Pathways

Aztreonam is a 1-β methyl-carbapenem that is structurally related to beta-lactam antibiotics.5 It was first authorized for use in the US in November 2001 and in Europe in April 2002. Shown to be effective against a wide range of Gram-positive and Gram-negative aerobic and anaerobic bacteria, ertapenem is used to treat various bacterial infections. Aztreonam exhibits a bactericidal mode of action. It works by binding to and inhibiting bacterial penicillin-binding proteins (PBPs).5 In Escherichia coli, it has a strong affinity toward PBPs 1a, 1b, 2, 3, 4 and 5 with preferential binding to PBPs 2 and 3.5 Upon binding to PBPs, ertapenem inhibits bacterial cell wall synthesis by interfering with the lengthening and strengthening of the peptidoglycan portion of the cell wall, thereby inhibiting cell wall synthesis.

Aztreonam is a drug that is not metabolized by the human body as determined by current research and biotransformer analysis. Aztreonam passes through the liver and is then excreted from the body mainly through the kidney.

Bacampicillin is a s an ampicillin prodrug used to treat various susceptible bacterial infections in the body, such as respiratory infections and skin and subcutaneous tissue infections. Bacampicillin exhibits a bactericidal mode of action. It works by binding to and inhibiting bacterial penicillin-binding proteins (PBPs). Upon binding to PBPs, ertapenem inhibits bacterial cell wall synthesis by interfering with the lengthening and strengthening of the peptidoglycan portion of the cell wall, thereby inhibiting cell wall synthesis.

Bacilysin is a non-ribosomally synthesized dipeptide antibiotic consisting of l-alanine residue at the N terminus and non-proteinogenic amino acid, l-anticapsin, at the C terminus linked by a peptide bond. It is known to be active against a wide range of bacteria and fungi and is synthesized by proteins in the bac operon of the gene cluster of the bacterial species Bacillus subtilis. This pathway shows the synthesis of bacilysin by B.subtilis and starts with the compound prephenate - the anionic form of prephenic acid and is an intermediate in the metabolism of many secondary metabolites. Its decarboxylation leads to the formation of the intermediates that lead up to the synthesis of the L-anticapsin intermediate that is produced by some Bacillus species and functions as a Trojan Horse antibiotic. Once inside the victim's cell, L-anticapsin is liberated by the action of peptidases and attacks the host so in order to protect the producers against inactivation of their own glucosamine synthase, L-alanine is ligated to the amino group of L-anticapsin, producing the benign dipeptide bacilysin. Bacilysin is excreted out of the cell and imported by the dipeptide permeases of susceptible bacterial or fungal cells. Bacilysin is then transported out of the B. subtilis cell via the transporter putative bacilysin exporter BacE.

