Patent Application
20150173357
This document relates to materials and methods for stimulating or inhibiting germination of Paenibacillus larvae spores, including methods for inhibiting P. larvae germination to reduce foulbrood in honey bees.
Honey bee pollination is crucial to a variety of crops, but honey bees are afflicted by a variety of pests and diseases. American foulbrood (AFB), a disease caused by Paenibacillus larvae, is arguably the most devastating of these diseases. AFB results in multi-million losses to the beekeeping industry, and is a likely contributing factor to Colony Collapse Disorder.
AFB kills developing honey bee larvae (White, “The bacteria of the apiary, with special reference to bee diseases,” GPO, Washington, 1906; and Broodsgaard et al., Apidologie 29:569-578, 1998). P. larvae spores are the infectious agent for AFB, but it is the vegetative cells that cause disease (Genersch, J. Invertebr. Pathol., 103:S10-S19, 2010; and Tarr, Ann. Appl. Biol., 25:807-814, 1938).
In 2005, a survey of almond pollinating bee colonies indicated 4% of colonies had significant AFB load (Eischen and Graham, Am. Bee. J., 145:390-391, 2005). Once a beekeeping operation is contaminated, the bacterial spores are not easily removed (Shimanuki, “Identification and control of honey bee diseases,” U.S. Dept. of Agriculture, Washington D.C., 1983). Although autoclaving and high concentrations of chemical disinfectants effectively kill spores, these treatments are not viable for the bee keeping industry (Dobbelaere et al., J. Appl. Microbiol., 91:212-216, 2001). In the United States, AFB typically is treated with prophylactic use of antibiotics (e.g., terramycin). However, the spore stage of P. larvae is not affected by antibiotic treatment, and the use of antibiotics can lead to resistant strains (Alippi, Vet. Microbiol., 125:290-303, 2007; and Lodesani and Costa, Bee World, 86:102-109, 2005). Further, antibiotic treatment can leave residue in wax and honey, and many countries ban the use of antibiotics. Thus, burning of infected colonies and beekeeping equipment has been the only accepted practice for controlling the spread of AFB (Genersch, supra; and Shimanuki, supra).
This document is based in part on the discovery that compounds such as indole and phenol can inhibit germination of P. larvae spores. In addition, this document provides materials and methods that can be used to inhibit (e.g., reduce or prevent) germination of P. larvae spores. For example, the materials and methods described herein can be used to reduce or prevent the rate of P. larvae spore germination, and thus can reduce the risk of developing P. larvae-associated disease or reduce existing P. larvae-associated disease. In some cases, for example, the materials and methods provided herein can be used to treat or prevent foulbrood in honey bees.
In one aspect, this document features a method for preventing germination of a Paenibacillus larvae spore. The method can include contacting the spore with:
(a) a compound of Formula (I):
or a pharmaceutically acceptable salt thereof, where each R is independently selected from the group consisting of: substituted or unsubstituted (C1-C6)alkyl, substituted or unsubstituted (C2-C6)alkenyl, substituted or unsubstituted (C2-C6)alkynyl, halo, (C1-C6)haloalkyl, ORA, CORA, COORA, OCORA, CN, NO2, NRARB, and NRACORB; each RA and RB are independently selected from the group consisting of H and (C1-C6)alkyl; and n is an integer from 0 to 6; or
(b) a compound of Formula (II):
or a pharmaceutically acceptable salt thereof, where R1 is selected from the group consisting of H and (C1-C6)alkyl; each R2 is independently selected from the group consisting of: substituted or unsubstituted (C1-C6)alkyl, substituted or unsubstituted (C2-C6)alkenyl, substituted or unsubstituted (C2-C6)alkynyl, halo, (C1-C6)haloalkyl, ORA, CORA, COORA, OCORA, CN, NO2, NRARB, and NRACORB; each RA and RB are independently selected from the group consisting of H and (C1-C6)alkyl; and n is an integer from 0 to 5. The spore can be contacted with the compound in an amount effective to prevent germination of the spore.
The method can include contacting the spore with a compound of Formula (I), where n is 1; contacting the spore with a compound of Formula (I), where n is 1 and R is halo; contacting the spore with a compound of Formula (I), where R is a substituted or unsubstituted (C1-C6)alkyl; contacting the spore with a compound of Formula (I), where R is an unsubstituted (C1-C6)alkyl; or contacting the spore with a compound of Formula (I), where R is CH3. The method can include contacting the spore with a compound of Formula (I), where the compound of Formula (I) is selected from the group consisting of:
or a pharmaceutically acceptable salt thereof. The method can include contacting the spore with a compound of Formula (II), where n is 0, or contacting the spore with a compound of Formula (II), where R1 is H. The method can include contacting the spore with a compound of Formula (II), where the compound of Formula (II) is:
or a pharmaceutically acceptable salt thereof.
