Vaccine Development Laboratory, National Public Health Institute, Mannerheimintie 166, FIN-00300 Helsinki, Finland
Correspondence
Vesa P. Kontinen
Vesa.Kontinen{at}ktl.fi
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ABSTRACT |
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INTRODUCTION |
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Extracytoplasmic function (ECF) sigma factors are regulatory components by which bacteria control gene expression in response to environmental stress. There are seven different ECF-type sigma factors in the Gram-positive model bacterium B. subtilis (Helmann, 2002; Kunst et al., 1997
). Several stress conditions activate the SigW regulon. These include alkaline shock (Wiegert et al., 2001
), inhibition of the cell wall synthesis by antibiotics such as vancomycin and disturbance of the integrity of the cell membrane by detergents (Cao et al., 2002b
). The SigW sigma factor is associated with the membrane-bound SigW anti-sigma factor when the Bacillus cell is not exposed to environmental stress (Schobel et al., 2004
). Under stress conditions, the anti-sigma factor is proteolytically degraded, resulting in the release of SigW from the membrane and binding to gene promoters of the regulon (Schobel et al., 2004
). A similar pattern is anticipated for other ECF-type sigma factors. SigM is required for combating stress due to antibiotic effects on the cell wall, ethanol, heat, acid and superoxide (Thackray & Moir, 2003
). It is also essential for survival in environments containing high concentrations of salt (Horsburgh & Moir, 1999
), suggesting that it is required for maintaining the integrity of the cell envelope. Alternative sigma factors are not the only regulatory systems that are involved in stress tolerance: two-component systems (TCSs) also have a role in controlling gene expression in environmental changes. TCSs are signalling devices composed of a membrane-bound sensor kinase and a response regulator. B. subtilis two-component regulation has recently been reviewed (Ogura & Tanaka, 2002
).
We studied stress responses to two naturally occurring antimicrobial peptides, LL-37 and PG-1, and their synthetic analogue poly-L-lysine (PLL), using a DNA macroarray and real-time RT-PCR. Human LL-37 is 37 amino acid residues long and belongs to the cathelicidin family of antimicrobial peptides (Johansson et al., 1998; Turner et al., 1998
). It is an amphipathic and
-helical peptide that probably disrupts the lipid bilayer by a toroidal pore mechanism (Henzler Wildman et al., 2003
; Johansson et al., 1998
). The porcine protegrin PG-1 is composed of 18 amino acid residues, including four cysteines, and forms a two-stranded antiparallel
-sheet linked by a
-turn (Aumelas et al., 1996
; Fahrner et al., 1996
). The four cysteines of PG-1 form two disulphide bonds, which are important for the
-sheet conformation and antimicrobial activity (Harwig et al., 1996
). The synthetic peptide PLL differs from the natural peptides in that it is not amphipathic. Its mode of action on membranes is unclear.
It was found that the antimicrobial peptides induced ECF-type sigma factor regulons in a complex manner. Several genes that are regulated by two-component signal transduction systems were also induced. Most interestingly, LL-37 strongly upregulated, via the YxdJK TCS, the yxdLM genes encoding an ABC-type transporter of unknown function. The yvcRS and bceAB (ytsCD) genes, which encode ABC transporters highly homologous with YxdLM, were also moderately upregulated. Interestingly PG-1, PLL and Triton X-100 did not induce the expression of any of these ABC-transporter genes.
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METHODS |
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Mutant constructions.
The sigW : : neo and sigM : : pMUTIN4 mutations were introduced into strains by transformation with chromosomal DNA of the B. subtilis strains HB4247 (kindly supplied by J. D. Helmann) and MJH003 (Horsburgh & Moir, 1999), respectively. The sigW sigM double mutant was constructed by transforming the strain IH8342 containing the sigW : : neo mutation with the chromosome of MJH003. The yvcQ gene was inactivated with pMUTIN4, as described by Vagner et al. (1998)
. The yxdJ and yxdK null mutations were obtained from Naotake Ogasawara (Nara Institute of Science and Technology, Nara, Japan).
