Effect of nystatin on invasion of Serratia grimesii and Serratia proteamaculans bacteria into epithelial cells

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Abstract

BACKGROUND: Bacteria use various endocytic pathways during entering non-phagocytic cells. The involvement of caveolae/lipid rafts in bacterial invasion has been demonstrated for many bacterial pathogens. However, for bacteria of the genus Serratia, the involvement of membrane microdomains in the process of bacterial internalization has been poorly studied.

AIM: To evaluate the involvement of caveolae/lipid rafts in the invasion of S. grimesii and S. proteamaculans bacteria into non-phagocytic epithelial Caco-2 and M-HeLa cells using nystatin.

MATERIALS AND METHODS: M-HeLa and Caco-2 epithelial cells were incubated with 50 μM nystatin for 1 hour at 37 ºC, after which they were infected with the bacteria S. grimesii strain 30063 and S. proteamaculans strain 94, the multiplicity of infection was 100 bacteria per cell. The number of intracellular bacteria was assessed using gentamicin protection assay. The level of caveolin-1 in cells was visualized using confocal microscopy and Western blotting. The expression of Toll-like receptors genes were measured by real-time RT-PCR.

RESULTS: Treatment of epithelial cells with nystatin reduces the internalization of S. grimesii and S. proteamaculans into M-HeLa cells by 30% and does not affect penetration into Caco-2 cells. At the same time, nystatin does not affect the redistribution / the integrity impairment of lipid rafts and does not lead to the cytoskeleton reorganization of eukaryotic cells. The addition of nystatin increases the level of caveolin-1 in M-HeLa cells (caveolin-1 is not expressed in Caco-2), which leads to a change plasma membrane fluidity. Nystatin promotes the secretion of proinflammatory cytokines interleukin-6 and interleukin-8 in both cell lines. Infection of M-HeLa cells pretreated with nystatin with the studied bacteria leads to an increase in the expression of tlr2 and tlr4 genes, but does not exceed the level of their expression in control samples. Therefore, it is impossible to speak unambiguously about the participation of Toll-like receptors in the invasion of Serratia bacteria.

CONCLUSIONS: The results obtained suggest that the interaction of bacteria with eukaryotic cells induces the expression of caveolin-1, which leads to a change plasma membrane components mobility. This may be due to the fact that β1-integrin is involved in the invasion of the studied bacteria, which should be stabilized at the plasma membrane upon binding of the ligand due to the formation of a cholesterol- and sphingolipid-rich membrane microenvironment.

About the authors

Yuliya M. Berson

Institute of Cytology of the Russian Academy of Sciences

Author for correspondence.
Email: juletschka.ber@gmail.com
ORCID iD: 0000-0003-0548-3745
SPIN-code: 5562-1057
Scopus Author ID: 57224308883

postgraduate student of the group of molecular cytology of prokaryotes and bacterial invasion

