Selective Toxicity Alteration of a Highly Toxic Antibiotic by an Enzyme Catalyzing Antibiotic Modification

Actinomycetologica (2008) 22:50–55 Copyright 2008 The Society for Actinomycetes Japan VOL. 22, NO. 2 Award Lecture Selective toxicity alteration of a highly toxic antibiotic by an enzyme catalyzing antibiotic modiﬁcation

Yoshimitsu Hamano Department of Bioscience, Fukui Prefectural University, 4-1-1 Matsuoka-Kenjojima, Eiheiji-cho, Yoshida-gun, Fukui 910-1195, Japan. (Received Sep. 27, 2008 / Accepted Sep. 29, 2008 / Published Dec. 25, 2008)

INTRODUCTION moiety of -lysine(s) has been shown to play a crucial role in antibiotic activity. On the other hand, Inamori et al. Streptothricins (STs) (Fig. 1) are broad-spectrum (Inamori et al., 1988) and Taniyama et al. (Taniyama et al., antibiotics that were first isolated from Streptomyces 1971) have independently reported that ST-F-acid (Fig. 1, lavendulae in 1943 (Waksman, 1943). All STs consist of termed as racenomycin-A-acid in their studies)—chemi- a carbamoylated D-gulosamine to which the -lysine cally prepared from ST-F—did not exhibit antibiotic homopolymer (1 to 7 residues) and the amide form of the activity against bacteria, fungi, and plants; however, the unusual amino acid ‘‘streptolidine lactam’’ are attached. biological activity of ST-D-acid was not tested. This result STs inhibit protein biosynthesis in prokaryotic cells; in confirmed that streptolidine lactam is essential for antibiotic addition, they strongly inhibit the growth of eukaryotes activity. We therefore hypothesized that microorganisms such as yeasts (Goldstein & McCusker, 1999; Hentges showing resistance to STs through alternative resistance et al., 2005; Shen et al., 2005), fungi (Idnurm et al., 2004), mechanisms might produce an enzyme that hydrolyzes protozoa (Joshi et al., 1995), insects (Takemoto et al., streptolidine lactam, thereby inactivating STs. Actinomy- 1980) and plants (Chamberlain et al., 1994). Therefore, STs cetes are known to produce many natural products with are used as effective selective agents for recombinant DNA structural diversity occurring due to the unique substrate work in some of these organisms. However, STs are not specificities of the enzymes. Therefore, we focused on currently used therapeutically due to their nephrotoxicity Streptomyces, the representative strains belonging to acti- (Hoffmann et al., 1986a and 1986b; Hartl et al.). nomycetes, to efficiently identify our target enzyme. To date, many ST-resistance genes have been identified Here, we describe the cloning of a gene whose product in transposons such as Tn1825 and Tn1826, which have confers ST resistance through the modification of strepto- been isolated from bacteria that are resistant to ST lidine lactam, as expected (Hamano et al., 2006). Addi- (Partridge & Hall, 2005); such transposons have also been tionally, we used the recombinant enzyme of this gene isolated from human pathogens such as Shiga toxin- product to investigate its functions and properties. We also producing Escherichia coli (Singh et al., 2005) and the discuss an interesting observation regarding the selective Shigella strain (Peirano et al., 2005). Bacterial resistance to toxicity of an ST compound that was converted by the gene antibiotics that inhibit protein biosynthesis (e.g., amino- product. glycosides) can occur as a result of decreased antibiotic uptake and accumulation, modification of 16S RNA or Cloning and sequencing analysis of the ST-resistance ribosomal proteins, or enzymatic modification of the gene antibiotics (Vakulenko & Mobashery, 2003). However, in To obtain our target gene that confers ST resistance via a the case of bacterial resistance to STs, only one resistance novel mechanism, we focused on ST-nonproducing Strep- mechanism has yet been identified: the resistance is due to tomyces strains since we had anticipated that the isolation a modification of the ST molecule by monoacetylation at of our target gene could be hindered by genes encoding the -amino group of -lysine(s). In fact, in ST producers NAT in ST producers. Based on the present studies of such as S. lavendulae (Horinouchi et al., 1987), S. rochei MICs for STs in the Streptomyces strains that have not (Fernańdez-Moreno et al., 1997) and S. noursei (Krugel been accepted as ST producers, Streptomyces albulus et al., 1988; Grammel et al., 2002), the ST-resistance genes NBRC14147 was found to be more resistant to STs than encoding N-acetyltransferase (NAT) have been identified the ST producer S. lavendulae NBRC12789 (Table 1). and their role in self-resistance against their own STs has PCR using primers designed for genes that encode NATs been investigated. Based on this resistance mechanism for STs and the genomic DNA of the NBRC14147 strain as and the fact that streptothricin D (ST-D, Fig. 1) is a more a template did not show any amplified fragments, whereas a effective antibiotic than streptothricin F (ST-F, Fig. 1), the specific amplified fragment was detected when the genomic

