scholarly journals Brucella abortusinduces a Warburg shift in host metabolism that is linked to enhanced intracellular survival of the pathogen

2017 ◽  
Author(s):  
Daniel M. Czyż ◽  
Jonathan Willett ◽  
Sean Crosson
Keyword(s):  
Lactic Acid ◽  
Host Cell ◽  
Brucella Abortus ◽  
Cell Metabolism ◽  
Lactate Production ◽  
Human Macrophage ◽  
Metabolic Shift ◽  
Intracellular Infections ◽  
Host Metabolism

ABSTRACTIntracellular bacterial pathogens exploit host cell resources to replicate and survive inside the host. Targeting these host systems is one promising approach to developing novel antimicrobials to treat intracellular infections. We show that human macrophage-like cells infected withBrucella abortusundergo a metabolic shift characterized by attenuated tricarboxylic acid cycle metabolism, reduced amino acid consumption, altered mitochondrial localization, and increased lactate production. This shift to an aerobic glycolytic state resembles the Warburg effect, a change in energy production that is well-described in cancer cells, and also occurs in activated inflammatory cells.B. abortusefficiently uses lactic acid as its sole carbon and energy source and requires the ability to metabolize lactate for normal survival in human macrophage-like cells. We demonstrate that chemical inhibitors of host glycolysis and lactate production do not affectin vitrogrowth ofB. abortusin axenic culture, but decrease its survival in the intracellular niche. Our data support a model in which infection shifts host metabolism to a Warburg-like state, andB. abortususes this change in metabolism to promote intracellular survival. Pharmacological perturbation of these features of host cell metabolism may be a useful strategy to inhibit infection by intracellular pathogens.IMPORTANCEBrucellaspp. are intracellular bacterial pathogens that cause disease in a range of mammals, including livestock. Transmission from livestock to humans is common and can lead to chronic human disease. Human macrophage-like cells infected withBrucella abortusundergo a Warburg-like metabolic shift to an aerobic glycolytic state where the host cells produce lactic acid and have reduced amino acid catabolism. We provide evidence that the pathogen can exploit this change in host metabolism to support growth and survival in the intracellular niche. Drugs that inhibit this shift in host cell metabolism inhibit intracellular replication and decrease the survival ofB. abortusin anin vitroinfection model; these drugs may be broadly useful therapeutics for intracellular infections.

scholarly journals Brucella abortus Induces a Warburg Shift in Host Metabolism That Is Linked to Enhanced Intracellular Survival of the Pathogen

2017 ◽  
Vol 199 (15) ◽  
Author(s):  
Daniel M. Czyż ◽  
Jonathan W. Willett ◽  
Sean Crosson
Keyword(s):  
Host Cell ◽  
Brucella Abortus ◽  
Cell Metabolism ◽  
Lactate Production ◽  
Human Macrophage ◽  
Metabolic Shift ◽  
Content Type ◽  
Intracellular Infections ◽  
Host Metabolism

ABSTRACT Intracellular bacterial pathogens exploit host cell resources to replicate and survive inside the host. Targeting these host systems is one promising approach to developing novel antimicrobials to treat intracellular infections. We show that human macrophage-like cells infected with Brucella abortus undergo a metabolic shift characterized by attenuated tricarboxylic acid cycle metabolism, reduced amino acid consumption, altered mitochondrial localization, and increased lactate production. This shift to an aerobic glycolytic state resembles the Warburg effect, a change in energy production that is well described in cancer cells and also occurs in activated inflammatory cells. B. abortus efficiently uses lactic acid as its sole carbon and energy source and requires the ability to metabolize lactate for normal survival in human macrophage-like cells. We demonstrate that chemical inhibitors of host glycolysis and lactate production do not affect in vitro growth of B. abortus in axenic culture but decrease its survival in the intracellular niche. Our data support a model in which infection shifts host metabolism to a Warburg-like state, and B. abortus uses this change in metabolism to promote intracellular survival. Pharmacological perturbation of these features of host cell metabolism may be a useful strategy to inhibit infection by intracellular pathogens. IMPORTANCE Brucella spp. are intracellular bacterial pathogens that cause disease in a range of mammals, including livestock. Transmission from livestock to humans is common and can lead to chronic human disease. Human macrophage-like cells infected with Brucella abortus undergo a Warburg-like metabolic shift to an aerobic glycolytic state where the host cells produce lactic acid and have reduced amino acid catabolism. We provide evidence that the pathogen can exploit this change in host metabolism to support growth and survival in the intracellular niche. Drugs that inhibit this shift in host cell metabolism inhibit intracellular replication and decrease the survival of B. abortus in an in vitro infection model; these drugs may be broadly useful therapeutics for intracellular infections.

