1 Division of HematologyOncology, Mailstop 57, Childrens Hospital Los Angeles, Los Angeles, CA 90027, USA 2 Division of Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, ...MD, USA 3,4 Division of Infectious Diseases 3 and Department of Surgery 4 , Childrens Hospital Los Angeles, Los Angeles, CA 90027, USA
Correspondence Ambrose Y. Jong ajong{at}chla.usc.edu
Received August 28, 2002
Accepted December 6, 2002
Infection by the human opportunistic fungal pathogen Candida albicans has been increasing over recent years. In an attempt to understand the molecular mechanism of Candida invasion across host tissues, the relationship of C. albicans enolase to human plasminogen/plasmin was investigated. C. albicans enolase is a cell-surface protein and an immunodominant antigen in infected patients sera. Plasminogen is an abundant plasma protein. Several lines of evidence support the binding of C. albicans enolase to human plasminogen. Firstly, it was found that various Candida strains were able to bind to plasminogen and its active form, plasmin. Secondly, recombinant Candida enolase was retained in a nickel-chelating affinity column matrix that can bind 125 I-labelled plasminogen or plasmin in a dose-dependent manner. Plasmin(ogen)-specific inhibitors, such as -aminocaproic acid and aprotinin, can effectively block plasmin-binding activity. Thirdly, as with many plasminogen receptors, binding of Candida enolase to plasmin(ogen) is lysine-dependent, whereas little inhibition occurred with arginine, aspartate and glutamate. Fourthly, immobilized enolase enhanced plasminogen's affinity for streptokinase at least tenfold, as demonstrated by its activation of plasmin activity. To elucidate the biological significance of this result, it was demonstrated that the plasmin(ogen)-bound Candida cells were able to induce fibrinolysis activity in a matrix-gel assay. Furthermore, plasmin-bound Candida cells had an increased ability to cross an in vitro bloodbrain barrier system. The results given here indicate that Candida enolase is a plasminogen- and plasmin-binding protein and that the interaction of C. albicans enolase with the plasminogen system may contribute to invasion of the tissue barrier.
These authors contributed equally to this work.
Abbreviations: EACA, -aminocaproic acid; ECM, extracellular matrix; HBMEC, human brain microvascular endothelial cells; Ni-NTA, nickel nitrilotriacetic acid; SAP, secreted aspartyl proteinase; TEER, transendothelial electric resistance; tPA, tissue-type plasminogen activator.
Cryptococcus neoformans is a life-threatening pathogenic yeast that causes devastating meningoencephalitis. The mechanism of cryptococcal brain invasion is largely unknown, and recent studies suggest ...that its extracellular microvesicles may be involved in the invasion process. The 14-3-3 protein is abundant in the extracellular microvesicles of C. neoformans, and the 14-3-3-GFP fusion has been used as the microvesicle's marker. However, the physiological role of 14-3-3 has not been explored. In this report, we have found that C. neoformans contains a single 14-3-3 gene that apparently is an essential gene. To explore the functions of 14-3-3, we substituted the promoter region of the 14-3-3 with the copper-controllable promoter CTR4. The CTR4 regulatory strain showed an enlarged cell size, drastic changes in morphology, and a decrease in the thickness of the capsule under copper-enriched conditions. Furthermore, the mutant cells produced a lower amount of total proteins in their extracellular microvesicles and reduced adhesion to human brain microvascular endothelial cells in vitro. Proteomic analyses of the protein components under 14-3-3-overexpressed and -suppressed conditions revealed that the 14-3-3 function(s) might be associated with the microvesicle biogenesis. Our results support that 14-3-3 has diverse pertinent roles in both physiology and pathogenesis in C. neoformans. Its gene functions are closely relevant to the pathogenesis of this fungus.
The ibeA gene (ibe10) previously identified by TnphoA mutagenesis is part of a 50-kDa full-length open-reading frame (ORF) encoded by a 1.37-kb DNA fragment. An isogenic in-frame deletion mutant of ...ibeA (ZD1) was constructed by chromosomal gene replacement with a suicide plasmid pCVD442 carrying a 2.1-kb DNA fragment with an ibeA deletion. Similar to the previously described TnphoA insertion mutant of ibeA the isogenic ibeA deletion mutant ZD1 was significantly less invasive in human brain microvascular endothelial cells (BMECs) than the parent strain. The mutant ZD1 was fully complemented by the ibeA ORF. The ibeA gene was subcloned into pET28a(+) and was expressed as a recombinant protein with an N-terminal histidine tag. The recombinant IbeA protein had much greater activity (50 times) in blocking the invasion of BMECs by Escherichia coli K1 than did the partial protein fragment, which provides further evidence that ibeA is an important determinant for E. coli K1 invasion of BMECs
Cryptococcus neoformans is a life-threatening pathogenic yeast that causes devastating meningoencephalitis. The mechanism of cryptococcal brain invasion is largely unknown, and recent studies suggest ...that its extracellular microvesicles may be involved in the invasion process. The 14-3-3 protein is abundant in the extracellular microvesicles of C. neoformans, and the 14-3-3-GFP fusion has been used as the microvesicle’s marker. However, the physiological role of 14-3-3 has not been explored. In this report, we have found that C. neoformans contains a single 14-3-3 gene that apparently is an essential gene. To explore the functions of 14-3-3, we substituted the promoter region of the 14-3-3 with the copper-controllable promoter CTR4. The CTR4 regulatory strain showed an enlarged cell size, drastic changes in morphology, and a decrease in the thickness of the capsule under copper-enriched conditions. Furthermore, the mutant cells produced a lower amount of total proteins in their extracellular microvesicles and reduced adhesion to human brain microvascular endothelial cells in vitro. Proteomic analyses of the protein components under 14-3-3-overexpressed and -suppressed conditions revealed that the 14-3-3 function(s) might be associated with the microvesicle biogenesis. Our results support that 14-3-3 has diverse pertinent roles in both physiology and pathogenesis in C. neoformans. Its gene functions are closely relevant to the pathogenesis of this fungus.
