- Email: [email protected]
A novel family (QnrAS) of plasmid-mediated quinolone resistance determinant
578
Letters to the Editor / International Journal of Antimicrobial Agents 36 (2010) 573–580
[5] Clinical Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. Nineteenth informational supplement. Document M100S19. Wayne, PA: CLSI; 2009. [6] Damjanova I, Tóth A, Pászti J, Hajbel-Vékony G, Jakab M, Berta J, et al. Expansion and countrywide dissemination of ST11 ST15 and ST147 ciprofloxacin-resistant CTX-M-15-type -lactamase-producing Klebsiella pneumoniae epidemic clones in Hungary in 2005—the new ‘MRSAs’? J Antimicrob Chemother 2008;62:978–85. [7] Hrabák J, Empel J, Bergerová T, Fajfrlík K, Urbásková P, Kern-Zdanowicz I, et al. International clones of Klebsiella pneumoniae and Escherichia coli with extendedspectrum -lactamases in a Czech hospital. J Clin Microbiol 2009;47:3353–7.
Caterina Mammina ∗ Aurora Aleo Department of Sciences for Health Promotion ‘G. D’Alessandro’, Section of Hygiene, University of Palermo, Via del Vespro 133, I-90127 Palermo, Italy Celestino Bonura Cinzia Calà Department of Sciences for Health Promotion ‘G. D’Alessandro’, Section of Microbiology, University of Palermo, Palermo, Italy Roberto Degl’Innocenti Antonella Conti Hospital Infections Control Team, General Hospital of Prato, Prato, Italy Patrizia Pecile Laboratory of Microbiology and Virology, Azienda Ospedaliero–Universitaria ‘Careggi’, Florence, Italy Giovanna Pesavento Antonino Nastasi Department of Public Health, University of Florence, Florence, Italy ∗ Corresponding
author. Tel.: +39 091 655 3623; fax: +39 091 655 3641. E-mail address: [email protected] (C. Mammina)
doi:10.1016/j.ijantimicag.2010.08.004
A novel family (QnrAS) of plasmid-mediated quinolone resistance determinant Sir, Since the plasmid-mediated quinolone resistance gene (qnr) was first described in a ciprofloxacin-resistant strain of Klebsiella pneumoniae in 1998 [1], many additional qnr genes have been discovered on plasmids or the bacterial chromosome [2]. Qnr proteins belong to the pentapeptide repeat protein (PRP) family and protect DNA gyrase and topoisomerase IV from quinolone attack [3]. Qnr proteins currently comprise five families (QnrA, QnrB, QnrC, QnrD and QnrS), which differ from each other in amino acid sequence by ≥39%. To reduce the chaos that developed from new sequences being given the same QnrB number or having the number changed frequently in an attempt to avoid duplication, a Qnr numbering scheme was recently proposed by Jacoby et al. on the basis of eight priority rules [2]. Furthermore, to fundamentally and continuously prevent misleading designations of Qnr alleles, we most importantly need a central clearing website with bioinformatics capabilities to serve as a repository for all sequences and name designations for Qnr alleles. Fortunately, there is currently a website (http://www.lahey.org/qnrStudies/) on which sequences, literature references and database accession numbers for Qnr alleles are posted and numbers are assigned. Whether this system succeeds or not depends on the co-operation of each researcher and the volun-
tary participation of the keeper(s) of the website. Therefore, allele designations should be assigned upon application, before new Qnr alleles are submitted to the GenBank database. Here, we focus on the characterisation of a new Qnr family (QnrAS, the gene of which is located on the chromosome of Aliivibrio salmonicida), which can help researchers to designate correctly new Qnr alleles and make the website effective and successful. To ensure that the Lahey website is effective and successful, the information (GenBank accession no., location and amino acid substitutions) for Qnr alleles should be correct. Because there were some misleading data, information for Qnr alleles shown on the Lahey website was updated recently through personal communication between the current authors and Dr Jacoby. Furthermore, we evaluated Qnr alleles in the GenBank database as of July 2010 to identify