Advertisement

  • Loading metrics

Open Access

Pearls

Pearls provide concise, practical and educational insights into topics that span the pathogens field.

See all article types »

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

Citation: Zeden MS, Burke Ó, Vallely M, Fingleton C, O’Gara JP (2021) Exploring amino acid and peptide transporters as therapeutic targets to attenuate virulence and antibiotic resistance in Staphylococcus aureus. PLoS Pathog 17(1): e1009093. https://doi.org/10.1371/journal.ppat.1009093

Editor: John M. Leong, Tufts Univ School of Medicine, UNITED STATES

Published: January 14, 2021

Copyright: © 2021 Zeden et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: Sources of funding: Health Research Board grants HRA-POR-2015-1158 and ILP-POR-2019-102 to JPOG (www.hrb.ie), Irish Research Council grants GOIPG/2016/36 to CF and JPOG; GOIPG/2018/943 to MV and JPOG; and GOIPG/2019/2011 to OB and JPOG (www.research.ie); and Science Foundation Ireland grant 19/FFP/6441 to JPOG (www.sfi.ie). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

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.

Why investigate amino acid and peptide transporters as potential therapeutic targets?

Amino acids are essential for sustaining cell integrity and metabolic homeostasis. In addition to protein synthesis, amino acids are also precursors for biosynthesis of nucleotides, lipids and cell wall components. S. aureus can synthesize many of these amino acids but will often preferentially transport them into the cell from the external environment [2].

Limited glucose availability (for example in an abscess) represents an environment in which catabolism of peptides or amino acids is important for S. aureus growth [3]. Bioinformatic analysis reveals several pathways that enable S. aureus to catabolize multiple amino acids, which in turn can generate key central metabolic intermediates such as pyruvate, oxaloacetate and 2-oxoglutarate. Reflecting this importance of amino acids in metabolism, S. aureus has multiple oligopeptide permeases, free amino acid transporters and proteases to degrade host proteins.

An analysis of 64 S. aureus strains revealed that amino acid metabolism genes are disproportionately associated with the pangenome [4] indicating that targeting transporters associated with core amino acid metabolism is likely to have broader therapeutic potential against diverse S. aureus isolates. The diversity and redundancy of amino acid, peptide, osmolyte and nucleoside uptake systems also presents a significant challenge. There are at least 292 genes in the USA300_FPR3757 genome predicted to encode membrane transporters, of which 120 appear to be associated with amino acid, osmolyte or nucleoside transport.

Bioinformatic tools are generally helpful in identifying and predicting the functions of putative transporters, but experimental work is required to verify the substrates transported by permeases and their physiological roles. Historically, studies on bacterial membrane transport 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 [5–19]. 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 [24–26]. When GTP and branched-chain 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.

PheP.

Mutation of predicted phenylalanine permease gene pheP impacted growth [19] and attenuated virulence [18]. The lysine analogue S-(β-aminoethyl)-L-cysteine (thiosine) can inhibit lysine transport by the E. coli PheP homologue, LysP, suggesting that inhibition of PheP with a phenylalanine analogue may attenuate S. aureus virulence.

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.

