Genome-wide expression profiling of intracellular bacteria: The interaction of Mycobacterium tuberculosis with macrophages

7 Genome-wide Expression Profiling of Intracellular Bacteria: The Interaction of

Mycobacterium tuberculosis with Macrophages Sabine Ehrt and Dirk Schnappinger Department of Microbiology and Immunology, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 1002 I, USA

Martin I Voskuil and Gary K Schoolnik Department of Microbiology and Immunology, Stanford University, Beckman Center, 300 Pasteur Drive, Stanford, CA 94305, USA I-

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CONTENTS Introduction The Design of Genome-wide Expression Profiling Experiments Infection of Macrophages with M. tuberculosis Preparation and Labelling of RNA from Intracellular M. tuberculosis Conclusions

INTRODUCTION The number of fully sequenced bacterial genomes is large and increasing. The value of genome sequences is, however, limited by the fact that functional information is not available for 30% or more of the open reading frames (orfs) in any given genome. Genes are usually transcribed only during conditions under which their function is beneficial to the survival of a bacterium. The conditions that stimulate expression of a gene can therefore be used to characterize its function. In a typical microarray expression experiment two different RNAs (or their corresponding cDNAs), which have been prepared from a micro-organism under two different conditions of growth and were labelled with

different fluorescent dyes, are hybridized to a single microarray. Binding to all hybridization probes on the array is quantified with a laser scanner that separately reports fluorescent intensities of the two dyes used for labelling. The fluorescence ratio of individual hybridization probes indicates the relative abundance of gene-specific RNAs and identifies genes, which are differentially expressed during the two conditions of growth. The capacity of microarrays readily accommodates the number of hybridization probes necessary to represent every individual orf of a bacterial genome. The relative expression status of every orf can therefore be determined in a single microarray expression experiment. In this chapter we focus on the application of microarrays for the analysis of M. tuberculosis inside host cells. More general methods used in microarray expression profiling have recently been described in detail elsewhere (Schoolnik et al., 2001).

~.~.4,4,~,~. T H E D E S I G N O F G E N O M E - W I D E EXPRESSION PROFILING EXPERIMENTS The genome-wide expression profile of a cell is a complex description of its physiological status that includes most of the cell's metabolic activities and, therefore, gives access to an unusually holistic approach to the analysis of biological systems. In contrast, related but more reductionistic experiments such as reporter-gene assays only describe a narrow aspect of the cellular physiology. While genome-wide expression profiling contributes to a more complete understanding of cellular physiology it also demands a careful experimental design. All steps of an experimental protocol have to be investigated with respect to their impact on the expression profile because even routine procedures, such as the concentration of bacteria by centrifugation, can change the expression profile. Control samples are essential for the interpretation of an expression profile. Controls that are prepared at the same time and in the same manner as the experimental samples, except for the exposure to the stimulus or change of condition that is under investigation, will identify gene regulation occurring in response to the unavoidable handling of the bacteria during the experiment. In addition it may be necessary to perform control experiments to distinguish between direct and indirect changes of the expression profile. Antimicrobial drugs, for example, directly affect the genome-wide expression profile due to the inhibition of a particular biochemical pathway. The initial alteration of the expression profile can therefore be used to identify the pathway inhibited by a drug (Wilson et al., 1998). After longer exposure to the drug, however, the expression profile changes more broadly, for example, in consequence of the cessation of growth. Time course experiments are helpful to separate direct from indirect changes of the expression profile. In this example control experiments in which the growth rate is changed by nutrient limitation may also identify indirectly regulated genes. Expression profiles are currently generated for a number of well-characterized growth conditions. The comparison of profiles 170

