ATP-Binding Cassette Transporters Are Targets for the Development of Antibacterial Vaccines and Therapies

INTRODUCTION TO ABC TRANSPORTERS

ATP-binding cassette (ABC) transporters are widespread among living organisms and comprise one of the largest protein families. For example, components of ABC transporters are encoded by approximately 5% of the Escherichia coli and Bacillus subtilis genomes (47, 80). These transporters are found in all species and are evolutionarily related. However, they are functionally diverse and have roles in a wide range of important cellular functions. The different ABC transporters can be assigned to classes, families and subfamilies on the basis of phylogenetic analyses (19).

Structure of ABC transporters.

ABC transporters are remarkably conserved in terms of the primary sequence and the organization of the domains or subunits. Characteristically, ABC transporters have a highly conserved ATPase domain (the ABC, also known as the nucleotide-binding domain) which binds and hydrolyzes ATP to provide energy for the import and export of a wide variety of substrates (or allocrites [80]). The ABC contains two highly conserved motifs, the Walker A and Walker B motifs, which together form a structure for binding ATP. However, these motifs may also be found in ATP-binding proteins not associated with transport. ABC transporters also contain a specific motif, the consensus signature sequence LSGGQ/R/KQR, which is highly conserved and is specific to the ABC superfamily. This sequence is also known as the linker peptide or C motif and is located N-terminal with respect to the Walker B motif. Further details of the functional and structural aspects of ABCs can be found in recent reviews by Jones and George (38), Kerr (41), and Schneider and Hunke (65).

The ABC transporters consist of ABCs associated with hydrophobic membrane-spanning domains (MSDs), which are also known as membrane domains, transmembrane domains, or integral membrane domains. It is thought that ABC transporters typically have a common four-domain arrangement consisting of two ABCs and two MSDs that may be fused in various ways into multidomain polypeptides (32). In most ABC transporters, an MSD typically forms six putative α-helical transmembrane segments (i.e., a total of 12 segments per ABC transporter) that constitute a channel through which allocrites may be transported (32). In addition, ABC transporters may also include additional proteins for specific functions (Fig. 1). Bacterial importers, also known as permeases, typically include periplasmic solute-binding proteins (SBPs) that bind to incoming allocrites and deliver them to the import complex in the inner membrane of gram-negative bacteria. Import across the outer membrane may involve outer membrane proteins (OMPs), such as porins. The importers of gram-positive bacteria, which have no periplasm or outer membrane, include equivalent allocrite-binding proteins anchored to the outside of the cell via lipid groups. This structural difference may mean that much smaller amounts of ligand-associated SBPs are needed to saturate the transporter complex than are needed for the corresponding interaction with soluble SBPs in gram-negative bacteria. SBPs are believed to confer affinity and specificity to the importers, as well as directionality, since they are not present in exporters. Instead, some gram-negative bacterial exporters include other accessory factors (AFs) that are required when the allocrite is destined for release into the extracellular medium. Other gram-negative bacterial exporters that do not require AFs include some of the exporters involved in the transport of allocrites to the periplasm or outer membrane only. In some cases, additional OMPs are required for full export of the allocrites. Some gram-positive bacterial exporters also have proteins similar to these AFs, although their role in export is not clear.

FIG. 1.

Schematic diagram showing typical organization of ABC transporters in gram-negative bacteria. IMP, inner membrane protein.

Physiological roles of bacterial ABC transporters.

In bacteria, ABC transporters have a diverse range of functions that may be required in response to the environments in which different bacteria find themselves. They import a variety of allocrites, including sugars and other carbohydrates (64), amino acids (33), peptides (20), polyamines (35), metal ions (16), sulfate (42), iron (44), and molybdate (66). ABC transporters are also responsible for the targeted export of other allocrites across the cytoplasmic membrane (for example, capsular polysaccharide [67] in gram-negative bacteria). Other exporters are responsible for the secretion of antibiotics in some antibiotic-producing bacteria (53) and in drug-resistant bacteria (77) or for the export of extracellular toxins. Members of another class of ABC systems have roles in cellular processes, such as translational regulation (14) and DNA repair (25).

