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1.
Vaccine ; 30(1): 9-13, 2011 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-22044742

RESUMO

The emergence of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) is a serious health concern worldwide that requires new therapeutic approaches that extend beyond the development and use of new antibiotics. In this study, a conformationally biased, response-selective agonist of human C5a, known as EP67, was used to induce host innate immunity as a therapeutic method of reducing CA-MRSA infections. Using a murine model of dermonecrosis we show that EP67 treatment effectively limits CA-MRSA infection by promoting cytokine synthesis and neutrophil influx. In contrast, EP67 was ineffective in reducing lesion formation in C5a receptor (CD88(-/-)) knockout mice, indicating that EP67 activates host innate immunity by engagement of CD88 bearing cells. These results suggest that EP67 may serve as a novel immunotherapeutic for prevention and treatment of CA-MRSA dermal infection.


Assuntos
Fatores Imunológicos/administração & dosagem , Staphylococcus aureus Resistente à Meticilina/imunologia , Peptídeos/administração & dosagem , Infecções Cutâneas Estafilocócicas/tratamento farmacológico , Infecções Cutâneas Estafilocócicas/microbiologia , Animais , Complemento C5a/agonistas , Modelos Animais de Doenças , Feminino , Camundongos , Resultado do Tratamento
2.
Infect Immun ; 79(7): 2510-8, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21518787

RESUMO

Bacillus anthracis is a Gram-positive spore-forming bacterium that causes anthrax disease in humans and animals. Systemic infection is characterized by septicemia, toxemia, and meningitis, the main neurological complication associated with high mortality. We have shown previously that B. anthracis Sterne is capable of blood-brain barrier (BBB) penetration, establishing the classic signs of meningitis, and that infection is dependent on the expression of both major anthrax toxins, lethal toxin (LT) and edema toxin (ET). Here we further investigate the contribution of the individual toxins to BBB disruption using isogenic toxin mutants deficient in lethal factor, ΔLF, and edema factor, ΔEF. Acute infection with B. anthracis Sterne and the ΔLF mutant resulted in disruption of human brain microvascular endothelial cell (hBMEC) monolayer integrity and tight junction protein zona occludens-1, while the result for cells infected with the ΔEF mutant was similar to that for the noninfected control. A significant decrease in bacterial invasion of BBB endothelium in vitro was observed during infection with the ΔLF strain, suggesting a prominent role for LT in promoting BBB interaction. Further, treatment of hBMECs with purified LT or chemicals that mimic LT action on host signaling pathways rescued the hypoinvasive phenotype of the ΔLF mutant and resulted in increased bacterial uptake. We also observed that toxin expression reduced bacterial intracellular survival by inducing the bulk degradative autophagy pathway in host cells. Finally, in a murine model of anthrax meningitis, mice infected with the ΔLF mutant exhibited no mortality, brain bacterial load, or evidence of meningitis compared to mice infected with the parental or ΔEF strains.


Assuntos
Antraz/microbiologia , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Bacillus anthracis/patogenicidade , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Barreira Hematoencefálica/microbiologia , Meningites Bacterianas/microbiologia , Animais , Antraz/mortalidade , Antraz/patologia , Antígenos de Bactérias/biossíntese , Autofagia , Bacillus anthracis/genética , Toxinas Bacterianas/biossíntese , Barreira Hematoencefálica/patologia , Encéfalo/irrigação sanguínea , Encéfalo/patologia , Células Endoteliais/patologia , Humanos , Proteínas de Membrana/metabolismo , Meningites Bacterianas/mortalidade , Meningites Bacterianas/patologia , Camundongos , Microvasos/patologia , Mutação , Fosfoproteínas/metabolismo , Junções Íntimas/ultraestrutura , Proteína da Zônula de Oclusão-1
3.
J Mol Med (Berl) ; 88(6): 633-9, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20419283

