Polypeptides for biofilm treatment

Abstract

Compositions and methods are provided for the treatment and degradation of biofilms. In some aspects, engineered polypeptides are provided that can both degrade extracellular polysaccharides (EPS) in biofilm matrix and breaks down quorum-sensing signal molecules that can contribute to formation of biofilms. The engineered polypeptides may synergize with antibiotics such as tobramycin to reduce biofilms. Uses of the engineered polypeptides to treat biofilm infections in medical and other settings are also provided.

Description

DESCRIPTIONPOLYPEPTIDES FOR BIOFILM TREATMENTBACKGROUND

[0001] This application claims the benefit of United States Provisional Patent Application Nos. 63 / 688,574, filed August 29, 2024, and 63 / 805,512, filed May 14, 2025, the entirety of which are incorporated herein by reference.

[0002] This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said XML Sequence Listing, created on August 21, 2025, is named UTFBP1377WO.xml and is 8,636 bytes in size.1. Field

[0003] The present disclosure relates generally to the field of molecular biology and medicine. More particularly, it concerns engineered enzymes for treatment of biofilms.2. Description of Related Art

[0004] Bacterial biofilms present a significant clinical problem. Bacterial biofilms can protect bacteria and are extremely challenging and costly to treat because they are notoriously resistant to antibiotics and host defenses1,2. It is estimated that 65 to 80% of all human bacterial infections are related to biofilms6. Biofilms are complex communities of bacteria embedded in an extracellular matrix composed of proteins, extracellular DNA (eDNA), and exopolysaccharides. The exopolysaccharide component of the biofilm matrix can function to impair antibiotic penetration7,8and provide a barrier against phagocytosis by host immune cells9. Given the rise of antibiotic resistance and the discovery that subinhibitory concentrations of antibiotics and antimicrobial compounds can promote biofilm formation, there is an urgent need for novel and effective treatments that target and disrupt biofilms10.

[0005] Pseudomonas aeruginosa is a ubiquitous, Gram-negative, opportunistic pathogen that is commonly associated with nosocomial infections13. Mortality associated with P. aeruginosa infections is high14, and the emergence of multidrug resistance and even pandrug resistance to antimicrobials has been reported15. The ability of P. aeruginosa to form biofilms is thought to be an important factor underlying the success of this organism in causing persistent infections in humans. P. aeruginosa utilize N-acyl-L-homoserine lactones (AHLs)- 1 -4919-9898-7617, v 1as signals to trigger coordinated initiation of biofilm formation and production of virulence factors. This inter-cellular coordination, known as quorum sensing improves resistance to and evasion of host immune response. Because the biofilm matrix is critical to the persistence of and resistance to antimicrobial agents7 8, efforts have been made to try to identify prophylactic treatments that inhibit biofilm formation through the activation of intrinsic bacterial responses18-23. While some improvements have been made, most compounds are unable to disrupt established biofilms. Biofilms also present significant problems in industrial settings, such as in industrial water systems. Clearly, there is a need for new compounds and methods for treating or removing biofilms, particularly biofilms made by P. aeruginosa.- 2 -4919-9898-7617, v 1SUMMARY

[0007] The present disclosure overcomes limitations in the art by providing new compositions and methods for the treatment and prevention of biofilms. Engineered polypeptides are provided herein that can be used to degrade biofilms including biofilms including those produced by P. aeruginosa. As shown in the below examples, fusion proteins were generated that include both (i) a truncated variant (SEQ ID NO:2) of the PsIG enzyme and (ii) the AiiA polypeptide (SEQ ID NO:3), in the hope that the fusion protein would be able to both degrade the extracellular polysaccharides (EPS) matrix and quorum sensing molecules of P. aeruginosa. However, the resulting fusion protein was insoluble under all conditions tested. Additional efforts were required to address this unexpected problem. The inventors discovered that further expressing a thioredoxin in the fusion construct, preferably at the N- terminus of the fusion protein (SEQ ID NO:4), greatly increased the solubility and stability of the resulting fusion protein and dramatic improvements in the degradation of P. aeruginosa biofilm by the fusion protein was observed. By including a cleavable linker before the thioredoxin portion of the fusion protein, the thioredoxin portion of the fusion protein could be removed by cleaving this linker, thus providing the active fusion protein comprising SEQ ID NO:2 and SEQ ID NO:3 (e.g., together as SEQ ID NO:1), which displayed the ability to both degrade P. aeruginosa biofilms and break down quorum-sensing signal molecules. The ability of the fusion construct to degrade quorum sensing signal molecules was dramatically increased by using cobalt to bind the metal ion active site of the lactonase portion of SEQ ID NO: 3 in the fusion protein. The resulting fusion protein can be used to provide multiple beneficial effects for the treatment of biofilms including P. aeruginosa biofilms that can be clinically particularly difficult and / or life-threatening. It is anticipated that a variety of thioredoxins and / or glutaredoxins can be used to allow for the generation of functional, soluble, and stable fusion proteins comprising SEQ ID NO:2 and SEQ ID NO:3 (e.g., SEQ ID NO:1, SEQ ID NO:4), or polypeptides having at least 90%, 95%, or 99% sequence identity thereto, that can degrade P. aeruginosa biofilm EPS and quorum-sensing signal molecules. The thioredoxin or glutaredoxin portion of the fusion protein may be located at the N-terminal region of the fusion protein via a cleavable linker. Fusion protein constructs and polypeptides provided herein may be used to degrade biofilms in a variety of settings including both clinical (e.g., hospital setting, indwelling device in patient such as a catheter, or infection of a patient) and industrial (e.g., industrial water systems). Additionally, synergistic results were observed for biomass reduction (reduction of microbial biomass within a biofilm) when used in combination with- 3 -4919-9898-7617, v 1tobramycin, and it is anticipated that similar synergistic results for biofilm reduction can be observed with other antibiotics including aminoglycoside antibiotics.

[0008] Data is provided in the below examples for the biofilm degrading properties of fusion proteins provided herein, as well as methods of generating the fusion proteins. Bifunctional enzyme biologies are provided herein that can degrade biofilms and prevent biofilm formation, empowering antibiotic treatment and host immunity. P. aeruginosa produces A-acyl-L-homoserine lactones (AHLs) to coordinate biofilm formation was generated. Quorum-quenching lactonases degrade AHLs, blocking biofilm formation. However, quorum quenching is less effective against mature biofilms. To overcome this limitation, AiiA, an AHL lactonase from B. thuringiensis, was coupled with PslG, an amylase from P. aeruginosa that cleaves Psi, the main polysaccharide component of PAO1 biofilms. Fused PslG- AiiA is well expressed in E. coli with a yield of 4mg per liter of culture. AiiA is a Zinc-metalloprotein with a Kcat / Km of l.bxlO4M ’s1for C6-HSL, a representative AHL. Substitution of Cobalt for Zinc increases catalytic efficiency 100-fold in the monomeric lactonase (Kcat / km = 1.4xl06M-1s-1) and fusion protein (Kcat / km = 1.1 xlO6M-1s-1). Using a Crystal violet assay to measure biofilm biomass, 300nM fusion reduces biomass by 85% following 1-h treatment of PAO1 biofilms compared with a 45% reduction from treatment with monomeric amylase. A checkerboard assay shows a synergistic effect between the fusion protein and tobramycin for biomass disruption. Further testing will define a dose-response relationship between biofilm disruption and fusion protein concentration alone and in combination with antibiotics as measured by CV and XTT. Reported herein is the development of a bifunctional biologic that acts as a quorum quencher and disrupter of biofilm matrices in P. aeruginosa. When co-administered with antibiotics, it can improve treatment of biofilm- embedded P. aeruginosa infections. As shown in the below examples, the fusion protein (PslG- AiiA; SEQ ID NO:1 without affinity tag, SEQ ID NO:4 with affinity tag) exhibited bifunctional action against P. aeruginosa biofilms and quorum sensing signal molecules. This fusion protein was well expressed in E. coli. Hyperactive lactonase activity was retained in the fusion protein. The fusion protein outperformed amylase monomer at biofilm clearance. Further experiments can be performed to (i) characterize activity of fusion protein in cystic fibrosis lung sputum model in vitro and / or in vivo, and / or (ii) characterize activity of fusion protein in rat lung infection model, and it is anticipated that the fusion polypeptides provided herein (e.g. , PslG- AiiA) will be therapeutic in these models of disease.- 4 -4919-9898-7617, v 1

