Summary
The high sequence identity among the Mycobacterium bovis and Mycobacterium tuberculosis genomes contrasts with the physiological differences reported between these pathogens, suggesting that variations in gene expression may be involved. In this study, microarray hybridization was used to compare the total transcriptome of M. bovis and M. tuberculosis, during the exponential phase of growth. Differential expression was detected in 258 genes, representing a 6% of the total genome. Variable genes were grouped according to functional categories. The main variations were found in genes encoding proteins involved in intermediary metabolism and respiration, cell wall processes, and hypothetical proteins. It is noteworthy that, compared to M. tuberculosis, the expression of a higher number of transcriptional regulators were detected in M. bovis. Likewise, in M. tuberculosis we found a higher expression of the PE/PPE genes, some of which code for cell wall related proteins. Also, in both pathogens we detected the expression of a number of genes not annotated in the M. tuberculosis H37Rv or M. bovis 2122 genomes, but annotated in the M. tuberculosis CDC1551 genome.
Our results provide new evidence concerning differences in gene expression between both pathogens, and confirm previous hypotheses inferred from genome comparisons and proteome analysis. This study may shed some new light on our understanding of the mechanisms relating to differences in gene expression and pathogenicity in mycobacteria.
Keywords: Mycobacterium bovis, Mycobacterium tuberculosis, Transcriptome, Gene expression, Microarray
Introduction
Mycobacterium bovis and Mycobacterium tuberculosis are closely related pathogens, responsible for bovine and human tuberculosis, respectively. Bovine tuberculosis is enzootic in most developing countries, causing great economic losses. Lately, this zoonosis has raised in importance within the HIV infected population.1,2 The M. bovis and M. tuberculosis genomes show 99.95% identity at the nucleotide level.3 However, distinct phenotypes, virulence and host tropism differentiate both pathogens.4 Indeed, M. bovis has shown increased virulence upon infection in mice, rabbits, calves and guinea pigs.5,6 Moreover, it causes disease in bovines, humans and a broad range of mammals. In comparison, man is the main natural host of M. tuberculosis.1,4,7,8 The high genome sequence homology and the lack of species-specific genes for M. bovis, suggest that distinctive mechanisms of gene expression might be involved in determining the differences among these bacilli.3 Genome features such as deletions, SNPs and hypervariable regions are important sources for polymorphism, but their contribution needs to be correlated with gene expression studies in order to explain the differences observed. Transcriptional analysis using microarrays has become a useful tool to study whole-genome expression and to identify changes in gene expression in cells exposed to different environmental conditions.9 Using this approach, diversity in gene expression has recently been reported among clinical isolates of M. tuberculosis.10
In this work, we used microarray hybridization to compare the total in vitro transcriptome of M. bovis and M. tuberculosis H37Rv. Our aim was to get a global view on the differences of gene expression among these pathogens as a mean to identify the genetic basis for their distinct phenotypes.
Materials and methods
Bacterial strains and culture conditions
The M. tuberculosis H37Rv (ATCC 27294) and M. bovis Ravenel (TMC 401) strains were used throughout and all cultures were grown under the same conditions. Bacteria were grown in 7H9 broth supplemented with 10% ADS (0.5% bovine serum albumin, fraction V, 0.2% glucose and 0.085% NaCl) and 0.05% Tween 80. For M. tuberculosis, 0.2% glycerol was also added to the media, while for M. bovis the media was supplemented with 0.4% sodium pyruvate. Liquid cultures were maintained at 37°C in plastic bottles in a roller apparatus and optical density (OD540) was registered daily to measure growth.
Isolation of bacterial RNA
To minimize variations in gene expression analysis, all cultures were collected at the same growth phase. To prepare bacterial RNA, procedures previously published were followed.11,12 Bacterial cell pellets were recovered from 30 ml of exponential phase culture (OD540 ∼0.25) by 3 min centrifugation and then quickly frozen on dry ice. Frozen pellets were then resuspended in 1 ml of TRI reagent (Molecular Research Center) and immediately transferred to a 2 ml screw cap microcentrifuge tube containing zirconia beads (0.1mm diameter). Samples were disrupted in a Mini-BeadBeater (BioSpec Products), and the RNA was extracted according to the TRI manufacturer's instructions. To remove residual DNA, samples were treated with Turbo™ DNAse (Ambion) and cleaned up using RNeasy columns (Qiagen). The integrity of all RNA samples was checked by non-denaturing agarose gel electrophoresis, with RNA concentration quantified by spectrophotometry. RNA samples were kept at −80°C until further use.
DNA microarray analysis
The M. tuberculosis microarray chips used in these assays were printed at the Center for Applied Genomics at the Public Health Research Institute. This array consists of 4,295 70-mer oligonucleotides representing 3,924 predicted open reading frames of the M. tuberculosis H37Rv strain, 371 non-redundant probes designed to the M. tuberculosis CDC-1551 strain, and 25 controls. Microarray analyses were performed according to previously described protocols 13 with modifications.14 cDNA was synthesized and fluorescently labeled by a direct procedure. Two micrograms of total RNA extracted from either M. tuberculosis or M. bovis was reverse transcribed in a reaction mix with a final concentration of 0.17 ug/ul random hexamers, 0.96x first strand buffer, 9.6 mM DTT, 0.44 mM dATP, dCTP and dGTP, 0.02 mM dTTP, 0.06mM Cyanine 3 or Cyanine 5 dUTP and 9.4 units Superscript II. The combination of reaction mix and total RNA was incubated for 10 minutes at 25°C followed by 90 minutes at 42°C. The labeled cDNA probes were then purified and concentrated using the MinElute Cleanup kit (Qiagen). The total purified cDNA probe was added to the arrays in a hybridization solution containing a final concentration of 0.5 ug/ul tRNA, 2.0x SSC, 0.25% formamide and 0.1% SDS. For each array, cDNA prepared from the M. tuberculosis RNA was mixed with cDNA from M. bovis. The slides were covered by a flat 22 × 22 mm coverslip and hybridized in sealed hybridization chambers for sixteen hours at 50°C in a water bath.
