Distinct host-immune response toward species related intracellular mycobacterial killing : a transcriptomic study

CITATION: Madhvi Abhilasha et al. 2020. Distinct host-immune response toward species related intracellular mycobacterial killing : a transcriptomic study. Virulence, 11(1):170-182, doi:10.1080/21505594.2020.1726561.The original publication is availablle at: https://www.ncbi.nlm.nih.govThe comparison of the host immune response when challenged with pathogenic and nonpatho- genic species of mycobacteria can provide answers to the unresolved question of how pathogens subvert or inhibit an effective response. We infected human monocyte derived macrophages (hMDMs) with different species of mycobacteria, in increasing order of pathogenicity, i.e. M. smegmatis, M. bovis BCG, and M. tuberculosis R179 that had been cultured in the absence of detergents. RNA was isolated post-infection and transcriptomic analysis using amplicons (Ampliseq) revealed 274 differentially expressed genes (DEGs) across three species, out of which we selected 19 DEGs for further validation. We used qRT-PCR to confirm the differential expression of 19 DEGs. We studied biological network through Ingenuity Pathway Analysis® (IPA) which revealed up-regulated pathways of the interferon and interleukin family related to the killing of M. smegmatis. Apart from interferon and interleukin family, we found one up-regulated (EIF2AK2) and two down-regulated (MT1A and TRIB3) genes as unique potential targets found by Ampliseq and qRT-PCR which may be involved in the intracellular mycobacterial killing. The roles of these genes have not previously been described in tuberculosis. Multiplex ELISA of culture supernatants showed increased host immune response toward M. smegmatis as compared to M. bovis BCG and M.tb R179. These results enhance our understanding of host immune response against M.tb infection.Publisher's versio

The association of OASL and type I interferons in the pathogenesis and survival of intracellular replicating bacterial species

CITATION: Leisching, G., Wiid, I. & Baker, B. 2017. The association of OASL and type I interferons in the pathogenesis and survival of intracellular replicating bacterial species. Frontiers in Cellular and Infection Microbiology, 7:196, doi:10.3389/fcimb.2017.00196.The original publication is available at https://www.frontiersin.orgThe type I IFN response quickly became associated with its role in the innate immune response to viral infection. The past few years have seen the significance of IFNs expand in breadth to include non-viral pathogens. Previous work has identified that following viral infection, type I IFN signaling induces the production of the 2′-5′-oligoadenylate synthetase (OAS) family, which include OAS1, OAS2, OAS3, and OAS-like (OASL) protein. OASL was identified to be strongly induced following viral infection through engaging the RNA sensor RIG-I and increasing signaling through this pathway to enhance the anti-viral type I IFN response. Surprisingly, infection with viral dsDNA revealed an IFN inhibitory role and therefore pro-viral function of OASL through the inhibition of the cGAS cytosolic DNA sensing mechanism. Intracellular bacteria are able to activate the cytosolic DNA sensing pathway, however the role of OASL during bacterial infection is largely unknown. Vacuolar pathogenic microbes such as mycobacteria induce OASL early post infection, where it functions in a prosurvival fashion by inhibiting autophagic mechanisms and antimicrobial peptide expression. This suggests an underestimated role of OASL in the innate immune response to infection with a variety of pathogens and points to OASL-associated modulation of the type I IFN response. OASL may therefore play a critical role in defining the outcome of infection. We provide a brief update on the recent developments of the OAS family of proteins in response to DNA and RNA virus infections, as well as discuss evidence of Oasl expression in response to a number of cytosolic and vacuolar replicating bacterial pathogens.https://www.frontiersin.org/articles/10.3389/fcimb.2017.00196/fullPublisher's versio

OAS1, OAS2 and OAS3 restrict intracellular M. tb replication and enhance cytokine secretion

The 2′,5′ (OASs) are known as mediators of the antiviral response system through activation of the RNA cleavage pathway. Interestingly, we observe OAS1, OAS2 and OAS3 upregulation in a number of gene expression signatures which discriminate active TB from latent TB infection, however their biological role during bacterial infection has not yet been elucidated. We observed that the expression of these genes was associated with pathogenicity and virulence of mycobacteria as infection with Mycobacterium bovis BCG failed to significantly induce OAS expression. Further, we observed that after silencing of these genes, M. tb CFU counts increased significantly 96 h post-infection in comparison to the respective controls. Luminex revealed that OAS silencing significantly decreased IL-1β, TNF-α and MCP-1 and had no effect of IL-10 secretion. We show for the first time that OAS1, 2 and 3 restrict intracellular pathogenic mycobacterial replication and enhance pro-inflammatory cytokine secretion. Keywords: M. tuberculosis, OAS, Host-defence, Anti-microbial, Pathogenesis, Virulenc

