Bacterial Anti-adhesives: Inhibition of Staphylococcus aureus Nasal

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Bacterial anti-adhesives: inhibition of Staphylococcus aureus nasal colonization Allison C Leonard, Laurenne E Petrie, and Georgina Cox ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.9b00193 • Publication Date (Web): 02 Aug 2019 Downloaded from pubs.acs.org on August 3, 2019

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ACS Infectious Diseases

Bacterial anti-adhesives: inhibition of Staphylococcus aureus nasal colonization

Allison C. Leonard1, Laurenne E. Petrie1 & Georgina Cox1*

1College

of Biological Sciences, Department of Molecular and Cellular Biology, University of

Guelph, 50 Stone Rd E, Guelph, Ontario, Canada N1G 2W1. *Corresponding author:

[email protected]

ACS Paragon Plus Environment

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Bacterial adhesion to the skin and mucosa is often a fundamental and early step in host colonization, the establishment of bacterial infections, and pathology. This process is facilitated by adhesins on the surface of the bacterial cell that recognize host cell molecules. Interfering with bacterial host cell adhesion—so-called anti-adhesive therapeutics—offers promise for the development of novel approaches to control bacterial infections. In this review, we focus on the discovery of anti-adhesives targeting the high priority pathogen Staphylococcus aureus. This organism remains a major clinical burden and S. aureus nasal colonization is associated with poor clinical outcomes. We describe the molecular basis of nasal colonization and highlight potentially efficacious targets for the development of novel nasal decolonization strategies.

Keywords Anti-adhesives; anti-virulence; Staphylococcus aureus; MRSA; antibiotic resistance; prophylactic therapeutics; colonization; ClfB; IsdA; WTA


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Antibiotics are life-saving molecules that have been relied upon for >70 years to control bacterial infections. Unfortunately, we are now experiencing a global ‘antibiotic resistance crisis’ that seriously threatens our ability to treat infections with these molecules1. This crisis has been spurred by the emergence of multi-drug resistant pathogenic bacteria and is compounded by our failure to discover new classes of antibiotics2. Due to the selective pressure imparted by antibacterial chemotherapy, antibiotic use and antibiotic resistance are intricately connected. Despite initiatives to encourage antibiotic stewardship, there continues to be a steady increase in global antibiotic consumption, which is expected to increase a further 200% by 20303-4. In addition to the burden of resistance, antibiotics negatively disrupt the ecology of our microbiota, which has been associated with a wide variety of health problems5. In particular, antibiotics are extensively administered to the pediatric population and up to 50% of infants under 1 year of age will have been exposed6. Early life antibiotic exposure has been linked to a number of negative health outcomes later in life such as metabolic syndrome, obesity, and asthma7. Addressing the antibiotic resistance crisis requires a multi-faceted approach involving the identification of new antibiotic classes and/or vaccines, improved antibiotic stewardship, and global surveillance programs. The development of therapeutic alternatives to antibiotics is also prominent among the solutions to this crisis8. One novel alternative approach is the attenuation of bacterial virulence, which has gained increased attention over the last 10 years and there is a growing pipeline for such drug candidates9. The rationale behind this strategy is to disarm a pathogen’s infectious capability without impacting growth, rendering the bacteria more susceptible to the host’s immune response. An attractive and validated anti-virulence approach involves inhibiting the attachment of bacteria to the host’s skin and mucosa10-11, which is enabled by bacterial adhesins12. Bacterial attachment to the skin and mucosa is often a fundamental and early step in host colonization, the establishment of infections, and host pathology10. By adhering to the host, bacteria prevent physical clearance by cleansing mechanisms and attain proximity to the host cell, which allows


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for the delivery of toxins, access to nutrients, colonization, biofilm formation and host cell invasion13. Adhesion is a common prerequisite for pathogenesis; initially, bacteria reversibly associate with host cells via weak non-specific interactions14. These transient interactions are reinforced by high-affinity interactions mediated by host cell specific bacterial adhesins. Bacteria possess a wide repertoire of these appendages, ranging from glycopolymers to monomeric proteins and complex multimeric proteinaceous assemblies12. From a drug discovery perspective, numerous stages of bacterial host cell adhesion could be targeted for the development of anti-adhesives and there are a number of factors to consider in the development of such compounds. Primarily, anti-adhesives are ‘precision therapeutics’, whereby they target bacterial specific virulence factors, leaving the remainder of the microbiota intact15. Indeed, the appendages used by bacteria to adhere to host tissues vary considerably, even amongst species. For example, virulent strains of Escherichia coli can cause a wide range of diseases including gastrointestinal and urinary tract infections (UTIs), which is dependent on the strain’s repertoire of virulence factors16. Some of the most promising examples of small molecule anti-adhesives target the carbohydrate recognition domains of uropathogenic E. coli fimbriae17-21, which enable adherence to the uroepithelium15, 22. These anti-adhesives form the basis of an orthogonal, or complementary, group of therapeutics to control UTIs15, 22. Clearly, anti-adhesives need to be tailored for specific pathogens and for use in different clinical contexts. A major advantage of anti-adhesive therapeutics is that they could alleviate some of the negative side effects associated with antibiotic use. Namely, antibiotics are intended to inhibit the growth of bacteria. As such, when a mixed population of bacteria are exposed, antibiotics naturally select for mutations that confer an advantage. The antibiotic resistant population of bacteria can then proliferate and outnumber the susceptible organisms. In contrast, antiadhesives are not intended to inhibit bacterial growth. Therefore, in principle, this approach ought to be less likely to promote and give rise to bacterial resistance due to reduced selective


