A Pilot Single Cell Analysis of the Zebrafish Embryo Cellular Responses to Uropathogenic Escherichia coli Infection

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Ashley Rawson
Vijay Saxena
Hongyu Gao
Jenaya Hooks
Xiaoling Xuei
Patrick McGuire
Takashi Hato
David S. Hains
Ryan M. Anderson
Andrew L Schwaderer

Abstract

Background: Uropathogenic Escherichia coli (UPEC) infections are common and when they disseminate can be of high morbidity.


Methods: We studied the effects of UPEC infection using single cell RNA sequencing (scRNAseq) in zebrafish. Bulk RNA sequencing has historically been used to evaluate gene expression patterns, but scRNAseq allows gene expression to be evaluated at the single cell level and is optimal for evaluating heterogeneity within cell types and rare cell types. Zebrafish cohorts were injected with either saline or UPEC,and scRNAseq and canonical pathway analyses were performed.


Results: Canonical pathway analysis of scRNAseq data provided key information regarding innate immune pathways in the cells determined to be thymus cells, ionocytes, macrophages/monocytes, and pronephros cells. Pathways activated in thymus cells included interleukin 6 (IL-6) signaling and production of reactive oxygen species. Fc receptor-mediated phagocytosis was a leading canonical pathway in the pronephros and macrophages. Genes that were downregulated in UPEC vs saline exposed embryos involved the cellular response to the Gram-negative endotoxin lipopolysaccharide (LPS) and included Forkhead Box O1a (Foxo1a), Tribbles Pseudokinase 3 (Trib3), Arginase 2 (Arg2) and Polo Like Kinase 3 (Plk3).


Conclusions: Because 4-day post fertilization zebrafish embryos only have innate immune systems, the scRNAseq provides insights into pathways and genes that cell types utilize in the bacterial response. Based on our analysis, we have identified genes and pathways that might serve as genetic targets for treatment and further investigation in UPEC infections at the single cell level.

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References

Raszka WV, Jr., Khan O. Pyelonephritis. Pediatr Rev. 2005;26(10):364-70. doi: 10.1542/pir.26-10-364. PubMed PMID: 16199590.

Foxman B. Epidemiology of urinary tract infections: incidence, morbidity, and economic costs. Am J Med. 2002;113 Suppl 1A:5S-13S. doi: 10.1016/s0002-9343(02)01054-9. PubMed PMID: 12113866.

Al-Hasan MN, Eckel-Passow JE, Baddour LM. Bacteremia complicating gram-negative urinary tract infections: a population-based study. J Infect. 2010;60(4):278-85. doi: 10.1016/j.jinf.2010.01.007. PubMed PMID: 20114061; PMCID: PMC2841447.

Levy MM, Artigas A, Phillips GS, Rhodes A, Beale R, Osborn T, Vincent JL, Townsend S, Lemeshow S, Dellinger RP. Outcomes of the Surviving Sepsis Campaign in intensive care units in the USA and Europe: a prospective cohort study. Lancet Infect Dis. 2012;12(12):919-24. doi: 10.1016/S1473-3099(12)70239-6. PubMed PMID: 23103175.

Gupta K, Donnola SB, Sadeghi Z, Lu L, Erokwu BO, Kavran M, Hijaz A, Flask CA. Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis. J Vis Exp. 2017(125). doi: 10.3791/54649. PubMed PMID: 28745629; PMCID: PMC5612360.

Tittel AP, Heuser C, Kurts C. Mouse model for pyelonephritis. Curr Protoc Immunol. 2013;Chapter 15:Unit 15 23 1-9. doi: 10.1002/0471142735.im1523s101. PubMed PMID: 23564687.

Nielsen TK, Petersen NA, Staerk K, Gronnemose RB, Palarasah Y, Nielsen LF, Kolmos HJ, Andersen TE, Lund L. A Porcine Model for Urinary Tract Infection. Front Microbiol. 2019;10:2564. doi: 10.3389/fmicb.2019.02564. PubMed PMID: 31824442; PMCID: PMC6882375.

