Use of bacteriophage outer membrane breaching proteins expressed in plants for the control of gram-negative bacteria

The current invention provides compositions and methods for killing or controlling development of Gram-negative bacteria that infect, infest or cause disease in plants, such as pathogenic, saprophytic and opportunistic microbes that cause disease in crops and food borne illness in people or in animal feed.


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All publications and patent applications herein are incorporated by reference to the exact same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The following description includes information that might be useful in understanding the current invention. It’s not an admission that some of the information provided herein is past art or relevant to the currently claimed inventions, or thatany book specifically or implicitly referenced is prior art.

Plants developed for commercial agricultural functions are nearly always implanted as uniform monocultures; this is, single varieties of a certain harvest are daunted by vegetative propagation or from seed and also are planted on a very large scale. When apathogen or insect arrives that may conquer the natural disease or pest resistance of a certain variety, severe financial losses may occur due to the practice of monoculture, sometimes involving loss of the entire harvest in a specific area. Control ofdiseases and pests employing massive programs of agricultural chemicals is expensive, environmentally unsound and frequently impossible. For example, citrus canker disease, brought on by a quarantined Gram-negative bacterial pathogen, Xanthomonas citri, hasspread uncontrollably throughout Florida. As a second example, the Gram-negative bacterial pathogen Ca. Liberibacter asiaticus is a USDA Select Agent (possible bioterrorist agent) that has been introduced to Florida in 2005 and has spread uncontrollablythroughout Florida. This pathogen threatens world citrus production. As a third example, the Gram negative bacterial pathogen Ralstoma solanacearum Race 3 Biovar 2 has been introduced in the U.S. numerous occasions and is such a critical hazard to U.S.potato production it is also a recorded USDA Select Agent. This pathogen has been introduced in the U.S. by infecting geranium plants, but asymptomatically, so that detection of this pathogen is postponed.

As a fourth and final instance, severe human illness and even deaths have been reported as a result of this Gram-negative bacterium Escherichia coli, which is capable of internally infecting–not just contaminating–particular crop plants like spinach,alfalfa sprouts and mung bean sprouts. Many outbreaks of Salmonella and E. coli O157:H7 related to organically grown sprouts and mesclun lettuce have been reported (Doyle, M. P. 2000. Nutrition 16: 647-9). According to the FDA in its webreport of this 2006 outbreak of E. coli in polluted salmon”To date, 204 cases of illness due to E. coil O157:H7 infection have been reported on the CDC including 31 cases between a kind of kidney failure known as Hemolytic Uremic Syndrome (HUS), 104hospitalizations, along with three deaths. The first departure was an elderly lady in Wisconsin; the next departure, a two-year-old in Idaho; along with the third death, an elderly woman in Nebraska.” Traditional plant breeding to control such diseases of plants orfood-borne contamination has proven to be impossible. There is therefore an urgent and pressing need for gene engineering techniques to provide plants, such as carrier plants such as geraniums, together with disease and pest resistance against diseases andpests that they naturally are vulnerable to, or tolerant of.

A vast array of antifungal and antibacterial proteins are identified and their genes isolated from the plants and animals. Due to the key differences in the structures of bacterial, Gram-positive bacterial and Gram-negativebacterial cell partitions, many of these proteins attack just fungi or Gram positive bacteria, which have cell walls that are exposed directly to the environment. Gram negative bacteria don’t have cell walls that are exposed directly to the environment.Instead, their walls are enveloped and protected by a distinctive outer membrane arrangement, the lipopolysaccharide (LPS) barrier, which offers a very effective additional barrier to protect their cell walls from most eukaryotic defenses,especially plant guards. The excellent bulk of the pathogens recorded by the USDA as Select Agents are bacterial pathogens, and all these are Gram negative.

The LPS provides an effective defense to Gram negative bacteria against externally generated enzymes that could effectively degrade the bacterial cell wall (also referred to as the murein layer), including the comparatively thick but exposed cell walls ofGram-positive bacteria and fungi. For example, lysozymes are antimicrobial agents found in mammalian cells, insects, plants, bacteria and viruses that violate fungal and bacterial cell walls, specifically cleaving bonds between the amino sugars of therecurring muropeptides (C-1 of N-acetylmuramic acid and C-4 of N-acetylglucosamine of microbial cell walls (Ibrahim et al. 2001 and references therein). Some lysozymes also are pleiotropically lytic proteins, meaning they’re active in killingGram-negative and Gram-positive bacteria, but this action isn’t due to the enzymatic activity of lysozyme, but specifically due to a brief, linear peptide fragment that is a degradation product of several lysozymes; it’s the linear degradation product ofthe lysozyme that penetrates the LPS barrier and the cell wall (but without damaging either), reaching the inner tissues and permeabilizing the internal membrane, resulting in lysis (During et al, 1999; Ibrahim et al. 2001). But this linear peptideactivity does not operate well in plants (see below).

