Staphylococcal Antimicrobial Peptides: Relevant ... - IngentaConnect

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Current Pharmaceutical Biotechnology, 2009, 10, 38-61

Staphylococcal Antimicrobial Peptides: Relevant Properties and Potential Biotechnological Applications M.C.F. Bastos1,*, H. Ceotto1,3,¶, M.L.V. Coelho1,¶ and J.S. Nascimento1,2,¶ 1

Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Góes, CCS, UFRJ, Rio de Janeiro, RJ, 2Centro Federal de Educação Tecnológica de Química de Nilópolis, Unidade Maracanã, RJ, and 3Instituto de Biologia, UFF, Niterói, RJ, Brazil Abstract: Bacteriocins are bacterial antimicrobial peptides with bactericidal activity against other bacteria. Staphylococcins are bacteriocins produced by staphylococci, which are Gram-positive bacteria with medical and veterinary importance. Most bacteriocins produced by staphylococci are either lantibiotics (e.g., Pep5, epidermin, epilancin K7, epicidin 280, staphylococcin C55/BacR1, and nukacin ISK-1) or class II bacteriocins (e.g., aureocins A70 and 53). Only one staphylococcin belonging to class III, lysostaphin, has been described so far. Production of staphylococcins is a selfprotection mechanism that helps staphylococci to survive in their natural habitats. However, since these substances generally have a broad spectrum of activity, inhibiting several human and animal pathogens, they have potential biotechnological applications either as food preservatives or therapeutic agents. Due to the increasing consumer awareness of the risks derived not only from food-borne pathogens, but also from the artificial chemical preservatives used to control them, the interest in the discovery of natural food preservatives has increased considerably. The emergence and dissemination of antibiotic resistance among human and animal pathogens and their association with the use of antibiotics constitute a serious problem worldwide requiring effective measures for controlling their spread. Staphylococcins may be used, solely or in combination with other chemical agents, to avoid food contamination or spoilage and to prevent or treat bacterial infectious diseases. The use of combinations of antimicrobials is common in the clinical setting and expands the spectrum of organisms that can be targeted, prevents the emergence of resistant organisms, decreases toxicity by allowing lower doses of both agents and can result in synergistic inhibition.

Keywords: Bacteriocins, staphylococcins, staphylococci, Staphylococcus, antimicrobial peptides, antimicrobial agents. INTRODUCTION Antimicrobial peptides constitute a large variety of ribosomally synthesized substances widely distributed in nature, being produced by prokaryotes and eukaryotes [1, 2]. Amongst the most studied antimicrobial peptides there are the bacteriocins produced by bacteria. Bacteriocins (Bac) are classically defined as proteinaceous compounds with bactericidal activity against other bacteria [3, 4]. The bacteriocin producing strains have developed a protection system against their own bacteriocin, named immunity. Each bacteriocin has its immunity system, which is generally expressed concomitantly with the bacteriocin structural genes [3, 4]. The ribosomal synthesis and the presence of an immunity system differ bacteriocins from peptide antibiotics produced by modular non-ribosomal peptide synthetases and various other secondary metabolites produced by bacteria that possess antibacterial activity [3]. Bacteriocins produced by Gram-positive bacteria form a heterogeneous group of peptides and proteins. Recently, *Address correspondence to this author at the Instituto de Microbiologia – UFRJ, Departamento de Microbiologia Geral, CCS - Bloco I – sala I-1-059 Cidade Universitária, 21941-590, Rio de Janeiro, RJ, Brazil; Tel: 55 -212560-8344, branch line 149; Fax: 55 -21-2560-8344; E-mail: [email protected] ¶ All three authors contributed equally to this work.

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Heng et al. [4] proposed a revised classification scheme for them based on their biochemical characteristics, which is a modification of the classification scheme presented by Cotter et al. in 2005 [5]. This revised scheme is summarized in Table 1 and, according to it, bacteriocins produced by Grampositive bacteria can be grouped into four classes, most of them with subdivisions. Bacteriocins from classes I and II are the most studied and with better elucidated mode of action, since they occur more frequently and possess potential industrial and clinical applications [3-5]. A large number of bacteriocins from both classes have been characterized at the biochemical level due to the development of efficient and standardized protocols for purification of these peptides. Members of the two classes are clearly different, both in terms of the structure of the bacteriocin itself and in terms of the machinery involved in production and processing. Knowledge of both aspects of bacteriocin biology is required when considering bacteriocin applications. Although various reviews on bacteriocins produced by Gram-positive bacteria have been published over the years [3, 6-11], they generally cover either only or mostly the aspects related to those antimicrobial peptides produced by lactic acid bacteria, which are of eminent economic importance because of their widespread use in food and feed fermentation. Limited if any information is included on staphylococcal bacteriocins although they have proven to possess

© 2009 Bentham Science Publishers Ltd.

Staphylococcal Antimicrobial Peptides

Table 1.

Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 1

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Classification Scheme for Bacteriocins Produced by Gram-Positive Bacteria Proposed by Heng and Co-Workers [4]

Classification

Characteristics

Groups/Subclasses*

Examples

Class I (lantibiotics)

Small peptides (50 μg/ml of nukacin ISK-1). The expression of either nukFEG or nukH alone conferred only partial immunity against nukacin ISK-1 (MICs of 6.25 μg/ml and 3.13 μg/ml, respectively) [76]. Quantitative analysis using dot blot hybridization showed that the difference in immunity levels was not due to a difference in the transcriptional level of nukF, -E, -G, and –H. These findings suggest that NukH contributes cooperatively to host immunity with NukFEG, as previously described for epidermin and gallidermin [76]. Since expression of nukFEG resulted in higher degree of immunity than did expression of nukH, this finding suggests that the NukFEG complex plays a major role in the self-protection system and that NukH might function as an accessory protein for NukFEG [76]. The nukacin ISK-1 immunity system also conferred immunity to lacticin 481, but not to nisin A, the prototype typeA(I) lantibiotic, which is produced by Lactococcus lactis subsp. lactis [76]. Western blot analysis showed that NukH was located at the membrane when expressed in Staphylococcus carnosus TM300. Quantitative peptide release/bind assays using L. lactis NZ9000 recombinant expressing nuk FEGH, nukFEG, and nukH revealed that the NukFEG complex extruded nukacin ISK-1 from the cell membrane and that the NukH expression contributed to cell-binding of nukacin ISK-1 even in the presence of NukFEG. The nukHexpressing strain bound nukacin ISK-1 and lacticin 481, but not nisin A [76]. Topological analysis indicated that NukH possesses two external loops, small (L1) and large (L2), and three transmembrane helices (H1, H2, and H3). The N terminus and L2 contain almost all positively-charged amino acids found in NukH. Therefore, these segments were predicted to be localized in the cytoplasm according to the positive-inside rule. Such prediction was confirmed by NukHPhoA in-frame fusion analyses combined with protease K susceptibility experiments [77]. Deletion of either the N or C terminus of NukH did not affect its binding activity and immunity function. Amino acid substitutions in either L1 or L2 abolished its immunity function; the binding activity was also decreased compared to NukH. The substitutions in L1, which is localized at the cytoplasmic membrane surface, resulted in a lower degree of binding activity than those in L2, suggesting that L1 rather than L2 would play a major role in the binding activity. Deletion of H3 and/or H2 abolished the immunity function, however, deletion of H3 did not affect the binding activity. These findings suggested that the whole structure of NukH, except for its N and C termini, is essential for its full immunity function, and that NukH inactivates nukacin ISK-1 after binding of the lantibiotic [77]. Recently, the cooperative mechanism between NukFEG and NukH was characterized by using fluorescein-4isothiocyanate (FITC)-labeled nukacin ISK-1 (FITC-nuk) to clarify the localization of nukacin ISK-1 in the immunity process [78]. L. lactis recombinants expressing nukFEGH, nukFEG, or nukH showed immunity against FITC-nuk, suggesting that FITC-nuk was recognized by the self-protection system against nukacin ISK-1. Interaction analysis between FITC-nuk and energy-deprived cells of the L. lactis recom-


