Prion protein and the molecular features of transmissible spongiform encephalopathy agents.

Transmissible spongiform encephalopathy (TSE) diseases, or prion diseases, are neurodegenerative diseases found in a number of mammals, including man. Although they are generally rare, TSEs are always fatal, and as of yet there are no practical therapeutic avenues to slow the course of disease. The epidemic of bovine spongiform encephalopathy (BSE) in the UK greatly increased the awareness of TSE diseases. Although it appears that BSE has not spread to North America, chronic wasting disease (CWD), a TSE found in cervids, is causing significant concern. Despite decades of investigation, the exact nature of the infectious agent of the TSEs is still controversial. Although many questions remain, substantial efforts have been made to understand the molecular features of TSE agents, with the hope of enhancing diagnosis and treatment of disease, as well as understanding the fundamental nature of the infectious agent itself. This review summarizes the current understanding of these molecular features, focusing on the role of the prion protein (PrP(c)) and its relationship to the disease-associated isoform (PrP(Sc)).

Factors affecting interactions between prion protein isoforms.

Interactions between normal, protease-sensitive prion protein (PrP-sen or PrP(C)) and its protease-resistant isoform (PrP-res or PrP(Sc)) are critical in transmissible spongiform encephalopathy (TSE) diseases. To investigate the propagation of PrP-res between cells we tested whether PrP-res in scrapie brain microsomes can induce the conversion of PrP-sen to PrP-res if the PrP-sen is bound to uninfected raft membranes. Surprisingly, no conversion was observed unless the microsomal and raft membranes were fused or PrP-sen was released from raft membranes. These results suggest that the propagation of infection between cells requires transfer of PrP-res into the membranes of the recipient cell. To assess potential cofactors in PrP conversion, we used cell-free PrP conversion assays to show that heparan sulphate can stimulate PrP-res formation, supporting the idea that endogenous sulphated glycosaminoglycans can act as important cofactors or modulators of PrP-res formation in vivo. In an effort to develop therapeutics, the antimalarial drug quinacrine was identified as an inhibitor of PrP-res formation in scrapie-infected cell cultures. Confirmation of the latter result by others has led to the initiation of human clinical trials as a treatment for Creutzfeldt-Jakob disease. PrP-res formation can also be inhibited using a variety of other types of small molecule, specific synthetic PrP peptides, and an antiserum directed at the C-terminus of PrP-sen. The latter results help to localize the sites of interaction between PrP-sen and PrP-res. Disruption of those interactions with antibodies or peptidomimetic drugs may be an attractive therapeutic strategy. The likelihood that PrP-res inhibitors can rid TSE-infected tissues of PrP-res would presumably be enhanced if PrP-res formation were reversible. However, our attempts to measure dissociation of PrP-sen from PrP-res have failed under non-denaturing conditions. Finally, we have attempted to induce the spontaneous conversion of PrP-sen into PrP-res using low concentrations of detergents. A conformational conversion from alpha-helical monomers into high-beta-sheet aggregates and fibrils was induced by low concentrations of the detergent sarkosyl; however, the aggregates had neither infectivity nor the characteristic protease-resistance ofPrP-res.

Inhibition of interactions and interconversions of prion protein isoforms by peptide fragments from the C-terminal folded domain.

The formation of protease-resistant prion protein (PrP-res or PrP(Sc)) involves selective interactions between PrP-res and its normal protease-sensitive counterpart, PrP-sen or PrP(C). Previous studies have shown that synthetic peptide fragments of the PrP sequence corresponding to residues 119-136 of hamster PrP (Ha119-136) can selectively block PrP-res formation in cell-free systems and scrapie-infected tissue culture cells. Here we show that two other peptides corresponding to residues 166-179 (Ha166-179) and 200-223 (Ha200-223) also potently inhibit the PrP-res induced cell-free conversion of PrP-sen to the protease-resistant state. In contrast, Ha121-141, Ha180-199, and Ha218-232 were much less effective as inhibitors. Mechanistic analyses indicated that Ha166-179, Ha200-223, and peptides containing residues 119-136 inhibit primarily by binding to PrP-sen and blocking its binding to PrP-res. Circular dichroism analyses indicated that Ha117-141 and Ha200-223, but not non-inhibitory peptides, readily formed high beta-sheet structures when placed under the conditions of the conversion reaction. We conclude that these inhibitory peptides may mimic contact surfaces between PrP-res and PrP-sen and thereby serve as models of potential therapeutic agents for transmissible spongiform encephalopathies.

