Proteomic analysis of the sarcosine‐insoluble outer membrane fraction of the bacterial pathogen Bartonella henselae

Abstract

Bartonella henselae is an emerging zoonotic pathogen causing a wide range of disease manifestations in humans. In this study, we report on the analysis of the sarcosine-insoluble outer membrane fraction of B. henselae ATCC 49882 Houston-1 by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1-D SDS-PAGE) and two-dimensional nonequilibrium pH gradient polyacrylamide gel electrophoresis (2-D NEPHGE). Protein species were identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and subsequent database query against the B. henselae genome sequence. Subcellular fractionation, application of the ionic detergent lauryl sarcosine, assessment of trypsin sensitivity, and heat modifiability of surface-exposed proteins represented valuable tools for the analysis of the outer membrane subproteome of B. henselae. 2-D NEPHGE was applied to display and catalogue a substantial number of proteins associated with the B. henselae sarcosine-insoluble outer membrane fraction, resulting in the establishment of a first 2-D reference map of this compartment. Thus, 53 distinct protein species associated with the outer membrane subproteome fraction were identified. This study provides novel insights into the membrane biology and the associated putative virulence factors of this pathogen of increasing medical importance.

Directed shotgun proteomics guided by saturated RNA-seq identifies a complete expressed prokaryotic proteome

“…We chose B. henselae as a model system for several reasons: (1) Its relatively small genome (1.93 Mbp) comprises 1488 predicted protein-coding genes (Alsmark et al 2004); (2) it is a facultative intracellular pathogen that can be grown in pure culture; (3) protocols for subcellular fractionation have been described (Rhomberg et al 2004); and (4) in vitro conditions that mimic the pH-dependent induction of virulence genes required for the successful interaction with host endothelial cells, the likely primary niche for B. henselae (Harms and Dehio 2012), have been established (Quebatte et al 2010). The availability of a model system that eliminates the need for coculture with human endothelial cells is critical to achieve complete coverage of an expressed proteome.…”

“…Second, our expressed proteome encompassed all proteins identified in three previous B. henselae proteomics studies (Rhomberg et al 2004;Eberhardt et al 2009;Li et al 2011), while adding many more low-abundance proteins (Supplemental Fig. S6A-C).…”

“…MQB307 was grown on Columbia blood agar (CBA) plates supplemented with 30 mg/L kanamycin with (induced condition) or without (uninduced condition) 500 mM IPTG at 35°C and 5% CO 2 for 60 h. The subcellular fractionation was performed as previously described (Rhomberg et al 2004;Supplemental Methods). To maximize the recovery of membrane proteins, the total membrane fraction (TM) was further separated into inner membrane (IM) and outer membrane (OM) fraction.…”

Comparative Proteome Analysis of the Outer Membrane Proteins of in Vitro‐Induced Multi‐Drug Resistant Neisseria gonorrhoeae

“…This is because whole-genome sequencing of gonococci was completed very recently and a 1175 COMPARATIVE PROTEOME ANALYSIS OF N. GONORRHOEAE OMP (18). Recent improvements in the solubilization and separation of membrane fractions from gram-negative bacteria allow excellent resolution of the OMPs, and many different methods have been tried to solubilize the OMPs optimally according to species (9,10,15,16). Detergents have been used to mimic a lipid-like environment on the inside of the micelles, but the quality of this simulation is highly variable from one detergent to another, so that solubilization performance also varied widely.…”

“…For OMP extraction we used a modified Sarkosyl method (9,10,15,16). Mutants and the parent strain were grown on the chocolate agar at 37 C, 5% CO 2 for 18 hr, then harvested and suspended in 50 mM Tris-HCl buffer (pH 7.4).…”