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pressure11, 23. While it is conceivable (and likely) that bacteria could mutate, becoming resistant to anti-adhesives, resistant isolates will likely not infect the host and will be expelled by cleansing mechanisms10-11. Therefore, these organisms will not be continually exposed to a sustained selective pressure that disrupts the balance between resistant and susceptible organisms24. In addition, it is anticipated that the spread of anti-adhesive resistance amongst bacterial populations will occur at much lower frequencies10. Finally, since anti-adhesives target specific pathogens, their therapeutic use should not cause dysbiosis of our microbiota15. However, these potentially promising factors need to be experimentally validated in vivo, under physiologically relevant conditions. There are a number of well-described strategies for the development of anti-adhesive therapeutics 10-11, 24. Anti-adhesives can be broadly grouped into major categories depending on their mechanism of action (Figure 1)10-11, 24: competitive inhibitors that can mimic bacterial adhesins17-21 (Class I) and host cell receptors (Class II), molecules that inhibit the biosynthesis or surface presentation of adhesins25-26 (Class III) or host receptors (Class IV)27. Finally, the use of antibodies targeting bacterial adhesins has been well described (Class V)10-11, 24. This review will discuss clinical targets for the development of small molecule antiadhesive therapeutics aimed at the Gram-positive pathogen Staphylococcus aureus. Antibiotic resistant S. aureus is a major clinical burden, which is exacerbated by the commensal lifestyle of this organism28. Despite efforts to develop a S. aureus vaccine, none have progressed past phase III clinical trials29, signifying a critical need for alternative innovative approaches. Adhesion is an important facet of S. aureus pathogenesis and the adhesins facilitating this process are well understood29-30. Targeting adhesion could offer an orthogonal and efficacious approach to control S. aureus infections.


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Figure 1: Schematic overview of bacterial anti-adhesives. The first panel shows a simplistic representation of S. aureus successfully adhering to host cells using cell wall-anchored (CWA) adhesins. The following panels depict the different classes of anti-adhesives. Methicillin-resistant S. aureus (MRSA): the need for new treatment options S. aureus is a colonizing opportunistic pathogen causing infections when the skin is breached and/or the host’s immune system is weakened. This organism is capable of causing a variety of diseases ranging from skin and soft tissue infections to severe systemic infections31. Antibiotic resistant S. aureus isolates, in particular methicillin-resistant S. aureus (MRSA), are associated with poorer clinical outcomes32. The World Health Organization reported global antibiotic resistance statistics showing 31-50% of S. aureus isolates are methicillin resistant33. The same report identified MRSA as a high priority for new antibiotic research and development efforts33. High proportions of MRSA-associated infections require treatment with second-line antibiotics, which are expensive and need monitoring during treatment due to undesirable side effects34. S. aureus persistently colonizes ~20% of the adult population and intermittently colonizes a further 30%; the nasal cavity is the natural niche/primary S. aureus reservoir in the human body35-36. S. aureus is considered to favour a dispersed—rather than biofilm-like—mode of growth in the nose37-38. It is important to note that the majority (~80%39) of invasive


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nosocomial infections arise from endogenous S. aureus isolates40-41. The association between S. aureus nasal carriage and wound infections was observed as early as the 1920s, which led investigations into ‘chemotherapeutic nasal snuffs’ to reduce colonization36. Currently, preoperative nasal decolonization with the antibiotic mupirocin is routinely used in the hospital setting and reduces the incidence of infections36, 42. However, mupirocin resistance is increasing and in some instances levels as high as 80% have been reported, threatening decolonization efficacy42. Indeed, there is a direct correlation between resistance emerging following exposure to mupirocin; increased use predisposes patients to resistance42. This observation is problematic since patients eradicated of S. aureus often become recolonized with isolates exhibiting decreased sensitivity to mupirocin36, 42. A strategy to decolonize that does not provide a strong selection for resistance is needed and anti-adhesives could be well-suited to this application. In summary, asymptomatic nasal carriage of S. aureus is a major risk factor for infections43-46 and for transmission in both the community and hospital settings. This review will discuss the molecular basis of this phenomenon, followed by an in-depth discussion of clinical targets to eradicate colonization. We will conclude with a review of promising known inhibitors of S. aureus adhesion.

The molecular basis of S. aureus nasal colonization: the host perspective Given that the nose is an important ecological niche, S. aureus tightly adheres to avoid mucociliary clearance, cell shedding and to compete with other commensal bacteria that make up the nasal microbiota28. There are two distinct S. aureus niches: the nasal vestibule (anterior nasal cavity; the nostril opening) and the inner nasal cavity, also known as the posterior nasal cavity (Figure 2). The epithelium of the nasal cavity in these two regions is distinct, which alters the molecular basis of S. aureus host cell adhesion, as described below.

Composition of the nasal vestibule and the inner nasal cavity


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The nasal vestibule is lined with the same stratified and keratinized epithelium of the skin (Figure 2)47. During desquamation (skin shedding), proliferating keratinocytes within the basal layer begin their migration to the surface of the skin. Through this process they change morphology, increase keratinization and lose their nucleus, eventually forming the stratum corneum (the outermost layer of the skin) (Figure 2). The keratins are the major structural proteins on the surface of these epithelial cells; the K1 and K10 cytokeratin molecules are present in large quantities on the both the interior and exterior surface of these dead keratinized cells (Figure 2)48. The stratum corneum is surrounded by a proteinaceous structure referred to as the cornified envelope, whereby the cytoplasmic membrane of the mature squamous cells is replaced by the protein loricrin (~80% of the total protein content)49 and other proteins such as involucrin (Figure 2). This enveloping structure is strengthened by transglutination and decorated by the covalent linkage of ceramides and fatty acids50; the latter exhibits antimicrobial activity and is a component of the innate immune response51-52. S. aureus has the highest affinity for the mature squames on the outermost layer of the epithelium (i.e., the stratum corneum)53-54. Indeed, recent studies investigating the location of S. aureus in the nasal vestibule of healthy individuals confirmed the stratum corneum as the primary source (Figure 2)47. However, lower quantities of bacterial cells were also identified in both the upper and lower layers of the epidermis and were found intracellular and in the different layers of the skin47. Overall, cytokeratin K1, K10 and loricrin proteins within the stratum corneum are integral S. aureus host ligands enabling colonization of the nasal vestibule48, 55. The second S. aureus niche resides within the inner cavity vestibule (Figure 2). The epithelium within this region transitions toward the morphology of ciliated and pseudostratified upper airway epithelium, losing the skin’s characteristic keratinization. These cells have been shown to express the scavenger receptor expressed by endothelial cells I (SREC-I; also termed SR-F1), an F-type scavenger receptor56. This host cell membrane protein has been identified as a key receptor for S. aureus adhesion within the inner nasal cavity56.


