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1.
PLoS Pathog ; 15(5): e1007800, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31116795

RESUMO

Staphylococcus aureus is a leading cause of endovascular infections. This bacterial pathogen uses a diverse array of surface adhesins to clump in blood and adhere to vessel walls, leading to endothelial damage, development of intravascular vegetations and secondary infectious foci, and overall disease progression. In this work, we describe a novel strategy used by S. aureus to control adhesion and clumping through activity of the ArlRS two-component regulatory system, and its downstream effector MgrA. Utilizing a combination of in vitro cellular assays, and single-cell atomic force microscopy, we demonstrated that inactivation of this ArlRS-MgrA cascade inhibits S. aureus adhesion to a vast array of relevant host molecules (fibrinogen, fibronectin, von Willebrand factor, collagen), its clumping with fibrinogen, and its attachment to human endothelial cells and vascular structures. This impact on S. aureus adhesion was apparent in low shear environments, and in physiological levels of shear stress, as well as in vivo in mouse models. These effects were likely mediated by the de-repression of giant surface proteins Ebh, SraP, and SasG, caused by inactivation of the ArlRS-MgrA cascade. In our in vitro assays, these giant proteins collectively shielded the function of other surface adhesins and impaired their binding to cognate ligands. Finally, we demonstrated that the ArlRS-MgrA regulatory cascade is a druggable target through the identification of a small-molecule inhibitor of ArlRS signaling. Our findings suggest a novel approach for the pharmacological treatment and prevention of S. aureus endovascular infections through targeting the ArlRS-MgrA regulatory system.


Assuntos
Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Endotélio Vascular/microbiologia , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/fisiologia , Animais , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Feminino , Fibrinogênio/genética , Fibrinogênio/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/patologia
2.
Tuberculosis (Edinb) ; 116S: S118-S122, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31072690

RESUMO

Mycobacterium tuberculosis (MTB) is a pathogen that infects and kills millions yearly. The mycobacterium's cell wall glycolipid trehalose 6,6'-dimycolate (TDM) has been used historically to model MTB induced inflammation and granuloma formation. Alterations to the model can significantly influence the induced pathology. One such method incorporates intraperitoneal pre-exposure, after which the intravenous injection of TDM generates pathological damage effectively mimicking the hypercoagulation, thrombus formation, and tissue remodeling apparent in lungs of infected individuals. The purpose of these experiments is to examine the histological inflammation involved in the TDM mouse model that induces development of the hemorrhagic response. TDM induced lungs of C57BL/6 mice to undergo granulomatous inflammation. Further histological examination of the peak response demonstrated tissue remodeling consistent with hypercoagulation. The observed vascular occlusion indicates that obstruction likely occurs due to subendothelial localized activity leading to restriction of blood vessel lumens. Trichrome staining revealed that associated damage in the hypercoagulation model is consistent with intra endothelial cell accumulation of innate cells, bordered by collagen deposition in the underlying parenchyma. Overall, the hypercoagulation model represents a comparative pathological instrument for understanding mechanisms underlying development of hemorrhage and vascular occlusion seen during MTB infection.


Assuntos
Fatores Corda/metabolismo , Endotélio Vascular/patologia , Granuloma do Sistema Respiratório/patologia , Pulmão/irrigação sanguínea , Mycobacterium tuberculosis/metabolismo , Pneumonia/patologia , Tuberculose Pulmonar/patologia , Animais , Coagulação Sanguínea , Modelos Animais de Doenças , Endotélio Vascular/microbiologia , Feminino , Granuloma do Sistema Respiratório/sangue , Granuloma do Sistema Respiratório/induzido quimicamente , Granuloma do Sistema Respiratório/microbiologia , Pulmão/microbiologia , Camundongos Endogâmicos C57BL , Pneumonia/sangue , Pneumonia/induzido quimicamente , Pneumonia/microbiologia , Tuberculose Pulmonar/sangue , Tuberculose Pulmonar/induzido quimicamente , Tuberculose Pulmonar/microbiologia , Remodelação Vascular
3.
PLoS Pathog ; 15(5): e1007737, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31071198

RESUMO

Streptococcus equi subsp. zooepidemicus (SEZ) is a zoonotic pathogen capable of causing meningitis in humans. The mechanisms that enable pathogens to traverse the blood-brain barrier (BBB) are incompletely understood. Here, we investigated the role of a newly identified Fic domain-containing protein, BifA, in SEZ virulence. BifA was required for SEZ to cross the BBB and to cause meningitis in mice. BifA also enhanced SEZ translocation across human Brain Microvascular Endothelial Cell (hBMEC) monolayers. Purified BifA or its Fic domain-containing C-terminus alone were able to enter into hBMECs, leading to disruption of monolayer barrier integrity. A SILAC-based proteomic screen revealed that BifA binds moesin. BifA's Fic domain was required for its binding to this regulator of host cell cytoskeletal processes. BifA treatment of hBMECs led to moesin phosphorylation and downstream RhoA activation. Inhibition of moesin activation or moesin depletion in hBMEC monolayers abrogated BifA-mediated increases in barrier permeability and SEZ's capacity to translocate across monolayers. Thus, BifA activation of moesin appears to constitute a key mechanism by which SEZ disrupts endothelial monolayer integrity to penetrate the BBB.