References

  1. 1. Kourtis AP, Hatfield K, Baggs J, Mu Y, See I, Epson E, et al. Vital Signs: Epidemiology and Recent Trends in Methicillin-Resistant and in Methicillin-Susceptible Staphylococcus aureus Bloodstream Infections—United States. MMWR Morb Mortal Wkly Rep. 2019;68(9):214–9. Epub 2019/03/08. pmid:30845118; PubMed Central PMCID: PMC6421967 potential conflicts of interest. No potential conflicts of interest were disclosed.
  2. 2. Alreshidi MM, Dunstan RH, Macdonald MM, Gottfries J, Roberts TK. The Uptake and Release of Amino Acids by Staphylococcus aureus at Mid-Exponential and Stationary Phases and Their Corresponding Responses to Changes in Temperature, pH and Osmolality. Front Microbiol. 2019;10:3059. Epub 2020/02/11. pmid:32038532; PubMed Central PMCID: PMC6990410.
  3. 3. Halsey CR, Lei S, Wax JK, Lehman MK, Nuxoll AS, Steinke L, et al. Amino Acid Catabolism in Staphylococcus aureus and the Function of Carbon Catabolite Repression. MBio. 2017;8(1). pmid:28196956; PubMed Central PMCID: PMC5312079.
  4. 4. Bosi E, Monk JM, Aziz RK, Fondi M, Nizet V, Palsson BO. Comparative genome-scale modelling of Staphylococcus aureus strains identifies strain-specific metabolic capabilities linked to pathogenicity. Proc Natl Acad Sci U S A. 2016;113(26):E3801–9. Epub 2016/06/12. pmid:27286824; PubMed Central PMCID: PMC4932939.
  5. 5. Kaiser JC, Sen S, Sinha A, Wilkinson BJ, Heinrichs DE. The role of two branched-chain amino acid transporters in Staphylococcus aureus growth, membrane fatty acid composition and virulence. Mol Microbiol. 2016;102(5):850–64. Epub 2016/09/03. pmid:27589208; PubMed Central PMCID: PMC6225994.
  6. 6. Kaiser JC, Omer S, Sheldon JR, Welch I, Heinrichs DE. Role of BrnQ1 and BrnQ2 in branched-chain amino acid transport and virulence in Staphylococcus aureus. Infect Immun. 2015;83(3):1019–29. Epub 2014/12/31. pmid:25547798; PubMed Central PMCID: PMC4333469.
  7. 7. Kaiser JC, King AN, Grigg JC, Sheldon JR, Edgell DR, Murphy MEP, et al. Repression of branched-chain amino acid synthesis in Staphylococcus aureus is mediated by isoleucine via CodY, and by a leucine-rich attenuator peptide. PLoS Genet. 2018;14(1):e1007159. Epub 2018/01/23. pmid:29357354; PubMed Central PMCID: PMC5794164.
  8. 8. Kaiser JC, Heinrichs DE. Branching Out: Alterations in Bacterial Physiology and Virulence Due to Branched-Chain Amino Acid Deprivation. MBio. 2018;9(5). Epub 2018/09/06. pmid:30181248; PubMed Central PMCID: PMC6123439.
  9. 9. Zeden MS, Kviatkovski I, Schuster CF, Thomas VC, Fey PD, Grundling A. Identification of the main glutamine and glutamate transporters in Staphylococcus aureus and their impact on c-di-AMP production. Mol Microbiol. 2020;113(6):1085–100. Epub 2020/01/31. pmid:31997474; PubMed Central PMCID: PMC7299772.
  10. 10. Gallagher LA, Shears RK, Fingleton C, Alvarez L, Waters EM, Clarke J, et al. Impaired Alanine Transport or Exposure to d-Cycloserine Increases the Susceptibility of MRSA to beta-lactam Antibiotics. J Infect Dis. 2020;221(6):1000–16. Epub 2019/10/20. pmid:31628459; PubMed Central PMCID: PMC7050987.
  11. 11. Lensmire JM, Dodson JP, Hsueh BY, Wischer MR, Delekta PC, Shook JC, et al. The Staphylococcus aureus Cystine Transporters TcyABC and TcyP Facilitate Nutrient Sulfur Acquisition during Infection. Infect Immun. 2020;88(3). Epub 2019/12/18. pmid:31843961; PubMed Central PMCID: PMC7035926.
  12. 12. Zhu Y, Xiong YQ, Sadykov MR, Fey PD, Lei MG, Lee CY, et al. Tricarboxylic acid cycle-dependent attenuation of Staphylococcus aureus in vivo virulence by selective inhibition of amino acid transport. Infect Immun. 2009;77(10):4256–64. Epub 2009/08/12. pmid:19667045; PubMed Central PMCID: PMC2747957.
  13. 13. Bayer AS, Coulter SN, Stover CK, Schwan WR. Impact of the high-affinity proline permease gene (putP) on the virulence of Staphylococcus aureus in experimental endocarditis. Infect Immun. 1999;67(2):740–4. Epub 1999/01/23. pmid:9916085; PubMed Central PMCID: PMC96381.
  14. 14. Waters NR, Samuels DJ, Behera RK, Livny J, Rhee KY, Sadykov MR, et al. A spectrum of CodY activities drives metabolic reorganization and virulence gene expression in Staphylococcus aureus. Mol Microbiol. 2016;101(3):495–514. Epub 2016/04/27. pmid:27116338; PubMed Central PMCID: PMC5007081.
