Exploring amino acid and peptide transporters as therapeutic targets to attenuate virulence and antibiotic resistance in Staphylococcus aureus

Staphylococcus aureus continues to pose a major threat to public health and is responsible for 20,000 deaths each year in the US [1]. The problem is exacerbated by methicillin-resistant S. aureus (MRSA) which, according to the World Health Organisation’s 2014 Antimicrobial Resistance Global Report on Surveillance is associated with a>60% increase in mortality compared to antibiotic susceptible S. aureus. Membrane transport systems can control both virulence and antibiotic resistance and represent novel targets for therapeutic agents. Here we discuss how efforts to overcome antimicrobial drug resistance could include novel agents targeting important metabolic processes dependent on membrane transporters, which have the potential to augment existing antibiotics.

systems focused on their contribution to growth and physiology in vitro. However, new data on nutrient availability and metabolism in niche-specific models of S. aureus infection suggests that amino acid transporters may also contribute to virulence and antibiotic resistance [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19]. In this context, renewed effort to understand how, why and when amino acids are taken up by S. aureus cells under controlled laboratory conditions, and in vivo, may identify specific membrane transporters as novel and potentially druggable therapeutic targets. Such studies need to focus on the role of these transport systems in metabolism, as well as their contribution to virulence, and resistance to existing antimicrobial drugs. Analogues of substrates for membrane transporters implicated in virulence and/or drug resistance may provide a starting point for novel antimicrobial agents, which are less likely to impose a selective pressure for the emergence of resistance. Future clinical trials would be needed to investigate the relevance of this therapeutic approach in humans or animals.

What effect does the environment have on amino acid transport, and what does this mean for virulence and resistance?
S. aureus can alter the total intracellular amino acid concentration and relative abundance of individual amino acids in response to external osmotic and pH stress [2]. Mild ethanol stress was shown to be associated with impaired acetate catabolism and ammonia accumulation and directed the specific uptake of individual amino acids from the culture medium [20]. This selective transport of amino acids may be driven by specific requirements for stress-responsive protein synthesis, and/or stress-driven redirection of metabolic activity [21]. Nutrient limitation and oxygen availability also impact amino acid uptake and catabolism, including during planktonic and biofilm growth [22,23].
Intracellular amino acid concentrations influence physiology and virulence levels by controlling the activity of the stringent response (ppGpp) and c-di-AMP nucleotide signaling systems, and the global transcriptional regulator CodY. Amino acid starvation-mediated activation of the stringent response and increased c-di-AMP levels have pleiotropic effects including expression of high-level β-lactam resistance [24][25][26]. When GTP and branchedchain amino acid (BCAA) levels are reduced under nutrient-limiting conditions, genes normally repressed by CodY are activated and collectively play a role in adaption to starvation. CodY also regulates several virulence factors [5,7], highlighting a link between environmental conditions, metabolism, and virulence.

Do all amino acid transporters have the same effects on growth, virulence and resistance?
Transport of several amino acids has been shown to be important for S. aureus in vivo survival, virulence and drug resistance. Interplay between these transporters in different growth media or host niches reveals both the complexity of amino acid transport mechanisms, as well as possible therapeutic opportunities to manipulate their activity in order to affect changes in virulence and/or antibiotic resistance.
BcaP and BrnQ. S. aureus transports BCAAs using BcaP and BrnQ1-2-3 [5,7,8]. In vitro, BrnQ1 is the primary transporter of leucine and valine, whereas BcaP plays a more significant role in vivo. Interestingly, a brnQ2 mutant was hypervirulent in a systemic infection model, potentially due to impaired isoleucine transport causing de-repression of CodY-controlled virulence genes, highlighting the importance of experimentally verifying the suitability of membrane transporters as drug targets.
PutP. Mutation of high-affinity proline transporter PutP, which is up-regulated by nutrient depletion [14], attenuates virulence in a rabbit endocarditis model [13] even though OpuD remains functional as a low-affinity proline transporter [15], suggesting that drug-mediated inhibition of PutP-mediated proline transport may have therapeutic potential.
GltT. The major aspartate transporter GltT is important for S. aureus survival in a mouse model of osteomyelitis. Although GltT does not transport glutamate [16], high concentrations of glutamate in bone tissue block GltT activity including GltT-mediated aspartate transport, making de novo aspartate biosynthesis essential for S. aureus survival and persistence in this niche [16]. A gltT mutant exhibited increased sensitivity to heat shock, acetic acid and gentamicin stress [17].
GlnPQ and AlsT. Mutation of the predicted glutamine transporter GlnPQ increased TCA cycle activity, decreased polysaccharide intercellular adhesin biosynthesis, and significantly reduced virulence in a rabbit endocarditis model [12]. Recently it was demonstrated that GlnPQ does not transport glutamine and that AlsT is instead the main glutamine transporter [9]. Moreover, AlsT-mediated glutamine uptake decreased c-di-AMP levels, which is known to effect cell envelope homeostasis, virulence and β-lactam resistance [9,25].
CycA. Impaired transport of alanine in a cycA mutant increased MRSA susceptibility to oxacillin and D-cycloserine (DCS), an alanine analogue antibiotic that interferes with peptidoglycan (PG) biosynthesis [10]. Mutation of cycA mutant or exposure to DCS had similar effects on PG structure [10], revealing interplay between alanine transport and susceptibility to β-lactam antibiotics. β-lactam/DCS combinations acted synergistically against MRSA in a mouse bacteremia model [10], suggesting that drug-mediated interference with CycA might ameliorate β-lactam resistance in MRSA.
TycP and TycABC. The low-affinity transporter TcyABC and the high-affinity permease TcyP, which transport the sulfur-containing amino acids cysteine and cystine, were recently implicated in virulence using a mouse model of systemic infection [11]. However, a double TcyP-TcyABC mutant still established infection, indicating that an alternative sulfur transporter(s) may facilitate glutathione transport, potentially complicating putative strategies to target sulfur transport using cysteine/cystine analogues.

How can amino acid and peptide transport activity be exploited to develop new therapeutic approaches for S. aureus infections?
Amino acid transporters are among the most abundant membrane proteins in S. aureus, with niche-and environment-specific roles in maintaining cell integrity and metabolic homeostasis in infected host tissue. Further advances in our understanding of the mechanisms underpinning the contribution of amino acid permeases to virulence and antibiotic resistance may identify new drug targets for which the natural substrates can be identified. The therapeutic potential of amino acid and peptide analogues has been explored for diseases ranging from diabetes to cancer. For example the glycine analogues, glyphosate and aminomethylphosphonic acid successfully inhibited growth in eight human cancer cell lines, but not two immortalized human normal prostatic epithelial cell lines [27].
An attractive feature of this strategy is the possibility of using amino acid analogues or new drugs derived from amino acid analogues as lead compounds in studies to evaluate the physiological impact of interfering with amino acid transport systems on the metabolism, growth and virulence of S. aureus. The activity of amino acid analogue antibiotics such as D-cycloserine and β-chloro-D-alanine against S. aureus demonstrates the effectiveness of drugs based on amino acid analogues [10,28]. Combining new drugs targeting amino acid transporters may also enable re-purposing of other antibiotics as part of efforts to overcome resistance in S. aureus and MRSA. Nevertheless, translation of this anti-transporter approach into clinical practice will encounter several obstacles. Multiple substrates for some transporters may complicate this approach. In addition, drugs identified in vitro may have limited activity in vivo or unwanted activity against host cell membrane transporters and the beneficial microbiota. Strategies to mitigate potential off-target side effects in a clinical setting will be needed to realize the therapeutic potential of this approach.