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binants showed that FITC-nuk specifically binds to cells expressing nukH. The interaction between FITC-nuk and nukH-expressing cells was inhibited by the addition of nonlabeled nukacin ISK-1 and its derivatives with deletions of the N-terminal tail region, but not by the addition of synthesized N-terminal tail region. This suggests that the NukH protein recognizes the C-terminal ring region of nukacin ISK-1. Addition of glucose to nukFEGH-expressing cells treated with FITC-nuk resulted in a time-dependent decrease in the fluorescence intensity, indicating that FITC-nuk was transported from the cell membrane by the NukFEG protein complex. These results revealed that after being captured by NukH in an energy-independent manner, nukacin ISK-1 was transported to the extracellular space by NukFEG in an energy-dependent manner [78]. Since Nukadoko contains a large amount of salts, S. warneri ISK-1 is adapted to grow under this condition. The effects of NaCl, KCl and sorbitol concentration on nukacin ISK-1 production were then analyzed in batch fermentation [74]. In addition, the relationship between the osmolarity of the culture broth and nukacin ISK-1 production was examined at the level of transcription of the nuk genes. The lantibiotic production was stimulated by osmotic stress [74]. Under these conditions, the transcriptional levels of the nukA gene and the nukM-H operon were increased by 2.5 fold and 4.0 fold, respectively [73, 74]. To understand the mechanism of transcriptional enhancement, Aso et al. [73] examined the effects of nukacin ISK-1 on the transcriptional level of nukA, which was not significantly affected by addition of the partially-purified bacteriocin to the growth medium. Therefore, production of nukacin ISK-1 is not under the control of an autoregulatory circuit. The putative orf1 product exhibited homology to LanR proteins (RR). As already mentioned, the lanR genes, together with lanK, are part of two-component regulatory systems found in some lantibiotic gene clusters. However, no ORF corresponding to a putative lanK gene was found in the vicinity of the nukacin ISK-1 gene cluster [73, 76]. The orf1 product has an N-terminal RR receiver domain, but lacks the C-terminal transcriptional regulator domain generally found in these molecules. Moreover, the residues Asp-13, Asp-57 and Lys-109, conserved among all RR sequences, were not found in the deduced amino acid sequence of the orf1 product. The regulatory function of orf1 has not been elucidated yet [71, 73]. A staphylococcin closely related to nukacin ISK-1 is warnericin RB4, produced by S. warneri RB4. The purified mature peptide exhibited an optimal pH range between 2.0 and 6.0 and a remarkable resistance to heat treatment, loosing only 50% of its activity after 15 min at 121 ºC. It contained also 27 residues and its Mr was estimated to be 2,958.2 Da [79]. Simulancin 3299 Simulancin 3299 is the first BLIS produced by S. simulans described in the literature [80]. Its producing strain, S. simulans 3299, was associated with bovine mastitis in Brazil. Simulancin 3299 has been purified by standard techniques and its Mr has been estimated to be 3.0 kDa by mass spectrometry analysis [unpublished data]. Amino acid sequencing

46 Current Pharmaceutical Biotechnology, 2009, Vol. 10, No. 1

by Edman degradation was blocked after the seventh amino acid, which suggested that simulancin 3299 might be a lantibiotic. Its primary structure and analysis of its gene cluster are currently under investigation by our group. This BLIS has been proven to exhibit potential practical applications as discussed below. STAPHYLOCOCCINS PRODUCED LASE-POSITIVE STAPHYLOCOCCI

BY

COAGU-

Bacteriocin production has been reported in the literature for S. aureus strains. Some of them are class II bacteriocins. Class II bacteriocins are heat-stable and unmodified peptides. Most of them are also synthesized with an N-terminal leader sequence which is removed upon secretion. In most cases, the double-glycine type of leader is present [6]. However, it has also been found that some class II bacteriocins are translated with sec-dependent leaders, while others are not processed at all [6, 81, 82]. In most cases, at least four genes are required for the production of class II bacteriocins [6]: (i) the structural gene encoding the prepeptide; (ii) the immunity gene; (iii) a gene encoding an ABC transporter necessary for bacteriocin externalization concomitant with processing of the leader sequence; and (iv) a gene encoding an accessory protein, whose role is not entirely clear, although it may function to aid the passage of the bacteriocin precursor through the ABC transporter [4]. The class II bacteriocin genes are most often arranged in one or two operons [3, 6]. Production of some class II bacteriocins is also regulated and this regulation is generally achieved through threecomponent regulatory systems, which encode a regulatory signal, a PHK and a RR [6, 8, 11]. In most cases, the regulatory signal is a bacteriocin-like peptide which is generally encoded by the first ORF of the regulatory operon. The regulatory peptide, also called induction peptide (IP), shares most of the physico-chemical properties of a regular class II bacteriocin. The IP is required not only for the production of bacteriocin synthesis but is also needed to maintain bacteriocin production [6]. Class II bacteriocins, with emphasis to those substances produced by lactic acid bacteria, have also been reviewed extensively over the years [3, 6, 8, 11]. Bacteriocins Produced by S. aureus but Only Partially Characterized The staphylococcins described below have not been studied in detail. Thus, only limited biochemical and genetic information is available about them. However, most of them have been proven to possess potential practical applications. Staphylococcin 414 Staphylococcin 414 is one of the first staphylococcins described with a broad spectrum of activity [17]. Since it was not found in the supernatant of a broth culture of the producing strain, a partial purification of this bacteriocin involved fragmentation of the producer cells at high pressure, differential centrifugation and Sephadex G-200 gel filtration. Upon isolation, staphylococcin 414 appeared to be a complex with a Mr larger than 200 kDa. However, the complex could be dissociated by SDS into smaller subunits of about 12.5 kDa, which still resulted in activity. The proteinaceous nature of this bacteriocin was confirmed due to its inactiva-

Bastos et al.

