The antifungal mechanism of action of zinc pyrithione.

BACKGROUND: Zinc pyrithione (ZPT) is the active ingredient most commonly used in many antidandruff treatments. Despite decades of successful use to treat human scalps, little is understood about the antifungal mechanism of action of ZPT. OBJECTIVES: The objective of this study is to understand the molecular mechanism by which ZPT inhibits fungal growth, the underlying basis for its therapeutic activity. METHODS: Modern systems biology approaches, such as deletion library screening and microarray analysis, were used in combination with traditional measures of metal content, microbial growth and enzyme assays. RESULTS: It was shown that ZPT inhibits fungal growth through increased cellular levels of copper, damaging iron-sulphur clusters of proteins essential for fungal metabolism. CONCLUSIONS: The molecular basis for the antifungal activity of the commonly used active ZPT has been elucidated, more than 50 years since its introduction, as utilizing a copper toxicity mechanism that targets critical iron-sulphur proteins.

Isolation of a deet-insensitive mutant of Drosophila melanogaster (Diptera: Drosophilidae).

Despite the widespread use of N,N,-diethyl-3-methylbenzamide (deet) in insect repellent products, nothing is known about the molecular basis for the repellency of deet, we initiated a molecular genetics program to elucidate the molecular mechanism of deet repellency in Drosophila melanogaster (Meigen). Deet repellency was apparently due to airborne vapors, as wild type flies were repelled by a deet-treated surface in the absence of physical contact and in the dark. A mutant was isolated using chemical mutagenesis and at choice assay. In a choice assay, mutant flies entered 82 +/- 1% of deet-containing tubes, whereas wild type flies entered only 6 +/- 2% of deet-containing tubes. The mutant was repelled by other repellents, benzaldehyde and citronellal. The mutation was recessive and located on the X chromosome.

Thermodynamic criterion for the conformation of P1 residues of substrates and of inhibitors in complexes with serine proteinases.

Eglin c, turkey ovomucoid third domain, and bovine pancreatic trypsin inhibitor (Kunitz) are all standard mechanism, canonical protein inhibitors of serine proteinases. Each of the three belongs to a different inhibitor family. Therefore, all three have the same canonical conformation in their combining loops but differ in their scaffoldings. Eglin c (Leu45 at P1) binds to chymotrypsin much better than its Ala45 variant (the difference in standard free energy changes on binding is -5.00 kcal/mol). Similarly, turkey ovomucoid third domain (Leu18 at P1) binds to chymotrypsin much better than its Ala18 variant (the difference in standard free energy changes on binding is -4.70 kcal/mol). As these two differences are within the +/-400 cal/mol bandwidth (expected from the experimental error), one can conclude that the system is additive. On the basis that isoenergetic is isostructural, we expect that within both the P1 Ala pair and the P1 Leu pair, the conformation of the inhibitor's P1 side chain and of the enzyme's specificity pocket will be identical. This is confirmed, within the experimental error, by the available X-ray structures of complexes of bovine chymotrypsin Aalpha with eglin c () and with turkey ovomucoid third domain (). A comparison can also be made between the structures of P1 (Lys+)15 of bovine pancreatic trypsin inhibitor (Kunitz) ( and ) and of the P1 (Lys+)18 variant of turkey ovomucoid third domain (), both interacting with chymotrypsin. In this case, the conformation of the side chains is strikingly different. Bovine pancreatic trypsin inhibitor with (Lys+)15 at P1 binds to chymotrypsin more strongly than its Ala15 variant (the difference in standard free energy changes on binding is -1.90 kcal/mol). In contrast, turkey ovomucoid third domain variant with (Lys+)18 at P1 binds to chymotrypsin less strongly than its Ala18 variant (the difference in standard free energies of association is 0.95 kcal/mol). In this case, P1 Lys+ is neither isostructural nor isoenergetic. Thus, a thermodynamic criterion for whether the conformation of a P1 side chain in the complex matches that of an already determined one is at hand. Such a criterion may be useful in reducing the number of required X-ray crystallographic structure determinations. More importantly, the criterion can be applied to situations where direct determination of the structure is extremely difficult. Here, we apply it to determine the conformation of the Lys+ side chain in the transition state complex of a substrate with chymotrypsin. On the basis of kcat/KM measurements, the difference in free energies of activation for Suc-AAPX-pna when X is Lys+ and X is Ala is 1.29 kcal/mol. This is in good agreement with the corresponding difference for turkey ovomucoid third domain variants but in sharp contrast to the bovine pancreatic trypsin inhibitor (Kunitz) data. Therefore, we expect that in the transition state complex of this substrate with chymotrypsin, the P1 Lys+ side chain is deeply inserted into the enzyme's specificity pocket as it is in the (Lys+)18 turkey ovomucoid third domain complex with chymotrypsin.

