Regulation of hemolysin in uropathogenic Escherichia coli fine-tunes killing of human macrophages

ABSTRACT Uropathogenic E. coli (UPEC) causes the majority of urinary tract infections (UTIs), which are a major global public health concern. UPEC uses numerous mechanisms to subvert the innate immune system, including targeting macrophage functions. We recently showed that some UPEC strains rapidly kill human macrophages via an NLRP3-independent pathway, and also trigger NLRP3-dependent IL-1β processing. In this study, we used random transposon mutagenesis in the reference strain CFT073 to identify UPEC genes that mediate human macrophage cell death. Our approach revealed that the hemolysin A (HlyA) toxin is essential for triggering both cell death and NLRP3 inflammasome-mediated IL-1β release in human macrophages. Random transposon mutagenesis also identified the cof gene, which encodes a poorly characterized phosphatase, as a novel hemolysin regulator; a CFT073 mutant deleted for the cof gene secreted significantly reduced levels of HlyA, had diminished hemolytic activity, and was impaired in its capacity to trigger human macrophage cell death and IL-1β release. Together, our findings reveal that Cof fine-tunes production of hemolysin, an important determinant of both UPEC-mediated inflammasome activation and human macrophage cell death.

[1]  S. Beatson,et al.  Novel genes associated with enhanced motility of Escherichia coli ST131 , 2017, PloS one.

[2]  G. Ulett,et al.  Protein-based profiling of the immune response to uropathogenic Escherichia coli in adult patients immediately following hospital admission for acute cystitis. , 2016, Pathogens and disease.

[3]  J. Lieberman,et al.  Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores , 2016, Nature.

[4]  A. Wieser,et al.  Uropathogenic Escherichia coli strain CFT073 disrupts NLRP3 inflammasome activation. , 2016, The Journal of clinical investigation.

[5]  R. Welch.  Uropathogenic Escherichia coli-Associated Exotoxins , 2016, Microbiology spectrum.

[6]  K. Schroder,et al.  Strain- and host species-specific inflammasome activation, IL-1β release, and cell death in macrophages infected with uropathogenic Escherichia coli , 2015, Mucosal Immunology.

[7]  Allison J. Greaney,et al.  Bacterial Exotoxins and the Inflammasome , 2015, Front. Immunol..

[8]  H. Mobley,et al.  Virulence and Fitness Determinants of Uropathogenic Escherichia coli , 2015, Microbiology spectrum.

[9]  S. Hultgren,et al.  Urinary tract infections: epidemiology, mechanisms of infection and treatment options , 2015, Nature Reviews Microbiology.

[10]  K. Schroder,et al.  A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases , 2015, Nature Medicine.

[11]  S. Hultgren,et al.  Dysregulation of Escherichia coli α-hemolysin expression alters the course of acute and persistent urinary tract infection , 2015, Proceedings of the National Academy of Sciences.

[12]  Y. Wannemuehler,et al.  FNR Regulates Expression of Important Virulence Factors Contributing to Pathogenicity of Uropathogenic Escherichia coli , 2014, Infection and Immunity.

[13]  J. Tschopp,et al.  The neutrophil NLRC4 inflammasome selectively promotes IL-1β maturation without pyroptosis during acute Salmonella challenge. , 2014, Cell reports.

[14]  J. Bernhagen,et al.  Crosstalk between Sentinel and Helper Macrophages Permits Neutrophil Migration into Infected Uroepithelium , 2014, Cell.

[15]  M. Sweet,et al.  An mRNA atlas of G protein-coupled receptor expression during primary human monocyte/macrophage differentiation and lipopolysaccharide-mediated activation identifies targetable candidate regulators of inflammation. , 2013, Immunobiology.

[16]  S. Beatson,et al.  The Serum Resistome of a Globally Disseminated Multidrug Resistant Uropathogenic Escherichia coli Clone , 2013, PLoS genetics.

[17]  Eun-Kyeong Jo,et al.  NLRP3 Inflammasome and Host Protection against Bacterial Infection , 2013, Journal of Korean medical science.

[18]  D. Hunstad,et al.  YbcL of Uropathogenic Escherichia coli Suppresses Transepithelial Neutrophil Migration , 2012, Infection and Immunity.

