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WO1993016172A1 - Mycobacteriophages reporters specifiques a des especes mycobacteriennes - Google Patents

Mycobacteriophages reporters specifiques a des especes mycobacteriennes Download PDF

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Publication number
WO1993016172A1
WO1993016172A1 PCT/US1993/000913 US9300913W WO9316172A1 WO 1993016172 A1 WO1993016172 A1 WO 1993016172A1 US 9300913 W US9300913 W US 9300913W WO 9316172 A1 WO9316172 A1 WO 9316172A1
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WIPO (PCT)
Prior art keywords
reporter
genes
mycobacteriophage
mycobacteriophages
specific
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PCT/US1993/000913
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English (en)
Inventor
William R. Jacobs, Jr.
Barry R. Bloom
Graham F. Hatfull
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Albert Einstein College Of Medicine Of Yeshiva University
University Of Pittsburgh
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Application filed by Albert Einstein College Of Medicine Of Yeshiva University, University Of Pittsburgh filed Critical Albert Einstein College Of Medicine Of Yeshiva University
Priority to BR9305855A priority Critical patent/BR9305855A/pt
Priority to EP93905782A priority patent/EP0625191A1/fr
Priority to CA002129034A priority patent/CA2129034A1/fr
Publication of WO1993016172A1 publication Critical patent/WO1993016172A1/fr
Priority to KR1019940702695A priority patent/KR950700404A/ko

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10311—Siphoviridae
    • C12N2795/10322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention relates to mycobacter i a l species-specific reporter mycobacteriophages (reporter mycobacteriophages), methods of making such reportermycobacteriophages, and the use of such reporter mycobacteriophages, for example, to rapidly diagnose mycobacterial infection and to assess drug susceptibilities of mycobacterial strains in clinical samples.
  • this invention relates to the use of mycobacterial species-specific luciferase reporter mycobacteriophages to diagnose tuberculosis and to assess the drug susceptibilities of the various strains of Mycobacterium tuberculosis
  • transcriptional promoters and reporter genes are introduced into the genomes of mycobacterial species-specific mycobacteriophages.
  • These reporter genes may be the genes for luciferase or the ⁇ -galactosidase gene, and provide the DNA which encodes production of a gene product.
  • the reporter mycobacteriophages may be used by incubating same with samples which may contain the specific mycobacteria of interest. If the mycobacteria of interest is present, then the reporter mycobacteriophages introduce the recombinant nucleic acids which encode expression of the gene product into the mycobacteria of interest, and the mycobacteria then express the gene product.
  • the expressed reporter gene product may be detected by a suitable assay, for example, through the detection of photons or the conversion of an easily assayable chemical reaction.
  • the presence of such gene product indicates that the sample contains the mycobacteria of interest, and hence the mycobacterial species-specific reporter mycobacteriophages may be used to detect and thereby diagnose the specific mycobacterial
  • the mycobacteria species-specific reporter mycobacteriophages of this invention may be used to assess the drug
  • mycobacteria If antibiotic drugs are added to the sample containing the reporter mycobacteriophages and the gene product is detected, the mycobacteria is metabolically active and hence resistant to the antibiotic drug.
  • a mycobacterial strain be assessed for drug resistance rapidly because a patient infected with a strain of M. tuberculosis or another mycobacteria must be treated immediately with the particular antibiotic drug(s) to which the strain is not resistant, and not with antibiotic drug(s) to which the strain is resistant, or the patient may die.
  • Mycobacterium avium (M. avium) is a mycobacteria which is often found in
  • This mycobacteria is typically disseminated throughout the bodies of
  • immunosuppressed patients such as AIDS patients, and causes M. avium infection. Because this mycobacteria often causes death in immunosuppressed patients, it is necessary to be able to diagnose and assess the drug susceptibilities of the various strains of M. avium.
  • This invention relates to broad host range and mycobacterial species-specific reporter
  • reporter mycobacteriophages are then incubated with a clinical sample which may contain the mycobacteria of interest, such as M. tuberculosis.
  • the reporter mycobacteriophages are specific for the mycobacteria which is sought to be detected.
  • the reporter mycobacteriophages efficiently introduce the recombinant nucleic acids which encode the expression of the reporter gene's gene product into the
  • mycobacteria of interest and the mycobacteria then express the gene product.
  • a substrate or other means capable of allowing for the detection of the gene product is then added to the sample. If the gene product or the signal generated by the gene product is detected, the presence of the infectious mycobacteria is known, thereby diagnosing the disease.
  • drugs such as antibiotics ma, be added to a sample containing the reporter mycobacteriophages of this invention. If the mycobacteria are susceptible to a drug after exposure to the drug, the mycobacteria will be killed.
  • drug-resistant mycobacteria will continue to be metabolically active in the presence of the drug, and will continue to express the detectable gene product of the reporter genes.
