BACKGROUND OF THE INVENTION
Mycoplasmas are very small microorganisms (Class Mollicutes) without cell walls that can cause infections in humans, animals, and plants. Mycoplasmas are also commonly found contaminating buffer solutions, and tissue culture media used in life science research. The Mycoplasma and Acholeplasma species, Acholeplasma laidlawii, M. hyorhinis, M. orale, M. salivarium, M. arginini, and M. hominis, account for about 98% of the tissue culture contaminants (McGarrity, G. J., & Carson, D. A., Adenosine phosphorylase-mediated nucleoside toxicity. Application towards the detection of mycoplasmal infection in mammalian cell cultures. Exp Cell Res. 1982 May; 139(1):199-205). As used herein, “mycoplasma” or “mycoplasmas” refers generally to members of the Class Mollicutes, including Mycoplasma and Acholeplasma species.
There is a clear unmet need for the real time detection of mycoplasmas for infection control monitoring in hospitals and for quality control of buffers and tissue culture media used in clinical laboratory testing and life science research.
SUMMARY OF THE INVENTION
The present invention provides biosensors and methods of use for detecting the presence or absence of mycoplasma contamination through the detection of hydrolytic enzymes that are conserved among Mycoplasma species. Such hydrolytic enzymes include, but are not limited to, proteases, reductases and nucleases
In preferred embodiments, the present invention provides a biosensor for detecting the presence or absence of Mycoplasma contamination comprising a support and a detectably labeled substrate for an enzyme produced and/or secreted by a mycoplasma, wherein the substrate is attached to the support. Typically, the enzyme is a Mycoplasma-specific hydrolytic enzyme selected from the group consisting of proteases, reductases and nucleases. In certain preferred embodiments, the enzyme is a Mycoplasma-specific protease selected from the group consisting of the gene product of pepA1 (MCAP—0157), pepA2 (MCAP—0195), pepA (leucyl aminopeptidase, such as MHP7448—0464), MCAP—0267 (metalloendopeptidase), pepP (Xaa-Pro endopeptidase, such as MCAP—0341 or MHP7448—0649), MCAP—0509, mapP (methionine amino peptidase, MCAP—0675 or MHP7448—0173), mixtures thereof and homologous enzymes with at least 40% sequence identity. When the enzyme is a mycoplasma-specific protease, preferred substrates include leucine-(7-methoxycoumarin-4-yl)acetyl (leu-MCA), arginine-(7-methoxycoumarin-4-yl)acetyl (arg-MCA), methionine-(7-methoxycoumarin-4-yl)acetyl (met-MCA), an acetoxymethyl ester or maleimide derivative of blue dye number 1 coupled to a peptide substrate of the mycoplasma-specific protease.
In other preferred embodiments, the enzyme is a Mycoplasma-specific reductase selected from the group consisting of the gene product of nrdE (such as MCAP—0101), MCAP—0427, trxB (thioredoxin reductase, such as MCAP—0779 or MHP7448—0098), MCAP—0858 and mixtures thereof. When enzyme is a mycoplasma-specific reductase, suitable substrates include reactive black 5,5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB), BODIPY®FL L-cystine, 2′,7′-difluoro-4′-(2-(5-((dimethyl amino phenyl)azo)pyridyl)dithiopropionyl aminomethyl)fluorescein (DFDMAP-fluorescein), or an azo dye that is sensitive to decolorization by microbial reductases.
In yet other preferred embodiments, the enzyme is a mycoplasma-specific nuclease selected from the group consisting of the 5′-3′ exonuclease encoded by MCAP—0047 or MHP7448—0581, the gene product of nfo (such as MCAP—0060 or MHP7448—0062), vacB (such as MCAP—0097 or MHP7448—0037), uvrC (such as MCAP—0252 or MHP7448—0066), mc (ribonuclease III, such as MCAP—0492 or MHP7448—0398), MCAP—0768, uvrB (such as MCAP—0773 or MHP7448—0648), uvrA (such as MCAP—0774 or MHP7448—0091) and mixtures thereof. When the enzyme is a mycoplasma-specific nuclease, a preferred substrate is an acetoxymethyl ester or maleimide derivative of blue dye number 1 coupled to an aminoallyl-dNTP labeled nucleic acid substrate of the mycoplasma-specific nuclease. Typically the substrate is a reagent container, a culture medium container or a cell culture container.
In other aspects, the present invention provides a method of detecting mycoplasma contamination of a cell culture comprising the steps of providing a cell-permeable detectable label coupled to a cell-impermeant carrier in the culture medium wherein cleavage of the detectable label by a mycoplasma-specific enzyme is followed by uptake of the detectable label into cells; and detecting labeled cells, thereby detecting mycoplasma contamination of the cell culture. In certain embodiments, the mycoplasma-specific enzyme is a protease and the detectable label is an acetoxymethyl ester of derivative of blue dye number 1 coupled to a peptide substrate of the mycoplasma-specific protease. Preferred proteases can be selected from the group consisting of the gene product of pepA1 (MCAP—0157), pepA2 (MCAP—0195), pepA (leucyl aminopeptidase, such as MHP7448—0464), MCAP—0267 (metalloendopeptidase), pepP (Xaa-Pro endopeptidase, such as MCAP—0341 or MHP7448—0649), MCAP—0509, mapP (methionine amino peptidase, MCAP—0675 or MHP7448—0173), and mixtures thereof. In other preferred embodiments, the mycoplasma-specific enzyme is a nuclease and the detectable label is an acetoxymethyl ester of derivative of blue dye number 1 coupled to a nucleic acid substrate of the mycoplasma-specific nuclease Preferred nucleases can be selected from the group consisting of the 5′-3′ exonuclease encoded by MCAP—0047 or MHP7448—0581, the gene product of nfo (such as MCAP—0060 or MHP7448—0062), vacB (such as MCAP—0097 or MHP7448—0037), uvrC (such as MCAP—0252 or MHP7448—0066), mc (ribonuclease III, such as MCAP—0492 or MHP7448—0398), MCAP—0768, uvrB (such as MCAP—0773 or MHP7448—0648), uvrA (such as MCAP—0774 or MHP7448—0091) and mixtures thereof.
In other aspects, the present invention provides a method of determining the presence or absence of mycoplasma in a sample, comprising the steps of contacting the sample with a detectably labeled substrate for an enzyme produced and/or secreted by a mycoplasma under conditions that result in the modification of the substrate by the enzyme; and detecting the modification or the absence of the modification of the substrate wherein modification of the substrate indicates the presence of mycoplasma in the sample, and wherein the absence of modification of the substrate indicates the absence of mycoplasma in the sample. Preferably, the level of the detectable label is quantitatively related to the presence or amount of mycoplasma in the sample.
