Ambrose Jong

Traversal of Candida albicans across Human Blood-Brain Barrier In Vitro

ABSTRACT Candida albicans is an opportunistic pathogen, which primarily affects neonates and immunocompromised individuals. The pathogen can invade the central nervous system, resulting in meningitis. At present, the pathogenesis of C. albicansmeningitis is unclear. We used an in vitro model of the human blood-brain barrier to investigate the interaction(s) of C. albicans with human brain microvascular endothelial cells (BMEC). Binding of C. albicans to human BMEC was time and inoculum dependent. Invasion of C. albicans into human BMEC was demonstrated by using an enzyme-linked immunosorbent assay based on fluorescent staining of C. albicans with calcoflour. In contrast, avirulent Candida mutant strains and nonpathogenic yeast Saccharomyces cerevisiae were not able to bind and invade human BMEC. Morphological studies revealed that on association with human BMEC, C. albicans formed germ tubes and was able to bud intracellularly. Transmission electron microscopy showed various stages of C. albicans interactions with human BMEC, e.g., pseudopod-like structures on human BMEC membrane and intracellular vacuole-like structures retaining C. albicans. Of interest, C. albicans was able to bud and develop pseudohyphae inside human BMEC without apparent morphological changes of the host cells. In addition, C. albicans penetrates through human BMEC monolayers without a detectable change in transendothelial electrical resistance and inulin permeability. This is the first demonstration that C. albicans is able to adhere, invade, and transcytose across human BMEC without affecting monolayer integrity. A complete understanding of the interaction(s) of C. albicans with human BMEC should contribute to the understanding of the pathogenic mechanism(s) ofC. albicans meningitis.

Cellular mechanisms of microbial proteins contributing to invasion of the blood-brain barrier.

One of the least understood issues in the pathogenesis and pathophysiology of microbial infection of the central nervous system (CNS) is how microorganisms cross the blood–brain barrier (BBB), which separates brain interstitial space from blood and is formed by the tight junctions of brain microvascular endothelial cells (BMEC). BMEC monolayer and bilayer culture systems have been developed as in vitro models to dissect the mechanisms of adhesion and invasion involved in pathogenesis of CNS infection caused by microbes. Viral, bacterial, fungal and parasitic pathogens may breach the BBB and enter the CNS through paracellular, transcellular and/or Trojan horse mechanisms. Conceivable evidence suggests that microbial proteins are the major genetic determinants mediating penetration across the BBB. Several bacterial proteins including IbeA, IbeB, AslA,YijP, OmpA, PilC and InlB contribute to transcellular invasion of BMEC. Viral proteins such as gp120 of HIV have been shown to play a role in penetration of the BBB. Fungal and parasitic pathothogens may follow similar mechanisms. SAG1 of Toxoplasma gondii has been suggested as a ligand to mediate host‐cell invasion. Understanding the fundamental mechanisms of microbial penetration of the BBB may help develop novel approaches to prevent the mortality and morbidity associated with central nervous system (CNS) infectious diseases.

Exosome-Mediated Transfer of microRNAs Within the Tumor Microenvironment and Neuroblastoma Resistance to Chemotherapy

BACKGROUND How exosomic microRNAs (miRNAs) contribute to the development of drug resistance in the context of the tumor microenvironment has not been previously described in neuroblastoma (NBL). METHODS Coculture experiments were performed to assess exosomic transfer of miR-21 from NBL cells to human monocytes and miR-155 from human monocytes to NBL cells. Luciferase reporter assays were performed to assess miR-155 targeting of TERF1 in NBL cells. Tumor growth was measured in NBL xenografts treated with Cisplatin and peritumoral exosomic miR-155 (n = 6 mice per group) CD163, miR-155, and TERF1 levels were assessed in 20 NBL primary tissues by Human Exon Arrays and quantitative real-time polymerase chain reaction. Students t test was used to evaluate the differences between treatment groups. All statistical tests were two-sided. RESULTS miR-21 mean fold change (f.c.) was 12.08±0.30 (P < .001) in human monocytes treated with NBL derived exosomes for 48 hours, and miR-155 mean f.c. was 4.51±0.25 (P < .001) in NBL cells cocultured with human monocytes for 48 hours. TERF1 mean luciferase activity in miR-155 transfected NBL cells normalized to scrambled was 0.36 ± 0.05 (P <.001). Mean tumor volumes in Dotap-miR-155 compared with Dotap-scrambled were 322.80±120mm(3) and 76.00±39.3mm(3), P = .002 at day 24, respectively. Patients with high CD163 infiltrating NBLs had statistically significantly higher intratumoral levels of miR-155 (P = .04) and lower levels of TERF1 mRNA (P = .02). CONCLUSIONS These data indicate a unique role of exosomic miR-21 and miR-155 in the cross-talk between NBL cells and human monocytes in the resistance to chemotherapy, through a novel exosomic miR-21/TLR8-NF-кB/exosomic miR-155/TERF1 signaling pathway.

