1976. virulence factor, released in significant amounts in the presence of glycerol in the growth medium, consists of toxic by-products such as H2O2 formed by l–glycerophosphate oxidase (GlpO), a membrane-located enzyme that is involved in the metabolism of glycerol. When embryonic calf nasal epithelial cells are infected with subsp. SC in the presence of physiological amounts of glycerol, H2O2 is usually released inside the cells prior to cell death. This process Lomitapide can be inhibited with monospecific anti-GlpO antibodies. species represent the smallest self-replicating organisms detected on earth. Their genomes range from the 580 kb of (24) to the 1,358 kb of (40) and serve as a blueprint for the design of synthetic living organisms (18). This prospects to the drastic economizing of genetic resources and to an obligate parasite way of life. Pathogenic species cause mainly atypical pneumonia, urogenital infections, and arthritis in humans and in animals (7, 8, 25). In contrast to other pathogenic bacteria where virulence is mostly determined by toxins, invasins, and cytolysins, pathogenic species appear to have no such typical main virulence Rabbit Polyclonal to MRPS30 factors, as revealed by the genomic sequence analysis of the eight species that have been completely sequenced (17, 24, 26, 28, 32, 35, 40, 48). Although diagnosis of mycoplasmal infections has improved significantly since the introduction of PCR methods, and antigenic variability has been studied in detail in several species (20, 39), there is currently very little knowledge available on the molecular mechanisms and the effectors that allow pathogenic mycoplasmas to cause host cell damage, inflammation, and disease. We have studied subsp. Small Colony (SC), the etiological agent of contagious bovine pleuropneumoniae (CBPP), a severe infectious disease causing major losses of livestock, as a model to investigate the molecular basis of mycoplasmal virulence. subsp. SC is an extracellular pathogen with a genome size of 1 1,211 kb (48) that lives in close association with the host cells. The rationale for the use of this species as a model is the high virulence of this species as well as the fact that it is clearly established as the etiological agent of CBPP. Furthermore, this severe cattle disease is usually of remarkable socioeconomic importance to livestock production in countries that currently suffer CBPP outbreaks (23). In addition, countries that are free of this epidemic are constantly threatened by reemerging infections. Recently, we suggested that subsp. SC might use particular, highly efficient metabolic pathways and the release of harmful intermediates or side products as virulence determinants (46). This hypothesis is based on the observation that highly virulent strains of subsp. SC that cause CBPP in Africa possess a particularly active ATP-binding cassette (ABC) transport system for the assimilation of glycerol, which is usually metabolized to dihydroxyacetone-phosphate (DHAP) with the release of hydrogen peroxide (H2O2), while less virulent strains from recent European outbreaks lacked part of the glycerol uptake genes Lomitapide due to a deletion. These latter consequently produced less H2O2 in the presence of glycerol compared to the highly virulent African strains. It has been proposed that H2O2 production of certain pathogenic species causes lysis of erythrocytes, peroxidation of lipids of infected fibroblasts, and inhibition of ciliary movement in infected tracheal organs (34, 45). Using catalase-deficient mice, H2O2 production of was suggested as a mycoplasmal virulence factor (15). Thus, since subsp. SC, and mycoplasmas in general, are devoid of catalase and dismutase activities as determined by genome sequence analysis (48), targeted release of H2O2 and other accompanying reactive oxygen species (ROS) to host cells may be responsible for inflammation and tissue damage. The current study investigates the function Lomitapide of l–glycerophosphate oxidase (GlpO) and the impact of release of H2O2/ROS, resulting from glycerol metabolism of subsp. SC, on cytotoxicity toward host cells. For this purpose, we have developed an in vitro system using main embryonic calf nasal epithelial cells (ECaNEp cells) to assess mycoplasma-induced cytotoxicity. MATERIALS AND METHODS Strains, cells, growth conditions, and DNA extraction. subsp. SC strain Afad, a highly virulent field strain isolated in 1968 at Farcha Laboratory, NDjamna, Chad, was utilized for the virulence studies unless otherwise noted. This strain causes CBPP under natural and experimental conditions (2). A less virulent strain, subsp. SC strain L2, which lacks the active glycerol uptake system GtsABC (46), has been used where stated specifically. Furthermore, the type strain PG1 and 10 other strains from African and European outbreaks were also used in this study for genetic analyses. Mycoplasmal cultures were produced in mycoplasma broth medium to a density of 108 to 109 CFU/ml or on solid mycoplasma agar medium (Axcell Biotechnologies, St. Genis l’Argentire, France). Growth.
1976