Part One: Identifying types of bacteria

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Identifying types of Bacteria
Depending on Structure of their CELL WALLS, THE BACTERIA ABSORB EITHER THE PURPLE DYE OR THE PINK DYE
Modern Biology. New York: Holt, Rinehart & Winston 2009 chapter 24
GRAM POSITIVE BACTERIA 1. GRAM-POSITIVE BACTERIA HAVE A THICKER LAYER OF PEPTIDOGLYCAN IN THEIR CELL WALLS, MADE OF A PROTEIN-SUGAR COMPLEX THAT TAKES ON THE PURPLE COLOR DURING GRAM STAINING. (Figure 24-4)
GRAM-NEGATIVE BACTERIA 1. GRAM-NEGATIVE BACTERIA HAVE AN EXTRA LAYER OF LIPID ON THE OUTSIDE OF THE CELL WALL AND APPEAR PINK AFTER GRAM STAINING. (Figure 24-4) 2. The extra lipid layer stops the PURPLE Stain from entering the CELL WALL. They do absorb the PINK Stain, so they are easily distinguished with a microscope. 3. The extra lipid layer also stops many ANTIBIOTICS from entering the bacteria. Treatment for these requires a different ANTIBIOTIC than those used for infections caused by Grampositive bacteria.
Although gram negatives have a more difficult outer layer, most hospital infections tend to be gram positive. So it is important to have ways in which to address both
http://www.hhmi.org/biointeractive/Antibiotics_Attack/bb_1.html
Aerobic and anaerobic bacteria can be identified by growing them in liquid culture: 1: Obligate aerobic bacteria gather at the top of the test tube in order to absorb maximal amount of oxygen. 2: Obligate anaerobic bacteria gather at the bottom to avoid oxygen. 3: Facultative bacteria gather mostly at the top, since aerobic respiration is the most beneficial one; but as lack of oxygen does not hurt them, they can be found all along the test tube. 4: Microaerophiles gather at the upper part of the test tube but not at the top. They require oxygen but at a low concentration. 5: Aerotolerant bacteria are not affected at all by oxygen, and they are evenly spread along the test tube.
Selected Conditions Associated With Clostridial Infections Condition Soft-tissue infection: crepitant cellulitis, myositis, clostridial myonecrosis Hemolysis Agent C. perfringens C. perfringens Toxin α-Toxin (others) Phospholipase C α-Toxin Muscle necrosis Enteric diseases Food poisoning Enteritis necroticans Antibiotic-associated colitis Neutropenic enterocolitis Colorectal malignancy Hemolysis by septicolysine Tissue necrosis DNA lysis by DNase Hyaluronan lysis by hyaluronilase Neurologic syndromes Tetanus Botulism Abdominal infections: Cholecystitis, peritonitis, ruptured appendix, bowel perforation, neutropenic enterocolitis C. tetani C. botulinum C. perfringens , C. ramosum (many others) Tetanospasmin Botulinal toxins AG β-Toxin C. perfringens type A C. perfringens type C C. difficile C. septicum (others) C. septicum C. septicum C. septicum C. septicum C. septicum δ-Toxin α-Toxin β-Toxin γ-Toxin Enterotoxin β-Toxin Toxin A Unknown, possibly β-toxin C. perfringens θ-Toxin
Important to understand the difference between addressing issues with toxin/toxoid and anaerobic bacterial infections
The DPT vaccine is made with toxoid, which is toxin that has been inactivated.
Safety testing is done in animals
toxoid
safe
Immunocompetent animals
toxoid
?
MTHFr mutation, T cells…
When testing is done in vitro (in a test tube) with NO immune system
Paul-Ehrlich-Institute, Federal Agency for Sera and Vaccines, Langen, Germany
“Neurotransmitters are stored in synaptic vesicles at neuron terminals that connect other neurons. When the synaptic vesicle membrane fuses with the plasma membrane, the neurotransmitter is released into the synaptic cleft.”
18 JUNE 2004
VOL 304
SCIENCE www.sciencemag.org
• Tetanus toxin blocks the release of GABA and glycine • Tetanus toxin affects the hypothalamus, decreases hormone levels (such as testosterone), • Tetanus toxin causes excessive sympathetic discharge with urinary excretion of catecholamines (i.e.dopamine) and can cause chronic seizure activity. • Antibodies against the GAD enzyme (the enzymes that converts glutamate to GABA) have been reported in some cases of tetanus toxicity. • Tetanus toxin can also inhibit the release of norepinephrine, enkephalins and acetylcholine.
• Tetanus toxin can also inhibit the release of acetylcholinesterase. • Excess levels of acetylcholine are regulated by the enzyme acetylcholinesterase which causes the breakdown of acetylcholine • Lack of acetylcholinesterase will result in a down-regulation of muscarinic receptors. • Tetanus toxin is known to cause sympathetic overactivity.
