Patent Application
20200199554
The present invention relates to the in vivo and in vitro use of previously uncharacterized viral proteins as DNA degrading enzymes.
DNases find their application in the medical field and in industrial applications. These enzymes typically work in a narrow pH range, are temperature sensitive and require specific buffer conditions. There is a need for more robust DNases. Genomic sequencing of phage genomes and analysis of Open Reading Frames (ORFs) has been performed. See for example Lavigne et al. (2003) Virology 312, 49-59; Karumidze et al. (2012) Appl Microbiol Biotechnol. 94, 1609-1617 and Ceyssens et al. (2006) J. Bacteriol. 188, 6924-6931. However, many of the characterized open reading frames have unknown functions. LUZ19_Gp5 [SEQ ID NO:1] is an uncharacterized ORF of 74 amino acids sharing sequence similarity only with other hypothetical sequences of Pseudomonas phages. In silico sequence analysis shows no significant sequence identity with other proteins and reveals no sequence motifs or protein domains which give a hint of the function of this protein.
The invention is summarized in the following statements:
1. The in vitro use of a polypeptide comprising a sequence with at least 60% identity with any one of the sequences with SEQ ID NO:1 to SEQ ID NO:9 or comprising a fragment thereof, wherein said polypeptide or fragment thereof has nuclease activity, as a DNA degrading enzyme.
2. The in vitro use according to statement 1, wherein the polypeptide is a fusion protein with said polypeptide or fragment thereof having nuclease activity.
3. The in vitro use of a polypeptide comprising a sequence with at least 60% identity with any one of the sequences with SEQ ID NO:1 to SEQ ID NO:9 or a fragment thereof, wherein said polypeptide or fragment thereof has nuclease activity, for the degradation of biofilms.
4. An in vitro method of degrading DNA comprising the step of contacting a DNA comprising sample with a polypeptide comprising a sequence at least 60% identity with any one of the sequences with SEQ ID NO:1 to SEQ ID NO:9 or a fragment thereof, wherein said polypeptide or fragment thereof has nuclease activity.
5. The method according to statement 4, wherein the method is performed at a pH below 4, below 3 or below 2, or above 8, above 9 or above 10, e.g. between ph 1 to 4, between 2 to 4, between 3 to 4, between 8 to 10 or between 8 to 9.
6. The method according to statement 4 or 5, wherein the method is performed at a temperature above 7° C., above 80° C. or above 90° C.
7. The in vitro use of a polypeptide comprising a sequence with at least 60% identity with any one of the sequences with SEQ ID NO:1 to SEQ ID NO:9 or a fragment thereof, wherein said polypeptide or fragment thereof has nuclease activity, as an antimicrobial agent.
8. The in vitro use according to any one of statements 1 to 7, wherein the polypeptide comprises a sequence with at least 70 or 75% identity with any one of the sequences with SEQ ID NO:1 to SEQ ID NO:9 or a fragment thereof wherein said polypeptide or fragment thereof has nuclease activity.
9. The in vitro use according to any one of statements 1 to 7, wherein the polypeptide comprises a sequence with at least 80 or 85% identity with any one of the sequences with SEQ ID NO:1 to SEQ ID NO:9 or a fragment thereof wherein said polypeptide or fragment thereof has nuclease activity.
10. The in vitro use according to any one of statements 1 to 7, wherein the polypeptide comprises a sequence with at least 90, 95, 97, 98 or 99% identity with any one of the sequences with SEQ ID NO:1 to SEQ ID NO:9 or a fragment thereof wherein said polypeptide or fragment thereof has nuclease activity.
11. A polypeptide comprising a sequence with at least 60% identity with any one of the sequences with SEQ ID NO:1 to SEQ ID NO:9 or a fragment thereof, wherein said polypeptide or fragment thereof has nuclease activity for use as medicament.
12. A polypeptide comprising a sequence with at least 60% identity with any one of the sequences with SEQ ID NO:1 to SEQ ID NO:9 or a fragment thereof, wherein said polypeptide or fragment thereof has nuclease activity for use in the treatment of cystic fibrosis.
13. A polypeptide comprising a sequence with at least 60% identity with any one of the sequences with SEQ ID NO:1 to SEQ ID NO:9 or a fragment thereof, wherein said polypeptide or fragment thereof has nuclease activity for use in the treatment or prevention of bacterial infections.
14. The polypeptide for use according to any one of statements 11 to 13, wherein the polypeptide comprises a sequence with at least 70 or 75% identity with any one of the sequences with SEQ ID NO:1 to SEQ ID NO:9 or a fragment thereof wherein said polypeptide or fragment thereof has nuclease activity.
15. The polypeptide for use according to any one of statements 11 to 13, wherein the polypeptide comprises a sequence with at least 80 or 85% identity with any one of the sequences with SEQ ID NO:1 to SEQ ID NO:9 or a fragment thereof wherein said polypeptide or fragment thereof has nuclease activity.
16. The polypeptide for use according to any one of statements 11 to 13, wherein the polypeptide comprises a sequence with at least 90, 95, 98 or 99% identity with any one of the sequences with SEQ ID NO:1 to SEQ ID NO:9 or a fragment thereof wherein said polypeptide or fragment thereof has nuclease activity.
17. A polypeptide comprising a sequence with at least 60% identity with any one of the sequences with SEQ ID NO:1 to SEQ ID NO:9 or a fragment thereof, wherein said polypeptide or fragment thereof has nuclease activity, with the proviso that said polypeptide is not a polypeptide consisting of a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9.
18. A polypeptide according to statement 17, with the proviso that said sequence does not comprise a sequence consisting of any one of SEQ ID NO:1 to SEQ ID NO:9.
19. The polypeptide according to statement 17 or 18, with at least 70% identity, at least 80%, at least 90% identity with any one of the sequences with SEQ ID NO:1 to SEQ ID NO:9 or a fragment thereof, wherein said polypeptide or fragment thereof has nuclease activity.
20. The polypeptide according to any one of statement 17 to 19, with at least 70% identity, at least 80%, at least 90% identity with any one of the sequences with SEQ ID NO:11 to SEQ ID NO:19 or a fragment thereof, wherein said polypeptide or fragment thereof has nuclease activity.
21. The polypeptide according to any one of statements 17 to 20, which comprises the sequence RVGLVNYSDRYLGAD [SEQ ID NO:20].
22. The polypeptide according to any one of statement 17 to 21, wherein said fragment is at least 50, at least 60 or at least 70 amino acids.
23. A polynucleotide comprising a sequence encoding a polypeptide according to any one of statements 17 to 22.
24. A vector comprising a polynucleotide according to statement 24.
25. An antibody specifically binding to a polypeptide comprising a sequence with at least 60% identity with any one of the sequences with SEQ ID NO:1 to SEQ ID NO:9 or a fragment thereof, wherein said polypeptide or fragment thereof has nuclease activity.