Interscaffolding additivity. Association of P1 variants of eglin c and of turkey ovomucoid third domain with serine proteinases.

Standard mechanism protein inhibitors of serine proteinases share a common mechanism of interaction with their cognate enzymes. The P1 residue of the inhibitor interacts with the enzyme in a substrate-like manner. Its side chain becomes imbedded in the S1 cavity of the enzyme. The nature of P1, the primary specificity residue, greatly affects the strength and specificity of the enzyme inhibitor association. In canonical inhibitors, residues P4-P2'(P3'), where P1-P1' is the reactive site, share a common main chain conformation that does not change on complex formation. The remainder of the inhibitor's structure, the scaffolding, is not always common. Instead, there are at least 20 inhibitor families, each with a different scaffolding. In this paper, we ask whether the differences in standard free energy of association of enzyme-inhibitor complexes upon P1 mutations are independent of the nature of the scaffolding. We have already reported on 25 P1 variants of turkey ovomucoid third domain, a member of the Kazal inhibitor family, interacting with six different serine proteinases. Here, we report on seven different P1 variants of eglin c, a potato I family member, interacting with the same six serine proteinases under the same conditions. The differences in standard free energy on P1 mutations in the eglin c system agree very well, when P1 Pro is omitted. Complete agreement indicates that these P1 residues are interscaffolding additive. This is consistent with the superimposition of the high-resolution structures of eglin c and of turkey ovomucoid third domain with chymotrypsin. In both cases, the P1 Leu side chain is similarly oriented in almost indistinguishable specificity pockets of the enzyme.

Optimization of the signal-sequence cleavage site for secretion from Bacillus subtilis of a 34-amino acid fragment of human parathyroid hormone.

We have effected the secretion from Bacillus subtilis of a 34-amino acid (aa) fragment of human parathyroid hormone (PTH,1-34), using a Bacillus amyloliquefaciens neutral protease signal sequence. The secretion efficiency depended on the aa sequence near the signal-sequence cleavage site. We constructed a series of gene fusions encoding different pairs of aa between the signal sequence and PTH,1-34. There was a correlation between those polypeptides which were efficiently secreted and the potential for a beta-turn in the region just beyond the signal-sequence cleavage site. Based on this correlation, we constructed a gene fusion which specified Gly rather than Ala at the C terminus of the signal sequence, thus creating a beta-turn potential at the end of the signal sequence. The change provided a slight increase in secretion efficiency.

Secretion of human serum albumin from Bacillus subtilis.

We have fused the structural gene (hsa) for human serum albumin (HSA) to the expression elements and signal sequence coding region of each of two genes from Bacillus amyloliquefaciens P, an alpha-amylase gene (amyBamP) and a neutral protease gene (nprBamP). Bacillus subtilis strains harboring either of these gene fusions synthesized a protein with the antigenic characteristics and size (68 kilodaltons) of HSA. Results from pulse-labeling studies indicated that the bacterially produced HSA was secreted from cells which had been converted to protoplasts. Results from similar studies with intact cells suggested that the signal sequence was removed from the hybrid protein, providing further evidence that B. subtilis can translocate this foreign protein across the cell membrane. Signal sequence removal was efficient when the level of HSA synthesis was low. However, in strains which synthesized HSA at a high level, signal sequence removal was less efficient.

Transformation of Bacillus subtilis by single-stranded plasmid DNA.

The single-stranded form of a pE194-based plasmid transformed Bacillus subtilis protoplasts at least as efficiently as did the double-stranded plasmid, but the single-stranded form did not detectably transform B. subtilis competent cells.

Expression of the staphylococcal protein A gene in Bacillus subtilis by integration of the intact gene into the B. subtilis chromosome.

Staphylococcal protein A was synthesized at high levels and was secreted efficiently into the culture medium by strains of Bacillus subtilis in which the cloned gene (spa) from Staphylococcus aureus 8325-4 was inserted into the chromosome. The spa gene could not be established in B. subtilis on multicopy plasmids.

A Bacillus subtilis plasmid that can be packaged as single-stranded DNA in Escherichia coli: use for oligodeoxynucleotide-directed mutagenesis.

A Bacillus subtilis/Escherichia coli shuttle vector was modified to contain the origin of DNA replication of the E. coli filamentous phage f1, in both orientations. Upon superinfection with and f1-related phage of an E. coli strain containing either of the modified vectors, the single-stranded (ss) form of the plasmid was packaged in virions and released to the culture medium. Each of these ss DNAs has been purified from the virions and used as a template for oligodeoxynucleotide-directed mutagenesis. The resulting mutations were demonstrated by DNA sequencing. The capacity of these vectors to be isolated as phage ss DNA from E. coli and to replicate as plasmids in B. subtilis makes them convenient substrates for the production of oligodeoxynucleotide-directed mutations for studies in B. subtilis.