[0009] An aspect of the present disclosure relates to a polypeptide comprising :(i) SEQ ID NO:2, or a polypeptide having at least 90% or at least 95% sequence identity, and (ii) SEQ ID NO:3, or a polypeptide having at least 90% or at least 95% sequence identity; wherein the polypeptide can both degrade a quorum- sensing molecule via hydrolysis and a biofilm extracellular polysaccharide (EPS). The may polypeptide comprise, in a C-terminal to N- terminal direction: (a) the SEQ ID NO:3, or polypeptide having at least 90% or at least 95% sequence identity, and (b) the SEQ ID NO:2, or polypeptide having at least 90% or at least 95% sequence identity. The polypeptide may further comprise a thioredoxin or a glutaredoxin. The polypeptide may comprise a thioredoxin. The thioredoxin or glutaredoxin may be located at the N-terminal end of the polypeptide. The thioredoxin or glutaredoxin may be attached to the polypeptide via a linker (e.g. , a cleavable linker, a flexible linker, a linker that is both cleavable and a flexible linker). The thioredoxin may be an E. coli thioredoxin (trxA), which may comprise or consist of SEQ ID NO:5. As shown in the Examples herein, inclusion of the thioredoxin or glutaredoxin may advantageously benefit the solubility and stability of the polypeptide. In some aspects, the polypeptide does not comprise a thioredoxin or a glutaredoxin. In some aspects, the polypeptide can be generated by expressing the polypeptide in a fusion protein further comprising a thioredoxin or glutaredoxin, and cleaving the thioredoxin or glutaredoxin from the fusion protein, thus producing the polypeptide. The polypeptide may further comprise an affinity tag. The affinity tag may be an affinity tag for protein purification (e.g., Strep-tag ii). The affinity tag may be located at the C-terminal end of the polypeptide. The polypeptide may comprise one or more non-natural amino acids (e.g. , D-amino acid, fluorinated amino acid, or non-natural amino acid to reduce degradation). The polypeptide may comprise or have bound one or more metal ions, preferably wherein the metal ions are Zinc (Zn2+) or Cobalt (Co2+). In some aspects, the metal ions are Cobalt (Co2+). Preferably two metal ions are bound to the polypeptide, preferably cobalt (Co2+). In some aspects, the metal ion is bound to an active site in SEQ ID NO:3. The polypeptide may have a Kcat / Kmfor hydrolysis of C6-HSL of at least about 1.OxlO6M1*sec-1. The biofilm extracellular polysaccharide (EPS) may be a P. aeruginosa EPS. In some aspects, the biofilm is a mucoid or non-mucoid biofilm from P. aeruginosa , and wherein the extracellular polysaccharide (EPS) is degraded by hydrolysis. The polypeptide may comprise or consist of SEQ ID NO:1 or SEQ ID NO:4, or a polypeptide having at least 90% or at least 95% sequence identity thereto.

[0010] Another aspect of the present disclosure relates to a nucleic acid encoding the polypeptide described above or herein. The nucleic acid may be comprised in a vector. The- 5 -4919-9898-7617, v 1nucleic acid may be operatively linked to a promoter. The promoter may be active in eukaryotic or prokaryotic cells.

[0011] Yet another aspect of the present disclosure relates to a host cell comprising the nucleic acid described above or herein. The host cell may be a eukaryotic or prokaryotic cell. The host cell may be a bacterial cell (e.g., E. coli).

[0012] Another aspect of the present disclosure relates to a composition comprising the polypeptide described above or herein. The composition may be further defined as a pharmacological composition comprising an excipient or diluent. The polypeptide may be comprised in an aqueous solution or saline. The composition may be further defined as a catheter lock solution. The composition may comprise an antibiotic. The antibiotic may be an aminoglycoside antibiotic, fluoroquinolone, rifampin, or ampicillin. In some aspects, the antibiotic is piperacillin, imipenem, ofloxacin, ciprofloxacin, levofloxacin, sparfloxacin, gentamicin, amikacin, tobramycin, amoxicillin-clavulanic acid, fosfomycin, or clarithromycin. The antibiotic may be an aminoglycoside antibiotic. The aminoglycoside antibiotic may be gentamicin, tobramycin, amikacin, plazomicin, streptomycin, neomycin, or paromomycin. In some aspects, the antibiotic is tobramycin. The composition may be a pharmaceutical composition that is formulated for topical administration. The pharmaceutical composition may be formulated for administration to the skin. The pharmaceutical composition may comprise a non-ionic surfactant (e.g., 0.05-l%(w / w) or 0.1%(w / w)), trehalose (e.g., 35- 45%(w / w)), polymer (e.g., 5-15%(w / w)), optionally wherein the polypeptide may comprise about 20-60%(w / w) or about 45-55%(w / w) of the pharmaceutical composition. The pharmaceutical composition may comprise carboxymethylcellouse, microcrystalline cellulose, hydroxypropylmethyl cellulose, or chitosan. The pharmaceutical composition may be formulated in for administration to the lungs or in an aerosol formulation. The pharmaceutical composition may comprise a non-ionic surfactant (e.g., 0.05-l%(w / w) or 0. l%(w / w)), albumin (e.g., 40-60%(w / w)), optionally wherein the polypeptide may comprise about 20-60%(w / w) or about 45-55%(w / w) of the pharmaceutical composition.

[0013] Yet another aspect of the present disclosure relates to a method of treating a biofilm infection in a mammalian subject, comprising contacting a pharmaceutically effective amount of the polypeptide described above or herein to at least a portion of the biofilm. In some aspects, the mammalian subject is preferably a human. The biofilm may be present on at least a portion of a medical device or indwelling device in the subject. The medical device- 6 -4919-9898-7617, v 1may be a catheter. The catheter may be a urinary catheter, a cardiac catheter, or an intravenous catheter; and the method may comprises administering the polypeptide into the catheter in a catheter lock solution. The subject may have a topical infection comprising a biofilm. The topical infection may be on the lungs or skin of the subject. The method may comprise contacting the polypeptide to a topical surface of the subject. The method may comprise contacting the polypeptide to skin or lungs of the subject. The method comprises contacting the polypeptide to a wound or infection on the subject (e.g.. on the skin of the subject). The method may comprise administering an antibiotic to the subject. The antibiotic may be an aminoglycoside antibiotic, fluoroquinolone, rifampin, or ampicillin. The antibiotic may be piperacillin, imipenem, ofloxacin, ciprofloxacin, levofloxacin, sparfloxacin, gentamicin, amikacin, tobramycin, amoxicillin-clavulanic acid, fosfomycin, or clarithromycin. The antibiotic may be an aminoglycoside antibiotic. The aminoglycoside antibiotic may be gentamicin, tobramycin, amikacin, plazomicin, streptomycin, neomycin, or paromomycin. In some aspects, the antibiotic is tobramycin. The antibiotic may be administered intravenously, orally, topically (e.g., to the lungs or skin of the subject), in a catheter lock solution, or onto a surface of a catheter or indwelling device in the subject. The biofilm may be a biofilm from P. aeruginosa. The biofilm may be a mucoid or non-mucoid biofilm from P. aeruginosa. The biofilm may be an established biofilm. The subject may be a human with cystic fibrosis.

[0014] Another aspect of the present disclosure relates to a method of degrading a biofilm on an industrial or medical surface comprising contacting the polypeptide described above or herein with the surface. The method may further comprise contacting the surface with ultrasonic waves, heat, chlorine, bromine, or ozone. The biofilm may be a biofilm from P. aeruginosa (e.g. , a mucoid or non-mucoid biofilm from P. aeruginosa). The biofilm may be an established biofilm.

[0015] Yet another aspect of the present disclosure relates to a kit comprising the polypeptide described above or herein or the composition described above or herein in a container means. The polypeptide may be comprised in an aqueous solution or saline.

[0016] Another aspect of the present disclosure relates to a method of making a polypeptide comprising: (i) expressing a polypeptide comprising: (a) SEQ ID NO:2, or polypeptide having at least 90% sequence identity; (b) SEQ ID NO:3, or polypeptide having at least 90% sequence identity, and (c) a thioredoxin or a glutaredoxin; wherein the thioredoxin or glutaredoxin is attached to the polypeptide via a cleavable linker; and (ii) cleaving the- 7 -4919-9898-7617, v 1cleavable linker, thus releasing the thioredoxin or glutaredoxin from the polypeptide comprising (a) and (b); and wherein the resulting polypeptide comprising (a) and (b) can degrade a quorum-sensing molecule via hydrolysis and / or degrade a biofilm extracellular polysaccharide (EPS). The method may further comprises purifying or substantially isolating the polypeptide. In some aspects, the polypeptide comprises an affinity tag, and wherein the purifying comprises affinity tag purification. The polypeptide may be the polypeptide described above or herein (e.g., SEQ ID NO:1 or SEQ ID NO:4).

[0017] The terms “subject,” “host,” “patient,” and “individual” are used interchangeably herein to refer to any mammalian subject for whom therapy is desired, particularly humans. Other subjects may include cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and so on.

[0018] The term “unit dose” when used in reference to a therapeutic composition refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent, i.e. carrier, or vehicle.

[0019] The terms “cell,” and “cells,” and “cell population,” used interchangeably, intend one or more mammalian cells. The term includes progeny of a cell or cell population. Those skilled in the art will recognize that “cells” include progeny of a single cell, and there are variations between the progeny and its original parent cell due to natural, accidental, or deliberate mutation or change.

[0020] The term “effective amount” is an amount sufficient to effect beneficial or desired clinical results. An effective amount can be administered in one or more administrations. For purposes of this application, an effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse, slow or delay the progression of the disease state. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease. For example, treatment of cancer may involve, for example, a reduction in the invasiveness of a tumor, reduction in the growth rate of the cancer, or prevention of metastasis. Treatment of cancer may also refer to prolonging survival of a subject with cancer.

[0021] An effective response of a patient or a patient’s “responsiveness” to treatment refers to the clinical or therapeutic benefit imparted to a patient at risk for, or suffering from, a- 8 -4919-9898-7617, v 1disease or disorder. Such benefit may include cellular or biological responses, a complete response, a partial response, a stable disease (without progression or relapse), or a response with a later relapse. For example, an effective response can be reduced tumor size or progression-free survival in a patient diagnosed with cancer.

[0022] The terms “in operable combination”, “in operable order”, and “operably linked” refer to a linkage wherein the components so described are in a relationship permitting them to function in their intended manner, for example, a linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene or the synthesis of desired protein molecule, or a linkage of amino acid sequences in such a manner so that a fusion protein is produced.

[0023] “Treatment” and “treating” refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition.

[0024] As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.

[0025] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” As used herein “another” may mean at least a second or more.

[0026] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the inherent variation in the method being employed to determine the value, the variation that exists among the study subjects, or a value that is within 10% of a stated value.