In preferred embodiments, the enzyme is a hydrolytic enzyme selected from a protease, a nuclease or a reductase. In certain embodiments, enzyme is a protease selected from group consisting of the gene product of pepA1 (MCAP—0157), pepA2 (MCAP—0195), pepA (leucyl aminopeptidase, such as MHP7448—0464), MCAP—0267 (metalloendopeptidase), pepP (Xaa-Pro endopeptidase, such as MCAP—0341 or MHP7448—0649), MCAP—0509, mapP (methionine amino peptidase, such as MCAP—0675 or MHP7448—0173), and mixtures thereof. In other preferred embodiments, the enzyme is a reductase selected from the group consisting of the gene product of nrdE (such as MCAP—0101), MCAP—0427, trxB (thioredoxin reductase, such as MCAP—0779 or MHP7448—0098), MCAP—0858 and mixtures thereof. In yet other preferred embodiments, the enzyme is a nuclease selected from the group consisting of the 5′-3′ exonuclease encoded by MCAP—0047 or MHP7448—0581, the gene product of nfo (such as MCAP—0060 or MHP7448—0062), vacB (such as MCAP—0097 or MHP7448—0037), uvrC (such as MCAP—0252 or MHP7448—0066), mc (ribonuclease III, such as MCAP—0492 or MHP7448—0398), MCAP—0768, uvrB (such as MCAP—0773 or MHP7448—0648), uvrA (such as MCAP—0774 or MHP7448—0091) and mixtures thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.
FIG. 1 is a photograph of the decolorization of an azo dye, reactive black 5, with supernatants of cultured bacteria. Each well was incubated with 10 μg of reactive black 5 plus 190 μl of culture supernatant from the following bacterium: E. coli, E. faecalis, S. aureus, P. aeruginosa, S. pyogenes, and S. marcescens. Such azo dyes were decolorized by most bacteria after incubation with the dye for about 18 hours. The decolorization is indicative of reductases produced by the bacteria.
FIG. 2A is a diagrammatic illustration of an embodiment of a contamination biosensor 200 placed on a container 100 for a reagent or culture medium.
FIG. 2B a diagrammatic illustration of an embodiment of a contamination biosensor 210 placed on a container 110 for tissue culture.
FIG. 3 is a diagrammatic illustration of an embodiment of a mycoplasma contamination detection system for cell culture, showing in FIG. 3A a cell 300 in an uncontaminated culture, and in FIG. 3B, a cell 300 in a contaminated culture containing a dye deposit 360 that is indicative of mycoplasma contamination.
FIG. 4A shows RNA samples used in RT-PCR. The lanes are: a) 1 kB DNA Ladder, b) BHK-21 cells infected with Mycoplasma hyorhinis as a monolayer, c) the pellet of BHK-21 cells medium infected with Mycoplasma hyorhinis, and d) the pellet of Mycoplasma hyorhinis from mycoplasma enrichment broth (not from tissue culture cells).
FIG. 4B is a graphical representation of the expression of several Mycoplasma hyorhinis genes under conditions A-J: A) lon, 3T3 cells growing as a monolayer, B) lon, BHK-21 cells growing in DMEM, C) map, 3T3 cells growing as a monolayer, D) map, BHK-21 cells growing in DMEM, E) pepA, 3T3 cells growing as a monolayer, F) pepA, BHK-21 cells growing in DMEM, G) trxB, 3T3 cells growing as a monolayer, H) trxB, BHK-21 cells growing in DMEM, I) vacB, 3T3 cells growing as a monolayer, J) vacB, BHK-21 cells growing in DMEM.
FIG. 5 shows the results of testing of primers with genomic DNA from Mycoplasma hyorhinis. The PCR products were run on a 1.5% agarose gel. We performed 35 cycles of hot start PCR with an initial melt of 95° C. for 4 minutes, followed by a melt at 95° C. for 45 seconds, an annealing at 50° C. for 45 seconds, and a final extension step 72° C. for 7 minutes. The lane order includes a 1 kB DNA ladder (a), 5′-3′ exonuclease (b &k), gcp (c & l), lon (d & m), map (e & n), nfo (f & o), nox, (g&p), trxB (h &q), uvrA (i &r), p37 (j&s), all using 0.5 μl of DNA template (B-J) or 1 μl of DNA diluted 1:10.
FIG. 6 shows the results of testing of further PCR primers with genomic DNA from Mycoplasma hyorhinis. Lanes include 1 kB DNA ladder (a), gcp (b), hrcA (c), lgt (d), pepA (e), pepP pth (g), mc (h), uvrB (i), uvrC (j), vacB (k), 5′-3′ (1), nox (m), p37 (n). The PCR product for pepF amplified the correct size product as well (not shown).
FIG. 7 shows the results of testing by RT-PCR of all primers that worked under 50° C. AT. The RNA template used was the BHK-21 DMEM pellet that had been previously treated with DNase. For each 41 of a 1:10 dilution of template. RT, 45° C. for 10 minutes, 95° C. for 15 minutes, amp. cycled 35 times 95° C. 15 s 50° C. 45 s, final extension at 72° C. for 7 minutes. Lanes include 1 kB DNA ladder (a), 5′-3′(b), lgt (c), lon (d), map (e), nfo (f), nox (g), pepA (h), pepF (i), pth (j), rnc (k), trxB (1), uvrA (m), uvrB (n), vacB (O), p37 (p), no primer control.
FIG. 8 shows an agarose gel loaded with double stranded DNA (dsDNA, lanes b-i) or double stranded RNA (ribosomal RNA, lanes j-q) treated with M. hyorhinis extract from a cell culture infection or supernantants from infected or uninfected cell cultures. The respective lanes contain: a) 1 kb DNA ladder, b) dsDNA exposed to an aliquot of M. hyorhinis extract for 30 minutes, c) dsDNA exposed to an aliquot of the supernatant of an infected cell culture for 30 minutes, d) dsDNA exposed to an aliquot of the supernatant of an uninfected cell culture for 30 minutes, e) dsDNA exposed to H2O for 30 minutes, f) dsDNA exposed to an aliquot of M. hyorhinis extract for 0 minutes, g) dsDNA exposed to an aliquot of the supernatant of an infected cell culture for 0 minutes, h) dsDNA exposed to an aliquot of the supernatant of an uninfected cell culture for 0 minutes, i) dsDNA exposed to H2O for 0 minutes, j) ribosomal RNA exposed to an aliquot of M. hyorhinis extract for 30 minutes, k) ribosomal RNA exposed to an aliquot of the supernatant of an uninfected cell culture for 30 minutes, 1) ribosomal RNA exposed to an aliquot of the supernatant of an uninfected cell culture for 30 minutes, m) ribosomal RNA exposed to exposed to H2O 30 minutes, n) ribosomal RNA exposed to an aliquot of M. hyorhinis extract for 0 minutes, o) ribosomal RNA exposed to an aliquot of the supernatant of an infected cell culture for 0 minutes, p) ribosomal RNA exposed to an aliquot of the supernatant of an uninfected cell culture for 0 minutes, and q) ribosomal RNA exposed to an aliquot of H2O for 0 minutes.
FIG. 9 is a graph of the results of testing the thioredoxin reductase activities of M. hyorhinis, E. coli and S. aureus. There was no significant activity from 104-106 CFU/ml of S. aureus or E. coli using the DTNB substrate.
FIG. 10A and FIG. 10B are schematic diagrams of a substrate-linked enzymatic reporter reagent. In FIG. 10A, a bead 400 is covalently linked to horseradish peroxidase 420 by a molecule DTSSP 410 that is a substrate for a reductase such as trxB. In FIG. 10B, a bead 400 is covalently linked to luciferase 440 by a molecule Leu 430 that is a substrate for a protease such as pepA.
FIG. 11 is a graph showing the effect of 1 mM DTT in enhancing the fluorescence of trxB substrates 2′,7′-difluoro-4′-(2-(5-((dimethylaminophenyl) azo)pyridyl)dithiopropionyl aminomethyl)fluorescein (DFDMAP) and BODIPY®FL L-cystine.
FIG. 12 is a graph showing the effect of digitonin on the trxB assay.