[19]  D. Hilbert,et al.  Clinical Escherichia coli isolates utilize alpha-hemolysin to inhibit in vitro epithelial cytokine production. , 2012, Microbes and infection.

[20]  S. Hultgren,et al.  Host-pathogen checkpoints and population bottlenecks in persistent and intracellular uropathogenic Escherichia coli bladder infection. , 2012, FEMS microbiology reviews.

[21]  O. Sokolova,et al.  Role of Uropathogenic Escherichia coli Virulence Factors in Development of Urinary Tract Infection and Kidney Damage , 2012, International journal of nephrology.

[22]  B. Dhakal,et al.  The UPEC pore-forming toxin α-hemolysin triggers proteolysis of host proteins to disrupt cell adhesion, inflammatory, and survival pathways. , 2012, Cell host & microbe.

[23]  Y. Carmi,et al.  IL-1α and IL-1β Recruit Different Myeloid Cells and Promote Different Stages of Sterile Inflammation , 2011, The Journal of Immunology.

[24]  M. Sweet,et al.  Intramacrophage survival of uropathogenic Escherichia coli: differences between diverse clinical isolates and between mouse and human macrophages. , 2011, Immunobiology.

[25]  J. Valle,et al.  Molecular Characterization of UpaB and UpaC, Two New Autotransporter Proteins of Uropathogenic Escherichia coli CFT073 , 2011, Infection and Immunity.

[26]  C. Svanborg,et al.  Genetics of innate immunity and UTI susceptibility , 2011, Nature Reviews Urology.

[27]  D. Hunstad,et al.  Attenuation of human neutrophil migration and function by uropathogenic bacteria. , 2011, Microbes and infection.

[28]  Betsy Foxman,et al.  The epidemiology of urinary tract infection , 2010, Nature Reviews Urology.

[29]  Abdulrasheed Abdu,et al.  Antimicrobial resistance in Escherichia coli strains from urinary tract infections. , 2009, Journal of the National Medical Association.

[30]  Leopold Parts,et al.  Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants. , 2009, Genome research.

[31]  R. Conran,et al.  Hemolysin of Uropathogenic Escherichia coli Evokes Extensive Shedding of the Uroepithelium and Hemorrhage in Bladder Tissue within the First 24 Hours after Intraurethral Inoculation of Mice , 2008, Infection and Immunity.

[32]  T. J. Wiles,et al.  Inactivation of host Akt/protein kinase B signaling by bacterial pore-forming toxins. , 2008, Molecular biology of the cell.

[33]  J. Pinkner,et al.  LeuX tRNA‐dependent and ‐independent mechanisms of Escherichia coli pathogenesis in acute cystitis , 2007, Molecular microbiology.

[34]  B. Molitoris,et al.  Real‐time studies of the progression of bacterial infections and immediate tissue responses in live animals , 2007, Cellular microbiology.

[35]  Liran Carmel,et al.  Genome-wide Analysis of Substrate Specificities of the Escherichia coli Haloacid Dehalogenase-like Phosphatase Family* , 2006, Journal of Biological Chemistry.

[36]  Yarery C. Smith,et al.  Novel Three-Dimensional Organoid Model for Evaluation of the Interaction of Uropathogenic Escherichia coli with Terminally Differentiated Human Urothelial Cells , 2006, Infection and Immunity.

[37]  S. Hultgren,et al.  Suppression of Bladder Epithelial Cytokine Responses by Uropathogenic Escherichia coli , 2005, Infection and Immunity.

[38]  S. Gerdes,et al.  A Genetic Screen for the Identification of Thiamin Metabolic Genes* , 2004, Journal of Biological Chemistry.

[39]  S. Hultgren,et al.  Intracellular Bacterial Biofilm-Like Pods in Urinary Tract Infections , 2003, Science.

[40]  R. Hotchkiss,et al.  The pathophysiology and treatment of sepsis. , 2003, The New England journal of medicine.

[41]  J. Hacker,et al.  Loss of Regulatory Protein RfaH Attenuates Virulence of Uropathogenic Escherichia coli , 2002, Infection and Immunity.

[42]  A. Siitonen,et al.  Variations in 10 putative uropathogen virulence genes among urinary, faecal and peri-urethral Escherichia coli. , 2002, Journal of medical microbiology.