Russian Federation, 4 Tikhoretsky Ave., Saint Petersburg, 194064

References

  1. Buccini DF, Cardoso MH, Franco OL. Antimicrobial peptides and cell-penetrating peptides for treating intracellular bacterial infections. Front Cell Infect Microbiol. 2020;10:612931. doi: 10.3389/fcimb.2020.612931
  2. Khanna A, Khanna M, Aggarwal A. Serratia marcescens – a rare opportunistic nosocomial pathogen and measures to limit its spread in hospitalized patients. J Clin Diagn Res. 2013;7(2):243–246. doi: 10.7860/JCDR/2013/5010.2737
  3. Bozhokina ES, Tsaplina OA, Efremova TN, et al. Bacterial invasion of eukaryotic cells can be mediated by actin-hydrolysing metalloproteases grimelysin and protealysin. Cell Biol Int. 2011;35(2):111–118. doi: 10.1042/CBI20100314
  4. Efremova T, Ender N, Brudnaja M, et al. Specific invasion of transformed cells by Escherichia coli A2 strain. Cell Biol Int. 2001;25(6):557–561. doi: 10.1006/cbir.2001.0670
  5. de Laurentiis A, Donovan L, Arcaro AL. Lipid rafts and caveolae in signaling by growth factor receptors. Open Biochem J. 2007;1:12–32. doi: 10.2174/1874091X00701010012
  6. Bagam P, Singh DP, Inda ME, Batra S. Unraveling the role of membrane microdomains during microbial infections. Cell Biol Toxicol. 2017;33(5):429–455. doi: 10.1007/s10565-017-9386-9
  7. Monjarás Feria J, Valvano MA. An overview of anti-eukaryotic T6SS effectors. Front Cell Infect Microbiol. 2020;10:584751. doi: 10.3389/fcimb.2020.584751
  8. Machado FS, Rodriguez NE, Adesse D, et al. Recent developments in the interactions between caveolin and pathogens. Adv Exp Med Biol. 2012;729:65–82. doi: 10.1007/978-1-4614-1222-9_5
  9. Konkel ME, Samuelson DR, Eucker TP, et al. Invasion of epithelial cells by Campylobacter jejuni is independent of caveolae. Cell Commun Signal. 2013;11:100. doi: 10.1186/1478-811X-11-100
  10. Eierhoff T, Bastian B, Thuenauer R, et al. A lipid zipper triggers bacterial invasion. Proc Natl Acad Sci USA. 2014;111(35):12895–12900. doi: 10.1073/pnas.1402637111
  11. Ishii M, Fukuoka Y, Deguchi S, et al. Energy-dependent endocytosis is involved in the absorption of indomethacin nanoparticles in the small intestine. Int J Mol Sci. 2019;20(3):476. doi: 10.3390/ijms20030476
  12. Kruger K, Schrader K, Klempt M. Cellular response to titanium dioxide nanoparticles in intestinal epithelial Caco-2 cells is dependent on endocytosis-associated structures and mediated by EGFR. Nanomaterials (Basel). 2017;7(4):79. doi: 10.3390/nano7040079
  13. Zhu XD, Zhuang Y, Ben JJ, et al. Caveolae-dependent endocytosis is required for class A macrophage scavenger receptor-mediated apoptosis in macrophages. J Biol Chem. 2011;286(10):8231–8239. doi: 10.1074/jbc.M110.145888
  14. Rosello-Busquets C, Hernaiz-Llorens M, Soriano E, Martínez-Mármol R. Nystatin regulates axonal extension and regeneration by modifying the levels of nitric oxide. Front Mol Neurosci. 2020;13:56. doi: 10.3389/fnmol.2020.00056
  15. Tsai YH, Chen WL. Host lipid rafts as the gates for listeria monocytogenes infection: a mini-review. Front Immunol. 2020;11:1666. doi: 10.3389/fimmu.2020.01666
  16. Zaas DW, Duncan M, Rae Wright J, Abraham SN. The role of lipid rafts in the pathogenesis of bacterial infections. Biochim Biophys Acta. 2005;1746(3):305–313. doi: 10.1016/j.bbamcr.2005.10.003
  17. Chaudhary N, Gomez GA, Howes MT, et al. Endocytic crosstalk: cavins, caveolins, and caveolae regulate clathrin-independent endocytosis. PLoS Biol. 2014;12(4):e1001832. doi: 10.1371/journal.pbio.1001832
  18. Silva LND, Garcia IJP, Valadares JMM, et al. Evaluation of cardiotonic steroid modulation of cellular cholesterol and phospholipid. J Membr Biol. 2021;254(5–6):499–512. doi: 10.1007/s00232-021-00203-z
  19. Lim JY, Barnett TC, Bastiani M, et al. Caveolin 1 restricts Group A Streptococcus invasion of nonphagocytic host cells. Cell Microbiol. 2017;19(12). doi: 10.1111/cmi.12772
  20. Fadeyibi O, Rybalchenko N, Mabry S, et al. The Role of lipid rafts and membrane androgen receptors in androgen’s neurotoxic effects. J Endocr Soc. 2022;6(5):bvac030. doi: 10.1210/jendso/bvac030
  21. Foster LJ, De Hoog CL, Mann MCL. Unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors. Proc Natl Acad Sci USA. 2003;100(10):5813–5818. doi: 10.1073/pnas.0631608100
  22. Lamberti Y, Alvarez Hayes J, Perez Vidakovics ML, Rodriguez ME. Cholesterol-dependent attachment of human respiratory cells by Bordetella pertussis. FEMS Immunol Med Microbiol. 2009;56(2):143–150. doi: 10.1111/j.1574-695X.2009.00557.x
  23. Berson Y, Khaitlina S, Tsaplina O. Involvement of lipid rafts in the invasion of opportunistic bacteria serratia into eukaryotic cells. Int J Mol Sci. 2023;24(10):9029. doi: 10.3390/ijms24109029