Those antimicrobial proteins demonstrated to kill Gram-negative germs are largely tiny peptides (proteins of less than 50 amino acids in length) that are amphipathic and positively charged, so they are drawn to the negatively chargedGram negative outer membrane, are small enough to permeate can penetrate the vulnerable LPS, and also the comparatively sparse Gram negative cell wall. These peptides generally behave to permeabilize the inner membrane, directly causing cell death. Throughout the lasttwo decades, over 500 antimicrobial peptides have been discovered in viruses, viruses, plants and animals (Jaynes et al, 1987; Mitra and Zhang, 1994; Broekaert et al. 1997; Nakajima et al, 1997; Vunnam et al, 1997). The best clarified of those arepeptides having broad spectrum action in the source receptor and in artificial media against viruses, bacteria, parasites, viruses as well as tumor cells (Hancock and Lehrer, 1998).

The biggest described group by far of these antimicrobial peptides are linear (eg., cecropins, attacins and magainins). However, linear peptides aren’t found naturally in plants and most linear peptides are rapidly degraded by plant proteases. As an example, cecropin B can be quickly degraded when incubated with intercellular plant fluid, using a half-life ranging from about three minutes from potato to approximately 25 hours in rice (Owens & Heutte, 1997). Transgenic tobacco plants expressing cecropins haveonly slightly increased immunity to (Gram-negative) Pseudomanas syringae pv. Tabaci, the cause of tobacco wildfire (Huang et al 1997). Artificial cecropin analogs Shiva-1 and SB-37, expressed from transgenes in potato plants, just slightly reducedbacterial infection caused by (Gram-negative) Erwinia carotovora (Arce et al 1999). Transgenic apple expressing the SB-37 peptide showed just slightly increased immunity to (Gram-negative) E. amylovora in area evaluations (Norelli et al 1998). Similarly,transgenic potatoes expressing attacin demonstrated resistance to fungal infection by E. carotovora (Arce et al 1999) and transgenic apple and pear expressing attacin genes also have shown slightly enhanced immunity to E. amylovora (Norelli et al 1994;Reynoird et al 1999). Attacin E has been also proven to be rapidly degraded by plants (Ko et al 2000). Transgenic tobacco plants expressing a synthetic magainin analog which was modified to be sensitive to extracellular plant proteases were onlyslightly immune to the bacterial pathogen E. carotovora (Li et al 2001).

The disulfide-linked peptides (e.g. defensins, prophenins and thaumatins) reveal more promise of equilibrium when expressed in plants, but resistance has either been weak, not revealed, or cytotoxicity problems have emerged. Hen egg-whitelysozyme genes (with lytic ability) have been utilized to confer weak Gram-negative bacterial disease resistance to transgenic tobacco plants (Trudel et al 1995; Kato et al 1998). Bacteriophage T4 lysozyme has also been reported to slightly enhanceresistance in transgenic potato against E. carotovora (During et al 1993; Ahrenholz et al., 2000) and in transgenic apple plants against E. amylovora (Ko 1999). However, as mentioned before, the activity of lysozyme against Gram-negative bacteria isspecifically because of a short lytic peptide fragment (Ibrahim et al. 2001) that’s sensitive to protease. Thaumatins exhibit the broadest range of antimicrobial activity so far characterized, but also exhibit potent cytotoxic effects oneukaryotic cells (Taguchi et al 2000). Defensins, made by plants, insects and mammals, are characterized by complicated .beta. -sheet structures with several disulfide bonds that bind and disrupt microbial plasma membranes. An plant defensin from alfalfagave strong resistance to a fungal pathogen (Guo et al 2000) and defensins from spinach were also active in vitro against Gram positive and Gram negative bacteria (Segura et al. 1998). However, human disorders have led from both alfalfa and spinachinfected with enteric bacteria; evidently these defensins are either not triggered by these bacteria or they are ineffective against these bacteria. More effective antibacterial agents are desperately needed to protect crop plants.

Nonenzymatic, antimicrobial peptides are plentiful in character but of limited value in transgenic crops, primarily due to degradation from plant proteases. In addition, some Gram-negative germs are resistant to antimicrobial peptides even inculture media, due to variations from the chemical structure of the LPS (Gutsmann et al., 2005). This can help explain why plant pathogenic germs can defeat host plant defensins. To date, no antimicrobial peptide has proved over marginallyeffective from Gram-negative germs when expressed in crops. More efficacious methods to control plant disease are urgently needed.

