Eimeria bovis Macromeront Formation Induces Glycolytic Responses and Mitochondrial Changes in Primary Host Endothelial Cells

Eimeria bovis is an intracellular apicomplexan parasite that causes considerable economic losses in the cattle industry worldwide. During the first merogony, E. bovis forms large macromeronts with >140,000 merozoites I in host endothelial cells. Because this is a high-energy demanding process, E. bovis exploits the host cellular metabolism to fulfill its metabolic requirements. We here analyzed the carbohydrate-related energetic metabolism of E. bovis–infected primary bovine umbilical vein endothelial cells during first merogony and showed that during the infection, E. bovis–infected culture presented considerable changes in metabolic signatures, glycolytic, and mitochondrial responses. Thus, an increase in both oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were found in E. bovis–infected host cells indicating a shift from quiescent to energetic cell status. Enhanced levels of glucose and pyruvate consumption in addition to increased lactate production, suggesting an important role of glycolysis in E. bovis–infected culture from 12 days p.i. onward. This was also tested by glycolytic inhibitors (2-DG) treatment, which reduced the macromeront development and diminished merozoite I production. As an interesting finding, we observed that 2-DG treatment boosted sporozoite egress. Referring to mitochondrial activities, intracellular ROS production was increased toward the end of merogony, and mitochondrial potential was enhanced from 12 d p. i. onward in E. bovis–infected culture. Besides, morphological alterations of membrane potential signals also indicated mitochondrial dysfunction in macromeront-carrying host endothelial culture.

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Meningococcal Internalization into Human Endothelial and Epithelial Cells Is Triggered by the Influx of Extracellular l-Glutamate via GltT l-Glutamate ABC Transporter inNeisseria meningitidis

Infection and Immunity ◽

10.1128/iai.00497-10 ◽

2010 ◽

Vol 79 (1) ◽

pp. 380-392 ◽

Cited By ~ 10

Author(s):

Hideyuki Takahashi ◽

Kwang Sik Kim ◽

Haruo Watanabe

Keyword(s):

Endothelial Cells ◽

Abc Transporter ◽

Glutamate Uptake ◽

Umbilical Vein ◽

Glutamate Transporter ◽

Amino Acid Sequences ◽

Host Cells ◽

Epithelial Cell Line ◽

Neighboring Gene ◽

Experimental Conditions

ABSTRACTMeningococcal internalization into human cells is likely to be a consequence of meningococcal adhesion to human epithelial and endothelial cells. Here, we identified three transposon mutants ofNeisseria meningitidisthat were primarily defective in the internalization of human brain microvascular endothelial cells (HBMEC), with insertions occurring in thegltT(a sodium-independentl-glutamate transporter) gene or its neighboring gene,NMB1964(unknown function). NMB1964 was tentatively namedgltMin this study because of the presence of a mammalian cell entry (MCE)-related domain in the deduced amino acid sequences. The null ΔgltT-ΔgltM N. meningitidismutant was also defective in the internalization into human umbilical vein endothelial cells and the human lung carcinoma epithelial cell line A549, and the defect was suppressed by transcomplementation of the mutants withgltT+-gltM+genes. The intracellular survival of the ΔgltT-ΔgltMmutant in HBMEC was not largely different from that of the wild-type strain under our experimental conditions. Introduction of a1-bp deletion and amber or ochre mutations ingltT-gltMgenes resulted in the loss of efficient internalization into HBMEC. The defect in meningococcal internalization into HBMEC andl-glutamate uptake in the ΔgltT-ΔgltMmutant were suppressed only in strains expressing both GltT and GltM proteins. The efficiency of meningococcal invasion to HBMEC decreased underl-glutamate-depleted conditions. Furthermore, ezrin, a key membrane-cytoskeleton linker, accumulated beneath colonies of thegltT+-gltM+N. meningitidisstrain but not of the ΔgltT-ΔgltMmutant. These findings suggest thatl-glutamate influx via the GltT-GltMl-glutamate ABC transporter serves as a cue forN. meningitidisinternalization into host cells.

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Exoproteome from Leptospira interrogans and Host Cells during Infection: Leptospiral Virulence Factors and Cellular Proteins Involved in Stress and Inflammation

10.21203/rs.3.rs-37165/v1 ◽

2020 ◽

Author(s):

Weilin Hu ◽

Muhammad Imran ◽

Kai-Xuan Li ◽

David M. Ojcius ◽

Ai-Hua Sun ◽

...

