Patent Grant
7371571
The present invention relates to recombinant mycobacterial strains overexpressing essential biosynthetic enzymes of pathogenic mycobacteria and to methods for using these strains. More specifically, the present invention relates to recombinant mycobacteria strains overexpressing
The bacterial cell wall is an ideal target for drug design since similar structures and biosynthetic pathways are absent from mammalian hosts. The lipid-rich mycobacterial cell wall acts as an efficient permeability barrier (Brennan and Nikaido, 1995). Peptidoglycan, the backbone of this structure, contains the
DCS is effective against mycobacteria and is recommended to treat multidrug-resistant M. tuberculosis in the DOTS-Plus management plan (Farmer, 2001; World Health Organization, 2000). However, undesirable side effects restrict its use in human chemotherapy (Yew et al., 1993). Nonetheless, the potent bactericidal effect of DCS against mycobacteria makes this drug an attractive prototype compound to develop novel antimycobacterial agents. In addition, identification of the lethal target(s) of DCS action would allow for the rational design of new antimycobacterial drugs, structurally related or unrelated to DCS, targeting enzymes of the
Recently, we observed that M. smegmatis alr null mutants are not dependent on
In order to develop novel antimicrobial agents structurally related to DCS, the lethal target(s) of DCS need to be identified and methods of screening for inhibition need to be developed.
In this study, we investigated the roles of both Alr and Ddl in the mechanisms of action of and resistance to DCS in M. smegmatis. We demonstrate that Ddl activity is inhibited by DCS in a concentration-dependent manner. Overexpression of the ddl gene confers resistance to DCS but not to β-chloro-
The present invention is directed to recombinant mycobacterial strains which overproduce
In one aspect of the present invention, recombinant M. smegmatis strains expressing heterologous or overexpressing native Ddl are provided. In one embodiment of this aspect, the Ddl is the product of M. smegmatis ddl gene. In another embodiment, the Ddl is the product of M. tuberculosis ddl gene. In yet another embodiment, the Ddl is the product of both M. smegmatis and M. tuberculosis ddl genes.
In another aspect of the invention, purified recombinant Ddl is provided.
In a further aspect of the invention, cell-free methods for screening for antimicrobial agents which target the
In another aspect of the invention, in vitro methods for detecting bactericidal activity of compounds against pathogenic mycobacteria are provided. The methods comprise the use of overexpressing recombinant mycobacterial strains In one embodiment of this aspect, the recombinant mycobacterial strain is GPM259. In another embodiment, the recombinant strain is GPM260. In a further embodiment, the recombinant strain is GPM265.
The publications and other materials used herein to illuminate the background of the invention, and in particular cases, to provide additional details respecting the practice, are incorporated by reference and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended List of References.
The present invention employs the following definitions:
“alrA” and “alr” refer to the
“Alr” and “Alr” refer to
“Displays increased susceptibility to antimycobacterial agent” refers to a reduction in the minimal inhibitory concentration of the mutant strain when compared with the wild type strain.
“DCS” and “
“ddl” refers to the
“Ddl” refers to
“Isolated polypeptide” refers to a polypeptide produced as an expression product of an isolated and manipulated genetic sequence, even if expressed in a homologous cell type. Synthetically made forms or molecules expressed by heterologous cells are inherently isolated molecules.
In previous studies, a genomic library from a DCS-resistant mutant constructed in a multicopy plasmid was introduced into the wild-type M. smegmatis strain and clones resistant to 300 μg of DCS ml−1 were selected and isolated (Caceres et al., 1997). Using this strategy, we did not identify a recombinant clone carrying the ddl gene. Since target overproduction determines a drug resistance phenotype, this outcome was unexpected. To exclude any possible bias in this library, we also screened a M. smegmatis cosmid library in a similar manner. Surprisingly, a DCS resistant clone carrying the ddl gene was still not identified. Lowering the DCS concentration resulted in a high background of DCS-sensitive colonies. These data suggest that either the overexpression of the ddl gene is toxic to the host or that the level of overexpression is not sufficient to confer a selectable resistance phenotype under the selection conditions described. To test these hypotheses, the M. smegmatis and M. tuberculosis ddl genes were amplified from genomic DNA and cloned into the E. coli-Mycobacterium shuttle vector pMV262, carrying a kanamycin resistance marker. Recombinant plasmids were introduced into M. smegmatis, and kanamycin-resistant transformants were isolated.