26. The antibody according to statement 25, binding to a polypeptide comprising a sequence with at least 70 or 75% identity with any one of the sequences with SEQ ID NO:1 to SEQ ID NO:9 or a fragment thereof, wherein said polypeptide or fragment thereof has nuclease activity.
27. The antibody according to statement 25, binding to a polypeptide comprising a sequence with at least 80 or 85% identity with any one of the sequences with SEQ ID NO:1 to SEQ ID NO:9 or a fragment thereof, wherein said polypeptide or fragment thereof has nuclease activity.
28. The antibody according to statement 25, binding to a polypeptide comprising a sequence with at least 90, 95, 98 or 99% identity with any one of the sequences with SEQ ID NO:1 to SEQ ID NO:9 or a fragment thereof, wherein said polypeptide or fragment thereof has nuclease activity.
29. The antibody according to any one of statements 25 to 28, wherein the antibody specifically binds to the sequence RVGLVNYSDRYLGAD [SEQ ID NO:20].
30. A method of identifying modified versions of a polypeptide selected from the group consisting of SEQ NO: 1 to 9: with nuclease activity, comprising the steps of:
introducing one or more changes in the nucleotide sequence of a polynucleotide encoding a sequence selected from the group consisting of SEQ NO: 1 to 9, the one or more changes resulting in a modified amino acid sequence;
expressing the protein with the modified amino acid sequence;
assaying the expressed protein for nuclease activity.
31. The method according to statement 30, wherein the one or more changes are substitutions, insertion or deletions.
32. The method according to statement 30 or 31, wherein the one or more changes are a deletion at the 5′ or the 3′ end of the nucleotide sequence resulting in truncated form of a polypeptide selected from the group consisting of SEQ NO: 1 to 9.
The present invention discloses the identification of a class of viral proteins as DNA degrading enzymes. The proteins have a remarkably low Mr and are active in a broad pH range, at high temperatures and in denaturing conditions such as 200 mM guanidium chloride.
In the context of the present invention a protein has “nuclease activity” if after 30 minutes incubation at least 10% of double stranded bacterial DNA is degraded, measured as % decrease of the intensity on an agarose gel.
In the context of the present invention “miniDNase” is a polypeptide comprising a sequence with at least 60% identity with any one of the sequences with SEQ ID NO:1 to SEQ ID NO:9 or comprising a fragment thereof, wherein said polypeptide or fragment thereof has nuclease activity.
Based on SEQ ID NO:1, eight other highly similar protein sequences have been identified in protein sequence databases and in translated DNA sequences databases. These sequences as such are known. Since their function is not known is it summited that proteins with a protein sequence depicted by any of SEQ ID NO:1 to 9 are accidental disclosures of a group of broader group of compounds with DNase activity which are proteins with similarity to SEQ ID NO: 1 to 8, or fragments thereof. Proteins with SEQ ID NO: 1 to 8, as such are accidental disclosures and are disclaimed from compound claims.
Database screening was performed up to Jul. 14, 2017 in patent and non-patent databases, and it is believed that all publicly available sequences have been retrieved. However any another sequence will be disclaimed as an undisclosed disclaimer of accidental prior art which is irrelevant for inventive step.
It is emphasized that prior art sequences are only relevant for the novelty of a compound claim on proteins or polynucleotides. Any use of this group of enzymes, including those with published protein and DNA sequences, which involves DNA degradation is novel and invention, making a disclaimer in a use or method claim unnecessary.
The sequences of SEQ ID NO:1 to 9 are translated sequences of DNA of viral genomes. The sequencing of these genomes is believed to be accurate, although sequence errors may occur. The sequence information contained in the database allows to design primers to re-sequence the genomic sequence and to correct for eventual sequences. The invention thus discloses variants of SEQ ID NO:1 to 9 and their use, wherein the variants are obtainable by sequencing Pseudomonas phage DNA using primers adjacent to or within the ORF of the miniDNase sequence. e.g. SEQ ID NO 10: is a sequence which comes into account for resequencing.
The present invention describes the characterization of proteins originally identified merely as open reading frames with unknown function from bacterial viruses which infect Pseudomonas aeruginosa. These proteins are represented by a group of sequence with SEQ ID NO:1 to 9.
These proteins have a length of between 72 to 75 amino acids.
Within the sequence databases a protein sequence can be found which has a length of 123 amino acids [SEQ ID NO: 10]. The C terminal part of this sequence is identical to the sequence of SEQ ID NO:4.
Depending on the expression system the amino terminal methionine may be removed by amino peptidase.
A sequence alignment (see
Within the groups of SEQ ID NO: 1 to 8, pairwise sequence identity ranges from 78 to 99%. SEQ ID NO: 8 has a pairwise sequence identity with any of SEQ ID NO: 1 to 7 of between 78 and 84%.
Despite a sequence identity of only 78% between SEQ ID NO:1 and SEQ ID NO: 8, both proteins show nuclease activity as explained in the examples.
Within the groups of SEQ ID NO: 1 to 7, pairwise sequence identity ranges from 90 to 99%.
In the group of SEQ ID NO:1 to SEQ:9, sequence variation especially occurs in the N terminal and C terminal parts.
Sequence identity ranges from 93 to 100% within SEQ ID NO 11: to 17.
Within the full length sequences of SEQ ID:NO 1 to 9 and the truncated version of SEQ ID NO: 11 to 19 all sequences contain the sequence RVGLVNYSDRYLGAD [SEQ ID NO: 20].
Accordingly, the below described in vivo and in vitro uses can be performed with a protein comprising a sequence as described in any one of SEQ ID NO:1 to 9, or a sequence with at least 60, 65, 70, 75, 80, 85, 90, 95, 97% sequence identity with any of these sequences, and having nuclease activity.
The below described in vivo and in vitro uses can be performed with a protein comprising a sequence as described in any one of SEQ ID NO:1 to 8, or a sequence with at least 70, 75, 80, 85, 90, 95, 97% sequence identity with any of these sequences, and having nuclease activity.
The below described in vivo and in vitro uses can be performed with a protein comprising a sequence as described in any one of SEQ ID NO:1 to 7, or a protein comprising a sequence with at least 90, 95, 97% sequence identity with any of these sequences, and having nuclease activity.
Accordingly, the below described in vivo and in vitro uses can be performed with a protein comprising a sequence as described in any one of SEQ ID NO:11 to 19, or a sequence with at least 65, 70, 75, 80, 85, 90, 95, or 97% sequence identity with any of these sequences, and having nuclease activity.
The below described in vivo and in vitro uses can be performed with a protein comprising a sequence as described in any one of SEQ ID NO:11 to 18, or a sequence with at least 75, 80, 85, 90, 95, or 97% sequence identity with any of these sequences, and having nuclease activity.
The below described in vivo and in vitro uses can be performed with a protein comprising a sequence a described in any one of SEQ ID NO:11 to 17, or a sequence with at least 65, 70, 75, 80, 85, 90, 95, or 97% sequence identity with any of these sequences, and having nuclease activity.