Bacterial sepsis begins when bacteria activate the Toll-like receptor TLR4 on the membranes of macrophages, T-cells and dendritic cells. TLR4 activates the production of interferon regulatory factor 3 (IRF3), TIR-domain-containing adapter-inducing interferon-β (TRIF), signal transducer and activator of transcription 1 (STAT1) and nuclear factor kappa B (NF-kB) in the cytoplasm [1]. The NF-kB protein then goes to nucleus and activates expression of nitric oxide synthase (iNOS) which generates nitric oxide (NO). It also activates aconitate decarboxylase (Irg1), tumor necrosis factor (TNF), interleukin 6 (IL-6) and interleukin 1 beta (IL-1β). These are the pro-inflammatory proteins while nitric oxide (NO) is also a pro-inflammatory molecule that can lead to the production of oxidized tyrosines (i.e., nitrotyrosine). Similarly, the newly expressed IRF3 goes to the nucleus and activates the production of interferon beta (IFN- β), which is another pro-inflammatory cytokine. The whole collection of cytokines, TNF, IL-6, IL-1β and IFN-β move into the bloodstream and head to the brain and into the hypothalamus, leading to release of the hypothalamic corticotropin releasing hormone (CRH) [2]. CRH, in turn, activates the release of pituitary adrenocorticotropic hormone (ACTH), which then moves down through the blood stream towards the adrenal glands (located at the top of the kidneys) to produce cortisol and epinephrine. Cortisol and epinephrine stimulate the ”flight or fight” response, leading to the increased production of glucose from the liver (via glycogen breakdown) and the release of short-chain acylcarnitines (also from the liver) to help support beta-oxidation of fatty acids. These compounds support cell synthesis and growth of the macrophages and neutrophils used in the innate immune response. The liver also produces more IL-6, more TNF and more NO to further stimulate the innate immune response. Higher nitric oxide (NO) levels lead to blood vessel dilation and reduced blood pressure, which in its most extreme form, can be a major problem in sepsis. Higher iNOS expression in macrophages, neutrophils and dendritic cells consumes the amino acid arginine to produce more NO which disrupts the mitochondrial TCA cycle leading to the accumulation of citrate and the production of fatty acids and acylcarnitines (needed for lipid synthesis). Increased Irg1 (actonitate decarboxylase) expression leads to accumulation of succinate, which results in the succinylation of phosphofructokinase M2 (PKM2) [3]. Succinate also leads to the release of hypoxia inducible factor 1-alpha (HIF-1α) from its PHD-mediated inhibition. HIF-1α interacts with succinylated PKM2 and induces the expression of glycolytic genes such as Glut1 (the glucose transporter) and the pro-inflammatory cytokine IL-1β [3]. As a result of these metabolic changes and the deactivation of the oxidative phosphorylation pathway in their mitochondria, macrophages, neutrophils, T-cells and dendritic cells shift to aerobic glycolysis [4]. This leads to the production of more reactive oxygen species (ROS) which results in the oxidation of certain amino acids, such as methionine. This leads to the increased production of methionine sulfoxide (Met-SO). As the inflammatory response continues, more glucose and arginine in the bloodstream are consumed by dividing white blood cells to produce more lactate and more NO to further push the aerobic glycolytic pathway [4]. This aerobic glycolysis occurs primarily in white blood cells leading to active cell division and rapid white cell propagation (growing by a factor of three to four in a few hours). Hexokinase (HK) along with increased levels of lactate from aerobic glycolysis activate the inflammasome inside macrophages and dendritic cells, leading to the secretion of IL-1β. This cytokine further drives the aerobic glycolysis pathway for these white blood cells. All these signals and effects combine to lead to the rapid and sustained production of large numbers of macrophages, neutrophils, dendritic cells and T-cells to fight the bacterial infection. This often leads to a reduction in essential amino acids (threonine, lysine, tryptophan, leucine, isoleucine, valine, arginine) and a mild reduction in gluconeogenic acids (glycine, serine) in the bloodstram. The reduction in essential amino acids is intended to “starve” the invading bacteria (and other pathogens) of the amino acids they need to reproduce [4]. Some of the reduction in amino acid levels is moderated by the proteolysis of myosin in the muscle and the proteolysis of serum albumin in the blood (the most abundant protein in the blood, which is produced by the liver). These proteins act as amino acid reservoirs to help support rapid immune cell production. The loss of serum albumin in the blood to help support amino acid synthesis elsewhere can lead to hypoalbuminemia, a common feature of infections, inflammation, late-stage cancer and sepsis. At some point during the innate immune response, the kynurenine pathway becomes dysregulated, potentially through over-stimulation by interferon gamma (IFNG). This hyperstimulation leads to large reductions in tryptophan levels as the indole dioxygenase (IDO) enzyme becomes more active. IDO activation results in the generation (from tryptophan) of large amounts of kynurenine (and its other metabolites) through a self-stimulating autocrine process. Kynurenine binds to the arylhydrocarbon receptor (AhR) found in most immune cells [5-7]. In addition to increased kynurenine production via IDO mediated synthesis, hyopalbuminemia can also lead to the release of bound kynurenine (and other immunosuppressive LysoPCs) into the bloodstream to fuel this kynurenine-mediated process. Regardless of the source of kynurenine, the kynurenine-bound AhR will migrate to the nucleus to bind to NF-kB which leads to more production of the IDO enzyme, which leads to more production of kynureneine and more loss of tryptophan. High kynurenine levels and low tryptophan levels leads to a shift in T-cell differentiation from a TH1 response (pro-inflammatory) to the production of Treg cells and an anti-inflammatory response [5-7]. High kynurenine levels also lead to the production of more IL10R (the interluekin-10 receptor) via binding of kynurenine to the arylhydrocarbon receptor (AhR). Activated AhR effectively increases the anti-inflammatory response from interleukin 10 (an anti-inflammatory cytokine). Low tryptophan levels also lead to the activation of the general control non-depressible 2 kinase (GCN2K) pathway, which inhibits the mammalian target of rapamycin (mTOR), and protein kinase C signaling. This leads to T cell autophagy and anergy. High levels of kynurenine also lead to the inhibition of T cell proliferation through induction of T cell apoptosis [5-7]. In other words, kynurenine leads to a blunted immune response as neither sufficient B-cells, macrophages nor T-cells (which are needed for B-cell production) are produced, leading to further immune suppression. This allows for uncontrolled viral propagation. As a result, the invading viruses are NOT successfully cleared. This leads to a “vicious” or futile cycle where the growing virus population pushes the body to produce more B-cells and T-cells and various organs (muscles, heart, liver) exhaust themselves to produce a more metabolites to fuel the pro-inflammatory response, while the kynurenine/tryptophan cycle keeps on killing off T-cells and blunting the immune response [5-7]. This “futile” cycle of producing ineffective B and T cells, leads to heightened lactate production resulting in lactic acidosis. Likewise, as more NO is produced, this leads to a further loss of blood pressure – both lactic acidosis and hypotension can lead to organ failure. The continuous release of proinflammatory cytokines through the failed fight to eliminate the virus can also damage the alveolar-capillary barrier in the lungs. Loss of integrity of this lung barrier leads to influx of pulmonary edema fluid and lung injury or fluid in the lungs. Excessive, long-term release of glucose, short-chain acylcarnitines and fatty acids from the liver along with higher amino acid production from the blood and liver via proteolysis of albumin (leading to more extreme hypoalbuminemia), results in reduced uremic toxin clearance and increased levels of uremic solutes in the blood. High levels of uremic toxins lead to liver, heart, brain and kidney injury [8]. Likewise excessive release of acylcarnitines from the heart and liver leads to heart and liver injury. Organ failure often develops in end-stage sepsis, leading to death.