In this study, we demonstrated that Ddl enzyme activities in crude extracts were inhibited by DCS in a concentration-dependent manner, similar to the effect observed for the Alr enzyme (Caceres et al., 1997). Approximately 50% of Ddl enzyme activity is inhibited by DCS at a concentration of 200 μg ml, while only 10 to 15% of the Ddl activity is inhibited at 50 μg ml, near the MIC. The apparent discrepancy reflects the difference between in vivo and in vitro conditions. In live bacilli, DCS also inhibits Alr, resulting in a limited supply of
Overexpression of either the M. smegmatis or M. tuberculosis ddl gene, using the same expression vectors as those used to overexpress Alr (Caceres et al., 1997), confers an intermediate level of resistance to DCS. Furthermore, GPM260, a recombinant strain of M. smegmatis overproducing both Alr and Ddl, was constructed and characterized. GPM260 displayed a higher level of resistance to DCS than its parent strain, GPM14, which is consistent with the increased DCS resistance levels of S. gordonii mutants with elevated Alr and Ddl activities (Reitz et al., 1967). To test the hypothesis that inhibition of Alr by DCS decreases the intracellular pool of
DCS targets both Alr and Ddl enzymes, but the lethal target for its bactericidal effect has not been identified M. smegmatis alr null mutants are not dependent on
The practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and genetics. See, e.g., Maniatis et al. (1982); Sambrook et al. (1989); Ausubel et al. (1992); Guthrie and Fink (1991); Weissbach and Weissbach (1986); Zaitlin et al. (1985) and Gelvin et al. (1990).
Method of Use: Drug Susceptibility Assays
The bactericidal activity of lead compounds are evaluated in vitro. Conventional protocols for determination of drug minimum inhibitory concentration (MIC) may be used. Example ID. employs one example of such assays. For example, the mycobacterial cells taken from glycerol stock, or preferably from a single colony, are grown in either M-ADC-TW (for faster growing strains such as M. smegmatis) or MO-ADC-TW (for slow growing strains such as M. avium or M. bovis BCG). Growth is continued in the absence of test compound until the optical density of 600 nm reaches approximately 0.1 to 1.0. Medium is supplemented with nutrients, if necessary.
Approximately 105 CFU in 0.1 ml are inoculated in triplicate onto 96-well microplates containing either serial twofold dilutions of the test compound or without, for controls. Plates are incubated at 37° C. and checked periodically for growth. MIC is determined as the lowest concentration at which there is no visible growth of bacteria. MIC is confirmed by consistent results from at least three independent cultures.
Alternatively, the bactericidal activity of the test compound can be evaluated by measuring
Method of Use: Inhibition Assays
The present invention is based on the evidence, reported in further detail herein: 1) that Ddl is a binding target of DCS and is inhibited in a concentration-dependent manner; 2) that Ddl is not significantly affected by DCS at the concentration that inhibits Alr; 3) that Alr overproduction contributes to the maintenance of the internal
Preferably, purified Ddl is obtained from M. tuberculosis ddl gene by PCR amplification and cloning into a proper expression shuttle plasmid vector, such as an E. coli expression vector endowed with a mycobacterial replication origin and/or promoter, and overexpressed in M. smegmatis. This host strain is modified by inactivation of its endogenous ddl gene, so that it only expresses the M. tuberculosis counterpart. The M. tuberculosis Ddl enzyme is purified as a polyhistidine-tagged fusion protein through a single-step affinity chromatography in large amount and its identity is verified with biochemical methods. Ddl catalyzes the reaction 2
The thin-layer chromatography (TLC)-based
Promising candidate antimycobacterial agents are further tested in vitro using M. smegmatis and M. tuberculosis reference strains (See Methods of Use: Drug Susceptibility Assays). Preferably, the overproducing strains of the present invention are used at this step to evaluate the potential bactericidal activity of the lead compounds compared with
Method of Use: Analysis of Drug Action
According to the present invention a method is provided for analyzing the in vivo metabolic change in mycobacteria upon drug treatment. The free amino acid pool is indicative of the pathways which are either shut down or activated by the test drug. In addition, the alteration of free amino acid pool, especially the abundance of L- or
Method of Use: Vaccine Development
The present invention is also useful for the development of a novel vaccine against pathogenic mycobacteria, such as, but not limited to, M. tuberculosis, M. bovis, M. africanum, M. microti, M. leprae, M. avium, M. intracellular, M. paratuberculosis, M. ulcerans, M. marinum, and subspecies and genetic variants thereof. A mycobacteria can be attenuated by selection of mutants that are better suited to growth in abnormal culture conditions and are therefore less capable of growth in the host. The attenuated mycobacterium may have inserted therein one or more DNA molecules for stimulation of an immune response directed against polypepticles encoded by the inserted nucleic acid molecule. Vaccines of the present invention may be formulated with conventional carriers and/or adjuvants.