Microarray data analysis
Microarray slides were scanned using a GenePix 4000A scanner (Axon Instruments). Images were processed and the fluorescent intensity of each spot was quantified using the GenePix Pro 4.0 software. Four independent biological replicates were analyzed for each strain, and one swap-dye experiment was included. Median intensity values were corrected by background subtraction and negative corrected intensities were set to +1.15 Further analysis was performed using GeneSpring 7.2 software (Silicon Genetics). Data was normalized using the locally weighted linear regression (Lowess) method, to remove the fluorescence-intensity dependent, dye-specific effect of low fluorescence intensity spots.16 20% of the data was used to calculate the Lowess fit at each point. Cy5/Cy3 intensity ratios were determined using normalized values and then log transformed. For each gene, the geometric mean was calculated from the intensity ratios of the four replicates and the resulting value was used to determine differences in mRNA abundance between both strains. Genes were classified as differentially expressed if they fulfilled both of the following criteria: a minimum 3-fold regulation difference and a p-value <0.05. Statistical significance of the chosen genes was verified by a t-test with the Benjamini Hochberg false discovery rate correction method17 implemented in GeneSpring.
The data discussed in this publication have been deposited in NCBIs Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE6889.
Reverse transcription and real time PCR
RT and PCR primers were designed with the Primer3 web software18 and purchased from IDT (Coralville, IA). Primer sequences used in this study are provided in supplementary Table S1. Reverse transcription was carried out at 60°C, using 100 ng of RNA, 0.3 μM antisense gene-specific primers (including sigA control primer) and the RNA Transcriptor First Strand cDNA Synthesis Kit (Roche), in a final volume of 20 μl, following the manufacturer's instructions. Control reactions, lacking reverse transcriptase, were performed for every RNA sample. Real time PCR reactions were accomplished using a LightCycler instrument (Roche), in a total volume of 10 μl, using 2 μl of diluted cDNA, 0.3 μM gene-specific primers and the LightCycler FastStart DNA Master SYBR Green I kit (Roche). After 10 min at 90°C, the PCR program consisted in 40-45 cycles at 95°C for 6 s, 62°C for 8 s and 72°C for 8 s. Fluorescence was measured at the end of the extension step. Reactions were run in triplicate for each gene and the specificity of the PCR products was verified by gel electrophoresis and melting curve analysis. For every PCR run, a standard curve was performed using serial dilutions of chromosomal DNA and the corresponding gene-specific primer set. These standard curves were used by the LightCycler software to calculate the initial amount of mRNA for each gene tested in the different samples. In order to compare gene expression between M. bovis and H37Rv strain, calculated values for each gene were normalized to the corresponding sample's sigA mRNA value,19 and normalized values were used to calculate the M. bovis/M. tuberculosis gene ratio.11 Ratios from 3 biological replicates were then averaged to give a final fold difference value.
Results and Discussion
Several lines of evidence have supported the fact that although slight differences are found in the genome sequence of M. tuberculosis and M. bovis, the physiology and host range spectrum is different. Variations in transcript abundance, even minor changes, may cause distinctive adaptive responses to changes in environmental conditions. To identify differences in the pattern of gene expression among M. tuberculosis and M. bovis, we used here a chip with all annotated M. tuberculosis genes. The comparative transcriptome profile was determined for both strains, grown to mid-exponential phase.
The results show that out of 4,295 genes that were represented on the chip, 6 % were differentially expressed. Of these genes, 95 exhibited higher expression in M. bovis compared to M. tuberculosis, while 163 genes were more highly expressed in M. tuberculosis compared to M. bovis. The list of differentially regulated genes in M. bovis and M. tuberculosis are shown in Table 1 and Table 2, respectively. The difference in transcript abundance for some of the selected genes was further confirmed by RT-qPCR (Table 3). Variations in transcript abundance were higher in RT-qPCR compared to microarray results, but followed the same pattern. Similar results have been observed in previous reports.20,21
Table 1.
Genes with higher expression level in M. bovis compared with M. tuberculosis H37Rv
| Mtb CDS | Mb CDS | Gene | Fold differencea |
p-value | RvD region |
