Topic: Osp C...(outer surface protein C....for newbies) and flagellin
Marnie
Frequent Contributor (5K+ posts)
Member # 773
posted
In immunocompetent nonhuman primates, spirochetes are present in low numbers in tissues. For this reason, it has been difficult to study their localization and changes in expression of surface proteins.
To further investigate this, we inoculated four immunosuppressed adult Macaca mulatta with 1 million spirochetes of the N40 strain of B. burgdorferi, and compared them with three infected immunocompetent animals and two uninfected controls.
The brain, spinal cord, peripheral nerves, skeletal muscle, heart, and bladder were obtained at necropsy 4 months later.
The spirochetal tissue load was first studied by polymerase chain reaction (PCR)-ELISA of the outer surface protein A (ospA) gene.
Immunohistochemistry was used to study the localization and numbers of spirochetes in tissues and the expression of spirochetal proteins and to characterize the inflammatory response.
Hematoxylin and eosin and trichrome stains were used to study inflammation and tissue injury.
The results showed that the number of spirochetes was significantly higher in immunosuppressed animals .
B. burgdorferi in the CNS localized to the leptomeninges, nerve roots, and dorsal root ganglia, but not to the parenchyma.
Outside of the CNS, B. burgdorferi localized to endoneurium and to connective tissues of peripheral nerves, skeletal muscle, heart, aorta, and bladder.
Although ospA, ospB, ospC, and flagellin were present at the time of inoculation,
***only flagellin was expressed by spirochetes in tissues 4 months later.***
Significant inflammation occurred only in the heart, and only immunosuppressed animals had cardiac fiber degeneration and necrosis. Plasma cells were abundant in inflammatory foci of steroid-treated animals.
We concluded that B. burgdorferi has a tropism for the meninges in the CNS and for connective tissues elsewhere in the body.
Flagellin is a protein that forms the flagella, or long tail, in bacterial flagellum. Due to its natural molecular structure, flagellin arranges itself into a hollow cylinder in a helical shape , which is essential to its function.
In flagellated bacteria, flagellin is one of the most common proteins in the cell. The flagella in a bacterium moves in order to propel the cell forward. It can do this because of certain unique properties of flagellin.
Flagellin forms into tubules to create a flagella, and a cross-section of the tubules shows that it looks a little like a spoked wheel.
The "spokes," actually a short protein called dinein, react to ATP in such a way that they force the sides of the tubule to expand and contract - which results in the wiggling thrash of the flagella.
Mammals tend to have a strong immune system response to flagellin since it's one of the most common proteins in some disease-causing bacteria .
In mammals, there is a specific T-cell receptor for flagellin. Bacteria, of course, respond by trying to overcome the response; some can switch between different flagellin genes to try to trick mammalian immune cells.
***Flagellin acting via TLR5 is the major activator of key signaling pathways leading to NF-κB and proinflammatory gene program activation in intestinal epithelial cells.***
We explored the mechanism by which Salmonella activates NF-κB during infection of cultured intestinal epithelial cells and found that flagellin produced by the bacteria and contained on them leads to NF-κB activation in all the cells ; invasion of cells by the bacteria is not required to activate NF-κB ."
"analysis of flagellins obtained by glycine extraction by Western immunoblot using a polyclonal anti-flagellin antibody."
"Glycine has a stabilizing effect on IgM"
Where does "lowly" glycine come from? Collagen.
``Polyclonal IgM is critical for clearance
To determine whether this IgM directly kills bacteria--a possibility that goes against current dogma in the field--or mediates its effects through complement C1q protein-induced phagocytosis, Connolly repeated infection experiments in mice lacking functional C1q.
C1q was not critical to IgM activity.
Finally, the team isolated monoclonal IgM from an infected animal and added it to an in vitro culture of spirochetes.
The classical bactericidal activity was observed, including formation of membrane blebs, exposure of periplasmic flagella, and stripping of outer membranes.
Based on these experiments, Connolly and Benach concluded that IgM was necessary and sufficient for clearance of the spirochetes .
Exactly how the antibody induces cell death remains unclear, but other researchers in Benach's lab are pursuing the question.''
IgM antibodies recognizing a glycine-alanine epitope are induced during acute infection with Epstein-Barr virus and cytomegalovirus.
IgM is the class of antibodies found in circulating body fluids.
IgM antibodies are the first antibodies to appear in response to an initial exposure to an antigen."
"in OspA (outer surface protein A), isoleucine and alanine,
and in OspB (outer surface protein B), isoleucine and glycine,
has been found around the peptidase cleavage site of the signal peptide. This is contemplated to apply for other outer surface proteins of B. burgdorferi as well."
Above...difference between OspA and B is alanine (plus isoleucine) instead of glycine (plus isoleucine).
