Microarray analysis of

Mycobacterium avium subspecies paratuberculosis (MAP) intracellular and extracellular transcriptomes

 

 

                              Contents

 

*  Introduction

*  Experimental Aims

*  Microarray construction and validation

*  cDNA generation and hybridization to microarrays

*  Differential expression analysis results

*  Conclusions and further work

 

 

 

 

Introduction

 

These experiments form part of the Department of Surgery, SGHMS research programme investigating the molecular biology of MAP and its involvement as a causative agent of Crohn's disease in humans. Contributors to this work include Dr Tim Bull, Professor John Hermon-Taylor, Dr Joe Sheridan, Dr Jason Hinds *, Dr Phil Butcher *.
            These results are preliminary ONLY, have not as yet been submitted for publication and must not be published, referenced or copied without permission from Dr. Tim Bull (tim.bull@sghms.ac.uk).
(* Microarray Facility, Department of Medical Microbiology, SGHMS)


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Experimental Aims

 

  1. To extract mRNA populations representational of the complete transcriptome from MAP cultured in extracellular and intracellular environments in sufficient quantity and quality for microarray analysis.
  2. To evaluate the MAP sub-microarray.
  3.  To determine MAP specific genes that are differentially expressed in an intracellular environment.


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Microarray construction and validation

 

  1. Generation of PCR products
    Primers (21mers) designed with Primer3 [Primer3 Input (primer3.cgi v 0.2c)] as specific for MAP or MTB were used to amplify products from 250ng MAP total genome DNA per sample extracted from MAP (strain 989) or MTB (strain H37Rv) respectively. MAP products were amplified using the following conditions: 1 cycle (96°C: 3min); 40 cycles (94°C : 30sec; 58°C : 30sec; 72°C : 90sec) ; 1 cycle (72 °C : 5 min). All MAP amplification products were cleaned once with Qiagen PCR Cleanup kit (Qiagen, UK) and purity checked (see Figure 1). MTB products were amplified and purified as described (http://www.sghms.ac.uk/depts/medmicro/bugs/Mtuberculosis/mtuberculosis.htm).
  2. Microarray gridding
    25 Arrays were printed onto Poly-L-lysine coated slides using the BioRobotics MicroGrid II split pin technology generating a 4 x 4 meta grid containing 256 spots delivered using a 16 pin tool. To eliminate background bias and reduce pipetting fluctuations, gene products from MAP genes were spotted four times onto the same array. The array contained 46 MAP gene products including GS cassette genes, 16SrDNA, sodA, katG, 65Kd heat shock Ag, IS900 transposase p43 and ORF’s immediately adjacent to fourteen IS900 loci. Also included were a random selection of 100 MTB gene products also spotted singularly or in duplicate. Each 4 x 4 grid also contained a dilution series of 16SrDNA, Cy5, Cy3 dCTP fluorescent spot controls.
  3. Validation
    To validate the quality of the gridding and to identify MTB products with sufficient homology to generate a significant signal, two array slides were hybridised at 65°C for 16hr with 2.5mg MAP 989 DNA labelled with Cy3 dCTP (see Figure 2). This showed that all 46 MAP specific genes and 40% of MTB genes (18 of 45 genes) were significantly reactive and gave acceptably consistent signals over each of the spots.

 

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Amoebic culture and mRNA extraction

 

  1. Growth of MAP in amoebae
    Acanthamoebae polyphaga cultures were grown to 80% confluency in a 750cm3 tissue culture flask at room temperature using Proteose Peptone Glucose (PPG) medium. MAP cultures grown on Middlebrooks 7H11 agar supplemented with glycerol and mycobactin J (MAP medium) were harvested and diluted to 1 x 107 cfu/ml (as estimated by nephelometry). Cultures were washed once in sterile PBS, passed slowly through a small gauge needle  (25G) in PBS to separate aggregates and then inoculated into a single flask of A. polyphaga and incubated at RT for 48 hrs. Samples were then treated with 100mg/ml Amikacin for 2 hours and the PPG media carefully poured off and replaced. MAP extracellular control cultures contained PPG only and were not treated with Amikacin. Cultures were then incubated at RT for 8-10 weeks without changing the PPG media.
  2. Viability of MAP in amoebic cultures
    MAP cultures were stained for acid-fast bacilli (Auramine/Rhodamine) and sub-cultured on MAP medium for viability. MAP could not be recovered from amoebic flasks on MAP medium. However further experiments using long term cultures of MAP in amoebae (not shown here) suggest that MAP, although now unable to grow on solid media, are still capable of replication within an intracellular environment and can survive in amoebic cultures for at least 12 months. Microscopy estimated an MOI of 3-5 bacilli per cell with over 70% of amoebae infected after 8 weeks. Each flask contained approximately 1 x 106 amoebae after 8 weeks incubation.
  3. mRNA extraction
    Five flasks of MAP infected amoebae and five of MAP in PPG controls were harvested and combined into test and control pools. Amoebae were then differentially lysed with 1% Triton-X100 for 20 min and the intact MAP pelleted and washed in Tris HCl (pH8.0). Pellets were then mixed with 0.6ml TRIZOL and ribolysed @ 6.5 for 45secs (Hybaid Ribolyser). mRNA populations were cold extracted with chloroform, precipitated with isopropanol and washed in 70% ethanol. Samples were further treated with DNAseI for 15mins, purified through an mRNA extraction column (Qiagen) and concentrations estimated against RNA standards on a 1.5% agarose gel  (see Figure 3).


