A flow cytometry method for quantitative measurement and molecular investigation of the adhesion of bacteria to yeast cells.

Publisher: Nature Publishing Group Country of Publication: England NLM ID: 101563288 Publication Model: Electronic Cited Medium: Internet ISSN: 2045-2322 (Electronic) Linking ISSN: 20452322 NLM ISO Abbreviation: Sci Rep Subsets: MEDLINE

Original Publication: London : Nature Publishing Group, copyright 2011-

Flow Cytometry*/methods ; Bacterial Adhesion* ; Escherichia coli*/genetics ; Escherichia coli*/metabolism ; Saccharomyces cerevisiae*/genetics ; Saccharomyces cerevisiae*/metabolism

The study of microorganism interactions is important for understanding the organization and functioning of microbial consortia. Additionally, the interaction between yeast and bacteria is of interest in the field of health and nutrition area for the development of probiotics. To investigate these microbial interactions at the cellular and molecular levels, a simple, reliable, and quantitative method is proposed. We demonstrated that flow cytometry enables the measurement of interactions at a single-cell level by detecting and counting yeast cells with bound fluorescent bacteria. Imaging flow cytometry revealed that the number of bacteria attached to yeast followed a Gaussian distribution whose maximum reached 14 bacterial cells using a clinical Escherichia coli strain E22 and the laboratory yeast strain BY4741. We found that the dynamics of adhesion resemble a Langmuir adsorption model, albeit it is a rapid and almost irreversible process. This adhesion is dependent on the mannose-specific type 1 fimbriae, as E. coli mutants lacking these appendages no longer adhere to yeast. However, this type 1 fimbriae-dependent adhesion could involve additional yeast cell wall factors, since the interaction between bacteria and yeast mutants with altered mannan content remained comparable to that of wild-type yeast. In summary, flow cytometry is an appropriate method for studying bacteria-yeast adhesion, as well as for the high-throughput screening of candidate molecules likely to promote or counteract this interaction.
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Pontier-Bres, R. et al. Saccharomyces boulardii modifies Salmonella typhimurium traffic and host immune responses along the intestinal tract. PLoS ONE 9, e103069. https://doi.org/10.1371/journal.pone.0103069 (2014). (PMID: 10.1371/journal.pone.0103069251185954145484)
Mourão, J. L. et al. Effect of mannan oligosaccharides on the performance, intestinal morphology and cecal fermentation of fattening rabbits. Anim. Feed Sci. Technol. 126, 107–120. https://doi.org/10.1016/j.anifeedsci.2005.06.009 (2006). (PMID: 10.1016/j.anifeedsci.2005.06.009)
Terré, M., Calvo, M. A., Adelantado, C., Kocher, A. & Bach, A. Effects of mannan oligosaccharides on performance and microorganism fecal counts of calves following an enhanced-growth feeding program. Anim. Feed Sci. Technol. 137, 115–125. https://doi.org/10.1016/j.anifeedsci.2006.11.009 (2007). (PMID: 10.1016/j.anifeedsci.2006.11.009)
Spring, P., Wenk, C., Dawson, K. A. & Newman, K. E. The effects of dietary mannaoligosaccharides on cecal parameters and the concentrations of enteric bacteria in the ceca of salmonella-challenged broiler chicks. Poult. Sci. 79, 205–211. https://doi.org/10.1093/ps/79.2.205 (2000). (PMID: 10.1093/ps/79.2.20510735748)