Use of chromosomal integration in the establishment and expression of blaZ, a Staphylococcus aureus beta-lactamase gene, in Bacillus subtilis.

With several different vectors, attempts were made to establish blaZ, a Staphylococcus aureus beta-lactamase gene, in Bacillus subtilis. Stable establishment of blaZ in B. subtilis was achieved by use of a vector that allowed the integration of a single copy of the gene into the chromosome of that host. blaZ was expressed in the heterologous host since B. subtilis strains carrying integrated blaZ produced beta-lactamase and were more resistant to ampicillin than was wild-type B. subtilis. blaZ was stably inherited in such strains, as no loss of the ability to produce beta-lactamase was observed after growth in nonselective liquid medium or on solid medium. In contrast, a blaZ-containing restriction fragment could not be established in B. subtilis with either pUB110- or pC194-based vectors. Similarly, a pC194-based shuttle vector (pGX318) containing the 5' end of blaZ (including the promoter and the coding region for the signal sequence and the first few amino acids of the mature protein) was unable to transform B. subtilis. Two derivatives of pGX318 that could be stably established in B. subtilis were isolated. The structures of these derivatives suggested that inactivation of the blaZ promoter was associated with the acquisition of the ability to be established.

Pathway of plasmid transformation in Pneumococcus: open circular and linear molecules are active.

We have extended the analysis of plasmid transformation in Streptococcus pneumoniae by finding that monomeric and dimeric open circular and linear forms of pMV158 were active in transformation. Their efficiencies were at least 35-fold lower than those of the corresponding closed circular forms. The evidence came largely from analysis of S1 nuclease-digested plasmid deoxyribonucleic acid by combinations of dye-buoyancy, gel electrophoresis, and sedimentation velocity methods. As with closed circular forms, monomer open circular forms gave second-order kinetics and dimer forms gave first-order kinetics. Unique linear products of digestion by either of two restriction enzymes were inactive, but a mixture of the two digests was active, as was the mixture of linear monomer deoxyribonucleic acids produced by S1 nuclease. Absolute efficiencies of transformation were low even for closed circular donors. All of the results, including the low efficiencies, were consistent with the interpretation that plasmid replicons were assembled in the recipient cell by pairing of fragments of single strands that had entered the cell separately from duplex donors that had been cut on the cell surface.

Monomer plasmid DNA transforms Streptococcus pneumoniae.

The covalently closed (CC) monomer form of plasmid pMV158 was found to transform pneumococcus (Streptococcus pneumoniae) and to do so with two-hit kinetics. The evidence came from analysis of the behavior of the transforming activity in fractions from preparative gel electrophoresis. Activity in the first major peak to elute (i) co-eluted with monomer CC as detected on analytical gels, (ii) banded as CC in dye-buoyancy gradients, (iii) sedimented with the velocity expected for monomer CC, and (iv) gave two-hit kinetics as functions of both concentration and time of exposure of the cells to DNA. A second major peak of activity behaved physically as though mostly due to dimer CC forms and gave single-hit response curves. Because almost no dimer was detectable optically on analytical gels of starting preparations, its specific activity was high relative to that of the monomers.

Properties and transforming activities of two plasmids in Streptococcus pneumoniae.

Two plasmids from group B streptococcus were introduced into pneumococcus (Streptococcus pneumoniae) and examined for copy number, stability, and some features of the process by which they transform pneumococcal recipients. The 3.6 Mdal pMV158 (tet) was present at a minimum of 12 to 16 copies per chromosome and was never observed to be cured. The 20 Mdal pIP501 (cat erm) had a minimum copy number of 3 to 4 per chromosome and was lost spontaneously at a frequency near 0.03 per division. The presence of novobiocin increased this frequency 2 to 3-fold. Competence for chromosomal transformation and the membrane endonuclease needed for normal DNA entry were required for plasmid transformation. Plasmid transformants segregated transformed cells one generation ahead of chromosomal transformants. Both single and multiple hit components of the transformation reaction kinetics were observed, but the latter could not be seen in the presence of competing chromosomal DNA. The major of the transforming activity behaved as covalently closed circular DNA in dye-buoyancy gradients. Although most of the activity for both plasmids sedimented in sucrose gradients more rapidly than did monomeric closed circular DNA, a significant fraction was found at a position suggesting that it may have been due to monomeric plasmids.