FIG. 13 is a graph showing the effect of acid pH levels on the leu-MCA assay.
FIG. 14 is a graph showing the effect of basic pH levels on the leu-MCA assay.
FIG. 15 is a graph showing the sensitivity of the leu-MCA assay.
DETAILED DESCRIPTION OF THE INVENTION
Many of the genomes of the genus Mycoplasma have been sequenced. It is apparent that the microorganism has few biosynthetic genes, and the microorganism can only thrive in very rich growth mediums. Using a genomic approach in which we compared the genomes of 10 different mycoplasma (M. gallisepticum, M. capricolum, M. genitalium, M. hyoppneumonia, M. mobile, M. mycoides, M. penetrans, M. pneumonia, M. pulmonis, and M. synoviae) at 40% sequence identity, we have identified 243 genes that are conserved in all Mycoplasma species studied to date. Mycoplasma species use an array of hydrolytic enzymes to uptake materials that compensates for having very few internal biosynthetic processes. The identified genes are listed in Table 1, below, using the M. capricolum notation.
The common genes included a variety of enzymes that can be grouped into seven classes: synthetic enzymes, hydrolytic enzymes, chaperones, permeases, kinases, transcription factors, and ribosomal proteins. The presence of acetate kinase has been disclosed as an assay for the presence of Mycoplasma (U.S. published patent application No. 2004/0265942). However, this assay is an enzyme cascade assay requiring luciferase and is not amenable to a simple and direct method for measuring contamination in culture and in vivo.
The hydrolytic enzymes are interesting targets both for diagnosis and the treatment of a mycoplasma infection because they are secreted and likely involved in infection and virulence. The common hydrolytic enzymes of Mycoplasma species include: proteases, such as the gene products of MCAP—0157, MCAP—0195, MCAP—0267, MCAP—0341, MCAP—0509, MCAP—0675, nucleases, such as the gene products of MCAP—0047, MCAP—0060, MCAP—0097, MCAP—0252, MCAP—0492, MCAP—0768, MCAP—0773, MCAP—0774, and reductases, such as the gene products of MCAP—0101, MCAP—0427, MCAP—0779, and MCAP—0858.
Reductase activity can be measured through a Azo dye that gets decolorized by the release of reductases from many bacterial cells. An azo dye such as reactive black 5 or DABCYL (4-((4-(dimethylamino)phenyl)azo)benzoic acid) is completely decolorized by many bacterial cultured supernatant after just 18 hours on incubation. A sensor placed on the bottom of a culture dish, buffer container or even on a swab for measuring the presence of mycoplasma in human fluids can be used to ascertain bacterial contamination or infection. The benefit of a simple azo dye sensor is low cost although it may not specifically detect different bacteria. FIG. 1 is a photograph of a microtiter plate containing reactive black 5 decolorized by incubation with different pathogenic bacteria. Each well was incubated with 10 μg of reactive black 5 plus 190 μl of filtered culture supernatant from the following bacterium: E. coli, E. faecalis, S. aureus, P. aeruginosa, S. pyogenes, and S. marcescens. Such azo dyes are decolorized by most bacteria after incubation with the dye for about 18 hours. The decolorization is indicative of reductases produced by the bacteria. Mycoplasma reductases such as the gene products of MCAP—0101. MCAP—0427, MCAP—0779, and MCAP—0858 can also decolorize such substrates.
In other embodiments, substrates for reductases are reagents that produce a fluorescent signal. Suitable such reagents include DTNB (5,5′-Dithio-bis-(2-nitrobenzoic acid), also known as Ellman's reagent.
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Two other suitable fluorogenic compounds are BODIPY®FL L-cystine,
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and 2′,7′-difluoro-4′-(2-(5-((dimethylaminophenyl)azo)pyridyl) dithiopropionyl aminomethyl)fluorescein,
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Specific peptidase substrates can be used to identify a specific bacterium. Published patent applications disclosing both specific and broad-spectrum targets for detection of pathogens include WO 2005/042770, WO2005/012556 and WO2004/087942, which are incorporated herein by reference. Mycoplasmas secrete a lysine-specific endopeptidase, an aminopeptidase and a carboxypeptidase that make it possible to specifically detect the presence of mycoplasma by using a substrate that is specific for these enzymes. Suitable aminopeptidases and carboxypeptidases have been purified by Watanabe and colleagues (Watanabe, T. (1988), Proteolytic activities of Mycoplasma salivarium, Adv Dent Res 2(2):297-300; Watanabe, T (1985) Proteolytic activity of mycoplasmas and ureaplasmas isolated freshly from human saliva, Medical Microbiology and Immunology 173(5): 251-255; Watanabe, T. et al., (1984) Aminopeptidase and caseinolytic activities of Mycoplasma salivarium Medical Microbiology and Immunology, 172 (4): 257-264). In a preferred embodiment, these purified or partially purified enzymes are used in a high-throughput screen to identify potential novel substrates.
Mycoplasmas produce both secreted and membrane-bound nucleases that are involved in obtaining nucleotides for DNA synthesis. See Minion, C. J. D. Goguen (1986) Identification and Preliminary Characterization of External Membrane-Bound Nuclease Activities in Mycoplasma pulmonis, Infection And Immunity, 51(1):352-354; Kannan, T. R.,& Baseman, J. B., (2006) ADP-ribosylating and vacuolating cytotoxin of Mycoplasma pneumoniae represents unique virulence determinant among bacterial pathogens. PNAS, 103:6724-6729; Bendjennat, M., et al., (1997) Purification and Characterization of Mycoplasma penetrans Ca2+/Mg2+-Dependent Endonuclease, Journal of Bacteriology 179:2210-2220; Minion, C. F., et al., (1993) Membrane-Associated Nuclease Activities in Mycoplasmas. Journal of Bacteriology 175:7842-7847.
RNA or DNA sequences that are efficiently hydrolyzed by Mycoplasma nucleases that labeled with a detectable colorimetric or fluorescent dye can be used to detect the presence of mycoplasma contamination. A dye such as blue dye number 1 is not decolorized by microorganisms and would be a good choice for a colorimetric reporter. The dye is labeled with a reactive aminoallyl-dUTP via a Klenow reaction using techniques known to one skilled in the art to covalently attach the dye to a nucleic acid. See Hasseman, J. J., et al., 2006 Microbial Genomic DNA Aminoallyl Labeling For Microarrays, The Institute For Genomic Research Standard Operating Procedure # M009. The aminoallyl groups on the nucleic acid would then be available for labeling with a reactive fluorescent or chromogenic dye molecule. The dye-labeled nucleic acid can be attached to the surface of a sterile bottle. If the bottle after opening became contaminated with mycoplasmas, the spot of color on the inner surface of the bottle would be released, indicating that the bottle is contaminated. FIG. 2A is a diagrammatic illustration of a contamination biosensor 200 placed on a container 100 for a reagent or culture medium. FIG. 2B a diagrammatic illustration of a contamination biosensor 210 placed on a container 110 for tissue culture.
Azo dyes such as reactive black 5 and DABCYL are decolorized by bacteria and can be used as a broad spectrum sensor for microbial contamination. Blue dye number 1, which is not decolorized by bacteria, can be used as a label of nucleic acids or a peptide to give a specific probe for mycoplasmas or other contaminating microorganism. Fluorescent probes or the release of fluorescent micro-spheres can be used to indicate contamination. Contamination can be measured by eye, using a fluorimeter or colorimeter or on a microscope stage.
