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

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.

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Hypoxic-response elements in the oncolytic parvovirus Minute virus of mice do not allow for increased vector production at low oxygen concentration

Journal of General Virology ◽

10.1099/vir.0.81754-0 ◽

2006 ◽

Vol 87 (5) ◽

pp. 1197-1201 ◽

Cited By ~ 6

Author(s):

Charlotte Servais ◽

Perrine Caillet-Fauquet ◽

Marie-Louise Draps ◽

Thierry Velu ◽

Yvan de Launoit ◽

...

Keyword(s):

Cell Metabolism ◽

Consensus Sequence ◽

High Titre ◽

Host Cells ◽

Low Oxygen ◽

Minute Virus Of Mice ◽

Minute Virus ◽

Responsive Element ◽

Low Oxygen Concentration

Vectors derived from the autonomous parvovirus Minute virus of mice, MVM(p), are promising tools for the gene therapy of cancer. The validation of their in vivo anti-tumour effect is, however, hampered by the difficulty to produce high-titre stocks. In an attempt to increase vector titres, host cells were subjected to low oxygen tension (hypoxia). It has been shown that a number of viruses are produced at higher titres under these conditions. This is the case, among others, for another member of the family Parvoviridae, the erythrovirus B19 virus. Hypoxia stabilizes a hypoxia-inducible transcription factor (HIF-1α) that interacts with a ‘hypoxia-responsive element’ (HRE), the consensus sequence of which (A/GCGTG) is present in the B19 and MVM promoters. Whilst the native P4 promoter was induced weakly in hypoxia, vector production was reduced dramatically, and adding HRE elements to the P4 promoter of the vector did not alleviate this reduction. Hypoxia has many effects on cell metabolism. Therefore, even if the P4 promoter is activated, the cellular factors that are required for the completion of the parvoviral life cycle may not be expressed.

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Response of C2C12 Myoblasts to Hypoxia: The Relative Roles of Glucose and Oxygen in Adaptive Cellular Metabolism

BioMed Research International ◽

10.1155/2013/326346 ◽

2013 ◽

Vol 2013 ◽

pp. 1-10 ◽

Cited By ~ 6

Author(s):

Wei Li ◽

Zhen-Fu Hu ◽

Bin Chen ◽

Guo-Xin Ni

Keyword(s):

Oxygen Consumption ◽

Glucose Concentration ◽

Cellular Response ◽

Cell Function ◽

Cell Metabolism ◽

Lactate Production ◽

Cellular Metabolism ◽

Glucose Consumption ◽

C2c12 Cells ◽

Low Oxygen

Background. Oxygen and glucose are two important nutrients for mammalian cell function. In this study, the effect of glucose and oxygen concentrations on C2C12 cellular metabolism was characterized with an emphasis on detecting whether cells show oxygen conformance (OC) in response to hypoxia.Methods. After C2C12 cells being cultured in the levels of glucose at 0.6 mM (LG), 5.6 mM (MG), or 23.3 mM(HG) under normoxic or hypoxic (1% oxygen) condition, cellular oxygen consumption, glucose consumption, lactate production, and metabolic status were determined. Short-term oxygen consumption was measured with a novel oxygen biosensor technique. Longer-term measurements were performed with standard glucose, lactate, and cell metabolism assays.Results. It was found that oxygen depletion in normoxia is dependent on the glucose concentration in the medium. Cellular glucose uptake and lactate production increased significantly in hypoxia than those in normoxia. In hypoxia the cellular response to the level of glucose was different to that in normoxia. The metabolic activities decreased while glucose concentration increased in normoxia, while in hypoxia, metabolic activity was reduced in LG and MG, but unchanged in HG condition. The OC phenomenon was not observed in the present study.Conclusions. Our findings suggested that a combination of low oxygen and low glucose damages the viability of C2C12 cells more seriously than low oxygen alone. In addition, when there is sufficient glucose, C2C12 cells will respond to hypoxia by upregulating anaerobic respiration, as shown by lactate production.

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A Single-Pass Type I Membrane Protein from the Apicomplexan Parasite Cryptosporidium parvum with Nanomolar Binding Affinity to Host Cell Surface

Microorganisms ◽

10.3390/microorganisms9051015 ◽

2021 ◽

Vol 9 (5) ◽

pp. 1015

Author(s):

Tianyu Zhang ◽

Xin Gao ◽

Dongqiang Wang ◽

Jixue Zhao ◽

Nan Zhang ◽

...