In another aspect, this document features a composition containing a carrier and:
(a) a compound of Formula (I):
or a pharmaceutically acceptable salt thereof, where each R is independently selected from the group consisting of: substituted or unsubstituted (C1-C6)alkyl, substituted or unsubstituted (C2-C6)alkenyl, substituted or unsubstituted (C2-C6)alkynyl, halo, (C1-C6)haloalkyl, ORA, CORA, COORA, OCORA, CN, NO2, NRARB, and NRACORB; each RA and RB are independently selected from the group consisting of H and (C1-C6)alkyl; and n is an integer from 0 to 6; or
(b) a compound of Formula (II):
or a pharmaceutically acceptable salt thereof, where R1 is selected from the group consisting of H and (C1-C6)alkyl; each R2 is independently selected from the group consisting of: substituted or unsubstituted (C1-C6)alkyl, substituted or unsubstituted (C2-C6)alkenyl, substituted or unsubstituted (C2-C6)alkynyl, halo, (C1-C6)haloalkyl, ORA, CORA, COORA, OCORA, CN, NO2, NRARB, and NRACORB; each RA and RB are independently selected from the group consisting of H and (C1-C6)alkyl; and n is an integer from 0 to 5.
The composition can include a compound of Formula (I), where n is 1; a compound of Formula (I), where n is 1 and R is halo; a compound of Formula (I), where R is a substituted or unsubstituted (C1-C6)alkyl; a compound of Formula (I), where R is an unsubstituted (C1-C6)alkyl, or a compound of Formula (I), where R is CH3. The composition can contain a compound of Formula (I), where the compound of Formula (I) is selected from the group consisting of:
or a pharmaceutically acceptable salt thereof. The composition can contain a compound of Formula (II), where n is 0; or a compound of Formula (II), where R1 is H. The composition can contain a compound of Formula (II), where the compound of Formula (II) is:
or a pharmaceutically acceptable salt thereof.
In another aspect, this document features a method for reducing germination of Paenibacillus larvae spores in honey bee larvae. The method can include administering to the honey bee larvae a composition comprising:
(a) a compound of Formula (I):
or a pharmaceutically acceptable salt thereof, where each R is independently selected from the group consisting of: substituted or unsubstituted (C1-C6)alkyl, substituted or unsubstituted (C2-C6)alkenyl, substituted or unsubstituted (C2-C6)alkynyl, halo, (C1-C6)haloalkyl, ORA, CORA, COORA, OCORA, CN, NO2, NRARB, and NRACORB; each RA and RB are independently selected from the group consisting of H and (C1-C6)alkyl; and n is an integer from 0 to 6; or
(b) a compound of Formula (II):
or a pharmaceutically acceptable salt thereof, where R1 is selected from the group consisting of H and (C1-C6)alkyl; each R2 is independently selected from the group consisting of: substituted or unsubstituted (C1-C6)alkyl, substituted or unsubstituted (C2-C6)alkenyl, substituted or unsubstituted (C2-C6)alkynyl, halo, (C1-C6)haloalkyl, ORA, CORA, COORA, OCORA, CN, NO2, NRARB, and NRACORB; each RA and RB are independently selected from the group consisting of H and (C1-C6)alkyl; and n is an integer from 0 to 5. The composition can be administered in an amount effective to reduce germination of P. larvae spores in the honey bee larvae.
The method can include administering a compound of Formula (I), where n is 1; administering a compound of Formula (I), where n is 1 and R is halo; administering a compound of Formula (I), where R is a substituted or unsubstituted (C1-C6)alkyl; administering a compound of Formula (I), where R is an unsubstituted (C1-C6)alkyl, or administering a compound of Formula (I), where R is CH3. The method can include administering a compound of Formula (I), where the compound of Formula (I) is selected from the group consisting of:
or a pharmaceutically acceptable salt thereof. The method can include administering a compound of Formula (II), where n is 0; or administering a compound of Formula (II), where R1 is H. The method can include administering a compound of Formula (II), where the compound of Formula (II) is:
or a pharmaceutically acceptable salt thereof.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
FIGS. 6A-6D are graphs plotting the inhibition activity of 5-chloroindole on P. larvae cells. P. larvae cells were grown in medium containing different concentrations of 5-chloroindole, and cellular growth was monitored over a 24 hour period. Graphs are shown for 6 hours (
AFB occurs when first or second instar larvae (within 48 hours after the egg hatches) ingest food contaminated with bacterial spores (Crailsheim and Riessberger-Galle, Apidologie, 32:91-103, 2001). Twelve hours after ingestion, P. larvae spores germinate, and the new vegetative cells begin proliferating inside the larval gut (Yue et al., Environ. Microbiol., 10:1612-1620, 2008). Several days post-infection, bacteremia causes the death of the honey bee larva (Davidson, J. Invertebr. Pathol., 21:53-61, 1973; Genersch et al., Appl. Environ. Microbiol., 71:7551-7555, 2005; and Genersch et al., Int. J. Syst. Evol. Microbiol., 56:501-511, 2006). After nutrients are depleted, P. larvae cells stop dividing and sporulate. As a result, billions of spores are found in the remains of each bee larva (Lindstrom et al., J. Invertebr. Pathol., 99:82, 2008; and Sturtevant, J. Agric. Res., 45:257, 1932). Spores are transmitted within the colony by adult bees that eat larval remains (Fries and Camazine, Apidologie, 32:199-214, 2001; and Gillard et al., J. Invertebr. Pathol., 99:92-95, 2008). In addition, P. larvae spores can be transmitted between colonies by contaminated beekeeping equipment, and by robbing of honey from infected colonies by neighboring bees (Fries et al., Vet. Microbiol., 114:269-274, 2006).