[0027] As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and / or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.- 9 -4919-9898-7617, v 1

[0028] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0029] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.- 10 -4919-9898-7617, v 1BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0031] FIG. 1. StreptactinXT purification of tPSLg-AiiA with pKJE8 chaperone set. cell lysate: tubes 1-2. Column wash: tubes 3-10. Elution with 50mM biotin, lOmM Biotin 5mg / mL B-casein, tube 11. Elution treated with ATP to remove chaperone. Tube 12. Untreated elution.

[0032] FIG. 2. Left: soluble and insoluble fraction s of tPSLg_AiiA expression. Right: Coelution of tPSLg-AiiA (~70kDa) with DnaKJ chaperone (~50kDa) in 1:1 ratio from StreptactinXT column.

[0033] FIG. 3. Alphafold prediction of lOOkDa fusion protein: thioredoxin (upper left) tPSlg (center) AiiA (upper right) C-terminal strep tag (far right). Also reproduced in Mirdita et al. (2022).

[0034] FIG. 4. Affinity Chromatography purification of fusion protein. Left to right: Clarified Cell lysate, affinity chromatography final wash fraction, elution fractions.

[0035] FIG. 5. Anion exchange purification of fusion protein, samples containing the low molecular weight contaminant are discarded.

[0036] FIG. 6. Crystal violet stain of P. aeruginosa PAO1 biofilm following coadministration of fusion protein or mixed monomers with 32pM Tobramycin.

[0037] FIG. 7. Kinetics of degradation of C6-HSL by tPSLg-AiiA.

[0038] FIG. 8. Quorum quenching bioassay: PAO1 cells expressing |3-Galactosidase under the control of a pC-HSL dependent promoter are mixed into LB agar containing pC-HSL and X-gal. Sample wells are filled with varying concentrations of Fusion protein or monomers. Quenching of pC-HSL signaling results in halos of reduced color due to reduced LacZ activity.- 11 -4919-9898-7617, v 1

[0039] FIG. 9. Results from the Quorum quenching bioassay described in FIG. 8 are shown for the AiiA monomer, the tPsIG monomer, and tPSLg-AiiA (“Fusion Protein”).

[0040] FIG. 10. SDS-PAGE visualization of fusion protein following expression without chaperone (top), expression & affinity purification with chaperone (middle), and expression of thioredoxin fused variant (bottom).

[0041] FIG. 11. Steady-state kinetics of hydrolysis of the quorum sensing involved molecule N-hexanoyl-(S)-homoserine lactone (C6-HSL) by monomeric Zn-AiiA (bottom line) monomeric Co-AiiA (top curve), dicobalt AiiA fused to tPSLg amylase (middle curve).- 12 -4919-9898-7617, v 1DESCRIPTION OF ILLUSTRATIVE EMBODIMENTSI. Degradation of Biofilms with Engineered Polypeptides

[0043] Biofilm embedded bacteria communicate via a system known as quorum sensing, using small molecule signals, N-acyl-homoserine-lactones (AHLS) to coordinate biofilm construction / maintenance as well as expression of virulence factors. This invention consists of a tethered quorum quenching lactonase enzyme which degrades AHL signals, with an amylase enzyme which degrades biofilm biopolymers. The lactonase, natively a zinc- metalloprotein is substituted with cobalt, increasing activity 100-fold. The engineered polypeptides provided herein can be used to treat infections by the gram-negative pathogen Pseudomonas aeruginosa.

[0044] Engineered fusion proteins of existing enzymes into a single stable protein are provided. These fusion proteins can provide advantages over administering a lactonase by itself, and formulations that stabilize this complex biologic and allow it to be delivered to the lungs and wound infections.

[0045] The present disclosure describes improved effectiveness against established biofilms compared to the lactonase monomer. Biofilms, once established, are typically extremely difficult or not possible to eradicate. The bifunctional enzyme provided herein can break down or degrade the biofilm and, at the same time, can interfere with the bacterial communication system that prevents biofilm production. The engineered enzymes provided herein can be used or administered to a patient, optionally in combination with one or more antibiotics to treat a biofilm infection. As shown in the below examples, synergistic results for the treatment of biofilms were observed when the engineered fusion protein was used in combination with the antibiotic tobramycin.

[0046] Engineered polypeptides provided herein can be used to disrupt established biofilms and / or blocking the establishment or restablishment of biofilms from planktonic bacteria. The engineered polypeptides can synergize with existing antibiotics. Some strains of pseudomonas alter the composition of the biofilm matrix which may reduce efficacy of the previously generated amylase, and engineered fusion proteins provided herein can provide advantages over individual enzymes. This can be overcome by incorporation of alternate amylases including PelA-h and Alginate Lyase.- 13 -4919-9898-7617, v 1

[0047] The current fusion composition is intended for treatment of P. aeruginosa biofilms. However, AHLs are widely used in quorum sensing by a variety of gram-negative bacteria. The super-active lactonase used in this invention is highly promiscuous, enabling degradation of a wide array of AHL molecules. Incorporation of other amylase components may allow degradation of biofilms produced by other gram-negative pathogens.

[0048] Design of the fusion protein is shown in (FIG. 10):• PslG: Known potent degrader of P. aeruginosa biofilms• Truncated variant (tPSLg) removes transmembrane domain, is well characterized• AiiA: Known to degrade key P. aeruginosa quorum sensing molecules• Resultant protein was insoluble under all tested conditions• Chaperone protein results in accumulation of proteimchaperone complex• protein crashes out on chaperone removal• Addition of N-terminal thioredoxin fusion greatly improves solubility and stability

[0049] Bacteria competing against biofilm-forming bacteria have evolved strategies to overcome biofilms by producing enzymes that block or degrade biofilm formation. The inventors have utilized these enzymes to develop a bifunctional biofilm degrading biologic. The instant invention consists of an amylase targeting the extracellular polymeric substance (EPS) of the biofilm, combined with a lactonase, which degrades AHL quorum sensing molecules.SEQUENCES

[0050] An engineered polypeptide of the present disclosure may comprise one or more of SEQ ID NOs: 1-5. The following polypeptide sequences were generated: di-CQ tPSLg-Aiia Fusion protein, without affinity tag:MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGK LTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAG SGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTENLYFQGAMEIQ VLKAPRAVVWKDFLGVNAQFLWFSPERYNKQIDRLQDLGLEWVRLDLHWDRLETA- 14 -4919-9898-7617, v 1EDQYQLASLDQLVKDLEARQLKSVFYLVGSARFITTAPFYSPFQDQYPPRDPEVFAR RMAMLSQRYPSVAAWQVWNEPNLIGFWRPKADPEGYAKLLQASTIALRMVDPEKP VVSAGMAFFSEMPDGRTMFDALGHLGVESLGTIATYHPYTQLPEGNYPWNLDFVSH ANQINRALRNAGVPAIWSTEWGWSAYKGPKELQDIIGVEGQADYVLRRLALMSALD YDRIFLFTLSDLDQRASVRDRDYGLLDLDANPKPVYLALQRFLKVTGPKLRPADPPV TEDLPDGSFSIGWTREDGRNVWLFWSARGGNVRLPKLKEATLHDPLSGKVTPLSGS DGLEVPVKSSLQMLVWEGGGGSGGGGSGGSAMTVKKLYFIPAGRCMLDHSSVNSA LTPGKLLNLPVWCYLLETEEGPILVDTGMPESAVNNEGLFNGTFVEGQILPKMTEED RIVNILKRVGYEPDDLLYIISSHLHFDHAGGNGAFTNTPIIVQRTEYEAALHREEYMK ECILPHLNYKIIEGDYEVVPGVQLLYTPGHSPGHQSLFIETEQSGSVLLTIDASYTKENF EDEVPFAGFDPELALSSIKRLKEVVKKEKPIIFFGHDIEQEKSCRVFPEYI (SEQ IDNO:1) tPSLg Sequence:EIQVLKAPRAVVWKDFLGVNAQFLWFSPERYNKQIDRLQDLGLEWVRLDLH WDRLETAEDQYQLASLDQLVKDLEARQLKSVFYLVGSARFITTAPFYSPFQDQYPPR DPEVFARRMAMLSQRYPSVAAWQVWNEPNLIGFWRPKADPEGYAKLLQASTIALR MVDPEKPVVSAGMAFFSEMPDGRTMFDALGHLGVESLGTIATYHPYTQLPEGNYPW NLDFVSHANQINRALRNAGVPAIWSTEWGWSAYKGPKELQDIIGVEGQADYVLRRL ALMSALDYDRIFLFTLSDLDQRASVRDRDYGLLDLDANPKPVYLALQRFLKVTGPKL RPADPPVTEDLPDGSFSIGWTREDGRNVWLFWSARGGNVRLPKLKEATLHDPLSGK VTPLSGSDGLEVPVKSSLQMLVWE (SEQ ID N0:2)AHA Sequence:MTVKKLYFIPAGRCMLDHSSVNSALTPGKLLNLPVWCYLLETEEGPILVDTG MPESAVNNEGLFNGTFVEGQILPKMTEEDRIVNILKRVGYEPDDLLYIISSHLHFDHA GGNGAFTNTPIIVQRTEYEAALHREEYMKECILPHLNYKIIEGDYEVVPGVQLLYTPG HSPGHQSLFIETEQSGSVLLTIDASYTKENFEDEVPFAGFDPELALSSIKRLKEVVKKE KPIIFFGHDIEQEKSCRVFPEYI (SEQ ID NO: 3) di-CQ tPSLg-Aiia Fusion protein, with affinity tag:MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGK LTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAG SGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTENLYFQGAMEIQ- 15 -4919-9898-7617, v 1VLKAPRAVVWKDFLGVNAQFLWFSPERYNKQIDRLQDLGLEWVRLDLHWDRLETA EDQYQLASLDQLVKDLEARQLKSVFYLVGSARFITTAPFYSPFQDQYPPRDPEVFAR RMAMLSQRYPSVAAWQVWNEPNLIGFWRPKADPEGYAKLLQASTIALRMVDPEKP VVSAGMAFFSEMPDGRTMFDALGHLGVESLGTIATYHPYTQLPEGNYPWNLDFVSH ANQINRALRNAGVPAIWSTEWGWSAYKGPKELQDIIGVEGQADYVLRRLALMSALD YDRIFLFTLSDLDQRASVRDRDYGLLDLDANPKPVYLALQRFLKVTGPKLRPADPPV TEDLPDGSFS1GWTREDGRNVWLFWSARGGNVRLPKLKEATLHDPLSGKVTPLSGS DGLEVPVKSSLQMLVWEGGGGSGGGGSGGSAMTVKKLYFIPAGRCMLDHSSVNSA LTPGKLLNLPVWCYLLETEEGPILVDTGMPESAVNNEGLFNGTFVEGQILPKMTEED RIVNILKRVGYEPDDLLYIISSHLHFDHAGGNGAFTNTPIIVQRTEYEAALHREEYMK ECILPHLNYKIIEGDYEVVPGVQLLYTPGHSPGHQSLFIETEQSGSVLLTIDASYTKENF EDEVPFAGFDPELALS SIKRLKEVVKKEKPIIFFGHDIEQEKSCRVFPEYIGGGGS GGS AWSHPQFEK (SEQ ID N0:4)Thioredoxin Sequence:MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGK LTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLA (SEQ ID NO:5)IL Polypeptides