unique sequences, excluding functionally silent variants and alleles having partial amino acid sequences, and found that there are 7 QnrA, 24 QnrB, 2 QnrC, 1 QnrD and 4 QnrS alleles. Unfortunately, we found that there has been current confusion on QnrC family designations [two different sequences (ACK75961 and CAQ79275) named as the same QnrC]. The sequence CAQ79275 had not been assigned as QnrC on the Lahey website; the sequence ACK75961 was designated QnrC. The unassigned sequence (218 amino acids) has been compared with those of five known Qnr families and aligned using CLUSTAL W (http://align.genome.jp/) for comparison. The sequence showed 65% amino acid identity to QnrA1, 40% identity to QnrB1, 65% identity to QnrS1, 67% identity to QnrC and 45% identity to QnrD. Therefore, the unassigned sequence (CAQ79275) was designated QnrAS (a new family) according to the rule proposed by Jacoby et al. [2], which states that a new family should differ substantially from existing families (≥30% difference suggested in nucleotides or derived amino acids). Phylogenetic analysis (http://align.genome.jp/) showed that QnrAS clustered independently from the known Qnr families but shared the highest identity to the QnrC family (data not shown). In silico analysis showed the QnrAS shared 59–80% amino acid identity with Qnr-like determinants of Vibrio parahaemolyticus CIP71.2 (59%), Vibrio vulnificus CIP03196 (60%), Vibrio splendidus 12B01 (66%), Vibrio cholerae O1 strain VC627 (69%), Photobacterium profundum CIP106289 (77%) and Vibrio fischeri ES114 (80%), which may be a reservoir for Qnr-like quinolone resistance determinants [4–6]. Furthermore, QnrAS belonged to the PRP family [3] on the basis of analysis using the Motif Scan program (http://hits.isb-sib.ch/cgi-bin/PFSCAN) and the National Center for Biotechnology Information Conserved Domain Search program (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). QnrAS contained two domains, of 9 and 29 tandem pentapeptide repeat units each, connected by a single glycine (G56) (data not shown), which is conserved in five Qnr families that affect quinolone susceptibility. To investigate whether or not QnrAS can affect quinolone susceptibility, the DNA sequence containing the coding region (GenBank accession no. FM178379; 657 bp region from 1699484 to 1700140 on chromosome I of A. salmonicida LFI1238) of the qnrAS gene [7] was chemically synthesised using GenScriptTM technology (GenScript Corp., Piscataway, NJ). Following digestion with ¯ SacI and XbaI (Takara Bio Inc., Otsu, Japan), the synthetic qnrAS gene was cloned into a pHSG398 vector (Takara Bio Inc.) and was transformed into the Escherichia coli TOP10 host strain (Invitrogen, Karlsruhe, Germany). Minimum inhibitory concentrations (MICs) were determined by Etest (AB BIODISK, Solna, Sweden) and were interpreted according to Clinical and Laboratory Standards Institute (CLSI) guidelines [8]. In the presence of the cloned qnrAS gene, an 8-fold increase in the MIC of nalidixic acid and a >8- to 83-fold increase in the MICs of fluoroquinolones were detected (Table 1), as observed with transformants harbouring qnrA1, qnrS1, qnrC and
Letters to the Editor / International Journal of Antimicrobial Agents 36 (2010) 573–580
579
Table 1 Susceptibility to various quinolones of transformants harbouring qnr genes as well as host or reference strains. Strain
qnr gene
TrfQnrASb TrfHSG398c Escherichia coli TOP10 E. coli ATCC 25922d
qnrAS
MIC (g/mL) (fold increasea ) NAL 8 (8) 1 1 2
OFX
NOR
LVX
CIP
0.25 (83) 0.003 0.003 0.016
0.125 (>8) <0.016 <0.016 0.032
0.064 (10) 0.006 0.006 0.008
0.047 (24) 0.002 0.002 <0.002
MIC, minimum inhibitory concentration; NAL, nalidixic acid; OFX, ofloxacin; NOR, norfloxacin; LVX, levofloxacin; CIP, ciprofloxacin. a Compared with MICs of the E. coli TOP10 host strain. b Escherichia coli TOP10 (host strain) transformant harbouring pHSG398::qnrAS (expressing the QnrAS determinant). c Escherichia coli TOP10 (host strain) transformant harbouring pHSG398. d MIC reference strain.