  15. 15. Wetzel KJ, Bjorge D, Schwan WR. Mutational and transcriptional analyses of the Staphylococcus aureus low-affinity proline transporter OpuD during in vitro growth and infection of murine tissues. FEMS Immunol Med Microbiol. 2011;61(3):346–55. pmid:21231964; PubMed Central PMCID: PMC3055929.
  16. 16. Potter AD, Butrico CE, Ford CA, Curry JM, Trenary IA, Tummarakota SS, et al. Host nutrient milieu drives an essential role for aspartate biosynthesis during invasive Staphylococcus aureus infection. Proc Natl Acad Sci U S A. 2020;117(22):12394–401. Epub 2020/05/18. pmid:32414924; PubMed Central PMCID: PMC7275739.
  17. 17. Yee R, Feng J, Wang J, Chen J, Zhang Y. Identification of Genes Regulating Cell Death in Staphylococcus aureus. Front Microbiol. 2019;10:2199. Epub 2019/10/22. pmid:31632363; PubMed Central PMCID: PMC6779855.
  18. 18. Needham AJ, Kibart M, Crossley H, Ingham PW, Foster SJ. Drosophila melanogaster as a model host for Staphylococcus aureus infection. Microbiology 2004;150(Pt 7):2347–55. Epub 2004/07/17. pmid:15256576.
  19. 19. Horsburgh MJ, Wiltshire MD, Crossley H, Ingham E, Foster SJ. PheP, a putative amino acid permease of Staphylococcus aureus, contributes to survival in vivo and during starvation. Infect Immun. 2004;72(5):3073–6. Epub 2004/04/23. pmid:15102825; PubMed Central PMCID: PMC387907.
  20. 20. Chatterjee I, Somerville GA, Heilmann C, Sahl HG, Maurer HH, Herrmann M. Very low ethanol concentrations affect the viability and growth recovery in post-stationary-phase Staphylococcus aureus populations. Appl Environ Microbiol. 2006;72(4):2627–36. Epub 2006/04/07. pmid:16597967; PubMed Central PMCID: PMC1449072.
  21. 21. Gaupp R, Schlag S, Liebeke M, Lalk M, Gotz F. Advantage of upregulation of succinate dehydrogenase in Staphylococcus aureus biofilms. J Bacteriol. 2010;192(9):2385–94. Epub 2010/03/09. pmid:20207757; PubMed Central PMCID: PMC2863491.
  22. 22. Zhu Y, Weiss EC, Otto M, Fey PD, Smeltzer MS, Somerville GA. Staphylococcus aureus biofilm metabolism and the influence of arginine on polysaccharide intercellular adhesin synthesis, biofilm formation, and pathogenesis. Infect Immun. 2007;75(9):4219–26. Epub 2007/06/20. pmid:17576756; PubMed Central PMCID: PMC1951195.
  23. 23. Ammons MC, Tripet BP, Carlson RP, Kirker KR, Gross MA, Stanisich JJ, et al. Quantitative NMR metabolite profiling of methicillin-resistant and methicillin-susceptible Staphylococcus aureus discriminates between biofilm and planktonic phenotypes. J Proteome Res. 2014;13(6):2973–85. Epub 2014/05/09. pmid:24809402; PubMed Central PMCID: PMC4059261.
  24. 24. Dordel J, Kim C, Chung M, Pardos de la Gandara M, Holden MT, Parkhill J, et al. Novel determinants of antibiotic resistance: identification of mutated loci in highly methicillin-resistant subpopulations of methicillin-resistant Staphylococcus aureus. MBio. 2014;5(2):e01000. Epub 2014/04/10. pmid:24713324; PubMed Central PMCID: PMC3993859.
  25. 25. Corrigan RM, Abbott JC, Burhenne H, Kaever V, Gründling A. c-di-AMP is a new second messenger in Staphylococcus aureus with a role in controlling cell size and envelope stress. PLoS Pathog. 2011;7(9):e1002217. pmid:21909268; PubMed Central PMCID: PMC3164647.
  26. 26. Pozzi C, Waters EM, Rudkin JK, Schaeffer CR, Lohan AJ, Tong P, et al. Methicillin resistance alters the biofilm phenotype and attenuates virulence in Staphylococcus aureus device-associated infections. PLoS Pathog. 2012;8(4):e1002626. pmid:22496652; PubMed Central PMCID: PMC3320603.
  27. 27. Li Q, Lambrechts MJ, Zhang Q, Liu S, Ge D, Yin R, et al. Glyphosate and AMPA inhibit cancer cell growth through inhibiting intracellular glycine synthesis. Drug Des Devel Ther. 2013;7:635–43. Epub 2013/08/29. pmid:23983455; PubMed Central PMCID: PMC3749059.
  28. 28. Sieradzki K, Tomasz A. Suppression of beta-lactam antibiotic resistance in a methicillin-resistant Staphylococcus aureus through synergic action of early cell wall inhibitors and some other antibiotics. J Antimicrob Chemother. 1997;39 Suppl A:47–51. Epub 1997/05/01. pmid:9511062.