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ABSTRACTCryptococcus neoformansis a pathogenic yeast that often causes devastating meningoencephalitis in immunocompromised individuals. We have previously identified the C. neoformans CPS1gene, which is required for a capsular layer on the outer cell wall. In this report, we investigate the function of the CPS1gene and its pathogenesis. We demonstrated that treatment of yeast with either 4-methylumbelliferone or hyaluronidase resulted in a reduction of the level of C. neoformansbinding 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 elegansdemonstrated that the CPS1wild-type strain is more virulent than the cps1? strain. When CPS1is expressed in Saccharomyces cerevisiaeand 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. neoformanscapsule. The size of hyaluronic acid molecules is evaluated by gel filtration and transmission electron microscopy studies. Together, our results support that C. neoformans CPS1encodes hyaluronic acid synthase and that its product, hyaluronic acid, plays a role as an adhesion molecule during the association of endothelial cells with yeast.

ABSTRACTEscherichia coliis 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. coliinteracts 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. colibinding 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. coliinvasion efficiently by binding directly to the bacteria. These results suggest that OmpA of E. coliK1 interacts with a gp96-like molecule on HBMECs for invasion.

Infectious diseases are one of the leading causes of death in the world. This major medical concern is due to the continual emergence of new infectious diseases and reemergence of old pathogens, together with an increasing number of pathogens resistant to antimicrobial drugs. The generation of new anti-infective agents for resolving these medical problems has emerged as an urgent issue in modern medicine. The availability of genome sequences, and the development of various high-throughput techniques and computational tools offer holistic and integrative strategies for dissecting the interplay between microbial pathogens and their hosts (infectomics). Both microbial and host signatures of infectomes, which mirror the interplay between pathogens and their hosts, provide invaluable fountains in the search for novel antimicrobial therapies. There are several major issues in the discovery and development of new drugs, from target discovery and validation to animal models and clinical trials. However, infectomic approaches integrating genomics, proteomics and glycomics will become increasingly important. Essential Therapeutics Inc. has successfully used functional genomic methods to identify several hundred antimicrobial targets in representative bacterial, fungal and viral pathogens. Recent advances in chemical genomics and proteomics have revolutionized drug discovery research by allowing the parallel processing of multiple genomic targets against large numbers of diverse compounds. The identification of small-molecule modulators of biological functions that are usually executed by large molecules (DNAs, RNAs, proteins and polysaccharides), and the process of transforming these into high-content lead series, are key activities in modern drug discovery. This review focuses on infectomics and its applications to antimicrobial drug discovery.

ABSTRACTCandida albicansis 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. albicanswith human brain microvascular endothelial cells (BMEC). Binding of C. albicansto human BMEC was time and inoculum dependent. Invasion of C. albicansinto human BMEC was demonstrated by using an enzyme-linked immunosorbent assay based on fluorescent staining of C. albicanswith calcoflour. In contrast, avirulent Candidamutant strains and nonpathogenic yeast Saccharomyces cerevisiaewere not able to bind and invade human BMEC. Morphological studies revealed that on association with human BMEC, C. albicansformed germ tubes and was able to bud intracellularly. Transmission electron microscopy showed various stages of C. albicansinteractions with human BMEC, e.g., pseudopod-like structures on human BMEC membrane and intracellular vacuole-like structures retaining C. albicans. Of interest, C. albicanswas able to bud and develop pseudohyphae inside human BMEC without apparent morphological changes of the host cells. In addition, C. albicanspenetrates through human BMEC monolayers without a detectable change in transendothelial electrical resistance and inulin permeability. This is the first demonstration that C. albicansis able to adhere, invade, and transcytose across human BMEC without affecting monolayer integrity. A complete understanding of the interaction(s) of C. albicanswith human BMEC should contribute to the understanding of the pathogenic mechanism(s) ofC. albicansmeningitis.

