Induction of b-lactamase in Enterobacter cloacae

Transcription

1 S42 Induction of b-lactamase in Enterobacter cloacae Bernd Wiedemann, Helgard Dietz, and Dieter Pfeifle From Pharmazeutische Mikrobiologie, University of Bonn, Bonn, Germany b-lactamase induction in Enterobacter cloacae, which is linked to peptidoglycan recycling, was investigated with use of high-performance liquid chromatography of cell wall fragments in genetically defined cells of Escherichia coli. After treatment of cells with b-lactams, we detected in the periplasm an increase of D-tripeptide (N-acetylglucosaminyl-1,6 anhydro N-acetylmuramyl-Lalanyl-D-glutamyl-meso-diaminopimelic acid), D-tetrapeptide (N-acetylglucosaminyl-1,6 anhydro N-acetylmuramyl-L-alanyl-D-glutamyl-meso-diaminopimelic acid-d-alanine), and a yet-unknown anhydromuropeptide. We identified this anhydromuramylpeptide by fast atom bombardment mass spectrometry as anhydromuramyl-pentapeptide. The amount of these molecules did not alter after treatment with cell wall active non b-lactams. The transmembrane protein AmpG transports not only D-tripeptide but also D-pentapeptide into the cell. In the cytoplasm these molecules are degraded into the corresponding monosaccharide peptides M-tripeptide (N-acetylmuramyl-L-alanyl- D-glutamyl-meso-diaminopimelic acid) and M-pentapeptide (N-acetylmuramyl-L-alanyl-D-glutamyl-meso-diaminopimelic acid-d-alanine-d-alanine). These findings indicate that besides M-tripeptide and D-tripeptide, probably M-tetrapeptide, D-tetrapeptide, M-pentapeptide, and D-pentapeptide are also signal muropeptides for b-lactamase induction. The emergence of resistance during a course of antimicrobial Recent studies demonstrated that AmpD, a cytosolic protein, therapy can be a serious therapeutic problem. In general, this functions as a negative regulator of AmpC expression [9 11], is an extremely rare event. However, in a study of Enterobacter as an N-acetyl-anhydromuramyl-L-alanine amidase [12]. Mutations cloacae infections, even with combination therapy with an in the ampd gene result in constitutive b-lactamase overcloacae aminoglycoside and a third-generation cephalosporin, resis- production. Simultaneously, large quantities of anhydro N-acetylmuramyl-L-alanyl-D-glutamyl-meso-diaminopimelic tance to all b-lactams except the carbapenems developed in acid nearly 50% of all patients investigated [1]. This is a result of (M-tripeptide) accumulate in the cytoplasm of the ampd-negative the specific complex regulation of the chromosomally mediated strain JRG, indicating that this compound is an AmpRthe cephalosporinase. Although it is now generally accepted that activating ligand [7]. Moreover, this molecule is transported the induction itself is not a real clinical problem, the regulation into the periplasm [13]. of the b-lactamase production is of great interest because of AmpG, a transmembrane protein, is required for b-lactamase the high mutation rate of [2], which can be easily induction [14]. This protein acts as a permease for the muropep- selected for and can result in an overproduction of b-lactamase. tide, N-acetylglucosaminyl-1,6 anhydro N-acetylmuramyl-Lalanyl-D-glutamyl-meso-diaminopimelic In these mutants the ampd gene is frequently modified. acid (D-tripeptide), as Nearly all Enterobacteriaceae and Pseudomonas aeruginosa a precursor molecule for M-tripeptide [7]. strains produce a chromosomal AmpC b-lactamase, which in In this article we study the impact of antibiotics on the most cases can be induced by b-lactam antibiotics [3 6]. Fur- formation of anhydromuropeptides and analyze whether AmpG thermore, this process is linked to the peptidoglycan recycling, transports other anhydromuramyl-peptides in addition to D- which is a general feature in eubacteria [7]. tripeptide into the cytoplasm. Inhibition of the penicillin-binding proteins involved in the