Patent Application
20230406892
The present invention provides proteins capable of exerting a cytotoxic effect on Salmonella, referred to as salmocins. The invention also provides compositions, including pharmaceutical compositions, comprising one or more of said proteins. Also provided is a method of preventing or reducing infection or contamination of an object with Salmonella, a method of treating infection with Salmonella of a subject or patient in need thereof, and a process of producing a composition comprising the protein.
Salmonella is a rod-shaped Gram-positive bacterium of Enterobacteriaceae family. Salmonella enterica is the type species and is further divided into six subspecies with S. enterica ssp. enterica as subspecies that includes over 2500 serovars. Salmonella infections are common and can result in protean clinical manifestations, ranging from an asymptomatic state to very severe diseases. Salmonella enterica causes an estimated 1 million illnesses in the United States each year, resulting in an estimated 19,000 hospitalizations and 380 deaths. Over the last 5 years, 46 Salmonella outbreaks have been recorded in USA, most of the food poisonings being due to contaminated poultry or vegetables, but also red meats and fish (CDC website).
Prevention Salmonella infections or reducing contamination of food with Salmonella requires control measures at all stages of the food chain, from agricultural production on the farm to processing, manufacturing and preparation of foods in both commercial establishments and household kitchens. Good hygienic practices reduce contamination of food with Salmonella, but do not guarantee the absence of Salmonella from products. Preventive measures for Salmonella in the home are similar to those used against other foodborne bacteria. Basic food hygiene practices, such as “cook thoroughly”, are recommended as a preventive measure against salmonellosis, cf. WHO at www.who.int/mediacentre/factsheets/fs139/en/.
Antimicrobial therapy may be used to treat humans or animals suffering from Salmonella infections. However, antimicrobial resistance is a global public health concern and Salmonella is one of the microorganisms in which some resistant serotypes have emerged, affecting the food chain.
Most of the above mentioned methods of preventing or treating Salmonella infections or reducing contamination with Salmonella are methods that are essentially independent from a particular pathogenic bacterium or from a particular serotype of Salmonella. This has the advantage that little prior knowledge of the specific Salmonella strain or Salmonella enterica serotype in question is necessary before counter-measures are taken. However, the above mentioned methods of preventing Salmonella infection or reducing contamination with Salmonella such as heating are not always applicable or change the treated good or food in undesirable ways. Other methods may have turned out non-effective with a particular patient. There is therefore a need for further methods of preventing or treating Salmonella infections or contamination, or methods for reducing or preventing contamination of objects with Salmonella, notably with Salmonella enterica ssp. enterica.
Some proteins active against Salmonella were previously described (WO 2018/172065) that were referred to as “salmocins”. While the salmocins described in WO 2018/172065 show high activity against Salmonella species and strains, there are limitations in that their toxic activities are similar, whereby the spectrum of activity is limited even if different ones of the known salmocins are combined. Further, mid- or long-term use of the same or similar salmocin types may select Salmonella strains that are resistant to the salmocins used. Therefore, additional salmocins that act by other mechanisms are highly desired. Notably, compositions comprising two or more salmocins of different types are desired for broadening the range of activity against many different Salmonella species.
For proteins for technical applications, ease of production, purification, and storage are important aspects that can be decisive for the question whether the technical applications are feasible.
It is an object of the invention to provide methods for preventing or treating Salmonella infections such as food-borne Salmonella infections. It is another object to provide methods for preventing or reducing contamination of objects, notably food, with Salmonella. It is a further object to provide methods for preventing or treating Salmonella infections and/or methods for reducing contamination of objects with Salmonella, that are effective against a wide range of Salmonella serogroups. It is another object to provide further salmocins that act by different or additional mechanisms against Salmonella. It is a further object of the invention to provide agents active against Salmonella that can be produced and/or purified and/or stored conveniently with high stability. Further, compounds, agents and compositions for such methods are desired.
Accordingly, the invention provides the subject-matter defined in the claims. The invention also provides:
(10) The protein according to (9), wherein the protein is as defined in (B-x), (C-x), (D-x), or (E-x), preferably as defined in (B-xi), (C-xi), (D-xi), (E-xi), (B-xii), (C-xii), (D-xii), or (E-xii), and the amino acid residue (of said protein) corresponding to residue 155 of SEQ ID NO: 33 is Pro and/or the amino acid residue corresponding to residue 246 of SEQ ID NO: 33 is Arg or Lys, preferably Arg.
Salmonella bacteriocins, herein together with derivatives thereof referred to as “salmocins” (herein abbreviated as “Scol” or “Sal”), are natural non-antibiotic antimicrobial proteins produced by certain Salmonella strains that kill or inhibit the growth of other Salmonella strains. Unlike relatively well studied Escherichia coli protein analogues termed colicins, salmocins have been given little attention. There is a number of Salmonella sequences with similarity to colicin sequences in the publicly available genome databases, most of them showing high identity to colicins M, Ia, Ib, 5 and 10. The inventors have identified salmocins similar to but different from colicins that can be used to prevent or reduce infection or contamination with Salmonella, notably with Salmonella enterica ssp. enterica.
The inventors have found that salmocins can be expressed efficiently in plants. Expression processes as those used in this study have already been brought to the level of GMP compliance, and are currently being used in different clinical trials as manufacturing processes. Most salmocins are expressed at high yields (up to 1.7 g active protein per kilogram of fresh green biomass), meaning low commercially viable manufacturing costs. Production can be made using, inter alia, tobacco and edible plants such as leaf beets or spinach. Among different salmocins, salmocins Ma (ScolMa), Mb (ScolMb), and Mc (ScolMc) are preferred, and compositions of these M-type salmocins with Ela (ScolE1a) and (more preferably) E1b (ScolE1b) or their derivatives are more preferred, since they were found to possess very broad antimicrobial activity against major pathogenic Salmonella strains. Each of the two salmocins ScolE1a and ScolE1b shows also a very high activity against all 36 major pathogenic strains tested. Also, ScolMa was shown to have broad antimicrobial activity against major pathogenic Salmonella strains (cf.
Treatments with low amounts of colicins (e.g. less than 10 mg colicin per kg of treated food product) reduce the bacterial load of different pathogenic strains by 3 to >6 logs in the assay employed. In spike experiments using poultry meats spiked with two to four pathogen serovars, colicins (colicins M, Ia and 5) efficiently reduced the titer of pathogenic bacteria. Therefore, it is expected that salmocins possessing higher antimicrobial activity towards S. enterica ssp. enterica serovars mentioned above than the colicins mentioned will reduce the titer of a Salmonella contamination on poultry even more effectively.
The experimental data of the present invention demonstrate that the non-antibiotic antibacterial salmocins can be expressed at very high levels in plants such as Nicotiana benthamiana, a standard manufacturing host for multiple biopharmaceuticals currently undergoing clinical trials, and the plant-expressed proteins are apparently fully active. The expression levels in most cases reach 37% of total soluble protein or 1.74 g/kg of fresh leaf biomass without process optimization, meaning that salmocins are not toxic to plants and that optimized industrial procedures of transfection or induction in transgenic hosts could be developed that are inexpensive. In contrast, attempts to express bacteriocin proteins in bacterial hosts at a high level were usually met with general toxicity of this bacteriocin class even in species other than homologous bacterial species (e.g. Medina et al., PLoS One, 2011; 6(8):e23055; Diaz et al., 1994). Thus, plants are excellent hosts for manufacturing salmocins.
The data of this invention demonstrate that salmocins can efficiently control most or all major pathogenic serotypes of Salmonella enterica ssp. enterica under actual exposure modelling. There is a limited variety among salmocins produced by Salmonella. Studied salmocins have quite diverse structure within the general three-domain (translocation, receptor and cytotoxic domains) architecture, similar to more studied E. coli colicins. Surprisingly, practically all tested salmocins and colicins, alone or together with antitoxin (immunity protein), are expressed very well in plants. This may be explained by a low toxicity of salmocins and colicins to plant cells and by the fact that these bacteriocin proteins, being classical representatives of ‘inherently disordered proteins’ (a feature essential for ability to unfold/refold during the bacterial cell wall and membrane translocation), probably do not impose unusual requirements on translation and post-translational machinery of the plant cell.
Unlike the list of major E. coli strains defined by FDA based on a historical analysis of food poisoning due to E. coli, a list of major foodborne Salmonella strains has not been defined by regulatory agencies, primarily due to higher diversity of the pathovars responsible for the outbreaks. Being confronted with this lack of guidance in the prior art, the inventors decided to pool three existing major studies that ranked the pathovars based on their prevalence and poisoning severity. In our study, 36 serovars have been selected to be analyzed, 29 of them caused at least 100 incidences reported to Centers for Disease Control in 2003-2012 (National Enteric Disease Surveillance: Salmonella Annual Report, 2013 (CDC, June 2016): laboratory-confirmed human Salmonella infections (US) reported to CDC 2003-2012) with 17 most notorious pathovars on CDCs top 20 list; the number being five times higher than the number of E. coli pathovars defined by the FDA (seven).
The data presented herein show that, based on their ability to control major pathogenic Salmonella strains, the five different Salmonella salmocins ScolE1a, ScolE1b, ScolE2, ScolE3, and ScolE7 can be divided into three groups. Salmonella salmocins E1a and E1b turned out to be universally active, each being able to kill all tested pathovars and showing the highest average activity. Average activity of the two salmocins on all tested strains was over 107 AU/μg. For example, the individual activity of salmocin Ela was: >103 AU/μg for 35 out of 36 strains, >104 AU/μg for 24 out of 36 strains and >106 AU/μg for 13 out of 36 strains. The remaining salmocins fell into two groups with Salmocins E2 and E7 being inhibitory to over 80% of strains but having a 100-fold lower average activity (less than 105 AU/μg), whereas salmocin E3 inhibited approx. 60% of strains at lower average activity (about 102 AU/μg). The inventors have further found that the salmocins ScolE1c, ScolE1d, ScolE1e, and ScolMa demonstrated significant antimicrobial activities. ScolE1b and ScolE1d were found to be surprisingly better in handling properties, notably for purification. ScolE1d was surprisingly found to have an exceptional storage stability as a solution (notably an aqueous solution when cooled below room temperature, such as at 0° to 10° C., preferably between 3° to 7° C., or at about 4° C.). Among the ScolM salmocins, ScolMb and ScolMc, in particular ScolMc, were found to be surprisingly better in handling properties, notably for purification, and high in antimicrobial activity against Salmonella species.
These results are unexpected, because colicins (salmocin analogues produced by E. coli cells) exhibit a much narrower spectrum of antimicrobial activity against seven E. coli pathovars, and mixtures of two to five colicins had to be preferably used to efficiently inhibit all seven STEC serotypes defined by FDA. Colicins also demonstrated much lower average activity against ‘Big Seven’ STEC strains (average <103 AU/μg), although much higher activity has been observed on strain H104:H4 (>105 AU/μg) that caused a major outbreak in 2011 in Europe, and a common laboratory strain.
The inventors' analysis of cross specific activity of salmocins and colicins on E. coli and Salmonella, respectively, demonstrates low activity against bacteria of different genus/species. In particular, activity of salmocins against ‘Big Seven’ STEC strains was low (less than 102 AU/μg) although some salmocins (such as E2, E7 and E1b, but surprisingly not Ela) were fairly active against H104:H4 (103 AU/μg) and laboratory strain DH10B (105 AU/μg). Similarly, activity of colicins on Salmonella pathovars was found to be low, with colicins Ia and Ib being active on over 80% of strains, but with average activity of only colicin Ia being higher than 3×103 AU/μg (or three to four orders of magnitude less than salmocin E1a/b). The inventors conclude from these studies that, to combat both pathogenic species, one has to use mixtures of colicins and salmocins. These results are also in seeming partial disagreement with the recent studies of ecological efficacy of colicin-like proteins in competitions between bacteria of different genera (Nedialkova et al., PLoS Pathog. 2014 January; 10(1):e1003844).
The invention provides new agents and compositions for controlling Salmonella. The salmocins of the invention have the advantage that marketing authorization can be obtained in an uncomplicated manner. For example, the FDA recently granted plant-produced colicins GRAS (Generally Regarded As Safe) status (GRN573, FDA website). Because of the unmet need for natural non-antibiotic antibacterials for Salmonella control, the inventors conceived exploring Salmonella bacteriocins (“salmocins”). Thereby, the present invention was accomplished.
