Segmentation in the Borrelia Genome Genetic Implications

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inmacdonald
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Segmentation in the Borrelia Genome Genetic Implications

Postby inmacdonald » Sun 19 Feb 2012 20:19

Bacterial Genetics of Borrelia burgdorferi and related Borrelia Species
is being refined and the complexities of Borrelia genetic gymnastics challenges many
long held textbook based beliefs about the repertoire of Borrelia proteins.
There are problem areas in the arena of Serology "assisted" diagnosis of Lyme borreliosis.

An appreciation of the intrinsic Borrelia Genome in Flux concepts facilitates an improved
respect for the vast numbers of structural variation in Borrelia protein epitopes.
Genome in flux and its consequent Proteins in flux offers a pathway to the appreciation
of the intrinsic shortcomings of Serology methods based on reagents prepared from the
original B31 strain of Borrelia burgdorferi.

I offer some images for your consideration:
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The textbook cartoon of the chromosome and the Plasmids of Borrelia burgdorferi....

[ from Stewart, p., et al, 2005, Plasmid 53:1-13 and reproduced from Chaconas,G., Molecular Microbiology,2005,58(3): 625-35.

Borrelia burgdorferi Genome  Chromosome and Plasmids  from Stewart 2005 Plasmid V53 pp1 13.jpg
Borrelia burgdorferi - Genome elements -Chromosome and 21 Plasmids from Stewart,P. 2005 Plasmid 53:1-3 and reproduced from Chaconas,G., Molecular Microbiology, 2005, 58(3) 625-635.
Borrelia burgdorferi Genome Chromosome and Plasmids from Stewart 2005 Plasmid V53 pp1 13.jpg (35.84 KiB) Viewed 4015 times




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Segmentation of DNA and RNA containing regions within a Borrelia burgdorferi Spirochete strain B31
Acridine orange stain with Calcium Chloride differentiation - 1000x oil immersion magnifiction
DNA regions in yellow and green,RNA regions in Orange/red [scroll the image to see all ]


Borrelia DNA  RNA Distribution in B31 Borrelia burgdorferi Acridine Orange.jpg
Zonation of DNA and RNA within the intact Borrelia burgdorferi spirochete
Borrelia DNA RNA Distribution in B31 Borrelia burgdorferi Acridine Orange.jpg (107.36 KiB) Viewed 4015 times




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Physical segmentation of Borrelia burgdorferi (B31) stained with Oligreen , Epifluorescence, magnification 1000x, oil immersion microscopy (scroll the image to see the segmented spirochete at the bottom)

2 Segmented Borrelia burgdorferi.jpg
segmented and nonsegmented Borrelia burgdorferi (B31) from Log phase culture stained with Oligreen (Invitrogreen) Epifluoresce microscopy ,1000x oild immersion magnification
2 Segmented Borrelia burgdorferi.jpg (53.82 KiB) Viewed 4015 times




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"COPY NUMBER" issues ---1. How many copies of the chromosome of Bb reside within each spirochetal cell?
___2. How many copies of each of the Plasmids reside within each spirochetal cell?

The answser to question 1. from my reading of the current literature is that
16 copies of the chromosome
reside within each Borrelia burgdorferi cell. ( This is in line with 16 chromosomal copies present
within each cell of Borrelia hermsii)
Correction: In the Monograph Borrelia Molecular Biology Host Interactiuns and Pathogenesis Radolf,j, and Samuels D.Scott ...Dr George Chaconas speaks of ONE chromsome per B. burgdorferi bacterium (page 55)

Second Correction and Update on The number of Chromosomes per Borrelia Cell:
Dr. Joseph Hinneusch and Dr. Alan Barbour have a Correction on the "Copy Number" of
Chromosomes per individual borrelia cell as follows;
TEN Chromosome copies per cell for Short borrelia forms ( Usually seen in Primary cultures)
Thirty Chromosome copies per cell for Long borrelia forms ( usually seen in long term cultures)
References for this revision : Dr Hinnenusch's article on Nulceoids of Borrelia published in 1997
Journal of Bacteriology.


The answer to question number 2...from my readiing of the current literature is that there is ONE copy of EACH plasmid per copy of the chromosome of Borrelia burgdorferi. Corrrection: In the Radolf and Samuels monograph Page 55, Dr S. Casjens speaks of copy numbers of 1 -2 per Chromosome.