  • the preferred reporter genes of the present invention are the Firefly luciferase lux gene (FFlux). the luciferase lux genes of Vibrio fischeri, the luciferase lux genes of Xenorhabdus luminescens and the E. coli ⁇ -galactosidase gene (lacZ).
  • FFlux Firefly luciferase lux gene
  • lacZ E. coli ⁇ -galactosidase gene
  • preferred promoters of the present invention are hsp60 and L5 gene 62 promoter, and the preferred mycobacteriophages are L5, TM4 and D56A. These reporter mycobacteriophages are preferably used for the rapid diagnosis of tuberculosis and M. avium
  • FIGURE 1 represents the genome organization of mycobacteriophage L5
  • FIGURE 2 represents a luciferase shuttle plasmid pYUB180 wherein reporter gene FFlux is fused to the BCG hsp60 promoter;
  • FIGURE 3 represents the amount of luciferase activity of M. smegmatis which contains the pYUB180 shuttle plasmid and the FFlux gene;
  • FIGURE 4 represents the effect of various antibiotic drugs on the metabolic activity of control mycobacteria and drug resistant mycobacteria in the
  • FIGURE 5 represents shuttle plasmid phAE39 wherein the reported gene is FFlux, the promoter is hsp60, the phage is TM4 and the cosmid is pYUB216.
  • FIGURE 6 represents luciferase activity
  • FIGURE 7 represents a flow chart for cloning different promoters into TM4 : : lux shuttle phasmid phAE39.
  • This invention is directed to mycobacterial species-specific reporter mycobacteriophages, (reporter mycobacteriophages), methods of producing such reporter mycobacteriophages and the use of such reporter mycobacteriophages for the rapid diagnosis of mycobacterial infections and the accurate assessment of mycobacterial drug susceptibilities.
  • mycobacterial species-specific mycobacteriophage genomes are modified by introducing therein transcriptional promoters and reporter genes whose gene product can be sensitively detected.
  • the reporter mycobacteriophages may then be incubated with clinical samples suspected of containing the mycobacteria of interest, either directly of after culture, and the samples tested for the presence of the reporter gene product, thereby diagnosing mycobacterial infection.
  • the method of this invention allows for rapid diagnosis because only the amount of time necessary for the reporter mycobacteriophages to infect their host cells and the amount of time necessary for the host cells to synthesize the reporter gene product are required to allow for diagnosis.
  • the amount of time required for the reporter mycobacteriophages to infect their host cells and for the host cells to synthesize the reporter gene product is between ten minutes and sixteen hours.
  • mycobacterial organisms the presence of which metabolic activity indicates that a drug has not killed the mycobacteria and that the mycobacteria is resistant to the drug.
  • Mycobacteriophage L5 a temperate virus with a broad host-range among mycobacteria, is the most thoroughly characterized of the mycobacteriophages.
  • L5 particles are morphologically similar to the family of phages that includes phage g and contain a linear dsDNA genome with cohesive ends. The inventors have determined the DNA sequence of the entire gene as well as several gene functions. The DNA sequence of the L5 mycobacteriophage is as follows:
  • AttP Integration-proficient plasmid vectors have been constructed which efficiently transform both fast-growing and slow-growing mycobacteria through stable integration of the plasmid sequences into the bacterial chromosomal attachment site (attB).
  • Figure 1 represents the genome organization of the entire L5 genome. DNA analysis has indicated that the L5 genome is organized into a right and left arm with the attachment site and integrase at the center of the genome. The integration functions have been successfully employed to construct integration-proficient vectors for mycobacteria.
  • L5 genome is not essential for mycobacteriophage growth. It has been demonstrated that all or most of the gene 62-61-60 can be deleted without affecting the cycle of the L5 phage. Therefore, there is a suitable region in the L5 mycobacteriophage for the insertion of reporter genes. It is critical that reporter genes be inserted into non-essential regions of the mycobacteirophage. Otherwise, the mycobacteriophage will be unable to survive and replicate.
  • the L5 mycobacteriophage may have introduced therein promoter gene 62 fused to reporter gene lacZ, and this reporter mycobacteriophage will be capable of rapid diagnosis of mycobacterial infection and accurate assessment of mycobacterial strain drug susceptibilities.
  • mycobaceriophages is the mycobacteriophage TM4.
  • TM4 has been used to construct a first generation reporter mycobaceriophage, and has the ability to discriminate between M. tuberculosis and BCG.
  • a shuttle plasmid may be employed with TM4, and may be useful in the construction of recombinant and other mycobacteriophages.
  • L5 which is a broad host-range mycobacteriophage
  • TM4 is a species-specific mycobacteriophage.
  • TM4 is not as well characterized as the L5 mycobacteriophage, and therefore it is more difficult to analyze its functions.
