Synthetic Immunobiotics - American Chemical Society

Perspective Cite This: ACS Infect. Dis. XXXX, XXX, XXX−XXX

pubs.acs.org/journal/aidcbc

Synthetic Immunobiotics: A Future Success Story in Small MoleculeBased Immunotherapy? Mary J. Sabulski Feigman and Marcos M. Pires* Department of Chemistry, Lehigh University, 6 E. Packer Ave., Bethlehem, Pennsylvania 18015, United States ABSTRACT: Drug resistance to our current stock of antibiotics is projected to increase to levels that threaten our ability to reduce and eliminate bacterial infections, which is now considered one of the primary health care crises of the 21st century. Traditional antibiotic agents (e.g., penicillin) paved the way for massive advances in human health, but we need novel strategies to maintain the upper hand in the battle against pathogenic bacteria. Nontraditional strategies, such as targeted immunotherapies, could prove fruitful in complementing our antibiotic arsenal. KEYWORDS: antibiotics, small molecules, immunotherapy, haptens, surface remodeling, bacteria

T

of disease.17−20 Today, cancer immunotherapeutics are considered a major breakthrough in how cancer patients are treated. In fact, more robust and sustained anticancer responses have been observed when compared to traditional cancer chemotherapeutics.21−25 By training the immune system to attack cancer cells or removing proteins that shield cancer cells, these agents can overcome the inherent resistance displayed by cancer cells in avoiding detection and eradication by the immune system. Critically, these agents set forth the precedence that a boost in immune response is potent enough to give the upper hand back to the human immune system. The incredible success in cancer immunotherapy invites the next logical question: can these results be translated to bacterial infections? Although the answer to this question can only be fully established once patients afflicted with serious bacterial infections are effectively cured in clinical settings, innovative approaches could make this dream a reality. Despite physiological differences (e.g., division rates) between human cancers and infectious agents, there are reasons to project that immunotherapy can be operable against bacterial pathogens. Successful vaccination against bacteria and virus indicates that the human immune system can fend off fast growing pathogens. Immunomodulatory strategies against bacterial infections (synthetic immunobiotics) can come in many flavors (Figure 1). In this Perspective, the primary focus is in one facet of immunotherapy: the use of small molecules to trigger the recruitment of antibodies onto bacterial cell surfaces. The translation of immunomodulatory molecules against pathogenic bacteria to the bedside would represent a major change in direction in how infections are treated today. Antibiotics being used in the clinic are almost exclusively traditional drugs, agents that directly lyse bacterial cells or interfere with cell

he rising threat of antibiotic-resistant bacteria may be the ultimate catalyst for the development of new strategies to fight pathogenic bacteria.1−5 These efforts may include systematic stewardship of existing antibiotics,6,7 improved diagnostics tools,8 discovery of effective vaccines,9−11 and development of nontraditional therapeutics.12,13 Within the class of nontraditional therapeutics, agents that target pathogens for host immune clearance have the potential to selectively stimulate immune responses against bacterial pathogens. The human immune system evokes a myriad of cellular responses in the defense against dangerous pathogens.14−16 Once foreign entities, such as viruses, bacteria, and fungi, manage to get past the primary physical and chemical barriers (e.g., skin and the intestinal epithelium), the human immune system must act rapidly to neutralize these foreign invaders. For most healthy humans, the immune system properly inactivates invading pathogens. In other instances, pathogens manage to escape detection or destruction on the way to cause systemic illness. This critical transition, from the initial invasion to colonization, often signals the point that the human immune system can no longer overcome and effectively clear the pathogen from the system. This is illustrated in the case of transformed cells growing into an aggressive tumor, an HIV-1 infection progressing to AIDS, and a bacterial infection leading to sepsis. In all of these cases, the disease propagates mostly unchecked by the immune system beyond a certain point and eventually becomes lethal without medical intervention. These observations call into question the role of the immune system in reversing diseases that have progressed to advanced stages for specific pathogens, even in immunocompetent patients. Given these scenarios, it is important to ask a fundamental question: can the immune system f ight back against pathogens that have extensively colonized to advanced stages? The advent of immunotherapeutics against several types of cancer in clinical use has definitively answered this question, at least for one type © XXXX American Chemical Society

Received: December 6, 2017 Published: February 12, 2018 A

DOI: 10.1021/acsinfecdis.7b00261 ACS Infect. Dis. XXXX, XXX, XXX−XXX

ACS Infectious Diseases

Perspective

Figure 1. Top: General description of two types of agents that can be deployed against bacterial infections. Bottom: Schematic representation of the primary goal of synthetic immunobiotic agents, to decorate the surface of pathogenic bacterial with hapten epitopes.

Figure 2. Primary mode of clearance of tagged pathogenic bacteria. Upon the opsonization of target cell, downstream initiation of complement dependent cytotoxicity (CDC) and antibody dependent cell mediated cytotoxicity (ADCC) result in the clearance of the pathogen.

