L-Sistein ve SigB'nin Listeria monocytogenes 10403S'nin termal toleransı üzerindeki oksijene bağlı etkileri

Abstract

Listeria monocytogenes, ısı, oksidatif ve ozmotik stresler dahil olmak üzere çok çeşitli çevresel koşullara dayanabilen önemli bir gıda kaynaklı patojendir. Alternatif sigma faktörü SigB, bakterinin stres adaptasyonunda merkezi bir rol oynamaktadır. Ancak oksijenin sınırlı olduğu koşullar ve besin açısından zengin ortamlardaki işlevi henüz tam olarak aydınlatılamamıştır. Et ve et ürünleri, süt ürünleri ve baklagillerinde yaygın olarak bulunan kükürt içeren bir amino asit olan L-sistein, hem metabolik bir sinyal molekülü hem de hücresel antioksidan sistemlerin öncüsü olarak stres toleransını etkileyebileceği önceki çalışmalarda gösterilmiştir. Bu çalışmada, hücre dışı L-sistein takviyesinin L. monocytogenes'in ısı direnci üzerindeki etkisi incelenmiş ve aerobik ile anaerobik büyüme koşulları altında SigB'nin rolü değerlendirilmiştir. Yabanıl tip L. monocytogenes 10403S suşu ile izojenik ΔsigB mutantı, 1,57 mM L-sistein ile takviye edilmiş tanımlı besiyerinde, kontrollü aerobik ve anaerobik koşullar altında 56°C'de ısı stresine maruz bırakılmıştır. Bakteriyel hayatta kalma oranı kantitatif olarak belirlenmiş ve elde edilen veriler farklı suşlar ve çevresel koşullar arasında karşılaştırmalı olarak analiz edilmiştir. Elde edilen bulgular, L-sistein takviyesinin özellikle anaerobik koşullarda uygulanan ısı stresi altında bakteriyel hayatta kalmayı anlamlı düzeyde artırdığını göstermiştir. Ayrıca, ΔsigB mutantının bu koşullarda yabanıl tip suşa kıyasla ısıl strese karşı daha yüksek direnç sergilediği belirlenmiştir. Bu sonuçlar, L-sistein metabolizması veya ilişkili biyokimyasal yolakların, SigB aracılı stres yanıtının yokluğunu kısmen telafi edebileceğini düşündürmektedir. Sonuç olarak, L-sistein mevcudiyeti, oksijen düzeyi ve SigB faktörü, L. monocytogenes'in ısı stresine karşı hayatta kalmasını bağlama bağlı olarak birlikte şekillendirmektedir. Bu etkileşimlerin daha iyi anlaşılması, gıda ürünleri ve işleme ortamlarında L. monocytogenes'in kontrolüne yönelik daha etkili ve hedefe yönelik stratejilerin geliştirilmesine katkı sağlayabilir.

Keywords

• Listeria monocytogenes
• Isı stresi direnci
• SigB (σ^B)
• L-sistein
• Anaerobik koşullar

Oxygen-dependent effects of L-Cysteine and SigB on thermal tolerance of Listeria monocytogenes 10403S

Abstract

Listeria monocytogenes is a resilient foodborne pathogen capable of surviving diverse environmental stresses, including heat, oxidative, and osmotic conditions. The alternative sigma factor SigB plays a central role in mediating stress adaptation. However, its function under oxygen-limited conditions and in nutrient-rich environments remains insufficiently understood. L-cysteine, commonly present in food matrices, may influence bacterial stress tolerance by acting as both a metabolic signal and a precursor for antioxidant systems. This study investigated the effect of extracellular L-cysteine supplementation on the heat resistance of L. monocytogenes and evaluated the contribution of SigB under both aerobic and anaerobic conditions. Wild-type L. monocytogenes 10403S and an isogenic ΔsigB mutant were subjected to heat stress in defined medium supplemented with 1.57 mM L-cysteine. Bacterial survival was quantified and compared across strains and environmental conditions. L-cysteine supplementation significantly enhanced bacterial survival under anaerobic heat stress. Notably, the ΔsigB mutant exhibited greater resistance than the wild-type strain under these conditions. This observation suggests that L-cysteine-associated metabolic pathways may compensate, at least partially, for the absence of SigB-mediated stress regulation. The enhanced survival in the mutant strain points to alternative protective mechanisms, potentially linked to redox balance or sulphur metabolism. Overall, the findings demonstrate that L-cysteine availability, oxygen conditions, and SigB interact in a complex and context-dependent manner to influence heat stress survival in L. monocytogenes. These results highlight the importance of metabolic state in shaping bacterial stress responses and suggest that sulphur metabolism may serve as a key compensatory pathway under oxygen-limited conditions. A deeper understanding of these interactions could support the development of more effective strategies for controlling L. monocytogenes in food systems and processing environments.

