An essential mycolate remodeling program for mycobacterial adaptation in host cells

Abstract

One Sentence Summary: Novel technology (Path-seq) discovers cell wall remodeling program 24 during Mycobacterium tuberculosis infection of macrophages 26 Abstract:The success of Mycobacterium tuberculosis (MTB) stems from its ability to remain hidden from the 28 immune system within macrophages. Here, we report a new technology (Path-seq) to sequence miniscule amounts of MTB transcripts within up to million-fold excess host RNA. Using Path-seq we have 30 discovered a novel transcriptional program for in vivo mycobacterial cell wall remodeling when the pathogen infects alveolar macrophages in mice. We have discovered that MadR transcriptionally 32 modulates two mycolic acid desaturases desA1/A2 to initially promote cell wall remodeling upon in vitro macrophage infection and, subsequently, reduces mycolate biosynthesis upon entering dormancy. We 34 demonstrate that disrupting MadR program is lethal to diverse mycobacteria making this evolutionarily conserved regulator a prime antitubercular target for both early and late stages of infection. 36 38 40 Main Text: 42Mycobacterium tuberculosis (MTB) infection occurs by inhalation of bacilli-containing aerosols. 44Alveolar macrophages, which line the airway, are the first host cells to phagocytize the bacteria. This initial contact of MTB with alveolar macrophages begins a complex battle between bacterial virulence 46 and host immunity, orchestrated in large part by intricate gene regulatory pathways(1, 2). As such, measuring gene expression in vivo is central to our understanding of TB disease control and 48 progression(3).RNA-seq provides a sensitive method for global gene expression analysis. Specific for infection 50 biology, dual RNA-seq methods have allowed simultaneous profiling of host and pathogen RNA. However, the striking excess of eukaryotic over bacteria RNA limits the coverage of pathogen transcripts 52 in dual RNA-seq studies(4-8), and methods to partially enrich for bacterial transcripts have had limited success(9, 10). It is clear more sensitive approaches are needed to profile the transcriptional state of the 54 pathogen during infection, especially in vivo.To improve the coverage of pathogen transcripts, we made use of biotinylated oligonucleotide 56 baits that are complementary to the pathogen transcriptome. The baits are hybridized to mixed hostpathogen RNA and used to enrich pathogen transcripts for sequencing. We applied our pathogen-58 sequencing (Path-seq) method to explore transcriptional changes in MTB following infection in mice.Here Path-seq has led to discovery that MTB transcriptionally regulate mycolic acids during infection of 60 host cells, influencing virulence and persistence of the pathogen. 62RESULTS and DISCUSSION 64Development of Path-seq To enrich the bacterial pathogen transcripts, we used Agilent eArray(11) to create a custom bait library 66 that covers all MTB transcripts at even intervals. Our MTB library contains 35,624 probes, each with biotinylated oligonucleotides of 120 base lengths. The bait library composition is modul...

Alveolar macrophages up-regulate a non-classical innate response toMycobacterium tuberculosisinfectionin vivo

“…The finding that AMs do not mount an inflammatory and bactericidal response to Mtb infection is supported by transcriptional profiling we performed in collaboration with Peterson et al on Mtb residing within infected AMs in vivo using a novel bead-based method, Path-Seq, to enrich for bacterial transcripts (under revision, Molecular Systems Biology ) ( 56 ). These studies show that infection of AMs in vivo fails to induce the robust Mtb stress response that is observed after infection of BMDMs in vitro .…”