It may be presumed that bacterial species that live in diverse environments need to adapt to different conditions and may therefore require numerous ABC systems, whereas other species may require fewer systems if they have a more restricted lifestyle. We have carried out bioinformatic analyses that show that the predicted numbers of ABC transporters encoded in bacterial genomes are different for different bacteria (D. N. Harland, H. S. Garmory, K. A. Brown, and R. W. Titball, unpublished data). The findings of these analyses are complex and cannot be covered sufficiently within the limitations of this review. However, our results indicate that the numbers of ABC transporters encoded in bacterial genomes correlate with the physiological niches in which the bacteria live and that this correlation is not merely a function of genome size. It is anticipated that the details of these analyses will be published elsewhere. In general, it was predicted that significantly more ABC transporters are present in environmental or extracellular bacteria than in intracellular bacteria. The finding that bacterial genomes encode different numbers of ABC transporters, which correlate with their lifestyles, suggests that bacterial ABC transporters are likely to be necessary for growth and/or survival of the bacteria in their ecological niches.

ABC TRANSPORTERS HAVE ROLES IN BACTERIAL VIRULENCE

The purpose of this review is to demonstrate the possibility of targeting ABC transporters for the development of antibacterial vaccines and therapies. Traditionally, bacterial ABC transporters have been considered to play roles in nutrient uptake and drug resistance. However, there is increasing evidence that these transport systems play either direct or indirect roles in the virulence of bacteria.

Virulence associated with uptake of nutrients.

The roles of ABC transporters in virulence of pathogenic bacteria have been attributed to the requirement for uptake of various nutrients. Several examples have been identified by using signature-tagged mutagenesis (STM), a technique designed to enable large-scale screening of bacterial mutants for organisms that have an attenuated phenotype (30). For example, STM of Streptococcus pneumoniae allowed identification of virulence-attenuated clones with mutations in genes homologous to E. coli potA and potF (involved in polyamine transport), B. subtilis glnH and glnQ (involved in glutamine transport), and Streptococcus mutans msmK (involved in sugar transport), among others (61). Similarly, Yersinia spp. with mutations in genes encoding putative ABC importers of phosphate (18) or nitrogen (40) were identified by using STM and mouse models of infection. Multiple animal infection models have also been used for STM to identify genetic loci that may be universally important for bacterial survival in vivo. When this approach was used with a number of Staphylococcus aureus models of infection, the largest gene class identified included genes encoding amino acid or oligopeptide transporters (17). For example, an oppC mutant was attenuated in multiple animal models. The oppC gene encodes a permease component of an oligopeptide transport system, Opp. In a separate study, STM was used to identify clones of S. aureus with mutations in genes with homology to oppD and oppF, which are genes that encode the ABC-containing proteins of the same oligopeptide ABC transport system (52).

Virulence associated with uptake of metal ions.

ABC transporters associated with the uptake of metal ions, such as iron, zinc, and manganese, have been shown to play roles in bacterial virulence in disease models (Table 1). These ABC transporters are classified in the MET family of ABC systems specific for metallic cations (19). These metals are considered vital elements required for bacterial growth, and removing the function of the ABC transporters responsible for metal import often has deleterious effects on the virulence of the microorganisms.

TABLE 1.