[43]  M. Pfaffl,et al.  A new mathematical model for relative quantification in real-time RT-PCR. , 2001, Nucleic acids research.

[44]  J. Shabanowitz,et al.  Escherichia coli α-Hemolysin (HlyA) Is Heterogeneously Acylated in Vivo with 14-, 15-, and 17-Carbon Fatty Acids* , 2000, The Journal of Biological Chemistry.

[45]  M. J. Bailey,et al.  RfaH and the ops element, components of a novel system controlling bacterial transcription elongation , 1997, Molecular microbiology.

[46]  Jennifer A. Leeds,et al.  Enhancing transcription through the Escherichia coli hemolysin operon, hlyCABD: RfaH and upstream JUMPStart DNA sequences function together via a postinitiation mechanism , 1997, Journal of bacteriology.

[47]  R. Welch,et al.  RfaH enhances elongation of Escherichia coli hlyCABD mRNA , 1996, Journal of bacteriology.

[48]  P. Stanley,et al.  Fatty acylation of two internal lysine residues required for the toxic activity of Escherichia coli hemolysin. , 1994, Science.

[49]  E V Koonin,et al.  Computer analysis of bacterial haloacid dehalogenases defines a large superfamily of hydrolases with diverse specificity. Application of an iterative approach to database search. , 1994, Journal of molecular biology.

[50]  M. Ceska,et al.  Interleukin-8 and the neutrophil response to mucosal gram-negative infection. , 1993, The Journal of clinical investigation.

[51]  P. Krammer,et al.  Cytocidal effects of Escherichia coli hemolysin on human T lymphocytes , 1993, Infection and immunity.

[52]  M. J. Bailey,et al.  Escherichia coli HIyT protein, a transcriptional activator of haemolysin synthesis and secretion, is encoded by the rfaH (sfrB) locus required for expression of sex factor and lipopolysaccharide genes , 1992, Molecular microbiology.

[53]  R. Welch.  Pore‐forming cytolysins of Gram‐negative bacteria , 1991, Molecular microbiology.

[54]  P. Anderson,et al.  Interleukin-6 response to deliberate colonization of the human urinary tract with gram-negative bacteria , 1991, Infection and immunity.

[55]  W. Seeger,et al.  Effects of Escherichia coli hemolysin on endothelial cell function , 1990, Infection and immunity.

[56]  P. Delepelaire,et al.  TolC, an Escherichia coli outer membrane protein required for hemolysin secretion. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[57]  H. Mobley,et al.  Pyelonephritogenic Escherichia coli and killing of cultured human renal proximal tubular epithelial cells: role of hemolysin in some strains , 1990, Infection and immunity.

[58]  R. Welch,et al.  Characterization of monoclonal antibodies against the Escherichia coli hemolysin , 1990, Infection and immunity.

[59]  S. Bhakdi,et al.  Potent leukocidal action of Escherichia coli hemolysin mediated by permeabilization of target cell membranes , 1989, The Journal of experimental medicine.

[60]  S. Larsen,et al.  In vitro cytotoxic effect of α‐hemolytic Escherichia coli on human blood granulocytes , 1988 .

[61]  S. Larsen,et al.  In vitro cytotoxic effect of alpha-hemolytic Escherichia coli on human blood granulocytes. Correlation with size of alpha-hemolysin production. , 1988, APMIS : acta pathologica, microbiologica, et immunologica Scandinavica.

[62]  R. Welch,et al.  Nucleotide sequence of an Escherichia coli chromosomal hemolysin , 1985, Journal of bacteriology.

[63]  R. Welch,et al.  Escherichia coli hemolysin is released extracellularly without cleavage of a signal peptide , 1985, Journal of bacteriology.

[64]  I. Orskov,et al.  In vitro cytotoxic effect of alpha-hemolytic Escherichia coli on human blood granulocytes , 1984, Infection and immunity.

[65]  T. Mosmann.  Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. , 1983, Journal of immunological methods.

[66]  I. Snyder,et al.  Effect of Escherichia coli alpha-hemolysin on human peripheral leukocyte function in vitro , 1982, Infection and Immunity.

[67]  Cooke Em,et al.  Properties of Strains of Escherichia Coli Isolated from a Variety of Sources , 1975 .