The environmental cues required to trigger P. larvae spore germination have not previously been elucidated. As described herein, experiments conducted to test the ability of various compounds to promote or inhibit P. larvae spore germination showed that P. larvae spores can recognize L-tyrosine and uric acid as co-germinants, while indole and phenol (the side chains of tryptophan and tyrosine), and several derivatives thereof, can inhibit germination.
This document provides, inter alia, materials and methods that can be used to inhibit (e.g., reduce or prevent) germination of P. larvae spores. For example, the materials and methods described herein can be used to reduce or prevent the rate of P. larvae spore germination, and thus can reduce the risk of developing P. larvae-associated disease or reduce existing P. larvae-associated disease. In some cases, such materials and methods can be used to treat or prevent foulbrood in honey bees, and can include administering to a subject (e.g., a honey bee larva) an effective amount of a germination-inhibiting compound derived from phenol or indole. The compound derived from indole or phenol can have a central core structure of the formula:
As used herein, the term “disease” refers to any abnormal condition that impairs physiological function and is associated with specific symptoms. The term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition or syndrome in which physiological function is impaired, irrespective of the nature of the etiology (or even whether the etiological basis for the disease is established).
As used herein, the term “P. larvae-associated disease” refers to any disease that involves (e.g., is caused, exacerbated, associated with, or characterized by the presence of) P. larvae residing and/or replicating in the body of a subject. Thus, the term encompasses any disease, disorder, pathology, symptom, clinical condition, or syndrome in which bacteria of the species P. larvae act as etiological agents or in which infection with one or more strains of P. larvae is implicated, detected or involved.
The terms “treatment” and “treating” refer to an intervention (e.g., the administration of an agent to a subject) that prevents, slows, or delays the onset or progression of a disease or reduces (e.g., eradicates) its incidence within a treated subject (e.g., a honey bee).
The term “minimum inhibitory concentration” or “MIC” defines the lowest concentration of a test compound that is needed to inhibit germination of a spore in vitro or in vivo. A common method for determining the MIC of an antibiotic is to prepare several tubes containing serial dilutions of the test compound that are then inoculated with the bacterial isolate of interest. Following incubation at appropriate atmosphere and temperature, the MIC of an antibiotic can be determined from the tube with the lowest concentration that shows no turbidity.
As used herein, an “effective amount,” “therapeutically effective amount,” or “prophylactically effective amount” of a compound is an amount that is sufficient to provide the desired effect (e.g., treatment or prophylaxis manifested by a permanent or temporary improvement in a subject's condition, such as reduced germination of P. larvae spores in honey bee larvae), and that can be administered without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Standard dosing techniques can be used to determine an appropriate “effective” amount for use in a subject or population of subjects. In some cases, for example, an effective amount of a compound as described herein can include the MIC of that compound or an amount at least 10 percent higher than the MIC (e.g., an amount 10 to 150 percent, 10 to 125 percent, 10 to 100 percent 10 to 75 percent, 20 to 150 percent, 30 to 150 percent, 30 to 100 percent, 50 to 150 percent, or 50 to 100 percent higher than the MIC). In some embodiments, an effective amount can include at least the IC50 for that compound (see, e.g., the Examples below), or an amount at least 10 percent higher than the IC50 (e.g., an amount 10 to 150 percent, 10 to 125 percent, 10 to 100 percent 10 to 75 percent, 20 to 150 percent, 30 to 150 percent, 30 to 100 percent, 50 to 150 percent, or 50 to 100 percent higher than the IC50).
Pharmaceutically acceptable derivatives as applied to the compounds provided herein are compounds that are obtained (or obtainable) by chemical derivatization of a parent compound (e.g., indole or phenol). Pharmaceutically acceptable derivatives are therefore suitable for administration to or use in contact with mammalian tissues without undue toxicity, irritation or allergic response (i.e., commensurate with a reasonable benefit/risk ratio). Derivatives can be obtained (or obtainable) by, for example, alkylation, esterification, or acylation of parent compounds. Derivatives may be active per se, or may be inactive until processed in vivo. In the latter case, the derivatives can act as prodrugs (e.g., covalently bonded compounds that release the active parent drug after cleavage of the covalent bond(s) in vivo). Pharmaceutically acceptable derivatives typically retain some or all of the activity of the parent compound. In some cases, activity can be increased by derivatization. Derivatization also can augment other biological activities of the parent compound, such as bioavailability.
The term “pharmaceutically acceptable salt” refers to the relatively non-toxic, inorganic and organic acid addition salts of a compound provided herein. These salts can be prepared in situ during the final isolation and purification of a compound provided herein, or by separately reacting the compound in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, laurylsulphonate salts, and amino acid salts, and the like. See, for example, Berge et al., J. Pharm. Sci., 66:1-19, 1977.