In specific embodiments the below described in vivo and in vitro uses can be performed with a protein comprising a sequence a described in SEQ ID NO: 1, SEQ ID NO:11, SEQ ID NO: 8 or SEQ ID NO:18 or a sequence with at least 65, 70, 75, 80, 85, 90, 95, or 97% sequence identity with any of these sequences, and having nuclease activity.
Accordingly, the below described in vivo and in vitro uses can be performed with a protein comprising a sequence as described in any one of SEQ ID NO:11 to 19, or a sequence with at least 65, 70, 75, 80, 85, 90, 95, or 97% sequence identity with any of these sequences, and having nuclease activity.
As indicated above, the protein sequences reveal the presence of a conserved sequence RVGLVNYSDRYLGAD [SEQ ID NO: 20]. Accordingly, the below described in vivo and in vitro uses can be performed with a protein comprising SEQ ID NO: 20 and having nuclease activity.
The alignment of the proteins with SEQ ID NO:1 to 9 shows 41 amino acids positions which are conserved in all sequences, and various positions with “similar” amino acids (H/R/K; V/I/L/M, F/W/Y; D/E/Q/N; G/A/S/T/C). Preliminary mutagenesis experiments have been performed (see examples) which indicate that even mutations at conserved positions wherein size and charge of the side chain are significantly changed (Gly to Arg and Glu to Gly), retain nuclease activity. Accordingly the present invention provides methods to identify whether or not a sequence with a given percentage to any one of SEQ ID NO: 1 to 9 is a protein having nuclease activity.
Specific embodiments of mutation are mutations which prevent degradation by sequence specific proteases, and mutations in the potential Asn17XaaSer19 glycosylation site upon expression in eukaryotic systems. Mutations of one or both of Cys43 and Cys51 in SEQ ID NO:1 to 8 may reveal the effect on activity, and inter and intramolecular disulfide formation. Equally Cys51 of SEQ ID NO:9 may be mutated and/or Met 43 may be modified into Cysteine.
The present invention further provides methods to identify whether truncated forms of any one of SEQ ID NO:1 to 9 retain nuclease activity. Herein, truncated forms of any one of SEQ ID NO:1 to 9 can be cloned and expressed in an expression system and the expressed truncated protein is tested for nuclease activity.
Thus the present invention envisages truncated version at the amino terminus and/or carboxyterminus of any of SEQ ID NO:1 to 9, with a length of at least 40, at least 50, at least 55, at least 60, at least 65 amino acids.
Specific truncations are fragments comprising the conserved motif with SEQ ID NO:20 and comprising the conserved cysteine at position 51, such as miniDNase fragments corresponding to amino acid 1 up to and including 51, miniDNase fragments corresponding to amino acid 8 up to and including 51, or miniDNase fragments corresponding to amino acid 12 up to and including 51.
Other fragments are C term truncations up to but excluding truncation of V65, truncations up to but excluding truncation of R66, truncations up to but excluding truncation of F67, or truncations up to but excluding truncation of I68, Other fragments are N term truncations up to but excluding truncation of R8. Other fragments are truncations at the N terminus up to but excluding truncation of R8 end at the C terminus as defined above.
Apart from proteins comprising fragments of SEQ ID NO:1 to 9 and modified versions thereof, proteins as used in the present invention may contains further polypeptide sequence such as signal peptides for secretion of the peptide, tags for antibody binding, such as HA-tags, protein sequences for affinity purification such as His Tags, MPB, GST and the like, polypeptides encoding fluorescent enzymes, or polypeptides with another enzymatic function or polypeptides with a pharmaceutical activity.
The function of the protein sequences of SEQ ID NO:1 to 9 have not been studied in the prior art. The present invention identified these sequences as nucleases. This allows to engineer modified versions of these sequences by mutagenesis and/or truncation of one of the sequences with SEQ ID NO:1 to 9, and to assay these modified versions for their DNase activity.
In the present invention, proteins with DNase activity comprising a sequence with at least 60% sequence identity with SEQ ID NO:1 to 9, or comprising fragments thereof with DNase activity, are described as miniDNase.
Aspect of the present invention relates to in vitro uses of the proteins of the present invention.
MiniDNase can be obtained in high amounts via recombinant expression in bacteria. Although this enzyme would be assumed to be toxic to the expression host, it is believed without being bound by theory that miniDNase becomes active only in the periplasm of the bacteria when disulfide bridges are formed. Expression systems wherein a signal peptide is used for transport to the periplasm and subsequent secretion are envisaged. Alternatively host strains with cytoplasmatic disulfide bridge formation are envisaged. A tightly controlled promoter and a subsequent strong induction may be needed if the miniDNase has strong activity within the bacterial host.
The fact that the miniDNase can be expressed with aminoterminal or carboxyterminal tags allows purification at a high purity. Furthermore in view of its thermal stability, eventual contaminating proteins can be heat-inactivated. The use of eukaryotic expression system such as yeast, insect cells or mammalian cells is equally envisaged.
In embodiments of the present invention the miniDNases are used in molecular biology techniques as an alternative to commercially available enzymes such as DNase I. MiniDNase is thus provides in a kit with suitable reaction buffers and optionally a component such as citrate to stop nuclease activity.
In other embodiments, miniDNase is used in in vitro applications wherein DNA degradation is needed. An example hereof is the reduction of viscosity in cell lysates of bacterial, yeast, or eukaryotic cell preparations.
Indeed, while some fermentation products are secreted by the cells and therefore automatically present in the extracellular medium, others remain intracellular, requiring cell lysis to access the desired products. However, cell lysis not only releases the fermentation products, but also other intracellular components, which can interfere with the downstream processing. In this regard, the release of the chromosomal DNA is responsible for an increase of the lysate viscosity that may reach 75% and thereby severely complicates further recovery steps.
Since the released chromosomal DNA is responsible for most of the lysate viscosity increase, it is the principal target for viscosity reduction to improve the downstream processing efficiency and production costs. In fermentation industry, this is currently achieved by an external addition of nucleases, which are relatively large nucleic acid-degrading enzymes, like Benzonase® [see table 11] (Benedik & Strych (1998) FEMS Microbiol. Lett. 165: 1-18). Furthermore, this degradation step also prevents the undesired spread of genetic material into the environment. Unfortunately, the addition of foreign biological substances, like enzymes, in the production stream is subject to stringent regulations, making this a very costly practice. Current research focusses on the production of fermentation strains that secrete nucleic acid degrading enzymes themselves to prevent an external addition step [Cooke et al. (2003) J. Biotech. 101: 229-239].
The present invention provides an alternative solution for reducing the viscosity of cell lysates, by providing a DNase that is active in wide pH and temperature range, The miniDNase can be coated on the walls of a recipient, can be immobilized on a bead, or can be added as soluble protein.
Another aspect of the present invention relates to the degradation of biofilms using a miniDNase of the present invention. These biofilms may contain, apart from extracellular matrix, extracellular DNA (eDNA).