generated in a pilot experiment with these reference profiles will identify stimuli that are applied in any given experiment and aid the selection of the most informative control experiments. Spotted microarrays are generally used to compare two differently labelled mixtures of nucleic acids. In expression profiling experiments RNA that was prepared from the control or experimental sample is transcribed into cDNA and compared with a reference nucleic acid mixture. The most commonly used reference is derived from RNA that has been prepared from the bacterial culture before it was exposed to the experimental or control stimuli. This design allows the immediate identification of regulated genes from a single microarray experiment. Alternatively, chromosomal DNA can be used to prepare a reference that contains labelled fragments for every individual open reading frame of a genome. If chromosomal DNA is used the comparison of two microarray experiments is necessary to identify regulated genes. On the other hand, chromosomal DNA provides a constant reference sample whereas a reference prepared from RNA will vary depending on the exact culture conditions of individual experiments. When microarrays were used for the first time, regulated genes were selected based on the magnitude of their change in expression. Recently, several laboratories demonstrated that this purely fold-change based approach has led to the incorrect identification of regulated genes. Moreover, other genes that failed to meet a particular fold-change criterion were misclassified as not having been significantly regulated (Ehrt et al., 2001; Long et al., 2001; Tusher et al., 2001). Microarray experiments should, therefore, be designed to include statistical analysis. Several statistical procedures have been used to analyse microarray data. All statistical procedures depend on repeated measurements; however, given the cost of microarray experiments, an experimental design that allows statistical analysis may seem expensive. Fortunately, permutations of a limited number of repeated measurements as generated by the SAM (_statistical _analysis of microarrays) procedure minimize the number of experiments necessary for a statistical analysis. SAM has been demonstrated to reliably identify regulated genes in an expression profile, providing the data set contains four arrays per experimental condition representing two biological and two technical replicates (Tusher ef al., 2001). For the purpose of this discussion a biological replicate requires the use of RNA derived from a de novo experiment, whereas a technical replicate uses a second microarray to study the same RNA. Control samples that are generated at the same time as the experimental samples can further increase the sensitivity of most statistical procedures since they allow pairing of experimental and control samples. While the use of appropriate microarray-specific instruments will select a gene set that is significantly regulated in a statistical sense, it does not prove that such regulation is biologically significant. Proof that the regulation of a gene is biologically significant may require a combination of mutational, biochemical and animal model studies. Expression profile libraries often contain millions of data points and much of the information they contain is likely to be overlooked. 171

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Multivariate statistical analysis, such as clustering or linear decomposition (Eisen et al., 1998; Alter et al., 2000), can overcome this problem by associating genes of unknown function (annotated as genes coding for hypothetical proteins in the genome database) with functionally annotated genes that subserve a common cellular process or pathway. Clustering the expression profiles of cancer cells, for example, has been used for cancer typing and predicting the success of a particular cancer therapy (Bittner et al., 2000; Scherf et al., 2000). Multivariate statistical methods should be included in every analysis of microarray data. Therefore, it is important that microarray experimental design be compatible with the statistical tools the investigator plans to use during the data mining and analysis phase of the project. The bioinformafics aspect of microarray experimentation is a rapidly developing field and further discussion of it is beyond the scope of this article; additional information can be found elsewhere (Sherlock, 2000; Altman and Raychaudhuri, 2001).

I N F E C T I O N OF M A C R O P H A G E S W I T H M. TUBERCULOSIS Basic considerations One of the most basic considerations when designing the infection experiment is that the amount of bacterial RNA extracted from infected tissue must be sufficient to perform a microarray experiment. Currently 0.5-1 btg of bacterial RNA is necessary for a single microarray experiment. Therefore, 2 btg should be available to allow analysis of the RNA for at least two independent hybridizations. At a multiplicity of infection of two to five bacilli, about 2 x 10s macrophages are necessary to allow the purification of a sufficient amount of M. tuberculosis RNA. This rather large number of cells imposes two restrictions on the experiment. First, primary human macrophages are often not available in the necessary quantity. Therefore, murine primary macrophages or human macrophage-like cell lines are typically used. Secondly, at very early time points, i.e. less than 4 h after infection, the numbers of intracellular bacteria are too low, even when 2 x l0 s macrophages are used, to allow purification of sufficient amounts of bacterial RNA. Another potential problem is that M. tuberculosis can form multicellular aggregates that, due to their size, interfere with their ingestion by macrophages. Therefore, in many infection experiments M. tuberculosis is exposed to rather harsh procedures, such as sonication, to reduce the size of the bacterial particles. These procedures should be avoided as they most likely change the expression profile of M. tuberculosis. Instead, growth conditions can be optimized to reduce the size of the bacterial particles. The particle size of M. tuberculosis is influenced by many factors including growth phase, culture density, medium components and strain characteristics. Many of the commonly used M. tuberculosis strains form 172

