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Finding Gluten Peptides Inside Bacteria - Part 2
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Examples of Immunogold Electron Microscopy
The following articles contain micrograpic images of immunogold-labelled
ultrathin sections of bacteria as well as discussions of the methods used
to prepare the specimens. The older articles are stored as PDF files of
scanned pages which take a long time to download. (Paste web addresses
together on one line):
Kruger E, Witt E, Ohlmeier S, Hanschke R, Hecker M. The clp proteases of
Bacillus subtilis are directly involved in degradation of misfolded
proteins. J Bacteriol. 2000 Jun;182(11):3259-65.
http://jb.asm.org/cgi/content/full/182/11/3259?view=full&pmid=10809708
Newman G, Crooke E. DnaA, the initiator of Escherichia coli chromosomal
replication, is located at the cell membrane. J Bacteriol. 2000 May;182
(9):2604-10.
http://jb.asm.org/cgi/content/full/182/9/2604?view=full&pmid=10762265
Walderich B, Holtje JV. Subcellular distribution of the soluble lytic
transglycosylase in Escherichia coli. J Bacteriol. 1991 Sep;173(18):5668-
76.
http://www.pubmedcentral.nih.gov/articlerender.fcgi?
tool=pubmed&pubmedid=1885544
Kroncke KD, Orskov I, Orskov F, Jann B, Jann K. Electron microscopic study
of coexpression of adhesive protein capsules and polysaccharide capsules in
Escherichia coli. Infect Immun. 1990 Aug;58(8):2710-4.
http://www.pubmedcentral.nih.gov/articlerender.fcgi?
tool=pubmed&pubmedid=1973415
Bayer MH, Keck W, Bayer ME. Localization of penicillin-binding protein 1b
in Escherichia coli: immunoelectron microscopy and immunotransfer studies.
J Bacteriol. 1990 Jan;172(1):125-35.
http://www.pubmedcentral.nih.gov/articlerender.fcgi?
tool=pubmed&pubmedid=2403537
Paques M, Teppema JS, Beuvery EC, Abdillahi H, Poolman JT, Verkleij AJ.
Accessibility of gonococcal and meningococcal surface antigens: immunogold
labeling for quantitative electron microscopy. Infect Immun. 1989 Feb;57
(2):582-9.
http://www.pubmedcentral.nih.gov/articlerender.fcgi?
tool=pubmed&pubmedid=2492264
Diaz E, Garcia E, Ascaso C, Mendez E, Lopez R, Garcia JL. Subcellular
localization of the major pneumococcal autolysin: a peculiar mechanism of
secretion in Escherichia coli. J Biol Chem. 1989 Jan 15;264(2):1238-44.
http://www.jbc.org/cgi/reprint/264/2/1238
Acker G, Bitter-Suermann D, Meier-Dieter U, Peters H, Mayer H.
Immunocytochemical localization of enterobacterial common antigen in
Escherichia coli and Yersinia enterocolitica cells. J Bacteriol. 1986
Oct;168(1):348-56.
http://www.pubmedcentral.nih.gov/articlerender.fcgi?
tool=pubmed&pubmedid=3531175
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Microscopy Links:
Advanced Electron Microscopy & Imaging
http://www.hei.org/research/depts/aemi/tec.htm
Optical Microscopy Primer
http://microscopy.fsu.edu/primer/index.html
SPI-Mark Colloidal Gold Conjugates for Immunogold Labelling - SPI Supplies
Introductory articles on gold labelling
http://www.2spi.com/catalog/chem/gold.html
SPI Microscopy Library - Books
http://www.2spi.com/catalog/books/bookmain.html
Tokuyasu KT. A technique for ultracryotomy of cell suspensions and tissues.
J Cell Biol. 1973 May;57(2):551-65.
http://www.jcb.org/cgi/reprint/57/2/551
Keller GA, Tokuyasu KT, Dutton AH, Singer SJ. An improved procedure for
immunoelectron microscopy: ultrathin plastic embedding of immunolabeled
ultrathin frozen sections. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5744-
7.
http://www.pubmedcentral.nih.gov/articlerender.fcgi?
tool=pubmed&pubmedid=6435119
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Dealing with Fecal Samples:
Here are some examples where bacteria from fecal samples are studied,
illustrating techiniques used to separate bacteria from feces:
Wang RF, Cao WW, Cerniglia CE. PCR detection and quantitation of
predominant anaerobic bacteria in human and animal fecal samples. Appl
Environ Microbiol. 1996 Apr;62(4):1242-7.
http://aem.asm.org/cgi/reprint/62/4/1242?view=reprint&pmid=8919784
MacKay WG, Williams CL, McMillan M, Ndip RN, Shepherd AJ, Weaver LT.
