ABC transporters of staphylococci

Res. Microbiol. 152 (2001) 351–356  2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S0923-2508(01)01206-2/FLA

ABC transporters of staphylococci Michael Otto∗ , Friedrich Götz Mikrobielle Genetik, Universität Tübingen, Waldhäuserstr. 70/8, 72076 Tübingen, Germany Received 10 October 2000; accepted 6 December 2000

Abstract – Some members of the genus Staphylococcus are important human pathogens, such as Staphylococcus aureus and Staphylococcus epidermidis. ATP binding cassette (ABC) transporters of pathogenic staphylococci are often responsible for antibiotic resistance and for the uptake of essential solutes and are therefore being considered as targets for novel antistaphylococcal drugs. Other ABC transporters are involved in the biosynthesis of a special class of peptide antibiotics, the lantibiotics, and in self-protection of the producer against them.  2001 Éditions scientifiques et médicales Elsevier SAS Staphylococcus / resistance / lantibiotics / ABC transporters

1. Introduction Staphylococci are Gram-positive bacteria that form grape-like clusters, after which the genus name was coined (from Greek staphylos, grape). Two major groups can be distinguished by the ability to produce coagulase. The most important member of the coagulase-positive staphylococci is Staphylococcus aureus, a human pathogen that can cause many diseases and that is also a common food-poisoning agent. Among coagulase-negative staphylococci (CNS), opportunistic pathogens are found, such as Staphylococcus epidermidis, Staphylococcus saprophyticus, and Staphylococcus haemolyticus [23]. However, the CNS group also contains innocuous species, e.g., Staphylococcus carnosus and Staphylococcus xylosus. Host-vector systems using these nonpathogens have been developed with the aim of producing heterologous proteins [15]. The growing interest in staphylococci lies in their being the most important pathogens involved in nosocomial infections [1]. The enormous threat that S. aureus infections pose to public health has been recognized for several years. More recently, the dramatic increase in S. epidermidis infections has convinced scientists that this normal inhabitant of the human skin also represents a serious problem, especially dur-

∗ Correspondence and reprints.

E-mail address: [email protected] (M. Otto).

ing the treatment of patients with indwelling medical devices [31]. The difficulty in treating S. aureus and S. epidermidis infections is due to the two main mechanisms that render the bacteria resistant to antibiotic treatment: (i) the production of an extracellular slime matrix, which decreases the efficiency of antibiotics by forming a diffusion barrier [9] and (ii) the acquisition of plasmids carrying genes for multiple antibiotic resistance. Multiple-antibiotic-resistant strains are distributed within hospitals mainly by the clinical personnel. World-wide distribution of these strains is constantly increasing in parallel to increasing tourism. Furthermore, the deliberate use of antibiotics has led to the selection of resistant strains in many countries. The most serious situation is in the United States of America, where more than 50% of S. aureus strains isolated from nosocomial infections are resistant to all commonly used antibiotics, leaving only vancomycin as the antibiotic of last resort [1, 11]. However, strains with intermediate resistance to vancomycin have already been isolated [18]. This situation makes the search for new antistaphylococcal agents one of the most important tasks in current drug development. One of the strategies pursued in modern drug development is the direct targeting of molecules responsible for the resistance to antibiotics. Bacteria can acquire resistance to antibiotics, among other possible mechanisms, by removing the antibiotics from their place of action, often by using drug exporters. In staphylococci, many drug exporters are known, some

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of which are ABC transporters. This article will focus on staphylococcal ABC transporters involved in the export of antistaphylococcal drugs, and will also consider the ABC transporters responsible for the export of antibacterial substances produced by staphylococci and for the self-protection of the producer. Furthermore, ABC transporters of staphylococci with other physiological tasks, such as molybdenum and iron uptake, will be briefly discussed. 2. Staphylococcal ABC transporters that confer resistance to antibiotics 2.1. MsrA