RNA isolation, labelling with 33P and DNA macroarray analysis.
For RNA isolations, strains were grown in the BFA minimal medium containing 100 mM NaCl in shake-flask cultures. Cell densities were measured with a Klett colorimeter. Antimicrobial peptides were added at 60 Klett units and samples for RNA isolation were harvested after 20 min from 4 ml cell culture by centrifugation. Control samples without peptides were treated in a similar manner. Cells were resuspended in 400 µl ice-cold culture medium and transferred to screw-capped Eppendorf tubes containing 1·5 g glass beads, 50 µl 10 % SDS, 50 µl 3 M sodium acetate and 500 µl phenol/chloroform/isoamylalcohol (25 : 24 : 1 by vol.). The tubes were frozen in liquid nitrogen, followed by vigorous shaking for 6 min with a face-grinding machine and centrifugation at 10 000 r.p.m. for 5 min. The water phase was mixed (Vortex) with 1 vol. chloroform and centrifuged at 14 000 r.p.m. for 2 min. Next, the water phase was mixed with 2 vols Roche lysis/binding buffer, and the RNA extraction was continued with the Roche High Pure RNA Isolation Kit according to the manufacturer's instructions.
DNA macroarray analysis was carried out using Panorama B. subtilis gene array filters and specific cDNA labelling primers (Sigma Genosys). The Panorama B. subtilis gene array contains duplicate spots of PCR products representing currently known B. subtilis genes. Prior to cDNA synthesis, the quality of RNA was confirmed using Northern blotting. For cDNA synthesis, 10 µg RNA was used, and the synthesis was performed as described by Wiegert et al. (2001). The SuperScript II reverse transcriptase was purchased from Gibco-BRL. cDNA was purified with MicroSpin G-25 columns (Amersham Pharmacia Biotech) and the labelling efficiency was determined with a liquid scintillation counter. Prehybridization, hybridization and washing of the filters were performed according to the manufacturer's instructions. The DNA array filters were exposed overnight on phosphor screens and the screens were scanned with a Fluorescent Image Analyser FLA-2000 (Fujifilm). Hybridization signal intensities were quantified with the ArrayVision software (Imaging Research), as described by Wiegert et al. (2001)
. Data were filtered to avoid false positives by excluding genes with a signal-to-noise ratio <3 (Array Vision software) and normalized by dividing the intensity of each spot by the mean intensity of all the spots. Each experiment was carried out twice with RNA isolated from two independent cultures. Genes were regarded as induced when the induction ratio was >2 in both experiments.
Quantitative real-time RT-PCR.
For real-time RT-PCR, RNA was isolated similarly as for the DNA array. RT reactions were carried out with the Omniscript Reverse Transcriptase Kit (Qiagen) according to the manufacturer's instructions with the exception of an additional DNase I (Roche) treatment. An equal amount of RNA (2 µg) was used in each RT reaction. Primers used in RT reactions were random hexamers (0·15 µg ml1) provided by Roche. The absence of chromosomal DNA in the RNA preparations was verified with a control sample that was not treated with RT, but was otherwise treated in a similar manner to the RT-treated samples. Real-time PCR reactions were carried out with specific primer pairs using the SYBR green PCR master mix (Applied Biosystems). Primers were designed with the Primer Express software (Applied Biosystems) and purchased from Sigma Genosys or TAGC Copenhagen. Sequences of the PCR primers for the genes studied are shown in Table 1. The amplification and detection of PCR products were performed with the ABI PRISM 5700 sequence detection system (Applied Biosystems). The cycling conditions were: 1 cycle at 50 °C for 2 min, 1 cycle at 95 °C for 10 min, 40 cycles at 95 °C for 15 s and at 60 °C for 1 min. The threshold cycle (Ct) is the first cycle at which the fluorescence becomes detectable above the background and is inversely proportional to the logarithm of the initial number of template molecules. Ct values of known quantities of B. subtilis chromosomal DNA were plotted for each primer pair to obtain standard curves. The standard curves allowed us to convert the Ct values of each amplified gene in the cDNA preparations to relative numbers of cDNA molecules. These cDNA values were normalized with the value of gyrA, which was constant in different growth conditions and phases (data not shown).