In some embodiments, a compound provided herein may contain one or more acidic functional groups and, thus, is capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term “pharmaceutically acceptable salts” in these instances refers to the relatively non-toxic inorganic and organic base addition salts of a compound provided herein. These salts can likewise be prepared in situ during the final isolation and purification of the compound, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary, or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts, and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like (see, for example, Berge et al., supra).
The term “pharmaceutically acceptable solvate” as applied to the compounds provided herein defines any pharmaceutically acceptable solvate form of a specified compound that retains the biological effectiveness of such compound. Examples of solvates include compounds in combination with water (hydrates), isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, ethanolamine, or acetone. Also included are miscible formulations of solvate mixtures, such as a compound in combination with an acetone and ethanol mixture. In some embodiments, a solvate can include a compound in combination with about 20% ethanol and about 80% acetone. Thus, the structural formulae include compounds having the indicated structure, including the hydrated as well as the non-hydrated forms.
The term “pharmaceutically acceptable prodrug” as applied to the compounds described herein defines any pharmaceutically acceptable compound that can be converted under physiological conditions or by solvolysis to the specified compound, to a pharmaceutically acceptable salt of such compound, or to a compound that shares at least some of the activity of the specified compound (e.g., exhibiting activity against germination of P. larvae spores).
The term “pharmaceutically acceptable metabolite” as applied to the compounds described herein defines a pharmacologically active product produced through metabolism in the body of the specified compound or salt thereof
Prodrugs and active metabolites of the compounds useful in the methods described herein can be identified using routine techniques known in the art (see, for example, Bertolini et al., J. Med. Chem., 40:2011-2016, 1997).
The term “pharmaceutically acceptable complex” as applied to the compounds provided herein defines compounds or compositions in which the compound forms a component part. Such complexes include derivatives in which a compound as described herein is physically associated (e.g., by covalent or non-covalent bonding) to another moiety or moieties. The term therefore includes multimeric forms of the compounds provided herein. Such multimers can be generated by linking or placing multiple copies of a compound in close proximity to each other (e.g., via a scaffolding or a carrier moiety).
In some embodiments, this document provides methods for preventing germination of a P. larvae spore. The methods can include contacting a P. larvae spore with an amount of indole, phenol, or a derivative thereof (e.g., a compound of Formula (I) or Formula (II) as described herein) that is effective to prevent germination of the spore. In some embodiments, the spore can be contacted in vitro, such as by including indole, phenol, or a derivative thereof in a culture medium containing the spore. In some embodiments, the methods provided herein can be used for inhibiting germination of P. larvae spores in vivo (e.g., in honey bee larvae). Thus, in some cases, the method can include providing honey bee larvae with a composition (e.g., a nutritional medium such as worker jelly or royal jelly) containing an amount of indole, phenol, or a derivative thereof, that is effective to prevent germination of P. larvae spores. Nutritional media for honey bees can be obtained commercially, for example, and supplemented with a compound as described herein. In some embodiments, a method for inhibiting germination of P. larvae spores can include contacting one or more spores with a compound of Formula (I):
or a pharmaceutically acceptable salt thereof. Each R can be independently selected from the group consisting of: substituted or unsubstituted (C1-C6)alkyl; substituted or unsubstituted (C2-C6)alkenyl; substituted or unsubstituted (C2-C6)alkynyl; halo; (C1-C 6)haloalkyl; ORA; CORA; COORA; OCORA; CN; NO2; NRARB; and NRACORB. Each RA and RB can be independently selected from the group consisting of H and (C1-C6)alkyl. N can be an integer from 0 to 6 (e.g., 0, 1, 2, 3, 4, 5, or 6). In some embodiments, for example, R can be a substituted or unsubstituted (C1-C6)alkyl (e.g., CH3). Thus, in some embodiments, the methods provided herein can include contacting one or more P. larvae spores with a compound selected from the group consisting of:
or a pharmaceutically acceptable salt thereof.
In some embodiments, a method for inhibiting germination of P. larvae spores can include contacting one or more spores with a compound of Formula (II):
or a pharmaceutically acceptable salt thereof. R1 can be selected from the group consisting of H and (C1-C6)alkyl. Each R2 can be independently selected from the group consisting of: substituted or unsubstituted (C1-C6)alkyl; substituted or unsubstituted (C2-C6)alkenyl; substituted or unsubstituted (C2-C6)alkynyl; halo; (C1-C6)haloalkyl; ORA; CORA; COORA; OCORA; CN; NO2; RARB; and NRACORB. Each RA and RB can be independently selected from the group consisting of H and (C1-C6)alkyl. N can be an integer from 0 to 5 (e.g., 0, 1, 2, 3, 4, or 5). In some embodiments, the methods provided herein can include contacting one or more P. larvae spores with the compound:
or a pharmaceutically acceptable salt thereof.
As used herein, the term “Cx-yalkyl” refers to saturated hydrocarbon groups, including straight-chain alkyl and branched-chain alkyl groups that contain from x to y carbons in the chain. The terms “C2-yalkenyl” and “C2-yalkynyl” refer to unsaturated aliphatic groups analogous in length to the alkyls described above, but that contain at least one double or triple bond, respectively.
As used herein, “halo” is a chloro, bromo, fluoro or iodo atom radical.
