Mucus layer modeling of human colonoids during infection with enteroaggragative E. coli.
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
29 06 2020
29 06 2020
Historique:
received:
14
02
2020
accepted:
02
06
2020
entrez:
1
7
2020
pubmed:
1
7
2020
medline:
16
12
2020
Statut:
epublish
Résumé
EAEC is a common cause of diarrheal illness worldwide. Pathogenesis is believed to occur in the ileum and colon, where the bacteria adhere and form a robust aggregating biofilm. Among the multiple virulence factors produced by EAEC, the Pic serine protease has been implicated in bacterial colonization by virtue of its mucinolytic activity. Hence, a potential role of Pic in mucus barrier disruption during EAEC infection has been long postulated. In this study, we used human colonoids comprising goblet cells and a thick mucin barrier as an intestinal model to investigate Pic's roles during infection with EAEC. We demonstrated the ability of purified Pic, but not a protease defective Pic mutant to degrade MUC2. Western blot and confocal microscopy analysis revealed degradation of the MUC2 layer in colonoids infected with EAEC, but not with its isogenic EAECpic mutant. Wild-type and MUC2-knockdown colonoids infected with EAEC strains exposed a differential biofilm distribution, greater penetration of the mucus layer and increased colonization of the colonic epithelium by Wild-type EAEC than its isogenic Pic mutant. Higher secretion of pro-inflammatory cytokines was seen in colonoids infected with EAEC than EAECpic. Although commensal E. coli expressing Pic degraded MUC2, it did not show improved mucus layer penetration or colonization of the colonic epithelium. Our study demonstrates a role of Pic in MUC2 barrier disruption in the human intestine and shows that colonoids are a reliable system to study the interaction of pathogens with the mucus layer.
Identifiants
pubmed: 32601325
doi: 10.1038/s41598-020-67104-4
pii: 10.1038/s41598-020-67104-4
pmc: PMC7324601
doi:
Substances chimiques
Escherichia coli Proteins
0
Mucins
0
Pic protein, E coli
EC 3.4.21.-
Serine Endopeptidases
EC 3.4.21.-
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
10533Subventions
Organisme : NIDDK NIH HHS
ID : K01 DK106323
Pays : United States
Organisme : NIAID NIH HHS
ID : P01 AI125181
Pays : United States
Références
Huang, D. B. et al. Enteroaggregative Escherichia coli is a cause of acute diarrheal illness: a meta-analysis. Clin Infect Dis 43, 556–563, https://doi.org/10.1086/505869 (2006).
doi: 10.1086/505869
pubmed: 16886146
Huang, D. B., Mohanty, A., DuPont, H. L., Okhuysen, P. C. & Chiang, T. A review of an emerging enteric pathogen: enteroaggregative Escherichia coli. J Med Microbiol 55, 1303–1311, https://doi.org/10.1099/jmm.0.46674-0 (2006).
doi: 10.1099/jmm.0.46674-0
pubmed: 17005776
Nataro, J. P. et al. Diarrheagenic Escherichia coli infection in Baltimore, Maryland, and New Haven, Connecticut. Clin Infect Dis 43, 402–407, https://doi.org/10.1086/505867 (2006).
doi: 10.1086/505867
pubmed: 16838226
Imdad, A. et al. Diarrheagenic Escherichia coli and Acute Gastroenteritis in Children in Davidson County, Tennessee, United States: A Case-control Study. Pediatr Infect Dis J 37, 543–548, https://doi.org/10.1097/INF.0000000000001908 (2018).
doi: 10.1097/INF.0000000000001908
pubmed: 29341983
pmcid: 5962020
Taylor, D. N. et al. A randomized, double-blind, multicenter study of rifaximin compared with placebo and with ciprofloxacin in the treatment of travelers’ diarrhea. Am J Trop Med Hyg 74, 1060–1066 (2006).