B



Perspective

proliferation (e.g., β-lactams, macrolides, tetracyclines, quinolones, aminoglycosides, and glycopeptides). As approval of new antibiotics curtails and investment in this area shrinks, medical care as we know it could be in jeopardy. Aside from direct treatment of bacterial infections, antibiotics have underpinned major medical advances throughout the last several decades. Innovation in this arena is desperately needed to reinvigorate drug discovery.2,26−29 We propose that nontraditional therapies can complement traditional modalities. The combined success of cancer immunotherapy and the lack of approval of traditional antibiotics with novel mechanisms of action may usher a new era that includes the pursuit of immunotherapeutic agents against bacterial pathogens. Small molecule-based immunotherapeutics should, in theory, operate by provoking a localized response to the infection site. The role of the small molecule is to decorate the surface of pathogenic bacteria with highly antigenic epitopes, which are usually heterobifunctional molecules (Figure 2). One end is composed of a bacteria-homing moiety, and the other, an antigenic fragment. The homing beacon is important to endow the agent with selectivity toward bacterial cells. After all, in designing immunotherapeutics, it is critically important to consider the potential off-target toxicity effects that may come from inducing an immune response against healthy patient tissues. The other benefit of having a homing beacon is that the immune response becomes concentrated at the site of infection. A generalized and systemic activation without guiding the molecules to the diseased area can either dampen the overall response against the pathogens or directly attack the host. The role of the hapten is to induce antibody recruitment onto the surface of bacterial cells, thus tagging them for clearance. By opsonizing bacterial cells, these agents should mimic the natural defense mechanisms used by both the innate and adaptive arms of the immune system. It is worthwhile to consider whether simply inducing the recruitment of antibodies to target cells is sufficient to produce a reversal in the course of the disease. After all, for immunocompetent patients, the immune system has presumably not been fundamentally compromised. Instead, the pathogen manages to escape detection and/or clearance. The success of “one size fits all” cancer vaccines30,31 and, more recently, personalized neoantigen-based cancer vaccines32 teaches us that bringing antibodies to target cells can be a potent mode of enlisting the immune system to clear pathogenic cells. Therefore, these developments cement the concept that opsonization alone can be a central framework for therapeutic intervention. Along these lines, we will highlight several milestones in the area of small molecule-based bacterial immunotherapeutics.

Escherichia coli (E. coli) (Figure 3A). The grafting of biotin handles onto bacterial cell surfaces led to avidin binding, followed by adsorption of antiavidin antibodies and induction of complement killing.

■

POLYMERIC SCAFFOLDS Targeting of mannose receptors on the surface of Gramnegative pathogens was further elaborated by Wang and coworkers.35 Instead of a small molecule, this research team synthesized an acrylamide-based polymer that was conjugated

■

RECEPTOR-BASED TARGETING The early work by Shokat and Schultz set forth the guidelines that are still operable today in small molecule-based immunotherapy against a variety of pathogenic cells including cancer.33 They described how chemical modification of the soluble protein CD4 with the hapten 2,4-dinitrophenol (DNP) could mediate the recruitment of anti-DNP antibodies onto gp120, the envelope protein of HIV virus. Soon after, Bertozzi and Bednarski laid the initial foundations to the idea of building heterobifunctional small molecules that decorate the surface of bacterial cells with immunogenic epitopes.34 In their seminal work, they synthesized a biotinylated C-glycoside mannose, which bound avidly to the mannose receptor on the surface of

Figure 3. Methods described to graft haptens onto the surface of pathogens. Agents outlined in (A−D) noncovalently associate with the surface of target cells. (E) FITC-modified sortase A peptide (LPETG) was shown to induce the recruitment of anti-FITC antibodies to the surface of S. aureus. Reprinted in part from Guatam et al.44 Copyright 2016 American Chemical Society. (F) Azido-modified (Az) Nacetylglucosamine led to the recruitment of FITC-labeled anti-DNP antibodies to the surface of H. pylori cells, and treatment with the control acetylated (Ac) sugar led to lower fluorescence levels. Reprinted in part with permission from Kaewsapsak et al.47 Copyright 2013 John Wiley and Sons. C