Salmocin expression vectors include pNMD28161, pNMD28151 and pNMD28172 for the expression of salmocins ScolE2, ScolE3 and ScolE7, respectively (
RB and LB indicate the right and left borders of T-DNA of binary vectors. Pact2: promoter of Arabidopsis actin2 gene; o: 5′ end from TVCV (turnip vein clearing virus); RdRp: RNA-dependent RNA polymerase open reading frame (ORF) from cr-TMV (crucifer-infecting tobamovirus); MP: movement protein ORF from cr-TMV; ScolE2: salmocin ScolE2 coding sequence; ScolE3: salmocin ScolE3 coding sequence; ScolE7: salmocin ScolE7 coding sequence; ScolE1a: salmocin ScolE1a coding sequence; ScolE1b: salmocin ScolE1b coding sequence; Spst: salmocin Spst coding sequence; N: 3′-non-translated region from cr-TMV; T: Agrobacterium nopaline synthase terminator; white segments interrupting grey segments in the RdRp and MP ORFs indicate introns inserted into these ORFs for increasing the likelihood of RNA replicon formation in the cytoplasm of plant cells, which is described in detail in WO2005049839. An intron was also inserted into ScolE2, ScolE3 and ScolE7 ORFs for preventing the cytotoxic effect of these proteins on E. coli cells used for plasmid cloning.
PVX-based vectors for the expression of immunity proteins include pNMD28222 and pNMD28232 for the expression of salmocin ScolE2 and ScolE7 immunity proteins, respectively (
RB and LB indicate the right and left borders of T-DNA of binary vectors. Pact2: promoter of Arabidopsis actin2 gene; o: 5′ end from TVCV (turnip vein clearing virus); RdRp: RNA-dependent RNA polymerase open reading frame (ORF) from cr-TMV (crucifer-infecting tobamovirus); MP: movement protein ORF from cr-TMV; colS4: colicin S4 coding sequence; col5: colicin 5 coding sequence; col10: colicin 10 coding sequence; colIa: colicin Ia coding sequence; colIb: colicin Ib coding sequence; coIM: colicin M coding sequence; N: 3′-non-translated region from cr-TMV; T: Agrobacterium nopaline synthase terminator; white segments interrupting grey segments in the RdRp and MP ORFs indicate introns inserted into these ORFs for increasing the likelihood of RNA replicon formation in the cytoplasm of plant cells, which is described in detail in WO2005049839.
ScolE1c: salmocin ScolE1c coding sequence; ScolE1d: salmocin ScolE1d coding sequence; ScolE1e: salmocin ScolE1e coding sequence; ScolMa: salmocin ScolMa coding sequence.
L—PageRuler™ Prestainded Protein Ladder (Thermo Fisher Scientific Inc. (Waltham, USA), #SM0671); 1—the extract obtained with the extraction buffer of pH 7.0; 2—the extract obtained with the extraction buffer of pH 5.0; 3—the extract obtained with the extraction buffer of pH 4.0.
The proteins of the invention are proteins that have a cytotoxic effect on Salmonella and are referred to herein as “salmocins”. The salmocins generally have at least a binding domain (also referred to as “receptor binding domain”) that allows binding of the salmocin to a surface receptor structure of cells of the target Salmonella. Salmocins further have a cytotoxic domain that may be a catalytic or a pore-forming domain. The catalytic domain may have an RNase or DNase catalytic activity, an inhibitory activity against cell wall peptidoglycan (murein) biosynthesis, or may degrade cell wall structures of Salmonella. Further, the salmocins may have a translocation domain that may interact with membrane proteins of cells of the target Salmonella so that the salmocin is translocated to a compartment where the salmocin exerts its cytotoxic function.
The inventors assume that the M-type salmocins (ScolM or SalM) of the invention are peptidoglycanases that specifically cleave the bond between the lipid moiety and the pyrophosphoryl group of the peptidoglycan lipid I and lipid II intermediates, located at the periplasmic side of the inner membrane (by analogy with Gross and Braun, Mol. Gen. Genet. 251 (1996) 388-396; Barreteau et al., Microbial Drug Resistance 18 (2012), 222-229). The released C55-polyisoprenol no longer translocates MurNAc-pentapeptide-GlcNAc across the cytoplasmic membrane. The ScolM salmocins kill sensitive Salmonella strains after it has been taken up across the outer membrane into the periplasm. The mode of action of ScolM is believed to involve the steps of adsorption to the FhuA outer membrane receptor, energy-dependent translocation through the outer cell membrane into the periplasm by the TonB import machinery (TonB, ExbB and ExbD), and catalytic action of its substrate. Each of these steps is performed by a specific protein domain. Accordingly, also the ScolM salmocins share a three-domain structural organization and a narrow antibacterial spectrum in that the antibacterial activity against bacteria other than Salmonella is limited.
For the specificity to Salmonella, the binding domain is of importance and (inter alia) distinguishes the salmocins from otherwise similar colicins. Thus, the protein of the invention may be defined by having at least a binding domain that comprises or consists of or is contained in any one of the amino acid sequence segments as defined in item (1) above or in claim 1. Items (a-i) to (a-v) of item (1) or claim 1 define binding domains of the salmocins ScolE2, ScolE3, ScolE7, ScolE1a, and ScolE1b, respectively. Items (a-vii) to (a-x) and (a-xi) and (a-xii) of item (1) above define binding domains of the salmocins ScolE1c, ScolE1d, ScolE1e, ScolMa, ScolMb, and ScolMc, respectively. The binding domain of Spst is contained in the amino acid sequence segment defined in item (a-vi) of item (1) above. The amino acid sequences of salmocins ScolE2, ScolE3, ScolE7, ScolE1a, ScolE1b, and Spst are given as SEQ ID NO: 1 to 6, respectively. The amino acid sequence of the salmocins ScolE1c, ScolE1d, ScolE1e, ScolMa, ScolMb, and ScolMc are given as SEQ ID NO: 25 to 28, 33, and 34, respectively. Items (b) to (d) of item (1) above define derivatives of ScolE2, ScolE3, ScolE7, ScolE1a, ScolE1b, Spst, ScolE1c, ScolE1d, ScolE1e, ScolMa, ScolMb, and ScolMc, having or containing derivative binding domains (or amino acid sequence segments). Analogously, items (B) to (E) and items (a) to (8) (defined below) define derivatives of ScolE2, ScolE3, ScolE7, ScolE1a, ScolE1b, Spst, ScolE1c, ScolE1d, ScolE1e, ScolMa, ScolMb, and ScolMc. Derivatives of ScolE2, ScolE3, ScolE7, ScolE1a, ScolE1b, Spst, ScolE1c, ScolE1d, ScolE1e, ScolMa, ScolMb, and ScolMc are preferably capable of exerting a cytotoxic effect on Salmonella.
In the present invention, salmocins ScolE1c, ScolE1d, ScolE1e, ScolMa, ScolMb, and ScolMc (and their derivatives as defined) are preferred; ScolMa, ScolMb, and ScolMc (and their derivatives as defined herein) are more preferred. ScolMb and in particular ScolMc (and their derivatives as defined herein) are most preferred due to their exceptional ease of purification after expression and their antimicrobial activity. ScolMc (and its derivatives as defined herein) is also preferred due to its exceptionally high antimicrobial activity (see e.g. Example 23). ScolE1a and ScolE1b as well as ScolE1D and derivatives thereof as defined herein are preferred for use in combination with a ScolM salmocin (such as ScolMa, ScolMb, and ScolMc) and their derivatives as defined herein.
Herein, an amino acid sequence segment (or, briefly, segment) refers to a plurality of contiguous amino acid residues of a protein or polypeptide having a larger number of amino acid residues than the segment. Domains are also referred to herein as “amino acid sequence segments” or briefly “segments”. The terms “protein” and “polypeptide” are used interchangeabley herein.
The protein of the invention comprises at least a binding domain. The following items (i) to (v), (vii) to (x), (xi), and (xii) of each of items (b) to (d) define preferred binding domains. Most preferred binding domains are those of items (a-i) to (a-v), (a-vii) to (a-x), (a-xi), and (a-xii). Items (vi) of each of the following items (b) to (d), i.e. sub-items (b-vi), (c-vi) and (d-vi), define preferred amino acid sequence segments that contain a binding domain and are derivatives of salmocin Spst. The protein of the invention preferably comprises any one of the following amino acid sequence segments:
Preferably, alternatively or additionally, the protein is as defined in any one of (b-x), (c-x), (d-x), (b-xi), (c-xi), (d-xi), (b-xii), (c-xii), (d-xii), and the amino acid residue corresponding to residue 155 of SEQ ID NO: 33 is Pro and/or the amino acid residue corresponding to residue 246 of SEQ ID NO: 33 is Arg or Lys, preferably Arg. Alternatively or additionally, the amino acid residues corresponding to residues 76 and 84 of SEQ ID NO: 33 may be Gln.
Herein, the wording “the amino acid residue corresponding to residue xx . . . of SEQ ID NO: yy” means that the amino acid sequence of said protein has, at the position corresponding to residue xx of SEQ ID NO: yy, the indicated amino acid residue. Here, xx stands for the number (from the N-terminus) of an amino acid residue in the amino acid sequence of the protein, and yy stands for the indicated SEQ ID NO:.
Corresponding amino acid residues may be determined by aligning the protein with the amino acid sequence of SEQ ID NO:33 to give the best alignment (as done and shown e.g.
Where a protein is defined herein by a number or number range of amino acid substitutions, additions, insertions or deletions, amino acid substitutions, additions, insertions or deletions may be combined, but the given number or number range refers to the sum of all amino acid substitutions, additions, insertions and deletions. Among amino acid substitutions, additions, insertions and deletions, amino acid substitutions, additions, and deletions are preferred. The term “insertion” relates to insertions within the amino acid sequence of a reference sequence, i.e. excluding additions at the C- or N-terminal end. The term additions means additions at the C- or N-terminal end of the amino acid sequence of a reference sequence. A deletion may be a deletion of a terminal or an internal amino acid residue of a reference sequence. Herein, where the protein or any domain thereof is defined by a number or number range of amino acid substitutions, additions, insertions or deletions relative to an indicated amino acid sequence of segment, in a further embodiment, the protein or domain may have from 1 to several amino acid substitutions, additions, insertions or deletions relative to the indicated amino acid sequence of segment.
The cytotoxic or catalytic domain of the protein of the invention may be as defined in item (3) above. In preferred embodiments, the protein of the invention comprises a cytotoxic or catalytic domain that comprises or consists of any one of the following amino acid sequence segments:
In more preferred embodiments, the protein of the invention comprises a cytotoxic or catalytic domain that comprises, or consists of, any one of the sequence segments (a-vii)′ to (a-x)′, (a-xi)′ or (a-xii)′.
The protein may be as defined in any one of (b-x)′, (c-x)′, (d-x)′, (b-xi)′, (c-xi)′, (d-xi)′, (b-xii)′, (c-xii)′, or (d-xii)′, and the amino acid residue of said protein corresponding to residue 155 of SEQ ID NO: 33 is Pro and/or the amino acid residue corresponding to residue 246 of SEQ ID NO: 33 is Arg or Lys, preferably Arg. Alternatively or additionally, the amino acid residues corresponding to residues 76 and 84 of SEQ ID NO: 33 may be Gln.
Herein, in any item (x-y)′ (wherein x stands for any one of a, b, c, or d, and y stands for any roman numeral i to xii), the prime ′ indicates catalytic domains or segments. Items (x-y) lacking the prime indicates binding domains or segments. Items (x-y)″ carrying the double prime ″ indicates translocation domains or segments. Among items (a) to (d), those of items (a), (b) and (d) are preferred and items (a) and (d) are more preferred. Similarly, among items (a)′ to (d)′, those of items (a)′, (b)′ and (d)′ are preferred and items (a)′ and (d)′ are more preferred. Similarly, among items (a)″ to (d)″, those of items (a)″, (b)″ and (d)″ are preferred and items (a)″ and (d)″ are more preferred:
Where the protein of the invention comprises a binding domain as defined herein and a catalytic domain as defined herein, any binding domain (or segment) as defined above may be combined with any catalytic domain (or segment). In a preferred embodiment, a binding domain of any sub-item from (i) to (x) is combined, in a protein of the invention, with a catalytic domain of sub-item (i)′ to (x)′, respectively (e.g. a binding domain of item (iii) is combined with a catalytic domain of item (iii)′), whereby the catalytic domain may be on the C-terminal side of the protein. In one embodiment, a binding domain of any item (a) to (d) is combined with a catalytic domain of item (a)′ to (d)′, respectively, whereby the catalytic domain may be on the C-terminal side of the protein.
