The fungal pathogen Cryptococcus neoformans has a predilection for the central nervous system (CNS), resulting in devastating meningoencephalitis. At present, it is unclear how C. neoformans traverses the blood-brain barrier (BBB) and causes CNS infection. The present study has examined and characterized the interaction of C. neoformans with human brain microvascular endothelial cells (HBMEC), which constitute the BBB. Adhesion of and transcytosis of HBMEC by C. neoformans was inoculum-and time-dependent and occurred with both encapsulated and acapsulated strains. C. neoformans induced marked morphological changes in HBMEC, for example membrane ruffling, irregular nuclear morphology and swelling of the mitochondria and the ER. These findings suggest that C. neoformans induced actin cytoskeletal reorganization of the host cells. In addition, it was observed that the dephosphorylated form of cofilin was increased during cryptococcal adherence to HBMEC, concomitant with the actin rearrangement. Cryptococcal binding to HBMEC was increased in the presence of Y27632, a Rho kinase (ROCK)-specific inhibitor. Since ROCK activates LIM kinase (LIMK), which phosphorylates cofilin (inactive form), this suggests the involvement of the ROCK!LIMK!cofilin pathway. In contrast, the phosphatase inhibitor sodium orthovanadate decreased adherence of Cryptococcus to HBMEC, concomitant with the increase of phosphorylation of cofilin. Furthermore, the tight junction marker protein occludin became Tritonextractable, indicating alteration of tight junctions in brain endothelial cells. This is the first demonstration that C. neoformans is able to adhere to and transcytose across the HBMEC monolayer and alter the cytoskeleton morphology in HBMEC. Further characterization of the interactions between C. neoformans and HBMEC should help the development of novel strategies to prevent cryptococcal meningitis and its associated morbidity.

Escherichia coli is the most common gram-negative bacteria causing meningitis during the neonatal period, but it is unclear what microbial factors mediate traversal of E. coli across the blood-brain barrier. Outer membrane protein A (OmpA), a highly conserved 35-kDa protein, was examined for its role in E. coli K1 invasion of brain microvascular endothelial cells (BMEC). The invasive capability of the OmpA ؉ strains was 25-to 50-fold greater than that of OmpA ؊ strains, and the invasive capability of OmpA ؊ strains was restored to the level of the OmpA ؉ strain by complementation with the ompA gene. Purified OmpA proteins and polyclonal anti-OmpA antibodies inhibited the invasion of OmpA ؉ E. coli into BMEC. Two short synthetic peptides (a hexamer, Asn-27-Glu-32, and a pentamer, Gly-65-Asn-69) generated from the N-terminal amino acid sequence of OmpA exhibited significant inhibition of OmpA ؉ E. coli invasion, suggesting that these two sequences represent the OmpA domains involved in E. coli invasion of BMEC. These findings suggest that OmpA is the first microbial structure identified to enhance E. coli invasion of BMEC, an important event in the pathogenesis of E. coli meningitis.

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.

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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. Escherichia coli K1 is the most frequent causative agent of neonatal meningitis. The pathogenic mechanisms of E. coli have been studied by utilizing brain microvascular endothelial cells (BMEC) as an in vitro blood-brain barrier (BBB) model (17,19,20). These studies suggest that S fimbriae mediate attachment to BMEC via NeuAc2,3-Gal epitopes of BMEC surface glycoproteins; however, they do not play a significant role in invasion (25). More-intimate attachment by the bacterial outer membrane protein A (OmpA) mediates the invasion process (15). A similar phenomenon has been identified in the pathogenesis of Neisseria gonorrhoeae, where pili promote initial adherence followed by Opa-mediated interaction for invasion (10). In addition to OmpA, other bacterial factors such as IbeA, IbeB, TraJ, and CNF also play roles in E. coli invasion of BMEC; however, OmpA appears to be the most important factor (2, 6, 7, 9). OmpA ϩ E. coli induces actin rearrangements at the site of bacterial entry, which are completely abolished by treatment of the bacteria with GlcNAc␤1-4GlcNAc polymers, which are receptor analogs (16,17). Computer simulation studies of the interactions between OmpA and GlcNAc␤1-4GlcNAc epitopes indicate that these sugars have more favorable energy than any other sugar molecule tested in our experiments (4). These results are in good agreement with earlier studies in which GlcNAc␤1-4GlcNAc moieties showed significant blocking of E. coli invasion both in vitro and in vivo (16).In support of the role of OmpA in E. coli K1 invasion, studies have also demonstrated that OmpA ϩ E. coli induces the phosphorylation of focal adhesion kinase (FAK) and its interaction with phosphatidylinositol 3-kinase (PI3K) (20, 21). Furthermore, GlcNAc␤1-4GlcNAc polymers blocked the activation of FAK, although at higher concentrations, indicating the role of the human BMEC (HBMEC) receptor for OmpA in transducing signals for internalization of E. coli. In addition, it was shown that E. coli also induces the activation of protein kinase C alpha (PKC-␣) in an OmpA-dependent manner (27). The activated PKC-␣ is recruited to the plasma membrane, where it interacts with caveolin-1, a protein marker for caveolae, for internalization of E. coli (28). Several receptors, such as epidermal growth factor and fibroblast growth factor (14), have been shown to accumulate in caveolae, suggesting that the OmpA receptor could be part of caveolae during...