ABC transporters involved in virulence of bacteria

ABC transporter or protein	Transported metal(s)	Details of virulence	Reference(s)
Yersinia pestis YbtPQ	Iron	ybtP mutant avirulent in mice	22
Yersinia pestis YfeABCD	Iron	yfeAB mutant shows reduced virulence in mice	4
Salmonella enterica serovar Typhimurium FeoABC	Iron	feoB mutant attenuated for virulence in mice	7
Erwinia chrysanthemi SufABCD	Iron	sufA and sufC mutants show reduced ability to cause maceration of chicory leaves; sufC needed for systemic invasion of Saintpaulia ionantha	54, 55
Streptococcus pneumoniae Pit1 and Pit2 (known as Piu and Pia)	Iron	pit1/pit2 mutant attenuated for virulence in mouse models of pulmonary and systemic infection	9
Salmonella enterica serovar Typhimurium SitABCD	Iron, manganese	sitABCD required for full virulence in mice	7, 36
Streptococcus pyogenes MtsABC	Iron, manganese	mtsABC mutant shows attenuated virulence in mice	37
Streptococcus mutans SloABC	Iron, manganese	sloC and sloA mutants show attenuated virulence in rat model of endocarditis	43, 57
Enterococcus faecalis EfaCBA	Manganese	efaCBA mutant shows attenuated virulence in mouse peritonitis model	68
Streptococcus pneumoniae PsaABC	Manganese	psaA mutant shows attenuated virulence in mice	5
Haemophilus ducreyi ZnuA	Zinc	znuA mutant shows decreased virulence in rabbit model of experimental chanchroid	46
Streptococcus parasanguis FimA		fimA mutant shows attenuated virulence in rat model of endocarditis	11

For example, iron is an essential nutrient for most bacteria, and the ability to obtain iron is essential to support bacterial growth in vivo and to provide cofactors for components of microbial antioxidative stress defense. In different niches in the host, pathogenic bacteria must be able to utilize different sources of iron. For this purpose, bacteria have a number of strategies to acquire iron for growth, including the use of iron-binding siderophores, hemoproteins, or glycoproteins (44). Yersinia pestis, like some other bacteria, possesses several iron acquisition systems. One way in which Y. pestis obtains iron is through the use of the siderophore yersiniabactin (Ybt) (21). Alternatively, two proteins encoding ABCs, designated YbtP and YbtQ, have been shown to be required for the uptake of iron (22). It is thought that these proteins function together in the transport of iron-siderophore complexes into Y. pestis. Furthermore, a ybtP mutant of Y. pestis showing reduced iron accumulation was avirulent in mice by the subcutaneous route of infection, mimicking flea transmission of the bacteria (22), which reflected the importance of this ABC transporter in bubonic plague. A second ABC transporter required for Y. pestis virulence is the Yfe system encoded by yfeABCD (4). This system, which transports manganese as well as iron, is thought to be important for iron uptake during later stages of the disease than the Ybt system (for example, during dissemination of Y. pestis to deep tissues and organs). A yfeAB mutant of Y. pestis showed reduced virulence via the subcutaneous route, and mutants lacking both the Ybt and Yfe transport systems were shown to be avirulent when they were administered by the intravenous route (4).

Similar iron uptake systems are also important in Salmonella enterica serovar Typhimurium virulence, in which the FeoABC transporter of iron (7) and the SitABCD transporter of iron and manganese (7, 36) are required for full virulence. Similar to Y. pestis mutants, S. enterica serovar Typhimurium mutants with deletions in both iron ABC transporter systems show reduced iron uptake and are less virulent than single mutants (7).

Collectively, the data in Table 1 underline the importance of ABC transporters involved in metal ion uptake in growth and survival of bacteria in host tissues, where the concentrations of metal ions are generally low.

Virulence associated with cell attachment.

Attachment of pathogenic bacteria to host cells is a critical step in the pathogenesis of many infections, particularly when the pathogens are confined to mucosal surfaces. For example, in Agrobacterium tumefaciens, attachment of the bacteria is an attribute required for virulence. In this bacterium, a mutant with a transposon mutation in an operon showing homology to operons encoding ABC transporters was shown to be deficient in attachment to host cells (51). Similarly, a glnQ mutant of group B streptococcus showed decreased adherence to and invasion of respiratory epithelial cells in vitro and decreased virulence in vivo, indicating the importance of the glutamine transporter in group B streptococcus virulence (73).