In certain embodiments, the protein of the invention may be capable of exerting a cytotoxic effect on Salmonella, and the protein comprises at least any one of the following combinations of amino acid sequence segments, preferably in the given order from N-terminus to the C-terminus of the protein:
The protein comprises preferably at least any one of the combinations of amino acid sequence segments defined in subclasses (vii) to (xii), even more preferably (x), (xi), or (xii).
All these embodiments may be combined with the preferred values for minimum sequence identities or similarities or preferred numbers of amino acid substitutions, additions, insertions or deletions of the respective segments defined herein.
Item (4) above (of the Summary of the Invention) defines translocation domains and derivatives thereof of ScolE2, ScolE3, Scol E7, ScolE1a, ScolE1b, ScolE1e, ScolE1d, ScolE1e, ScolMa, ScolMb, and ScolMc. The definitions of the translocation domains and derivatives thereof may be combined with the definitions of the cytotoxic and binding domains or derivatives thereof. The definitions of the translocation domains and derivatives thereof may be combined with the definitions of the protein as inter alia defined below.
A protein of the invention may have a binding domain (or binding segment) according to any one of items (a-i) to (a-x), (a-xi) and (a-xii) or according to any of the derivatives of items (b-i) to (b-x), (b-xi) and (b-xii), (c-i) to (c-x), (c-xi) and (c-xii), or (d-i) to (d-x), (d-xi) and (d-xii). Preferably, a protein of the invention may have a binding domain (or binding segment) according to any one of items (a-vii) to (a-x), (a-xi) and (a-xii) or according to any of the derivatives of items (b-vii) to (b-x), (b-xi) and (b-xii), (c-vii) to (c-x), (c-xi) and (c-xii), or (d-vii) to (d-x), (d-xi) and (d-xii). Any such binding domain may be combined with a catalytic/cytotoxic domain according to any one items (a-i)′ to (a-x)′, (a-xi)′, (a-xii)′, (b-i)′ to (b-x)′, (b-xi)′, (b-xii)′, (c-i)′ to (c-x)′, (c-xi)′, (c-xii)′, or (d-i)′ to (d-x), (d-xi)′, or (d-xii)′. Preferably, any such binding domain may be combined with a catalytic/cytotoxic domain according to any one items (a-vii)′ to (a-x)′, (a-xi)′, (a-xii)′, (b-vii)′ to (b-x)′, (b-xi)′, (b-xii)′, (c-vii)′ to (c-x)′, (c-xi)′, (c-xii)′, or (d-i)′ to (d-x), (d-xi)′, (d-xii)′, whereby this preferred embodiment may preferably be combined with the preferred binding domains given above in this paragraph.
The domain structure of the salmocins allows establishing artificial salmocins wherein domains from different salmocins of the invention, or derivatives thereof as defined herein, are combined to form novel salmocins (chimeric salmocins). In such chimeric salmocins, the domain sequence of natural salmocins, from the N-terminus to the C-terminus, of a translocation domain (if present), a binding domain, and a catalytic or activity domain may or may not be maintained; preferably, it is maintained. Thus, the protein of the invention may comprise, from the N-terminus to the C-terminus, a binding domain of any one of items (a-i) to (a-x), (a-xi), and (a-xii) or according to any of the derivatives of items (b-i) to (b-x), (b-xi), (b-xii), (c-i) to (c-x), (c-xi), (c-xii), or (d-i) to (d-x), (d-xi), (d-xii), and a catalytic domain (segment) of any one of items (a-i)′ to (a-x)′, (a-xi)′, (a-xii)′, (b-i)′ to (b-x)′, (b-xi)′, (b-xii)′, (c-i)′ to (c-x)′, (c-xi)′, (c-xii)′ or (d-i)′ to (d-x)′, (d-xi)′, (d-xii)′. In a preferred embodiment, the protein of the invention may comprise, from the N-terminus to the C-terminus, a translocation domain of any one of items (a-i)″ to (a-ix)″, (b-i)″ to (b-ix)″, (c-i)″ to (c-ix)″ or (d-i)″ to (d-ix)″, a binding domain of any one of items (a-i) to (a-x), or according to any of the derivatives of items (b-i) to (b-x), (c-i) to (c-x) or (d-i) to (d-x), and a catalytic domain (segment) of any one of items (a-i)′ to (a-x)′, (b-i)′ to (b-x)′, (c-i)′ to (c-x)′ or (d-i)′ to (d-x)′.
Within the three cytotoxic activities of the salmocins nuclease, pore-forming and muramidase (Table 1), domains may be exchanged between salmocins of the same type of cytotoxic activity. For example, a new salmocin with RNase-type cytotoxicity may be formed from the translocation and binding domains of ScolE2 or ScolE7 (or derivatives of these domains) and the cytotoxic domain of ScolE3. Preferably, however, a binding domain of any one of sub-items (i) to (x) is combined with a catalytic domain of any one of sub-items (i)′ to (x)′, respectively, for increased similarity to natural salmocins, preferably each of any of items (a) to (d). More preferably, however, a binding domain of any one of sub-items (i) to (v) or (vii) to (x) may be combined with a catalytic domain of any one of sub-items (i)′ to (v)′ or (vii)′ to (x)′, respectively, and a translocation domain of any one of sub-items (i)″ to (v)″ or (vii)″ to (x)″, respectively, preferably of any of items (a) to (d), for increased similarity to natural salmocins.
In another embodiment, a binding domain of sub-items (i) to (v) is combined with a catalytic domain of sub-items (i)′ to (vi)′ (preferably (i)′ to (v)′), respectively, for increased similarity to natural salmocins, preferably each of any of items (a) to (d). In a further embodiment, a binding domain of any one of sub-items (i) to (v) is combined with a catalytic domain of any one of sub-items (i)′ to (v)′, respectively, and a translocation domain of any one of sub-items (i)″ to (v)″, respectively, preferably of any of items (a) to (d), for increased similarity to natural salmocins.
The invention also provides a protein that is preferably capable of exerting a cytotoxic effect on Salmonella, said protein comprising or consisting of the following amino acid sequences:
As is generally understood and for avoiding any doubt, the wording “protein comprising any one of the following amino acid sequences” means that the amino acid sequence of said protein may comprise additional amino acid residues or sequence stretches than those defined (e.g. a purification tag or other tag). The wording “protein consisting of any one of the following amino acid sequences” means that the amino acid sequence of said protein does not have additional amino acid residues than those defined. As above, among the above proteins (or polypeptides), those of subclasses identified by numerals (vii) to (xii) are preferred (in all classes (a) to (d)) and those of subclass (x), (xi) and (xii) are more preferred. This also applies to the preferred embodiments described below.
In another embodiment, the invention provides a protein that is preferably capable of exerting a cytotoxic effect on Salmonella, wherein the amino acid sequence of said protein is as defined in any one of items (A-i) to (A-x), (A-xi), (A-xii), (B-i) to (B-x), (B-xi), (B-xii), (C-i) to (C-x), (C-xi), (C-xii), (D-i) to (D-x), (D-xi), (D-xii), or (E-i) to (E-x), (E-xi), or (E-xii). In a preferred embodiment, the invention provides a protein that is preferably capable of exerting a cytotoxic effect on Salmonella, wherein the amino acid sequence of said protein is as defined in any one of items (A-vii) to (A-x), (A-xi), (A-xii), (B-vii) to (B-x), (B-xi), (B-xii), (C-vii) to (C-x), (C-xi), (C-xii), (D-vii) to (D-x), (D-xi), (D-xii), or (E-vii) to (E-x), (E-xi), or (E-xii).
The above definitions of the proteins with respect to the entire sequence of SEQ ID NOs 1 to 6, 25 to 28, 33 or 34 may be combined with the above definitions of the protein based on one or more particular domains such as a binding and/or catalytic or cytotoxic domains and/or translocation domain where available.
Herein, the determination of sequence identities and similarities is done using Align Sequences Protein BLAST (BLASTP 2.6.1+) (Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schäffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25:3389-3402.).
The derivatives of domains and/or protein of the invention as defined above in items (b) to (d), (b)′ to (d)′, (b)″ to (d)″, or items (B) to (D) or (E) may, notwithstanding the sequence varieties allowed by the embodiments defined above, preserve amino acid residues as defined in the following. In preferred embodiments, the amino acid residue(s) corresponding to residue 125 of SEQ ID NO: 4 is Asn or Ser;
residue 181 of SEQ ID NO: 4 is Glu or Asp, preferably Glu;
residue 184 of SEQ ID NO: 4 is Arg or Gln, preferably Arg;
residue 192 of SEQ ID NO: 4 is Ala or Thr;
residue 195 of SEQ ID NO: 4 is Ala or Val;
residue 196 of SEQ ID NO: 4 is Glu or Gln, preferably Glu;
residue 198 of SEQ ID NO: 4 is Ala or Thr;
residue 209 of SEQ ID NO: 4 is Leu or Ile, preferably Leu;
residue 273 of SEQ ID NO: 4 is Leu or Ile;
residue 280 of SEQ ID NO: 4 Arg;
residue 283 of SEQ ID NO: 4 Lys;
residue 286 of SEQ ID NO: 4 Gln or Lys;
residue 290 of SEQ ID NO: 4 Ala, or Thr;
residue 299 of SEQ ID NO: 4 Asp, Asn or Glu;
residue 301 of SEQ ID NO: 4 Leu;
residue 302 of SEQ ID NO: 4 Asn or Asp;
residue 346 of SEQ ID NO: 4 Asn, Asp, or Glu;
residue 363 of SEQ ID NO: 4 Lys, Asn or Arg;
residue 364 of SEQ ID NO: 4 Lys or Gln.
The wording “amino acid residue(s) corresponding to the amino acid residue . . . ” refers to the alignments shown in
In derivatives of ScolE1a and ScolE1b, and/or in derivative domains of ScolE1a and ScolE1b, corresponding amino acid residues that are the same in the alignment of ScolE1a and ScolE1b of
In derivatives of ScolE2 and ScolE7, and/or in derivative domains of ScolE2 and ScolE7, corresponding amino acid residues that are the same in the alignment of ScolE2 and ScolE7 of
A salmocin according to the invention may comprise an additional N- or C-terminal amino acid sequence stretch such as purification tags, e.g. as a His-tag of 6 or more contiguous histidine residues; the derivative has, preferably, no N-terminal amino acid residue addition.
The protein (salmocin) of the invention is preferably capable of exerting a cytotoxic effect on Salmonella, notably of Salmonella enterica and more preferably Salmonella enterica ssp. enterica. Whether this condition is fulfilled can be tested experimentally using a radial diffusion assays via spot-on-lawn-method. The cytotoxicity of a protein to be tested against Salmonella enterica is such that it and the protein of SEQ ID NO: 1 produce spots free of viable bacteria of Salmonella enterica ssp. enterica serovar Newport strain ATCC® 6962™* of the same diameter 12 hours after spotting 5 microliters of a solution of said protein to be tested and the protein of SEQ ID NO: 1 onto a softagar overlay plate seeded with 0.14 mL bacterial solution of 1×107 cfu/mL per cm2 of the sensitive Salmonella enterica strain and subsequent incubation of the agar plate at 37° C., wherein the concentration of the protein to be tested is at most 5 times that of the comparative solution of the protein SEQ ID NO: 1. In a preferred embodiment, the point of reference is not the protein of SEQ ID NO: 1, but the protein of SEQ ID NO: 4 or 5 under otherwise identical conditions.
