CYP121, CYP51 and associated redox systems in Mycobacterium tuberculosis: towards deconvoluting enzymology of P450 systems in a human pathogen.

An extraordinary array of P450 (cytochrome P450) enzymes are encoded on the genome of the human pathogen Mycobacterium tuberculosis (Mtb) and in related mycobacteria and actinobacteria. These include the first characterized sterol 14alpha-demethylase P450 (CYP51), a known target for azole and triazole drugs in yeasts and fungi. To date, only two Mtb P450s have been characterized in detail: CYP51 and CYP121. The CYP121 P450 shows structural relationships with P450 enzymes involved in synthesis of polyketide antibiotics. Both P450s exhibit tight binding to a range of azole drugs (e.g. clotrimazole and fluconazole) and the same drugs also have potent effects on growth of mycobacteria (but not of e.g. Escherichia coli). Atomic structures are available for both Mtb CYP51 and CYP121, revealing modes of azole binding and intriguing mechanistic and structural aspects. This paper reviews our current knowledge of these and the other P450 systems in Mtb including recent data relating to the reversible conversion of the CYP51 enzyme between P450 (thiolate-co-ordinated) and P420 (thiol-co-ordinated) species on reduction of the haem iron in the absence of a P450 substrate. The accessory flavoprotein and iron-sulfur proteins required to drive P450 catalysis are also discussed, providing an overview of the current state of knowledge of Mtb P450 redox systems.

The effect of base/liner use on restoration leakage.

Central to the success of a restoration is the quality of the restoration-dentin interfacial seal; any compromise of the seal can lead to secondary or recurrent decay. Class V restorations have a high leakage propensity and this study evaluates the effect of base/liner placement on leakage behavior. Class V intracoronal half enamel/half dentin preparations (3.0 x 2.0 x 2.0 mm) were cut in four groups (n = 10) of extracted human teeth with a new bur used for each cavity preparation. All teeth were single-rooted, single-canal anterior teeth. Base/liner usage differed between each group. The first group of teeth had no liner or base, while a liner was placed in the second group of teeth prior to conditioning and restoration. A base was placed in the third group of cavity preparations and both the base and liner were placed in the fourth group. After preparation, a small diameter bare-end PVC-insulated copper wire was inserted within the root canal of each tooth from the apex to firm contact with the pulp chamber roof. The tooth-wire interface and root surface was sealed and leakage was followed electrochemically for 35 days in 0.9% NaCl solution. All of the teeth leaked to some degree; however, teeth that were restored without liner or base demonstrated the smallest amount of leakage. The greatest leakage was noted in teeth restored with both a base and a liner; teeth restored with only a base showed greater leakage than those restored with only a liner. The findings indicate that the presence of a base and/or a liner results in greater leakage compared with intracoronal Class V preparations that were conditioned and restored only. The data suggest that placing both a base and a liner increases restoration leakage significantly.

Biodiversity of cytochrome P450 redox systems.

P450s (cytochrome P450 mono-oxygenases) are a superfamily of haem-containing mono-oxygenase enzymes that participate in a wide range of biochemical pathways in different organisms from all of the domains of life. To facilitate their activity, P450s require sequential delivery of two electrons passed from one or more redox partner enzymes. Although the P450 enzymes themselves show remarkable similarity in overall structure, it is increasingly apparent that there is enormous diversity in the redox partner systems that drive the P450 enzymes. This paper examines some of the recent advances in our understanding of the biodiversity of the P450 redox apparatus, with a particular emphasis on the redox systems in the pathogen Mycobacterium tuberculosis.

The TB structural genomics consortium: a resource for Mycobacterium tuberculosis biology.

The TB Structural Genomics Consortium is an organization devoted to encouraging, coordinating, and facilitating the determination and analysis of structures of proteins from Mycobacterium tuberculosis. The Consortium members hope to work together with other M. tuberculosis researchers to identify M. tuberculosis proteins for which structural information could provide important biological information, to analyze and interpret structures of M. tuberculosis proteins, and to work collaboratively to test ideas about M. tuberculosis protein function that are suggested by structure or related to structural information. This review describes the TB Structural Genomics Consortium and some of the proteins for which the Consortium is in the progress of determining three-dimensional structures.

Cytochromes P450: novel drug targets in the war against multidrug-resistant Mycobacterium tuberculosis.

Novel drug strategies are desperately needed to combat the global threat posed by multidrug-resistant strains of Mycobacterium tuberculosis (Mtb). The genome sequence of Mtb has revealed an unprecedented number of cytochrome P450 enzymes in a prokaryote, suggesting fundamental physiological roles for many of these enzymes. Several azole drugs (known inhibitors of cytochromes P450) have been shown to have potent anti-mycobacterial activity, and the most effective azoles have extremely tight binding constants for one of the Mtb P450s (CYP121). The structure of CYP121 has been determined at atomic resolution, revealing novel features of P450 structure, including mixed haem conformations and putative proton-relay pathways from protein surface to haem iron. The structure provides both a platform for investigation of structure/mechanism of cytochrome P450, and for design of inhibitor molecules as novel anti-tubercular agents.

Long-term transgene expression in mice infected with a herpes simplex virus type 1 mutant severely impaired for immediate-early gene expression.