Assuntos
Proteínas de Bactérias/metabolismo , Barreira Hematoencefálica/patologia , Encéfalo/patologia , Endotélio Vascular/patologia , Proteínas dos Microfilamentos/metabolismo , Streptococcus/fisiologia , Virulência , Animais , Proteínas de Bactérias/genética , Transporte Biológico , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/microbiologia , Encéfalo/metabolismo , Encéfalo/microbiologia , Permeabilidade da Membrana Celular , Células Cultivadas , Endotélio Vascular/metabolismo , Endotélio Vascular/microbiologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C
4.
Methods Mol Biol ; 1969: 135-148, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30877675

RESUMO

Bacterial meningitis is a serious, life-threatening infection of the central nervous system (CNS). To cause meningitis, bacteria must interact with and penetrate the meningeal blood-cerebrospinal fluid barrier (mB/CSFB), which comprises highly specialized brain endothelial cells. Neisseria meningitidis (meningococcus) is a leading cause of bacterial meningitis, and examination meningococcus' interaction with the BBB is critical for understanding disease progression. To examine specific interactions, in vitro mB/CSFB models have been developed and employed and are of great importance because in vivo models have been difficult to produce considering Neisseria meningitidis is exclusively a human pathogen. Most in vitro blood-brain barrier and mB/CSF models use primary and immortalized brain endothelial cells, and these models have been used to examine bacterial-mB/CSFB interactions by a variety of pathogens. This chapter describes the use of past and current in vitro brain endothelial cells to model Neisseria meningitidis interaction with the mB/CSFB, and inform on the standard operating procedure for their use.


Assuntos
Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Endotélio Vascular/metabolismo , Interações Hospedeiro-Patógeno , Infecções Meningocócicas/metabolismo , Neisseria meningitidis/fisiologia , Fatores de Virulência/metabolismo , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/microbiologia , Encéfalo/citologia , Encéfalo/microbiologia , Endotélio Vascular/citologia , Endotélio Vascular/microbiologia , Humanos , Infecções Meningocócicas/microbiologia , Ligação Proteica
5.
Emerg Microbes Infect ; 8(1): 413-425, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30898074

RESUMO

Candidatus (Ca.) Neoehrlichia mikurensis is the cause of neoehrlichiosis, an emerging tick-borne infectious disease characterized by fever and vascular events. The bacterium belongs to the Anaplasmataceae, a family of obligate intracellular pathogens, but has not previously been cultivated, and it is uncertain which cell types it infects. The goals of this study were to cultivate Ca. N. mikurensis in cell lines and to identify possible target cells for human infection. Blood components derived from infected patients were inoculated into cell lines of both tick and human origin. Bacterial growth in the cell cultures was monitored by real-time PCR and imaging flow cytometry. Ca. N. mikurensis was successfully propagated from the blood of immunocompromised neoehrlichiosis patients in two Ixodes spp. tick cell lines following incubation periods of 7-20 weeks. Human primary endothelial cells derived from skin microvasculature as well as pulmonary artery were also susceptible to infection with tick cell-derived bacteria. Finally, Ca. N. mikurensis was visualized within circulating endothelial cells of two neoehrlichiosis patients. To conclude, we report the first successful isolation and propagation of Ca. N. mikurensis from clinical isolates and identify human vascular endothelial cells as a target of infection.


Assuntos
Infecções por Anaplasmataceae/microbiologia , Anaplasmataceae/crescimento & desenvolvimento , Anaplasmataceae/isolamento & purificação , Células Endoteliais/microbiologia , Endotélio Vascular/microbiologia , Tropismo Viral , Animais , Técnicas de Cultura de Células , Citometria de Fluxo , Humanos , Ixodes , Reação em Cadeia da Polimerase em Tempo Real
6.
Microbiol Spectr ; 7(2)2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30848239

RESUMO

A wide variety of pathogens reach the circulatory system during viral, parasitic, fungal, and bacterial infections, causing clinically diverse pathologies. Such systemic infections are usually severe and frequently life-threatening despite intensive care, in particular during the age of antibiotic resistance. Because of its position at the interface between the blood and the rest of the organism, the endothelium plays a central role during these infections. Using several examples of systemic infections, we explore the diversity of interactions between pathogens and the endothelium. These examples reveal that bacterial pathogens target specific vascular beds and affect most aspects of endothelial cell biology, ranging from cellular junction stability to endothelial cell proliferation and inflammation.


