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Search for Microbial Signatures within Human and Microbial Calcifications Using Soft X-Ray Spectromicroscopy
  1. Karim Benzerara,
  2. Virginia M. Miller,
  3. Gerard Barell,
  4. Vivek Kumar,
  5. Jennyfer Miot,
  6. Gordon E. Brown Jr,
  7. John C. Lieske
  1. From the Institut de Minéralogie et de Physique des Milieux Condensés, UMR 7590 and Institut de Physique du Globe de Paris (K.B., J.M.), Paris Cedex, France; Surface and Aqueous Geochemistry Group (K.B., G.E.B.), Department of Geological and Environmental Sciences, Stanford University, Stanford, CA; Departments of Surgery and Physiology and Biomedical Engineering (V.M.M.) and Division of Nephrology (G.B., V.K. J.C.L.), Mayo Clinic College of Medicine, Rochester, MN.
  1. We gratefully acknowledge the support of National Science Foundation Grant CHE-0431425 (Stanford Environmental Molecular Science Institute) (G.E.B.), as well as support from the Stanford Institute for the Environment (G.E.B. and K.B.) and the France-Stanford Center for Interdisciplinary Studies (K.B. and G.E.B.). The work at the Advanced Light Source beam line 11.0.2 was supported in part by the Office of Science, Office of Basic Energy Sciences, Division of Materials Sciences, and Division of Chemical Sciences, Geosciences, and Biosciences of the US Department of Energy at Lawrence Berkeley National Laboratory under contract No. DE-AC03-76SF00098. We acknowledge the support of the National Institutes of Health (DK 62021), The Ralph C. Wilson, Sr., and Ralph C. Wilson, Jr. Medical Research Foundation, and the Mayo Clinic and Foundation.
  2. Presented at Experimental Biology 2006, San Francisco, CA, April 1-5, 2006.
  3. Address correspondence to: Dr. Karim Benzerara, IMPMC, Bat 7, 140 Rue de Lourmel, 75015 Paris, France. e-mail: karim.benzerara{at}impmc.jussieu.fr.

Abstract

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 L2,3-edge on a single-cell scale from calcified C. crescentus and R. tataouinensis displayed unique spectral signatures different from that of nonbiologic hydroxyapatite (Ca10(PO4)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.

Key words
  • nanobacteria
  • hydroxyapatite
  • atherosclerosis
  • aneurysm
  • stone formation

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