qnrD [9,10]. Like other Qnr families, QnrAS provided low-level resistance to all quinolones tested (Table 1). Acknowledgment The authors would like to thank Dr Gian Maria Rossolini for helpful discussions. Funding: This work was supported by research grants from the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (KRF-2008-313-C00790 and 2010-0014572), as well as the Marine & Extreme Genome Research Center Program of the Ministry of Land, Transport, and Maritime Affairs, Republic of Korea. Competing interests: None declared. Ethical approval: Not required. References [1] Martínez-Martínez L, Pascual A, Jacoby GA. Quinolone resistance from a transferable plasmid. Lancet 1998;351:797–9. [2] Jacoby GA, Cattoir V, Hooper D, Martínez-Martínez L, Nordmann P, Pascual A, et al. qnr gene nomenclature. Antimicrob Agents Chemother 2008;52: 2297–9. [3] Sánchez MB, Hernández A, Rodríguez-Martínez JM, Martínez-Martínez L, Martínez JL. Predictive analysis of transmissible quinolone resistance indicates Stenotrophomonas maltophilia as a potential source of a novel family of Qnr determinants. BMC Microbiol 2008;8:148. [4] Poirel L, Liard A, Rodríguez-Martínez JM, Nordmann P. Vibrionaceae as a possible source of Qnr-like quinolone resistance determinants. J Antimicrob Chemother 2005;56:1118–21. [5] Cattoir V, Poirel L, Mazel D, Soussy CJ, Nordmann P. Vibrio splendidus as the source of plasmid-mediated QnrS-like quinolone resistance determinants. Antimicrob Agents Chemother 2007;51:2650–1. [6] Fonseca EL, Dos Santos Freitas F, Vieira VV, Vicente AC. New qnr gene cassettes associated with superintegron repeats in Vibrio cholerae O1. Emerg Infect Dis 2008;14:1129–31. [7] Hjerde E, Lorentzen MS, Holden MTG, Seeger K, Paulsen S, Bason N, et al. The genome sequence of the fish pathogen Aliivibrio salmonicida strain LFI1238 shows extensive evidence of gene decay. BMC Genomics 2008;9:616. [8] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. Twentieth informational supplement. Document M100-S20. Wayne, PA: CLSI; 2010. [9] Wang M, Guo Q, Xu X, Wang X, Ye X, Wu S, et al. New plasmid-mediated quinolone resistance gene, qnrC, found in a clinical isolate of Proteus mirabilis. Antimicrob Agents Chemother 2009;53:1892–7. [10] Cavaco LM, Hasman H, Xia S, Aarestrup FM. qnrD, a novel gene conferring transferable quinolone resistance in Salmonella enterica serovar Kentucky and Bovismorbificans strains of human origin. Antimicrob Agents Chemother 2009;53:603–8.
Ha Ik Sun 1 Drug Resistance Proteomics Laboratory, Department of Biological Sciences, Myongji University, Yongin, Gyeonggi-do, Republic of Korea Da Un Jeong a,b,1 Drug Resistance Proteomics Laboratory, Department of Biological Sciences, Myongji University, Yongin, Gyeonggi-do, Republic of Korea b Department of Chemistry, Korea University, Seoul, Republic of Korea a
Jung Hun Lee 1 Xing Wu Kwang Seung Park Jae Jin Lee Byeong Chul Jeong Sang Hee Lee ∗ Drug Resistance Proteomics Laboratory, Department of Biological Sciences, Myongji University, Yongin, Gyeonggi-do, Republic of Korea ∗ Corresponding
author. Tel.: +82 31 330 6195; fax: +82 31 335 8249. E-mail address: [email protected] (S.H. Lee) 1
These three authors contributed equally to this work.