99% of the world's bacterial population is estimated to reside in biofilms. These are surface-associated bacterial communities embedded within a matrix of self-produced extracellular polymeric substances (EPS), like polysaccharides, proteins, lipids and eDNA. This three-dimensional matrix provides unique properties to the biofilm, including efficient resource trapping and pour penetration of the biofilm by antibacterials. In addition, biofilms are characterized by the efficient spread of resistance genes, the presence of persisters and a decreased growth rate, additionally contributing to an increased resistance to antibacterials compared to their free-living counterparts. Besides occupying natural environments, biofilms are often found in an industrial and medical context, where they are usually unwanted.
Biofilms are common threats in many industrial branches. The presence of biofilms involves operational problems in many different industrial branches. For example, they are ubiquitous at the inner surface of heat exchanging tubes in cooling water systems, being able to reduce the heat change capacity up to 90%. Furthermore, surface properties in pipelines are often altered due to the adhesion of biofilms, causing not only clogging, but also an increased fluid frictional resistance, which results in substantial energy losses. In addition, biofilms can actively accelerate the corrosion of metal surfaces up to 10,000 times, referred to as biocorrosion. Finally, the presence of biofilms involves additional issues in industries directly related to animal and human health, like the drinking water and food industry. Free-living bacteria and their spores are continuously released from plant-associated biofilms into the product streams, leading to food spoilage and contamination with pathogens.
Since eDNA is ubiquitous in the biofilm matrix and crucial to the structural integrity, its degradation with nucleases is an attractive strategy for biofilm control [Okshevski et al. (2015) Current Opinion Biotechnology 33, 73-80]. On the other hand, proactive strategies focus on the prevention biofilm formation. This can be achieved by interfering with cell signalling systems that are responsible for biofilm formation, or applying coatings on medical or industrial surfaces with bactericidal, matrix-degrading or attachment-preventing properties. For example, the coating of a surface with the nuclease DNase I has been shown to reduce the adhesion of the bacteria Pseudomonas aeruginosa and Staphylococcus aureus with 99% and 95% respectively [Swartjes et al. (2013) Advanced Functional Materials. 23, 2843-2849]. Finally, also bacteriophages are currently examined as an innovative strategy for biofilm eradication. They are viruses that specifically infect bacteria, making them natural enemies. Besides being excellent killers of bacteria, some bacteriophages also efficiently penetrate the biofilm matrix by the use of matrix-degrading enzymes [Harper et al. (2014) Antibiotics 3, 270-284]. For example, Salmonella biofilms have been shown to be prevented and reduced for 90% and 66% respectively upon bacteriophage treatment [Gong & Jiang (2017) Poultry Sci. 96, 1838-1848].
The present invention thus relates to the use of a miniDNase, alone or in combination with other microbial agents to combat biofilms of bacteria or yeast. Biofilm formation can be prevented by immobilizing DNase on surfaces prone to biofilm growth. Biofilms also grow e.g. on cloths, accordingly the use of MiniDNase in washing powders and detergents is equally envisaged.
The invention thus also relates to the use of miniDNase as an antimicrobial agent. The miniDNase can be used as an antimicrobial agent in non-medical applications whereby the DNase is formulated as a soluble protein or encapsulated in liposomes. Such liposomes are e.g. described in Jones (2005) Methods Enzymol. 391, 211-228.
In certain embodiments the miniDNase is cloned in the genome of a bacteriophage such that the miniDNase protein is presented at the surface of the phage. Suitable cloning methods are known from phage display technology.
Another embodiment is the medical use of a miniDNase of the present invention. Due to their high resistance to antibacterials, as well as to the host immune system, biofilms are a major concern to animal and human health. Since 80% of the human infections are estimated to result from biofilms, there is a high demand for the development of effective control strategies. Two types of infection-causing biofilms can be distinguished. On the one hand, biofilms can attach to the surface of medical devices, like catheters, prostheses, pacemakers and contact lenses. When these devices are brought into contact with the human body, the biofilms gain access to human tissues and cause infection. On the other hand, many pathogens can cause native biofilm-associated infections in body parts that are normally germ-free.
Human DNaseI (hDNAseI) is used since decades for improving the respiratory function of cystic fibrosis patients, commercialized as Pulmozyme® [see table 11]. Despite its success, still a significant group of patients are non-responding to a hDNAse treatment or developed antibodies to the hDNAse.
Thus the present invention envisages miniDNase for the use in the alleviation of the symptoms of cystic fibrosis as monotherapy or in combination with hDNAse. Specific patients groups are non-responders to hDNase or patients who have developed an antibody response against hDNAse.
Other disorders which can be treated with the miniDNases of the present invention include lung infections outside the context of CF, bone infections and persistent wound infections (Burmolle M et al. (2010) FEMS Immunol. Medical Microbiol. 59, 324-336).
Due to its robust properties it is believed that miniDNase has advantageous properties as well in the production of aerosols as in the pharmaceutical activity of the compound. In addition, due to its low molecular weight, the protein is likely not immunogenic.
The use of phages to treat bacterial infection is an emerging field, and is applicable to e.g. skin infections, infections of the respiratory systems (such as bacterial pneumonia), but is also for the prevention and treatment of bacterial infections caused by contamination of catheters or other surgical materials such as implants. A review can be found in Barbu (2016) Cold Spring Harb Perspect Biol 8(10). An aspect of the invention is the use of a miniDNase in such phage therapy. This can be done by cloning the miniDNase as a fusion protein with a structural protein of the phage such that the phage already by entry acts on the DNA of the infected cells. Alternatively the miniDNase is cloned as a late gene in the viral genome and is released as a soluble protein.
Other embodiments of antibacterial therapy are combination therapies with miniDNase and antibacterial proteins such as the cell membrane degrading endolyins or artilysins (Briers et al. (2014) mBio. 5, e01379-14).
Other embodiments of antibacterial therapy are combination therapies with miniDNase and antibacterial peptides or antibiotics.
The invention is illustrated in the following examples.
LUZ19_Gp5 (74 amino acids long, MW=8451 Da) is a hypothetical protein originating from Pseudomonas aeruginosa phage LUZ19 (Leuven UZ phage isolate 19).
The open reading frame [SEQ ID NO:21 ] and the encoded amino acid sequence [SEQ ID NO:1 ] is shown below:
Purified genomic DNA of phage LUZ19 was used as a template for the amplification of the open reading frame (ORF5) encoding the hypothetical protein LUZ19_Gp5 in standard PCR reactions with Pfu polymerase (Thermo Scientific, Waltham, Mass., USA) or DreamTaq polymerase (Thermo Scientific). The following parameters were used:
Forward (Primer 1) and reverse (Primer 2) primers for these PCRs are shown in Table 1.
