Benzerara, Karim

The microbiologic nature of self‐replicating, calcifying entity nanoparticles (NPs) isolated from calcified human tissues, including blood vessels and kidney stones is debated because of the difficulty in discriminating minerals precipitated on microbes from minerals nucleated on macromolecules and cell membranes. To begin to resolve this controversy, calcifying bacteria of aquatic origin ( Caulobacter crescentus ) and NPs cultured from human samples were examined using a Scanning Transmission X‐ray Microscope (STXM). The monochromated soft X‐ray beam (150–2000 eV) produced by synchrotron radiation yields microscopic and spectroscopic information on both organic compounds and minerals at the 25 nm scale. High‐spatial and energy resolution Near‐Edge X‐ray Absorption Fine Structure (NEXAFS) spectra indicative of elemental speciation acquired at the C K‐edge and Ca L 2,3 ‐edge on a single‐cell scale from calcified C. crescentus displayed a unique spectral signature different from non‐biologic hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 ). Preliminary NEXAFS measurements of calcium and carbon functional groups of cultured NPs revealed organics associated with hydroxyapatite minerals and in one case, spectroscopic signatures similar to those identified in C. crescentus . Using NEXAFS, it is possible to define a biochemical signature of proteins, polysaccharides, nucleic acids, and hydroxyapatite for cultured calcified bacteria suggesting that NPs isolated from human samples share spectroscopic characteristics with calcifying bacteria.

Background The origin of advanced arterial and renal calcification remains poorly understood. Self-replicating, calcifying entities have been detected and isolated from calcified human tissues, including blood vessels and kidney stones, and are referred to as nanobacteria. However, the microbiologic nature of putative nanobacteria continues to be debated, in part because of the difficulty in discriminating biomineralized microbes from minerals nucleated on anything else (eg, macromolecules, cell membranes). To address this controversy, the use of techniques capable of characterizing the organic and mineral content of these self-replicated structures at the submicrometer scale would be beneficial. Methods Calcifying gram-negative bacteria ( Caulobacter crescentus, Ramlibacter tataouinensis) used as references and self-replicating calcified nanoparticles cultured from human samples of calcified aneurysms were examined using a scanning transmission x-ray microscope (STXM) at the Advanced Light Source at Lawrence Berkeley National Laboratory. This microscope uses a monochromated and focused synchrotron x-ray beam (80-2,200 eV) to yield microscopic and spectroscopic information on both organic compounds and minerals at the 25 nm scale. Results High-spatial and energy resolution near-edge x-ray absorption fine structure (NEXAFS) spectra indicative of elemental speciation acquired at the C K-edge, N K-edge, and Ca L 2,3 -edge on a single-cell scale from calcified C. crescentus and R. tataouinensis displayed unique spectral signatures different from that of nonbiologic hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 ). Further, preliminary NEXAFS measurements of calcium, carbon, and nitrogen functional groups of cultured calcified nanoparticles from humans revealed evidence of organics, likely peptides or proteins, specifically associated with hydroxyapatite minerals. Conclusion Using NEXAFS at the 25 nm spatial scale, it is possible to define a biochemical signature for cultured calcified bacteria, including proteins, polysaccharides, nucleic acids, and hydroxyapatite. These preliminary studies suggest that nanoparticles isolated from human samples share spectroscopic characteristics with calcified proteins.