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. 2014 Apr 22;9(4):e94939.
doi: 10.1371/journal.pone.0094939. eCollection 2014.

Transcriptional profiling of Mycobacterium tuberculosis replicating ex vivo in blood from HIV- and HIV+ subjects

Michelle B Ryndak  1 , Krishna K Singh  1 , Zhengyu Peng  2 , Susan Zolla-Pazner  3 , Hualin Li  4 , Lu Meng  2 , Suman Laal  3
Affiliations

Transcriptional profiling of Mycobacterium tuberculosis replicating ex vivo in blood from HIV- and HIV+ subjects

Michelle B Ryndak et al. PLoS One. .

Abstract

Hematogenous dissemination of Mycobacterium tuberculosis (M. tb) occurs during both primary and reactivated tuberculosis (TB). Although hematogenous dissemination occurs in non-HIV TB patients, in ∼80% of these patients, TB manifests exclusively as pulmonary disease. In contrast, extrapulmonary, disseminated, and/or miliary TB is seen in 60-70% of HIV-infected TB patients, suggesting that hematogenous dissemination is likely more common in HIV+ patients. To understand M. tb adaptation to the blood environment during bacteremia, we have studied the transcriptome of M. tb replicating in human whole blood. To investigate if M. tb discriminates between the hematogenous environments of immunocompetent and immunodeficient individuals, we compared the M. tb transcriptional profiles during replication in blood from HIV- and HIV+ donors. Our results demonstrate that M. tb survives and replicates in blood from both HIV- and HIV+ donors and enhances its virulence/pathogenic potential in the hematogenous environment. The M. tb blood-specific transcriptome reflects suppression of dormancy, induction of cell-wall remodeling, alteration in mode of iron acquisition, potential evasion of immune surveillance, and enhanced expression of important virulence factors that drive active M. tb infection and dissemination. These changes are accentuated during bacterial replication in blood from HIV+ patients. Furthermore, the expression of ESAT-6, which participates in dissemination of M. tb from the lungs, is upregulated in M. tb growing in blood, especially during growth in blood from HIV+ patients. Preliminary experiments also demonstrate that ESAT-6 promotes HIV replication in U1 cells. These studies provide evidence, for the first time, that during bacteremia, M. tb can adapt to the blood environment by modifying its transcriptome in a manner indicative of an enhanced-virulence phenotype that favors active infection. Additionally, transcriptional modifications in HIV+ blood may further accentuate M. tb virulence and drive both M. tb and HIV infection.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. M. tb replication in whole blood from HIV- and HIV+ subjects.
(A) Total (intra- and extra-cellular) M. tb CFU counts after 2 and 96 hr in blood from 10 HIV- donors or in blood from 15 HIV+ patients. (Wilcoxon Mann-Whitney two-tailed analysis; P values≤0.05 statistically significant) (B) M. tb CFU fold change between 2 hr and 96 hr in blood from HIV- donors and HIV+ patients (same as in panel A). (Mann-Whitney two-tailed analysis; P value<0.05 statistically significant).
Figure 2
Figure 2. Differentially expressed M. tb genes in whole blood from HIV- and HIV+ donors.
Heat map denoting upregulated genes in red and down-regulated genes in green in M. tb grown in blood from 6 HIV- donors (1-6) and 6 HIV+ patients (7-12) with dye flip (12 samples for each condition, HIV- and HIV+). Genes are listed as upregulated or down-regulated in blood from both HIV- and HIV+ donors or in HIV- donor blood only or HIV+ patient blood only. HIV+ blood sample codes (H#) are listed at the bottom for comparison with CD4+ T cell counts and viral loads in Table S1.
Figure 3
Figure 3. Differential M. tb gene expression in blood from HIV+ patients versus HIV- donors.
(A) Heat map of M. tb genes upregulated ≥2 fold during replication in blood from HIV+ patients (7–12) versus HIV- donors (1-6). (B) Heat map of M. tb genes down-regulated ≥2 fold during replication in blood from HIV+ patients (7-12) versus HIV- donors (1-6).
Figure 4
Figure 4. Percentages of genes within functional categories differentially expressed in blood from HIV- and/or HIV+ donors.
(A) Upregulated genes arranged by functional category and corresponding blood environments. (B) Down-regulated genes arranged by functional category and corresponding blood environments. (Functional categories as designated in the Tuberculist website http://tuberculist.epfl.ch/)
Figure 5
Figure 5. Specific categories of M. tb genes affected during growth in the blood environment.
(A) DosR regulon genes (B) Genes encoding heat shock proteins (8) and stimulators of heat shock proteins (2) (Total 10 genes assessed). (C) Genes involved in PDIM and PGL synthesis and transport. Venn diagrams indicate numbers of genes differentially expressed in blood from HIV- and/or HIV+ donors with common affected genes in the overlap. (Green indicates down-regulated. Red indicates upregulated.) Numbers in parentheses next to headings indicate the total number of genes in the designated category encoded in the M. tb genome. Tables below include the Hypergeometric Probability P-values from Fisher's Exact testing of differentially expressed genes represented in each category and each blood environment (HIV- or HIV+) (significant P-values in bold), as well as the ranges of expression fold change.
Figure 6
Figure 6. Upregulation of ESX loci and esat-6-like genes.
(A) Genes upregulated within ESX-1 locus. (B) Genes upregulated within ESX-5 locus. (C) Total genes encoding ESAT-6-like proteins upregulated in the M. tb genome. (D) Genes encoding ESAT-6-like proteins which are upregulated and located outside of ESX loci. Venn diagrams indicate numbers of genes upregulated in blood from HIV- and/or HIV+ donors. Genes that are upregulated in both environments are in the overlap. Numbers in parentheses next to headings indicate the total number of genes in the designated category encoded in the M. tb genome. Tables below include the Hypergeometric Probability P-values from Fisher's Exact testing of differentially expressed genes represented in each category and each blood environment (HIV- or HIV+) (significant P-values in bold), as well as the ranges of expression fold change. Note: Genes located within ESX-2, ESX-3, and ESX-4 loci were not differentially expressed in blood from either HIV- or HIV+ donors.
Figure 7
Figure 7. qRT-PCR validation of microarray results.
Log2 fold-changes in expression of seven selected genes in M. tb replicating in blood from HIV- donors (black dots) or from HIV+ patients (open black circles) at 96 hr compared to M. tb logarithmically growing in 7H9 broth media. (A) Microarray results from 6 biological replicates (6 different donors) per condition (blood from HIV- or HIV+ donors) with 2 technical replicates (Cy5/Cy3 dye swap) (total 12 values per condition are shown). (B) qRT-PCR results from M. tb replicating in blood from 6 different donors per condition (HIV- or HIV+) (total 6 values per condition are shown). Y-axis values (Log2 fold change) of ≥1 indicate upregulation and values ≤-1 indicate down-regulation. (For qRT-PCR, numbers of transcripts were normalized to copies of 16S rRNA.) P-values indicate the statistical difference between the average log2 fold-change of a gene in blood from HIV- subjects compared to HIV+ patient blood by Mann-Whitney two-tailed test.
Figure 8
Figure 8. Effect of M. tb on HIV-1 p24 production by U1 cells.
(A) Effect of gamma-irradiated, intact M. tb on p24 production by U1 cells over 5 days at increasing MOI (M. tb:cell). (B) Effect of M. tb subcellular fractions- cell wall, lysate, and culture filtrate proteins (CFP) at 20 ug/ml on U1 p24 production over 5 days. U1 culture medium alone represents the negative control in both assays. Shown are representatives of two or more experiments.
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