Four strains of Bordetella bronchiseptica (CSU-P-1, 64-C-0406, 1120-A-83-013, and B205BT) with defined virulence for neonatal swine were examined, and an attempt was made to correlate the presence of certain in vitro phenotypic characteristics with the ability of a particular B. bronchiseptica strain to produce turbinate and lung lesions in piglets. All of the strains except CSU-P-1 colonized the nasal passages of the pigs heavily, and strains 1120-A-83-013 and B205BT produced moderate to severe nasal and lung lesions in experimentally infected piglets. All of the strains attached equally well to porcine tracheal ring explant cultures, and all of the strains except CSU-P-1 produced smooth, hemolytic colonies on Bordet-Gengou medium, agglutinated porcine erythrocytes, and possessed adenylate cyclase activity. Strains 1120-A-83-013 and B205BT produced considerably higher levels of dermonecrotic toxin activity than did strains CSU-P-1 and 64-C-0406. These results indicate that production of nasal and lung lesions in neonatal piglets by the B. bronchiseptica strains tested can be directly correlated with their level of dermonecrotic toxin production.

Lipid flippases of the P4-ATPase family are ATP-driven transporters that translocate lipids from the exoplasmic to the cytosolic leaflet of biological membranes. In the encapsulated fungal pathogen Cryptococcus neoformans, the P4-ATPase Apt1p is an important regulator of polysaccharide secretion and pathogenesis, but its biochemical characterization is lacking. Phylogenetic analysis revealed that Apt1p belongs to the subclade of P4A-ATPases characterized by the common requirement for a β-subunit. Using heterologous expression in S. cerevisiae, we demonstrate that Apt1p forms a heterodimeric complex with the C. neoformans Cdc50 protein. This association is required for both localization and activity of the transporter complex. Lipid flippase activity of the heterodimeric complex was assessed by complementation tests and uptake assays employing fluorescent lipids and revealed a broad substrate specificity, including several phospholipids, the alkylphospholipid miltefosine, and the glycolipids glucosyl- and galactosylceramide. Our results suggest that transbilayer lipid transport in C. neoformans is finely regulated to promote fungal virulence, which reinforces the potential of Apt1p as a target for antifungal drug development.

Reconstitution of membrane proteins into model membranes is an essential approach for their functional analysis under chemically defined conditions. Established model-membrane systems used in ensemble average measurements are limited by sample heterogeneity and insufficient knowledge of lipid and protein content at the single vesicle level, which limits quantitative analysis of vesicle properties and prevents their correlation with protein activity. Here, we describe a versatile total internal reflection fluorescence microscopy-based bleaching protocol that permits parallel analysis of multiple parameters (physical size, tightness, unilamellarity, membrane protein content, and orientation) of individual proteoliposomes prepared with fluorescently tagged membrane proteins and lipid markers. The approach makes use of commercially available fluorophores including the commonly used nitrobenzoxadiazole dye and may be applied to deduce functional molecular characteristics of many types of reconstituted fluorescently tagged membrane proteins.

Lipid flippases of the P4-ATPase family actively transport phospholipids across cell membranes, an activity essential for key cellular processes such as vesicle budding and membrane trafficking. Members of this transporter family have also been implicated in the development of drug resistance in fungi. The encapsulated fungal pathogen Cryptococcus neoformans contains four P4-ATPases, among which Apt2-4p are poorly characterized. Using heterologous expression in the flippase-deficient S. cerevisiae strain dnf1Δdnf2Δdrs2Δ, we tested their lipid flippase activity in comparison to Apt1p using complementation tests and fluorescent lipid uptake assays. Apt2p and Apt3p required the co-expression of the C. neoformans Cdc50 protein for activity. Apt2p/Cdc50p displayed a narrow substrate specificity, limited to phosphatidylethanolamine and –choline. Despite its inability to transport fluorescent lipids, the Apt3p/Cdc50p complex still rescued the cold-sensitive phenotype of dnf1Δdnf2Δdrs2Δ, suggesting a functional role for the flippase in the secretory pathway. Apt4p, the closest homolog to Saccharomyces Neo1p, which does not require a Cdc50 protein, was unable to complement several flippase-deficient mutant phenotypes, neither in the presence nor absence of a β-subunit. These results identify C. neoformans Cdc50 as an essential subunit for Apt1-3p and provide a first insight into the molecular mechanisms underlying their physiological functions.

Reconstitution of membrane proteins into liposomal membranes represents a key technique in enabling functional analysis under well-defined conditions. In this review, we provide a brief introduction to selected methods that have been developed to determine membrane protein orientation after reconstitution in liposomes, including approaches based on proteolytic digestion with proteases, site-specific labeling, fluorescence quenching and activity assays. In addition, we briefly highlight new strategies based on single vesicle analysis to address the problem of sample heterogeneity.

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Reconstitution of membrane proteins into model membranes is an essential approach for their functional analysis under chemically defined conditions. Established model-membrane systems used in ensemble average measurements are limited by sample heterogeneity and insufficient knowledge of lipid and protein content at the single vesicle level, which limits quantitative analysis of vesicle properties and prevents their correlation with protein activity. Here, we describe a versatile total internal reflection fluorescence microscopy-based bleaching protocol that permits parallel analyses of multiple parameters (physical size, tightness, unilamellarity, membrane protein content and orientation) of individual proteoliposomes prepared with fluorescently tagged membrane proteins and lipid markers. The approach makes use of commercially available fluorophores including the commonly used nitrobenzoxadiazole (NBD) dye and may be applied to deduce functional molecular characteristics of many types of reconstituted fluorescently tagged membrane proteins.