doi: 10.4269/ajtmh.2006.74.1060
Olesen, B. et al. Etiology of diarrhea in young children in Denmark: a case-control study. J Clin Microbiol 43, 3636–3641, https://doi.org/10.1128/JCM.43.8.3636-3641.2005 (2005).
doi: 10.1128/JCM.43.8.3636-3641.2005
pubmed: 16081890
pmcid: 1234006
Nuesch-Inderbinen, M. T., Hofer, E., Hachler, H., Beutin, L. & Stephan, R. Characteristics of enteroaggregative Escherichia coli isolated from healthy carriers and from patients with diarrhoea. J Med Microbiol 62, 1828–1834, https://doi.org/10.1099/jmm.0.065177-0 (2013).
doi: 10.1099/jmm.0.065177-0
pubmed: 24008499
Cohen, M. B. et al. Prevalence of diarrheagenic Escherichia coli in acute childhood enteritis: a prospective controlled study. J Pediatr 146, 54–61, https://doi.org/10.1016/j.jpeds.2004.08.059 (2005).
doi: 10.1016/j.jpeds.2004.08.059
pubmed: 15644823
Steiner, T. S., Lima, A. A., Nataro, J. P. & Guerrant, R. L. Enteroaggregative Escherichia coli produce intestinal inflammation and growth impairment and cause interleukin-8 release from intestinal epithelial cells. J Infect Dis 177, 88–96, https://doi.org/10.1086/513809 (1998).
doi: 10.1086/513809
pubmed: 9419174
Rogawski, E. T. et al. Epidemiology of enteroaggregative Escherichia coli infections and associated outcomes in the MAL-ED birth cohort. Plos Negl Trop Dis 11, e0005798, https://doi.org/10.1371/journal.pntd.0005798 (2017).
doi: 10.1371/journal.pntd.0005798
pubmed: 28742106
pmcid: 5542697
Boll, E. J. et al. Enteroaggregative Escherichia coli Adherence Fimbriae Drive Inflammatory Cell Recruitment via Interactions with Epithelial MUC1. mBio 8, https://doi.org/10.1128/mBio.00717-17 (2017).
Vial, P. A. et al. Characterization of enteroadherent-aggregative Escherichia coli, a putative agent of diarrheal disease. J Infect Dis 158, 70–79, https://doi.org/10.1093/infdis/158.1.70 (1988).
doi: 10.1093/infdis/158.1.70
pubmed: 2899125
Harrington, S. M., Dudley, E. G. & Nataro, J. P. Pathogenesis of enteroaggregative Escherichia coli infection. FEMS Microbiol Lett 254, 12–18, https://doi.org/10.1111/j.1574-6968.2005.00005.x (2006).
doi: 10.1111/j.1574-6968.2005.00005.x
pubmed: 16451173
Hicks, S., Candy, D. C. & Phillips, A. D. Adhesion of enteroaggregative Escherichia coli to pediatric intestinal mucosa in vitro. Infect Immun 64, 4751–4760 (1996).
doi: 10.1128/IAI.64.11.4751-4760.1996
Henderson, I. R., Czeczulin, J., Eslava, C., Noriega, F. & Nataro, J. P. Characterization of pic, a secreted protease of Shigella flexneri and enteroaggregative Escherichia coli. Infect Immun 67, 5587–5596 (1999).
doi: 10.1128/IAI.67.11.5587-5596.1999
Ruiz-Perez, F. & Nataro, J. P. Bacterial serine proteases secreted by the autotransporter pathway: classification, specificity, and role in virulence. Cell Mol Life Sci https://doi.org/10.1007/s00018-013-1355-8 (2013).
doi: 10.1007/s00018-013-1355-8
pubmed: 23689588
pmcid: 3871983
Parham, N. J. et al. PicU, a second serine protease autotransporter of uropathogenic Escherichia coli. FEMS Microbiol Lett 230, 73–83, doi:S0378109703008620 [pii] (2004).
