Molecular characterization of the 28- and 31-kilodalton subunits of the Legionella pneumophila major outer membrane protein

Abstract

The major outer membrane protein of LegioneHla pneumophila exhibits an apparent molcular mass of 100 kDa. Previous studies revealed the oligomer to be composed of 28-and 31-kDa subunits; the latter subunit is covalently bound to peptidoglycan. These proteins exhibit cross-reactivity with polyclonal anti-31-kDa protein serum. In this study, we present evidence to confirm that the 31-kDa subunit is a 28-kDa subunit containing a bound fragment of peptidoglycan. Peptide maps of purified proteins were generated following cyanogen bromide cleavage or proteolysis with staphylococcal V8 protease. A comparison of the banding patterns resulting from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a common pattern. Selected peptide fragments were sequenced on a gas phase microsequencer, and the sequence was compared with the sequence obtained for the 28-kDa protein. While the amino terminus of the 31-kDa protein was blocked, peptide fragments generated by cyanogen bromide treatment exhibited a sequence identical to that of the amino terminus of the 28-kDa protein, but beginning at amino acid four (glycine), which is preceded by methionine at the third position. This sequence, (Gly-Thr-Met)-Gly-Pro-Val-Trp-ThrP Gly-Asn .... confirms that these proteins have a common amino terminus. An oligonucleotide synthesized from the codons of the common N-terminal amino acid sequence was used to establish by Southern and Northern (RNA) blot analyses that a single gene coded for both proteins. With regard to the putative porin stucture, we have identified two major bands at 70 kDa and at approximately 120 kDa by nonreducing SDS-PAGE. The former may represent the typical trimeric motif, while the latter may represent either a double trimer or an aggregate. Analysis of these two forms by two-dimensional SDS-PAGE (first dimension, nonreducing; second dimension, reducing) established that both were composed of 31-and 28-kDa subunits cross-linked via interchain disulfide bonds. These studies confirm that the novel L. pneumophila major outer membrane protein is covalently bound to peptidoglycan via a modified 28-kDa subunit (31-kDa anchor protein) and cross-linked to other 28-kDa subunits via interchain disulfide bonds.

Cloning and nucleotide sequence of a gene (ompS) encoding the major outer membrane protein of Legionella pneumophila

“…This subunit is covalently crosslinked to 28-kDa subunits via interchain disulfide bonds (5,27). Progress in assessing the role of this protein in pathogenesis as well as in characterizing the novel structure of this putative porin has been hindered by an inability to clone the structural gene.…”

“…We found that both subunits had a common N-terminal amino acid sequence (27). On the basis of this sequence (GTMGPVWT), a series of oligonucleotides were generated, one of which hybridized to a single site of restricted genomic DNA, as judged by Southern blot analysis.…”

“…Preparation of genomic DNA from these strains has been described previously (24). Purification of the 31-and 28-kDa proteins, peptide maps, and peptide sequencing were as described elsewhere (5,27). Oligonucleotides used in this study were synthesized at the Molecular Resource Center, Department of Microbiology and Immunology, University of Tennessee, Memphis, and at the Molecular Gene Probe Laboratory, Dalhousie University.…”

“…MATERIALS AND METHODS Bacterial strains, protein sequence, and oligonucleotide reagents. A streptomycin-resistant strain (SVir) of L. pneumophila Philadelphia 1 (serogroup 1) was used as the source of the 28-and 31-kDa proteins and chromosomal DNA as described elsewhere (24,27). Other serogroups of L. pneumophila and various Legionella species used in this study included L. pneumophila serogroups 1, 2, 3, 4, 5, and 7; L. micdadei (HEBA and Tatlock); L. jordanis serogroup 1; and L. oakridgensis OR10.…”

Automated Immunomagnetic Processing and Separation of Legionella Pneumophila with Manual Detection by Sandwich ELISA and PCR Amplification of the ompS Gene

“…For PCR assay, we use the gene of the major outer membrane protein (ompS), which encodes for a surface protein composed of two subunits (28 and 31 kDa). 16,17 It was shown that the ompS DNA sequence is highly conserved among the different serogroups of L. pneumophila. This makes the gene a perfect target for detection of this pathogen.…”