sufficiently small particles in early log phase of growth if cultivated in the presence of detergent in a slowly moving (1.5 rpm) roller bottle. Because genome-wide expression profiles are influenced by a large number of stimuli, nearly any difference in the experimental conditions in repeated measurements will lead to different expression profiles. To minimize these differences conditions of every individual experiment should be monitored as closely as possible. In the case of macrophage infection experiments the activation status of macrophages is one source of experimental variation. Accordingly, the activation status should be determined by measuring the generation of reactive oxygen and nitrogen intermediates and by monitoring the survival of intracellular M. tuberculosis (Ehrt et al., 2001). Macrophages used for infection with M. tuberculosis should form adherent monolayers of about 95% confluency. Murine bone marrow derived macrophages are readily obtained in large numbers, adhere well to tissue culture flasks even in large volumes and are not pre-activated during preparation. They are, therefore, well suited for microarray expression profiling of intracellular M. tuberculosis. A protocol for the isolation and maturation of murine bone marrow derived macrophages has been described previously in detail (Rhoades and Orme 1998; Roberts et al., 1998). A brief summary of the method is given in Protocol 1. Protocol I

Isolation of m u r i n e bone m a r r o w m a c r o p h a g e s

1. Isolate bone marrow cells from mouse femurs by flushing the femurs with DMEM (Invitrogen). Expect about 107 cells per femur. Harvest the cells by centrifugation at 1000 rpm for 10 min at 4°C. 2. To lyse the red blood cells, resuspend the ceils in 5 ml cold 0.2% NaC1; after 30 s add 5 ml cold 1.6% NaC1 and 10 ml PBS. Centrifuge cells as above. 3. Resuspend cells in complete cell culture medium containing 20% L929 fibroblast conditioned medium and count cell number with a haematocytometer. 4. Seed 5 x 107 cells per 175 cm ~triple layer flask (Nunc) and incubate at 37°C in 5% CO2. Induced by M-CSF from the L-cell conditioned medium, the cells will differentiate into macrophages over a period of 6-7 days. The cell number will approximately double over that time period and the monolayer should reach a confluency of more than 90%. 5. At day 4 after isolation feed the cells with one fourth volume complete cell culture medium. 6. At day 7 after isolation remove the medium and any non-adherent cells, wash the monolayers once with prewarmed PBS, then add complete cell culture medium containing 10% L-cell conditioned medium and no antibiotics. Add 100 U / m l recombinant murine interferon- 7 (Genentech) if activation is desired. The macrophages are ready for infection 24 h or 48 h after the medium change.

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Protocol 2 Preparation of M. tuberculosis culture and infection of macrophages 1. M. tuberculosis is grown in 7H9 medium with 10% ADNaC1 and

0.05% Tween80 in 11 roller bottles rotated at 1.5 rpm. The bacteria are sub-cultured every 3-4 days (once they reach an ODss0 between 0.6 and 0.8) at a 1 : 100 dilution into fresh medium. After about 2 months a new aliquot of M. tuberculosis is thawed and cultured. 2. For infection of macrophages, M. tuberculosis cultures are used at an ODss0 of 0.3-0.4. Collect the appropriate amount of bacteria by centrifugation for 10 min at 3500 rpm. 3. Resuspend the bacteria in prewarmed complete cell culture medium and add to the macrophage monolayer at a multiplicity of infection of 5.' Determine the actual titre of the bacteria by plating serial dilutions onto 7Hll agar plates (BD Diagnostic Systems).

Protocol 3 Harvesting of M . tuberculosis grown in liquid culture for the preparation of R N A 2

1. Transfer an appropriate volume of the liquid culture (for example 20-25 ml of a logarithmically growing culture of an OD580 of 0.2-0.4) to a 50 ml centrifugation tube containing the same volume of GTC solution. 3Mix immediately and centrifuge for 10 min at 4000 rpm. 2. Pour off the supernatant; freeze the pellet on dry ice and store at -80°C.

