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Growth of Chlamydia pneumoniae Is Enhanced in Cells with Impaired Mitochondrial Function
This study massively confirms what Ray Peat has been writing about. Maintaining high respiration (oxidative phosphorylation) and low anaerobic glycolysis is the key to prevent chronic diseases. Focus on having good mitochondrial energy production and you will become resistant to many diseases. As everyone who follows Ray Peat knows, endotoxin is one of the biggest factors inhibiting good mitochondrial function. Endotoxin increases all the stress mediators like estrogen, cortisol, serotonin, lactate, nitric oxide etc. One source of chronic overproduction of endotoxin which is chronic infection with gram negative bacteria.
Similar to Lyme disease, the pathogen Chlamydia Pneumoniae is a gram-negative intracellular bacteria that causes chronic infection and is a constant source of endotoxin. It is strongly implicated with autoimmune conditions like Multiple Sclerosis, Chronic fatigue, Cardiac disease, Interstitial cystitis, Prostatitis, Crohn's disease, Inflammatory bowel disease, Alzheimer's Disease, Asthma, Arthritis, Fibromyalgia, Chronic refractory sinusitis, Macular Degeneration, and others for which medicine claims “there is no cause, no cure, we can only manage the symptoms with cortisone”. Not only does cortisone not cure these autoimmune conditions, it actually makes them worse by suppressing the immune system and causing mitochondrial dysfunction and hypoxia, which have been shown to greatly stimulate the growth of the pathogen. Estrogen is ofcourse known to cause hypoxia and low oxygen consumption, so you can see that both estrogen and cortisol are things which increase the chances of developing autoimmune conditions through the growth of intracellular bacteria like Chlamydia Pneumoniae. It also appears that having good mitochondrial function protects against the growth of this pathogen. I think Ray Peat would recommend B-vitamins, Methylene blue, Pregnenolone, Progesterone, DHEA, Magnesium, Caffeine, Aspirin, Quinones like K2, etc. to protect the mitochondrial system and maintain high ATP levels which protect the cell from invading pathogens. Now, if you already have an autoimmune disease and established Chlamydia Pneumonia infection, I think you should first try the things Ray Peat recommends and when it fails to eradicate the disease (because bacterial count is very high) the only option is to take antibiotics to eradicate the pathogen alongside stimulating good mitochondrial function (preventing hypoxia). Also other studies have found Iron and Tryptophan greatly stimulate the growth of Chlamydia Pneumoniae, which again confirms the work of Ray Peat in minimizing these nutrients.
Abstract
Effective growth and replication of obligate intracellular pathogens depend on host cell metabolism. How this is connected to host cell mitochondrial function has not been studied so far. Recent studies suggest that growth of intracellular bacteria such as Chlamydia pneumoniae is enhanced in a low oxygen environment, arguing for a particular mechanistic role of the mitochondrial respiration in controlling intracellular progeny. Metabolic changes in C. pneumoniae infected epithelial cells were analyzed under normoxic (O2 ≈ 20%) and hypoxic conditions (O2 < 3%). We observed that infection of epithelial cells with C. pneumoniae under normoxia impaired mitochondrial function characterized by an enhanced mitochondrial membrane potential and ROS generation. Knockdown and mutation of the host cell ATP synthase resulted in an increased chlamydial replication already under normoxic conditions. As expected, mitochondrial hyperpolarization was observed in non-infected control cells cultured under hypoxic conditions, which was beneficial for C. pneumoniae growth. Taken together, functional and genetically encoded mitochondrial dysfunction strongly promotes intracellular growth of C. pneumoniae.
Intro
Mitochondria play a major role in the generation of energy but also regulate cell death, production of reactive oxygen species (ROS), and sensing of glucose and oxygen (Heikal, 2010). Thus, mitochondrial function is a prerequisite for cell homeostasis and mitochondrial dysfunction has been linked to various pathologies e.g., cancer cell formation that is characterized by inhibition of cytochrome c induced apoptosis (Vaughn and Deshmukh, 2008). Moreover, mitochondrial dysfunction is a consequence of low oxygen concentrations referred to as hypoxia arising at pathophysiological sites. Thereby, cells need to metabolically adapt to hypoxia by switching the metabolism from oxidative phosphorylation (OXPHOS) to anaerobic glycolysis to maintain ATP supply. Mitochondrial dysfunction can be defined as abnormality in generation of ATP by OXPHOS, ROS production, regulation of apoptosis, regulation of cytoplasmic and mitochondrial matrix calcium, synthesis and catabolism of metabolites as well as mitochondrial trafficking (Brand and Nicholls, 2011).