  • DS6A is a mycobaceriophage that has been found to be specific for the M. tuberculosis complex of mycobacteria. It has been shown to infect both M. tuberculosis and BCG. It has been demonstrated that DS6A can infect over 3,000 different types of M. tuberculosis strains. Current efforts are under way to develop DS6A shuttle phasmids containing
  • mycobacteriophages may be needed to increase specificity and then increase the ability to distinguish drug susceptibilities.
  • DS6A grows on BCG and M. tuberculosis, but does not grow on M. smegmatis.
  • the host range of the mycobacteriophages varies greatly, some being able to infect only M. smegmatis and others being able to infect M. smegmatis. BCG and M. tuberculosis. but not M. avium.
  • These mycobacteriophages may be developed into reporter mycobacteriophages and cosmid cloning systems, and may provide a source of useful transcriptional translation initiating sequences, transcriptional terminators, or host-range specificity genes.
  • Luciferase reporter genes have been used in many diversified biological systems, including E. coli. cyanobacteria, phytopathogenic bacteria and Bacillus. The presence of luciferase reporter genes can be detected by the emission of photons in the presence of a substrate, such as luciferin or
  • Luciferin and decanal can permeate mycobacteria, and thereby allow for the detection of gene products, such as photons. Since one molecule of the luciferase gene product can yield 0.85 photons of light, it is the most sensitive biological reporter molecule known.
  • the preferred reporter genes of this invention are luciferase reporter genes, such as the Firefly lux gene (FFlux), the Vibrio fischeri lux genes and the Xenorhabdus luminescens lux genes, as well as the E. coli ⁇ -galactosidase (lacZ) genes. Luciferase genes, especially the Firetly lux gene, geneate a high amount of luminescence activity.
  • promoters In choosing transcriptional promoters to be introduced into the mycobacteriophages, it is desirable to use strong promoters since this will increase the sensitivity of the system. In addition, it is important that the promoter be active following mycobacteriophage infection.
  • the best promoter candidates currently available are the BCG hsp60 promoter and the L5 gene 62 promoter, which are of comparable strength.
  • the hsp60 promoter gives good levels of luciferase expression from plasmid recombinants, but lower levels of luciferase expression where the mycobacteriophage is TM4.
  • the hsp60 promoter is shut off by the TM4 enzymes following infection, thus producing only a modest level of luciferase.
  • the gene 62 promoter may behave in a similar manner with the TM4 phage since the gene 62 product is a good candidate for the L5 repressor and is expressed at high levels in the absence of other mycobacteriophage functions. Knowing the sequence of the mycobacteriophage used will help in identifying, characterizing and cloning the appropriate promoter to be used in the reporter mycobacteriophages of this invention.
  • the shuttle phasmids which consist of the E. coli cosmid, the reporter genes and mycobacteriophage promoters, may then be characterized.
  • Shuttle phasmids can be propagated in E. coli as plasmids, and propagated in mycobacteria as mycobacteriophages.
  • mycobacteriophages is by homologous recombination or PCR.
  • mycobacteriophage must be determined. Again, in order to do this, it is necessary to know the sequence of the mycobacteriophage. Consequently, L5 is an ideal phage to use with this method as its genome has
  • plasmids are constructed wherein reporter genes hooked to transcriptional promoters are flanked by mycobacteriophage non-essential region sequences in mycobacterial plasmids. Then, homologous recombination systems or PCR may be utilized in
  • transposons For example, transposon IS1096 may be utilized. In order to use this
  • reporter genes and transcriptional promoters are put into transposons, and the
  • transposons containing the reporter genes and transcriptional promoters are delivered on plasmids in mycobacteria.
  • plasmids in mycobacteria.
  • M. smegmatis which strain contains the transposons.
  • the transposons will hop into non-essential regions of the mycobacteriophages, thereby introducing themselves therein.
  • mycobacteriophages are still viable, and contain the reporter genes and transcriptional promoters.
  • a fourth method of introducing reporter genes and transcriptional promoters into mycobacteriophages is by debilitated phages packaged into phage heads and tails (phage particles).
  • phage particles phage heads and tails
  • helper phage systems which allow for pieces of DNA containing pac sites to be packaged.
  • Helper phage systems may be generated from the L5 mycobacteriophage. The genome of the helper phage is put into the mycobacterial chromosome, at which time the mycobacteria are grown up. Next, pacmids which
  • L5 genome has cohesive (cos) termini. This suggests the possibility of constructing L5 cosmid vectors, which could be packaged through the cos sites into L5 particles either in vivo or in vitro. Then, a large number of genes could be easily and efficiently delivered to mycobacteria.
  • Packaging into phage heads and tails may also be utilized in a fifth methodology wherein the pacmid is a plasmid.