The composition of the invention comprises a protein (salmocin) as described above and optionally further components as the case requires such as a carrier. The composition preferably comprises ScolE1a and/or ScolE1b or a derivative thereof as described above and optionally further components as the case requires such as a carrier. The composition may comprise one or more different proteins (salmocins) as defined herein, such as two, three or four different proteins (salmocins) as defined herein. “Different” means that the proteins differ in at least one amino acid residue. The composition may comprise two, three or more salmocins from the same subclass represented by any one of items (i) to (x), (xi) or (xii) above or, preferably, from different classes represented by any one of items (i) to (x) above. The composition may further comprise one or more E. coli colicin or a derivative thereof e.g. as described in EP 3 097 783 A1, e.g. for concomitantly controlling pathogenic E. coli such as EHEC.
As the protein of the invention is preferably produced by expression in plants or cells thereof, the composition may be a plant material or extract thereof, wherein the plant material is a material from a plant having expressed the protein, preferably Nicotiana or an edible plant having expressed said protein. An extract of plant material is an aqueous solution containing water-soluble proteins including a salmocin of the invention that is present or expressed in said plant material, or a dried product of such aqueous solution. The extract preferably has water-insoluble components of the plant material removed e.g. by filtration or centrifugation. The plant material may be a material from a plant selected from the group consisting of spinach, chard, beetroot, carrot, sugar beet, leafy beet, amaranth, Nicotiana, and/or said plant material is one or more leaves, roots, tubers, or seeds, or a crushed, milled or comminuted product of said leaves, roots, tubers, or seeds.
The composition or said extract from a plant material may be a solid or liquid composition, such as a solution or a dispersion, containing said salmocin(s). The liquid composition may be aqueous, such as an aqueous solution. The concentration of said protein in said aqueous dispersion or solution may be from 0.0001 to 1 mg/ml, preferably from 0.001 to 0.1 mg/ml, more preferably from 0.005 to 0.05 mg/ml. If more than one salmocin capable of exerting a cytotoxic effect on Salmonella is employed, these concentrations relate to the total concentration of all such salmocins.
The aqueous solution may, apart from the one or more salmocin, contain a buffer. The buffer may be an inorganic or organic acid or salts thereof. An example of an inorganic acid is phosphoric acid or salts thereof. Examples of the organic acid are HEPES, acetic acid, succinic acid, tartaric acid, malic acid, benzoic acid, cinnamic acid, glycolic acid, lactic acid, citric acid, and ascorbic acid. Preferred organic acids are malic acid, lactic acid, citric acid, and ascorbic acid. The pH of the solution may generally be from 4 to 8, preferably from 5 to 8, more preferably from 6.0 to 7.5. If the object to which the composition is applied is meat, the pH of the solution may generally be from 4 to 8, preferably from 4.5 to 7, more preferably from 5.0 to 6.5, and even more preferably from 5.0 to 6.0. Further, the solution may contain isotonic agents such as glycerol or a salt. A preferred salt to be used is sodium chloride. The aqueous solution containing the one or more salmocin may be a buffered aqueous solution that may contain further solutes e.g. salts such as from 50 to 400 mM NaCl, preferably from 100 to 200 mM NaCl. The aqueous solution may further contain a sulfhydryl compound such as dithiothreitol (DTT), dithioerythritol, thioethanol or glutathione, preferably DTT. The concentration of the total of sulfhydryl compounds in the aqueous solution may be from 1 to 50 mM, preferably from 2 to 20 mM and more preferably from 4 to 10 mM.
If the composition of the invention is a solid composition, it may be a powder such as a lyophilized solid composition obtained by lyophilization of the extract or solution mentioned above. The powder may contain additional solid components such as those mentioned above for the aqueous solution. Before use, it may be reconstituted with a suitable liquid, such as water or buffer. The solid composition may contain buffer, salts or other components as mentioned above, such that the concentrations given above may be achieved upon reconstitution or dissolution of the solid composition.
Examples of carriers of the composition are solvents such as water or an aqueous buffer (as described above), salts, sugars such as monosaccharides and disaccharides, sugar alcohols, and other carriers such as those known from pharmaceutical compositions. Examples of the latter are starch, cellulose and other proteins such as albumin. Examples of sugars are glucose, fructose, lactose, sucrose, and maltose.
The composition of the invention may contain at least 10, preferably at least 20, more preferably at least 30, even more preferably at least 50, even more preferably at least 75% by weight of one or more salmocins of the invention based on the total weight of protein in the composition. The content of salmocin(s) in the composition may be determined by subjecting the composition to SDS-PAGE and analyzing the obtained gel, after staining, by determining the intensity of bands on the gel. Thereby, intensity of bands due to salmocins can be determined in relation to the sum of intensities of bands due to all proteins in the composition. The total protein content in the composition may be determined using the well-known Bradford protein assay.
In one embodiment, the composition of the invention is a pharmaceutical composition. The pharmaceutical composition may, apart from one or more salmocin(s) of the invention, optionally contain an E. coli colicin, and/or one or more suitable pharmaceutically acceptable excipients.
The invention provides a method of preventing or reducing infection or contamination of an object with Salmonella, comprising contacting said object with one or more proteins (salmocins) as described above or a composition as described above. The object may be a surface of any non-organic object or an organic object such as food. Contamination of an object with Salmonella means adhesion of viable Salmonella cells to the object. Reducing contamination with Salmonella means reducing the number of viable Salmonella cells adhering to the object. Determining contamination of objects with Salmonella is part of the general knowledge. For example, dilution plating of solutions or dispersions of homogenized food as done in the Examples or dilution plating of a rinsing solution of other objects may be used, followed by counting bacterial colonies. Preferably, the object is food or animal feed. The food may be meat such as whole poultry carcasses, raw meat, cooked meat, and minced meat, eggs such raw eggs, whole eggs, peeled cooked eggs, scrambled eggs, fried eggs, raw fruit, or raw or cooked vegetable.
For treating or contacting the object with the protein or composition, a solution of the protein or a liquid composition as described above is generally contacted with the object. For example, said object is sprayed with an aqueous solution or is immersed into the aqueous solution as a composition of the invention. The object may be immersed for at least 10 seconds, preferably for at least 1 minute, preferably for at least 5 minutes into the aqueous solution. Contacting the object with a liquid composition helps to distribute the composition over the surface of the object. Where sufficiently even distribution can be achieved, it is possible to contact the object with a solid composition according to the invention, e.g. upon mincing meat.
The invention also provides a method of treating infection with Salmonella of a subject in need thereof, comprising administering to said subject one or more proteins (salmocins) as described above or a composition as described above. The subject may be a human being or a mammal such as a farm animal. Examples of farm animals are poultry and cattle. Generally, a liquid or solid pharmaceutical composition containing the salmocin(s) and optionally further components as described above is prepared for administration to the animal or human. Liquid compositions may be aqueous solutions as described above. Solid compositions may be powder containing the at least one salmocin(s) e.g. in freeze-dried form, or tablets obtained from such powder or capsules filled with such powder. Administration may be oral. In this case, the pharmaceutical preparation is one that allows passage through the stomach without being attacked by the acid medium in the stomach. The salmocin(s) should then be released from the pharmaceutical preparation in the intestine. Such pharmaceutical preparations are known in the art. Examples are tablets and capsules resistant to the acid medium in the stomach. It is further possible to administer orally a biological material such as E. coli or plant material containing expressed salmocin(s) to a patient. The salmocin(s) may be administered to a human adult in amounts of 1 mg to 1000 mg per day, preferably of from 10 mg to 250 mg per day to a human patient. Such amounts may also be administered to an animal. In a probiotic approach, a patient may be treated by administering to the patient a genetically-modified microorganism expressing the at least one salmocin(s). The genetically-modified microorganism may be a genetically-modified non-pathogenic E. coli or a lactic acid-producing microorganism as commonly employed in fermentation of milk products. Examples of lactic acid-producing microorganism are bacteria from the genera Lactobacillus such as Lactobacillus lactis and Bifidobacterium such as Bifidobacterium bifidum or Bifidobacterium breve. Another route of administration is by injection into the blood stream of a patient for preventing infection with Salmonella. For this purpose, the salmocin(s) may be dissolved in a physiological saline and the solution be sterilized.
In the methods described above, the Salmonella is Salmonella enterica, preferably Salmonella enterica ssp. enterica.
Salmocins ScolE1a and ScolE1b have a particularly wide activity against many different serovars of Salmonella, notably of Salmonella enterica, preferably of Salmonella enterica ssp. enterica, as demonstrated in the Examples below. Therefore, ScolE1a and ScolE1b, or derivatives thereof, are preferably used for treating infection or for preventing or reducing contamination with any Salmonella enterica, preferably any Salmonella enterica ssp. enterica. Salmocins E2, E3, E7 and Spst also have a wide activity against target Salmonella. However, ScolE2 and derivatives thereof may be preferably used against strains 1, 3, 4, 15, 20, 22 to 30 as defined in Tables 5A and 5B. ScolE3 and derivatives thereof may be preferably used against strains 1, 3, 4, 17, and 20 to 25 as defined in Tables 5A and 5B. ScolE7 and derivatives thereof may be preferably used against strains 1, 3, 4, 5, 15, 20, 22 to 30 and 32 as defined in Tables 5A and 5B.
A salmocin according to the invention may be produced by known methods of protein expression in a standard expression system. For producing the salmocin, a nucleotide sequence encoding it may be expressed in a suitable host organism. Methods usable for producing and purifying a protein of interest have been described in the prior art and any such methods may be used. An E. coli expression system as generally known in the art may, for example, be used. If a eukaryotic expression system is used, one or more introns may be inserted in the coding sequence of the salmocin to prevent toxicity on the bacterial organism used for cloning.
Particularly efficient expression methods are plant expression systems that are also known in the prior art. Plant expression systems usable for expressing a salmocin according to the invention are described in the Examples. A possible way of achieving expression of a nucleotide sequence of interest in plants is the use of self-replicating (viral) replicons containing the nucleotide sequence encoding the salmocin. The coding sequence of the salmocin may be codon optimized for expression in plants or in the particular plant used as expression host. Plant viral expression systems have been described in many publications, such as in WO2012019660, WO2008028661, WO2006003018, WO2005071090, WO2005049839, WO2006012906, WO02101006, WO2007137788 or WO02068664 and many more publications are cited in these documents. Various methods for introducing a nucleic acid molecule, such as a DNA molecule, into a plant or plant part for transient expression are known. Agrobacteria may be used for transfecting plants with the nucleic acid molecule (vector) or nucleic acid construct e.g. by agroinfiltration or spraying with agrobacterial suspensions. For references, see WO 2012019660, WO 2014187571, or WO 2013149726.
In embodiments wherein strong expression of a salmocin as a protein of interest is desired, a nucleic acid construct containing a nucleotide sequence encoding the salmocin may encode a viral vector that can replicate in plant cells to form replicons of the viral vector. In order to be replicating, the viral vector and the replicons may contain an origin of replication that can be recognized by a nucleic acid polymerase present in plant cells, such as by the viral polymerase expressed from the replicon. In case of RNA viral vectors (referred to as “RNA replicons”), the replicons may be formed by transcription under the control of a promoter active in plant cells, from the DNA construct after the latter has been introduced into plant cell nuclei. In case of DNA replicons, the replicons may be formed by recombination between two recombination sites flanking the sequence encoding the viral replicon in the DNA construct, e.g. as described in WO00/17365 and WO 99/22003. If the replicon is encoded by the DNA construct, RNA replicons are preferred. Use of DNA and RNA viral vectors (DNA or RNA replicons) has been extensively described in the literature over the years. Some examples are the following patent publications: WO2008028661, WO2007137788, WO 2006003018, WO2005071090, WO2005049839, WO02097080, WO02088369, WO02068664. Examples of DNA viral vectors are those based on geminiviruses. For the present invention, viral vectors or replicons based on plant RNA viruses, notably those based on plus-sense single-stranded RNA viruses may be preferably used. Accordingly, the viral replicon may be a plus-sense single-stranded RNA replicon. Examples of such viral vectors are those based on tobacco mosaic virus (TMV) and potexvirus X (PVX). “Based on” means that the viral vector uses the replication system such as the replicase and/or other proteins involved in replication of these viruses. Potexvirus-based viral vectors and expression systems are described in EP2061890 or WO2008/028661.