Keyword(s):

Endothelial Cells ◽

Virulence Factors ◽

Inflammatory Cytokines ◽

Vascular Endothelial Cells ◽

Umbilical Vein ◽

Host Cells ◽

Leptospira Interrogans ◽

Vascular Endothelial ◽

Macrophage Infection ◽

Blood Coagulation Factors

Abstract Background: Leptospirosis, caused mainly by Leptospira interrogans, is a global zoonotic infectious disease. Macrophages and vascular endothelial cells are the main host cells for L. interrogans during infection, but the proteins released from the pathogen and the two host cells during infection remain mostly unknown.Results: Cellular supernatant proteins (CSPs) from human THP-1 macrophages or umbilical vein endothelial cells (HUVECs) infected with L. interrogans strain Lai were extracted by TCA/FASP methods. The exoproteins in the CSPs were identified by LC-MS/MS. Viability of the leptospires and host cells during infection was confirmed by confocal microscopy and MTT. The results showed that higher co-culture temperature (from 28°C to 37°C) and different biochemical environments cause a large change in the exoproteome of the spirochete. L. interrogans increased levels of leptospiral exoproteins related to stress, signal transduction and virulence factors, while the lipoprotein antigens LipL41, LipL21 and/or Loa22 were not detected. During infection of macrophages and endothelial cells, there was a large increase in host-cell exoproteins involved in stress response, complement pathways (C4/5/7/8), inflammatory cytokines (IL-6, TNF-α, MIF, MCP-1 and GM-CSF), extracellular matrix proteins (FN, LN and COLs), and blood coagulation factors. One-third of the leptospires and infected THP-1 macrophages died during macrophage infection, but nearly all the leptospires and endothelial cells remained viable during endothelial cell infection.Conclusions: Infection causes stress reponses for both leptospires and human macrophages and vascular endothelial cells and release of virulence factors, alteration of surface leptospiral lipoprotein antigens and secretion of complement components and inflammatory cytokines from host cells.

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Proteasome-Independent Activation of Nuclear Factor κB in Cytoplasmic Extracts from Human Endothelial Cells byRickettsia rickettsii

Infection and Immunity ◽

10.1128/iai.66.5.1827-1833.1998 ◽

1998 ◽

Vol 66 (5) ◽

pp. 1827-1833 ◽

Cited By ~ 41

Author(s):

Sanjeev K. Sahni ◽

Daniel J. Van Antwerp ◽

Marina E. Eremeeva ◽

David J. Silverman ◽

Victor J. Marder ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Umbilical Vein ◽

Spotted Fever ◽

Nuclear Factor Κb ◽

Host Cells ◽

Nuclear Factor ◽

Rocky Mountain Spotted Fever ◽

Free System

ABSTRACT Interaction of many infectious agents with eukaryotic host cells is known to cause activation of the ubiquitous transcription factor nuclear factor κB (NF-κB) (U. Siebenlist, G. Franzoso, and K. Brown, Annu. Rev. Cell Biol. 10:405–455, 1994). Recently, we reported a biphasic pattern of NF-κB activation in cultured human umbilical vein endothelial cells consequent to infection withRickettsia rickettsii, an obligate intracellular gram-negative bacterium and the etiologic agent of Rocky Mountain spotted fever (L. A. Sporn, S. K. Sahni, N. B. Lerner, V. J. Marder, D. J. Silverman, L. C. Turpin, and A. L. Schwab, Infect. Immun. 65:2786–2791, 1997). In the present study, we describe activation of NF-κB in a cell-free system, accomplished by addition of partially purified R. rickettsii to endothelial cell cytoplasmic extracts. This activation was rapid, reaching maximal levels at 60 min, and was dependent on the number ofR. rickettsii organisms added. Antibody supershift assays using monospecific antisera against NF-κB subunits (p50 and p65) confirmed the authenticity of the gel-shifted complexes and identified both p50-p50 homodimers and p50-p65 heterodimers as constituents of the activated NF-κB pool. Activation occurred independently of the presence of endothelial cell membranes and was not inhibited by removal of the endothelial cell proteasome. Lack of involvement of the proteasome was further confirmed in assays using the peptide-aldehyde proteasome inhibitor MG 132. Activation was not ATP dependent since no change in activation resulted from addition of an excess of the unhydrolyzable ATP analog ATPγS, supplementation with exogenous ATP, or hydrolysis of endogenous ATP with ATPase. Furthermore, Western blot analysis before and after in vitro activation failed to demonstrate phosphorylation of serine 32 or degradation of the cytoplasmic pool of IκBα. This lack of IκBα involvement was supported by the finding that R. rickettsii can induce NF-κB activation in cytoplasmic extracts prepared from T24 bladder carcinoma cells and human embryo fibroblasts stably transfected with a superrepressor phosphorylation mutant of IκBα, rendering NF-κB inactivatable by many known signals. Thus, evidence is provided for a potentially novel NF-κB activation pathway wherein R. rickettsii may interact with and activate host cell transcriptional machinery independently of the involvement of the proteasome or known signal transduction pathways.

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Eimeria bovis infections induce G1 cell cycle arrest and a senescence-like phenotype in endothelial host cells

Parasitology ◽

10.1017/s0031182020002097 ◽

2020 ◽

pp. 1-13

Author(s):

Zahady D. Velásquez ◽

Sara López-Osorio ◽

Daniel Waiger ◽

Carolina Manosalva ◽

Learta Pervizaj-Oruqaj ◽

...