ABSTRACTNeonatal Escherichia colimeningitis 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. coliinvasion of the blood-brain barrier, a cerebrospinal fluid isolate of E. coliK1 was mutagenized with TnphoA. TnphoAmutant 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 TnphoAinsertion in 27A-6 was cloned and sequenced and determined to be homologous toE. coliK-12 aslA(arylsulfatase-like gene). The deduced amino acid sequence of the E. coliK1aslAgene product shows homology to a well-characterized arylsulfatase family of enzymes found in eukaryotes, as well as prokaryotes. Two additional aslAmutants were constructed by targeted gene disruption and internal gene deletion. Both of these mutants demonstrated decreased invasion phenotypes, similar to that of TnphoAmutant 27A-6. Complementation of the decreased-invasion phenotypes of these mutants was achieved whenaslAwas supplied in trans. This is the first demonstration that this locus contributes to invasion of the blood-brain barrier by E. coliK1.

ABSTRACTMost cases of Escherichia colimeningitis develop as a result of hematogenous spread, but it is not clear how circulatingE. colicrosses the blood-brain barrier. A TnphoAmutant of E. coliK1 RS218 was shown to be significantly less invasive than its parent strain in bovine and human brain microvascular endothelial cells (BMEC), which constitute the blood-brain barrier. More importantly, traversal of the blood-brain barrier was significantly less with this mutant than with the parent strain in newborn rats with experimental hematogenous meningitis. A DNA segment containing the TnphoAinsertion site was cloned from RS218, and the cloned DNA complemented the TnphoAmutant in invasion of BMEC. Nucleotide sequence revealed a near identity to that of a hypothetical yijPgene (also calledf577) in the E. coliK-12 genome. Sequence analysis indicated that the E. coliK1 yijPgene likely encodes a 66.6-kDa membrane protein. Deletion and complementation experiments indicated that the yijPgene was involved in E. coliK1 invasion of BMEC, i.e., the invasive ability of E. coliK1 was significantly reduced after yijPwas deleted and was restored by complementation with a plasmid containing the yijPopen reading frame. This is the first demonstration that the yijPgene locus plays a role in the pathogenesis of E. coliK1 meningitis.

ABSTRACTEscherichia coliK1 is the most common gram-negative organism causing neonatal meningitis, but the mechanism by whichE. coliK1 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. coliK1 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 TnphoAmutant 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. colimeningitis than the parent strain. The TnphoAinsert 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. coliK-12 genome. However, no homology was observed between ibeBand other known invasion genes when DNA and protein databases in GenBank were searched. Like the TnphoAinsertion mutant 7A-33, an isogenic ibeBdeletion mutant (IB7D5) was unable to invade BMEC. A 7.0-kb locus containing ibeBwas isolated from a LambdaGEM-12 genomic library of E. coliRS218 and subcloned into a pBluescript KS vector (pKS7-7B). pKS7-7B was capable of completely restoring the BMEC invasion of the noninvasive TnphoAmutant 7A-33 and the ibeBdeletion mutant IB7D5 to the level of the parent strain. More importantly, the ibeBdeletion mutant IB7D5 was fully complemented by pFN476 carrying the ibeBORF (pFN7C), indicating thatibeBis required for E. coliK1 invasion of BMEC. Taken together, these findings indicate that severalE. colideterminants, including ibeAandibeB, contribute to crossing of the blood-brain barrier.

ABSTRACTThe molecular basis of Escherichia colitraversal of the blood-brain barrier in the development of E. colimeningitis is not well understood. We have previously shown that a novel Ibe10 protein found in cerebrospinal fluid isolates of E. coliis necessary for invasion of the brain microvascular endothelial cells (BMEC) that constitute the blood-brain barrier both in vitro and in a newborn rat model of hematogenous meningitis. Here we identified a novel Ibe10 binding molecule/receptor (Ibe10R) on both bovine BMEC (HBMEC) and human BMEC (HBMEC) that is responsible for invasion by E. coli. Ibe10R, an approximately 55-kDa protein, was purified from BBMEC by Ibe10-Ni-Sepharose affinity chromatography. Bovine Ibe10R, as well as polyclonal antibodies to Ibe10R, blocked E. coliinvasion of BBMEC very effectively. The N-terminal amino acid sequence of Ibe10R showed 75% homology to serum albumin. However, the amino acid sequence of an Ibe10R fragment generated by limited enzymatic digestion did not reveal homology to any other proteins, suggesting that Ibe10R represents a novel albumin-like protein. Immunocytochemical analysis of BBMEC using anti-Ibe10R antibody suggested that only a subset of cultured BBMEC express Ibe10R on their surface. Enrichment of Ibe10R-positive BBMEC by fluorescence-activated cell sorting with anti-Ibe10R antibody resulted in enhanced invasion by E. coli. The anti-Ibe10R antibody raised against bovine Ibe10R also blocked E. coliinvasion of HBMEC very effectively. Interestingly, anti-Ibe10R antibody affinity chromatography of HBMEC membrane proteins revealed a smaller protein with an approximate molecular mass of 45 kDa. These results suggest that the Ibe10 of E. coliinteracts with a novel BMEC surface protein, Ibe10R, for invasion of both BBMEC and HBMEC.