Predicted function |
|---|---|---|---|---|---|---|
| Rv0065 | Mb0066 | 14.8 | 0.00384 | Conserved hypothetical protein | ||
| Rv0077c | Mb0079c | 4.6 | 0.0415 | Probable oxidoreductase | ||
| Rv0215c | Mb0221c | fadE3 | 4.1 | 0.00384 | Probable acyl-coa dehydrogenase | |
| Rv0216 | Mb0222 | 10.8 | 0.0086 | Conserved hypothetical protein | ||
| Rv0260c | Mb0266c | 4.3 | 0.0133 | Posible transcriptional regulatory protein | ||
| Rv0448c | Mb0456c | 11.1 | 0.0111 | Conserved hypothetical protein | ||
| Rv0449c | Mb0457c | 27.5 | 0.00568 | Conserved hypothetical protein | ||
| Rv0465c | Mb0474c | 6 | 0.0141 | Posible transcriptional regulatory protein | ||
| Rv0549c | Mb0563c | 4.3 | 0.0179 | Conserved hypothetical protein | ||
| Rv0550c | Mb0565c | 3.8 | 0.027 | Hypothetical protein | ||
| Rv0591 | Mb0606 | mce2C | 9.2 | 0.0219 | Thought to be involved in host cell invasion | |
| Rv0592 | Mb0607 | mce2Da | 8 | 0.0045 | Thought to be involved in host cell invasion | |
| Rv0619 | Mb0635 | galT | 17.3 | 0.00073 | Probable galactose 1-phosphate uridyl transferase | |
| Rv0769 | Mb0792 | 9.7 | 0.00384 | Probable dehydrogenase/reductase | ||
| Rv0770 | Mb0793 | 3.4 | 0.0111 | Probable dehydrogenase/reductase | ||
| Rv0771 | Mb0794 | 3.7 | 0.00802 | Possible 4-carboxymuconolactone decarboxylase | ||
| Rv0782 | Mb0804 | ptrBb | 25.5 | 0.00384 | Probable protease II (oligopeptidase b) | |
| Rv0892 | Mb0916 | 5.5 | 0.00389 | Probable monooxygenase | ||
| Rv0928 | Mb0951 | pstS3 | 4.5 | 0.00562 | Periplasmic phosphate-binding lipoprotein | |
| Rv0929 | Mb0952 | pstC2 | 3 | 0.0129 | Phosphate-transport integral membrane ABC transporter | |
| Rv0930 | Mb0953 | pstA1 | 3 | 0.0049 | Phosphate-transport integral membrane ABC transporter | |
| Rv1135c | Mb1166c | PPE16 | 7 | 0.00664 | PPE family protein | |
| Rv1421 | Mb1456 | 3.6 | 0.00523 | Conserved hypothetical protein | ||
| Rv1493 | Mb1530 | mutB | 3.1 | 0.0224 | Probable methylmalonyl-coa mutase | |
| Rv1588c | Mb1614c | 3.3 | 0.0164 | Partial REP13E12 repeat protein | ||
| Rv1652 | Mb1680 | argC | 4.1 | 0.0366 | Probable n-acetyl-gamma-glutamyl-phoshate reductase | |
| Rv1653 | Mb1681 | argJ | 3.1 | 0.0147 | Probable glutamate n-acetyltransferase | |
| Rv1656 | Mb1684 | argF | 3 | 0.0152 | Probable ornithine carbamoyltransferase | |
| MT1802 | Mb1787 | mmpL14 | 27.7 | 0.00562 | 2 | Probable conserved transmembrane transport protein |
| Rv1758 | Mb1788 | cut1 | 28 | 0.0289 | Probable cutinase | |
| Rv1947 | Mb1982 | 3.3 | 0.0426 | Hypothetical protein | ||
| Rv2004c | Mb2027c | 3.7 | 0.0266 | Conserved hypothetical protein | ||
| Rv2005c | Mb2028c | 3.7 | 0.0105 | Conserved hypothetical protein | ||
| MT2081 | Mb2048c | 20.6 | 0.0166 | 1 | Conserved hypothetical protein | |
| Rv2025c | Mb2050c | 5.4 | 0.00802 | Possible conserved membrane protein | ||
| Rv2277c | Mb2300c | 21.3 | 0.00384 | Possible glycerolphosphodiesterase | ||
| Rv2386c | Mb2407c | mbtI | 7 | 0.00476 | Putative isochorismate synthase | |
| Rv2421c | Mb2444c | nadD | 3.1 | 0.0119 | Probable nicotinate-nucleotide adenylyltransferase | |
| Rv2431c | Mb2457c | PE25 | 3 | 0.0336 | PE family protein | |
| Rv2435c | Mb2461c | 3.2 | 0.0134 | Probable cyclase | ||
| Rv2562 | Mb2591 | 5.8 | 0.0166 | Conserved hypothetical protein | ||
| Rv2619c | Mb2652c | 3.8 | 0.0255 | Conserved hypothetical protein | ||
| Rv2620c | Mb2653c | 10 | 0.00384 | Probable conserved transmembrane protein | ||
| Rv2621c | Mb2654c | 13.1 | 0.00362 | Possible transcriptional regulatory protein | ||
| Rv2622 | Mb2655 | 13.4 | 0.00137 | Possible methyltransferase | ||
| Rv2623 | Mb2656 | TB31.7 | 4.1 | 0.0105 | Conserved hypothetical protein | |
| Rv2629 | Mb2662 | 3.1 | 0.00384 | Conserved hypothetical protein | ||
| Rv2779c | Mb2801c | 3.2 | 0.0181 | Possible transcriptional regulatory protein | ||
| Rv2789c | Mb2812c | fadE21 | 3.5 | 0.0206 | Probable acyl-coa dehydrogenase | |
| Rv2873 | Mb2898 | mpb83 | 30.9 | 0.00361 | Cell surface lipoprotein | |
| Rv2874 | Mb2899 | dipz | 11.5 | 0.00362 | Possible membrane cytochrome biogenesis protein | |
| Rv2875 | Mb2900 | mpb70 | 118 | 0.00384 | Major secreted immunogenic protein | |
| Rv2876 | Mb2901 | 5.7 | 0.0049 | Possible conserved transmembrane protein | ||
| Rv2917 | Mb2941 | 7.9 | 0.0086 | Conserved hypothetical alanine and arginine rich protein | ||
| Rv2930 | Mb2955 | fadD26 | 5.8 | 0.0086 | Fatty-acid-coa ligase | |
| Rv2931 | Mb2956 | ppsA | 10.7 | 0.00384 | Phenolpthiocerol synthesis type-I polyketide synthase | |
| Rv2932 | Mb2957 | ppsB | 6.9 | 0.0166 | Phenolpthiocerol synthesis type-I polyketide synthase | |
| Rv2933 | Mb2958 | ppsC | 7.9 | 0.0049 | Phenolpthiocerol synthesis type-I polyketide synthase | |
| Rv2934 | Mb2959 | ppsD | 3.9 | 0.0166 | Phenolpthiocerol synthesis type-I polyketide synthase | |
