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1.
Microbes Infect ; 8(2): 460-9, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16243562

RESUMO

Escherichia coli K1 invasion of human brain microvascular endothelial cells (HBMEC) requires the reorganization of host cytoskeleton at the sites of bacterial entry. Both actin and myosin constitute the cytoskeletal architecture. We have previously shown that myosin light chain (MLC) phosphorylation by MLC kinase is regulated during E. coli invasion by an upstream kinase, p21-activated kinase 1 (PAK1), which is an effector protein of Rac and Cdc42 GTPases, but not of RhoA. Here, we report that the binding of only Rac1 to PAK1 decreases in HBMEC upon infection with E. coli K1, which resulted in increased phosphorylation of MLC. Overexpression of a constitutively active (cAc) form of Rac1 in HBMEC blocked the E. coli invasion significantly, whereas overexpression of a dominant negative form had no effect. Increased PAK1 phosphorylation was observed in HBMEC expressing cAc-Rac1 with a concomitant reduction in the phosphorylation of MLC. Immunocytochemistry studies demonstrated that the inhibition of E. coli invasion into cAc-Rac1/HBMEC is due to lack of phospho-MLC recruitment to the sites of E. coli entry. Taken together the data suggest that E. coli modulates the binding of Rac1, but not Cdc42, to PAK1 during the invasion of HBMEC.


Assuntos
Encéfalo/microbiologia , Regulação para Baixo , Endotélio Vascular/microbiologia , Escherichia coli/patogenicidade , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Encéfalo/irrigação sanguínea , Encéfalo/citologia , Células Cultivadas , Células Endoteliais/microbiologia , Endotélio Vascular/citologia , Humanos , Cadeias Leves de Miosina/metabolismo , Fosforilação , Quinases Ativadas por p21
2.
Infect Immun ; 71(5): 2787-97, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12704153

RESUMO

Cytoskeletal dynamics, modulated by actin-myosin interactions, play an important role in Escherichia coli K1 invasion of human brain microvascular endothelial cells (HBMEC). Herein, we show that inhibitors of myosin function, butanedione monoxide and ML-7, significantly blocked the E. coli invasion of HBMEC. The invasive E. coli induces myosin light-chain (MLC) phosphorylation during the invasion process, which gets recruited to the site of actin condensation beneath the bacteria. We also show that invading E. coli downregulates the activity of p21-activated kinase 1 (PAK1), which is an upstream regulator of MLC kinase (MLCK). Overexpression of wild-type PAK1 and constitutively active PAK1 in HBMEC inhibits E. coli invasion significantly with a concomitant decrease in MLC phosphorylation. The inhibition of E. coli invasion by these PAK1 mutants is due to the absence of phospho-MLC at the actin condensation points. In contrast, the dominant-negative PAK1 shows no effect either on the invasion or on MLC phosphorylation or phospho-MLC recruitment to the actin focal points, suggesting that activated PAK1 inactivates MLCK. Taken together, these results suggest that E. coli invasion of HBMEC induces MLC phosphorylation by inhibiting the activity of PAK1 and the recruitment of phosphorylated MLC to the site of actin condensation beneath the bacteria for efficient internalization of E. coli into HBMEC.


Assuntos
Encéfalo/microbiologia , Endotélio Vascular/microbiologia , Escherichia coli/patogenicidade , Cadeias Leves de Miosina/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Actinas/metabolismo , Encéfalo/irrigação sanguínea , Células Cultivadas , Regulação para Baixo , Endotélio Vascular/citologia , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Humanos , Fosforilação , Proteína Quinase C/fisiologia , Proteína Quinase C-alfa , Proteínas Tirosina Quinases/fisiologia , Quinases Ativadas por p21
3.
Infect Immun ; 71(4): 1680-8, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12654781

RESUMO

Escherichia coli is one of the most common gram-negative bacteria that cause meningitis in neonates. Our previous studies have shown that outer membrane protein A (OmpA) of E. coli interacts with a 95-kDa human brain microvascular endothelial cell (HBMEC) glycoprotein, Ecgp, for invasion. Here, we report the identification of a gene that encodes Ecgp by screening of an HBMEC cDNA expression library as well as by 5' rapid amplification of cDNA ends. The sequence of the Ecgp gene shows that it is highly similar to gp96, a tumor rejection antigen-1, and contains an endoplasmic reticulum retention signal, KDEL. Overexpression of either Ecgp or gp96 in both HBMECs and CHO cells increases E. coli binding and invasion. We further show that Ecgp gene-transfected HBMECs express Ecgp on the cell surface despite the presence of the KDEL motif. Northern blot analysis of total RNA from various eukaryotic cells indicates that Ecgp is significantly expressed in HBMECs. Recombinant His-tagged Ecgp blocked E. coli invasion efficiently by binding directly to the bacteria. These results suggest that OmpA of E. coli K1 interacts with a gp96-like molecule on HBMECs for invasion.


Assuntos
Antígenos de Neoplasias/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Clonagem Molecular , Escherichia coli/patogenicidade , Proteínas do Tecido Nervoso/metabolismo , Receptores de Superfície Celular/metabolismo , Homologia de Sequência de Aminoácidos , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/genética , Proteínas da Membrana Bacteriana Externa/genética , Sequência de Bases , Encéfalo/irrigação sanguínea , Células CHO , Células Cultivadas , Cricetinae , Endotélio Vascular/citologia , Endotélio Vascular/microbiologia , Escherichia coli/metabolismo , Biblioteca Gênica , Humanos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Análise de Sequência de DNA , Transfecção
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