Search for microbial signatures within human and microbial calcifications using soft x-ray spectromicroscopy

Karim Benzerara, Virginia M Miller, Gerard Barell, Vivek Kumar, Jennyfer Miot, Gordon E. Brown, John C Lieske

Research output: Contribution to journalArticle

38 Citations (Scopus)

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 (Ca 10(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.

Original languageEnglish (US)
Pages (from-to)367-379
Number of pages13
JournalJournal of Investigative Medicine
Volume54
Issue number7
DOIs
StatePublished - Nov 2006

Fingerprint

X-Rays
Minerals
Calcifying Nanoparticles
X rays
Durapatite
Caulobacter crescentus
Nanoparticles
Bacteria
Microscopes
Organic minerals
Synchrotrons
Proteins
Kidney Calculi
Blood vessels
Cell membranes
Gram-Negative Bacteria
Macromolecules
Organic compounds
Nucleic Acids
Functional groups

Keywords

  • Aneurysm
  • Atherosclerosis
  • Hydroxyapatite
  • Nanobacteria
  • Stone formation

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Search for microbial signatures within human and microbial calcifications using soft x-ray spectromicroscopy. / Benzerara, Karim; Miller, Virginia M; Barell, Gerard; Kumar, Vivek; Miot, Jennyfer; Brown, Gordon E.; Lieske, John C.

In: Journal of Investigative Medicine, Vol. 54, No. 7, 11.2006, p. 367-379.

Research output: Contribution to journalArticle

Benzerara, Karim ; Miller, Virginia M ; Barell, Gerard ; Kumar, Vivek ; Miot, Jennyfer ; Brown, Gordon E. ; Lieske, John C. / Search for microbial signatures within human and microbial calcifications using soft x-ray spectromicroscopy. In: Journal of Investigative Medicine. 2006 ; Vol. 54, No. 7. pp. 367-379.
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AU - Miot, Jennyfer

AU - Brown, Gordon E.

AU - Lieske, John C

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AB - 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 (Ca 10(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.

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