Perspective

to both α-mannosyl and the hapten Galα1 → 3Gal (α-Gal) (Figure 3B). The α-mannosyl units were included with the goal of binding to mannose receptors on cell surfaces. The use of αGal as the hapten unit took advantage of components that are already present in the human immune system (even in the absence of vaccination). Approximately 1−2% of the total pool of human IgGs are composed of anti-α-Gal antibodies.36 In this initial report, the strategy was established in vitro by assessing binding of α-Gal antibodies to their polymeric scaffold via an ELISA assay. Whitesides and co-workers developed polymers that targeted pathogenic bacteria by decorating the end groups with the potent antibiotic vancomycin as a homing beacon and fluorescein as the hapten unit (Figure 3C).37,38 Vancomycin associates with bacterial cell walls, thus endowing this polymeric scaffold with a mode of surface binding. The construction of vancomycin/fluorescein polyvalent polymers promoted the targeting of the several Gram-positive bacteria including Staphylococcus aureus (S. aureus), Staphylococcus epidermidis, and Streptococcus pneumoniae. Vancomycin drove the binding of the polymer onto newly synthesized peptidoglycan (PG) on bacterial surfaces, and fluorescein epitopes triggered the recruitment of antifluorescein antibodies. This work demonstrated that the dually modified polymer could induce phagocytosis of opsonized bacteria by macrophages in the presence of antifluorescein antibodies. While humans do not naturally have antifluorescein antibodies in circulation, the use of an exogenous antigen leaves open the possibility of controlling the levels of therapeutic antibody by administration or vaccination. Synthetic polymeric scaffolds may face some challenges in terms of in vivo use, which may have spurred the pursuit of other modes of targeting based on large biomacromolecules. More recently, Nizet and co-workers developed a DNA aptamer that bound to group A Streptococcus bacteria by binding to a well-conserved patch on the surface-anchored M protein (Figure 3D).39 The aptamer was modified at the 5′-end with α-Gal to graft antigens onto bacterial cell surfaces. Incubation of bacteria with the modified aptamer led to the recruitment of anti-Gal antibodies to the streptococcal surface and induction of uptake and killing of bacteria by human phagocytes. The display of antigens on bacterial surfaces for which humans have a standing pool of antibodies against should eliminate the need to preimmunize patients against the antigenic epitopes. Most importantly, the use endogenous antibodies may result in a single agent generalizable strategy.

expected to persist longer compared to a noncovalent surface modification method. Cell surfaces are constantly remodeled by both Grampositive and -negative bacteria. In Gram-positive bacteria, one of the most prominent modes of surface alteration occurs by the sortase machinery. Sortases are membrane-anchored enzymes that catalyze the covalent attachment of proteins onto the penta-glycine PG precursor. As the PG is further assembled into the mature polymeric scaffold, the “sorted” proteins become displayed on bacterial cell surfaces. Interestingly, it was discovered early on that a short sorting peptide sequence was sufficient to designate proteins for sortase-based transpeptidation onto a complementary pentaglycine peptide.42 In other words, diverse peptidic and nonpepditic molecules when displayed with the minimum 5 amino acid sequence (LPXTG) were competent substrates of sortase in vitro. Spiegel and co-workers showed that the same chemistry was also operable on the surface of live bacteria.43,44 They synthesized fluorescein-tagged sortase sorting peptides and found that bacterial cell surfaces were metabolically labeled in a sortasespecific manner (Figure 3E). Moreover, S. aureus cells tagged with fluorescein were opsonized by antifluorescein antibodies. The use of sortase sorting peptides to decorate bacterial cell surfaces provides a powerful and versatile mode of surface tagging. However, high concentrations (0.5−1 mM) were required to achieve adequate surface labeling levels. The Bertozzi laboratory has pioneered the technique of metabolic oligosaccharide engineering (MOE).45,46 In this strategy, cells are treated with an unnatural sugar that is metabolically processed by living cells and click chemsitry handles are incorporated within the cellular glycan. Dube and co-workers showed that treatment of Helicobacter pylori (Hp) with N-azidoacetylglucosamine, a synthetic analog of the Nacetylglucosamine precursor within lipopolysaccharide (LPS) chains in Gram-negative bacteria, yielded surface labeling with azido handles (Figure 3F).47 The azido group was subsequently reacted with DNP epitopes via a Staudinger ligation. They confirmed that anti-DNP antibodies were successfully recruited to bacterial cell surfaces. In the absence of the azido group within the sugar unit, lower antibody recruitment levels were observed. The Pires research team first devised a strategy to metabolically label bacterial cell surfaces for immunomodulation by synthesizing an analog of a PG building block modified with the hapten DNP.48 By using this novel modality, we combined the features of metabolic labeling and immune response activation in a single agent. Similar to α-Gal, humans have high levels of anti-DNP antibodies in circulation that can be leveraged to opsonize potentially pathogenic entities.49−51 A distinguishing feature of Gram-positive bacteria is the presence of a thick PG scaffold found on the outside of the cytoplasmic membrane. Exposure of the PG layer to the extracellular milieu, where antibodies and immune cells are located, provides an opportunity for the PG scaffold to engage various components of the immune system. During active points in cell wall remodeling (cellular growth and division), PG undergoes a series of chemical modifications. An essential and highly conversed PG modification is the crosslinking of neighboring PG stem peptide chains by D,D- and L,Dtranspeptidases. We, and others, have recently demonstrated that unnatural D-amino acids from the surrounding medium can displace D-alanine from bacterial PG, thus resulting in the swapping of the terminal stem peptide amino acid with