Heimer, S. R., Rasko, D. A., Lockatell, C. V., Johnson, D. E. & Mobley, H. L. Autotransporter genes pic and tsh are associated with Escherichia coli strains that cause acute pyelonephritis and are expressed during urinary tract infection. Infect Immun 72, 593–597 (2004).
doi: 10.1128/IAI.72.1.593-597.2004
Boisen, N., Ruiz-Perez, F., Scheutz, F., Krogfelt, K. A. & Nataro, J. P. Short report: high prevalence of serine protease autotransporter cytotoxins among strains of enteroaggregative Escherichia coli. Am J Trop Med Hyg 80, 294–301 (2009).
doi: 10.4269/ajtmh.2009.80.294
Rasko, D. A. et al. Origins of the E. coli strain causing an outbreak of hemolytic-uremic syndrome in Germany. N Engl J Med 365, 709–717, https://doi.org/10.1056/NEJMoa1106920 (2011).
doi: 10.1056/NEJMoa1106920
pubmed: 21793740
pmcid: 3168948
Hosseini Nave, H., Mansouri, S., Taati Moghadam, M. & Moradi, M. Virulence Gene Profile and Multilocus Variable-Number Tandem-Repeat Analysis (MLVA) of Enteroinvasive Escherichia coli (EIEC) Isolates From Patients With Diarrhea in Kerman, Iran. Jundishapur J Microbiol 9, e33529, https://doi.org/10.5812/jjm.33529 (2016).
doi: 10.5812/jjm.33529
pubmed: 27635212
pmcid: 5013238
Xu, Y. et al. High Prevalence of Virulence Genes in Specific Genotypes of Atypical Enteropathogenic Escherichia coli. Front Cell Infect Microbiol 7, 109, https://doi.org/10.3389/fcimb.2017.00109 (2017).
doi: 10.3389/fcimb.2017.00109
pubmed: 28421169
pmcid: 5378719
Abreu, A. G. et al. The serine protease Pic as a virulence factor of atypical enteropathogenic Escherichia coli. Gut Microbes 7, 115–125, https://doi.org/10.1080/19490976.2015.1136775 (2016).
doi: 10.1080/19490976.2015.1136775
pubmed: 26963626
pmcid: 4856457
Bhullar, K. et al. The Serine Protease Autotransporter Pic Modulates Citrobacter rodentium Pathogenesis and Its Innate Recognition by the Host. Infect Immun 83, 2636–2650, https://doi.org/10.1128/IAI.00025-15 (2015).
doi: 10.1128/IAI.00025-15
pubmed: 25895966
pmcid: 4468532
Abreu, A. G. et al. The Serine Protease Pic From Enteroaggregative Escherichia coli Mediates Immune Evasion by the Direct Cleavage of Complement Proteins. J Infect Dis 212, 106–115, https://doi.org/10.1093/infdis/jiv013 (2015).
doi: 10.1093/infdis/jiv013
pubmed: 25583166
Navarro-Garcia, F. et al. Pic, an autotransporter protein secreted by different pathogens in the Enterobacteriaceae family, is a potent mucus secretagogue. Infect Immun 78, 4101–4109, https://doi.org/10.1128/IAI.00523-10 (2010).
doi: 10.1128/IAI.00523-10
pubmed: 20696826
pmcid: 2950354
Harrington, S. M. et al. The Pic protease of enteroaggregative Escherichia coli promotes intestinal colonization and growth in the presence of mucin. Infect Immun 77, 2465–2473, https://doi.org/10.1128/IAI.01494-08 (2009).
doi: 10.1128/IAI.01494-08
pubmed: 19349428
pmcid: 2687332
Ruiz-Perez, F. et al. Serine protease autotransporters from Shigella flexneri and pathogenic Escherichia coli target a broad range of leukocyte glycoproteins. Proc Natl Acad Sci U S A 108, 12881–12886, https://doi.org/10.1073/pnas.1101006108 (2011).