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RESULTS |
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Altogether, the LL-37 treatment induced 96 genes (Table 2, only the first gene of an operon is listed), including several genes that are regulated by the SigW and SigM ECF sigma factors (Huang et al., 1999
; Cao et al., 2002a
; Thackray & Moir, 2003
; Asai et al., 2003
). Of the 30 verified promoters of the SigW regulon (Cao et al., 2002a
), only 10 were expressed at elevated levels (greater than twofold) in LL-37-treated cells, with the fold-induction ratios ranging from 2·4 to 14·7, suggesting that LL-37 is a weak SigW inducer. The promoters that are directly regulated by SigM are less well known, but it was observed that 15 candidate promoters of the SigM regulon (Asai et al., 2003
), including sigM itself, were upregulated by LL-37 (Table 2
). Many of the induced genes are involved in extracytoplasmic functions such as synthesis of the cell wall. These genes included pbpE, encoding a penicillin-binding protein (4·5-fold induction), wapA, encoding a cell wall-associated protein (2·5-fold induction), murG, involved in cell wall formation (6·1-fold-induction) and maf, required for septum formation (2·5-fold induction). The upregulated genes also included bcrC (ywoA), which is dependent on several sigma factors and is required for bacitracin resistance (Cao & Helmann, 2002
) (4·1-fold induction), and the gene encoding the penicillin-binding protein ponA (2·3-fold induction). The most strongly induced gene was liaI (yvqI) (Mascher et al., 2004
), which was induced 58-fold (Table 2
); other genes of the liaIHGFSR (yvqIHGFEC) gene cluster were also upregulated, but to a lesser extent (not shown). liaI (yvqI) is not known to be dependent on any ECF sigma factor. Furthermore, yrhH (14·7-fold), encoding a putative methyltransferase, yxdL (22·7-fold) and yhcG (14·7-fold), encoding putative ATP-binding components of ABC transporters, and yoeB (9·1-fold), encoding a putative exported protein of unknown function, were strongly upregulated.
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Genes that were induced by both peptides were araE, bcrC (ywoA), dltB, liaI, murG, pbpE, pspA, spoOM, wprA, yceC, yeaA, yjbC, yoeB, yqeZ and yuaG (Table 2). Interestingly, the genes of the yxdLM operon, yrhH and yhcG, which were highly induced by LL-37, were not induced by PG-1 (see also below).
PLL was also an ECF inducer and the gene induction pattern resembled that of the other antimicrobial peptides, but characteristic differences were also observed (Table 2). Among 86 upregulated genes, there were 23 and 8 genes that belonged to the SigW and SigM regulons, respectively. Thus, compared to the response to LL-37 (above), it seems that PLL is clearly a stronger activator of the SigW regulon. There were also several genes that were induced at high levels by PLL, but not at all by LL-37 or PG-1, including csfB (6·6-fold), yaaN (5·1-fold), yfhL (5·6-fold), yocA (4·5-fold) and yrzI (12·4-fold), encoding proteins of unknown function. In addition, genes involved in purine, pyrimidine and ribosomal protein synthesis were strongly induced, a phenomenon not seen with the natural peptides. Furthermore, some genes that were induced at high levels by either or both of the natural peptides were not induced by PLL, notably liaI (yvqI) and other genes of the lia (yvq) cluster, yxdL, which was strongly induced by LL-37, and yhcG and yoeB.
Decreased expression of several genes was also observed. However, the experimental setup, short time of exposure to the peptides, and very different degradation rates of mRNAs hampered the interpretation of these results and they were not analysed in this study.