Bacterial sepsis occurs when viral coated proteins bind and activate Toll-like receptors (TLR) 2 and 4 on white blood cells and is taken up by macrophages. Due to this ingestion by these macrophages activating TLR receptors which are directly correlated to the activation of the innate immune response. This causes the activation and production of many interleukins, transcription factors and cytokines. One of these interactions is the NF-kβ protein that enters the nucleus and activates nitric oxide synthase (iNOS), Irg1, tumor necrosis factor (TNF), interleukin 6 (IL-6) and interleukin 1 beta (IL-1β). These are pro inflammatory cytokines that can oxidize tyrosines such as nitrotyrosine, and move into the bloodstream which transports them to the brain. At the brain the proinflammatory cytokines activate the hypothalamus, releasing hypothalamic corticotropin releasing hormone (CRH) into the hypophyseal portal system. CRH acts on the anterior pituitary releasing adrenocorticohormone (ACTH), this hormone travels in the bloodstream to the adrenal glands releasing cortisol and epinephrine. This stimulates the sympathetic nervous system into a "fight or flight" response, increasing glucose production, release short chain acylcarnitines, beta oxidation of fatty acids in order to allow cells to produce and develop immune cells such as macrophages and neutrophils. In turn also stimulates the liver to produce even more proinflammatory cytokines like IL-6, TNF and NO to strengthen the innate immune response. The increased concentration of nitric oxide causes blood vessels to dilate, reducing blood pressure, disrupting mitochondrial tricarboxylic acid (TCA) cycle this leads to accumulation of byproducts of the cycle such as citrate and production of acylcarnitines and fatty acids. Succinate also begins to accumulate resulting in a downstream effect production of pro inflammatory cytokine. This deactivates oxidative phorphorylation in mitochondria and white blood cells shifting them to aerobic glycolysis leading to more reactive oxygen species (ROS) being produced as a byproduct and oxidation of amino acids. Aerobic glycolysis in white blood cells lead to its division and propagation in a quick time frame, causing a highly exagerrated response. This highly inflammatory response leads to a harmful postive feedback leading to more glucose and arginine consumed and more lactate and NO produced further exacerbating this aerobic glycolytic pathway. Ultimately causing a reduction in amino acids and gluconeogenic acids causing the body to act on amino acid reservoirs such as myosin in the muscle or serum albumin in the blood. Detrimental in some cases as low levels of serum albumin can lead to hypoalbuminemia leading to swelling as albumin is responsible for keeping fluid within the blood vessels. Throughout this activation of the innate immune response the pathway for kynurenine thrown into dysregulation, that is suspected to be due to stimulation from interferon gamma. Hyperstimulation leads to activation of indole dioxygenase (IDO) enzyme leading to reductions of tryptophan. Subsequently activation of IDO leads to increased concentration of kynurenine and its metabolites leading to a self stimulating autocrine process. Kynurenine then binds to arylhydrocarbon receptor (AhR) on immune cells, this bounded compound will travel to the nucleus to bind NF-kβ causing more production of IDO enzyme, reduction of tryosine and production of kynurenine. High levels of kynurenine and low levels of tryptophan leads T cell differentiation to shift to an anti-inflammtory response, inhibition of T cell proliferation and T cell apoptosis. Overall leading to a blunted immune response, causing the infection to continue to spread and contributing to a futile cycle. As the energy needed to sustain the immune response exhausts the body and ends up being damaged by the kynurenine pathway. This ultimately can result in hypotension, lactic acidosis, damaging of barriers, pulmonary edema, hypoalbuminemia, build up of uremic toxins, organ injury, organ failure and in extreme cases death.