1 Department of Biochemistry, University of Stellenbosch, Private Bag X1, 7602 Matieland, South Africa
2 Laboratoire de Microbiologie Fondamentale et Appliquée, CNRS FRE 2224, IBMIG, UFR Sciences, 40 Avenue du Recteur Pineau, 86022 Poitiers Cedex, France
3 Department of Dairy and Food Science, Centre for Advanced Food Studies, LMC, The Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark
Correspondence
Marina Rautenbach
mra{at}sun.ac.za
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ABSTRACT |
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INTRODUCTION |
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Reports of resistance development by listerial strains against class IIa bacteriocins in laboratory systems have implied that increased resistance may compromise the potential role of these antimicrobial compounds in biopreservation. This has in turn spurred interest into understanding resistance phenomena displayed by the pathogen. In strains of the food-borne pathogen Listeria monocytogenes, there is cross-resistance between different class IIa bacteriocins, indicating common or similar resistance mechanisms (Vadyvaloo et al., 2002). A prevalent mechanism involving the loss of the enzyme IIAB subunit of a mannose-specific phosphotransferase system (EIIABMan PTS, encoded by mptA) was recently described (Dalet et al., 2001
; Gravesen et al., 2002
). An up-regulation of two
-glucoside-specific PTS genes, possibly associated with the absent EIIABMan, was also demonstrated (Gravesen et al., 2000
, 2002
). In a previous study, we investigated the compositional changes in the cell membrane associated with intermediate resistant and highly resistant strains. We observed an overall increase in short-acyl-chain and unsaturated phosphatidylglycerol (PG) species for all resistant strains, which indicated greater fluidity of the cell membranes. In this paper we report on a continuation of that study to characterize other factors that contribute to resistance of L. monocytogenes.
Studies on structure and activity of class IIa bacteriocins suggest that the cationic nature of the peptides enables interactions with negatively charged cell surfaces, while the hydrophobic region of the peptide induces membrane permeabilization (Chen et al., 1997; Ennahar et al., 2000
). Modification of the bacterial surface charge could, therefore, be expected to affect the initial electrostatic interaction between the peptide and the membrane that is required for pore formation, with or without mediation by a membrane-bound receptor-type molecule (Dalet et al., 2001
; Ennahar et al., 2000
). Two important options for modulating the charge of the cell envelope are D-alanylation of teichoic acid (TA) and lipoteichoic acid (LTA) in the cell wall, and lysinylation of the cell-membrane phospholipids. Previous reports have indicated a role of D-alanine-esterified TA and LTA in conferring sensitivity to cationic peptides and other cationic antimicrobial compounds in various Gram-positive pathogenic bacteria, including induced nisin sensitivity in D-alanine-deficient LTA of L. monocytogenes (Abachin et al., 2002
; Ganz, 2001
; Mantovani & Russell, 2001
; Peschel & Collins, 2001
; Scott et al., 1999
). The description of lysine-deficient phospholipid due to insertional inactivation of the mprF (multiple peptide resistance factor) gene in Staphylococcus aureus was the first study indicating an influential role of charge modification of membrane phospholipids in resistance to antimicrobial peptides (Peschel et al., 2001
). The lysinylated phospholipids L-lysyl-pg and L-lysyl-cardiolipin are among the four major phospholipids of the Listeria spp. (Fischer & Leopold, 1999
), and it is conceivable that these phospholipids could display differences in lysinylation in the different strains. Changes of the cell-surface charge by D-alanyl esterification of TA and lysinylation of membrane phospholipid were investigated, as well as the expression of genes that potentially influence cell-surface/cell-membrane charge (dltA, lmo1695).
The effect of the cell-wall-acting antibiotic D-cycloserine (DCS) was also analysed, since the cell wall of nisin-resistant Listeria innocua has been shown to be thickened and more resistant to cell-wall-acting antibiotics, such as DCS (Maisnier-Patin & Richard, 1996). In addition, alanine that is incorporated in TA is also the substrate for DCS enzyme targets (Wargel et al., 1971
) dal (Scott et al., 1999
) and ddlA (Peteroy et al., 2000
). The dal gene product plays a role in converting L-alanine to D-alanine, which is channelled into TA and cell-wall biosynthesis (Thompson et al., 1998
). The ddlA gene product catalyses amide-bond formation between two D-alanines for incorporation into peptidoglycan (Peteroy et al., 2000
). Furthermore, the expression of genes that potentially influence DCS inhibitory activity (dal, ddlA) was analysed by real-time PCR.
