Phylogenetic profile of bacterial genera for uninfected and <i>M. tuberculosis</i> H37Rv infected mice.

<p>Stacked bar charts for uninfected and H37Rv-infected mice of the 16 main genera identified based on ≥1% abundance present in at least two samples. Unclassified sequences are not shown. The black colored bar along x-axis indicates the five uninfected mice, while the red colored bar indicates mice infected with H37Rv.</p

Community structure of individual <i>M. tuberculosis</i> CDC1551 infected mice over time.

<p>(<b>A</b>) Survival time in days post-infection for each mouse. (<b>B</b>) Phylogenetic profile of bacterial genera. Stacked bar charts in chronological order for each mouse of the 18 main genera identified based on ≥1% abundance present in at least two samples. Unclassified sequences are not shown. Black colored bars along x-axis indicate samples taken prior to infection, while red colored bars indicate post-infection. Each group represents an individual mouse, followed to death. The mice are represented sequentially, with mouse 1 on the left, and mouse 5 on the right. (<b>C</b>) Community diversity in each sample as measured by the Shannon diversity index, plotted against the percent survival time.</p

Differentially abundant OTUs identified between pre-infection and post-infection.

<p>OTUs are ordered by consensus taxonomic classification, with OTUs scaled by relative abundances for each row ranging from low relative abundance (blue) to high relative abundance (red).</p

Differentially abundant OTUs identified between uninfected and <i>M. tuberculosis</i> H37Rv infected mice.

<p>OTUs are ordered by consensus taxonomic classification, with OTUs scaled by relative abundances for each row ranging from low relative abundance (blue) to high relative abundance (red).</p

Aerosol <i>Mycobacterium tuberculosis</i> Infection Causes Rapid Loss of Diversity in Gut Microbiota

<div><p><i>Mycobacterium tuberculosis</i> is an important human pathogen, and yet diagnosis remains challenging. Little research has focused on the impact of <i>M. tuberculosis</i> on the gut microbiota, despite the significant immunological and homeostatic functions of the gastrointestinal tract. To determine the effect of <i>M. tuberculosis</i> infection on the gut microbiota, we followed mice from <i>M. tuberculosis</i> aerosol infection until death, using 16S rRNA sequencing. We saw a rapid change in the gut microbiota in response to infection, with all mice showing a loss and then recovery of microbial community diversity, and found that pre-infection samples clustered separately from post-infection samples, using ecological beta-diversity measures. The effect on the fecal microbiota was observed as rapidly as six days following lung infection. Analysis of additional mice infected by a different <i>M. tuberculosis</i> strain corroborated these results, together demonstrating that the mouse gut microbiota significantly changes with <i>M. tuberculosis</i> infection.</p></div

Gut microbiota composition of <i>M. tuberculosis</i> H37Rv infected mice is significantly different from uninfected mice.

<p>(<b>A</b>) Unweighted and (<b>B</b>) weighted Unifrac measures of beta-diversity visualized using Principle Coordinate Analysis (PCoA) for the comparison of H37Rv-infected mice to uninfected mice at a single time point. Blue dots indicate samples collected pre-infection. Red dots indicate samples collected post-infection. Variance for first two component axes is shown as percent of total variance. In both unweighted and weighted Unifrac measures, there was a statistically significant difference (AMOVA p≤0.005). (<b>C</b>) Network analysis of OTUs partitioned among samples, using a five sequence cutoff, and colored by phylum.</p

Composition of the gut microbiota significantly changes with <i>M. tuberculosis</i> CDC1551 infection.

<p>(<b>A</b>) Unweighted and (<b>B</b>) weighted Unifrac measures of beta-diversity visualized using Principle Coordinate Analysis (PCoA) following individual mice over time with <i>M. tuberculosis</i> CDC1551 infection. Blue dots indicate samples collected pre-infection. Red dots indicate samples collected post-infection. Variance for first two component axes is shown as percent of total variance. An analysis of molecular variance (AMOVA) was performed to test whether the separation of uninfected and TB-infected samples was statistically significant. In both unweighted and weighted Unifrac measures, there was a statistically significant difference (p<0.001). (<b>C</b>) Network analysis of OTUs partitioned among samples, using a five sequence cutoff, and colored by phylum.</p

Immunogenic properties of the Rv2190c protein.

<p>N-terminal truncated Rv2190c was purified and analyzed for immunogenic properties by Dot-Blot ELISA against various sera. <b>A.</b> Immunogenic response in the human serum of an individual with latent TB (PPD-Pos.) (left panel) as opposed to a negligible response in a PPD negative human serum (right panel). <b>B.</b> Immunogenic response in serum from an <i>M. bovis</i> infected rabbit (left panel) and weak responses from sera raised against WhiB6 protein (middle panel) or pre-bleeds (right panel). i) 2ng of Rv2190c and ii) 2ng of BSA.</p

The <i>M. tuberculosis Rv2190c</i> mutant is attenuated <i>in vivo</i>.

<p><b>A. </b><i>M. tuberculosis</i> aerosol implantation in the lungs of BALB/c mice for the <i>Rv2190c</i> mutant, complement and WT (CDC1551) strains for the time-to-death experiment, determined day 1 post-infection. <b>B.</b> Survival curves for the time-to-death experiment. <b>C–D.</b> Lung (C) and spleen (D) CFU counts for BALB/c mice infected via aerosol with ∼3.4 log₁₀CFUs of WT, <i>Rv2190c</i> mutant and complement <i>M. tuberculosis</i> strains. <b>E.</b> Gross pathology of infected mouse lungs at 112 days post-infection.</p

<i>Rv2190c</i> expression peaks at log phase and is involved in the response to cell wall disruption.

<p><b>A.</b> Fold change of <i>Rv2190c</i> expression (relative to level at the start of the growth curve) over increasing cell density in 7H9 broth in wild-type <i>M. tuberculosis</i> CDC1551, normalized to <i>sigA</i> expression. pc: post-clumping. <b>B.</b> Fold-change of <i>Rv2190c</i> expression after exposure to oxidative (cumene), disulfide (diamide) and detergent (SDS) stress conditions, relative to expression levels prior to addition of stressors and normalized to <i>sigA</i>. <b>C.</b> Survival of WT, the <i>Rv2190c</i> mutant and complement <i>M. tuberculosis</i> strains following 24 hours of lysozyme exposure. <b>D.</b> PDIMs for <i>M. tuberculosis</i> WT and Rv2190c mutant strains analyzed by thin layer chromatography using hexane∶diethyl ether∶acetic acid solvent. <b>E.</b> PDIMs analyzed using petroleum ether∶diethyl ether solvent.</p