doi:10.1016/j.ijantimicag.2010.08.009
Disulphide bonds of the peptide protegrin-1 are not essential for antimicrobial activity and haemolytic activity Sir, In its natural state, the antimicrobial peptide protegrin-1 (PG1) has two disulphide bonds, with the four cysteines linked at positions 6–15 and 8–13 [1]. Linear protegrin analogues, or analogues containing only one of the two disulphide bonds, have been made either by (a) reducing and then alkylating cysteines on intact protegrins with iodoacetamide, (b) synthesising protegrins using cysteines blocked with triphenylmethyl or acetamidomethyl groups, (c) synthesising protegrins with cysteine to alanine substitutions [2] or (d) synthesising protegrins with cysteine to threonine substitutions and arginine to d-proline substitution at position 10 of the sequence [3]. The linear forms of the peptide have been reported to be considerably less active than the native form as well as being sensitive to physiological salt concentrations; both of the single disulphide variants have intermediate activity. However, these claims have overlooked the study of Mangoni et al. [4] who reported that linear PG-1 itself (no disulphide bonds) was at least equally as active as natural PG-1 (two disulphide bonds) against Escherichia coli. The current paper presents a comparison of the antimicrobial activities of cyclic and unmodified linear PG-1 against one Gram-negative (E. coli K91BK) and two Gram-positive bacteria [Bacillus anthracis Sterne strain 34F2 (a veterinary vaccine) and Bacillus globigii] in the absence and presence of added salt. Haemolytic activity of the two forms was also studied. Linear PG-1 (free sulphydryl on the four cysteine amino acids) and cyclic PG-1 (two disulphide bonds) were synthesised by SBS Genetech Co. Ltd. (Beijing, China) with a modified Nterminus (acetate) and C-terminus (amide). Antimicrobial and haemolytic assays were performed as described previously [5].
Letters to the Editor / International Journal of Antimicrobial Agents 36 (2010) 573–580
[5] Clinical Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. Nineteenth informational supplement. Document M100S19. Wayne, PA: CLSI; 2009. [6] Damjanova I, Tóth A, Pászti J, Hajbel-Vékony G, Jakab M, Berta J, et al. Expansion and countrywide dissemination of ST11 ST15 and ST147 ciprofloxacin-resistant CTX-M-15-type -lactamase-producing Klebsiella pneumoniae epidemic clones in Hungary in 2005—the new ‘MRSAs’? J Antimicrob Chemother 2008;62:978–85. [7] Hrabák J, Empel J, Bergerová T, Fajfrlík K, Urbásková P, Kern-Zdanowicz I, et al. International clones of Klebsiella pneumoniae and Escherichia coli with extendedspectrum -lactamases in a Czech hospital. J Clin Microbiol 2009;47:3353–7.