Keywords

• Listeria monocytogenes
• Heat stress resistance
• SigB (σ^B)
• L-cysteine
• Anaerobic conditions

References

1. Amezaga, M.-R., Davidson, I., Mclaggan, D., Verheul, A., Abee2, T., & Booth’, I. R. (1995). The role of peptide metabolism in the growth of Listeria monocytogenes ATCC 23074 at high osmolarity. Microbiology, 141(1), 41-49. https://doi.org/10.1099/00221287-141-1-41.
2. Aryal, B., Kwakye, J., Ariyo, O. W., Ghareeb, A. F. A., Milfort, M. C., Fuller, A. L., Khatiwada, S., Rekaya, R., & Aggrey, S. E. (2025). Major Oxidative and Antioxidant Mechanisms During Heat Stress-Induced Oxidative Stress in Chickens. Antioxidants, 14(4), 471. https://doi.org/10.3390/antiox14040471
3. Berude, J. C., Kennouche, P., Reniere, M. L., & Portnoy, D. A. (2024). Listeria monocytogenes utilizes glutathione and limited inorganic sulfur compounds as sources of essential cysteine. Infection and Immunity, 92(3), 1-18. https://doi.org/10.1128/iai.00422-23
4. Boura, M., Keating, C., Royet, K., Paudyal, R., O’Donoghue, B., O’Byrne, C. P., & Karatzas, K. A. G. (2016). Loss of sigb in listeria monocytogenes strains egde and 10403s confers hyperresistance to hydrogen peroxide in stationary phase under aerobic conditions. Applied and Environmental Microbiology, 82(15), 4584–4591. https://doi.org/10.1128/AEM.00709-16
5. Bucur, F. I., Grigore-Gurgu, L., Crauwels, P., Riedel, C. U., & Nicolau, A. I. (2018). Resistance of Listeria monocytogenes to Stress Conditions Encountered in Food and food processing environments. Frontiers in Microbiology, 9, 2700. https://doi.org/10.3389/fmicb.2018.02700
6. Burguière, P., Fert, J., Guillouard, I., Auger, S., Danchin, A., & Martin-Verstraete, I. (2005). Regulation of the Bacillus subtilis ytmI operon, involved in sulfur metabolism. Journal of Bacteriology, 187(17), 6019–6030. https://doi.org/10.1128/JB.187.17.6019-6030.2005
7. Caballero Cerbon, D. A., Gebhard, L., Dokuyucu, R., Ertl, T., Härtl, S., Mazhar, A., & Weuster-Botz, D. (2024). Challenges and Advances in the Bioproduction of L-Cysteine. Molecules, 29(2), 486. https://doi.org/10.3390/molecules29020486
8. Clemente-Carazo, M., Cebrian, G., Garre, A., & Palop, A. (2020). Variability in the heat resistance of Listeria monocytogenes under dynamic conditions can be more relevant than that evidenced by isothermal treatments. Food Research International, 137, 109538. https://doi.org/10.1016/j.foodres.2020.109538