Corresponding author: Phone: +81-776-61-6000. Fax: +81-776-61-6015. E-mail address: [email protected]

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carbamoylated streptolidine streptolidine D-gulosamine lactam O O O OH H O OH H N N NH hydrolysis of OH H2N O O N H2N O O N streptolidine lactam N N NH2 H H OH SttH OH OH OH HN H HN H N N ST-F (n=1) H ST-F-acid (n=1) H ST-D (n=3) O NH2 n ST-D-acid (n=3) O NH2 n

β-lysine

Fig. 1. Chemical structure of streptothricins (STs).

DNA of S. lavendulae NBRC12789 was used (data not shown). Therefore, since the NBRC14147 strain was Table 1. ST resistance proﬁles of the Streptomyces strains suggested to have no gene encoding NAT, this strain was MIC (mg/mL) ST selected as a source of the novel ST-resistance gene. To Streptomyces strains of STs production obtain the novel ST-resistance gene, the NBRC14147 S. lavendulae NBRC12789 400 yes strain genomic library was constructed with the pWHM3 S. albulus NBRC14147 >400 no plasmid carrying the thiostrepton-resistance gene. This S. lividans TK23 6.25 no library was introduced into Streptomyces lividans TK23, + pWHM3 6.25y — which is sensitive to STs and thiostrepton, and trans- + pWHM3-st11 >400y — formants resistant to both thiostrepton (20 mg/ml) and + pWHM3-orf2-3 >400y — STs (>400 mg/ml, a mixture of ST-F and ST-D with + pWHM3-orf1 6.25y — ST-F:ST-D ratio of approximately 5:1) were isolated. From ATCC medium No. 5 plates containing STs (0–400 mg/mL) these transformants, we selected one which harbored the were used. yATCC medium No. 5 plates containing STs pWHM3 plasmid carrying a 2.9-kb fragment (pWHM3- (0–400 mg/mL) and thiostrepton (20 mg/mL) were used. The st11, Fig. 2 and Table 1) for further experiments because MICs were determined after incubation for 3 days at 30 C. Southern blotting using this fragment as a probe revealed that all plasmids isolated from these transformants carried

0.5-kb ORF1 sttH (ORF2) ORF3

Eco RI Kpn I Bam HI/Sau 3AI Sau 3AI/Bam HI pWHM3-st11 pWHM3 1 2913-bp pWHM3-orf2-3 pWHM3 pWHM3-orf1 pWHM3

amino acid molecular ORFs homologous proteins (identity, accession no. in the UniProt database) residues weight