tion after treatment with proteolytic enzymes. Staphylococcin 414 proved to be stable when heated to temperatures lower than 70 ºC. However, when submitted to 100 ºC for 30 min, half of its activity was lost. Staphylococcin 462 In 1973, Hale and Hinsdill reported the isolation of staphylococcin 462 [83]. This BLIS is produced by S. aureus 462, a strain selected amongst 200 staphylococcal isolates of animal origin [17]. No appreciable staphylococcin activity was found in the culture supernatant fluids obtained from strain 462. The activity remained with the pelleted cells. However, extraction of the cells with 7M urea resulted in liberation of much of the activity. The material was then purified by chromatography on Sephadex G-200 gel [83]. Staphylococcin 462 was shown to be sensitive to heating. Treatment of staphylococcin 462 with proteolytic enzymes resulted in a partial loss of activity. Its Mr was estimated to be 9 kDa [83]. Staphylococcin IYS2 Nakamura et al. [21] have studied the inhibitory potential of staphylococcal strains isolated from human saliva. S. aureus strain IYS2 exhibited the largest inhibitory zone against the indicator strain. The inhibitory substance was then purified by ammonium sulfate precipitation, fractionation with ethanol, ion-exchange chromatography and gel filtration. Its Mr and pI were estimated to be 5 kDa and 10.0, respectively. Although the primary structure of this BLIS was not determined, its amino acid composition revealed the predominance of Lys, His, Asp, Val and Phe residues. No reduction in activity was detected even when staphylococcin IYS2 was heated at 100ºC for 15 min. However, lost of 75% activity was detected when this BLIS was submitted to heating at 120ºC, for 15 min. Staphylococcin IYS2 was completely inactivated by proteolytic enzymes. Staphylococcin Au-26 Staphylococcin Au-26 is a BLIS produced by a vaginal S. aureus strain [84]. It was obtained from liquid cultures containing 0.1% of Tween 80 and purified by chromatographic fractionation on XAD-2, carboxymethyl Sephadex and reversed-phase HPLC. Its Mr was estimated by SDS-PAGE to be 2.7 kDa. Lan residues were detected in the molecule, indicating that staphylococcin Au-26 is a lantibiotic. Other characteristics presented by staphylococcin Au-26, such as the high stability to heating at acidic pH and its inactivation by proteases, are also shared by lantibiotic peptides. The Nterminus of the molecule is represented by an Ile residue, a characteristic also displayed by the lantibiotics epidermin and gallidermin. According to the authors, this bacteriocin was the first lantibiotic reported to be produced by a S. aureus strain [84]. Bac1829 Bac1829 is a BLIS produced by S. aureus KSI1829 [25]. The optimized procedure for Bac1829 purification involved ammonium sulfate precipitation, isoelectric focusing and HPLC using a propyl hydrophobic-interaction column [85]. The peptide was heat stable. Analysis of the purified preparation of Bac1829 by mass spectrometry revealed that the peptide had a Mr of 6,418 Da. Amino acid analysis showed a


high concentration of Ala (14), Gly (11) and Thr (six) residues. As expected, high levels of hydrophobic amino acids were also present, accounting for the hydrophobic nature of this BLIS [25, 85]. Bac201 This BLIS is produced by S. aureus AB201, a clinical strain isolated from a wound [86]. The substance was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and reversed-phase HPLC [87]. Bac201 was sensitive to proteolytic enzymes, resistant to heat and organic solvents, and active over a wide range of pH (2.5-10.0). It could also be stored at –20oC without loss of activity. The purified Bac201 migrated as a single band on SDS-PAGE with an estimated Mr of 41 kDa [86, 87]. The amino acid composition revealed the predominance of Gly (39%), Pro (13%) and Ala (8%) residues [87]. Staphylococcin 188 Staphylococcin 188 was isolated from S. aureus AB188, also a clinical isolate from a wound [88]. After partial purification, it showed a remarkable stability over a wide range of pH (pH 2.0–14.0) and temperatures (37-121oC and +4 to 20oC) and sensitivity to proteolytic enzymes [24]. This BLIS was purified to homogeneity by 80% ammonium sulfate precipitation and conventional size exclusion gel chromatography using a Sephadex G-75 column [89]. Although the separation profile resulted in two major and well separated peaks, the major antimicrobial activity was detected in peak II. Staphylococcin 188 had an estimated Mr of 4 kDa [89]. Fully-Characterized Bacteriocins Produced by CPS Staphylococcin C55/BacR1 Several groups have demonstrated that bacteriocin production is especially common in S. aureus belonging to phage group II, particularly in strains of phage type 71 [19, 90-93]. Dajani et al. partially purified an inhibitory agent from cultures of S. aureus C55, a skin isolate that they adopted as the prototype of the phage group II bacteriocin producers [90, 91]. The purification protocol essentially involved ammonium sulfate precipitation followed by gel filtration on Sephadex G-100. The antimicrobial substance could be stored at -20 oC for several weeks without appreciable reduction in activity. Only a slight reduction occurred when the substance was boiled for 15 min. Activity was comparable in various samples in which the pH was adjusted over a range of 4.0 to 8.5. Proteolytic enzymes totally abolished the inhibitory activity [90]. Later, Navaratna et al. [94] re-examined the nature of the bactericidal activity of S. aureus C55 and demonstrated that this strain produces a bacteriocin named staphylococcin C55. Staphylococcin C55 became the first S. aureus bacteriocin to have its primary structure completely elucidated and it was also the first twocomponent bacteriocin described in staphylococci. Staphylococcin C55 is composed of two distinct peptide components, staphylococcins C55 and C55. These peptides were identified during C8 reversed-phase chromatography, where the inhibitory activity against the target microorganism, Micrococcus luteus, was detected in two well-separated fractions. When combined in approximately equimolar amounts, staphylococcins C55 and C55 acted synergistically to kill