  • the methodology is similar to the methodology wherein a debilitated phage is used, however, instead of using phage pacmids, the pacmids comprise plasmids which have pac sites, reporter genes, transcriptional promoters, and plasmid replicons.
  • mycobacteriophage with the unique restriction enzyme sites and the reporter gene cassette.
  • the result is a circular DNA molecule which consists of the mycobacteriophage, the reporter genes and the transcriptional promoters.
  • the circular DNA may then be electroporated directly into mycobacteria.
  • a promoter probe vector was constructed which incorporated a truncated E. coli ⁇ -galactosidase
  • lacZ lacZ gene as a reporter probe into a shuttle plasmid vector that replicated in either mycobacteria or
  • E. coli Random DNA fragments from the three mycobacteriophages L1, TM4 and Bxbl were cloned into a unique BamHl site immediately upstream of the lacZ gene and screened for their ability to produce ⁇ -galactosidase.
  • lacZ could be used as a reporter gene in the mycobacteria, and identified the DNA sequences which could effectively express foreign genes in both M. smegmatis and M. tuberculosis.
  • ⁇ -galactosidase activity could be detected from lysed cells using OMPG, or from unlysed cells using either X-gal or a fluorescent methylumbelliferyl ⁇ -galactosidase derivative.
  • the promoter hsp60 gene highly expressed the lacZ gene in both M. smegmatis and BCG.
  • the FFlux gene was cloned into pMV261 downstream from the hsp60 promoter in plasmid pYUB180 (see Figure 2), which plasmid was shown to express the FFlux gene in M. smegmatis, BCG and M. tuberculosis H37Ra.
  • the expression of the FFlux gene was detected by observing luminescence of mycobacterial clones containing the cloned gene in the dark room, and verified use in photographic film. This demonstrated that the luciferase was expressed in the mycobacteria, and that luciferin, the substrate used, was able to penetrate mycobacterial cell walls and yield photons expressed by the mycobacteria.
  • M. smegmatis The expression of FFlux from the plasmid pYUB180 in M. smegmatis provided a model with which to determine a minimal number of individual cells detectable with the luciferase assay.
  • M. smegmatis containing pYUB180 were grown in the presence of kanamycin to ensure that every cell contained the plasmid.
  • the cells were diluted 10-fold serially and the amount of luciferase activity was determined using a luminometer.
  • Figure 3 shows that the amount of luciferase activity from 5 ⁇ 10 7 cells approached
  • luciferase is a powerful indicator of the metabolic abilities of a bacterial cell. Since anti-tuberculosis drugs are likely to significantly decrease the metabolic activity of a cell, the measurement of luciferase activity should provide a sensitive means of distinguishing drug-resistant mycobacteria from drug-sensitive mycobacteria.
  • the first vectors developed to introduce recombinant DNA into mycobacteria were shuttle phasmid phage vectors.
  • Shuttle phasmids have the ability to replicate in E. coli as cosmids and then replicate in mycobacteria as phages.
  • Shuttle phasmids of TM4 which contained the FFlux and lacZ genes transcribed from hsp60 and L1 promoters, respectively, were constructed (see Figure 5).
  • TM4 lux mycobacteriophage is capable of introducing the FFlux gene into mycobacterial cells, and that the FFlux gene can be expressed in mycobacteriophage-infected cells.
  • Figure 7 represents a flow chart for cloning different promoters into the TM4 : : lux shuttle phasmid phAE39.
  • L5 mycobacteriophage Strategies for construction of the recombinant L5 mycobacteriophage may be investigated.
  • Another approach would be to attempt to introduce genes by homologous recombination with plasmids.
  • Still another approach would be to transpose lux genes onto L5 using either the mini-Mu in vitro transposition system or a mycobacterial transposon such as IS1096.
  • Recombining reporter genes from recombinant plasmids onto L5 using a double recombination event may also be performed. This involves first constructing a recombinant plasmid that carries a reporter gene (lacZ may be more suitable) inserted into gene 62 such that both the upstream and downstream parts of gene 62 are present. Advantages of this approach are that lacZ can be easily detected in agar media, that gene 62 is not an essential gene, and that lacZ is efficiently expressed from a promoter immediately upstream of gene 62.
  • An L5 mycobacteriophage lysate may be prepared by growth of the plasmid-containing strain and recombinant mycobacteriophage progeny identified by plating the lysate on wild-type M. smegmatis for individual plaques on agar containing the indicator X-gal.
  • This recombination approach may be expanded to introduce other gene or DNA segments of the L5 genome.
  • inclusion of polylinker containing restriction enzyme sites unique for L5 would open the way for construction of L5 recombinants in vitro. Similar genetic strategies may be used to systematically reduce the size of the L5 genome by deletion of non-essential sequences.
  • Transposition offers an alternative method for the construction of reporter mycobacteriophages.