doi: 10.1073/pnas.1101006108
pubmed: 21768350
pmcid: 3150873
Munera, D. et al. Autotransporters but not pAA are critical for rabbit colonization by Shiga toxin-producing Escherichia coli O104:H4. Nat Commun 5, 3080, https://doi.org/10.1038/ncomms4080 (2014).
doi: 10.1038/ncomms4080
pubmed: 24445323
pmcid: 3905246
Bellini, E. M. et al. Antibody response against plasmid-encoded toxin (Pet) and the protein involved in intestinal colonization (Pic) in children with diarrhea produced by enteroaggregative Escherichia coli. FEMS Immunol Med Microbiol 43, 259–264, https://doi.org/10.1016/j.femsim.2004.08.008 (2005).
doi: 10.1016/j.femsim.2004.08.008
pubmed: 15681156
Zachos, N. C. et al. Human Enteroids/Colonoids and Intestinal Organoids Functionally Recapitulate Normal Intestinal Physiology and Pathophysiology. J Biol Chem 291, 3759–3766, https://doi.org/10.1074/jbc.R114.635995 (2016).
doi: 10.1074/jbc.R114.635995
pubmed: 26677228
Stelzner, M. et al. A nomenclature for intestinal in vitro cultures. Am J Physiol Gastrointest Liver Physiol 302, G1359–1363, https://doi.org/10.1152/ajpgi.00493.2011 (2012).
doi: 10.1152/ajpgi.00493.2011
pubmed: 22461030
pmcid: 3378093
Rajan, A. et al. Novel Segment- and Host-Specific Patterns of Enteroaggregative Escherichia coli Adherence to Human Intestinal Enteroids. mBio 9, https://doi.org/10.1128/mBio.02419-17 (2018).
Ayala-Lujan, J. L. et al. Broad spectrum activity of a lectin-like bacterial serine protease family on human leukocytes. Plos One 9, e107920, https://doi.org/10.1371/journal.pone.0107920 (2014).
doi: 10.1371/journal.pone.0107920
pubmed: 25251283
pmcid: 4176022
In, J. G., Foulke-Abel, J., Clarke, E. & Kovbasnjuk, O. Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium. J Vis Exp, https://doi.org/10.3791/59357 (2019).
Noel, G. et al. A primary human macrophage-enteroid co-culture model to investigate mucosal gut physiology and host-pathogen interactions. Sci Rep 7, 45270, https://doi.org/10.1038/srep45270 (2017).
doi: 10.1038/srep45270
pubmed: 28345602
pmcid: 5366908
Hews, C. L. et al. The StcE metalloprotease of enterohaemorrhagic Escherichia coli reduces the inner mucus layer and promotes adherence to human colonic epithelium ex vivo. Cell Microbiol 19, https://doi.org/10.1111/cmi.12717 (2017).
Luo, Q. et al. Enterotoxigenic Escherichia coli secretes a highly conserved mucin-degrading metalloprotease to effectively engage intestinal epithelial cells. Infect Immun 82, 509–521, https://doi.org/10.1128/IAI.01106-13 (2014).
doi: 10.1128/IAI.01106-13
pubmed: 24478067
pmcid: 3911403
Kumar, P. et al. EatA, an immunogenic protective antigen of enterotoxigenic Escherichia coli, degrades intestinal mucin. Infect Immun 82, 500–508, https://doi.org/10.1128/IAI.01078-13 (2014).
doi: 10.1128/IAI.01078-13
pubmed: 24478066
pmcid: 3911389
Ranganathan, S. et al. Evaluating Shigella flexneri Pathogenesis in the Human Enteroid Model. Infect Immun 87, https://doi.org/10.1128/IAI.00740-18 (2019).
Koestler, B. J. et al. Human Intestinal Enteroids as a Model System of Shigella Pathogenesis. Infect Immun 87, https://doi.org/10.1128/IAI.00733-18 (2019).
