Role of the twin-arginine translocase (tat) system in iron uptake in Listeria monocytogenes.

The twin-arginine translocase (Tat) complex is a unique system that translocates folded proteins across the cytoplasmic membrane. In this study, the Tat transporter system in Listeria monocytogenes was characterized to determine the role of Tat in the iron uptake pathway. A putative tatAC operon, containing conserved Fur-binding sequences in the promoter region, has been predicted to encode Tat-translocase components. Another operon, fepCAB, with a putative Fur-binding sequence in the promoter, close to TatAC, was identified in the complementary strands of L. monocytogenes. Electrophoretic mobility shift assay showed that the listerial Fur-repressor binds to the promoter of the tatAC operon, suggesting that tat is under Fur regulation. Using a heterologous system in a reporter assay, FepB was translocated across the membrane. Mutations in tatC and fepB were constructed to determine the roles of Tat and FepB, respectively. In a whole-cell ferric reductase assay, the fepB and tatC mutants were found to have reduced levels of ferric reductase activities compared with those of the isogenic parent strain. Although ferric reductase activity has been demonstrated in Listeria, a conventional ferric reductase encoding sequence does not appear to be present in its genome. Hence, we propose that fepB encodes a ferric reductase enzyme, which is translocated by the Tat-translocase system onto the bacterial cell surface, and plays an important role in the reductive iron uptake process in L. monocytogenes.

Tea tree oil-induced transcriptional alterations in Staphylococcus aureus.

Tea tree oil (TTO) is a steam distillate of Melaleuca alternifolia that demonstrates broad-spectrum antibacterial activity. This study was designed to document how TTO challenge influences the Staphylococcus aureus transcriptome. Overall, bioinformatic analyses (S. aureus microarray meta-database) revealed that both ethanol and TTO induce related transcriptional alterations. TTO challenge led to the down-regulation of genes involved with energy-intensive transcription and translation, and altered the regulation of genes involved with heat shock (e.g. clpC, clpL, ctsR, dnaK, groES, groEL, grpE and hrcA) and cell wall metabolism (e.g. cwrA, isaA, sle1, vraSR and vraX). Inactivation of the heat shock gene dnaK or vraSR which encodes a two-component regulatory system that responds to peptidoglycan biosynthesis inhibition led to an increase in TTO susceptibility which demonstrates a protective role for these genes in the S. aureus TTO response. A gene (mmpL) encoding a putative resistance, nodulation and cell division efflux pump was also highly induced by TTO. The principal antimicrobial TTO terpene, terpinen-4-ol, altered ten genes in a transcriptional direction analogous to TTO. Collectively, this study provides additional insight into the response of a bacterial pathogen to the antimicrobial terpene mixture TTO.

Application of orange essential oil as an antistaphylococcal agent in a dressing model.

BACKGROUND: Staphylococcus aureus is the pathogen most often and prevalently involved in skin and soft tissue infections. In recent decades outbreaks of methicillin-resistant S. aureus (MRSA) have created major problems for skin therapy, and burn and wound care units. Topical antimicrobials are most important component of wound infection therapy. Alternative therapies are being sought for treatment of MRSA and one area of interest is the use of essential oils. With the increasing interest in the use and application of natural products, we screened the potential application of terpeneless cold pressed Valencia orange oil (CPV) for topical therapy against MRSA using an in vitro dressing model and skin keratinocyte cell culture model. METHODS: The inhibitory effect of CPV was determined by disc diffusion vapor assay for MRSA and vancomycin intermediate-resistant S. aureus (VISA) strains. Antistaphylococcal effect of CPV in an in vitro dressing model was tested on S. aureus inoculated tryptic soya agar plate. Bactericidal effect of CPV on MRSA and VISA infected keratinocyte cells was examined by enumeration of extra- and intra-cellular bacterial cells at different treatment time points. Cytotoxic effects on human skin cells was tested by adding CPV to the keratinocyte (HEK001) cells grown in serum free KSFM media, and observed by phase-contrast microscope. RESULTS: CPV vapour effectively inhibited the MRSA and VISA strains in both disc diffusion vapour assay and in vitro dressing model. Compared to untreated control addition of 0.1% CPV to MRSA infected keratinocyte decreased the viable MRSA cells by 2 log CFU/mL in 1 h and in VISA strain 3 log CFU/mL reduction was observed in 1 h. After 3 h viable S. aureus cells were not detected in the 0.2% CPV treatment. Bactericidal concentration of CPV did not show any cytotoxic effect on the human skin keratinocyte cells in vitro. CONCLUSIONS: At lower concentration addition of CPV to keratinocytes infected with MRSA and VISA rapidly killed the bacterial cells without causing any toxic effect to the keratinocytes. Therefore, the results of this study warrant further in vivo study to evaluate the potential of CPV as a topical antistaphylococcal agent.

Fatty acids regulate stress resistance and virulence factor production for Listeria monocytogenes.