  • a transposition system which is available is the mini-Mu in vitro transposition system. This is a defined biochemical reaction in which a mini-Mu transposon carrying the desired gene is transposed onto the phage genome using purified MuA and MuB proteins. Similar transposition experiments have been tried with L5, but few L5 mini-Mu derivatives have been isolated. It is possible that this is due to the relatively large size of the transposon used. It is necessary to first construct a small Mu transposon which contains the reporter gene, a promoter and the two Mu in order for these experiments to be successful.
  • g cosmids and packaging systems provide the efficiency of mycobacteriophage infection with the ability to inject large segments of non-mycobacteriophage DNA.
  • Analogous mycobacterial systems would overcome packaging constraints encountered with recombinant mycobacteriophage genomes and allow the introduction of multiple copies or types of reporter genes into mycobacteria, potentially enhancing the sensitivity of the assay. In addition, they would help overcome any problems with host synthesis inhibition.
  • L5 cosmids and packaging systems are dependent on the finding that the L5 genome contains cohesive termini.
  • the g paradigm suggests that a relatively small region of DNA (approximately
  • the first series of experiments with L5 would therefore be to identify the segment of the genome required for packaging by constructing a series of plasmids containing the L5 cos site and surrounding sequences.
  • Cos activity may be determined by preparation of an L5 lysate on plasmid-containing M. smegmatis strains, followed by the identification of antibiotic-resistant transductants in the lysate, by transduction of M. smegmatis. This assay assumes that plasmid multimers of a total size of approximately 50kb are present in the cell and will be packaged.
  • cosmid vectors which contain both L5 g cos sites may be constructed. Insertion of 40-45kb of DNA (as in the construction of cosmid libraries) followed by g packaging in vitro and infection with E. coli will generate 50kb sized molecules containing L5 cos site. These should be isolated from E. coli and introduced by electroporation into M. smegmatis. Assuming that one of these approaches is successful, it would then be possible to define a small segment of L5 DNA required for packaging.
  • in vivo cosmid packaging systems The construction of in vivo cosmid packaging systems is a particularly attractive idea since it has proven very useful in E. coli.
  • Thermoinducible lysogens of L5 may be suitable for in vivo packaging of L5 cosmids without further modification, since prophage excision may be a temperature-sensitive event.
  • Efficient packaging of extrachromosomal cosmids present in the lysogen may be achieved by simple induction and growth at 42°C.
  • Extracts may be prepared from thermoinducible strains with non-packagable prophages and assessed for their ability to package exogenously added L5 cosmid or mycobacteriophage DNA. Optimization of conditions should follow both empirical biochemical approaches and the well-established g systems. For example, it may be necessary to supplement the extracts with purified mycobacteriophage products such as the terminase or the tape-measure analogues (genes A/Nu and H of g respectively), neither of which have yet been identified.
  • mycobacteriophages L5 and TM4 can be used in the development of diagnostic luciferase and ⁇ -galactosidase shuttle phasmids
  • mycobacteriophages DS6A which only infects BCG and M. tuberculosis strains, that might prove to have a more useful host range for clinical isolates.
  • Diagnostic luciferase mycobacteriophages from these other mycobacteriophages may be developed by using the shuttle phasmid methodology described herein that has been proven successful for constructing mycobacteriophage vectors from both TM4 and phage L1.
  • TM4 mycobacteriophages that can efficiently infect any clinical isolate and possibly distinguish M. tuberculosis from M. avium and BCG.
  • Both mycobacteriophages TM4 and L5 appear to have the ability to infect a large number of M. tuberculosis isolates.
  • TM4 is very closely related to phage 33D, a mycobacteriophage that has been found not to infect every M. tuberculosis isolate used to define the mycobacteriophage typing schemes for M. tuberculosis isolates. However, this mycobacteriophage does not infect BCG.
  • TM4 has been found to be almost identical by DNA hybridization and restriction analysis to 33D, and it shares the host-specificity with 33D in that it infects M. tuberculosis, but fails to infect BCG.
  • mycobacteriophage L5 appears to share the same receptor as mycobacteriophage D29 which receptor has been previously shown to infect a very large number of M. tuberculosis isolates. L5, unlike 33D or TM4, infects all three morphotypes of M. avium including a wide range of serovariants.
  • TM4 mycobacteriophages which plaque on the particular isolate.
  • the inability to plaque on a particular isolate could result from the lack of a mycobacteriophage receptor or be the result of lysogenization of the isolate with a homoimmune phage.
  • Phage mutants with altered host range specificities or mutants which no longer bind a repressor (equivalent to virulent mutant of g) have been isolated in other systems.
  • Variants of TM4 which can efficiently infect BCG have been isolated at frequencies of 10 7 . Previous work has demonstrated that 33D, similarly to TM4 , can not adsorb to BCG cells.