[0051] As used herein, the term "polypeptide" refers to a linear or branched polymer of amino acid residues linked by peptide bonds. The term encompasses naturally occurring proteins, recombinant polypeptides, synthetic polypeptides, and derivatives thereof. Polypeptides may comprise a full-length protein or biologically active fragments, analogs, variants, or fusion constructs thereof.

[0052] A polypeptide may consist of a single amino acid chain or multiple chains, which may be identical or different and which may be linked by disulfide bridges or other covalent interactions. The term "polypeptide" is used interchangeably with "protein" unless otherwise specified. The amino acid residues of the polypeptides disclosed herein may be naturally occurring L-amino acids, D-amino acids, or a combination thereof. In certain embodiments, one or more amino acids may be substituted with non-natural or non-canonical amino acids, including but not limited to amino acid analogs, chemically modified residues, or synthetically derived residues that impart desired physicochemical or biological properties.- 16 -4919-9898-7617, v 1Such non-natural amino acids may be introduced by chemical synthesis or by engineered biosynthetic pathways utilizing orthogonal tRNA and aminoacyl-tRNA synthetase pairs.

[0053] The disclosed polypeptides may further comprise one or more post-translational modifications or chemically engineered modifications. Non-limiting examples include: Glycosylation (N-linked, O-linked, or other forms); Phosphorylation of serine, threonine, tyrosine, or histidine residues; Acetylation of lysine or N-terminal residues; Methylation (mono-, di-, or tri) of lysine or arginine; Ubiquitination, sumoylation, or neddylation; Lipidation (e.g., myristoylation, prenylation, palmitoylation); PEGylation or conjugation to polymers, dyes, drugs, or nanoparticles. Such modifications may be performed enzymatically, chemically, or through incorporation of modified amino acids during synthesis or expression. The polypeptides may further include one or more disulfide bonds, cross-links, or cyclized regions to stabilize tertiary or quaternary structure.

[0054] The polypeptides of the present disclosure may be isolated and purified from a variety of expression systems, including but not limited to Escherichia coli, yeast (e.g., Saccharomyces cerevisiae, Pichia pastoris), insect cells, mammalian cells, or in vitro expression systems. Purification of polypeptides may be accomplished using standard techniques known in the art. These may include, without limitation: Affinity chromatography, Ion-exchange chromatography (IEX), Size exclusion chromatography (SEC), Hydrophobic interaction chromatography (HIC), Reverse-phase high performance liquid chromatography (RP-HPLC), dialysis, Ultrafiltration and diafiltration, Ammonium sulfate or solvent-based precipitation, Immunoaffinity chromatography using monoclonal or polyclonal antibodies or any combination thereof.

[0055] In certain embodiments, the polypeptides may be expressed as fusion constructs comprising one or more affinity tags to facilitate purification, detection, or localization. Nonlimiting examples of affinity tags include: Polyhistidine tag (His-tag), typically comprising 6 to 10 histidine residues; enables purification by immobilized metal affinity chromatography (IMAC) using Ni2+or Co2+chelating resins; Glutathione S-transferase (GST) tag, binds to immobilized glutathione; enhances solubility and allows purification using glutathione-agarose columns; Maltose-binding protein (MBP), facilitates purification via amylose resin and enhances solubility and folding of fusion partners; Strep-tag and Twin-Strep-tag, binds specifically to engineered streptavidin or Strep-Tactin resins; FLAG-tag, a short, hydrophilic peptide (e.g. , DYKDDDDK (SEQ ID NO:6)) that can be captured using anti-FLAG antibodies- 17 -4919-9898-7617, v 1or resins; HA tag, derived from the influenza hemagglutinin protein; detectable using anti-HA antibodies; Myc tag, a short epitope from the c-Myc protein, suitable for detection and immunoprecipitation; or HaloTag, SNAP-tag, and CLIP-tag, covalently bind to specific ligands or substrates for purification or labeling.

[0056] The affinity tag may be positioned at the N-terminus, C-terminus, or internal region of the polypeptide, and may optionally include a protease recognition site (<?.g., TEV, thrombin, enterokinase, HRV-3C site) to allow removal of the tag following purification, thereby yielding a native or near-native protein product.III. Nucleic Acids

[0057] Also provided are nucleic acid molecules that encode a protein described herein. These nucleic acid molecules include DNA, cDNA, and RNA sequences. Nucleic acid molecules that encode only the ectodomain of the protein are also contemplated. The nucleic acid molecule can be incorporated into a vector, such as an expression vector.

[0058] The nucleic acid may be a self-replicating RNA molecule. The nucleic acid may include a modified RNA molecule. Also provided are compositions comprising a nucleic acid described herein.

[0059] A nucleic acid encoding a polypeptide often comprises an open reading frame that encodes the polypeptide. Unless otherwise indicated, a particular nucleic acid sequence also includes degenerate codon substitutions.

[0060] Nucleic acids can include one or more expression control or regulatory elements operably linked to the open reading frame, where the one or more regulatory elements are configured to direct the transcription and translation of the polypeptide encoded by the open reading frame in a mammalian cell. Non-limiting examples of expression control / regulatory elements include transcription initiation sequences (e.g., promoters, enhancers, a TATA box, and the like), translation initiation sequences, mRNA stability sequences, poly A sequences, secretory sequences, and the like.

[0061] In a bacterial system, expression control / regulatory elements may include bacterial promoters, which can be constitutive or inducible depending on the desired level and timing of expression. Bacterial promoters that may be utilized include, but are not limited to: Constitutive promoters (e.g., lac promoter, araBAD promoter, T7 promoter or P_BAD- 18 -4919-9898-7617, v 1promoter) or inducible promoters (e.g. , inducible lac promoter, TET promoter, P con promoter or N25 promoter).

[0062] In a mammalian system, expression control / regulatory elements may include mammalian promoters, which can be constitutive or inducible depending on the desired level, tissue specificity, and timing of expression. Mammalian promoters that may be utilized include, but are not limited to: Constitutive promoters (e.g., cytomegalovirus (CMV) immediate early promoter, elongation factor-1 alpha (EF-la) promoter, SV40 early promoter, or ubiquitin C (UbC) promoter) or inducible promoters (e.g., tetracycline (Tet) -responsive promoter, mifepristone-inducible promoter, ecdysone-inducible promoter, or heat shock protein (HSP) promoter).

[0063] Expression control / regulatory elements can be obtained from the genome of any suitable organism, including bacterial species such as Escherichia coli, Salmonella enterica, Shigella flexneri, Vibrio cholerae, and others, as well as from phages or synthetic sources. The choice of promoter, whether constitutive or inducible, can be tailored based on the desired application, ensuring that the expression of the polypeptide is optimized for the system in use.

[0064] In various embodiments, additional regulatory elements, such as mRNA stability sequences (e.g. , ribosomal binding sites or untranslated regions), may also be included to enhance expression or to allow for fine-tuning of translation efficiency in the host cell.IV. Methods of Disinfecting Surfaces and Medical Devices

[0065] It is anticipated that an engineered polypeptide provided herein can be used to treat, reduce, or prevent biofilms on a variety of medical devices. For example, the medical device may be a central venous catheter, urinary catheter, contact lens, intrauterine device. It is anticipated that water lines (e.g. , dental chair water lines, etc.) or other surfaces can be treated with the engineered polypeptide. The surface may also be exposed to ionizing radiation prior to or after contact with the engineered polypeptide. The surface may optionally be treated with ultrasonic waves, heat, chlorine, bromine, or ozone before or after contact with the engineered polypeptide.- 19 -4919-9898-7617, v 1V. Pharmaceutical Compositions

[0066] The pharmaceutical formulations provided herein may further include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type that are available in the art. Examples of such substances include normal saline solutions such as physiologically buffered saline solutions and water. Specific non-limiting examples of the carriers and / or diluents that are useful in the pharmaceutical formulations of the present invention include water and physiologically acceptable buffered saline solutions such as phosphate buffered saline solutions pH 7, 0-8.0. Suitable pharmaceutical carriers include, but are not limited to sterile water, salt solutions (such as Ringer's solution), alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, tale, silicic acid, viscous paraffin, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, etc. The pharmaceutical preparations can be mixed with auxiliary agents, e.g., lubricants, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and / or aromatic substances and the like which do not deleteriously react with the active compounds. They can also be combined where desired with other active substances, e.g. , ileal brake hormone regulatory substances to improve metabolism and ameliorate metabolic syndromes.