Assuntos
Bactérias/patogenicidade , Infecções Bacterianas/microbiologia , Endotélio/microbiologia , Animais , Infecções Bacterianas/sangue , Farmacorresistência Bacteriana , Endotélio Vascular/microbiologia , Interações Hospedeiro-Patógeno , Humanos
7.
Cell Mol Neurobiol ; 38(7): 1349-1368, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30117097

RESUMO

This review aims to elucidate the different mechanisms of blood brain barrier (BBB) disruption that may occur due to invasion by different types of bacteria, as well as to show the bacteria-host interactions that assist the bacterial pathogen in invading the brain. For example, platelet-activating factor receptor (PAFR) is responsible for brain invasion during the adhesion of pneumococci to brain endothelial cells, which might lead to brain invasion. Additionally, the major adhesin of the pneumococcal pilus-1, RrgA is able to bind the BBB endothelial receptors: polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM-1), thus leading to invasion of the brain. Moreover, Streptococcus pneumoniae choline binding protein A (CbpA) targets the common carboxy-terminal domain of the laminin receptor (LR) establishing initial contact with brain endothelium that might result in BBB invasion. Furthermore, BBB disruption may occur by S. pneumoniae penetration through increasing in pro-inflammatory markers and endothelial permeability. In contrast, adhesion, invasion, and translocation through or between endothelial cells can be done by S. pneumoniae without any disruption to the vascular endothelium, upon BBB penetration. Internalins (InlA and InlB) of Listeria monocytogenes interact with its cellular receptors E-cadherin and mesenchymal-epithelial transition (MET) to facilitate invading the brain. L. monocytogenes species activate NF-κB in endothelial cells, encouraging the expression of P- and E-selectin, intercellular adhesion molecule 1 (ICAM-1), and Vascular cell adhesion protein 1 (VCAM-1), as well as IL-6 and IL-8 and monocyte chemoattractant protein-1 (MCP-1), all these markers assist in BBB disruption. Bacillus anthracis species interrupt both adherens junctions (AJs) and tight junctions (TJs), leading to BBB disruption. Brain microvascular endothelial cells (BMECs) permeability and BBB disruption are induced via interendothelial junction proteins reduction as well as up-regulation of IL-1α, IL-1ß, IL-6, TNF-α, MCP-1, macrophage inflammatory proteins-1 alpha (MIP1α) markers in Staphylococcus aureus species. Streptococcus agalactiae or Group B Streptococcus toxins (GBS) enhance IL-8 and ICAM-1 as well as nitric oxide (NO) production from endothelial cells via the expression of inducible nitric oxide synthase (iNOS) enhancement, resulting in BBB disruption. While Gram-negative bacteria, Haemophilus influenza OmpP2 is able to target the common carboxy-terminal domain of LR to start initial interaction with brain endothelium, then invade the brain. H. influenza type b (HiB), can induce BBB permeability through TJ disruption. LR and PAFR binding sites have been recognized as common routes of CNS entrance by Neisseria meningitidis. N. meningitidis species also initiate binding to BMECs and induces AJs deformation, as well as inducing specific cleavage of the TJ component occludin through the release of host MMP-8. Escherichia coli bind to BMECs through LR, resulting in IL-6 and IL-8 release and iNOS production, as well as resulting in disassembly of TJs between endothelial cells, facilitating BBB disruption. Therefore, obtaining knowledge of BBB disruption by different types of bacterial species will provide a picture of how the bacteria enter the central nervous system (CNS) which might support the discovery of therapeutic strategies for each bacteria to control and manage infection.


Assuntos
Bactérias/metabolismo , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/microbiologia , Encéfalo/metabolismo , Encéfalo/microbiologia , Permeabilidade Capilar/fisiologia , Animais , Bactérias/crescimento & desenvolvimento , Bactérias/patogenicidade , Endotélio Vascular/metabolismo , Endotélio Vascular/microbiologia , Interações Hospedeiro-Parasita/fisiologia , Humanos
8.
Sci Rep ; 8(1): 12708, 2018 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-30139948

RESUMO

Sulforaphane (SFN) has been shown to protect the brain vascular system and effectively reduce ischemic injuries and cognitive deficits. Given the robust cerebrovascular protection afforded by SFN, the objective of this study was to profile these effects in vitro using primary mouse brain microvascular endothelial cells and focusing on cellular redox, metabolism and detoxification functions. We used a mouse MitoChip array developed and validated at the FDA National Center for Toxicological Research (NCTR) to profile a host of genes encoded by nuclear and mt-DNA following SFN treatment (0-5 µM). Corresponding protein expression levels were assessed (ad hoc) by qRT-PCR, immunoblots and immunocytochemistry (ICC). Gene ontology clustering revealed that SFN treatment (24 h) significantly up-regulated ~50 key genes (>1.5 fold, adjusted p < 0.0001) and repressed 20 genes (<0.7 fold, adjusted p < 0.0001) belonging to oxidative stress, phase 1 & 2 drug metabolism enzymes (glutathione system), iron transporters, glycolysis, oxidative phosphorylation (OXPHOS), amino acid metabolism, lipid metabolism and mitochondrial biogenesis. Our results show that SFN stimulated the production of ATP by promoting the expression and activity of glucose transporter-1, and glycolysis. In addition, SFN upregulated anti-oxidative stress responses, redox signaling and phase 2 drug metabolism/detoxification functions, thus elucidating further the previously observed neurovascular protective effects of this compound.