  • TM4 Host-range variants of TM4 which not only plaque BCG, but also still plaque M. tuberculosis have been isolated. Similar strategies for M. tuberculosis isolates which are uninfected by L5 or TM4 may be used.
  • the combined sensitivities of luciferase and mycobacteriophage infections should permit the detection of previously undetectable levels of M. tuberculosis cells in sputum, blood samples, or cerebral spinal fluid.
  • a number of preliminary studies to optimize the detection of M. tuberculosis cells in a variety of body samples will be performed.
  • M. tuberculosis H37Ra As a model system for optimizing detection of M. tuberculosis in infected monocytes and macrophages, primary human monocytes which have been purified by adherence for 1 hour or primary macrophages which have been cultured for 6 days in microwells will be infected with M. tuberculosis H37Ra at varying multiplicities. The number of cells initially infected will be determined microscopically, and then at various periods of time from 2 hours to 30 days, the cells will by lysed by non-ionic detergent NP40 which has no effect on viability of mycobacteria, concentrated by centrifugation, plated for viable organisms and infected with the luciferase plasmids. Quantitative studies at different moi's and with varying numbers of infected cells will indicate how few bacilli/cell and bacilli/specimen can be detected.
  • M. tuberculosis cells isolated from macrophages could result from either the absence of the expression of the mycobacteriophage-receptor or the masking of the receptor with a membrane from a phagosome of the macrophage.
  • the level of expression of phage receptors may be regulated by the environment in which the host cell is grown. For example, the g repressor of E. coli is induced by maltose and repressed by glucose. Studies to identify the receptors for mycobacteriophage L5 have been initiated. Similar studies for mycobacteriophage TM4 will also be performed.
  • the receptor By identifying the genes encoding the receptor, it is possible to assay gene repression of the mycobacteriophage receptor of M. tuberculosis cells when grown in macrophages by hybridization for the mRNA synthesis. If the receptor is not expressed in macrophages, it may be necessary to use a mycobacteriophage which recognizes a receptor that is constitutively expressed.
  • the cells isolated from macrophages may be treated with a variety of different detergents to find a treatment that would allow infection of the M. tuberculosis cells with the mycobacteriophages. Again, it may be necessary to cultivate the detergent-treated macrophages in broth for a few generations to gain expression of the receptors.
  • the assays to determine the infectability of macrophages from mycobacteria include not only the luciferase assay for the TM4 : : lux mycobacteriophages, but also infectious centers assays in which free mycobacteriophages are removed and mycobacteriophage-producing cells are scored by a mixed plating on a lawn of M.
  • smegmatis This assay would be useful since infectability can be scored even if there are insufficient M. tuberculosis cells to form a bacterial lawn. It is important to re-evaluate the host range specificities of all of the mycobacteriophages in this assay. Free mycobacteriophages can simply be removed through the use of specific anti-mycobacteriophage antibodies.
  • M. tuberculosis in Sputum Samples Sputum from a patient infected with M. tuberculosis contains a mixture of mucoploysaccharide, free M. tuberculosis cells, macrophages containing M. tuberculosis cells and a variety of cellular debris. Sputum samples from patients thought to have pulmonary tuberculosis may be used for a study in which various numbers of M. tuberculosis cells are added to sputum samples found to have no or few organisms by acid-fast staining.
  • a variety of methods can be used to treat sputum samples so as to liquify the mucous and decontaminate the specimen under conditions in which all bacteria other than mycobacteria are killed. Because of the specificity of the phasmids, decontamination may not be as important as preserving the mycobacteriophage receptors. Nonetheless, the sputum samples may be treated initially with 2% w/v NaOH for 30 minutes at 37°C or with 0.5% N-acetyl cysteine + 1% NaOH. Alternatively, the sample may be treated with a variety of hydrolytic enzymes, such as collagenase, to help dissolve the sputum sample. If mycobacteriophage receptors are carbohydrates possibly sensitive to these conditions, other conditions may be utilized or the cells will be cultured 3-16 hours to allow recovery of infectivity before mycobacteriophage infection. Detecting Mycobacteria In Blood Samples
  • Tuberculosis has been known to have a bacteremia. If the sensitivity necessary to detect 100 to 200 M. tuberculosis cells in a ml of sample can be obtained, levels of bacteremia in tuberculosis patients which were not previously observable may be observed.
  • White cells should be purified over Ficoil-hypaque and lysed with 2% NP40, 1% SDS or freeze-thawing in the presence of DNAse to liberate intracellular mycobacteria. The pellet should then be infected with the diagnostic luciferase mycobacteriophage, or if only few organisms are present they can be concentrated by filtration onto filters, and filter areas cut out and infected.
  • the luciferase assay may be optimized such that positive correlations of M. tuberculosis infections as indicated in the clinical lab may be obtained.