The plasmid-encoded efflux pump MsrA, first found in S. epidermidis [33], is widespread among staphylococcal strains, predominantly in CNS [24]. MsrA confers resistance to erythromycin, other 14and 15-membered ring macrolides, and streptogramin B after induction with erythromycin. MsrA is a protein of 488 amino acids and harbors two ATP-binding motifs. In contrast to homologous ATP-dependent transporters from Gram-negative bacteria and eukaryotes, the two ATP-binding motifs are separated by a linker of exceptional length. This linker is rich in glutamine and other hydrophilic amino acids and shows a specific spacing pattern of hydrophobic amino acids, both of which are characteristic of so-called Q-linkers, flexible interdomain fusion junctions. Heterologous expression of the msrA gene from S. epidermidis in S. aureus RN4220 confers resistance to erythromycin. It is, however, not clear whether additional factors are necessary for the erythromycinresistant phenotype. It has been suggested that additional transmembrane domains encoded on the chromosome might be necessary [33]. Two open reading frames on the chromosome of S. aureus that are highly similar to two genes adjacent to msrA on the original S. epidermidis plasmid have been shown not to be required for erythromycin resistance [32]. In S. xylosus, a plasmid-encoded msrA homologue coding only for the C-terminal fragment causes resistance when expressed in Bacillus subtilis or S. aureus [25]. In contrast, constructed N- or C-terminal parts of S. epidermidis MsrA do not confer resistance to S. aureus [32]. Obviously, further work is required to identify additional components necessary for the effluxpump-driven resistance to erythromycin in staphylococci. An MsrA homologue, MsrC, has also recently

been found in Enterococcus faecium, indicating that a similar type of macrolide resistance might be present in other Gram-positive bacteria [30]. With regard to new antistaphylococcal drugs on the market, a novel antibiotic against Gram-positive infections, the oxazolidinone linezolid, is active against staphylococci harboring the msrA gene [14]. 2.2. VgaB

In S. aureus, the plasmid-encoded vgaB gene confers resistance to streptogramin and related compounds. vgaB has never been found on the chromosome. On the plasmid, vgaB is adjacent to the vat gene, which encodes a streptogramin A acetyltransferase. vgaB and vat, or similar loci from S. simulans and S. cohnii, together with another gene conferring resistance to streptogramin, vgb, which encodes a streptogramin B lactonase, render S. simulans and S. cohnii resistant to synergistic mixtures of streptogramins [2 – 4]. The ABC transporter VgaB consists of 552 amino acids with a calculated molecular mass of 61 kDa. The mechanism of VgaB has not yet been elucidated. It harbors Walker A and B boxes, but no transmembrane domains, and might therefore also be dependent on additional transmembrane proteins encoded on the chromosome [2]. VgaB does not confer resistance towards the novel drug linezolid [14]. 2.3. Methicillin resistance

Many of the proteins involved in cell wall metabolism have been found via their ability to confer resistance to methicillin. Adjacent to the gene for penicillin-binding protein 4 (pbpD), an ABC transporter is encoded, which is thought to influence the resistance phenotype by regulatory means and not by serving as an efflux pump [12]. 3. ABC transporters as targets for immunotherapy Recently, by using a phage-antibody-display library of human mRNA from antibody-producing cells, an immunodominant ABC transporter was found in an epidemic methicillin-resistant S. aureus (EMRSA) strain [7]. The protein, named EMRSA-15 ABC, has a molecular mass of 61 kDa and is highly similar to the YkpA ABC transporter of Bacillus subtilis. It shows lower similarity to other ABC trans-

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porters, such as MsrA and VgaB. The common features of EMRSA-15 ABC, MsrA, and VgaB are the Walker A and B sites and the lack of transmembrane domains. The two Walker sites in EMRSA-15 ABC are far apart, which is typical of the subfamily 3 of dimeric ATPases [7]. While the potential therapeutic use of antisera directed against ABC transporters must be more thoroughly investigated, it is noteworthy that in the first systematic approach to detect immunodominant proteins in EMRSA, an ABC transporter was found. 4. ABC transporters found in lantibiotic gene clusters of staphylococci Lantibiotics are ribosomally synthesized peptide antibiotics harboring unusual amino acids, such as lanthionine and methyl-lanthionine, after which the term lantibiotic was coined [19]. Some lantibiotics are produced by S. epidermidis strains: epidermin [13], epicidin 280 [17], and Pep5 [21]. Gallidermin, which is Leu6-epidermin, is produced by Staphylococcus gallinarum [22]. Based on the S. aureus genome sequence information available in the GenBank database, there is a gene cluster very similar to the epidermin gene cluster of S. epidermidis, but the genes have not yet been investigated in detail (figure 1, unpublished results). The target of these substances, all of which belong to the A subtype of lantibiotics, is the cytoplasmic membrane, where they form pores. Recently, an additional mechanism has been found for epidermin, which might also apply to other similar lantibiotics, and is based on the interaction of epidermin with the cell wall precursor lipid II [6]. The genetic loci governing expression of many lantibiotics have been sequenced and characterized. Generally, two classes of ABC transporters have been found in these clusters: (i) those putatively involved in the export of the lantibiotic precursor to the surrounding medium and (ii) those responsible for producer self-protection against the lantibiotic [19]. 4.1. ABC transporters involved in lantibiotic precursor export