| Rv2945c | Mb2970c | lppX | 3.3 | 0.0226 | Probable conserved lipoprotein | |
| Rv2955c | Mb2979c | 4.5 | 0.00568 | Conserved hypothetical protein | ||
| Rv2972c | Mb2997c | 3.8 | 0.0172 | Possible conserved membrane or exported protein | ||
| Rv2973c | Mb2998c | recG | 7.3 | 0.0166 | Probable atp-dependent dna helicase | |
| Rv2974c | Mb2999c | 5.3 | 0.0119 | Conserved hypothetical alanine rich protein | ||
| Rv2987c | Mb3011c | leuD | 4.3 | 0.015 | Probable 3-isopropylmalate dehydratase (small subunit) | |
| Rv2988c | Mb3012c | leuC | 3.4 | 0.0336 | Probable 3-isopropylmalate dehydratase (large subunit) | |
| Rv2989 | Mb3013 | 6 | 0.00568 | Probable transcriptional regulatory protein | ||
| Rv2998A | Mb3023c | 3.8 | 0.00384 | Conserved hypothetical protein | ||
| Rv3054c | Mb3080c | 5.4 | 0.0331 | Conserved hypothetical protein | ||
| Rv3082c | Mb3109c | virS | 4.2 | 0.019 | Virulence-regulating transcriptional regulator | |
| MT3248 | Mb3184c | PPE70 | 7.5 | 0.0166 | PPE family protein | |
| Rv3324c | Mb3353c | moaC3 | 13 | 0.00612 | Probable molybdenum cofactor biosynthesis protein C3 | |
| MT3427 | Mb3355c | moaA3 | 10.3 | 0.0133 | 5 | Probable molybdenum cofactor biosynthesis protein A |
| MT3427.1 | Mb3356 | 22.6 | 0.0119 | 5 | Hypothetical protein | |
| MT3428 | Mb3358 | embR2 | 8 | 0.027 | 5 | Possible transcriptional regulatory protein |
| Rv3331 | Mb3364 | sugI | 15.5 | 0.00401 | Probable sugar-transport integral membrane protein | |
| Rv3332 | Mb3365 | nagA | 5.2 | 0.0413 | Probable n-acetylglucosamine-6-phosphate deacetylase | |
| Rv3340 | Mb3372 | metC | 3.1 | 0.015 | Probable o-acetylhomoserine sulfhydrylase | |
| Rv3397c | Mb3430c | phyA | 3.4 | 0.0219 | Probable phytoene synthase | |
| Rv3398c | Mb3431c | idsA | 4.4 | 0.0086 | Probable geranylgeranyl pyrophosphate synthetase | |
| Rv3453 | Mb3483 | 6.2 | 0.0184 | Possible conserved transmembrane protein | ||
| Rv3454 | Mb3483 | 3.2 | 0.0086 | Probable conserved integral membrane protein | ||
| Rv3466 | Mb3495 | 3.5 | 0.0086 | Conserved hypothetical protein | ||
| Rv3530c | Mb3560c | 3.9 | 0.0224 | Possible oxidoreductase | ||
| Rv3651 | Mb3675 | 3.2 | 0.0131 | Conserved hypothetical protein | ||
| Rv3697c | Mb3723c | 3.1 | 0.00641 | Possible conserved membrane protein | ||
| Rv3862c | Mb3892c | whiB6 | 30.4 | 0.00663 | WhiB-like possible transcriptional regulatory protein | |
| Rv3897c | Mb3927c | 5.7 | 0.00523 | Conserved hypothetical protein | ||
| MT0573.1 | 3.5 | 0.0105 | Hypothetical protein | |||
| MT0915.1 | 6.3 | 0.00568 | Hypothetical protein | |||
| MT1812 | 55.5 | 0.00384 | Hypothetical protein | |||
| MT1813 | 62.5 | 0.0266 | Hypothetical protein | |||
| MT2083 | 5.1 | 0.00568 | Hypothetical protein | |||
| MT2941 | 42.6 | 0.00861 | Hypothetical protein | |||
| MT3718.1 | 6.6 | 0.0179 | Hypothetical protein |
Fold differences are the average of normalized intensity ratios from 4 microarray experiments using 4 independent biological replicates.
Table 2.
Genes with higher expression level in M. tuberculosis H37Rv compared with M. bovis
| Mtb CDC |
Gene | Fold differencea |
p-value | RD region |
Predicted function |
|---|---|---|---|---|---|
| Rv0032 | bioF2 | 3.7 | 0.0383 | Possible 8-amino-7-oxononanoate synthase | |
| Rv0040c | mtc28 | 3.1 | 0.0365 | Secreted proline rich protein | |
| Rv0112 | gca | 3.1 | 0.0146 | GDP-D-mannose dehydratase | |
| Rv0166 | fadD5 | 3.3 | 0.0157 | Fatty-acid-coa ligase | |
| Rv0167 | yrbe1a | 3.6 | 0.0233 | Conserved hypothetical integral membrane protein | |
| Rv0221 | 42.6 | 0.00818 | Conserved hypothetical protein | ||
| Rv0222 | echA1 | 10.4 | 0.0235 | 10 | Probable enoyl-coa hydratase |
| Rv0223c | 12.9 | 0.0227 | Probable aldehyde dehydrogenase | ||
| Rv0232 | 4 | 0.0196 | Probable transcriptional regulatory protein (tetr/acrr-family) | ||
| Rv0276 | 40.5 | 0.00818 | Conserved hypothetical protein | ||
| Rv0520 | 7.4 | 0.0487 | Possible methyltransferase/methylase | ||
| Rv0544c | 6.1 | 0.00659 | Possible Conserved transmembrane protein | ||
| Rv0794c | 4 | 0.0487 | Probable oxidoreductase | ||
| Rv0796 | 6.5 | 0.00268 | Putative transposase for insertion sequence IS6110 | ||
| Rv0888 | 4.7 | 0.0108 | Probable exported protein | ||
| Rv0931c | pknD | 10.6 | 0.00659 | Transmembrane serine/threonine-protein kinase | |
| Rv0934 | pstS1 | 7.3 | 0.0108 | Periplasmic phosphate-binding lipoprotein | |
| Rv0935 | pstC1 | 4.3 | 0.016 | Phosphate-transport integral membrane ABC transporter | |
| Rv0936 | pstA2 | 4.2 | 0.032 | Phosphate-transport integral membrane ABC transporter | |
| Rv1038c | esxJ | 3.2 | 0.00707 | Esat-6 like protein | |
| Rv1076 | lipU | 5.2 | 0.0235 | Possible lipase | |
| Rv1130 | 28.1 | 0.00659 | Conserved hypothetical protein | ||