The role of glutamine oxoglutarate aminotransferase and glutamate dehydrogenase in nitrogen metabolism in Mycobacterium bovis BCG

CITATION: Viljoen, A. J. et al. 2013. The role of glutamine oxoglutarate aminotransferase and glutamate dehydrogenase in nitrogen metabolism in Mycobacterium bovis BCG. PLoS ONE, 8(12):e84452, doi:10.1371/journal.pone.0084452.The original publication is available at http://journals.plos.org/plosoneRecent evidence suggests that the regulation of intracellular glutamate levels could play an important role in the ability of pathogenic slow-growing mycobacteria to grow in vivo. However, little is known about the in vitro requirement for the enzymes which catalyse glutamate production and degradation in the slow-growing mycobacteria, namely; glutamine oxoglutarate aminotransferase (GOGAT) and glutamate dehydrogenase (GDH), respectively. We report that allelic replacement of the Mycobacterium bovis BCG gltBD-operon encoding for the large (gltB) and small (gltD) subunits of GOGAT with a hygromycin resistance cassette resulted in glutamate auxotrophy and that deletion of the GDH encoding-gene (gdh) led to a marked growth deficiency in the presence of L-glutamate as a sole nitrogen source as well as reduction in growth when cultured in an excess of L-asparagine.http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0084452Publisher's versio

Growth of <i>Δgdh</i> in 7H9 with different nitrogen sources.

<p>Growth of wt BCG, the <i>Δgdh</i> mutant and the <i>Δgdh</i> complemented strain in (A) standard 7H9 containing approximately 4 mM ammonium sulphate (AS, (NH₄)₂SO₄) and 3 mM L-Glu, (B) ‑N7H9 (nitrogen-depleted 7H9) + 3 mM L-Glu, (C) 7H9 + 30 mM L-Glu, (D) 7H9 + 30 mM L-Asn, (E) -N7H9 + 30 mM L-Asn, (F) 7H9 + 3 mM L-Asn, (G) -N7H9 + 3 mM L-Asn, or (H) 7H9 + 30 mM L-Asp. Growth of the <i>Δgdh</i> mutant in (I) ‑N7H9 + 3 mM L-Glu supplemented with increasing concentrations of AS. Cultures for cfu/ml determinations were inoculated to OD₆₀₀ = 0.0005 (cfu/ml of approximately 10⁵). Log₁₀(cfu/ml) of <i>Δgdh</i> cultured in -N7H9 + 3 mM Glu was different from log₁₀(cfu/ml) of <i>Δgdh</i> cultured in -N7H9 + 3 mM Glu supplemented with 1 mM AS at day 9 and 14 (p < 0.01). Log₁₀(cfu/ml) of <i>Δgdh</i> cultured in 7H9 + 30 mM L-Asn was different from log₁₀(cfu/ml) of <i>Δgdh</i> cultured in -N7H9 + 3 mM Glu supplemented with 1 mM AS at day 6, 9 and 14 (p < 0.01). (J) Growth of the <i>Δgdh</i> mutant cultured in ‑N7H9 + 3 mM L-Glu for three weeks when sub-cultured in fresh 7H9 or –N7H9 + 3 mM L-Glu. Aliquots of three week old <i>Δgdh</i> mutant ‑N7H9 + 3 mM L-Glu cultures were washed once with ‑N7H9 and used to inoculate fresh 7H9 or ‑N7H9 + 3 mM L-Glu to an OD₆₀₀ = 0.020. (K) Determination of growth of single colonies obtained from three week old <i>Δgdh</i> mutant ‑N7H9 + 3 mM L-Glu cultures in fresh –N7H9 + 3 mM L-Glu. A1 and A2 were obtained from the first growth curve experiment, B1 and B2 from the second and C1-C3 from the third. Mean OD measurements with standard deviations presented in panels A-H and J and mean log₁₀(cfu/ml) with standard errors presented in panel I were calculated with growth curve data obtained from three independent experiments performed for each condition tested. In some instances error bars were smaller than the symbols used to depict the means. AS, ammonium sulphate.</p

Growth of <i>M. bovis</i> BCG in standard 7H9, 7H9 lacking nitrogen sources (‑N7H9) and 7H9 containing alanine as sole nitrogen source (‑N7H9 + 3 mM L-Ala).

<p>Mean OD measurements with standard deviations were calculated with growth curve data obtained from three independent experiments performed for each condition tested.</p

Replacement of the <i>gltBD</i> operon with a hygromycin cassette and gene deletion of <i>gdh</i>.