Second Correction: See above Statement
Note the Ratio of Chromosomes to Plasmids approximately 1::21 remains the same

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Minimal INFECTIVE unit for Borrelia burgdorferi in various hosts.????
There is no data in the literature to answer this question,

Perhaps a borrelia burgdorferi spirochete single genome - chrosome and corresponding complement
of ( 21 times ??) plasmids might enable a single Borrelia burgdorfei spirochete to transmit infection
from vector to host ( or for that matter from infected mouse to feeding nymphal tick ) This is merely speculation on my part.
Please recall that when a single borrelia burgdorferi spirochetes undergoes Segmentation - as in the image
above, ... each segment could potentially contain all of the genetic material ( chromosome and full plasmid complement) as is illustrated in the Genomic cartoon, which is the first image from Dr P. Stewart's paper
in 2005.
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Respectfully submitted,

Alan B. MacDonald, MD
Feb 19,2012

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Last edited by inmacdonald on Mon 7 May 2012 13:02, edited 2 times in total.

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Re: Segmentation in the Borrelia Genome Genetic Implications

Postby X-member » Mon 20 Feb 2012 12:05

Alan B MacDonald MD, you wrote:

Minimal INFECTIVE unit for Borrelia burgdorferi in various hosts.????
There is no data in the literature to answer this question,

Perhaps all 16 chromosomes of a borrelia burgdorferi spirochete and their corresponding complement
of (16 times 21) plasmids might enable a single Borrelia burgdorfei spirochete to transmit infection
from vector to host ( or for that matter from infected mouse to feeding nymphal tick ) This is merely speculation on my part.
Please recall that when a single borrelia burgdorferi spirochetes undergoes Segmentation - as in the image
above, ... each segment could potentially contain all of the genetic material ( chromosome and full plasmid complement) as is illustrated in the Genomic cartoon, which is the first image from Dr P. Stewart's paper
in 2005.


I think that (at least in special circumstances) it could be enough with only one "segment" for a transmission of the infection.

This is very interesting! 8-)

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inmacdonald
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Re: Segmentation in the Borrelia Genome Genetic Implications

Postby inmacdonald » Mon 20 Feb 2012 15:37

Atomic force Microscopy (AFM) - Borrelia burgdorferi- Dr Judith Miklossy_

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Scroll image to view complete

Borrelia burgdorferi  segmentation AFM image  Miklossy.jpg
Borrelia burgorferi (AFM) Atomic force microscopy - Intact Borrelia burgdorferi
Borrelia burgdorferi segmentation AFM image Miklossy.jpg (91.89 KiB) Viewed 3948 times





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Borrelia burgdorf segmentation - AFM Miklossy.jpg
Dr Judith Miklossy - Borrelia burgdorferi - Atomic force microscopy
Borrelia burgdorf segmentation - AFM Miklossy.jpg (50.22 KiB) Viewed 3948 times




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Borrelia burgdorferi zonation pre segmentation AFM Miklossy.jpg
Borrelia burgdorferi - AFM Image - Showing zonation (light /dark) within spiral forms of Borrelia burgdorferi - Dr Judith Miklossy
Borrelia burgdorferi zonation pre segmentation AFM Miklossy.jpg (30.7 KiB) Viewed 3948 times






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MacDonald's comment:(1) Transmission electron microscopy (TEM) demonstrates the cross section
microscopic anatomy of Borrelia. To my knowledge, no images of "nucleoid-like" zonation have ever been
published inside Borrelia spiral forms which would explain the organization of DNA.
Correction: Hinnenbusch and Bendich, 1997 have described filamentous nucleoid regions in
Borrelia whch are structuraly different from the Nucleoids of E. Coli.
Link: http://www.ncbi.nlm.nih.gov/pmc/article ... 792228.pdf
...." Instead of a rosette of loops emanating from a central bright node, the borrelia nucleoid appeared to be composed of a loose network of strands with no obvious central organizing node...."

MacDonald's comment:(2) In Contrast with borrelia spiral (vegetative froms), The work of Dr Oystein Brorson and Dr S. H. Brorson have indeed demonstrated in Cystic forms of Borrelia that electron dense nucleus-like areas (Nucleoid) are present.

[ Mention has been made on this forum that the DNA of Borrelia is attached to the inner cell wall, but I have never seen the publication citation or image data to illustrate this concept[/i].]