Further Characterization of Escherichia coli Brain Microvascular Endothelial Cell Invasion Gene ibeA by Deletion, Complementation, and Protein Expression

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.

Involvement of human CD44 during Cryptococcus neoformans infection of brain microvascular endothelial cells

Pathogenic yeast Cryptococcus neoformans causes devastating cryptococcal meningoencephalitis. Our previous studies demonstrated that C. neoformans hyaluronic acid was required for invasion into human brain microvascular endothelial cells (HBMEC), which constitute the blood–brain barrier. In this report, we demonstrate that C. neoformans hyaluronic acid interacts with CD44 on HBMEC. Our results suggest that HBMEC CD44 is a primary receptor during C. neoformans infection, based on the following observations. First, anti‐CD44 neutralizing antibody treatment was able to significantly reduce C. neoformans association with HBMEC. Second, C. neoformans association was considerably impaired using either CD44‐knock‐down HBMEC or C. neoformans hyaluronic acid‐deficient strains. Third, overexpression of CD44 in HBMEC increased their association activity towards C. neoformans. Fourth, confocal microscopic images showed that CD44 was enriched at and around the C. neoformans association sites. Fifth, upon C. neoformans and HBMEC engagement, a subpopulation of CD44 and actin translocated to the host membrane rafts. Our results highlight the interactions between C. neoformans hyaluronic acid and host CD44 and the dynamic results of these interactions, which may represent events during the adhesion and entry of C. neoformans at HBMEC membrane rafts.

Down-regulation of growth arrest DNA damage-inducible gene 45β expression is associated with human hepatocellular carcinoma

In this study, we describe the growth arrest DNA damage-inducible gene 45beta (GADD45beta), whose expression was significantly down-regulated in the hepatocellular carcinoma (HCC) microarray study and confirmed by Northern blot analysis. The results suggested that expression of GADD45beta was decreased in human liver cancer cell lines HepG2 and Hep3B, but not in normal human embryonic liver cell line CL-48 or normal liver tissue. Histochemistry study and real-time PCR further confirmed that GADD45beta staining in HCC was significantly decreased when compared to surrounding non-neoplastic liver tissue. In further studies of multiple human cancer tissues, GADD45beta strongly stained tissues such as colon cancer, breast cancer, prostate cancer, squamous cell cancer, lymphoma, and leiomyosarcoma, suggesting that the decreased expression of GADD45beta is specific to HCC. Eighty-five cases of primary HCC were further examined by immunohistochemistry and statistical analyses demonstrated that HCC scored lower than matched non-neoplastic liver tissues consistently and significantly. No staining occurred in 12.94% of HCC cases (score = 0, n = 11); 42.35% had weak staining (score = 1, n = 36); 27.06% had moderate staining (score = 2, n = 23); and 17.65% had staining as strong as normal tissue (score = 3, n = 15). Overall, surrounding non-neoplastic liver tissue was highly positive for GADD45beta compared to adjacent neoplastic liver tissues (P < 0.01). We further observed that down-regulation of GADD45beta expression was strongly correlated with differentiation (P < 0.01) and high nuclear grade (P < 0.01). Moreover, we found that expression of GADD45beta was inversely correlated to the presence of mutant p53 in HCC tissue (P < 0.05). Thus, the results of our study suggest that GADD45beta, which is down-regulated in most cases of HCC, remains an ideal candidate for development as a molecular marker in the diagnosis of HCC and as a potential therapeutic target.