As used herein, “haloalkyl” means a hydrocarbon substituent, which is a linear or branched alkyl substituted with one or more chloro, bromo, fluoro, and/or iodo atom(s). In some embodiments, haloalkyls are of 1 to about 3 carbons in length. For example, a haloalkyl can be CF3.
The term “substituted” refers to moieties having substituents replacing a hydrogen on one or more non-hydrogen atoms of the molecule. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. Substituents can include, for example, an alkyl, an alkene, an alkyne, a halo, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), an alkoxyl, an amino, an amido, an amidine, an imine, a cyano, a nitro, or an azido, moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate.
This document also provides compositions containing one or more compounds described herein (e.g., indole, phenol, or a derivative thereof) in combination with a carrier. Suitable carriers include, without limitation, water, buffered water, saline (e.g., 0.8% saline), glycine (e.g., 0.3% glycine), hyaluronic acid, and the like. The compositions also may contain auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, sodium hydroxide, etc.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
Materials: Chemicals were purchased from Sigma Aldrich Corporation (St. Louis, Mo.). Dehydrated culture media was purchased from BD Difco (Franklin Lakes, NJ.). Paenibacillus larvae subsp. pulvifaciens strain ATCC 49843 was purchased from the American Tissue Culture Collection (ATCC). Environmental American Foulbrood scales (the remains of infected larvae collected from infected hives) were kindly donated by Dr. Jay D. Evans at the USDA Bee Research Facility in Beltsville, Md. The environmental strain was identified as a strain of Paenibacillus larvae subsp. larvae based on its phenotypic characteristics and 16S rRNA analysis (Piccini et al., World J. Microb. Biot., 18:761-765, 2002).
P. larvae spore preparation: P. larvae strains were grown on BD tryptic soy agar plates for 7 days in a 5% CO2 incubator at 37° C. The resulting bacterial lawns were collected by flooding with ice-cold deionized water. Spores were pelleted by centrifugation and resuspended in fresh deionized water. After three washing steps, spores were separated from vegetative and partially sporulated forms by centrifugation through a 20%-50% HistoDenz gradient. The spore pellet was washed five times with water and stored at 4° C. (Akoachere et al., J. Biol. Chem., 282:12112-12118, 2007). Spore preparations were more than 90% pure as determined by microscopic observation of Schaeffer-Fulton stained samples (Schaeffer and Fulton, Science 77:194-194, 1933).
Preparation of germinant solution: Sixteen complex media (MYPGP, TSB, BHI, Nutrient, LB, TMYGP, NZ amine, NZCYM, Lactobacillus, SOC, Bailey, Clostridium, Michael, Terrific, MD, and J broths) were prepared (Bailey and Lee, J. Gen. Microbiol., 29:711-717, 1962; Dingman and Stahly Appl. Environ. Microbiol., 46:860-869, 1983; and Zimbro, Difco & BBL manual : Manual of microbiological culture media. Becton, Dickinson and Company, Sparks, Md., 2009). A defined medium was prepared as previously described (Ramirez and Abel-Santos, J. Bacteriol., 192:418-425, 2010). An artificial worker jelly (AWJ) medium was prepared based on modifications to the published composition of worker jelly (Rembold and Dietz, Vitam. Horm., 23:359-382, 1966). For AWJ, the following stock solutions were prepared: 100 mM inosine in 220 mM NaOH, 400 mM for each sugar (fructose, glucose, and arabinose) in water, 30 mM for each of the 20 proteinogenic L-amino acids in 0.36 N HCl, 100 mM uric acid in 220 mM NaOH and 0.2 mg/ml vitamins (thiamine, riboflavin, pyridoxine, β-alanine, para-aminobenzoic acid, nicotinic acid, pantothenic acid, biotin, folic acid, and inositol) in water. To prepare AWJ, inosine, uric acid, sugars, and amino acids were dissolved to 3 mM final concentration in 0.1 M sodium phosphate buffer (0.06 mM Na2HPO4 and 0.04 mM NaH2PO4) and adjusted to pH 7.0. This solution was supplemented with vitamins to 1 μg/ml final concentration.
Determination of germinants for P. larvae spores: The decrease in optical density is proportional to spore germination (Powell, J. Gen. Microbiol., 4:330-338, 1950). Changes in light diffraction during spore germination were monitored at 580 nm (OD580) on a Biomate 5 (ThermoElectron Corporation, Waltham, Mass.) or a Tecan Infinite m200 (Tecan group, Männedorf, Switzerland) spectrophotometer. Experiments were carried out in 96-well plates (200 μL/well). In preparation for germination assays, P. larvae spore suspensions were washed three times with water. Spores were then heat activated at 70° C. for 30 minutes. The heat-activated spores were allowed to reach room temperature and transferred to 0.1 M sodium phosphate buffer (pH 7.0) to an approximate OD580 of 0.70. Spores were monitored for auto-germination for 30 minutes. Germination experiments were carried out with spores that did not auto-germinate. Putative germinants were added individually or in combinations to a final concentration of 3 mM. Experiments were performed in triplicate with at least two different spore preparations. After germinant addition, OD580 of the spore suspension was measured every minute for an hour. Relative OD values were derived by dividing each OD580 reading by the initial OD580. Spore germination rates (v) were calculated from the initial linear decrease in relative OD (Akoachere et al., supra). Germination rates were set to 100% for P. larvae spores that had the fastest germination rate in an assay. Germination rates for other conditions were divided by the maximum germination rate for that assay and are reported as percent germination. Standard deviations were calculated from at least six independent measurements and are typically below 10%. Spore germination was confirmed in selected samples by microscope observation of Schaeffer-Fulton stained aliquots (Schaeffer and Fulton, supra).