It is also possible that the attenuation of virulence in bacteria caused by the inactivation of ABC transporters involved in nutrient or metal ion uptake may result indirectly from attachment of the bacteria to host cells. For example, S. pneumoniae PsaA, an SBP of the PsaABC manganese transporter, also exhibits amino acid sequence similarity to several streptococcal lipoproteins believed to have direct or indirect roles in promoting bacterial adhesion to host cells. Indeed, a psaA mutant of S. pneumoniae showed only 9% adherence to pneumocytes in vitro compared to the adherence of wild-type bacteria (5). Although the direct role of PsaA as an adhesin is unclear, it is possible that PsaA and other SBPs could be indirectly implicated in virulence through their involvement in metal ion transport (16).

BACTERIAL ABC TRANSPORTER PROTEINS ARE IMMUNOGENIC

The components of ABC transporters that may be expected to be most immunogenic in gram-negative bacteria are the associated OMPs. However, components that are predicted to be located in the periplasmic space or inner membrane may also be immunogenic. The finding that there are proteins which are apparently not surface located yet can induce good antibody responses may appear to be paradoxical. However, there are many examples of antibodies in convalescent sera which are directed to cytoplasmic proteins (28, 39, 48, 78). In the case of some proteins, such as Hsp60, this may reflect the surface location of at least part of the protein population (24, 29). Another possibility is that bacteria with disrupted cell walls or bacteria which are taken up by antigen-presenting cells display cytoplasmic or periplasmic protein to the immune system. Reported examples of immunogenic ABC transporter proteins from both gram-positive and gram-negative bacteria are listed in Table 2.

TABLE 2.

Immunogenic ABC transporter proteins

Protein(s)	Protein type	Details of immunogenicity	Reference(s)
Campylobacter jejuni CjaA and CjaC	SBP	React with rabbit antiserum raised against inactivated bacterial cells	58, 59
Actinobacillus pleuropneumoniae ApaA	SBP	Homologue of C. jejuni CjaA identified by screening genomic expression library by immunoblotting with convalescent sera taken from a pig infected with A. pleuropneumoniae	50
Lactobacillus fermentum BspA	SBP	Identified by screening DNA library with antiserum raised against L. fermentum	76
Bacillus anthracis protein	SBP	Encoded by the B. anthracis virulence plasmid pXO1; shown to be reactive with rabbit hyperimmune sera	2
Mycobacterium tuberculosis PstS	SBP	Identified from a phage M. tuberculosis expression library by screening with antibody raised against culture filtrate antigens; three different forms of PstS shown to be immunogenic	45
Streptococcus pneumoniae PsaA	SBP	Induces specific antibody responses in humans following pneumococcal exposure	62
Methicillin-resistant Staphylococcus aureus protein	ABC	Identified by screening genomic expression library by immunoblotting with sera from patients with septicemia due to methicillin-resistant S. aureus; homology with B. subtilis YpkA	12
Vancomycin-resistant Enterococcus faecium protein	ABC	Two amino acid sequences identified in a genomic expression library by immunoblotting with serum and peritoneal dialysate from a patient surviving septicemia due to vancomycin-resistant E. faecium; homology with B. subtilis YbxA and YbaE	13
Enterococcus faecalis EfaA	ABC transporter component	Loss of a 37-kDa immunoreactive band demonstrated in Western blots following disruption of the E. faecalis antigen A (EfaA) gene	68
Brucella ovis protein	ABC transporter component	Identified by two-dimensional polyacrylamide gel electrophoresis and immunoblotting of B. ovis proteins with serum from a naturally infected ram	75
Treponema pallidum TroA	ABC transporter component	Antigenic protein in experimentally infected rabbits and syphilitic patients	6, 27
Streptococcus agalactiae protein	ABC transporter component	Antisera raised against protein recognized clinically important serotypes of group B streptococcus	34
Chlamydia trachomatis TroA and TroB	ABC transporter components	Identified by differentially screening an expression library with antisera from monkeys infected with live or killed C. trachomatis; homology with T. pallidum TroA	3

One way in which immunogenic SBPs have been identified as immunodominant proteins is through screening with immune sera. For example, a plasmid DNA clone encoding the immunoreactive BspA protein was isolated from a gene library by screening with antiserum raised against whole Lactobacillus fermentum cells (76). Similarly, the CjaC and CjaA proteins of Campylobacter jejuni were identified by screening with antiserum raised against whole C. jejuni cells (58, 59). The CjaC and CjaA proteins are considered to be potential vaccine candidates. A protein with homology to CjaA, Actinobacillus pleuropneumoniae ApaA, also has been shown to be strongly reactive with sera from convalescent swine (50).