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Interestingly, nasal colonization is considered to be a multifactorial process involving these two different S. aureus niches (Figure 2)54, 57. SREC-I mediated adhesion to the inner nasal cavity is thought to be important in the initial stages of colonization38, 57. In contrast, longterm persistent colonization, which is observed in ~20% of the adult population, implicates the squamous keratinized epithelium within the nasal vestibule38, 54, 57.

frontal sinus

sphenoid sinus

nasal turbinates

nasopharynx

nasal cavity

vestibulum nasi

eustachian tube orifice

anterior naris

hard palate

IsdA

ClfB WTA

Mucus

loricrin, involucrin and filaggrin

N N

N

N

N

N

N

SREC-1

stratum corneum

Goblet cell

stratum granulosum

cytokeratin K1/K10

stratum spinosa

cytokeratin K5/K14

stratum basal

ClfB and IsdA CWA proteins facilitate adhesion to the nasal vestibule

WTAs mediate adhesion to the host cell receptor SREC-I within the inner nasal cavity

Figure 2: S. aureus colonization of the human nose. S. aureus adheres to the anterior vestibulum nasi using the CWA proteins ClfB and IsdA, which bind to host cell proteins within the cornified envelope of the stratum corneum. S. aureus also colonizes the inner nasal cavity due to the interaction of WTAs with the host cell receptor SREC-I, which is expressed on the surface of the ciliated epithelial cells lining this region of the nose. The molecular basis of S. aureus nasal colonization: the bacterial perspective


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Given the differences of the epithelium in these two different S. aureus niches (Figure 2), S. aureus adhesins facilitating colonization are also distinct. The CWA proteins clumping factor B (ClfB)48, 58-59 and the iron-regulated surface determinant A (IsdA)38, 60-61 have been identified as the major S. aureus adhesins that enable persistent colonization of the nasal vestibule, via adhesion to proteinaceous host molecules within the squamous epithelium (Figure 2). On the other hand, the S. aureus glycopolymer wall teichoic acid (WTA) mediates adhesion to ciliated epithelial cells expressing SREC-I within the inner nasal cavity (Figure 2).

Cell wall-anchored (CWA) proteins mediate adhesion to the nasal vestibule There are four major staphylococcal cell wall-anchored (CWA) protein families, comprising 25 different proteins30. These surface proteins play various roles in pathogenesis30 and are covalently anchored to the cell wall by sortases (see Foster et al., 2014 for a comprehensive review30). CWA proteins significantly contribute to the virulence of S. aureus by facilitating immune evasion, cell invasion, nutrient acquisition, and adhesion to host cells30. Historically, the names of CWA proteins were assigned in regard to the function they were first associated. However, this nomenclature can be misleading, and some have been re-named since many of these proteins display numerous functions and possess different ligand binding sites. The CWA proteins ClfB and IsdA are the primary adhesins facilitating colonization of the nasal vestibule via interaction with the host cell receptor molecules cytokeratin K10 and/or loricrin. The genes encoding these determinants are highly conserved amongst S. aureus isolates, indicating that anti-adhesives targeting these proteins could enjoy clinical success62. While additional CWA proteins—namely SdrC, SdrD, and SasG60, 63—have been implicated in nasal colonization, ClfB and IsdA are considered the major determinants of S. aureus colonization of the nasal vestibule. Specifically, IsdA and ClfB devoid S. aureus strains are defective in colonizing the nares of rodents61, 64. In human volunteers, a ClfB mutant was also shown to be impaired in nasal colonization over an extended time period, highlighting the


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importance of this protein in persistent colonization59. Of note, the latter study used the S. aureus strain 8325-4 defective in the alternative sigma factor B (σB) 59. Therefore, this study has limitations since this global regulator is involved in the upregulation of clfB expression, which is described in more detail below. Finally, elevated expression of isdA and clfB has been observed in a cotton rat model of nasal colonization (a common and efficacious model to study S. aureus nasal colonization65) several days after bacterial instillation38. ClfB was originally identified as a fibrinogen-binding protein (hence the designation clumping factor B) but has since been shown to be susceptible to proteolytic cleavage by the S. aureus metalloprotease aureolysin66. The truncated form is unable to bind fibrinogen but has been shown to retain affinity for cytokeratin K1048. ClfB is the predominant keratin-binding adhesin expressed in the exponential phase of growth58. This adhesin interacts with repeats of the amino acid sequence Y[GS]nY in the C-terminal tail region of cytokeratin K10, which is a highly variable region of the keratins58. The Y[GS]nY motifs form omega loops within the keratin molecule: the GS sequences project out due to hydrophobic interactions between the Y residues55. Cytokeratin K10 binds in a hydrophobic trench between the N2 and N3 subdomains of ClfB (Figure 3A & B)67-68 via the ‘dock, lock and latch’ binding mechanism described for other staphylococcal adhesins69. Specifically, by interacting with the hydrophobic ClfB trench, a conformational change is induced in the C-terminal extension of the N3 domain, covering the peptide ligand, while also interacting with residues located in the N2 domain. This interaction effectively locks the peptide ligand in place and the strength of the interaction has been shown to be enhanced by force70. The latter indicates S. aureus adheres more firmly under high shear stress induced by physical stresses, such as mucociliary clearance70. It was originally assumed that cytokeratin K10 is the primary ClfB ligand. However, subsequent studies revealed that ClfB also binds loricrin, which has been shown to be critical for nasal colonization55. Loricrin is composed of cytokeratin K10 similar omega loops mediating this interaction. Loricrin is the major component of the cornified envelope; the interaction of ClfB


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with loricrin is also crucial to the establishment of S. aureus skin infections71. In summary, loricrin is considered the major ClfB ligand in the nose, signifying that the loricrin-ClfB interaction is integral to nasal colonization and is a key target for the development of nasal decolonization strategies.