The respiratory burst-inhibiting acid phosphatase AcpA is not essential for the intramacrophage growth or virulence of Francisella novicida.

Acid phosphatases capable of inhibiting the respiratory burst of neutrophils have been identified in certain intracellular pathogens. Here we evaluate the role of AcpA, a respiratory burst-inhibiting acid phosphatase of Francisella, in the virulence and intracellular growth of this organism. An F. novicida acpA null mutant was created and found to exhibit wild-type growth kinetics in both cell-line and inflammatory mouse macrophages. The acpA mutant also shows wild-type replication in the spleens of experimentally infected mice. These data suggest that AcpA is not essential for the intracellular growth or virulence of F. novicida.

An erythromycin resistance cassette and mini-transposon for constructing transcriptional fusions to cat.

A new cassette (Er-Cm cassette) and mini-transposon (mTn) (TnMaxErCm) based on the previously described mTn, TnMax2 [Haas et al., Gene 130, 23-31.], have been constructed. The cassette and mTn make use of an erythromycin resistance (ErR) marker encoded by ermC'. Both the Er-Cm cassette and TnMaxErCm also carry a promoterless cat gene to allow the construction of transcriptional fusions and the measurement of transcriptional activity by assaying for chloramphenicol acetyltransferase. We show the function of these genetic elements by analyzing the regulation of expression of the mglA gene of Francisella novicida and by using TnMaxErCm to probe for promoter activity within an F. novicida recombinant clone. The reporter cassette and mTn described here further expand the family of TnMax transposons and facilitate the study of gene expression in organisms where direct Tn mutagenesis methods are unavailable.

MglA and MglB are required for the intramacrophage growth of Francisella novicida.

Francisella novicida is a facultative intracellular pathogen capable of growing in macrophages. A spontaneous mutant of F. novicida defective for growth in macrophages was isolated on LB media containing the chromogenic phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate (X-p) and designated GB2. Using an in cis complementation strategy, four strains were isolated that are restored for growth in macrophages. A locus isolated from one of these strains complements GB2 for both the intracellular growth defect and the colony morphology on LB (X-p) media. The locus consists of an apparent operon of two genes, designated mgIAB, for Macrophage Growth Locus. Both mglA and mglB transposon insertion mutants are defective for intracellular growth and have a phenotype similar to GB2 or LB (X-p) media. Sequencing on mglA cloned from GB2 identified a missense mutation, providing evidence that both mglA and mglB are required for the intramacrophage growth of F. novicida. mglB expression in GB2 was confirmed using antiserum against recombinant MglB. Cell fractionation studies revealed several differences in the protein profiles of mgI mutants compared with wild-type F. novicida. The deduced amino acid sequences of mglA and mglB show similarity to the SspA and SspB proteins of Escherichia coli and Haemophilus spp. In E. coli, SspA and/or SspB influence the levels of multiple proteins under conditions of nutritional stress, and SspA can associate with the RNA polymerase holoenzyme. Taken together, these observations suggest that in Francisella MglA and MglB may affect the expression of genes whose products contribute to survival and growth within macrophages.

Mycobacterium tuberculosis efpA encodes an efflux protein of the QacA transporter family.