The nucleoid has also been demonstrated in Gemmae by Burgdorfer and Hayes.
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Alan B. MacDonald MD
Feb 20, 2012
Last edited by inmacdonald on Tue 21 Feb 2012 20:55, edited 1 time in total.

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Re: Segmentation in the Borrelia Genome Genetic Implications

Postby inmacdonald » Mon 20 Feb 2012 17:37

Borrelia Segmentation - Images from Log phase growth - Borrelia burgdorferi (B31)
Copyright AlanB. MacDonald MD 2006 all rights reserved
Note: Sausage shaped segments leave the parent spiral(vegetative) Borrelia spirochetes
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7 Segmented Borrelia burgdorferi.jpg
Segmented Borrelia Burgdorferi - Log phase growth - B31 strain - Sausage shaped segments from parent Borrelia - Epifluorescent microscopy 1000x magnification - Copyright Alan B MacDonald MD 2006 - all rigts reserved
7 Segmented Borrelia burgdorferi.jpg (38.7 KiB) Viewed 3936 times




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Scroll to see the entire image


4 Segmented Borrelia burgdorferi.jpg
Borrelia burgdorferi (B31) log phase growth - Oligreen stain - Epifluorescent microscopy - 1000x magnification- Copyright Alan B. MacDonald MD 2006- all rights reserved
4 Segmented Borrelia burgdorferi.jpg (32.19 KiB) Viewed 3936 times




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5 segmented Borrelia burgdorferi.jpg
Segmented Borrelia burgdorferi (B31) log phase growth - Oligreen stain- Epifluorescent microscopy- 1000x magnification - Copyright Alan B. MacDonald MD 2006 all rights reserved
5 segmented Borrelia burgdorferi.jpg (37.94 KiB) Viewed 3936 times





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MacDonald comment: I believe that the sausage shaped and rounded shaped separate segments
which are derived from the parent spiral Borrelia spirochetes are vital, viable, and capable of
regenerating spiral vegetative forms.

Alan B. MacDonald MD
Feb 20, 2012

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inmacdonald
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Re: Segmentation in the Borrelia Genome Genetic Implications

Postby inmacdonald » Mon 20 Feb 2012 18:09

Schematic diagram - illustrating the formation of round profiles from Sausage shaped segmnts
shed from Borrelia burgdorferi
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Scroll image to view complete
Sausage to granular transformation Borrelia schematic.jpg
Sausage to Granular interconvrsion - Schematic- Copyright Alan B. MacDonald MD 2012
Sausage to granular transformation Borrelia schematic.jpg (79.85 KiB) Viewed 3932 times



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MacDonald comment: Note that the length of the sausage segments determines the Diameter of the
Rounded segments derived from Borrelia burgdorferi. Not all granular forms have the same diameter.

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For Comparison : Treponema Pallidum - spiral to granular (so called Constellation forms by MacDonald)
Note that the diameter of the rounded granular forms derived from Treponema pallidum is variable.
Illustration from Jacquet and Sezary 1907, Bulletin Soc Med Hosp Paris ..Paris France..
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Jacquet and Sezary composite image.jpg
1907 Treponema pallidum - Jacquet and Sezary
Jacquet and Sezary composite image.jpg (35.25 KiB) Viewed 3932 times



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Jacquet and Sezary 1907.jpg
Treponema Pallidum 1907 granular forms Jacquet and Sezary
Jacquet and Sezary 1907.jpg (22.44 KiB) Viewed 3932 times



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There is image data fro the time of epidemic syphilis, which is informative in the emerging pathobiology
repertoire of Borrelia species.

Respectfully submitted,

Alan B. MacDonald MD
Feb 20,2012

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Re: Segmentation in the Borrelia Genome Genetic Implications

Postby inmacdonald » Mon 20 Feb 2012 19:16

Granular forms of Borrelia burgdorferi in Human Autopsy Brain - Stained with H5332 [monoclonal antobody
specific for the Outer surface protein a (OspA) of Borrelia burgdorferi, and which by definition
from Dr Alan G Barbour ( then of the Rocky Mtn Laboratory, National Institutes of Health, National Institut
of Allergy and Infectious Disease----Now Professor of Medicine and Microbiology , University of California
school of Medicine, Irvine, California -- See Dr Barbour's website "Barbour Lab" for recent information)