Important to understand the difference between addressing issues with toxin/toxoid and anaerobic bacterial infections
Biofilms
Why we want to look at: SNPs Gut bugs Gut Environment
Actually identifying organisms
CSA DNA Stool
Indications that there are bacterial issues
DHPPA tyrosinase FIGLU ß hydroxybutyrate
•DHPPA elevated due to bacteria •Can use OAT/MAP/CSA/DNA stool test to determine organisms and approach to addressing (flow chart) •DHPPA may deplete tyrosinase. This is a back up for pathway to dopamine. HVA levels on a MAP are an indication this may be an issue. •Phenols and salicylc acid also inactivate tyrosinase. So, combination of bacteria (genetic susceptibility) along with phenols and salicylic acid can deplete back up route
Think about BH4, bacteria, oxidative stress
Relates back to DHPPA and tyrosinase later
BH4 BH4
serotonin GABA
BH2 Inhibited by lead Inhibited by aluminum BH4
Methylation cycle: purines T cell activation/cytokines GTP Macrophage infection H2NTP
BH4
neopterin
Immune response
Krebs cycle Methylation pathway
TNF/cytokines
BH4
aluminum
Relationship between BH4 and infection
Think about BH4, bacteria, oxidative stress
Relates back to DHPPA and tyrosinase later
BH4 BH4
serotonin GABA
In a moment we will look at the effect of infection on the back up route to dopamine
Indications that bacterial infection is an issue: Bacterial production of beta hydroxybutyrate
Indications that bacterial infection is an issue: Increased FIGLU due to bacterial infection
High Figlu or high glutamate, check succinate levels and think about role of aluminum/mitochondria in terms of Krebs cycle that we already discussed. Catch 22 bacteria->aluminum->dec succinate->inc figlu and glutamate
succinate
Indications that bacterial infection is an issue: Bacterial production of DHPPA
•Relationship between tyrosinase and DHPPA •Does the action of tyrosinase on DHPPA deplete tyrosinase levels? •Think about role of tyrosinase as the back up route to dopamine, especially after BH4 depletion (due in part to bacterial/aluminum issues)
In addition to depletion by DHPPA Tyrosinase can be inhibited by salicylates and phenols
Possible Relationship Between Tyrosinase Activity and Phenol Sensitivity
• Tyrosinase is also commonly called polyphenol oxidase.
• Polyphenol oxidase, or tyrosinase usually catalyzes the conversion of
monophenols to o-diphenols and oxidation of diphenols to the corresponding quinones. (Biochemistry. 1986 Aug 12;25(16):4489-92. )
• Phenol oxidase inhibitors such as phenylthiourea, potassium cyanide,
and sodium azide inhibit the reaction drastically.
92. ) (Biochemistry. 1986 Aug 12;25(16):4489-
In the event that tyrosinase (poly phenol oxidase) is used for dopamine synthesis rather than TH, it is possible that the level of tyrosinase available to detoxify phenols is reduced. Conversely phenols may deplete tyrosinase.
Picture 2
Recall the role of Tyrosinase as alternate route to dopamine
First lets review why we might need an alternate route to dopamine aside from tyrosine hydroxylase route
Tyrosine Hydroxylase
• First step in the conversion of tyrosine to dopamine. • Subject to feedback inhibition by dopamine, norepinephrine and epinephrine.
Variety of Compounds Affect Tyrosine Hydroxylase Biochem Biophys Levels ResIICommun. 2004 Jan 16;313(3): hydroxylase in the rat adrenal medulla. Central angiotensin increases biosynthesis of tyrosine
Dogan MD, Sumners C, Broxson CS, Clark N, Tumer N.
Angiotensin II acting centrally contributes to the regulation of blood pressure and water intake and stimulates the
release of catecholamines from the adrenal medulla. We hypothesized that the central angiotensin II is one mediator of biosynthesis of catecholamines in the adrenal medulla. Rats were administered i.c.v. angiotensin II or saline, and TH mRNA and protein levels in adrenal medulla were measured 1 or 3 h later. Angiotensin II did not change TH mRNA or protein 1 h later. However, by 3 h, angiotensin
II increased TH mRNA and protein levels. Centrally administered angiotensin II elevates TH mRNA expression and protein levels in the adrenal medulla. In conclusion, one component of central angiotensin II
elevation of blood pressure may be the result of increased catecholamine synthesis in the adrenal gland and elevated TH synthesis represents one underlying mechanism.
Neuropharmacology. 1989 May;28(5):521-7 Central regulation of adrenal tyrosine hydroxylase: interaction between dopamine and GABA systems. Gabor R, Regunathan S, Sourkes TL.