9. Couvert, O., Divanac’h, M. L., Lochardet, A., Thuault, D., & Huchet, V. (2019). Modelling the effect of oxygen concentration on bacterial growth rates. Food Microbiology, 77, 21–25. https://doi.org/10.1016/j.fm.2018.08.005
10. Crespo Tapia, N., Dorey, A. L., Gahan, C. G. M., den Besten, H. M. W., O’Byrne, C. P., & Abee, T. (2020). Different carbon sources result in differential activation of sigma B and stress resistance in Listeria monocytogenes. International Journal of Food Microbiology, 320. https://doi.org/10.1016/j.ijfoodmicro.2019.108504
11. Doyle, M. E., Mazzotta, A. S., Wang, T., Wiseman, D. W., and, §, & Scott, V. N. (2001). Heat Resistance of Listeria monocytogenes. Journal of Food Protection, 64(3), 410-429. https://doi.org/10.4315/0362-028x-64.3.410.
12. Even, S., Burguière, P., Auger, S., Soutourina, O., Danchin, A., & Martin-Verstraete, I. (2006). Global control of cysteine metabolism by CymR in Bacillus subtilis. Journal of Bacteriology, 188(6), 2184–2197. https://doi.org/10.1128/JB.188.6.2184-2197.2006
13. Ferreira, A., O’Byrne, C. P., & Boor, K. J. (2001). Role of σB in Heat, Ethanol, Acid, and Oxidative Stress Resistance and during Carbon Starvation in Listeria monocytogenes. Applied and Environmental Microbiology, 67(10), 4454–4457. https://doi.org/10.1128/AEM.67.10.4454-4457.2001
14. Fu, H., Yuan, J., & Gao, H. (2015). Microbial oxidative stress response: Novel insights from environmental facultative anaerobic bacteria. Archives of Biochemistry and Biophysics, 584, 28–35. https://doi.org/10.1016/j.abb.2015.08.012
15. George, S. M., & Peck, M. W. (1998). Redox potential affects the measured heat resistance of Escherichia coli O157:H7 independently of oxygen concentration. Letters in Applied Microbiology, 27(6), 313-317. https://doi.org/10.1046/j.1472-765x.1998.00466.x.
16. Gnanou Besse, N., Dubois Brissonnet, F., Lafarge, V., & Leclerc, V. (2000). Effect of various environmental parameters on the recovery of sublethally salt-damaged and acid-damaged Listeria monocytogenes. Journal of Applied Microbiology, 89(6), 944–950. https://doi.org/10.1046/j.1365-2672.2000.01195.x
17. Górska-Warsewicz, H., Laskowski, W., Kulykovets, O., Kudlińska-Chylak, A., Czeczotko, M., & Rejman, K. (2018). Food products as sources of protein and amino acids—The case of Poland. Nutrients, 10(12), 1977. https://doi.org/10.3390/nu10121977
18. Ji, Q., Zhang, L., Sun, F., Deng, X., Liang, H., Bae, T., & He, C. (2012). Staphylococcus aureus CymR is a new thiol-based oxidation-sensing regulator of stress resistance and oxidative response. Journal of Biological Chemistry, 287(25), 21102–21109. https://doi.org/10.1074/jbc.M112.359737
19. Karatzas, K. A. G., Brennan, O., Heavin, S., Morrissey, J., & O’Byrne, C. P. (2010). Intracellular accumulation of high levels of γ-aminobutyrate by Listeria monocytogenes 10403S in response to low pH: Uncoupling of γ-aminobutyrate synthesis from efflux in a chemically defined medium. Applied and Environmental Microbiology, 76(11), 3529–3537. https://doi.org/10.1128/AEM.03063-09
20. Knabel, S. J., Walker, H. W., Hartman, P. A., & Mendonca, A. F. (1990). Effects of Growth Temperature and Strictly Anaerobic Recovery on the Survival of Listeria monocytogenes during Pasteurization. Applied and Environmental Microbiology, 56(2), 370-376. https://doi.org/10.1128/aem.56.2.370-376.1990.
21. Kouassi, Y., & Shelef, L. A. (1995). Listeriolysin 0 secretion by Listeria monocytogenes in the presence of cysteine and sorbate. Letters in Applied Microbiology, 20(5), 295-299. https://doi.org/10.1111/j.1472-765x.1995.tb00449.x.