By comparison with bacterial pathogens of animals, the vast majority of bacterial pathogens of plants are all Gram negative. As stated above, the distinguishing feature of Gram-negative bacteria is the existence of the LPS, which forms an outermembrane that completely encircles the wall. Mutations affecting the structure of the LPS of a (Gram-negative) bacterial plant pathogen of citrus caused the pathogen to expire quite quickly on citrus, but not on bean (Kingsley et al., 1993),indicating the significance of the LPS arrangement in evading particular plant phytochemical protects. Additionally, mutations affecting multidrug efflux in Gram-negative bacteria cause the bacteria to expire quickly in plants, highlighting the role of lowmolecular fat plant defense compounds (phytoalexins) in plant defense, and further indicating the value of the intact LPS of Gram-negative in resisting plant defense compounds (Reddy et al., 2007). Multidrug efflux requires a complete LPS forfunction.

Animals possess a unique set of inherent defenses against microbial invasion that’s independent of previous exposure to pathogens (Hoffman et al., 1999). Among these are the lytic peptides discussed above, and additionally the neutrophil, a white blood cellthat a part of the innate immune system. Neutrophils make a variety of protein and peptide antibiotics which kill microorganisms. Among these is your bactericidal/permeability increasing (BPI) protein, which can be a potent antimicrobial protein which isprimarily active towards Gram negative bacteria (Levy, 2000). BPI is not toxic to Gram positive bacteria, fungi or animal cells, but rather attacks the LPS layer of Gram negative cells, disrupting its structure, and finally attacking the innermembrane and inducing lysis (Mannion et al., 1990). A hallmark of BPI proteins is their strongly cationic, lysine rich nature and their opsonic or immune system activation capability (Levy et al., 2003). Participants of the BPI protein household includelipopolysaccharide binding protein (LBP), lung particular X protein (LUNX), palate, lung and nasal epithelial clone (PLUNC) and parotid secretory protein (PSP), many of which were identified by bioinformatics methods with as much as 43% id betweenfamily members (Wheeler et al. 2003). There are numerous patents covering utilization of BPI and particular smaller peptide derivatives (by way of instance, U.S. Pat. No. 5,830,860 and U.S. Pat. No. 5,948,408).


Antimicrobial Bacteriophage Proteins.


All bacteriophages have to escape from bacterial host cells, either by extrusion from the host cell, much like filamentous phages, or from host cell lysis from inside. Host cell lysis from inside requires two occasions: capability to permeate the innermembrane of both gram negative and gram positive bacteria, and ability to depolymerize the murein layer, which is relatively thick in gram positive cell walls.

Bacteriophage penetration of, and egress through, the inner membrane is achieved in many, but evidently not , phage by usage of small membrane-localized proteins known as”holins” which appear to collect in the bacterial internal membraneuntil attaining a specific concentration, at which time they’re believed to self-assemble to permeabilize the internal tissues (Grundling et al., 2001; Wang et al. 2000; Young et al., 2000). The terms”holin” and”holin-like” aren’t biochemically or evenfunctionally accurate provisions, but rather as used herein refer to any phage protein with at least one transmembrane domain that is capable of permeabilizing the internal membrane, hence allowing molecules other than holins which are typically sequestered inthe cyctoplasm by the internal tissue, such as proteins including endolysins, to breach or penetrate the internal tissue to get to the cell wall. The biochemical function(s) of holins is speculative; most, if not all of the curent knowledge on holins isbased on the .lamda. phage S protein (Haro et al. 2003).

Holins are encoded by genes at at least 35 distinct households, having at least one transmembrane domain and categorized to three topological classes (classes I, II, and III, together with three, two and one transmembrane domains [TMD], respectively),all without discovered orthologous relationships (Grundling et al., 2001). At least two holins are known to be hemolytic and this hemolytic function has been demonstrated to play a role in the pathogenesis of certain bacteria towards insects and nematodes(Brillard et al., 2003). Only a few have been partially characterized with regard to in vivo function, leading to two quite different notions of how they may operate. The most widely accepted theory is that holins operate to form oligomericmembrane pores (Graschopf & Blasi, 1999; Young et al., 2000).

