約80 % 確診的泌尿道感染病患中皆發現到泌尿道致病性大腸桿菌。近期已表明泌尿道致病性大腸桿菌可存在於食品中。一些研究顯示在泌尿道感染病患尿液及糞便中採集到相同的泌尿道致病性大腸桿菌。也有腸道上皮細胞模型的證據指出泌尿道致病性大腸桿菌可從腸道轉移至血液。因此，食源性泌尿道感染一詞已常作為描述與食品源相關的泌尿道感染。雖然一些研究已表示泌尿道致病性大腸桿菌具備與腸道 (血腸障壁) 交互作用及轉移的能力，但鮮少資訊關於導致這種交互作用的細菌風險因子。此研究目的為透過泌尿道致病性大腸桿菌 (UTI89) 蛋白質體微陣列鑑定泌尿道致病性大腸桿菌蛋白質與腸道間交互作用並確定涉及泌尿道致病性大腸桿菌從腸內轉移之蛋白。我們建構泌尿道致病性大腸桿菌蛋白質體微陣列並使用螢光標記的腸道上皮細胞 (Colo 205) 作為蛋白質結合試驗的探針。在篩選細胞與泌尿道致病性大腸桿菌蛋白質間交互作用後，針對此細胞結合蛋白進行一系列生物資訊分析。生物資訊分析篩選出最有機會與上皮細胞結合之外膜及分泌性蛋白質後，進一步以流式細胞儀驗證其與人類腸道上皮細胞 (Colo 205) 之結合。泌尿道致病性大腸桿菌蛋白質體微陣列找出37種泌尿道致病性大腸桿菌蛋白質與人類腸道上皮細胞結合。而生物資訊分析預測17種分泌性蛋白質。其中1種蛋白質也被預測為外膜蛋白質。在17種泌尿道致病性大腸桿菌蛋白質中，以流式細胞儀驗證12種蛋白質能與Colo 205細胞結合。接著，藉由膠體電泳，在12種結合蛋白質中驗證出YgdD蛋白的分泌性。因此，這些驗證指出YgdD蛋白具有分泌性且具有與腸道上皮細胞結合的能力。在MTT試驗中，YgdD降低細胞存活率，意味YgdD具有細胞毒性。侵入試驗結果中指出YgdD促進細菌侵入Colo205細胞。因此，這些結果揭露新興的毒性因子YgdD可能抑制腸道上皮復原及誘導泌尿道致病性大腸桿菌入侵腸道。

Uropathogenic Escherichia coli (UPEC) strains are found to be present in about 80% of diagnosed urinary tract infection (UTI) patients and the knowledge about the UPEC pathogenesis of UTI had not been enough. Recently, it has been showed that food can be a reservoir for UPEC. Some studies revealed that the UPEC strains in UTI patients’ urine are the same as the UPEC strains in UTI patients’ fecal. Also, the evidence of gut epithelial cell model indicated that UPEC strains translocate to the bloodstream from gut. Thus, the term foodborne urinary tract infections (FUTIs) has been commonly used due to the UTIs from food. Although some studies have indicated that UPEC had translocation ability from gut to bloodstream due to interaction with gut epithelial cells (gut-blood barrier), little information is available on the bacterial risk factors are involved in this interaction. The purpose of this study is to investigate the interaction between UPEC proteins and intestine by UPEC (UTI89) proteome microarrays and determine the role of this interaction with intestine. We constructed the proteome microarrays consisting of entire UTI89 proteome and used the human intestinal epithelial cell line (Colo 205) labelled with fluorescence to be the probe for the protein binding assay. After screening the interaction between cell and UTI89 proteins, these cell-binding proteins were analyzed by a serial of bioinformatics. After bioinformatic analysis selected outer membrane and secretory proteins as only they have the chance to interact with epithelial cells, the flow cytometric assay was conducted to further validate their binding to the human intestinal epithelial cells (Colo 205). UTI89 proteome microarrays screened 37 epithelial cell-binding proteins between UPEC proteins and human intestinal epithelial cell (Colo 205). Bioinformatic analysis predicted 17 secretory proteins. One of them also predicted outer membrane protein. Among these UTI89 proteins, there were 12 secretory proteins bound to Colo 205 cells in the binding validation by flow cytometric assay. Subsequently, YgdD was validated secretory function by gel electrophoresis. Thus, these validation indicate YgdD protein have secretory in the bacteria and ability of intestinal epithelial cell binding. In the MTT assay, YgdD decreased cell viability, suggesting YgdD had cell toxicity. The result of invasion assay indicated that YgdD promote bacterial invasion to Colo 205 cells. Thus, these results reveal the novel virulence factor which may cause the suppression of intestinal epithelium recovery and induce UPEC invasion.