For obvious practical and moral reasons, initial work in humans to determine the efficacy o experimental compositions with regard to such afflictions is infeasible. Accordingly, in the early development of any drug or vaccine it is standard procedure to employ appropriate animal models for reasons of safety and expense. The success of implementing laboratory animal models is predicted on the understanding that immunogenic epitopes are frequently active in different host species. Thus, an immunogenic determinant in one species, for example a rodent or guinea pig, will generally be immunoreactive in a different species such as in humans. Only after the appropriate animal models are sufficiently developed will clinical trials in humans be carried out to further demonstrate the safety and efficacy of a vaccine in man.
With regard to alveolar or pulmonary infections by M. tuberculosis, the guinea pig model closely resembles the human pathology of the disease in many respects. Accordingly, it is well understood by those skilled in the art that it is appropriate to extrapolate the guinea pig model of this disease to humans and other mammals. As with humans, guinea pigs are susceptible to tubercular infection with low doses of the aerosolized human pathogen M. tuberculosis. Unlike humans where the initial infection is usually controlled, guinea pigs consistently develop disseminated disease upon exposure to the aerosolized pathogen, facilitating subsequent analysis. Further, both guinea pigs and humans display cutaneous delayed-type hypersensitivity reactions characterized by the development of a dense mononuclear cell induration or rigid area at the skin test site. Finally, the characteristic tubercular lesions of humans and guinea pigs exhibit similar morphology including the presence of Langhans giant cells. As guinea pigs are more susceptible to initial infection and progression of the disease than humans, any protection conferred in experiments using this animal model provides a strong indication that the same protective immunity may be generated in man or other less susceptible mammals. Those skilled in the art will appreciate that the present invention may be practiced with various mammalian hosts including humans and domesticated animals.
A strain of mycobacteria having a regulatable promoter to control the expression of the ddl gene, whose product is responsible for the synthesis of
The mycobacterial strain used may be a wild-type mycobacterial strain or, alternatively, an alr null mutant mycobacterial may be used, such as the alr mutant described in Appl'n Ser. No. U.S. 2003/0133952.
The present invention is further described in the following examples, which are offered by way of illustration and are not intended to limit the invention in any manner. Standard techniques known in the art or the techniques specifically described below are utilized.
Materials and Methods
A. Bacterial Strains, Plasmids, and Culture Conditions
Bacterial strains and plasmids used in this example are listed in Table 1. E. coli strains were grown at 37° C. in Luria-Bertani broth or agar. M. smegmatis strains were grown at 37° C. with shaking (200 rpm; Innova 4300 incubator shaker; New Brunswick Scientific, Edism, N.J.) in Middlebrook 7H9 broth (BBL Microbiology Systems, Cockeysville, Md.) supplemented with albumin-dextrose complex (ADC) and 0.05% Tween 80 (M-ADC-TW broth). For analysis of the intracellular amino acid pools, M. smegmatis was grown in a minimal medium based on the formulation of Zygmunt (1963) and modified as previously described (Chacon et al., 2002). Tryptic soy agar base (Difco Laboratories, Detroit, Mich.) was used for growth of M. smegmatis on solid media. Transformations of E. coli and M. smegmatis were performed by electroporation with a Gene Pulser (Bio-Rad Laboratories, Richmond, Calif.) as described previously (Foley-Thomas et al., 1995). The following antibiotics were added to the media when necessary: kanamycin (Sigma Chemical Co., St. Louis, Mo.) at 50 μg ml−1 or carbenicillin (Sigma) at 50 μg ml−1 for E. coli strains and kanamycin at 10 μg ml−1 for M. smegmatis strains.
