Introduction

It is now well acknowledged that during pathogen penetration, the host organism triggers the defense system by recognizing and responding to microbial exposure. For its part, the pathogenic microorganism also responds to the signals generated by its interaction with the defense system by changing the gene expression pattern to neutralize (or at least decrease) the destroying potential of the host defense. The understanding of host-pathogen interactions not only requires an investigation of host innate and adaptive immune responses to the infection, but also knowledge of parasite factors that provide adaptation of the pathogen to the host environment. At the moment, this understanding is limited, and new experimental approaches to analyze these complex interactions are needed. The sequencing of hundreds of microbial genomes stimulated development of novel approaches for functional studies at the genome-wide level. Modern technologies, such as serial analysis of gene expression (1), subtractive hybridization (2), and DNA microarray analysis (3,4) enable the assessment of bulk gene expression profiles in a single experiment, and provide a deeper insight into host-pathogen interactions (for review, see References (5) and (6). However, the efficacy of these technologies critically depends upon the availability of bacterial RNA samples that precisely reflect the real ratios of individual bacterial mRNAs in the infected host tissues. This is very challenging, given the paucity of bacterial mRNA compared with the amounts of mammalian RNA in samples. To resolve this problem, several experimental approaches were proposed, including differential lysis for bacterial RNA extraction (5,7), enrichment of bacterial RNA by cDNA-RNA subtractive hybridization (8), the DECAL method (9), and hybridization-based positive cDNA selection (selective capture of transcribed sequences, or SCOTS) (10). At present, the most popular ways of preparing cDNA probes to be further used for hybridization with microarrays are (i) selective amplification of the corresponding cDNA with primers against ORFs of a given bacterial genome on a template of previously enriched bacterial RNA isolated from the infected tissue (11,12), and (ii) polyadenylation of the bacterial RNA followed by further amplification using T7 oligo(dT) primer and T7 RNA polymerase (13). However, these approaches are not universal, and since they are based on a known strain's genome sequence, they do not take into account genomic variability of bacteria due to insertion-deletion polymorphisms, which is known to be rather high (14,15). Here we propose an easy-to-implement hybridization method based upon the coincidence cloning (CC) approach (16) that allows isolation of representative bacterial cDNA pools from infected organs. Co-denaturation and co-renaturation of the excess of bacterial genomic DNA with the cDNA transcribed from total RNA of the infected tissue enabled selective isolation of the bacterial cDNA fraction from the sample, and a single round of coincidence cloning resulted in >1000-fold enrichment of bacterial transcripts. As a model organism, we chose Mycobacterium tuberculosis, the intracellular pathogen responsible for tuberculosis in millions of people per year. We used this approach to selectively isolate M. tuberculosis cDNA from the lung tissue of A/Sn mice after aerosol challenge, the infection model developed in A.S.A.'s laboratory (17). To make an exhaustive description of the enriched bacterial cDNA sample, we performed high-throughput pyrosequencing of this sample, which is less biased and more reliable than other methods including microarrays. 454 pyrosequencing is an extremely powerful sequencing technique that allows for gathering sequence data of hundreds of millions of nucleotides in one experiment (18). A major advantage of the proposed technique over other methods of transcriptome analysis is that it does not require prior knowledge of the pathogen's genome structure, requires only the genomic DNA, and permits analysis of even small amounts of tissue from the infected organism.

Materials and methods

Basic protocols are given in the Supplementary Materials.

RNA isolation and cDNA synthesis

RNA was isolated from the lung tissue of mice at week 9 of infection with M. tuberculosis H37Rv (strain was cultivated at the Central Institute for Tuberculosis, Moscow, Russia) using the SV Total RNA Isolation System (Promega, Madison, WI, USA) according to the manufacturer's recommendations. RNA samples were treated with 1 U/µL DNaseI (MBI Fermentas, Vilnius, Lithuania) to remove residual DNA. The first cDNA strand was synthesized using BR and SMART primers (synthesized at the Institute of Bioorganic Chemistry, Moscow, Russia) (Supplementary Table S1). The primers (12 pM each) were annealed in 11 µL of a mixture containing 2 µg total RNA. The mixture was heated for 2 min at 70°C and then chilled on ice for 10 min. cDNA synthesis was performed according to the manufacturer's protocol with (RT+) or without (RT-) the addition of Power-Script II reverse transcriptase (Clontech, Mountain View, CA, USA). The RT+ and RT- reaction mixtures were then incubated at 37° and 42°C for 10 and 120 min, respectively. Preparative synthesis of cDNA was performed with 5S primer for 30 cycles (95°C for 20 s, 64°C for 20 s, and 72°C for 2 min). cDNA was cleaned with a QIAquick PCR Purification kit (Qiagen, Valencia, CA, USA).