Defining the Ail Ligand-Binding Surface: Hydrophobic Residues in Two Extracellular Loops Mediate Cell and Extracellular Matrix Binding To Facilitate Yop Delivery.
Yersinia pestis, the causative agent of plague, binds host cells to deliver cytotoxic Yop proteins into the cytoplasm that prevent phagocytosis and generation of proinflammatory cytokines. Ail is an eight-stranded β-barrel outer membrane protein with four extracellular loops that mediates cell binding and resistance to human serum. Following the deletion of each of the four extracellular loops that potentially interact with host cells, the Ail-Δloop 2 and Ail-Δloop 3 mutant proteins had no cell-binding activity while Ail-Δloop 4 maintained cell binding (the Ail-Δloop 1 protein was unstable). Using the codon mutagenesis scheme SWIM (selection without isolation of mutants), we identified individual residues in loops 1, 2, and 3 that contribute to host cell binding. While several residues contributed to the binding of host cells and purified fibronectin and laminin, as well as Yop delivery, three mutations, F80A (loop 2), S128A (loop 3), and F130A (loop 3), produced particularly severe defects in cell binding. Combining these mutations led to an even greater reduction in cell binding and severely impaired Yop delivery with only a slight defect in serum resistance. These findings demonstrate thatY. pestisAil uses multiple extracellular loops to interact with substrates important for adhesion via polyvalent hydrophobic interactions.
Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan, USA.
Tiffany M
TM
Tsang
Department of Biomedical and Diagnostic Sciences, University of Detroit Mercy School of Dentistry, Detroit, Michigan, USA.
Jeffrey S
JS
Wiese
Department of Biomedical and Diagnostic Sciences, University of Detroit Mercy School of Dentistry, Detroit, Michigan, USA.
Jamal A
JA
Alhabeil
Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, Michigan, USA.
Lisa D
LD
Usselman
Department of Biomedical and Diagnostic Sciences, University of Detroit Mercy School of Dentistry, Detroit, Michigan, USA.
Joshua J
JJ
Thomson
Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, Michigan, USA.
Rafla
R
Matti
Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, Michigan, USA.
Malte
M
Kronshage
Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan, USA.
Natalie
N
Maricic
Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, Michigan, USA.
Shanedah
S
Williams
Department of Biomedical and Diagnostic Sciences, University of Detroit Mercy School of Dentistry, Detroit, Michigan, USA.
Naama H
NH
Sleiman
Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, Michigan, USA.
Suleyman
S
Felek
Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan, USA krukones@udmercy.edu.|Department of Biomedical and Diagnostic Sciences, University of Detroit Mercy School of Dentistry, Detroit, Michigan, USA.|Department of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, Michigan, USA.
Eric S
ES
Krukonis
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001422:Bacterial Adhesion; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D001665:Binding Sites; D005109:Extracellular Matrix / Q000378:metabolism; D054884:Host-Pathogen Interactions; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D009154:Mutation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017384:Sequence Deletion; D015010:Yersinia pestis / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D000596:Amino Acids; D001425:Bacterial Outer Membrane Proteins
plague; serum resistance; type III secretion