E. coli XL10-
M. smegmatis
M. smegmatis
M. smegmatis
M. smegmatis
M. smegmatis
M. smegmatis
M. smegmatis
E. coli-Mycobacterium shuttle plasmid
M. tuberculosis ddl gene
M. smegmatis ddl gene
B. Oligonucleotide primers, nucleic acid manipulations, and primer extension analysis.
All oligonucleotide primers were from Integrated DNA Technologies, Inc., Coralville, Iowa. For PCR amplification of the complete ddl gene from M. smegmatis were SMDDLCF (5′-CGC ATA AGG CCA GGT CAG-3′(SEQ ID NO:1) and SMDDLCR (5′-CGA AAA ACC CGT CGT GC-3′(SEQ ID NO:2). The primers for PCR amplification of the ddl gene from M. tuberculosis were DDLATBU (5′-GCT AAG TGC CGA TCG CAA G-3′(SEQ ID NO:3) and DDLATBD (5′-ATA ACG CTG CTG CTG GGT C-3′(SEQ ID NO:4) and TBDDLEXF (5′-CGG GAT CCG TGA GTG CTA ACG AC-3′(SEQ NO:5) and TBDDLEXR (5′-CGG AAG CTT GTG CCG ATC GCA AGC-3′(SEQ ID NO:6). The primers SMDDLPE (5′-AAA CGC TCC GGA TCG AGG TTG-3′(SEQ ID NO:7) and TBDDLPE (5′-GAG ATG GCG TGC TCG TTG-3′(SEQ ID NO:8) were used in primer extension analysis for the ddl mRNA of M. smegmatis and M. tuberculosis, respectively. PCR amplifications were performed in a Perkin-Elmer GeneAmp 9600 thermal cycler (Roche Molecular Systems, Branchburg, N.J.) by using the Expand high-fidelity PCR system (Roche Molecular Biochemicals, Indianapolis, Ind.). For restriction digestions, ligations, and agarose gel electrophoresis, standard procedures previously described (Sambrook et al., 1989) were followed. Total RNA from M. smegmatis strains was isolated by using RNAWIZ (Ambion, Inc., Austin, Tex.) with minor modifications (Bashyam and Tyagi, 1994). Primer extension analysis of the ddl mRNA was carried out as described previously (Davis et al., 1994). The oligonucleotide was radiolabeled with [y-32P]ATP by using T4 polynucleotide kinase (Promega, Madison, Wis.), and the reactions were extended with Moloney murine leukemia virus reverse transcriptase (Promega). Radioactivity in primer extension products was quantified with a Phosphorlmager by using ImageQuant, version 3.3 (Molecular Dynamics, Sunnyvale, Calif.).
C. M. smegmatis genomic library construction and cloning of the M. smegmatis ddl gene.
Chromosomal DNA from M. smegmatis mc2155 was prepared as described previously (Whipple et al., 1987). For library construction, chromosomal DNA was partially digested with Sau3AI, and fragments of 3.0 to 4.0 kb were purified from 0.8% agarose. This fraction was ligated with the E. coli-Mycobacterium shuttle plasmid pMV262 (Connell et al., 1993), linearized with BamHI, and dephosphorylated. The ligation mixture was transformed into E. coli XL10-GOLD (Stratagene, La Jolla, Calif.), and approximately 6,000 recombinants were obtained for a theoretical representation of P as >99% of the M. smegmatis genome.
A recombinant clone carrying the M. smegmatis ddl gene was identified from a genomic library by colony hybridization using a species-specific probe. An internal fragment of the M. smegmatis ddl gene was amplified by PCR using a pair of degenerate primers, DDLF and DDLR, based on two signature peptides of bacterial Ddl enzymes (Dutka-Malen et al., 1992). This amplified fragment was verified and radiolabeled with the Rediprime II labeling system (Amersham Pharmacia Biotech, Piscataway, N.J.). For screening the library, about 10,000 colonies from the library pool were plated, transferred to the NYTRAN nylon membrane (Midwest Scientific, Valley Park, Mo.), and screened with the labeled probe as described previously (Sambrook et al., 1989). After three rounds of screening, the recombinant plasmid pBUN172 was identified and confirmed to contain the full-length ddl gene. This sequence is identical to the sequence at GenBank with accession no. AF077728 (Belanger et al., 2000) and the sequence from the unfinished M. smegmatis mc2155 genome [(J. Craig Center Institute)].
