Development of flow cytometry based adherence assay for Neisseria gonorrhoeae using 5′-carboxyfluorosceinsuccidyl ester

Thakur, Sidharath Dev; Obradović, Milan; Dillon, Jo-Anne R.; Ng, Siew Hon; Wilson, Heather L.

doi:10.1186/s12866-019-1438-2

Cited by 5 publications

(3 citation statements)

References 17 publications

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“…Flow cytometry-based adherence assay. Flow cytometric analysis of bacterial adhesion was performed as previously described 72 . Briefly, E. coli cultures were stained with CFSE (Cell-Trace CFSE Cell Proliferation Kit, Thermofisher, C34554) before infection of HCT116 and HT-29 cells at MOI 100, spun down for 4 min 300 x g to synchronize adhesion and incubated at 37°C, 5% CO2 for 3h.…”

Section: Isolation Ofmentioning

confidence: 99%

Colibactin-induced genotoxicity and colorectal cancer exacerbation critically depends on adhesin-mediated epithelial binding

Jans,

Kolata,

Blancke

et al. 2023

Preprint

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Various bacteria are suggested to contribute to colorectal cancer (CRC) development, including pks+ E. coli which produce the genotoxin colibactin that induces characteristic mutational signatures in host epithelial cells. It remains unclear how the highly unstable colibactin molecule is able to access host epithelial cells and its DNA to cause harm. Using the microbiota-dependent ZEB2-transgenic mouse model of invasive CRC, we found that pks+ E. coli drives CRC exacerbation and tissue invasion in a colibactin-dependent manner. Using isogenic mutant strains, we further demonstrate that CRC exacerbation critically depends on expression of the E. coli type-1 pilus adhesin FimH and the F9-pilus adhesin FmlH. Blocking bacterial adhesion using a pharmacological FimH inhibitor attenuates colibactin-mediated genotoxicity and CRC exacerbation. Together, we show that the oncogenic potential of pks+ E. coli critically depends on bacterial adhesion to host epithelial cells and is critically mediated by specific bacterial adhesins. Adhesin-mediated epithelial binding subsequently allows production of the genotoxin colibactin in close proximity to host epithelial cells, which promotes DNA damage and drives CRC development. These findings present promising therapeutic avenues for the development of anti-adhesive therapies aiming at mitigating colibactin-induced DNA damage and inhibiting the initiation and progression of CRC, particularly in individuals at risk for developing CRC.

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Section: Isolation Ofmentioning

confidence: 99%

Colibactin-induced genotoxicity and colorectal cancer exacerbation critically depends on adhesin-mediated epithelial binding

Jans,

Kolata,

Blancke

et al. 2023

Preprint

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“…To adhere to cells, bacteria use a range of factors, from pili, fimbria, flagella, and other adhesins ( 62 – 64 ). Conventional adherence assays are laborious and based on counting bacteria on agar plates or from stained cells ( 28 , 65 ). Genetic methods, such as multi-plex PCR, test for the presence of multiple adhesin genes in a single reaction but do not provide evidence of functionality ( 29 ).…”

Section: Adhesion and Invasionmentioning

confidence: 99%

High-throughput functional trait testing for bacterial pathogens

Stromberg,

Phillips,

Omberg

et al. 2023

mSphere

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Functional traits are characteristics that affect the fitness and metabolic function of a microorganism. There is growing interest in using high-throughput methods to characterize bacterial pathogens based on functional virulence traits. Traditional methods that phenotype a single organism for a single virulence trait can be time consuming and labor intensive. Alternatively, machine learning of whole-genome sequences (WGS) has shown some success in predicting virulence. However, relying solely on WGS can miss functional traits, particularly for organisms lacking classical virulence factors. We propose that high-throughput assays for functional virulence trait identification should become a prominent method of characterizing bacterial pathogens on a population scale. This work is critical as we move from compiling lists of bacterial species associated with disease to pathogen-agnostic approaches capable of detecting novel microbes. We discuss six key areas of functional trait testing and how advancing high-throughput methods could provide a greater understanding of pathogens.

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“…Breeuwer, Drocourt, Rombouts, and Abee (1994) and Breeuwer et al (1995) showed that FDA and carboxyfluorescein diacetate (CFDA) penetrated yeast rapidly but that esterase activity was limiting and an energy-dependent efflux of carboxyfluorescein from viable cells also impacted negatively on the efficacy of the staining method. Success has been reported with 5 -carboxyfluoroscein succidyl ester (CFDA-SE) with Neisseria gonorrhoeae (Thakur, Obradovic, Dillon, Ng, & Wilson, 2019).…”

Section: Metabolic Activitymentioning

confidence: 99%

Estimation of Microbial Viability Using Flow Cytometry

Davey

Guyot

2020

CP Cytometry

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For microorganisms in particular, viability is a term that is difficult to define and a state consequently difficult to measure. The traditional (and gold standard) usage equates viability and culturability (i.e., the ability to multiply) but the process of determining culturability is often too slow. Flow cytometry provides the opportunity to make rapid and quantitative measurements of dye uptake in large numbers of cells and we can therefore exploit the flow cytometric approach to evaluate so-called viability stains and to develop protocols for more routine assessments of microbial viability. This article provides a commentary and several protocols have been included to ensure that users have a firm basis for attempting these reasonably difficult assays on traditional flow cytometer instruments. What is clear is that each assay must be carefully validated with the particular microorganism of interest before being applied in any research, clinical, or service form.

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Development of flow cytometry based adherence assay for Neisseria gonorrhoeae using 5′-carboxyfluorosceinsuccidyl ester

Cited by 5 publications

References 17 publications

Colibactin-induced genotoxicity and colorectal cancer exacerbation critically depends on adhesin-mediated epithelial binding

Colibactin-induced genotoxicity and colorectal cancer exacerbation critically depends on adhesin-mediated epithelial binding

High-throughput functional trait testing for bacterial pathogens

Estimation of Microbial Viability Using Flow Cytometry

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