In, J. G. et al. Human mini-guts: new insights into intestinal physiology and host-pathogen interactions. Nat Rev Gastroenterol Hepatol 13, 633–642, https://doi.org/10.1038/nrgastro.2016.142 (2016).
doi: 10.1038/nrgastro.2016.142
pubmed: 27677718
pmcid: 5079760
Bu, X. D., Li, N., Tian, X. Q. & Huang, P. L. Caco-2 and LS174T cell lines provide different models for studying mucin expression in colon cancer. Tissue Cell 43, 201–206, https://doi.org/10.1016/j.tice.2011.03.002 (2011).
doi: 10.1016/j.tice.2011.03.002
pubmed: 21470648
Augeron, C. & Laboisse, C. L. Emergence of permanently differentiated cell clones in a human colonic cancer cell line in culture after treatment with sodium butyrate. Cancer Res 44, 3961–3969 (1984).
pubmed: 6744312
Johansson, M. E., Larsson, J. M. & Hansson, G. C. The two mucus layers of colon are organized by the MUC2 mucin, whereas the outer layer is a legislator of host-microbial interactions. Proc Natl Acad Sci U S A 108(Suppl 1), 4659–4665, https://doi.org/10.1073/pnas.1006451107 (2011).
doi: 10.1073/pnas.1006451107
pubmed: 20615996
Szentkuti, L., Riedesel, H., Enss, M. L., Gaertner, K. & Von Engelhardt, W. Pre-epithelial mucus layer in the colon of conventional and germ-free rats. Histochem J 22, 491–497, https://doi.org/10.1007/bf01007234 (1990).
doi: 10.1007/bf01007234
pubmed: 1702088
Johansson, M. E. et al. The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria. Proc Natl Acad Sci U S A 105, 15064–15069, https://doi.org/10.1073/pnas.0803124105 (2008).
doi: 10.1073/pnas.0803124105
pubmed: 18806221
pmcid: 2567493
Johansson, M. E. & Hansson, G. C. Preservation of mucus in histological sections, immunostaining of mucins in fixed tissue, and localization of bacteria with FISH. Methods Mol Biol 842, 229–235, https://doi.org/10.1007/978-1-61779-513-8_13 (2012).
doi: 10.1007/978-1-61779-513-8_13
pubmed: 22259139
Valeri, M. et al. Pathogenic E. coli exploits SslE mucinase activity to translocate through the mucosal barrier and get access to host cells. Plos One 10, e0117486, https://doi.org/10.1371/journal.pone.0117486 (2015).
doi: 10.1371/journal.pone.0117486
pubmed: 25789808
pmcid: 4366376
Greenberg, D. E., Jiang, Z. D., Steffen, R., Verenker, M. P. & DuPont, H. L. Markers of inflammation in bacterial diarrhea among travelers, with a focus on enteroaggregative Escherichia coli pathogenicity. J Infect Dis 185, 944–949, https://doi.org/10.1086/339617 (2002).
doi: 10.1086/339617
pubmed: 11920319
Harrington, S. M., Strauman, M. C., Abe, C. M. & Nataro, J. P. Aggregative adherence fimbriae contribute to the inflammatory response of epithelial cells infected with enteroaggregative Escherichia coli. Cell Microbiol 7, 1565–1578, https://doi.org/10.1111/j.1462-5822.2005.00588.x (2005).
doi: 10.1111/j.1462-5822.2005.00588.x
pubmed: 16207244
Iversen, M. B. et al. An innate antiviral pathway acting before interferons at epithelial surfaces. Nat Immunol 17, 150–158, https://doi.org/10.1038/ni.3319 (2016).
doi: 10.1038/ni.3319
pubmed: 26595890
Shin, W. & Kim, H. J. Intestinal barrier dysfunction orchestrates the onset of inflammatory host-microbiome cross-talk in a human gut inflammation-on-a-chip. Proc Natl Acad Sci U S A 115, E10539–E10547, https://doi.org/10.1073/pnas.1810819115 (2018).