The role of viral immediate-early (IE) gene expression in herpes simplex virus type 1 (HSV-1) latency was investigated. The HSV-1 multiple mutant in1312, defective for the expression of the virion transactivator VP16 and the IE proteins ICP0 and ICP4, was used as the parent for these studies. The coding sequences of the Escherichia coli lacZ gene, preceded by the encephalomyocarditis virus internal ribosome entry site, were inserted into the region of in1312 that encodes the latency-associated transcripts (LATs) such that transcription of the transgene was controlled by the LAT promoter. This insert has previously been shown to direct long-term latent-phase expression of beta-galactosidase in a wild-type HSV-1 genome (R. H. Lachmann and S. Efstathiou, J. Virol. 71, 3197-3207, 1997). The resulting recombinant, in1388, was apathogenic after inoculation into mice via the footpad and did not detectably replicate in dorsal root ganglia (DRG) or footpads. Mutant in1388 established latency in DRG, and beta-galactosidase was expressed in increasing numbers of neurons over the first 25 days of infection. During latency, more than 1% of neurons in ganglia that innervate the footpad expressed beta-galactosidase, with the number of positive cells remaining constant for at least 5 months. Rescue of the VP16, ICP0, or ICP4 mutations of in1388 did not affect the number of beta-galactosidase-expressing neurons detected during latency. The results demonstrate that HSV-1 mutants severely impaired for IE gene expression are capable of establishing latency and efficiently expressing a foreign gene product under control of the LAT promoter.

A non-cytotoxic herpes simplex virus vector which expresses Cre recombinase directs efficient site specific recombination.

The coding sequences for the bacteriophage P1 recombinase Cre were cloned into the genome of a herpes simplex virus type 1 (HSV-1) mutant which is severely impaired for the synthesis of immediate early (IE) proteins. The resulting recombinant, virus in1372, expressed functional Cre which mediated the excision in trans of loxP-flanked sequences located in the HSV-1 genome, both in tissue culture cells and in vivo in mouse sensory neurons. Infection with in1372 also resulted in recombination, at high efficiency, between loxP sequences in the cellular genome without causing detectable cytotoxicity. Mutant in1372 is a versatile vector for the delivery of Cre in tissue culture and in vivo.

An equine herpesvirus-1 gene 71 deletant is attenuated and elicits a protective immune response in mice.

The pathogenesis of pulmonary infection and the immune response following intranasal inoculation of mice with two equine herpesvirus type 1 (EHV-1) deletion mutants have been assessed. The mutants, ED71 and ED75, have deletions in genes 71 (EUS4) and 75 (10K), respectively. Deletions were replaced by the Escherichia coli lacZ gene driven by the simian virus 40 (SV40) early promoter. It has previously been shown that the protein products of genes 71 and 75 are dispensable in vitro but that removal of gene 71 results in a defect in virus maturation and capsid envelopment which impairs the ability of mutant virus to spread via release and readsorption. This study demonstrated that the 192-kDa gene 71 product is required for full expression of virulence in mice, whereas the putative 10-kDa product of gene 75 has minimal effect. Both mutants exhibited the same tissue and cytotropism as wild-type EHV-1 and induced both humoral and cell-mediated immune responses indistinguishable from those induced by the parental strain. Irrespective of the reduced pathogenicity of the gene 71 mutant, infected mice were protected against a challenge with wild-type EHV-1. These findings highlight the potential of ED71 as a vaccine candidate.

Demonstration of equine herpesvirus-1 neuronal latency in murine olfactory bulbs using a novel combined in situ PCR and protein synthesis method.

Equine herpesvirus-1 (EHV-1) latency in murine olfactory bulbs was demonstrated by a novel combined in situ PCR and in vitro protein synthesis method (in situ PS-PCR). The Escherichia coli lacZ gene replacing a deletion in EHV-1 gene 71 (EUS4) was thus amplified and transcribed/translated in situ followed by enzymatic detection using X-Gal (5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside). beta-Galactosidase was found to be concentrated over mitral/tufted neurons indicating those to be the sites of latency. Our results suggest that, in common with other alpha-herpesviruses, EHV-1 can establish latency in central nervous system neurons and that the unique membrane glycoprotein encoded by EHV-1 gene 71 is nonessential for infection of neural tissues.

Investigation of the stability of medicinal additives in animal feedingstuffs to prepare reference feeds.

The stability of several medicinal additives in cattle, pig and poultry feeds has been monitored. The feeds were stored at various temperatures under different conditions; processes such as freeze-drying, gamma-irradiation and pelletization were also applied. The medicinal additives appeared to be more stable in the feeds stored at reduced temperatures and under conditions that totally exclude light. Processing of feeds and storage at elevated temperature appeared to reduce the content of the medicinal additives examined.

Evaluation of an inactivated rabies virus vaccine in domestic ferrets.

Efficacy of an SC-administered commercial inactivated vaccine for prevention of rabies was evaluated in domestic ferrets. Ferret immunity was challenged by the IM inoculation of street rabies virus. All ferrets developed titers of rabies virus-neutralizing antibodies within 30 days of vaccination (geometric mean titer [GMT] = 154, n = 41) that were maintained for at least one year (GMT = 106, n = 36), compared with no seroconversion in controls (GMT less than 5, n = 39). Following rabies virus challenge inoculation, 89% (32/36) of vaccinated ferrets survived vs less than 6% (2/38) survival in control ferrets. These results demonstrate the protective efficacy of a commercial, inactivated rabies vaccine of at least one year's duration for domestic ferrets.