■

METABOLIC LABELING An alternative route to grafting antigens onto the surface of bacterial cells involves the hijacking of endogenous biosynthetic pathways. During the past several decades, it has become evident that in select cases there are steps in the biosynthesis of bacterial natural building blocks (e.g., lipids, proteins, nucleic acids, metabolites, etc.) that tolerate synthetic analogs.40,41 This enzymatic promiscuity can, in turn, provide a versatile mode for studying fundamental cellular processes and developing novel therapeutic modalities. By mimicking structural features of endogenous building blocks, synthetic analogs can be metabolically incorporated into live target cells. A major advantage of metabolic-based surface labeling is that it provides a covalent attachment within the surface matrix. The irreversible installment of the antigenic handle would be D



Perspective

Figure 4. Left: the three designs described by the Pires laboratory in metabolically modifying bacterial cell surfaces with DNP epitopes. Right: Recruitment of FITC-labeled anti-DNP antibodies was demonstrated directly from pooled serum. Higher recruitment levels were found in D-amino acids displaying a C-terminus carboxamide (DK-Amide) relative to carboxylic acid (DK-Acid). Reprinted in part with permission from Fura et al.62 Copyright 2015 John Wiley and Sons.

unnatural D-amino acids.52−54 PG transpeptidases have unique promiscuity in tolerating diverse amino acid side chains (as long as the backbone is a D-stereocenter). The extent of this promiscuity was revealed when D-amino acids displaying entirely unnatural side chains were also found to be metabolically incorporated onto bacterial PG scaffolds.55 In the first design, the Pires laboratory successfully developed an approach to re-engage components of the immune system by tagging PG from Gram-positive organisms with hapten conjugated D-amino acids (Figure 4). More specifically, we synthesized a series of D-amino acids with a variable polyethylene glycol spacer connected to DNP epitopes. Synthetic D-amino acids were metabolically incorporated as cell wall building blocks in live cells, which led to cell surface presentation of unnatural side chains modified with haptens. We further demonstrated that cells treated with DNPconjugated D-amino acids triggered the recruitment of endogenous antibodies (existing antibodies in human serum) and induced phagocytosis by macrophages in several pathogenic Gram-positive organisms such as Enterococcus faecalis (E. faecalis) and S. aureus. Critically, we showed that D-amino acid agents specifically tagged bacterial cell surfaces

but not mammalian cell surfaces, thus establishing a strong selectivity toward bacteria. More recently, we showed that Damino acids can also tag S. aureus in live host organisms, which establishes a first step toward advanced animal studies.56 During further investigations, it became evident that a fraction of the DNP-tagged PG was actively removed by surface-bound carboxypeptidases. Carboxypeptidases play a role in tailoring the length and structure of the overall PG scaffold by removing terminal amino acids from the PG stem peptide.57 The terminal site is also where the DNP epitopes are incorporated and, therefore, carboxypeptidase activity can potentially lower the overall level of available haptens on cell surfaces. To counter this effect, we sought an alternative point of entry into the PG biosynthetic pathway (Figure 4). We and others had previously shown that D-amino acid dipeptides can hijack PG biosynthesis at the MurF ligase stage, leading to greater control on where the DNP is displayed within the stem peptide.58,59 MurF is the enzyme responsible for ligating D-AlaD-Ala dipeptide units to UDP-MurNAc-tripeptide. Indeed, using this strategy, we found that DNP labeling on bacterial cell surfaces increased relative to the use of single amino acids.60 E



Perspective

Figure 5. Chemical structures of DNP-modified vancomycin in the absence (A) and presence of a sortase A linker (B). (C) Induction of phagocytosis by macrophages of S. aureus target cells upon treatment with Sort3, which combines both vancomycin and sortase. (D) Labeling of live Caenorhabditis elegans infected with S. aureus (constitutively expressing mCherry) with a sortase−vancomycin conjugate. The sortase−vancomycin conjugate was derivatized with FITC. Scale bar presents 20 μm. (C, D) Reproduced from ref 64 with permission from the Royal Society of Chemistry.

required to adequately tag bacterial cell surfaces. For these reasons, a complementary strategy was explored aimed at labeling bacterial cell surfaces by combining conventional antibiotics with immunomodulation. The major advantages of this novel strategy are 2-fold. First, it may ultimately provide a dual-action therapeutic agent. If the inherent antimicrobial activity is maintained, then enlistment of the immune system serves to add a second and distinct mode of bacterial clearance. Second, these agents would likely operate at the same concentrations that are already employed in clinical settings and thus may provide more robust candidates for future clinical testing. There are several widely used FDA-approved antibiotics that home to bacterial cell surfaces as a prerequisite for targeting bacterial pathogens. For Gram-positive bacteria, the potent glycopeptide antibiotic vancomycin is a prime example. Vancomycin inactivates bacteria by associating with the D-AlaD-Ala terminal end of lipid II (a critical PG precursor), which effectively halts PG biosynthesis.63 While lipid II is the lethal target of vancomycin, D-Ala-D-Ala displayed on mature PG scaffolds will also associate with vancomycin. We reasoned that