A decrease in maximal respiration indicates a mitochondrial dysfunction (Brand and Nicholls, 2011). Impairment of the respiratory chain leads to an increased glycolysis, supporting tumor cell growth, and proliferation (Warburg, 1956). Further, an increased mitochondrial membrane potential, termed mitochondrial hyperpolarization, prevents apoptosis (Michelakis, 2008). Mitochondria mediate innate immune responses at different levels by supporting cellular metabolic reprogramming and the cytosolic immune signaling cascades (Monlun et al., 2016). Activation of macrophages and dendritic cells by pro-inflammatory stimuli causes a metabolic switch away from OXPHOS toward glycolysis (Kelly and O'Neill, 2015).
Numerous studies have described apoptosis regulation through the mitochondrial pathway in Chlamydia (Sharma and Rudel, 2009), but still there is a lack of information regarding other mitochondrial functions involved in chlamydial infection. It has previously been shown that Chlamydia trachomatis induces ROS production, which is beneficial for their development (Abdul-Sater et al., 2010; Boncompain et al., 2010; Chumduri et al., 2013). Further, C. trachomatis infection preserved the mitochondrial network (Chowdhury et al., 2017). Besides the production of ROS in Chlamydia pneumoniae, mitochondrial dysfunction is implicated with inflammasome activation in C. pneumoniae infected macrophages (Shimada et al., 2011).
Although the obligate intracellular bacterium C. pneumoniae relies on host cell metabolism, little is known about the influence of mitochondrial respiration on intracellular growth and progeny of C. pneumoniae.
While chlamydiae have a limited metabolic capacity due to their small genome size, they are still able to perform certain metabolic steps through the interconversion of metabolites obtained from host cells (Stephens et al., 1998; Kalman et al., 1999). Whole genome sequencing revealed metabolic genes of glycolysis and pentose phosphate pathway (PPP) (Stephens et al., 1998; Iliffe-Lee and McClarty, 1999; Kalman et al., 1999; McClarty, 1999). Furthermore, the chlamydial energy generation is maintained by functional components of the electron transport chain (ETC) and their own V-ATP synthase (McClarty, 1999; Gerard et al., 2002; Skipp et al., 2005). Finally, the chlamydial genome contains a cytochrome bd oxidase, which has been associated with microaerobic respiration in Coxiella burnettii under low oxygen conditions (Omsland et al., 2009).
In our model we focused on infections with C. pneumoniae, which showed a significantly enhanced growth under hypoxic conditions (Juul et al., 2007; Rupp et al., 2007; Szaszak et al., 2013). In this study we aimed to assess the mitochondrial activity during intracellular C. pneumoniae infection and investigated how mitochondrial dysfunction interferes with chlamydial growth and progeny. We utilized hypoxia as a model for analyzing mitochondrial dysfunction in a physiological condition and further targeted the F0-subunit of the host cell ATP synthase to validate the findings in a more defined setting.
Results
Abnormalities in the function of mitochondria are defining a mitochondrial dysfunction, such as impairment of the respiratory chain or mitochondrial hyperpolarization leading to increased mitochondrial membrane potential, preventing apoptosis (Michelakis, 2008; Brand and Nicholls, 2011). Further, mitochondrial dysfunction induces ROS (Murphy, 2009). ROS production during C. trachomatis infection is beneficial for bacterial development through activation of caspase-1 and hypoxia-inducible factor-1α (HIF-1α) (Prusty et al., 2012). Therefore, we hypothesize that these features would be beneficial for chlamydial growth. It has been previously shown that C. pneumoniae infection of mouse macrophages induced mitochondrial dysfunction (Shimada et al., 2011). Here we show that C. pneumoniae infected HEp-2 cells exhibit a mitochondrial dysfunction, characterized by decreased maximal respiration, hyperpolarization of the mitochondrial membrane potential and enhanced ROS generation. The decrease in maximal respiration was specific for active chlamydial growth since heat-inactivated C. pneumoniae did not affect the maximal respiration (data not shown).