P R E P A R A T I O N A N D L A B E L L I N G OF R N A FROM I N T R A C E L L U L A R M. TUBERCULOSIS The use of microarrays to analyse the expression profile of bacteria that are co-cultivated with host cells is obviously complicated by the eukaryotic RNA that is present in excess. Two approaches have been used to minimize the interference of eukaryotic RNA during hybridization. Talaat et al. (2000) designed a small number of short primers that can be used preferentially to label mycobacterial RNA that is present in a mixture with eukaryotic RNA. Butcher and colleagues instead took advantage of the very stable mycobacterial cell wall to develop a differential lysis procedure that allows the separation of mycobacteria from the eukaryotic tissue prior to purification of the mycobacterial RNA (Monahan et al., 2001). The key step of this procedure is the incubation of the infected tissue with a guanidinium thiocyanate (GTC) solution that besides GTC contains detergent and [3-mercaptoethanol as active ingredients. GTC is a highly denaturing salt, which has been used for the preparation of RNA especially from samples that contain high concen174

trations of RNase (Chirgwin et al., 1979). Incubation of infected macrophages with the GTC solution immediately lyses the macrophages but does not lyse M. tuberculosis. Although it does not lyse M. tuberculosis the GTC solution kills the bacteria and stabilizes all transcripts that are present at the time the solution is added. After treatment with the GTC solution the bacteria can be safely exposed to procedures that would normally alter their expression profile. Because this procedure allows purification of bacterial particles prior to their lysis a selective labelling protocol is not needed. Protocol 4

Harvesting intracellular M. tuberculosis

1. Inspect the macrophage culture for integrity of the monolayer and location of bacteria. Discard the medium and quickly wash with 50 ml of prewarmed (37°C) complete medium if a large number of obviously extracellular bacteria are present (relevant for early time points after infection). Remove medium before proceeding to step 2. 2. Pour 50 ml of GTC solution into the tissue culture flask and lyse the cells by shaking. The solution will become viscous due to the release of eukaryotic chromosomal DNA. Continue shaking till the viscosity is reduced to a point allowing transfer of the solution. 3. Transfer the GTC solution from the tissue culture flask into two 50 ml V-bottom plastic tubes and vortex for I min. At the end of the vortexing the viscosity of the solution should be about the same as fresh GTC solution. 4. Centrifuge for 15 min at 4000 rpm and room temperature to harvest the bacteria. 5. Decant the GTC solution and resuspend the cell pellet in 10 ml of fresh GTC solution. 6. Centrifuge as above and remove as much GTC solution as possible. The cell pellets can now be stored at -80°C or be used directly for the preparation of RNA.

Protocol 5

Preparation of R N A

1. Resuspend the bacterial pellets in 1 ml Trizol (Invitrogen) and transfer the suspension into a 2 ml screw cap tube containing 0.4 ml 0.1 mm silica beads (Biospec). 2. Shake the screw cap tube for 30 s in a bead beater (Biospec) at maxim u m speed and repeat this procedure twice. Cool the sample in between bead beating incubations if the temperature exceeds 37°C. 3. Centrifuge the sample for 20 s at 13 000 rpm at room temperature to separate the Trizol solution from the beads and bacterial debris. 4. Transfer the Trizol solution into a fresh 2 ml tube containing 350 ~1 chloroform: isoamylalcohol (24 : 1). invert rapidly for 15 s and continue inverting periodically for 2 min. (contd.)

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5. Centrifuge for 5 min at maximum speed and room temperature. Remove the aqueous, upper layer and transfer it into a 1.5 ml tube containing 700 gl isopropanol and mix well. 6. Incubate for 2 h at -20°C and centrifuge for 20 min at 13 000 rpm and 4°C to precipitate nucleic acids. Remove isopropanol, add 1 ml of 75% ethanol, invert several times and centrifuge for 5 min at 13 000 rpm and 4°C. Remove ethanol and air dry for 5 min. 7. Resuspend the precipitated nucleic acids in water, determine their concentration and degrade DNA with DNase for 30 min at 37°C. Inactivate DNase for 15 min at 65°C or remove it by column purification using the RNeasy procedure (Qiagen). Store RNA at -80°C.