Effect of temperature and pH on P. larvae germination: For temperature experiments, P. larvae spores were germinated in 3 mM L-tyrosine and 3 mM uric acid. Germination rates were determined as described above, except that the germination temperature was varied between 25° C. and 42° C. The germination rate was set to 100% for spores germinated at 42° C. Germination rates for other conditions were divided by the maximum germination rate at 42° C., and are reported as percent germination. Germination rate differences were analyzed using ANOVA followed by a Tukey-Kramer procedure (SigmaPlot v.9).
For pH experiments, P. larvae spores were re-suspended in 0.1 M sodium phosphate, potassium/sodium phosphate, or citrate phosphate buffer. The pH of the buffers was adjusted between 3.0 and 9.0. Spores were germinated in the presence of 3 mM L-tyrosine and 3 mM uric acid. Germination rates were determined as described above. The germination rate was set to 100% for spores germinated at pH 7.0. Germination rates for other conditions were divided by the maximum germination rate at pH 7, and are reported as percent germination. As above, germination rate differences were analyzed using ANOVA followed by a Tukey-Kramer procedure (SigmaPlot v.9).
Activation of P. larvae spore germination by L-tyrosine and uric acid: P. larvae spore germination was tested with different combinations of L-tyrosine and uric acid. For L-tyrosine titrations, spores were exposed to varying concentrations of L-tyrosine and a constant 3 mM uric acid. For uric acid titrations, spores were exposed to varying concentrations of uric acid and a constant 3 mM L-tyrosine. Germination rates were determined as above. The germination rate was set to 100% for P. larvae spores germinated in the presence of 3 mM L-tyrosine/3 mM uric acid. Germination rates for other conditions were divided by the maximum germination rate obtained with 3 mM L-tyrosine/3 mM uric acid, and are reported as percent germination. Percent germination was plotted against compound concentrations. The resulting sigmoidal curves were fitted using the four parameter logistic function of the SigmaPlot v.9 software to calculate EC50 values (for enhancers of spore germination). EC50 is defined as the concentration of a germinant required to increase the germination rate to 50% of the maximal value (Rodbard et al., Clin. Chem., 22:350-358, 1976; and Sebaugh, Pharm. Stat., 10:128-134, 2011).
Agonists of P. larvae spore germination: To test for possible agonists of P. larvae spore germination, spores were individually supplemented with 3 mM of a purine analog and 3 mM L-tyrosine. Separately, P. larvae spores were incubated with 3 mM of an amino acid analog and 3 mM uric acid. Spore germination was monitored as described above. Germination rates for other conditions were divided by the maximum germination rate obtained with 3 mM L-tyrosine/3 mM uric acid, and are reported as percent germination.
Antagonists of P. larvae spore germination: To test for possible antagonists of P. larvae spore germination, spores were individually supplemented with 3 mM of a purine analog or 3 mM of an amino acid analog. Spore suspensions were incubated for 15 minutes at room temperature while monitoring OD580. If no germination was detected, L-tyrosine and uric acid were added to 3 mM final concentrations, and germination was monitored as described above. Germination rates for other conditions were divided by the uninhibited maximum germination rate obtained with 3 mM L-tyrosine/3 mM uric acid, and are reported as percent germination.
Inhibition of P. larvae spore germination by indole and phenol: P. larvae spores were individually incubated with varying concentrations of indole, phenol, 1-N-methylindole, 3-mehtylindole, 5-methylindole, or 7-methylindole. After 15 minutes of incubation, spores were treated with 3 mM L-tyrosine/3 mM uric acid. The germination rate was set to 100% for P. larvae spores germinated in the absence of inhibitor. Germination rates for other conditions were divided by the uninhibited maximum germination rate obtained with 3 mM L-tyrosine/3 mM uric acid, and are reported as percent germination. Percent germination was plotted against inhibitor concentrations. The resulting sigmoidal curves were fitted using the four parameter logistic function in
SigmaPlot v.9 to calculate IC50 values. IC50 is the concentration of a germination inhibitor required to reduce the germination rate to 50% of the maximal value (Akoachere et al., supra; and Rodbard et al., supra).