We have combined an ex vivo whole blood and a Transwell insert system to evaluate the biological relevance of antibiotic-induced lipopolysaccharide (LPS) release, and have shown that this event results in a brisk release of TNFα in whole blood (Arditi M., Kabat W., Yogev R. Antibiotic-induced bacterial killing stimulates tumor necrosis factor-α release in whole blood. J Infect Dis1993; 167:240-244). We now investigated the activation of vascular endothelial cells in response to antibiotic-induced bacterial killing and free LPS release. We have previously shown that the endothelial cells respond to LPS in a serum- or soluble CD14-dependent manner. We used LPS-induced IL-6 release from human umbilical vein endothelial cells (HUVEC) and human blood cells as two readout systems. Escherichia coliO111:B4 (106CFU/ml) was placed into the upper chamber of a Transwell filter insert (0.1 μm) and incubated for 6 h with or without ceftriaxone (40x MIC) in 24-well plates containing HUVEC monolayers in the presence of 5% serum, or diluted human blood cells in the lower chamber. Filter inserts were removed at 6 h and plates were incubated for an additional 12 h and supernatants were collected for IL-6 measurement. Bacterial kill curves were generated. Antibiotic-induced killing of E. coliresulted in activation of human endothelial and blood cells as assessed by IL-6 release. Addition of 5 μg/ml of bactericidal permeability factor (BPI) to the media containing ceftriaxone did not influence the kill curve but resulted in over 96% decrease in IL-6 release from both endothelial and blood cells, suggesting that free LPS was the predominant stimulus for IL-6 release. Furthermore, when the endothelial cell experiments were conducted in serum-free conditions, antibiotic-induced bacterial killing did not induce IL-6 release from HUVEC, and addition of recombinant human soluble CD14 into the media reconstituted the serum effect, consistent with the concept that serum or soluble CD14 is absolutely required for LPS-induced endothelial cell responses, including LPS-induced IL-6 release. IL-10 was able to significantly inhibit LPS-induced IL-6 production by human blood cells but had no effect on LPS-mediated IL-6 release from HUVEC. These data suggest that antibiotic-induced free LPS release leads to activation of both human vascular endothelial cells and blood cells, which can be readily inhibited by the addition of BPI. The results support the hypothesis that free endotoxin released from E. colifollowing bacteriolysis is the major contributor to the enhancement of the inflammation associated with the initiation of antibiotic therapy. The dichotomy of IL-10 effects on LPS-induced cytokine production in blood mononuclear cells versus endothelial cells may play a role in the pathophysiology of TH-2-directed responses.

The most virulent newborn meningitis-associated Escherichia coli are of the serotype O18: K1: H7. We previously isolated a large number of E. coli O18:K1:H7 mutants resulting from transposon TnphoA mutagenesis that fail to invade brain microvascular endothelial cells. We have now determined the locations of 45 independent insertions. Twelve were localized to the 98 min region, containing a 120 kb segment that is characteristic of E. coli O18:K1:H7. Another, the previously described insertion ibe-10::TnphoA, was localized to the 87 min region, containing a 20 kb segment found in this E. coli. These noninvasion mutations may define new O18:K1:H7 pathogenicity islands carrying genes for penetration of the blood-brain barrier of newborn mammals.