[0067] Polypeptides provided herein may be formulated in a pharmaceutical composition, which may include pharmaceutically acceptable carriers, thickeners, duents, buffers, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients and the like in addition to the cells.

[0068] Pharmaceutically acceptable carriers or excipients that can be used in the pharmaceutical composition include, but are not limited to, aqueous solutions, saline, phosphate-buffered saline (PBS), or other buffered solutions, as well as inert organic solvents such as dimethyl sulfoxide (DMSO), ethanol, and polyethylene glycol (PEG). The composition may also contain stabilizers, such as sugars (e.g., sucrose, trehalose), amino acids (e.g., glycine), or proteins (e.g., serum albumin), to prevent aggregation or degradation of the polypeptide.

[0069] Pharmaceutical compositions may also include one or more active ingredients such as, anti-inflammatory agents, anesthetics, and the like. Formulations for oral or topical administration may include buffers, liposomes, diluents and other suitable additives. The- 20 -4919-9898-7617, v 1compositions provided herein may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional compatible pharmaceutically- active materials such as, e.g. , statins, linaclotide, ileal brake hormone releasing substances, anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the composition of present invention, such as dyes, flavoring agents, antioxidants, opacifiers, thickening agents and stabilizers. Depending on the particular active ingredients, the formulations may be administered in the same pill or tablet or as a distinct pill or tablet as part of a co-administration protocol. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions provided herein.

[0070] In preferred embodiments, the pharmaceutical composition of the invention is administered orally, or topically. Dosing can be dependent on a number of factors, including severity and responsiveness of the disease state to be treated, and with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Toxicity and therapeutic efficacy of compounds provided herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.

[0071] "Pharmaceutically acceptable salt" refers to salts of the compounds of the present invention derived from the combination of such compounds and an organic or inorganic acid (acid addition salts) or an organic or inorganic base (base addition salts). The compounds of the present invention may be used in either the free base or salt forms, with both forms being considered as being within the scope of the present invention.

[0072] However, pharmaceutical compositions provided herein may be in any form which allows for the composition to be administered to a patient by the oral and topical route. The pharmaceutical composition is formulated so as to allow the active ingredients contained therein to be bioavailable at the site targeted upon administration of the composition to a patient. Compositions that will be administered to a patient take the form of one or more dosage units, where tablet may be a single dosage unit, and a container of one or more compounds of the invention in oral form may hold a plurality of dosage units.

[0073] A liquid pharmaceutical composition as used herein, whether in the form of a solution, suspension or other like form, may include one or more of the following adjuvants:- 21 -4919-9898-7617, v 1diluents such as water, preferably fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glyc-erin, propylene glycol or other solvents; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.

[0074] For topical or dermal application, the pharmaceutical composition may be formulated as a cream, gel, ointment, lotion, spray, patch, or transdermal delivery system. The composition may further include additional excipients such as emollients, gelling agents, thickeners, or penetration enhancers to improve the viscosity, stability, or spreadability of the formulation and to facilitate absorption of the active ingredient, including the polypeptide, through the skin or mucosal membranes. Such penetration enhancers may include fatty acids, alcohols, surfactants, urea, dimethyl sulfoxide (DMSO), or cyclodextrins, which can transiently alter the skin barrier to allow for improved delivery of the active ingredients.

[0075] In certain embodiments, the pharmaceutical composition may include tobramycin, an aminoglycoside antibiotic, which is included to provide antimicrobial properties in the treatment of topical infections. Tobramycin may be combined with the polypeptide to achieve synergistic effects in managing infectious conditions or inflammation, such as those caused by Gram-negative bacteria. The formulation may incorporate tobramycin in an amount sufficient to provide a therapeutically effective dose while minimizing potential irritation or systemic absorption.

[0076] The pharmaceutical composition may also include one or more stabilizing agents to maintain the integrity and activity of the polypeptide during storage and use. Such stabilizers may include antioxidants (e.g., ascorbic acid, sodium metabisulfite), chelating agents e.g., EDTA), or pH adjusters (e.g., hydrochloric acid, sodium hydroxide). The composition may be stored under conditions that prevent degradation, such as at refrigerated temperatures or under freeze-dried conditions, depending on the stability profile of the polypeptide.

[0077] The pharmaceutical composition is administered in an effective amount, which is an amount sufficient to produce the desired therapeutic or prophylactic effect, while minimizing potential side effects or toxicity. The effective dosage will depend on various factors, including the specific polypeptide, the condition being treated, the patient’s age, weight,- 22 -4919-9898-7617, v 1and health status, as well as the route of administration. The dosage may be adjusted by the skilled artisan based on clinical judgment and relevant pharmacological data.VI. Combination therapies

[0078] An engineered polypeptide as disclosed herein can be used to treat an infection or remove a biofilm from a surface in combination with one or more additional antimicrobial or therapeutic agents. For example, the engineered polypeptide may be applied to a surface (e.g. , the surface of a medical device, hospital or medical environment surface, skin, etc.) in combination with an antibiotic. As shown in the Examples, engineered polypeptides provided herein can synergize with antibiotics e.g., tobramycin) to kill bacteria and reduce biofilms. If desired, the engineered polypeptide may be applied to a medical device prior to or during use.

[0079] In some aspects, the engineered polypeptide, optionally in combination with one or more additional antimicrobial, anti-inflammatory, or therapeutic agents, is administered to a patient to treat a biofilm associated with a disease. The disease may comprise or be associated with a bacterial infection, wherein the infection contributes to or exacerbates the biofilm formation. For example, the disease may include but is not limited to cystic fibrosis (e.g., infection by Pseudomonas aeruginosa), otitis media (e.g., infection by Haemophilus influenzae), periodontitis (e.g., infection by Prevotella intermedia, Porphyromonas gingivalis, P. aerobicus, or Fusobacterium nucleatum), or infective endocarditis (e.g., infection by Staphylococcus aureus, Streptococcus viridans, or Enterococcus faecal is).

[0080] In certain embodiments, the engineered polypeptide may be administered in combination with one or more antibiotics selected from the group consisting of fluoroquinolones, rifampin, ampicillin, piperacillin, imipenem, ofloxacin, ciprofloxacin, levofloxacin, sparfloxacin, gentamicin, amikacin, tobramycin, amoxicillin-clavulanic acid, fosfomycin, or clarithromycin. These antibiotics may be used to enhance antibacterial activity, disrupt biofilm architecture, or increase susceptibility of biofilm-embedded bacteria to treatment. The agents may be administered systemically, topically, or locally at the site of infection, and may be delivered concurrently or sequentially with the engineered polypeptide.

[0081] In some embodiments, the therapeutic combination may further comprise antiinflammatory agents (e.g., corticosteroids, non-steroidal anti-inflammatory drugs (NSAIDs)), mucolytics (e.g., domase alfa, N-acetylcysteine), or biofilm-disrupting agents such as quorum- 23 -4919-9898-7617, v 1sensing inhibitors, enzymatic dispersants, or chelating agents (e.g., EDTA, lactoferrin), which may aid in penetration of the therapeutic agents and clearance of biofilms.

[0082] The antibiotic may be an aminoglycoside antibiotic, including but not limited to gentamicin, tobramycin, amikacin, plazomicin, streptomycin, neomycin, and paromomycin. In some embodiments, the aminoglycoside antibiotic may be selected based on its activity against Gram-negative or Gram-positive bacteria implicated in biofilm-associated infections. The aminoglycoside may be administered in free form or encapsulated in a delivery system such as liposomes, micelles, or nanoparticles, and may be formulated for topical, inhalational, oral, or parenteral delivery. In certain embodiments, the aminoglycoside is co-administered with the engineered polypeptide to achieve synergistic disruption of biofilms and enhanced bactericidal activity.VII. Kits

[0083] In certain embodiments, the present disclosure provides a kit or article of manufacture comprising one or more components necessary or useful for practicing the methods described herein or for the preparation, detection, analysis, administration, or application of the compositions disclosed herein.

[0084] In some embodiments, the kit comprises one or more containers, vials, tubes, or receptacles containing one or more of the following components: a polypeptide, nucleic acid, small molecule, reagent, buffer, enzyme, substrate, probe, detection agent, therapeutic agent, or other biologically or chemically active compound. The kit may further include one or more auxiliary agents, including but not limited to stabilizers, preservatives, excipients, carriers, diluents, surfactants, or solubilizing agents.

[0085] In certain embodiments, the kit may additionally comprise one or more instructional materials, which may include printed or electronic documentation, labels, or inserts describing the contents of the kit, instructions for use, storage conditions, safety guidelines, and / or regulatory compliance information. Such instructional materials may be provided in any tangible or electronically accessible format.

[0086] In some embodiments, the kit may further comprise one or more detection means, which may include fluorescent, luminescent, chromogenic, radioactive, or enzymatic detection reagents, substrates, or readout systems.- 24 -4919-9898-7617, v 1

[0087] In certain embodiments, the kit may be configured for use in research, clinical, diagnostic, therapeutic, prophylactic, or industrial settings. The components of the kit may be provided in solution, in lyophilized form, as tablets, powders, or in any other suitable formulation. Reagents and other kit components may be packaged individually or in combination and may be supplied in unit dosage or bulk formats.