No significant P. larvae spore germination was detected in any of the 16 different complex media tested, even after 24 hours of incubation. In comparison, spores of Bacillus anthracis and Bacillus cereus germinate within two hours in rich medium (Johnson et al., J. Food Sci., 48:286-287, 1983; and Sanz et al., Infect. Immun., 76:1036-1047, 2008). Similarly, P. larvae spores failed to germinate in defined medium containing metabolites commonly used as germinants by Bacillus and Clostridia species (
Honey bee larvae are fed royal or worker jelly, which can be contaminated with P. larvae spores (41. Winston, The biology of the honey bee, Harvard University Press, Cambridge, Mass., 1987). P. larvae spores were resuspended in a chemically defined medium (AWJ) that differs from worker jelly only in its pH value. The optical density of P. larvae spores suspended in AWJ decreased, indicating that the spores were germinating (
To identify compounds necessary to trigger P. larvae spore germination, groups of compounds were systematically omitted from the AWJ medium. P. larvae spores germinated well in the absence of sugars and vitamins, suggesting that germination onset required uric acid and proteinogenic amino acid(s). The testing of individual amino acids showed that only L-tyrosine was able to synergize with uric acid to produce a strong germination response in P. larvae spores (
To determine the effects of temperature on P. larvae spore germination, a range of 25° C. to 42° C. was tested (
Titration of L-tyrosine at a saturating uric acid concentration yielded an EC50 of 1.2 mM for L-tyrosine activation of P. larvae spore germination (
Since uric acid is a degradation product of purine catabolism, the ability of purine analogs to act as co-germinants of P. larvae spores was tested. As L-tyrosine is the only amino acid able to trigger P. larvae spore germination, its stereoisomer (D-tyrosine) and its side chain (phenol) also were tested as co-germinants with uric acid. In these experiments, however, none of the compounds tested was able to activate P. larvae spore germination.
L-tyrosine and purine analogs were also tested for their ability to inhibit P. larvae germination. None of the purine analogs tested inhibited uric acid/L-tyrosine-induced germination of P. larvae spores. Similarly, D-tyrosine did not inhibit P. larvae spore germination. In contrast, indole (the side chain of tryptophan) and phenol (the side chain of tyrosine) both were able to inhibit P. larvae spore germination. Titrations of indole and phenol yielded an IC50 of 0.37 mM for indole (
To test the generality of P. larvae spore response, spores were prepared from P. larvae subsp. pulvifaciens strain ATCC 49843, and also from an environmental AFB sample. Based on phenotypic characteristics, the environmental sample was identified as P. larvae subsp. larvae (Genersch et al., Appl. Environ. Microbiol., 71:7551-7555, 2005; and Genersch et al., Int. J. Syst. Evol. Microbiol., 56:501-511, 2006). Spores of both P. larvae sub-species responded identically to L-tyrosine and uric acid, and their germination was similarly inhibited by indole and phenol.
P. larvae spores were incubated with various concentrations of indole analogs for 15 minutes prior to addition of 3 mM urea and 3 mM L-tyrosine. IC50 was calculated by plotting percent germination vs. indole analog concentration. Standard deviations are shown in parentheses. N/A, No activity under the conditions tested.
Materials: Chemicals were from the Sigma-Aldrich Corporation (St. Louis, Mo.) and VWR International (Radnor, Pa.). The dehydrated culture medium was obtained from BD Difco (Franklin Lakes, N.J.). Paenibacillus larvae subsp. pulvifaciens strain
ATCC 49843 was from the American Tissue Culture Collection (ATCC). Lyophilized royal jelly from GloryBee Foods (Eugene, Oreg.) was stored at −20° C. until used for diet preparation.
P. larvae spore preparation: P. larvae strains were grown on BD tryptic soy agar plates for 7 days in a 5% CO2 incubator at 37° C. The resulting bacterial lawns were collected by flooding with ice-cold deionized water. Spores were pelleted by centrifugation and resuspended in fresh deionized water. After three washing steps, the spores were separated from their vegetative and partially sporulated forms by centrifugation through a 20%-to-50% HistoDenz gradient. The spore pellet was washed five times with water and stored at 4° C. Spore preparations were 90% pure as determined by microscopic observation of Schaeffer-Fulton-stained samples.
Antagonists of P. larvae spore germination: To test for possible antagonists of P. larvae spore germination, spores were individually supplemented with 0.4 mM indole analog or 0.4 mM phenol analog. Spore suspensions were incubated for 15 minutes at room temperature while monitoring the OD580. If no germination was detected, L-tyrosine and uric acid were added to final concentrations of 3 mM, and germination was monitored as described above. Germination rates for other conditions were divided by the uninhibited maximum germination rate obtained with 3 mM L-tyrosine-3 mM uric acid and are reported as percent germination.
Inhibition of P. larvae spore germination by indole and phenol: P. larvae spores were individually incubated with various concentrations of indole, phenol, 1-N-methylindole, 3-methylindole, 5-methylindole, or 7-methylindole. After a 15 minute incubation, the spores were treated with 3 mM L-tyrosine-3 mM uric acid. The germination rate was set to 100% for P. larvae spores germinated in the absence of inhibitor. Germination rates for other conditions were divided by the uninhibited maximum germination rate obtained with 3 mM L-tyrosine-3mM uric acid, and are reported as percent germination. Percent germination was plotted against the inhibitor concentrations. The resulting sigmoidal curves were fitted using the four-parameter logistic function in SigmaPlot v.9 to calculate the germination 50% inhibitory concentrations (IC50 ).
