Nanobacteria: An alternative mechanism for pathogenic intra- and extracellular calcification and stone formation

Abstract

Calcium phosphate is deposited in many diseases, but formation mechanisms remain speculative. Nanobacteria are the smallest cell-walled bacteria, only recently discovered in human and cow blood and commercial cell culture serum. In this study, we identified with energydispersive x-ray microanalysis and chemical analysis that all growth phases of nanobacteria produce biogenic apatite on their cell envelope. Fourier transform IR spectroscopy revealed the mineral as carbonate apatite. The biomineralization in cell culture media resulted in biofilms and mineral aggregates closely resembling those found in tissue calcification and kidney stones. In nanobacteria-infected fibroblasts, electron microscopy revealed intra-and extracellular acicular crystal deposits, stainable with von Kossa staining and resembling calcospherules found in pathological calcification. Previous models for stone formation have led to an hypothesis that elevated pH due to urease and͞or alkaline phosphatase activity is a lithogenic factor. Our results indicate that carbonate apatite can be formed without these factors at pH 7.4, at physiological phosphate and calcium concentrations. Nanobacteria can produce apatite in media mimicking tissue f luids and glomerular filtrate and provide a unique model for in vitro studies on calcification.The formation of discrete and organized inorganic crystalline structures within macromolecular extracellular matrices is a widespread biological phenomenon generally referred to as biomineralization. Mammalian bone and dental enamel are examples of biomineralization involving apatite minerals. The molecular basis of mineralization remains largely unknown (1). Recently, bacteria have been implicated as factors in biogeochemical cycles for mineral formation in aqueous sediments (2, 3). The principal constituent of modern authigenic phosphate minerals in marine sediments is carbonate (hydroxy)fluorapatite Ca 10 (PO 4 ) 6-x (CO 3 ) x (F,OH) 2ϩx . Microorganisms are capable of depositing apatite outside thermodynamic equilibrium in sea water. They can segregate Ca from Mg and actively nucleate carbonate apatite by means of specific oligopeptides under conditions pH Ͻ 8.5 and [Mg]:[Ca] Ͼ 0.1 (4). Such conditions are also present in the human body.We have discovered nanobacteria in human and cow blood that are cytotoxic in vitro (5) and in vivo (6). They have been deposited in DSM (no. 5819-5821; Braunschweig, Germany). Nanobacteria possess unusual properties, making their detection difficult with standard microbiological methods. Although they typically had diameters of 0.2-0.5 m, they passed through 0.1-m filters probably because tiny forms (0.05-0.2 m) were also observed in transmission electron microscopy (TEM) (7). Nanobacteria were poorly disruptable, stainable, fixable, and exceptionally resistant to heat (8, 9). Their doubling time was about 3 days. High doses of ␥-irradiation or aminoglycoside antibiotics prevented their multiplication. According to the 16S rRNA gene sequence (EMBL X98418 and X98419), na...

Hierarchical Role of Fetuin-A and Acidic Serum Proteins in the Formation and Stabilization of Calcium Phosphate Particles

“…2B). The former particles were initially described as calcified nanobacteria (41). A recent biochemical analysis, however, challenged the "calcifying nanobacteria theory" in stating that the alleged nanobacteria are actually fetuinmineral complexes (42).…”

“…Furthermore, the CPPs synthesized in vitro may serve as a model for entities present in human pathology like nanoscopic calcifications (41) or colloidal protein-mineral composites from the ascites of a dialysis patient (Fig. 1B) that are similar to secondary CPPs (Fig.…”

Nanobacteria-like calcite single crystals at the surface of the Tataouine meteorite

“…Nanometerscale spherical-, rod-, or ovoid-shaped objects in the 25-to 300-nm range have been considered by many authors as bacteria and named nanobacteria (10). They have been observed in a wide variety of samples: for example, terrestrial carbonates (10,11), sulfides (12), mammalian blood (13), and Martian meteorites (6). They thus could be an important component on Earth (14) and could be related to life on Mars (6).…”

An infectious origin of extraskeletal calcification

“…However, nanobacterial infections do occur in people. An analysis of 30 demineralized kidney or bladder stones with two different monoclonal antibodies revealed nanobacterial antigens in every specimen (2). In some instances, sterile filtered extracts of the stones also grew pure nanobacteria cultures, which when injected intravenously into rabbits, localized preferentially to the kidneys (8).…”

“…In this issue of the Proceedings, Kajander and Çiftçioglu (2) show that a new class of bacteria, designated nanobacteria because of their small size (0.05-0.5 m in diameter), produce sufficient calcium apatite to initiate pathologic calcification and stone formation. The nanobacteria were discovered in white films sticking to the surfaces of tissue culture vessels containing mammalian cells and media supplemented with bovine serum (3).…”