peptidoglycan metabolism with b-lactams leads to increased degradation of the murein sacculus. Thus, degradation products Materials and Methods accumulate in the periplasm [8]. They are transported into the Bacteria, Plasmids, and Culture Conditions cytoplasm, where they can act as an inducer, converting the transcriptional regulator AmpR into an activator of b-lactamase The Escherichia coli K12 strains used in this study were expression [9]. JRG582 and its derivative, JRG JRG582 is a knock-out mutant for the ampd and ampe genes [15]. JRG58201 carries an aminoglycoside phosphotransferase cassette inserted in the ampg gene [16]. Grant support: Deutsche Forschungsgemeinschaft (WI 361/15-2). Reprints or correspondence: Dr. Bernd Wiedemann, Pharmazeutische Mikrobiologie, Plasmids pbp21 and pbp22 are derivatives of puc-19 University of Bonn, Meckenheimer Allee 168, Bonn, Ger- (pbp19; [17]) carrying kanamycin resistance and the E. cloacae many. ampe gene and the ampd and ampe genes, respectively. Plas- Clinical Infectious Diseases 1998;27(Suppl 1):S42 7 mid pbp21 was constructed as follows. The 1.1-kb SphI/XbaI 1998 by the Infectious Diseases Society of America. All rights reserved /98/ $03.00 fragment of the E. cloacae 14 wild-type strain carrying the

2 CID 1998;27 (Suppl 1) Induction of b-lactamase S43 ampe gene was obtained by PCR. The XbaI site of this PCR phate (ph, 4.95) containing 15% methanol (buffer B) over 100 fragment was blunt-ended by the DNA polymerase T4, and the minutes at 38.5 C, with a flow rate of 0.5 ml/min. resulting fragment was inserted into the unique SmaI and SphI sites of pbp19. pbp22 was constructed by subcloning the 600- bp BamHI/EcoRI fragment of pbp 21 into pbp20 [10]. HPLC Analyses of Imipenem-Treated Cells Recombinant DNA techniques and transformation of DNA The prepared anhydromuropeptides of the ampg- and ampdwere performed as described in [18]. The E. coli strains were negative strain JRG58201 were separated by a gradient com- grown at 37 C in M9 medium supplemented with glucose posed of two linear gradients. The first part of this gradient (0.2%), casamino acids (0.1%), thiamine (1 mg/ml), uracil (50 was the one described above. The second part was a linear mg/ml), nicotinamide (5 mg/ml), and MgSO 4 (1 mm). gradient of the same buffers, from 36% to 100% of buffer B, over 25 minutes. Preparation of Hot Water Extracts After 0.25-mL fractions of the column effluent were collected, their radioactivity was determined by liquid scintillation This method is based on a previously described method [7]. counting. Peak areas and the area percentage for every peak A 1l Erlenmeyer flask containing 200 ml of minimal medium were calculated with the Microsoft (Seattle) Excel program. M9 was inoculated with 2 ml of overnight culture growth and grown to mid-log phase (optical density 456 Å 0.5), with the N-acetylmuramyl-L-alanine Amidase Assay addition of 15 mci of ( 3 H) diaminopimelic acid (0.35 Ci/mmol; Amersham, Braunschweig). Cells were harvested by centrifudissolved The lyophilized hot water extract of JRG582/pBP21 was gation at 4 C and washed once with one-half volume of 10- in 1 mm of sodium phosphate buffer (ph, 6.8) con- mm Tris-HCL (ph, 8.0). The bacteria were suspended in 20 taining 0.02% sodium azide. To one-half of this, the MalE- ml of boiling water and heated at 100 C for 20 minutes. After AmpD fusion protein (about 1 mg; [10]) was added, and then removal of particulate matter by centrifugation at 16,000g for we incubated the samples for 12 hours at 30 C. 10 minutes, the supernatant (hot water extract) was recovered and lyophilized. Results Methanol Precipitation A rapid method for the removal of proteins is methanol precipitation [13]. The lyophilized samples of the hot water extracts and the culture media were resuspended in 400 ml of water and chilled on ice for 15 minutes. Then, ice