47

S. aureus and M. luteus, but not S. epidermidis [94]. The Nterminal amino acid sequences of both peptides were determined and staphylococcins C55 and C55 were shown to be lantibiotics with Mr of 3,339 Da and 2,993 Da, respectively (Table 2) [94]. A plasmid of 32 kb carries the genetic determinants for staphylococcin C55 production and immunity to its action [95, 96]. A fragment of 6,276 bp from this plasmid was cloned and sequenced. Computer analysis revealed the presence of four genes in the same orientation, sacA, sac A, sacM1, and sacT (Fig. (2)). The previously determined C55 peptide sequence was consistent with the deduced amino acid sequence of the sacA gene. A second lantibiotic structural gene, sacA, was found immediately downstream of sacA, and its deduced amino acid sequence agrees with the C55 sequence obtained by N-terminal sequencing. When compared with the lantibiotics produced by CNS, marked differences were found, showing that neither staphylococcin C55 nor C55 was related to any of those lantibiotics. However, a very high homology was detected with the twocomponent lantibiotic system of lacticin 3147, a bacteriocin produced by L. lactis [97]. sacM1 encodes a putative protein with homology with the lanM gene found in gene clusters of some lantibiotics. sacT encodes a protein with strong homologies with the genes for several ABC transporters, which have been shown to be involved in both transport and processing of this type of lantibiotic [96, 97]. Additional genes involved in staphylococcin C55 production, sacM2 and sacJ, were recently reported by O’Connor et al. [98]. The staphylococcin C55 ORFs (Fig. (2)) are arranged identically to the corresponding ORFs which encode lacticin 3147 [96, 98]. The two structural peptides of lactacin 3147 are LtnA1 (3,306 Da) and LtnA2 (2,847 Da) [99]. Staphylococcin C55 and LtnA1 resemble natural variants with a difference of only 4 aa (86% identity) between the two peptides, while the complementary peptides, C55 and LtnA2, share less identity (55%) [98]. When the lacticin 3147 and the staphylococcin C55 peptides were cross combined, complementation was observed in the single nanomolar range, comparing well the native pairings [98]. In addition to the structural peptides, the proposed modification and transport proteins exhibit significant homology. The lacticin 3147 modification peptides, LtnM1 and LtnM2, share 44.7 and 40.7% identity to their staphylococcin counterparts SacM1 and SacM2, respectively. The transport peptides lacticin LtnT and staphylococcin SacT are 48.9 identical, while lacticin LtnJ and staphylococcin SacJ share 47% identity [98]. The lanJ gene is found in a minority of lantibiotic gene clusters. The presence of the sacJ gene in staphylococcin C55 gene cluster raised concerns with respect to the identity of the amino acid at position 7 of C55 [98]. It may be that this residue is a D-Ala, as is the case for LtnA1, rather than the Dha reported previously [94]. This suggestion is based on the fact that amino acid analysis predicts the presence of three Ala residues in the mature form of C55 and that the staphylococcin C55 operon contains an LtnJ homolog [98]. D-Ala has already been detected in other lantibiotics such as lactocin S [100] and lacticin 3147 [99]. Analysis of the genetic sequence predicted Ser residues in certain positions in the propeptide of these lantibiotics, as observed for


C55 (Table 2), whereas D-Ala was detected in the mature peptide, suggesting the conversion of L-Ser into D-Ala. The D-Ala residue is proposed to arise from the stereospecific hydrogenation of a Dha residue, probably a function of the LanJ protein [99]. BacR1 is produced by S. aureus UT0007, a phage group II clinical isolate that also produces the exfoliative toxin B [92]. BacR1 was obtained from a culture supernatant of the producing strain after precipitation by ammonium sulfate, cation-exchange column and C4 reversed-phase chromatography [26]. The mass of the purified peptide was reported to be 3,338 Da, a size close to that of staphylococcin C55. Purified BacR1 was resistant to a wide range of environmental conditions, including extremes of temperature and maintained full activity even after exposure for 15 min to temperatures from –20 to 95 ºC and pH from 3.0 to 11.0 [19, 26]. Amino acid sequence analysis revealed a high molar concentration of Gly, Ala and Pro residues [26]. In 1999, Navaratna et al. established that staphylococcin C55 and BacR1 are identical and that this type of bacteriocin is widely distributed in phage group II S. aureus [96]. The gene cluster involved in BacR1 production is also carried on a large plasmid, pRW001, associated with the production of exfoliative toxin B [26, 92, 96]. Using a codon usage table made from the staphylococcal genomic sequence information, the peptide amino acid sequence was backtranslated to construct an oligonucleotide probe. The probe was then used to identify a fragment of the pRW001 plasmid believed to contain the BacR1 structural gene. The BacR1 gene cluster was identified by homology between the putative gene products and entries in the GenBank database. As expected, the BacR1 gene cluster is quite similar to that of staphylococcin C55 [101]. Aureocin A70 Aureocin A70 is a class II bacteriocin produced by S. aureus A70, a strain isolated from commercial milk [102]. The genetic determinants for aureocin A70 production are located on a 8.0-kb plasmid, pRJ6 [102, 103]. There are at

Bastos et al.

least two transcriptional units involved in bacteriocin activity (Fig. (3)). The first operon is composed of four genes, aurABCD, which represent the aureocin A70 structural genes. This operon encodes four peptides that are small (3031residues), strongly cationic (with pI of 9.85 to 10.04), and also highly hydrophobic (Table 2). These peptides have also a high content of small amino acid residues like Gly and Ala, and no Cys residue. Purification of these peptides, followed by mass spectrometry analysis, revealed that these peptides are not processed. Aureocin is the first and unique fourcomponent bacteriocin described in the literature [81]. The individual inhibitory activity of each chemically-synthesized peptide was tested on solid medium and depended on the target microorganism. When they were tested against Listeria innocua and S. aureus strains, inhibition was only observed when all four peptides were combined together (25 μg each). Against M. luteus, AurA, AurB, and AurC exhibited inhibitory activity when tested alone (60 μg) with a decreasing effectiveness as follows, AurA>AurB>AurC; AurD alone was not bioactive. However, combination of all four peptides resulted in larger inhibition zones [unpublished results]. The second transcriptional unit contains only one gene, aurT, which encodes a 571-aa protein that resembles an ATP-dependent transporter, similar to that involved in Pep5 export [81]. Consistent with the absence of a leader sequence in aureocin A70 peptides is the finding that AurT does not contain the N-terminally located proteolytic domain responsible for cleavage at the processing sites generally found in class II bacteriocins. AurT is essential for aureocin A70 production [81]. A gene encoding an accessory protein and generally found in class II bacteriocin gene clusters is missing in pRJ6. A second putative operon is also found on pRJ6 and seems to contain two ORFs: the first gene, orfA, is predicted to encode a hydrophilic 82-aa protein similar to small repressor proteins and to CylR2 (46% identity and 66% similarity). CylR2 is a 66-aa peptide that, together with CylR1, is part of a quorum sensing system involved in the regulation

Fig. (3). Representative biosynthetic gene clusters of the class II bacteriocins produced by S. aureus: aureocin A70 [81] and aureocin A53 [82]. The structural genes are represented in black. The number under each gene designation indicates the number of amino acid residues of each gene product. Promoters for the transcriptional units in these clusters are indicated by arrows and transcriptional terminators by lollipops, where known. The genes are not drawn to scale.