Protocol 6

Labelling of R N A

1. Bring 0.5-2 ~tg of RNA to a volume of 10 gl and add 2 gl (2 gg/gl) random hexamers (Roche). 2. Denature RNA for 10 min at 65°C. Chill on ice and centrifuge for 1 min. 3. Add 5.0 gl first-strand buffer (5X), 2.5 gl DTT (100 mM), 2.3 gl dNTP mix (5 mM dATP, dCTP, dGTP and 0.2 mM dTTP), 1.5 gl Cy3-dUTP or Cy5-dUTP (Amersham) and 1.0 gl reverse transcriptase (Invitrogen). Incubate for 5 min at room temperature followed by 90 rain at 42°C. The labelled sample can be stored at -20°C; concentrate the sample using a microcon-10 (Ambion) or other concentration method before hybridization.

CONCLUSIONS A differential lysis procedure that stabilizes the bacterial RNA as soon as the lysis solution is added to the infected tissue is an ideal method to study the genome-wide expression profile of pathogens in contact with their host cells. Changes of the expression profile due to the collection of the bacteria are prevented and pathogen- and host-derived RNA are separated, significantly simplifying specific labelling of the pathogen's RNA. Unfortunately, the GTC solution used for the preparation of RNA from intracellular M. tuberculosis disrupts most other bacterial species. Thus, the GTC method described here cannot be used to study, for example, the interaction of Gram-negative bacteria with eukaryotic cells. Nonetheless, it may be possible to design alternative differential lysis solutions that can be used for the analysis of bacteria more fragile than M. tuberculosis. In contrast, a differential labelling procedure, using a small number of genome directed primers (Talaat et al., 2000) or a large number of primers specifically designed for every orf in a genome, should 176

be applicable for the analysis with its host. It should be kept differential labelling is used, stabilized as early as possible expression profile.

of the interaction of almost any pathogen in mind though, even when this method of that the RNA of the pathogen must be to avoid procedure-related changes of the

Acknowledgements Work by the authors has been supported by the Walter and Idun Berry Foundation (M.V.), National Institutes of Health grant AI44826 (G. K. S.), the Action TB Program of GlaxoWellcome (G. K. S.) and the Deutsche Forschungsgemeinschaft (D.S.). We thank Heran Darwin for her valuable comments on the manuscript.

Notes 1. We recommend determining the relationship of CFU and ODss0 for the M. tuberculosis strain under study and spectrophotometer. In our hands an OD~s0of 0.1 corresponds to 5 x 107 CFU. 2. This protocol should be used for the preparation of the reference samples if RNA is used as a reference in the microarray hybridization. 3. Use GTC at a concentration of 5 M instead of 4 M.

Reagents Complete cell culture medium DMEM (Invitrogen) with 4.5 g/1 glucose, 0.584 g/1 L-glutamine, 1 mM Na-pyruvate, 10% heat-inactivated FBS (HyClone), 10 mM Hepes buffer, 20% L929-fibroblast conditioned medium (contains M-CSF) and 100 U m l ' penicillin G, 100 btg m l ' streptomycin. Reduce L-cell conditioned medium to 10% and leave out the antibiotics, after differentiation of macrophages and for infection with bacteria.

L-929 conditioned medium L-929 cells from American type culture collection (ATCC) are grown in DMEM (Invitrogen) with 4.5 g 1 ' glucose, 0.584 g 1 ' L-glutamine, 1 mM Na-pyruvate, 10% heat-inactivated FBS (HyClone) until confluent. Collect supernatant, filter through a 0.22-~m filter or centrifuge for 10 min at 1000 rpm and collect the supernatant. Freeze conditioned medium at -20°C in 50-ml aliquots.

GTC solution (4M) Dissolve 50 g guanidine thiocyanate (Sigma) in 70 ml deionized water. Incubate the solution in a 50°C water bath until clear and add 0.5 g 177

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s o d i u m N-lauryl sarcosine (0.5%), 0.75 g tri-sodium citrate (50 raM) and 0.7 ml 2-mercaptoethanol (0.1M). Adjust p H to 7.0 and v o l u m e to 100 ml. Keep the solution at r o o m t e m p e r a t u r e and store it for no longer than 48 h.

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