Bakthavatchalu V, Wert KJ, Feng Y, Mannion A, Ge Z, Garcia A, Scott KE, Caron TJ, Madden CM, Jacobsen JT, Victora G, Jaenisch R, Fox JG. Cytotoxic Escherichia coli strains encoding colibactin isolated from immunocompromised mice with urosepsis and meningitis. PLoS One. 2018;13(3):e0194443. doi: 10.1371/journal.pone.0194443. PubMed PMID: 29554148; PMCID: PMC5858775.

Novoa B, Figueras A. Zebrafish: model for the study of inflammation and the innate immune response to infectious diseases. Adv Exp Med Biol. 2012;946:253-75. doi: 10.1007/978-1-4614-0106-3_15. PubMed PMID: 21948373.

Kolodziejczyk AA, Kim JK, Svensson V, Marioni JC, Teichmann SA. The technology and biology of single-cell RNA sequencing. Mol Cell. 2015;58(4):610-20. doi: 10.1016/j.molcel.2015.04.005. PubMed PMID: 26000846.

Shapiro E, Biezuner T, Linnarsson S. Single-cell sequencing-based technologies will revolutionize whole-organism science. Nat Rev Genet. 2013;14(9):618-30. doi: 10.1038/nrg3542. PubMed PMID: 23897237.

Kulkarni AA, Conteh AM, Sorrell CA, Mirmira A, Tersey SA, Mirmira RG, Linnemann AK, Anderson RM. An In Vivo Zebrafish Model for Interrogating ROS-Mediated Pancreatic beta-Cell Injury, Response, and Prevention. Oxid Med Cell Longev. 2018;2018:1324739. doi: 10.1155/2018/1324739. PubMed PMID: 29785241; PMCID: PMC5896207.

Mastracci TL, Robertson MA, Mirmira RG, Anderson RM. Polyamine biosynthesis is critical for growth and differentiation of the pancreas. Sci Rep. 2015;5:13269. doi: 10.1038/srep13269. PubMed PMID: 26299433; PMCID: PMC4547391.

Mora-Bau G, Platt AM, van Rooijen N, Randolph GJ, Albert ML, Ingersoll MA. Macrophages Subvert Adaptive Immunity to Urinary Tract Infection. PLoS Pathog. 2015;11(7):e1005044. doi: 10.1371/journal.ppat.1005044. PubMed PMID: 26182347; PMCID: PMC4504509.

Butler A, Hoffman P, Smibert P, Papalexi E, Satija R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol. 2018;36(5):411-20. doi: 10.1038/nbt.4096. PubMed PMID: 29608179; PMCID: PMC6700744.

Satija R, Farrell JA, Gennert D, Schier AF, Regev A. Spatial reconstruction of single-cell gene expression data. Nat Biotechnol. 2015;33(5):495-502. doi: 10.1038/nbt.3192. PubMed PMID: 25867923; PMCID: PMC4430369.

Stuart T, Butler A, Hoffman P, Hafemeister C, Papalexi E, Mauck WM, 3rd, Hao Y, Stoeckius M, Smibert P, Satija R. Comprehensive Integration of Single-Cell Data. Cell. 2019;177(7):1888-902 e21. doi: 10.1016/j.cell.2019.05.031. PubMed PMID: 31178118; PMCID: PMC6687398.

McCarthy DJ, Campbell KR, Lun AT, Wills QF. Scater: pre-processing, quality control, normalization and visualization of single-cell RNA-seq data in R. Bioinformatics. 2017;33(8):1179-86. doi: 10.1093/bioinformatics/btw777. PubMed PMID: 28088763; PMCID: PMC5408845.

Hafemeister C, Satija R. Normalization and variance stabilization of single-cell RNA-seq data using regularized negative binomial regression. Genome Biol. 2019;20(1):296. doi: 10.1186/s13059-019-1874-1. PubMed PMID: 31870423; PMCID: PMC6927181.

Farnsworth DR, Saunders LM, Miller AC. A single-cell transcriptome atlas for zebrafish development. Dev Biol. 2020;459(2):100-8. doi: 10.1016/j.ydbio.2019.11.008. PubMed PMID: 31782996; PMCID: PMC7080588.