of the expression of a lantibiotic with cytolysin properties produced by Enterococcus faecalis [104]. The second putative gene, orfB, codes for a protein with 138 residues which presents a high pI and a hydrophobicity profile similar to those of proteins associated with bacteriocin immunity [81]. Another putative gene, orfM, was recently found in the vicinity of the aureocin A70 structural genes, preceding the aurABCD operon (Fig. (3)). It encodes a 102-aa putative protein, with a pI of 10.02. Although cationic as most immunity proteins, it lacks transmembrane domains generally found in these proteins. No homology was detected between the orfB or orfM products and other proteins in databases. The role of OrfA, OrfB, and OrfM is presently under investigation. Aureocin A70 and its variants seem to be the most frequently produced bacteriocin by Bac+ S. aureus strains [16]. Among 363 strains of S. aureus tested by our group, 21 were shown to produce bacteriocins. This collection includes strains either isolated from food, patients and healthy cattle or involved in sub-clinical bovine mastitis [102, 103, 105107]. From these 21 strains, 17 were shown to produce bacteriocins either identical or similar to aureocin A70 [108]. Its production has also been detected in CNS [80]. The high dissemination of aureocin A70 production in the staphylococcal Bac+ population is consistent with our previous data which showed that pRJ6 and its closely-related plasmids can be mobilizable by staphylococcal conjugative plasmids [103, 105]. Aureocin A53 Aureocin A53 is a bacteriocin produced by S. aureus A53, a strain also isolated from commercial milk [102]. Aureocin A53 is a highly cationic peptide (pI of 10.5) of 51 aa containing ten Lys and five Trp residues (Table 2) [82]. Aureocin A53 was purified and mass spectrometry analysis yielded a Mr of 6,021.5 Da, which was 28 Da higher than predicted from the structural gene sequence of the bacteriocin. The mass increment resulted from an N-formylmethionine residue, indicating that aureocin A53 is synthesized and secreted without a typical bacteriocin leader sequence or sec-dependent signal peptide. Further unique features that distinguish aureocin A53 from other peptide bacteriocins include remarkable protease stability and a defined, rigid structure in aqueous solution [82, 102]. An ordered structure in aqueous solution consisting of 36% ± 5% helical and 18% ± 4% -sheet conformation was observed by circular-dichroism spectroscopy [82]. Aureocin A53 is encoded by a 10.4-kb plasmid, pRJ9 [102]. Few ORFs identified in the vicinity of the bacteriocin structural gene, aucA, showed similarity to genes typically found in bacteriocin gene clusters previously described (Fig. (3)). Three of them, aucF aucE and aucG, constitute an operon, and their products possess similarity to threecomponent ABC transporters [82]. Cloning experiments using heterologous hosts showed the involvement of these three genes in conferring partial immunity to aureocin A53 [109]. Since no other dedicated transporter can be found in the aureocin A53 gene cluster, it is assumed that aucFEG is also involved in bacteriocin secretion. Two ORFs, orf10 and orf11, probably arranged as an operon, are found between aucA and the operon aucFEG. They encode putative proteins


49

of 96 aa and 94 aa, respectively, both with unknown functions. Their role in complementing the partial immunity conferred by AucFEG is currently under investigation. Upstream of aucA, two ORFs, orf7 and orf8, are found with an organization which resembles an operon [82]. Genes homologous to orf7 and orf8 were found in context with the alanineracemase operon [82]. In S. aureus, the alanine-racemase operon is essential to the alanylation of lipoteichoic acids and teichoic acids of the bacterial cell wall. The degree of alanylation of the anionic cell wall polymers determines the overall negative charge in the cell wall and has a significant impact on the susceptibility of Gram-positive bacteria for cationic peptides [3, 6, 7, 9, 10]. Recent experiments suggest that neither orf7 nor orf8 gene products seem to be essential for aureocin A53 production in a heterologous host [unpublished data]. Recently, new bacteriocins with homology to aureocin A53 were discovered. The first one is lacticin Z, produced by the L. lactis strain QU 14 isolated from horse intestinal tract [110]. After purification, mass spectrometry assays have determined the Mr of lacticin Z to be 5,968.9 Da. The second bacteriocin homologous to aureocin A53, termed lacticin Q, is produced by L. lactis strain QU 5, isolated from corn [111]. Its Mr was determined to be 5,926.50 Da. Both bacteriocins share 45% identity to aureocin A53, consist of 53 residues and the initiation amino acid of the purified peptides is also a N-formylated Met residue [110, 111]. CLASS III BACTERIOCINS STAPHYLOCOCCI

PRODUCED

BY

Class III bacteriocins include large peptides (M r 25 kDa) which are generally heat-labile. This class of bacteriocins was further subdivided by Heng et al. into two distinct groups: (i) the bacteriolytic enzymes (or bacteriolysins) and (ii) the non-lytic antimicrobial proteins [4]. Staphylococci have been shown to produce bacteriolysins, from which lysostaphin is considered to be the prototype. Lysostaphin is an extracellular enzyme secreted by S. simulans biovar staphylolyticus ATCC1362 [112]. The peptidoglycan of Gram-positive microorganisms, an important component of the bacterial cell wall, is hydrolyzed at specific times and sites during physiological growth of the exoskeleton [113]. This is accomplished by murein hydrolases. Lysostaphin is one example of such enzymes. Most of these enzymes display a domain structure [114]. In general, murein hydrolases harbor an Nterminal signal peptide followed by a second domain containing the enzymatic activity. In addition, these proteins harbor repeated sequences that flank either the N- or Cterminal side of the enzymatic domain. Lysostaphin is a zinc-containing metallo enzyme with a molecular weight of 25 kDa, a pI of 9.5 and a pH optimum of 7.5 [115, 116], synthesized as a preproenzyme of 493 aa and initiated into the secretory pathway by an Nterminal leader peptide of 36 aa (Fig. (4)). The proenzyme is released into the culture medium and contains 15 tandem repeats of 13 aa length at the N-terminal end [117]. Prolysostaphin is 4.5-fold less active than mature lysostaphin, and the N-terminal repeats are removed in a growth phase-dependent manner by a secreted cystein protease to


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Fig. (4). A. Domain structure of the preprolysostaphyn (493 aa). B. Lysostaphin is synthesized as a preproenzyme, and after signal peptide removal, soluble prolysostaphin is released into the extracellular medium. Proteolytic cleavage of the propeptide of 211 aa, from which 195 aa are organized in 15 tandem repeats of 13-aa length, generate mature lysostaphin, which is biologically active, functioning as a glycilglycine endopeptidase against susceptible cells [118]. The lysostaphin molecule is predicted to consist of two distinct domains: a N-terminal peptidase domain (PD) responsible for its catalytic activity and the C-terminal wall targeting domain (CWT), which directs lysostaphin to its receptor on the staphylococcal surface [119].

yield the fully-activated lysostaphin molecule [118].The lysostaphin molecule seems to consist of two distinct domains: (i) an N-terminal peptidase domain responsible for the catalytic activity of the protein and (ii) a C-terminal targeting domain involved in binding to the peptidoglycan substrate [119]. Unlike peptide-bacteriocins, bacteriolysin producers do not always have specific immunity genes, but might rely on resistance mechanisms which result in modifications of the producer cell wall (see below). The lysostaphin endopeptidase gene (lss) and the gene involved in lysostaphin resistance (lif) reside on plasmid pACK1 [118, 120]. lss and lif are flanked by insertion sequences, suggesting that S. simulans biovar staphylolyticus received these genes by horizontal gene transfer [118]. A homolog of lysostaphin, ALE-1 from Staphylococcus capitis, has also been described and characterized [121]. The molecular structure and targeting mechanism of this enzyme, as well as the producer resistance, appear to be identical to those described for lysostaphin [121, 122]. MODE OF ACTION OF STAPHYLOCOCCINS Peptide Bacteriocins Type-A lantibiotics and unmodified bacteriocins exert their activity by disruption of the barrier function of mi-