Fatty acids (FAs) are the major structural component of cellular membranes, which provide a physical and chemical barrier that insulates intracellular reactions from environmental fluctuations. The native composition of membrane FAs establishes the topological and chemical parameters for membrane-associated functions and is therefore modulated diligently by microorganisms especially in response to environmental stresses. However, the consequences of altered FA composition during host-pathogen interactions are poorly understood. The food-borne pathogen Listeria monocytogenes contains mostly saturated branched-chain FAs (BCFAs), which support growth at low pH and low temperature. In this study, we show that anteiso-BCFAs enhance bacterial resistance against phagosomal killing in macrophages. Specifically, BCFAs protect against antimicrobial peptides and peptidoglycan hydrolases, two classes of phagosome antimicrobial defense mechanisms. In addition, the production of the critical virulence factor, listeriolysin O, was compromised by FA modulation, suggesting that FAs play a key role in virulence regulation. In summary, our results emphasize the significance of FA metabolism, not only in bacterial virulence regulation but also in membrane barrier function by providing resistance against host antimicrobial stress.

Metabolomes of the psychrotolerant bacterium Listeria monocytogenes 10403S grown at 37 °C and 8 °C.

Listeria monocytogenes is a food-borne pathogen with the ability to grow at refrigeration temperatures. Knowledge of the mechanisms involved in low temperature growth is incomplete and here we report the results of a metabolomics investigation of this. The small molecule contents of L. monocytogenes 10403S grown at 37 °C and 8 °C were compared by gas chromatography/mass spectrometry (GC/MS). Over 500 peaks were detected in both 37 °C and 8 °C-grown cells, and 103 were identified. Of the identified metabolites, the concentrations of 56 metabolites were increased (P<0.05), while the concentrations of 8 metabolites were decreased at low temperature. Metabolites increasing in concentration at 8 °C included amino acids, sugars, organic acids, urea cycle intermediates, polyamines, and different compatible solutes. A principal component analysis (PCA) was used to visualize and compare the matrix containing the data in 6 samples, and this clearly identified the 37 °C and 8 °C metabolomes as different. The results indicated that an increase in solute concentrations in the cytoplasm was associated with low temperature adaptation, which may be a response to chill stress with the effect of lowering the freezing point of intracellular water and decreasing ice crystal formation.

Transcriptomic response of Listeria monocytogenes to iron limitation and Fur mutation.

Iron is required by almost all bacteria, but concentrations above physiological levels are toxic. In bacteria, intracellular iron is regulated mostly by the ferric uptake regulator, Fur, or a similar functional protein. Iron limitation results in the regulation of a number of genes, especially those involved in iron uptake. A subset of these genes is the Fur regulon under the control of Fur. In the present study, we have identified Fur- and iron-regulated genes in Listeria monocytogenes by DNA microarray analysis using a fur mutant and its isogenic parent. To identify genes regulated exclusively in response to iron limitation, the whole-genome transcriptional responses to the iron limitation of a fur mutant and its isogenic parent were compared. Fur-regulated genes were identified by comparing the transcriptional profile of the parent with the transcriptional profile of the isogenic fur mutant. Our studies have identified genes regulated exclusively in response to iron and those that are negatively regulated by Fur. We have identified at least 14 genes that were negatively regulated directly by Fur. Under iron-limited conditions, these genes were upregulated, while the expression of fur was found to be downregulated. To further investigate the regulation of fur in response to iron, an ectopic fur promoter-lacZ transcriptional fusion strain was constructed, and its isogenic fur and perR mutant derivatives were generated in L. monocytogenes 10403S. Analysis of the iron limitation of the perR mutant indicated that the regulation of genes under the negative control of Fur was significantly inhibited. Our results indicate that Fur and PerR proteins negatively regulate fur and that under iron-limited conditions, PerR is required for the negative regulation of genes controlled by Fur.

Insertional inactivation of branched-chain alpha-keto acid dehydrogenase in Staphylococcus aureus leads to decreased branched-chain membrane fatty acid content and increased susceptibility to certain stresses.

Staphylococcus aureus is a major community and nosocomial pathogen. Its ability to withstand multiple stress conditions and quickly develop resistance to antibiotics complicates the control of staphylococcal infections. Adaptation to lower temperatures is a key for the survival of bacterial species outside the host. Branched-chain alpha-keto acid dehydrogenase (BKD) is an enzyme complex that catalyzes the early stages of branched-chain fatty acid (BCFA) production. In this study, BKD was inactivated, resulting in reduced levels of BCFAs in the membrane of S. aureus. Growth of the BKD-inactivated mutant was progressively more impaired than that of wild-type S. aureus with decreasing temperature, to the point that the mutant could not grow at 12 degrees C. The growth of the mutant was markedly stimulated by the inclusion of 2-methylbutyrate in the growth medium at all temperatures tested. 2-Methylbutyrate is a precursor of odd-numbered anteiso fatty acids and bypasses BKD. Interestingly, growth of wild-type S. aureus was also stimulated by including 2-methylbutyrate in the medium, especially at lower temperatures. The anteiso fatty acid content of the BKD-inactivated mutant was restored by the inclusion of 2-methylbutyrate in the medium. Fluorescence polarization measurements indicated that the membrane of the BKD-inactivated mutant was significantly less fluid than that of wild-type S. aureus. Consistent with this result, the mutant showed decreased toluene tolerance that could be increased by the inclusion of 2-methylbutyrate in the medium. The BKD-inactivated mutant was more susceptible to alkaline pH and oxidative stress conditions. Inactivation of the BKD enzyme complex in S. aureus also led to a reduction in adherence of the mutant to eukaryotic cells and its survival in a mouse host. In addition, the mutant offers a tool to study the role of membrane fluidity in the interaction of S. aureus with antimicrobial substances.