In another embodiment, a peptide or nucleic acid can be labeled with an acetoxymethyl ester of a dye, such as blue dye number 1, that upon hydrolytic cleavage would be taken up by cells in culture and thereby turn them blue to indicate the presence of mycoplasmas in the culture medium. FIG. 3 is a diagrammatic illustration of an embodiment of such a mycoplasmas contamination detection system for cell culture, showing in FIG. 3A a cell 300 in an uncontaminated culture, and in FIG. 3B, a cell 300 in a contaminated culture containing a dye deposit 360 that is indicative of mycoplasmas contamination. An acetoxymethyl ester derivative of blue dye number 1 coupled to a peptide or nucleic acid carrier would be impermeable to tissue culture cells until contamination with mycoplasmas. The proteases or nucleases from Mycoplasma spp. would cleave the carrier from the acetoxymethyl ester derivative of blue dye number 1, thereby allowing the acetoxymethyl ester derivative of blue dye number 1 to be taken up by the tissue culture cells. Tissue culture cells that become colored blue indicate that the culture was contaminated with mycoplasmas. The colored cells can be observed with a light microscope. Alternatively a cell permeable fluorescent dye can be used and the fluorescing cells can be detected with a fluorescence microscope.
TABLE 1
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Genes characterized by sequences common to Mycoplasma spp. (40% identity)
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Gene
Symbol
Common Name
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MCAP_0001
dnaA
chromosomal replication initiator protein DnaA
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MCAP_0002
dnaN
DNA polymerase III, beta subunit
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MCAP_0004
ksgA
dimethyladenosine transferase
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MCAP_0008
dnaX
DNA polymerase III gamma-tau subunits
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MCAP_0010
tmk
thymidylate kinase
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MCAP_0011
DNA polymerase III, delta prime subunit
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MCAP_0017
ftsH
ATP-dependent metalloprotease FtsH
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MCAP_0022
acyl carrier protein phosphodiesterase, putative
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MCAP_0026
rpsR
30S ribosomal protein S18
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MCAP_0035
metG
methionyl-tRNA synthetase
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MCAP_0038
ABC transporter, permease protein
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MCAP_0039
ABC transporter, permease protein
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MCAP_0040
gyrA
DNA gyrase, A subunit
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MCAP_0041
gyrB
DNA gyrase, B subunit
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MCAP_0045
secA
preprotein translocase, SecA subunit
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MCAP_0047
5-3 exonuclease family protein
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MCAP_0060
endonuclease IV
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MCAP_0065
rplK
50S ribosomal protein L11
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MCAP_0066
rplA
ribosomal protein L1
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MCAP_0067
rplJ
50S ribosomal protein L10
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MCAP_0068
rplL
50S ribosomal protein L7/L12
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MCAP_0070
rpoB
DNA-directed RNA polymerase, beta subunit
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MCAP_0071
rpoC
DNA-directed RNA polymerase beta subunit
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MCAP_0074
ribose 5-phosphate isomerase B, putative
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MCAP_0075
glyA
serine hydroxymethyltransferase
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MCAP_0076
upp
uracil phosphoribosyltransferase
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MCAP_0078
atpB
ATP synthase F0, subunit A
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MCAP_0079
atpE
ATP synthase F0, subunit c
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MCAP_0082
atpA1
ATP synthase F1, alpha subunit
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MCAP_0083
atpG
ATP synthase F1, gamma subunit
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MCAP_0084
atpD1
ATP synthase F1, beta subunit
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MCAP_0094
ptsG
PTS system, glucose-specific IIABC component
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MCAP_0096
smpB
SsrA-binding protein
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MCAP_0097
Rnase R (VacB) and RNase II family 3-5 exoribonucleases
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MCAP_0101
nrdE
ribonucleoside-diphosphate reductase 2, alpha subunit
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MCAP_0104
prs
phosphoribosylpyrophosphate synthase
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MCAP_0105
pth
peptidyl-tRNA hydrolase
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MCAP_0107
dnaC
replicative DNA helicase
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MCAP_0110
cysS
cysteinyl-tRNA synthetase
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MCAP_0111
RNA methyltransferase, TrmH family, group 3
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MCAP_0114
nusG
transcription antitermination protein NusG
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MCAP_0119
oligopeptide ABC transporter, ATP-binding protein
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MCAP_0120
oligopeptide ABC transporter, ATP-binding protein
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MCAP_0124
hydrolase, TatD family
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MCAP_0130
gltX
glutamyl-tRNA synthetase
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MCAP_0136
fba
fructose-1,6-bisphosphate aldolase, class II
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MCAP_0140
rpmE
ribosomal protein L31
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MCAP_0142
DHH phosphoesterase family protein, putative
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MCAP_0143
tdk
thymidine kinase
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MCAP_0144
prfA
peptide chain release factor 1
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MCAP_0145
modification methylase, HemK family
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MCAP_0151
rpsL
30S ribosomal protein S12
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MCAP_0152
rpsG
30S ribosomal protein S7
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MCAP_0153
fusA
translation elongation factor G
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MCAP_0154
tuf
translation elongation factor Tu
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MCAP_0157
pepA1
cytosol aminopeptidase
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MCAP_0159
alaS
alanyl-tRNA synthetase
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MCAP_0163