crobial cytoplasmic membrane [3]. It is generally assumed that the peptides associate in oligodynamic aggregates that can form transient pores when oriented across the membrane [123]. Most peptide staphylococcins from classes I and II analyzed so far have been shown to be bactericidal to sensitive cells without a bacteriolytic effect [19, 21, 25, 124127]. Incubation of sensitive cells with increasing amounts of purified bacteriocin results in a typical dosedependent reduction in cell growth [25, 26, 127]. They cause rapid efflux of low M r compounds such as ions, amino acids and ATP [65, 125-130], thus causing a rapid breakdown of the membrane potential and a complete cessation of biosynthesis of DNA, RNA, protein, and polysaccharides. Subsequent studies showed that membrane depolarization occurs in a voltage-dependent manner upon treatment with Pep5 [131], epidermin and gallidermin [132]. The membrane disruption is initiated by the formation of aqueous membrane pores through which low molecular weight cytoplasmic organic compounds and inorganic ions equilibrate with the external medium [130]. Using intact bacterial cells and artificial membrane systems, pore formation was shown to require a transnegative membrane potential of 50-100 mV. The peptide induced voltage-gated short-living channels with varying pore sizes of up to 2 nm in planar membranes [131]. This mode of action has also been established for other staphylococcins such as epilancin K7 [123]. Pep5 forms pores


that work only in one direction (rectifying) [131], whereas gallidermin and epidermin form nonrectifying channels that are also more stable [132]. Additionally, Pep5 was shown to induce autolysis of staphylococci leading to cell wall breakdown at the septa between dividing cells. It is though that the interaction of the cationic peptide with the negatively-charged teichoic and lipoteichoic acids displaces and activates cell-wall hydrolyzing enzymes usually associated with the teichoic acids [133, 134]. Gallidermin and epidermin are able to kill bacteria through complex mechanisms of action which involve pore formation and cell wall inhibition [135]. Generally, their pore formation capacity is reduced when compared to other lantibiotics and depends on membrane thickness. They interact in a highly specific manner with the cell wall precursor lipid II [undecaprenyl-pyrophosphorylMurAc-(pentapeptide)-GlcNAc] for docking onto the membrane and subsequent pore formation [123, 135]. Since lipid II is the essential membrane-bound precursor for bacterial cell wall formation, these lantibiotics also inhibit peptidoglycan biosynthesis. Hence, the binding to lipid II alone constitutes a very potent antimicrobial activity in vivo. Although the possibility can be excluded that lipid II serves a similar function for Pep5 and epilancin K7, there are significant indications leading to the assumption that Pep5 similarly uses a different high-affinity receptor/docking molecule for its biological activity, which is presently unknown [10, 35, 123]. The type-A(II) lantibiotic nukacin ISK-1, whose structure is shown in Fig. (1), has three net positive charges and the anionic bacterial membrane is also the target for action of this staphylococcin [136]. The antibacterial activity and membrane binding of either nukacin ISK-1 or its fragments (tail region and ring region) and mutants were evaluated to delineate the determinants governing structure-function relationships [136]. No antibacterial activity of its ring or tail region (Fig. (1)) was observed; these regions showed no synergistic effect in combination, and neither of them had any effect on the activity of nukacin ISK-1. Both a fragment with three Lys in the N terminus deleted (nukacin4-27 ) and a mutant with these three Lys replaced by Ala (K1-3A nukacin) imparted very low activity (32-fold lower than nukacin ISK-1). In this same study, SPR determined by a BIAcore biosensor was used to investigate the binding behavior of nukacin ISK-1, its fragments and mutants carrying deletions, insertions or substitutions of amino acids to model membranes. Nukacin ISK-1 exhibited a remarkably higher binding affinity to an anionic model membrane than to a zwitterionic model membrane, but there was no binding of tail region, ring region, nukacin 4-27 , and K1-3A nukacin to the anionic model membrane. Binding affinities shown by SPR sensorgrams were significantly correlated with the observed antibacterial activities. Taken together, these results suggest that the complete structure of nukacin ISK-1 is necessary for its full activity, and that the first three Lys residues in the tail region [N-terminus; Fig. (1)] play the vital role in its antibacterial activity, in which the positive charges are the key determinant for strong membrane binding of nukacin ISK-1. Its antibacterial action might, therefore, be dependent on the Cterminus [136].


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Based on the similarities detected between staphylococcin C55 and lacticin 3147, both two-component lantibiotics, it is speculated that the staphylococcin C55 peptides display a dual mode of action similar to lacticin peptides [98]. The mechanism of action of lacticin 3147 has recently been shown to result from the sequential action of the two peptides, on condition that LtnA1 be added prior to LtnA2 [137]. It is predicted that, like LtnA1, C55 binds to lipid II, thereby inhibiting cell wall biosynthesis through the prevention of transglycosylation. The second function of pore formation and rapid efflux of ions is assigned to C55. C55 would interact with C55-lipid II complex to bring about more effective insertion into the target membrane and pore formation. Since C55 and LtnA2, sharing only 55% identity, were exchanged for each other in complementation studies with no loss of activity, such findings suggest that the region responsible for peptide:peptide interaction spans the 16-aa region at the C terminus of both peptides (81% identity), while the less conserved N-terminus of both C55 and LtnA2 is responsible for membrane insertion and pore formation [98]. Netz et al. [138] have investigated the mode of action of aureocin A53 on living bacterial cells and model membranes. Aureocin A53 acted bactericidally against S. simulans 22 with >90% of the cells killed within a few minutes. Cell death was followed by lysis. Aureocin A53 rapidly dissipated the membrane potential and simultaneously stopped the biosynthesis of DNA, polysaccharides, and proteins. When model membranes were employed, aureocin A53 provoked significant leakage of carboxyfluorescein exclusively from acidic liposomes but only at relatively high concentrations (0.5 to 8 mol %). Since aureocin A53 requires micromolar concentrations to kill bacteria and induce efflux from cells and negatively-charged liposomes, it is likely to produce membrane permeabilization through generalized membrane disruption rather than through formation of defined pores [138]. Lysostaphin Lysostaphin is a cell-wall lytic enzyme. The cell-wall degrading activity of lysostaphin is due to a glycil-glycine endopeptidase activity (Fig. (5)), which lyses many staphylococcal strains [139]. The target of the lysostaphin is the pentaglycine crossbridge of the peptidoglycan, which in S. aureus, S. simulans, S. carnosus, and other staphylococcal strains is composed of five Gly residues [140, 141]. Lysostaphin seems to cleave specifically between the third and the fourth Gly residues of the pentaglycine cross-bridge [142]. Therefore, by hydrolyzing the cell wall, lysostaphin kills the sensitive cells. S. simulans biovar staphylolyticus peptidoglycan is resistant to the hydrolytic activity of lysostaphin, since the cells elaborate a resistance factor that causes the incorporation of Ser residues into the cell wall crossbridge [118]. If one or more Gly residues of the interpeptide bridge are replaced by Ser residues, the cell wall becomes less susceptible to lysostaphin [118, 143]. Lysostaphin is unable to hydrolyze glycilserine and serylglycine peptide bonds [143]. It was also shown that lysostaphin binds much


more avidly to S. aureus, which possesses a pentaglycine cross-bridge, than to S. simulans cells. When added to mixed bacterial populations, purified lysostaphin kills 1,000 S. aureus cells for every S. simulans cell [144].