22. Lee, B. H., Cole, S., Badel-Berchoux, S., Guillier, L., Felix, B., Krezdorn, N., & Piveteau, P. (2019). Biofilm formation of Listeria monocytogenes strains under food processing environments and pan-genome-wide association study. Frontiers in Microbiology, 10, 2698. https://doi.org/10.3389/fmicb.2019.02698
23. Lin, Y. D., & Chou, C. C. (2004). Effect of heat shock on thermal tolerance and susceptibility of Listeria monocytogenes to other environmental stresses. Food Microbiology, 21(5), 605-610. https://doi.org/10.1016/j.fm.2003.10.007.
24. Liu, Y., Orsi, R. H., Gaballa, A., Wiedmann, M., Boor, K. J., & Guariglia-Oropeza, V. (2019). Systematic review of the Listeria monocytogenes σ regulon supports a role in stress response, virulence and metabolism. Future Microbiology, 14(9), 801–828. https://doi.org/10.2217/fmb-2019-0072
25. Manso, B., Melero, B., Stessl, B., Jaime, I., Wagner, M., Rovira, J., & Rodríguez-Lázaro, D. (2020). The response to oxidative stress in Listeria monocytogenes is temperature dependent. Microorganisms, 8(4), 521. https://doi.org/10.3390/microorganisms8040521.
26. Mols, M., Pier, I., Zwietering, M. H., & Abee, T. (2009). The impact of oxygen availability on stress survival and radical formation of Bacillus cereus. International Journal of Food Microbiology, 135(3), 303–311. https://doi.org/10.1016/j.ijfoodmicro.2009.09.002
27. Müller-Herbst, S., Wüstner, S., Mühlig, A., Eder, D., Fuchs, T. M., Held, C., Ehrenreich, A., & Scherer, S. (2014). Identification of genes essential for anaerobic growth of Listeria monocytogenes. Microbiology, 160(4), 752–765. https://doi.org/10.1099/mic.0.075242-0
28. NicAogáin, K., & O’Byrne, C. P. (2016). The role of stress and stress adaptations in determining the fate of the bacterial pathogen Listeria monocytogenes in the food chain. Frontiers in Microbiology, 7, 1865. https://doi.org/10.3389/fmicb.2016.01865
29. O’Byrne, C. P., & Karatzas, K. A. G. (2008). The Role of Sigma B (σB) in the Stress Adaptations of Listeria monocytogenes: Overlaps Between Stress Adaptation and Virulence. Advances in Applied Microbiology, 65, 115-140. https://doi.org/10.1016/S0065-2164(08)00605-9
30. Pieniazek Danuta, Grabarek Z., & Rakowska Maria. (1975). Quantitative Determination of the Content of Available Methionine and Cysteine in Food Proteins. Nutrition & Metabolism, 18, 16–22. https://doi.org/10.1159/000175569. PMID: 1172609.
31. Poimenidou, S. V., Chatzithoma, D. N., Nychas, G. J., & Skandamis, P. N. (2016). Adaptive response of Listeria monocytogenes to heat, salinity and low pH, after habituation on cherry tomatoes and lettuce leaves. PLoS One, 11(10), e0165746. https://doi.org/10.1371/journal.pone.0165746.
32. Roberts, B. N., Chakravarty, D., Gardner, J. C., Ricke, S. C., & Donaldson, J. R. (2020). Listeria monocytogenes response to anaerobic environments. Pathogens, 9(3). https://doi.org/10.3390/pathogens9030210
33. Rodionova, I. A., Lim, H. G., Gao, Y., Rodionov, D. A., Hutchison, Y., Szubin, R., & Palsson, B. O. (2024). CyuR is a dual regulator for L-cysteine dependent antimicrobial resistance in Escherichia coli. Communications Biology, 7(1), 1160. https://doi.org/10.1038/s42003-024-06831-0
34. Shen, Q., Jangam, P. M., Soni, K. A., Nannapaneni, R., Schilling, W., & Silva, J. L. (2014). Low, medium, and high heat tolerant strains of Listeria monocytogenes and increased heat stress resistance after exposure to sublethal heat. Journal of Food Protection, 77(8), 1298–1307. https://doi.org/10.4315/0362-028X.JFP-13-423