| Rv1131 | gltA1 | 28.4 | 0.00899 | Probable citrate synthase | |
| Rv1172c | PE12 | 9.1 | 0.00659 | PE family protein | |
| Rv1181 | pks4 | 3.9 | 0.00707 | Probable polyketide beta-ketoacyl synthase | |
| Rv1182 | papA3 | 5.4 | 0.0146 | Probable polyketide synthase associated protein | |
| Rv1183 | mmpL10 | 4.3 | 0.0121 | Probable Conserved transmembrane transport protein | |
| Rv1184c | 7.6 | 0.00268 | Possible exported protein | ||
| Rv1195 | PE13 | 7.7 | 0.00818 | PE family protein | |
| Rv1196 | PPE18 | 14.8 | 0.00268 | PPE family protein | |
| Rv1197 | esxK | 3.2 | 0.00268 | Esat-6 like protein | |
| Rv1200 | 3.1 | 0.0235 | Probable Conserved integral membrane transport protein | ||
| Rv1220c | 3 | 0.0105 | Probable methyltransferase | ||
| Rv1257c | 28.4 | 0.0183 | 13 | Probable oxidoreductase | |
| Rv1361c | PPE19 | 11.7 | 0.00659 | PPE family protein | |
| Rv1369c | 4.2 | 0.0156 | Probable transposase | ||
| Rv1370c | 7.9 | 0.0227 | Putative transposase for insertion sequence IS6110 | ||
| Rv1386 | PE15 | 6 | 0.0108 | PE family protein | |
| Rv1387 | PPE20 | 8.5 | 0.00966 | PPE family protein | |
| Rv1396c | PE PGRS25 | 6.2 | 0.0356 | PE PGRS family protein | |
| Rv1397c | 7.1 | 0.0369 | Conserved hypothetical protein | ||
| Rv1398c | 5.6 | 0.0248 | Conserved hypothetical protein | ||
| Rv1497 | lipL | 3.2 | 0.0477 | Probable esterase | |
| Rv1506c | 15 | 0.0149 | 4 | hypothetical protein | |
| Rv1507c | 31.9 | 0.0217 | 4 | Conserved hypothetical protein | |
| Rv1508c | 44.4 | 0.0252 | 4 | Probable membrane protein | |
| Rv1509 | 5.6 | 0.0486 | 4 | hypothetical protein | |
| Rv1510 | 4.3 | 0.0365 | 4 | Probable Conserved membrane protein | |
| Rv1511 | gmdA | 40.5 | 0.016 | 4 | GDP-D-mannose dehydratase |
| Rv1512 | epiA | 22.5 | 0.0393 | 4 | Probable nucleotide-sugar epimerase |
| Rv1513 | 8.7 | 0.00351 | 4 | Conserved hypothetical protein | |
| Rv1515c | 69.4 | 0.00739 | 4 | Conserved hypothetical protein | |
| Rv1516c | 15.2 | 0.0235 | 4 | Probable sugar transferase | |
| Rv1535 | 3.4 | 0.0208 | Hypothetical protein | ||
| Rv1563c | treY | 6.7 | 0.0435 | Maltooligosyltrehalose synthase | |
| Rv1611 | trpC | 6 | 0.00659 | Probable indole-3-glycerol phosphate synthase | |
| Rv1612 | trpB | 6.1 | 0.00268 | Probable tryptophan synthase, beta subunit | |
| Rv1613 | trpA | 7 | 0.0183 | Probable tryptophan synthase, alpha subunit | |
| Rv1614 | lgt | 11.1 | 0.00651 | Possible prolipoprotein diacylglyceryl transferases | |
| Rv1639c | 5.4 | 0.00268 | Conserved hypothetical membrane protein | ||
| Rv1646 | PE17 | 3.2 | 0.0291 | PE family protein | |
| Rv1651c | PE PGRS30 | 16.2 | 0.0421 | PE PGRS family protein | |
| Rv1730c | 5 | 0.00268 | Possible Penicillin-binding protein | ||
| Rv1757c | 6.9 | 0.016 | Putative transposase for insertion sequence IS6110 | ||
| Rv1763 | 7.1 | 0.0162 | Putative transposase for insertion sequence IS6110 | ||
| Rv1764 | 5.4 | 0.0116 | Putative transposase for insertion sequence IS6110 | ||
| Rv1792 | esxM | 3.2 | 0.00707 | Esat-6 like protein | |
| Rv1809 | PPE33 | 11.2 | 0.00659 | PPE family protein | |
| Rv1872c | lldD2 | 4.5 | 0.0296 | Possible l-lactate dehydrogenase | |
| Rv1884c | rpfC | 5.5 | 0.0288 | Probable resuscitation-promoting factor | |
| Rv1925 | fadD31 | 3.1 | 0.00984 | Possible dehydrogenase | |
| Rv1965 | yrbE3B | 14.1 | 0.0217 | 7 | Conserved hypothetical integral membrane protein |
| Rv1976c | 29.3 | 0.0176 | 7 | Conserved hypothetical protein | |
| Rv1977 | 6.9 | 0.0452 | 7 | Conserved hypothetical protein | |
| Rv2071c | cobM | 3.7 | 0.00765 | Precorrin-4 c11-methyltransferase | |
| Rv2073c | 4.8 | 0.032 | 9 | Probable shortchain dehydrogenase | |
| Rv2074 | 83.3 | 0.00268 | 9 | Conserved hypothetical protein | |
| Rv2077c | 13 | 0.0108 | Possible Conserved transmembrane protein | ||
| Rv2105 | 8 | 0.0157 | Putative transposase for insertion sequence IS6110 | ||
| Rv2106 | 5.2 | 0.016 | Probable transposase for insertion sequence IS6110 | ||
| Rv2137c | 5.7 | 0.0157 | Conserved hypothetical protein | ||
| Rv2159c | 24.3 | 0.00965 | Conserved hypothetical protein | ||
| Rv2160c | 44.4 | 0.0121 | Conserved hypothetical protein (putative tetr family) | ||
| Rv2161c | 9.8 | 0.00268 | Conserved hypothetical protein | ||
| Rv2162c | PE PGRS38 | 17.9 | 0.00659 | PE PGRS family protein | |
| Rv2167c | 4.3 | 0.00268 | Probable transposase | ||
| Rv2168c | 9.2 | 0.0156 | Putative transposase for insertion sequence IS6110 | ||
| Rv2187 | fadD15 | 5.6 | 0.016 | Probable long-chain-fatty-acid-coa ligase | |
| Rv2278 | 9.9 | 0.0103 | Putative transposase for insertion sequence IS6110 | ||
| Rv2279 | 6.6 | 0.0121 | Probable transposase | ||
| Rv2346c | esxO | 3.1 | 0.0121 | 5 | Putative Esat-6 like protein |
| Rv2347c | esxP | 3.4 | 0.00268 | 5 | Putative Esat-6 like protein |