Assuntos
Encéfalo/metabolismo , Endotélio Vascular/microbiologia , Genômica/métodos , Isotiocianatos/farmacologia , Proteômica/métodos , Trifosfato de Adenosina/metabolismo , Animais , Antioxidantes/metabolismo , Western Blotting , Encéfalo/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Imunofluorescência , Imuno-Histoquímica , Camundongos , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
9.
Tuberculosis (Edinb) ; 111: 1-7, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30029892

RESUMO

Mycobacterium tuberculosis (Mtb) has plagued humanity for tens of thousands of years, yet still remains a threat to human health. Its pathology is largely associated with pulmonary tuberculosis with symptoms including fever, hemoptysis, and chest pain. Mtb, however, also manifests in other extrapulmonary organs, such as the pleura, bones, gastrointestinal tract, central nervous system, and lymph nodes. Compared to the knowledge of pulmonary tuberculosis, extrapulmonary pathologies of Mtb are quite understudied. Lymph node tuberculosis is one of the most common extrapulmonary manifestations of tuberculosis, and presents significant challenges in its diagnosis, management, and treatment due to its elusive etiologies and pathologies. The objective of this review is to overview the current understanding of the tropism and pathogenesis of Mtb in endothelial cells of the extrapulmonary tissues, particularly, in lymph nodes. Lymphatic endothelial cells (LECs) are derived from blood vascular endothelial cells (BECs) during development, and these two types of endothelial cells demonstrate substantial molecular, cellular and genetic similarities. Therefore, systemic comparison of the differential and common responses of BECs vs. LECs to Mtb invasion could provide new insights into its pathogenesis, and may promote new investigations into this deadly disease.


Assuntos
Linhagem da Célula , Células Endoteliais/microbiologia , Endotélio Linfático/microbiologia , Endotélio Vascular/microbiologia , Mycobacterium tuberculosis/patogenicidade , Tuberculose/microbiologia , Animais , Antituberculosos/uso terapêutico , Biomarcadores/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Endotélio Linfático/efeitos dos fármacos , Endotélio Linfático/imunologia , Endotélio Linfático/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/imunologia , Endotélio Vascular/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/metabolismo , Fenótipo , Transdução de Sinais , Tuberculose/tratamento farmacológico , Tuberculose/imunologia , Tuberculose/metabolismo
10.
PLoS Pathog ; 14(4): e1006981, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29630665

RESUMO

Purpura fulminans is a deadly complication of Neisseria meningitidis infections due to extensive thrombosis of microvessels. Although a Disseminated Intra-vascular Coagulation syndrome (DIC) is frequently observed during Gram negative sepsis, it is rarely associated with extensive thrombosis like those observed during meningococcemia, suggesting that the meningococcus induces a specific dysregulation of coagulation. Another specific feature of N. meningitidis pathogenesis is its ability to colonize microvessels endothelial cells via type IV pili. Importantly, endothelial cells are key in controlling the coagulation cascade through the activation of the potent anticoagulant Protein C (PC) thanks to two endothelial cell receptors among which the Endothelial Protein C Receptor (EPCR). Considering that congenital or acquired deficiencies of PC are associated with purpura fulminans, we hypothesized that a defect in the activation of PC following meningococcal adhesion to microvessels is responsible for the thrombotic events observed during meningococcemia. Here we showed that the adhesion of N. meningitidis on endothelial cells results in a rapid and intense decrease of EPCR expression by inducing its cleavage in a process know as shedding. Using siRNA experiments and CRISPR/Cas9 genome edition we identified ADAM10 (A Disintegrin And Metalloproteinase-10) as the protease responsible for this shedding. Surprisingly, ADAM17, the only EPCR sheddase described so far, was not involved in this process. Finally, we showed that this ADAM10-mediated shedding of EPCR induced by the meningococcal interaction with endothelial cells was responsible for an impaired activation of Protein C. This work unveils for the first time a direct link between meningococcal adhesion to endothelial cells and a severe dysregulation of coagulation, and potentially identifies new therapeutic targets for meningococcal purpura fulminans.


Assuntos
Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Receptor de Proteína C Endotelial/metabolismo , Endotélio Vascular/patologia , Proteínas de Membrana/metabolismo , Infecções Meningocócicas/complicações , Microvasos/patologia , Proteína C/metabolismo , Púrpura Fulminante/etiologia , Proteína ADAM10/genética , Secretases da Proteína Precursora do Amiloide/genética , Aderência Bacteriana , Coagulação Sanguínea/fisiologia , Células Cultivadas , Receptor de Proteína C Endotelial/genética , Endotélio Vascular/metabolismo , Endotélio Vascular/microbiologia , Humanos , Proteínas de Membrana/genética , Infecções Meningocócicas/microbiologia , Microvasos/metabolismo , Microvasos/microbiologia , Neisseria meningitidis/fisiologia , Proteína C/genética , Púrpura Fulminante/metabolismo , Púrpura Fulminante/patologia
11.
Sci Rep ; 8(1): 4705, 2018 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-29549320

