Peter J. Coote

A study of the minimum inhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol.

Aims: The minimum inhibitory concentration (MIC) of oregano essential oil (OEO) and two of its principle components, i.e. thymol and carvacrol, against Pseudomonas aeruginosa and Staphylococcus aureus was assessed by using an innovative technique. The mechanism of action of the above substances was also investigated.

Acid tolerance in Listeria monocytogenes : the adaptive acid tolerance response (ATR) and growth-phase-dependent acid resistance

Listeria monocytogenes acquired increased acid tolerance during exponential growth upon exposure to sublethal acid stress, a response designated the acid tolerance response (ATR). Maximal acid resistance was seen when the organism was exposed to pH 5.0 for 1 h prior to challenge at pH 3.0, although intermediate levels of protection were afforded by exposure to pH values ranging from 4.0 to 6.0. A 60 min adaptive period was required for the development of maximal acid tolerance; during this period the level of acid tolerance increased gradually. Full expression of the ATR required de novo protein synthesis; chloramphenicol, a protein synthesis inhibitor, prevented full induction of acid tolerance. Analysis of protein expression during the adaptive period by two-dimensional gel electrophoresis revealed a change in the expression of at least 23 proteins compared to the non-adapted culture. Eleven proteins showed induced expression while 12 were repressed, implying that the ATR is a complex response involving a modulation in the expression of a large number of genes. In addition to the exponential phase ATR, L.monocytogenes also developed increased acid resistance upon entry into the stationary phase; this response appeared to be independent of the pH-dependent ATR seen during exponential growth.

Evidence of a New Role for the High-Osmolarity Glycerol Mitogen-Activated Protein Kinase Pathway in Yeast: Regulating Adaptation to Citric Acid Stress

ABSTRACT Screening the Saccharomyces cerevisiae disruptome, profiling transcripts, and determining changes in protein expression have identified an important new role for the high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway in the regulation of adaptation to citric acid stress. Deletion of HOG1, SSK1, PBS2, PTC2, PTP2, and PTP3 resulted in sensitivity to citric acid. Furthermore, citric acid resulted in the dual phosphorylation, and thus activation, of Hog1p. Despite minor activation of glycerol biosynthesis, the inhibitory effect of citric acid was not due to an osmotic shock. HOG1 negatively regulated the expression of a number of proteins in response to citric acid stress, including Bmh1p. Evidence suggests that BMH1 is induced by citric acid to counteract the effect of amino acid starvation. In addition, deletion of BMH2 rendered cells sensitive to citric acid. Deletion of the transcription factor MSN4, which is known to be regulated by Bmh1p and Hog1p, had a similar effect. HOG1 was also required for citric acid-induced up-regulation of Ssa1p and Eno2p. To counteract the cation chelating activity of citric acid, the plasma membrane Ca2+ channel, CCH1, and a functional vacuolar membrane H+-ATPase were found to be essential for optimal adaptation. Also, the transcriptional regulator CYC8, which mediates glucose derepression, was required for adaptation to citric acid to allow cells to metabolize excess citrate via the tricarboxylic acid (TCA) cycle. Supporting this, Mdh1p and Idh1p, both TCA cycle enzymes, were up-regulated in response to citric acid.

Parallel and comparative analysis of the proteome and transcriptome of sorbic acid-stressed Saccharomyces cerevisiae.

Exposure of Saccharomyces cerevisiae to 0.9 mM sorbic acid at pH 4.5 resulted in the upregulation of 10 proteins; Hsp42, Atp2, Hsp26, Ssa1 or Ssa2, Ssb1 or Ssb2, Ssc1, Ssa4, Ach1, Zwf1 and Tdh1; and the downregulation of three proteins; Ade16, Adh3 and Eno2. In parallel, of 6144 ORFs, 94 (1.53%) showed greater than a 1.4‐fold increase in transcript level after exposure to sorbic acid and five of these were increased greater than two‐fold; MFA1, AGA2, HSP26, SIP18 and YDR533C. Similarly, of 6144 ORFs, 72 (1.17%) showed greater than a 1.4‐fold decrease in transcript level and only one of these, PCK1, was decreased greater than two‐fold Functional categories of genes that were induced by sorbic acid stress included cell stress (particularly oxidative stress), transposon function, mating response and energy generation. We found that proteomic analysis yielded distinct information from transcript analysis. Only the upregulation of Hsp26 was detected by both methods. Subsequently, we demonstrated that a deletion mutant of Hsp26 was sensitive to sorbic acid. Thus, the induction of Hsp26, which occurs during adaptation to sorbic acid, confers resistance to the inhibitory effects of this compound. Copyright

