Antibiotics produced by Bacteria activate Human Oocytes, creating Healthy Babies: AMPK links the Creation of Human Life with HIV, Progeria, & Cancer

CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0/)], via Wikimedia Commons;By De Wood, Pooley, USDA, ARS, EMU. [Public domain], via Wikimedia Commons 

A recent study published online in the journal Fertility and Sterility in September of 2017 systematically reviewed for the first time evidence for the effect of two compounds, ionomycin and A23187 (also known as calcimycin), on fertilization rates and pregnancy outcomes in infertile couples undergoing an in vitro fertilization procedure known as intracytoplasmic sperm injection (ICSI) [1]. ICSI involves the direct deposition of sperm into the oocyte cytoplasm, which typically leads to high rates of fertilization. However, fertilization failure despite repeated ICSI is likely caused by a failure of the oocyte to activate [1]. Physiological oocyte activation is accomplished by the delivery of a sperm-borne oocyte activating factor called phospholipase C zeta (PLCζ). PLCζ activates human oocytes by inducing an intracellular signaling cascade that ultimately results in increased calcium (Ca2+) oscillations in the oocyte, which drives oocyte activation to completion [1]. As oocyte activation is an indispensable prerequisite for the creation of all human life, every human being alive today and any human being that has ever lived began their existence as an activated oocyte [2]. Ionomycin and A23187 increase the levels of intracellular Ca2+ and are thus commonly known as Ca2+ ionophores [1]. The authors of the Fertility and Sterility study showed that over a total of 1,521 ICSI cycles, calcium ionophores including ionomycin and A23187 led to a statistically significant improvement in fertilization, cleavage, blastulation, implantation rates, overall pregnancy, and live-birth rates [1]. Ionomycin and A23187 have also been shown in several independent studies to effectively induce human oocyte activation, leading to the birth of normal, healthy children [3,4].

Strikingly, as described further below, both ionomycin and A23187 are antibiotics that are naturally produced by certain species within the bacterial genus Streptomyces [5,6]. Other structurally distinct compounds and methods have also been shown to induce human oocyte activation, including ethanol, puromycin (an antibiotic and protein synthesis inhibitor produced by Streptomyces alboniger), as well as mechanical manipulation and electrical stimulation, both of with have been reported to result in the creation of normal children [7-11]. Interestingly, as mouse oocytes are considered models for human oocytes, ionomycin, A23187, anisomycin (an antibiotic and protein synthesis inhibitor produced by Streptomyces griseolus), mycophenolic acid (an immunosuppressant produced by the fungus Penicillium brevicompactum), cycloheximide (a protein synthesis inhibitor produced by Streptomyces griseus), carvacrol (a secondary plant metabolite produced by Origanum vulgare{oregano}), and phorbol 12-myristate 13-acetate (PMA, a secondary plant metabolite produced by Croton tiglium) each induce activation of mouse oocytes [12-22]. Ionomycin, A23187, PMA, and reactive oxygen species (ROS) also induce the acrosome reaction in human sperm, a process characterized by the release of hydrolytic enzymes from the head of sperm which is necessary for oocyte penetration and thus indispensable for the creation of all human life outside of a clinical setting (ICSI bypasses the need for oocyte penetration) [23,24]. Additionally, although an over-production of ROS, similar to Ca2+, may lead to deleterious effects including cell death/apoptosis, low levels of ROS have been shown to act as signaling molecules and ROS is significantly increased on or immediately following mouse oocyte activation [25,26].

Furthermore, the master metabolic regulator AMPK is critical for oocyte meiotic resumption and maturation (a process that precedes and is essential for oocyte activation), is found located across the entire acrosome in the head of human sperm, and is activated by increases in ROS and Ca2+ [27-29]. Ionomycin, A23187, ethanol, puromycin, mechanical force, electrical stimulation, anisomycin, mycophenolic acid, carvacrol, and PMA also induce AMPK activation, indicating that a common mechanism of action links chemically distinct compounds with the creation of human life [30-39]. This common mechanism of action likely centers on the induction of cellular stress, mediated by indirect increases in intracellular Ca2+, ROS, and/or the AMP(ADP)/ATP ratio, etc. as I originally proposed in 2016 [40]. Because the bacterial-derived antibiotics ionomycin and A23187 induce both the acrosome reaction in human sperm and human oocyte activation, producing normal, healthy children, it can be said that “non-human organisms have the power to create human life or the power to end life.” As explained below, the beneficial effects of cellular stress induction (i.e. a “shock”) crosses species boundaries and may indeed play a role in facilitating natural selection, a process that underlies and drives evolution.  