Acquisition of 1st instar larvae: First instar honey bee larvae were obtained from two colonies headed by naturally mated queens (Honey Bee Genetics, Vacaville Calif.). Queens were trapped on empty frames within a cage constructed with queen excluder. Worker bees can fit through the excluder but the queen cannot exit the cage. The frame was placed within the brood chamber to stimulate egg laying by the queen overnight. Eggs were incubated in the hive for 4 days to allow newly hatched larvae to be fed by nurse bees. First instar honey bee larvae were brought into the laboratory and stored in a 35° C. 85% relative humidity chamber before grafting.
Sterile six well plates with a well volume of 17.0 ml were filled with 2.5 ml of pre-warmed artificial worker jelly. Honey bee larvae were transferred into each well using the thin tongue made of flexible plastic from a Chinese grafting tool. The plastic grafting tool was decontaminated with 70% ethanol throughout usage. Grafting was performed in the sterile environment of a honey bee laboratory, and honey bee larvae were then transferred to a bacterial spore laboratory for control and spore exposure treatments.
Artificial worker jelly preparation: Artificial worker jelly was prepared using a lyophilized royal jelly, D-glucose, D-fructose, yeast extract, and autoclaved double distilled water. Ingredients minus lyophilized royal jelly were dissolved in autoclaved double distilled water. Lyophilized royal jelly was added to this sugar-yeast extract solution and mixed thoroughly with a vortex mixer. Artificial diet was prepared an hour prior to use for larval rearing. To reduce the development of worker-queen hybrids because of nutrition, the composition and amounts of artificial worker jelly were controlled in every larval stage.
Incubation conditions: Honey bee larvae were incubated in an air tight plastic container placed inside an incubator. The desired relative humidity (95%) and temperature (35° C.) was verified with an iButton data logger (Maxim Integrated; San Jose, Calif.). The appropriate relative humidity was obtained by placing a solution of 20% glycerol by weight. The plastic containers were was decontaminated throughout usage with detergent, 70% ethanol, and treated with an ultraviolet lamp overnight.
Rearing procedure: Feeding of diets was performed daily in different amounts according to Table 2. The amount of diet consumed by larvae was assumed based on published literature. A micropipette was used to place a food drop next to the mouth of the larvae. Drowning was avoided by maintaining the appropriate humidity and water content of diet. Forty first and second instar larvae were placed into one well of a six well plate. Third instar larvae were transferred individually to 48 well plates filled with fresh food.
Larval survival was determined daily by observing signs of respiration, disease symptoms, and other abnormalities with a stereo microscope. The number of dead larvae was recorded, and surviving larvae were fed fresh food. The start of pupation was indicated by the appearance of uric acid crystals and light yellow excretions in the remaining food. Any larvae that failed to pupate were considered to have died on day seven.
Indole analog application: The concentration of indole analogs applied to the larval diet was selected based on preliminary experiments with indole. When 0.5 mM indole was incorporated into the larval diet, larvae were protected from development of AFB disease. Each indole analog was dissolved in warm autoclaved water to provide a stock solution that could be used to produce artificial worker jelly. Artificial worker jelly containing indole analogs was not frozen, because the solubility of solutes is dependent on temperature.
Survival analysis: The larval survival data was analyzed using the LogRank test in SigmaPlot. The LogRank test determines if there are significant differences between survival curves and then identifies pairs of curves that are significantly different.
Indole (IC50 0.4 mM) and phenol (IC50 0.5 mM) were previously shown to inhibit P. larvae spore germination. To identify functional groups that enhance inhibitor binding to germination receptor, and potentially identify stronger inhibitors, the effect of 33 indole and phenol structural analogues on P. larvae spore germination was tested in vitro (
The screen included indole structural analogues that consisted of a six-membered benzene ring fused to a five-membered ring. Substitution of the nitrogen at position 1 of indole with a sulfur group failed to significantly change P. larvae spore germination. The addition of nitrogen to position 2, 2+3, 3, or 7 also did not significantly affect spore germination. Further, addition of functional groups to the above-mentioned analogues did not significantly alter P. larvae spore germination. Similar effects were observed with the phenol structural analogues tested.
P. larvae spore germination was, however, significantly affected by indole analogs with electron withdrawing groups. Titration of these indole analogues at saturating uric acid/L-tyrosine concentrations were performed to obtain IC50 values (TABLE 2). The maximum effect on P. larvae spore germination was observed on indole with halides groups. The dose response assays resulted in sigmoidal curves, which passed the Durbin-Watson statistical test for autocorrelation.
5-Chloroindole was then tested in vitro, using P. larvae cells or spores. The inhibitory effect of 5-chloroindole on P. larvae cells is shown in
The inhibitory effect of 5-chloroindole on P. larvae spores is shown in
The effectiveness of various halo indole compounds were then tested in vivo by assessing their effects on survival of honey bee larvae contacted with P. larvae spores. For example,
The effect of 5-chloroindole was further studied by testing various concentrations of the compound. As shown in
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims benefit of priority from U.S. Provisional Application No. 61/919,224, filed on Dec. 20, 2013.
This invention was made with government support under NEVR-2010-03755 awarded by the United States Department of Agriculture. The government has certain rights in the invention. 2011-67013-30169 from the USDA/NIFA
| Number | Date | Country | |
|---|---|---|---|
| 61919224 | Dec 2013 | US |
