Transcriptional responses of Listeria monocytogenes to low-temperature stress reveal distinct adaptive mechanisms in the absence of BKD and PrfA activation.

Listeria monocytogenes, a foodborne pathogen capable of growth at refrigeration temperatures, represents a significant public health challenge. Its ability to grow at low temperatures is mediated by incorporation of high levels of branched-chain fatty acids (BCFAs) into its membrane. This adaptation is crucial for maintaining optimal membrane fluidity and function under harsh conditions. L. monocytogenes synthesizes BCFAs through a pathway that is linked to branched-chain amino acids (BCAAs) degradation, a process critically dependent on the branched-chain α-keto acid dehydrogenase (BKD) enzyme, which catalyzes a key step in converting BCAAs into precursors for BCFA synthesis. While BKD contributes to membrane integrity and supports virulence, the master regulator of virulence factors in L. monocytogenes is PrfA. This study investigated the transcriptional response of L. monocytogenes to low temperatures under two distinct genetic conditions: deletion of bkdA1 (ΔbkdA1), which encodes a subunit of BKD, and constitutive activation of PrfA (F2365PrfA∗ strain). The results indicate that while PrfA activation does not affect BCFA biosynthesis, or phospholipid composition, the BKD enzyme remains crucial for BCFA production and maintaining normal phospholipid profiles. RNA-seq analysis revealed contrasting adaptive strategies in the ΔbkdA1 and F2365PrfA∗ strains compared to the wild-type (WT). A key finding was the upregulation of BCAA biosynthesis in the absence of BKD and downregulation under active PrfA conditions. The upregulation observed in the ΔbkdA1 strain may reflect a compensatory mechanism for the loss of BKD function, while the downregulation in the PrfA-activated strain could be part of a broader metabolic shift associated with virulence genes expression. Additionally, the ΔbkdA1 strain showed significant upregulation of fabH encoding β-ketoacyl-ACP synthase III, despite general downregulation of other fatty acid biosynthesis genes. This suggests a compensatory mechanism for BCFA synthesis in the absence of BKD complex activity. Conversely, the F2365PrfA∗ strain exhibited upregulation of several fatty acid biosynthesis genes. Both strains showed altered expression of genes involved in amino acid metabolism, cell wall structure, and transport systems, reflecting comprehensive cellular reprogramming. These findings provide new insights into adaptive mechanisms of L. monocytogenes under low temperatures and highlight the intricate interplay between metabolism, stress response, and virulence in this medically important pathogen.