***SEE the difference between glycine and alanine here:
You will see CH3 instead of Hydrogen. Alanine (in OspA) has CH3.
CH3 - methyl?
To determine the structure of OspC, the researchers used a technique at Brookhaven's National Synchrotron Light Source (NSLS) known as multiple wavelength anomalous diffraction.
First, researchers grew crystals of the protein that could withstand the intense x-rays at the NSLS. To make large quantities of OspC, the team used the T7 gene-expression system, which was developed at Brookhaven.
Then the crystal was illuminated with beams of x-rays at different energies, and diffraction patterns were recorded on a detector. With the aid of powerful computers, the researchers then analyzed the diffraction patterns to gain the vital information needed to create an image of the protein structure.
John Dunn, a member of the research team from Brookhaven, explains that
the structure of OspC is predominantly helical, and very different from OspA (alanine + isoleucine), which is flat.
Also, a region on the surface of OspC has a strong negative charge.
Dunn says the negatively charged region may be attracted to a positively charged site on the surface of human cells, helping the bacterium to cause infection.
This feature is only found in the OspC protein derived from bacterial strains that cause human disease.
The scientists believe that a vaccine based on OspC will be more effective than the current OspA-based vaccine because the OspA protein is only present in the bacteria while they are in the cold-blooded deer tick's stomach, and not in the host.
After the tick bites the warm-blooded mammalian host, the injected bacteria produce OspC in the host's bloodstream.
When the host is vaccinated solely with OspA, antibodies to this protein can only kill the bacterium inside the tick if it ingests these antibodies with its blood-meal .
If the bacterium finds its way into the host, it changes into several other forms for which the vaccine offers no protection.
In contrast, an OspC-based vaccine would enable the host to make antibodies to kill the Lyme disease bacteria within the host's body.
Another member of the Brookhaven team, Subramanyam Swaminathan, added, "In order to develop an effective OspC-based vaccine, we'll have to know the three-dimensional structures of at least a few variants of OspC, especially those from invasive strains.
Since we've solved the structure of OspC based on two infectious strains of the Lyme disease bacterium, we now have a prototype for determining the structure of OspC from other strains."
Another source says:
OspC is a major surface protein produced by B. burgdorferi when infected ticks feed but whose synthesis decreases after transmission to a mammalian host .
We have previously shown that spirochetes lacking OspC are competent to replicate in and migrate to the salivary glands of the tick vector but do not infect mice .
Here we assessed the timing of the requirement for OspC by using an ospC mutant complemented with an unstable copy of the ospC gene and show that B. burgdorferi's requirement for OspC is specific to the mammal and limited to a critical early stage of mammalian infection .
By using this unique system, we found that most bacterial reisolates from mice persistently infected with the initially complemented ospC mutant strain no longer carried the wild-type copy of ospC. Such spirochetes were acquired by feeding ticks and migrated to the tick salivary glands during subsequent feeding.
Despite normal behavior in ticks, these ospC mutant spirochetes did not infect naive mice.
ospC mutant spirochetes from persistently infected mice also failed to infect naive mice by tissue transplantation. We conclude that OspC is indispensable for establishing infection by B. burgdorferi in mammals but is not required at any other point of the mouse-tick infection cycle ."
"The findings confirmed that sera from patients with early Lyme disease contain high concentrations of IgM or IgG borreliacidalthat bind a conserved region of OspC .
For example, borreliacidal OspC antibodies are produced shortly after infection, but the antibodies are not detected when B. burgdorferi 297 spirochetes are used for testing.
The 297 spirochetes express OspC,
but
the concurrent expression of OspA and OspB hinders the attachment of the OspC borreliacidal antibodies (30).
In contrast, B. burgdorferi 50772 is highly susceptible to OspC borreliacidal antibodies (7, 30), because the spirochetes lack the plasmid containing ospA and ospB (2) and the absence of the Osps enables the OspC borreliacidal antibodies to bind."
"In this study, borreliacidal activity against B. burgdorferi 50772 was used to determine the region of OspC recognized by the immunoglobulin M (IgM) and IgG borreliacidal antibodies in sera from patients with early Lyme disease.
Several investigators have shown that borreliacidal OspC antibodies are formed shortly after infection with B. burgdorferi.
Our results confirmed these findings and showed that OspC borreliacidal antibodies in early Lyme disease sera were predominantly IgM or IgG. Moreover, the borreliacidal OspC antibodies bound specifically to a region within the 50 amino acids nearest the C terminus of the protein.
It was extraordinary that adsorbing the early Lyme disease sera with OspC-Dra removed the entire concentration of OspC antibodies .
Outer surface lipoprotein [?] (Osp) C is a virulence factor required for transmission of the Lyme disease agent, Borrelia burgdorferi.