In previous studies it was shown that a mutation in the ATP synthase 8 (ATP8) alters the mitochondrial performance and increases ROS (Yu et al., 2009). Further, Schröder et al. defined that ATP8 mutation leads to a mitochondrial dysfunction, characterized by increased ROS generation and low ATP (Schroder et al., 2016). For additional characterization of ATP8 mutant cells, we tried to establish Seahorse XF Cell Mito Stress Test. It was difficult to maintain a homogenous cell monolayer, which is necessary to perform this technique. Therefore, the respiration rate was too low for validation. We found that C. pneumoniae growth is indeed promoted by a predominant mitochondrial dysfunction in fibroblasts carrying ATP8 mutation. Likewise, knockdown of ATP5G1 of the ATP synthase led to an increased infectious progeny and genome copy numbers. As the generation time during exponential log phase was comparable between ATP5G1 siRNA treated and control cells we assume that primarily a shorter lag phase accounts for the increase in infectious progeny. A shortened lag phase was also described by Juul et al. during C. pneumoniae growth under hypoxic conditions accounting for enhanced infectious progeny (Juul et al., 2007).
Inhibition of host ATP synthase increases the mitochondrial membrane potential (Rego et al., 2001) and switches ATP production to glycolysis (Brand and Nicholls, 2011). Recently, Chowdhury et al. reported that the increased energy demand of C. trachomatis-infected cells is provided by elongated mitochondria as a result of higher fusion/fission ratio and that mitochondrial ATP is essential for C. trachomatis growth (Chowdhury et al., 2017). In contrast, our data indicate that in C. pneumoniae infection mitochondrial dysfunction seems to be the preferred phenotypic state. Different metabolic characteristics might occur due to the different tissue tropism and differences in their genome, which requires an individual adaption of the two species. So far, no data is available to demonstrate if a mitochondrial dysfunction is involved in the growth of C. pneumoniae under in vivo conditions.
We used hypoxia as physiological model since tissue displays different oxygen concentrations varying from 14.5% in the alveolar space to 5.6% in the peribronchial tissue (Ryan and Hickam, 1952; Herold et al., 1998). Moreover, oxygen concentration further declines due to growth of intracellular pathogens and inflammatory processes (Kempf et al., 2005; Eltzschig and Carmeliet, 2011; Campbell et al., 2014). In previous studies it was demonstrated that low oxygen conditions are beneficial for the intracellular growth of C. pneumoniae (Juul et al., 2007; Rupp et al., 2007; Szaszak et al., 2013). Mitochondrial dysfunction could be of relevance for enhanced intracellular growth under hypoxia since an inadequate oxygen supply leads to a defective OXPHOS. ETC activity is altered under hypoxia due to the oxygen-dependent regulation of a cytochrome c oxidase (COX) subunit (Semenza, 2007). Low oxygen inhibits COX (Chandel et al., 1998), resulting in a premature electron transfer at complex III (Semenza, 2007), thereby increasing the amount of ROS under hypoxia (Bell et al., 2007). Further, hypoxia impairs the activity of the ATP synthase, leading to a hyperpolarization of mitochondrial membrane potential, thus causing a switch to glycolysis (Chandel et al., 1997; Gao and Wolin, 2008). The here induced changes in mitochondrial hyperpolarization and ROS by hypoxia might play a crucial role for chlamydial growth, since they promote glycolysis and increase cellular longevity.