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METHODS |
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Separation of TA from peptidoglycan cell wall was done according to the method described by Kaya et al. (1985). The lyophilized material (0·2 ml mg1 in 25 mM glycine/HCl, pH 2·5) was heated to 100 °C for 10 min. The suspension was cooled to room temperature and centrifuged at 20 000 g at 4 °C for 45 min. The TA extraction from the cell wall was repeated once. The supernatant was ultrafiltered using a polyethersulfone membrane (Millipore) with a molecular mass limit of 10 000. The ultrafiltrate or TA suspension was lyophilized and resuspended in analytical-grade water.
Phospholipid isolation.
Phospholipid was isolated according to the method described by Bligh & Dyer (1959) as previously modified by Vadyvaloo et al. (2002)
. The chloroform fraction containing the phospholipid was dried using vacuum rotary evaporation.
Quantification of phosphorus.
Organic phosphate was quantified using the method described by Ames (1966). Phosphorus present in the ribitol-phosphate backbone of TA, and in the phospholipid extracts, forms a phosphomolybdate complex with ammonium phosphomolybdate in 1 M H2SO4, after ashing under a direct flame. The phosphomolybdate complex is reduced by ascorbic acid to yield a blue colour complex with absorbance maximum at 820 nm.
Quantification of alanine and lysine.
Alanine and lysine were quantified by HPLC (Breeze System, Waters) using the PicoTag method (Bidlingmeyer et al., 1984). Alanine was liberated from TA by base hydrolysis in 4 M NaOH at 112 °C for 24 h. Lysine was liberated from phospholipid by acid hydrolysis in 6 M HCl containing 0·5 % phenol at 112 °C for 18 h.
Cationic cytochrome c binding to whole cells.
This method was carried out as described by Peschel et al. (1999) with some modifications. Cells were harvested at mid-exponential phase (OD600 approx. 0·5) and washed twice by centrifugation in 20 mM MOPS buffer, pH 7. The cells were resuspended in the neutral pH MOPS buffer to a final OD600 of 0·150 before interaction with the positively charged cytochrome c to counteract pH influence on charge. Cationic cytochrome c was added to the cells at a concentration of 0·5 mg ml1 and the mixture was incubated at room temperature for 10 min. The solution was centrifuged at 8000 r.p.m. in an Eppendorf benchtop centrifuge for 5 min, and unbound cationic cytochrome c in the supernatant was quantified photometrically at 530 nm.
DCS inhibition assay.
A 1 % inoculum of the bacterial culture was added to a tube containing 10 ml medium, and growth at OD600 was monitored. At mid-exponential phase (OD600 approx. 0·5), DCS was added to the culture at approximately 100 µg ml1. After addition of DCS, the OD600 was monitored over 10 h for the L. monocytogenes B73 and 412 families, and 6 h for the L. monocytogenes EGDe family. Control cultures were made without antibiotic addition. All data were analysed on Graphpad Prism version 3.0 for Windows (GraphPad Software). A sigmoidal plot (percentage relative survival versus log time) was constructed to calculate the 50 % survival/inhibition time. The sigmoidal curve with variable slope, constant top of 100 and bottom of 0 was fitted to each of the datasets. For curve fitting only, the mean value of each data point, without weighting, was considered. The time taken to achieve 50 % survival was calculated from the 50 % inhibition value halfway between top and bottom. The intersection between the tangent across the top of each curve and the tangent of the plateau of a loglog plot was used to calculate the onset time of DCS inhibition. Curve fitting was carried out as described above, but with no top and bottom constraints.
cDNA synthesis.
Total RNA was isolated from a mid-exponential-phase culture (10 ml) using the RNAwiz kit (Ambion), according to the manufacturer's instructions. The extracted RNA was treated with DNase-RNase free (Invitrogen) and its quality was assessed by running samples on a 1 % formaldehyde agarose gel. RNA was quantified spectrophotometrically. cDNA was synthesized from 2 µg total RNA using random hexamers and the Superscript II Kit (Gibco), according to the manufacturer's instructions. A reaction containing all the components but omitting reverse transcriptase was included in order to assess DNA contamination.
Real-time PCR.