Caterina Mammina ∗ Aurora Aleo Department of Sciences for Health Promotion ‘G. D’Alessandro’, Section of Hygiene, University of Palermo, Via del Vespro 133, I-90127 Palermo, Italy Celestino Bonura Cinzia Calà Department of Sciences for Health Promotion ‘G. D’Alessandro’, Section of Microbiology, University of Palermo, Palermo, Italy Roberto Degl’Innocenti Antonella Conti Hospital Infections Control Team, General Hospital of Prato, Prato, Italy Patrizia Pecile Laboratory of Microbiology and Virology, Azienda Ospedaliero–Universitaria ‘Careggi’, Florence, Italy Giovanna Pesavento Antonino Nastasi Department of Public Health, University of Florence, Florence, Italy ∗ Corresponding
author. Tel.: +39 091 655 3623; fax: +39 091 655 3641. E-mail address: [email protected] (C. Mammina)
doi:10.1016/j.ijantimicag.2010.08.004
A novel family (QnrAS) of plasmid-mediated quinolone resistance determinant Sir, Since the plasmid-mediated quinolone resistance gene (qnr) was first described in a ciprofloxacin-resistant strain of Klebsiella pneumoniae in 1998 [1], many additional qnr genes have been discovered on plasmids or the bacterial chromosome [2]. Qnr proteins belong to the pentapeptide repeat protein (PRP) family and protect DNA gyrase and topoisomerase IV from quinolone attack [3]. Qnr proteins currently comprise five families (QnrA, QnrB, QnrC, QnrD and QnrS), which differ from each other in amino acid sequence by ≥39%. To reduce the chaos that developed from new sequences being given the same QnrB number or having the number changed frequently in an attempt to avoid duplication, a Qnr numbering scheme was recently proposed by Jacoby et al. on the basis of eight priority rules [2]. Furthermore, to fundamentally and continuously prevent misleading designations of Qnr alleles, we most importantly need a central clearing website with bioinformatics capabilities to serve as a repository for all sequences and name designations for Qnr alleles. Fortunately, there is currently a website (http://www.lahey.org/qnrStudies/) on which sequences, literature references and database accession numbers for Qnr alleles are posted and numbers are assigned. Whether this system succeeds or not depends on the co-operation of each researcher and the volun-
tary participation of the keeper(s) of the website. Therefore, allele designations should be assigned upon application, before new Qnr alleles are submitted to the GenBank database. Here, we focus on the characterisation of a new Qnr family (QnrAS, the gene of which is located on the chromosome of Aliivibrio salmonicida), which can help researchers to designate correctly new Qnr alleles and make the website effective and successful. To ensure that the Lahey website is effective and successful, the information (GenBank accession no., location and amino acid substitutions) for Qnr alleles should be correct. Because there were some misleading data, information for Qnr alleles shown on the Lahey website was updated recently through personal communication between the current authors and Dr Jacoby. Furthermore, we evaluated Qnr alleles in the GenBank database as of July 2010 to identify unique sequences, excluding functionally silent variants and alleles having partial amino acid sequences, and found that there are 7 QnrA, 24 QnrB, 2 QnrC, 1 QnrD and 4 QnrS alleles. Unfortunately, we found that there has been current confusion on QnrC family designations [two different sequences (ACK75961 and CAQ79275) named as the same QnrC]. The sequence CAQ79275 had not been assigned as QnrC on the Lahey website; the sequence ACK75961 was designated QnrC. The unassigned sequence (218 amino acids) has been compared with those of five known Qnr families and aligned using CLUSTAL W (http://align.genome.jp/) for comparison. The sequence showed 65% amino acid identity to QnrA1, 40% identity to QnrB1, 65% identity to QnrS1, 67% identity to QnrC and 45% identity to QnrD. Therefore, the unassigned sequence (CAQ79275) was designated QnrAS (a new family) according to the rule proposed by Jacoby et al. [2], which states that a new family should differ substantially