Upon gaining further insight into PG remodeling by synthetic D-amino acids, we were able to show that PG-linked transpeptidases tolerate not only unnatural side chains but also diverse C-terminal functional groups.61 One specific variation (carboxamide) led to more pronounced labeling than the native carboxylic acid C-terminus, as measured by a fluorescently labeled D-amino acid. With this information in hand, the DNPmodified D-amino acid was modified with a carboxamide at the C-terminus (Figure 4). The second generation agents showed a 10-fold improvement in antibody recruitment levels against B. subtilis and E. faecalis, and we showed the recruitment of antiDNP antibodies onto cell surfaces by direct treatment with human serum.62

■

ANTIBIOTIC-BASED TARGETING While metabolic labeling provides an irreversible tagging modality, there are some potential disadvantages in relying on metabolic labeling for surface tagging. The primary roadblock is the competition with endogenous substrates, which can often exist at high concentrations (or high effective concentrations). Therefore, high concentrations of exogenous agents are F



Perspective

Small-Molecule Immunotherapeutics. Angew. Chem., Int. Ed. 56 (42), 13036−13040. (2) Levy, S. B., and Marshall, B. (2004) Antibacterial resistance worldwide: causes, challenges and responses. Nat. Med. 10 (12 Suppl), S122−S129. (3) Gold, H. S., and Moellering, R. C., Jr. (1996) Antimicrobial-drug resistance. N. Engl. J. Med. 335 (19), 1445−53. (4) Czaplewski, L., Bax, R., Clokie, M., Dawson, M., Fairhead, H., Fischetti, V. A., Foster, S., Gilmore, B. F., Hancock, R. E., Harper, D., Henderson, I. R., Hilpert, K., Jones, B. V., Kadioglu, A., Knowles, D., Olafsdottir, S., Payne, D., Projan, S., Shaunak, S., Silverman, J., Thomas, C. M., Trust, T. J., Warn, P., and Rex, J. H. (2016) Alternatives to antibiotics-a pipeline portfolio review. Lancet Infect. Dis. 16 (2), 239−51. (5) Hauser, A. R., Mecsas, J., and Moir, D. T. (2016) Beyond Antibiotics: New Therapeutic Approaches for Bacterial Infections. Clin. Infect. Dis. 63 (1), 89−95. (6) Kaki, R., Elligsen, M., Walker, S., Simor, A., Palmay, L., and Daneman, N. (2011) Impact of antimicrobial stewardship in critical care: a systematic review. J. Antimicrob. Chemother. 66 (6), 1223−30. (7) Schuts, E. C., Hulscher, M., Mouton, J. W., Verduin, C. M., Stuart, J., Overdiek, H., van der Linden, P. D., Natsch, S., Hertogh, C., Wolfs, T. F. W., Schouten, J. A., Kullberg, B. J., and Prins, J. M. (2016) Current evidence on hospital antimicrobial stewardship objectives: a systematic review and meta-analysis. Lancet Infect. Dis. 16 (7), 847− 856. (8) Burnham, C. D., Leeds, J., Nordmann, P., O’Grady, J., and Patel, J. (2017) Diagnosing antimicrobial resistance. Nat. Rev. Microbiol. 15 (11), 697−703. (9) Coffman, R. L., Sher, A., and Seder, R. A. (2010) Vaccine adjuvants: putting innate immunity to work. Immunity 33 (4), 492− 503. (10) Curtiss, R., 3rd (2002) Bacterial infectious disease control by vaccine development. J. Clin. Invest. 110 (8), 1061−6. (11) Mishra, R. P., Oviedo-Orta, E., Prachi, P., Rappuoli, R., and Bagnoli, F. (2012) Vaccines and antibiotic resistance. Curr. Opin. Microbiol. 15 (5), 596−602. (12) Zumla, A., Rao, M., Wallis, R. S., Kaufmann, S. H., Rustomjee, R., Mwaba, P., Vilaplana, C., Yeboah-Manu, D., Chakaya, J., Ippolito, G., Azhar, E., Hoelscher, M., and Maeurer, M. (2016) Host-directed therapies for infectious diseases: current status, recent progress, and future prospects. Lancet Infect. Dis. 16 (4), e47−63. (13) Hancock, R. E., Nijnik, A., and Philpott, D. J. (2012) Modulating immunity as a therapy for bacterial infections. Nat. Rev. Microbiol. 10 (4), 243−54. (14) Kieser, K. J., and Kagan, J. C. (2017) Multi-receptor detection of individual bacterial products by the innate immune system. Nat. Rev. Immunol. 17 (6), 376−390. (15) Philpott, D. J., Sorbara, M. T., Robertson, S. J., Croitoru, K., and Girardin, S. E. (2014) NOD proteins: regulators of inflammation in health and disease. Nat. Rev. Immunol. 14 (1), 9−23. (16) Royet, J., Gupta, D., and Dziarski, R. (2011) Peptidoglycan recognition proteins: modulators of the microbiome and inflammation. Nat. Rev. Immunol. 11 (12), 837−51. (17) Pardoll, D. M. (2012) The blockade of immune checkpoints in cancer immunotherapy. Nat. Rev. Cancer 12 (4), 252−64. (18) Rosenberg, S. A., Yang, J. C., and Restifo, N. P. (2004) Cancer immunotherapy: moving beyond current vaccines. Nat. Med. 10 (9), 909−15. (19) Restifo, N. P., Dudley, M. E., and Rosenberg, S. A. (2012) Adoptive immunotherapy for cancer: harnessing the T cell response. Nat. Rev. Immunol. 12 (4), 269−81. (20) Schumacher, T. N., and Schreiber, R. D. (2015) Neoantigens in cancer immunotherapy. Science 348 (6230), 69−74. (21) Grupp, S. A., Kalos, M., Barrett, D., Aplenc, R., Porter, D. L., Rheingold, S. R., Teachey, D. T., Chew, A., Hauck, B., Wright, J. F., Milone, M. C., Levine, B. L., and June, C. H. (2013) Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N. Engl. J. Med. 368 (16), 1509−18.

