Bacterial Interference With Lactate Dehydrogenase Assay Leads to an Underestimation of Cytotoxicity

Overview

Journal Front Cell Infect Microbiol

Specialties Cell Biology
Infectious Diseases
Microbiology

Date 2020 Oct 12

PMID 33042868

Citations 12

Authors

Sara Van den Bossche

Eva Vandeplassche

Lisa Ostyn

Tom Coenye

Aurelie Crabbe

Affiliations

Soon will be listed here.

Abstract

Models to study host-pathogen interactions are an important tool for investigating the infectious disease process and evaluating the efficacy of antimicrobial compounds. In these models, the viability of mammalian cells is often determined using the lactate dehydrogenase (LDH) cytotoxicity assay. In the present study we evaluated whether bacteria could interfere with the LDH assay. As a model for host-pathogen interactions, we co-cultured lung epithelial cells with eight bacteria encountered in the lower respiratory tract. We show that LDH activity is affected by , and , and that this depends on the density of the start inoculum and the duration of infection. Two different mechanisms were discovered through which bacteria interfered with LDH activity, i.e., acidification of the cell culture medium (by and ) and protease production (by and ). In addition, we developed and validated a modified protocol to evaluate cytotoxicity using the LDH assay, where bacterial interference with LDH quantification is avoided.

Citing Articles

Echinacoside reduces intracellular c-di-GMP levels and potentiates tobramycin activity against Pseudomonas aeruginosa biofilm aggregates.

Cai Y, Hong F, De Craemer A, Malone J, Crabbe A, Coenye T NPJ Biofilms Microbiomes. 2025; 11(1):40.

PMID: 40055321 PMC: 11889090. DOI: 10.1038/s41522-025-00673-2.

An Overview of the Current State of Cell Viability Assessment Methods Using OECD Classification.

Madorran E, Ambroz M, Knez J, Sobocan M Int J Mol Sci. 2025; 26(1.

PMID: 39796074 PMC: 11719996. DOI: 10.3390/ijms26010220.

Shining a light on -induced epithelial damage with a luciferase reporter.

Tesfamariam M, Vij R, Trumper V, Hube B, Brunke S mSphere. 2024; 9(11):e0050924.

PMID: 39412273 PMC: 11580449. DOI: 10.1128/msphere.00509-24.

Aggregatibacter is inversely associated with inflammatory mediators in sputa of patients with chronic airway diseases and reduces inflammation in vitro.

Goeteyn E, Taylor S, Dicker A, Bolle L, Wauters M, Joossens M Respir Res. 2024; 25(1):368.

PMID: 39395980 PMC: 11471032. DOI: 10.1186/s12931-024-02983-z.

-chalcones obtained one-pot synthesis as the anti-neurodegenerative agents and their effect on the HT-22 cell line.

Olender D, Kujawski J, Skora B, Baranowska-Wojcik E, Sowa-Kasprzak K, Pawelczyk A Heliyon. 2024; 10(17):e37147.

PMID: 39286165 PMC: 11403034. DOI: 10.1016/j.heliyon.2024.e37147.

References

Karaba S, White R, Cianciotto N . Stenotrophomonas maltophilia encodes a type II protein secretion system that promotes detrimental effects on lung epithelial cells. Infect Immun. 2013; 81(9):3210-9. PMC: 3754218. DOI: 10.1128/IAI.00546-13. View

Gay R, MCCOMB R, BOWERS Jr G . Optimum reaction conditions for human lactate dehydrogenase isoenzymes as they affect total lactate dehydrogenase activity. Clin Chem. 1968; 14(8):740-53. View

Hawdon N, Aval P, Barnes R, Gravelle S, Rosengren J, Khan S . Cellular responses of A549 alveolar epithelial cells to serially collected Pseudomonas aeruginosa from cystic fibrosis patients at different stages of pulmonary infection. FEMS Immunol Med Microbiol. 2010; 59(2):207-20. DOI: 10.1111/j.1574-695X.2010.00693.x. View

Dabrowska A, Czapinska E . Limited proteolysis of bovine muscle and heart lactate dehydrogenase is inhibited by phospholipid liposome interaction. Biochim Biophys Acta. 1990; 1027(3):301-3. DOI: 10.1016/0005-2736(90)90322-f. View

Marquart M, OCallaghan R . Infectious keratitis: secreted bacterial proteins that mediate corneal damage. J Ophthalmol. 2013; 2013:369094. PMC: 3556867. DOI: 10.1155/2013/369094. View

