Nanobacteria are mineralo fetuin complexes

Abstract

"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.

Figure 1. Morphological Analysis of Nanons

(A) Transmission electron microscopy of nanons featuring a dense core surrounded by a loose halo (bar = 500 nm). (B) Immunofluorescence staining of nanons with anti-nanon antibodies (1:160). Magnification ×100.

Figure 2. Silver-Stained SDS-PAGE and Western Blot Analysis of Nanon Proteins

(A) Silver staining of 2.5 μg (lane 1) or 5 μg (lane 2) of nanon extract subjected to 10% SDS-PAGE. (B) Western blot performed with two distinct mouse anti-nanon antibodies (1:2,500). Lanes 3 and 5, pre-immune sera; lane 4, mice 1; lane 6, mice 2. Molecular weight markers (MW) are on the left side.

Figure 3. Identification of Nanon Proteins by a Proteomic Approach

(A) MALDI-TOF chromatogram obtained from the tryptic digest of the 65-kDa protein excised from silver-stained gel. (B) Mass and sequences of tryptic peptides mapping to the identified sequence are underlined. This spectrum is representative for 2 distinct experiments.

Figure 4. Comparative SDS-PAGE and Western Blot Analysis of Nanon Proteins and Fetuin

Nanon proteins and bovine fetuin resolved by 10% SDS-PAGE were visualized by silver staining (A) or transferred to nitrocellulose before probing with the mouse anti-bovine fetuin antibodies (1:10,000) (B). Lane 1, nanons (5 μg); lane 2, bovine fetuin (5 μg). Molecular weight markers (MW) are indicated on the left.

Figure 5. EM Analysis of Nanons Following Immunogold Staining

(A) Post-embedding staining of nanons with either the anti-nanon antibodies (1:200, bar 500 nm) or (B) with the anti-fetuin antibodies (1:400, bar 200 nm). (C) Whole mount preparation of nanons stained with anti-fetuin antibodies (1:400, bar 200 nm).

Figure 6. SDS-PAGE and Western Blot Analysis of Proteins Extracted from Human Kidney Stones

50 μl of proteic extracts obtained from human kidney stones of 2 distinct patients were subjected to 10% SDS-PAGE and silver-stained (A). Alternatively, obtained gels were transferred on a nitrocellulose membrane before probing with anti-nanon ([B], 1:2,500) or anti-human fetuin ([C], 1:10,000) antibodies. Molecular weight markers (MW) are on the left side.