cold methanol was added, and after an additional 15 minutes on ice the samples were centrifuged at 10,000g for 10 minutes to yield a clear supernatant. Accumulation of Two Anhydromuropeptides in ampd-negative Cells Hot water extracts of JRG582/pBP21 (ampd-negative) and JRG582/pBP22 (ampd-positive) were analyzed by HPLC with use of the described linear gradient. In the ampd-negative strain, two more peaks than in the ampd-positive strain were detected (figure 1; table 1), with an area percentage of 65 (peak High-Performance Liquid Chromatography Analyses After methanol precipitation, the samples were lyophylized and suspended in 100 ml of water. The muropeptides were then reduced with sodium borohydride [19]. Prior to injection, the samples were centrifuged for 10 minutes at 6,000g. Highperformance liquid chromatography (HPLC) was performed with a Pharmacia (Piscataway, NJ) gradient system consisting of a model 2152 LC controller, model 2150 pumps, and model ultragrade mixer-driver. A C18 Hypersil ODS reversephase column ( mm; 3-mm particle size) from Bischoff (Leonberg, Germany), together with a guard column containing mbondapack C18 Corasil (Waters, Germany), was used to separate the muropeptides. Optimal separation of muropeptide-monomers with this HPLC system was achieved by a linear gradient from methanolfree 50-mM sodium phosphate (ph, 4.31) containing 0.8- mg/l sodium azide (buffer A) to 35% 75-mM sodium phos- Figure 1. High-performance liquid chromatography (HPLC) analysis of hot water extracts from JRG582/pBP22 (ampd-positive) and JRG582/pBP21 (ampd-negative). The chromatogram of the ampd- negative strain shows two additional peaks, the M-tripeptide and the M-pentapeptide.

3 S44 Wiedemann, Dietz, and Pfeifle CID 1998;27 (Suppl 1) Table 1. Amount of M-tripeptide, M-pentapeptide, D-tripeptide, and D-tetrapeptide in the hot water extract and the culture medium of JRG582 (ampd-negative). Source M-tripeptide D-tripeptide M-pentapeptide D-tetrapeptide Hot water extract* Culture medium NOTE. The values indicate the area percentage of the corresponding peak in the HPLC chromatogram. * These values resulted from double extracts. These values resulted from one analysis. 1) and 5.6 (peak 2). Peak 1 and 2 were identified by mass peptide can be transported into the cytoplasm, although the D- spectrometry as the M-tripeptide. Furthermore, we could demonstrate that the corresponding compounds were substrates for of ampg-positive cells. pentapeptide could not be detected in the cytoplasmic fractions AmpD. The peaks of interest disappeared upon AmpD treatment. JRG58201 contains four times as much D-tetrapeptide as D- The culture medium of the ampd- and ampg-negative strain tripeptide [1], which reflects the normal distribution between these two muropeptides in the cell wall [7]. The same relative The Role of AmpG in Peptidoglycan Recycling amounts of these muropeptides are present in the hot water If M-pentapeptide as well as M-tripeptide is formed in the extract of JRG58201 (table 2). cytoplasm, AmpG would transport both precursor molecules into the cytoplasm. To investigate this hypothesis, we used an Effect of Cell Wall Active Antibiotics ampd- and ampg-negative strain (JRG58201). Since neither In contrast to b-lactams, other cell wall active antibiotics M-tripeptide nor M-pentapeptide is generated in this strain like D-cycloserine and vancomycin do not induce the produc- (figure 2), we proved that in fact AmpG is also the permease tion of b-lactamase (data not shown). Therefore, we tried to for the precursor molecule of M-pentapeptide. Thus, AmpG is figure out if b-lactams like imipenem, cefoxitin, and cefotaxnecessary for the formation of M-tripeptide as well as M- ime change the peptidoglycan metabolism in a different way pentapeptide in the cytoplasm. in comparison with D-cycloserine and vancomycin. To investigate