A

N

SP

N

B

197 63

SD repeats 542

MS 812

C 927

C

NEAT

SP 1

N3

N2

N1

1

180

350

C

Figure 3: Domain organization and structures of the nasal cell adhesins ClfB and IsdA. (A) Schematic model depicting the domain organization of S. aureus ClfB (top) and IsdA (bottom). For both proteins, the N and C terminus are labelled, and the amino acid numbering indicates the domain boundaries. SP corresponds to the signal peptides targeting the proteins for secretion. For ClfB, the cytokeratin K10/loricrin binding interface is located within the N2 and N3 region, followed by the serine-aspartate (SD) repeat region and finally, the membrane spanning portion of the protein (MS). Of note, there is an aureolysin66 protease cleavage site located between the N1 and N2 region that truncates the protein, abolishing fibrinogen binding. The heme binding NEAr iron Transporter (NEAT) domain of IsdA is proposed to also mediate binding to host cell ligands such as cytokeratin and loricrin. (B) Ribbon diagram of the N2 and N3 domains of ClfB (PDB accession number: 4F1Z) bound to a cytokeratin K10 peptide (shown as green sticks). ClfB is coloured blue and residues that are critical for ligand binding—the ClfB ‘hotspot’—are colored red. (C) Ribbon diagram of the IsdA NEAT domain (shown in dark blue) in complex with heme (shown as sticks and colored green). The NEAT domain is proposed to mediate affinity to the nasal epithelium (PDB accession number: 2ITF).


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The iron-regulated CWA IsdA protein is the second adhesin implicated in nasal cell adhesion55. The nose is a unique habitat characterized by low nutrient and iron availability72. Iron is essential for bacterial growth and vertebrates have evolved systems to sequester iron intracellularly to prevent the proliferation of pathogenic bacteria73. In S. aureus, the NEAr iron Transporter (NEAT) domain family encompasses four iron-regulated surface determinants (Isd): IsdA, IsdB, IsdC and IsdH. The expression of these CWA proteins is induced when iron is deficient and these proteins have been shown to be key components of a system facilitating iron acquisition via a relay system involving heme binding and subsequent degradation in the cytoplasm of the bacterial cell74. As the nose represents an iron-limited environment, there is increased expression of Isd CWA proteins in nasal isolates72. Expression of IsdA has been shown to maximize nasal colonization in cotton rats and promote adherence to human nasal epithelial cells in vitro61. IsdA has broad ligand binding capacity, including components of the extra cellular matrix (ECM) such as fibrinogen and fibronectin75, serum proteins76 and heme77. However, the role of these interactions in nasal colonization remain unknown. The IsdA NEAT domain spans ~120 amino acids forming an immunoglobulin-like βsandwich fold78. Heme binds in a large hydrophobic pocket (Figure 3C) formed between one sheet and a loop, residing in a region of the protein opposite the chain termini78. The NEAT domain is responsible for the broad ligand binding capacity of IsdA and this domain can only interact with one ligand at a time, indicating that the different ligands likely have overlapping binding sites79. The ability of the NEAT domain to bind cytokeratin K10, loricrin, and involucrin, is thought to underlie the proteins ability to promote S. aureus nasal colonization79. The precise binding interface and the molecular details underlying the proteins ability to bind cytokeratin K10, loricrin and involucrin is unknown. In analogy to ClfB, loricrin is likely the major ligand for IsdA in the nose. Finally, in addition to host cell ligand binding mediated by the NEAT domain, the hydrophilic C-terminal domain of IsdA (Figure 3A) provides resistance against hydrophobic


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fatty acids present on the surface of the cornified envelope80. In addition, IsdA was shown to protect S. aureus from the antibacterial activities of the protease apolactoferrin, an abundant polypeptide in nasal secretions81. Therefore, this protein promotes adhesion to the nasal epithelium whilst also mediating resistance to the host’s innate defences80-81. To summarize, ClfB and IsdA are integral for long-term nasal colonization. Based on these findings and the observation that the genes encoding ClfB and IsdA are present in the vast majority of S. aureus isolates62, interfering with these elements offers promise for the development of novel anti-adhesive nasal decolonization strategies.

The glycopolymer WTA mediates adhesion to the inner nasal cavity during the initial stages of colonization WTAs are situated within the S. aureus cell wall and extend beyond the peptidoglycan of the cell; the polymer is composed of ~40 repeats of glycosylated and D-alanylated ribitol-phosphate units82-83. For some time, WTAs have been implicated in nasal colonization84. SREC-I was identified as the S. aureus WTA receptor expressed at the surface of ciliated epithelial cells lining the inner nasal cavity (Figure 2)56. SREC-I is a membrane protein composed of a large extracellular domain comprising multiple epidermal growth factor (EGF) and EGF-like repeats, followed by transmembrane and cytoplasmic domains (Figure 4)85. WTA possesses high affinity for the SREC-I receptor and the binding site was located to domains 3 and 4 of the EFG-like repeats (Figure 4). This interaction is charge-dependent, relying on the free amino groups of the D-alanine moieties decorating the WTA polymer56. The alanine residues counter the predominant negative charge of the phosphate groups within WTA, rendering the polymer more zwitterionic in nature. More recently, glycosylation of WTA (Figure 4) has also been shown to be a key factor in nasal colonization of cotton rats86. However, the receptor for WTA glycosyl residues is unknow86. Glycosylation is mediated by the enzymes TarM87 and TarS88, which


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decorate WTA with -N-acetylglucosamine (-GlcNAc) and -GlcNAc, respectively. The gene encoding TarS is highly conserved amongst S. aureus, however, tarM is less prevalent86. Considering the importance of WTA in the early stages of S. aureus nasal colonization, inhibiting the WTA-SREC-I interaction could offer promise in the development of anti-adhesive nasal decolonizing strategies. Given the multifactorial process of S. aureus nasal colonization, it is conceivable that such therapeutics could be combined with anti-adhesives targeting the interaction of ClfB and/or IsdA with the nasal vestibule, providing a combinatorial therapy that removes S. aureus from both nasal niches. Taking these factors into consideration, here we highlight candidate anti-adhesive targets (Figure 1) to enable the development of novel S. aureus nasal decolonization strategies. Table 1 summarises the anti-adhesive targets discussed in this review.