1. Spencer JD, Schwaderer A, McHugh K, Hains DS. 2010. Pediatric urinary tract infections: an analysis of hospitalizations, charges, and costs in the USA. Pediatr. Nephrol. 25:2469-75
2. Kaper JB, Nataro JP, Mobley HL. 2004. Pathogenic Escherichia coli. Nature reviews microbiology 2:123-40
3. Foxman B. 2003. Epidemiology of urinary tract infections: incidence, morbidity, and economic costs. Dis. Mon. 49:53-70
4. Olson PD, Hruska KA, Hunstad DA. 2016. Androgens enhance male urinary tract infection severity in a new model. J. Am. Soc. Nephrol. 27:1625-34
5. Russo TA, Johnson JR. 2003. Medical and economic impact of extraintestinal infections due to Escherichia coli: focus on an increasingly important endemic problem. Microbes and infection 5:449-56
6. Foxman B. 2010. The epidemiology of urinary tract infection. Nature Reviews Urology 7:653-60
7. Manges A. 2016. Escherichia coli and urinary tract infections: the role of poultry-meat. Clin. Microbiol. Infect. 22:122-9
8. Poolman JT, Wacker M. 2016. Extraintestinal pathogenic Escherichia coli, a common human pathogen: challenges for vaccine development and progress in the field. The Journal of infectious diseases 213:6-13
9. Lüthje P, Brauner A. 2014. Virulence factors of uropathogenic E. coli and their interaction with the host. Adv. Microb. Physiol. 65:337-72
10. Moreno E, Andreu A, Pigrau C, Kuskowski MA, Johnson JR, Prats G. 2008. Relationship between Escherichia coli strains causing acute cystitis in women and the fecal E. coli population of the host. J. Clin. Microbiol. 46:2529-34
11. Magruder M, Sholi AN, Gong C, Zhang L, Edusei E, et al. 2019. Gut uropathogen abundance is a risk factor for development of bacteriuria and urinary tract infection. Nature communications 10:1-9
12. Stamm WE, Norrby SR. 2001. Urinary tract infections: disease panorama and challenges. The Journal of infectious diseases 183:S1-S4
13. Vincent C, Boerlin P, Daignault D, Dozois CM, Dutil L, et al. 2010. Food reservoir for Escherichia coli causing urinary tract infections. Emerg. Infect. Dis. 16:88
14. Nordstrom L, Liu CM, Price LB. 2013. Foodborne urinary tract infections: a new paradigm for antimicrobial-resistant foodborne illness. Front. Microbiol. 4:29
15. Mortensen S, Johansen AE, Thøfner I, Christensen JP, Pors SE, et al. 2019. Infectious potential of human derived uropathogenic Escherichia coli UTI89 in the reproductive tract of laying hens. Vet. Microbiol. 239:108445
16. Mora A, Viso S, López C, Alonso MP, García-Garrote F, et al. 2013. Poultry as reservoir for extraintestinal pathogenic Escherichia coli O45: K1: H7-B2-ST95 in humans. Vet. Microbiol. 167:506-12
17. Markland S, LeStrange K, Sharma M, Kniel K. 2015. Old friends in new places: exploring the role of extraintestinal E. coli in intestinal disease and foodborne illness. Zoonoses Public Health 62:491-6
18. Vollmerhausen TL, Woods JL, Faoagali J, Katouli M. 2014. Interactions of uroseptic Escherichia coli with renal (A-498) and gastrointestinal (HT-29) cell lines. J. Med. Microbiol. 63:1575-83
19. Owrangi B, Masters N, Kuballa A, O’Dea C, Vollmerhausen TL, Katouli M. 2018. Invasion and translocation of uropathogenic Escherichia coli isolated from urosepsis and patients with community-acquired urinary tract infection. Eur. J. Clin. Microbiol. Infect. Dis. 37:833-9
20. Wang Z-x, Deng R-p, Jiang H-W, Guo S-J, Le H-y, et al. 2012. Global identification of prokaryotic glycoproteins based on an Escherichia coli proteome microarray. PLoS One 7:e49080
21. Tu Y-H, Ho Y-H, Chuang Y-C, Chen P-C, Chen C-S. 2011. Identification of lactoferricin B intracellular targets using an Escherichia coli proteome chip. PLoS One 6:e28197