ORF1 401 42328 esterase from; - Streptomyces avermitilis MA4680 (46%, Q82NJ9) - Streptomyces chrysomallus (44%, O87861) - Streptomyces coelicolor A3(2) (34%, Q9RKC5) β-lactamase from; - Mycobacterium paratuberculosis (44%, Q73XM6 ) - Pseudomonas fluorescens PfO-1 (34%, Q3KGY6) - Anaeromyxobacter dehalogenans 2CP-C (33%, Q4NPE1) ORF2 (sttH ) 234 23567 isochorismatase-like - Shewanella amazonensis SB2B (31%, Q3QE02) hydrolase from; - Burkholderia cenocepacia AU 1054 (30%, Q44WW2) - Pseudomonas putida F1 (28%, Q2XEN3) ORF3 -- lipase from; - Trichodesmium erythraeum IMS101 (29%, Q3H7D3) (partial) - Rhodopirellula baltica (28%, Q7UQZ0 )

Fig. 2. Schematic organization of the cloned 2.9-kb fragment involved in ST resistance, and ORFs deduced by sequencing analysis. The hatched boxes represent the cloned fragments in the pWHM3 plasmid.

51 ACTINOMYCETOLOGICA VOL. 22, NO. 2 the 2.9-kb fragment (data not shown). (mV) ST-F The sequencing analysis of the 2.9-kb DNA fragment 100 (A) and frame analysis with the codon usage for Streptomyces strains revealed two ORFs (ORF1 and 2) and one partial 50 ORF (ORF3) (Fig. 2). In order to predict the functions of 0 the individual ORFs, we searched the relevant databases (UniProt) with their translated products using BLAST; the ST-F (B) results are summarized in Fig. 2. In brief, the three ORFs 100 showed similarity to esterase and -lactamase (ORF1), 50 isochorismatase-like hydrolase (ORF2) and lipase (ORF3). Thus, this fragment contained no gene homologous to the 0 nat gene. Since the amino acid sequence of ORF1 was 100 (C) similar to those of the -lactamases, this ORF was thought product to be our target. To address this, we constructed plasmids pWHM3-orf1 and pWHM3-orf2-3, which carried ORF1 50 and ORF2-ORF3, respectively (Fig. 2), and introduced 0 them into S. lividans TK23. However, unexpectedly, MIC 0 5 10 15 20 25 (min) studies conﬁrmed that the transformant harboring pWHM3- orf2-3 showed ST resistance (Table 1). Considering the Fig. 3. HPLC analysis of the product formed by rSttH. ST-F was fact that the pWHM3-orf2-3 plasmid carried only a partial incubated with (C) or without (B) rSttH, and the reaction mixtures form of ORF3, ORF2 was found to confer STs resistance and ST-F standard (A) were then analyzed by reverse-phase and was designated as sttH, a novel ST-resistance gene. HPLC.

