Nanobacteria Are Mineralo Fetuin Complexes

Citation: Raoult D, Drancourt M, Azza S, Nappez C, Guieu R, et al. ( Nanobacteria Are Mineralo Fetuin Complexes Didier Raoult Didier.Raoult@medecine 0 1 2 Michel Drancourt 0 1 2 Sad Azza 0 1 2 Claude Nappez 0 1 2 Re gis Guieu 0 1 2 Jean-Marc Rolain 0 1 2 Patrick Fourquet 0 1 2 Bernard Campagna 0 1 2 Bernard La Scola 0 1 2 Jean-Louis Mege 0 1 2 Pascal Mansuelle 0 1 2 Eric Lechevalier 0 1 2 Yvon Berland 0 1 2 Jean-Pierre Gorvel 0 1 2 Patricia Renesto 0 1 2 0 Current address: IGS UPR 2589, Parc Scientifique et Technologique de Luminy , Marseille , France 1 Editor: John A. T. Young, The Salk Institute for Biological Studies , United States of America 2 1 Unite des Rickettsies, Centre National de la Recherche Scientifique UMR 6020, IFR 48, Faculte de Me decine , Marseille , France , 2 Centre National de la Recherche Scientifique FRE2738, Faculte de Me decine Secteur Nord , Marseille , France , 3 Centre d'Immunologie de Marseille-Luminy, Aix Marseille Universite , Faculte de Sciences de Luminy , Marseille, France, 4 INSERM, U631, Marseille , France , 5 Centre National de la Recherche Scientifique UMR 6102, Marseille, France, 6 Service d'Urologie, H opital de la Conception, Assistance Publique-H opitaux de Marseille, France, 7 Service de Nephrologie, Ho pital de la Conception, Assistance Publique-Hopitaux de Marseille , France 3 www.plospathogens.org ''Nanobacteria'' are nanometer-scale spherical and ovoid particles which have spurred one of the biggest controversies in modern microbiology. Their biological nature has been severely challenged by both geologists and microbiologists, with opinions ranging from considering them crystal structures to new life forms. Although the nature of these autonomously replicating particles is still under debate, their role in several calcification-related diseases has been reported. In order to gain better insights on this calciferous agent, we performed a large-scale project, including the analysis of ''nanobacteria'' susceptibility to physical and chemical compounds as well as the comprehensive nucleotide, biochemical, proteomic, and antigenic analysis of these particles. Our results definitively ruled out the existence of ''nanobacteria'' as living organisms and pointed out the paradoxical role of fetuin (an anti-mineralization protein) in the formation of these self-propagating mineral complexes which we propose to call ''nanons.'' The presence of fetuin within renal calculi was also evidenced, suggesting its role as a hydroxyapatite nucleating factor. - Nanobacteria are mysterious particles that have spurred one of the biggest controversies in modern microbiology [1 3]. First discovered by geologists as 100 nm coccoid particles present on mineral surfaces [4], such structures were later found in human and cow blood as well as in commercial cell culture serum [5]. The culturability of nanobacteria was then reported by Kajanders team [6] who established a link between these particles and kidney stone formation [7]. The data described by Cisars group reached completely opposite conclusions as Kajanders original assertion considering nanobacteria as living microorganisms [2]. In contrast to what would be expected from growth of a living entity, Cisar et al. failed to detect nucleic acids and suggested that observed biomineralization may be initiated by non living macromolecules generating self propagating microcrystalline apatite. In the last few years, these calcifying nanoparticles have been associated with several human diseases including polycystic kidney disease, renal calculi, and chronic prostatitis [8]. However, despite the various pathological disorders they cause, whether nanobacteria are living or nonliving cells is still under debate [9]. Here, a comprehensive analysis was undertaken with the Nanobacterium sp. strain Seralab 901045 provided by O. Kajander (Nanobac Oy, Kuopio, Finland), in order to gain better insights on such a propagating calcifying agent putatively endowed with pathogenic properties. To address this question, several features of the nanobacteria previously reported were examined in detail including their propagation conditions, susceptibility to various chemical and physical treatments and their effect on eukaryotic cell viability. Their nucleic acid and proteomic content was also carefully analyzed. The antigenic properties of nanobacteria were investigated by immunization of mice with such particles and the specificity of the response was determined against bacteria from Rickettsia, Coxiella and Bartonella genera. Our data provided evidence that the particles previously attributed to nanobacteria are self-propagating mineralfetuin complexes that we propose to call nanons. Growth of Nanons on Axenic Media After a 10-d incubation in DMEM supplemented with 10% heat-decomplemented fetal calf serum, nanons formed a clearly visible film at the bottom of culture flask. Subculturing was done every 14 d at a 1:10 dilution. Also, serum-free DMEM sustained both growth and subculture of nanons following the same procedure. Under these conditions, the ability to form particles was transferable for 3 to 5 passages. In contrast, nanons did not grow in vitamin-free DMEM. However, addition of any one of the 11 vitamins comprised in this medium restored growth. Growth on Loeffler medium was not successful. Analysis of Nucleotidic Content of Nanons Hoechst 33342 did not stain nanons. In contrast, small fluorescent particles were visible after acridine orange and DAPI staining. Among the 22 PCR assays targeting 16S rRNA In the last decade, the exact nature of nanobacteria was one of the most controversial of scientific questions. An audacious theory proposed the existence of nanobacteria, initially discovered in Italian hot spring deposits, as a new life form responsible for a wide range of diseases in humans, thus qualifying them as new agents of emerging infectious diseases. The community of microbiologists remained therefore skeptical about the fact that such structures, 100 times smaller than bacteria and highly resistant to heat and other treatments that would normally kill the latter, could be living entities fully capable of self-replication. Other scientists wondered if they might be an unusual form of crystal rather than micro-organisms. The comprehensive characterization of nanobacteria was the focus of our study. Our results definitively ruled out the existence of nanobacteria as living entities and revealed that they correspond to self-propagating mineral-fetuin complexes that we called nanons. gene, 8 yielded amplicons. Their DNA sequence was found to be 99% similar to that of Arthrobacter luteolus in 2 cases and 97% similar to that of Bacillus sp. in other ones. Two amplicons exhibited similarities with Stenotrophomonas maltophila (99%) and Pseudomonas sp. (99%), respectively. Mixed sequences were obtained in the remaining 2 cases. All attempts to extract RNA failed (not shown). Susceptibility of (...truncated)