Posadas, G. A. et al. Yeast pro- and paraprobiotics have the capability to bind pathogenic bacteria associated with animal disease. Transl. Anim. Sci. 1, 60–68. https://doi.org/10.2527/tas2016.0007 (2017). (PMID: 10.2527/tas2016.0007320644607011128)
Sharon, N. & Ofek, I. Safe as mother’s milk: carbohydrates as future anti-adhesion drugs for bacterial diseases. Glycoconj. J. 17, 659–664. https://doi.org/10.1023/a:1011091029973 (2000). (PMID: 10.1023/a:101109102997311421356)
Snellings, N. J., Tall, B. D. & Venkatesan, M. M. Characterization of Shigella type 1 fimbriae: expression, FimA sequence, and phase variation. Infect. Immun. 65, 2462–2467. https://doi.org/10.1128/iai.65.6.2462-2467.1997 (1997). (PMID: 10.1128/iai.65.6.2462-2467.19979169792175344)
Knight, S. D. & Bouckaert, J. Structure, function, and assembly of type 1 fimbriae. Top. Curr. Chem. 288, 67–107. https://doi.org/10.1007/128_2008_13 (2009). (PMID: 10.1007/128_2008_1322328027)
Feenstra, T. et al. Adhesion of Escherichia coli under flow conditions reveals potential novel effects of FimH mutations. Eur. J. Clin. Microbiol. Infect. Dis. 36, 467–478. https://doi.org/10.1007/s10096-016-2820-8 (2017). (PMID: 10.1007/s10096-016-2820-827816993)
McLay, R. B. et al. Level of fimbriation alters the adhesion of Escherichia coli bacteria to interfaces. Langmuir 34, 1133–1142. https://doi.org/10.1021/acs.langmuir.7b02447 (2018). (PMID: 10.1021/acs.langmuir.7b0244728976770)
Klis, F. M., Mol, P., Hellingwerf, K. & Brul, S. Dynamics of cell wall structure in Saccharomyces cerevisiae. FEMS Microbiol. Rev. 26, 239–256 (2002). (PMID: 10.1111/j.1574-6976.2002.tb00613.x12165426)
Orlean, P. Architecture and biosynthesis of the Saccharomyces cerevisiae cell wall. Genetics 192, 775–818 (2012). (PMID: 10.1534/genetics.112.144485231353253522159)
Gow, N. A. R. & Lenardon, M. D. Architecture of the dynamic fungal cell wall. Nat. Rev. Microbiol. 21, 248–259. https://doi.org/10.1038/s41579-022-00796-9 (2023). (PMID: 10.1038/s41579-022-00796-936266346)
Xu, X., Qiao, Y., Peng, Q., Gao, L. & Shi, B. Inhibitory effects of YCW and MOS from Saccharomyces cerevisiae on Escherichia coli and Salmonella pullorum adhesion to Caco-2 cells. Front. Biol. 12, 370–375. https://doi.org/10.1007/s11515-017-1464-0 (2017). (PMID: 10.1007/s11515-017-1464-0)
Sauvaitre, T. et al. Lentils and yeast fibers: A new strategy to mitigate enterotoxigenic Escherichia coli (ETEC) strain H10407 virulence?. Nutrients https://doi.org/10.3390/nu14102146 (2022). (PMID: 10.3390/nu14102146356312879144138)
Ageorges, V. et al. Genome-wide analysis of antigen 43 (Ag43) variants: New insights in their diversity, distribution and prevalence in bacteria. Int. J. Mol. Sci. https://doi.org/10.3390/ijms24065500 (2023). (PMID: 10.3390/ijms240655003698258010058404)
Mammarappallil, J. G. & Elsinghorst, E. A. Epithelial cell adherence mediated by the enterotoxigenic Escherichia coli Tia protein. Infect. Immun. 68, 6595–6601. https://doi.org/10.1128/iai.68.12.6595-6601.2000 (2000). (PMID: 10.1128/iai.68.12.6595-6601.20001108377097755)
Ganner, A. & Schatzmayr, G. Capability of yeast derivatives to adhere enteropathogenic bacteria and to modulate cells of the innate immune system. Appl. Microbiol. Biotechnol 95, 289–297. https://doi.org/10.1007/s00253-012-4140-y (2012). (PMID: 10.1007/s00253-012-4140-y22615053)
