N-Terminal Gly224–Gly411 Domain in Listeria Adhesion Protein Interacts with Host Receptor Hsp60

Background Listeria adhesion protein (LAP) is a housekeeping bifunctional enzyme consisting of N-terminal acetaldehyde dehydrogenase (ALDH) and C-terminal alcohol dehydrogenase (ADH). It aids Listeria monocytogenes in crossing the epithelial barrier through a paracellular route by interacting with its host receptor, heat shock protein 60 (Hsp60). To gain insight into the binding interaction between LAP and Hsp60, LAP subdomain(s) participating in the Hsp60 interaction were investigated. Methods Using a ModBase structural model, LAP was divided into 4 putative subdomains: the ALDH region contains N1 (Met1–Pro223) and N2 (Gly224–Gly411), and the ADH region contains C1 (Gly412–Val648) and C2 (Pro649–Val866). Each subdomain was cloned and overexpressed in Escherichia coli and purified. Purified subdomains were used in ligand overlay, immunofluorescence, and bead-based epithelial cell adhesion assays to analyze each domain's affinity toward Hsp60 protein or human ileocecal epithelial HCT-8 cells. Results The N2 subdomain exhibited the greatest affinity for Hsp60 with a K D of 9.50±2.6 nM. The K D of full-length LAP (7.2±0.5 nM) to Hsp60 was comparable to the N2 value. Microspheres (1 µm diameter) coated with N2 subdomain showed significantly (P<0.05) higher binding to HCT-8 cells than beads coated with other subdomains and this binding was inhibited when HCT-8 cells were pretreated with anti-Hsp60 antibody to specifically block epithelial Hsp60. Furthermore, HCT-8 cells pretreated with purified N2 subdomain also reduced L. monocytogenes adhesion by about 4 log confirming its involvement in interaction with epithelial cells. Conclusion These data indicate that the N2 subdomain in the LAP ALDH domain is critical in initiating interaction with mammalian cell receptor Hsp60 providing insight into the molecular mechanism of pathogenesis for the development of potential anti-listerial control strategies.

[1]  V. Fischetti,et al.  A major surface protein on group A streptococci is a glyceraldehyde-3- phosphate-dehydrogenase with multiple binding activity , 1992, The Journal of experimental medicine.

[2]  M. Naujokas,et al.  InlB-Dependent Internalization of Listeria Is Mediated by the Met Receptor Tyrosine Kinase , 2000, Cell.

[3]  A. Bhunia,et al.  Heat Shock Protein 60 Acts as a Receptor for the Listeria Adhesion Protein in Caco-2 Cells , 2004, Infection and Immunity.

[4]  P. Cossart,et al.  Conjugated action of two species-specific invasion proteins for fetoplacental listeriosis , 2008, Nature.

[5]  J. Vandekerckhove,et al.  Characterization of a Listeria monocytogenes Protein Interfering with Rab5a , 2008, Traffic.

[6]  P. Cossart,et al.  LapB, a novel Listeria monocytogenes LPXTG surface adhesin, required for entry into eukaryotic cells and virulence. , 2010, The Journal of infectious diseases.

[7]  P. Cossart,et al.  Bacterial Adhesion and Entry into Host Cells , 2006, Cell.

[8]  W. Goebel,et al.  Listeria Pathogenesis and Molecular Virulence Determinants , 2001, Clinical Microbiology Reviews.

[9]  V. Pancholi,et al.  Plasminogen-mediated group A streptococcal adherence to and pericellular invasion of human pharyngeal cells. , 2003, Microbial pathogenesis.

[10]  J. Content,et al.  Listeria monocytogenes Possesses Adhesins for Fibronectin , 1999, Infection and Immunity.

[11]  P. Cossart,et al.  A Transgenic Model for Listeriosis: Role of Internalin in Crossing the Intestinal Barrier , 2001, Science.

[12]  J. Kumaran,et al.  Listeria monocytogenes Internalin B Activates Junctional Endocytosis to Accelerate Intestinal Invasion , 2010, PLoS pathogens.

[13]  W. Goebel,et al.  The gene coding for protein p60 of Listeria monocytogenes and its use as a specific probe for Listeria monocytogenes , 1990, Infection and immunity.

