Transcriptome Analysis Reveals Novel Entry Mechanisms and a Central Role of SRC in Host Defense during High Multiplicity Mycobacterial Infection

Citation: Zhang J (2013) Transcriptome Analysis Reveals Novel Entry Mechanisms and a Central Role of SRC in Host Defense during High Multiplicity Mycobacterial Infection. PLoS ONE 8(6): e65128. doi:10.1371/journal.pone.0065128 Transcriptome Analysis Reveals Novel Entry Mechanisms and a Central Role of SRC in Host Defense during High Multiplicity Mycobacterial Infection Jay Zhang 0 Anil Kumar Tyagi, University of Delhi, India 0 Genomics Research Centre, Griffith Health Institute, Gold Coast Campus, Griffith University , Southport, Queensland , Australia Mycobacterium tuberculosis (MTB) infects an estimated one-third of the global population and is one of the main causes of mortality from an infectious agent. The characteristics of macrophages challenged by MTB with a high multiplicity of infection (MOI), which mimics both clinical disseminated infection and granuloma formation, are distinct from macrophages challenged with a low MOI. To better understand the cross talk between macrophage host cells and mycobacteria, we compared the transcription patterns of mouse macrophages infected with bacille Calmette-Gue rin, H37Ra and M. smegmatis. Attention was focused on the changes in the abundance of transcripts related to immune system function. From the results of a transcriptome profiling study with a high mycobacterial MOI, we defined a pathogen-specific host gene expression pattern. The present study suggests that two integrins, ITGA5 and ITGAV, are novel cell surface receptors mediating mycobacterium entry into macrophages challenged with high MOI. Our results indicate that SRC likely plays a central role in regulating multiple unique signaling pathways activated by MTB infection. The integrated results increase our understanding of the molecular networks behind the host innate immune response and identify important targets that might be useful for the development of tuberculosis therapy. - Funding: This work was funded by a postgraduate research account (No. MSC1010) from Griffith University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The author has declared that no competing interests exist. As a primary response to Mycobacterium tuberculosis (MTB) infection, activated macrophages increase the expression of a panel of phagocytic receptors, termed pattern recognition receptors (PRRs), which regulate inflammation-mediated microbial clearance [1]. Such receptors include mannose receptor, Tolllike receptors (TLRs), NOD-like receptors (NLRs), and complement receptors [2]. Activated PRRs activate cytoplasmic tyrosine kinase SRC, which has been linked to many intracellular signaling pathways in macrophages. Activated SRC is known to be involved in regulating many downstream inflammatory signaling pathways [3,4]. Several gene expression profiling studies comparing pathogenic and non-pathogenic mycobacterial infection under relatively low multiplicity of infection (MOI) (MOI , = 10) have revealed significant differences in the expression of genes involved in a wide range of processes [5,6]. In addition, different response patterns are also seen at different MOIs. Macrophage apoptosis depends, in part, on intracellular bacillary load, and rapid cytotoxicity occurs when a MOI threshold of 25 is exceeded [7]. We hypothesized that high intracellular loads of mycobacterial exposure would generate a disease-relevant gene expression profile. A system-wide analysis of these profiles would yield clinically specific pathways for diseases [8] and avenues for drug development. Our present study suggests the integrins (ITGA5 and ITGAV) as possible novel PRRs for mycobacterium entry into macrophages. We also reveal that SRC plays a central role in the host defense network. The host targets identified could be sound candidates for host-directed anti-mycobacterial therapies. Materials and Methods Cells, Cultures, and Media The murine macrophage cell line J774A.1 (American Type Culture Collection, ATCC) was used in this study. J774A.1 cells were cultured in DMEM (HyClone from Thermo Scientific) medium containing 10% (v/v) fetal calf serum, 50 mg/ml of penicillin/streptomycin and 2 mM glutamine. Cells were used to conduct experiments when they reached ,70% confluence. All treatments were performed in serum-free medium. All mycobacteria were grown on Middlebrook 7H11 agar at 37uC, 5% CO2-95% air atmosphere. For broth cultures, H37Ra (ATCC 25177, the lab strain of MTB) and bacillus CalmetteGuerin (ATCC 35734, BCG, the vaccine strain of M. Bovis) were grown in 7H9 medium supplemented with glycerol (0.5%, vol/vol) and OADC supplement. M. smegmatis (ATCC 700084, mc2-155,) was grown in 7H9 medium supplemented with glycerol (0.5%, vol/vol) and ADS supplement. All liquid cultures were supplemented with 0.05% Tween 80. Macrophage Infections In order to obtain a single cell suspension for an infection assay, the following procedure was performed as previously described [54]. Briefly, bacteria were centrifuged and washed twice in PBS, re-suspended in media (no additives), and sonicated at 30% power for 10 sec in a cuphorn sonicator, twice. Sonicated bacteria were dispersed by aspiration five times each with a 24-gauge needle, followed by an additional dispersion 5 times through a 30-gauge needle. This was then vortexed until no bacterial clumps were detectable, and the dispersed bacteria were allowed to stand for 5 min. The upper half of the suspension was then used for the experiments. Quantification of bacteria was done by taking absorbance at a 600-nm wavelength (0.6 OD corresponds to,1006106 bacteria). Cells were infected with mycobacterium species at a multiplicity of infection (MOI) of 50 in antibiotic-free DMEM (HyClone) 37C for 2 hours and then washed 3 times with fresh media to remove extracellular bacteria and further incubated for an additional 2 hours in DMEM. After the infection, cells grown on cover slips infected with various mycobacteria were stained using TB Quick Stain Kit (BD Diagnostic Systems). Twenty randomly-infected mouse macrophage cells were counted for intracellular bacterial load as well as infection rate under a Nikon microscope. RNA Isolation and Microarray Experiments Total RNA was isolated from 26106 J774A.1 cells 4 hours after infection with various mycobacterial species and from un-infected cells. Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturers protocol, followed by on-column digestion of DNA using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). RNA quantity and quality were assessed with a Qubit RNA Assay Kit using a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) and Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). 500 ng of total RNA was amplified using the GeneChip 39 IVT Express Kit. Standard Affymetrix protocols were used to process and scan Affymetrix MOE430_2 microarrays (A (...truncated)