| Rv2348c | 87.7 | 0.00268 | 5 | hypothetical protein | |
| Rv2349c | plcC | 67.6 | 0.00659 | 5 | Probable phospholipase C3 |
| Rv2350c | plcB | 64.5 | 0.00966 | 5 | Probable membrane-associated phospholipase C2 |
| Rv2351c | plcA | 19 | 0.016 | 5 | Probable membrane-associated phospholipase C |
| Rv2352c | PPE38 | 69.4 | 0.00565 | 5 | PPE family protein |
| Rv2354 | 9.5 | 0.016 | Probable transposase for insertion sequence IS6110 | ||
| Rv2355 | 4.2 | 0.0217 | Probable transposase | ||
| Rv2395 | 3.9 | 0.00588 | Probable Conserved integral membrane protein | ||
| Rv2479c | 4 | 0.0266 | Probable transposase | ||
| Rv2480c | 6.5 | 0.0208 | Possible transposase for insertion sequence IS6110 | ||
| Rv2490c | PEPGRS43 | 3.7 | 0.00707 | PE PGRS family protein | |
| Rv2600 | 3.8 | 0.0268 | Probable Conserved integral membrane protein | ||
| Rv2648 | 9.6 | 0.0126 | 11 | Probable transposase for insertion sequence IS6110 | |
| Rv2649 | 6.7 | 0.0162 | 11 | Probable transposase for insertion sequence IS6110 | |
| Rv2651c | 6 | 0.0108 | 11 | Possible phirv2 prophage protease | |
| Rv2657c | 29.1 | 0.0188 | 11 | Probable phirv2 prophage protein | |
| Rv2814c | 4.8 | 0.0176 | Probable transposase | ||
| Rv2815c | 8.6 | 0.0108 | Probable transposase for insertion sequence IS6110 | ||
| Rv2822c | 3.7 | 0.0216 | Hypothetical protein | ||
| Rv2823c | 13.6 | 0.0472 | Conserved hypothetical protein | ||
| Rv2877c | merT | 54.1 | 0.00659 | Probable Conserved integral membrane protein | |
| Rv3061c | fadE22 | 3.1 | 0.0236 | Probable acyl-coa dehydrogenase | |
| Rv3083 | 5.3 | 0.0121 | Probable monooxygenase | ||
| Rv3084 | lipR | 3.6 | 0.00966 | Probable acetyl-hydrolase/esterase | |
| Rv3086 | adhD | 3.9 | 0.0299 | Aldehyde reductase | |
| Rv3088 | 3.7 | 0.0277 | Conserved hypothetical protein | ||
| Rv3093c | 6.6 | 0.0176 | Hypothetical oxidoreductase | ||
| Rv3094c | 10.9 | 0.0176 | Conserved hypothetical protein | ||
| Rv3136 | PPE51 | 47.2 | 0.00707 | PPE family protein | |
| Rv3184 | 10.4 | 0.00707 | Probable transposase for insertion sequence IS6110 | ||
| Rv3185 | 38.5 | 0.032 | Probable transposase | ||
| Rv3186 | 10.4 | 0.00818 | Probable transposase for insertion sequence IS6110 | ||
| Rv3187 | 5.6 | 0.0108 | Probable transposase | ||
| Rv3325 | 6.5 | 0.024 | Probable transposase for insertion sequence IS6110 | ||
| Rv3326 | 7.4 | 0.00872 | Probable transposase | ||
| Rv3354 | 4.2 | 0.00707 | Conserved hypothetical protein | ||
| Rv3380c | 5 | 0.0162 | Probable transposase | ||
| Rv3381c | 8.5 | 0.0234 | Probable transposase for insertion sequence IS6110 | ||
| Rv3390 | lpqD | 3.1 | 0.0121 | Probable Conserved lipoprotein | |
| Rv3407 | 15.9 | 0.00818 | Conserved hypothetical protein | ||
| Rv3408 | 13 | 0.00659 | Conserved hypothetical protein | ||
| Rv3426 | PPE58 | 30.5 | 0.0272 | 6 | PPE family protein |
| Rv3429 | PPE | 45.7 | 0.00659 | PPE family protein | |
| Rv3474 | 11 | 0.016 | Possible transposase for insertion sequence IS6110 | ||
| Rv3475 | 4.4 | 0.0133 | Possible transposase for insertion sequence IS6110 | ||
| Rv3477 | PE31 | 20.4 | 0.00659 | PE family protein | |
| Rv3478 | PPE60 | 16.6 | 0.00268 | PPE family protein | |
| Rv3479 | 11.2 | 0.0235 | Possible transmembrane protein | ||
| Rv3487c | lipF | 12.9 | 0.00965 | Probable esterase/lipase | |
| Rv3618 | 65.8 | 0.00659 | 8 | Possible monooxygenase | |
| Rv3620c | esxW | 3.9 | 0.0108 | 8 | Putative Esat-6 like protein |
| Rv3623 | lpqG | 75.8 | 0.00659 | Probable Conserved lipoprotein | |
| Rv3633 | 3 | 0.0104 | Conserved hypothetical protein | ||
| Rv3726 | 7.2 | 0.00818 | Possible dehydrogenase | ||
| Rv3749c | 5.2 | 0.0171 | Conserved hypothetical protein | ||
| Rv3750c | 9.3 | 0.00899 | Possible excisionase | ||
| Rv3760 | 3.2 | 0.0255 | Possible Conserved membrane protein | ||
| Rv3822 | 4.4 | 0.016 | No description | ||
| Rv3823c | mmpL8 | 4.2 | 0.0291 | Probable Conserved integral membrane transport protein | |
| Rv3824c | papA1 | 10 | 0.0119 | Probable polyketide synthase associated protein | |
| Rv3825c | pks2 | 11.9 | 0.0116 | Probable polyketide synthase | |
| MT0291.3 | 3.7 | 0.0104 | Hypothetical protein | ||
| MT1178 | 3.9 | 0.0176 | Hypothetical protein | ||
| MT2420 | 17.7 | 0.0116 | Conserved hypothetical protein | ||
| MT2421 | 6 | 0.0237 | Conserved hypothetical protein | ||
| MT2466 | 6.8 | 0.00659 | Hypothetical protein | ||
| MT2467 | 3.6 | 0.0149 | Hypothetical protein | ||
| MT2880.1 | 3.8 | 0.0267 | Hypothetical protein | ||
| MT3580.2 | 22.2 | 0.00659 | Hypothetical protein | ||
| MT3846 | 25.2 | 0.0307 | Hypothetical protein | ||
| MT4026.1 | 8.9 | 0.00659 | Hypothetical protein |
Fold differences are the average of normalized intensity ratios from 4 microarray experiments using 4 biological replicates.