RESUMO

Protein glycosylation has been described as the most abundant and complex post-translational modification occurring in nature. Recent studies have enhanced our view of how this modification occurs in bacteria highlighting the role of protein glycosylation in various processes such as biofilm formation, virulence and host-microbe interactions. We recently showed that the collagen- and laminin-binding adhesin Cnm of the dental pathogen Streptococcus mutans is post-translationally modified by the PgfS glycosyltransferase. Following this initial identification of Cnm as a glycoprotein, we have now identified additional genes (pgfM1, pgfE and pgfM2) that are also involved in the posttranslational modification of Cnm. Similar to the previously characterized ΔpgfS strain, inactivation of pgfM1, pgfE or pgfM2 directly impacts Cnm by altering its migration pattern, proteolytic stability and function. In addition, we identified the wall-associated protein A (WapA) as an additional substrate of Pgf-dependent modification. We conclude that the pgS-pgfM1-pgfE-pgfM2 operon encodes for a protein machinery that can modify, likely through the addition of glycans, both core and non-core gene products in S. mutans.


Assuntos
Proteínas de Bactérias/metabolismo , Glicosiltransferases/metabolismo , Proteínas de Membrana/metabolismo , Óperon , Processamento de Proteína Pós-Traducional , Infecções Estreptocócicas/microbiologia , Streptococcus mutans/metabolismo , Adesinas Bacterianas/química , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Sequência de Aminoácidos , Aderência Bacteriana , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Colágeno/metabolismo , Endotélio Vascular/metabolismo , Endotélio Vascular/microbiologia , Regulação Bacteriana da Expressão Gênica , Glicosilação , Glicosiltransferases/química , Glicosiltransferases/genética , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Streptococcus mutans/genética , Streptococcus mutans/crescimento & desenvolvimento , Virulência
12.
Biochem Biophys Res Commun ; 497(2): 742-748, 2018 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-29462613

RESUMO

Migration of monocytes into the subendothelial layer of the intima is one of the critical events in early atherosclerosis. Chlamydia pneumoniae (C. pneumoniae) infection has been shown to promote monocyte transendothelial migration (TEM). However, the exact mechanisms have not yet been fully clarified. In this study, we tested the hypothesis that C. pneumoniae infection increases vascular endothelial cell (VEC) permeability and subsequent monocyte TEM through stimulating the tyrosine phosphorylation of vascular endothelial-cadherin (VE-cadherin). Here, we demonstrated that C. pneumoniae infection promoted monocyte TEM in a TEM assay possibly by increasing the permeability of a VEC line EA.hy926 cell as assessed by measuring the passage of FITC-BSA across a VEC monolayer. Subsequently, Western blot analysis showed that C. pneumoniae infection induced VE-cadherin internalization. Our further data revealed that Src-mediated VE-cadherin phosphorylation at Tyr658 was involved in C. pneumoniae infection-induced internalization of VE-cadherin, VEC hyperpermeability and monocyte TEM. Taken together, our data indicate that C. pneumoniae infection promotes monocyte TEM by increasing VEC permeability via the tyrosine phosphorylation and internalization of VE-cadherin in VECs.


Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Permeabilidade Capilar , Infecções por Chlamydophila/metabolismo , Chlamydophila pneumoniae/fisiologia , Monócitos/microbiologia , Migração Transendotelial e Transepitelial , Células Cultivadas , Infecções por Chlamydophila/microbiologia , Infecções por Chlamydophila/patologia , Endotélio Vascular/metabolismo , Endotélio Vascular/microbiologia , Endotélio Vascular/patologia , Interações Hospedeiro-Patógeno , Humanos , Monócitos/citologia , Monócitos/patologia , Fosforilação
13.
Cardiovasc Res ; 114(6): 907-918, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29361046

RESUMO

Aims: TREM-1 (Triggering Receptor Expressed on Myeloid cells-1) is an immunoreceptor expressed on neutrophils and monocytes/macrophages whose role is to amplify the inflammatory response driven by Toll-Like Receptors engagement. The pharmacological inhibition of TREM-1 confers protection in several pre-clinical models of acute inflammation. In this study, we aimed to decipher the role of TREM-1 on the endothelium. Methods and results: We first showed by qRT-PCR, flow cytometry and confocal microscopy that TREM-1 was expressed in human pulmonary microvascular endothelial cells as well as in mouse vasculature (aorta, mesenteric artery, and pulmonary vessels). TREM-1 expression was upregulated following septic insult. We next observed that TREM-1 engagement impaired mouse vascular reactivity and promoted vascular inflammation. The pharmacological inhibition of TREM-1 (using the synthetic inhibitory peptide LR12) prevented these disorders both in vitro and in vivo. We generated endothelium-conditional Trem-1 ko mice (EndoTREM-1-/-) and submitted them to a caecal ligation and puncture-induced septic shock. As compared with wild-type littermates, targeted endothelial Trem-1 deletion conferred protection during septic shock in modulating inflammatory cells mobilization and activation, in restoring vasoreactivity, and in improving the survival. Conclusion: We reported that TREM-1 is expressed and inducible in endothelial cells and plays a direct role in vascular inflammation and dysfunction. The targeted deletion of endothelial Trem-1 conferred protection during septic shock in modulating inflammatory cells mobilization and activation, restoring vasoreactivity, and improving survival. The effect of TREM-1 on vascular tone, while impressive, deserves further investigations including the design of endothelium-specific TREM-1 inhibitors.


Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Sepse/prevenção & controle , Receptor Gatilho 1 Expresso em Células Mieloides/deficiência , Animais , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/microbiologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/microbiologia , Endotélio Vascular/fisiopatologia , Endotoxinas/farmacologia , Humanos , Mediadores da Inflamação/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/metabolismo , Infiltração de Neutrófilos , Neutrófilos/metabolismo , Oligopeptídeos/farmacologia , Sepse/metabolismo , Sepse/microbiologia , Sepse/fisiopatologia , Transdução de Sinais , Receptor Gatilho 1 Expresso em Células Mieloides/antagonistas & inibidores , Receptor Gatilho 1 Expresso em Células Mieloides/genética , Receptor Gatilho 1 Expresso em Células Mieloides/metabolismo , Vasoconstrição , Vasodilatação
14.
J Vet Sci ; 19(2): 207-215, 2018 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-28693312

RESUMO

Pasteurella multocida serotype B:2 causes hemorrhagic septicemia in cattle and buffalo. The invasion mechanism of the bacterium when invading the bloodstream is unclear. This study aimed to characterize the effects of immunomodulatory molecules, namely dexamethasone and lipopolysaccharide, on the invasion efficiency of P. multocida serotype B:2 toward bovine aortic endothelial cells (BAECs) and the involvement of actin microfilaments in the invasion mechanism. The results imply that treatment of BAECs with lipopolysaccharide at 100 ng/mL for 24 h significantly increases the intracellular bacteria number per cell (p < 0.01) compared with those in untreated and dexamethasone-treated cells. The lipopolysaccharide-treated cells showed a significant decrease in F-actin expression and an increase in G-actin expression (p < 0.001), indicating actin depolymerization of BAECs. However, no significant differences were detected in the invasion efficiency and actin filament reorganization between the dexamethasone-treated and untreated cells. Transmission electron microscopy showed that P. multocida B:2 resided in a vacuolar compartment of dexamethasone-treated and untreated cells, whereas the bacteria resided in cellular membrane of lipopolysaccharide-treated cells. The results suggest that lipopolysaccharide destabilizes the actin filaments of BAECs, which could facilitate the invasion of P. multocida B:2 into BAECs.


Assuntos
Aorta/microbiologia , Endotélio Vascular/microbiologia , Lipopolissacarídeos/farmacologia , Pasteurella multocida/patogenicidade , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/microbiologia , Animais , Aorta/efeitos dos fármacos , Bovinos , Dexametasona/farmacologia , Endotélio Vascular/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Técnicas In Vitro , Microscopia Eletrônica de Transmissão , Pasteurella multocida/efeitos dos fármacos
15.
Mol Oral Microbiol ; 33(1): 29-37, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28984079

RESUMO

Epidemiological studies have established a clinical association between periodontal disease and atherosclerosis. Bacteremia and endotoxemia episodes in patients with periodontitis appear to link these two diseases by inducing a body-wide production of cardiovascular markers. The presence of oral bacteria in atherosclerotic lesions in patients with periodontitis suggests that bacteria, or their antigenic components, induce alterations in the endothelium associated with atherosclerosis. Therefore, a causal mechanism explaining the association between both diseases can be constructed using in vitro models. This review presents current experimental approaches based on in vitro cell models used to shed light on the mechanism by which periodontal pathogenic microorganisms, and their antigenic components, induce proatherosclerotic endothelial activity. Monolayer cultures of endothelial vascular or arterial cells have been used to assess periodontal pathogenic bacteria and their antigenic compounds and endothelial activation. However, these models are not capable of reflecting the physiological characteristics of the endothelium inside vascularized tissue. Therefore, the shift from two-dimensional (2D) cellular models toward three-dimensional (3D) models of endothelial cells resembling an environment close to the physiological environment of the endothelial cell within the endothelium is useful for evaluating the physiological relevance of results regarding the endothelial dysfunction induced by periodontopathogens that are currently obtained from 2D models. The use of in vitro 3D cellular models can also be relevant to the search for therapeutic agents for chronic inflammatory diseases such as atherosclerosis. Here, we present some strategies for the assembly of 3D cultures with endothelial cells, which is useful for the study of periodontopathogen-mediated disease.