The salmocin may be expressed in a multi-cellular plant or a part thereof, notably a higher plant or parts thereof. Both monocot and dicot (crop) plants can be used. Common plants usable for expressing the protein of interest include Nicotiana benthamiana, Nicotiana tabacum, spinach, Brassica campestris, B. juncea, beets (Beta vulgaris), cress, arugula, mustard, strawberry, Chenopodium capitatum, lettuce, sunflower, cucumber, chinese cabbage, cabbage, carrot, green onion, onion, radish, lettuce, field peas, cauliflower, broccoli, burdock, turnip, tomato, eggplant, squash, watermelon, prince melon, and melon. Preferred plants are spinach, chard, beetroot, carrot, sugar beet, Nicotiana tabacum, and Nicotiana benthamiana. Expression in edible plants may be used for preventing contamination of the plants or food made therefrom with Salmonella. In one embodiment, plants are used that do not normally enter the human or animal food chain such as Nicotiana species such as N. tabacum and N. benthamiana.
Generally, the salmocin as a protein of interest is expressed in the cytosol of cells of the plants or plant parts. In this case, no signal peptide directing the protein of interest into a particular compartment is added to the protein. Alternatively, the protein of interest can be expressed in or targeted into chloroplasts of the plants; in the latter case, an N-terminal pre-sequence, generally referred to as plastid transit peptide or chloroplast targeting peptide, is added to the N-terminal or C-terminal end, preferably the N-terminal end, of the salmocin as the protein of interest.
The salmocin may be co-expressed together with an immunity protein as described in the experimental section, notably if the salmocin has nuclease activity, for preventing toxicity on plant tissue. Suitable immunity proteins that may be co-expressed are those given in Table 2 below.
In the process of producing a composition comprising at least one salmocin, a salmocin is, in the first step, expressed in a plant or cells of a plant, such as an edible plant. In the next step, plant material containing expressed salmocin from a plant having expressed the salmocin is harvested. Plant material may e.g. be leaves, roots, tubers, or seeds, or a crushed, milled or comminuted product of leaves, roots, tubers, or seeds. In step (iii), the salmocin is extracted from the plant material using an aqueous buffer. This may include that the plant material is homogenized and insoluble material may be removed by centrifugation or filtration. Soluble components including the salmocin will be extracted into the aqueous buffer to produce a salmocin solution in the aqueous buffer. The aqueous buffer may contain an inorganic or organic acid or salts thereof and may have a pH as defined above for the aqueous solution as a composition of the invention. Further, the aqueous buffer may contain salt and/or a sulfhydryl compound as also described above for the aqueous solution as a composition of the invention. If a relatively pure salmocin composition is desired, the salmocin solution in the aqueous buffer may be further purified by removing undesired components in step (iv) according to known methods of protein purification.
Accordingly, the invention provides a process of producing a composition comprising a protein according to the invention, said process comprising the following steps:
If a salmocin is expressed in plants, the plants or tissue thereof having expressed protein is harvested, the tissue may be homogenized and insoluble material may be removed by centrifugation or filtration. If relatively pure salmocin is desired, the salmocin may be further purified by generally known method of protein purification such as by chromatographic methods which can remove other host-cell proteins and plant metabolites such as alkaloids and polyphenols. Purified salmocin solutions may be concentrated and/or freeze-dried.
If salmocins are expressed in edible plants, crude protein extracts from the edible plants or semi-purified concentrates may be used for preventing or reducing contamination of an object such as food with Salmonella.
A protein the amino acid sequence of which comprises or consists of any one of the amino acid sequences (A-x), (B-x), (C-x), (D-x), or (E-x).
A protein the amino acid sequence of which comprises or consists of any one of the amino acid sequences (A-xi), (B-xi), (C-xi), (D-xi), or (E-xi).
A protein the amino acid sequence of which comprises or consists of the amino acid sequences (A-xii), (B-xii), (C-xii), (D-xii), or (E-xii).
A protein according to any one of the previous three preferred embodiments (sentences), wherein the protein is as defined in any one of (B-x), (C-x), (D-x), (E-x), (B-xi), (C-xi), (D-xi), (E-xi), (B-xii), (C-xii), (D-xii), or (E-xii), preferably as defined in any one of (B-xi), (C-xi), (D-xi), (E-xi), (B-xii), (C-xii), (D-xii), or (E-xii), and
A protein (ScolE1d of SEQ ID NO:26 or its derivative) the amino acid sequence of which comprises or consists of any one of the amino acid sequences (A-viii), (B-viii), (C-viii), (D-viii), or (E-viii), optionally as further defined above regarding amino acid residues corresponding to SEQ ID NO: 4.
A composition comprising a protein the amino acid sequence of which comprises or consists of any one of the amino acid sequences (A-x), (B-x), (C-x), (D-x), or (E-x), optionally as further defined above with regard to specific amino acid residues, and a protein the amino acid sequence of which comprises or consists of any one of the amino acid sequences (A-v), (B-v), (C-v), (D-v), or (E-v);
A composition comprising a protein the amino acid sequence of which comprises or consists of any one of the amino acid sequences (A-xi), (B-xi), (C-xi), (D-xi), or (E-xi), optionally as further defined above with regard to specific amino acid residues, and a protein the amino acid sequence of which comprises or consists of any one of the amino acid sequences (A-v), (B-v), (C-v), (D-v), or (E-v);
A composition comprising a protein the amino acid sequence of which comprises or consists of any one of the amino acid sequences (A-xii), (B-xii), (C-xii), (D-xii), or (E-xii), optionally as further defined above with regard to specific amino acid residues, and a protein the amino acid sequence of which comprises or consists of any one of the amino acid sequences (A-v), (B-v), (C-v), (D-v), or (E-v);
A composition comprising a protein the amino acid sequence of which comprises or consists of any one of the amino acid sequences (A-x), (B-x), (C-x), (D-x), or (E-x), optionally as further defined above with regard to specific amino acid residues, and a protein the amino acid sequence of which comprises or consists of any one of the amino acid sequences (A-iv), (B-iv), (C-iv), (D-iv), or (E-iv);
A composition comprising a protein the amino acid sequence of which comprises or consists of any one of the amino acid sequences (A-xi), (B-xi), (C-xi), (D-xi), or (E-xi), optionally as further defined above with regard to specific amino acid residues, and a protein the amino acid sequence of which comprises or consists of any one of the amino acid sequences (A-iv), (B-iv), (C-iv), (D-iv), or (E-iv);
A composition comprising a protein the amino acid sequence of which comprises or consists of any one of the amino acid sequences (A-xii), (B-xii), (C-xii), (D-xii), or (E-xii), optionally as further defined above with regard to specific amino acid residues, and a protein the amino acid sequence of which comprises or consists of any one of the amino acid sequences (A-iv), (B-iv), (C-iv), (D-iv), or (E-iv).
The protein or composition of any one of the preceding sentences for use in a method of treating infection of a subject with Salmonella.
A method of preventing or reducing infection or contamination of an object with Salmonella, comprising contacting said object with a protein as defined in any one of the preceding sentences or with a composition as defined in any one of the preceding sentences.
A method of preventing or reducing infection or contamination of an object with Salmonella, comprising contacting said object with a protein comprising or consisting of any one of the amino acid sequences (A-x), (B-x), (C-x), (D-x), or (E-x) (optionally as further defined above with regard to specific amino acid residues), or contacting said object with a composition comprising a protein the amino acid sequence of which comprises or consists of any one of the amino acid sequences (A-x), (B-x), (C-x), (D-x), or (E-x) (optionally as further defined above with regard to specific amino acid residues) and a protein the amino acid sequence of which comprises or consists of any one of the amino acid sequences (A-v), (B-v), (C-v), (D-v), or (E-v).
A method of preventing or reducing infection or contamination of an object with Salmonella, comprising contacting said object with a protein the amino acid sequence of which comprises or consists of any one of the amino acid sequences (A-xi), (B-xi), (C-xi), (D-xi), or (E-xi) (optionally as further defined above with regard to specific amino acid residues), or contacting said object with a composition comprising a protein the amino acid sequence of which comprises or consists of any one of the amino acid sequences according to (A-xi), (B-xi), (C-xi), (D-xi), or (E-xi) (optionally as further defined above with regard to specific amino acid residues) and a protein the amino acid sequence of which comprises or consists of any one of the amino acid sequences (A-v), (B-v), (C-v), (D-v), or (E-v).
A method of preventing or reducing infection or contamination of an object with Salmonella, comprising contacting said object with a protein the amino acid sequence of which comprises or consists of any one of the amino acid sequences (A-xii), (B-xii), (C-xii), (D-xii), or (E-xii) (optionally as further defined above with regard to specific amino acid residues), or contacting said object with a composition comprising a protein the amino acid sequence of which comprises or consists of any one of the amino acid sequences (A-xii), (B-xii), (C-xii), (D-xii), or (E-xii) (optionally as further defined above with regard to specific amino acid residues) and a protein the amino acid sequence of which comprises or consists of any one of the amino acid sequences (A-v), (B-v), (C-v), (D-v), or (E-v).
Methods of treating infection with Salmonella of a subject in need thereof, comprising administering to said subject a protein as defined in any one of the preceding sentences or a composition as defined in any one of the preceding sentences.
These preferred embodiments can be combined with other embodiments or preferred embodiments described herein.
Six salmocins representing four activity groups were selected (Table 1).
The list comprises salmocins ScolE2, ScolE3, ScolE7, ScolE1a, ScolE1b and Spst. Respective amino acid sequences were retrieved from GenBank; corresponding nucleotide sequences with codon usage optimized for Nicotiana benthamiana were synthesized by Thermo Fisher Scientific Inc. In case of salmocins ScolE2, ScolE3 and ScolE7, the coding sequence was interrupted by insertion of the cat 1 intron (the first intron from Ricinus communis cat1 gene for catalase CAT1 (GenBank: D21161.1, nucleotide positions between 679 and 867)) to prevent the cytotoxicity in Escherichia coli cells used for cloning. Salmocin coding sequences were inserted into TMV-based assembled viral vector pNMD035 (described in detail in WO2012/019660) resulting in plasmid constructs depicted in
In preliminary expression studies, it was found that bacteriocins with nuclease (RNase and DNase) activities are usually highly toxic for plant tissues where they are expressed. Their expression resulted in tissue necrosis and poor accumulation of recombinant protein. However, co-expression with appropriate immunity proteins reduced the toxic effect and increased the accumulation of these bacteriocins dramatically. Salmocin immunity proteins used in our studies are listed in the Table 2.
Immunity proteins SImmE2 and SImmE7 for salmocins ScolE2 and ScolE7, respectively. Amino acid sequences of immunity proteins were retrieved from GenBank; corresponding nucleotide sequences with codon usage optimized for Nicotiana benthamiana were synthesized by Thermo Fisher Scientific Inc. and subcloned into PVX-based assembled viral vector pNMD670 as described in WO2012/019660. Resulting plasmid constructs are shown in
6 week-old Nicotiana benthamiana plants were infiltrated using needleless syringe with diluted Agrobacterium tumefaciens cultures carrying TMV-based assembled vectors for cytosolic salmocin expression. In case of salmocins ScolE2 and ScolE7, Agrobacterium cultures carrying TMV-based vector for salmocin expression were mixed in equal proportions with other cultures carrying PVX-based vectors for the expression of the corresponding immunity proteins. Individual overnight cultures were adjusted to OD600=1.5 and further diluted 1:100 with infiltration buffer containing 10 mM MES, pH 5.5 and 10 mM MgSO4. Plasmid constructs used in this experiment are summarized in Table 3. For determination of optimal harvesting timepoint, plant material was harvested at several timepoints post infiltration and used for protein extraction with 5 volumes of buffer containing 50 mM HEPES (pH 7.0), 10 mM potassium acetate, 5 mM magnesium acetate, 10% (v/v) glycerol, 0.05% (v/v) Tween-20 and 300 mM NaCl. Total soluble protein (TSP) concentration was determined using the Bradford assay, and TSP extracts were analyzed using SDS-PAGE with Coomasssie staining. In our experiment, all tested salmocins were expressed on reasonably high levels varying between 1.2 and 1.8 mg recombinant colicin/g FW or between 18 and 47% of TSP (Table 4) as determined by comparison with bovine serum albumin (BSA) protein.
Nicotiana benthamiana plants.
We analyzed the antimicrobial activity of plant-made recombinant salmocins against 36 strains of 33 different serotypes of S. enterica ssp. enterica. Details of strains used in the experiments are given in Tables 5A and 5B.
