ABSTRACT
Human neutrophil elastase (NE) is a key host defense protease that cleaves virulence factors of Gram-negative bacteria. NE and cathepsin G (CG) are chymotrypsin-like serine proteases with sequence and structural similarities, and both are abundant in neutrophil granules. Unlike NE, CG does not cleave virulence factors of enteric bacteria. Through structure-function analysis, we identified regions in NE that are essential for cleaving Shigella virulence proteins. NE residues at eight different positions were replaced with analogous amino acids in CG or with alanine. Functional analysis of recombinant mutant proteins showed that a single residue at position 98 and multiple amino acid stretches in the three different regions 58A-61, 163-181, and 216-224 determine NE specificity. These NE mutants cleaved the CG-specific, but not the NE-specific, synthetic peptide substrate and did not degrade Shigella virulence factors. Interestingly, exchanging the amino acid at position 98 in CG for the NE equivalent enabled this CG mutant to cleave Shigella virulence factors. Analysis of the NE proteolytic products of the Shigella virulence factor IpaB shows that NE has specific cleavage sites. These results indicate that Shigella virulence factor specificity maps to a distinct region close to NE's active site.
Subject(s)
Amino Acids/metabolism , Leukocyte Elastase/metabolism , Shigella flexneri , Virulence Factors/metabolism , Amino Acid Sequence , Amino Acids/genetics , Amino Acids/physiology , Bacterial Proteins/metabolism , Binding Sites/genetics , Catalytic Domain/genetics , Cathepsin G , Cathepsins/genetics , Cathepsins/metabolism , Enzyme Activation , Humans , Leukocyte Elastase/genetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutant Proteins/chemical synthesis , Mutant Proteins/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Substrate SpecificityABSTRACT
Many pathogens can manipulate macrophages after phagocytosis yet are efficiently killed by neutrophils. This poses the question of whether neutrophils have mechanisms that enable them to specifically recognise pathogens and have pathogens evolved mechanisms to modulate neutrophil function? Here, we review recent work on neutrophils and their interaction with four different bacteria: Staphylococcus aureus, Helicobacter pylori, Anaplasma phagocytophilum and members of the Enterobacteriae family.