Saccharomyces cerevisiae Cdc6 is a protein required for the initiation of DNA replication. The biochemical function of the protein is unknown, but the primary sequence contains motifs characteristic of nucleotidebinding sites. To study the requirement of the nucleotide-binding site for the essential function of Cdc6, we have changed the conserved Lys 114 at the nucleotidebinding site to five other amino acid residues. We have used these mutants to investigate in vivo roles of the conserved lysine in the growth rate of transformant cells and the complementation of cdc6 temperature-sensitive mutant cells. Our results suggest that replacement of Lys with Glu (K114E) and Pro (K114P) leads to lossof-function in supporting cell growth, replacement of the Lys with Gln (K114Q) or Leu (K114L) yields partially functional proteins, and replacement with Arg yields a phenotype equivalent to wild-type, a silent mutation. To investigate what leads to the growth defects derived from the mutations at the nucleotide-binding site, we evaluated its gene functions in DNA replication by the assays of the plasmid stability and chromosomal DNA synthesis. Indeed, the K114P and K114E mutants showed the complete retraction of DNA synthesis. In order to test its effect on the G 1 /S transition of the cell cycle, we have carried out the temporal and spatial studies of yeast replication complex. To do this, yeast chromatin fractions from synchronized culture were prepared to detect the Mcm5 loading onto the chromatin in the presence of the wild-type Cdc6 or mutant cdc6(K114E) proteins. We found that cdc6(K114E) is defective in the association with chromatin and in the loading of Mcm5 onto chromatin origins. To further investigate the molecular mechanism of nucleotidebinding function, we have demonstrated that the Cdc6 protein associates with Orc1 in vitro and in vivo. Intriguingly, the interaction between Orc1 and Cdc6 is disrupted when the cdc6(K114E) protein is used. Our results suggest that a proper molecular interaction between Orc1 and Cdc6 depends on the functional ATPbinding of Cdc6, which may be a prerequisite step to assemble the operational replicative complex at the G 1 /S transition.Cell cycle regulation is a complicated but highly coordinated process. It has a conserved mechanism among eukaryotes from yeast to human. The primary control of the eukaryotic cell cycle is provided by a family of cyclin-dependent kinases (CDKs) 1 and their associated cyclins, which regulate kinase activity. In unicellular yeast cells, a single prototype CDK gene, CDC28 in the budding yeast Saccharomyces cerevisiae or cdc2 ϩ in the fission yeast Schizosaccharomyces pombe functions at different cell cycle stages. Different cyclins activate the same kinase as different points in the cycle. It is now known that the central control of cell cycle progression by CDK complexes is regulated positively and negatively to monitor each step of the progression (1, 2). This regulatory control, associated with checkpoints, orchestrates various types of cell cycle ge...

Escherichia coli K1 is the most common gram-negative organism causing neonatal meningitis, but the mechanism by whichE. coli K1 crosses the blood-brain barrier is incompletely understood. We have previously described the cloning and molecular characterization of a determinant, ibeA (also called ibe10), from the chromosome of an invasive cerebrospinal fluid isolate of E. coli K1 strain RS218 (O18:K1:H7). Here we report the identification of another chromosomal locus, ibeB, which allows RS218 to invade brain microvascular endothelial cells (BMEC). The noninvasive TnphoA mutant 7A-33 exhibited <1% the invasive ability of the parent strain in vitro in BMEC and was significantly less invasive in the central nervous system in the newborn rat model of hematogenousE. coli meningitis than the parent strain. The TnphoA insert with flanking sequences was cloned and sequenced. A 1,383-nucleotide open reading frame (ORF) encoding a 50-kDa protein was identified and termed ibeB. This ORF was found to be 97% identical to a gene encoding a 50-kDa hypothetical protein (p77211) and located in the 13-min region of the E. coli K-12 genome. However, no homology was observed between ibeB and other known invasion genes when DNA and protein databases in GenBank were searched. Like the TnphoA insertion mutant 7A-33, an isogenic ibeBdeletion mutant (IB7D5) was unable to invade BMEC. A 7.0-kb locus containing ibeB was isolated from a LambdaGEM-12 genomic library of E. coli RS218 and subcloned into a pBluescript KS vector (pKS7-7B). pKS7-7B was capable of completely restoring the BMEC invasion of the noninvasive TnphoA mutant 7A-33 and the ibeB deletion mutant IB7D5 to the level of the parent strain. More importantly, the ibeB deletion mutant IB7D5 was fully complemented by pFN476 carrying the ibeB ORF (pFN7C), indicating thatibeB is required for E. coli K1 invasion of BMEC. Taken together, these findings indicate that severalE. coli determinants, including ibeA andibeB, contribute to crossing of the blood-brain barrier.