Purification of an intracellular bacterial β-lactam by affinity chromatography

Journal o f Mtcrobtologwal Methods 12 (1990) 8 3 - 89 Elsevier

83

MIMET 00389

Purification of an intracellular bacterial fl-lactam by affinity chromatography David G. Allison Pharmacy Department, Manchester Umverstty, Manchester, UK (Received 9 April 1990; revision received 6 June 1990; accepted 6 June 1990)

Summary The purification of naturally occurring B-lactams by conventional separation techniques is often complex and time consuming. The method described here permits the concentration and rapid isolation of B-lactams from large sample volumes by affinity chromatography. Using aztreonam as the hgand, the isolation of an intracellular monobactam from a strain of Pseudomonas aerugmosa is reported The method employed is quick and simple to run and as such increases the diversity of bacterial samples that can be screened for novel/3-1actam activity.

Key words: Affinity chromatography; Intracellular; Monobactam; Pseudomonas aerugmosa

Introduction

/3-1actam antibiotics comprise one of the major chemotherapeutic means for the control of bacterial infections. The widespread use of ~-lactams, however, has led to an increased incidence of bacterial resistance to such treatments. Consequently, a greater need to isolate and develop novel antibiotics has arisen. Traditionally, high throughput screening methods have been employed by the Pharmaceutical Industry to detect natural/3-1actams in fungal and actinomycete culture broths [1]. Indeed, such studies were based on the precept that a bicyclic fused-ring system was necessary for significant antibacterial activity. Additional support for this theory was provided with the identification of the monocyclic nocardicins which exhibit insignificant antimicrobial activity [2]. An important development of natural product research was the discovery of bacterially produced ~-lactams. These include carbapenems [3], cephalosporins [4] and monocyclic ~-lactams (monobactams) [5, 6]. The aptly named monobactams constitute a structurally novel class of/5-1actam antibiotics, which, with side-chain variation, are potently inhibitory to pathogenic bacteria. Synthesis of such compounds has subsequently been shown to occur in a number of Correspondence to: D.G. Allison, Pharmacy Department, Manchester University, Oxford Road, Manchester MI3 9PL, UK.

0167-7012/90/$ 3.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

84 dlstlnct species, including Flavobacterium, Agrobactenum, Chromobactenum, Flextbacter and Pseudomonas [7]. Given their considerable potential as novel chemotherapeutic agents, of immense value would be the development of screening and isolation procedure which would generate high yields of purified monobactam from original sources. Currently, purification of ~-lactams by conventional techniques is extremely complex, time consuming and requires large quantities of culture broth [7, 8]. There is a need therefore to develop a rapid and simple method of isolation. This report describes that the use of aztreonam-sepharose affinity columns fulfils this need. Furthermore, the ~-lactam activities described are all intracellular bacterial products and as such provide a novel source of activity. Materials and Methods

Bacteria andgrowth conditions. All the strains listed in Table 1 were obtained from the culture collection at ICI Pharmaceuticals, Cheshire, UK. Cultures were grown at 30 °C until the onset o f stationary phase ( -~ 16. h - l; E470 10.0) in GN broth (. 11: yeast extract 5 g, peptone 5 g, beef extract 5 g, sucrose 5 g) prior to harvesting (20 min, 10000 × g) and sample preparation. Unless otherwise stated, all preparative steps were performed at 4 °C. Preparation ofintracellular material. Bacterial cell pellets were washed three times in 0.85% NaCl, resuspended in distilled water (20 mg dry wt. ml -l) and lysed by sonication. After removal of cell debris by ultracentrifugation (40 min, 100000×g), the supernatant (intracellular) material was concentrated 50-fold by lyophilization and subsequent resuspension in distilled water. Affmtty chromatography. 1 g cyanogen-bromide-activated Sepharose 4B was washed thoroughly with distilled water (1 l) by vacuum filtration before immediately resuspending in 10 ml 0.1 M bicarbonate buffer (pH 10.3) containing 2.5 mM aztreoham. After allowing the coupling reaction to continue for 48 h, the liganded resin was washed free of any unbound aztreonam with distilled water. Packed columns (7 × 0.5 cm) were washed and equilibrated with 10 column volumes of 0.04 M phosphate buffer (pH 6.5) before applying 300/A of the reconstituted intracellular concentrate and eluting at a linear flow rate of 5 c m . h -~. Fraction volumes were 850 #l. When all proteinaceous material had passed through the column, elution was halted for 16 h. On resumption, ~-lactam-containing fractions were collected. Endogenous /3-1actamase bound to the column was then eluted by addition of 0.5 M KCl to the loading buffer. HPLC/Diode-array analysis. ~lactam-containing fractions were pooled, concentrated five-fold, then subjected to HPLC/Diode-array analysis. Aliquots (500 gl) were injected onto a reverse-phase column (Lichrosorb 10rpl8) using 0.1 M ammonium bicarbonate buffer (pH 6.7), methanol acting as the organic modifier, l-ml fractions were collected at a flow rate of 2 ml.min -1. 13-lactamase homogeneity. Purity of the eluted/3-1actamase enzyme was confirmed

85 by sodium-dodecyl-sulphate (SDS) polyacrylamide gel electrophoresis as previously described [9]. Proteins were visualized by Coomassie Blue staining.