A cold-sensitive Listeria monocytogenes mutant has a transposon insertion in a gene encoding a putative membrane protein and shows altered (p)ppGpp levels.

A cold-sensitive Listeria monocytogenes mutant designated cld-14 was obtained by transposon Tn917 mutagenesis. The gene interrupted by Tn917 in cld-14 was the L. monocytogenes LMOf2365_1485 homolog, which exhibits 45.7% homology to the Bacillus subtilis yqfF locus. LMOf2365_1485, here designated pgpH, encodes a putative integral membrane protein with a predicted molecular mass of 81 kDa. PgpH is predicted to contain a conserved N-terminal signal peptide sequence, seven transmembrane helices, and a hydrophilic C terminus, which likely extends into the cytosol. The Tn917 insertion in pgpH is predicted to result in production of a premature polypeptide truncated at the fifth transmembrane domain. The C terminus of PgpH, which is probably absent in cld-14, contains a highly conserved HD domain that belongs to a metal-dependent phosphohydrolase family. Strain cld-14 accumulated higher levels of (p)ppGpp than the wild type accumulated, indicating that the function of PgpH may be to adjust cellular (p)ppGpp levels during low-temperature growth. The cld-14pgpH(+) complemented strain was able to grow at a low temperature, like the parent strain, providing direct evidence that the activity of PgpH is important in low-temperature adaptation. Because of its predicted membrane location, PgpH may play a critical role in sensing the environmental temperature and altering cellular (p)ppGpp levels to allow the organism to adapt to low temperatures.

Intact mutS in laboratory-derived and clinical glycopeptide-intermediate Staphylococcus aureus strains.

The mutS gene of the methyl-directed mismatch repair system was sequenced in 10 parent and glycopeptide-intermediate Staphylococcus aureus strains. The mutS gene was intact in all strains studied. Hence, mutations in this gene had played no role in the development of vancomycin resistance in these strains.

Pine oil cleaner-resistant Staphylococcus aureus: reduced susceptibility to vancomycin and oxacillin and involvement of SigB.

Mutants of Staphylococcus aureus strain COL resistant to a household pine oil cleaner (POC) were isolated on laboratory media containing POC. S. aureus mutants expressing the POC resistance (POC(r)) phenotype also demonstrate reduced susceptibility to the cell wall-active antibiotics vancomycin and oxacillin. The POC(r) phenotype is reliant on the S. aureus alternative transcription factor SigB, since inactivation of sigB abolished expression of elevated POC resistance and the reductions in vancomycin and oxacillin susceptibilities. The isolation of suppressor mutants of COLsigB::kan, which maintain the sigB::kan allele, indicates that the POC(r) phenotype can also be expressed to a lesser degree via a sigB-independent mechanism. These results bolster a growing body of reports suggesting that common disinfectants can select for bacteria with reduced susceptibilities to antibiotics. A series of in vitro-selected glycopeptide-intermediate S. aureus (GISA) isolates also expressed reductions in POC susceptibility compared to parent strains. Viewed collectively, our evidence suggests that mutations leading to the POC(r) phenotype may also be involved with the mechanism that leads to the GISA phenotype.

Purification and characterization of methionine sulfoxide reductases from mouse and Staphylococcus aureus and their substrate stereospecificity.

Many organisms have been shown to possess a methionine sulfoxide reductase (MsrA), exhibiting high specificity for reduction the S form of free and protein-bound methionine sulfoxide to methionine. Recently, a different form of the reductase (referred to as MsrB) has been detected in several organisms. We show here that MsrB is a selenoprotein that exhibits high specificity for reduction of the R forms of free and protein-bound methionine sulfoxide. The enzyme was partially purified from mouse liver and a derivative of the mouse MsrB gene, in which the codon specifying selenocystein incorporation was replaced by the cystein codon, was prepared, cloned, and overexpressed in Escherichia coli. The properties of the modified MsrB protein were compared directly with those of MsrA. Also, we have shown that in Staphylococcus aureus there are two MsrA and one nonselenoprotein MsrB, which demonstrates the same substrate stereospecificity as the mouse MsrB.