  • the recombinant mycobacteriophages may be tested to ascertain the range of specificity that they have for other mycobacteria, and for the closely related genera Norcardia, Corynebacterium, and Actinomycetes strains. These strains may be obtained from the ATCC.
  • a number of blinded tests including negative controls, M. tuberculosis-infected patients, samples from patients infected with M. avium, and samples infected with other non-mycobacterial pathogens may be performed to ascertain the range of specificity.
  • M. tuberculosis isolates to isoniazid, ethambutol, rifampicin, pyrazinamide and other antibiotics will have a major impact on the treatment of tuberculosis patients.
  • the assessment of drug-susceptibilities may take an additional 2 to 9 weeks. Diagnostic reporter mycobacteriophages may allow for evaluations of drug-susceptibilities at the time a sputum sample is collected. Alternatively, this approach would shorten the time necessary to assess drug-susceptibilities of purified M. tuberculosis colonies grown up from clinical samples.
  • the choice of the promoter for expressing luciferase may provide a needed parameter to more readily assess drug action.
  • gyrase promoters are greatly stimulated in the presence of gyrase inhibitors.
  • Clinical isolates of M. tuberculosis may be transformed with PYUB160 and tested for luciferase activity in the presence and absence of drugs.
  • the luciferase assays with mycobacteriophage infections with lux mycobacteriophages on in vitro-grown M. tuberculosis cells will first be optimized, and then extended to M. tuberculosis cells grown in macrophages or isolated from sputum samples.
  • the luciferase assay may be optimized so that the drug-susceptibility patterns for any clinical isolate may be obtained. It may be possible to add diagnostic mycobacteriophages to a single clinical specimen, aliquot the mixture into various tubes and add antibiotic drugs. Thus every experiment would have an internal control and each drug-treated sample could be compared to an untreated control. The critical parameter to conclude drug-resistance or sensitivity lies in the comparison.

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Abstract

L'invention se rapporte à des mycobactériophages reporters spécifiques à des espèces mycobactériennes (mycobactériophages reporters), à des procédés de production de ces mycobactériophages reporters et à leur utilisation dans le diagnostic rapide d'infections mycobactériennes et l'évaluation des sensibilités aux médicaments de souches mycobactériennes dans des échantillons cliniques. L'invention concerne notamment la production et l'utilisation de mycobactériophages reporters de la luciférase afin de diagnostiquer la tuberculose. Les mycobactériophages reporters spécifiques à des espèces mycobactériennes comportent des mycobactériophages spécifiques à des espèces mycobactériennes qui contiennent des gènes reporters et des promoteurs de transcription. Lorsque les mycobactériophages reporters sont incubés avec des échantillons cliniques qui peuvent contenir les mycobactéries d'intérêt, le produit génique des gènes reporters sera exprimé si l'échantillon contient les mycobactéries d'intérêt, diagnostiquant ainsi l'infection mycobactérienne.
PCT/US1993/000913 1992-02-07 1993-02-02 Mycobacteriophages reporters specifiques a des especes mycobacteriennes WO1993016172A1 (fr)

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BR9305855A BR9305855A (pt) 1992-02-07 1993-02-02 Micobacteriófagos repórteres de espécies micobacterianas especificas
EP93905782A EP0625191A1 (fr) 1992-02-07 1993-02-02 Mycobacteriophages reporters specifiques a des especes mycobacteriennes
CA002129034A CA2129034A1 (fr) 1992-02-07 1993-02-02 Mycobacteriophages marqueurs de certaines especes de mycobacteries
KR1019940702695A KR950700404A (ko) 1992-02-07 1994-08-05 마이코박테리아 종 특이적 리포터마이코박테리오파지(mycobacterial species-specific reporter mycobacteriophages)

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US83343192A 1992-02-07 1992-02-07
US07/833,431 1992-02-07

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EP0743366A1 (fr) * 1995-05-18 1996-11-20 MERCK PATENT GmbH Méthode de détermination pour Listeria utilisant des bactériophages récombinants
US5612182A (en) * 1995-03-10 1997-03-18 Becton, Dickinson And Company Mycobacteriophage specific for the mycobacterium tuberculosis complex
EP0784700A1 (fr) * 1994-10-03 1997-07-23 Pathogenesis Corporation Souches reporteurs mycobacteriennes et leurs utilisations
US5656424A (en) * 1995-02-15 1997-08-12 Albert Einstein College Of Medicine, A Division Of Yeshiva University Identification of mycobacterium tuberculosis complex species