The purpose of this study was to examine prophylactic efficacy of probiotics in neonatal sepsis and meningitis caused by E. coli K1. The potential inhibitory effect of Lactobacillus rhamnosus GG ...(LGG) on meningitic E. coli K1 infection was examined by using (i) in vitro inhibition assays with E44 (a CSF isolate from a newborn baby with E. coli meningitis), and (ii) the neonatal rat model of E. coli sepsis and meningitis. The in vitro studies demonstrated that LGG blocked E44 adhesion, invasion, and transcytosis in a dose-dependent manner. A significant reduction in the levels of pathogen colonization, E. coli bacteremia, and meningitis was observed in the LGG-treated neonatal rats, as assessed by viable cultures, compared to the levels in the control group. In conclusion, probiotic LGG strongly suppresses meningitic E. coli pathogens in vitro and in vivo. The results support the use of probiotic strains such as LGG for prophylaxis of neonatal sepsis and meningitis.
To study the effect of HIV-1 gp41 ectodomain (gp41-I90) on the cytoskeletal changes in human brain microvascular endothelial cells (HBMECs) induced by Cryptococcus neoformans.
HBMECs were cultured on ...collagen-coated chamber slide or transwell to allow the formation of cell monolayers. After pre-treatment with gp41-I90 and infection with Cryptococcus neoformans, the HBMECs were examined for the expression of actin or filamin by immunofluorescence assay. HRP permeability of the HBMECs treated with gp41-I90 was detected by ELISA. Transcytosis of Cryptococcus neoformans through the gp41-I90-treated HBMECs was detected by direct counting from a hemocytometer.
gp41-I90 obviously enhanced the cytoskeletal changes of the HBMECs infected by Cryptococcus neoformans, causing curved and sparse filamentous arrangement of actin and filamin. gp41-I90 treatment also resulted in obviously increased HRP permeability of the cells and transcytosis of Cryptococcus neoformans.
gp41- I90 enhances Cryptococcus neoformans binding to
IbeR is a regulator present in meningitic Escherichia coli strain E44 that carries a loss-of-function mutation in the stationary-phase (SP) regulatory gene rpoS. In order to determine whether IbeR is ...an SP regulator in E44, two-dimensional gel electrophoresis and LC-MS were used to compare the proteomes of a noninvasive ibeR deletion mutant BR2 and its parent strain E44 in the SP. Four up-regulated (TufB, GapA, OmpA, AhpC) and three down-regulated (LpdA, TnaA, OpmC) proteins in BR2 were identified when compared to E44. All these proteins contribute to energy metabolism or stress resistance, which is related to SP regulation. One of the down-regulated proteins, tryptophanase (TnaA), which is regulated by RpoS in other E. coli strains, is associated with SP regulation via production of a signal molecule indole. Our studies demonstrated that TnaA was required for E44 invasion, and that indole was able to restore the noninvasive phenotype of the tnaA mutant. The production of indole was significantly reduced in BR2, indicating that ibeR is required for the indole production via tnaA. Survival studies under different stress conditions indicated that IbeR contributed to bacteria stress resistance in the SP. Taken together, IbeR is a novel regulator contributing to the SP regulation.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, OILJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK, VSZLJ
The cell division controller Cdc6 plays a central role in the initiation of DNA replication. It was found that elevated levels of Cdc6 were present in many of human cancer cells, and the accumulation ...of Cdc6 is required for cell proliferation. In this study, we have investigated the control of Cdc6 expression and its effect on cell proliferation and death in human neuroblastoma cells. Elevated levels of Cdc6 are found in the LA-N-2, CHLA255, and other cell lines that grow fast. Cdc6 knockdown via a Cdc6 short hairpin RNA lentivirus causes the accumulation of sub-G1 populations with the decrease of S contents in the LA-N-2 and CHLA255 cells. Expression profile from the selected genes shows the reduction of cyclin E, cyclin A, and Cdc25C, with a boosted increase of the CDK inhibitor p27Kip¹, indicating the suppression of tumor cell proliferation. Further, Cdc6 knockdown causes the increase of pro-apoptotic Bax accompanied with the decrease of anti-apoptotic Bcl-2, resulting in the increased cell death. Furthermore, Cdc6 knockdown causes a sharp reduction of tumor suppressor protein p53, and Cdc6 overexpression renders a boosted p53 expression; and this regulation is at p53 posttranscriptional level. Our study indicates that human Cdc6 functions in several pathways to control the cell proliferation and the cell death.
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