The Mycobacterium tuberculosis H37Rv efpA gene encodes a putative efflux protein, EfpA, of 55,670 Da. The deduced EfpA protein was similar in secondary structure to Pur8, MmrA, TcmA, LfrA, EmrB, and other members of the QacA transporter family (QacA TF) which mediate antibiotic and chemical resistance in bacteria and yeast. The predicted EfpA sequence possessed all transporter motifs characteristic of the QacA TF, including those associated with proton-antiport function and the motif considered to be specific to exporters. The 1,590-bp efpA open reading frame was G+C rich (65%), whereas the 40-bp region immediately upstream had an A+T bias (35% G+C). Reverse transcriptase-PCR assays indicated that efpA was expressed in vitro and in situ. Putative promoter sequences were partially overlapped by the A+T-rich region and by a region capable of forming alternative secondary structures indicative of transcriptional regulation in analogous systems. PCR single-stranded conformational polymorphism analysis demonstrated that these upstream flanking sequences and the 231-bp, 5' coding region are highly conserved among both drug-sensitive and multiply-drug-resistant isolates of M. tuberculosis. The efpA gene was present in the slow-growing human pathogens M. tuberculosis, Mycobacterium leprae, and Mycobacterium bovis and in the opportunistic human pathogens Mycobacterium avium and Mycobacterium intracellular. However, efpA was not present in 17 other opportunistically pathogenic or nonpathogenic mycobacterial species.

Molecular characterization of the kinetoplastid membrane protein-11 from African trypanosomes.

The kinetoplastid membrane protein-11 molecule was purified from Trypanosoma brucei rhodesiense and an internal peptide sequence was obtained. This sequence information was used with cosmid library screening and polymerase chain reaction amplifications of both genomic DNA and cDNA to obtain the entire DNA sequence of the encoding gene and the corresponding translated amino acid sequence of 92 residues. The sequence showed 18% divergence from the corresponding molecule of the related kinetoplastid Leishmania donovani, including one key amino acid at position 45 which may be of functional relevance. The protein had a calculated molecular mass of 11078 Da, a pI of 6.0 and an overall net charge of -2 at physiological pH. The secondary structure of the molecule was predicted to consist of two amphipathic helices connected by a random-coil segment, and suggests that it would interact with lipid bilayers in the trypanosome cell membrane. Northern and Southern blot analyses showed that the trypanosome kinetoplastid membrane protein-11 molecule was translated from a single transcript and was transcribed from a single gene copy, thus making this molecule an attractive target for knockout mutagenesis.

Characterization and sequencing of a respiratory burst-inhibiting acid phosphatase from Francisella tularensis.

Acid phosphatases (Acp) of intracellular pathogens have recently been implicated as virulence factors that enhance intracellular survival through suppression of the respiratory burst. We describe here the identification, purification, characterization, and sequencing of a novel burst-inhibiting acid phosphatase from the facultative intracellular bacterium, Francisella tularensis. Similar to other the burst-inhibiting Acps, F. tularensis Acp (AcpA) is tartrate-resistant and has broad substrate specificity. The AcpA enzyme is unique, however, in that it is easily released from the bacterial cell in soluble form, is a basic enzyme, suppresses the respiratory burst of not only fMet-Leu-Phe but also phorbol 12-myristate 13-acetate-stimulated neutrophils and does not fit into any of the three currently recognized classes of acid phosphatase. We also report the complete nucleotide sequence of the gene acpA, encoding AcpA, and the deduced primary structure of its encoded polypeptide. Comparative sequence analyses of AcpA is discussed. To our knowledge, this is the first report describing the cloning and sequencing of a burst-inhibiting acid phosphatase.

Serum-sensitive mutation of Francisella novicida: association with an ABC transporter gene.

Francisella novicida is a facultative intracellular pathogen that can survive and grow in macrophages by preventing phagolysosomal fusion. In this study in vitro cassette mutagenesis was used to generate a library of insertion mutants of F.novicida. Two related mutants, KM14 and KM14S, initially identified as defective for growth in macrophages, were found to be sensitive to serum. These mutants were also found to grow approximately 1000-fold less well in the livers and spleens of infected mice. We cloned a genetic locus that was presumably mutagenized in these mutants and found that it included genes that had high similarity in their deduced amino acid sequence to those of msbA and orfE of Escherichia coli. The former is a member of the superfamily of ABC transporter proteins. We named the corresponding genes in F. novicida, valAB. Integration of a cloned valAB locus into the chromosome of KM14S partially restored the serum resistance phenotype found in wild-type F. novicida.