Assay for ~-lactam activity. Samples were incubated in the presence of Bacillus licheniformis as previously described [10]. Induction of B. licheniformis ~3-1actamase enzyme was assayed using the chromogenic cephalosporin nitrocefin (1 mg-ml-I), the results expressed qualitatively in terms of a 1 : 1 plate dilution assay.

~-lactamase assay. 13-1actamase was assayed as previously described [11]. Results and Discussion

It is becoming well recognized that a wide range of bacteria are capable of Blactamase production. Indeed, such findings are qualified in Table 1. However, what is less clear, is the incidence of 13-1actam synthesis in bacteria. The results depicted in Table 1 clearly demonstrate, that for every strain assayed,/3-1actam synthesis occurs. As such, the B-lactam activity was found only as an intracellular product and not as an extracellular metabolite. Since the assay system employed is extremely sensitive and highly specific, detecting levels as low as 0.1 pg-m1-1, it is assumed that no extracellular ~3-1actam secretion occurs. Although ~-lactam synthesis has previously been exploited, they along with other classes of antibiotics, have not been ascribed any clearly recognizable role in the producer organisms. Suggestions have included differentiation effectors [12], regulatory molecules [13] and sporulation factors [14]. Until more is known about the control and regulation of B-lactam biosynthesis, the debate regarding TABLE 1 RELATIVE B-LACTAM A N D B - L A C T A M A S E ACTIVITY D E T E C T E D AS E I T H E R I N T R A C E L L U L A R OR E X T R A C E L L U L A R P R O D U C T S . ACTIVITIES W E R E D E T E C T E D AS DESCRIBED. P E N I C I L L I N G ( l / ~ g . m l - l ) W A S ASSAYED TO P R O V I D E A N I N D I C A T I O N OF S T A N D A R D / 3 L A C T A M ACTIVITY Intracellular ~-lactam Bacdlus cereus 2230 Bacdlus subtdus 12321 Mzcrococcus luteus 7016 Serratla marcescens 10152 P s e u d o m o n a s aerugmosa 13234 Enterobact er cloacae 1719 Staphylococcus aureus 601065 Staphylococcus aureus 601074 Escher~ch~a cob 2197 Escherwhta coil 2203 Streptococcus faecahs 60119 Flavobacterium lutescens

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86 possible functional roles remains very much alive. O f the bacterial isolates screened (Table 1), P. aeruginosa strain ACC 13234 produced the greatest amount of B-lactam actwity. Subsequent experiments were performed with the objective of ~solatmg and purifying this activity in a quick and reliable manner. Since endogenous/3-1actamase appeared to be associated with the ~-lactam activity, a prerequisite was the removal of any such enzymic activity. Initial experiments indicated that m a x i m u m separation in a non-destructive manner was achieved by affinity chromatography. The widespread use of affinity chromatography reflects its success in obtaining rapid separations which are often time consuming using conventional methods. Moreover, the technique provides opportunities for the isolation of substances according to their biological function, differing radically from conventional chromatographic procedures which depend on gross physical and chemical differences between molecules. Lysates of P.. aeruginosa concentrated 50-fold, containing a ratio of 15 : 7 wells of Blactam : ~-lactamase activity, respectively, were applied to a Sepharose 4B affinity chromatography column in which aztreonam was the ligand. No spacer arm was used on this matrix. Aztreonam was chosen as the ligand as it had previously been shown to possess a high enzyme affinity but low hydrolysis rate (data not shown). Studies on the capacity of the resin to bind B-lactamase indicated that I g of the affinity resin was capable of binding 87/zg. This value was calculated from known amounts of enzyme added to the resin before activity was detected in the eluate. Fig. 1 illustrates the elution profile of/~-lactam activity following application of P s e u d o m o n a s intracellular material to the column. Repeat experiments showed conclusively that enhanced Blactam elution only occurred after incubation for 16 h in the presence of a mobile phase containing 0.04 M phosphate buffer. Increased B-lactam elution did not occur following longer periods of incubation. A possible explanation may be the existence of a putative B-lactam/~-lactamase complex, which in the continued presence of phosphate buffer is allowed to dissociate. This would enable the B-lactamase enzyme to associate with the immobilised aztreonam, to which it demonstrates a greater affinity, and the/3-1actam moiety to be subsequently eluted. Further studies need to be per0.5 ~16 0.4

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Fig. 1. Chromatography of cell-bound /3-1actam ([]) from P. aerugmosa using an aztreonam-affimty column. (i) Elution halted for 16 h to allow complete dissociation of a possible/3-1actam//3-1actamasecomplex. (iQEndogenous/~-lactamase( m) eluted by addition of 0.5 M KCI. Protein was measured by absorbance at 280 nm.