vancomycin modified with haptens may graft haptens onto surfaces of Gram-positive bacteria by tagging the terminal end of PG (Figure 5A). Indeed, we found that treatment of several types of Gram-positive bacteria with DNP conjugated vancomycin led to anti-DNP recruitment.64 With this initial design in mind, we sought to improve incorporation efficiency and persistence on the cell surface by combining the surface homing properties of vancomycin to target lipid II with the covalent tag provided by bacterial sortases. In this second generation class of agents, a sortase sorting peptide was incorporated within the vancomycin−DNP conjugate (Figure 5B).64 The goal was to improve the availability of the synthetic sortase substrate by increasing its effective concentration, a process mediated by vancomycin due to their similar lipid II target. The combination led to high levels of anti-DNP recruitment at low concentrations and observable recruitment at submicromolar cell treatment. Most importantly, surface tagging with these agents led to an induction in phagocytosis by macrophages (Figure 5C). Finally, we showed, for the first time, that bacterial cell surfaces can be specifically modified in a live host (Caenorhabditis elegans) using synthetic cell wall analogs (Figure 5D).

■

CONCLUDING REMARKS While the emerging field of bacterial immunotherapy is still in its early stages, there are exciting new developments on the horizon. An important lesson from the cancer immunotherapy domain is that slow progress and stumbles along the way do not reflect the ultimate potential in harnessing the immune system to fight off pathogens. There will be many challenges in applying the principles of immunotherapy against bacterial infections. Yet, we as a field are much better positioned to lean on the insights from the past decade to propel us forward. There will always be a place for conventional antibiotics in clinical use. The addition of complementary immunotherapies can potentially be another weapon in the arsenal of antibiotics.

■

AUTHOR INFORMATION

Corresponding Author

*Tel: 610 758 2887. E-mail: [email protected]. ORCID

Marcos M. Pires: 0000-0002-5676-0725 Author Contributions

M.J.S.F. and M.M.P. contributed equally. Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS We would like to thank the Pires group for their suggestions during the editing phase of this manuscript. ABBREVIATIONS DNP, 2,4-dinitrophenol; E. coli, Escherichia coli; α-Gal, Galα1 → 3Gal; S. aureus, Staphylococcus aureus; PG, peptidoglycan; MOE, metabolic oligosaccharide engineering; Hp, Helicobacter pylori; LPS, lipopolysaccharide; E. faecalis, Enterococcus faecalis; CDC, complement dependent cytotoxicity; ADCC, antibody dependent cell mediated cytotoxicity

■

REFERENCES

(1) Chirkin, E., Muthusamy, V., Mann, P., Roemer, T., Nantermet, P. G., and Spiegel, D. A. (2017) Neutralization of Pathogenic Fungi with G