Ruzicka L, Howe DG, Ramachandran S, Toro S, Van Slyke CE, Bradford YM, Eagle A, Fashena D, Frazer K, Kalita P, Mani P, Martin R, Moxon ST, Paddock H, Pich C, Schaper K, Shao X, Singer A, Westerfield M. The Zebrafish Information Network: new support for non-coding genes, richer Gene Ontology annotations and the Alliance of Genome Resources. Nucleic Acids Res. 2019;47(D1):D867-D73. doi: 10.1093/nar/gky1090. PubMed PMID: 30407545; PMCID: PMC6323962.

La Manno G, Soldatov R, Zeisel A, Braun E, Hochgerner H, Petukhov V, Lidschreiber K, Kastriti ME, Lonnerberg P, Furlan A, Fan J, Borm LE, Liu Z, van Bruggen D, Guo J, He X, Barker R, Sundstrom E, Castelo-Branco G, Cramer P, Adameyko I, Linnarsson S, Kharchenko PV. RNA velocity of single cells. Nature. 2018;560(7719):494-8. doi: 10.1038/s41586-018-0414-6. PubMed PMID: 30089906; PMCID: PMC6130801.

Barber AE, Fleming BA, Mulvey MA. Similarly Lethal Strains of Extraintestinal Pathogenic Escherichia coli Trigger Markedly Diverse Host Responses in a Zebrafish Model of Sepsis. mSphere. 2016;1(2). doi: 10.1128/mSphere.00062-16. PubMed PMID: 27303721; PMCID: PMC4894679.

Wiles TJ, Bower JM, Redd MJ, Mulvey MA. Use of zebrafish to probe the divergent virulence potentials and toxin requirements of extraintestinal pathogenic Escherichia coli. PLoS Pathog. 2009;5(12):e1000697. doi: 10.1371/journal.ppat.1000697. PubMed PMID: 20019794; PMCID: PMC2785880.

Saxena V, Fitch J, Ketz J, White P, Wetzel A, Chanley MA, Spencer JD, Becknell B, Pierce KR, Arregui SW, Nelson RD, Schwartz GJ, Velazquez V, Walker LA, Chen X, Yan P, Hains DS, Schwaderer AL. Whole Transcriptome Analysis of Renal Intercalated Cells Predicts Lipopolysaccharide Mediated Inhibition of Retinoid X Receptor alpha Function. Sci Rep. 2019;9(1):545. doi: 10.1038/s41598-018-36921-z. PubMed PMID: 30679625; PMCID: PMC6345901.

Terlizzi ME, Gribaudo G, Maffei ME. UroPathogenic Escherichia coli (UPEC) Infections: Virulence Factors, Bladder Responses, Antibiotic, and Non-antibiotic Antimicrobial Strategies. Front Microbiol. 2017;8:1566. doi: 10.3389/fmicb.2017.01566. PubMed PMID: 28861072; PMCID: PMC5559502.

Dong G, Wang Y, Xiao W, Pacios Pujado S, Xu F, Tian C, Xiao E, Choi Y, Graves DT. FOXO1 regulates dendritic cell activity through ICAM-1 and CCR7. J Immunol. 2015;194(8):3745-55. doi: 10.4049/jimmunol.1401754. PubMed PMID: 25786691; PMCID: PMC4390509.

Brown J, Wang H, Suttles J, Graves DT, Martin M. Mammalian target of rapamycin complex 2 (mTORC2) negatively regulates Toll-like receptor 4-mediated inflammatory response via FoxO1. J Biol Chem. 2011;286(52):44295-305. doi: 10.1074/jbc.M111.258053. PubMed PMID: 22045807; PMCID: PMC3247956.

Smith SM, Moran AP, Duggan SP, Ahmed SE, Mohamed AS, Windle HJ, O'Neill LA, Kelleher DP. Tribbles 3: a novel regulator of TLR2-mediated signaling in response to Helicobacter pylori lipopolysaccharide. J Immunol. 2011;186(4):2462-71. doi: 10.4049/jimmunol.1000864. PubMed PMID: 21220698.