35. Smirnova, G., Tyulenev, A., Sutormina, L., Kalashnikova, T., Muzyka, N., Ushakov, V., & Oktyabrsky, O. (2024). Regulation of cysteine homeostasis and its effect on Escherichia coli sensitivity to ciprofloxacin in LB medium. International Journal of Molecular Sciences, 25(8), 4424. https://doi.org/10.3390/ijms25084424.
36. Soutourina, O., Poupel, O., Coppée, J. Y., Danchin, A., Msadek, T., & Martin-Verstraete, I. (2009). CymR, the master regulator of cysteine metabolism in Staphylococcus aureus, controls host sulphur source utilization and plays a role in biofilm formation. Molecular Microbiology, 73(2), 194–211. https://doi.org/10.1111/j.1365-2958.2009.06760.x
37. Turnbull, A. L., & Surette, M. G. (2010). Cysteine biosynthesis, oxidative stress and antibiotic resistance in Salmonella typhimurium. Research in Microbiology, 161(8), 643–650. https://doi.org/10.1016/j.resmic.2010.06.004
38. UK Health Security Agency. (2025, May 8). Listeriosis in England and Wales: Summary for 2022 (Surveillance report). UK Health Security Agency. https://www.gov.uk/government/publications/listeria-monocytogenes-surveillance-reports/listeriosis-in-england-and-wales-summary-for-2022
39. Wang, Y., Wang, H. L., Xing, G. D., Qian, Y., Zhong, J. F., & Chen, K. L. (2021). S-allyl cysteine ameliorates heat stress-induced oxidative stress by activating Nrf2/HO-1 signaling pathway in BMECs. Toxicology and Applied Pharmacology, 416. https://doi.org/10.1016/j.taap.2021.115469
40. Wang, Z., Tao, X., Liu, S., Zhao, Y., & Yang, X. (2021). An update review on Listeria infection in pregnancy. Infection and drug resistance, 1967-1978. https://doi.org/10.2147/IDR.S313675.
41. Wemekamp-Kamphuis, H. H., Wouters, J. A., De Leeuw, P. P. L. A., Hain, T., Chakraborty, T., & Abee, T. (2004). Identification of sigma factor σB-controlled genes and their impact on acid stress, high hydrostatic pressure, and freeze survival in Listeria monocytogenes EGD-e. Applied and Environmental Microbiology, 70(6), 3457–3466. https://doi.org/10.1128/AEM.70.6.3457-3466.2004
42. Wiedmann, M., Arvik, T. J., Hurley, R. J., & Boor, K. J. (1998). General Stress Transcription Factor B and Its Role in Acid Tolerance and Virulence of Listeria monocytogenes. Journal of Bacteriology, 180(14), 3650-3656. https://doi.org/10.1128/JB.180.14.3650-3656.1998.
43. Wiktorczyk-Kapischke, N., Skowron, K., Grudlewska-Buda, K., Wałecka-Zacharska, E., Korkus, J., & Gospodarek-Komkowska, E. (2021). Adaptive Response of Listeria monocytogenes to the Stress Factors in the Food Processing Environment. Frontiers in Microbiology, 12, 710085. https://doi.org/10.3389/fmicb.2021.710085
44. Xayarath, B., Marquis, H., Port, G. C., & Freitag, N. E. (2009). Listeria monocytogenes CtaP is a multifunctional cysteine transport-associated protein required for bacterial pathogenesis. Molecular Microbiology, 74(4), 956–973. https://doi.org/10.1111/j.1365-2958.2009.06910.x
45. Yang, J., Duan, X., Landry, A. P., & Ding, H. (2010). Oxygen is required for the l-cysteine-mediated decomposition of protein-bound dinitrosyl-iron complexes. Free Radical Biology and Medicine, 49(2), 268–274. https://doi.org/10.1016/j.freeradbiomed.2010.04.012
46. Yilmaz Topcam, M. M., Balagiannis, D. P., & Karatzas, K. A. G. (2025). Impact of c-di-AMP Accumulation, L-cysteine, and Oxygen on Catalase Activity and Oxidative Stress Resistance of Listeria monocytogenes 10403S. Microorganisms, 13(6). https://doi.org/10.3390/microorganisms13061400