RESUMO

Staphylococcus aureus is one of the most prevalent organisms responsible for nosocomial infections, and cases of community-acquired S. aureus infection have continued to increase despite widespread preventative measures. Pathologies attributed to S. aureus infection are diverse; ranging from dermal lesions to bacteremia, abscesses, and endocarditis. Reported cases of S. aureus-associated meningitis and brain abscesses have also increased in recent years, however, the precise mechanism whereby S. aureus leave the bloodstream and gain access to the central nervous system (CNS) are not known. Here we demonstrate for the first time that S. aureus efficiently adheres to and invades human brain microvascular endothelial cells (hBMEC), the single-cell layer which constitutes the blood-brain barrier (BBB). The addition of cytochalasin D, an actin microfilament aggregation inhibitor, strongly reduced bacterial invasion, suggesting an active hBMEC process is required for efficient staphylococcal uptake. Furthermore, mice injected with S. aureus exhibited significant levels of brain bacterial counts and histopathologic evidence of meningeal inflammation and brain abscess formation, indicating that S. aureus was able to breech the BBB in an experimental model of hematogenous meningitis. We found that a YpfP-deficient mutant, defective in lipoteichoic acid (LTA) membrane anchoring, exhibited a decreased ability to invade hBMEC and correlated to a reduced risk for the development of meningitis in vivo. Our results demonstrate that LTA-mediated penetration of the BBB may be a primary step in the pathogenesis of staphylococcal CNS disease.


Assuntos
Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/microbiologia , Membrana Celular/química , Lipopolissacarídeos/metabolismo , Staphylococcus aureus/patogenicidade , Ácidos Teicoicos/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Parede Celular/metabolismo , Células Endoteliais/citologia , Células Endoteliais/microbiologia , Humanos , Masculino , Camundongos , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/citologia , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo
4.
J Bacteriol ; 191(23): 7165-73, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19820089

RESUMO

Anthrax is a zoonotic disease caused by the gram-positive spore-forming bacterium Bacillus anthracis. Human infection occurs after the ingestion, inhalation, or cutaneous inoculation of B. anthracis spores. The subsequent progression of the disease is largely mediated by two native virulence plasmids, pXO1 and pXO2, and is characterized by septicemia, toxemia, and meningitis. In order to produce meningitis, blood-borne bacteria must interact with and breach the blood-brain barrier (BBB) that is composed of a specialized layer of brain microvascular endothelial cells (BMEC). We have recently shown that B. anthracis Sterne is capable of penetrating the BBB in vitro and in vivo, establishing the classic signs of meningitis; however, the molecular mechanisms underlying the central nervous system (CNS) tropism are not known. Here, we show that attachment to and invasion of human BMEC by B. anthracis Sterne is mediated by the pXO1 plasmid and an encoded envelope factor, BslA. The results of studies using complementation analysis, recombinant BslA protein, and heterologous expression demonstrate that BslA is both necessary and sufficient to promote adherence to brain endothelium. Furthermore, mice injected with the BslA-deficient strain exhibited a significant decrease in the frequency of brain infection compared to mice injected with the parental strain. In addition, BslA contributed to BBB breakdown by disrupting tight junction protein ZO-1. Our results identify the pXO1-encoded BslA adhesin as a critical mediator of CNS entry and offer new insights into the pathogenesis of anthrax meningitis.


Assuntos
Antraz/microbiologia , Bacillus anthracis/patogenicidade , Proteínas de Bactérias/genética , Barreira Hematoencefálica/microbiologia , Adesinas Bacterianas/genética , Animais , Bacillus anthracis/genética , Aderência Bacteriana/genética , Linhagem Celular , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Microscopia , Plasmídeos/genética
5.
J Innate Immun ; 1(5): 494-506, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-20375606