D. Drug Susceptibility Assays.
MICs were determined by a microdilution method described previously (Takiff et al., 1996) with minor modifications. Briefly, M. smegmatis cells were grown in M-ADC-TW to mid-exponential phase (optical density at 600 nm, 0.6 to 1.0). Approximately 105 CFU in 0.1 ml were inoculated in triplicate onto 96-well microplates containing serial twofold dilutions of inhibitory compounds. Plates were incubated at 37° C. and examined daily. The MIC was defined as the minimal concentration of the drug or inhibitor that prevented visible bacterial growth after 48 h. Each MIC represents the consistent result from at least three independent cultures. Amikacin, DCS, βCDA, ethambutol, and vancomycin (all from Sigma) were prepared in sterile deionized water. Rifabutin (Amersham Pharmacia) was prepared in dimethyl sulfoxide (Fisher Scientific, St. Louis, Mo.). All further dilutions of each antibiotic were prepared in growth medium. Inhibition of colony formation by DCS was evaluated as described previously in Caceres et al., (1997), which is incorporated herein by reference. Appropriate dilutions of exponentially growing M. smegmatis cells were plated onto agar containing 0 to 1,200 μg of DCS ml−1. Colonies were counted after 5 days of incubation at 37° C. Statistical analysis was conducted using the SAS general linear model procedure (SAS Institute, Cary, N.C.).
E. Preparation of Crude Cell Extracts from M. smegmatis Strains.
M. smegmatis cells were harvested at exponential phase and concentrated 50-fold in ice-cold 50 mM Tris-HCl (pH 8.0). Cells were disrupted with a French pressure cell (Thermo Spectronic US, Rochester, N.Y.) at 14,000 lb/in2. The lysate was centrifuged at 4° C. for 30 min at 30,000×g to remove cell debris. The supernatant was subjected to ultracentrifugation at 4° C. for 4 h at 110,000×g to remove the membrane fraction. The recovered supernatant was dialyzed twice against 50 mM Tris-HCl (pH 8.0) at 4° C. and sterilized by filtration through 0.22-μm-poresize filters. The protein concentration was determined by using the DC protein assay (Bio-Rad) as recommended by the manufacturer.
F. Enzyme Assays.
The crude extract was assayed for Ddl activity by a modified thin-layer chromatography (TLC)-based method described previously (Marshall and Wright, 1998). This procedure can quantitatively determine the amount of
G. Analysis of Intracellular Pools of Amino Acids.
M. smegmatis cells were grown in minimal medium until exponential phase and split into two subcultures, and DCS was added to a final concentration of 75 μg ml−1 to one of the subcultures. After 2 h of incubation, cells were harvested at 4° C. by centrifugation, washed twice with ice-cold double-distilled water, and concentrated 50-fold. Cells were sonicated in a salt-ice-water bath with a Vibra-Cell model VC600 sonicator (Sonic and Materials, Inc., Danbury, Conn.) for 10 min at 80% power output and 50% duty cycle. The lysate was centrifuged at 4° C. for 30 min at 30,000×g to remove bacterial debris. Protein was removed from the supernatant by serial passages through YM-10 and YM-3 Centricon concentrators (Millipore Corp., Bedford, Mass.). Determination of the abundance of individual amino acids was performed at the Amino Acid Geochronology Laboratory of Northern Arizona University (Flagstaff, Ariz.) by a reverse-phase high-performance liquid chromatography (HPLC) procedure described previously (Kaufman and Manley, 1998). This procedure is able to detect nine pairs of L- and
H. Overproduction and Purification of M. tuberculosis Ddl Enzyme
Introduction. The development of assays to screen for and characterize novel inhibitors of the M. tuberculosis Ddl enzyme requires large amounts of purified protein. Although a preliminary biochemical characterization of inhibitors could be undertaken with crude cell extracts from M. tuberculosis, a more thorough analysis of biochemical parameters require purified products. Furthermore, large amounts of the purified protein are required for crystallographic studies. One possibility is to purify the protein directly from M. tuberculosis cell extracts. However, the purification yield is low, necessitating large cultures of a serious human pathogen. Fortunately, recombinant DNA technology provides the means to produce large amount of recombinant products using hosts such as Escherichia coli, which are safe to handle, and easy to grow and manipulate genetically. In general, the E. coli system is able to synthesize the proper mycobacterial protein; however, this is confirmed for the protein of interest. The M. tuberculosis Ddl in E. coli is designated herein as r-Ddl. The corresponding biochemical properties of the native M. tuberculosis Ddl are compared with those of r-Ddl. The purify proteins are used to determine basic biochemical parameters including the inhibitory parameters for the well-known inhibitor DCS and derivatives thereof.
Likewise, the determination of biochemical parameters are performed as a means to compare the properties of r-Ddl and the native enzyme, as well as to correlate the values obtained in our study with those previously reported in classic studies of mycobacteria (David et al., 1969) and other microbial systems.