We have constructed an inducible promoter system to study the function and regulation of OspC by integrating regulatory elements from the Escherichia coli lac operon into the B. burgdorferi genome.
(Slipping in a note...it appears flacp is a flagellin promoter)
An inducible promoter (flacp) was constructed by inserting a synthetic lac operator sequence between the transcriptional start site and the ribosomal binding site of the B. burgdorferi flgB [?] promoter;
flacp was then used to replace the native ospC and rpoS [?] promoters in B. burgdorferi derivatives that constitutively express the E. coli Lac repressor protein (LacI).
***In vitro, the expression of ospC and rpoS [?] from flacp***
was dependent on the inducer isopropyl beta-D-thiogalactopyranoside and was unaffected by temperature or pH, conditions commonly used to mimic different aspects of the B. burgdorferi life cycle.
Our results suggest that OspC is essential immediately upon injection into a mouse and OspC expression must be maintained during the early stages of infection.
In addition, the mouse infectivity experiment indicates that this system can be used to regulate B. burgdorferi genes in vivo, within the context of an experimental tick-mouse infectious cycle.
RpoS is an alternative sigma factor that is required for ospC transcription . However, the role of other temperature-dependent factors has not previously been addressed.
Our results with the inducible rpoS [?] strain demonstrate that RpoS alone is sufficient to activate OspC expression , even at 23 degrees C.
This is the first functional inducible promoter system developed for use in B. burgdorferi and, for the first time, will provide researchers with the ability to artificially regulate the expression of genes in this pathogenic spirochaete. Mol. Microbiol. (2007) PMID: 17257307
If Bb uses rpoS to make its outer surface protein C (ospC) which leads to infection...
What can prevent it from forming?
"Osmotic induction of rpoS [?] can be triggered by addition of NaCl or sucrose and is alleviated by glycine betaine .
Glycine betaine?
Let's talk about glycine first:
"As one of the genetically coded amino acids, glycine stands alone as a protein constituent that lacks a center of chirality, meaning it is not optically active and lacks stereoisomers.
(Translation...it does not have a mirror image.)
Known also as glycin, glycine, the simplest of the amino acids is soluble in both water and alcohol and helps release oxygen required for cell production and the biosynthesis of hormones .
One of the most ubiquitous of the amino acids found in humans, glycine has the ability to readily combine with many toxic substances, rendering them harmless for subsequent excretion, and the biochemical may play a role in the process of tissue repair and wound healing.
The primary source of natural glycine is collagen , but it is a constituent of all proteins and helps in fat storage, synthesis of hemoglobin, and reduction of high blood fat and uric acid levels in the body .
Other than human-produced glycine, the amino acid may be taken dietarily in fish, meat, beans, and dairy foods that are high in protein .
Beyond a surgical use as an irrigant, other uses for glycine include as a photographic developer, flavor enhancer and masker, pH buffer and stabilizer , and an acid indicator in bacteriology .
Glycine, alternatively known as glycocol, aminoethanoic acid, or aminoacetic acid, is considered a "non-essential amino acid" since it can usually be manufactured by the well-nourished body, but has been synthesized in the laboratory.
The non-prescription food supplement is marketed in a purified powder form as a growth hormone releaser , for its brain calming effect including treating schizophrenia , as a general detoxifier , and for possible benefits to men with prostrate gland enlargement .
Dietary precursors such as glycine may help control the rates of synthesis of serotonin, acetylcholine, dopamine, and norepinephrine in the brain , lending credence to the food-supplement manufacturers' claims for its soothing powers.
Crystals of glycine are white, odorless, and form an acidic solution of pH 4.0. In the past, doctors and nutritionists warned against consuming too many eggs in the diet or risk the chance of heart disease, but that was before some discovered the benefits of a little-known nutrient,
betaine.
Found naturally not only in eggs and liver, but also in fish, beets, and legumes, betaine is able to reduce the levels of harmful homocysteine in human plasma.
Just as the better known vitamins folic acid, B-6, and B-12, betaine in the diet lessens the chances for cardiovascular disease and reduces the risk of giving birth to children with neural tube defects.
However, betaine re-methylation is restricted to the liver while folic acid acts throughout the body.
Known also as
trimethylglycine ,
betaine is produced in the human body from choline and the amino acid glycine .
Similar in function to the nutritional supplements marketed as SAMe (S-adenosylmethionine), vitamin B-12, and choline, betaine acts as a methyl group donor , which play an important role in normal liver function, detoxification, cellular replication, and probably act in protecting the kidneys from damage .
Not to be outdone by nature, pharmaceutical companies have sythesized betaine as betaine hydrochloride, betaine citrate, and betaine aspartate for the health-food market and as Cystadane, for controlling homocysteine in the blood and urine, particularly for people genetically lacking a needed enzyme."
Time for a review?