oligopeptide ABC transporter, ATP-binding protein
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MCAP_0195
pepA2
cytosol aminopeptidase
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MCAP_0200
spermidine/putrescine ABC transporter, permease protein
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and spermidine/putrescine-binding protein
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MCAP_0201
spermidine/putrescine ABC transporter, permease protein
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MCAP_0203
rplT
50S ribosomal protein L20
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MCAP_0205
infC
translation initiation factor IF-3
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MCAP_0208
gmk
guanylate kinase
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MCAP_0213
eno
enolase4.2.1.11
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MCAP_0216
hpt1
hypoxanthine phosphoribosyltransferase
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MCAP_0220
pfkA
Phosphofructokinase
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MCAP_0221
pyk
pyruvate kinase
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MCAP_0222
thrS
threonyl-tRNA synthetase
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MCAP_0223
NADH oxidase
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MCAP_0224
lipoate-protein ligase
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MCAP_0225
pdhA
pyruvate dehydrogenase complex, El component,
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alpha subunit
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MCAP_0226
pdhB
pyruvate dehydrogenase complex, E1 component,
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beta subunit
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MCAP_0228
pdhD
dihydrolipoamide dehydrogenase
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MCAP_0229
pta
phosphate acetyltransferase
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MCAP_0230
ackA
acetate kinase
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MCAP_0233
ptsI
phosphoenolpyruvate-protein phosphotransferase
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MCAP_0234
crr
PTS system, glucose-specific IIA component
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MCAP_0237
rpsD
30S ribosomal protein S4
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MCAP_0245
GTP-binding conserved hypothetical protein
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MCAP_0251
greA
transcription elongation factor GreA
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MCAP_0252
uvrC
excinuclease ABC, C subunit
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MCAP_0255
conserved hypothetical protein
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MCAP_0258
valS
valyl-tRNA synthetase
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MCAP_0260
rpe
ribulose-phosphate 3-epimerase
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MCAP_0261
rsgA
ribosome small subunit-dependent GTPase A
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MCAP_0267
metalloendopeptidase, putative
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MCAP_0318
proS
prolyl-tRNA synthetase
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MCAP_0321
lepA
GTP-binding protein LepA
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MCAP_0323
aspS
aspartyl-tRNA synthetase
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MCAP_0324
hisS
His-tRNA synthetase 6.1.1.21
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MCAP_0330
rpsO
30S ribosomal protein S15
|
MCAP_0333
infB
translation initiation factor IF-2
|
MCAP_0336
transcription elongation protein nusA, putative
|
MCAP_0339
polC
DNA polymerase III, alpha subunit, Gram-positive type
|
MCAP_0340
cdsA
phosphatidate cytidylyltransferase
|
MCAP_0341
Xaa-Pro peptidase
|
MCAP_0342
trpS
tryptophanyl-tRNA synthetase
|
MCAP_0358
atpA2
ATP synthase F1, alpha subunit
|
MCAP_0359
atpD2
ATP synthase F1, beta subunit
|
MCAP_0364
RNA methyltransferase, TrmH family
|
MCAP_0365
hydrolase of the HAD superfamily, putative
|
MCAP_0367
hrcA
heat-inducible transcription repressor HrcA
|
MCAP_0369
dnaK
Chaperone protein dnaK (Heat shock protein 70)
|
(Heat shock 70 kDaprotein) (HSP70)
|
MCAP_0371
rpsB
30S ribosomal protein S2
|
MCAP_0372
tsf
translation elongation factor Ts
|
MCAP_0374
pyrH
uridylate kinase
|
MCAP_0375
frr
ribosome recycling factor
|
MCAP_0376
argS
arginyl-tRNA synthetase
|
MCAP_0383
pheS
phenylalanyl-tRNA synthetase, alpha subunit
|
MCAP_0384
pheT
phenylalanyl-tRNA synthetase, beta subunit
|
MCAP_0388
mraW
S-adenosyl-methyltransferase MraW 2.1.1.- 479149
|
MCAP_0393
ileS
isoleucyl-tRNA synthetase
|
MCAP_0395
ribosomal large subunit pseudouridine synthase, RluA family
|
MCAP_0410
conserved hypothetical protein, TIGR00096
|
MCAP_0412
rplU
50 ribosomal protein L21
|
MCAP_0414
rpmA
50S ribosomal protein L27
|
MCAP_0423
ImpB/MucB/SamB family protein
|
MCAP_0427
pyridine nucleotide-disulphide oxidoreductase
|
MCAP_0439
ldh
L-lactate/malate dehydrogenase
|
MCAP_0445
triacylglycerol lipase
|
MCAP_0446
triacylglycerol lipase, putative
|
MCAP_0449
lipoate-protein ligase
|
MCAP_0454
gtsA
glycerol ABC transporter, ATP-binding protein
|
MCAP_0456
parC
DNA topoisomerase IV, A subunit
|
MCAP_0457
parE
DNA topoisomerase IV, B subunit
|
MCAP_0462
RNA methyltransferase, TrmH family
|
MCAP_0465
pgi
glucose-6-phosphate isomerase
|
MCAP_0469
aminotransferase, class V
|
MCAP_0472
HIT family protein
|
MCAP_0474
ung
uracil-DNA glycosylase
|
MCAP_0476
gid
glucose inhibited division protein
|
MCAP_0478
metK
S-adenosylmethionine synthetase
|
MCAP_0479
conserved hypothetical protein TIGR00282
|
MCAP_0481
ftsY
signal recognition particle-docking protein FtsY
|
MCAP_0488
rpmB
50S ribosomal protein L28
|
MCAP_0492
ribonuclease III
|
MCAP_0495
structural maintenance of chromosomes
|
(SMC) superfamily protein
|
MCAP_0497
apt
adenine phosphoribosyltransferase
|
MCAP_0503
rpoD
RNA polymerase sigma factor RpoD
|
MCAP_0504
dnaG
DNA primase
|
MCAP_0505
glyS
glycyl-tRNA synthetase
|
MCAP_0507
era
GTP-binding protein Era
|
MCAP_0509
Peptidase C39 family protein
|
MCAP_0510
DJ-1 family protein
|
MCAP_0516
lon
ATP-dependent protease La
|
MCAP_0517
tig
trigger factor
|
MCAP_0519
efp
translation elongation factor P
|
MCAP_0521
conserved hypothetical protein
|
MCAP_0523
trmU
tRNA (5-methylaminomethyl-2-thiouridylate)-
|
methyltransferase
|
MCAP_0529
nicotinate (nicotinamide) nucleotide adenylyltransferase/
|
conserved hypothetical domain
|
MCAP_0532
Spo0B-associated GTP-binding protein, putative
|
MCAP_0544
rplS
50S ribosomal protein L19
|
MCAP_0545
trmD
tRNA(guanine-N1)-methyltransferase
|
MCAP_0547
rpsP
30S ribosomal protein S16
|
MCAP_0549
ffh
signal recognition particle protein
|
MCAP_0551
recA
recA protein
|
MCAP_0577
engA
GTP-binding protein engA
|
MCAP_0578
cmk
cytidylate kinase
|
MCAP_0581
ppa
inorganic pyrophosphatase
|
MCAP_0589
ribulose-phosphate 3-epimerase, putative
|
MCAP_0601
scpB
chromosomal segregation and condensation protein B
|
MCAP_0606
hydrolase, alpha/beta fold family
|
MCAP_0609
Uncharacterised membrane protein, UPF0154 family
|
MCAP_0610
tkt
transketolase
|
MCAP_0613
glucose-inhibited division protein, putative
|
MCAP_0616
fructose/tagatose bisphosphate aldolase, class II
