The Combined Effect of Cold and Copper Stresses on the Proliferation and Transcriptional Response of Listeria monocytogenes

Abstract

Listeria monocytogenes is a foodborne pathogen that can cause severe disease in susceptible humans. This microorganism has the ability to adapt to hostile environmental conditions such as the low temperatures used by the food industry for controlling microorganisms. Bacteria are able to adjust their transcriptional response to adapt to stressful conditions in order to maintain cell homeostasis. Understanding the transcriptional response of L. monocytogenes to stressing conditions could be relevant to develop new strategies to control the pathogen. A possible alternative for controlling microorganisms in the food industry could be to use copper as an antimicrobial agent. The present study characterized three L. monocytogenes strains (List2-2, Apa13-2, and Al152-2A) adapted to low temperature and challenged with different copper concentrations. Similar MIC-Cu values were observed among studied strains, but growth kinetic parameters revealed that strain List2-2 was the least affected by the presence of copper at 8°C. This strain was selected for a global transcriptional response study after a 1 h exposition to 0.5 mM of CuSO 4 × 5H 2 O at 8 and 37°C. The results showed that L. monocytogenes apparently decreases its metabolism in response to copper, and this reduction is greater at 8°C than at 37°C. The most affected metabolic pathways were carbohydrates, lipids and nucleotides synthesis. Finally, 15 genes were selected to evaluate the conservation of the transcriptional response in the other two strains. Results indicated that only genes related to copper homeostasis showed a high degree of conservation between the strains studied, suggesting that a low number of genes is implicated in the response to copper stress in L. monocytogenes . These results contribute to the understanding of the molecular mechanisms used by bacteria to overcome a combination of stresses. This study concluded that the application of copper in low concentrations in cold environments may help to control foodborne pathogens as L. monocytogenes in the industry.

Cobalamin cbiP mutant shows decreased tolerance to low temperature and copper stress in Listeria monocytogenes

“…The genome sequence analysis of List 2-2 strain allowed us to identify the 22 genes previously described involved in the anaerobic pathway of cobalamin biosynthesis. We have observed that the growth of L. monocytogenes in low temperature and copper activates mechanisms in response to stress that allow the bacterium to tolerate these conditions [ 36 , 37 ]. Recently, Ahn et al .…”

“…Previously we had observed that the transcriptional response of L. monocytogenes differed when stress factors were presented separately or together. In particular, exposure of List2-2 to low temperature + copper activated different cellular processes from those regulated by cold or copper [ 36 ]. In this study, we observed an increase in the expression of genes associated with the cobalamin pathway when L. monocytogenes was cultured at different times at low temperatures with copper in aerobic conditions.…”

“…Although copper has been described as an essential micronutrient that participates as an enzyme cofactor [ 32 , 33 ], its intracellular level is strictly regulated because its excess is toxic [ 34 , 35 ]. We previously observed that the antibacterial effect of copper in L. monocytogenes was enhanced at low temperatures [ 35 ] and we determined that the minimal inhibitory concentration of copper at 37 °C was 10–12 mM and at 8 °C was 4–6 mM of CuSO 4 × 5H 2 O [ 36 ].…”

“…We analyzed the proliferation rate of different strains of L. monocytogenes and identified that the List2-2 strain had the highest growth rate at 8 °C [ 37 ]. This strain was the least affected by the combination of low temperature + copper according to their growth kinetic parameters [ 36 ]. List2-2 was selected to evaluate the effect of the global transcriptional response to low temperature and copper.…”

“…List2-2 was selected to evaluate the effect of the global transcriptional response to low temperature and copper. This strain modified the expression of 263 genes in response to a sub-inhibitory copper concentration (0.5 mM of CuSO 4 × 5H 2 O) + low temperature (8 °C), including some genes encoding for proteins involved in the cobalamin biosynthesis pathway [ 36 ]. Based on this information, we hypothesized that cobalamin biosynthesis was involved in the tolerance of L. monocytogenes List2-2 to the simultaneous exposure to low temperature (8 °C) + copper (0.5 mM) as a stress condition.…”