Both obtained PCR fragments were purified using the GeneJet™ PCR purification kit (Thermo Scientific) according to the manufacturer's protocol. The purified fragment of the amplified ORF for C-terminal His-tag fusion was then ligated in the commercial available pEXP5-CT/TOPO® expression vector (Invitrogen, Carlsbad, Calif., USA) following the TA-cloning protocol provided by the manufacturer, causing a fusion on the 3′/C-terminal side of the phage protein to the 6×Histidine tag (His-tag) necessary for purification. The purified fragment for N-terminal GST-tag fusion was directionally cloned in the commercial available pGEX-6P-1 vector (GE Healthcare, Little Chalfont, UK) using restriction enzymes BamHI (Thermo Scientifc) and EcoRI (Thermo Scientific), causing a fusion on the 5′/N-terminal side of the phage protein to the PreScission cleavage site and the glutathione S-transferase tag (GST-tag). After restriction of the PCR fragment and the vector in the condition suggested by the manufacturer, the linearized vector was dephosphorylated by adding 5 μl FastAP (thermosensitive alkaline phosphatase, Thermo Scientific) and incubation for 10 min at 37° C. Following inactivation (10 min, 75° C.) and purification, the DNA fragment was ligated using T4 DNA ligase (Thermo Scientific) according to the manufacturer's protocol. A 10-fold access of the insert DNA compared to the linearized vector was used. The obtained DNA and amino acid sequences for the recombinant LUZ19_Gp5 protein are shown in Table 2.
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFP
NLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGV
SRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDAL
DVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGG
GDHPPKSDLEVLFQGPLGSMSSRDPYRIGHRVGLVNYSDRYLGADAAGTKGTI
Recombinant expression of LUZ19_Gp5 was performed in exponentially growing E. coli BL21 (DE3) cells after induction with 1 mM IPTG (isopropylthiogalactoside) at 16° C. (GST-tagged Gp5) or 30° C. (His-tagged Gp5) overnight. The phage protein was then purified using a 5 ml GSTrap HP column (GE Healthcare) or 1 ml HisTrap HP column (GE Healthcare), depending on the fused tag, on an Äkta Fast Protein Liquid Chromatograph (FPLC, GE Healthcare). The affinity chromatography is performed in four subsequent steps, all at room temperature:
For the C-terminal His-tagged protein (1 ml HisTrap HP column)
1. Equilibration of the column with 10 column volumes of Lysis Buffer (20 mM NaH2PO4, 500 mM NaCl on pH7.4) at a flow rate of 1 ml/min.
2. Loading of the total lysate (with the desired phage protein) on the HisTrap HP column at a flow rate of 0.5 ml/min.
3. Washing of the column with 10 column volumes of Washing buffer (50 mM imidazole, 20 mM NaH2PO4, 500 mM NaCl on pH7.4) at a flow rate of 1 ml/min.
4. Elution of bounded Gp5 from the column with 10 column volumes of Elution Buffer (500 mM imidazole, 20 mM NaH2PO4, 500 mM NaCl on pH7.4) at a flow rate of 0.5 ml/min.
For the N-terminal GST-tagged protein (5 ml GSTrap HP column)
1. Equilibration of the column with 2 column volumes of PBS Buffer (10 mM Na2HPO4, 1.8 mM KH2PO4, 140 mM NaCl, 10 mM KCl on pH 7.3) at a flow rate of 2 ml/min.
2. Loading of the total lysate (with the desired phage protein) on the GSTrap HP column at a flow rate of 1 ml/min.
3. Washing of the column with 5 column volumes of PBS Buffer at a flow rate of 2 ml/min.
4. Elution of bounded Gp5 from the column with 2 column volumes of Elution Buffer (50 mM Tris, 10 mM glutathione reduced on pH 8) at a flow rate of 1 ml/min.
The yields for the purifications of recombinant LUZ19_Gp5 are shown in Table 3. The protein concentration was determined spectrophotometrically at a wavelength of 280 nm. Purified stock solutions of both proteins were analyzed visually on an 16% SDS-PAGE gel (
To remove the glutathione S-transferase tag, the elution fractions containing the protein of interest were first concentrated and dialysed to a Digestion Buffer (50 mM Tris, 100 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol on pH 8) using Microsep™ Advance Centrifugal Device 3K (Pall Corporation, Port Washington, N.Y., USA) and Slide-A-Lyzer® MINI Dialysis Devices 3.5K 2 ml (Thermo Scientific) respectively, according to the manufacturer's protocols. Next, 1 U of the human rhinovirus 3C (HRV3C) Protease (TURBO3C, 2 U/μl, MoBiTec GmbH, Göttingen, Germany) was added per 400 μg of GST-tagged protein and incubated overnight at 4° C. to cleave the protein from the GST-tag. The final purification was done with size-exclusion chromatography using HiLoad 16/600 Superdex 75 Prep grade gel filtration column (GE Healthcare), which was calibrated using the Gel Filtration Calibration Kit LMW (GE Healthcare). After an equilibration with one column volume (124 ml) of Gel Filtration Buffer (50 mM Tris, 100 mM NaCl on pH 8) at 1 ml/min, the protein sample was loaded at the same speed and 120 1 ml fractions were collected. The samples containing the recombinant protein were finally concentrated with Amicon Ultra 3K 0.5 ml (Millipore, Ontario, Canada) and analyzed visually on an SDS-PAGE gel (
The nuclease activity was qualitatively demonstrated by using agarose gel electrophoresis and ethidium bromide staining to detect the loss of nucleic acids after exposure to the phage protein. The gels contained 1% agarose and the running buffer was TAE (40 mM Tris, 0.5 mM sodium acetate, 50 mM EDTA on pH 7.2).
First, the degradation capacity of LUZ19_Gp5 for deoxyribonucleic acids (DNA) and ribonucleic acids (RNA) was studied. Increasing concentrations of the untagged phage protein (0-15 μM) were incubated with 100 ng host DNA (P. aeruginosa genome) or host RNA (isolated P. aeruginosa mRNA, rRNA and tRNA) in DNase I buffer+MgCl2 (Thermo Scientific) for 30 min at 37° C. As positive controls, 1 U of the commercial DNase I (Thermo scientific) and 10 μM of the commercial RNase A (Thermo Scientific) were used. The results showed that DNA was completely degraded, even at the lowest concentration tested (2.5 μM). In addition, the phage-encoded nuclease interacted with the RNA, but no digestion was observed (for details see
To evaluate its specificity for various DNA molecules, the purified LUZ19_Gp5 protein was incubated with diverse deoxyribonucleic acids including the double-stranded (ds) P. aeruginosa genome, the ds E. coli genome, the ds linearized and circular pUC18-mini-Tn7-Lac-GW plasmid and the single-stranded (ss) φX174 virion genome. For this, 100 ng of nucleic acids were respectively incubated with 10 μM of the purified LUZ19_Gp5 protein in 20 μl of reaction mixture including DNase I buffer+MgCl2 at 37° C. for 1 h. Moreover, the influence of a fusion protein on its activity was tested by using the N-terminal GST-tagged variant. The commercial DNase I was also included as a positive control and the purified GST protein as a negative control. Since all the deoxyribonucleic acids could be degraded by LUZ19_Gp5, the nuclease was described as non-specific. In addition, the nuclease was still active in presence of a N-terminal fusion protein, even when this protein was three times its size (for details see
Finally, the specificity of LUZ19_Gp5 for various bacteriophage genomes was tested. All the tested phages are Caudovirales, but belong to different families: LUZ19, φKMV, LUZ24 (Podoviridae), YuA (Siphoviridae), φKZ and 14-1 (Myoviridae). 100 ng of purified phage genomes were incubated with 10 μM of the purified LUZ19_Gp5 protein in 20 μl of reaction mixture including DNase I buffer+MgCl2 at 37° C. for 1 h. Pure water (milliQ) and the commercial DNase I were used as a negative and a positive control respectively. The results showed that all the phage genomes were completely degraded, even the modified YuA genome and the LUZ19 genome, which contains the LUZ19_gp5 gene (for details see
MilliQ and the commercial DNase I were used as negative and positive control respectively.