22. Tsai C-H, Ho Y-H, Sung T-C, Wu W-F, Chen C-S. 2017. Escherichia coli proteome microarrays identified the substrates of ClpYQ protease. Mol. Cell. Proteomics 16:113-20
23. Sutandy FR, Qian J, Chen CS, Zhu H. 2013. Overview of protein microarrays. Current protocols in protein science 72:27.1. 1-.1. 16
24. Ho Y-H, Shah P, Chen Y-W, Chen C-S. 2016. Systematic analysis of intracellular-targeting antimicrobial peptides, bactenecin 7, hybrid of pleurocidin and dermaseptin, proline–arginine-rich peptide, and lactoferricin B, by using Escherichia coli proteome microarrays. Mol. Cell. Proteomics 15:1837-47
25. Chen C-S, Sullivan S, Anderson T, Tan AC, Alex PJ, et al. 2009. Identification of novel serological biomarkers for inflammatory bowel disease using Escherichia coli proteome chip. Mol. Cell. Proteomics 8:1765-76
26. Hsiao FS-H, Sutandy FR, Syu G-D, Chen Y-W, Lin J-M, Chen C-S. 2016. Systematic protein interactome analysis of glycosaminoglycans revealed YcbS as a novel bacterial virulence factor. Sci. Rep. 6:1-10
27. Chen Y-W, Teng C-H, Ho Y-H, Ho TYJ, Huang W-C, et al. 2014. Identification of bacterial factors involved in type 1 fimbria expression using an Escherichia coli K12 proteome chip. Mol. Cell. Proteomics 13:1485-94
28. Orfanoudaki G, Economou A. 2014. Proteome-wide subcellular topologies of E. coli polypeptides database (STEPdb). Mol. Cell. Proteomics 13:3674-87
29. Apweiler R, Bairoch A, Wu CH, Barker WC, Boeckmann B, et al. 2004. UniProt: the universal protein knowledgebase. Nucleic Acids Res. 32:D115-D9
30. Keseler IM, Collado-Vides J, Santos-Zavaleta A, Peralta-Gil M, Gama-Castro S, et al. 2010. EcoCyc: a comprehensive database of Escherichia coli biology. Nucleic Acids Res. 39:D583-D90
31. Yu CS, Lin CJ, Hwang JK. 2004. Predicting subcellular localization of proteins for Gram‐negative bacteria by support vector machines based on n‐peptide compositions. Protein Sci. 13:1402-6
32. Chou K-C, Shen H-B. 2008. Cell-PLoc: a package of Web servers for predicting subcellular localization of proteins in various organisms. Nat. Protoc. 3:153
33. Bendtsen JD, Kiemer L, Fausbøll A, Brunak S. 2005. Non-classical protein secretion in bacteria. BMC Microbiol. 5:1-13
34. Rose R, Häuser S, Stump-Guthier C, Weiss C, Rohde M, et al. 2018. Virulence factor-dependent basolateral invasion of choroid plexus epithelial cells by pathogenic Escherichia coli in vitro. FEMS Microbiol. Lett. 365:fny274
35. Hazes B, Frost L. 2008. Towards a systems biology approach to study type II/IV secretion systems. Biochimica et Biophysica Acta (BBA)-Biomembranes 1778:1839-50
36. Bohn E, Sonnabend M, Klein K, Autenrieth IB. 2019. Bacterial adhesion and host cell factors leading to effector protein injection by type III secretion system. Int. J. Med. Microbiol. 309:344-50
37. Green ER, Mecsas J. 2016. Bacterial secretion systems: an overview. Virulence mechanisms of bacterial pathogens:213-39
38. Yunck R, Cho H, Bernhardt TG. 2016. Identification of MltG as a potential terminase for peptidoglycan polymerization in bacteria. Mol. Microbiol. 99:700-18
39. Kobayashi K, Masuda T, Tajima N, Wada H, Sato N. 2014. Molecular phylogeny and intricate evolutionary history of the three isofunctional enzymes involved in the oxidation of protoporphyrinogen IX. Genome Biol. Evol. 6:2141-55
40. Paulsen VS, Mardirossian M, Blencke H-M, Benincasa M, Runti G, et al. 2016. Inner membrane proteins YgdD and SbmA are required for the complete susceptibility of Escherichia coli to the proline-rich antimicrobial peptide arasin 1 (1–25). Microbiology 162:601-9
41. Lai PK, Tresnak DT, Hackel BJ. 2019. Identification and elucidation of proline‐rich antimicrobial peptides with enhanced potency and delivery. Biotechnol. Bioeng. 116:2439-50