Ganner, A., Stoiber, C., Uhlik, J. T., Dohnal, I. & Schatzmayr, G. Quantitative evaluation of E. coli F4 and Salmonella Typhimurium binding capacity of yeast derivatives. AMB Express 3, 62. https://doi.org/10.1186/2191-0855-3-62 (2013). (PMID: 10.1186/2191-0855-3-62241483083816590)
Tiago, F. C. P. et al. Adhesion to the yeast cell surface as a mechanism for trapping pathogenic bacteria by Saccharomyces probiotics. J. Med. Microbiol. 61, 1194–1207. https://doi.org/10.1099/jmm.0.042283-0 (2012). (PMID: 10.1099/jmm.0.042283-022580913)
Martyniak, A., Medyńska-Przęczek, A., Wędrychowicz, A., Skoczeń, S. & Tomasik, P. J. Prebiotics, probiotics, synbiotics, paraprobiotics and postbiotic compounds in IBD. Biomolecules https://doi.org/10.3390/biom11121903 (2021). (PMID: 10.3390/biom11121903349445468699341)
Mirelman, D., Altmann, G. & Eshdat, Y. Screening of bacterial isolates for mannose-specific lectin activity by agglutination of yeasts. J. Clin. Microbiol. 11, 328–331. https://doi.org/10.1128/jcm.11.4.328-331.1980 (1980). (PMID: 10.1128/jcm.11.4.328-331.19806989854273398)
Pérez-Sotelo, L. S. et al. In vitro evaluation of the binding capacity of Saccharomyces cerevisiae Sc47 to adhere to the wall of Salmonella spp. Rev. Latinoam. Microbiol. 47, 70–75 (2005). (PMID: 17061530)
Becker, P. M., Galletti, S., Roubos-van den Hil, P. J. & van Wikselaar, P. G. Validation of growth as measurand for bacterial adhesion to food and feed ingredients. J. Appl. Microbiol. 103, 2686–2696. https://doi.org/10.1111/j.1365-2672.2007.03524.x (2007). (PMID: 10.1111/j.1365-2672.2007.03524.x17850303)
Millsap, K. W., van der Mei, H. C., Bos, R. & Busscher, H. J. Adhesive interactions between medically important yeasts and bacteria. FEMS Microbiol. Rev. 21, 321–336. https://doi.org/10.1111/j.1574-6976.1998.tb00356.x (1998). (PMID: 10.1111/j.1574-6976.1998.tb00356.x9532746)
Popolo, L. & Vai, M. The Gas1 glycoprotein, a putative wall polymer cross-linker. Biochim. Biophys. Acta 1426, 385–400 (1999). (PMID: 10.1016/S0304-4165(98)00138-X9878845)
Latour, R. A. The Langmuir isotherm: a commonly applied but misleading approach for the analysis of protein adsorption behavior. J. Biomed. Mater. Res. A 103, 949–958. https://doi.org/10.1002/jbm.a.35235 (2015). (PMID: 10.1002/jbm.a.3523524853075)
Han, M.-J. Exploring the proteomic characteristics of the Escherichia coli B and K-12 strains in different cellular compartments. J. Biosci. Bioeng. 122, 1–9. https://doi.org/10.1016/j.jbiosc.2015.12.005 (2016). (PMID: 10.1016/j.jbiosc.2015.12.00526777236)
Thoma, J. et al. Protein-enriched outer membrane vesicles as a native platform for outer membrane protein studies. Commun. Biol. 1, 23. https://doi.org/10.1038/s42003-018-0027-5 (2018). (PMID: 10.1038/s42003-018-0027-5302719106123736)
Schembri, M. A., Sokurenko, E. V. & Klemm, P. Functional flexibility of the FimH adhesin: Insights from a random mutant library. Infect. Immun. 68, 2638–2646. https://doi.org/10.1128/iai.68.5.2638-2646.2000 (2000). (PMID: 10.1128/iai.68.5.2638-2646.20001076895597470)
Eshdat, Y., Speth, V. & Jann, K. Participation of pili and cell wall adhesion in the yeast agglutination activity of Escherichia coli. Infect. Immun. 34, 980–986. https://doi.org/10.1128/iai.34.3.980-986.1981 (1981). (PMID: 10.1128/iai.34.3.980-986.19816120898350964)