In addition, the nuclease activity was confirmed by zymography. First, samples of purified untagged LUZ19_Gp5 (4.86 and 8.74 μM) diluted in 4× SDS-PAGE loading dye (200 mM Tris HCl pH 6.8, 8 mM EDTA, 40% (v/v) glycerol, 4% (w/v) SDS, 0.4% (w/v) bromophenol blue) were separated on an 16% SDS-PAGE gel containing 100 μg/ml DNA from fish sperm (Sigma Aldrich, St. Louis, Mo., USA). Then, the gel was washed three times for 5 min in mQ and incubated for 48 h in zymogram renaturation buffer (150 mM NaH2PO4 pH 7, 10 mM MgCl2 and 0.1% Triton X-100), which allowed the proteins to refold to their native state. Finally, the gel was stained for 20 min in 0.5 μg/ml ethidium bromide and destained for 20 min in the zymogram renaturation buffer. After staining with ethidium bromide, the enzymes with nuclease activity were visible as non-fluorescent bands using an UV transilluminator. The impact of boiling on the renaturing capacity of Gp5 was tested by loading both preboiled (10 min, 95° C.) and non-preboiled samples. Clear bands were observed around 8-9 kDa, indicating the nuclease activity of Gp5 (MW=8.5 KDa) (for details see
The effect of the pH on enzymatic stability and activity was tested over a pH range of 3 to 10, using a Tris/HCl buffer system. First, 10 μM of the purified LUZ19_gp5 was pre-incubated in a buffer mixture containing DNase I buffer+MgCl2 and a Tris/HCl solution to obtain the appropriate pH, for 30 min at 37° C. Then, 100 ng of P. aeruginosa genome was added and the reaction mixtures were incubated for another 30 min at 37° C. Finally, agarose gel electrophoresis and ethidium bromide staining were used to detect the loss of nucleic acids at every pH tested. The results showed that DNase I was found to be inactive in acidic solutions, in contrast to LUZ19_Gp5 which showed enzymatic activity in all solutions tested (
To evaluate the thermostability of the nuclease, the purified LUZ19_Gp5 protein was first pre-incubated for 30 min at various temperatures ranging from 10° C. to 100° C. in the absence of DNA, followed by incubation of the nuclease with DNA for 30 min at the corresponding temperature. Each reaction mixture consisted of 10 μM of the purified LUZ19_Gp5 protein in 20 μl DNase I buffer+MgCl2. 100 ng of P. aeruginosa genome was added after 30 min of pre-incubation. As a negative and a positive control, milliQ water and the commercial DNase I were used respectively. In addition to the inactivation of DNase I at a temperature of 80° C., LUZ19_Gp5 remained active after incubation at every temperature tested. This demonstrated its high thermostability (
Even the DNA which has been treated at 100° C. and tends to aggregates, since it remains in the loading well (
Knowing that LUZ19_Gp5 displays a substrate specificity similar to DNase I, the similarities and differences between Gp5 and DNase I are of interest. For this, agarose gel electrophoresis and ethidium bromide staining were used to detect the loss of nucleic acids under different conditions in presence of Gp5 or DNase I. Each test started with a pre-incubation period of the nuclease in DNase I buffer+MgCl2 for 1 h at 37° C. (unless stated differently) before the substrate 5 ng/μl λ DNA (Thermo Scientific) was added. After addition, the samples were incubated another 1 h at 37° C. (unless stated differently) and then loaded on an 1% agarose gel.
The commercially available DNase I from Thermo Scientific is quantified in Units (U), while the amount of LUZ19_Gp5 that match 1 U has not been determined yet. 1 U is defined as the amount of DNase I that completely degrades 1 μg plasmid DNA in 10 min at 37° C. (Thermo Scientific). This makes it difficult to quantitatively compare both enzymes. Therefore, a qualitative method was designed to compare the activity of Gp5 to DNase I under different conditions. First, a series of four two-fold dilutions was prepared for each nuclease and an activity test was performed under standard conditions, in a way that the degradation of 5 ng/μl λ DNA was completed at the second dilution (5 μM for Gp5 and 1/256 U/20 μl for DNase I, see
After developing the scoring system, the nuclease activity of both Gp5 and DNase I was evaluated under various experimental conditions. These conditions were obtained by varying the (pre)incubation temperature and time, changing the pH of the samples or adding different compounds to the samples, like metal chelators, reducing agents, denaturants, detergents and ions. To improve the reliability, each activity test was done in triplicate. The results of the activity tests under the different conditions are summarized in table 4.
The possibility of protein refolding after heat treatment was considered as well, as some heat denatured proteins can refold to their active conformation when cooled down slowly. To investigate the possibility that LUZ19_Gp5 is reversibly denatured at 90° C., an additional nuclease activity test in which the samples were cooled down rapidly on ice followed by the addition of the Gp5 inhibitor citrate (final concentration: 20 mM, see Table 4: chelators) after 10 seconds was performed. The results indicate that Gp5 retains its capacity to degrade the DNA under these conditions, which indicates that Gp5 is not denatured at 90° C. and nuclease activity is still effective at high temperatures.