The primers used for the real-time PCR are listed in Table 2. They were designed from the gene sequences of the L. monocytogenes EGDe genome (http://genolist.pasteur.fr/Listilist/) using Primer Express software version 1.0 (Applied Biosystems). The real-time PCR was carried out using the SYBR Green PCR Core Reagent kit (Applied Biosystems), as recommended. PCR reactions were run on the ABI Prism 7700 sequence detector (Applied Biosystems) under the following conditions: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles at 95 °C for 10 s and 60 °C for 1 min. Each assay included, in triplicate, a serial twofold dilution of L. monocytogenes genomic DNA, a control without template or the cDNA from the same sample.
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RESULTS |
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We considered a threefold difference in expression () as a significant change (i.e.
CT>1·6 or <1·6) in expression. There was thus no change in expression of dltA, dal, ddlA and lmo1695 in any of the resistant mutants (see fold expression change results in Table 4
). Results for the expression analysis of the mptA gene for all the spontaneous resistant strains appear in Fig. 6
.
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DISCUSSION |
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We were unable to correlate the increase in D-alanine content of the resistant strains with expression levels of the genes dltA and dal, which are related to D-alanine and TA metabolism. However, Gravesen et al. (2004) recently showed, using different growth conditions, that 412P had significantly reduced expression of dltA, dal and dat, which would lead to lower availability of D-alanine for incorporation into not only LTA and TA, but also peptidoglycan. Their results, in the light of our observation of the higher content of D-alanine in TA, thus indicate that strict regulation of the D-alanine utilization and channelling most probably exists at a post-translational level. Also, our finding of a more positive cell surface for the resistant 412P, which is probably due to the D-alanine in the TA, could explain the reduced nisin sensitivity in 412P observed by Gravesen et al. (2004)
.
Membrane charge is influenced by L-lysine in the phospholipids and will influence the susceptibility to cationic antimicrobial compounds (Ganz, 2001; Peschel et al., 2001
). Lysinylation of phospholipids has not yet been explored in L. monocytogenes. Cardiolipin and PG in L. monocytogenes are normally negatively charged, whereas the lysinylated forms of these two phospholipids bear a net positive charge (Peschel et al., 1999
). Our results indicate, in addition to the increase in D-alanine in cell wall TA, a possible increase in the lysine content of the membrane phospholipid in highly resistant strains (Figs 2 and 4
). We also found that B73-V2 had the lowest lysine : phosphorus ratio of all strains tested, which differed statistically from both B73-V1 and B73-MR1 (Fig. 2
). This lower lysinylated PG content in B73-V2 possibly eliminates some of the resistance benefits that the high D-alanine content in its TA affords it.
For the highly resistant strains, the increase in positive charge due to alanine and possibly also lysine incorporation would decrease the anionic property of the cell permeability barriers. Furthermore, this combined increase in charge from both alanine and possibly lysine (from cell wall and membrane) explains why there was least binding of the cytochrome c to the highly resistant cells (Fig. 3). Additionally, other factors could influence net surface charge, such as the zwitterionic phosphatidylethanolamine content of L. monocytogenes phospholipid (Crandall & Montville, 1998
). The genes involved in D-alanylation and lysinylation (dltA and lmo1695) did not display increased expression in any of the resistant mutant strains studied. This may suggest that the regulation of the activity of these enzymes is not at the transcriptional level. It is, however, quite clear from our results that the highly resistant strains have a more positive cell surface than the susceptible strains, and that this would interfere with the initial and subsequent electrostatic interaction of the cationic bacteriocin with the resistant cells. It is important to consider at this point that electrostatic interactions have been identified to play a central role in the interaction of nisin with its docking molecule, lipid II (Hsu et al., 2003
).
In Fig. 4, a summary of the data concerned with cell surface charge is given, and from this comparison it is clear that more than one factor does influence cell surface charge. For example, modulation of the incorporation of D-alanine in TA and lysine in membrane phospholipids could negate the influence of each other, as we observed for B73-V2 that had a 14±4 % lower lysine : phosphorus ratio and a 29±4 % higher alanine : phosphorus ratio than the sensitive B73 strain, but a similar tendency to bind cytochrome c. Factors other than lysine and alanine incorporation do influence cell surface charge, as is clear from the results of the insertional mutant EGK54, which has a large D-alanine content (91±24 % higher than 412), but only marginally lower cytochrome c binding ability (17±4 % lower than 412). B73-MR1, on the other hand, has significantly reduced cytochrome c binding (59±1 % lower than B73), with only 13±1 % higher alanine content and 23±8 % higher lysine content than B73. However, we found a good correlation between cytochrome c and D-alanine content in TA for 412P.