Korhonen, T. K., Leffler, H. & Svanborg Eden, C. Binding specificity of piliated strains of Escherichia coli and Salmonella typhimurium to epithelial cells, Saccharomyces cerevisiae cells, and erythrocytes. Infect. Immun. 32, 796–804. https://doi.org/10.1128/iai.32.2.796-804.1981 (1981). (PMID: 10.1128/iai.32.2.796-804.19816114036351515)
Russell, P. W. & Orndorff, P. E. Lesions in two Escherichia coli type 1 pilus genes alter pilus number and length without affecting receptor binding. J. Bacteriol. 174, 5923–5935. https://doi.org/10.1128/jb.174.18.5923-5935.1992 (1992). (PMID: 10.1128/jb.174.18.5923-5935.19921355769207130)
Jones, C. H. et al. FimH adhesin of type 1 pili is assembled into a fibrillar tip structure in the Enterobacteriaceae. Proc. Natl. Acad. Sci. USA 92, 2081–2085. https://doi.org/10.1073/pnas.92.6.2081 (1995). (PMID: 10.1073/pnas.92.6.2081789222842427)
Uscanga, B. Influence de paramètres de croissance et des conditions de mise en oeuvre sur la compoistion et l'architecture de la paroi cellulaire de la levure. Ph D Thesis, no from the "Institut National Des Sciences Appliquées", Toulouse, 174 (2003).
Schiavone, M. et al. A combined chemical and enzymatic method to determine quantitatively the polysaccharide components in the cell wall of yeasts. FEMS Yeast Res. 14, 933–947. https://doi.org/10.1111/1567-1364.12182 (2014). (PMID: 10.1111/1567-1364.1218225041403)
Lussier, M., Sdicu, A. M., Ketela, T. & Bussey, H. Localization and targeting of the Saccharomyces cerevisiae Kre2p/Mnt1p alpha 1,2-mannosyltransferase to a medial-Golgi compartment. J. Cell Biol. 131, 913–927 (1995). (PMID: 10.1083/jcb.131.4.9137490293)
Nakayama, K., Nagasu, T., Shimma, Y., Kuromitsu, J. & Jigami, Y. OCH1 encodes a novel membrane bound mannosyltransferase: Outer chain elongation of asparagine-linked oligosaccharides. EMBO J. 11, 2511–2519 (1992). (PMID: 10.1002/j.1460-2075.1992.tb05316.x1628616556726)
Ishihara, S. et al. Homologous subunits of 1,3-beta-glucan synthase are important for spore wall assembly in Saccharomyces cerevisiae. Eukaryot. Cell 6, 143–156. https://doi.org/10.1128/ec.00200-06 (2007). (PMID: 10.1128/ec.00200-0617158736)
Dallies, N., Francois, J. & Paquet, V. A new method for quantitative determination of polysaccharides in the yeast cell wall. Application to the cell wall defective mutants of Saccharomyces cerevisiae. Yeast 14, 1297–1306 (1998). (PMID: 10.1002/(SICI)1097-0061(1998100)14:14<1297::AID-YEA310>3.0.CO;2-L9802208)
Martin-Yken, H., Francois, J. M. & Zerbib, D. Knr4: A disordered hub protein at the heart of fungal cell wall signalling. Cell Microbiol. 18, 1217–1227. https://doi.org/10.1111/cmi.12618 (2016). (PMID: 10.1111/cmi.1261827199081)
Jorgensen, P., Nishikawa, J. L., Breitkreutz, B. J. & Tyers, M. Systematic identification of pathways that couple cell growth and division in yeast. Science 297, 395–400 (2002). (PMID: 10.1126/science.107085012089449)
Osiro, D., Filho, R. B., Assis, O. B., Jorge, L. A. & Colnago, L. A. Measuring bacterial cells size with AFM. Braz. J. Microbiol. 43, 341–347. https://doi.org/10.1590/s1517-838220120001000040 (2012). (PMID: 10.1590/s1517-838220120001000040240318373768968)