Perspective

by metabolic labeling of the bacterial cell surface. FEMS Microbiol Rev. 39 (2), 184−202. (41) Hudak, J. E., Alvarez, D., Skelly, A., von Andrian, U. H., and Kasper, D. L. (2017) Illuminating vital surface molecules of symbionts in health and disease. Nat. Microbiol 2, 17099. (42) Popp, M. W., Antos, J. M., Grotenbreg, G. M., Spooner, E., and Ploegh, H. L. (2007) Sortagging: a versatile method for protein labeling. Nat. Chem. Biol. 3 (11), 707−8. (43) Nelson, J. W., Chamessian, A. G., McEnaney, P. J., Murelli, R. P., Kazmiercak, B. I., and Spiegel, D. A. (2010) A biosynthetic strategy for re-engineering the Staphylococcus aureus cell wall with non-native small molecules. ACS Chem. Biol. 5 (12), 1147−1155. (44) Gautam, S., Kim, T., Lester, E., Deep, D., and Spiegel, D. A. (2016) Wall teichoic acids prevent antibody binding to epitopes within the cell wall of Staphylococcus aureus. ACS Chem. Biol. 11 (1), 25−30. (45) Prescher, J. A., Dube, D. H., and Bertozzi, C. R. (2004) Chemical remodelling of cell surfaces in living animals. Nature 430 (7002), 873−7. (46) Luchansky, S. J., Hang, H. C., Saxon, E., Grunwell, J. R., Yu, C., Dube, D. H., and Bertozzi, C. R. (2003) Constructing azide-labeled cell surfaces using polysaccharide biosynthetic pathways. Methods Enzymol. 362, 249−72. (47) Kaewsapsak, P., Esonu, O., and Dube, D. H. (2013) Recruiting the host’s immune system to target Helicobacter pylori’s surface glycans. ChemBioChem 14 (6), 721−6. (48) Fura, J. M., Sabulski, M. J., and Pires, M. M. (2014) D-amino acid mediated recruitment of endogenous antibodies to bacterial surfaces. ACS Chem. Biol. 9 (7), 1480−9. (49) Lu, Y., Sega, E., and Low, P. S. (2005) Folate receptor-targeted immunotherapy: induction of humoral and cellular immunity against hapten-decorated cancer cells. Int. J. Cancer 116 (5), 710−9. (50) Jakobsche, C. E., Parker, C. G., Tao, R. N., Kolesnikova, M. D., Douglass, E. F., Jr., and Spiegel, D. A. (2013) Exploring binding and effector functions of natural human antibodies using synthetic immunomodulators. ACS Chem. Biol. 8 (11), 2404−11. (51) Sheridan, R. T., Hudon, J., Hank, J. A., Sondel, P. M., and Kiessling, L. L. (2014) Rhamnose glycoconjugates for the recruitment of endogenous anti-carbohydrate antibodies to tumor cells. ChemBioChem 15 (10), 1393−8. (52) Kuru, E., Hughes, H. V., Brown, P. J., Hall, E., Tekkam, S., Cava, F., de Pedro, M. A., Brun, Y. V., and VanNieuwenhze, M. S. (2012) In Situ probing of newly synthesized peptidoglycan in live bacteria with fluorescent D-amino acids. Angew. Chem., Int. Ed. 51 (50), 12519−23. (53) Shieh, P., Siegrist, M. S., Cullen, A. J., and Bertozzi, C. R. (2014) Imaging bacterial peptidoglycan with near-infrared fluorogenic azide probes. Proc. Natl. Acad. Sci. U. S. A. 111 (15), 5456−61. (54) Siegrist, M. S., Whiteside, S., Jewett, J. C., Aditham, A., Cava, F., and Bertozzi, C. R. (2013) D)-Amino acid chemical reporters reveal peptidoglycan dynamics of an intracellular pathogen. ACS Chem. Biol. 8 (3), 500−5. (55) Fura, J. M., Kearns, D., and Pires, M. M. (2015) D-Amino Acid Probes for Penicillin Binding Protein-based Bacterial Surface Labeling. J. Biol. Chem. 290 (51), 30540−50. (56) Pidgeon, S. E., and Pires, M. M. (2017) Cell Wall Remodeling of Staphylococcus aureus in Live Caenorhabditis elegans. Bioconjugate Chem. 28 (9), 2310−2315. (57) Popham, D. L., and Young, K. D. (2003) Role of penicillinbinding proteins in bacterial cell morphogenesis. Curr. Opin. Microbiol. 6 (6), 594−9. (58) Sarkar, S., Libby, E. A., Pidgeon, S. E., Dworkin, J., and Pires, M. M. (2016) In Vivo Probe of Lipid II-Interacting Proteins. Angew. Chem., Int. Ed. 55 (29), 8401−4. (59) Liechti, G. W., Kuru, E., Hall, E., Kalinda, A., Brun, Y. V., VanNieuwenhze, M., and Maurelli, A. T. (2014) A new metabolic cellwall labelling method reveals peptidoglycan in Chlamydia trachomatis. Nature 506 (7489), 507−10. (60) Fura, J. M., Pidgeon, S. E., Birabaharan, M., and Pires, M. M. (2016) Dipeptide-Based Metabolic Labeling of Bacterial Cells for Endogenous Antibody Recruitment. ACS Infect. Dis. 2 (4), 302−309.