this further, we fractionated the cells from the We treated the bacteria with an antibiotic concentration of ampg-positive strain JRG582 into periplasmic and cytoplasmic MIC to prevent lysis of the cells. With use of an ampdfractions by treatment with lysozyme and EDTA [20]. Formapositive strain, the monosaccharide-peptides as well as the dition of spheroplasts could be seen by osmotic sensitivity and saccharide-peptides are under limit of detection and hardly conversion into spheres after dilution of an aliquot of the susdetectable upon imipenem treatment. Therefore, we analyzed pension (1:5) in distilled water [21]. Spheroplasts contained the ampd- and ampg-negative strain JRG D-tetrapeptide [8], which again shows that this anhydromuro- We separated the muropeptides by a gradient composed of two linear gradients (see Materials and Methods). As can be seen in table 3, the amount of D-tripeptide and D-tetrapeptide increased only upon treatment with b-lactams. Since we analyzed an ampg-negative strain, D-tripeptide as well as D-tetrapeptide must be of periplasmic origin. Furthermore, in contrast to D-cycloserine and vancomycin, another peak appears after treatment with b-lactams (D-pentapeptide; figure 3, table 3). The amount of this peak is much higher with imipenem and cefoxitin strong inducers of b-lactamase production than with cefotaxime, a weak inducer. Since this peak disappears after incubation with AmpD overnight at 30 C, it is also a substrate an anhydromuropeptide of AmpD. We identified this compound by fast-atom bombardment mass spectroscopy as the anhydromuramylpentapeptide [8]. Figure 2. HPLC analysis of the hot water extracts from JRG582 Discussion Murein recycling in E. coli can use a pathway [7] that in- (ampd-negative) and JRG58201 (ampd- and ampg-negative). volves the products of the genes ampd [11] and ampg [13].

4 CID 1998;27 (Suppl 1) Induction of b-lactamase S45 Table 2. Amount of M-tripeptide, M-pentapeptide, D-tripeptide, and D-tetrapeptide in the hot water extract and the culture medium of JRG58201 (ampd- and ampg-negative). Source M-tripeptide D-tripeptide M-pentapeptide D-tetrapeptide Hot water extract* 0.4 (0.3) 0.5 (0.2) (0.15) Culture medium NOTE. The values indicate the area percentage of the corresponding peak in the HPLC chromatogram; 0 Å under limit detection. * These values resulted from six extracts. SD is in parentheses. These values resulted from one analysis. AmpD and AmpG play an important role also in b-lactamase 4 5 times as much D-tetrapeptide as D-tripeptide, AmpG should induction. Recently, it could be shown that the cytosolic protein transport D-tetrapeptide into the cell too. However, we could not AmpD is an N-acetyl-anhydromuramyl-L-alanine amidase [12]. detect anhydromuramyl-tetrapeptide in the cytoplasm. Therefore, it is assumed that AmpD exerts its negative effect It seems that AmpG is not specific for one anhydromuropepon AmpC expression by hydrolyzing anhydromuropeptides, tide. AmpG is probably also the permease for other anhydromuropeptides positive signal molecules for b-lactamase induction. Furthermore, released from the cell wall. These anhydromuropeppositive M-tripeptide is accumulated in the cytoplasm of an ampd tides might also be recycled. This is in agreement with the fact mutant [7]. that AmpD is an amidase for many anhydromuropeptides [12]. Here we demonstrate that in an ampd mutant, not only M- We found that b-lactams, in contrast to other cell wall tripeptide accumulates, but also, to a smaller extent, another active non-b-lactam antibiotics like D-cycloserine and vanco- anhydromuropeptide, M-pentapeptide. This has not been dem- mycin, lead to an increased release of D-tripeptide and D- onstrated by previous investigators (table 1) [7]. Furthermore, tetrapeptide from the peptidoglycan sacculus (table 3, figure we show that in an ampd-ampg double