Keyword(s):

Cell Cycle ◽

Endothelial Cells ◽

Cell Cycle Arrest ◽

Host Cell ◽

Cyclin B1 ◽

Host Cells ◽

Phenotypic Change ◽

Cyclin E1 ◽

Cycle Arrest ◽

Eimeria Bovis

Abstract Apicomplexan parasites are well-known to modulate their host cells at diverse functional levels. As such, apicomplexan-induced alteration of host cellular cell cycle was described and appeared dependent on both, parasite species and host cell type. As a striking evidence of species-specific reactions, we here show that Eimeria bovis drives primary bovine umbilical vein endothelial cells (BUVECs) into a senescence-like phenotype during merogony I. In line with senescence characteristics, E. bovis induces a phenotypic change in host cell nuclei being characterized by nucleolar fusion and heterochromatin-enriched peripheries. By fibrillarin staining we confirm nucleoli sizes to be increased and their number per nucleus to be reduced in E. bovis-infected BUVECs. Additionally, nuclei of E. bovis-infected BUVECs showed enhanced signals for HH3K9me2 as heterochromatin marker thereby indicating an infection-induced change in heterochromatin transition. Furthermore, E. bovis-infected BUVECs show an enhanced β-galactosidase activity, which is a well-known marker of senescence. Referring to cell cycle progression, protein abundance profiles in E. bovis-infected endothelial cells revealed an up-regulation of cyclin E1 thereby indicating a cell cycle arrest at G1/S transition, signifying a senescence key feature. Similarly, abundance of G2 phase-specific cyclin B1 was found to be downregulated at the late phase of macromeront formation. Overall, these data indicate that the slow proliferative intracellular parasite E. bovis drives its host endothelial cells in a senescence-like status. So far, it remains to be elucidated whether this phenomenon indeed reflects an intentionally induced mechanism to profit from host cell-derived energy and metabolites present in a non-dividing cellular status.

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Invasion of Aortic and Heart Endothelial Cells byPorphyromonas gingivalis

Infection and Immunity ◽

10.1128/iai.66.11.5337-5343.1998 ◽

1998 ◽

Vol 66 (11) ◽

pp. 5337-5343 ◽

Cited By ~ 264

Author(s):

Rajashri G. Deshpande ◽

Mahfuz B. Khan ◽

Caroline Attardo Genco

Keyword(s):

Electron Microscopy ◽

Endothelial Cells ◽

Protein Synthesis ◽

De Novo ◽

Umbilical Vein ◽

Host Cells ◽

Periodontal Pathogen ◽

Host Immune System ◽

Umbilical Vein Endothelial Cells ◽

De Novo Protein Synthesis

ABSTRACT Invasion of host cells is believed to be an important strategy utilized by a number of pathogens, which affords them protection from the host immune system. The connective tissues of the periodontium are extremely well vascularized, which allows invading microorganisms, such as the periodontal pathogen Porphyromonas gingivalis, to readily enter the bloodstream. However, the ability of P. gingivalis to actively invade endothelial cells has not been previously examined. In this study, we demonstrate that P. gingivalis can invade bovine and human endothelial cells as assessed by an antibiotic protection assay and by transmission and scanning electron microscopy. P. gingivalis A7436 was demonstrated to adhere to and to invade fetal bovine heart endothelial cells (FBHEC), bovine aortic endothelial cells (BAEC), and human umbilical vein endothelial cells (HUVEC). Invasion efficiencies of 0.1, 0.2, and 0.3% were obtained with BAEC, HUVEC, and FBHEC, respectively. Invasion of FBHEC and BAEC by P. gingivalis A7436 assessed by electron microscopy revealed the formation of microvillus-like extensions around adherent bacteria followed by the engulfment of the pathogen within vacuoles. Invasion of BAEC by P. gingivalisA7436 was inhibited by cytochalasin D, nocodazole, staurosporine, protease inhibitors, and sodium azide, indicating that cytoskeletal rearrangements, protein phosphorylation, energy metabolism, andP. gingivalis proteases are essential for invasion. In contrast, addition of rifampin, nalidixic acid, and chloramphenicol had little effect on invasion, indicating that bacterial RNA, DNA, and de novo protein synthesis are not required for P. gingivalisinvasion of endothelial cells. Likewise de novo protein synthesis by endothelial cells was not required for invasion by P. gingivalis. P. gingivalis 381 was demonstrated to adhere to and to invade BAEC (0.11 and 0.1% efficiency, respectively). However, adherence and invasion of the corresponding fimA mutant DPG3, which lacks the major fimbriae, was not detected. These results indicate thatP. gingivalis can actively invade endothelial cells and that fimbriae are required for this process. P. gingivalisinvasion of endothelial cells may represent another strategy utilized by this pathogen to thwart the host immune response.

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