A number of bacterial species residing within the genus Streptomyces have proven to be extremely important and medicinally valuable as approximately 70% of clinically useful antibiotics are derived from Streptomyces [41]. The antibiotics ionomycin and A23187 are naturally produced by Streptomyces conglobatus and Streptomyces chartreusensis, respectively [5,6]. Other important examples include the antibiotic tetracycline (produced by Streptomyces aureofaciens), the immunosuppressant rapamycin (produced by Streptomyces hygroscopicus), and the anti-helminthic avermectins (produced by Streptomyces avermitilis) [42]. Many soil and aquatic-dwelling species of Streptomyces can be found in harsh environments and are characterized by a unique life cycle, including spore germination followed by vegetative mycelium production, aerial hyphae formation, sporulation (i.e. spore formation), and antibiotic production [43,44]. Curiously, just as cellular stress induction leads to the creation of human life and other beneficial effects in human cells (see below), stress induction also promotes the induction of aerial hyphae formation, sporulation, and antibiotic production in many Streptomyces species (spp.). Indeed, a decrease in the levels of ATP and bacterial growth is associated with sporulation, aerial hyphae formation, and antibiotic production [42,45]. A reduction in glucose/nutritional deprivation, the preferred sugar/carbon source for many Streptomyces spp., also significantly increases antibiotic production [46]. An increase in intracellular ROS and Ca2+ is associated with spore germination, aerial hyphae formation, and antibiotic production [47-49]. Other cellular stressors, including heat shock and ethanol, also significantly increase antibiotic production, provocatively indicating that the effects of cellular stress crosses species boundaries, enhancing bacterial survival and facilitating the creation of human life [50,51].

The beneficial effects of low-level cellular stress induction also extends to plants, as many plants produce secondary metabolites partly for the purpose of self-defense, analogous to antibiotics. Similar to the harsh, stressful environments often inhabited by Streptomyces spp., the Great Basin Bristlecone Pine (Pinus Longaeva), considered the oldest living non-clonal organism on the planet ( >5000 years old), thrives in an exceptionally harsh environment, characterized by increased elevations and exposure to UV radiation, nutritionally-deprived soils, harsh temperatures, and mechanical stress due to wind variances, leading early researchers to conclude that it’s longevity is intimately associated with adversity [52-54]. Conversely, Pinus Longaeva species that are located in less stressful environments (i.e. lower elevations) are strongly associated with younger age classes (<875 years) [55].  Similarly, the Creosote bush (Larrea tridentate), considered one of the oldest living clonal organisms on the planet (>11,000 years old), also thrives in harsh environments including the Mohave Desert [56]. AMPK, which increases lifespan and healthspan in several model organisms, is the primary sensor of cellular stress in eukaryotic organisms (e.g. plants and humans) and the plant AMPK orthologue SnRK1 as well as Ca2+ and ROS are critical for seed germination, fertilization, root gravitropism, and secondary metabolite production [57-64]. The secondary plant metabolites PMA (which activates mouse oocytes and promotes the acrosome reaction in human sperm) and artemisinin (an anti-malarial drug) both activate AMPK and the antibiotic A23187 also increases production of the secondary metabolite resveratrol in grape cell cultures, again indicating that exposure to low-level stressors may promote extension of lifespan and initiate the creation of human life [17,23,39,65,66].