(22) Hamid, O., Robert, C., Daud, A., Hodi, F. S., Hwu, W. J., Kefford, R., Wolchok, J. D., Hersey, P., Joseph, R. W., Weber, J. S., Dronca, R., Gangadhar, T. C., Patnaik, A., Zarour, H., Joshua, A. M., Gergich, K., Elassaiss-Schaap, J., Algazi, A., Mateus, C., Boasberg, P., Tumeh, P. C., Chmielowski, B., Ebbinghaus, S. W., Li, X. N., Kang, S. P., and Ribas, A. (2013) Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma. N. Engl. J. Med. 369 (2), 134−44. (23) Khalil, D. N., Smith, E. L., Brentjens, R. J., and Wolchok, J. D. (2016) The future of cancer treatment: immunomodulation, CARs and combination immunotherapy. Nat. Rev. Clin. Oncol. 13 (6), 394. (24) Morgensztern, D., and Herbst, R. S. (2016) Nivolumab and Pembrolizumab for Non-Small Cell Lung Cancer. Clin. Cancer Res. 22 (15), 3713−7. (25) Rosenberg, S. A., Yang, J. C., Sherry, R. M., Kammula, U. S., Hughes, M. S., Phan, G. Q., Citrin, D. E., Restifo, N. P., Robbins, P. F., Wunderlich, J. R., Morton, K. E., Laurencot, C. M., Steinberg, S. M., White, D. E., and Dudley, M. E. (2011) Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell transfer immunotherapy. Clin. Cancer Res. 17 (13), 4550−7. (26) Payne, D. J., Gwynn, M. N., Holmes, D. J., and Pompliano, D. L. (2007) Drugs for bad bugs: confronting the challenges of antibacterial discovery. Nat. Rev. Drug Discovery 6 (1), 29−40. (27) Koul, A., Arnoult, E., Lounis, N., Guillemont, J., and Andries, K. (2011) The challenge of new drug discovery for tuberculosis. Nature 469 (7331), 483−90. (28) Silver, L. L. (2011) Challenges of antibacterial discovery. Clin Microbiol Rev. 24 (1), 71−109. (29) Coates, A., Hu, Y., Bax, R., and Page, C. (2002) The future challenges facing the development of new antimicrobial drugs. Nat. Rev. Drug Discovery 1 (11), 895−910. (30) Pijpers, F., Faint, R., and Saini, N. (2005) Therapeutic cancer vaccines. Nat. Rev. Drug Discovery 4 (8), 623−4. (31) Mullard, A. (2016) The cancer vaccine resurgence. Nat. Rev. Drug Discovery 15 (10), 663−5. (32) Yarchoan, M., Johnson, B. A., 3rd, Lutz, E. R., Laheru, D. A., and Jaffee, E. M. (2017) Targeting neoantigens to augment antitumour immunity. Nat. Rev. Cancer 17 (4), 209−222. (33) Shokat, K. M., and Schultz, P. G. (1991) Redirecting the Immune-Response - Ligand-Mediated Immunogenicity. J. Am. Chem. Soc. 113 (5), 1861−1862. (34) Bertozzi, C., and Bednarski, M. (1992) C-glycosyl compounds bind to receptors on the surface of Escherichia coli and can target proteins to the organism. Carbohydr. Res. 223, 243−253. (35) Li, J., Zacharek, S., Chen, X., Wang, J., Zhang, W., Janczuk, A., and Wang, P. G. (1999) Bacteria targeted by human natural antibodies using alpha-Gal conjugated receptor-specific glycopolymers. Bioorg. Med. Chem. 7 (8), 1549−1558. (36) Rother, R. P., and Squinto, S. P. (1996) The alpha-galactosyl epitope: a sugar coating that makes viruses and cells unpalatable. Cell 86 (2), 185−188. (37) Krishnamurthy, V. M., Quinton, L. J., Estroff, L. A., Metallo, S. J., Isaacs, J. M., Mizgerd, J. P., and Whitesides, G. M. (2006) Promotion of opsonization by antibodies and phagocytosis of Grampositive bacteria by a bifunctional polyacrylamide. Biomaterials 27 (19), 3663−3674. (38) Metallo, S. J., Kane, R. S., Holmlin, R. E., and Whitesides, G. M. (2003) Using Bifunctional Polymers Presenting Vancomycin and Fluorescein Groups To Direct Anti-Fluorescein Antibodies to SelfAssembled Monolayers Presenting d-Alanine-d-Alanine Groups. J. Am. Chem. Soc. 125 (15), 4534−4540. (39) Kristian, S. A., Hwang, J. H., Hall, B., Leire, E., Iacomini, J., Old, R., Galili, U., Roberts, C., Mullis, K. B., Westby, M., and Nizet, V. (2015) Retargeting pre-existing human antibodies to a bacterial pathogen with an alpha-Gal conjugated aptamer. J. Mol. Med. (Heidelberg, Ger.) 93 (6), 619−31. (40) Siegrist, M. S., Swarts, B. M., Fox, D. M., Lim, S. A., and Bertozzi, C. R. (2015) Illumination of growth, division and secretion H



Perspective

(61) Pidgeon, S. E., Fura, J. M., Leon, W., Birabaharan, M., Vezenov, D., and Pires, M. M. (2015) Metabolic Profiling of Bacteria by Unnatural C-terminated D-Amino Acids. Angew. Chem., Int. Ed. 54 (21), 6158−62. (62) Fura, J. M., and Pires, M. M. (2015) D-amino carboxamidebased recruitment of dinitrophenol antibodies to bacterial surfaces via peptidoglycan remodeling. Biopolymers 104 (4), 351−9. (63) Barna, J. C. J., and Williams, D. H. (1984) The Structure and Mode of Action of Glycopeptide Antibiotics of the Vancomycin Group. Annu. Rev. Microbiol. 38, 339−357. (64) Sabulski, M. J., Pidgeon, S. E., and Pires, M. M. (2017) Immuno-targeting of Staphylococcus aureus via surface remodeling complexes. Chem. Sci. 8 (10), 6804−6809.

I