Experimental design. The M. tuberculosis ddl gene is subcloned into an E. coli overexpression vector, so that a polyhistidine-tagged Ddl protein is synthesized by the E. coli recombinant clone. The fusion protein is purified from E. coli Ddl and other endogenous components by one-step affinity chromatography based on the properties of the histidine tag. Following removal of polyhistidine, polyclonal antibodies are raised against the r-Ddl and used to purify the native M. tuberculosis Ddl from semi-purified extracts. Using biochemical assays, the kinetic parameters of Vmax, Km for
To obtain large amount of M. tuberculosis Ddl enzyme, the ddl gene is PCR amplified based on the genome sequence and cloned into an E. coli expression vector to achieve a high level of expression. Preferably, the commercially available pET system (Novagen) is used. The advantage of this system is that the inserted gene will be under the control of the strong T7 promoter, allowing large quantities of protein to be produced. In the pET system, the T7 RNA polymerase gene BL21 (DE3), and is itself under the control of the lac promoter. In the presence of the inducer IPTG, T7 RNA polymerase is expressed and will initiate transcription of the ddl gene located in the plasmid. The M. tuberculosis ddl gene is expressed as a fusion protein with a poly-His tag at either the N- or C-terminus, for use in purification of the resulting protein by a single-step affinity chromatography. After optimization of protein expression, a large-scale experiment is performed to obtain milligram levels of purified protein. The purity and identity of the recombinant Ddl protein is verified by two-dimensional SDS-PAGE, mass spectroscopy and N-terminal sequencing, based on the deduced protein sequence and computer-predicted characteristics, including molecular weight and isoelectric point.
Primers for the M. tuberculosis ddl gene are designed so that the 5′ ends contain the proper restriction enzyme sites to facilitate cloning. These restriction sites will be chosen using the following criteria: the sites are not present in the ddl gene; and they will produce an in-frame fusion with the His tag in the vector after cloning. Thermostable high-fidelity DNA polymerase is used to amplify the ddl gene from M. tuberculosis genomic DNA. The resulting PCR produce is then digested with the appropriate restriction enzymes and cloned into pET 15b following standard cloning procedures. Restriction digestion analysis and DNA sequencing of the insertional junctions is performed to ensure the correct in-frame fusion. After verification, the plasmid is transformed into E. coli BL21 (DE3), which carries the T7 RNA polymerase gene under the control of the lac promoter. Protein expression is induced by adding 1 mM IPTG to cultures of recombinant E. coli when the OD600 is between 0.5 and 1.0. Cells are harvested at various time points after induction and whole-cell crude extracts are analyzed by SDS-PAGE to detect the expression of the Ddl protein. Optimal conditions for protein production are obtained from small-scale analysis of variations in initial culture density, inducer concentration, growth temperature, agitation speed and cell density at harvest. Optimized conditions are adopted for large-scale preparation. Cells are lysed by French Press and the resulting cell lysate passed through a Ni affinity column. Recombinant protein with the His tag bound to the column is eluted and the process repeated until the protein is purified close to homogeneity. The His tag in the recombinant protein is cleaved by thrombin prior to analysis. The purity and identity of the recombinant protein is confirmed by 2-D SDS-PAGE, mass spectrometry and N-terminal sequencing.
Purification of M. tuberculosis native Ddl protein. The Ddl from E. coli and M. tuberculosis is purified to determine the identity of the recombinant and native proteins. Given the cytoplasmic localization of the Ddl enzyme, we plan to prepare crude cell extracts of M. tuberculosis H37Rv are prepared and the cytosolic fractions are separated as described above for M. smegmatis. If necessary, further partial purification of this crude cell extract is performed by treating the cytosolic fraction with 0.3 mg of RNase and 0.3 mg of DNase, followed by centrifugation at 100,000×g for 2 h at 4° C. in a Beckman Ultracentrifuge by using a Ti 40 rotor. To the precooled new supernatant fraction, distilled acetone is added to precipitate the lipopolyssacharide-like components. The supernatant of the acetone precipitation is centrifuged a 100,000×g for one hour to precipitate proteins. The pellet is redissolved in buffer, and the Ddl protein is further purified by affinity chromatography using the polyclonal antibodies raised against the purified r-Ddl. Most of these steps are similar to those described in the classic studies of the M. tuberculosis Ddl by David et al. (1969), except for the last affinity chromatography step. Alternatively, the crude cell-free extract will be concentrated and loaded on the affinity chromatography column, without additional purification steps.