Cross-talk in signal transduction pathways mediating stress responses induced by a cationic peptide
We also carried out the DNA array analysis with sigM and sigW knockout mutants using LL-37 for the induction. The gene induction patterns of the sigma mutants and wild-type strain were compared in scatter plots (Fig. 2). The LL-37 treatment elicited a significantly lower number of induced genes in both sigma mutants than in the wild-type. The genes induced in the sigM and sigW mutants are listed in Table 3
. This reduced stress response was not due to decreased stress in the sigma mutants, since the effective concentration of LL-37 causing the growth inhibition was the same in all three strains (see below). In the sigM mutant, as expected, the genes of the SigM regulon were not induced. Surprisingly, the array data also showed either that the SigW-regulated genes were not induced or that their induction levels were clearly lower than in the wild-type strain. Analogous results were obtained with the sigW mutant. Interestingly, numerous genes which are not known to belong to these two sigma factor regulons were also induced in a SigM- or SigW-dependent manner. These results suggest significant cross-talk between the SigM and SigW regulons, and some other regulon(s) responding to LL-37. Furthermore, a significant observation was the clearly higher level of expression of several genes in the sigW mutant compared to that of the wild-type (Fig. 2b
). This set included (Table 3
) genes encoding endo-1,4-
-glucanase (bglC; 4·6-fold), a protein synthesizing
-1,4-glucan using ADP-glucose (glgA; 5·1-fold) and 6-phospho-
-glucosidase (glvA/malA; 3·9-fold).
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In the wild-type strain treated with LL-37, liaI and yxdL were the most highly induced genes (Table 4), as was also the case in the array analysis. Higher induction ratios were seen in the 10 min samples than in the 20 min samples, indicating that the induction was fast and transient. As shown in Fig. 3
, expression was dependent on the dosage of LL-37, as demonstrated with the liaI gene (Fig. 3a
), and decreased (also that of liaH) from the maximal level (10 min time point) back to the uninduced level in about 2 h (Fig. 3b
). Consistent with the induction of the liaIHGFSR gene cluster, the LiaH protein appeared in the proteome of cytoplasmic proteins, as demonstrated by two-dimensional gel electrophoresis and spot identification by matrix-associated laser desorption ionizationtime of flight mass spectrometry (MALDI-TOF) (data not shown). The other genes were induced to a lesser extent, and in only half of them was the induction transient (Table 4
). The normalized mRNA levels (not shown) indicated that liaI, yxdL and yuaG were expressed at a very low level in non-treated cells (basal expression level). The basal expression level of wprA, yjbC, ypuA and radC was fairly high, and bcrC (ywoA) and racX were expressed at intermediate levels. The genes that were expressed at a low level in non-treated cells exhibited the strongest induction in peptide-treated cells. Normalized mRNA levels of the genes varied approximately twofold from one experiment to another and between non-treated wild-type and ECF sigma mutant cells (not shown). Considerable experimental variation was especially observed in the induction ratios of the liaI and yxdL genes (Table 4
). The RT-PCR displayed clearly higher induction ratios than the DNA array, but there was a good overall consistency of the induction pattern in these two types of assay.
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The induction ratios of all genes studied were significantly lower in PG-1-treated cells than in LL-37-treated cells (Table 5), suggesting that the stress caused by the PG-1 treatment was less severe; for example, the induction ratio of liaI with LL-37 was several-fold higher than with PG-1. Despite the lower induction level in PG-1-treated cells, the sigM and sigW mutations reduced the induction of liaI as in LL-37-treated cells (Table 5
). The concentration of PG-1 used induced the yjbC and bcrC (ywoA) genes very weakly in all three strains. PG-1 treatment induced yuaG expression in a similar manner to LL-37 treatment, and the strong SigW dependency and moderate SigM dependency of yuaG were also observed with PG-1 (Table 5
). In the wild-type, racX was induced four- to ninefold, whereas no induction was observed in the sigW mutant, suggesting SigW-dependent regulation of racX. In the sigM mutant, racX was induced two- to sixfold by PG-1. The inactivation of the sigma factors did not impair the induction of wprA, consistent with an induction mechanism that is independent of SigW and SigM.