Keyword(s):

Membrane Protein ◽

Cell Surface ◽

Host Cell ◽

Binding Affinity ◽

Cryptosporidium Parvum ◽

Host Cells ◽

Watery Diarrhea ◽

Type I ◽

Single Pass ◽

Host Cell Surface

Cryptosporidium parvum is a globally recognized zoonotic parasite of medical and veterinary importance. This parasite mainly infects intestinal epithelial cells and causes mild to severe watery diarrhea that could be deadly in patients with weakened or defect immunity. However, its molecular interactions with hosts and pathogenesis, an important part in adaptation of parasitic lifestyle, remain poorly understood. Here we report the identification and characterization of a C. parvum T-cell immunomodulatory protein homolog (CpTIPH). CpTIPH is a 901-aa single-pass type I membrane protein encoded by cgd5_830 gene that also contains a short Vibrio, Colwellia, Bradyrhizobium and Shewanella (VCBS) repeat and relatively long integrin alpha (ITGA) N-terminus domain. Immunofluorescence assay confirmed the location of CpTIPH on the cell surface of C. parvum sporozoites. In congruence with the presence of VCBS repeat and ITGA domain, CpTIPH displayed high, nanomolar binding affinity to host cell surface (i.e., Kd(App) at 16.2 to 44.7 nM on fixed HCT-8 and CHO-K1 cells, respectively). The involvement of CpTIPH in the parasite invasion is partly supported by experiments showing that an anti-CpTIPH antibody could partially block the invasion of C. parvum sporozoites into host cells. These observations provide a strong basis for further investigation of the roles of CpTIPH in parasite-host cell interactions.

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

Microorganisms ◽

10.3390/microorganisms9061144 ◽

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.

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Cellular and immunological basis of the host-parasite relationship during infection withNeospora caninum

Parasitology ◽

10.1017/s0031182006000485 ◽

2006 ◽

Vol 133 (3) ◽

pp. 261-278 ◽

Cited By ~ 76

Author(s):

A. HEMPHILL ◽

N. VONLAUFEN ◽

A. NAGULESWARAN

Keyword(s):

Host Cell ◽

Cell Biology ◽

Neospora Caninum ◽

Host Cells ◽

Term Survival ◽

Prevention Of Infection ◽

Parasite Relationship ◽

Potential Applications

Neospora caninumis an apicomplexan parasite that is closely related toToxoplasma gondii, the causative agent of toxoplasmosis in humans and domestic animals. However, in contrast toT. gondii, N. caninumrepresents a major cause of abortion in cattle, pointing towards distinct differences in the biology of these two species. There are 3 distinct key features that represent potential targets for prevention of infection or intervention against disease caused byN. caninum. Firstly, tachyzoites are capable of infecting a large variety of host cellsin vitroandin vivo. Secondly, the parasite exploits its ability to respond to alterations in living conditions by converting into another stage (tachyzoite-to-bradyzoite orvice versa). Thirdly, by analogy withT. gondii, this parasite has evolved mechanisms that modulate its host cells according to its own requirements, and these must, especially in the case of the bradyzoite stage, involve mechanisms that ensure long-term survival of not only the parasite but also of the host cell. In order to elucidate the molecular and cellular bases of these important features ofN. caninum, cell culture-based approaches and laboratory animal models are being exploited. In this review, we will summarize the current achievements related to host cell and parasite cell biology, and will discuss potential applications for prevention of infection and/or disease by reviewing corresponding work performed in murine laboratory infection models and in cattle.

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LncRNA GHET1 Promotes Hypoxia-Induced Glycolysis, Proliferation, and Invasion in Triple-Negative Breast Cancer Through the Hippo/YAP Signaling Pathway

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.643515 ◽

2021 ◽

Vol 9 ◽

Author(s):

Yu Wang ◽

Shuwei Liu

Keyword(s):

Breast Cancer ◽

Signaling Pathway ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Nuclear Translocation ◽