Interestingly, ABC-containing proteins of ABC transporters, located in the inner membrane of gram-negative bacteria, are also able to stimulate specific immune responses. Examination of the antibody responses in convalescent-phase sera from individuals that had been infected by vancomycin-resistant Enterococcus faecium led to selection of plasmid DNA clones coding for two amino acid sequences containing ABCs (13). A similar immunoblotting approach with sera from patients with septicemia due to methicillin-resistant S. aureus also resulted in identification of a protein showing homology to ABC transporters (12). In both studies, recombinant antibodies raised against the proteins gave reductions in organ colony counts in mouse models of infection (12, 13). Thus, it is likely that some inner membrane-located proteins may be exposed to the immune system to stimulate antibody responses. Together, the findings described here suggest a potential role of ABC transporters as targets for immunotherapy. The value of such immunotherapy might be enhanced when it is used in combination with antibiotics. For example, antibiotics which disrupt the cell wall might fully expose components of the ABC transporter system to the immune system, and antibodies which block antibiotic efflux ABC transporters might potentiate the effects of antibiotics. In support of these suggestions, the antibody fragment with activity against the methicillin-resistant S. aureus ABC transporter described above (12) is now in phase III clinical trials as the drug Aurograb (NeuTec Pharma), where it is being used in combination with vancomycin.

DEVELOPMENT OF VACCINES

The roles of some ABC transporters in bacterial virulence indicate that components of the transporters may be suitable targets for mutation for the development of live attenuated antibacterial vaccines. For example, mice immunized with a Brucella abortus mutant having a deletion in a virulence gene encoding the ABC-containing protein ExsA exhibited superior protective immunity against virulent B. abortus challenge compared to mice immunized with commercial vaccine strains (63). Furthermore, since ABC transporters may be immunogenic, they might also be exploited as candidate subunits for vaccination against pathogenic bacteria. Vaccines of this nature have been evaluated for Mycobacterium tuberculosis and S. pneumoniae. The PstS SBP of M. tuberculosis has been shown to be involved in phosphate transport and to be surface exposed on mycobacteria (45). When intramuscularly administered to mice as a DNA vaccine, PstS was highly immunogenic and showed protective efficacy against intravenous infection with M. tuberculosis (74). The PsaA metal-binding protein of S. pneumoniae has been used to immunize mice against nasopharyngeal carriage of S. pneumoniae (8). Finally, recombinant PiuA and PiaA, which are SBPs involved in iron uptake in S. pneumoniae, have also been shown to immunize mice against systemic S. pneumoniae infection (10, 56). PsaA, PiuA, and PiaA are all lipoprotein components of ABC transporters and are probably attached to the extracellular surface of the bacterial membrane, beneath both the cell wall and the capsule of S. pneumoniae. It is unlikely that any of these lipoproteins are exposed on the outer surface of the bacteria (44, 70), so it is considered unlikely that antibodies raised against the proteins are opsonic (54). It is presumed that antibodies must diffuse through the capsule and cell wall to bind to the lipoproteins and prevent their biological functions (54) (i.e., uptake of manganese [PsaA] or iron [PiuA and PiaA]). It is also possible that phase variation of the bacteria, which alters levels of polysaccharide production, may render the lipoproteins more accessible to antibody (54). Since conditions in vivo are likely to be restricted in terms of iron and manganese content, inhibiting these metal ion uptake systems may have a significant effect on the in vivo growth of S. pneumoniae. Further studies are required to confirm the process by which proteins that are not surface exposed are able to stimulate antibody responses. Such studies may allow a more rational approach to the selection of ABC transporter proteins as candidate vaccine antigens.