87

formed to confirm this theory. Released/3-1actam activity was not due to synthesis by viable cells attached to the column, however, since each fraction and a sample of the affinity matrix recovered from the column after use were assayed for bacterial growth by plating out onto the surface of pre-dried plates of nutrient agar and incubating at 37 °C for 24 h. Furthermore, preliminary studies (data not shown) indicate that the endogenous/3-1actam is bound, but not hydrolysed, by the cellular ~-lactamase. Elution of the bound/3-1actamase was achieved by simply increasing the ionic strength of the loading buffer by the addition of 0.5 M KCI. Further increases in salt concentration resulted in no additional elution of the/3-1actamase (data not shown). SDS polyacrylamide gel electrophoresis indicated the eluted ~-lactamase to have a molecular size of --~34 kDa [15]. Purity of the/3-1actam recovered from the Pseudornonas lysate was assessed by HPLC/Diode-array analysis (Fig. 2). A single, uncontaminated active fraction was identified, with a maximum absorption peak at 230 nm. Furthermore, proton NMR spectrographic analysis indicated the presence of a monobactam. The structure suggested by the available physical data was of a basic unsubstituted 3-aminomonobactamic acid nucleus (Fig. 3). Control experiments demonstrated no loss or degradation of the immobilized ligand during sample preparation. Moreover, whilst the isolated monobactam had a retention time of 33 min on the reverse-phase HPLC column (Fig. 2), aztreonam was retained for only 24 min (data not shown). Whilst the currently available naturally occurring monobactams are generally poor antibiotics [8], the simplicity of the Pseudomonas monobactam is such that the azetidinone ring is readily accessible to a variety of substitutions. In this respect, similar II Ill

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Structure of P. aerugmosa lntracellular monobactam.

studies have been conducted on a number of monobactams, resulting in the production of semi-synthetic analogs possessing a high level of antimicrobial activity [8]. The inherent synthetic flexibility of monobactams therefore permits the development of additional clinically significant antibacterial agents. Thus, by increasing the nature and diversity of bacterial samples screened, this potential is further enhanced. Although monobactams have previously been detected in the culture broths o f Pseud o m o n a s species [5-7], this is the first report detailing the presence of a cell-bound activity. The results depicted in Table 1 clearly indicate that intracellular/3-1actam synthesis occurs in many bacteria. Indeed, preliminary evidence suggests the presence of a monobactam in the cytoplasm of S. aureus strain 601065 (Allison, unpubl, data). Affinity chromatography provides a simple and very rapid method for the homogeneous purification of bacterial monobactams. An important application of this particular technique is the ability to concentrate trace amounts of/3-1actam activity present in large sample volumes. In this manner, ~lactams which might otherwise be rejected on the basis of insufficient material, are subsequently eluted from the affinity column as a sharp peak of active material. In addition, this technique has not only the potential for the isolation of monobactams from novel sources, but also for the isolation of specific penicillin-binding proteins. Acknowledgements

Sincere thanks are expressed to R.D. Nolan for invaluable discussions, to P.M. Roberts for excellent technical assistance and to A. Bramwell for critically reading the manuscript.

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89 Wells, J. S (1981) Monocychc B-lactam antibiotics produced by bacteria. Nature (London) 291,489-491. 6 lmada, A., Kitano, K., Kmtaka, K., Murol, M. and Asal, M. (1981) Sulfazacin and isosulfazacm, novel ~-lactam anubloucs of bactertal origin. Nature (London) 289, 590-591. 7 Parker, W.L., O'Sullivan, J. and Sykes, R.B. (1986) Naturally occurring monobactams. Adv. Appl. Mierobiol. 31, 181-205. 8 Sykes, R B. and Bonner, D. P. (1985) Discovery and development of the monobactams. Rev. Infect. Dis 7, Suppl. 4, $579-$593. 9 Anwar, H , Brown, M. R. W., Day, A. and Weller, P H. (1984) Outer membrane antigens of mucold Pseudomonas aerugmosa isolated directly from the sputum of a cystic fibrosis pauent. FEMS Microb~ol Lett. 24, 235-239. 10 Alhson, D.G. and Roberts, P M. (1988) Protoplast formauon and reversion m Actinomadurae. Appl. Mlcroblol. Biotechnol. 28, 580-582 11 Rogers, M. E., Adlard, M. W, Saunders, G and Holt, G. (1985) High-performance hqutd affimty chromatography of~-lactamase J Chromatogr 326, 163-172 12 Krumphanzl, V., Slkyta, B. and Vanek, Z. (1982) Overproducuon of Microbial Metabohtes. Academic Press, London. 13 Hutter, R. (1982) Design of culture medm capable of provokmg wide gene expression In- Bioactwe M~crobial Products (Bu'Lock, J D., N~sbet, L. J. and Wmstanley, D. J, eds.), pp. 37-50, Academic Press, New York. 14 Demam, A. L. (1974) How do antibiotic-producing mlcroorgamsms avmd smclde Ann. N.Y. Acad Scl. 235, 601-612. 15 Alhson, D. G. and Nolan, R. D. (1986) Separation of B-lactamase from/3-1actam by affimty chromatography XIV lnt Cong. Mlcroblol. Abs. PG10-10.