Rapid microprocedure for isolating detergent-insoluble outer membrane proteins from Haemophilus species

Carlone, George M.; Thomas, Matthew L.; Rumschlag, H S; Sottnek, F O

doi:10.1128/jcm.24.3.330-332.1986

Cited by 198 publications

(85 citation statements)

References 22 publications

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“…Female Balb/c mice were immunized with His-FsaP in order to generate anti-FsaP antibodies. Sarkosyl-insoluble outer membrane proteins were extracted from plate-grown bacteria by a rapid procedure described elsewhere (Carlone et al, 1986). Whole-cell preparations and Western blot experiments were performed as reported by others (Timpe et al, 2003).…”

Section: Antibodies Protein Preparation and Analysismentioning

confidence: 99%

Identification of aFrancisella tularensisLVS outer membrane protein that confers adherence to A549 human lung cells

Melillo

Sledjeski

Lipski

et al. 2006

FEMS Microbiology Letters

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Francisella tularensis is a highly pathogenic bacterium; however, little is known about its initial interactions with mucosal surfaces of the human respiratory tract. To investigate these interactions, we tested whether two Francisella strains could adhere to A549 human lung epithelial cells. We found that LVS adhered well to these cells while Francisella novicida adhered poorly. We used surface biotinylation to identify bacterial proteins that might mediate this adherence. We report the identification of the F. tularensis surface protein FsaP, which, when expressed in nonadherent Escherichia coli, confers recombinant bacteria with the ability to bind to A549 cells.

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Section: Antibodies Protein Preparation and Analysismentioning

confidence: 99%

Identification of aFrancisella tularensisLVS outer membrane protein that confers adherence to A549 human lung cells

Melillo

Sledjeski

Lipski

et al. 2006

FEMS Microbiology Letters

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“…Detergent-insoluble outer membrane proteins were prepared from Haemophilus strains by a method based on that of Carlone et al (1986). Bacteria from one Petri dish were resuspended in 1 ml of PBSB containing a protease inhibitor cocktail [PIC; 1 mM phenylmethylsulphonyl fluoride (PMSF), 1 mM E-64, 1 mM pepstatin A, 6 nM bestatin and 100 mM EDTA] and sonicated (six bursts, 10 s at 50% maximum output) with a W-375 model sonicator (Heat Systems-Ultrasonics).…”

Section: Preparation Of Detergent-insoluble Haemophilus Outer Membranmentioning

confidence: 99%

The variable P5 proteins of typeable and non‐typeable Haemophilus influenzae target human CEACAM1

Hill

Toleman

Evans

et al. 2001

Molecular Microbiology

105

115

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SummaryHaemophilus influenzae, a commensal of the human respiratory mucosa, is an important cause of localized and systemic infections. We have recently shown that numerous strains of capsulate (typeable) and acapsulate (non-typeable) H. influenzae target the carcinoembryonic antigen (CEA) family of cell adhesion molecules (CEACAMs). Moreover, the ligands appeared to be antigenically variable and, when using viable typeable bacteria, their adhesive functions were inhibited by the presence of capsule. In this report, we show that the antigenically variable outer membrane protein, P5, expressed by typeable and non-typeable H. influenzae targets human CEA-CAM1. Variants and mutants lacking the expression of P5 of all strains tested were unable to target purified soluble receptors. A non-typeable strain that did not interact with CEACAM1 was made adherent to both the soluble receptors and CEACAM1-transfected Chinese hamster ovary cells by transformation with the P5 gene derived from the adherent typeable strain Rd. However, several H. influenzae mutants lacking P5 expression continued to bind the cell-bound CEACAM1 receptors. These observations suggest that (i) CEACAM1 alone can support P5 interactions and (ii) some strains contain additional ligands with the property to target CEACAM1 but require the receptor in the cellular context. The identification of a common ligand in diverse strains of H. influenzae and the presence of multiple ligands for the same receptor suggests that targeting of members of the CEACAM family of receptors may be of primary significance in colonization and pathogenesis of H. influenzae strains.

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“…Strains were grown to an optical density at 600 nm of 0.8. Sarcosyl-insoluble outer membrane proteins were isolated by the method of Carlone et al (1986), and secreted proteins were precipitated with 10% trichloroacetic acid as described previously . Outer membrane fractions were resuspended in 12.5 ml of 10 mM HEPES, pH 7.4, plus 12 l of 2 × Laemmli buffer, while precipitated extracellular proteins were resuspended in 10 ml of 1 M Tris, pH 9.0, plus 10 l of 2 × Laemmli buffer.…”

Section: Analysis Of Secreted Proteins and Outer Membrane Proteinsmentioning

confidence: 99%

Structural determinants of processing and secretion of the Haemophilus influenzae Hap protein

Hendrixson

Morena

Stathopoulos

et al. 1997

Molecular Microbiology

102

138

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SummaryHaemophilus influenzae elaborates a surface protein called Hap, which is associated with the capacity for intimate interaction with cultured epithelial cells. Expression of hap results in the production of three protein species: outer membrane proteins of approximately 155 kDa and 45 kDa and an extracellular protein of approximately 110 kDa. The 155 kDa protein corresponds to full-length mature Hap (without the signal sequence), and the 110 kDa extracellular protein represents the N-terminal portion of mature Hap (designated Hap s ). In the present study, we examined the mechanism of processing and secretion of Hap. Site-directed mutagenesis suggested that Hap is a serine protease that undergoes autoproteolytic cleavage to generate the 110 kDa extracellular protein and the 45 kDa outer membrane protein. Biochemical analysis confirmed this conclusion and established that cleavage occurs on the bacterial cell surface. Determination of N-terminal amino acid sequence and mutagenesis studies revealed that the 45 kDa protein corresponds to the Cterminal portion of Hap, starting at N1037. Analysis of the secondary structure of this protein (designated Hap ␤ ) predicted formation of a ␤-barrel with an N-terminal transmembrane ␣-helix followed by 14 transmembrane ␤-strands. Additional analysis revealed that the final ␤-strand contains an amino acid motif common to other ␤-barrel outer membrane proteins. Upon deletion of this entire C-terminal consensus motif, Hap could no longer be detected in the outer membrane, and secretion of Hap s was abolished. Deletion or complete alteration of the final three amino acid residues had a similar but less dramatic effect, suggesting that this terminal tripeptide is particularly important for outer membrane localization and/or stability of the protein. In contrast, isolated point mutations that disrupted the amphipathic nature of the consensus motif or eliminated the C-terminal tryptophan had no effect on outer membrane localization of Hap or secretion of Hap s . These results provide insight into a growing family of Gram-negative bacterial exoproteins that are secreted by an IgA1 protease-like mechanism; in addition, they contribute to a better understanding of the structural determinants of targeting of ␤-barrel proteins to the bacterial outer membrane.

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Rapid microprocedure for isolating detergent-insoluble outer membrane proteins from Haemophilus species

Cited by 198 publications

References 22 publications

Identification of aFrancisella tularensisLVS outer membrane protein that confers adherence to A549 human lung cells

Identification of aFrancisella tularensisLVS outer membrane protein that confers adherence to A549 human lung cells

The variable P5 proteins of typeable and non‐typeable Haemophilus influenzae target human CEACAM1

Structural determinants of processing and secretion of the Haemophilus influenzae Hap protein

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