Comparison of the inhibitory effect of sorbic acid and amphotericin B on Saccharomyces cerevisiae: is growth inhibition dependent on reduced intracellular pH?

D. BRACEY, C D. HOLYOAK AND P J. COOTE. 1998. The effects of sorbic acid and amphotericin B on the growth and intracellular pH (pH1) of Saccharomyces cerevisiae were studied and compared. Past evidence has suggested that the inhibitory action of sorbic acid on yeast is due to reduction of pH1 per se. However, using a novel method to measure pH1 in growing cells, little correlation was found between reduced growth rate on exposure to sorbic acid and reduction of pHi. In fact, growth inhibition correlated with an increase in the intracellular ADP/ATP ratio due to increased ATP consumption by the cells. This was partly attributed to the activation of protective mechanisms, such as increased proton pumping by the membrane H+‐ATPase, which ensured that pH1 did not decline when cells were exposed to sorbic acid. Therefore, the available evidence suggested that the inhibitory action of sorbic acid was due to the induction of an energetically expensive protective mechanism that compensated for any disruption of pH1 homeostasis but resulted in less available energy for normal growth. In contrast to sorbic acid, with amphotericin B there was a direct correlation between growth inhibition and reduction of pH1 due to the uncoupling effect of this compound on the plasma membrane. The inhibitory effect of amphotericin B was consistent with membrane disruption, or ‘proton‐uncoupling’ leading to growth inhibition due to proton influx, decline in pHi and partial dissipation of the proton gradient.

Wax moth larva (Galleria mellonella): an in vivo model for assessing the efficacy of antistaphylococcal agents

OBJECTIVES To investigate whether the wax moth larva, Galleria mellonella, is a suitable host for assessing the in vivo efficacy of antistaphylococcal agents against Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) infections. METHODS Wax moth larvae were infected with increasing doses of S. aureus to investigate the effect of inoculum size on larval survival. In addition, infected wax moth larvae were treated with daptomycin, penicillin or vancomycin to examine whether these agents were effective against S. aureus and MRSA infections in vivo. RESULTS Increasing inoculum doses of live S. aureus cells resulted in greater larval mortality, but heat-killed bacteria and cell-free culture filtrates had no detrimental effects on survival. Larval mortality rate also depended on the post-inoculation incubation temperature. After larvae were infected with S. aureus, larval survival was enhanced by administering the antistaphylococcal antibiotics daptomycin or vancomycin. Larval survival increased with increasing doses of the antibiotics. Moreover, penicillin improved survival of larvae infected with a penicillin-susceptible methicillin-susceptible S. aureus (MSSA) strain, but it was ineffective at similar doses in larvae infected with MRSA (penicillin resistant). Daptomycin and vancomycin were also effective when administered to the larvae prior to infection with bacteria. CONCLUSIONS This is the first report to demonstrate that antibiotics are effective in the wax moth larva model for the treatment of infections caused by Gram-positive bacteria. The new wax moth larva model is a useful preliminary model for assessing the in vivo efficacy of candidate antistaphylococcal agents before proceeding to mammalian studies, which may reduce animal experimentation and expense.