RESUMO

Bacillus anthracis is a National Institute of Allergy and Infectious Diseases Category A priority pathogen and the causative agent of the deadly disease anthrax. We applied a transposon mutagenesis system to screen for novel chromosomally encoded B. anthracis virulence factors. This approach identified ClpX, the regulatory ATPase subunit of the ClpXP protease, as essential for both the hemolytic and proteolytic phenotypes surrounding colonies of B. anthracis grown on blood or casein agar media, respectively. Deletion of clpX attenuated lethality of B. anthracis Sterne in murine subcutaneous and inhalation infection models, and markedly reduced in vivo survival of the fully virulent B. anthracis Ames upon intraperitoneal challenge in guinea pigs. The extracellular proteolytic activity dependent upon ClpX function was linked to degradation of cathelicidin antimicrobial peptides, a front-line effector of innate host defense. B. anthracis lacking ClpX were rapidly killed by cathelicidin and alpha-defensin antimicrobial peptides and lysozyme in vitro. In turn, mice lacking cathelicidin proved hyper-susceptible to lethal infection with wild-type B. anthracis Sterne, confirming cathelicidin to be a critical element of innate defense against the pathogen. We conclude that ClpX is an important factor allowing B. anthracis to subvert host immune clearance mechanisms, and thus represents a novel therapeutic target for prevention or therapy of anthrax, a foremost biodefense concern.


Assuntos
Adenosina Trifosfatases/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bacillus anthracis/efeitos dos fármacos , Bacillus anthracis/patogenicidade , Farmacorresistência Bacteriana , Endopeptidase Clp/metabolismo , Adenosina Trifosfatases/genética , Animais , Antraz/microbiologia , Bacillus anthracis/enzimologia , Bacillus anthracis/genética , Elementos de DNA Transponíveis , Endopeptidase Clp/genética , Cobaias , Hemólise , Humanos , Imunidade Inata , Camundongos , Mutagênese , Fenótipo , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
6.
PLoS One ; 3(8): e2964, 2008 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-18698416

RESUMO

BACKGROUND: Anthrax meningitis is the main neurological complication of systemic infection with Bacillus anthracis approaching 100% mortality. The presence of bacilli in brain autopsies indicates that vegetative bacteria are able to breach the blood-brain barrier (BBB). The BBB represents not only a physical barrier but has been shown to play an active role in initiating a specific innate immune response that recruits neutrophils to the site of infection. Currently, the basic pathogenic mechanisms by which B. anthracis penetrates the BBB and causes anthrax meningitis are poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: Using an in vitro BBB model, we show for the first time that B. anthracis efficiently invades human brain microvascular endothelial cells (hBMEC), the single cell layer that comprises the BBB. Furthermore, transcriptional profiling of hBMEC during infection with B. anthracis revealed downregulation of 270 (87%) genes, specifically key neutrophil chemoattractants IL-8, CXCL1 (Gro alpha) and CXCL2 (Gro beta), thereby strongly contrasting hBMEC responses observed with other meningeal pathogens. Further studies using specific anthrax toxin-mutants, quantitative RT-PCR, ELISA and in vivo assays indicated that anthrax toxins actively suppress chemokine production and neutrophil recruitment during infection, allowing unrestricted proliferation and dissemination of the bacteria. Finally, mice challenged with B. anthracis Sterne, but not the toxin-deficient strain, developed meningitis. CONCLUSIONS/SIGNIFICANCE: These results suggest a significant role for anthrax toxins in thwarting the BBB innate defense response promoting penetration of bacteria into the central nervous system. Furthermore, establishment of a mouse model for anthrax meningitis will aid in our understanding of disease pathogenesis and development of more effective treatment strategies.


Assuntos
Antígenos de Bactérias/farmacologia , Toxinas Bacterianas/farmacologia , Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/fisiopatologia , Circulação Cerebrovascular/efeitos dos fármacos , Endotélio Vascular/fisiopatologia , Meningites Bacterianas/induzido quimicamente , Meningites Bacterianas/patologia , Microcirculação/efeitos dos fármacos , Neutrófilos/fisiologia , Bacillus anthracis , Encéfalo/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Humanos , Modelos Neurológicos , Neutrófilos/efeitos dos fármacos , Transdução de Sinais/fisiologia
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