DCS influence on class IIa bacteriocin-resistant L. monocytogenes strains
DCS affected the strains differently, with the highly resistant stains, except B73-MR1, being very sensitive to this inhibitor (Fig. 5, Table 3
). The whole B73 family was more resistant to DCS than the other two families, which may partially explain the lower sensitivity of B73-MR1. The differences between the wild-type and intermediate resistant B73 strains were negligible, but the onset of DCS inhibition was earlier for these strains compared to the slower-growing highly resistant strain B73-MR1. There were no significant alterations in the levels of transcription of the dal and ddlA genes between the resistant and sensitive strains. This indicated that the DCS targets were not implicated in the variances in DCS sensitivity unless there is interference with regulation at the post-transcriptional level. DCS sensitivity could well be affected by differences in ability to transport DCS into the cells. DCS is transported into the cell by the same transporter as alanine (Wargel et al., 1971
), which probably links the DCS sensitivity of the resistant 412P and EGK54 strains to their much higher D-alanine content in their TA.
Expression of mptA in class IIa bacteriocin resistance
Present evidence suggests that prevention of mptACD expression confers resistance in L. monocytogenes strains, because sensitivity could be due to a potential interaction or docking of the class IIa bacteriocin on the MptCMptD membrane complex of the mannose PTS (Dalet et al., 2001; Gravesen et al., 2002
). Additionally, the requirement of stereospecificity for leucocin A activity supports the presence of a docking molecule (Yan et al., 2000
). Our results correlated well with previous work by our group (Gravesen et al., 2000
, 2002
), as we observed a clear correlation between levels of transcription of the mptA gene and levels of resistance to class IIa bacteriocins. A decrease of greater than 1000-fold in transcription levels of the mptA gene was found for the highly resistant spontaneous mutants B73-MR1 and 412P. We also present here the first evidence of the involvement of mptA down-regulation in intermediate resistance following exposure to leucocin A, a class IIa bacteriocin. B73-V1 and B73-V2, which were observed to be two and four times more resistant than the wild-type respectively (Vadyvaloo et al., 2002
), showed an approximate fourfold decrease in transcription level of this gene.
Conclusions
In a previous study on the PG composition of L. monocytogenes, we found an overall increase in unsaturated and short-acyl-chain PG species in all class IIa bacteriocin-resistant strains of L. monocytogenes (Vadyvaloo et al., 2002), which indicated greater fluidity of the cell membranes in the resistant strains. The increase of unsaturated acyl chains was, however, significantly greater in the intermediate resistant strains B73-V1 and B73-V2, while the highly resistant B73-MR1 showed significantly greater increase in short-acyl-chain PG species (Vadyvaloo et al., 2002
).
From this study and our previous work, we can thus deduce that the highly resistant strains have: (1) a more positively charged cell wall, (2) possibly a more neutral cell membrane, (3) a more fluid cell membrane (Vadyvaloo et al., 2002), and (4) a highly decreased mptA expression. Initial binding of the cationic bacteriocin to the cell will be hampered by (1), transfer to the cell membrane by (2), self-association in pores by (3), and if the bacteriocin action is receptor mediated by mannose PTS EIIAB, (4) will have the ultimate influence on resistance. The intermediate resistance is unclear in terms of the role of cell-wall charge, but we can deduce that this type of resistance is possibly the result of both a more fluid cell membrane and decreased mptA expression.
In another study we also found that the highly resistant B73-MR1 and the defined EGDe mutant showed a shift from a predominantly lactic acid of their wild-strains to a mixed acid fermentation (Vadyvaloo et al., 2004). Therefore, apart from the cell-wall and cell-membrane changes, a change in metabolism also correlates with resistance and the absence (or possibly with lower expression) of the enzyme IIAB subunit of a mannose-specific phosphotransferase system. The change in metabolism may directly impact the composition of the cell wall and cell membrane via different channelling of key metabolites.
Our results therefore strongly indicate that at least four cell-surface changes contribute to class IIa bacteriocin resistance, and that the combination and contribution of each of these changes determine the level of bacteriocin resistance.
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ACKNOWLEDGEMENTS |
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Received 27 January 2004;
revised 11 May 2004;
accepted 9 June 2004.
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