mutant, both anhy- 3). Furthermore, another anhydromuropeptide emerges only dromuropeptides are absent. Thus, both anhydromuropeptides upon treatment with b-lactams. We identified this anhydromur- are formed only in the cytoplasm. amylpeptide as anhydromuramyl-pentapeptide [8]. The amount Since M-pentapeptide also is formed only in the cytoplasm, of this anhydromuropeptide is much higher with imipenem and AmpG must be the permease for the precursor molecule D- cefoxitin, strong inducers of b-lactamase production, than with pentapeptide (figure 4). By forming spheroplasts of the ampgpositive cefotaxime, a weak inducer (table 3). These findings indicate strain JRG582, we showed that D-pentapeptide is present that this anhydromuramyl-pentapeptide perhaps plays a pre- in the cytoplasm, in contrast to the ampg-negative strain dominant role in b-lactamase induction, as do D-tripeptide and JRG Since in exponentially growing cells the wall contains D-tetrapeptide [8]. Table 3. Influence of cell wall active antibiotics on the peptidoglycan metabolism of JRG58201 (ampd- and ampg-negative). Value in experiments 1 and 2 D-tripeptide D-tetrapeptide D-pentapeptide Antibiotic* (mg/ml) 1 2 Mean 1 2 Mean 1 2 Mean None D-cycloserine (8) Vancomycin (16) Cefotaxime (0.25) Cefoxitin (2) Imipenem (0.25) NOTE. In the first line (data without antibiotics), the values are linked as 1. The absolute values are D-tripeptide, 0.55 and 1.1 (mean Å 0.83); D-tetrapeptide, 2.4 and 7.4 (mean Å 4.9); and D-pentapeptide, 0 and * The antibiotic was added for 25 minutes after the cells reached an OD 465 of 0.5. The concentration of each antibiotic was half the MIC. The values for the anhydromuropeptides indicate the area of the corresponding peak in the HPLC chromatogram relative to the untreated cells.

5 S46 Wiedemann, Dietz, and Pfeifle CID 1998;27 (Suppl 1) The results presented in this paper led us to a modified model of the recycling of anhydromuropeptides and their role in the regulation of class I chromosomal b-lactamase (figure 4, based on [7]). D-tripeptide, D-tetrapeptide, and D-pentapeptide accumulate in the periplasm upon degradation of the murein sacculus. D-tripeptide and D-pentapeptide are then transported via AmpG into the cytoplasm, where they are converted into the corresponding monosaccharide-peptides by the b-n-acetylglucosaminidase. Probably all four anhydromuropeptides can induce b-lactamase production. In the presence of AmpD, they can be further degraded. The resulting oligopeptides can then be recycled [22]. The role of the third anhydromuropeptide, the anhydromuramyl-pentapeptide, is probably specific for the Figure 3. HPLC analysis of the hot water extract from JRG58201 induction of b-lactamase, but this has to be investigated fur- (ampd- and ampg-negative). The effect of imipenem (0.25 mg/ml) ther [8]. is shown. Values on the y axis are disintegrations per minute. Probably all three anhydromuropeptides serve as a signal for b-lactamase induction, since their amount in the periplasm is increased only upon treatment with b-lactams (table 3). D- cycloserine and vancomycin do not change the amount of these three anhydromuropeptides in the periplasm and simultaneously do not induce b-lactamase production. Acknowledgment The authors thank Prof. J.-V. Höltje for his active support and discussion. References 1. Füssle R, Biscoping J, Behr R, Sziegoleit A. Development of resistance by Enterobacter cloacae during therapy of pulmonary infections in intensive care patients. Clin Invest 1994;72: Livermore DM. Clinical signification of beta lactamase induction and stable derepression in gram-negative rods. Eur J Clin Microbiol 1987; 6: Kopp U, Wiedemann B. Induktion der Beta-Lactamase in Enterobacter cloacae. Chemotherapy Journal 1993;4: Lindberg F, Normark S. Common mechanism of ampc b-lactamase induction in enterobacteria: regulation