LUZ19_gp5 was amplified with Pfu polymerase using specific primers (Table 5) and phage LUZ19 genome as a template, and cloned in a Gateway entry vector using the pENTR/SD/D-TOPO cloning kit (Invitrogen) according to the manufacturer's protocol. The gene was then transferred to the E. coli-P. aeruginosa shuttle expression vector pUC18-mini-Tn7T-Lac, which first was made Gateway compatible, using the Gateway® LR Clonase® enzyme mix (Thermo Scientific) following the protocol provided by the manufacturer. Co-transformation of 300 ng of the pUC18-mini-Tn7T-Lac-GW construct and 500 ng pTNS2 plasmid by electroporation to P. aeruginosa PAO1 allowed single-copy integration of the phage gene in the Pseudomonas genome under the control of an IPTG-inducible lac promotor. Finally, three 100-fold dilution series (100, 10−2, 10−4 and 10−6), each of an independent overnight culture, were spotted in parallel on LB medium (1% (w/v) Bacto tryptone, 1% (w/v) NaCl and 0.5% (w/v) yeast extract) with and without 1 mM IPTG. As a negative control, a P. aeruginosa PAO1 strain, encoding an empty pUC18-mini-Tn7T-Lac-GW expression cassette, was used (
To investigate the effect on the host morphology, the cell growth of the P. aeruginosa PAO1 cells with the phage gene integrated in their genome was analyzed over time using time-lapse microscopy. For this, overnight cultures were prepared in LB medium supplemented with 30 μg/ml Gentamicin (Gm30; VWR international Ltd). Next, 2 μl of a thousand-fold dilution of the overnight culture was spotted on LB agar (LB with 1.5% (w/v) agar) supplemented with 1 mM IPTG. Growth was recorded at 37° C. (Okolab Ottaviano, Italy) in real time for 5 h with a Ti-Eclipse inverted microscope (Nikon, Champi-gny-sur-Marne, France) connected to a Nikon DS-Qi2 camera (
The in vivo nuclease activity of LUZ19_Gp5 was examined under a microscope using the P. aeruginosa PAO1 cells with the LUZ19_gp5 gene integrated in their genome (see example 6) and SYBR Green I to stain the P. aeruginosa genome.
First, two overnight cultures were prepared in LB medium supplemented with Gm30. The next day, 80 μl of these cultures were transferred to fresh 4 ml LB medium supplemented with Gm30 and the cells were grown at 37° C. until an OD600 nmof 0.2 was reached. Then, 1 mM IPTG was added two one culture and both cultures were incubated for 2 h at 37° C. before 0.01% (v/v) SYBR Green I was added to both samples. The samples were incubated 20 min while shaking, and 2 μl of each sample was spotted on AB medium (3 mM (NH4)2SO4, 18 mM Na2HPO4, 4 mM KH2PO4, 10 mM NaCl, 0,1 mM CaCl2, 1 mM MgCl2 and 3 μM FeCl3). Microscopic images were taken with a Ti-Eclipse inverted microscope connected to a Nikon DS-Qi2 camera.
The toxicity of LUZ19_Gp5 prevents bacterial growth. However, the presence of a mutation in the gene can result in loss of toxicity. Such mutations were identified by plating P. aeruginosa PAO1 cells containing the pUC18-mini-Tn7T-Lac-GW expression cassette with LUZ19_gp5 gene inserted in their genome overnight on LB with 1 mM IPTG. Next, the pUC18-mini-Tn7T-Lac-GW cassettes of present colonies were amplified using vector primers (Table 6, primers 1-2) and the sequences of both promotor region and the phage gene were determined.
Three different mutations were identified in the LUZ19_gp5 gene potentially neutralizing its toxicity (
The obtained PCR fragments were purified using the GeneJet™ Gel extraction kit (Thermo Scientific) according to the manufacturer's protocol. 25 ng of the purified PCR mix was then incubated with 0.5 μl Quick T4 ligase (New England BioLabs) in the corresponding buffer following the protocol provided by the manufacturer. If the sequence of the obtained plasmid was correct, a co-transformation of 300 ng of this pUC18-mini-Tn7T-Lac-GW construct and 500 ng pTNS2 was performed that allow single-copy integration of the mutated phage gene in the Pseudomonas genome. Finally, three 100-fold dilution series (100, 10−2, 10−4 and 10−6), each of an independent overnight culture, were spotted in parallel on LB medium with and without 1 mM IPTG. As a negative and positive control, a P. aeruginosa PAO1 strain encoding an empty pUC18-mini-Tn7T-Lac-GW expression cassette and one with integrated wild-type LUZ19_gp5 gene were used respectively (
Since size exclusion chromatography failed for each mutant, the mutant proteins fused to a GST-tag were selected for the nuclease tests. Purified stock solutions of the three mutant proteins were analyzed visually on an 16% SDS-PAGE gel (
The Basic Local Alignment Search Tool (BLAST) was used to find homologous proteins of LUZ19_Gp5. Both the nucleotide and protein sequence were compared to sequence databases using the standard nucleotide BLAST (blastn) and the translated BLAST (tblastx), and the results are shown in Table 7 and Table 8.
agenera belonging to the Podoviridae family. All these viruses have P. aeruginosa as host
One homolog of LUZ19_Gp5, namely phiKMV_Gp5 [SEQ ID NO: 8], was selected and tested on nuclease activity. Compared to LUZ19_Gp5, this protein has an E-value of 4e-36 and 78% of the amino acids are identical between both proteins (table 8).
First, the open reading frame (ORF5) encoding the hypothetical protein phiKMV_Gp5 was directionally cloned into a pGEX-6P-1 vector similar to the cloning of LUZ19_gp5 gene into the same vector (see example 1). The used primers for PCR are shown in Table 9.
Recombinant expression and purification of the protein was performed similar to the purification of the LUZ19_Gp5 protein (see example 1). The yields after affinity-based purification (N-terminal GST-tag), and removal of the GST-tag followed by size-exclusion chromatography (untagged) are shown in Table 10. The protein concentration was determined spectrophotometrically at a wavelength of 280 nm. The purified stock solution of the protein was analyzed visually on an 16% SDS-PAGE gel (
The nuclease activity of phiKMV_Gp5 was studied similar to the nuclease activity of LUZ19_Gp5 (See example 5). The test started with a pre-incubation period of the nuclease in DNase I buffer+MgCl2 for 1 h at 37° C. before the substrate 5 ng/μl λ DNA was added. After addition, the samples were incubated another 1 h at 37° C. and then loaded on an 1% agarose gel. The result showed that DNA was completely degraded, even at the lowest concentration tested (
Table 11 compares the properties of the miniDNase of the present invention with those of commercially available products.
Besides phiKMV_Gp5, an additional similar protein of LUZ19_Gp5, namely LKD16_Gp5, was tested for nuclease activity. This protein has the lowest blast similarity to LUZ19_Gp5 of all available proteins (Table 8) with an E-value of 1e-25 and 72% identical amino acids between both proteins (table 8). Furthermore, LKD16_Gp5 (SEQ ID NO: 9) consists of only 72 amino acids, compared to LUZ19_Gp5 (SEQ ID NO: 1) and phiKMV_Gp5 (SEQ ID NO: 8) which consist of 74 and 75 amino acids respectively.
Like with phiKMV_gp5, the open reading frame (ORF5) encoding the hypothetical protein LKD16_Gp5 was directionally cloned into a pGEX-6P-1 vector similar to the cloning of LUZ19_gp5 gene into the same vector (see example 1). The used primers for PCR are shown in Table 12.