scholarly journals Metabolic Signatures of Cryptosporidium parvum-Infected HCT-8 Cells and Impact of Selected Metabolic Inhibitors on C. parvum Infection under Physioxia and Hyperoxia

Biology ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 60
Author(s):  
Juan Vélez ◽  
Zahady Velasquez ◽  
Liliana M. R. Silva ◽  
Ulrich Gärtner ◽  
Klaus Failing ◽  
...  
Keyword(s):  
Host Cell ◽  
Cryptosporidium Parvum ◽  
Cell Metabolism ◽  
Lactate Production ◽  
Glucose Consumption ◽  
Host Cells ◽  
Metabolic Inhibitors ◽  
Low Oxygen ◽  
Metabolic Signatures

Cryptosporidium parvum is an apicomplexan zoonotic parasite recognized as the second leading-cause of diarrhoea-induced mortality in children. In contrast to other apicomplexans, C.parvum has minimalistic metabolic capacities which are almost exclusively based on glycolysis. Consequently, C. parvum is highly dependent on its host cell metabolism. In vivo (within the intestine) infected epithelial host cells are typically exposed to low oxygen pressure (1–11% O2, termed physioxia). Here, we comparatively analyzed the metabolic signatures of C. parvum-infected HCT-8 cells cultured under both, hyperoxia (21% O2), representing the standard oxygen condition used in most experimental settings, and physioxia (5% O2), to be closer to the in vivo situation. The most pronounced effect of C. parvum infection on host cell metabolism was, on one side, an increase in glucose and glutamine uptake, and on the other side, an increase in lactate release. When cultured in a glutamine-deficient medium, C. parvum infection led to a massive increase in glucose consumption and lactate production. Together, these results point to the important role of both glycolysis and glutaminolysis during C. parvum intracellular replication. Referring to obtained metabolic signatures, we targeted glycolysis as well as glutaminolysis in C. parvum-infected host cells by using the inhibitors lonidamine [inhibitor of hexokinase, mitochondrial carrier protein (MCP) and monocarboxylate transporters (MCT) 1, 2, 4], galloflavin (lactate dehydrogenase inhibitor), syrosingopine (MCT1- and MCT4 inhibitor) and compound 968 (glutaminase inhibitor) under hyperoxic and physioxic conditions. In line with metabolic signatures, all inhibitors significantly reduced parasite replication under both oxygen conditions, thereby proving both energy-related metabolic pathways, glycolysis and glutaminolysis, but also lactate export mechanisms via MCTs as pivotal for C. parvum under in vivo physioxic conditions of mammals.

scholarly journals Revisiting Ehrlichia ruminantium Replication Cycle Using Proteomics: The Host and the Bacterium Perspectives

2021 ◽  
Vol 9 (6) ◽  
pp. 1144
Author(s):  
Isabel Marcelino ◽  
Philippe Holzmuller ◽  
Ana Coelho ◽  
Gabriel Mazzucchelli ◽  
Bernard Fernandez ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Host Cell ◽  
Cell Metabolism ◽  
Replication Cycle ◽  
Host Cells ◽  
Ehrlichia Ruminantium ◽  
Host Interaction ◽  
Infected Cells ◽  
Related Proteins

The Rickettsiales Ehrlichia ruminantium, the causal agent of the fatal tick-borne disease Heartwater, induces severe damage to the vascular endothelium in ruminants. Nevertheless, E. ruminantium-induced pathobiology remains largely unknown. Our work paves the way for understanding this phenomenon by using quantitative proteomic analyses (2D-DIGE-MS/MS, 1DE-nanoLC-MS/MS and biotin-nanoUPLC-MS/MS) of host bovine aorta endothelial cells (BAE) during the in vitro bacterium intracellular replication cycle. We detect 265 bacterial proteins (including virulence factors), at all time-points of the E. ruminantium replication cycle, highlighting a dynamic bacterium–host interaction. We show that E. ruminantium infection modulates the expression of 433 host proteins: 98 being over-expressed, 161 under-expressed, 140 detected only in infected BAE cells and 34 exclusively detected in non-infected cells. Cystoscape integrated data analysis shows that these proteins lead to major changes in host cell immune responses, host cell metabolism and vesicle trafficking, with a clear involvement of inflammation-related proteins in this process. Our findings led to the first model of E. ruminantium infection in host cells in vitro, and we highlight potential biomarkers of E. ruminantium infection in endothelial cells (such as ROCK1, TMEM16K, Albumin and PTPN1), which may be important to further combat Heartwater, namely by developing non-antibiotic-based strategies.