  24. Pridmore AC, Jarvis GA, John CM, et al. Activation of toll-like receptor 2 (TLR2) and TLR4/MD2 by Neisseria is independent of capsule and lipooligosaccharide (LOS) sialylation but varies widely among LOS from different strains. Infect Immun. 2003;71(7):3901–3908. doi: 10.1128/IAI.71.7.3901-3908.2003
  25. Furrie E, Macfarlane S, Thomson G, et al. Toll-like receptors-2, -3 and -4 expression patterns on human colon and their regulation by mucosal-associated bacteria. Immunology. 2005;115(4):565–574. doi: 10.1111/j.1365-2567.2005.02200.x
  26. Amemiya K, Dankmeyer JL, Bernhards RC, et al. Activation of toll-like receptors by live gram-negative bacterial pathogens reveals mitigation of TLR4 responses and activation of TLR5 by flagella. Front Cell Infect Microbiol. 2021;11:745325. doi: 10.3389/fcimb.2021.745325
  27. Hellwing C, Schoeniger A, Roessler C, et al. Lipid raft localization of TLR2 and its co-receptors is independent of membrane lipid composition. Peer J. 2018;6:e4212. doi: 10.7717/peerj.4212
  28. Triantafilou M, Miyake K, Golenbock DT, Triantafilou K. Mediators of innate immune recognition of bacteria concentrate in lipid rafts and facilitate lipopolysaccharide-induced cell activation. J Cell Sci. 2002;115(Pt 12):2603–2611. doi: 10.1242/jcs.115.12.2603
  29. Wong SW, Kwon MJ, Choi AM, et al. Fatty acids modulate Toll-like receptor 4 activation through regulation of receptor dimerization and recruitment into lipid rafts in a reactive oxygen species-dependent manner. J Biol Chem. 2009;284(40):27384–27392. doi: 10.1074/jbc.M109.044065
  30. Soong G, Reddy B, Sokol S, et al. TLR2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells. J Clin Invest. 2004;113(10):1482–1489. doi: 10.1172/JCI20773
  31. Salyer AC, Caruso G, Khetani KK, et al. Identification of adjuvantic activity of amphotericin B in a novel, multiplexed, poly-TLR/NLR high-throughput screen. PLoS One. 2016;11(2):e0149848. doi: 10.1371/journal.pone.0149848
  32. Razonable RR, Henault M, Watson HL, Paya CV. Nystatin induces secretion of interleukin (IL)-1beta, IL-8, and tumor necrosis factor alpha by a toll-like receptor-dependent mechanism. Antimicrob Agents Chemother. 2005;49(8):3546–3549. doi: 10.1128/AAC.49.8.3546-3549.2005
  33. Song J, Bishop BL, Li G, et al. TLR4-initiated and cAMP-mediated abrogation of bacterial invasion of the bladder. Cell Host Microbe. 2007;1(4):287–298. doi: 10.1016/j.chom.2007.05.007
  34. Mittal R, Debs LH, Patel AP, et al. Otopathogenic Staphylococcus aureus invades human middle ear epithelial cells primarily through cholesterol dependent pathway. Sci Rep. 2019;9(1):10777. doi: 10.1038/s41598-019-47079-7
  35. Goluszko P, Nowicki B. Membrane cholesterol: a crucial molecule affecting interactions of microbial pathogens with mammalian cells. Infect Immun. 2005;73(12):7791–7796. doi: 10.1128/IAI.73.12.7791-7796.2005
  36. Harush-Frenkel O, Rozentur E, Benita S, Altschuler Y. Surface charge of nanoparticles determines their endocytic and transcytotic pathway in polarized MDCK cells. Biomacromolecules. 2008;9(2):435–443. doi: 10.1021/bm700535p
  37. Zemljic Jokhadar S, Božič B, Kristanc L, Gomišček G. Osmotic effects induced by pore-forming agent nystatin: from lipid vesicles to the cell. PLoS One. 2016;11(10):e0165098. doi: 10.1371/journal.pone.0165098
  38. Zhang X, Li T, Chen X, et al. Nystatin enhances the immune response against Candida albicans and protects the ultrastructure of the vaginal epithelium in a rat model of vulvovaginal candidiasis. BMC Microbiol. 2018;18(1):166. doi: 10.1186/s12866-018-1316-3
  39. Cai C, Zhu H, Chen J. Overexpression of caveolin-1 increases plasma membrane fluidity and reduces P-glycoprotein function in Hs578T/Dox. Biochem Biophys Res Commun. 2004;320(3):868–874. doi: 10.1016/j.bbrc.2004.06.030
  40. Hoffmann C, Berking A, Agerer F, et al. Caveolin limits membrane microdomain mobility and integrin-mediated uptake of fibronectin-binding pathogens. J Cell Sci. 2010;123(24):4280–4291. doi: 10.1242/jcs.064006
  41. Bonazzi M, Veiga E, Pizarro-Cerdá J, Cossart P. Successive post-translational modifications of E-cadherin are required for InlA-mediated internalization of Listeria monocytogenes. Cell Microbiol. 2008;10(11):2208–2222. doi: 10.1111/j.1462-5822.2008.01200.x
  42. Tsaplina O, Bozhokina E. Bacterial outer membrane protein ompx regulates beta1 integrin and epidermal growth factor receptor (EGFR) involved in invasion of M-HeLa cells by Serratia proteamaculans. Int J Mol Sci. 2021;22(24):13246. doi: 10.3390/ijms222413246

