John Irvin Glass

Synergistic Antiviral Activity of Human Interferon Combinations in the Hepatitis C Virus Replicon System

The use of type I interferon (IFN), in combination with ribvirin, to treat chronic hepatitis C virus (HCV) infection has many drawbacks that prevent widespread application, ultimately leading to a significant unmet clinical need. Potential improvements in IFN therapy through targeted delivery, molecular alteration, and combination with other agents are ongoing in an attempt to decrease adverse effects and increase efficacy. In this report, the HCV replicon cell culture system was used to assess potential synergistic antiviral effects of multiple IFN species when administered in combination. Quantitative analysis of HCV replicon RNA by TaqMan (PE Applied Biosystems, Foster City, CA) and qualitative analysis of HCV protein expression were used to measure the antiviral efficacy of individual and combination IFN treatments, and synergistic responses of IFN combinations were determined through statistical analysis of the TaqMan results. We found that when administered simultaneously, type I/II IFN combinations (IFN-alpha2b + IFN-gamma or IFN-beta + IFN-gamma) resulted in dramatic antiviral synergy, whereas a type I/I combination (IFN-alpha2b + IFN-beta) demonstrated a slightly antagonistic profile. The synergistic effect is likely due to differential cell surface receptors and signaling pathways employed by types I and II IFNs. Conversely, all type I IFN species bind the same receptor and signal through similar pathways, possibly accounting for the nearly additive response observed. In support of this hypothesis, IFN treatment resulted in differential induction of Stat1 phosphorylation at Tyr 701. In conclusion, simultaneous type I/II IFN combination treatment may allow an overall decreased effective IFN dose, which may reduce the side effect profiles that hinder current therapy.

Streptococcus pneumoniae as a genomics platform for broad-spectrum antibiotic discovery

Streptococcus pneumoniae is a useful tool for the discovery of broad-spectrum antibiotics because of its genetic malleability and importance as a pathogen. Recent publications of complete chromosomal DNA sequences for S. pneumoniae facilitate rapid and effective use of genomics-based technology to identify essential genes encoding new targets for antibacterial drugs. These methods include computational comparative genomics, gene disruption studies to determine essentiality or identify essential genes, and gene expression analysis using microarrays and gel-based proteomics. We review how genomics has transformed the use of the pneumococcus for the pursuit of new antibiotics, and made it the best species for the identification and validation of new antibiotic targets.

Ureaplasma urealyticum: an opportunity for combinatorial genomics.

In his article on pp. 169–175, Dennis Pollack has elegantly fused genomics and enzymology, a technique he calls combinatorial genomics, to model purine and pentose-phosphate metabolism in Ureaplasma urealyticum. Dr Pollack combined our predictions for the functions of the proteins encoded by U. urealyticum, which we generated by a comparative genomics analysis of the complete genome sequence of the bacterium 1xThe complete sequence of the mucosal pathogen Ureaplasma urealyticum. Glass, J.I et al. Nature. 2000; 407: 757–761Crossref | PubMed | Scopus (247)See all References1, with three decades of enzymology on U. urealyticum and other members of the class Mollicutes. The results are several plausible hypotheses for how U. urealyticum functions with a set of biosynthetic enzymes remarkably different from that of other bacteria. In particular, Dr Pollack offers reasonable explanations for the absence of identifiable genes for nucleotide diphosphate kinase and nucleoside diphosphate reductase, two enzymes that are supposedly essential.At present, only the U. urealyticum urease gene has been shown to encode a protein with a specific enzymatic activity 2xOrganization of Ureaplasma urealyticum urease gene cluster and expression in a suppressor strain of Escherichia coli. Neyrolles, O et al. J. Bacteriol. 1996; 178: 647–655PubMedSee all References2; however, almost 30 different enzymatic activities have been detected in U. urealyticum. In the absence of tractable genetic systems for this bacterium, we must resort to comparative genomics, an indirect approach, to associate genes with specific enzymatic functions. As Dr Pollack emphasized, our comparative-genomics-based annotation of the U. urealyticum gene set is largely in agreement with enzymatic characterizations of the bacterium; however, there are exceptions. We predicted some U. urealyticum open reading frames encode enzymes that could not be detected using biochemical analyses, and for some demonstrated enzymatic activities we identified no genes. Dr Pollacks article offers several explanations for these inconsistencies.One of the possible reasons that gene content and enzymology do not agree is that the studies analyzed different organisms. We sequenced the genome of a clinical isolate of serovar 3, the serovar most commonly isolated from humans 1xThe complete sequence of the mucosal pathogen Ureaplasma urealyticum. Glass, J.I et al. Nature. 2000; 407: 757–761Crossref | PubMed | Scopus (247)See all References1, whereas most biochemical studies of U. urealyticum have used the serovar 8 type-strain T-960. The genome of strain T-960 is estimated to be 17–21% larger than the serovar 3 genome we sequenced, and would probably encode at least 100 more genes 3xA physical map of the genome of Mycoplasma mycoides subspecies mycoides Y with some functional loci. Pyle, L.E et al. Nucleic Acids Res. 1988; 16: 6015–6025Crossref | PubMed | Scopus (46)See all References, 4xPulse-field electrophoresis indicates full-length Mycoplasma chromosomes range widely in size. Neimark, H.C and Lange, C.S. Nucleic Acids Res. 1990; 18: 5443–5448Crossref | PubMed | Scopus (35)See all References, 5xHuman ureaplasmas show diverse genome sizes by pulsed-field electrophoresis. Robertson, J.A et al. Nucleic Acids Res. 1990; 18: 1451–1455Crossref | PubMed | Scopus (36)See all References. There is a proposal to reclassify the 14 U. urealyticum serovars into two species. Serovar 3, and the other three small-genome serovars (1, 6 and 14) will comprise Ureaplasma parvum; serovar 8 will retain the name U. urealyticum 6xInternational committe on Systematic Bacteriology Subcommittee on Mollicutes: minutes of the interim meeting 12 and 18 July 1996, Orlando, Florida, USA. Bradbury, J.M. Intl. J. System. Bacteriol. 1997; 47: 911–914CrossrefSee all References6. Dr Pollack listed ten enzymatic activities reported for U. urealyticum that we could not annotate genes for in serovar 3. Possibly the proposed U. urealyticum serovars but not the proposed U. parvum serovars express those enzymes.Dr Pollack also suggests that some genes whose predicted functions are incongruous with what is known about U. urealyticum metabolism might be vestigial and ‘metabolically useless’ 7xThe comparative metabolism of the mollicutes (Mycoplasmas): the utility for taxonomic classification and the relationship of putative gene annotation and phylogeny to enzymatic function in the smallest free-living cells. Pollack, J.D et al. Crit. Rev. Microbiol. 1997; 23: 269–354Crossref | PubMedSee all References7. Although that is possible, we believe that the mutational pressure in U. urealyticum to convert G+C base pairs to A+T base pairs (the genome is only 25.5% G+C), would rapidly render any truly useless gene unrecognizable 1xThe complete sequence of the mucosal pathogen Ureaplasma urealyticum. Glass, J.I et al. Nature. 2000; 407: 757–761Crossref | PubMed | Scopus (247)See all References1. Every gene in the chromosome might not be essential, but if it does not confer some advantage to the ureaplasmas, it would soon be lost.Finally, we wish to conclude our reply by applauding Dr Pollacks creative integration of genomic with enzymatic data. We concur with his view that such analysis of whole biochemical pathways could be an effective way to discover metabolic alternatives available to bacteria and could thus better identify suitable enzymatic targets for novel antibiotic therapeutics.