It has been previously demonstrated that nigrostriatal dopaminergic fibres participate in the neural regulation of the activity of adrenal tyrosine hydroxylase, specifically in its induction. To determine whether activation or inhibition of these fibres is responsible for this induction, the role of presynaptic dopamine receptors was investigated. Apomorphine (0.2 mg/kg), (+)3-PPP (10 mg/kg) and BHT 920 (1-3 mg/kg), drugs that are reported to bind to presynaptic dopamine receptors and thereby inhibit the release of that neurotransmitter, caused significant increases in the activity of the enzyme. As a central GABA (gamma-aminobutyric acid) system is believed to exert inhibitory control over the release of dopamine, GABA agonists were also tested for their effects. Muscimol (3 mg/kg), gamma-hydroxybutyrate (500 mg/kg) and HA-966 (150 mg/kg) produced significant induction of the adrenal enzyme; this induction was not blocked by dopamine postsynaptic receptor antagonists. After intraventricular administration (5 micrograms/rat) in normal animals, HA-966 produced significant induction of tyrosine hydroxylase. Its systemic administration did not induce the enzyme in animals with the adrenal denervated. When administered together at submaximal doses, HA-966 and BHT 920 produced an additive effect in the induction
of adrenal tyrosine hydroxylase.
Variety of Compounds Affect Tyrosine Hydroxylase Biochem Biophys Levels ResIICommun. 2004 Jan 16;313(3): hydroxylase in the rat adrenal medulla. Central angiotensin increases biosynthesis of tyrosine
Dogan MD, Sumners C, Broxson CS, Clark N, Tumer N.
Angiotensin II acting centrally contributes to the regulation of blood pressure and water intake and stimulates the
release of catecholamines from the adrenal medulla. We hypothesized that the central angiotensin II is one mediator of biosynthesis of catecholamines in the adrenal medulla. Rats were administered i.c.v. angiotensin II or saline, and TH mRNA and protein levels in adrenal medulla were measured 1 or 3 h later. Angiotensin II did not change TH mRNA or protein 1 h later. However, by 3 h, angiotensin
II increased TH mRNA and protein levels. Centrally administered angiotensin II elevates TH mRNA expression and protein levels in the adrenal medulla. In conclusion, one component of central angiotensin II
elevation of blood pressure may be the result of increased catecholamine synthesis in the adrenal gland and elevated TH synthesis represents one underlying mechanism.
Neuropharmacology. 1989 May;28(5):521-7 Central regulation of adrenal tyrosine hydroxylase: interaction between dopamine and GABA systems. Gabor R, Regunathan S, Sourkes TL.
It has been previously demonstrated that nigrostriatal dopaminergic fibres participate in the neural regulation of the activity of adrenal tyrosine hydroxylase, specifically in its induction. To determine whether activation or inhibition of these fibres is responsible for this induction, the role of presynaptic dopamine receptors was investigated. Apomorphine (0.2 mg/kg), (+)3-PPP (10 mg/kg) and BHT 920 (1-3 mg/kg), drugs that are reported to bind to presynaptic dopamine receptors and thereby inhibit the release of that neurotransmitter, caused significant increases in the activity of the enzyme. As a central GABA (gamma-aminobutyric acid) system is believed to exert inhibitory control over the release of dopamine, GABA agonists were also tested for their effects. Muscimol (3 mg/kg), gamma-hydroxybutyrate (500 mg/kg) and HA-966 (150 mg/kg) produced significant induction of the adrenal enzyme; this induction was not blocked by dopamine postsynaptic receptor antagonists. After intraventricular administration (5 micrograms/rat) in normal animals, HA-966 produced significant induction of tyrosine hydroxylase. Its systemic administration did not induce the enzyme in animals with the adrenal denervated. When administered together at submaximal doses, HA-966 and BHT 920 produced an additive effect in the induction
of adrenal tyrosine hydroxylase.
Glutamate and Tyrosine hydroxylase
• TH activity is stimulated by phosphorylation • Glutamate via NMDA receptors and dopamine via D2 receptors decreases TH phosphorylation by decreasing cAMP. • Decreased TH phosphorylation leads to decreased activity and lower levels of dopamine.
NMDA Glutamate receptors Dopamine D2 receptors
Tyrosine hydroxylase
“This work indicates that, in the striatum, glutamate via NMDA receptors and dopamine via D2 receptors, decrease TH phosphorylation …provide a mechanism explaining the ability of NMDA to decrease TH activity.”
Regulation of tyrosine hydroxylase via activation of NMDA and Dopamine D2 Receptors Lindgren, Xu, Haycock et al
tyrosine hydroxylase
Role of BH4 levels at this point in the pathway secretin increases A1298C and CBS up regulations decrease
aromatic amino acid decarboxylase
This enzyme is also shared by the tryptophan pathway for conversion of tryptophan to serotonin Role of metals and excess sulfur groups at this point in the pathway
dopamine/tyramine-bhydroxylase
phenylethanolamine-N-methyl transferase
Role of methylation and SAMe at this point in the pathway.
A number of factors can impair TH levels or this pathway so it is important to have an alternate route to dopamine synthesis