doi: 10.1073/pnas.1810819115
pubmed: 30348765
pmcid: 6233106
Van der Sluis, M. et al. Muc2-deficient mice spontaneously develop colitis, indicating that MUC2 is critical for colonic protection. Gastroenterology 131, 117–129, https://doi.org/10.1053/j.gastro.2006.04.020 (2006).
doi: 10.1053/j.gastro.2006.04.020
Heazlewood, C. K. et al. Aberrant mucin assembly in mice causes endoplasmic reticulum stress and spontaneous inflammation resembling ulcerative colitis. PLoS Med 5, e54, https://doi.org/10.1371/journal.pmed.0050054 (2008).
doi: 10.1371/journal.pmed.0050054
pubmed: 18318598
pmcid: 2270292
Chichlowski, M. & Hale, L. P. Bacterial-mucosal interactions in inflammatory bowel disease: an alliance gone bad. Am J Physiol Gastrointest Liver Physiol 295, G1139–1149, https://doi.org/10.1152/ajpgi.90516.2008 (2008).
doi: 10.1152/ajpgi.90516.2008
pubmed: 18927210
pmcid: 2604805
Foulke-Abel, J. et al. Human Enteroids as a Model of Upper Small Intestinal Ion Transport Physiology and Pathophysiology. Gastroenterology 150(638–649), e638, https://doi.org/10.1053/j.gastro.2015.11.047 (2016).
doi: 10.1053/j.gastro.2015.11.047
Heijmans, J. et al. ER stress causes rapid loss of intestinal epithelial stemness through activation of the unfolded protein response. Cell Rep 3, 1128–1139, https://doi.org/10.1016/j.celrep.2013.02.031 (2013).
doi: 10.1016/j.celrep.2013.02.031
pubmed: 23545496
Sato, T. et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 141, 1762–1772, https://doi.org/10.1053/j.gastro.2011.07.050 (2011).
doi: 10.1053/j.gastro.2011.07.050
pubmed: 21889923
Nataro, J. P. et al. Heterogeneity of enteroaggregative Escherichia coli virulence demonstrated in volunteers. J Infect Dis 171, 465–468, https://doi.org/10.1093/infdis/171.2.465 (1995).
doi: 10.1093/infdis/171.2.465
pubmed: 7844392
Levine, M. M. et al. Escherichia coli strains that cause diarrhoea but do not produce heat-labile or heat-stable enterotoxins and are non-invasive. Lancet 1, 1119–1122, https://doi.org/10.1016/s0140-6736(78)90299-4 (1978).
doi: 10.1016/s0140-6736(78)90299-4
pubmed: 77415
In, J. et al. Enterohemorrhagic Escherichia coli reduce mucus and intermicrovillar bridges in human stem cell-derived colonoids. Cell Mol Gastroenterol Hepatol 2(48–62), e43, https://doi.org/10.1016/j.jcmgh.2015.10.001 (2016).
doi: 10.1016/j.jcmgh.2015.10.001
Johansson, M. E. et al. Bacteria penetrate the inner mucus layer before inflammation in the dextran sulfate colitis model. PLoS One 5, e12238, https://doi.org/10.1371/journal.pone.0012238 (2010).
doi: 10.1371/journal.pone.0012238
pubmed: 20805871
pmcid: 2923597
Hayashi, N. et al. Extracellular Signals of a Human Epithelial Colorectal Adenocarcinoma (Caco-2) Cell Line Facilitate the Penetration of Pseudomonas aeruginosa PAO1 Strain through the Mucin Layer. Front Cell Infect Microbiol 7, 415, https://doi.org/10.3389/fcimb.2017.00415 (2017).
doi: 10.3389/fcimb.2017.00415
pubmed: 28983473
pmcid: 5613098