Figure 4: Schematic diagram of the interaction between S. aureus wall teichoic acid (WTA) and the nasal cell scavenger receptor expressed by endothelial cells I (SREC-I; also termed SR-F1). S. aureus WTA is covalently linked to the peptidoglycan (shown as yellow circles and blue hexagons, representing repeating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid, respectively). The polymer is composed of ~40-60 ribitol-phosphate repeats (yellow triangles) that can be decorated with cationic D-alanine esters (shown as green circles) and glycosylated with /-GlcNAc (blue hexagons). The interaction between S. aureus WTA and SREC-I is charge dependent, involving the free amines of D-alanine residues (green


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circles) decorating the polymer. SREC-I is a transmembrane protein (as shown), comprising an extracellular region of epidermal growth factor (EFG) and EFG-like repeats. WTA has been shown to interact with the EFG 3 and 4 repeats of the receptor. Loss of WTA glycosylation (blue hexagons), mediated by the enzymes TarS and TarM, also interferes with WTA-mediated adhesion to the inner nasal cavity. Inhibiting S. aureus nasal colonization: the development of anti-adhesives targeting ClfB Anti-adhesives targeting ClfB would interfere with the protein’s ability to bind loricrin. ClfB interacts with G/S-rich omega loops within proteinaceous ligands using the ‘dock, lock and latch’ binding mechanism67-68 (Figure 3B). The molecular details of this interaction were revealed by crystallization of the protein in complex with its peptide ligands, identifying a GSSGXGXXG binding motif67-68. Importantly, ClfB residues implicated in binding to this motif are highly conserved amongst S. aureus lineages67-68. The development of a direct, ClfB-targeting, antiadhesive requires the identification of a molecule interfering with the ClfB-loricrin protein-protein interaction. Small-molecule inhibition of protein-protein interactions, which is generally more cost effective than other therapeutics89, is challenging. In contrast to the deep cavities often recognized by small molecules, protein-protein interfaces are frequently large and flat90. However, not all residues within these interfaces are critical for binding and there has been significant progress into small molecule inhibitors of protein-protein interactions, many of which have been found to bind their targets with high affinity89-90. In particular, fragment-based lead discovery (FBLD) offers promise in this field89-90. This approach uses biophysical methods to identify molecules that bind weakly to protein surfaces89-90. These molecules can be evolved to bind with a higher affinity, increasing potency. The ClfB-ligand binding interface is 1463 Å2 and ligand recognition resides within amino acid residues (526-542) in the ‘lock’ region67. Despite this large contact surface area, substitution of either N526Q, Q235A, S236Q, F328A, and W522V (Figure 3B) residues completely abrogate the ability of ClfB to bind protein ligands67; this ‘hotspot’ could be the target of small molecules. Despite the challenges associated with the


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inhibition of protein-protein interactions, the ClfB hotspot could be an efficacious target for the development of anti-ClfB therapeutics. An alternate approach involves the indirect inhibition of adhesion by interfering with the biosynthesis or surface assembly/presentation of ClfB (Figure 1). The vast majority of CWA adhesins are anchored to the cell surface by the enzyme sortase A (SrtA). Deletion of the gene encoding this enzyme inhibits the presentation of cell surface proteins and significantly reduces the virulence of S. aureus, without impacting growth74, 91-92. Therefore, SrtA has been the focus of a number of drug discovery endeavours, resulting in the identification of promising pre-clinical inhibitors9. Since this has been an active area of research for some time, SrtA inhibitors are described in more detail later in this review. Here we will discuss alternate ClfB anti-adhesive targets. A number of CWA proteins—including ClfB—harbour a long stretch of Ser-Asp dipeptide repeats (referred to as the SD region) shown to be glycosylated by the SD-repeat glycosyltransferases (Sdg) A and B 93 (Figure 5A). The Ser residues in these repeats are modified with O-linked GlcNAc by the SdgB enzyme94. SdgA glycosylates sites that have already been modified by SdgB94. Glycosylation of the SD region prevents proteolytic degradation of adhesins, such as ClfB, by human neutrophil-derived cathepsin93 (Figure 5A). Indeed, the virulence of SdgB defective S. aureus is significantly impaired in a murine model of sepsis94. Inhibiting SdgB-mediated glycosyltransferase activity could render ClfB more susceptible to proteolytic cleavage in the host environment, reducing the affinity of S. aureus for the nasal epithelium (Figure 5A). However, whether deletion of the gene encoding SdgB, and thus inhibition of ClfB glycosylation, impacts nasal decolonization has not been investigated. Furthermore, the discovery of bacterial glycosyltransferase inhibitors can be challenging95-96. With the exception of pyranose scaffold compounds inhibiting glycosyltransferases involved in peptidoglycan polymerization97, there is a lack of potent and specific bacterial


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glycosyltransferase inhibitors98. The efficacy of SdrB as an anti-adhesive target awaits to be established and would be enhanced by increased mechanistic and structural insight. Another tactic in the search for indirect anti-adhesives entails the inhibition of global regulators of clfB expression (Figure 5B). S. aureus virulence factors are controlled by complex networks of regulators including two-component systems, sigma factors, regulatory RNAs and DNA-binding proteins99-101. Indeed, a single virulence gene can be influenced by several regulators that ‘cross talk’; regulators can impact expression directly via promoter recognition or indirectly through the action of additional regulators102. There is a biphasic distinction between the expression of CWA proteins earlier in the growth cycle followed by the expression of secreted toxins in the stationary phase100. In regard to ClfB, a subset of studies show production is positively regulated by the global regulator Rot (the repressor of toxins)102-103. Analysis of Rotmediated global regulation revealed that the only other S. aureus surface proteins positively regulated by Rot are SdrC and protein A102. Of note, this study was conducted with S. aureus harvested at the late-exponential phase of growth, which could impart a limitation since ClfB is maximally expressed at the mid-exponential phase66. An additional study using S. aureus strain 8325-4, describes Rot directly binding to the clfB promoter, enhancing expression103. However, as described, the use of this strain in the study of virulence gene expression is significantly compromised due to impaired σB (also referred to as SigB) activity104. Rot exhibits homology with the global transcriptional regulator SarA (the staphylococcal accessory regulator), which recognizes a specific motif in the promoter of its target genes105. All members of the SarA family share an KXRXXXDER amino acid motif implicated in this recognition99. However, it has been suggested that rot expression is repressed by SarA binding to the rot promoter106. Rot is a dimer with subunits forming the characteristic winged helix-turn-helix DNA-binding motif107. Mutagenesis of specific Rot residues inhibit the proteins activity, highlighting the DNA binding site and thus, the target of potential anti-adhesives107. Transcription factors have long been considered unsuitable targets for drug discovery, partly due to the lack of appropriate methods


