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Comprehensive insights into transcriptional adaptation of intracellular mycobacteria by microbe-enriched dual RNA sequencing.

ABSTRACT: The human pathogen Mycobacterium tuberculosis has the capacity to escape eradication by professional phagocytes. During infection, M. tuberculosis resists the harsh environment of phagosomes and actively manipulates macrophages and dendritic cells to ensure prolonged intracellular survival. In contrast to other intracellular pathogens, it has remained difficult to capture the transcriptome of mycobacteria during infection due to an unfavorable host-to-pathogen ratio.We infected the human macrophage-like cell line THP-1 with the attenuated M. tuberculosis surrogate M. bovis Bacillus Calmette-Guérin (M. bovis BCG). Mycobacterial RNA was up to 1000-fold underrepresented in total RNA preparations of infected host cells. We employed microbial enrichment combined with specific ribosomal RNA depletion to simultaneously analyze the transcriptional responses of host and pathogen during infection by dual RNA sequencing. Our results confirm that mycobacterial pathways for cholesterol degradation and iron acquisition are upregulated during infection. In addition, genes involved in the methylcitrate cycle, aspartate metabolism and recycling of mycolic acids were induced. In response to M. bovis BCG infection, host cells upregulated de novo cholesterol biosynthesis presumably to compensate for the loss of this metabolite by bacterial catabolism.Dual RNA sequencing allows simultaneous capture of the global transcriptome of host and pathogen, during infection. However, mycobacteria remained problematic due to their relatively low number per host cell resulting in an unfavorable bacterium-to-host RNA ratio. Here, we use a strategy that combines enrichment for bacterial transcripts and dual RNA sequencing to provide the most comprehensive transcriptome of intracellular mycobacteria to date. The knowledge acquired into the pathogen and host pathways regulated during infection may contribute to a solid basis for the deployment of novel intervention strategies to tackle infection.

SUBMITTER: Rienksma RA

PROVIDER: S-EPMC4334782 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Comprehensive insights into transcriptional adaptation of intracellular mycobacteria by microbe-enriched dual RNA sequencing.

Rienksma Rienk A RA Suarez-Diez Maria M Mollenkopf Hans-Joachim HJ Dolganov Gregory M GM Dorhoi Anca A Schoolnik Gary K GK Martins Dos Santos Vitor Ap VA Kaufmann Stefan He SH Schaap Peter J PJ Gengenbacher Martin M

BMC genomics 20150205

<h4>Background</h4>The human pathogen Mycobacterium tuberculosis has the capacity to escape eradication by professional phagocytes. During infection, M. tuberculosis resists the harsh environment of phagosomes and actively manipulates macrophages and dendritic cells to ensure prolonged intracellular survival. In contrast to other intracellular pathogens, it has remained difficult to capture the transcriptome of mycobacteria during infection due to an unfavorable host-to-pathogen ratio.<h4>Result ...[more]

PMID: 25649146

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Project description:Long-term survival of bacterial pathogens during persistent bacterial infections can be associated with antibiotic treatment failure and poses a serious public health problem. Infections caused by the Gram-negative pathogen Pseudomonas aeruginosa, which can cause both acute and chronic infections, are particularly challenging due to its high intrinsic resistance to antibiotics. The ineffectiveness of antibiotics is exacerbated when bacteria reside intracellularly within host cells where they can adopt a drug tolerant state. While the early steps of adherence and entry of P. aeruginosa into mammalian cells have been described, the subsequent fate of internalized bacteria, as well as host and bacterial molecular pathways facilitating bacterial long-term survival, are not well defined. In particular, long-term survival within bladder epithelial cells has not been demonstrated and this may have important implications for the understanding and treatment of UTIs caused by P. aeruginosa. Here, we demonstrate and characterize the intracellular survival of wild type (WT) P. aeruginosa inside bladder epithelial cells and a mutant with a disruption in the bacterial two-component regulator AlgR that is unable to survive intracellularly. Using simultaneous dual RNA-seq transcriptional profiling, we define the transcriptional response of intracellular bacteria and their corresponding invaded host cells. The bacterial transcriptional response demonstrates that WT bacteria rapidly adapt to the stress encountered in the intracellular environment in contrast to ΔalgR bacteria. Analysis of the host transcriptional response to invasion suggests that the NF-κB signaling pathway, previously shown to be required for extracellular bacterial clearance, is paradoxically also required for intracellular bacterial survival. Lastly, we demonstrate that intracellular survival is important for pathogenesis of P. aeruginosa in vivo using a model of murine urinary tract infection. We propose that the unappreciated ability of P. aeruginosa to survive intracellularly may play an important role in contributing to the chronicity and recurrence of P. aeruginosa in urinary tract infections.

Project description:BackgroundThe cell-surface enzyme carbonic anhydrase IX (CAIX/CA9) promotes tumor growth, survival, invasion, and metastasis, mainly via its pH-regulating functions. Owing to its tumor-specific expression, CAIX-targeting antibodies/chemicals are utilized for therapeutic and diagnostic purposes. However, mechanisms of CAIX trafficking, which affects such CAIX-targeting modalities remain unclear. In this study, roles of the AMAP1-PRKD2 pathway, which mediates integrin recycling of invasive cancer cells, in CAIX trafficking were investigated.MethodsUsing highly invasive MDA-MB-231 breast cancer cells, the physical association and colocalization of endogenous proteins were analyzed by immunoprecipitation and immunofluorescence, protein/mRNA levels were quantified by western blotting/qPCR, and cell-surface transport and intracellular/extracellular pH regulation were measured by biotin-labeling and fluorescent dye-based assays, respectively. The correlation between mRNA levels and patients' prognoses was analyzed using a TCGA breast cancer dataset.ResultsAMAP1 associated with the CAIX protein complex, and they colocalized at the plasma membrane and tubulovesicular structures. AMAP1 knockdown reduced total/surface CAIX, induced its lysosomal accumulation and degradation, and affected intracellular/extracellular pH. PRKD2 knockdown excluded AMAP1 from the CAIX complex and reduced total CAIX in a lysosome-dependent manner. Unexpectedly, AMAP1 knockdown also reduced CAIX mRNA. AMAP1 interacted with PIAS3, which stabilizes HIF-1α, a transcriptional regulator of CA9. AMAP1 knockdown inhibited the PIAS3-HIF-1α interaction and destabilized the HIF-1α protein. High-ASAP1 (AMAP1-encoding gene) together with high-PIAS3 correlated with high-CA9 and an unfavorable prognosis in breast cancer.ConclusionThe AMAP1-PRKD2 pathway regulates CAIX trafficking, and modulates its total/surface expression. The AMAP1-PIAS3 interaction augments CA9 transcription by stabilizing HIF-1α, presumably contributing to an unfavorable prognosis.

Dataset Information

Comprehensive insights into transcriptional adaptation of intracellular mycobacteria by microbe-enriched dual RNA sequencing.

Publications

Comprehensive insights into transcriptional adaptation of intracellular mycobacteria by microbe-enriched dual RNA sequencing.

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