Mitchell, L

Mitchell, L. one of the most prevalent life-threatening tick-borne zoonoses in North America (2, 3, 6). is an obligate intracellular bacterium in the order have limited biosynthetic capability due to the loss of many genes present in the closely related free-living -during reductive genome development (4). These bacteria therefore cannot survive extracellularly and are obliged to import large varieties of nutrients and metabolic products from their host cells. As gram-negative bacteria, rickettsiae have two lipid bilayer membranes, i.e., inner and outer membranes. In order for small hydrophilic compounds, such as sugars, amino acids, or ions, to pass through the outer membrane, most gram-negative bacteria have special proteins, called porins, around the outer membrane which function as passive diffusion channels (20, 22). Porins also allow the diffusion of antibiotics. Despite the potential importance of porins for rickettsial physiology, SB 242084 hydrochloride pathogenesis, and chemotherapy, the porin activities of these pathogens have not been reported until now. The best-studied outer membrane protein of is usually P44. P44 is the immunodominant, most abundant outer membrane protein and is encoded by a polymorphic multigene family (40). Rabbit Polyclonal to NT Notably, 113 genes are present in the genome, including 22 full-length, 64 silent/reserve (without a start codon), 21 fragmented (made up of only the 5 or 3 conserved region), and 6 truncated (made up of a portion of the hypervariable region and a portion of either the 5 or 3 conserved region) genes (4). There is one major expression site for (gene occurs after the hypervariable region of that gene replaces the gene currently occupying the expression locus by nonreciprocal recombination in a RecF-dependent manner (16, 17). Each expressed gene can be recognized by its signature central hypervariable sequence. Among all of the genes, 65 (71 if duplicated genes in the genome are included) have been found thus far to be expressed in cell culture, HGA patients, experimentally infected mice and horses, or ticks (5, 14-16, 18, 36, 40, 42). Growth of the P44 family is thought to provide a diverse antigenic populace for establishing new infections in regions where is usually endemic. It also allows antigenic variance to persist in immunocompetent mammals to SB 242084 hydrochloride allow effective tick transmission, because cannot be transmitted transovarially in the vector tick. However, the biological functions of P44 for the bacterium have been unknown. Therefore, in the present study, we first examined the pore-forming activity of the isolated outer membrane portion of physiology and pathogenesis. MATERIALS AND METHODS Bacterial strains. strain HZ (29) was propagated in HL-60 cells (American Type Culture Collection, Manassas, VA) in RPMI 1640 SB 242084 hydrochloride medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (U.S. Biotechnologies, Parker Ford, PA) and 2 mM l-glutamine (Invitrogen). Cultures were incubated at 37C in a humidified 5% CO2-95% air flow atmosphere. Outer membrane portion preparation. for 5 min, and both the SB 242084 hydrochloride pellet and the supernatant were saved. The pellet was resuspended in RPMI medium and SB 242084 hydrochloride sonicated twice for 7 s each at setting 2.5, using a W-380 sonicator (Heat Systems, Farmingdale, NY) with a microtip. After sonication, the suspension was centrifuged at 400 for 10 min to remove cell debris and unbroken host cells. The supernatant was combined with the culture supernatant before sonication and exceeded sequentially through syringe-driven 5-m nylon and 2.7-m glass fiber membrane filters (Millipore, Billerica, MA). The filtrate was centrifuged at 10,000 for 10 min at 4C to pellet the organism. The outer membrane portion was obtained from the isolated organism by using 0.1% for 10 min to remove residual Sarkosyl. The pellet was resuspended in 2% for 10 min. The supernatant was utilized for an in vitro proteoliposome swelling assay. The outer membrane portion solubilized with OG was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with GelCode blue according to the manufacturer’s.