[0088] The kit may optionally be contained within a packaging material, including but not limited to a box, pouch, blister pack, vacuum-sealed enclosure, or other suitable form of containment that maintains the stability and integrity of the kit components during storage and transport. The packaging material may be labeled with identifiers, barcodes, or other machine- readable indicia for inventory, tracking, or automated use.IV. Examples

[0089] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.Example 1 - Lactonase- Amylase fusion protein summary

[0090] Purification of fusion protein: The original confirmation of the protein consisted of PSIG31-442 (tPslG) omitting the transmembrane domain24, fused with the AiiA AHL lactonase which has been previously characterized25. Expression of this fusion protein resulted in the accumulation of insoluble inclusion bodies which were not investigated for solubilization and refolding. Coexpression with chaperone protein sets (Takara bio) resulted in purification of soluble fusion protein in a 1 : 1 complex with the DnaK / J chaperone complex. Incubation with ATP to promote GrpE mediated release of the chaperone complex from the fusion protein resulted in immediate crashout of the fusion protein in the chromatography system tubing. Incorporation of a thioredoxin fusion protein (Txr) on the N-terminus improves solubility, the resultant Txr_tPslG_AiiA fusion (97kDa) is soluble and purified from LB media following induction with 0.5mM 1PTG and addition of lOOpM Cobalt chloride, which has previously- 25 -4919-9898-7617, v 1been demonstrated to result in purification of AiiA lactonase with cobalt in the active site in place of the canonical Zinc, resulting in an 100-fold increase in KCat / Kmto ~10A6 M-1*sec’12. The maximum yield of the fusion protein is 4mg per litre of cell culture.

[0091] tPSLg Amylase activity: Activity of the Amylase domain was assayed by Crystal violet assay. Pseudomonas aeruginosa strain PAO1 cell culture was grown overnight at 37C in 96 well plates to allow biofilm formation. Growth media was removed and the well was washed 3x with sterile PBS. Fusion protein was added to the well in PBS to a final concentration of I -5 M. As a positive control, tPSLg monomer fused to an MBP fusion protein for solubility was tested at equal concentration. The plate was incubated for one hour at 25C and washed again with PBS. A 0.5% crystal violet solution was added and incubated for 20 additional minutes. After a final wash step the plate was read in a Tecan infinite 200 plate reader at 595nm. Each condition was measured in triplicate.

[0092] AiiA Lactonase activity: Activity of the cobalt substituted lactonase was assayed using a previously described colorimetric assay25,26. Briefly, hydrolysis of homoserine lactones results in the release of one equivalent proton at pH values significantly above the pKa of the product carboxylic acid (~4.0). Under weakly buffered conditions a colorimetric buffer phenol component allows quantitative measurement of proton release by monitoring absorbance at 557nm. The final conditions of the assay are: ImM HEPES pH7.5, 40pM Phenol-Red, 30pM CoC12, lOOmM Na2SO4, 25nM enzyme. Enzyme activity was assayed from 25pM to 2.0mM and data was fit to the Michaelis -Menten equation. Steady state parameters were calculated giving a Kmof 247pM and a Kcat 250 s’1. Consistent with values of Km= 360pM, Kcat=510 s’1 2and Km= 220pM Kcat =1100 s’1 27.Example 2 - Quorum Quenching Bioassay

[0093] Principle: P. aeruginosa PAO-MW 1 is a Iasi, rhll mutant that does not make acyl-HSL signals. The pRpal / R vector encodes 3- Galactosidase (LacZ) under control of the p-coumaroyl-l-homoserine lactone (pC-HSL) dependent rPaR transcription factor. When this cell line is exposed to exogenous pC-HSL, LacZ is expressed which can be detected via X-gal based blue-white screening. Treatment with Lactonases (Monomeric AiiA or Lactonaseamylase fusion) degrades pC-HSL, down-regulating LacZ expression.- 26 -4919-9898-7617, v 1

[0094] Methods: LB-Agar was prepared and cooled to 42°C. The agar was then supplemented with gentamicin (50pg / ml), pC-HSL (200pM), X-Gal (200pg / mL) and a 1:100 dilution of overnight culture of PAO-MW 1 cells containing the pRpal / R vector. 60mL of the mixture was then poured into a 15x100mm Petri plate and cooled to 4C to solidify. Once solidified 5mm sample wells were bored into the agar 2.5cm apart from each other. The bottom of the wells were capped with lOuL molten agar which was allowed to solidify. Sample wells were filled with 40pL of either buffer (25mM Tris pH7.4 5mM NaCl lOp M CoC12 or protein in the same buffer. The wells were overlayed with 15pL of mineral oil and the plates were incubated overnight at 30°C. Plates were photographed and the diameter of the halo was measured in millimeters.

[0095] Results: The fusion protein produces a halo of reduced blue / green color consistent with reduced X-gal hydrolysis resulting from reduced LacZ expression. An inner halo of dark blue occurs in some wells, this is likely due to drying out of the agar and denaturation of the protein. This halo has been previously observed (Momb et al. 2010). The diameter of the halo increases with increased concentration, with diameter doubling from lOOnM to lO M fusion protein. The diameter of the halo produced by incubation of the AiiA lactonase monomer appears to be slightly larger than for the fusion protein, but the difference is small. Incubation with the tPslG amylase monomer produces no halo effect. (FIGS. 8-9)

[0096] The AiiA lactonase derived from Bacillus thuringensis disrupts quorum sensing through hydrolysis of N-acyl-L-homoserine lactones (AHL). AiiA is natively a metalloprotein containing two zinc ions. Substitution of Zinc for cobalt produces a 90- to 100-fold increase in catalytic activity with no reduction in protein stability (FIGS. 11). However, lactonase effectiveness is greatly reduced in mature biofilms.

[0097] Table 1. Steady state kinetic parameters of Zn or Co metalated AiiA lactonase- 27 -4919-9898-7617, v 1

[0098] Table 2.

[0099] Table 3.

[0100] Fusion protein effectively breaks down quorum-sensing signal molecules. The cobalt metalated lactonase component of the fusion protein retains similar kinetic characteristics of the characterized monomer. Comparable substrate binding is observed as measured by substrate Km. Initial characterization indicates that incorporation into fusion protein reduces Kcat by -50%. Catalytic efficiency (Kcat / Km) of the fusion protein is 70% of the super-active cobalt substituted monomer.Example 3 - Method for purification of Thioredoxin tagged tPslG-AiiA fusion protein- 28 -4919-9898-7617, v 1

[0101] Protein construct: Thioredoxin-6His-ThrombinSite-Stag-TevSite-tPslG(31- 442)-flexibleLinker-AiiA-strepTag

[0102] Vector: pET32a(Tev) - TEV site added in house by SDM

[0103] Expression strain: B121(DE3) pLysS - do not use normal B121(DE3)

[0104] Buffers:Lactonase Amylase (LA) buffer: 50mM Tris pH 7.4 lOOmM NaCl lOpM C0CI2Co-Nta buffer: 50mM Tris pH 7.4 lOOmM NaCl lOpM C0CI2 +500mM ImidazoleDEAE buffer-A: 50mM Tris pH 7.4 lOpM C0CI2DEAE buffer-B: 50mM Tris pH 7.4 lOpM C0CI2500mM NaClStrep column buffer: 50mM Tris pH 7.4 lOpM C0CI2 lOOmM NaCl + lOOmM BiotinStorage buffer: 50mM Tris pH 7.4 lOOmM NaCl lOpM CoCh 20% Glycerol

[0105] Cell CultureDay 0• Prepare LB broth in 2.8L flasks - store at 4C• Inoculate starter culture from glycerol stock and grow overnight at 37C 180rpmDay 1• Prewarm LB to 37C - add ImL each lOOOx Ampicillin, Chloramphenicol• Add overnight culture - lOmL per 1 litre flask• Grow at 37C 210 rpm for 2.5 hours o Measure OD o Once OD = 0.5 -0.7 remove from incubator and place on ice 10 minutes o Cool incubator to 25 CPlace flasks in incubator, add IPTG to 0.25mM, CoC12 to lOOpM- 29 -4919-9898-7617, v 1Express for 16 hours, cool to 4C

[0106] Lysis• Pellet cells: lOmin @ 5000xg 4C• Resuspend in lOmL per gram lactonase-amylase buffer• Add lysozyme to 0.1 mg / ml• Add Dnasel• Disrupt large pellets with serological pipette• Lyse: 85% amplitude, 7.5sec on / off 8min total• Remove insoluble lysate: spin 40,000xg 45min @4C

[0107] Column 1: Cobalt-Nta affinity resin to enrich fusion protein• Wash 20mL CoNta with lOOmL LA buffer• Wash system with LA buffer until baseline and conductivity are stable for at least 5 minutes o Install Co-Nta buffer on line B• Apply clarified lysate to column at 3.0mL / min or overnight by batchwise incubation• Allow lysate to pass over resin at least once• Connect loaded column to system• Collecting 6mL fractions, wash the resin with LA buffer until the baseline returns to 0, allow at least 12mL at baseline• Elute with a gradient from 0 to 100% Co-Nta buffer over 180mLs• Collect 6mL fractions• Pool elution peaks and concentrate to a final volume of >25mL and place in dialysis tubing• Dialyze overnight against IL DEAE buffer-A- 30 -4919-9898-7617, v 1