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to screen for small molecule inhibitors108-109. However, approaches have now been described that could enable higher throughput screening of transcriptional targets108. Indeed, the development of approaches to target transcription factors in the treatment of cancer has seen a revival (referred to as transcription therapy) 110. Additionally, structure based drug design has been described for the identification of SarA inhibitors111 and this approach could be applied to Rot. More recently, the RNA-binding protein SpoVG was implicated in clfB expression112. The spoVG locus encodes an RNA binding regulatory protein positively regulated by the σB (Figure 5B). σB is a subunit of RNA polymerase providing promoter recognition specificity, enabling differential gene expression in response to changes in environmental conditions113. SpoVG was recently shown to positively regulate clfB expression by directly interacting with the clfB promoter and/or in a Rot-mediated fashion112. Therefore, inhibiting the action of SpoVG or the σB would interfere with the expression of ClfB, making these elements additional candidates for the development of indirect anti-adhesives (Figure 5B). The gene encoding σB is located within the sigB operon (composed of rsbU-V-W-sigB genes)114. The activity of this global regulator is controlled by so-called ‘partner-switching’ between the anti-σB kinase (encoded by rsbW), which associates and sequesters σB, and the non-phosphorylated form of RsbV (anti-anti-σB). RsbV competes for anti-σB binding and triggers release of σB 115. To add a further layer of detail, the rsbU gene in the sigB operon encodes a phosphatase that dephosphorylates RsbV, triggering the release of σB. Therefore, another attractive target for inhibition is the phosphatase RsbU (Figure 4B). Inhibition of this enzyme would result in a higher concentration of phosphorylated RsbV (anti-anti-σB), reducing the affinity of the protein for anti-σB. Thus, phosphorylated RsbV maintains anti-σB-SigB complexes, inhibiting the interaction of σB with RNA polymerase. Indeed, the overexpression of rsbU in S. aureus strongly activates σB 114 and the S. aureus 8325-4 strain has compromised σB activity due to mutation in the rsbU gene104. However, evidence supporting the efficacy of σB as an anti-virulence target are conflicting, with a subset of studies


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describing no impact to pathogenicity in animal models of infection when σB is compromised104, 116-117.

For this reason, decolonization strategies targeting σB and σB-related elements ought to

be postponed until there is more information supporting the efficacy of this target in relevant infection models. In summary, the development of anti-ClfB nasal decolonization strategies should focus on the identification of anti-adhesives that interfere with ClfB-mediated recognition of loricrin within the nasal vestibule (Figure 2). Such approaches are facilitated by a wealth of structural and molecular information that highlight the ClfB ligand binding ‘hotspot’ as a target for the development of protein-protein inhibitors. On the other hand, the development of indirect antiadhesive therapeutics would be enhanced by studies investigating the in vivo efficacy of the aforementioned targets.

A

B

Figure 5: Examples of indirect anti-adhesive targets to reduce the surface presentation of ClfB. (A) Schematic model of the CWA protein ClfB (as seen in Figure 3A). The glycosyltransferase SdgB covalently links N-acetylglucosamine (GlcNAc; shown as blue squares) to serine residues within the serine-aspartate repeat (SDR) region of ClfB. A second glycosyltransferase, SdgA, then adds an additional GlcNAc moiety. Glycosylation of the SDR region has been shown to protect SDR containing proteins from proteolytic cleavage by human neutrophil-derived cathepsin G. Inhibition of SdgB could render ClfB more susceptible to host proteolytic cleavage, reducing the surface presentation of the protein and thus, nasal


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colonization. (B) Schematic diagram of the regulatory cascades impacting clfB expression. The arrows represent positive interactions and the bars indicate repression. The red crosses depict favourable targets for anti-adhesives. Inhibiting S. aureus nasal colonization: the development of IsdA anti-adhesives The identification of directly acting IsdA anti-adhesives necessitates the need for molecules interfering with the adhesin’s affinity for loricrin. As described, the broad ligand binding capabilities of IsdA reside in its single-copy NEAT domain (spanning amino acids I69 to E177)75, which is solely composed of -sheets78 (Figure 3C). Non-specific electrostatic interactions are proposed to underlie the proteins ligand promiscuity78. With the exception of heme, the structures of IsdA bound to other ligands have not been reported and the IsdA-loricrin binding interface remains unknown. Therefore, we are unable to provide a detailed description of approaches to inhibit the IsdA-loricrin interaction. In the absence of structural and mechanistic knowledge, FBLD—as described for ClfB—could be used to identify IsdA-loricrin protein-protein interaction inhibitors using the NEAT domain as a target. Indirect IsdA anti-adhesives intend to interfere with the expression or presentation of IsdA on the cell surface. The isdA gene is negatively regulated by iron through the iron responsive regulatory protein Fur (ferric uptake regulator)75. Fur is a dimeric protein requiring metal cofactors, which binds a palindromic 19 base pair consensus sequence (the ‘Fur box’) within the promoters of target genes118. This interaction inhibits the transcription of downstream genes; in low iron conditions Fur dissociates allowing transcription to proceed118. In contrast to inhibition of ClfB mediated by interference with global regulators, inhibiting the action of Fur would result in increased isdA expression, and thus nasal colonization. Overall, the identification of IsdA anti-adhesives would be expedited by more insight into the molecular details underlying the broad ligand binding specificity of this protein. Akin to ClfB, the identification of a ligand binding ‘hotspot’ would enable the development of small molecule protein-protein interaction inhibitors. Furthermore, with the exception of Fur (and CWA protein


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common targets such as SrtA), it is unknown if other genetic and cellular elements are implicated in the presentation of this protein. Such studies could expand the target space for the development of anti-IsdA therapeutics.

