ABSTRACT
The gram-negative bacterium Pseudomonas aeruginosa secretes many proteins into its extracellular environment via the type I, II, and III secretion systems. In this study, a gene, chiC, coding for an extracellular chitinolytic enzyme, was identified. The chiC gene encodes a polypeptide of 483 amino acid residues, without a typical N-terminal signal sequence. Nevertheless, an N-terminal segment of 11 residues was found to be cleaved off in the secreted protein. The protein shows sequence similarity to the secreted chitinases ChiC of Serratia marcescens, ChiA of Vibrio harveyi, and ChiD of Bacillus circulans and consists of an activity domain and a chitin-binding domain, which are separated by a fibronectin type III domain. ChiC was able to bind and degrade colloidal chitin and was active on the artificial substrates carboxymethyl-chitin-Remazol Brilliant Violet and p-nitrophenyl-beta-D-N,N',N"-triacetylchitotriose, but not on p-nitrophenyl-beta-D-N-acetylglucosamine, indicating that it is an endochitinase. Expression of the chiC gene appears to be regulated by the quorum-sensing system of P. aeruginosa, since this gene was not expressed in a lasIR vsmI mutant. After overnight growth, the majority of the ChiC produced was found intracellularly, whereas only small amounts were detected in the culture medium. However, after several days, the cellular pool of ChiC was largely depleted, and the protein was found in the culture medium. This release could not be ascribed to cell lysis. Since ChiC did not appear to be secreted via any of the known secretion systems, a novel secretion pathway seems to be involved.
Subject(s)
Chitin/metabolism , Chitinases/metabolism , Pseudomonas aeruginosa/enzymology , Amino Acid Sequence , Bacterial Proteins , Cell Compartmentation , Chitinases/genetics , Enzyme Stability , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Enzymologic , Molecular Sequence Data , Protein Transport , Sequence Homology, Amino Acid , Subcellular Fractions/enzymology , Substrate SpecificityABSTRACT
We analyzed a genetic polymorphism of Fc gamma receptor IIIa (CD16) that is present on position 158 (Phe or Val) in the membrane-proximal, IgG-binding domain. With a polymerase chain reaction-based allele-specific restriction analysis assay we genotyped 87 donors and found gene frequencies of 0.57 and 0.43 for Fc gammaRIIIA-158F and -158V, respectively. A clear linkage was observed between the Fc gammaRIIIA-158F and -48L genotypes on the one hand and the Fc gammaRIIIA-158V and -48H or -48R genotypes on the other hand (chi2 test; P < .001). To determine the functional consequences of this Fc gammaRIIIa-158V/F polymorphism, we performed IgG binding experiments with natural killer (NK) cells from genotyped donors. All donors were also typed for the recently described triallelic Fc gammaRIIIa-48L/R/H polymorphism. NK cells were treated with lactic acid to remove cell-associated IgG. Fc gammaRIIIa(NK)-158F bound significantly less IgG1, IgG3, and IgG4 than did Fc gammaRIIIa(NK)-158V, irrespective of the Fc gammaRIIIa-48 phenotype. Moreover, freshly isolated NK cells from Fc gammaRIIIa-158VV individuals carried significantly more cytophilic IgG than did NK cells from Fc gammaRIIIa-158FF individuals. In addition, CD16 monoclonal antibody (MoAb) MEM154 bound more strongly to Fc gammaRIIIa-158V, compared with -158F, again independently of the Fc gammaRIIIa-48 phenotype. The binding of MoAb B73.1 was not influenced by the Fc gammaRIIIa-158V/F polymorphism, but proved to depend solely on the amino acid present at position 48 of Fc gammaRIIIa. In conclusion, the previously reported differences in IgG binding among the three Fc gammaRIIIa-48L/R/H isoforms are a consequence of the linked, biallelic Fc gammaRIIIa-158V/F polymorphism at amino-acid position 158.
