Unravelling the Molecular Mechanisms Underlying the Protective Effect of Lactate on the High-Pressure Resistance of Listeria monocytogenes

Abstract

Formulations with lactate as an antimicrobial and high-pressure processing (HPP) as a lethal treatment are combined strategies used to control L. monocytogenes in cooked meat products. Previous studies have shown that when HPP is applied in products with lactate, the inactivation of L. monocytogenes is lower than that without lactate. The purpose of the present work was to identify the molecular mechanisms underlying the piezo-protection effect of lactate. Two L. monocytogenes strains (CTC1034 and EGDe) were independently inoculated in a cooked ham model medium without and with 2.8% potassium lactate. Samples were pressurized at 400 MPa for 10 min at 10 °C. Samples were subjected to RNA extraction, and a shotgun transcriptome sequencing was performed. The short exposure of L. monocytogenes cells to lactate through its inoculation in a cooked ham model with lactate 1h before HPP promoted a shift in the pathogen's central metabolism, favoring the metabolism of propanediol and ethanolamine together with the synthesis of the B12 cofactor. Moreover, the results suggest an activated methyl cycle that would promote modifications in membrane properties resulting in an enhanced resistance of the pathogen to HPP. This study provides insights on the mechanisms developed by L. monocytogenes in response to lactate and/or HPP and sheds light on the understanding of the piezo-protective effect of lactate.

Keywords: HPP; Listeria monocytogenes; organic acids; piezo-resistance; pressurization.

Figure 1

Inactivation (Log N₀/N) of each biological replicate of the L. monocytogenes CTC1034 and EGDe strains observed after HPP (400 MPa for 10 min) in cooked ham model medium without (control) and with 2.8% (v/v) potassium lactate.

Figure 2

Venn diagrams of differentially expressed genes (DEGs) of L. monocytogenes strains CTC1034 (A) and EGDe (B) due to the exposure of cells to lactate, the application of the HPP (400 MPa for 10 min) and the application of both stresses compared to control conditions (exposed to CHMM without lactate).

Figure 3

Predicted carbon flux in L. monocytogenes CTC1034 and EGDe when exposed to lactate. Blue, red, and grey arrows and text indicate genes that were upregulated, downregulated, or were not differentially expressed, respectively. Genes and proteins: EutH, ethanolamine transporter; EutA, ethanolamine transporter protein EutA; EutB, ethanolamine ammonia-lyase large subunit; EutC, ethanolamine ammonia-lyase small subunit; EutG, alcohol dehydrogenase; EutE, aldehyde dehydrogenase; EutD, phosphotransacetylase; EutQ, ethanolamine utilization protein EutQ; Glo1, lactoylglutathione lyase; PduC, propanediol dehydratase large subunit; PduD, propanediol dehydratase medium subunit; PduE, propanediol dehydratase small subunit; PduP, propionaldehyde dehydrogenase; PduQ, 1-propanol dehydrogenase; PduL, phosphate propanoyltransferase; PduW, propionate kinase; TPI, triosephosphate isomerase; FruA, fructose PTS system EIIBC; FruK, 1-phosphofructokinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ppdK, pyruvate orthophosphate dikinase; pdhC, pyruvate dehydrogenase E2 component; pflD, formate C-acetyltransferase; adhE, acetaldehyde dehydrogenase/alcohol dehydrogenase; tktA, tktB, transkelotase; G6PD, glucose-6-phosphate 1-dehydrogenase; RpiB, ribose 5-phosphate isomerase B; DhaL, phosphoenolpyruvate-glycerone phosphotransferase subunit DhaL; GlpK, glycerol kinase; RhaB, rhamnulokinase; RhaA, L-rhamnose isomerase; gltB, glutamate synthase; gadB/A, glutamate descarboxylase; GABA-AT, GABA aminotransferase; SSADH, succinate semialdehyde dehydrogenase

Figure 4

Predicted activation of the methyl cycle in L. monocytogenes CTC1034 and EGDe strains when exposed to lactate and its potential role on the piezo-protective effect exerted by lactate on L. monocytogenes stress induced by HPP. Blue and red arrows and text indicate genes that were upregulated and downregulated, respectively. Genes and proteins: CysE, serine O-acetyltransferase; metX, homoserine O-acetyltransferase; metC, cysteine-S-conjugate beta-lyase; mmuM and metE, homocysteine S-methyltransferases; metK, S-adenosylmethionine synthetase; luxS, S-ribosylhomocysteine lyase; SAM, S-adenosyl-methionine; SAH, S-adenosyl-homocysteine.