[0108] Column 2: DEAE anion exchange column protein pl = 5.3 - protein elutes around 200mM [Can also use strong exchanger Q-Sepharose]• Wash system and column with 50-100mL Buffer- A at 4mL / min• Apply dialyzed sample to washed resin and let bind by gravity o Collect flowthrough, run through UV monitor to check binding o Wash column with 3CVS buffer- A• Set gradient program: 0 to 50% buffer-B (0 to 500mM NaCl) o Flowrate: 4ml / min o Total volume: 400mL o Collect 8mL fractions• Once program has run, wash column with 50mL buffer-B to remove any remaining protein• Check fractions by combining lOuL with 200uL Bradford dye, should line up with UV trace data• Visualize fractions around peak(s) by SDS-PAGE• Pool fractions containing significant fusion protein and buffer exchange by either dialysis or spin column back into LA-buffer

[0109] Column 3: streptactinXT flow (normal or High capacity)• Equilibrate streptactinXT column with 10C VS LA-buffer o Verify that resin is ready to use with ImM HABA, resin should turn dark orange / red o Install Strep column buffer on line-B• Apply sample to column at Iml / min or by batchwise incubation overnight o For batchwise, allow resin lOmin in column to settle- 31 -4919-9898-7617, v 1Wash column with > lOCVs of wash buffer / until baseline returns to normal• Elute with strep column buffer, collecting 2mL fractions until baseline returns to normal• Pool elution fractions and concentrated to 50-100pM, buffer exchange into storage buffer• Aliquot into 11 Op L volumes and snap-freeze in liquid nitrogen, store at -80CExample 4 - compositions

[0110] An examples of Lactonase-Amylase fusion protein formulations for administration to the lungs are provided here.Ingredient %w / wFusion Protein 50%Non-ionic surfactant 0.1%Albumin 49.9%

[0111] Formulations were prepared by mixing the components in aqueous solution and spray drying at low temperature conditions (<100 deg C). Non-ionic surfactants include tweens and pluronics. Albumin can also be substituted with other endogenous proteins such as lysozyme, lactoferrin, or IgG. Formulations may also be lyophilized and then milled to aerodynamic particle sizes suitable for lung delivery.Example 5 - compositions

[0112] An example of Lactonase-Amylase fusion protein formulations for administration to wound infections are provided here.Ingredient %w / wFusion Protein 50%- 32 -4919-9898-7617, v 1Non-ionic surfactant 0.1%Trehalose 40.9%Polymer 9%

[0113] Formulations were prepared by mixing the components in aqueous solution and lyophilized. These compositions could be reconstituted and used to treat wound infections. Additional excipients included in the formulation include polymers (carboxymethylcellouse, microcrystalline cellulose, hydroxypropylmethyl cellulose, chitosan, and other natural and synthetic polymers).Example 6 - compositions

[0114] Lactonase-Amylase fusion protein formulations can be combined with antibiotics used to treat Pseudomonas aeruginosa biofilm infections including tobramycin. Formulations in examples 4 and 5 include amounts of antibiotic.* * *

[0115] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.- 33 -4919-9898-7617, v 1REFERENCESThe following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.US5270181AMirdita M, et al. Nature Methods (2022) doi: 10.1038 / s41592-022-01488-lMomb J, Yoon DW, Fast W. Enzymic disruption of N-aroyl-L-homoserine lactone- based quorum sensing. Chembiochem. 2010 Jul 26; 11 ( 11): 1535-7. doi: 10.1002 / cbic.201000191. PMID: 20544776.1. J. W. Costerton, P. S. Stewart, E. P. Greenberg, Bacterial biofilms: A common cause of persistent infections. Science 284, 1318-1322 (1999).2. C. A. Fux, J. W. Costerton, P. S. Stewart, P. Stoodley, Survival strategies of infectious biofilms. Trends Microbiol. 13, 34-40 (2005).6. C. Potera, Forging a link between biofilms and disease. Science 283, 1837-1839 (1999).7. K. M. Colvin, V. D. Gordon, K. Murakami, B. R. Borlee, D. J. Wozniak, G. C. L. Wong, M. R. Parsek, The pel polysaccharide can serve a structural and protective role in the biofilm matrix of Pseudomonas aeruginosa. PLOS Pathog. 7, el001264 (2011).8. N. Billings, M. R. Millan, M. Caldara, R. Rusconi, Y. Tarasova, R. Stocker, K. Ribbeck, The extracellular matrix component Psi provides fast-acting antibiotic defense in Pseudomonas aeruginosa biofilms. PLOS Pathog. 9, el003526 (2013).9. M. Mishra, M. S. Byrd, S. Sergeant, A. K. Azad, M. R. Parsek, L. McPhail, L. S. Schlesinger, D. J. Wozniak, Pseudomonas aeruginosa Psi polysaccharide reduces neutrophil phagocytosis and the oxidative response by limiting complement-mediated opsonization. Cell. Microbiol. 14, 95-106 (2012).10. Y. Morita, J. Tomida, Y. Kawamura, Responses of Pseudomonas aeruginosa to antimicrobials. Front. Microbiol. 4, 422 (2014).13. M. D. Obritsch, D. N. Fish, R. MacLaren, R. Jung, Nosocomial infections due to multidrug-resistant Pseudomonas aeruginosa: Epidemiology and treatment options. Pharmacotherapy 25, 1353-1364 (2005).14. C.-I. Kang, S.-H. Kim, H.-B. Kim, S.-W. Park, Y.-J. Choe, M.-D. Oh, E.-C. Kim, K.- W. Choe, Pseudomonas aeruginosa bacteremia: Risk factors for mortality and- 34 -4919-9898-7617, v 1influence of delayed receipt of effective antimicrobial therapy on clinical outcome. Clin. Infect. Dis. 37, 745-751 (2003).18. M. Hentzer, M. Givskov, Pharmacological inhibition of quorum sensing for the treatment of chronic bacterial infections. J. Clin. Invest. 112, 1300-1307 (2003).19. M. Hentzer, K. Riedel, T. B. Rasmussen, A. Heydorn, J. B. Andersen, M. R. Parsek, S. A. Rice, L. Eberl, S. Molin, N. Hpiby, S. Kjelleberg, M. Givskov, Inhibition of quorum sensing in Pseudomonas aeruginosa biofilm bacteria by a halogenated furanone compound. Microbiology 148, 87-102 (2002).20. C. Kim, J. Kim, H.-Y. Park, H.-J. Park, J. H. Lee, C. K. Kim, J. Yoon, Furanone derivatives as quorum-sensing antagonists of Pseudomonas aeruginosa. Appl. Microbiol. Biotechnol. 80, 37-47 (2008).21. N. C. Cady, K. A. McKean, J. Behnke, R. Kubec, A. P. Mosier, S. H. Kasper, D. S.Burz, R. A. Musah, Inhibition of biofilm formation, quorum sensing and infection in Pseudomonas aeruginosa by natural products -inspired organosulfur compounds. PLOS One 7, e38492 (2012).22. H.-S. Kim, S.-H. Lee, Y. Byun, H.-D. Park, 6-Gingerol reduces Pseudomonas aeruginosa biofilm formation and virulence via quorum sensing inhibition. Sci. Rep. 5, 8656 (2015).23. C. T. O’Loughlin, L. C. Miller, A. Siryaporn, K. Drescher, M. F. Semmelhack, B. L. Bassler, A quorum-sensing inhibitor blocks Pseudomonas aeruginosa virulence and biofilm formation. Proc. Natl. Acad. Sci. U.S.A. 110, 17981-17986 (2013).24. Baker, P. et al. Exopolysaccharide biosynthetic glycoside hydrolases can be utilized to disrupt and prevent Pseudomonas aeruginosa biofilms. Sci Adv 2, (2016).25. Momb, J., Thomas, P. W., Breece, R. M., Tierney, D. L. & Fast, W. The Quorum- Quenching Metallo-y-lactonase from Bacillus thuringiensis Exhibits a Leaving Group Thio Effect f. (2006) doi:10.1021 / bi061238o.26. Thomas, P. W., Stone, E. M., Costello, A. L., Tierney, D. L. & Fast, W. The quorumquenching lactonase from Bacillus thuringiensis is a metalloprotein. Biochemistry 44, 7559-7569 (2005).27. Thomas, P. W. & Fast, W. Chapter 20 Heterologous Overexpression, Purification, and In Vitro Characterization of AHL Lactonases. doi: 10.1007 / 978-l-60761-971-0_20.- 35 -4919-9898-7617, v 1

Claims

WHAT IS CLAIMED IS:

1. A polypeptide comprising:(i) SEQ ID NO:2, or a polypeptide having at least 90% sequence identity, and(ii) SEQ ID NO:3, or a polypeptide having at least 90% sequence identity; wherein the polypeptide can both degrade a quorum-sensing molecule via hydrolysis and a biofilm extracellular polysaccharide (EPS).

2. The polypeptide of claim 1, wherein the polypeptide comprises, in a C-terminal to N- terminal direction:(a) the SEQ ID NO:3, or polypeptide having at least 90% sequence identity, and(b) the SEQ ID NO:2, or polypeptide having at least 90% sequence identity.

3. The polypeptide of any one of claims 1-2, wherein the polypeptide further comprises a thioredoxin or a glutaredoxin.

4. The polypeptide of claim 3, wherein the polypeptide comprises a thioredoxin.

5. The polypeptide of any one of claims 3-4, wherein the thioredoxin or glutaredoxin is located at the N-terminal end of the polypeptide.

6. The polypeptide of any one of claims 3-5, wherein the thioredoxin or glutaredoxin is attached to the polypeptide via a cleavable linker.

7. The polypeptide of any one of claims 3-6, wherein the thioredoxin or glutaredoxin is attached to the polypeptide via a flexible linker.

8. The polypeptide of any one of claims 3-6, wherein the thioredoxin is E. colt thioredoxin (trxA) or comprises or consists of SEQ ID NO:5.