Inhibiting S. aureus nasal colonization: interfering with WTA mediated nasal colonization Anti-adhesives targeting WTA mediated nasal colonization would interfere with the interaction of this polymer with the host cell receptor SREC-I. One interesting avenue of attack could be the inhibition of WTA biosynthesis. WTA biosynthesis is mediated by an enzyme cluster: the teichoic acid ribitol pathway (tar enzymes; referred to as tag enzymes in other organisms such as Bacillus subtilis)82. In contrast to the late stages of WTA biosynthesis, which are conditionally essential, the early-stages (involving the membrane-associated glycosyltransferases TarO and TarA) are non-essential for in vitro S. aureus growth119-121. However, deficiencies in these enzymes leads to an avirulent phenotype, highlighting these early steps as attractive antivirulence targets83, 119. Indeed, one of the first accounts of WTA-mediated virulence was observed in a cotton rat model of nasal colonization; tarO deficient S. aureus was unable to colonize the nares of rodents84. TarO reversibly catalyzes the transfer of GlcNAc phosphate—from UDP-GlcNAc—to a molecule of undecaprenyl phosphate in the bacterial membrane121. TarA then transfers NAcetyl-D-mannosamine (ManNAc)—from UDP-ManNAc—to the terminal GlcNAc, which serves as a substrate for the next enzyme in the pathway121. Inhibition of either of these enzymes renders the cell devoid of WTA. Therefore, TarO and/or TarA represent potentially efficacious anti-adhesive targets. Such approaches are supported by structural knowledge of the two proteins, which could facilitate the identification of enzyme-specific inhibitors. The crystal structure of TarO remains unknown, however, a recent study describes a three-dimensional model of the protein based on a structurally and functionally similar plasma membrane associated glycosyltransferase122. The crystal structure of a S. aureus TarA homolog from


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Thermoanaerobacter italicus was also recently reported (PDB accession codes: 5WB4 & 5WFG), highlighting a unique glycosyltransferase fold. Finally, since WTA is an attractive antivirulence therapeutic target, there are a number of known TarO-specific inhibitors that could be promising anti-adhesives122-124. A series of potent and recently described TarO inhibitors compounds are discussed in more detail in the proceeding section122. In addition to inhibiting WTA biosynthesis, a complementary approach could involve the inhibition of the enzymes that tailor the polymer. As described, TarS is a highly conserved WTA glycosyltransferase integral to nasal colonization in cotton rats86. While a proportion of strains do harbour both tarM and tarS, which would necessitate the need to compromise both enzymes, the majority only possess TarS86. Importantly, compromising TarS does not induce growth defects in vitro88. Combined, these factors make TarS an attractive anti-adhesive target (Figure 4). Such efforts could be enabled by a recent study depicting the structure and mechanism of action of this enzyme (PDB accession codes: 5TZ8 and 5TZE)125.

Promising known inhibitors of S. aureus host cell adhesion Inhibiting nasal colonization mediated by CWA proteins An attractive strategy to prevent the surface presentation of the majority of CWA proteins— including ClfB and IsdA—is the inhibition of the anchoring enzyme SrtA. As such, the identification of SrtA inhibitors has been an active research area for >15 years126. Such efforts involved a number of approaches including structure based mimetic substrate design, in silico virtual screening and the profiling of naturally derived and synthetic small molecules (see Guo et al. 2015 and Cascioferro et al. 2015 for detailed reviews). The in silico virtual screening approach was particularly successful, identifying one of the most promising classes of known SrtA inhibitors: the triazolothiadiazoles (Figure 6)127. This approach involved virtual screening of ~300,000 compounds to identify molecules that bind the SrtA active site, using a known natural product inhibitor (topsentin A128) as a model ligand127.


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The most efficacious compounds were 3,6-disubstituted triazolothiadiazoles (Figure 6), which lack antibacterial activity, exhibit potent and reversible inhibition of SrtA, and had efficacy in an animal model of sepsis127. These compounds could be potentially efficacious anti-adhesives and it would be advantageous to investigate their use in a cotton rat model of nasal colonization. An additional study, published simultaneously, describe a class of benzo[d]isothiazol3(2H)-one-adamantane amine derivatives (Figure 6) identified from an in vitro screen of >50,000 compounds against recombinant SrtA129. In contrast to the triazolothiadiazole compounds, these inhibitors show irreversible inhibition of SrtA by reaction with the active site cysteine residue, forming a covalent linkage between the cysteine thiol and the compounds benzothiazole129. However, unlike the triazolothiadiazoles, these compounds do exhibit modest S. aureus antibacterial activity (the most promising compounds have a minimum inhibitory concentration of 8-16 g/mL) and modest cytotoxicity129. Therefore, the triazolothiadiazoles may be more promising for the development of anti-adhesives that do not impart selective pressure.

Inhibiting WTA-mediated nasal colonization As indicated above, interfering with the biosynthesis and/or tailoring of WTAs could have potential for the development of anti-adhesives targeting the inner nasal cavity. Inhibiting the early stages of WTA biosynthesis renders S. aureus devoid of WTA, while not impacting growth in vitro. As such, this process has been the target of a recent large-scale (2.8 million compounds) drug discovery endeavour by Merck Research Laboratories122. Overall, two chemically distinct TarO inhibitor series were identified: tarocin A (oxazolidinone series) and tarocin B (benzimidazole series)122 (Figure 6). Within the tarocin A and B series, the most promising compounds did not inhibit the growth of S. aureus and/or other microorganisms tested, lacked cytotoxicity, and exhibited potent inhibition of WTA biosynthesis122. The inhibitors were shown to target TarO and it was proposed that they inhibit the interaction of this enzyme with its bactoprenol substrate122. To summarise, these compounds are strong therapeutic leads


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taking aim at WTA biosynthesis. Future studies could assess their ability to decolonize the nares of rodents to investigate their efficacy as potential anti-adhesive therapeutics. One potential limitation regarding anti-adhesives targeting the inner nasal cavity could be the presence of mucous within this region of the nose. Compounds targeting this niche will need to be experimentally investigated to determine if this is a physiochemical barrier that needs to be overcome.