Recombinant expression and purification of the protein was performed similar to the purification of the LUZ19_Gp5 protein (see example 1). The yields after affinity-based purification (N-terminal GST-tag), and removal of the GST-tag followed by size-exclusion chromatography (untagged) are shown in Table 13. The purified stock solution of the protein was analyzed visually on a 16% SDS-PAGE gel (
The nuclease activity of LKD16_Gp5 was analyzed similar to the nuclease activity of LUZ19_Gp5 (See example 5) and concentrations up to 2.5 μM have been tested. The DNA digestion after incubation with LKD16_Gp5 is shown in
Truncation mutants are variants of LUZ19_Gp5 that have a deletion of specific amino acids at the N- and/or C-terminus of the protein. Studying their activity in vitro and in vivo gives more insights into the protein region essential for its nuclease activity and toxicity, respectively. Based on the amino acid conservation of the homologs of LUZ19_Gp5 and on the predicted secondary structure, seven truncation mutants have been selected as examples (
To express the truncation mutants in E. coli for in vitro nuclease assessment, seven pGEX-6P-1 expression plasmids were constructed by site-directed mutagenesis, each containing the phage gene with a 5′ and/or 3′ deletion of coding nucleotides. To create N- or C-terminal truncations, the original pGEX-6P-1 plasmid expressing LUZ19_gp5_wt was used as template for the amplification (see example 1). First, primers were designed that amplify the entire pGEX-6P-1 expression plasmid, except for the corresponding 5′ or 3′ nucleotides that result in the desired truncation. For this, one primer annealed to the vector backbone right next to the 5′ or 3′ end of the coding sequence (referred to as backbone primer, Table 15A: primers 1-2), while the other primer annealed to the coding sequence (referred to as coding primers, table 15B: primers 1-6), causing a deletion of certain nucleotides. For the truncation with a deletion both at the N- and C-terminus (i.e. LUZ19_gp5R8−Q70), pGEX-6P-1 expressing LUZ19_gp5R8 was used as template. This template was then amplified with backbone primer 1 and coding primer 1 (Table 15A and B). Primer phosphorylation, site-directed mutagenesis and PCR fragment purification were performed exactly as in example 8. Next, the truncation mutants were expressed as N-terminal GST-tagged proteins, exactly as described in example 1. If protein yields >1 mg/l were obtained, the N-terminal GST-tag was removed to obtain untagged proteins, as described in example 1. The yields for the purifications of recombinant LUZ19_Gp5 truncation mutants are shown in Table 16. The protein concentration was determined spectrophotometrically at a wavelength of 280 nm.
The nuclease activity of the LUZ19_Gp5 truncation mutants was studied similar to the nuclease activity of the wild-type LUZ19_Gp5 (see example 5). If no untagged protein was available, the activity test was performed with the N-terminal GST-tagged protein. However, since these samples were less pure (only affinity purification and no size-exclusion purification), the pre-incubation step of 1 h was performed at 90° C., which cannot inactivate LUZ19_Gp5-GST as discovered earlier (see example 4). Each test was performed in triplicate. When nuclease activity was detected at concentrations <5 μM of truncation mutant protein, the truncation mutant was classified as ‘active’. The results showed that LUZ19_gp5Q70, LUZ19_gp5I68, LUZ19_gp5R8 and LUZ19_gp5R8−Q70 still had in vitro nuclease activity, while the other truncations mutants were inactive.
E. coli expression culture as determined by spectrophotometric measurement
To evaluate the impact of the truncations on the in vivo toxicity of the nuclease in P. aeruginosa, the eight LUZ19_gp5 truncation mutants were expressed in P. aeruginosa PAO1. First, the pUC18-mini-Tn7T-Lac-GW plasmids were constructed by site-directed mutagenesis similar to the above described construction of the pGEX-6P-1 expression plasmids with truncated phage gene. For the site-directed mutagenesis, the same coding primers (table 15B: primers 1-6), but different backbone primers (table 15A, primers 3-4) were used and the pUC18-mini-Tn7T-Lac-GW vector containing the wild-type LUZ19_gp5 gene served as the template (see example 6). Single copy chromosomal integration of the truncated phage genes, as well as the assessment of the in vivo toxicity in P. aeruginosa PAO1 were performed exactly as described in example 6. The results showed that LUZ19_gp5Q70 was still toxic to P. aeruginosa, while all other truncation mutants did not inhibit the growth (
P. putida is a metabolic versatile bacterium that is increasingly used as a microbial cell factory to produce various relevant products (e.g. polyhydroxyalkanoates). Engineering of production strains can be used to recover the products in a more cost-effective and efficient way. For example, expression of nucleases can be used to degrade genomic DNA and thereby reduce viscosity in the product stream. In this regard, the activity of LUZ19_gp5 in P. putida KT2440 has been assessed by intracellular expression.
The phenotypical effect of expression of LUZ19_gp5, one toxic truncation mutant (LUZ19_gp5Q70) and one non-toxic truncation mutant (LUZ19_gp5R8) (see example 13) on the growth of P. putida KT2440 was evaluated. Co-transformation of 300 ng of the pUC18-mini-Tn7T-Lac-GW constructs, constructed as in example 6 and 13, and 500 ng pTNS2 plasmid by electroporation to P. putida KT2440 allowed single-copy integration of the phage gene and the truncation mutants in the P. putida genome under the control of an IPTG-inducible lac promotor. Protein expression to evaluate the phenotypical effect on the bacterial growth was performed similar to the evaluation in P. aeruginosa (see example 6 and 13). As a negative control, a P. putida KT2440 strain, encoding an empty pUC18-mini-Tn7T-Lac-GW expression cassette, was used. Results indicated that LUZ19_gp5 and its truncation mutants display the same phenotypical effect on the growth of P. putida and P. aeruginosa, namely LUZ19_gp5 and LUZ19_gp5Q70 cause growth inhibition upon intracellular expression, while LUZ19_gp5R8 does not inhibit bacterial growth.
The degradation of dsDNA by nucleases causes an increase in UV absorption at 260 nm (ΔA260), referred to as hyperchromicity. This absorption increase allows to follow the degradation of dsDNA by nucleases in time and can therefore be used to quantitatively demonstrate the nuclease activity of LUZ19_Gp5. First, 38 μM of LUZ19_Gp5 was pre-incubated in DNase I buffer+MgCl2 for 30 min at room temperature. Next, 50 μg/ml λ DNA was added, resulting in a final volume of 400 μl, and ΔA260 was immediately assessed in a cuvette (UVette® 220 nm-1,600 nm, Eppendorf) using a spectrophotometer (BioPhotometer 6131, Eppendorf). A mixture containing only DNase I buffer+MgCl2 and λ DNA was used as blank and measurements were taken every 10 minutes. A clear linear increase in absorption at 260 nm was observed, confirming the biochemical nuclease activity of LUZ19_gp5 in vitro (
MKASTCCWSR AARCPATSPA RRTAGSTLNT TRAARTGARS STLQELRTMS SRDPYRIGHR
| Number | Date | Country | Kind |
|---|---|---|---|
| 1712145.0 | Jul 2017 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/070253 | 7/26/2018 | WO | 00 |