Fig. (5). Site of hydrolysis of lysostaphin on the staphylococcal peptidoglycan [142]. NacG, N-acetylglucosamine; NacM, N-acetylmuramic acid; A, L-alanine; D-iQ, D-isoglutamine; K, L-lysine; DA, D-alanine; G, L-glycine.

The S. simulans gene lif (lysostaphin immunity factor) is required for incorporation of Ser residues into the peptidoglycan [118]. Lif shows similarity to FemA and FemB proteins, which are involved in the biosynthesis of the Gly interbridge of the staphylococcal peptidoglycan [118]. POTENTIAL BIOTECHNOLOGICAL TIONS OF STAPHYLOCOCCINS

APPLICA-

The primary role of bacteriocins is to provide the producing bacteria with an advantage over their competitive organisms occupying the same ecological niche [3]. However, this antagonistic activity may be exploited for biotechnological applications. Most staphylococcins inhibit many bacterial species (Table 3), including several pathogens, and, therefore, may possess potential practical applications, either in the food industry, as alternatives to chemical preservatives, or in medicine, in the prevention or in the treatment of bacterial infectious diseases. The same procedures used to detect bacteriocinproducing strains can be used to determine the inhibitory spectrum of a given staphylococcin. Alternatively, when partially or totally purified bacteriocin preparations are available, the critical dilution assay may also be employed. In this method, a 100-μl volume of culture medium, bacteriocin fractions at two-fold dilutions, and the target strain are added to each well of a microtiter plate. After incubation at the adequate temperature, the growth of the target strain is measured spectrophotometrically at 600 nm. One bacteriocin arbitrary unit (AU) is defined as the amount of bacteriocin that inhibits the bacterial growth by 50% (50% of the turbidity of the control culture without bacteriocin) [145]. Food Preservation Utilization of the competitive activity of bacteria has practical applications especially in food industry. For instance, many lactic acid bacteria produce bacteriocins that prevent the growth of various food-spoilage organisms and

Bastos et al.

food-borne pathogens [5, 146-148] and have been commercially exploited for their application in food preservation. Nowadays, industrial food producers face a major challenge when it comes to food preservation against pathogen development. The current preservation methods based on potentially-toxic chemical compounds are being questioned by consumers. The demands for safe foods with long shelf-life, that do not contain chemical preservatives, can be perceived worldwide. Another food problem is the possible dissemination of important pathogens such as Listeria monocytogenes, the etiological agent of listeriosis [149]. L. monocytogenes is quite hardy and resists the deleterious effects of freezing, drying, and heat remarkably well for a bacterium that does not form spores. L. monocytogens is the cause of approximately 25% of deaths originated by food-borne pathogens in the USA annually [149]. In consequence of this alarming rate, this organism acquired the zero-tolerance standards for ready-to-eat (RTE) food [149], and, in 2003, the Food Safety and Inspection Service announced a ruling that required the manufacturers of RTE food to take further steps to address the problem posed by the presence of L. monocytogenes in consumables [150]. This ruling encourages the implementation of technologies that can kill the bacteria or prevent its growth after cooking or packaging. Some bacteriocins are able to inhibit this microorganism [5, 145, 147, 148]. Other factors that have contributed to the increasing in applied research on bacteriocins are: (i) the safe use of nisin as a food preservative for over 40 years and its approval by the FDA (Food and Drug Administration, USA) as a GRAS (generally recognized as safe) substance; (ii) realization that bacteriocinogenic strains have been isolated from a variety of food products of industrial and natural origins and, therefore, can be considered safe; and (iii) the availability of molecular biology tools to engineer genetic variants of bacteriocins to alter stability, host range or other properties [5, 145, 147, 148]. Staphylococcins have potential to be used as food preservatives. Aureocins A70 and A53, both produced by S. aureus strains originally isolated from commercial milk in Brazil, are bacteriocins that exhibit anti-listerial activity, although they do not contain the Tyr-Gly-Asn-Gly-Val/Leu motif highly conserved and generally found near the Nterminus of anti-listerial bacteriocins [81, 82]. Both aureocins seem to be produced constitutively and do not demand rich media to be produced, what makes them cheaper for a large scale production prior to their application in food industry [23]. Yet, both aureocins are quite stable at either room or high temperatures (15 min at 80 oC or 5 min at 100 °C), showing no detectable decrease in their activities under theses conditions [unpublished data], and exhibit resistance to high salt concentrations [23]. Moreover, the relatively low complexity of their gene clusters, associated to the fact that both staphylococcins do not need post-translational modifications to become active [81, 82], could facilitate cloning and expression of their genes into bacterial strains used as starter cultures in fermentative processes adopted in some food preparations. By this approach, the bacteriocins would be produced in situ, discarding the necessity of their further addition to the final product. Preliminary studies performed with aureocin A53 showed that its gene cluster can be ex-


pressed heterologously in lactobacilli strains, resulting in bacteriocin production [unpublished data]. Another staphylococcin with potential use in food industry is warnericin RB4, a lantibiotic closely related to nukacin ISK-1 [79]. This bacteriocin is capable of inhibiting the growth of Alicyclobacillus acidoterrestris, a novel Grampositive thermoacidophilic spore-former that has been recognized as a food-spoilage microorganism [79, 151, 152]. This microorganism is considered one of the most important target organisms in quality control of fruit juices and fruit juice-containing drinks, because of its ability to germinate and outgrow spores under highly acidic conditions (pH 91% of the strains. Pep5 inhibited only S. aureus strains (63%), but not the streptococcal ones. Interestingly, it was also found that a combination of aureocins A70 and A53 enhanced the inhibitory spectrum reaching 93% of all strains tested. This synergism between both aureocins was more profound in relation to the streptococcal strains, since the combination of aureocins A53 and A70 inhibited 92% of the strains, while each bacteriocin alone inhibited only 68% and 1.4% of the strains, respectively. The amount of each bacteriocin needed for the combined inhibitory activity was as low as 20 AU/ml [39]. Another staphylococcin currently under investigation and with great potential to be used to control streptococcal bovine mastitis is simulancin 3299, which was able to inhibit 80% of 74 strains of S. agalatiae tested [80]. Due to their ability to inhibit S. aureus strains, Bac1829 and BacR1/staphylococcin C55 may also find use in the prevention or treatment of bovine mastitis, although both staphylococcins have not been tested against bovine mastitis pathogens yet. To our knowledge, few bacteriocins have been described as having an effect against mastitis-causing pathogens. Nisin is used as an active agent in Wipe-Out, a teat wipe [5]. An-


Table 3.

Bastos et al.