Functional analysis of SttH We constructed a recombinant SttH (rSttH) as N- does not possess the nat gene homolog that is normally terminal 6 His-tagged fusion proteins. A highly purified clustered with the biosynthetic genes for ST in ST- rSttH obtained by Ni-affinity chromatography was incu- producing Streptomyces strains, and (ii) ST-related com- bated with ST-F. An rSttH-dependent product eluted with a pounds were undetectable in the fermentation broth. This retention time longer than that of ST-F on reverse-phase raises the possibility that the true role of SttH may not be its HPLC was specifically detected in the reaction mixture involvement in self-resistance against STs produced by without any additives such as cofactors or metal ions the organism; instead, it may catalyze the hydrolysis of (Fig. 3). Similarly, an rSttH-dependent product was also naturally occurring cyclic amide compounds in the metabo- detected when ST-D was used as a substrate (data not lism of S. albulus. In this study, it was shown that the shown). To determine the structures of the rSttH-dependent deduced primary structure of SttH resembles those of ether products thus obtained, these compounds were purified and hydrolases that belong to the isochorismatase superfamily analyzed by ESI-MS and NMR. The molecular masses of as described earlier. Isochorismatase (EC 3.3.2.1) is an ST-F and the ST-F-derived compounds were determined enzyme (EntB) occurring in E. coli and is produced during to be 503 (m=z ¼ 504 ½M þ Hþ) and 521 (m=z ¼ 522 the biosynthesis of enterobactin, an iron-chelating product ½M þ Hþ), respectively, by ESI-MS analysis (Figs. 4A and derived from chorismic acid and involved in the transport B). In addition, ESI-MS/MS spectra also revealed that of iron from the bacterial environment into the cell cyto- this change in the molecular mass (18 Da) had occurred in plasm (Young & Gibson, 1969; Nahlik et al., 1987; Nahlik the streptolidine lactam moiety. A correlative observation et al., 1989). The phzD gene encoding an isochorismatase- was also noted in the ST-D-derived compound (Figs. 4C related enzyme, which shares 46% identity with the EntB and D). This, together with the finding that the primary isochorismatase, is also known to participate in phena- structure of SttH is similar to that of isochorismatase-like zine biosynthesis in Pseudomonas strains (Parsons et al., hydrolase, indicated that rSttH catalyzes the hydrolysis of 2003). Parsons et al. have recently reported that the 3D the amide bond of streptolidine lactam. To confirm these structure of PhzD is remarkably similar to other structures predicted structures, we performed NMR analysis, finding from a subfamily of /-hydrolase fold enzymes, whose that the obtained 1H NMR spectral features of the strepto- members are known to hydrolyze amides, phosphates, lidine moiety were in complete agreement with those of a phosphonates, epoxides and C-X bonds (Parsons et al., chemically synthesized streptolidine reported by Jackson 2003). However, to the best of our knowledge, no amide et al. (Jackson et al., 2002). The rSttH products from ST-F hydrolysis reactions have been observed in EntB or PhzD. and ST-D were thus determined to be ST-F-acid and ST-D- On the other hand, the recent genome sequencing projects acid, respectively (Fig. 1). in bacteria have shown that the genes encoding proteins We believe S. albulus NBRC14147 to be an ST non- belonging to the isochorismatase-like hydrolase superfam- producer based on the following observations: (i) this strain ily exist in almost all bacteria including enterobactin- or

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(M+H)+ (M+H)+ (A) O (C) O H 504 H 170 100% N 100% m/z 170 NH + N NH N (M+H) m/z 170 N N 170 N H H

OH Intensity Intensity OH O OH O OH H N O 100 200 300 400 500 m/z 2 O H2N O O 100 200 300 400 m/z (M+2H)2+ MS/MS + (M+2H)2+ MS/MS OH 252 (M+H) 100% OH 380 504 100% (M+H)+ 760 Intensity Intensity 200 400 600 800m/z 200 400 600 800m/z

O + (M+H)+ (B) H (M+H) (D) O N OH 522 H 188 m/z 188 N 100% N 100% m/z 188 OH + N N NH2 (M+H) H N NH2 O OH 188 H OH O OH

Intensity OH Intensity H N O O 2 H N O m/z 2 O 100 200 300 400 m/z (M+2H)2+ 100 200 300 400 500 OH 261 MS/MS 2+ 100% OH (M+2H) MS/MS (M+H)+ 389 100% 522 (M+H)+ 778 Intensity Intensity 200 400 600 800m/z 200 400 600 800 m/z

Fig. 4. ESI-MS/MS analysis of the rSttH product from ST-F (ST-F-acid) and ST-D (ST-D-acid). The ESI-MS and -MS/MS spectra of ST-F (A), ST-F-acid (B), ST-D (C), and ST-D-acid (D), which were dissolved in 0.2% formic acid/50% acetonitrile, were obtained.

