Hypoxia Sensing and Persistence Genes Are Expressed during the Intragranulomatous Survival of Mycobacterium tuberculosis

Abstract

Although it is accepted that the environment within the granuloma profoundly affects Mycobacterium tuberculosis (Mtb) and infection outcome, our ability to understand Mtb gene expression in these niches has been limited. We determined intragranulomatous gene expression in human-like lung lesions derived from nonhuman primates with both active tuberculosis (ATB) and latent TB infection (LTBI). We employed a non-laser-based approach to microdissect individual lung lesions and interrogate the global transcriptome of Mtb within granulomas. Mtb genes expressed in classical granulomas with central, caseous necrosis, as well as within the caseum itself, were identified and compared with other Mtb lesions in animals with ATB (n = 7) or LTBI (n = 7). Results were validated using both an oligonucleotide approach and RT-PCR on macaque samples and by using human TB samples. We detected approximately 2,900 and 1,850 statistically significant genes in ATB and LTBI lesions, respectively (linear models for microarray analysis, Bonferroni corrected, P < 0.05). Of these genes, the expression of approximately 1,300 (ATB) and 900 (LTBI) was positively induced. We identified the induction of key regulons and compared our results to genes previously determined to be required for Mtb growth. Our results indicate pathways that Mtb uses to ensure its survival in a highly stressful environment in vivo. A large number of genes is commonly expressed in granulomas with ATB and LTBI. In addition, the enhanced expression of the dormancy survival regulon was a key feature of lesions in animals with LTBI, stressing its importance in the persistence of Mtb during the chronic phase of infection.

Keywords: Mycobacterium tuberculosis; granuloma; hypoxia; macaque; microdissection.

Figure 1.

(A and B) Representative hematoxylin and eosin (H&E) image of active tuberculosis (ATB; DF30) lung. (A) A ×25 magnification image of two coalescing granulomata, with extensive intervening and surrounding regions of inflammation. We termed this presentation as “representative pathology,” which includes classical granulomata and associated caseum. Arrows define the two coalescing lung granulomas. (B) A ×50 magnification image of an animal with ATB. (C and D) Representative H&E image of latent tuberculosis infection (LTBI) (FJ05) lung. (C) A ×25 magnification image of a solitary granuloma surrounded by regions of normal lung. (D) A ×50 magnification image of the same granuloma clearly describes the presence of a central necrotic region (the caseum). We termed such lesions as “classical granulomata.” (A,C, and D) Asterisks correspond to necrotic-hypoxic centers within granulomas. (C) The arrowheads define the boundary between the necrotic center and the cellular layer to the outside. (D) The arrows define the boundary between the granuloma and the normal lung as well as between the myeloid and the lymphocytic layers. Scale bars: 500 μm.

Figure 2.

Clinical correlates of disease state and associated lung bacterial burdens and pathology. (A) Two groups of seven macaques were classified with either ATB (red) or LTBI (blue). Peak serum C-reactive protein (CRP; mg/ml), (B) changes in body weight, (C) Mycobacterium tuberculosis (Mtb) bacterial burden per gram of lung tissue after killing, (D) percentage lung affected by tuberculosis pathology at necropsy and over course of infection, representative confocal staining of a section of lung with anti-Mtb antibody for an animal with LTBI (E) and with ATB (F). Mtb, red; To-Pro-3 iodide, green; quantification of Mtb-positive signal over area (mm²) (G); representative chromogen staining of a section of lung with anti-Mtb antibody for an animal with LTBI (H) and with ATB (I). Scale bar in panel H is 10 μm, and scale bar in panel I is 5 μm. *P < 0.05, **P < 0.01 and ***P < 0.001 using unpaired Student’s t test.

Figure 3.

Transcriptome profiles. Statistically significant genes whose fold change expression is positively induced within nonhuman primate (NHP) lung in (A) ATB (a) classical granuloma, (b) caseum of classical granuloma, (c) representative pathology; and (B) LTBI (a) classical granuloma, (b) caseum of classical granuloma. (C) Corresponding genetic similarity among genes with induced expression in (a) all ATB granulomatous pathology, (b) all LTBI granulomatous pathology and (ab) genes with induced expression in all NHP-derived samples. P < 0.05 using linear models for microarray analysis and Bonferroni correction.

Figure 4.

Expression of Mtb dormancy survival regulon (dosR regulon) and detection of hypoxia in ATB and LTBI lung granulomas. (A) Comparison of genes within the dosR regulon with induced expression in all NHPs with either LTBI or ATB in each granuloma sample subset. For all heat maps included here, genes within the regulon or gene family of interest were included based upon published association and statistically significant positively induced expression within at least one granuloma category. The least induction is seen in ATB representative pathology, and the most in LTBI. (B) Confocal image of pimonidazole hydrochloride (PIMO) conjugated with Daylight-546 (red) with To-Pro-3 iodide staining all nuclei (green). Signal in a representative ATB animal was much less than signal in a representative LTBI animal (C). Scale bars: 100 μm. In addition, PIMO signal within the lung as a percentage of total microscopic field was significantly greater in LTBI (blue squares) than ATB (red circles) (Student’s t test, **P < 0.005) (D). Finally, heat map of genes associated with host hypoxic response showed greater response during LTBI as compared with ATB within NHP granulomas (E).

Figure 5.

Mechanisms of persistence and validation. Comparison of genes within the (A) proline-glutamate/proline-proline-glutamate gene family, (B) toxin–antitoxin complexes, and (C) sigma factor family with induced expression in all NHPs with either LTBI or ATB in each granuloma sample subset.

Figure 6.

Real-time RT-PCR to validate macaque Mtb transcriptome results. Using cDNA derived from in vitro, log-phase–grown Mtb, and using the 16S gene as an internal reference, the fold changes of expression of vapB21 (A), sigE (B), sigH (C), and dosR (D) was assessed in classical granulomata derived from animals with ATB (red circles) or LTBI (blue squares) (Student’s t test, **P < 0.005). ns, not significant.