A substitution at His-120 in the LasA protease of Pseudomonas aeruginosa blocks enzymatic activity without affecting propeptide processing or extracellular secretion

被引:42
作者
Gustin, JK
Kessler, E
Ohman, DE
机构
[1] UNIV TENNESSEE,DEPT MICROBIOL & IMMUNOL,MEMPHIS,TN 38163
[2] VET AFFAIRS MED CTR,MEMPHIS,TN 38163
[3] TEL AVIV UNIV,SACKLER FAC MED,SHEBA MED CTR,MAURICE & GABRIELA GOLDSCHLEGER EYE RES INST,IL-52621 TEL HASHOMER,ISRAEL
关键词
D O I
10.1128/jb.178.22.6608-6617.1996
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
The LasA protease of Pseudomonas aeruginosa can degrade elastin and is an important contributor to the pathogenesis of this organism. LasA (20 kDa) is a member of the beta-lytic endopeptidase family of extracellular bacterial proteases, and it shows high-level staphylolytic activity. We sequenced the lasA gene from strain FRD1 and overexpressed it in Escherichia coli. The lasA gene encodes a precursor, known as pre-proLasA, of 45, 582 Da. Amino-terminal sequence analysis allowed the identification of the signal peptidase cleavage site and revealed that the 31-amino-acid signal peptide was removed in E. coli. The remaining proLasA (42 kDa) did not undergo autoproteolytic processing and showed little staphylolytic activity. However, it was readily processed to a 20-kDa active staphylolytic protease by incubation with trypsin or with the culture filtrate of a P. aeruginosa lasA Delta mutant. Thus, removal of the propeptide (22 kDa) was required to convert proLasA into an active protease. Although LasA protease was critical for staphylolytic activity, other proteases like elastase were found to enhance staphylolysis. Under the control of an inducible trc promoter, lasA was overexpressed in P. aeruginosa and the processing intermediates were examined. Compared with wild-type cells, the overproducing cells accumulated more 42-kDa LasA protease. Small amounts of a 25-kDa extracellular LasA-related protein, which could represent a potential processing intermediate, were also observed. To better understand the structure-function relationship in LasA protease, we tested whether His-120-X-His-122 in the mature portion of LasA plays a role in activity. This motif and surrounding sequences are conserved in the related beta-lytic protease of Achromobacter lyticus. Oligonucleotide-directed mutagenesis was used to change His-120 to Ala-120, thus forming the lasA5 allele. The product of lasA5 expressed from the chromosome of P. aeruginosa was processed to a stable, secreted 20-kDa protein (designated LasA-H120A) which was devoid of staphylolytic activity. This suggests that His-120 is essential for LasA activity and favors the possibility that proLasA processing and secretion in P. aeruginosa can proceed via mechanisms which do not involve autoproteolysis.
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页码:6608 / 6617
页数:10
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共 41 条
[1]   MOLECULAR CHARACTERIZATION AND NUCLEOTIDE-SEQUENCE OF THE PSEUDOMONAS-AERUGINOSA ELASTASE STRUCTURAL GENE [J].
BEVER, RA ;
IGLEWSKI, BH .
JOURNAL OF BACTERIOLOGY, 1988, 170 (09) :4309-4314
[2]   EVALUATION OF PSEUDOMONAS-AERUGINOSA EXOTOXIN-A AND ELASTASE AS VIRULENCE FACTORS IN ACUTE LUNG INFECTION [J].
BLACKWOOD, LL ;
STONE, RM ;
IGLEWSKI, BH ;
PENNINGTON, JE .
INFECTION AND IMMUNITY, 1983, 39 (01) :198-201
[3]   SYNTHESIS OF MULTIPLE EXOPRODUCTS IN PSEUDOMONAS-AERUGINOSA IS UNDER THE CONTROL OF RHLR-RHLI, ANOTHER SET OF REGULATORS IN STRAIN PAO1 WITH HOMOLOGY TO THE AUTOINDUCER-RESPONSIVE LUXR-LUXI FAMILY [J].
BRINT, JM ;
OHMAN, DE .
JOURNAL OF BACTERIOLOGY, 1995, 177 (24) :7155-7163
[4]   REVISED NUCLEOTIDE-SEQUENCE OF THE IASA GENE FROM PSEUDOMONAS-AERUGINOSA PAO1 [J].
DARZINS, A ;
PETERS, JE ;
GALLOWAY, DR .
NUCLEIC ACIDS RESEARCH, 1990, 18 (21) :6444-6444
[5]   PSEUDOMONAS-AERUGINOSA ALGG IS A POLYMER LEVEL ALGINATE C5-MANNURONAN EPIMERASE [J].
FRANKLIN, MJ ;
CHITNIS, CE ;
GACESA, P ;
SONESSON, A ;
WHITE, DC ;
OHMAN, DE .
JOURNAL OF BACTERIOLOGY, 1994, 176 (07) :1821-1830
[6]   PSEUDOMONAS-AERUGINOSA LASA GENE - DETERMINATION OF THE TRANSCRIPTION START POINT AND ANALYSIS OF THE PROMOTER REGULATORY REGION [J].
FRECKODONNELL, LC ;
DARZINS, A .
GENE, 1993, 129 (01) :113-117
[7]   STRUCTURAL GENE AND COMPLETE AMINO-ACID SEQUENCE OF PSEUDOMONAS-AERUGINOSA IFO-3455 ELASTASE [J].
FUKUSHIMA, J ;
YAMAMOTO, S ;
MORIHARA, K ;
ATSUMI, Y ;
TAKEUCHI, H ;
KAWAMOTO, S ;
OKUDA, K .
JOURNAL OF BACTERIOLOGY, 1989, 171 (03) :1698-1704
[8]   ACTIVATION OF AN ELASTASE PRECURSOR BY THE LASA GENE-PRODUCT OF PSEUDOMONAS-AERUGINOSA [J].
GOLDBERG, JB ;
OHMAN, DE .
JOURNAL OF BACTERIOLOGY, 1987, 169 (10) :4532-4539
[9]   CLONING AND TRANSCRIPTIONAL REGULATION OF THE ELASTASE LASA GENE IN MUCOID AND NONMUCOID PSEUDOMONAS-AERUGINOSA [J].
GOLDBERG, JB ;
OHMAN, DE .
JOURNAL OF BACTERIOLOGY, 1987, 169 (03) :1349-1351
[10]   CLONING AND EXPRESSION IN PSEUDOMONAS-AERUGINOSA OF A GENE INVOLVED IN THE PRODUCTION OF ALGINATE [J].
GOLDBERG, JB ;
OHMAN, DE .
JOURNAL OF BACTERIOLOGY, 1984, 158 (03) :1115-1121