Determination of kinetic parameters. Using both the TLC and the Pi release assay, the type of inhibition and the corresponding kinetic parameters are determined. The Vmax, Km and Ki for DCS will be evaluated by the double reciprocal plot method. Preferably, the Pi assay is used because it is readily adaptable to a high throughput screen procedure.
The M. smegmatis ddl gene was isolated from a genomic library as described in Example 1. The DNA fragment containing the ddl gene coding sequence, including 190 bp of the 5′ flanking region and 35 bp of the 3′ flanking region, was amplified by PCR and ligated into the EcoRI site of pMV262 to generate pBUN250. This plasmid was electroporated into M. smegmatis mc2155, yielding GPM259. Since pMV262 is present in M. smegmatis at 5 to 10 copies (Caceres et al., 1997), the ddl gene is expected to be overexpressed in the recombinant M. smegmatis strain due to a gene dosage effect. This was confirmed by primer extension analysis using total RNA isolated from mc2155 and GPM259. Radiometric quantitation of the primer extension bands confirmed that GPM259 overproduced the ddl transcript by 25-fold compared to mc2155. In both samples, two transcriptional start sites were detected, one immediately upstream and another 3 nucleotides upstream of the start codon (GTG) (
Following the same strategy, we constructed an M. smegmatis recombinant strain, GPM198A, carrying the plasmid pBUN128A, a pMV262-based plasmid containing the M. tuberculosis ddl gene coding sequence, including 140 bp of the 5′ flanking region and 130 bp of the 3′ flanking region. Total RNA isolated from GPM198A was subjected to primer extension analysis using oligonucleotides specific to either the M. smegmatis or the M. tuberculosis ddl gene. The results revealed that this strain overexpressed the endogenous M. smegmatis ddl gene rather than the plasmid-carried M. tuberculosis ddl gene. To circumvent this problem, a different strategy was followed. Briefly, the M. tuberculosis ddl gene was amplified by PCR and cloned into pMV262, yielding pBUN276. In this construct, the M. tuberculosis ddl gene is fused in frame with the first six codons of the Mycobacterium bovis BCG hsp60 gene and is under the control of its promoter. Total RNA isolated from the corresponding recombinant strain, GPM265, was subjected to primer extension analysis as described above. In this case, the chromosomally encoded ddl gene in GPM265 was expressed at a level similar to the level in wild-type mc2155 (
The overexpression of the M. smegmatis or M. tuberculosis ddl gene was further confirmed by measuring Ddl enzyme activities. For this, Ddl-specific activities in cell crude extracts from mc2155, GPM259, and GPM265 were determined by the TLC-based method described in Example 1. A typical autoradiogram of the TLC-based Ddl enzyme assays for strain GPM259 is shown in
Previously, David et al. (1969) reported that DCS competitively inhibits the M. tuberculosis Ddl enzyme. In this study, we performed in vitro inhibition assays to determine the Ddl-specific activities of crude extracts from mc2155, GPM259, and GPM265 in the presence of increasing concentrations of DCS. A typical autoradiogram of the inhibition assay for the crude extract of GPM259 is shown in
To test whether Ddl overproduction confers a DCS resistance phenotype, we evaluated the susceptibilities of M. smegmatis strains overproducing Ddl to DCS. The MICs of DCS for mc2155, GPM259, GPM265, and GPM14 were determined (Table 2). GPM14, overproducing Alr, is a spontaneous DCS-resistant mutant derived from mc2155 (Caceres et al., 1997). The MICs of DCS for strains GPM259 and GPM265 (both at 150 μg ml−1) were twofold greater than that for mc2155 (75 μg ml−1) but were lower than that for GPM14 (300 μg ml−1). Thus, recombinant strains overproducing Ddl showed increased levels of resistance to DCS but were not as resistant as the Alr-overproducing strain GPM14. The susceptibilities of these strains to 13CDA, which interferes with the incorporation of
It has been reported previously that a specific type of DCS-resistant Streptococcus gordonii mutants displayed elevated Alr and Ddl activities and that the level of resistance to DCS is higher than that for mutants with elevated Alr activity only (Reitz et al., 1967). To test the effect of the overproduction of both Alr and Ddl in M. smegmatis on DCS susceptibility, we introduced the multicopy plasmid pBUN250 carrying the ddl gene into the Alr-overproducing strain GPM14. As expected, the resulting strain, GPM260, overproduced both Alr and Ddl as demonstrated by enzymatic characterization (
aMICs were determined in M-ADC-TW media by a microdilution method as described in Materials and Methods.