Haase M, Fitze G. HSP90AB1: Helping the good and the bad. Gene. 2016;575(2 Pt 1):171-86. doi: 10.1016/j.gene.2015.08.063. PubMed PMID: 26358502; PMCID: PMC5675009.

Nishimura H, Emoto M, Kimura K, Yoshikai Y. Hsp70 protects macrophages infected with Salmonella choleraesuis against TNF-alpha-induced cell death. Cell Stress Chaperones. 1997;2(1):50-9. doi: 10.1379/1466-1268(1997)002<0050:hpmiws>2.3.co;2. PubMed PMID: 9250395; PMCID: PMC312980.

Vinokurov M, Ostrov V, Yurinskaya M, Garbuz D, Murashev A, Antonova O, Evgen'ev M. Recombinant human Hsp70 protects against lipoteichoic acid-induced inflammation manifestations at the cellular and organismal levels. Cell Stress Chaperones. 2012;17(1):89-101. doi: 10.1007/s12192-011-0288-0. PubMed PMID: 21845530; PMCID: PMC3227847.

Roy BC, Subramaniam D, Ahmed I, Jala VR, Hester CM, Greiner KA, Haribabu B, Anant S, Umar S. Role of bacterial infection in the epigenetic regulation of Wnt antagonist WIF1 by PRC2 protein EZH2. Oncogene. 2015;34(34):4519-30. doi: 10.1038/onc.2014.386. PubMed PMID: 25486432; PMCID: PMC4459936.

Zuniga-Traslavina C, Bravo K, Reyes AE, Feijoo CG. Cxcl8b and Cxcr2 Regulate Neutrophil Migration through Bloodstream in Zebrafish. J Immunol Res. 2017;2017:6530531. doi: 10.1155/2017/6530531. PubMed PMID: 28642884; PMCID: PMC5470028.

van Berkel PH, Geerts ME, van Veen HA, Mericskay M, de Boer HA, Nuijens JH. N-terminal stretch Arg2, Arg3, Arg4 and Arg5 of human lactoferrin is essential for binding to heparin, bacterial lipopolysaccharide, human lysozyme and DNA. Biochem J. 1997;328 ( Pt 1):145-51. doi: 10.1042/bj3280145. PubMed PMID: 9359845; PMCID: PMC1218898.

Starner TD, Barker CK, Jia HP, Kang Y, McCray PB, Jr. CCL20 is an inducible product of human airway epithelia with innate immune properties. Am J Respir Cell Mol Biol. 2003;29(5):627-33. doi: 10.1165/rcmb.2002-0272OC. PubMed PMID: 12748059.

Yuan J, Li Z, Li F, Lin Z, Yao S, Zhou H, Liu W, Yu H, Liu Y, Liu F, Li F, Ran H, Zhang J, Huang Y, Fu Q, Wang L, Liu J. Proteomics reveals the potential mechanism of Mrps35 controlling Listeria monocytogenes intracellular proliferation in macrophages. Proteomics. 2021;21(10):e2000262. doi: 10.1002/pmic.202000262. PubMed PMID: 33763969.

Wang S, Chen Z, Zhu S, Lu H, Peng D, Soutto M, Naz H, Peek R, Jr., Xu H, Zaika A, Xu Z, El-Rifai W. PRDX2 protects against oxidative stress induced by H. pylori and promotes resistance to cisplatin in gastric cancer. Redox Biol. 2020;28:101319. doi: 10.1016/j.redox.2019.101319. PubMed PMID: 31536951; PMCID: PMC6811995.

Jiang F, Gao Y, Dong C, Xiong S. ODC1 inhibits the inflammatory response and ROS-induced apoptosis in macrophages. Biochem Biophys Res Commun. 2018;504(4):734-41. doi: 10.1016/j.bbrc.2018.09.023. PubMed PMID: 30217446.

Wroblewski LE, Noble PJ, Pagliocca A, Pritchard DM, Hart CA, Campbell F, Dodson AR, Dockray GJ, Varro A. Stimulation of MMP-7 (matrilysin) by Helicobacter pylori in human gastric epithelial cells: role in epithelial cell migration. J Cell Sci. 2003;116(Pt 14):3017-26. doi: 10.1242/jcs.00518. PubMed PMID: 12808021.