US5750384A (en) * 1992-02-07 1998-05-12 Albert Einstein College Of Medicine Of Yeshiva University, A Division Of Yeshiva University L5 shuttle phasmids
US6737245B1 (en) 1999-09-08 2004-05-18 Xenogen Corporation Luciferase expression cassettes and methods of use
US7056728B2 (en) 2000-07-06 2006-06-06 Xenogen Corporation Compositions and methods for use thereof in modifying the genomes of microorganisms
WO2006075996A2 (fr) * 2004-04-07 2006-07-20 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Systeme d'encapsidation de phage de plasmide rapporteur utile pour detecter les bacteries
EP1404869B1 (fr) * 2001-03-20 2009-08-05 Norchip A/S Detection de micro-organismes utilisant des genes inductibles
EP2142667A1 (fr) * 2007-04-05 2010-01-13 Sequella, Inc. Procédés perfectionnés et compositions pour déterminer l'état pathogène des agents infectieux
US8501400B2 (en) 2007-04-05 2013-08-06 Sequella, Inc. Methods and compositions for determining the pathogenic status of infectious agents
WO2019070612A1 (fr) * 2017-10-02 2019-04-11 Quidel Corporation Procédé de détection à base de phages pour le test de susceptibilité antimicrobienne et l'identification d'espèces bactériennes
US11560584B2 (en) 2016-12-30 2023-01-24 Quidel Corporation Phage-mediated immunoassay and methods for determining susceptibility of bacteria to antibiotic or probiotic agents

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US4861709A (en) * 1985-05-31 1989-08-29 Technicon Research A.G. Detection and/or identification of microorganisms in a test sample using bioluminescence or other exogenous genetically-introduced marker

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Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5750384A (en) * 1992-02-07 1998-05-12 Albert Einstein College Of Medicine Of Yeshiva University, A Division Of Yeshiva University L5 shuttle phasmids
EP0784700A1 (fr) * 1994-10-03 1997-07-23 Pathogenesis Corporation Souches reporteurs mycobacteriennes et leurs utilisations
US5679515A (en) * 1994-10-03 1997-10-21 Pathogenesis Corporation Mycobacterial reporter strains and uses thereof
EP0784700A4 (fr) * 1994-10-03 1999-09-01 Pathogenesis Corp Souches reporteurs mycobacteriennes et leurs utilisations
US5656424A (en) * 1995-02-15 1997-08-12 Albert Einstein College Of Medicine, A Division Of Yeshiva University Identification of mycobacterium tuberculosis complex species
US5612182A (en) * 1995-03-10 1997-03-18 Becton, Dickinson And Company Mycobacteriophage specific for the mycobacterium tuberculosis complex
US5824468A (en) * 1995-05-18 1998-10-20 Merck Patent Gesellschaft Mit Beschrankkter Haftung Detection of listeria by means of recombinant bacteriophages
EP0743366A1 (fr) * 1995-05-18 1996-11-20 MERCK PATENT GmbH Méthode de détermination pour Listeria utilisant des bactériophages récombinants
US6737245B1 (en) 1999-09-08 2004-05-18 Xenogen Corporation Luciferase expression cassettes and methods of use
US7056728B2 (en) 2000-07-06 2006-06-06 Xenogen Corporation Compositions and methods for use thereof in modifying the genomes of microorganisms
EP1404869B1 (fr) * 2001-03-20 2009-08-05 Norchip A/S Detection de micro-organismes utilisant des genes inductibles
WO2006075996A2 (fr) * 2004-04-07 2006-07-20 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Systeme d'encapsidation de phage de plasmide rapporteur utile pour detecter les bacteries
WO2006075996A3 (fr) * 2004-04-07 2006-10-26 Us Gov Health & Human Serv Systeme d'encapsidation de phage de plasmide rapporteur utile pour detecter les bacteries
EP2142667A1 (fr) * 2007-04-05 2010-01-13 Sequella, Inc. Procédés perfectionnés et compositions pour déterminer l'état pathogène des agents infectieux
EP2142667A4 (fr) * 2007-04-05 2011-08-10 Sequella Inc Procédés perfectionnés et compositions pour déterminer l'état pathogène des agents infectieux
US8501400B2 (en) 2007-04-05 2013-08-06 Sequella, Inc. Methods and compositions for determining the pathogenic status of infectious agents
AU2008236615B2 (en) * 2007-04-05 2013-10-03 Sequella, Inc. Improved methods and compositions for determining the pathogenic status of infectious agents
US11560584B2 (en) 2016-12-30 2023-01-24 Quidel Corporation Phage-mediated immunoassay and methods for determining susceptibility of bacteria to antibiotic or probiotic agents
WO2019070612A1 (fr) * 2017-10-02 2019-04-11 Quidel Corporation Procédé de détection à base de phages pour le test de susceptibilité antimicrobienne et l'identification d'espèces bactériennes
JP2020535808A (ja) * 2017-10-02 2020-12-10 クイデル コーポレーション 細菌種の抗菌薬感受性試験及び同定のためのファージに基づく検出方法

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AU3605293A (en) 1993-09-03
KR950700404A (ko) 1995-01-16

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