Salmonella enterica ssp. enterica strains used for antimicrobial activity screen.
Enteritidis
Enteritidis
Typhimurium
Typhimurium
Newport
Javiana
Javiana
Montevideo
Infantis
Muenchen
Heidelberg
Bareilly
Thompson
Saintpaul
Oranienburg
Mississippi
Anatum
Agona
Salmonella enterica ssp. enterica strains used for antimicrobial activity screen.
Berta
Dublin
Derby
Cerro
Senftenberg
Kentucky
Mbandaka
Cholerasius
Tallahassee
Paratyphi A
Abony
Pullorum
Vellore
Bispebjerg
Poona
Gallinarum
Gallinarum
Braenderup
Antimicrobial activity of recombinant salmocin-containing plant extracts was tested in radial diffusion assays via spot-on-lawn-method. For this purpose, we prepared agar plates overlaid with soft agar containing cells of tested Salmonella strains. 10×10 cm quadratic petri dishes were poured with 15-20 ml LB agar medium (1.5% w/v agar). LB soft agar medium (0.8% (w/v) agar) was melted, 20 ml aliquots were transferred into 50 ml plastic tubes and their temperature was adapted to 50-55° C. Salmonella overnight cultures adjusted to OD600=1.0 with LB medium were added to the soft agar medium with a ratio of 1:100 resulting in the final OD600=0.01 or approximately 1×107 cells/ml and 20 ml LB softagar containing Salmonella test strain are poured on the pre-poured LB plate resulting in 0.14 mL bacterial solution of 1×107 cfu/mL per cm2.
Plant leaf material was extracted as described in Example 2. We prepared 1:1 dilution series of plant extracts starting with undiluted samples by using same extraction buffer. 5 μl aliquots of TSP dilution series were applied to agar plates; plates were incubated at 37° C. overnight. Antimicrobial activity was evaluated based on clearing zones.
Among the 6 tested salmocins, one demonstrated narrow antimicrobial activity (Spst—12% of strains inhibited), one salmocin had medium activity spectrum (ScolE3—60% of strains inhibited), and 4 others had broad activity spectrum: ScolE2 and ScolE7—inhibited about 90% of strains and ScolE1a and ScolE1b—inhibited 100% of strains (
Salmocins ScolE1 and ScolE1b demonstrated not only broad but also remarkably high activity against tested Salmonella strains (
For semi-quantitative comparison, we represented relative antimicrobial activity of recombinant colicins in arbitrary units (AU), calculated as a dilution factor for the highest dilution of protein extract causing a detectable clearing effect in the radial diffusion assay. Salmocin antimicrobial activity against Salmonella strains calculated in AU per mg FW of the plant tissue is shown in
Six colicins representing two activity groups were selected (Table 6). The list comprises colicins colS4, col5, col10, colIa, colIb and colM. Respective amino acid sequences were retrieved from GenBank; corresponding nucleotide sequences with codon usage optimized for Nicotiana benthamiana were synthesized by Thermo Fisher Scientific Inc. Colicin coding sequences were inserted into TMV-based assembled viral vector pNMD035 (described in detail in WO2012/019660) resulting in plasmid constructs depicted in
6 week-old Nicotiana benthamiana plants were infiltrated using needleless syringe with diluted Agrobacterium tumefaciens cultures carrying TMV-based assembled vectors for cytosolic colicin expression. Agrobacterium overnight cultures were adjusted to OD600=1.5 and further diluted 1:100 with infiltration buffer containing 10 mM MES, pH 5.5 and 10 mM MgSO4. Plasmid constructs used in this experiment are summarized in Table 7. For determination of optimal harvesting timepoint, plant material was harvested at several timepoints post infiltration and used for protein extraction with 5 volumes of buffer containing 50 mM HEPES (pH 7.0), 10 mM potassium acetate, 5 mM magnesium acetate, 10% (v/v) glycerol, 0.05% (v/v) Tween-20 and 300 mM NaCl. Total soluble protein (TSP) concentration was determined using the Bradford assay, and TSP extracts were analyzed using SDS-PAGE with Coomasssie staining. In our experiment, all tested colicins were expressed on reasonably high levels varying between 1.5 and 4.7 mg recombinant colicin/g FW or 16 and 41% of TSP (Table 8) as determined by comparison with bovine serum albumin (BSA) protein.
Nicotiana benthamiana plants.
We analyzed the antimicrobial activity of plant-made recombinant colicins against 35 strains of 32 different serotypes of S. enterica ssp. enterica. Details of strains used in our experiments are given in tables 5A and 5B (strain numbers 1-35).
Antimicrobial activity of recombinant colicin-containing plant extracts was tested in radial diffusion assays via spot-on-lawn-method as described in Example 3.
Among the 6 tested colicins, one demonstrated narrow antimicrobial activity (colS4—25% of strains inhibited), three colicins had medium activity spectrum (col5, col10 and colM—48%, 46% and 42% of strains inhibited, respectively), and two colicins had broad activity spectrum: colIa and colIb—inhibited 96% and 89% of strains, respectively (
Plant-produced colicins were tested for antibacterial activity on samples of chicken breast fillet contaminated with pathogenic Salmonella.
Evaluation of efficacy encompasses the analysis of pathogenic S. enterica ssp. enterica populations on contaminated meat samples subsequently treated with blends of plant-made recombinant colicins or a control carrier solution consisting of plant extract from the same production host but without colicins, and storage of treated meat samples for various time periods at 4° C.
No special sourcing of meat samples is used to ensure that bacteriocin activity is evaluated in representative consumer products. Raw chicken breast fillets are purchased at retail outlets (for these studies, ALDI supermarket, Halle, Germany), one day before the experiment. The meat is stored at 4° C. and the meat is not washed or pre-treated before experimental exposures.
The meat test matrices are experimentally contaminated with a 1:1 or 1:1:1:1 mixture of 2 or 4 Salmonella enterica ssp. enterica strains representing the serotypes Typhimurium and Enteritidis (ATCC®9270™*, ATCC®13076™*) or Typhimurium, Enteritidis, Newport and Anatum (ATCC®9270™*, ATCC®13076™*, ATCC®6962™* and ATCC®9270®*), respectively (
Contaminated meat is either treated with carrier or colicin blend solution (TSP extracts prepared 50 mM HEPES pH7.0, 10 mM K acetate, 5 mM Mg acetate, 10% (v/v) glycerol, 0.05% (v/v) Tween-20, 300 mM NaCl from N. benthamiana either non-treated plant material or plant material upon syringe-inoculation with Agrobacterium for colicin expression) by low-pressure spraying (2-4 bar) using atomizer flasks. Proposed application rates are 3 mg/kg for colicin M and 1 mg/kg for any other colicin used in the blend (colicin Ia and colicin 5). The meat is further incubated for 30 min at RT while inverted every 15 min.
Thirty minutes after colicin application, aliquots of ˜25 g chicken breast trims are placed into sterile sample bags (BagFilter®400 P) in replicates, the exact weight of each sample is recorded, and sample bags are closed using a closing clip (BagClip®400). In total, meat samples are incubated at room temperature for 1 h upon colicin treatment before the sealed meat samples are then stored at 4° C.
Meat samples are sampled at 1 h, 48 h and 72 h of storage at 4° C. for determination of on-matrix microbial contamination levels. For recovery of pathogenic Salmonella from meat samples, to each ˜25 g aliquot of meat sample ˜100 ml buffered peptone water is added. The samples are homogenized in a laboratory blender (BagMixer® 400CC®; settings: gap 0, time 30 s, speed 4). Microbial suspensions from filtered part of the storage bag are collected and a 1:10 dilution series is prepared. 100 μl aliquots of undiluted or diluted microbial suspensions are plated on XLD agar. The plates are incubated for 18-24 h at 37° C. and the CFU (colony forming units) are enumerated. The CFU number per g sample is calculated as follows:
The efficacy of the colicin treatment in reducing the number of viable pathogenic Salmonella in the experimentally contaminated meat samples is evaluated by comparing the data obtained with the carrier-treated control samples and colicin-treated samples by one-way ANOVA (Tukey's multiple comparisons test) and unpaired parametric t-test using GraphPad Prism v. 6.01.
The results of bacterial counts are shown in
In summary, statistically significant reduction of Salmonella populations on contaminated meat could be achieved by treatment of meat with a colicin blend.
Salmocin blends or salmocin/colicin blends will be tested for decontamination of food products from Salmonella and are planned to be used in food industry for reducing Salmonella contamination. Salmocins ScolE1a and ScolE1b show the broadest antimicrobial activity against tested Salmonella strains. Thus, they can be used as a main ingredient of salmocin cocktails for the control of Salmonella.
N. benthamiana was transformed by Agrobacterium-mediated leaf disk transformation using vectors for EtOH-inducible transgene expression and induction of detached leaves of TO generation transgenic plants for salmocin expression. This was done as described in Schulz et al. Proc. Natl. Acad. Sci. USA. 112, E5454-E5460 (2015).
Stable transgenic Nicotiana benthamiana plants containing the genomic insertion of TMV-based viral vector double-inducible with ethanol for ScolE1b expression (
Spinacia oleracea cv. Frühes Riesenblatt plants were grown in the greenhouse (day and night temperatures of 19-23° C. and 17-20° C., respectively, with 12 h light and 35-70% humidity). Six-week-old plants were used for syringe infiltration as described in Example 2. Expression of recombinant proteins was confirmed using SDS-PAGE with Coomassie staining (
We further analyzed the antimicrobial activity of plant-made recombinant salmocins against other strains of Salmonella as described in Example 6. To determine the salmocin antimicrobial activity spectrum, 109 strains representing 105 S. enterica ssp. enterica serotypes were selected and screened (Table 9). The screen included one strain each of all serotypes (except serotypes Typhi and I4,5:12:r:-) that are documented at the U.S. Centers for Disease Control and Prevention (CDC) (www.cdc.gov/nationalsurveillance/pdfs/salmonella-annual-report-2013-508c.pdf) as having caused at least 100 incidences of human Salmonella infection from 2003-2012, two strains of serotypes Typhimurium, Enteritidis and Javiana and 6 serotypes causing less than 100 incidences or not reported to CDC.
Enteritidis
Typhimurium
Newport
Javiana
Heidelberg
Montevideo
Muenchen
Saintpaul
Infantis
Oranienburg
Braenderup
Mississippi
Thompson
Agona
Paratyphi B var.
Bareilly
Poona
Hadar
Schwarzengrund
Berta
Anatum
Stanley
Litchfield
Hartfort
Mbandaka
Panama
Sandiego
Paratyphi A
Senftenberg
Norwich
Tennessee
Rubislaw
Derby
Give
Paratyphi B
Miami
Dublin
Kentucky
Brandenburg
Virchow
Gaminara
Weltevreden
Bovismorbisficans
Manhattan
Adelaide
Uganda
Pomona
Muenster
Kiambu
Blockley
Ohio
Hvittingfoss
Reading
Inverness
Urbana
London
Johannesburg
Chester
Havana
Bredeney
Telelkebir
Cerro
Albany
Agbeni
Minnesota
Worthington
Rissen
Oslo
Baildon
Cotham
Ealing
Lomalinda
Cubana
Carrau
Eastbourne
Monschaui
Alachua
Corvallis
Potsdam
Meleagridis
Indiana
Concord
Cholerasius
Altona
Pensacola
Othmarschen
Lovingstone
Grumpensis
Wandsworth
Kintambo
Edinburgh
Kottbus
Durban
Abony
Bispebjerg
Vellore
Pullorum
Tallahassee
Gallinarum
†The number of incidences refers to laboratory-confirmed human Salmonella infections (US) reported to CDC 2003-2012 published in National Enteric Disease Surveillance: Salmonella Annual June 2016; Report, 2013 (CDC, www.cdc.gov/nationalsurveillance/pdfs/salmonella-annual-report-2013-508c.pdf.