We could show that a metabolic switch associated with a mitochondrial dysfunction occurs during C. pneumoniae growth under hypoxia in HEp-2 cells. Oxygen is needed to produce energy and plays a fundamental role in cell metabolism (Carreau et al., 2011). Thus, our metabolic screen revealed metabolic alterations between normoxic and hypoxic conditions. We observed in non-infected cells that of all metabolites the carbohydrate metabolism was influenced to the greatest extent between normoxic and hypoxic conditions. Ojcius et al. showed that an enhanced carbohydrate metabolism was connected with C. psittaci infection, compensating the increased energy burden in infected cells (Ojcius et al., 1998). Therefore, the high variations in the carbohydrate metabolism seem to be beneficial for C. pneumoniae growth since C. pneumoniae takes up and utilizes these metabolites. Hence, the metabolic variations identified in non-infected cells between normoxia and hypoxia were abrogated when cells are infected with C. pneumoniae. Evidence for enhanced uptake of metabolites by C. pneumoniae is provided by transcriptomic data, where chlamydial transporters show enhanced expression under hypoxia. Since Chlamydia lack biosynthetic pathways for NAD and NAD(P), C. pneumoniae depends on host cell derived NAD uptake, maintained by the ATP/ADP translocase (Fisher et al., 2013). Indeed, we could show by transcriptome analysis that the ATP/ADP translocase (Cpn0614) was upregulated under hypoxic conditions. So far it is not possible to prove the involvement of the transporters in the enhanced growth since suitable techniques for genetic modification in this organism are missing. Thus, we suggest that the increased level of NAD(P)H in the chlamydial inclusion indicated an increased need for this metabolic coenzyme leading to enhanced metabolic activity of C. pneumoniae under hypoxic conditions. In addition, this was accompanied with enhanced transcription of chlamydial genes related to glycolysis and PPP. With the applied bioinformatic method used to get the probability of differentially expressed genes from transcriptome screen without replication and the validation of selected genes in multiple replicates by qRT-PCR we received a reliable picture of the chlamydial transcriptome. The relevance of chlamydial metabolic capabilities was indicated by Engström et al. where inhibition of chlamydial glucose-6-phosphate metabolism reduced C. trachomatis progeny (Engstrom et al., 2015).
We observed an increase of NAD(P)H intensity in the nucleus in non-infected as well as C. pneumoniae-infected cells under hypoxia-induced mitochondrial hyperpolarization. Enhanced NAD(P)H autofluorescence indicates high glycolytic activity since the autofluorescence increases in a glucose-dependent manner (Evans et al., 2003). These results suggest that the enhanced host cell glycolysis, caused by the hyperpolarization induced switch from OXPHOS to glycolysis, promotes the increased growth of C. pneumoniae under hypoxic conditions.
This study addresses for the first time that an increased growth of C. pneumoniae arises in cells with impaired mitochondrial function. We suggest that enhanced growth of C. pneumoniae under hypoxia is the result of hypoxia-induced mitochondrial dysfunction and the associated metabolic switch. However, we cannot exclude that the decrease in ROS under hypoxia during C. pneumoniae growth is responsible for the enhanced growth. Since the effects of our observations are moderate further characterization is still required to prove the biological relevance of our findings in upcoming studies using in vivo experiments.
This study massively confirms what Ray Peat has been writing about. Maintaining high respiration (oxidative phosphorylation) and low anaerobic glycolysis is the key to prevent chronic diseases. Focus on having good mitochondrial energy production and you will become resistant to many diseases. As everyone who follows Ray Peat knows, endotoxin is one of the biggest factors inhibiting good mitochondrial function. Endotoxin increases all the stress mediators like estrogen, cortisol, serotonin, lactate, nitric oxide etc. One source of chronic overproduction of endotoxin which is chronic infection with gram negative bacteria.
Similar to Lyme disease, the pathogen Chlamydia Pneumoniae is a gram-negative intracellular bacteria that causes chronic infection and is a constant source of endotoxin. It is strongly implicated with autoimmune conditions like Multiple Sclerosis, Chronic fatigue, Cardiac disease, Interstitial cystitis, Prostatitis, Crohn's disease, Inflammatory bowel disease, Alzheimer's Disease, Asthma, Arthritis, Fibromyalgia, Chronic refractory sinusitis, Macular Degeneration, and others for which medicine claims “there is no cause, no cure, we can only manage the symptoms with cortisone”. Not only does cortisone not cure these autoimmune conditions, it actually makes them worse by suppressing the immune system and causing mitochondrial dysfunction and hypoxia, which have been shown to greatly stimulate the growth of the pathogen. Estrogen is ofcourse known to cause hypoxia and low oxygen consumption, so you can see that both estrogen and cortisol are things which increase the chances of developing autoimmune conditions through the growth of intracellular bacteria like Chlamydia Pneumoniae. It also appears that having good mitochondrial function protects against the growth of this pathogen. I think Ray Peat would recommend B-vitamins, Methylene blue, Pregnenolone, Progesterone, DHEA, Magnesium, Caffeine, Aspirin, Quinones like K2, etc. to protect the mitochondrial system and maintain high ATP levels which protect the cell from invading pathogens. Now, if you already have an autoimmune disease and established Chlamydia Pneumonia infection, I think you should first try the things Ray Peat recommends and when it fails to eradicate the disease (because bacterial count is very high) the only option is to take antibiotics to eradicate the pathogen alongside stimulating good mitochondrial function (preventing hypoxia). Also other studies have found Iron and Tryptophan greatly stimulate the growth of Chlamydia Pneumoniae, which again confirms the work of Ray Peat in minimizing these nutrients.