ABC TRANSPORTERS AS TARGETS FOR NOVEL ANTIMICROBIALS

The association of some ABC transporters with virulence or survival in the host, their locations in the cell wall, and the fact that importers are not found in mammalian hosts make them ideal targets for novel antimicrobial compounds to treat disease (79). However, the successful development of antimicrobials which target ABC transporters is likely to be dependent on knowledge of their structures and molecular modes of action. To date, there are few structures available. In part, this reflects the difficulties associated with isolating and crystallizing proteins which are normally membrane located.

An alternative approach exploits ABC transporters as a system for the delivery of antimicrobials into the bacterial cell rather than as the target for antimicrobial compounds (1, 23). Many candidate antimicrobials fail because they are unable to cross the cell wall, so an active uptake system would greatly increase the number of compounds with potential value. Additionally, since mammalian host cells lack ABC import systems, an additional tier of specificity can be introduced by exploiting these bacterial import systems. Transporters such as the OppA system have the remarkable ability to bind to a wide range of peptide substrates (Fig. 2), reflecting the cavernous architecture of the binding site (71, 72). It is this broad specificity which suggests that they might be exploited for the uptake of novel antimicrobials. Proof of principle of this approach is provided by several naturally occurring systems. For example, antibiotics such as bialaphos, bacilysin, and phosphinothricyl are naturally synthesized with the toxic moiety attached to a peptide which directs entry into the cell via a peptide transporter (31, 69). Artificially created drugs, in which the antibacterial compound is linked to the natural substrate for the transporter, have been referred to as smugglins (60) and have been proposed for several decades. Several reports have indicated the feasibility of developing such drugs, and many of these drugs appear to exploit peptide ABC transporters, such as the OppA or DppA systems (15, 23, 26, 31, 69, 71). One of the most elegant examples of this approach involved the synthesis of an analogue of 3-deoxy-d-manno-2-octulosonic acid attached to a dipeptide. The resulting compound was active against gram-negative bacteria and had the ability to block the linking of lipid A to the core polysaccharide of lipopolysaccharide (15, 26). More recently, Marshall et al. reasoned that the potential antibacterial compound N³-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid might be linked to a peptide taken up by the DppA or OppA systems (49). Although the ability to bind to DppA or OppA was maintained, the complex consisting of the peptide and N³-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid appeared to be too large to be transported into the cell.

FIG. 2.

Crystal structure of S. enterica serovar Typhimurium OppA complexed with trilysine (green). The image was created by using Sybyl 6.8 (Tripos UK Ltd.) running on SG 02.

Although it is clear that this route of antimicrobial drug development is not universally exploitable, there can be no doubt that this approach does have significant potential. Bearing in mind that the first studies to exploit these systems were carried out over 25 years ago, it is both disappointing and surprising that drugs based on ABC import systems have not yet reached maturity.

CONCLUSIONS

It is clear that ABC transporters play an important role in bacteria, importing various nutrients required for survival in different niches and exporting substances toxic to the cell. Not surprisingly, disrupting the function of ABC transporters through mutagenesis demonstrates the roles in bacterial virulence that many ABC transporters play. In addition, the membrane location and surface exposure of many of the proteins forming ABC transporters raise the likelihood that these proteins may be immunogenic. Thus, the studies described in this review indicate that ABC transporter proteins may be suitable targets for the development of antibacterial vaccines, either through the development of live attenuated bacteria or through the development of protein- and DNA-based subunit vaccines. Furthermore, the finding that antibodies raised against ABC transporter proteins may clear bacterial infections in vivo suggests that these proteins may be suitable targets for the development of postinfection therapies. Finally, the potential for the development of novel antimicrobials which indirectly or directly exploit ABC transport systems has yet to be fully realized.