<p>A) Comparison of the wild-type <i>M. bovis</i> BCG and mutant <i>gltBD</i> regions. B) Southern blot analysis of wild-type <i>M. bovis</i> BCG (lane 1) and <i>ΔgltBD::hyg</i> (lane 2) with a probe that hybridises upstream of <i>gltB</i>. Southern blot analysis of wild-type <i>M. bovis</i> BCG (lane 3) and <i>ΔgltBD::hyg</i> (lane 4) with a probe that hybridises downstream of <i>gltD</i>. C) Comparison of the wild-type <i>M. bovis</i> BCG and mutant <i>gdh</i> regions. The GDH domain region is the sequence in <i>gdh</i> which aligned with a 98% query coverage (66% identity, 80% positives) in a blastp to the GDH domain sequence of the previously characterised Streptomyces <i>clavuligerus</i> L-180 GDH [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084452#B36" target="_blank">36</a>]. The NruI fragment spanning the GDH domain is deleted in the <i>Δgdh</i> chromosome. The probe which is complementary to fragments both upstream and downstream of the GDH domain does not hybridise across its full length with wild-type <i>M. bovis</i> BCG DNA, but does with <i>Δgdh</i> mutant DNA, as indicated in the figure. D) Southern blot analysis of wild-type <i>M. bovis</i> BCG (lane 1) and <i>Δgdh</i> (lane 2). <i>S</i>, SphI; <i>hyg</i>^<i>R</i>, hygromycin resitance cassette; <i>K</i>, KpnI; <i>N</i>, NruI; U, upstream; D, downstream.</p

Genes involved in nitrogen metabolism in the slow growing mycobacterium <i>M. bovis</i> BCG.

<p>Ammonia is assimilated in the production of L-glutamine and L-glutamate. Together, L-glutamine, L-glutamate, and L-aspartate act as precursors or nitrogen donors to most other nitrogenous compounds in the mycobacterium. The map was constructed from the combined PATRIC pathways for <i>M. bovis</i> BCG <i>str</i>. Pasteur 1743P2 nitrogen metabolism and alanine, aspartate and glutamate metabolism [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084452#B3" target="_blank">3</a>]. Genes were assigned to the EC numbers by PATRIC and/or Refseq and/or Legacy BRC.</p

Growth of <i>ΔgltBD</i> in 7H9 with different nitrogen sources.

<p>Growth of wt-BCG, the <i>ΔgltBD</i> mutant and the <i>ΔgltBD</i> complemented strain in (A) standard 7H9 containing approximately 4 mM ammonium sulphate (AS, (NH₄)₂SO₄) and 3 mM L-Glu, (B) 7H9 + 10 mM L-Glutamate, or (C) ‑N7H9 (nitrogen-depleted 7H9) + 4 mM AS. Growth of the <i>ΔgltBD</i> mutant in (D) ‑N7H9 + 4 mM AS supplemented with increasing concentrations of glutamate. Cultures for cfu/ml determinations were inoculated to OD₆₀₀ = 0.0005 (cfu/ml of approximately 10⁵). Log₁₀(cfu/ml) of <i>ΔgltBD</i> cultured in -N7H9 + 4 mM AS supplemented with 10 mM L-Glu was different from log₁₀(cfu/ml) of <i>ΔgltBD</i> cultured in -N7H9 + 4 mM AS at every time point after and including 3 days (P < 0.001). Log₁₀(cfu/ml) of <i>ΔgltBD</i> cultured in -N7H9 + 4 mM AS supplemented with 3 mM L-Glu was different from log₁₀(cfu/ml) of <i>ΔgltBD</i> cultured in -N7H9 + 4 mM AS at 3 days (p < 0.01) and every following time point (p < 0.001). Log₁₀(cfu/ml) of <i>ΔgltBD</i> cultured in -N7H9 + 4 mM AS supplemented with 0.3 mM L-Glu was different from log₁₀(cfu/ml) of <i>ΔgltBD</i> cultured in -N7H9 + 4 mM AS at 6 days (p < 0.01) and every following time point (p < 0.001). Growth of wt-BCG, the <i>ΔgltBD</i> mutant and the <i>ΔgltBD</i> complement strain in (E) –N7H9 + 3 mM L-Glu, (F) –N7H9 + 3 mM L-Asn, (G) ‑N7H9 + 3 mM L-Gln, (H) –N7H9 + 3 mM L-Asp, (I) unmodified –N7H9, or (J) 7H9 + 30 mM AS. Mean OD measurements with standard deviations presented in panels A-C and E-J and mean log₁₀(cfu/ml) with standard errors presented in panel D were calculated with growth curve data obtained from three independent experiments performed for each condition tested. In some instances error bars were smaller than the symbols used to depict the means. AS, ammonium sulphate.</p