Assuntos
Aterosclerose/microbiologia , Aterosclerose/patologia , Bactérias/patogenicidade , Doenças Periodontais/microbiologia , Antígenos de Bactérias/imunologia , Técnicas de Cultura de Células/métodos , Células Endoteliais/imunologia , Células Endoteliais/microbiologia , Células Endoteliais/patologia , Endotélio Vascular/imunologia , Endotélio Vascular/microbiologia , Endotélio Vascular/patologia , Humanos , Técnicas In Vitro , Lipopolissacarídeos/imunologia , Macrófagos , Monócitos , Doenças Periodontais/patologia , Periodontite/microbiologia , Periodontite/patologia , Porphyromonas gingivalis/imunologia , Porphyromonas gingivalis/patogenicidade
16.
J Infect Public Health ; 11(4): 592-593, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29277334

RESUMO

Actinomyces species are part of the commensal flora of the mucous membranes of the oropharynx, gastrointestinal tract and female genital tract. Actinomyces europaeus is a short, nonmotile, facultative anaerobic rod first described in 1997, susceptible in vitro to a wide range of b-lactam antibiotics which are regarded as first choice. In this report we described the case of A. europaeus infection in a young female patient admitted to Intensive Care Unit and the possible damage of vascular endothelium due to a chronic progressive actinomycosis that at first involved neck soft tissue, then cervical lymphnodes, and finally extended to the vascular structure.


Assuntos
Actinomicose/diagnóstico , Linfadenopatia/microbiologia , Doenças do Sistema Nervoso/complicações , Actinomyces/efeitos dos fármacos , Actinomyces/genética , Actinomyces/isolamento & purificação , Actinomicose/tratamento farmacológico , Actinomicose/imunologia , Antibacterianos/uso terapêutico , Encéfalo/diagnóstico por imagem , Endotélio Vascular/microbiologia , Endotélio Vascular/patologia , Feminino , Humanos , Unidades de Terapia Intensiva , Linfonodos/microbiologia , Linfadenopatia/diagnóstico , Linfadenopatia/diagnóstico por imagem , Linfadenopatia/tratamento farmacológico , Doenças do Sistema Nervoso/microbiologia , RNA Ribossômico 16S , Simbiose , Tomografia Computadorizada por Raios X , Resultado do Tratamento , Vasculite/microbiologia , Adulto Jovem
17.
Crit Care ; 21(1): 261, 2017 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-29058634

RESUMO

BACKGROUND: Sugar-protein glycocalyx coats healthy endothelium, but its ultrastructure is not well described. Our aim was to determine the three-dimensional ultrastructure of capillary endothelial glycocalyx in the heart, kidney, and liver, where capillaries are, respectively, continuous, fenestrated, and sinusoidal. METHODS: Tissue samples were processed with lanthanum-containing alkaline fixative, which preserves the structure of glycocalyx. RESULTS: Scanning and transmission electron microscopy revealed that the endothelial glycocalyx layer in continuous and fenestrated capillaries was substantially thicker than in sinusoids. In the heart, the endothelial glycocalyx presented as moss- or broccoli-like and covered the entire luminal endothelial cell surface. In the kidney, the glycocalyx appeared to nearly occlude the endothelial pores of the fenestrated capillaries and was also present on the surface of the renal podocytes. In sinusoids of the liver, glycocalyx covered not only the luminal side but also the opposite side, facing the space of Disse. In a mouse lipopolysaccharide-induced experimental endotoxemia model, the capillary endothelial glycocalyx was severely disrupted; that is, it appeared to be peeling off the cells and clumping. Serum concentrations of syndecan-1, a marker of glycocalyx damage, were significantly increased 24 h after administration of lipopolysaccharide. CONCLUSIONS: In the present study, we visualized the three-dimensional ultrastructure of endothelial glycocalyx in healthy continuous, fenestrated, and sinusoidal capillaries, and we also showed their disruption under experimental endotoxemic conditions. The latter may provide a morphological basis for the microvascular endothelial dysfunction associated with septic injury to organs.


Assuntos
Endotélio Vascular/anatomia & histologia , Glicocálix/patologia , Animais , Endotélio Vascular/microbiologia , Glicocálix/metabolismo , Glicocálix/fisiologia , Coração/anatomia & histologia , Estimativa de Kaplan-Meier , Rim/anatomia & histologia , Rim/irrigação sanguínea , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/metabolismo , Fígado/anatomia & histologia , Fígado/irrigação sanguínea , Camundongos/anatomia & histologia , Camundongos/microbiologia , Microscopia Eletrônica/métodos , Modelos de Riscos Proporcionais
18.
Traffic ; 18(11): 733-746, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28799243

RESUMO

Eukaryotic cells utilize multiple endocytic pathways for specific uptake of ligands or molecules, and these pathways are commonly hijacked by pathogens to enable host cell invasion. Escherichia coli K1, a pathogenic bacterium that causes neonatal meningitis, invades the endothelium of the blood-brain barrier, but the entry route remains unclear. Here, we demonstrate that the bacteria trigger an actin-mediated uptake route, stimulating fluid phase uptake, membrane ruffling and macropinocytosis. The route of uptake requires intact lipid rafts as shown by cholesterol depletion. Using a variety of perturbants we demonstrate that small Rho GTPases and their downstream effectors have a significant effect on bacterial invasion. Furthermore, clathrin-mediated endocytosis appears to play an indirect role in E. coli K1 uptake. The data suggest that the bacteria effect a complex interplay between the Rho GTPases to increase their chances of uptake by macropinocytosis into human brain microvascular endothelial cells.


