Disulfide-bonded outer membrane proteins in the genus Legionella

Abstract

Legionella pneumophila and related species were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for outer membrane proteins. Of the 10 species examined, 9 contained a 24-kilodalton (kDa) major outer membrane protein (MOMP) that was resolvable only when outer membrane material was heated in the presence of 2-mercaptoethanol. Labeling studies with [35S]cysteine indicated that the protein contained cysteine, and disulfide cross-linking of the unreduced complex was demonstrated by labeling with iodoacetamide. The unreduced outer membrane preparation contained peptidoglycan, and after treatment with lysozyme to remove peptidoglycan, a protein complex of 95 kDa was observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the absence of 2-mercaptoethanol. Reduction of the 95-kDa complex yielded 24-kDa monomers, suggesting that the 95-kDa complex was composed of four subunits. The 24-kDa MOMP from L. pneumophila was purified, and antibody produced to this protein cross-reacted with all species of Legionella as determined from an immunoblot of a sodium dodecyl sulfate gel. Only serogroup 1 strains of L. bozemanii lacked the 24-kDa MOMP and showed no cross-reactivity. These results suggest that the 24-kDa MOMP common to most species of Legionella contains a genus-specific epitope.

Amoeba host‐Legionella synchronization of amino acid auxotrophy and its role in bacterial adaptation and pathogenic evolution

“…Cysteine is an absolute requirement for L. pneumophila to grow in vitro, and rich media must be supplemented with ∼3 mM cysteine (Carlsson et al, 1979;Hoffman et al, 1983). Legionella pneumophila metabolizes both serine and cysteine to pyruvate then to acetyl coenzyme A to feed the tricarboxylic acid cycle (TCA), which is the main metabolic pathway for energy generation in L. pneumophila (Pine et al, 1979;Warren and Miller, 1979;Hoffman and Pine, 1982;Keen and Hoffman, 1984;Butler et al, 1985;Eylert et al, 2010).…”

DsbA2 (27 kDa Com1‐like protein) of Legionella pneumophila catalyses extracytoplasmic disulphide‐bond formation in proteins including the Dot/Icm type IV secretion system

“…In silico analysis of the primary amino acid sequence of DsbA2 identified a sec-dependent leader sequence typical of DsbA proteins (PSORTb v3.0) (Yu et al, 2010) and predicted with high confidence a periplasmic location (score of 9.84 and similar to that predicted for DsbA1). To confirm a periplasmic location, whole-cell extracts were subjected to cellular fractionation protocols routinely used to localize proteins in L. pneumophila (Butler et al, 1985;Lema et al, 1988;Matthews and Roy, 2000;Vincent et al, 2006). To identify DsbA2 in these fractions, monospecific mouse polyclonal antibodies raised against purified His 6-tagged DsbA2 protein (no cross-reactivity with DsbA1) was used.…”

“…To identify DsbA2 in these fractions, monospecific mouse polyclonal antibodies raised against purified His 6-tagged DsbA2 protein (no cross-reactivity with DsbA1) was used. As a control, antibodies against periplasmic protein LepB, cytosolic protein IHF and outer membrane protein OmpS were used (Butler et al, 1985;Vincent et al, 2006;Morash et al, 2009). As depicted in Fig.…”

“…Subcellular localization of DsbA2 protein was conducted using L. pneumophila harvested from 50 ml of cultures that were grown to stationary phase in BYE broth at 37°C. Bacterial cells were disrupted by sonication and lysates fractionated into soluble and membrane-associated proteins by ultracentrifugation at 100 000 g (Butler et al, 1985;Vincent et al, 2006). Total membrane fractions were treated with 1% Triton X-100 and the suspension subjected to ultracentrifugation to separate the soluble fraction (inner membrane and periplasmic proteins) from the outer membrane pellet.…”

Role of the 25 kDa major outer membrane protein ofLegionella pneumophilain attachment to U‐937 cells and its potential as a virulence factor for chick embryos

“…pneumophila and Legionella species, some Leg. pneumophila serogroups do contain a single major outer membrane protein (MOMP) that ranges in mass between 24 and 29 kDa (Ehret et al 1984 ;Butler et al 1985 ;Hindahl and Iglewski 1986). Butler and Hoffman (1990) have also reported the presence of 28 and 31 kDa outer membrane proteins (OMP) in Leg.…”