Wu, K.-H., Wang, K.-C., Lee, L.-W., Huang, Y.-N. & Yeh, K.-S. A constitutively mannose-sensitive agglutinating Salmonella enterica subsp. enterica Serovar Typhimurium strain, carrying a transposon in the fimbrial usher gene stbC, exhibits multidrug resistance and flagellated phenotypes. Sci. World J. 2012, 280264. https://doi.org/10.1100/2012/280264 (2012). (PMID: 10.1100/2012/280264)
Thomas, W. Catch bonds in adhesion. Annu. Rev. Biomed. Eng. 10, 39–57. https://doi.org/10.1146/annurev.bioeng.10.061807.160427 (2008). (PMID: 10.1146/annurev.bioeng.10.061807.16042718647111)
Forero, M., Yakovenko, O., Sokurenko, E. V., Thomas, W. E. & Vogel, V. Uncoiling mechanics of Escherichia coli type I fimbriae are optimized for catch bonds. PLoS Biol. 4, e298. https://doi.org/10.1371/journal.pbio.0040298 (2006). (PMID: 10.1371/journal.pbio.0040298169339771557399)
Yakovenko, O. et al. FimH forms catch bonds that are enhanced by mechanical force due to allosteric regulation. J. Biol. Chem. 283, 11596–11605. https://doi.org/10.1074/jbc.M707815200 (2008). (PMID: 10.1074/jbc.M707815200182920922431072)
Thomas, W. E. et al. Recombinant FimH adhesin demonstrates how the allosteric catch bond mechanism can support fast and strong bacterial attachment in the absence of shear. J. Mol. Biol. 434, 167681. https://doi.org/10.1016/j.jmb.2022.167681 (2022). (PMID: 10.1016/j.jmb.2022.167681356972939398990)
Francois, J. M. Cell surface interference with plasma membrane and transport processes in yeasts. Adv. Exp. Med. Biol 892, 11–31. https://doi.org/10.1007/978-3-319-25304-6_2 (2016). (PMID: 10.1007/978-3-319-25304-6_226721269)
Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: The Keio collection. Mol. Syst. Biol. https://doi.org/10.1038/msb4100050 (2006). (PMID: 10.1038/msb4100050167385541681482)
Brachmann, C. B. et al. Designer deletion strains derived from Saccharomyces cerevisiae S288C: A useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14, 115–132. https://doi.org/10.1002/(sici)1097-0061(19980130)14:2%3c115::Aid-yea204%3e3.0.Co;2-2 (1998). (PMID: 10.1002/(sici)1097-0061(19980130)14:2<115::Aid-yea204>3.0.Co;2-29483801)
Dagkesamanskaya, A. et al. Use of photoswitchable fluorescent proteins for droplet-based microfluidic screening. J Microbiol Methods. 147, 59–65. https://doi.org/10.1016/j.mimet.2018.03.001 (2018). (PMID: 10.1016/j.mimet.2018.03.00129518436)
Marches, O. et al. Role of tir and intimin in the virulence of rabbit enteropathogenic Escherichia coli serotype O103:H 2 . Infect. Immun. 68, 2171–2182. https://doi.org/10.1128/IAI.68.4.2171-2182.2000 (2000). (PMID: 10.1128/IAI.68.4.2171-2182.20001072261797401)
Johnson, J. R., Johnston, B., Kuskowski, M. A., Nougayrede, J. P. & Oswald, E. Molecular epidemiology and phylogenetic distribution of the Escherichia coli pks genomic island. J. Clin. Microbiol. 46, 3906–3911. https://doi.org/10.1128/jcm.00949-08 (2008). (PMID: 10.1128/jcm.00949-08189458412593299)
Martin, P. et al. Interplay between Siderophores and Colibactin Genotoxin biosynthetic pathways in Escherichia coli. PLOS Pathog. 9, e1003437. https://doi.org/10.1371/journal.ppat.1003437 (2013). (PMID: 10.1371/journal.ppat.1003437238535823708854)

SAIC2016/048 & SAIC/2018/010 Industrial grants

Keywords: Adhesion; Bacteria; Cell wall; Fimbriae; Flow cytometry; Yeast

Date Created: 20240909 Date Completed: 20240909 Latest Revision: 20240912

20240913

PMC11385505

10.1038/s41598-024-72030-w

39251857