Organismal exposure to beneficial levels of stress may also play a critical role in evolution. As first noted by Charles Darwin, evolution is driven by natural selection, a process characterized by environmentally-induced phenotypic changes that may lead to inheritable survival and reproductive advantages [67]. From “On the Origin of Species by Means of Natural Selection, or the Preservation of Favoured Races in the Struggle for Life”, Darwin explained that “if there be, owing to the high geometrical powers of increase of each species, at some age, season, or year, a severe struggle for life, and this certainly cannot be disputed;……But if variations useful to any organic being do occur, assuredly individuals thus characterised will have the best chance of being preserved in the struggle for life;” [67].  This “struggle for life” Darwin spoke of is embodied by selective pressures which may be abiotic (i.e. light, wind, temperature, etc.) or biotic (predation, disease, competition, etc.) [68,69]. As alluded to above, such selective pressures are indeed sources of cellular stress, sensed by both prokaryotes and eukaryotes, that induce beneficial responses (at appropriate levels), leading to the acquisition of phenotypes conducive for continued survival. Both biotic (e.g. infection) and abiotic (e.g. heat) stressors/selective pressures activate AMPK (which is evolutionarily conserved among eukaryotes) in human cells [70,71]. A phenomenon often cited as an example of natural selection on a readily observable timescale is the development of bacterial resistance to antibiotics, resulting in problematic mutant strains that may be life-threatening for some individuals (i.e. the elderly and immunocompromised) [72]. Intriguingly, lethal levels of bactericidal antibiotics have been shown to kill microorganisms via the induction of ROS, sub-lethal levels of bactericidal antibiotics however increase mutagenesis and bacterial resistance via induction of lower levels of ROS, and heat as well as nutritional stress increase bacterial resistance to antibiotics, providing compelling evidence that continuous exposure to low levels of stress likely plays a significant role in natural selection and evolution [73-75].

Moreover, gravity itself likely functions as a cellular stressor/selective pressure that has influenced the development of organisms on Earth since the emergence of the very first lifeform. Gravity exerts its effects on living organisms via the application of force, which is experienced by human cells in the form of mechanical loading or stress [76]. The application of force or a mechanical load has recently been shown to activate AMPK and simulated microgravity (i.e. hind limb unloading in mice) significantly decreases AMPK activation [77,78]. Spaceflight also inhibits the activation of T cells (immune cells essential for adaptive immunity), whereas the application of force and AMPK activation promote T cell activation [79-81]. Interestingly, spaceflight has recently been shown to decrease the levels of the master antioxidant transcription factor Nrf2 and the heat shock-inducible protein HSP90a but increase the levels of the growth-promoting kinase mTOR in mice [82]. AMPK however inhibits mTOR but increases the phosphorylation, nuclear retention, and transcriptional activity of Nrf2 [57,83,84]. Also, HSP90 interacts with and maintains AMPK activity and HSP90 is necessary for progesterone-induced human sperm acrosome reaction [85,86]. Interestingly, rapamycin, an immunosuppressant produced by Streptomyces hygroscopicus, extends lifespan in genetically heterogeneous mice, activates AMPK in vivo in normal aged mice, and increases human sperm motility [42,87,88]. Simulated microgravity via the use of NASA-designed rotating wall vessels (RWVs) however drastically reduces rapamycin production (~90%) whereas the antibiotic gentamycin increases rapamycin production by Streptomyces hygroscopicus, providing further evidence that cellular stress, in the form of mechanical loading induced by gravity, is essential for development, function, and survival of Earth-bound organisms [89,90].

The induction of cellular stress also links seemingly dissimilar physiological and pathological states with the activation of AMPK. As discussed above, both ionomycin and ROS activate AMPK and promote oocyte meiotic resumption, a process that is AMPK-dependent and is essential for efficient oocyte activation [27,30,91]. ROS is also critical for ovulation, PMA and ionomycin both activate mouse oocytes, and ionomycin is extensively used during ICSI procedures, creating normal healthy children, suggesting that cellular stress-induced AMPK activation is also essential for oocyte activation [3,4,12,17,92]. The activation of oocytes and T cells share strikingly similar intracellular signaling mechanisms (e.g. PLC-PIP2-DAG-PKC-IP3-Ca2+) and ionomycin combined with PMA are extremely effective in activating T cells and are often used as positive controls in HIV-1 latency reversal studies [93-95]. Reactivating latent/dormant HIV-1 in CD4+ T cells, potentially facilitating immune system detection and virus destruction, is currently being pursued as a method for the potential eradication of HIV-1 (called the “shock and kill” approach) [96]. Similar to oocyte activation, both Ca2+ and ROS are critical for T cell activation (and hence latent HIV-1 reactivation) and other cellular stress-inducing compounds, including NDGA derived from the Creosote bush, butyrate derived from bacteria, as well as ROS and HSP90 have been shown to reactivate latent HIV-1 [26,93,94,97-101]. Interestingly,  AMPK inhibition leads to cell death on T cell activation, knockdown of AMPK significantly decreases HIV-1 replication, and metformin (a well-studied AMPK activator derived from the French Lilac plant) increases butyrate production in human diabetic patients [81,102,103]. Perhaps most convincingly, early preliminary data showed that metformin significantly reduced several markers preferentially associated with cells latently infected with HIV-1 (e.g. PD-1, TIGIT, TIM-3) and also destabilized the latent HIV-1 reservoir in chronically-infected HIV patients, indicating that cellular-stress induced AMPK activation likely links the creation of human life with the potential eradication of HIV-1, as I originally proposed in 2016 [40,104,105].

