A new Technique for Obtaining Whole Pathogen Transcriptomes from Infected Host Tissues

Abstract

We propose a novel experimental approach based on coincidence cloning for analyzing sequences of bacterial intracellular pathogens specifically transcribed in affected tissues. Co-denaturation and co-renaturation of excess bacterial genomic DNA with the cDNA prepared on total RNA of the infected tissue allows one to select the bacterial fraction of the cDNA sample. We used this technique for preparing and characterizing the Mycobacterium tuberculosis cDNA pool, representing the transcriptome of infected mouse lungs in the chronic phase of infection. A cDNA pool enriched in fragments of mycobacterial cDNA was analyzed by the high-throughput 454 sequencing procedure. We demonstrated that its composition corresponded to what can be expected in the chronic phase of infection and, after the adaptation of M. tuberculosis to the host immune system, was characterized by an active lipid metabolism and switched from aerobic to anaerobic respiration. The technique is universal and requires no prior knowledge of the pathogen genome sequence. Pools of transcribed sequences obtained by this technique retain the main characteristics of the genome-wide gene transcription pattern within infected tissue, and can be used for in vivo analysis of gene expression of a wide spectrum of infection agents, such as viruses, bacteria, and protista.

Integrity of prokaryotic mRNA isolated from complex samples for in vivo bacterial transcriptome analysis

“…To address the issue of low relative prokaryotic yield, Azhikina et al (2010) proposed a novel experimental approach for studies on host-bacterium interactions. However, this method is very laborious, expensive, and can provide biases for the differential expression of genes.…”

Adaptive changes in gene expression of Mycobacterium tuberculosis during the development of the infection

“…The char acteristic features in this case are a steady decrease in an expression of NADH dehydrogenase genes of type I (nuoA N), and an increase in the expression of genes from the cluster narGHJI encoding nitrate reductase and the gene narK2, whose product is a nitrate trans porter protein [31,36,41]. This metabolic shift most likely means that the reorganization of the electron transport chain (ETC) results in the use of nitrate as a final electron acceptor.…”

“…One of the most characteristic changes in the expression of genes involved in lipid metabolism is the activation of expression of genes in clusters fadA, fadB, fadD, fadE and echA [31,36,41,45]. These genes encode enzymes involved in the process of β oxida tion of fatty acids and in the catabolism of cholesterol.…”

“…Transcription of genes papA and pks, the protein products of which are required for the synthesis of polyketides that are com ponents of the cellular membrane of M. tuberculosis [49][50][51], are almost always found when experimental dare analyzed. Nevertheless, these genes may have an increased or decreased expression in vivo; probably, it reflects the features of the lipid metabolism under cer tain conditions [33,[36][37][38]41]. Interestingly, the expression of these genes is reduced in avirulent strains M. bovis BCG and M. tuberculosis H37Ra compared to the virulent strain M. tuberculosis H37Rv [33,37].…”

Deep Sequencing in Pre- and Clinical Vaccine Research

“…After the introduction of deep sequencing technologies, a representative collection of epidemiologically relevant strains are sequenced and antigen selection is performed taking into consideration the level of conservation of the antigens. Another example is the use of RNA-seq to determine the expression level of antigens in different conditions, including infected tissues [13,14]. Increased gene expression during infection, if paralleled by a role in the pathogenesis, is generally considered an important indication that a protein is a potential vaccine candidate.…”