9. The polypeptide of any one of claims 1-2, wherein the polypeptide does not comprise a thioredoxin or a glutaredoxin.- 36 -4919-9898-7617, v 110. The polypeptide of claim 9, wherein the polypeptide was generated by expressing the polypeptide in a fusion protein further comprising a thioredoxin or glutaredoxin, and cleaving the thioredoxin or glutaredoxin from the fusion protein, thus producing the polypeptide.

11. The polypeptide of any one of claims 1-10, wherein the polypeptide further comprises an affinity tag.

12. The polypeptide of claim 11, wherein the affinity tag is an affinity tag for protein purification, optionally Strep-tag ii.

13. The polypeptide of any one of claims 11-12, wherein the affinity tag is located at the C-terminal end of the polypeptide.

14. The polypeptide of any one of claims 1-13, wherein the polypeptide comprises a nonnatural amino acid.

15. The polypeptide of any one of claims 1-13, wherein the polypeptide comprises or has bound a metal ion, wherein the metal ion is Zinc (Zn2+) or Cobalt (Co2+).

16. The polypeptide of claim 15, wherein the metal ion is Cobalt (Co2+).

17. The polypeptide of any one of claims 15-16, wherein the metal ion is bound to an active site in SEQ ID NO:3.

18. The polypeptide of any one of claims 1-17, wherein the polypeptide has a Kcat / Kmfor hydrolysis of C6-HSL of at least about l.OxlO6M’^sec’1.

19. The polypeptide of any one of claims 1-18, wherein the biofilm extracellular polysaccharide (EPS) is a P. aeruginosa EPS.

20. The polypeptide of any one of claims 1-19, wherein the biofilm is a mucoid or nonmucoid biofilm from P. aeruginosa , and wherein the extracellular polysaccharide (EPS) is degraded by hydrolysis.

21. The polypeptide of any one of claims 1-20, wherein the polypeptide comprises or consists of SEQ ID NO:1 or SEQ ID NO:4, or a polypeptide having at least 90% or at least 95% sequence identity thereto.

22. A nucleic acid encoding the polypeptide of any one of claims 1-21.- 37 -4919-9898-7617, v 123. The nucleic acid of claim 22, wherein the nucleic acid is comprised in a vector.

24. The nucleic acid of claim 23, wherein the nucleic acid is operatively linked to a promoter.

25. The nucleic acid of claim 24, wherein the promoter is active in eukaryotic or prokaryotic cells.

26. A host cell comprising the nucleic acid of any one of claims 22-25.

27. The host cell of claim 26, wherein the host cell is a eukaryotic or prokaryotic cell.

28. The host cell of claim 27, wherein the host cell is a bacterial cell, optionally E. coli.

29. A composition comprising the polypeptide of any one of claims 1-21.

30. The composition of claim 29, wherein the composition is further defined as a pharmacological composition comprising an excipient or diluent.

31. The composition of any one of claim 30, wherein the polypeptide is comprised in an aqueous solution or saline.

32. The composition of any one of claims 29-31 , wherein the composition is further defined as a catheter lock solution.

33. The composition of any one of claims 29-32, wherein the composition comprises an antibiotic.

34. The composition of claim 33, wherein the antibiotic is an aminoglycoside antibiotic, fluoroquinolone, rifampin, or ampicillin.

35. The composition of claim 34, wherein the antibiotic is piperacillin, imipenem, ofloxacin, ciprofloxacin, levolloxacin, sparfloxacin, gentamicin, amikacin, tobramycin, amoxicillin-clavulanic acid, fosfomycin, or clarithromycin.

36. The composition of claim 34, wherein the antibiotic is an aminoglycoside antibiotic.

37. The composition of claim 36, wherein the aminoglycoside antibiotic is gentamicin, tobramycin, amikacin, plazomicin, streptomycin, neomycin, or paromomycin.- 38 -4919-9898-7617, v 138. The composition of claim 36, wherein the antibiotic is tobramycin.

39. The composition of any one of claims 29-38, wherein the composition is a pharmaceutical composition that is formulated for topical administration.

40. The composition of claim 39, wherein the pharmaceutical composition is formulated for administration to the skin.

41. The composition of claim 40, wherein the pharmaceutical composition comprises a non-ionic surfactant (e.g., 0.05-l%(w / w) or 0.1%(w / w)), trehalose (e.g., 35-45%(w / w)), polymer (.e.g. , 5-15%(w / w)), optionally wherein the polypeptide comprises about 20- 60%(w / w) or about 45-55%(w / w) of the pharmaceutical composition.

42. The composition of any one of claims 40-41, wherein the pharmaceutical composition comprises carboxymethylcellouse, microcrystalline cellulose, hydroxypropylmethyl cellulose, or chitosan.

43. The composition of claim 39, wherein the pharmaceutical composition is formulated in for administration to the lungs or in an aerosol formulation.

44. The composition of claim 43, wherein the pharmaceutical composition comprises a non-ionic surfactant (e.g., 0.05-l%(w / w) or 0.1%(w / w)), albumin (e.g., 40-60%(w / w)), optionally wherein the polypeptide comprises about 20-60%(w / w) or about 45-55 %(w / w) of the pharmaceutical composition.

45. A method of treating a biofilm infection in a mammalian subject, comprising contacting a pharmaceutically effective amount of the polypeptide of any one of claims 1-21 to at least a portion of the biofilm.

46. The method of claim 45, wherein the mammalian subject is a human.

47. The method of claim 45, wherein the biofilm is present on at least a portion of a medical device or indwelling device in the subject.

48. The method of any one of claims 45-47, wherein the medical device is a catheter.- 39 -4919-9898-7617, v 149. The method of any one of claims 45-48, wherein the catheter is a urinary catheter, a cardiac catheter, or an intravenous catheter; and wherein the method comprises administering the polypeptide into the catheter in a catheter lock solution.

50. The method of any one of claims 45-46, wherein the subject has a topical infection comprising a biofilm.

51. The method of claim 50, wherein the topical infection is on the lungs or skin of the subject.

52. The method of any one of claims 45-51, wherein the method comprises contacting the polypeptide to a topical surface of the subject.

53. The method of claim 52, wherein the method comprises contacting the polypeptide to skin or lungs of the subject.

54. The method of claim 52, wherein the method comprises contacting the polypeptide to a wound or infection on the subject.

55. The method of any one of claims 45-52, wherein the method comprises administering an antibiotic to the subject.

56. The method of claim 55, wherein the antibiotic is an aminoglycoside antibiotic, fluoroquinolone, rifampin, or ampicillin.

57. The method of claim 55, wherein the antibiotic is piperacillin, imipenem, ofloxacin, ciprofloxacin, levofloxacin, sparfloxacin, gentamicin, amikacin, tobramycin, amoxicillin- clavulanic acid, fosfomycin, or clarithromycin.

58. The method of claim 55, wherein the antibiotic is an aminoglycoside antibiotic.

59. The method of claim 58, wherein the aminoglycoside antibiotic is gentamicin, tobramycin, amikacin, plazomicin, streptomycin, neomycin, or paromomycin.

60. The method of claim 59, wherein the antibiotic is tobramycin.

61. The method of any one of claims 55-60, wherein the antibiotic is administered intravenously, orally, topically, in a catheter lock solution, or onto a surface of a catheter or indwelling device in the subject.- 40 -4919-9898-7617, v 162. The method of any one of claims 55-60, wherein the biofilm is a biofilm from P. aeruginosa.

63. The method of claim 62, wherein the biofilm is a mucoid or non-mucoid biofilm from P. aeruginosa.

64. The method of any one of claims 62-63, wherein the biofilm is an established biofilm.

65. The method of any one of claims 55-64, wherein the subject is a human, wherein the human has cystic fibrosis.

66. A method of degrading a biofilm on an industrial or medical surface comprising contacting the polypeptide of any one of claims 1-21 with the surface.

67. The method of claim 66, wherein the method further comprises contacting the surface with ultrasonic waves, heat, chlorine, bromine, or ozone.

68. The method of any one of claims 66-67, wherein the biofilm is a biofilm from P. aeruginosa.

69. The method of claim 68, wherein the biofilm is a mucoid or non-mucoid biofilm from P. aeruginosa.

70. The method of any one of claims 62-63, wherein the biofilm is an established biofilm.

71. A kit comprising the polypeptide of any one of claims 1-21 or the composition of any one of claims 29-44 in a container means.

72. The kit of claim 71, wherein the polypeptide is comprised in an aqueous solution or saline.

73. A method of making a polypeptide comprising:(i) expressing a polypeptide comprising:(a) SEQ ID NO:2, or polypeptide having at least 90% sequence identity;(b) SEQ ID NO:3, or polypeptide having at least 90% sequence identity, and(c) a thioredoxin or a glutaredoxin;- 41 -4919-9898-7617, v 1wherein the thioredoxin or glutaredoxin is attached to the polypeptide via a cleavable linker; and(ii) cleaving the cleavable linker, thus releasing the thioredoxin or glutaredoxin from the polypeptide comprising (a) and (b); and wherein the resulting polypeptide comprising (a) and (b) can degrade a quorum-sensing molecule via hydrolysis and / or degrade a biofilm extracellular polysaccharide (EPS).

74. The method of claim 29, wherein the method further comprises purifying or substantially isolating the polypeptide.

75. The method of claim 30, wherein the polypeptide comprises an affinity tag, and wherein the purifying comprises affinity tag purification.

76. The method of any one of claims 73-75, wherein the polypeptide is the polypeptide of any one of claims 1-21.- 42 -4919-9898-7617, v 1

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