Lactate Production ◽

Glucose Consumption ◽

Breast Epithelial Cell ◽

Epithelial Cell Line

ObjectiveThis study was to assess the specific impacts and mechanism of lncRNA GHET1 in the development of triple-negative breast cancer (TNBC).MethodsThe lncRNA GHET1 expression in TNBC tissues and adjacent healthy tissues was detected by qRT-PCR, and its expression was then measured at the cellular level, including TNBC cells and human normal breast epithelial cell line MCF10A. On the completion of transfection of negative shRNA or lncRNA GHET1 shRNA, the TNBC cells, HCC1937 and MDA-MB-468, were then cultured in a normoxia or hypoxia environment, respectively. 5-Ethynyl-2′-deoxyuridine (EdU) assay, colony formation assay, and transwell assay were applicable to the determination of cell proliferation, cell viability, and invasion in each group, respectively. Reagent kits were used for testing glucose consumption and lactate production levels. HCC1937 cells with knockdown or overexpression of lncRNA GHET1 were injected into the nude mice, followed by the examination of resulting tumor volume and weight. The distribution and expression of Hippo/YAP signaling pathway-related proteins were probed using western blotting.ResultsHighly expressed lncRNA GHET1 in TNBC tissues and cells and induction of lncRNA GHET1 by hypoxia were proved. Knockdown of lncRNA GHET1 significantly reduced proliferation, viability, and invasion of TNBC cells, and decreased glucose consumption and lactate production levels under the hypoxia condition. Furthermore, lncRNA GHET1 knockdown decreased HIF-1α expression in hypoxia and significantly inhibited tumor development in vivo. Knockdown of lncRNA GHET1 increased the phosphorylation levels of LATS1 and Yes-associated protein (YAP) to retain YAP within the cytoplasm, while the overexpression of lncRNA GHET1 or hypoxia promoted nuclear translocation of YAP and TNBC development.ConclusionLncRNA GHET1 expression can be induced by hypoxia, which leads to excessive activation of the Hippo/YAP signaling pathway, thus promoting TNBC progression.

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Blocking circ-CNST suppresses malignant behaviors of osteosarcoma cells and inhibits glycolysis through circ-CNST-miR-578-LDHA/PDK1 ceRNA networks

Journal of Orthopaedic Surgery and Research ◽

10.1186/s13018-021-02427-0 ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Rui Hu ◽

Shan Chen ◽

Jianxin Yan

Keyword(s):

Colony Formation ◽

Lactate Production ◽

Glucose Consumption ◽

Pyruvate Dehydrogenase Kinase ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Physical Interaction ◽

U2os Cells

Abstract Background CircRNA CNST (circ-CNST) is a newly identified biomarker for prognosis of osteosarcoma (OS). However, its role in OS progression remains to be well documented. Methods Expression of circ-CNST, microRNA (miR)-578, lactate dehydrogenase A (LDHA), and pyruvate dehydrogenase kinase 1 (PDK1) was detected by quantitative real-time polymerase chain reaction and Western blotting. The physical interaction was confirmed by dual-luciferase reporter assay. Cell behaviors and glycolysis were measured by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay, colony formation assay, flow cytometry, transwell assays, xenograft experiment, and commercial kits. Results Circ-CNST was upregulated in human OS tissues and cells, accompanied with downregulation of miR-578 and upregulation of LDHA and PDK1. There were negative correlations between miR-578 expression and circ-CNST or LDHA/PDK1 in OS tissues. Moreover, high circ-CNST/LDHA/PDK1 or low miR-578 might predict shorter overall survival, advanced TNM stages, and lymph node metastasis. Physically, miR-578 was targeted by circ-CNST, and miR-578 could target LDHA/PDK1. Functionally, blocking circ-CNST and restoring miR-578 enhanced apoptosis rate and suppressed cell proliferation, colony formation, migration, and invasion in 143B and U2OS cells, accompanied with decreased glucose consumption, lactate production, and adenosine triphosphate (ATP)/adenosine diphosphate (ADP) ratio. Furthermore, in vivo growth of U2OS cells was retarded by silencing circ-CNST. Depletion of miR-578 could counteract the suppressive role of circ-CNST deficiency in 143B and U2OS cells, and restoring LDHA or PDK1 partially reversed the role of miR-578 inhibition as well. Conclusion Circ-CNST knockdown could antagonize malignant behaviors and glycolysis of OS cells by regulating miR-578-LDHA/PDK1 axes.

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

10.1101/120527 ◽

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.

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Fluorescence-Reported Allelic Exchange Mutagenesis-Mediated Gene Deletion Indicates a Requirement for Chlamydia trachomatis Tarp during In Vivo Infectivity and Reveals a Specific Role for the C Terminus during Cellular Invasion

Infection and Immunity ◽

10.1128/iai.00841-19 ◽

2020 ◽

Vol 88 (5) ◽

Author(s):

Susmita Ghosh ◽

Elizabeth A. Ruelke ◽

Joshua C. Ferrell ◽

Maria D. Bodero ◽

Kenneth A. Fields ◽

...