bA chi-square test was performed for MICs obtained from at least four independent cultures. Susceptibilities to βCDA can be divided into two groups (group I, mc2155, GPM2, GPM259, and GPM265; group II, GPM14 and GPM260). Significant differences (P < 0.001) between these two groups were detected, while no significant differences (P ≧ 0.537) within each group were detected.
cA chi-square test was performed for MICs obtained from at least seven independent cultures. Susceptibilities to DCS can be divided into four groups (group I, mc2155 and GPM2; group II, GPM259 and GPM265; group III, GPM14; group IV, GPM260). Significant differences between these two groups were detected (group I versus group II, P = 0.001; group II versus group III, P P = 0.001; group IIIversus group VI, P = 0.008), while no significant differences within each group were detected (group I, P = 0.881; group II, P = 0.893).
dMore than 95% of the Ddl activity in GPM265 is due to the expression of the episomal copy of the M. tuberculosis ddl gene (see text).
ewt, wild type.
The incorporation of
Comparison of the amino acid pools in each strain upon DCS exposure revealed that this treatment had a specific effect on the intracellular alanine pools (
The intracellular pools of
Promising lead drug candidates are further tested in vitro using reference mycobacterial strains, such as M. smegmatis and M. tuberculosis Ddl overproducing strains, GPM259, GPM260, and GPM265, to evaluate the potential mycobactericidal activity of the lead compounds compared to DCS.
Vaccine development starts with virulent M. tuberculosis microorganisms from which attenuated mutants are generated. These mutants preferably carry at least two attenuating deletion mutations and no antibiotic resistant markers, so as to avoid unwanted reversions or transfers of drug-resistance. Vaccine strains may include one or more additional mutations. The vaccine strain is optionally potentiated by co-administration of cytokines or by endowing the engineered strain with the capability to produce cytokines or phagosome membrane disrupters, such as lysteriolysin. Preferably, temporal expression of these genes is regulated by promoters solely active inside phagocytic cells.
A strain of M. tuberculosis is constructed which is merodiploid, with an episomal copy of the ddl gene under the control of a promoter that is not expressed in vivo. The chromosomal copy of the native ddl gene is inactivated by means known in the art, such as point mutation or by addition, deletion, or substitution of one or more base pairs, preferably by deletion of one or more base pairs. The resulting strain is able to grow in synthetic medium but is unable to grow in vivo, because
Such strains would be useful for the generation of attenuated live-attenuated vaccine candidates against tuberculosis and other human or animal diseases caused by mycobacteria such as leprosy, Johne's disease, and possibly Crohn's disease. Because such strains are impaired in the ability to synthesize the basic building block of cell walls, peptidoglycan, the strains undergo spontaneous lysis in vivo. Lysis provides improved antigen delivery and containment of the vaccine strain.
The plasmid pBUN276 has been deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, USA, on Jan. 26, 2007 in accordance with the Budapest Treaty and have been accorded accession number PTA-8190.
From the foregoing it will be seen that this invention is one well adapted to attain all ends and objectives herein-above set forth, together with the other advantages which are obvious and which are inherent to the invention.
Since many possible embodiments may be made of the invention without departing from the scope thereof, is to be understood that all matters herein set forth or shown in the accompanying drawings are to be interpreted as illustrative, and not in a limiting sense.
While specific embodiments have been shown and discussed, various modifications may of course be made, and the invention is not limited to the specific forms or arrangement of parts and steps described herein, except insofar as such limitations are included in the following claims. Further, it will be understood that certain features and sub-combinations are of utility and may be employed without reference to other features and sub-combinations. This is contemplated by and is within the scope of the claims.
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This application is related to U.S. Provisional Application Ser. No. 60/434,200, filed on Dec. 17, 2002, which is incorporated herein by reference.
The present invention was sponsored by the United States Department of Agriculture under USDA Cooperative State Research Service Project Grant No. NEB 14-108 and the National Institute of Health under contract number RO3 A1051176-01. The government may have certain rights in the invention.
| Number | Name | Date | Kind |
|---|---|---|---|
| 20030133952 | Barletta et al. | Jul 2003 | A1 |
| Number | Date | Country | |
|---|---|---|---|
| 20040241830 A1 | Dec 2004 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60434200 | Dec 2002 | US |