|
MCAP_0623
metallo-beta-lactamase superfamily protein
|
MCAP_0631
pgk
phosphoglycerate kinase
|
MCAP_0632
gap
glyceraldehyde-3-phosphate dehydrogenase
|
MCAP_0635
mutM
formamidopyrimidine-DNA glycosylase
|
MCAP_0636
polA
DNA polymerase I
|
MCAP_0637
dnaE
DNA-directed DNA polymerase III (polc)
|
MCAP_0639
tyrS
tyrosyl-tRNA synthetase
|
MCAP_0659
leuS
leucyl-tRNA synthetase
|
MCAP_0662
rpsI
30S ribosomal protein S9
|
MCAP_0663
rplM
50S ribosomal protein L13
|
MCAP_0666
cobalt ABC transporter, permease protein
|
MCAP_0667
cobalt ABC transporter, ATP-binding protein, putative
|
MCAP_0668
cobalt ABC transporter, ATP-binding protein, putative
|
MCAP_0669
rplQ
50S ribosomal protein L17
|
MCAP_0670
rpoA
DNA-directed RNA polymerase, alpha chain
|
MCAP_0671
rpsK
30S ribosomal protein S11
|
MCAP_0672
rpsM
30S ribosomal protein S13
|
MCAP_0675
map
methionine aminopeptidase, type I
|
MCAP_0676
adk
adenylate kinase
|
MCAP_0677
secY
preprotein translocase, SecY subunit
|
MCAP_0678
rplO
50S ribosomal protein L15
|
MCAP_0679
rpsE
30S ribosomal protein S5
|
MCAP_0680
rplR
50S ribosomal protein L18
|
MCAP_0681
rplF
50S ribosomal protein L6
|
MCAP_0682
rpsH
ribosomal protein S8
|
MCAP_0684
rplE
ribosomal protein L5
|
MCAP_0686
rplN
ribosomal protein L14
|
MCAP_0687
rpsQ
ribosomal protein S17
|
MCAP_0689
rplP
ribosomal protein L16
|
MCAP_0690
rpsC
ribosomal protein S3
|
MCAP_0691
rplV
ribosomal protein L22
|
MCAP_0692
rpsS
30S ribosomal protein S19
|
MCAP_0693
rplB
50S ribosomal protein L2
|
MCAP_0694
rplW
50S ribosomal protein L23
|
MCAP_0695
rplD
50S ribosomal protein L4
|
MCAP_0696
rplC
50 ribosomal protein L3
|
MCAP_0697
rpsJ
30S ribosomal protein S10
|
MCAP_0707
potassium uptake protein, TrkH family, putative
|
MCAP_0708
potassium uptake protein, TrkA family, putative
|
MCAP_0709
gatB
glutamyl-tRNA(Gln) amidotransferase, B subunit
|
MCAP_0710
gatA
glutamyl-tRNA(Gln) amidotransferase, A subunit
|
MCAP_0712
ligA
DNA ligase, NAD-dependent
|
MCAP_0714
ribosomal large subunit pseudouridine synthase, RluA family
|
MCAP_0716
ptsH
phosphocarrier protein hpr
|
MCAP_0750
tpiA
triosephosphate isomerase
|
MCAP_0751
HAD-superfamily hydrolase subfamily IIB, protein
|
MCAP_0752
gpmI
2,3-bisphosphoglycerate-independent
|
phosphoglycerate mutase
|
MCAP_0755
deoC
deoxyribose-phosphate aldolase
|
MCAP_0757
deoA
pyrimidine-nucleoside phosphorylase
|
MCAP_0761
trmB
tRNA (guanine-N(7)-)-methyltransferase
|
MCAP_0765
hpt2
hypoxanthine phosphoribosyltransferase
|
MCAP_0768
deoxyribonuclease, TatD family
|
MCAP_0773
uvrB
excinuclease ABC, B subunit
|
MCAP_0774
uvrA
excinuclease ABC, A subunit
|
MCAP_0778
lgt1
prolipoprotein diacylglyceryl transferase
|
MCAP_0779
trxB
thioredoxin reductase
|
MCAP_0780
lgt2
prolipoprotein diacylglyceryl transferase
|
MCAP_0781
conserved hypothetical protein
|
MCAP_0792
topA
DNA topoisomerase I
|
MCAP_0805
engD
GTP-dependent nucleic acid-binding protein engD
|
MCAP_0807
gidB
methyltransferase gidB
|
MCAP_0808
pgsA
CDP-diacylglycerol--glycerol-3-phosphate 3-
|
phosphatidyltransferase
|
MCAP_0814
transporter protein, putative
|
MCAP_0818
rpsT
30S ribosomal protein S20
|
MCAP_0819
trmE
tRNA modification GTPase TrmE
|
MCAP_0821
glycoprotease family protein
|
MCAP_0824
asnS
asparaginyl-tRNA synthetase
|
MCAP_0834
hydrolase, haloacid dehalogenase-like family, putative
|
MCAP_0836
lysS
lysyl-tRNA synthetase
|
MCAP_0839
serS
seryl-tRNA synthetase
|
MCAP_0844
PTS system glucose-specific IIBC component
|
MCAP_0849
deoD
purine nucleoside phosphorylase
|
MCAP_0856
gidA
glucose inhibited division protein A
|
MCAP_0858
pyridine nucleotide-disulphide oxidoreductase
|
MCAP_0860
membrane protein, putative
|
MCAP_0870
rpmH
50S ribosomal protein L34
|
|
Example 1
Mycoplasma hyorhinis Enzymes
The subset of Mycoplasma genes from the Mycoplasma hyorhinis genome that were selected for further study are listed in Table 2, below. DNA PCR primers were made for each of these genes and the PCR are shown in FIG. 5 and FIG. 6.
TABLE 2
|
|
Hydrolytic enzymes in common among Mycoplasma ssp.
|
Gene
Symbol
Common Name
|
|
1
MHP7448_0010
hrcA
heat-inducible transcription repressor
|
2
MHP7448_0037
vacB
VACB-like ribonuclease II
|
3
MHP7448_0062
nfo
endonuclease IV
|
4
MHP7448_0066
uvrC
excinuclease ABC subunit C
|
5
MHP7448_0082
nox
NADH oxidase
|
6
MHP7448_0091
uvrA
excinuclease ABC subunit A
|
7
MHP7448_0097
lgt
prolipoprotein diacylglyceryl transferase
|
8
MHP7448_0098
trxB
thioredoxin reductase
|
9
MHP7448_0173
map
methionine aminopeptidase
|
10
MPH7448_0204
pth
prptidyl-tRNA hydrolase
|
11
MHP7448_0398
rnc
ribonuclease III
|
12
MHP7448_0464
pepA
leucyl aminopeptidase
|
13
MHP7448_0521
pepF
oligoendopeptidase F
|
14
MHP7448_0524
lon
heat shock ATP-dependent protease
|
15
MHP7448_0581
5′-3′ exonuclease
|
16
MHP7448_0635
gcp
O-sialoglycoprotein endopeptidase
|
17
MHP7448_0648
uvrB
excinuclease ABC subunit B
|
18
MHP7448_0659
pepP
XAA-PRO aminopeptidase
|
|
Purification of Total RNA
Total RNA was isolated from Mycoplasma hyorhinis grown in BHK-21 and Swiss 3T3 tissue culture cells (FIG. 4A). RNA was purified both from the infected tissue culture media and culture cells (BHK-21 & Swiss 3T3) from the infected dish. RNA was purified using either the Invitrogen Triazol® Max bacterial RNA isolation kit or an acid phenol-guanidium thiocyanate and chloroform extraction procedure. Although the acid phenol-guanidium thiocyanate and chloroform extraction procedure had better quality RNA as judged by gel electrophoresis in a 1.5% agarose gel, the RNA from the Triazol® Max bacterial RNA kit had more mycoplasma RNA as judged by PCR of the p37 control gene. As a positive control for the RT-PCR reactions, we amplified the p37 gene from the total infected BHK-21 and 3T3 cell media. As a negative control, we also isolated total RNA from uninfected tissue culture media and uninfected BHK-21 or 3T3 cells and then performed RT-PCR using the p37 primer set. RT PCR was performed in a iCycler iQ PCR Detection System (Bio-Rad) using the SYBR Green One-Step Quantitative RT-PCR kit. Alternatively, for the preliminary studies RT PCR was performed with the Ambion Ag-Path kit. FIG. 4A shows an exemplary result of agarose gel electrophoresis of the RNA samples used in RT-PCR. The lanes are: a) 1 kB DNA Ladder, b) BHK-21 cells infected with Mycoplasma hyorhinis as a monolayer, c) the pellet of BHK-21 cells medium infected with Mycoplasma hyorhinis, and d) the pellet of Mycoplasma hyorhinis from mycoplasma enrichment broth (not from tissue culture cells).
The results of preliminary studies indicate that the following genes vacB, trxB, map, pepA, lon, and uvrB are transcribed at a high level. FIG. 4B is a graphical representation of the expression of several Mycoplasma hyorhinis genes under conditions A-J: A) lon, 3T3 cells growing as a monolayer, B) lon, BHK-21 cells growing in DMEM, C) map, 3T3 cells growing as a monolayer, D) map, BHK-21 cells growing in DMEM, E) pepA, 3T3 cells growing as a monolayer, F) pepA, BHK-21 cells growing in DMEM, G) trxB, 3T3 cells growing as a monolayer, H) trxB, BHK-21 cells growing in DMEM, I) vacB, 3T3 cells growing as a monolayer, J) vacB, BHK-21 cells growing in DMEM. The level of expression from strongest to weakest for these abundant mRNA is trxB>pepA>map>vacB>lon>uvrB.