Giri H. Plk3 Deficient Mice are Resistant to LPS-Induced Endotoxic Shock and Lethality. World Journal of Internal Medicine. 2019;1(1):1-2.

Bellizzi JJ, 3rd, Widom J, Kemp C, Lu JY, Das AK, Hofmann SL, Clardy J. The crystal structure of palmitoyl protein thioesterase 1 and the molecular basis of infantile neuronal ceroid lipofuscinosis. Proc Natl Acad Sci U S A. 2000;97(9):4573-8. doi: 10.1073/pnas.080508097. PubMed PMID: 10781062; PMCID: PMC18274.

Rogan MR, Patterson LL, Wang JY, McBride JW. Bacterial Manipulation of Wnt Signaling: A Host-Pathogen Tug-of-Wnt. Front Immunol. 2019;10:2390. doi: 10.3389/fimmu.2019.02390. PubMed PMID: 31681283; PMCID: PMC6811524.

Hwang PP. Ion uptake and acid secretion in zebrafish (Danio rerio). J Exp Biol. 2009;212(Pt 11):1745-52. doi: 10.1242/jeb.026054. PubMed PMID: 19448083.

Hwang PP, Chou MY. Zebrafish as an animal model to study ion homeostasis. Pflugers Arch. 2013;465(9):1233-47. doi: 10.1007/s00424-013-1269-1. PubMed PMID: 23568368; PMCID: PMC3745619.

Rose-John S, Winthrop K, Calabrese L. The role of IL-6 in host defence against infections: immunobiology and clinical implications. Nat Rev Rheumatol. 2017;13(7):399-409. doi: 10.1038/nrrheum.2017.83. PubMed PMID: 28615731.

Nunez V, Alameda D, Rico D, Mota R, Gonzalo P, Cedenilla M, Fischer T, Bosca L, Glass CK, Arroyo AG, Ricote M. Retinoid X receptor alpha controls innate inflammatory responses through the up-regulation of chemokine expression. Proc Natl Acad Sci U S A. 2010;107(23):10626-31. doi: 10.1073/pnas.0913545107. PubMed PMID: 20498053; PMCID: PMC2890831.

Ben Mkaddem S, Benhamou M, Monteiro RC. Understanding Fc Receptor Involvement in Inflammatory Diseases: From Mechanisms to New Therapeutic Tools. Front Immunol. 2019;10:811. doi: 10.3389/fimmu.2019.00811. PubMed PMID: 31057544; PMCID: PMC6481281.

Lerner EM, 2nd. Phagocytosis of bacteria in the absence of antibody and the effect of physical surface; a reinvestigation of surface phagocytosis. J Exp Med. 1956;104(2):233-43. doi: 10.1084/jem.104.2.233. PubMed PMID: 13345968; PMCID: PMC2136657.

Munn DH, Cheung NK. Antibody-independent phagocytosis of tumor cells by human monocyte-derived macrophages cultured in recombinant macrophage colony-stimulating factor. Cancer Immunol Immunother. 1995;41(1):46-52. doi: 10.1007/BF01788959. PubMed PMID: 7641219.

Ilie OD, Paduraru E, Robea MA, Balmus IM, Jijie R, Nicoara M, Ciobica A, Nita IB, Dobrin R, Doroftei B. The Possible Role of Bifidobacterium longum BB536 and Lactobacillus rhamnosus HN001 on Locomotor Activity and Oxidative Stress in a Rotenone-Induced Zebrafish Model of Parkinson's Disease. Oxid Med Cell Longev. 2021;2021:9629102. doi: 10.1155/2021/9629102. PubMed PMID: 34691361; PMCID: PMC8531778.

Qiu W, Liu X, Yang F, Li R, Xiong Y, Fu C, Li G, Liu S, Zheng C. Single and joint toxic effects of four antibiotics on some metabolic pathways of zebrafish (Danio rerio) larvae. Sci Total Environ. 2020;716:137062. doi: 10.1016/j.scitotenv.2020.137062. PubMed PMID: 32036144.