Abstract
Effective growth and replication of obligate intracellular pathogens depend on host cell metabolism. How this is connected to host cell mitochondrial function has not been studied so far. Recent studies suggest that growth of intracellular bacteria such as Chlamydia pneumoniae is enhanced in a low oxygen environment, arguing for a particular mechanistic role of the mitochondrial respiration in controlling intracellular progeny. Metabolic changes in C. pneumoniae infected epithelial cells were analyzed under normoxic (O2 ≈ 20%) and hypoxic conditions (O2 < 3%). We observed that infection of epithelial cells with C. pneumoniae under normoxia impaired mitochondrial function characterized by an enhanced mitochondrial membrane potential and ROS generation. Knockdown and mutation of the host cell ATP synthase resulted in an increased chlamydial replication already under normoxic conditions. As expected, mitochondrial hyperpolarization was observed in non-infected control cells cultured under hypoxic conditions, which was beneficial for C. pneumoniae growth. Taken together, functional and genetically encoded mitochondrial dysfunction strongly promotes intracellular growth of C. pneumoniae.
Intro
Mitochondria play a major role in the generation of energy but also regulate cell death, production of reactive oxygen species (ROS), and sensing of glucose and oxygen (Heikal, 2010). Thus, mitochondrial function is a prerequisite for cell homeostasis and mitochondrial dysfunction has been linked to various pathologies e.g., cancer cell formation that is characterized by inhibition of cytochrome c induced apoptosis (Vaughn and Deshmukh, 2008). Moreover, mitochondrial dysfunction is a consequence of low oxygen concentrations referred to as hypoxia arising at pathophysiological sites. Thereby, cells need to metabolically adapt to hypoxia by switching the metabolism from oxidative phosphorylation (OXPHOS) to anaerobic glycolysis to maintain ATP supply. Mitochondrial dysfunction can be defined as abnormality in generation of ATP by OXPHOS, ROS production, regulation of apoptosis, regulation of cytoplasmic and mitochondrial matrix calcium, synthesis and catabolism of metabolites as well as mitochondrial trafficking (Brand and Nicholls, 2011).
A decrease in maximal respiration indicates a mitochondrial dysfunction (Brand and Nicholls, 2011). Impairment of the respiratory chain leads to an increased glycolysis, supporting tumor cell growth, and proliferation (Warburg, 1956). Further, an increased mitochondrial membrane potential, termed mitochondrial hyperpolarization, prevents apoptosis (Michelakis, 2008). Mitochondria mediate innate immune responses at different levels by supporting cellular metabolic reprogramming and the cytosolic immune signaling cascades (Monlun et al., 2016). Activation of macrophages and dendritic cells by pro-inflammatory stimuli causes a metabolic switch away from OXPHOS toward glycolysis (Kelly and O'Neill, 2015).
Numerous studies have described apoptosis regulation through the mitochondrial pathway in Chlamydia (Sharma and Rudel, 2009), but still there is a lack of information regarding other mitochondrial functions involved in chlamydial infection. It has previously been shown that Chlamydia trachomatis induces ROS production, which is beneficial for their development (Abdul-Sater et al., 2010; Boncompain et al., 2010; Chumduri et al., 2013). Further, C. trachomatis infection preserved the mitochondrial network (Chowdhury et al., 2017). Besides the production of ROS in Chlamydia pneumoniae, mitochondrial dysfunction is implicated with inflammasome activation in C. pneumoniae infected macrophages (Shimada et al., 2011).
Although the obligate intracellular bacterium C. pneumoniae relies on host cell metabolism, little is known about the influence of mitochondrial respiration on intracellular growth and progeny of C. pneumoniae.
While chlamydiae have a limited metabolic capacity due to their small genome size, they are still able to perform certain metabolic steps through the interconversion of metabolites obtained from host cells (Stephens et al., 1998; Kalman et al., 1999). Whole genome sequencing revealed metabolic genes of glycolysis and pentose phosphate pathway (PPP) (Stephens et al., 1998; Iliffe-Lee and McClarty, 1999; Kalman et al., 1999; McClarty, 1999). Furthermore, the chlamydial energy generation is maintained by functional components of the electron transport chain (ETC) and their own V-ATP synthase (McClarty, 1999; Gerard et al., 2002; Skipp et al., 2005). Finally, the chlamydial genome contains a cytochrome bd oxidase, which has been associated with microaerobic respiration in Coxiella burnettii under low oxygen conditions (Omsland et al., 2009).