phenazine-producing bacteria. Although bacteria common- Table 2. Kinetic parameters for STs ly possess isochorismatase-like hydrolase(s), the functions K V V =K Substrate m max max m of these proteins remain unclear. Interestingly, an analysis (mM) (Uy/mg) (Uy/(mgmM)) using 3D-PSSM (Kelley et al., 2000) demonstrated that the ST-F 0:96 0:19 42:3 2:8 44.0 deduced 3D structure of SttH is also considerably similar ST-D 5:74 0:99 69:0 13:9 12.0 to isochorismatase-like hydrolases of unknown function Kinetic parameters were determined with 100 mM NaPB Enterococcus faecalis from v583 (PDB accession number, (pH 6.5) at 30 C. yU, mmol production of ST-acids/min. Each 2A67) and E. coli (PDB accession number, 1J2R). Thus, value is represented as the mean SD of three experiments. SttH is the first enzyme whose function was determined among these isochorismatase-like hydrolases that commonly exist in bacteria. These findings also suggest that some of the isochorismatase-like hydrolases of unknown function Although the enzyme activity was maximal at 45 C, an from other bacteria could hydrolyze cyclic amides. A activity level of approximately 90% was detected at 65 C. further in-depth enzymatic characterization of SttH and The enzyme dialyzed against sodium phosphate buffer sequencing analysis of the regions flanking the sttH gene (pH 6.5) containing 10 mM ethylenediaminetetraacetic acid should facilitate the understanding of the true biological (EDTA) showed no decrease in activity, demonstrating that role of SttH in S. albulus. Such investigation might also be this enzyme does not require metal ions for its activity (data able to provide insights into the biological role of these not shown). The kinetic parameters are summarized and isochorismatase-like hydrolases that commonly occur in shown in Table 2. The Km values of rSttH were calculated bacteria. to be 0:96 0:19 mM for ST-F and 5:74 0:99 mM for ST-D; this shows that the enzyme has a higher affinity for Enzymatic properties of rSttH the ST compound having a shorter chain of the -lysine The optimal pH was measured in two buffers (100 mM) polymer. However, the Vmax value of rSttH for ST-D was at various pH values: sodium phosphate, pH 4.5–7 and slightly higher than that for ST-F. The calculated Vmax=Km Tris-HCl, pH 7–10. Maximum enzyme activity was ob- value of the reaction with ST-F was four-fold higher served at pH 6.5 and was rapidly lost with a decrease in pH than that of the reaction with ST-D. The native molecular (around 4). The effect of temperature on the enzyme mass of rSttH was estimated to be 50 kDa by gel filtration activity was also investigated over the temperature range of (data not shown), suggesting that rSttH was present as a 25 C–75 C in 100 mM sodium phosphate buffer (pH 6.5). homodimer.