Schmeck B, Gross R, NGuessan P, Hocke A, Hammerschmidt S, Mitchell T . Streptococcus pneumoniae-induced caspase 6-dependent apoptosis in lung epithelium. Infect Immun. 2004; 72(9):4940-7. PMC: 517413. DOI: 10.1128/IAI.72.9.4940-4947.2004. View

Vallee R, Williams Jr R . The two-step reversible denaturation of lactate dehydrogenase at low pH. Biochemistry. 1975; 14(12):2574-80. DOI: 10.1021/bi00683a003. View

Hoskins J, Alborn Jr W, Arnold J, Blaszczak L, Burgett S, DeHoff B . Genome of the bacterium Streptococcus pneumoniae strain R6. J Bacteriol. 2001; 183(19):5709-17. PMC: 95463. DOI: 10.1128/JB.183.19.5709-5717.2001. View

Korzeniewski C, Callewaert D . An enzyme-release assay for natural cytotoxicity. J Immunol Methods. 1983; 64(3):313-20. DOI: 10.1016/0022-1759(83)90438-6. View

10.

Okuda J, Hayashi N, Tanabe S, Minagawa S, Gotoh N . Degradation of interleukin 8 by the serine protease MucD of Pseudomonas aeruginosa. J Infect Chemother. 2011; 17(6):782-92. DOI: 10.1007/s10156-011-0257-7. View

11.

Caccamo A, Scaltriti M, Caporali A, DArca D, Scorcioni F, Astancolle S . Cell detachment and apoptosis induction of immortalized human prostate epithelial cells are associated with early accumulation of a 45 kDa nuclear isoform of clusterin. Biochem J. 2004; 382(Pt 1):157-68. PMC: 1133926. DOI: 10.1042/BJ20040158. View

12.

Slevogt H, Seybold J, Tiwari K, Hocke A, Jonatat C, Dietel S . Moraxella catarrhalis is internalized in respiratory epithelial cells by a trigger-like mechanism and initiates a TLR2- and partly NOD1-dependent inflammatory immune response. Cell Microbiol. 2006; 9(3):694-707. DOI: 10.1111/j.1462-5822.2006.00821.x. View

13.

Wang J, Dong Y, Zhou T, Liu X, Deng Y, Wang C . Pseudomonas aeruginosa cytotoxicity is attenuated at high cell density and associated with the accumulation of phenylacetic acid. PLoS One. 2013; 8(3):e60187. PMC: 3612096. DOI: 10.1371/journal.pone.0060187. View

14.

Mantovani R, Levy C, Yano T . A heat-stable cytotoxic factor produced by Achromobacter xylosoxidans isolated from Brazilian patients with CF is associated with in vitro increased proinflammatory cytokines. J Cyst Fibros. 2012; 11(4):305-11. DOI: 10.1016/j.jcf.2012.02.002. View

15.

Mairpady Shambat S, Chen P, Nguyen Hoang A, Bergsten H, Vandenesch F, Siemens N . Modelling staphylococcal pneumonia in a human 3D lung tissue model system delineates toxin-mediated pathology. Dis Model Mech. 2015; 8(11):1413-25. PMC: 4631791. DOI: 10.1242/dmm.021923. View

16.

Cheng J, Zhang J, Han B, Barkema H, Cobo E, Kastelic J . Klebsiella pneumoniae isolated from bovine mastitis is cytopathogenic for bovine mammary epithelial cells. J Dairy Sci. 2020; 103(4):3493-3504. DOI: 10.3168/jds.2019-17458. View

17.

Grela E, Kozlowska J, Grabowiecka A . Current methodology of MTT assay in bacteria - A review. Acta Histochem. 2018; 120(4):303-311. DOI: 10.1016/j.acthis.2018.03.007. View

18.

Schaible B, McClean S, Selfridge A, Broquet A, Asehnoune K, Taylor C . Hypoxia modulates infection of epithelial cells by Pseudomonas aeruginosa. PLoS One. 2013; 8(2):e56491. PMC: 3572047. DOI: 10.1371/journal.pone.0056491. View

19.

Yang P, Chen W, Chang F, Chen H, Lin C, Hsu Y . HepG2 cells infected with Klebsiella pneumoniae show DNA laddering at apoptotic and necrotic stages. Apoptosis. 2011; 17(2):154-63. DOI: 10.1007/s10495-011-0666-1. View

20.

Mosmann T . Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983; 65(1-2):55-63. DOI: 10.1016/0022-1759(83)90303-4. View