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A similar induction of liaI expression (790-fold at the 10 min time point) and slightly reduced induction ratios in the sigM and sigW mutants, as with LL-37, were observed (Table 6). The strong dependency of yuaG on SigW was also demonstrated with the detergent, but the moderate SigM dependency was not observed. The induction of three other genes (radC, ypuA and bcrC) was also partially dependent on both sigma factors. radC was expressed in the wild-type strain at two- to fivefold, ypuA at two- to threefold and bcrC (ywoA) at threefold higher levels than in the sigma mutants (Table 6
). In the wild-type strain and sigM mutant, a clearly stronger induction of racX was observed when cells were treated with Triton X-100 (1961-fold) than when they were treated with the antimicrobial peptides (four- to ninefold). In a similar manner to that observed with PG-1, no induction was seen in the sigW mutant, consistent with the SigW dependency of racX. The sigma mutations did not impair the induction of yjbC in Triton X-100-treated cells, in contrast to LL-37-treated cells. Furthermore, the wprA gene was not induced by Triton-X-100.
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yxdL gene expression is specifically induced by LL-37
A highly interesting observation was that the yxdL gene, which was strongly induced by LL-37 (704-fold), was not induced by PG-1 (Table 5). The lack of induction was seen in both the DNA array and RT-PCR analyses, and it was true for both wild-type and sigM and sigW mutant cells. The yxdL gene was not induced by Triton X-100 either (Table 6
). These results suggest that there is a strong specificity in the induction mechanism of yxdL.
LL-37 causes upregulation of three paralogous ABC-transporter genes via TCS-mediated signalling dedicated to the regulation of transporter expression
The yxdL gene encodes the putative ATP-binding component of an ABC transporter of unknown function. The downstream gene yxdM, which most probably forms an operon with yxdL, encodes the permease component of the ABC transporter. Immediately upstream from yxdLM there are the yxdJ and yxdK genes, which encode the components of a TCS of unknown function (see the organization of the gene cluster in Fig. 4). The yxeA gene, which encodes a conserved protein of unknown function, is located downstream from the yxdM gene and most probably belongs to the same operon as yxdLM (not shown in Fig. 4
). The DNA array data also revealed that LL-37 strongly induced the yxeA gene (36-fold) in a similar manner to yxdLM, consistent with the operon organization. A sequence similarity search revealed that yxdL and yxdM are highly homologous with the corresponding genes of two other ABC transporters of B. subtilis, BceAB (formerly YtsCD) and YvcRS (Fig. 4
; see also Joseph et al., 2002
; Mascher et al., 2003
; Ohki et al., 2003b
). Interestingly, genes encoding TCSs are also located in the immediate upstream regions of the bceAB and yvcRS genes in a pattern similar to that of the yxdLM region. It has been shown that the BceRS TCS is able to sense extracellular bacitracin and induce the expression of the BceAB ABC transporter, which confers resistance to bacitracin (Mascher et al., 2003
; Ohki et al., 2003b
). The YxdLM ABC transporter as well as the YxdJK TCS exhibit homology with the corresponding proteins of the Yvc and Bce systems. The homology is highest between the ATP-binding components of the ABC transporters (about 50 % identity), and, in the following order, is less between the response regulators of TCS, the sensor kinases of TCS and the permease components of the ABC transporters (Fig. 4
). The Yxd proteins exhibit slightly higher similarity to the Yvc proteins than to the Bce proteins.