from existing families (≥30% difference suggested in nucleotides or derived amino acids). Phylogenetic analysis (http://align.genome.jp/) showed that QnrAS clustered independently from the known Qnr families but shared the highest identity to the QnrC family (data not shown). In silico analysis showed the QnrAS shared 59–80% amino acid identity with Qnr-like determinants of Vibrio parahaemolyticus CIP71.2 (59%), Vibrio vulnificus CIP03196 (60%), Vibrio splendidus 12B01 (66%), Vibrio cholerae O1 strain VC627 (69%), Photobacterium profundum CIP106289 (77%) and Vibrio fischeri ES114 (80%), which may be a reservoir for Qnr-like quinolone resistance determinants [4–6]. Furthermore, QnrAS belonged to the PRP family [3] on the basis of analysis using the Motif Scan program (http://hits.isb-sib.ch/cgi-bin/PFSCAN) and the National Center for Biotechnology Information Conserved Domain Search program (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). QnrAS contained two domains, of 9 and 29 tandem pentapeptide repeat units each, connected by a single glycine (G56) (data not shown), which is conserved in five Qnr families that affect quinolone susceptibility. To investigate whether or not QnrAS can affect quinolone susceptibility, the DNA sequence containing the coding region (GenBank accession no. FM178379; 657 bp region from 1699484 to 1700140 on chromosome I of A. salmonicida LFI1238) of the qnrAS gene [7] was chemically synthesised using GenScriptTM technology (GenScript Corp., Piscataway, NJ). Following digestion with ¯ SacI and XbaI (Takara Bio Inc., Otsu, Japan), the synthetic qnrAS gene was cloned into a pHSG398 vector (Takara Bio Inc.) and was transformed into the Escherichia coli TOP10 host strain (Invitrogen, Karlsruhe, Germany). Minimum inhibitory concentrations (MICs) were determined by Etest (AB BIODISK, Solna, Sweden) and were interpreted according to Clinical and Laboratory Standards Institute (CLSI) guidelines [8]. In the presence of the cloned qnrAS gene, an 8-fold increase in the MIC of nalidixic acid and a >8- to 83-fold increase in the MICs of fluoroquinolones were detected (Table 1), as observed with transformants harbouring qnrA1, qnrS1, qnrC and
Letters to the Editor / International Journal of Antimicrobial Agents 36 (2010) 573–580
579
Table 1 Susceptibility to various quinolones of transformants harbouring qnr genes as well as host or reference strains. Strain
qnr gene
TrfQnrASb TrfHSG398c Escherichia coli TOP10 E. coli ATCC 25922d
qnrAS
MIC (g/mL) (fold increasea ) NAL 8 (8) 1 1 2
OFX
NOR
LVX
CIP
0.25 (83) 0.003 0.003 0.016
0.125 (>8) <0.016 <0.016 0.032
0.064 (10) 0.006 0.006 0.008
0.047 (24) 0.002 0.002 <0.002
MIC, minimum inhibitory concentration; NAL, nalidixic acid; OFX, ofloxacin; NOR, norfloxacin; LVX, levofloxacin; CIP, ciprofloxacin. a Compared with MICs of the E. coli TOP10 host strain. b Escherichia coli TOP10 (host strain) transformant harbouring pHSG398::qnrAS (expressing the QnrAS determinant). c Escherichia coli TOP10 (host strain) transformant harbouring pHSG398. d MIC reference strain.
qnrD [9,10]. Like other Qnr families, QnrAS provided low-level resistance to all quinolones tested (Table 1). Acknowledgment The authors would like to thank Dr Gian Maria Rossolini for helpful discussions. Funding: This work was supported by research grants from the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (KRF-2008-313-C00790 and 2010-0014572), as well as the Marine & Extreme Genome Research Center Program of the Ministry of Land, Transport, and Maritime Affairs, Republic of Korea. Competing interests: None declared. Ethical approval: Not required. References [1] Martínez-Martínez L, Pascual A, Jacoby GA. Quinolone resistance from a transferable plasmid. Lancet 1998;351:797–9. [2] Jacoby GA, Cattoir V, Hooper D, Martínez-Martínez L, Nordmann P, Pascual A, et al. qnr gene nomenclature. Antimicrob Agents Chemother 2008;52: 2297–9. [3] Sánchez MB, Hernández A, Rodríguez-Martínez JM, Martínez-Martínez L, Martínez JL. Predictive analysis of transmissible quinolone resistance indicates Stenotrophomonas maltophilia as a potential