scholarly journals Intracellular localisation of Mycobacterium tuberculosis affects antibiotic efficacy

2020 ◽  
Author(s):  
Pierre Santucci ◽  
Daniel J. Greenwood ◽  
Antony Fearns ◽  
Kai Chen ◽  
Haibo Jiang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Host Cell ◽  
Combination Chemotherapy ◽  
Human Macrophage ◽  
Ion Microscopy ◽  
Antibiotic Efficacy ◽  
Subcellular Level ◽  
Intracellular Localisation

AbstractTo be effective, chemotherapy against tuberculosis (TB) must kill the intracellular population of Mycobacterium tuberculosis (Mtb). However, how host cell environments affect antibiotic accumulation and efficacy remains elusive. Pyrazinamide (PZA) is a key antibiotic against TB, yet its behaviour is not fully understood. Here, by using correlative light, electron, and ion microscopy to image PZA at the subcellular level, we investigated how human macrophage environments affect PZA activity. We discovered that PZA accumulates heterogeneously between individual bacteria in multiple host cell environments. Crucially, Mtb phagosomal localisation and acidification increase PZA accumulation and efficacy. By imaging two antibiotics commonly used in combined TB therapy, we showed that bedaquiline (BDQ) significantly enhances PZA accumulation by a host cell mediated mechanism. Thus, intracellular localisation and specific microenvironments affect PZA accumulation and efficacy; explaining the potent in vivo efficacy compared to its modest in vitro activity and the critical contribution to TB combination chemotherapy.

scholarly journals Characterization of increased mucus production of HT29-MTX-E12 cells grown under Semi-Wet interface with Mechanical Stimulation

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0261191
Author(s):  
Janneke Elzinga ◽  
Benthe van der Lugt ◽  
Clara Belzer ◽  
Wilma T. Steegenga
Keyword(s):  
Cell Line ◽  
Mechanical Stimulation ◽  
Cell Metabolism ◽  
Lactate Production ◽  
Intestinal Cell ◽  
Mucus Layer ◽  
Mucus Production ◽  
Intestinal Mucus ◽  
Cells Cultured

The intestinal mucus layer plays a crucial role in human health. To study intestinal mucus function and structure in vitro, the mucus-producing intestinal cell line HT29-MTX-E12 has been commonly used. However, this cell line produces only low amounts of the intestine-specific MUC2. It has been shown previously that HT29-MTX-E12 cells cultured under Semi-Wet interface with Mechanical Stimulation (SWMS) produced higher amounts of MUC2, concomitant with a thicker mucus layer, compared to cells cultured conventionally. However, it remains unknown which underlying pathways are involved. Therefore, we aimed to further explore the cellular processes underlying the increased MUC2 production by HT29-MTX-E12 cells grown under SWMS conditions. Cells grown on Transwell membranes for 14 days under static and SWMS conditions (after cell seeding and attachment) were subjected to transcriptome analysis to investigate underlying molecular pathways at gene expression level. Caco-2 and LS174T cell lines were included as references. We characterized how SWMS conditions affected HT29-MTX-E12 cells in terms of epithelial barrier integrity, by measuring transepithelial electrical resistance, and cell metabolism, by monitoring pH and lactate production per molecule glucose of the conditioned medium. We confirmed higher MUC2 production under SWMS conditions at gene and protein level and demonstrated that this culturing method primarily stimulated cell growth. In addition, we also found evidence for a more aerobic cell metabolism under SWMS, as shown previously for similar models. In summary, we suggest different mechanisms by which MUC2 production is enhanced under SWMS and propose potential applications of this model in future studies.