Substrates for the hydrolytic enzymes corresponding to these putative abundant mRNAs from Mycoplasma were identified using both literature and patent searches. Certain selected examples are provided in Table 3, below.
TABLE 3
|
|
Mycoplasma Hydrolase Substrates
|
Gene Symbol
Substrates
References
|
|
vacB
ds RNAse activity
J. Cell. Physiol. 143(3) 416-419
|
trxB
DTNB
Cayman Chemical Company
|
Map
Rhodamine based fluorogenic
J. Biomol. Screening 7(6)
|
substrates
531-540, 2002
|
pepA
Leucine at N-terminal of a peptide
Curr. Microbiol. 48(1)32-38, 2004
|
lon
Glutaryl-Ala-Ala-Phe-
J. Biol. Chem. 260(22) 1 1985
|
methoxynaphthylamine + ATP
|
uvrB
(UvrA)2(UvrB)1 com
Proc. Natl Soc. USA 86(14)
|
5237-5241, 1985.
|
|
The genes that were determined by quantitative RT-PCR results to be highly expressed in Mycoplasma hyorhinis when infecting 3T3 cells or BHK-21 cells: trxB, pepA, lon, vacB, map, and uvrB. The substrates for the enzymes produced by these gene products is reported in Table 4, below.
TABLE 4
|
|
Mycoplasma hyorhinis
|
Gene Encoding Enzymes and Enzyme Substrates
|
Gene
Enzyme
Substrate
|
|
trxB
Thioredoxin reductase
DTNB
|
pepA
Leucine aminopeptidase
leu-MCA
|
lon
ATP-dependent protease
glt-ala-ala-phe-MCA
|
map
Methionine aminopeptidase
met-MCA
|
vacB
exoribonuclease
dsRNA
|
uvrB
excinuclease
dsDNA
|
|
We examined the nuclease activities of VacB and UvrB by challenging extracts from M. hyorhinis isolated from a cell culture infection or medium from Mycoplasma-infected or uninfected cell culture with either double stranded (ds) RNA or dsDNA. The reaction was incubated for 30 minutes at 37° C. and the products were then analyzed by agarose gel electrophoresis, as shown in FIG. 8.
FIG. 8 shows an agarose gel loaded with double stranded DNA (dsDNA, lanes b-i) or double stranded RNA (ribosomal RNA, lanes j-q) treated with M. hyorhinis extract from a cell culture infection or supernatants from infected or uninfected cell cultures. The respective lanes contain: a) 1 kb DNA ladder, b) dsDNA exposed to an aliquot of M. hyorhinis extract for 30 minutes, c) dsDNA exposed to an aliquot of the supernatant of an infected cell culture for 30 minutes, d) dsDNA exposed to an aliquot of the supernatant of an uninfected cell culture for 30 minutes, e) dsDNA exposed to H2O for 30 minutes, f) dsDNA exposed to an aliquot of M. hyorhinis extract for 0 minutes, g) dsDNA exposed to an aliquot of the supernatant of an infected cell culture for 0 minutes, h) dsDNA exposed to an aliquot of the supernatant of an uninfected cell culture for 0 minutes, i) dsDNA exposed to H2O for 0 minutes, j) ribosomal RNA exposed to an aliquot of M. hyorhinis extract for 30 minutes, k) ribosomal RNA exposed to an aliquot of the supernatant of an uninfected cell culture for 30 minutes, l) ribosomal RNA exposed to an aliquot of the supernatant of an uninfected cell culture for 30 minutes, m) ribosomal RNA exposed to exposed to H2O 30 minutes, n) ribosomal RNA exposed to an aliquot of M. hyorhinis extract for 0 minutes, o) ribosomal RNA exposed to an aliquot of the supernatant of an infected cell culture for 0 minutes, p) ribosomal RNA exposed to an aliquot of the supernatant of an uninfected cell culture for 0 minutes, and q) ribosomal RNA exposed to an aliquot of H2O for 0 minutes.
There was no detectable dsDNAse activity (lanes b & c) in the M. hyorhinis extract or M. hyorhinis-infected medium suggesting that there is insufficient urvB activity for this enzyme to serve as a suitable basis for a diagnostic test for Mycoplasma contamination. Although dsRNAse activity was observed in the M. hyorhinis extract and M. hyorhinis-infected medium (lanes j and k), the uninfected tissue culture media control also had appreciable dsRNAse activity. This finding indicates that vacB activity would not be a suitable basis for a Mycoplasma diagnostic test due to cross-reactivity from ribonucleases present in the culture medium of uninfected cells.
TABLE 5
|
|
Mycoplasma hyorhinis
|
Assays for Proteolytic Activity
|
Gene
Enzyme
Substrate
Condition
Vmax
|
|
lon
ATP-dependent protease
glt-ala-ala-phe-MCA
Buffer
55.8
|
Uninfected medium
1460
|
Infected medium
1802
|
Mycoplasma
3354
|
positive control
|
pepA
Leucine aminopeptidase
leu-MCA
BufferA
2874
|
Uninfected medium
454
|
Infected medium
906
|
Mycoplasma
522
|
positive control
|
map
Methionine aminopeptidase
met-MCA
Buffer
19.4
|
Uninfected medium
430
|
Infected medium
627
|
Mycoplasma
733
|
positive control
|
|
AThis reading is probably artefactually high, and may indicate a bubble in the well. Subsequent experiments showed that Vmax in buffer was essentially zero.
|
Further studies examined the suitability of the aminopeptidases map or lon or the protease pepA for use in a diagnostic test. These studies used several fluorogenic substrates consisting of small chain amino acids coupled to a methoxy coumarin fluorescent probe (MCA, (7-methoxycoumarin-4-yl)acetyl). The results of the initial studies are provided in Table 5, above. The test conditions were “buffer,” phosphate-buffered saline (PBS), “uninfected medium,” medium from cell cultures not infected with Mycoplasma, “infected medium,” medium from cell cultures infected with Mycoplasma, and a Mycoplasma positive control derived from a Mycoplasma culture. In each case the uninfected media control had substantial background proteolytic activity, with pepA being the most candidate with a signal-to-noise ratio (S/N) of about 2, where S/N=(Vmax infected medium)/(Vmax uninfected medium).
Example 2
Detection of Mycoplasma Using trxB
In contrast to the protease and the double stranded nuclease markers, thioredoxin reductase (trxB) had significant activity specific to tissue culture cells co-infected with Mycoplasma hyorhinis. In earlier studies, we demonstrated that Mycoplasma thioredoxin reductase activity was measured in infected culture medium using the substrate DTNB (5,5′-Dithio-bis-(2-nitrobenzoic acid), also known as Ellman's reagent.
![]()
Other suitable fluorogenic thioredoxin reductase substrates have been reported in the literature.