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cDNA generation and hybridization to microarrays

 

  1. cDNA
    10mg mRNA samples were mixed with 3mg of a Random Primer Set (Invitrogen) heated to 95°C for 5 min then snap cooled on ice. cDNA was then generated by adding First Strand Buffer, DTT, 450U Superscript Reverse Transcriptase (Invitrogen) and dNTP’s  to the following final concentrations 0.5mM dATP, dTTP, dGTP, 0.2mM dCTP and Cy3 (test sample) or Cy5 (control sample) (Amersham Pharmacia) to 0.3mM in a final volume of 50ml and incubated in the dark @ 25°C for 10min followed by 42°C for 90min. Cy3 and Cy5 samples were then combined and purified through a MinElute column (Qiagen), eluted into 10.5ml H2O then mixed with filtered Stock Hyb Buffer (Final conc 4x SSC, 0.3% SDS) to a final volume of 16ml.
  2. Hybridization
    Microarrays were pre-hybridised in buffer (3.5x SSC, 0.1% SDS, 10mg/ml BSA) @ 65°C for 20min followed by thorough rinsing in dH2O and then propan-2-ol with subsequent drying by centrifugation @ 1500rpm for 5 min. 16ml purified Cy3/Cy5 labelled sample was heated to 95°C for 2min then added directly to the microarray slide. A coverslip was applied and the microarray hybridised in a hybridization cassette (Telechem International) @ 65°C in the dark for 18hr. Microarrays were then vigorously washed twice in 0.06x SSC, dried by centrifugation @1500 rpm for 5 mins and immediately read with a dual laser 428 Scanner (Affymetrix).

 

 

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Differential expression analysis results

 

  1. Analysis calculations
     Imagene V4.2 software (Biodiscovery, Inc) was used for array imaging, normalization and signal enhancement. Genespring (Silicon Genetics) software was used for preliminary cluster analysis and signal output manipulation (see Figure 4 Microarray Image.htm). Intracellular/extracellular signal ratios (Cy3/Cy5) were calculated by  (Data_Mean_signal – Data_Mean.background) / (Reference_Mean_signal – Reference_Mean.background). Replicate ratios for each gene spot were averaged and are displayed with mean and standard deviation
    (see Table 1).
  2. Controls and signal cutoffs
    16SrDNA (from MAP and MTB products) control dilution spots gave differential signal ratios of 1.42 ± 0.08 indicating that the proportion of RNA hybridized to the microarray from intracellular and extracellular samples was relatively equal. All spot blanks gave consistently low total signal values, which were significantly lower (5-500x lower) than spots with products. The cut-off total signal value was defined as Mean + 2SD of the total signal values (Ref signal value + Data signal value) from a set (x16) of 16SrDNA (diluted 1:125) spots distributed across the whole microarray. Signal data from spots giving total signal values below this figure (in this case 7700) were discarded from the analysis. The total signal values of 90% spots with MTB DNA products were significantly lower than in those with MAP DNA spots.
  3. Transcriptome analysis
    Gene clusters were arbitrarily assigned to 6 groups with mean ratios x0.5, x1, x2, x3, x4 and > x5 of the 16SrDNA control mean ratio.
    x1 group
    The majority of MTB homologues (117 gene products) and MAP gene products specific for IS900, p43 from IS900 and the 65Kd MAP heat-shock antigen showed no significant differential expression between intracellular and extracellular signal expression.
    x2 and x0.5 groups
    17 MTB homologues: 0 MAP genes in the extracellular culture and 2 MTB homologues: 24 MAP genes in the intracellular culture showed more than double the expression signal relative to16SrDNA.
    x3, x4 and >x5 groups
    12 MAP genes showed more than three fold increase in expression signal in the intracellular samples. Maximum increase in expression signal was seen in gsc and gsd genes from the GS cassette. The highest increase in expression signal from IS900 associated genes was associated with the des gene from Locus 5 and a tetR regulation gene from Locus 6.
    Differential expression signal enhancement associated with IS900 loci
    All signal ratios associated with IS900 Loci show at least some increase in intracellular expression signal. In 7 of 11 Loci with ORF interrupted by IS900, an increase (column Left-Right in Table 2) in expression signal of the interrupted region (Left side of IS900) was observed relative to the region immediately before the IS900 (Right side of IS900).

 

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Conclusions and further work

a)      The MAP microarray functioned well, especially with MAP specific PCR products but also with MAP cDNA hybridisation to MTB derived product targets.

b)      We were able to extract sufficient mRNA from MAP grown intracellularly (within amoebae for at least 8 weeks) and extracelluarly (in proteose peptone media) to generate cDNA populations suitable for microarray analysis.

c)      The following genes were demonstrated to have a significantly increased expression signal ( >2 fold) in MAP from intracellular samples
All GS cassette genes including gsa, gsbA, gsbB, gsc, gsd and mpa.
MAP specific genes sodA, hupB, drrC
MAP genes specific for both adjacent genomic regions of IS900 Loci 1, 2, 4-6, 8-11, 13, 14.
MAP genes specific for one adjacent genomic region of IS900 loci 3, 7, 12.
MAP homologues to MTB genes (especially homologues to Rv0046c and Rv0057)
The following genes were demonstrated to have a highly increased expression signal (>4 fold) in intracellular samples
MAP specific genes from the GS cassette: gsc and gsd.
MAP specific genes homologous to desA1 from IS900 locus 5 and tetR regulation genes from IS900 locus 6

d)      MAP signals for all MTB spots were relatively weak. The production of a microarray containing the whole MAP genome in individual PCR products would be most desirable.


 
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Last revised: 21.01.02