Supplementary files

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2. Fig. 1. The number of intracellular bacteria in Caco-2 and M-HeLa cells preincubated with 50 μM nystatin for 1 h relative to control cells preincubated with DPBS (control). Means and standard deviations are shown. * p < 0.05

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3. Fig. 2. Confocal fluorescence microscopy of M-HeLa cells without bacteria and after 2-h incubation with S. grimesii or S. proteamaculans. Nuclei are stained with DAPI (black), caveolin — indirect immunofluorescence (white)

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4. Fig. 3. The protein level of caveolin-1 in Caco-2 and M-HeLa cells treated with 50 µM nystatin (+) or DPBS (–) was detected by western blot analysis assays (a). The number of intracellular bacteria per M-HeLa cell preincubated with 50 μM nystatin or DPBS (control), after that bacteria were added in DMEM or DMEM containing 0,05% Tween20. Means and standard deviations are shown. * p < 0.05 (relative to corresponding control samples) (b)

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5. Fig. 4. Confocal fluorescence microscopy: monitoring of lipid rafts distribution. Caco-2 (a) and M-HeLa (b) cells incubated with 50 μM nystatin at 37 °C for 1 hour. GM1 was stained with CTxB-FITC (white), cell nuclei were stained with DAPI (black)

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6. Fig. 5. Visualization the cytoskeleton of Caco-2 (a) and M-HeLa (b) cells treated with 50 μM nystatin or DPBS for 1 h by confocal fluorescence microscopy. Nuclei are stained with DAPI (blue), F-actin is stained with RF (red), α-tubulin is indirect immunofluorescence (green)

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7. Fig. 6. Relative production of cytokines IL-6 and IL-8 by M-HeLa and Caco-2 cells after one-hour incubation with 50 μM nystatin, DPBS was added to control cells. Means and standard deviations are shown. Statistically significant differences between the control and treated groups are indicated by asterisks: * p < 0.05, *** p < 0.005

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8. Fig. 7. Relative normalized (to β-actin) expression of tlr2 and tlr4 genes. Caco-2 and M-HeLa cells were incubated with 50 μM nystatin or DPBS (control) for 1 h (a). M-HeLa cells were incubated with 50 µM nystatin or DPBS (control) for 1 h, and then bacterial invasion was performed (MOI 100) (b)

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