AMPK activation may also link the disparate disease states of HIV-1 latency and Hutchinson-Gilford progeria syndrome (HGPS). HGPS is a genetic disorder caused by aberrant alternative splicing of the LMNA gene, generating a toxic protein called progerin that induces an accelerated aging phenotype and premature death at approximately 14 years of age [106]. Excessive activity of the gene splicing factor SRSF1 has been shown to prevent reactivation of latent HIV-1 and contribute to aberrant splicing of the LMNA gene in HGPS [107-109]. Metformin however has recently been shown to ameliorate the accelerated aging phenotype in cells derived from children with HGPS by reducing the levels of both SRSF1 and progerin and activating AMPK, as I first proposed in 2014 [110-112]. Interestingly, both Ca2+ and ROS induce autophagy (a process of disposing of damaged/toxic proteins and organelles) and rapamycin, which activates AMPK in vivo and increases intracellular Ca2+ levels, improves accelerated aging in progeria cells by inducing autophagic degradation of progerin [87,113-116]. Temsirolimus, an analog of rapamycin, also alleviated accelerated aging defects in progeria cells but also increased the levels of ROS and superoxide within the first hour of treatment [117]. Such evidence strongly suggests that cellular stress-induced AMPK activation links the reversal of HIV-1 latency and alleviation of accelerated cellular aging defects in HGPS.

Cellular stress-induced AMPK activation also links the potential elimination of cancer stem cells (CSCs) with HIV-1 latency reversal and viral eradication. CSCs, which are largely resistant to chemoradiation therapy, are a subpopulation of cancer cells that exhibit characteristics similar to embryonic stem cells (ESCs), including self-renewal, multi-lineage differentiation, & the ability to initiate tumorigenesis [118,119]. Mechanisms that sustain quiescence & promote self-renewal in adult stem cells (ASCs) & CSCs likely also function to maintain latency of HIV-1 in CD4+ memory T cells. Indeed, HIV-1 has been found to establish long-lasting latency in a recently discovered subset of CD4+ T cells that exhibit stem cell-like properties known as T memory stem (TSCM) cells and increases in Ca2+, ROS, and AMPK activation have been shown to promote T cell activation and ESC, ASC, and CSC differentiation [119,120].  Additionally, A23187 and PMA have been shown to promote CSC differentiation (causing CSCs to become more susceptible to chemoradiation) and metformin induces CSC differentiation and/or apoptosis in an AMPK-dependent manner in the deadliest of cancers, including glioblastoma and pancreatic cancer, providing support for my publication in 2017 in which I first proposed that CSC differentiation and/or apoptosis and HIV-1 latency reversal/viral eradication may be linked by cellular stress-induced AMPK activation [119,121-124].

In conclusion, the ability of non-human organisms including certain Streptomyces spp. to initiate the creation of human life is predicated on the induction of cellular stress, mediated by increases in intracellular ROS, Ca2+, AMP(ADP)/ATP ratio increase, etc.  The beneficial effects of transient cellular stress induction, which may be likened to selective pressures, crosses species boundaries and may indeed play a role in facilitating natural selection, a process that underlies and drives evolution, as evidenced by stress-induced increases in antibiotic production by Streptomyces spp. and stress-induced mutagenesis and antibiotic resistance in various bacterial strains. Because AMPK, a primary sensor of cellular stress in eukaryotic cells that increases lifespan and healthspan, plays a critical role in oocyte meiotic resumption/maturation, T cell activation, and stem cell differentiation, the creation of human life, the potential eradication of HIV-1, amelioration of accelerated aging in HGPS cells, and CSC differentiation/apoptosis are likely linked by a “Shock to Live”, or a “Shock to Kill”.  

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