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Monoclonal Antibody H5332 stains OspA of Borrelia burdorferi, but does not stain epitopes of
othr Borrelia species. At the time that this photomicrograph - provided "bullet proof" evidence
that the fluorescence originated from elements of Borrelia Burgdorferi .
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Brain Imprint Granular H5332 Monoclonal Murine Antibody to OspA (bba 015) - Copyright 1984 Alan B. MacDonald MD all rights reserved..jpg
Borrelia burgdorferi derived granular segments derived from parent spiral (vegetative form)
as evidenced by binding of MAB H5332. Copyright Alan B. MacDonald, MD, 1984
Brain Imprint Granular H5332 Monoclonal Murine Antibody to OspA (bba 015) - Copyright 1984 Alan B. MacDonald MD all rights reserved..jpg (99.21 KiB) Viewed 3927 times



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MacDonald comment: Note the striking similaritites , almost 77 years apart, between silver stained
derivaives from Treponema pallidum ( line drawings 1907) and
Monoclonal Immunofluorescent staining with H5332 (epifluorescence microscopy 1000x magnification)
demonstrating granular derivatives from Borrelia burgdorferi.

MacDonald comment: The Fingerprint technique was used to obtain this unique image. The technique
is to Press the surface of a cleaned glass slide against the cut surface of FRESH tissue and use
the method that law enforcement personnel utilize to avoid "smearing or smudging" the imprint.
I suspect that Drs Jacquet and Sezary might have used a similar technique in obtaining their
images of granular spirochetes - (probably from direct slide contact with the open weeping lesions
of the Chancre in primary syphilis.)
If one attempts to use a microtome to section a tissue block which might contain a similar clearly demarcated granular profile without any "background noise", it is nearly impossible, because there is only one perfect plane of section in which all of the elements will be visible in a single plane of focus.
So I always recommend the Fingerprint Imprint technique from freshly cut tissues to obtain
perfectly FLAT infinitely Thin views of any spirochetes which might reside in the fresh tissues.
Imprint cytology is an important technique in hospital diagnostic pathology for diagnosis
of neoplastic diseases and infectious diseases.
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Alan B. MacDonald MD
Feb 20, 2012

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Re: Segmentation in the Borrelia Genome Genetic Implications

Postby inmacdonald » Mon 20 Feb 2012 20:35

Dr Judith Miklossy - PhD Thesis
link :http://www.miklossy.ch/media/83fcb06de359f83cffff87f8ffa86321.pdf

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MacDonald comment: There are many treasures to be found in Dr Judith Miklossy's thesis.

My personal favorite part is the Image data ( There it is again- Image Raw data) from
Figure 8, parts of which are reproduced here.
Granular forms of Borrelia turn up INSIDE of Rat Neuronal cells in tissue culture coincubated
with live Borrelia burgdorferi (Figure 8 j--( with Negative control Figure 8 K)

____________________________________________________________________________________________Scroll to see entire image

Figure legend from Dr Miklossy's thesis - Figure 8i, 8j, 8k


Scroll image to see entire field

Miklossy Thesis Fig 8 i,8j, 8k.jpg
Dr Judith Miklossy PhD thesis - Figure 8j- demonstrating OspA granular forms which have penetrated the rat neuronal cell
Miklossy Thesis Fig 8 i,8j, 8k.jpg (70.61 KiB) Viewed 3920 times
Miklossy thesis Fig 8 Legend.jpg
Miklossy, J PhD thesis - Figure legend for Fig 8i, 8j, 8k
Miklossy thesis Fig 8 Legend.jpg (117.59 KiB) Viewed 3920 times



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Comment: In Vitro evidence for:
1. Transformation of spiral Borrelia burgdorferi to granular Borrelia burgdorferi
2. Intracellular residence of OspA positive granular forms of borrelia burgdorferi
within tissue cultured Rat brain cells
3. No Osp A staining in negative control rat brain cells (8K)
Comment :
This is an important example of the viability of granular forms of Borrelia
in a living cell system, (Tissue culture)
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Comment: Bravo! Dr Judith Miklossy!