In order to estimate the breadth of the activity spectrum, all strains were tested at least once and 36 or 35 strains were subsequently re-screened in triplicate experiments with salmocins and colicins, respectively (
The five salmocins analysed were divided into four groups based on their ability to control major pathogenic Salmonella strains. Salmocins ScolE1a and ScolE1b were universally active, each being able to kill all tested pathovars and showing the highest average activity of higher than 105 AU/μg recombinant protein on all tested strains (
In contrast to the high potencies of salmocins in inhibiting enteropathogenic S. enterica strains, the specific activities of colicins Ia, Ib, M, 5, 10 and S4 (Table 6) were 2-4 orders of magnitude lower (2-3 logs AU/μg,
The bactericidal efficacy of plant-produced individual salmocin ScolE1a as well as salmocin blends for control of Salmonella-contaminated meat surfaces was analyzed in a simulation study.
Chicken breast fillet was purchased from a local supermarket. Nalidixic acid resistant mutants of strains of S. enterica ssp. enterica serovars Enteritidis (strain ATCC®13076™*), Typhimurium (strain ATCC®14028 ™*), Newport (strain ATCC®6962 ™*), Javiana (strain ATCC®10721®*), Heidelberg (strain ATCC®8326™*), Infantis (strain ATCC®BAA-1675™*) and Muenchen (strain ATCC®8388™*) were individually grown in LB medium supplemented with 25 μg/ml nalidixic acid to stationary phase, diluted with fresh LB and grown to exponential phase. For contamination of poultry, bacterial cultures were diluted with LB medium to OD600=0.001 (˜2×105 cfu/ml) and mixed 1:1:1:1:1:1:1. A pool of chicken breast fillets cut into pieces of about 20 g weight was inoculated with 1 ml of a mixture of 7 S. enterica strains at ˜2×105 CFU/ml density per 100 g of meat at room temperature resulting in an initial contamination level of meat matrices of about 3 log CFU/g of a 7-serotype mixture of pathogenic S. enterica; attachment of bacteria to meat surfaces was allowed for 30 min at room temperature. Subsequently, chicken breast trims were treated by spraying (10 ml/kg) with either plant extract control (TSP extract of WT N. benthamiana plant material with no salmocins, prepared with 50 mM HEPES pH 7.0, 10 mM K acetate, 5 mM Mg acetate, 10% (v/v) glycerol, 0.05% (v/v) Tween-20, 300 mM NaCl), or salmocin solutions (either individual or mixtures of TSP extracts of N. benthamiana plant material expressing salmocins ScolE1a, ScolE1b, ScolE2 and ScolE7 prepared with the same buffer as the plant extract control) at concentrations of 3 mg/kg ScolE1a, or 3 mg/kg ScolE1a, 1 mg/kg ScolE1b, 1 mg/kg ScolE2, 1 mg/kg ScolE7 or 0.3 mg/kg ScolE1a, 0.1 mg/kg ScolE1b, 0.1 mg/kg ScolE2, and 0.1 mg/kg ScolE7. Treated meat trims were further incubated at room temperature for 30 min. Aliquots of meat trims corresponding to ˜40 g were packed into BagFilter®400P sterile bags (Interscience) and stored for 1 h, 1 d and 3 d at 10° C., which represents realistic industrial meat processing conditions that are permissive but suboptimal for bacterial growth.
In total, meat samples were incubated at room temperature for 1.5 h during salmocin treatment before they were sealed and stored at 10° C. For analysis of bacterial populations, poultry aliquots were homogenized with 4 vol. peptone water using Bag Mixer®400CC® homogenizer (settings: gap 0, time 30 s, speed 4; Interscience) and colony forming units (CFU) of S. enterica were enumerated on XLD medium (Sifin Diagnostics) supplemented with 25 μg/ml nalidixic acid upon plating of serial dilutions of microbial suspensions. Samples were analysed in quadruplicate.
The efficacy of the salmocin treatment in reducing the number of viable pathogenic Salmonella in the experimentally contaminated meat samples was evaluated by comparing the data obtained with the carrier-treated control samples and salmocin-treated samples by two-tailed unpaired parametric t-test with 6 degrees of freedom using GraphPad Prism v. 6.01.
Efficacy of salmocin treatment was assessed for the extent of reduction in the pathogenic bacterial population level on salmocin-treated (individual ScolE1a at an application rate of 3 mg/kg meat and salmocin blend consisting of ScolE1a+ScolE1b+ScolE2+ScolE7 applied at 3+1+1+1 mg/kg meat, respectively), both in relation to plant extract control-treated, meat samples and statistically significant net reductions in viable counts of 2-3 logs CFU/g meat at all timepoints analysed were found (
The primary structure including post-translational modifications of the plant-expressed recombinant salmocins contained in plant TSP extracts was analysed by Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS).
For proteolytic digestion, TSP extracts prepared from plant material expressing salmocins with 5 vol. 20 mM Na citrate, 20 mM NaH2PO4, 30 mM NaCl, pH 5.5 were subjected to SDS-PAGE and Coomassie-stained SDS gel bands containing 5 μg of protein were excised and destained by consecutive washing with 100 mM NH4HCO3 and 100 mM NH4HCO3 in acetonitrile (ACN)/H2O (50; 50, v/v). Disulfide bonds were reduced with 10 mM DTT for 45 min at 50° C. followed by alkylation with 10 mg/ml of iodoacetamide for 60 min. Destained and alkylated gel bands were then subjected to proteolytic digestion with different sequencing grade endoproteinases (Promega, Madison, USA). Protease:protein ratio in the digestion solutions was adjusted to 1:20 (w/w) and digestions were carried out for 12 h at 25° C. (chymotrypsin) or 37° C. (Asp-N, Glu-C, Lys-C, trypsin). Proteolytic peptides were extracted by consecutive washing with H2O, ACN/H2O/trifluoroacetic acid (50; 45; 5, v/v/v) and ACN, respectively. Extraction solutions were combined, concentrated in a vacuum centrifuge and resolubilized in H2O/acetic acid (90; 10, v/v).
Proteolytic salmocin peptides obtained as described above or purified intact plant-produced salmocin ScolE1a, ScolE1b and ScolE7 proteins were purified for mass spectrometry by solid-phase extraction using C4 or C18 bonded silica material (ZipTip®, Millipore, Darmstadt, Germany) and elution solutions were co-crystallized on a MALDI ground steel target with 2,5-dihydroxyacetophenone as well as 2,5-dihydroxybenzoic acid matrix (Bruker Daltonics, Bremen, Germany).
Mass spectra were acquired on a MALDI-TOF/TOF mass spectrometer (Autoflex Speed™, Bruker Daltonics, Bremen, Germany) with positive polarity in linear mode for molecular mass determination and in reflector mode for protein sequencing by In-source decay (ISD) analysis. The matrix crystals were irradiated with a Nd:YAG laser (Smart beam-II™, Bruker Daltonics, Bremen, Germany) at an emission wavelength of 355 nm and set to a pulse rate of 1 kHz.
MS and MS/MS spectra were recorded with flexControl (version 3.4, Bruker Daltonics, Bremen, Germany) by accumulation of at least 5000 or 10000 laser shots (per sample spot), respectively. Laser energy was set slightly above the threshold for MS experiments and set to maximum for MS/MS analyses. Spectra processing was carried out with flexAnalysis (version 3.4, Bruker Daltonics, Bremen, Germany) by applying baseline subtraction with TopHat algorithm, smoothing with Savitzky-Golay algorithm and peak detection with SNAP algorithm.
The mass spectrometer was calibrated using a set of standard peptides and proteins with known masses (Peptide Calibration Standard II, Protein Calibration Standard I and II, Bruker Daltonics, Bremen, Germany).
Determination of the intact molecular mass was based on the mass-to-charge-ratios (m/z) of single and multiple charged molecular ions.
Sequencing of protein termini was carried out by ISD analysis. The annotation of ISD fragment spectra was carried using BioTools (version 3.2, Bruker Daltonics, Bremen, Germany) by in silico generation of m/z values for fragment ions and their comparison with the m/z values of the fragment signals observed within the acquired ISD spectra. This approach enabled the identification of the terminal amino acid sequences as well as of present modifications.
For protein sequencing analysis, only fragment (MS/MS) spectra were used for the identification of proteolytic peptides and the annotation was carried out with PEAKS Studio (version 7.5, Bioinformatics Solutions Inc., Waterloo, Canada). Identification of proteins and verification of their amino acid sequences was performed by searching the MS/MS data against the NCBI nr database and the UniProt/SwissProt database to which the sequences of the salmocins were appended, respectively. Database search was performed with a parent mass error tolerance of 50 ppm and a fragment mass error tolerance of 0.5 Da. The maximum number for both missed cleavages as well as post-translational modifications for one proteolytic fragment was set to 3. Non-specific cleavage was allowed for both protein termini.
Search results of each MS/MS dataset from proteolytic peptides of salmocins against the UniProt/SwissProt database confirmed the identity of each of the analysed salmocins (Table 11). The integrity of purified salmocins ScolE1a, ScolE1b and ScolE7 was further analysed by MS-based sequencing of protein termini using ISD and molecular mass determination methods, which confirmed that all salmocin proteins were intact upon plant expression. Post-translational modifications observed were restricted to cleavage of N-terminal methionine in case of ScolE2, ScolE7, ScolE1a and ScolE1b and N-terminal acetylation for ScolE7 and ScolE1a (Table 11).
As it was shown in Example 10, two pore-forming salmocins ScolE1a and ScolE1b demonstrated the highest and broadest antimicrobial activity against all tested Salmonella strains. To identify other salmocins to control Salmonella, we performed a homology search in NCBI database for Salmonella proteins similar to ScolE1a and ScolE1b but different to colicins in the N-terminal part. This search revealed three new sequences, which we called ScolE1c (SEQ ID NO: 25), ScolE1d (SEQ ID NO: 26) and ScolE1e (SEQ ID NO: 27) (Table 12). The CLUSTAL Omega alignment of these sequences is shown in
We also searched for Salmonella proteins similar to colicin M in order to have another functional domain expressing antimicrobial activity, which is not related to nuclease (as this often creates the need for co-expression of immunity proteins and most of these proteins were not easy to purify). This search resulted in ScolMa sequence (SEQ ID NO: 28) (Table 12).
Amino acid sequences of salmocins ScolE1c, ScolE1d, ScolE1e and ScolMa were retrieved from GenBank; corresponding nucleotide sequences with codon usage optimized for Nicotiana benthamiana were synthesized by Thermo Fisher Scientific Inc. SEQ ID NO: 29 encoded ScolE1c, SEQ ID NO: 30 encoded ScolE1d, SEQ ID NO: 31 encoded ScolE1e, and SEQ ID NO: 32 encoded ScolMa.
Salmocin coding sequences were inserted into TMV-based assembled viral vector pNMD035 (described in detail in WO2012/019660) resulting in plasmid constructs depicted in
Salmocin expression screen was performed as described in Example 2. The accumulation of salmocins ScolE1c, ScolE1d and ScolMa in Nicotiana benthamiana leaves was high. In contrast, the expression of ScolE1e was poor (
We compared the antimicrobial activity of plant-made recombinant salmocins ScolE1c, ScolE1d, ScolE1e and ScolMa against ScolE1a and ScolE1b. For this comparison, we used 10 Salmonella enterica ssp. enterica strains (Table 13). The evaluation of antimicrobial activity in plant extracts containing salmocins was performed using a radial diffusion spot-on-lawn assay as described in Example 6. The extract from untransfected plant tissue (Wt) was used as a negative control. All tested new salmocins demonstrated significant antimicrobial activities, although they were not superior of ScolE1a and ScolEb (Table 13).
Based on the expression and antimicrobial activity levels, we selected ScolE1d and ScolMa for generation of ethanol-inducible stable transgenic Nicotiana benthamiana hosts.
N. benthamiana was transformed by Agrobacterium-mediated leaf disk transformation using vectors for EtOH-inducible transgene expression (pNMD49621 for ScolE1d and pNMD49632 for ScolMa,
Antimicrobial activities of plant-made recombinant salmocins ScolE1b, ScolE1d and ScolMa were analyzed as described in Example 3. In these experiments, we tested the plant extracts containing recombinant salmocins against 36 strains of 33 different serotypes of S. enterica ssp. enterica using radial diffusion assay via spot-on-lawn-method. Bacterial strains are listed in Tables 5A and 5B.