Depolymerization of the murein layer is achieved by lytic enzymes known as endolysins. There are at least three functionally distinct classes of endolysins: 1) glucosaminidases (lysozymes) that attack the glycosidic linkages between the aminosugars of the peptidoglycan; 2) amidases that attack the N-acetylmuramoyl-L-alanine amide linkage between the glycan strand and also the cross-linking peptide, and 3) endopeptidases that attack the interpeptide bridge linkages (Sheehan et al., 1997).Endolysins are synthesized without an export signal sequence that would permit them access into the peptidoglycan (murein) coating, and they therefore usually accumulate in the cytoplasm of phage infected germs until they are released by the activity ofholins (Youthful and Blasi, 1995).

Lysozymes have been suggested as antibiotics that can be used as external agents against both Gram-positive and Gram-negative germs because at least some of them are multifunctional (Throughout et al., 1999). This dual operation isbased on the finding that both phage T4 and hen egg white lysozyme have both glucosaminidase activity as well as amphipathic helical stretches that allow them to penetrate and disrupt bacterial, fungal and plant membranes (Throughout et al., 1999). Themicrobicidal activity of lysozymes can be impacted by C-terminal improvements; developments of hydrophobic amino acids decreased activity against Gram positive bacteria, but increased activity against Gram negative E. coli (Arima et al., 1997; Ito et al.,1997). Additions of histidine, a hydrophilic amino acid, to T4 lysozyme doubled its antimicrobial activity against Gram-positive and Gram-negative germs (Throughout et al., 1999).

The nonenzymatic, microbicidal use of lysozymes seemed to be on account of amphipathic C-terminal domain names that could be mimicked by small synthetic peptides modeled after the C-terminal lysozyme domain names (During et al., 1999). As describedabove, transgenic plants have been created that extract lysozymes and provide some immunity to certain plant pathogens. Because most endolysins collect to elevated titers within the bacterial cell without inducing lysis, endolysins other than certainlysozymes like T4 wouldn’t be expected to attack Gram-negative germs if externally implemented, because Gram-negative germs are surrounded by an outer membrane comprised in LPS along with a lipid bilayer that will protect its murein layer from enzymaticattack equally as effectively as its inner membrane does.

Attempts have been made to deal with bacterial infections of both animals and plants using intact bacteriophage. All of these efforts have severe limitations in their own utility. For cases, U.S. Pat. No. 5,688,501 reveals a method fortreating an infectious disease of animals using intact bacteriophage specific for the bacterial causal agent of that disease. U.S. Pat. No. 4,957,686 reveals a way of preventing dental caries by using intact bacteriophage specific for thebacterial causal agent of dental caries. Flaherty et al. (2000) describe a way of treating an infectious disease of plants using intact bacteriophage specific for the bacterial causal agent of that disease. In these cases and in similar casesusing undamaged bacteriophage, the bacteriophage have to attach to the bacterial host, and that attachment is highly host specific, limiting the utility of these phage to particular bacterial host species, and at times particular bacterial host strains. Inaddition, for attachment to occur, the bacteria have to be in the ideal growth stage, along with the phage needs to have the ability to gain access to the germs, which are often buried deep within tissues of either animals or plants, or shielded by bacterial biofilms,formed in part by the secretion of bacterial extracellular polysaccharides (EPS).

Efforts are made to take care of Erwinia amylovora bacterial diseases of pear and apple trees through the use of transgenic plants expressing an extracellular polysaccharide (EPS) degrading enzyme, EPS-depolymerase, based on an E.amylovora phage. However, the amount of resistance achieved was weak, at best, along with the phage EPS-depolymerase was quite specific for the EPS out of E. amylovora. More efficacious, and more generally applicable, approaches are obviously needed.

Attempts have been made to treat gram-positive bacterial diseases of animals, but not plants, by use of lytic enzyme preparations extracted from bacteriophage infected bacteria or by bacteria expressing bacteriophage genes. These, too, haveserious limitations. By Way of Example, U.S. Pat. No. 5,985,271 reveals a method of treating an animal disease brought on by a particular gram positive bacterium, Streptococcus, by use of a crude specific endolysin preparation. Likewise U.S. Pat. No.6,017,528 reveals a method of preventing and treating Streptococcus infection of animals by use of a crude specific endolysin preparation. Similarly, WO 01/90331 and US 2002/0058027 disclose methods of treating and preventing Streptococcus infectionof animals by use of a purified preparation composed of a particular endolysin. In each of these scenarios, the enzyme preparations have to be processed, buffered, prepared for delivery to the goal areas and maintained at the target site. In addition, theenzyme has to be able to gain entry to the infecting bacteria, and be present in sufficient quantity to kill the bacteria that are growing. None of these approaches would be helpful in the treatment of gram negative bacteria, because the endolysins can notpenetrate the outer membrane of such bacteria.