[14]  A. Bhunia,et al.  Expression of LAP, a SecA2-dependent secretory protein, is induced under anaerobic environment. , 2009, Microbes and infection.

[15]  P. Cossart,et al.  Listeria monocytogenes Surface Proteins: from Genome Predictions to Function , 2007, Microbiology and Molecular Biology Reviews.

[16]  A. Carson,et al.  Staphylococcal Fibronectin Binding Protein Interacts with Heat Shock Protein 60 and Integrins: Role in Internalization by Epithelial Cells , 2000, Infection and Immunity.

[17]  A. Bhunia,et al.  A Listeria adhesion protein-deficient Listeria monocytogenes strain shows reduced adhesion primarily to intestinal cell lines , 2003, Medical Microbiology and Immunology.

[18]  L. Baldomà,et al.  Role of secreted glyceraldehyde-3-phosphate dehydrogenase in the infection mechanism of enterohemorrhagic and enteropathogenic Escherichia coli: interaction of the extracellular enzyme with human plasminogen and fibrinogen. , 2007, The international journal of biochemistry & cell biology.

[19]  B. Henderson,et al.  Stress Wars: the Direct Role of Host and Bacterial Molecular Chaperones in Bacterial Infection , 2006, Infection and Immunity.

[20]  K. Okuda,et al.  Role of lysine in interaction between surface protein peptides of Streptococcus gordonii and agglutinin peptide. , 2009, Oral microbiology and immunology.

[21]  M. Wilmanns,et al.  The Crystal Structure of the Allosteric Non-phosphorylating Glyceraldehyde-3-phosphate Dehydrogenase from the Hyperthermophilic Archaeum Thermoproteus tenax * , 2002, The Journal of Biological Chemistry.

[22]  Timo K. Korhonen,et al.  Bacterial metastasis: the host plasminogen system in bacterial invasion. , 2005, Trends in microbiology.

[23]  W. Goebel,et al.  The iap gene of Listeria monocytogenes is essential for cell viability, and its gene product, p60, has bacteriolytic activity , 1993, Journal of bacteriology.

[24]  Wen-gang Yang,et al.  Entamoeba histolytica has an alcohol dehydrogenase homologous to the multifunctional adhE gene product of Escherichia coli. , 1994, Molecular and biochemical parasitology.

[25]  Arun K. Bhunia,et al.  Listeria monocytogenes Uses Listeria Adhesion Protein (LAP) To Promote Bacterial Transepithelial Translocation and Induces Expression of LAP Receptor Hsp60 , 2010, Infection and Immunity.

[26]  Keith Ireton,et al.  Entry of the bacterial pathogen Listeria monocytogenes into mammalian cells , 2007, Cellular microbiology.

[27]  M. Rohde,et al.  α‐Enolase of Streptococcus pneumoniae is a plasmin(ogen)‐binding protein displayed on the bacterial cell surface , 2001, Molecular microbiology.

[28]  B. Henderson,et al.  Chaperonins are cell-signalling proteins: the unfolding biology of molecular chaperones , 2000, Expert Reviews in Molecular Medicine.

[29]  J. Belisle,et al.  Mycobacterium tuberculosis malate synthase is a laminin‐binding adhesin , 2006, Molecular microbiology.

[30]  P. Cossart,et al.  FbpA, a novel multifunctional Listeria monocytogenes virulence factor , 2004, Molecular microbiology.

[31]  P. Cossart,et al.  gC1q‐R/p32, a C1q‐binding protein, is a receptor for the InlB invasion protein of Listeria monocytogenes , 2000, The EMBO journal.

[32]  P. Cossart,et al.  E-Cadherin Is the Receptor for Internalin, a Surface Protein Required for Entry of L. monocytogenes into Epithelial Cells , 1996, Cell.

[33]  Marc A. Martí-Renom,et al.  MODBASE: a database of annotated comparative protein structure models and associated resources , 2005, Nucleic Acids Res..

[34]  P. Cossart,et al.  Subversion of cellular functions by Listeria monocytogenes , 2006, The Journal of pathology.

