Targeted RNA-Seq Reveals the M. tuberculosis Transcriptome from an In Vivo Infection Model

SIMPLE SUMMARY: High-throughput sequencing techniques such as RNA-seq allow a more detailed characterization of the gene expression profile during in vivo infections. However, using this strategy for intracellular pathogens such as Mycobacterium tuberculosis (Mtb) entails technical limitations. Some authors have resorted to flow cytometers to separate infected cells or significantly increase sequencing depth to obtain pathogens’ gene expression. However, these options carry additional expenses in specialized equipment. We propose an experimental protocol based on differential cell lysis and a probe-based ribosomal depletion to determine the gene expression of Mtb and its host during in vivo infection. This method allowed us to increase the number of observed expressed genes from 13 using a traditional RNA-seq approach to 702. In addition, we observed the expression of genes essential for establishing the infection, codifying proteins such as PE-PGRS, lipoproteins lppN and LpqH, and three ncRNAs (small RNA MTS2823, transfer-messenger RNA RF00023, and ribozyme RF00010). We believe our method represents a valuable alternative to current RNA-seq approaches to study host–pathogen interactions and will help explore host–pathogen mechanisms in tuberculosis and other similar models of intracellular infections. ABSTRACT: The study of host-pathogen interactions using in vivo models with intracellular pathogens like Mycobacterium tuberculosis (Mtb) entails technical limitations, such as: (i) Selecting an efficient differential lysis system to enrich the pathogen cells; (ii) obtaining sufficient high-quality RNA; and (iii) achieving an efficient rRNA depletion. Thus, some authors had used flow cytometers to separate infected cells or significantly increase the sequencing depth of host–pathogen RNA libraries to observe the pathogens’ gene expression. However, these options carry additional expenses in specialized equipment typically not available for all laboratories. Here, we propose an experimental protocol involving differential cell lysis and a probe-based ribosomal depletion to determine the gene expression of Mtb and its host during in vivo infection. This method increased the number of observed pathogen-expressed genes from 13 using the traditional RNA-seq approach to 702. After eliminating rRNA reads, we observed that 61.59% of Mtb sequences represented 702 genes, while 38.41% represented intergenic regions. Some of the most expressed genes codified for IS1081 (Rv2512c) transposase and eight PE-PGRS members, such as PGRS49 and PGRS50. As expected, a critical percent of the expressed genes codified for secreted proteins essential for infection, such as PE68, lppN, and LpqH. Moreover, three Mtb ncRNAs were highly expressed (small RNA MTS2823, transfer-messenger RNA RF00023, and ribozyme RF00010). Many of the host-expressed genes were related to the inflammation process and the expression of surfactant proteins such as the Sftpa and Sftpc, known to bind Mtb to alveolar macrophages and mi638, a microRNA with no previous associations with pulmonary diseases. The main objective of this study is to present the method, and a general catalog of the Mtb expressed genes at one point of the in vivo infection. We believe our method represents a different approach to the existing ones to study host–pathogen interactions in tuberculosis and other similar intracellular infections, without the necessity of specialized equipment.