Attempts have been made to treat both gram-positive and gram-negative bacterial infections of animals, but not plants, using lytic enzyme preparations extracted from bacteriophage infected germs or from bacteria expressing bacteriophagegenes. WO 01/51073, WO 01/82945, WO 01/019385, US 2002/0187136 and US 2002/0127215 disclose ways of preventing and treating a variety of gram positive and gram negative bacterial diseases of animals using lytic enzymes which may optionallyinclude specific”holin lytic enzymes” or”holin enzymes”.

Since holins are not proven to exhibit adrenal function, and because examples of these holin lytic enzymes are not shown or educated in WO 01/51073, WO 01/82945, WO 01/19385, US 2002/0187136 and US 2002/0127215, these enzymes seem torepresent a theoretical and undemonstrated receptor characterized by reference to some desired characteristic or property. As correctly stated elsewhere by the same inventors:”Holin doesn’t have any behavioral activity” (refer WO 01/90331, page 9 line 12). Lytic enzymes,which form the basis for the methods revealed in all of these PCT books, are internally defined:”The present invention is based upon the discovery which phage lytic enzymes specific to bacteria infected with a certain phage can effectively andefficiently break down the cell wall of the bacterium in question. At precisely the exact same time, the substrate for the enzyme isn’t present in mammalian cells” (WO 01/51073 paragraph 3, page 4). “The lytic enzymes created by bacterial phages are particular andeffective for killing pick bacteria.” (paragraph 2, page 7).

The term”holin enzyme” as used in Claim #3 WO 01/51073 describes the enzymes found in Claim #1 as”the category composed of lytic enzymes, altered lytic enzymes and combinations thereof.” Similar references in the promises of WO 01/82945, WO01/019385 and US 2002/0187136 and US 2002/0127215 may be found. None of those patent applications disclose or assert the usage of holin or other phage derived proteins which lack enzymatic action in any manner, including the formulation of a chemical ormethod of treatment of animal or plant diseases.

WO 02/102405 discloses a way of preventing food poisoning in animals by inclusion of a purified preparation composed of specific lytic enzymes and optionally, certain lytic”holin enzymes”. Again, since holins aren’t known to exhibitenzymatic work, it is unclear regarding what is educated or specified in the promises, other than a theoretical and undemonstrated enzyme defined by reference to a desired trait or property.

It has been suggested that a specific endolysin in the bacteriophage that strikes a gram negative bacterial plant pathogen may be successful in providing immunity to that pathogen if the endolysin gene were cloned and expressed in plants(Ozawa et al., 2001). This proposal is most unlikely, since endolysins aside from T4 lysozyme aren’t known to permeate bacterial membranes, and Gram-negative bacteria have a distinguishing outer membrane, the LPS barrier, that provides a strongenvironmental barrier that is impermeable to many atoms.

It’s been shown that a gene from a bacteriophage infecting Ralstoma solanacearum encodes a lytic peptide that is capable of lysing many R. solanacearum strains (Ozawa et al. 2001). These authors indicated that this lytic peptide ofundisclosed sequence may be utilised to enhance resistance against R. solanacearum in transgenic tobacco plants. But, there’s absolutely no teaching or suggestion this lytic peptide has bacteriocidal or bacteriostatic ability against some other germs otherthan certain strains of R. solanacearum. Indeed, this evidently species-specific lytic peptide was expressed in E. coli without report of harm to the producing E. coli strains (Ozawa et al. 2001. This isn’t surprising, since phage are highlyspecific due to their bacterial host strains, and are normally limited in host array to some small subset of strains within a given host species. Methods are urgently needed to enhance resistance of plants from a broader variety of pathogenic germs than afew breeds of one species that was parasitic.

In all previously published cases wherein phage genes are reported or suggested to be used in a transgenic approach, the phage genes either encoded enzymes or, in one case, a highly species specific lytic peptide. In all previously printed caseswherein phage preparations are incorporated, described or used, enzymes or enzyme preparations are included. These enzymes have to be refined, buffered, prepared for delivery to the target regions and maintained at the target website.

Thus, the prior art fails to teach or explain the identification or use of phage proteins with wide anti-microbial action against Gram-negative bacteria. The prior art fails to teach the usage genes encoding phage proteins with wideanti-microbial action against Gram-negative bacteria. In particular, the prior art fails to teach using phage proteins which are capable of destabilizing or permeabilizing the outer bacterial membrane (the bacterial lipopolysaccharide or LPSbarrier) for the control of Gram negative bacterial infections of plants.

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