The gene probably responsible for the export of the lantibiotic to the surrounding medium has been named lanT. Among the lantibiotic gene clusters of staphylococci, the Pep5, epidermin, and gallidermin

Figure 1. The gene cluster of S. aureus NCTC8325 encoding an epidermin-like biosynthetic system. Analysis of the S. aureus NCTC8325 genome sequence available in GenBank revealed the existence of lantibiotic biosynthesis genes on the chromosome. The genes are clustered as in the other lantibiotic biosynthesis loci studied to date. The encoded proteins show very high sequence similarity to the epidermin and gallidermin biosynthetic proteins. The possibly produced lantibiotic was therefore named aureodermin, and the corresponding genes were designated admA, admB, admC, admD, admP, admF, admE, and admG. Two similar structural genes, admA1 and admA2, are present. The gene cluster lacks a lanT gene (encoding the LanT ABC transporter), but harbors lanFEG-equivalent genes, admFEG, encoding an ABC transporter involved in producer self-protection (shown in black).

clusters harbor a lanT gene. LanT carries both the ATPase and transmembrane domains of ABC transporters in one protein [19]. Some LanT proteins may also carry a peptidase domain, which cleaves off the leader peptide of the lantibiotic precursor during export [16]. This is not the case among the LanT proteins found in staphylococci. In these bacteria, depending on the cellular location of the lantibiotic leader peptidase (LanP), the LanT transporter must deal with either the mature lantibiotic as substrate, such as in the case of Pep5, or the fully matured lantibiotic precursor with the leader peptide still attached, such as in the case of epidermin and gallidermin. Interestingly, the epiT gene, the lanT-equivalent in the epidermin gene cluster, is disrupted and therefore very likely nonfunctional. gdmT of the very similar gallidermin gene cluster can complement for the nonfunctional epiT [29]. All LanT proteins of staphylococcal lantibiotic biosynthetic systems are dispensable for lantibiotic production, but have been shown to increase the production when cloned in a homologous or heterologous host [5, 29]. In the epidermin producer S. epidermidis Tü3298, in which EpiT is nonfunctional, another exporter must be able to ac-

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cept the lantibiotic precursor as a substrate. It is possible that the LanFEG self-protection system (EpiFEG in the case of epidermin) may function as an exporter of the lantibiotic precursor, which can be speculated based on its putative mechanism [27]. However, to date, there is no experimental evidence for such a mechanism. In this respect, it is also worth mentioning that a lanT gene is not present in the newly identified epidermin-like gene cluster of S. aureus. 4.2. ABC transporters involved in self-protection of the lantibiotic producer

The lanF, lanE, and lanG genes have been shown to be involved in “immunity”, or self-protection, against the respective lantibiotic [28]. LanE and LanG represent the membrane-spanning domains, and LanF the ATPase-harboring domain. lanFEG genes can be found in the epidermin (epiFEG), gallidermin, and S. aureus epidermin-like gene cluster. Most of the work on LanFEG proteins has been carried out with EpiFEG. Each domain is indispensable for self-protection, and the system has a high substrate specificity. For example, nisin, a lantibiotic produced by Lactococcus lactis closely related to epidermin, is not a substrate of the EpiFEG transporter [27, 28]. The results of experiments in which the substrate of the transporter was applied to the extracellular fluid and the amount of remaining substrate measured after incubation, with and without the addition of glucose, suggest that EpiFEG works in an energy-dependent manner and expels its substrate to the surrounding medium rather than importing it for proteolytic degradation (figure 2). Expulsion of the lantibiotic thus removes it from its target, the cytoplasmic membrane [27]. 5. ABC transporters found to attenuate S. aureus growth in infection models