Keyword(s):

Chlamydia Trachomatis ◽

Host Cell ◽

Host Cells ◽

Actin Binding ◽

Wild Type ◽

Content Type ◽

Allelic Exchange ◽

Binding Domains

ABSTRACT The translocated actin recruiting phosphoprotein (Tarp) is a multidomain type III secreted effector used by Chlamydia trachomatis. In aggregate, existing data suggest a role of this effector in initiating new infections. As new genetic tools began to emerge to study chlamydial genes in vivo, we speculated as to what degree Tarp function contributes to Chlamydia’s ability to parasitize mammalian host cells. To address this question, we generated a complete tarP deletion mutant using the fluorescence-reported allelic exchange mutagenesis (FRAEM) technique and complemented the mutant in trans with wild-type tarP or mutant tarP alleles engineered to harbor in-frame domain deletions. We provide evidence for the significant role of Tarp in C. trachomatis invasion of host cells. Complementation studies indicate that the C-terminal filamentous actin (F-actin)-binding domains are responsible for Tarp-mediated invasion efficiency. Wild-type C. trachomatis entry into HeLa cells resulted in host cell shape changes, whereas the tarP mutant did not. Finally, using a novel cis complementation approach, C. trachomatis lacking tarP demonstrated significant attenuation in a murine genital tract infection model. Together, these data provide definitive genetic evidence for the critical role of the Tarp F-actin-binding domains in host cell invasion and for the Tarp effector as a bona fide C. trachomatis virulence factor.

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Core N-Glycan Structures Are Critical for the Pathogenicity of Cryptococcus neoformans by Modulating Host Cell Death

mBio ◽

10.1128/mbio.00711-20 ◽

2020 ◽

Vol 11 (3) ◽

Author(s):

Eun Jung Thak ◽

Su-Bin Lee ◽

Shengjie Xu-Vanpala ◽

Dong-Jik Lee ◽

Seung-Yeon Chung ◽

...

Keyword(s):

Cell Death ◽

Fungal Infection ◽

Host Cell ◽

Host Cells ◽

Glycan Structure ◽

Macrophage Cell ◽

Content Type ◽

Host Cell Death ◽

Intact Core

ABSTRACT Cryptococcus neoformans is a human-pathogenic fungal pathogen that causes life-threatening meningoencephalitis in immunocompromised individuals. To investigate the roles of N-glycan core structure in cryptococcal pathogenicity, we constructed mutant strains of C. neoformans with defects in the assembly of lipid-linked N-glycans in the luminal side of the endoplasmic reticulum (ER). Deletion of ALG3 (alg3Δ), which encodes dolichyl-phosphate-mannose (Dol-P-Man)-dependent α-1,3-mannosyltransferase, resulted in the production of truncated neutral N-glycans carrying five mannose residues as a major species. Despite moderate or nondetectable defects in virulence-associated phenotypes in vitro, the alg3Δ mutant was avirulent in a mouse model of systemic cryptococcosis. Notably, the mutant did not show defects in early stages of host cell interaction during infection, including attachment to lung epithelial cells, opsonic/nonopsonic phagocytosis, and manipulation of phagosome acidification. However, the ability to drive macrophage cell death was greatly decreased in this mutant, without loss of cell wall remodeling capacity. Furthermore, deletion of ALG9 and ALG12, encoding Dol-P-Man-dependent α-1,2-mannosyltransferases and α-1,6-mannosyltransferases, generating truncated core N-glycans with six and seven mannose residues, respectively, also displayed remarkably reduced macrophage cell death and in vivo virulence. However, secretion levels of interleukin-1β (IL-1β) were not reduced in the bone marrow-derived dendritic cells obtained from Asc- and Gsdmd-deficient mice infected with the alg3Δ mutant strain, excluding the possibility that pyroptosis is a main host cell death pathway dependent on intact core N-glycans. Our results demonstrated N-glycan structures as a critical feature in modulating death of host cells, which is exploited by as a strategy for host cell escape for dissemination of C. neoformans. IMPORTANCE We previously reported that the outer mannose chains of N-glycans are dispensable for the virulence of C. neoformans, which is in stark contrast to findings for the other human-pathogenic yeast, Candida albicans. Here, we present evidence that an intact core N-glycan structure is required for C. neoformans pathogenicity by systematically analyzing alg3Δ, alg9Δ, and alg12Δ strains that have defects in lipid-linked N-glycan assembly and in in vivo virulence. The alg null mutants producing truncated core N-glycans were defective in inducing host cell death after phagocytosis, which is triggered as a mechanism of pulmonary escape and dissemination of C. neoformans, thus becoming inactive in causing fatal infection. The results clearly demonstrated the critical features of the N-glycan structure in mediating the interaction with host cells during fungal infection. The delineation of the roles of protein glycosylation in fungal pathogenesis not only provides insight into the glycan-based fungal infection mechanism but also will aid in the development of novel antifungal agents.

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