source of a novel family of Qnr determinants. BMC Microbiol 2008;8:148. [4] Poirel L, Liard A, Rodríguez-Martínez JM, Nordmann P. Vibrionaceae as a possible source of Qnr-like quinolone resistance determinants. J Antimicrob Chemother 2005;56:1118–21. [5] Cattoir V, Poirel L, Mazel D, Soussy CJ, Nordmann P. Vibrio splendidus as the source of plasmid-mediated QnrS-like quinolone resistance determinants. Antimicrob Agents Chemother 2007;51:2650–1. [6] Fonseca EL, Dos Santos Freitas F, Vieira VV, Vicente AC. New qnr gene cassettes associated with superintegron repeats in Vibrio cholerae O1. Emerg Infect Dis 2008;14:1129–31. [7] Hjerde E, Lorentzen MS, Holden MTG, Seeger K, Paulsen S, Bason N, et al. The genome sequence of the fish pathogen Aliivibrio salmonicida strain LFI1238 shows extensive evidence of gene decay. BMC Genomics 2008;9:616. [8] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. Twentieth informational supplement. Document M100-S20. Wayne, PA: CLSI; 2010. [9] Wang M, Guo Q, Xu X, Wang X, Ye X, Wu S, et al. New plasmid-mediated quinolone resistance gene, qnrC, found in a clinical isolate of Proteus mirabilis. Antimicrob Agents Chemother 2009;53:1892–7. [10] Cavaco LM, Hasman H, Xia S, Aarestrup FM. qnrD, a novel gene conferring transferable quinolone resistance in Salmonella enterica serovar Kentucky and Bovismorbificans strains of human origin. Antimicrob Agents Chemother 2009;53:603–8.
Ha Ik Sun 1 Drug Resistance Proteomics Laboratory, Department of Biological Sciences, Myongji University, Yongin, Gyeonggi-do, Republic of Korea Da Un Jeong a,b,1 Drug Resistance Proteomics Laboratory, Department of Biological Sciences, Myongji University, Yongin, Gyeonggi-do, Republic of Korea b Department of Chemistry, Korea University, Seoul, Republic of Korea a
Jung Hun Lee 1 Xing Wu Kwang Seung Park Jae Jin Lee Byeong Chul Jeong Sang Hee Lee ∗ Drug Resistance Proteomics Laboratory, Department of Biological Sciences, Myongji University, Yongin, Gyeonggi-do, Republic of Korea ∗ Corresponding
author. Tel.: +82 31 330 6195; fax: +82 31 335 8249. E-mail address: [email protected] (S.H. Lee) 1
These three authors contributed equally to this work.
doi:10.1016/j.ijantimicag.2010.08.009
Disulphide bonds of the peptide protegrin-1 are not essential for antimicrobial activity and haemolytic activity Sir, In its natural state, the antimicrobial peptide protegrin-1 (PG1) has two disulphide bonds, with the four cysteines linked at positions 6–15 and 8–13 [1]. Linear protegrin analogues, or analogues containing only one of the two disulphide bonds, have been made either by (a) reducing and then alkylating cysteines on intact protegrins with iodoacetamide, (b) synthesising protegrins using cysteines blocked with triphenylmethyl or acetamidomethyl groups, (c) synthesising protegrins with cysteine to alanine substitutions [2] or (d) synthesising protegrins with cysteine to threonine substitutions and arginine to d-proline substitution at position 10 of the sequence [3]. The linear forms of the peptide have been reported to be considerably less active than the native form as well as being sensitive to physiological salt concentrations; both of the single disulphide variants have intermediate activity. However, these claims have overlooked the study of Mangoni et al. [4] who reported that linear PG-1 itself (no disulphide bonds) was at least equally as active as natural PG-1 (two disulphide bonds) against Escherichia coli. The current paper presents a comparison of the antimicrobial activities of cyclic and unmodified linear PG-1 against one Gram-negative (E. coli K91BK) and two Gram-positive bacteria [Bacillus anthracis Sterne strain 34F2 (a veterinary vaccine) and Bacillus globigii] in the absence and presence of added salt. Haemolytic activity of the two forms was also studied. Linear PG-1 (free sulphydryl on the four cysteine amino acids) and cyclic PG-1 (two disulphide bonds) were synthesised by SBS Genetech Co. Ltd. (Beijing, China) with a modified Nterminus (acetate) and C-terminus (amide). Antimicrobial and haemolytic assays were performed as described previously [5].