An In Vivo Model of Separate M. tuberculosis Phagocytosis by Neutrophils and Macrophages: Gene Expression Profiles in the Parasite and Disease Development in the Mouse Host

Abstract

The role of neutrophils in tuberculosis infection remains less well studied compared to that of the CD4+ T-lymphocytes and macrophages. Thus, alterations in Mycobacterium tuberculosis transcription profile following phagocytosis by neutrophils and how these shifts differ from those caused by macrophage phagocytosis remain unknown. We developed a mouse model that allows obtaining large amounts of either neutrophils or macrophages infected in vivo with M. tuberculosis for mycobacteria isolation in quantities sufficient for the whole genome RNA sequencing and aerosol challenge of mice. Here, we present: (i) the differences in transcription profiles of mycobacteria isolated from liquid cultures, neutrophils and macrophages infected in vivo; (ii) phenotypes of infection and lung inflammation (life span, colony forming units (CFU) counts in organs, lung pathology, immune cells infiltration and cytokine production) in genetically TB-susceptible mice identically infected via respiratory tract with neutrophil-passaged (NP), macrophage-passaged (MP) and conventionally prepared (CP) mycobacteria. Two-hour residence within neutrophils caused transcriptome shifts consistent with mycobacterial transition to dormancy and diminished their capacity to attract immune cells to infected lung tissue. Mycobacterial multiplication in organs did not depend upon pre-phagocytosis, whilst survival time of infected mice was shorter in the group infected with NP bacilli. We also discuss possible reasons for these phenotypic divergences.

Small RNA MTS1338 Configures a Stress Resistance Signature in Mycobacterium tuberculosis

“…Since macrophages are the first line of defense against mycobacterial infection, we compared the Mtb transcriptomic profiles obtained here with those observed in peritoneal macrophages of I/St mice [13], and found that about 50% of the DEGs identified in the stressed MTS1338-overexpressing strain in vitro overlapped with those activated in Mtb residing in peritoneal macrophages: 32 of 60 DEGs in normal conditions, 32 of 63 at low pH, 16 of 42 with ROS, and 54 of 83 with RNS. These results suggest that the upregulation of MTS1338 during Mtb infection triggers the adaptive mechanisms that support mycobacterial survival in the aggressive intra-macrophage environment.…”

“…For this, we assessed the effects of MTS1338 overexpression on Mtb survival during macrophage infection and under macrophage-like conditions modeled in vitro. Transcriptomics of the MTS1338-overexpressing strain revealed a reproducible transcriptional signature that corresponded to that of Mtb phagocytosed by macrophages [13], suggesting the involvement of MTS1338 in the Mtb stress response and its contribution to the adaptation and survival of mycobacteria in the hostile intracellular environment.…”

>b<Host adaptation of >i<Mycobacterium tuberculosis>/i< and >i<Mycobacterium bovis>/i<:>/b< a genomic and transcriptional approach

“…Difficulties in RNA extraction are expected when working with MTBC species due to its complex cell wall. Even though many papers are published following the protocols tested herein, there is lack of description about initial volumes of bacterial cultures and the bead beating instrument used (JEON et al, 2017;OH et al, 2017;GIBSON et al, 2021;KONDRATIEVA et al, 2022), which affect the reproducibility of new studies. In this study, small concentrations of RNA were generated using TissueLyser (QIAGEN), and the present work was only able to successfully obtain sufficient and high quality RNA using the protocol described by Rohde; Abramovitch; Russell, 2007, with the MagNA Lyser and some modifications in the reagent volumes and time of beat beating.…”