems. These alterations drove bacterial cells toward death rather than the formation of VBNC state. Collectively, results in this study proved that HPCD-induced asr expression promotes intracellular protein aggregation to establish a low-energy status, thereby facilitating VBNC formation in E. coli O157:H7.