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Table 3. MICs of the ST compound in prokaryotic and eukary- against various microorganisms, including Gram-positive otic cells and Gram-negative bacteria, clinically isolated pathogenic MICs (mM) bacteria and yeasts. The MIC studies revealed that in ST-F ST-D contrast to the almost complete loss of the antibacterial Strains ST-Fa ST-Db -acidc -acidd activity of ST-F-acid against both prokaryotic and eukary- (c/a)} (d/b)} otic cells, ST-D-acid showed a high activity against bacteria such as E. coli, B. subtilis and S. aureus, but not S. cerevisiae CKY8 against eukaryotic cells such as S. cerevisiae and S. pombe + pAD4 0.5 <0:03 NT NT (Table 3). In particular, the antibacterial activities of ST-D- + pAD4-nat >4 >4 NT NT acid have been found to be robust against the clinically + pAD4-sttH >4 >4 NT NT isolated pathogenic bacteria S. aureus AB (unpublished E. coli XL1-Blue MRF0y enterotoxin AB-producing strain) and S. aureus FIR1169 + pQE30 <0:03 <0:03 NT NT (toxic shock syndrome exotoxins-producing strain). STs + pQE30-nat 4 2 NT NT have not been clinically developed due to their toxicities in + pQE30-SHF6R 0.25 <0:03 NT NT mammals. However, in the present study, we found that S. cerevisiae S288Cz 0.062 0.004 >4 1 ST-D-acid exhibits a potent antibacterial activity even (>64) (250) when its toxicity against eukaryotic cells is reduced by S. pombe L972z 0.125 0.008 >4 1 SttH. This suggests that ST-D-acid has potential for clinical (>32) (125) development or for use as a new lead compound for drug E. coli W3110x 0.03 0.008 4 0.25 discovery. (133) (31.3) B. subtilis NBRC13169x 0.016 0.004 4 0.125 ACKNOWLEDGEMENT (250) (31.3) S. aureus ABx 0.016 0.004 2 0.06 It is my great honor to receive the Hamada Award of (126) (15) the Society for Actinomycetes, Japan (SAJ) in 2008. I S. aureus FIR1169x 0.016 0.004 2 0.06 would like to thank the past and present members of my (126) (15) laboratory who have carried out a substantial amount of the y z Microorganisms were cultivated in SC-Leu ,LB, YPD , and work described herein. I particularly thank Prof. Hiroshi Heart Infusion Brothx (Difco) medium. Yeasts and bacteria were grown for 2 days at 30 C and for 1 day at 37 C, Takagi for his generous support and for providing an respectively. }Inactivation ratio of ST-acids. NT, not tested. opportunity to accomplish my study, and the graduated students—Ine Nicchu and Miwa Kitamura—for working as experimental partners. I would further like to express my Investigation of the ST-D resistance profile in E. coli thanks to Prof. Nobuyasu Matsuura (Okayama University and yeast cells overexpressing the sttH or nat gene and of Science), Prof. Hiromichi Fujino (Chiba University), and in other microorganisms Ms. Chitose Maruyama. I would like to acknowledge the Although the biological activity of chemically prepared continuing encouragement from my former supervisors, ST-F-acid has been reported to be negligible in micro- namely, Prof. Nobuya Ito and Prof. Tohru Dairi (Toyama organisms and plants (Inamori et al., 1988; Taniyama et al., Prefectural University). Finally, I would like to thank the 1971), the biological activity of ST-D-acid remains unclear. members of the SAJ for their continuing interest and The sttH gene conferred resistance against the mixture of support. ST-F and ST-D (ST-F:ST-D ratio was approximately 5:1) in the S. lividans and E. coli strains as described above. REFERENCES However, it is still unclear whether SttH detoxified ST-D. Therefore, we determined the MICs of both ST-F and ST-D Chamberlain, D. A., Brettell, R. I. S., Last, D. I., Witrzens, B., for E. coli and Saccharomyces cerevisiae strains expressing McElroy, D., Dolferus, R. & Dennis, E. S. (1994). The use of SttH (Table 3). In this experiment, we also employed the the Emu promoter with antibiotic and herbicide resistance E. coli and S. cerevisiae strains expressing NAT as genes for the selection of transgenic wheat callus and rice controls. The E. coli (pQE-nat) and S. cerevisiae (pAD4- plants. Australian. J. Plant. Physiol. 21, 95–112. nat) strains expressing NAT showed resistance to both Fernańdez-Moreno, M. A., Vallıń, C. & Malpartida, F. (1997). ST-F and ST-D as reported previously. Interestingly, Streptothricin biosynthesis is catalyzed by enzymes related to nonribosomal peptide bond formation. J. Bacteriol. 179, 6929– the MIC value of ST-D was extremely low for E. coli 6936. (pQE30-SHF6R) expressing rSttH; this was in contrast to Goldstein, A. L. & McCusker, J. H. (1999). Three new dominant the case of S. cerevisiae (pAD4-sttH) and indicates that the drug resistance cassettes for gene disruption in Saccharomyces ST-D-acid formed by SttH was still active as an antibacte- cerevisiae. Yeast 15, 1541–1553. rial agent against prokaryotic cells. To confirm this, we Grammel, N., Pankevych, K., Demydchuk, J., Lambrecht, K., investigated the selective toxicity of ST-acids and STs Saluz, H. P. & Krugel, H. (2002). A -lysine adenylating

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