Sheng-He Huang

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.

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.

Escherichia coli K1 aslA contributes to invasion of brain microvascular endothelial cells in vitro and in vivo.

ABSTRACT Neonatal Escherichia coli meningitis remains a devastating disease, with unacceptably high morbidity and mortality despite advances in supportive care measures and bactericidal antibiotics. To further our ability to improve the outcome of affected neonates, a better understanding of the pathogenesis of the disease is necessary. To identify potential bacterial genes which contribute toE. coli invasion of the blood-brain barrier, a cerebrospinal fluid isolate of E. coli K1 was mutagenized with TnphoA. TnphoA mutant 27A-6 was found to have a significantly decreased ability to invade brain microvascular endothelial cells compared to the wild type. In vivo, 32% of the animals infected with mutant 27A-6 developed meningitis, compared to 82% of those infected with the parent strain, despite similar levels of bacteremia. The DNA flanking the TnphoA insertion in 27A-6 was cloned and sequenced and determined to be homologous toE. coli K-12 aslA (arylsulfatase-like gene). The deduced amino acid sequence of the E. coli K1aslA gene product shows homology to a well-characterized arylsulfatase family of enzymes found in eukaryotes, as well as prokaryotes. Two additional aslA mutants were constructed by targeted gene disruption and internal gene deletion. Both of these mutants demonstrated decreased invasion phenotypes, similar to that of TnphoA mutant 27A-6. Complementation of the decreased-invasion phenotypes of these mutants was achieved whenaslA was supplied in trans. This is the first demonstration that this locus contributes to invasion of the blood-brain barrier by E. coli K1.

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.

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.

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.

Invasion of Cryptococcus neoformans into Human Brain Microvascular Endothelial Cells Is Mediated through the Lipid Rafts-Endocytic Pathway via the Dual Specificity Tyrosine Phosphorylation-regulated Kinase 3 (DYRK3)

Cryptococcus neoformans is a neurotropic fungal pathogen, which provokes the onset of devastating meningoencephalitis. We used human brain microvascular endothelial cells (HBMEC) as the in vitro model to investigate how C. neoformans traverses across the blood-brain barrier. In this study, we present several lines of evidence indicating that C. neoformans invasion is mediated through the endocytic pathway via lipid rafts. Human CD44 molecules from lipid rafts can directly interact with hyaluronic acid, the C. neoformans ligand. Bikunin, which perturbs CD44 function in the lipid raft, can block C. neoformans adhesion and invasion of HBMEC. The lipid raft marker, ganglioside GM1, co-localizes with CD44 on the plasma membrane, and C. neoformans cells can adhere to the host cell in areas where GM1 is enriched. These findings suggest that C. neoformans entry takes place on the lipid rafts. Upon C. neoformans engagement, GM1 is internalized through vesicular structures to the nuclear membrane. This endocytic redistribution process is abolished by cytochalasin D, nocodazole, or anti-DYRK3 (dual specificity tyrosine-phosphorylation-regulated kinase 3) siRNA. Concomitantly, the knockdown of DYRK3 significantly reduces C. neoformans invasion across the HBMEC monolayer in vitro. Our data demonstrate that the lipid raft-dependent endocytosis process mediates C. neoformans internalization into HBMEC and that the CD44 protein of the hosts, cytoskeleton, and intracellular kinase-DYRK3 are involved in this process.

Invasion of Cryptococcus neoformans into human brain microvascular endothelial cells requires protein kinase C-alpha activation.

Pathogenic fungus Cryptococcus neoformans has a predilection for the central nervous system causing devastating meningoencephalitis. Traversal of C. neoformans across the blood–brain barrier (BBB) is a crucial step in the pathogenesis of C. neoformans. Our previous studies have shown that the CPS1 gene is required for C. neoformans adherence to the surface protein CD44 of human brain microvascular endothelial cells (HBMEC), which constitute the BBB. In this report, we demonstrated that C. neoformans invasion of HBMEC was blocked in the presence of G109203X, a protein kinase C (PKC) inhibitor, and by overexpression of a dominant‐negative form of PKCα in HBMEC. During C. neoformans infection, phosphorylation of PKCα was induced and the PKC enzymatic activity was detected in the HBMEC membrane fraction. Our results suggested that the PKCα isoform might play a crucial role during C. neoformans invasion. Immunofluorescence microscopic images showed that induced phospho‐PKCα colocalized with β‐actin on the membrane of HBMEC. In addition, cytochalasin D (an F‐filament‐disrupting agent) inhibited fungus invasion into HBMEC in a dose‐dependent manner. Furthermore, blockage of PKCα function attenuated actin filament activity during C. neoformans invasion. These results suggest a significant role of PKCα and downstream actin filament activity during the fungal invasion into HBMEC.