Dataset Information

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

ABSTRACT: Models to study host-pathogen interactions in vitro 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 Pseudomonas aeruginosa, Klebsiella pneumoniae, Stenotrophomonas maltophilia, and Streptococcus pneumoniae, 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 K. pneumoniae and S. pneumoniae) and protease production (by P. aeruginosa and S. maltophilia). In addition, we developed and validated a modified protocol to evaluate cytotoxicity using the LDH assay, where bacterial interference with LDH quantification is avoided.

SUBMITTER: Van den Bossche S

PROVIDER: S-EPMC7523407 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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

Van den Bossche Sara S Vandeplassche Eva E Ostyn Lisa L Coenye Tom T Crabbé Aurélie A

Frontiers in cellular and infection microbiology 20200915

Models to study host-pathogen interactions <i>in vitro</i> 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 ...[more]

PMID: 33042868

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Project description:UnlabelledPrevious studies of the oral pathogen Streptococcus mutans have determined that this Gram-positive facultative anaerobe mounts robust responses to both acid and oxidative stresses. The water-forming NADH oxidase (Nox; encoded by nox) is thought to be critical for the regeneration of NAD(+), for use in glycolysis, and for the reduction of oxygen, thereby preventing the formation of damaging reactive oxygen species. In this study, the free NAD(+)/NADH ratio in a nox deletion strain (Δnox) was discovered to be remarkably higher than that in the parent strain, UA159, when the strains were grown in continuous culture. This unanticipated result was explained by significantly elevated lactate dehydrogenase (Ldh; encoded by ldh) activity and ldh transcription in the Δnox strain, which was mediated in part by the redox-sensing regulator Rex. cDNA microarray analysis of S. mutans cultures exposed to simultaneous acid stress (growth at a low pH) and oxidative stress (generated through the deletion of nox or the addition of exogenous oxygen) revealed a stress response synergistically heightened over that with either stress alone. In the Δnox strain, this elevated stress response included increased glucose phosphoenolpyruvate phosphotransferase system (PTS) activity, which appeared to be due to elevated manL transcription, mediated in part, like elevated ldh transcription, by Rex. While the Δnox strain does possess a membrane composition different from that of the parent strain, it did not appear to have defects in either membrane permeability or ATPase activity. However, the altered transcriptome and metabolome of the Δnox strain were sufficient to impair its ability to compete with commensal peroxigenic oral streptococci during growth under aerobic conditions.ImportanceStreptococcus mutans is an oral pathogen whose ability to outcompete commensal oral streptococci is strongly linked to the formation of dental caries. Previous work has demonstrated that the S. mutans water-forming NADH oxidase is critical for both carbon metabolism and the prevention of oxidative stress. The results of this study show that upregulation of lactate dehydrogenase, mediated through the redox sensor Rex, overcompensates for the loss of nox. Additionally, nox deletion led to the upregulation of mannose and glucose transport, also mediated through Rex. Importantly, the loss of nox rendered S. mutans defective in its ability to compete directly with two species of commensal streptococci, suggesting a role for nox in the pathogenic potential of this organism.

Dataset Information

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

Publications

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

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