The metabolism of glucose and pyruvate in rat retina

1962 ◽  
Vol 156 (963) ◽  
pp. 139-143 ◽  
Keyword(s):  
Carbon Dioxide ◽  
Lactic Acid ◽  
Quantitative Data ◽  
Lactate Production ◽  
Anaerobic Conditions ◽  
Aerobic Conditions ◽  
Rat Retina ◽  
Adult Rat ◽  
Total Glucose

The metabolism of [U- 14 C]glucose and [3- 14 C]pyruvate in the adult rat retina is described. In vitro under aerobic conditions, in either phosphate or bicarbonate medium, glucose was converted into lactate, carbon dioxide, glutamate, γ -aminobutyrate, aspartate, glutamine and alanine. Under anaerobic conditions, total glucose metabolized was reduced to 60 to 70% of that under aerobic conditions, lactic acid being the only metabolic product detected. Under aerobic conditions [3- 14 C]pyruvate was converted by the retina into the same metabolites as was glucose. The quantitative data for oxygen uptake and 14 CO 2 formation were similar to those obtained with glucose as substrate; lactate production was lower and amino acid formation higher.

EFFECT OF LASALOCID, MONENSIN AND THIOPEPTIN ON LACTATE PRODUCTION FROM IN VITRO RUMEN FERMENTATION OF STARCH

1986 ◽  
Vol 66 (1) ◽  
pp. 129-139 ◽  
Author(s):  
T. G. NAGARAJA ◽  
S. J. GALITZER ◽  
D. L. HARMON ◽  
S. M. DENNIS
Keyword(s):  
Lactic Acid ◽  
Rumen Fluid ◽  
Lactate Concentration ◽  
Lactate Production ◽  
Starch Fermentation ◽  
Key Words Antibiotics ◽  
Lactate Fermentation ◽  
Set Up ◽  
In Vitro Rumen Fermentation

Starch fermentations with strained rumen fluid and centrifuged rumen fluid devoid of protozoa were set up to test the effect of lasalocid, monensin, and thiopeptin on L(+) and D(−) lactate production. Protozoa-free rumen fluid was the supernatant from low-speed centrifugation of strained rumen fluid. Starch fermentation in the control (no antibiotic) with centrifuged rumen fluid resulted in higher lactate concentration than the fermentation with strained rumen fluid. Decreased lactate production with strained rumen fluid was attributed to sequestration of starch by protozoa and to enhanced lactate fermentation. Addition of lasalocid or monensin (1.5–48.0 μg mL−1) to the fermentation enhanced L(+) and D(−) lactate production in the presence of protozoa. In the absence of protozoa, lasalocid and monensin inhibited L(+) lactate production; however, D(−) lactate concentration was unaffected. Increased lactate production by lasalocid and monensin in the presence of protozoa was possibly due to inhibition of protozoal engulfment of starch. Thiopeptin had no effect on lactate production in the presence of protozoa but in the absence of protozoa lactate production was inhibited. Similar antibiotic responses were observed at different starch amounts (0.5, 1.5 and 3.0 g) and with starch types (soluble, corn and wheat) and with rumen fluid collected from defaunated cattle. Key words: Antibiotics, cattle, rumen, starch, fermentation, lactic acid

scholarly journals The PFKFB3 Inhibitor AZ67 Inhibits Angiogenesis Independently of Glycolysis Inhibition

2021 ◽  
Vol 22 (11) ◽  
pp. 5970
Author(s):  
Besa Emini Veseli ◽  
Pieter Van Wielendaele ◽  
Mirela Delibegovic ◽  
Wim Martinet ◽  
Guido R. Y. De Meyer
Keyword(s):  
Biological Effects ◽  
Cell Metabolism ◽  
Protein A ◽  
Lactate Production ◽  
Tube Formation ◽  
Significant Inhibition ◽  
Titration Calorimetry ◽  
Inhibition Of Angiogenesis

Angiogenesis is the process of new blood vessel formation. In this complex orchestrated growth, many factors are included. Lately, focus has shifted to endothelial cell metabolism, particularly to the PFKFB3 protein, a key regulatory enzyme of the glycolytic pathway. A variety of inhibitors of this important target have been studied, and a plethora of biological effects related to the process of angiogenesis have been reported. However, recent studies have disputed their mechanism of action, questioning whether all the effects are indeed due to PFKFB3 inhibition. Remarkably, the most well-studied inhibitor, 3PO, does not bind to PFKFB3, raising questions about this target. In our study, we aimed to elucidate the effects of PFKFB3 inhibition in angiogenesis by using the small molecule AZ67. We used isothermal titration calorimetry and confirmed binding to PFKFB3. In vitro, AZ67 did not decrease lactate production in endothelial cells (ECs), nor ATP levels, but exhibited good inhibitory efficacy in the tube-formation assay. Surprisingly, this was independent of EC migratory and proliferative abilities, as this was not diminished upon treatment. Strikingly however, even the lowest dose of AZ67 demonstrated significant inhibition of angiogenesis in vivo. To our knowledge, this is the first study to demonstrate that the process of angiogenesis can be disrupted by targeting PFKFB3 independently of glycolysis inhibition.