For semi-quantitative comparison, we represented relative antimicrobial activity of recombinant salmocins in arbitrary units (AU), calculated as a dilution factor for the highest dilution of protein extract causing a detectable clearing effect in the radial diffusion assay. Concentration of salmocin proteins in TSP extracts was evaluated by visual comparison with BSA standard on Coomassie-stained polyacrylamide gels after protein electrophoresis. Specific antimicrobial activity was calculated in arbitrary units (AU) per μg of recombinant salmocin as an average of 3 independent experiments.
We have found that ScolE1d has an antibacterial activity spectrum very similar to ScolE1b, although in most cases the overall activity of ScolE1d was lower, and in just few cases it was comparable with ScolE1b (
Antimicrobial activity of ScolMa was lower than ScolE1b for 20 strains, comparable for 10 strains and higher for 6 strains (
For stability evaluation, purified salmocin protein samples were stored as dry lyophilized powder and as a solution at 4° C. and room temperature (20-25° C.). Protein stability was assessed on the basis of antimicrobial activity for 93 days (ScolE1a), 387 days (ScolE1b) and 200 days (ScolE1d). Antimicrobial activity against susceptible bacterium was evaluated by radial diffusion assay. All salmocins were tested with the same bacterial strain: Salmonella Typhimurium ATCC® 14028.
Lyophilized protein samples were resuspended in distilled water (0.2-0.4 mg/ml). Soluble protein concentration was measured for each sample using Bradford assay. For radial diffusion assay, serial 1:2 dilutions of proteins solubilized in PBS buffer were made. 5 μL of protein dilutions (1-1.8 μg of protein before dilution), were spotted on soft agar plates with susceptible bacterial strain. Residual activity of salmocins was evaluated after overnight incubation of plates. Antimicrobial activity was evaluated as specific activity units (AU)—highest dilution giving a difference to non-affected bacterial growth area determined by visual inspection of plates for bacterial growth inhibition by holding the plate in front of a light source. Highest dilution with growth inhibition was recorded as an activity in AU/mg of salmocins.
All three salmocins remained stable throughout the whole study period when stored as a dry powder either at room temperature or at 4° C. (
To identify additional salmocins with phosphatase M activity, we performed a homology search in NCBI database for Salmonella proteins similar to ScolMa (SEQ ID NO: 28). As a result of this search, we identified two proteins: ScolMb (SUF52254.1; SEQ ID NO: 33) and ScolMc (01N38022.1; SEQ ID NO: 34). The CLUSTAL Omega (1.2.4) multiple sequence alignment of ScolMa, ScolMb and ScolMc sequences is shown in
ScolMb and ScolMc encoding nucleotide sequences with codon usage optimized for Nicotiana benthamiana were synthesized by Thermo Fisher Scientific Inc. SEQ ID NO: 35 encoded ScolMb, and SEQ ID NO: 36 encoded ScolMc.
Salmocin coding sequences were inserted into TMV-based assembled viral vector pNMD035 (described in detail in WO2012/019660) resulting in plasmid constructs depicted in
ScolMb and ScolMc expression screen was performed as described in Example 2. At 7 dpi, plant tissue necrosis developed, suggesting earlier harvesting time points to be preferrable. ScolMb and ScolMc recombinant proteins accumulated in Nicotiana benthamiana leaves at comparable levels (
For comparison, antimicrobial activities of plant-made recombinant salmocins ScolMa, ScolMb and ScolMc were analyzed as described in Example 3. We tested recombinant-salmocin-containing plant extracts against 10 strains of 8 different serotypes of S. enterica ssp. enterica using radial diffusion assay via spot-on-lawn-method. Bacterial strains are listed in Tables 5A and 5B.
For semi-quantitative comparison, we represented relative antimicrobial activity of recombinant salmocins in arbitrary units (AU), calculated as a dilution factor for the highest dilution of protein extract causing a detectable clearing effect in the radial diffusion assay. Concentration of salmocin proteins in TSP extracts was evaluated by visual comparison with BSA standard on Coomassie-stained polyacrylamide gels after protein electrophoresis. Specific antimicrobial activity was calculated in arbitrary units (AU) per μg of recombinant salmocin as an average of 3 independent experiments.
All three tested salmocins had very similar antimicrobial pattern: they were active against Typhimurium, Javiana, Muenchen, Heidelberg, Dublin and Abony strains, and exerted no or very low activity against Enteritidis and Newport strains (
The Minimum Inhibitory Concentration (MIC) is defined as the lowest concentration of bacteriocin, which prevents visible growth of corresponding bacterial strain. We determined MICs of ScolE1b, ScolE7, ScolE1d, ScolMb and ScolMc salmocins by agar dilution method using MHB medium (Müller-Hinton Bouillon; Carl Roth GmbH& Co. KG, Karlsruhe, Germany). The tested Salmonella strains (Enteritidis ATCC13076, Typhimurium ATCC14028, Typhimurium ATCC13311 and Dublin ATCC14028) were streaked for single colony onto a MHB agar plate (MHB medium supplemented 0.75% (w/v) agar, bacteriology grade (AppliChem GmbH, Darmstadt, Germany)) and the plate was incubated for at least 16 h at 37° C. The main culture was prepared by picking 8 equally sized colonies for inoculation of 4 ml of MHB medium, which was further incubated for 2-3h at 37° C. and 180 rpm until an OD600 of about 0.2 was reached. The bacterial culture was pre-diluted to OD600=0.02 followed by further dilution to potential 2×106 cfu/ml.
For evaluation of the of the MIC, the tested lyophilized purified salmocin proteins were resuspended in PBS buffer (phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4)) supplemented with 0.1 mg/ml BSA (AppliChem GmbH, Darmstadt, Germany). The protein concentration was adjusted to the appropriate amount (100 fold higher than the concentration which will be tested) and 11 further 1:1 protein dilutions in PBS+0.1 mg/ml BSA were prepared. The different salmocin containing MHB agar plates were prepared using 6-well sterile cell culture plates (TPP Techno Plastics Products AG, Trasadingen, Switzerland) after melting of the MHB agar, dividing into 5 ml aliquots and cooling of the agar to 50° C. After addition of 50 μl salmocin/5 ml MHB agar the solution was filled into one well of the 6-well plate. After cooling of the plates to room temperature, 4 spots of 5μl diluted Salmonella culture was dropped in each well. The drops were dried and the plates were incubated for ˜16h at 37° C. After incubation, the plates were checked for bacterial growth at different concentrations. The MIC is defined as the lowest concentration of the antimicrobial substance that inhibits visible growth of the test microbe.
Salmonella enterica ssp. enterica strains
Enteritidis
Typhimurium
Typhimurium
Dublin
MICs determined for individual salmocins show certain batch-to-batch variation, which may be related to the batch quality, i.e. the presence of proteinaceous impurities or proteolytic degradation products. In our previous experiments, ScolE1b and ScolE1d showed the broadest antimicrobial activity (Example 3); they have also very low MICs for the tested strains. SalE1b has a broader high activity as one can also detect a low MIC for the second tested Typhimurium strain. ScolMc has a higher activity on the tested ATCC14028 strains in comparison to ScolMb. The lowest MIC (<0.98 ng/ml) was surprisingly detected for ScolMc, see e.g. data with the Typhimurium strain ATCC13311.
Salmocin ScolMa containing Nicotiana benthamiana leaf biomass was produced using Agrobacterium-mediated delivery of TMV-based viral vector pNMD47730 (
For ScolMa protein isolation, we tested various purification strategies. Extraction of recombinant protein of interest from N. benthamiana leaf biomass at pH 4.0-5.5 is preferable as in this pH range the RUBISCO, the most abundant plant host protein, is precipitated and can be further easily removed together with other solids. Unfortunately, at pH 4.0-5.5 the ScolMa extraction yield was very poor. The ScolMa yield was much higher at pH 6.0, although the resulting extracts contained high amounts of RUBISCO as well. Such extracts were further subjected to column chromatography purification using various approaches. All protein purification experiments were performed using the ÄKTA™ Pure chromatography system (Cytiva Europe GmbH, Freiburg im Breisgau, Germany).
For example, we tested Hydrophobic Interaction Chromatography (HIC) using HiTrap™ Phenyl FF (LS) and HiTrap™ Butyl FF resins (Cytiva Europe GmbH, Freiburg im Breisgau, Germany). In both cases, the buffer consisting of 20 mM citrate (pH6.0), 20 mM NaH2PO4 and 2 M NaCl was used for extraction. This buffer was also used for column equilibration and for column washing after the sample loading. The elution was performed using the same buffer with 0-100% NaCl gradient. For both resins, no efficient separation between ScolMa and host proteins was achieved (
Other numerous purification attempts using HiTrap™ Capto™ MMC, HiTrap™ Capto™ Adhere, HiTrap™ Capto™ Q, HiTrap™ Capto™ Adhere, HiTrap™ Capto™ MMC, HiTrap™ Capto™ MMC, HiTrap™ Capto™ S, HiTrap™ Capto™ MMC and HiTrap™ Capto™ DEAE resins (all Cytiva Europe GmbH, Freiburg im Breisgau, Germany) did not result in satisfying ScolMa yield and purity. The outcome of these experiments is summarized in the Table 15. As an example,
We tested the extraction of ScolMa, ScolMb and ScolMc proteins from Nicotiana benthamiana leaf biomass using the buffers with pH 4.0, pH 5.0 and pH 7.0. Leaf biomass containing recombinant proteins of interest was produced as described in Example 26. pNMD47730 (
The extraction was performed at 4° C. or at the room temperature (approx. 22° C.). For the extraction at 4° C., frozen ground plant material was thoroughly mixed with the extraction buffer and incubated for 30 min on ice. After 10 min centrifugation at 13000 rpm at 4° C., the supernatant was transferred into the new tube (repeated twice). Resulting supernatant was analyzed using SDS-PAGE (15% gel). For the extraction at room temperature, frozen ground plant material was thoroughly mixed with the extraction buffer and incubated for 15 min at room temperature. After 5 min centrifugation at 7100 g at 22° C., the supernatant was transferred into the new tube and centrifuged again for 20 min at 7100 g at 22° C. Resulting supernatant was analyzed using SDS-PAGE (15% gel).
ScolMb was efficiently purified using HiTrap™ Capto™ MMC resin. N. benthamiana leaf material was extracted with 5 volumes of the extraction buffer (15 mM Na-acetate (pH 4.0), 20 mM NaH2PO4, 30 mM NaCl, 0.05% Tween-80) at room temperature. The same buffer was used for the column equilibration and column washing. The protein elution was performed using a linear 0-100% gradient of the elution buffer consisting of 10 mM Na-acetate (pH 7.5), 20 mM NaH2PO4, 50 mM NaCl. Protein samples were analyzed using SDS-PAGE (
ScolMb was also efficiently purified using HiTrap™ Phenyl FF (LS) resin. Plant biomass was extracted with 5 volumes of extraction buffer (15 mM Na-acetate (pH 4.0), 20 mM NaH2PO4, 30 mM NaCl, 0.05% Tween-80) at room temperature. Filtered plant extract was mixed with 50 mM Na-acetate (pH 4.0), 30 mM NaCl, 3 M (NH4)2SO4 to get final 1 M (NH4)2SO4 concentration. The column was equilibrated and washed with the buffer containing 15 mM Na-acetate (pH 4.0), 20 mM NaH2PO4, 30 mM NaCl, 0.05% Tween 80, 1 M (NH4)2SO4. A linear 0-100% gradient of the elution buffer containing 10 mM Na-acetate (pH 7.5), 20 mM NaH2PO4, 50 mM NaCl was used for protein elution. Protein samples were analyzed using SDS-PAGE (
ScolMc was purified using HiTrap™ Capto™ MMC resin according to the same protocol as in case of ScolMb. SDS-PAGE analysis of protein purification fractions is shown in
Again, ScolMc was purified with HiTrap™ Phenyl FF (LS) resin using the same protocol as for ScolMc.
This patent application claims the priority of U.S. patent application Ser. No. 16/577,484, filed on Sep. 20, 2019, published as US-2020-0010517-A1, the content of which is incorporated herein by reference including entire description, claims, figures, and sequence listing.
| Number | Date | Country | Kind |
|---|---|---|---|
| 17 162 784.7 | Mar 2017 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/076334 | 9/21/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16577484 | Sep 2019 | US |
| Child | 17761854 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/EP2018/055479 | Mar 2018 | US |
| Child | 16577484 | US |