[35]  P. Cossart,et al.  The Listeria monocytogenes Virulence Factor InlJ Is Specifically Expressed In Vivo and Behaves as an Adhesin , 2008, Infection and Immunity.

[36]  A. Bhunia,et al.  Surface protein p104 is involved in adhesion of Listeria monocytogenes to human intestinal cell line, Caco-2. , 1999, Journal of medical microbiology.

[37]  Manfred Rohde,et al.  Glyceraldehyde-3-Phosphate Dehydrogenase of Streptococcus pneumoniae Is a Surface-Displayed Plasminogen-Binding Protein , 2004, Infection and Immunity.

[38]  J. Malda,et al.  Discovery and characterization of IGFBP-mediated endocytosis in the human retinal pigment epithelial cell line ARPE-19. , 2009, Experimental eye research.

[39]  G. S. Chhatwal,et al.  Housekeeping enzymes as virulence factors for pathogens. , 2003, International journal of medical microbiology : IJMM.

[40]  S. Romero-Steiner,et al.  Adherence of recombinant pneumococcal surface adhesin A (rPsaA)-coated particles to human nasopharyngeal epithelial cells for the evaluation of anti-PsaA functional antibodies. , 2006, Vaccine.

[41]  F. Fiedler,et al.  Formation of D‐alanyl‐lipoteichoic acid is required for adhesion and virulence of Listeria monocytogenes , 2002, Molecular microbiology.

[42]  P. Cossart,et al.  Sequence and Binding Activity of the Autolysin-Adhesin Ami from Epidemic Listeria monocytogenes 4b , 2004, Infection and Immunity.

[43]  Arun K. Bhunia,et al.  LAP, an alcohol acetaldehyde dehydrogenase enzyme in Listeria, promotes bacterial adhesion to enterocyte-like Caco-2 cells only in pathogenic species. , 2010, Microbiology.

[44]  Pascale Cossart,et al.  Invasion of mammalian cells by Listeria monocytogenes: functional mimicry to subvert cellular functions. , 2003, Trends in cell biology.

[45]  C. Weber,et al.  The lysine‐ and glutamic acid‐rich protein KERP1 plays a role in Entamoeba histolytica liver abscess pathogenesis , 2007, Cellular microbiology.

[46]  P. Cossart,et al.  Gp96 is a receptor for a novel Listeria monocytogenes virulence factor, Vip, a surface protein , 2005, The EMBO journal.

[47]  S. Rottem,et al.  α-Enolase Resides on the Cell Surface of Mycoplasma fermentans and Binds Plasminogen , 2007, Infection and Immunity.

[48]  J. Wehland,et al.  Structure of Internalin, a Major Invasion Protein of Listeria monocytogenes, in Complex with Its Human Receptor E-Cadherin , 2002, Cell.

[49]  Jamal Stie,et al.  Surface-Associated Plasminogen Binding of Cryptococcus neoformans Promotes Extracellular Matrix Invasion , 2009, PloS one.

[50]  M. Prevost,et al.  A lysine‐ and glutamic acid‐rich protein, KERP1, from Entamoeba histolytica binds to human enterocytes , 2005, Cellular microbiology.

[51]  P. Cossart,et al.  Internalin of Listeria monocytogenes with an intact leucine-rich repeat region is sufficient to promote internalization , 1997, Infection and immunity.

[52]  Arun K Bhunia,et al.  Adhesion characteristics of Listeria adhesion protein (LAP)-expressing Escherichia coli to Caco-2 cells and of recombinant LAP to eukaryotic receptor Hsp60 as examined in a surface plasmon resonance sensor. , 2006, FEMS microbiology letters.

[53]  J. Potempa,et al.  pH-regulated Secretion of a Glyceraldehyde-3-Phosphate Dehydrogenase from Streptococcus gordonii FSS2: Purification, Characterization, and Cloning of the Gene Encoding this Enzyme , 2001, Journal of dental research.

[54]  N. Freitag,et al.  Listeria monocytogenes CtaP is a multifunctional cysteine transport‐associated protein required for bacterial pathogenesis , 2009, Molecular microbiology.

[55]  P. Cossart,et al.  Internalin must be on the bacterial surface to mediate entry of Listeria monocytogenes into epithelial cells , 1996, Molecular microbiology.