Enhanced Antibacterial Activity Of Doxycycline And Rifampicin Combination Loaded In Nanoparticles Against Intracellular Brucella Abortus

2021 ◽  
Vol 18 ◽  
Author(s):  
Shilpa Dawre ◽  
Padma V. Devarajan ◽  
Abdul Samad
Keyword(s):  
Antibacterial Activity ◽  
Brucella Abortus ◽  
Colloidal Stability ◽  
Polymeric Nanoparticles ◽  
Entrapment Efficiency ◽  
Diffusion Method ◽  
Human Macrophage ◽  
Scanning Calorimetry ◽  
Macrophage Cells

Background: Brucellosis is a zoonotic disease and prevalent in livestock animals. The bacteria reside inside the macrophage cells of host. The WHO endorsed the combination treatment therapy for brucellosis as compared to monotherapy to avoid relapse and resistance. Therefore, we developed nanoparticles incorporating doxycycline and rifampicin in combination. Objective: The aim of the study is to develop polymeric nanoparticles incorporating doxycycline as well as rifampicin and investigate the antibacterial activity of nanoparticles in U937 human macrophage cells infected with B. abortus. Methods: Polymeric nanoparticles were developed by emulsion-solvent diffusion method and characterization was done. Results: The nanoparticles with high entrapment efficiency of both drugs were developed successfully. Scanning electron microscopy revealed spherical morphology with a size ranging ~450nm, which can be easily engulfed by macrophages. Zeta potential confirmed colloidal stability. Differential scanning calorimetry and X-ray diffraction suggested amorphization of doxycycline and rifampicin in nanoparticles. Fourier transfer infrared spectroscopy could not confirm interaction of drugs with AOT. In vitro haemolysis study confirmed safety of nanoparticles (<10%) for IV administration. Further, nanoparticles revealed the sustained release of both drugs, which followed diffusion kinetics. Nanoparticles were found stable for 6 months as per WHO guidelines. The internalization study revealed nanoparticles can be easily uptake by U-937 human macrophage cells. The efficacy study demonstrated significantly high antibacterial activity of nanoparticles as compared to free drug solution in U937 human macrophage cells infected with Brucella abortus. Conclusion: It can be concluded that developed nanoparticles entrapping doxycycline and rifampicin combination could be considered as a promising delivery system for enhancing the antibacterial activity against Brucella abortus.

scholarly journals Establishment of a reverse genetics system for Schmallenberg virus, a newly emerged orthobunyavirus in Europe

2013 ◽  
Vol 94 (4) ◽  
pp. 851-859 ◽  
Author(s):  
Richard M. Elliott ◽  
Gjon Blakqori ◽  
Ingeborg C. van Knippenberg ◽  
Elina Koudriakova ◽  
Ping Li ◽  
...  
Keyword(s):  
Host Cell ◽  
Recombinant Virus ◽  
Reverse Genetics ◽  
Cell Metabolism ◽  
Schmallenberg Virus ◽  
Wild Type Virus ◽  
Cell Protein ◽  
Virus Recombinant ◽  
Rescue System

Schmallenberg virus (SBV) is a newly emerged orthobunyavirus that has caused widespread disease in cattle, sheep and goats in Europe. Like other orthobunyaviruses, SBV is characterized by a tripartite negative-sense RNA genome that encodes four structural and two non-structural proteins. This study showed that SBV has a wide in vitro host range, and that BHK-21 cells are a convenient host for both SBV propagation and assay by plaque titration. The SBV genome segments were cloned as cDNA and a three-plasmid rescue system was established to recover infectious virus. Recombinant virus behaved similarly in cell culture to authentic virus. The ORF for the non-structural NSs protein, encoded on the smallest genome segment, was disrupted by introduction of translation stop codons in the appropriate cDNA, and when this plasmid was used in reverse genetics, a recombinant virus that lacked NSs expression was recovered. This virus had reduced capacity to shut-off host-cell protein synthesis compared with the wild-type virus. In addition, the NSs-deleted virus induced interferon (IFN) in cells, indicating that, like other orthobunyaviruses, NSs functions as an IFN antagonist, most probably by globally inhibiting host-cell metabolism. The development of a robust reverse genetics system for SBV will facilitate investigation of its pathogenic mechanisms as well as the creation of attenuated strains that could be candidate vaccines.

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