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Alan B. MacDonald MD
Feb 20, 2011

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Re: Segmentation in the Borrelia Genome Genetic Implications

Postby inmacdonald » Mon 20 Feb 2012 21:04

Miklossy, Judith
2011 Summary Research of Tertiary Neuroborreliosis with Dementa

Link: http://www.google.com/search?q=miklossy ... US470US471

Role of Granular forms of Borrelia in areas oj human brain where histologic evidence of
disease is present. (Partial Content of this comprehensive review article)
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Granular spirochetal forms in granulovacuolar degeneration:
Miklossy diagrams:

Diseased neurons with granular spirochetes in cytoplasm:

Miklossy 2011 Neuron.jpg
Granules in diseased neurons - Miklossy diagram
Miklossy 2011 Neuron.jpg (48.98 KiB) Viewed 3917 times


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Diseased Astroglial cells with granular spirochetes in cytoplasm:

Miklossy 2011 Astrocytes.jpg
Granules in diseased Astroglial cells- Miklossy diageam
Miklossy 2011 Astrocytes.jpg (30.18 KiB) Viewed 3917 times



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Hypothesis
The images speak for an infectious etiology for diseased Neurons and Astroglial cells
( I join with Dr Miklossy in embracing this hypothesis)
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The Category of Granulo-vacuolar degeneration is already established in the Neuropathology literature, but the Etiology of granulovacuolar degeneration is obscure.

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Respectfully submitted,

Alan B. MacDonald MD
Feb 20,2012

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Re: Segmentation in the Borrelia Genome Genetic Implications

Postby inmacdonald » Mon 20 Feb 2012 22:03

Granular spirochetes and their relationship to Granulovacuolar Degeneration ( MacDonald Images and original 1988 Hypothesis)

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GVB  Hypothesis 1986 Concurrent Necortical Borreliosis ..Spirochetal Cyst form- Annals NY Academy of Science- Alan B, MacDonald MD.jpg
Statement - 1988 -MacDonald in Concurrent Neocortical Borreliosis and Alzheimer's Disease - Demonstration of a Spirochetal Cyst form - Annals of the New York Academy of Science
GVB Hypothesis 1986 Concurrent Necortical Borreliosis ..Spirochetal Cyst form- Annals NY Academy of Science- Alan B, MacDonald MD.jpg (34.64 KiB) Viewed 3902 times



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GVB In Situ DNA Hybridization - Alan B. MacDonald MD.jpg
Human Alzheimer Brain Hippocampal Fingerprint imprint - with In Situ DNA Hybridization (Alexa Fluor) for the borrelia burgdorferi Flagellin B Open reading Frame BBO 0147
GVB In Situ DNA Hybridization - Alan B. MacDonald MD.jpg (66.88 KiB) Viewed 3902 times


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Comparison - GVB in Situ DNA Hybridization human nerve - (MacDonald)- GVB like change in Tissue Culture Rat neuron (Miklossy) OspA immunostain.jpg
Comparison Miklossy Image with MacDonald Image - Granulovacuolar degeneration
Comparison - GVB in Situ DNA Hybridization human nerve - (MacDonald)- GVB like change in Tissue Culture Rat neuron (Miklossy) OspA immunostain.jpg (94.73 KiB) Viewed 3902 times



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Respectfully submitted,

Alan B. MacDonald, MD
Last edited by inmacdonald on Thu 23 Feb 2012 22:03, edited 2 times in total.

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Re: Segmentation in the Borrelia Genome Genetic Implications

Postby inmacdonald » Mon 20 Feb 2012 22:14

Images of Granulovacuolar degeneration (human autopsy tissue) , and Granular Change in Borrelia burgdorferi

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GVB in Alzheimer's Disease - Textbook Image - Ellison - Elsevier Publishers.jpg
Textbook Image of Granulovacuolar Degeneration in human autopsy tissue- Ellison and Love- Neuropathology - Elsevier Publishers
GVB in Alzheimer's Disease - Textbook Image - Ellison - Elsevier Publishers.jpg (51.34 KiB) Viewed 3899 times




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Granular change in Borrelia spirochete with attached cell wall deficient form.jpg
Granular change in Borrelia burgdorferi (B31) in long term culture- with attached L form(cell wall deficient form) Alan B. MacDonald MD
Granular change in Borrelia spirochete with attached cell wall deficient form.jpg (43.67 KiB) Viewed 3899 times


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Attachments
Borrelia spirochete undergoing Granular Change - Monoclonal H5332- MacDonald 1984.jpg
Alzheimer brain fresh tissue Fingerprint Imprint - stained with MAB H5t332 for OspA- Alan B. MacDonald MD 1984
Borrelia spirochete undergoing Granular Change - Monoclonal H5332- MacDonald 1984.jpg (38.47 KiB) Viewed 3899 times


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