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DISCUSSION |
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The antimicrobial peptides induced expression of 96 (LL-37), 58 (PG-1) and 86 (PLL) genes in B. subtilis. In this complex response, some patterns were recognized. A prominent feature was a high proportion of induced genes belonging to the SigW and SigM ECF-type sigma factor regulons. However, only subsets of these sigma factor regulon genes were induced by the antimicrobial peptides. The non-amphipathic PLL was the most effective peptide in activating the SigW regulon, as evidenced by the induction of 23 out of the 30 verified promoters of the regulon [see Cao et al. (2002a) for the SigW regulon]. The amphipathic peptides LL-37 and PG-1 upregulated only about one-third of the 30 SigW-regulated genes. In a similar manner, only subsets of putative SigM-regulated promoters were induced in peptide-treated cells. These results suggest that SigW and SigM are involved in the stress responses to the antimicrobial peptides. However, the high numbers of induced genes that are expressed independently of SigW and SigM suggest that probably several other signal transduction pathways and regulators also mediate the stress responses.
It has been shown that the ECF sigma factor regulons are partly overlapping (Cao et al., 2002b; Huang et al., 1998
). Nevertheless, the effect of sigW or sigM mutation on the number of induced genes in cells treated with LL-37 was striking. Not only the genes belonging to the mutated sigma regulon but also those under the control of other ECF sigma factors and those expressed independently of these sigma factors were poorly induced after peptide treatment. This phenomenon may be partly due to the increased basal expression level of several genes in the sigW and sigM mutants. Consequently, the additional stress of cationic peptides may not have caused further induction. Yet this alone does not explain why so few genes were induced in the sigma factor mutants, since the basal expression level was elevated in the sigma mutants only in the case of about 30 % of the genes induced in the wild-type. The RT-PCR analysis revealed decreased induction ratios for bcrC, liaI, radC, racX, ypuA and yxdL in both the sigma mutants. However, it is apparent that not all the affected genes are directly regulated by the sigma factors. It has been shown that liaI is regulated by the LiaRS (YvqCE) TCS (Mascher et al., 2003
; H.-L. Hyyryläinen and others, unpublished results). The effects of ECF sigma factor mutations on liaI expression in cells treated with alkaline (Wiegert et al., 2001
) or cationic antimicrobial peptides (this study) are most probably indirect. It is possible that the inactivation of one ECF sigma factor results in disturbance of the sensory function of other membrane-associated stress sensors. The similarity of the lethal doses of the cationic peptides in the sigma factor mutants and the wild-type, however, suggests that the degree of stress in these strains was the same. These results suggest that the stress response to cationic antimicrobial peptides is very complex. The functional overlap of several sigma factors and other types of regulators may also explain why the inactivation of SigW, SigM or both of them did not make cells sensitive to antimicrobial peptides.
PLL is expected to interact with the negatively charged cell wall and head groups of the membrane phospholipids (carpet or detergent-like mechanism; Yeaman & Yount, 2003), but may not penetrate deeper into the membrane interior. LL-37 and PG-1 penetrate into the membrane and disturb its integrity by forming pores (Henzler Wildman et al., 2003
; Oren et al., 1999
; Yang et al., 2000
). We hypothesize that SigW-regulated promoters are activated by antimicrobial peptides by the interaction of the latter with the cell membrane surface and the cell wall, rather than by deeper effects inside the membrane. This conclusion is consistent with the strong induction of the SigW regulon by cell wall antibiotics (Cao et al., 2002b
).
The araE, bcrC (ywoA), dltB, pbpE, pspA (ydjF), yceC, spo0M, yeaA, yjbC, yqeZ and yuaG genes were induced by all three peptide treatments. Each of these genes belongs to at least one ECF sigma factor regulon (Table 2). Some of these genes are involved in interactions with antimicrobial compounds interfering with the cell wall or membrane: pbpE encodes a penicillin-binding protein, yceC is similar to the tellurium resistance proteins, and bcrC (ywoA) encodes a bacitracin permease (Cao & Helmann, 2002
; Podlesek et al., 1995
). The dlt operon including dltB is involved in the D-alanine esterification of lipoteichoic and wall teichoic acids (Perego et al., 1995
), which increases bacterial resistance to cationic antimicrobial peptides (Peschel et al., 1999
; Cao & Helmann, 2004
).