Assuntos
Encéfalo/microbiologia , Células Endoteliais/microbiologia , Escherichia coli/patogenicidade , Microvasos/microbiologia , Pinocitose/fisiologia , Actinas/metabolismo , Translocação Bacteriana , Encéfalo/irrigação sanguínea , Linhagem Celular , Colesterol/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Endotélio Vascular/microbiologia , Escherichia coli/fisiologia , Humanos , Microvasos/metabolismo , Virulência
19.
Microb Pathog ; 110: 352-358, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28711510

RESUMO

INTRODUCTION: Pneumococcal infections have caused morbidity and mortality globally. Streptococcus pneumoniae (pneumococci) are commensal bacteria that colonize the nasopharynx, asymptomatically. From there, pneumococci can spread in the lungs causing pneumonia and disseminate in the bloodstream causing bacteremia (sepsis) and reach the brain leading to meningitis. Endothelial cells are one of the most important components of the blood-brain barrier that separates the blood from the brain and plays the first protective role against pneumococcal entry. Thus this study aimed to investigate on the ability of non-meningitis pneumococcal clinical strains to adhere and invade a brain endothelium model. METHODS: Two pneumococcal Brunei clinical strains were serotyped by multiplex PCR method using oligonucleotide sequences derived from Centers for Disease Control and Prevention. A validated immortalised mouse brain endothelial cell line (bEnd.3) was used as a brain endothelium model for the study of the pneumococcal breach of the blood-brain barrier using an adherence and invasion assay. RESULTS: Both of the pneumococcal clinical strains were found to be serotype 19F, a common circulating serotype in Southeast Asia and globally and possess the ability to adhere and invade the brain endothelial cells. CONCLUSION: In addition, this is the first report on the serotype identification of pneumococci in Brunei Darussalam and their application on a brain endothelium model. Further studies are required to understand the virulence capabilities of the clinical strains.


Assuntos
Adesinas Bacterianas , Barreira Hematoencefálica/microbiologia , Células Endoteliais/microbiologia , Endotélio Vascular/microbiologia , Infecções Pneumocócicas/microbiologia , Sorotipagem , Streptococcus pneumoniae/genética , Animais , Ásia Sudeste , Barreira Hematoencefálica/metabolismo , Brunei , Linhagem Celular , DNA Bacteriano , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Humanos , Meningites Bacterianas/metabolismo , Meningites Bacterianas/microbiologia , Camundongos , Modelos Biológicos , Reação em Cadeia da Polimerase Multiplex , Sorogrupo , Streptococcus pneumoniae/metabolismo , Streptococcus pneumoniae/patogenicidade , Proteínas de Junções Íntimas/metabolismo , Virulência , Fator de von Willebrand/metabolismo
20.
Int J Mol Sci ; 18(7)2017 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-28698491

RESUMO

MicroRNAs (miRNAs) mediate gene silencing by destabilization and/or translational repression of target mRNA. Infection of human microvascular endothelial cells as primary targets of Rickettsiarickettsii, the etiologic agent of Rocky Mountain spotted fever, triggers host responses appertaining to alterations in cellular gene expression. Microarray-based profiling of endothelial cells infected with R.rickettsii for 3 or 24 h revealed differential expression of 33 miRNAs, of which miRNAs129-5p, 200a-3p, 297, 200b-3p, and 595 were identified as the top five up-regulated miRNAs (5 to 20-fold, p ≤ 0.01) and miRNAs 301b-3p, 548a-3p, and 377-3p were down-regulated (2 to 3-fold, p ≤ 0.01). Changes in the expression of selected miRNAs were confirmed by q-RT-PCR in both in vitro and in vivo models of infection. As potential targets, expression of genes encoding NOTCH1, SMAD2, SMAD3, RIN2, SOD1, and SOD2 was either positively or negatively regulated. Using a miRNA-specific mimic or inhibitor, NOTCH1 was determined to be a target of miRNA 200a-3p in R. rickettsii-infected human dermal microvascular endothelial cells (HMECs). Predictive interactome mapping suggested the potential for miRNA-mediated modulation of regulatory gene networks underlying important host cell signaling pathways. This first demonstration of altered endothelial miRNA expression provides new insights into regulatory elements governing mechanisms of host responses and pathogenesis during human rickettsial infections.


Assuntos
Células Endoteliais/metabolismo , Células Endoteliais/microbiologia , Endotélio Vascular/metabolismo , Endotélio Vascular/microbiologia , MicroRNAs/genética , Rickettsia rickettsii/patogenicidade , Proteínas de Transporte/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Receptor Notch1/genética , Transdução de Sinais/fisiologia , Proteína Smad2/genética , Proteína Smad3/genética , Superóxido Dismutase/genética
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