Evaluation of protocol using gene capture and PCR for detection of
Helicobacter pylori DNA in feces. J Clin Microbiol. 2003 Oct;41(10):4589-
93.
http://aem.asm.org/cgi/reprint/62/4/1242?view=reprint&pmid=8919784
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An example of incubating feces with an added substance to study bacterial
takeup:
J Agric Food Chem. 2004 May 5;52(9):2689-96.
Human fecal metabolism of soyasaponin I.
Hu J, Zheng YL, Hyde W, Hendrich S, Murphy PA.
Department of Food Science and Human Nutrition, and College of Veterinary
Medicine, Iowa State University, 2312 Food Science Building, Ames, Iowa
50011, USA.
The metabolism of soyasaponin I (3-O-[alpha-L-rhamnopyranosyl-beta-D-
galactopyranosyl-beta-D-glucuronopyranosyl]olean-12-ene-3beta,22beta,24-
triol) by human fecal microorganisms was investigated. Fresh feces were
collected from 15 healthy women and incubated anaerobically with 10 mmol
soyasaponin I/g feces at 37 degrees C for 48 h. The disappearance of
soyasaponin I in this in vitro fermentation system displayed apparent first-
order rate loss kinetics. Two distinct soyasaponin I degradation phenotypes
were observed among the subjects: rapid soyasaponin degraders with a rate
constant k = 0.24 +/- 0.04 h(-)(1) and slow degraders with a k = 0.07 +/-
0.02 h(-)(1). There were no significant differences in the body mass index,
fecal moisture, gut transit time, and soy consumption frequency between the
two soyasaponin degradation phenotypes. Two primary gut microbial
metabolites of soyasaponin I were identified as soyasaponin III (3-O-[beta-
D-galactopyranosyl-beta-D-glucuronopyranosyl]olean-12-ene-3beta,22beta,24-
triol) and soyasapogenol B (olean-12-ene-3beta,22beta,24-triol) by NMR and
electrospray ionized mass spectroscopy. Soyasaponin III appeared within the
first 24 h and disappeared by 48 h. Soyasapogenol B seemed to be the final
metabolic product during the 48 h anaerobic incubation. These results
indicate that dietary soyasaponins can be metabolized by human gut
microorganisms. The sugar moieties of soyasaponins seem to be hydrolyzed
sequentially to yield smaller and more hydrophobic metabolites.
PMID: 15113177 [PubMed - in process]
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Centrifugation Techniques:
Centrifugation
http://ntri.tamuk.edu/centrifuge/centrifugation.html
Percoll
http://www.cellseparation.nu/media/18-1115-69AC.pdf
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Book Review:
I have been considering the possibilities of carrying out an immunogold
electron microscope investigation to find gluten peptides within gut
bacteria myself, but I lack hands-on experience and access to laboratory
facilities. I do note that my former university, about 1.5 hours away, has
lab facilities available for use by NIH funded projects and does have a
transmission electron microscope available with quite a bit of time open on
its scheduling calender. Quite tempting...
I have gone so far as to purchase a couple of books on electron microscopy
which I can recommend to any one interested. These books are:
Electron Microscopy: Principles and Techniques for Biologists
by John J. Bozzola, Lonnie D. Russell
Hardcover: 670 pages ; Dimensions (in inches): 1.50 x 11.25 x 9.00
Publisher: Jones & Bartlett Pub; 2nd edition (January 1999)
List Price: $91.95 (WalMart Price: $83.38)
Principles and Techniques of Electron Microscopy: Biological Applications
by M. A. Hayat
Hardcover: 543 pages ; Dimensions (in inches): 1.08 x 10.32 x 8.33
Publisher: Cambridge University Press; 4th edition (April 15, 2000)
List Price: $110.00 (Amazon Price: $80.30)
There a number of good books on electron microscopy... all expensive.
These 2 stood out. These books complement each other very well, covering
material that neither one alone covers. Bozzola is an excellent
introductory book covering all the basics of both transmission and scanning
electron microscopes. Hayat goes into depth on the chemistry of specimen
preparation, staining, and labelling covering advanced techniques not found
in Bozzola including a full chapter on immunogold labelling. Hayat is also
the editor of the authorative (and expensive) 3 volume set, Colloidal Gold:
Principles, Methods and Applications.
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