Spectrum of Activity of Staphylococcins Against Human and Animal Bacterial Pathogens Relevant sensitive microorganisms

Staphylococcin(s) Gram-positive bacteria

Gram-negative bacteria and Mycobacteria

References

Bac1829, BacR1/Staphylococcin C55

S. aureus, Streptococcus suis, Corynebacterium diphteriae, Corynebacterium renale

Haemophilus parasuis, Bordetella pertussis, Bordetella bronchoseptica, Moraxella bovis, Pasteurella multocida

[25, 26, 94, 208]

BacR1/Staphylococcin C55

Enterococcus faecalis, Group-A haemolytic Streptococci, Streptococcus pneumoniae

Neisseria meningitidis, Neisseria gonorrhoeae,

[90, 98, 208]

Pep5

S. aureus, CNS, Corynebacterium spp.

[38, 39, 194, 198]

Epidermin/Staphylococcin 1580

S. aureus, S. epidermidis, Streoptoccus spp., Corynebacterium spp., Propionibacterium acnes

[38, 39, 51, 193, 194]

Staphylococcin T/Gallidermin

Staphylococcus spp., Streptococcus spp., Listeria spp., Corynebacterium spp., Clostridium spp.

Simulancin 3299

S. agalactiae, Corynebacterium spp.

[80]

Warnericin RB4

Alicyclobacillus acidoterrestris

[79]

S. warneri RK Bacteriocin

Neisseria spp., Moraxella spp.

Legionella pneumophila

[68]

[208]

Aureocin A70

L. monocytogenes, S. aureus, S. agalactiae, Corynebacterium spp.

Aureocin A53

L. monocytogenes, S. aureus, S. agalactiae, Corynebacterium spp.

M. bovis

[38, 39, 103]

Bac188

S. aureus, E. faecalis, S. pneumoniae, C. diphteriae

E. coli, Salmonella enterica serovar typhi, Shigella dysenteriae, Mycobacterium tuberculosis

[24, 87, 88,167]

Bac201

S. aureus, E. faecalis, S. agalactiae

N. meningitidis

[86]

Staphylococcin IYS2

S. aureus, Streptococcus salivarius, P. acnes, L. monocytogenes, Actinomyces israeli

-

[21]

Lysosthapin

S. aureus, S. epidermidis

-

[181, 182, 183, 184, 185, 190]

other bacteriocin successfully used in mastitis prevention is lacticin 3147, produced by L. lactis subsp. lactis strain DPC3147. A handful studies involving the use of this bacteriocin combined to teat seals revealed that it is very effective in protecting cows in the dry period from infections caused by the main agents involved in bovine mastitis [165, 166]. Bac188 was found to have an extremely broad spectrum of activity, being able to inhibit not only Gram-positive and Gram-negative pathogens (Table 3), as well as zoodermatophytes, including Microsporum canis and Microsporum gypseum [24, 88, 167]. Both Microsporum species are associated to skin, nail and hair infections in animals. Another interesting characteristic of Bac188, which makes it attractive for veterinary use, is its anti-viral activity against the avian New Castle Disease (NCD) virus [89]. The in vivo anti-NCD virus properties of Bac188 were investigated using the chick embryo technique. It was found that this BLIS was able to promote a marked reduction in the virus infectivity. For example, the ELD50 (50% egg lethal dose) in case of virus injected system was found to be 10-9 while in the presence of Bac188 the ELD50 of viruses was increased to 10-4 , indicating that in the presence of Bac188 a high viral load

[38, 39, 103]

was required to infect chick embryos. These preliminary results suggest that Bac188 possesses a strong inhibitory effect against the NCD virus, decreasing its infectivity at about 105 fold. New Castle Disease is a highly contagious viral disease of domestic poultry, cage and wild birds. NCD virus is infective for almost all avian species [168, 169]. The global economic impact of NCD is enormous. It certainly surpasses any other poultry virus disease and probably represents a bigger drain on the world’s economy than any other animal virus [168, 169]. In many developing countries, NCD is endemic and, therefore, represents an important limiting factor in the development of commercial poultry production and establishment of trade links. Many of these countries rely on village chickens to supply a significant portion of dietary protein in the form of eggs and meat. The constant losses from NCD severely affect the quantity and quality of the food of people on marginal diets. Therefore, the economic impact of NCD should not only be measured in direct commercial losses, but also, in some countries, in the effect on human health and loss of potential socioeconomic gain [170]. Given the disease importance, the use of Bac188 to


treat avian suffering from NCD could eliminated or minimize the heavy economic losses suffered by the poultry industry due to the infection of chickens by this virus. As a putative cheaper treatment, the use of this staphylococcin would be of great importance especially in the poor developing countries, where the vaccination of the poultry represents a huge expense for the small producers. Besides inhibiting the NCD virus, Bac188 inhibits also various human pathogens, being the first BLIS described which presents such broad potential clinical applications, including viral diseases [89]. Human Medical Use Among the most relevant human pathogens inhibited by staphylococcins, those involved in nosocomial infections deserve serious considerations. During the last century S. aureus has become the leading overall cause of nosocomial infections worldwide [12]. After introduction of the methicillin in 1961 to treat infections by penicillin-resistant S. aureus, there were reports of isolation of multi-resistant S. aureus [171]. Nowadays, strains of methicillin-resistant S. aureus (MRSA) are becoming increasingly difficult to combat because of emerging resistance to all current antibiotic classes [172]. MRSA has gradually disseminated and MRSA is now a problem in hospitals worldwide and is increasingly recovered from nursing homes and the community. CNS, such as S. epidermidis, S. haemolyticus, and S. saprophyticus, can also be found in association with human infections. They are the most frequently reported pathogens in nosocomial blood-stream infections and they are often associated with implanted medical devices [173, 174]. Increasing drugresistance among SCN has also become of concern [175]. Therefore, the emergence and dissemination of antibioticresistant in staphylococci constitute serious problems worldwide, requiring effective measures for controlling their spread [12, 172]. Multidrug-resistant staphylococci are potential targets for bacteriocin applications. Lysostaphin was probably the first staphylococcin discovered [112]. Lysostaphin is active primarily against CPS but retaining some residual activity against CNS, although CNS requires increased concentration of the enzyme and larger incubation times to be killed [176]. Lysostaphin is sold commercially by Sigma-Aldrich and is indispensable for staphylococcal genetic studies, being used for DNA isolation, formation of protoplasts, and differentiation of staphylococcal strains [118]. Moreover, due to its ability to kill human pathogenic staphylococci, such as S. aureus and S. epidermidis, various reports have recommended lysostaphin use in a variety of medical applications. Lysostaphin has many attractive features for use as an antimicrobial agent: it has activity against nondividing as well as dividing cells, it is digested by intestinal proteinases, it has no oral toxicity, and its fail to elicit a significant immune response following oral administration to rats [177]. The first relate of efficacy of lysostaphin against S. aureus was published by Harrison and Cropp in 1965 [178]. In this study, the efficacy of lysostaphin over 50 clinical isolates, most of them penicillinase producers, was compared to the antibiotics penicillin G, ampicillin, methicillin, ristocetin, vancomycin, and erythromycin. The results have shown that


55

the MIC values for lysostaphin ranged from