In our model we focused on infections with C. pneumoniae, which showed a significantly enhanced growth under hypoxic conditions (Juul et al., 2007; Rupp et al., 2007; Szaszak et al., 2013). In this study we aimed to assess the mitochondrial activity during intracellular C. pneumoniae infection and investigated how mitochondrial dysfunction interferes with chlamydial growth and progeny. We utilized hypoxia as a model for analyzing mitochondrial dysfunction in a physiological condition and further targeted the F0-subunit of the host cell ATP synthase to validate the findings in a more defined setting.
Results
Abnormalities in the function of mitochondria are defining a mitochondrial dysfunction, such as impairment of the respiratory chain or mitochondrial hyperpolarization leading to increased mitochondrial membrane potential, preventing apoptosis (Michelakis, 2008; Brand and Nicholls, 2011). Further, mitochondrial dysfunction induces ROS (Murphy, 2009). ROS production during C. trachomatis infection is beneficial for bacterial development through activation of caspase-1 and hypoxia-inducible factor-1α (HIF-1α) (Prusty et al., 2012). Therefore, we hypothesize that these features would be beneficial for chlamydial growth. It has been previously shown that C. pneumoniae infection of mouse macrophages induced mitochondrial dysfunction (Shimada et al., 2011). Here we show that C. pneumoniae infected HEp-2 cells exhibit a mitochondrial dysfunction, characterized by decreased maximal respiration, hyperpolarization of the mitochondrial membrane potential and enhanced ROS generation. The decrease in maximal respiration was specific for active chlamydial growth since heat-inactivated C. pneumoniae did not affect the maximal respiration (data not shown).
In previous studies it was shown that a mutation in the ATP synthase 8 (ATP8) alters the mitochondrial performance and increases ROS (Yu et al., 2009). Further, Schröder et al. defined that ATP8 mutation leads to a mitochondrial dysfunction, characterized by increased ROS generation and low ATP (Schroder et al., 2016). For additional characterization of ATP8 mutant cells, we tried to establish Seahorse XF Cell Mito Stress Test. It was difficult to maintain a homogenous cell monolayer, which is necessary to perform this technique. Therefore, the respiration rate was too low for validation. We found that C. pneumoniae growth is indeed promoted by a predominant mitochondrial dysfunction in fibroblasts carrying ATP8 mutation. Likewise, knockdown of ATP5G1 of the ATP synthase led to an increased infectious progeny and genome copy numbers. As the generation time during exponential log phase was comparable between ATP5G1 siRNA treated and control cells we assume that primarily a shorter lag phase accounts for the increase in infectious progeny. A shortened lag phase was also described by Juul et al. during C. pneumoniae growth under hypoxic conditions accounting for enhanced infectious progeny (Juul et al., 2007).
Inhibition of host ATP synthase increases the mitochondrial membrane potential (Rego et al., 2001) and switches ATP production to glycolysis (Brand and Nicholls, 2011). Recently, Chowdhury et al. reported that the increased energy demand of C. trachomatis-infected cells is provided by elongated mitochondria as a result of higher fusion/fission ratio and that mitochondrial ATP is essential for C. trachomatis growth (Chowdhury et al., 2017). In contrast, our data indicate that in C. pneumoniae infection mitochondrial dysfunction seems to be the preferred phenotypic state. Different metabolic characteristics might occur due to the different tissue tropism and differences in their genome, which requires an individual adaption of the two species. So far, no data is available to demonstrate if a mitochondrial dysfunction is involved in the growth of C. pneumoniae under in vivo conditions.