Figure 2. Two alternative models for the mechanism of the EpiFEG transporter. The model shows the EpiE and EpiG proteins situated within the cytoplasmic membrane and an EpiF dimer bound to the complex at the cytoplasmic side of the membrane and harboring the ATPase binding site as predicted by hydropathy plots and sequence comparisons. Black arrows illustrate mechanism model 1, the export of epidermin into the surrounding medium. Every exported molecule in model 1 is likely to be able to reintegrate into the membrane. Gray arrows illustrate model 2, the import of epidermin into the cytoplasm, where one would expect proteolytic degradation of the lantibiotic. Our results suggest that model 1 represents the mechanism of the EpiFEG transporter. It is not known whether the transporter recognizes the substrate in its monomeric or oligomeric form. Both possibilities are illustrated in the figure.

ATPases (oppD and oppF); a gene coding for a substrate binding protein (oppA) is found only within the opp1 operon. The opp system(s) might be responsible for the uptake of peptides for the salvage of essential amino acids. These peptides might originate from growing peptidoglycan; it should be noted that the opp2 system is in close proximity to the fem genes involved in the synthesis of the pentaglycine peptidoglycan cross-bridge. 6. Other ABC transporters of staphylococci

Several ABC transporters have been found in an approach through which genetic loci with an impact on growth and survival of S. aureus during infection were identified [10]. Multiple Tn917 insertions were mapped within the opp1 and opp2 operons, both of which encode oligopeptide permease systems. Each operon consists of two genes encoding putative channel-forming transmembrane proteins (oppB and oppC) and two genes encoding putative

6.1. SitABC

In S. epidermidis, an iron-regulated operon consisting of the genes sitA, sitB, and sitC has been found; these genes encode an ATPase, a cytoplasmic membrane protein, and a lipoprotein, respectively [8]. Work to date has focused on the 32-kDa lipoprotein. Lipoprotein components of Gram-positive solute up-

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take systems are the counterpart of periplasmic binding proteins of Gram-negative bacteria. Although the solute specificity of the lipoprotein has not yet been tested, the three proteins are believed to assemble into an ABC transporter complex involved in iron acquisition. The 28-kDa ATPase harbors classical ATP binding motifs, whereas the 35-kDa cytoplasmic membrane protein comprises seven putative membranespanning domains and lacks an ATP binding site. The lipoprotein harbors an ATP binding site and shows similarity to known bacterial lipoprotein components of ABC transporters. Interestingly, many of these lipoproteins have also been shown to function as adhesins [20]. 6.2. ModABC

Neubauer et al. have characterized a molybdate import system of S. carnosus, encoded by the modABC locus, and consisting of the ModA, ModB, and ModC proteins [26]. The configuration is analogous to the SitABC system, the ModABC system is also composed of a cytoplasmic ATPase (ModC), an integral membrane-channel-forming protein (ModB), and a solute-binding lipoprotein (ModA). Molybdate is needed for the synthesis of molybdopterin, an essential cofactor of the dissimilatory nitrate reductase. In contrast to the homologous molybdate uptake system of E. coli, expression of the S. carnosus system seems to be molybdate-independent. The investigations to date have focused on the lipoprotein part of the ABC transporter. The identity of ModA as a lipoprotein has been proven by 14 C-palmitate labeling [26]. 7. Conclusions Investigation of the S. aureus genome sequence data available in GenBank has demonstrated that there are a large number of putative ABC transporters present in staphylococci, most of which have not yet been characterized. Some have been detected in a signature-tag transposon-mutagenesis approach aiming at finding possible novel drug targets [10]. Furthermore, an ABC transporter has been identified as the immunodominant protein during S. aureus infections [7]. Both findings demonstrate that ABC transporters will play an important role as target molecules in the search for novel antistaphylococcal therapies.

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