Identification and Characterization of CPS1 as a Hyaluronic Acid Synthase Contributing to the Pathogenesis of Cryptococcus neoformans Infection

ABSTRACT Cryptococcus neoformans is a pathogenic yeast that often causes devastating meningoencephalitis in immunocompromised individuals. We have previously identified the C. neoformans CPS1 gene, which is required for a capsular layer on the outer cell wall. In this report, we investigate the function of the CPS1 gene and its pathogenesis. We demonstrated that treatment of yeast with either 4-methylumbelliferone or hyaluronidase resulted in a reduction of the level of C. neoformans binding to human brain microvascular endothelial cells (HBMEC). Yeast extracellular structures were also altered accordingly in hyaluronidase-treated cells. Furthermore, observation of yeast strains with different hyaluronic acid contents showed that the ability to bind to HBMEC is proportional to the hyaluronic acid content. A killing assay with Caenorhabditis elegans demonstrated that the CPS1 wild-type strain is more virulent than the cps1Δ strain. When CPS1 is expressed in Saccharomyces cerevisiae and Escherichia coli, hyaluronic acid can be detected in the cells. Additionally, we determined by fluorophore-assisted carbohydrate electrophoretic analysis that hyaluronic acid is a component of the C. neoformans capsule. The size of hyaluronic acid molecules is evaluated by gel filtration and transmission electron microscopy studies. Together, our results support that C. neoformans CPS1 encodes hyaluronic acid synthase and that its product, hyaluronic acid, plays a role as an adhesion molecule during the association of endothelial cells with yeast.

The essential role of Saccharomyces cerevisiae CDC6 nucleotide-binding site in cell growth, DNA synthesis, and Orc1 association.

Saccharomyces cerevisiaeCdc6 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 nucleotide-binding sites. To study the requirement of the nucleotide-binding site for the essential function of Cdc6, we have changed the conserved Lys114 at the nucleotide-binding 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 cdc6temperature-sensitive mutant cells. Our results suggest that replacement of Lys with Glu (K114E) and Pro (K114P) leads to loss-of-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 G1/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 nucleotide-binding 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 ATP-binding of Cdc6, which may be a prerequisite step to assemble the operational replicative complex at the G1/S transition.

CPS1, a Homolog of the Streptococcus pneumoniae Type 3 Polysaccharide Synthase Gene, Is Important for the Pathobiology of Cryptococcus neoformans

ABSTRACT The polysaccharide capsule is known to be the major factor required for the virulence of Cryptococcus neoformans. We have cloned and characterized a gene, designated CPS1, that encodes a protein containing a glycosyltransferase moiety and shares similarity with the type 3 polysaccharide synthase encoded by the cap3B gene of Streptococcus pneumoniae. Cps1p also shares similarity with hyaluronan synthase of higher eukaryotes. Deletion of the CPS1 gene from a serotype D strain of C. neoformans resulted in a slight reduction of the capsule size as observed by using an India ink preparation. The growth at 37°C was impaired, and the ability to associate with human brain endothelial cells in vitro was also significantly reduced by the deletion of CPS1. Using site-specific mutagenesis, we showed that the conserved glycosyltransferase domains are critical for the ability of the strain to grow at elevated temperatures. A hyaluronan enzyme-linked immunosorbent assay method demonstrated that CPS1 is important for the synthesis of hyaluronan or its related polysaccharides in C. neoformans. Comparisons between the wild-type and the cps1Δ strains, using three different transmission electron microscopic methods, indicated that the CPS1 gene product is involved in the composition or maintenance of an electron-dense layer between the outer cell wall and the capsule. These and the virulence studies in a mouse model suggested that the CPS1 gene is important in the pathobiology of C. neoformans.

Infectomics: genomics and proteomics of microbial infections

Abstract. The completion of genomic sequences is the greatest triumph of molecular reductionism since the discovery of the DNA double helix in 1953. However, the utility of reductionism is becoming limited and holistic approaches, including theories and techniques, are desperately needed in the postgenomic era. In the field of infectious diseases there is an urgent need for global approaches that can efficiently, precisely and integratively study structural and functional genomics and proteomics of microbial infections (infectomics). The combination of new (e.g. DNA and protein microarrays) and traditional approaches (e.g. cloning, PCR, gene knockout and knockin, and antisense) will help overcome the challenges we are facing today. We assume that the global phenotypic changes (infectomes) in microbes and their host during infections are encoded by the genomes of microbial pathogens and their hosts, expressed in certain environmental conditions devoted to specific microbe-host interactions. Global drug responses (pharmacomes) in microbes and their host can be detected by genomic and proteomic approaches. Genome-wide approaches to genotyping and phenotyping or expression profiling will eventually lead to global dissection of microbial pathogenesis, efficient and rapid diagnosis of infectious diseases, and the development of novel strategies to control infections. The key fundamental issue of infectious diseases is how to globally and integratively understand the interactions between microbial pathogens and their hosts by using infectomics. In this review, we focus on the events that are considered important in infectomics.