of the cloned Enterobacter cloacae P99 b-lactamase gene. J Bacteriol 1987;169: Lodge JM, Piddock LJV. The control of class 1 lactamase expression in Enterobacteriaceae and Pseudomonas aeroginosa. J Antimicrob Chemother 1991;28: Normark S, Bartowsky E, Erickson J, Jacobs C, Lindberg F. Mechanism of chromosomal b-lactamase induction in gram-negative bacteria. In: Ghuysen J-M, Hakenberg R, eds. Bacterial cell wall. Amsterdam: Elsevier, 1994: Jacobs C, Huang L, Bartowsky E, Normark S, Park JT. Bacterial cell wall recycling provides cytosolic muropeptides as effector for b-lactamase induction. EMBO 1994;13: Figure 4. Proposed model of the two pathways for recycling muro- 8. Dietz H, Pfeifle D, Wiedemann B. The signale molecule for b-lactamase peptides and their roles in regulation of class I chromosomal b- induction in Enterobacter cloacae is the anhydromuramyl-pentapeptide. lactamase ( Å GlucNAc; Å anhmurnac; Å MurNAc; O Å Antimicrob Agents Chemother 1997;41: Ala; Å Glu; Å DAP; DAP Å D-tri-, D-penta-, M-tri-, and M- 9. Lindquist S, Galleni M, Lindberg F, Normark S. Signaling proteins in pentapeptide; OM Å outer membrane; IM Å inner membrane; PG Å enterobacterial AmpC b-lactamase regulation. Mol Microbiol 1989;3: peptidoglycan; OPP Å oligopeptide permease; UDP Å uridinediphos phate). Murein is degraded in the periplasm. The resulting muropeptides, 10. Kopp U, Wiedemann B, Lindquist S, Normark S. Sequences of wild- D-tripeptide and D-tetrapeptide, are transported via AmpG into type and mutant ampd genes of Citrobacter freundii and Enterobacter the cytoplasm, where they are hydrolyzed by AmpD. The resulting cloacae. Antimicrob Agents Chemother 1993;37: tripeptide can be reused for murein synthesis. Alternatively, the N- 11. Lindberg F, Lindquist S, Normark S. Inactivation of the ampd gene causes acetylglucosamine can be removed first and the monosaccharide-peptides semiconstitutive overproduction of the inducible Citrobacter freundii degraded by AmpD. In an ampd-negative strain, the M-tripep- b-lactamase. J Bacteriol 1987;169: tide can also be transported out of the cell. The disaccharide- as well 12. Höltje JV, Kopp U, Ursinus A, Wiedemann B. The negative regulator of as the monosaccharide-peptides can act as inducers for b-lactamase b-lactamase induction AmpD is a N-acetyl-anhydromuramyl-L-alanine expression by binding to AmpR. amidase. FEMS Microbiol Lett 1994;122:

6 CID 1998;27 (Suppl 1) Induction of b-lactamase S Dietz H, Pfeifle D, Wiedemann B. Location of N-acetylmuramyl-L-alanylfusions 17. Broome-Smith J, Spratt BG. A vector for the construction of translational D-glutamyl meso diaminopimelic acid, presumed signal molecule for b- to TEM beta-lactamase and the analysis of protein export signals lactamase induction in the bacterial cell. Antimicrob Agents Chemother and membrane protein topology. Gene 1986;49: ;40: Maniatis T, Fritsch EF, Sambrook J. Molecular cloning: a laboratory man- 14. Korfmann G, Sanders CC. ampg is essential for high-level expression of ual. Cold Spring Harbour, New York: Laboratory Press, Glauner B, Höltje J-V, Schwarz U. The composition of the murein of AmpC b-lactamase in Enterobacter cloacae. Antimicrob Agents Che- Escherichia coli. J Biol Chem 1988;263: mother 1989;33: Spratt BG. Properties of the penicillin-binding proteins of Escherichia coli 15. Guest JR, Stephens PE. Molekular cloning of the pyruvate dehydrogenase K 12 Eur J Biochem 1977;72: complex genes of Escherichia coli. J Gen Microbiol 1980;121: 21. Birdsell DC, Cota-Robles EH. Production and ultrastructure of lysozyme and ethylenediaminetetraacetate-lysozyme spheroblasts of Escherichia 16. Lindquist S, Weston-Hafer K, Schmidt H, et al. AmpG, a signal transducer coli. J Bacteriol 1967;93: in chromosomal b-lactamase induction. Mol Microbiol 1993;9: 22. Goodell EW. Recycling of murein by Escherichia coli. J Bacteriol 1985; :