The DNA array and real-time RT-PCR analyses revealed that not only the ECF sigma factors but also TCSs have a major role in sensing antimicrobial peptides. Most importantly, LL-37 induced the genes of three ABC-type transporters; yxdLM was induced strongly (about 700-fold) and its close homologues yvcRS and bceAB were induced moderately (about sixfold). All these ABC-transporter genes are regulated by TCSs. The TCSs are encoded by genes in the immediate upstream regions of the ABC transporter genes, as evidenced by the lack/decrease of the induction in TCS mutants (Mascher et al., 2003; Ohki et al., 2003b
; this study) or demonstrated by primer extension and DNase protection experiments (Pascale et al., 2004
). In this study, the results suggested some low-level cross-talk between the three TCSs.
It has been shown that the expression of bceAB is induced more than 200-fold by bacitracin via BceRS TCS-mediated signalling (Mascher et al., 2003) and that the BceAB ABC transporter has a role in bacitracin resistance (Ohki et al., 2003b
). In contrast, the YxdLM and YvcRS ABC transporters are of unknown function. These TCSs may be involved in sensing conditions inside the cell membrane (Mascher et al., 2003
).
This study demonstrates for the first time an activator of the YxdJK TCS: LL-37. Our results also indicate that Triton X-100 and PLL do not activate YxdJK, suggesting distinctly different modes of action for LL-37 and detergents/detergent-like molecules, in contrast to what has been claimed (Oren et al., 1999). LL-37 is most probably a pore-forming peptide (Henzler Wildman et al., 2003
), and the penetration of this amphipathic molecule into the membrane is probably crucial for the activation of the YxdJK TCS. The very short extracytoplasmic loop of the YxdK sensor is consistent with the conclusion that YxdJK senses signals inside the membrane, possibly by direct interaction with LL-37, and not signals on the membrane surface or cell wall, or membrane disturbance as such.
PG-1 did not activate YxdJK either, being a pore-forming peptide (Yang et al., 2000), suggesting that the pore formation may not be required for the activation of YxdJK. It may be essential to YxdJK activation that a peptide interacts directly and appropriately with the YxdK sensor in the membrane. In contrast, the LiaRS (YvqCE) TCS, which regulates liaIHG expression (Mascher et al., 2004
), was strongly activated by both peptides (LL-37 and PG-1) as well as by Triton X-100. Several other stress treatments, such as alkaline shock (Wiegert et al., 2001
), vancomycin (Cao et al., 2002b
) and secretion stress (H.-L. Hyyryläinen and others, unpublished results) also activate LiaRS (YvqCE). This further confirms that YxdJK senses a narrow range of signals or peptides, while LiaRS broadly senses various stress conditions. Interestingly, however, PLL did not activate LiaRS. These peptide ligands of known structure with differences in their specificity give an excellent future opportunity to study the structurefunction relationships of these TCSs.
In addition to yxdLM and liaIH (yvqIH), some other genes of unknown function were also strongly induced by cationic antimicrobial peptides (DNA array). These included yrhH, which was induced by both LL-37 and PLL. The yrhH gene encodes a putative methyltransferase. yhcG was strongly upregulated by LL-37 (14·7-fold) and enhanced expression levels of several other genes of the yhc operon were also observed. yhcG encodes an ABC-transporter ATP-binding protein. The yhcH gene, which was induced 4·6-fold, also encodes a putative (second) ABC-transporter ATP-binding protein, and the yhcI gene, which was induced 5·1-fold, encodes a putative ABC-transporter permease homologous with bacitracin permeases. The putative roles of these ABC transporters in the removal of LL-37 from cells should be studied in the future.
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ACKNOWLEDGEMENTS |
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Received 10 November 2004;
revised 1 February 2005;
accepted 3 February 2005.
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