Table 3.
Validation of microarray results by RT-qPCR
| CDS | Gene | Fold differencea |
|---|---|---|
| M. bovis/M. tuberculosis | ||
| Mb0606 | mce2C | 16.2 |
| Mb0607 | mce2D | 23 |
| Mb2898 | mpb83 | 38.8 |
| Mb2900 | mpb70 | 166.9 |
| Mb2955 | fadD26 | 3.4 |
| Mb2956 | ppsA | 7.1 |
| Mb2957 | ppsB | 3.6 |
| Mb2958 | ppsC | 22 |
| Mb2959 | ppsD | 28.6 |
| Mb3358 | embR2 | 173.3b |
| Mb3450 | WhiB3 | 4.5 |
| Mb3706c | WhiB4 | 3.1 |
| Mb3892c | WhiB6 | 77.4 |
Fold differences are averaged ratios from 3 biological replicates. Each ratio was calculated between the numbers of cDNA copies for each gene in both strains, normalized to sigA.
The embR2 gene is not present in the M. tuberculosis H37Rv genome, therefore, the value represent the absolute number of cDNA copies detected in M. bovis.
Differentially regulated genes were grouped according to functional categories as described in TubercuList and BoviList (Table 4). The highest difference in gene expression was found in genes related to general metabolism, insertion sequences, hypothetical proteins and cell wall proteins. A significant proportion of genes coding for proteins associated with metabolic processes were identified for both pathogens. Using the Kyoto Encyclopedia of Genes and Genomes (KEGG) 22 to search for metabolic pathways assigned to these genes, we found that some of the differences observed within this category corresponded to genes encoding proteins related to amino acid, steroid and sugar metabolism. With the aim of support successful growth, the culture media was supplemented with different carbon sources. Keeping in consideration that this may have an effect on the expression of some genes, we particularly looked for variations on genes that are likely to be affected, such as the key glycolytic enzymes glpK, pykA and pdhA. Our results showed no significant differences using the 3-fold cut-off. On the other hand, Tween 80 was also added to both culture media. This oleic acid ester can be used as a carbon source in vitro by mycobacteria, and it has also been shown that fatty acids are the primary carbon source in vivo. 23 Therefore, taking together this data, we consider that although different carbon sources may influence gene expression to some extent, the results presented in this paper are representative of the in vitro differences on gene expression between M. bovis and M. tuberculosis.
Table 4.
Distribution of differentially expressed genes according to functional categories
| Functional categorya | M. bovis | M. tuberculosis | ||
|---|---|---|---|---|
| n | % | n | % | |
| Cell wall and cell processes | 17 | 17.9 | 31 | 19 |
| Conserved hypotheticals | 19 | 20 | 22 | 13.5 |
| Conserved hypotheticals with an orthologue in M. bovis/M. tuberculosis | 4 | 4.2 | 2 | 1.2 |
| Information pathways | 1 | 1.1 | - | - |
| Insertion seqs and phages | 2 | 2.1 | 33 | 20.2 |
| Intermediary metabolism and respiration | 22 | 23.2 | 30 | 18.4 |
| Lipid metabolism | 10 | 10.5 | 8 | 4.9 |
| PE/PPE | 3 | 3.2 | 18 | 11 |
| Regulatory proteins | 7 | 7.4 | 3 | 1.8 |
| Unknown | - | - | 3 | 1.8 |
| Virulence, detoxification, adaptation | 3 | 3.2 | 3 | 1.8 |
| Hypothetical proteins CDC1551b | 7 | 7.4 | 10 | 6.1 |
| Total | 95 | 100 | 163 | 100 |
Genes were grouped according to functional classifications as annotated in TubercuList.
ORFs annotated in the M. tuberculosis CDC1551 genome.
Interestingly, 20 % of the differentially expressed genes code for hypothetical proteins in M. bovis compared to 13.5% in M. tuberculosis. These results point to the need of assigning a function to these proteins, as they may be involved in determining the physiological differences described for both bacilli.
Within the category of cell wall proteins, 18 genes of the PE/PPE family were found to be higher expressed in M. tuberculosis compared to three genes identified in M. bovis. These genes code for surface exposed proteins involved in host-pathogen interactions.24-26 In addition, six ESAT6-like genes showed higher expression in M. tuberculosis whereas, in M. bovis, no increased expression of ESAT-6 genes was detected. Similar results were obtained in a previous report on proteome analysis that showed a differential pattern of expression for Rv2346c (esxO) and Rv3620c (esxW) between M. bovis (BCG) and M. tuberculosis.27 The ESAT6-like proteins have been a focus of attention as they are highly immunogenic, secreted proteins capable of inducing a strong T cell response in the host.28,29 The observed polymorphism in the expression pattern of genes encoding cell wall and secreted proteins correlates with the variation in their sequences and could be an important source of antigenic diversity.
The major difference in the secretome of both bacilli is the elevated expression in M. bovis of the two serodominant antigens MPB70 and MPB83.30 Behr and colleagues have recently reported that in both bacilli the mpb70/mpb83 genes are under the positive control of sigK. Lately, a mutation in the gene encoding anti-SigK has been shown to be responsible for the high level of expression of MPB70/MPB83 in M. bovis.31,32 Accordingly, in our array analysis mpb70 showed the highest fold difference value. In addition, a higher expression was also found in M. bovis for the gene mpb83 and the neighboring genes dipZ and Mb2901. The orthologous genes in M. tuberculosis have been described as part of a putative operon.33 Recent data showed that these genes are members of the SigK-RskA regulon.32 Interestingly, although the expression of mpb70 and mpb83 is low in M. tuberculosis, it greatly increases upon macrophage infection,32,34 suggesting an important in vivo function.