Identification and characterization of an ATP binding cassette L-carnitine transporter in Listeria monocytogenes

ABSTRACT We identified an operon in Listeria monocytogenes EGD with high levels of sequence similarity to the operons encoding the OpuC and OpuB compatible solute transporters from Bacillus subtilis, which are members of the ATP binding cassette (ABC) substrate binding protein-dependent transporter superfamily. The operon, designated opuC, consists of four genes which are predicted to encode an ATP binding protein (OpuCA), an extracellular substrate binding protein (OpuCC), and two membrane-associated proteins presumed to form the permease (OpuCB and OpuCD). The operon is preceded by a potential SigB-dependent promoter. An opuC-defective mutant was generated by the insertional inactivation of theopuCA gene. The mutant was impaired for growth at high osmolarity in brain heart infusion broth and failed to grow in a defined medium. Supplementation of the defined medium with peptone restored the growth of the mutant in this medium. The mutant was found to accumulate the compatible solutes glycine betaine and choline to same extent as the parent strain but was defective in the uptake ofl-carnitine. We conclude that the opuC operon in L. monocytogenes encodes an ABC compatible solute transporter which is capable of transporting l-carnitine and which plays an important role in osmoregulation in this pathogen.

Proteomic detection of PhoPQ- and acid-mediated repression of Salmonella motility.

Salmonella adaptation to low pH is a critical survival response and essential for virulence. Here, we show that another key virulence‐associated process, flagella‐mediated cell motility, is co‐regulated by low pH via the PhoPQ signal transduction system. Using a proteomic approach, we found that phase 1 and phase 2 flagellin were specifically down‐regulated when acid‐adapted (pH 5.0) Salmonella SL1344 cells were exposed to pH 3.0. Decreased flagellin expression and cell motility was dependent on activation of the PhoPQ pathway, which directly or indirectly negatively regulated transcription of the flagellin gene fliC. In contrast, the general stress sigma factor RpoS (σS) positively regulated flagellar gene expression. Low external pH had no effect on the level of H‐NS protein, a further regulator of flagellar gene expression. We suggest that flagellar repression at low pH conserves ATP for survival processes and helps to limit the influx of protons into the cytosol. These results highlight the power of proteomics to reveal unanticipated links between relatively well‐characterised regulatory systems in bacteria.

Interaction of amyloid binding alcohol dehydrogenase/Aβ mediates up-regulation of peroxiredoxin II in the brains of Alzheimer's disease patients and a transgenic Alzheimer's disease mouse model

Alzheimers patients have increased levels of both the 42 beta amyloid-beta-peptide (Abeta) and amyloid binding alcohol dehydrogenase (ABAD) which is an intracellular binding site for Abeta. The over-expression of Abeta and ABAD in transgenic mice has shown that the binding of Abeta to ABAD results in exaggerating neuronal stress and impairment of learning and memory. From a proteomic analysis of the brains from these animals we identified that peroxiredoxin II levels increase in Alzheimers diseased brain. This increase in peroxiredoxin II levels protects neurons against Abeta induced toxicity. We also demonstrate, for the first time in living animals, that the expression level of peroxiredoxin II is an indicator for the interaction of ABAD and Abeta as its expression levels return to normal if this interaction is perturbed. Therefore this indicates the possibility of reversing changes observed in Alzheimers disease and that the Abeta-ABAD interaction is a suitable drug target.

An amphibian‐derived, cationic, α‐helical antimicrobial peptide kills yeast by caspase‐independent but AIF‐dependent programmed cell death

The dermaseptins are a family of antimicrobial peptides from the tree‐frog Phyllomedusa sauvagii. Yeast exposed to dermaseptin S3(1‐16), a truncated derivative of dermaseptin S3 with full activity, showed diagnostic markers of yeast apoptosis: the appearance of reactive oxygen species and fragmentation of nuclear DNA. This process was independent of the yeast caspase, Yca1p. Screening of a non‐essential gene deletion collection in yeast identified genes that conferred resistance to dermaseptin S3(1‐16): izh2Δ, izh3Δ, stm1Δ and aif1Δ, all known to be involved in regulating yeast apoptosis. The appearance of apoptotic markers was reduced in these strains when exposed to the peptide. Dermaseptin S3(1‐16) was shown to interact with DNA, and cause DNA damage in vivo, a process known to trigger apoptosis. Supporting this, a dermaseptin S3(1‐16) affinity column specifically purified Stm1p, Mre11p and Htb2p; DNA‐binding proteins implicated in yeast apoptosis and DNA repair. Thus, amphibians may have evolved a mechanism to induce cell suicide in invading fungal pathogens.