Figure 6: Structures of sortase A (SrtA) and TarO inhibitors. The first two panels are SrtA inhibitors (A) 3,6-disubstituted triazolothiadiazoles and (B) SrtA benzo[d]isothiazol-3(2H)-oneadamantane amine derivatives. The bottom two panels are TarO inhibitors (C) tarocin A and (D) tarocin B. With the exception of the abovementioned compounds, there is a lack of known inhibitors of S. aureus adhesion. While the 3,6-disubstituted triazolothiadiazoles are promising pre-clinical candidates, other previously identified SrtA inhibitors often suffer from off-target effects and thus cytotoxicty126. Principally, CWA proteins contain an LPXTG motif that is cleaved by SrtA via an active site cysteine residue, prior to covalent linkage to the peptidoglycan. Previous studies have relied upon an in vitro assay that detects inhibition by monitoring


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cleavage of this motif126. Sortase inhibitors often react with the thiols of cysteine residues within other proteins, causing non-specific cytotoxicity. A whole-cell based assay would rule out such inhibitors at the beginning of the drug discovery process, circumventing this problem. However, one bottleneck in the discovery of anti-adhesives has been the lack of a high-throughput whole cell-based assay. Existing methods to study S. aureus adhesion are often not high-throughput, involving the laborious determination of viable remaining bacteria by culture-based methods. Additional methods describe the use of crystal violet to stain adhered bacteria. However, due to the lack of specificity of this dye, high-throughput assays are often hindered by poor sensitivity. Therefore, future efforts should include the development of innovative high-throughput assays to enable the rapid profiling of chemical libraries and the identification of small molecule inhibitors of S. aureus adhesion.

Concluding remarks Anti-virulence approaches have received increased attention over the last decade; as such, there have been a subset of drugs approved for clinical use that block exotoxins produced by bacteria9. Furthermore, it has been rigorously established that small molecule inhibition of myriad virulence factors attenuates and/or prevents disease9, which signifies that this research area offers significant clinical potential. Indeed, the numerous S. aureus virulence factors offers the promise for multiple avenues of attack. Given the high prevalence and increased risk factor associated with S. aureus nasal colonization, targeting the adhesins—namely ClfB, IsdA and WTA—facilitating nasal colonization could offer promise in reducing the health burden imposed by S. aureus. Prophylactic antibiotic use (e.g., mupirocin) in nasal decolonization has proven success in the clinic. However, the emergence of mupirocin-resistant isolates is comprising the efficacy of this approach. Anti-adhesives could be well-suited to correct this shortfall, providing an orthogonal and innovative approach to control bacterial infections that does not exacerbate the antibiotic resistance crisis. Overall, the development of high-throughput approaches to


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detect such molecules, in combination with increased insight into the molecular underpinnings of nasal colonization, would provide substantial benefits in the search for new ways to control S. aureus infections. Finally, we highlight the potential of a combinatorial anti-adhesive therapy targeting the two S. aureus niches within the nose; the nasal vestibule and the inner nasal cavity. Indeed, as discussed in the preceding section, there are a number of promising pre-clinical compounds that could be used as anti-adhesive decolonizing therapeutics. In particular, the 3,6-disubstituted triazolothiadiazole SrtA inhibitors (impacting ClfB/IsdA mediated adhesion) and the tarocins (inhibiting WTA biosynthesis and thus SREC-I adhesion) could represent a novel combinatorial therapy to eradicate S. aureus from the two respective niches. Establishing their anti-adhesive efficacy in vivo—in relevant animal models of nasal colonization—could highlight their potential as novel decolonizing agents, reducing the clinical burden of S. aureus infections.


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Table 1. Clinical targets for the development of S. aureus anti-adhesive nasal decolonizing strategies Target

Description

References

ClfB

Antiadhesive Group Class II

Nasal cell adhesin that binds loricrin and cytokeratin proteins

48, 55, 58, 61, 64, 66-68

IsdA

Class II

Nasal cell adhesin—expressed in low iron environments—that binds loricrin and cytokeratin proteins

38, 60, 61, 72, 78, 79

SrtA

Class III

Transpeptidase that covalently anchors cell-wall associated proteins to the peptidoglycan

74, 91, 92

SdgB

Class III

SD-repeat glycosyltransferase that protects ClfB from proteolytic cleavage within the host

93, 94

SigB (σB)

Class III

Subunit of RNA polymerase providing promoter recognition specificity, enhancing the expression of clfB

112-114

RsbU

Class III

Phosphatase targeting RsbV, triggering the release of σB from inhibitory proteins

104, 114

Rot

Class III

Global regulator enhancing the expression of clfB

102, 103

SpoVG

Class III

RNA binding regulatory protein that is positively regulated by the σB. SpoVG positively regulates clfB expression by directly interacting with the clfB promoter and/or in a Rot-mediated fashion

112, 113

TarO

Class III

Glycosyltransferase catalyzing the first step in WTA biosynthesis

83, 84, 119-124

TarA

Class III

Glycosyltransferase catalyzing the second step in WTA biosynthesis

120, 121

TarS

Class III

Glycosyltransferase catalyzing β-OGlcNAcylation of the WTA polymer

86, 88, 125


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Acknowledgements This work was supported by funding through the New Frontiers in Research Fund-Exploration Grant (NFRFE-2018-01058) and a Medical Research Grant from the J.P. Bickell Foundation.


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Bacterial Anti-adhesives: Inhibition of Staphylococcus aureus Nasal

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