A distinctive pattern of expression was also observed for the genes encoding for the phosphate-specific transport (Pst) system. This system comprises a periplasmic phosphate-binding protein (PstS), two transmembrane channel-forming proteins (PstA and PstC) and a cytoplasmic ATP binding protein (PstB) that probably interacts with PstA-PstC. The cluster of genes encoding for these proteins is formed by three putative operons: pstS3/pstC2/pstA1, pstS2/pknD and pstB/pstS1/pstC1/pstA2.35,36 Interestingly, we observed a higher expression of the pstS1/pstC1/pstA2 operon in M. tuberculosis, whereas the pstS3/pstC2/pstA1 genes were increased in M. bovis. Another gene highly expressed in M. tuberculosis, pknD, is a pseudogene in M. bovis, thought to be involved in phosphate transport regulation.37 Thus, these results suggest the use of different mechanisms controlling the transport of phosphate in each bacteria.
The family of genes involved in lipid metabolism and cell wall composition are of particular relevance as their proteins have been related to M. tuberculosis pathogenesis. In M. bovis, a higher expression was found for fadD26 and the ppsA-D genes, which codes for fatty-acid-coA ligase and polyketide synthases, respectively. These enzymes are involved in phtiocerol dimycocerosate (PDIM) and phenolphtiocerol glycolipids (PGLs) biosynthesis.38,39 PGLs are not produced by most strains of M. tuberculosis, includin H37Rv and CDC1551 strains, due to a frameshift mutation in the pks1 gene.40 Noteworthy, some strains of M. tuberculosis that produce PGLs display a hypervirulent phenotype,41,42 thus, supporting the role of these lipids in mycobacteria pathogenesis. Moreover, fadD26 and ppsA-E genes have been associated with virulence by signature-tagged transposon mutagenesis and bioinformatics analyses.43,44 We also detected an increased expression in M. tuberculosis of two clusters of genes with similar organization: pks4/papA3/mmpL10 and mmpL8/papA1/pks2. The pks2 and mmpL8 genes codes for a polyketide synthase and a lipid transporter, respectively. Both genes are required for the biosynthesis and transport of SL-1, a sulfolipid found exclusively in M. tuberculosis and thought to be implicated in host-pathogen interactions.45,46
Special attention was given to transcriptional regulators as they play a crucial role in the survival of the mycobacteria. Within this group, we observed higher expression for the M. tuberculosis Rv2160c and Rv0232 genes, which code for putative transcriptional regulators of the TetR/AcrR-family. In contrast, seven transcriptional regulatory genes were differentially expressed in M. bovis. Among these, whiB6 exhibited the highest degree of difference (30.5-fold). This gene is markedly up-regulated in M. tuberculosis cultured under SDS, ethanol, heat-shock or oxidative stress conditions.47 Due to the high expression observed for whiB6 and keeping in mind that minor changes in the expression of transcriptional regulators may cause significant changes to the phenotype, we searched for the other members of the whiB family. The data showed that in M. bovis, whiB3 and whiB4 expression were close to the cut-off level (2.7 and 2.8 fold difference, respectively). Whereas, in M. tuberculosis, a similar result was observed for whiB1 (2.6 fold difference). These results support the view that differential gene regulation might contribute to the physiological differences observed between these pathogens.
The presence of several deletions in one genome relative to the others have been proposed as key features for evolution in members of the M. tuberculosis complex.4,48 We detected the expression of 31 genes annotated in the RD 4, 5, 6, 7, 8, 9, 10, 11 and 13 regions, present in M. tuberculosis but deleted in the M. bovis genome. These groups of genes include potential candidates for virulence factors and phenotype variation such as phospholipase C, prophages, ESAT-6 and PE family of proteins. In comparison, the expression of five genes in the RvD regions 1, 2 and 5, were detected in M. bovis. These genes code for a trans-membrane transporter protein, two hypothetical proteins, a molybdenum cofactor biosynthesis protein and a putative transcriptional regulator. Further research is needed to assess the role of the deleted regions in the physiology of the pathogen. So far, only two deletions have been attributed function across the M. tuberculosis complex; RD1 plays a key role in the attenuation of BCG,49 while Wilkinson and colleagues recently described loss of RD750 as being involved in host interaction of the EAI clade of M. tuberculosis.50
An unexpected result was the finding of expression for some genes annotated in the M. tuberculosis CDC1551 genome whose annotation was missing from the M. bovis or M. tuberculosis H37Rv genomes. These genes hybridized to a set of probes designed for CDC1551 open reading frames. BLAST analysis of the probes sequences showed no significant identity with the annotated coding sequences of M. bovis and M. tuberculosis H37Rv genomes. Similar results were previously found at the proteome level by analysis of H37Rv supernatant proteins.27 One of the identified genes with higher expression in M. bovis is MT2941. This gene is located between mpb83 and dipZ, which suggest it might be co-transcribed as part of the mpb83/Mb2901 region. A previous report has shown that when comparing CDC1551 and H37Rv strain, important differences where found in gene prediction due to sequence polymorphism.51 In this respect, we detected a high expression in M. bovis of MT1812, a gene absent in M. tuberculosis H37Rv and non-predicted in M. bovis 2122. Although the genes detected in our study were not included in the re-annotation of the M. tuberculosis H37Rv genome,52 the data presented here suggests that gene prediction approaches used in the annotation of the M. tuberculosis H37Rv and M. bovis 2122 genomes have overlooked some coding sequences and hence, their annotation needs to be updated.
A 15 % variation in gene expression among M. tuberculosis clinical isolates10 has been previously reported. Due to differences in the experimental approach, it is not feasible to directly compare that report with our data. However, a similar tendency in the distribution pattern of differentially expressed genes can be depicted across functional categories.
The transcriptional analysis presented in this work showed a good correlation with previously reported differences at the proteome level and with differences predicted by comparative genomics, which supports the validity of our study. Mutational studies will help to understand the particular contribution of variable genes in the mechanisms underlying the differences between both pathogens.
Supplementary Material
Acknowledgments
We would like to thank Alejandro Araya (C.N.R.S., Bordeaux Cedex, France) for his helpful comments and review of the manuscript. We also thank Eugenie Dubnau (TB Center, P.H.R.I) for valuable discussions and Saleena Ghanny (CAG, P.H.R.I), for her advice with microarray processing. This work was funded by FONDEF grant D02I1111 (awarded to A.M.Z.) and NIH grant HL068513 (awarded to I.S.). G.R. was supported by MECESUP AUS0006 fellowship and DID-UACH grant D2004-08.
Footnotes
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