1.Bb uses rpoS to make its outer surface protein C (ospC) which leads to infection.
2. Osmotic induction of rpoS [?] can be triggered by addition of NaCl or sucrose and is
alleviated by glycine betaine .
We KNOW Bb uses NaCl for its MOTILITY...via flagella.
Also keep in mind:
All that flagellin triggers NFkB...which triggers IL1 B and TNF alpha.
The question is: does glycine betaine impact the proteins in Bb's flagella (flagellins) which look to be the "final" proteins left at the end of 4 months in non human primates intentionally infected with Bb for research purposes.
What breaks apart flagellin?
Apparently someone thinks:
"Neutrophil elastase also rips apart flagellin, report Yolanda S. Lopez-Boado of Wake Forest University School of Medicine in Winston-Salem, N.C., and her colleagues. In test-tube studies, the enzyme can destroy flagellin whether it's part of a bacterium's flagella or just free-floating in a solution."
Since our neutrophils (most abundant WBCs) are produced every 3 weeks...(herx cycle), it likely will take a considerable amt. of time to finish the job.
Elastase, an enzyme that also breaks apart elastin in our lungs is a rough way to go!
(Heparin inhibits elastase release from neutrophils.) Elastase is implicated in COPD...chronic obstructive pulmonary disease. Large numbers of neutrophils with unopposed neutrophil elastase (NE) proteolytic activity are found in lower respiratory tract secretions from most patients with advanced cystic fibrosis (CF)too. A substance called elastase destroys the alveoli's ability to expand and contract.
Looks like we need to find a safer way...
At least:
``Magnesium enhances human *pancreatic* elastase DIGESTION of elastin.'' PMID: 8292494
Posts: 9481 | From Sunshine State | Registered: Mar 2001
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treepatrol
Honored Contributor (10K+ posts)
Member # 4117
posted
So ospC is the one they should look for thats why they say it cross reacts just to remove the only true expression from beginning to end disease progression.
And take Magnesium.
-------------------- Do unto others as you would have them do unto you. Remember Iam not a Doctor Just someone struggling like you with Tick Borne Diseases.
Marnie
Frequent Contributor (5K+ posts)
Member # 773
posted
Don't forget Bb comes cloaked in the SALP protein it picks up in the saliva of the tick which buys it time to infect too.
I think that protein MIGHT BE skeletal alkaline phosphatase.
How long Bb remains cloaked, hiding its "real" protein cell walls (plural) is unknown.
I'm guessing not long...about as fast as the appearance of the RASH..."allergic reaction"...histAmine triggered.
(Like HIV, Bb has zinc fingers...cysteine and histIdine bound to zinc.)
Once uncovered of the SALP protein...we can finally react to Bb's actual various protein "packages" (proteins in its cell walls and flagella i.e., "tail").
The first step in eliminating ALL gram negative pathogens is to either stop the cell walls from forming in the first place
OR
Damage them...poke holes in them (which is how some abx. work).
The cell walls ARE crystaline which can be effected by sound waves. (Ya know how sound waves can shatter crystal.)
Our cell walls are NOT crystaline, so they are NOT impacted by the very specific frequencies used.
The frequencies should be pulsed and SCANNED to hit any mutations which Bb is very capable of doing!
Without flagella, Bb can't MOVE. If the experiment with other primates applies to humans too...we are reacting (ongoing) to Bb's flagellin proteins...all that "remain".
Copper (we need only TRACE amts.) impacts flagella negatively too. That sort of explains the popularity of copper bracelets years ago worn by those with arthritis. (We DO absorb things thru our skin!)
If one's copper levels are ALREADY high, it maybe a body "protective move".
Too much of any nutrient isn't good. It is restoring the balance that is so darned tricky!
BTW...copper CLEANER (Wright's cream) removes tannins. I use it to clean tannin stains (from Merlot wine) that happen to my plastic wine glasses (use plastic drink glasses around the pool). I don't know what is IN that cleaner that does it. The jar has no list of ingredients.
Curious though because the "Universal Remedy" years ago was:
Mg oxide, tannic ACID, and activated charcoal.
Tannins are in things that are supposed to be very good for us (dark colored fruits esp. - blueberries, blackberries, raspberries, etc.)
Posts: 9481 | From Sunshine State | Registered: Mar 2001
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treepatrol
Honored Contributor (10K+ posts)
Member # 4117
posted
Yep
-------------------- Do unto others as you would have them do unto you. Remember Iam not a Doctor Just someone struggling like you with Tick Borne Diseases.
sizzled
Frequent Contributor (1K+ posts)
Member # 1357
posted
Thank-you, Marnie!
If OspaC could be made in to a cheap feed we could give it to rodents and deer? I think it could be done and am not being sarcastic!
Posts: 4258 | From over there | Registered: Jul 2001
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