We used hypoxia as physiological model since tissue displays different oxygen concentrations varying from 14.5% in the alveolar space to 5.6% in the peribronchial tissue (Ryan and Hickam, 1952; Herold et al., 1998). Moreover, oxygen concentration further declines due to growth of intracellular pathogens and inflammatory processes (Kempf et al., 2005; Eltzschig and Carmeliet, 2011; Campbell et al., 2014). In previous studies it was demonstrated that low oxygen conditions are beneficial for the intracellular growth of C. pneumoniae (Juul et al., 2007; Rupp et al., 2007; Szaszak et al., 2013). Mitochondrial dysfunction could be of relevance for enhanced intracellular growth under hypoxia since an inadequate oxygen supply leads to a defective OXPHOS. ETC activity is altered under hypoxia due to the oxygen-dependent regulation of a cytochrome c oxidase (COX) subunit (Semenza, 2007). Low oxygen inhibits COX (Chandel et al., 1998), resulting in a premature electron transfer at complex III (Semenza, 2007), thereby increasing the amount of ROS under hypoxia (Bell et al., 2007). Further, hypoxia impairs the activity of the ATP synthase, leading to a hyperpolarization of mitochondrial membrane potential, thus causing a switch to glycolysis (Chandel et al., 1997; Gao and Wolin, 2008). The here induced changes in mitochondrial hyperpolarization and ROS by hypoxia might play a crucial role for chlamydial growth, since they promote glycolysis and increase cellular longevity.
We could show that a metabolic switch associated with a mitochondrial dysfunction occurs during C. pneumoniae growth under hypoxia in HEp-2 cells. Oxygen is needed to produce energy and plays a fundamental role in cell metabolism (Carreau et al., 2011). Thus, our metabolic screen revealed metabolic alterations between normoxic and hypoxic conditions. We observed in non-infected cells that of all metabolites the carbohydrate metabolism was influenced to the greatest extent between normoxic and hypoxic conditions. Ojcius et al. showed that an enhanced carbohydrate metabolism was connected with C. psittaci infection, compensating the increased energy burden in infected cells (Ojcius et al., 1998). Therefore, the high variations in the carbohydrate metabolism seem to be beneficial for C. pneumoniae growth since C. pneumoniae takes up and utilizes these metabolites. Hence, the metabolic variations identified in non-infected cells between normoxia and hypoxia were abrogated when cells are infected with C. pneumoniae. Evidence for enhanced uptake of metabolites by C. pneumoniae is provided by transcriptomic data, where chlamydial transporters show enhanced expression under hypoxia. Since Chlamydia lack biosynthetic pathways for NAD and NAD(P), C. pneumoniae depends on host cell derived NAD uptake, maintained by the ATP/ADP translocase (Fisher et al., 2013). Indeed, we could show by transcriptome analysis that the ATP/ADP translocase (Cpn0614) was upregulated under hypoxic conditions. So far it is not possible to prove the involvement of the transporters in the enhanced growth since suitable techniques for genetic modification in this organism are missing. Thus, we suggest that the increased level of NAD(P)H in the chlamydial inclusion indicated an increased need for this metabolic coenzyme leading to enhanced metabolic activity of C. pneumoniae under hypoxic conditions. In addition, this was accompanied with enhanced transcription of chlamydial genes related to glycolysis and PPP. With the applied bioinformatic method used to get the probability of differentially expressed genes from transcriptome screen without replication and the validation of selected genes in multiple replicates by qRT-PCR we received a reliable picture of the chlamydial transcriptome. The relevance of chlamydial metabolic capabilities was indicated by Engström et al. where inhibition of chlamydial glucose-6-phosphate metabolism reduced C. trachomatis progeny (Engstrom et al., 2015).
We observed an increase of NAD(P)H intensity in the nucleus in non-infected as well as C. pneumoniae-infected cells under hypoxia-induced mitochondrial hyperpolarization. Enhanced NAD(P)H autofluorescence indicates high glycolytic activity since the autofluorescence increases in a glucose-dependent manner (Evans et al., 2003). These results suggest that the enhanced host cell glycolysis, caused by the hyperpolarization induced switch from OXPHOS to glycolysis, promotes the increased growth of C. pneumoniae under hypoxic conditions.
This study addresses for the first time that an increased growth of C. pneumoniae arises in cells with impaired mitochondrial function. We suggest that enhanced growth of C. pneumoniae under hypoxia is the result of hypoxia-induced mitochondrial dysfunction and the associated metabolic switch. However, we cannot exclude that the decrease in ROS under hypoxia during C. pneumoniae growth is responsible for the enhanced growth. Since the effects of our observations are moderate further characterization is still required to prove the biological relevance of our findings in upcoming studies using in vivo experiments.