Since thioredoxin reductases are widely distributed in eukaryotes and prokaryotic cells, there is a possibility that thioredoxin reductases from other microbes may cross-react with this assay to give a false positive result. One possible approach would be to use gentle lysis buffers that disrupt Mycoplasma cells, which do not have a cell wall, but do not appreciably lyse other bacteria that possess a cell wall. Studies demonstrated that there was no appreciable hydrolysis of DTNB by up to 106 CFU/ml E. coli or S. aureus (FIG. 9). This result indicates that the thioredoxin reductases of other gram-positive and gram-negative bacteria, represented by E. coli and S. aureus, are either minor enzymatic components or have a much lower specific activity than that of M. hyorhinis. This finding is consistent with the previous demonstration that Mollicutes such as Mycoplasma have a very highly active thioredoxin reductase system (NTS) (0.09-0.25 SA units) in the presence of NADPH. This high NTS activity is presumed to be useful for Mycoplasma for the detoxification of reactive oxygen compounds, since the Mollicutes have simple genomes that lack the genes encoding enzymes such as catalase, peroxidase and oxygen dismutase that function to remove H2O2 and other oxygen radicals in other bacteria (Gibson, D. G., et al., Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science. 319(5867):1215-1220 (2008)).
Although initial attempts to detect trxB with DTNB were successful and the DTNB did not cross react with Staphylococcus aureus or Escherichia coli (FIG. 9), the sensitivity of the assay was unacceptable (>106 CFU/ml). Other fluorescent probes such as BODIPY®FL L-cystine and the 2′,7′-difluoro-4′-(2-(5-((dimethyl amino phenyl)azo)pyridyl)dithiopropionyl aminomethyl)fluorescein (abbreviated as DFDMAP-fluorescein) were studied to improve the sensitivity and the signal-to-noise ratio of the assay. Assays were performed with 40 mM Tris pH 7.2, 100 mM NaCl±detergent.
A detergent lysis buffer procedure that would hydrolyze the simple Mycoplasma cell membranes but not lyse the tissue culture cells was needed. Methyl-6-O—(N-heptylcarbamoyl)-α-D-glucopyranoside (HECAMEG) is a preferred detergent. We have found that 0.5% HECAMEG was sufficient to lyse Mycoplasma cells while not disrupting the membranes of the cells grown in the culture medium. In contrast, 0.25% Triton X-100°, 0.4% BriJ 35®, and digitonin resulted in either significant increase in the background or loss in the true level of trxB activity. The activity of the fluorescent substrates is low compared to reduction with DTT suggesting that they may not be ideal or specific for trxB (FIG. 11). As expected, 0.1 mM NADPH enhanced the activity of trxB (FIG. 12). As a substrate for trxB, Bodipy® FL L-cystine provided a signal that was 7500 times that of the buffer control, however this signal was an artifact of the digitonin. DFDMAP fluorescein had a moderately improved detection level of 10,000-20,000 RFU at 107-108 CFU/ml.
An alternative substrate to produce the thioredoxin reductase signal was to measure the release of horseradish peroxidase (HRP) from a chromatography bead tethered with the heterobifunctional crosslinking reagent 3,3′-dithiobis[sulfosuccinimidylpropionate] (DTSSP).
![]()
The HRP-DTSSP-BEAD conjugate is shown schematically in FIG. 10A.
It was expected that frxB would be able to reduce the disulfide bridge of DTSSP, thereby releasing the HRP to react with its substrate tetramethylbenzidine (TMB), and in the presence of Mycoplasma, produce a blue color. However, it was found that the HRP-DTSSP-BEAD conjugate also cross-reacted to the tissue culture uninfected medium sample.
Other alternative substrates can use fluorescence energy transfer (FRET) with a disulfide bridge between EDANS and DABSYL, as shown below:
![]()
Alternatively, DFDMAP and BODIPY® FL L-cysteine moieties could be coupled to the thioredoxin peptide or the central Gly-Ala residues to enhance the specificity of these fluorescent probes. Initial studies of these approaches have not shown improved sensitivity or reduction of background of the uninfected media control.
Example 3
Detection of Mycoplasma Using Proteases
The proteases pepA, lon, and map were evaluated for use in the detection of Mycoplasma. pepA and lon were found to be expressed at a higher level than map based on RT-qPCR results (FIG. 5 and FIG. 6). Arginine amino peptidase activity has also been reported in Mycoplasma species.
A presently preferred substrate is leu-MCA that had significant activity above the uninfected media control under the gentle conditions used to lyse the Mycoplasma (0.05% HECAMEG, 1 mM MgCl2, 100 mM NaCl, 40 mM Tris buffer, pH 8.5). The MCA-Leu substrate produces a signal level of 500 mOD in 30 minutes with M. hyorhinis, while M. hyorhinis has weak activity for arg-MCA. Results are provided in Table 6, below.
TABLE 6
|
|
Vmax Measured Using MCA Labeled Substrates
|
Under Different Conditions
|
Vmax
|
Condition
Arg-MCA
Leu-MCA
Met-MCA
Leu/Met
All 3
|
|
Buffer
0
236
125
0
193
|
Medium
676
2118
1731
2024
1443
|
Uninfected
29034
17422
19004
14937
25770
|
Medium
|
Infected
60903
415190
302082
373456
271170
|
Medium
|
Signal/Noise
2.10
23.83
15.90
25.00
10.52
|
|
In further studies, we determined that the background of the uninfected cells could be reduced even further by adjusting the pH, with an optimum at pH 8.5. FIG. 13 is a graph showing the effect of acid pH levels on the leu-MCA assay. FIG. 14 is a graph showing the effect of basic pH levels on the leu-MCA assay.
Detergent lysis of M. hyorhinis with HECAMEG gives better signal than sonication. Manganese or magnesium also improves the signal to noise ratio. In the studies on the effects of divalent cations, 1 mM MgCl2 of the standard mixture was replaced by 1 mM MnCl2, 1 mM MgSO4 or 1 mM EDTA, as indicated in Table 7, below.
TABLE 7
|
|
Effect of Divalent Cations
|
Vmax, MCA-Leu Substrate
|
Condition
EDTA
MnCl2
MgSO4
MnCl2
|
|
Buffer
0
0
483
84
|
Medium
1710
1703
—
—
|
Uninfected
3873
3819
5125
3032
|
Infected
45241
89467
90365
95120
|
Signal/Noise
11.68
23.43
17.63
31.37
|
|
Using the leu-MCA substrate, a sensitivity of 105 CFU/ml can be achieved (FIG. 15). Further increases in sensitivity may be obtained using a bis-leu rhodamine 110 labeled substrate, or using luciferase-leucine-bead complex, as shown schematically in FIG. 10B.
The sensitivity of the present assay using the leu-MCA substrate was compared to two commercially available Mycoplasma detection tests: a Mycoplasma PCR ELISA test (Roche cat #11 663 925 910), and the MycoAlert Sample Kit, (Lonza cat #LT37-618). The results are presented in Table 8, below.
TABLE 8
|
|
Test Sensitivity Comparison
|
Present leu-MCA
PCR ELISA
MycoAlert
|
Assay
test
Sample Kit
|
|
M. hyorhinis
105
80
106
|
(CFU/ml)
|
Time Required
20 minutes
2 Days
20 minutes
|
For Test
|
|
The cross reactivity of the present assay using the leu-MCA substrate was evaluated with the following microorganisms: two bacteria (S. aureus, E. coli) and three species of fungus (Candida albicans, Aspergillis niger, Saccharomyces cerevisiae). Only in the case of a completely turbid cultures was there weak low cross reactivity with the present assay using the leu-MCA substrate.
Patent Application
20100221758
