Whole transcriptomic and proteomic analyses of an isogenic <i>M</i>. <i>tuberculosis</i> clinical strain with a naturally occurring 15 Kb genomic deletion

<div><p>Tuberculosis remains one of the most difficult to control infectious diseases in the world. Many different factors contribute to the complexity of this disease. These include the ability of the host to control the infection which may directly relate to nutritional status, presence of co-morbidities and genetic predisposition. Pathogen factors, in particular the ability of different <i>Mycobacterium tuberculosis</i> strains to respond to the harsh environment of the host granuloma, which includes low oxygen and nutrient availability and the presence of damaging radical oxygen and nitrogen species, also play an important role in the success of different strains to cause disease. In this study we evaluated the impact of a naturally occurring 12 gene 15 Kb genomic deletion on the physiology and virulence of <i>M</i>. <i>tuberculosis</i>. The strains denominated ON-A WT (wild type) and ON-A NM (natural mutant) were isolated from a previously reported TB outbreak in an inner city under-housed population in Toronto, Canada. Here we subjected these isogenic strains to transcriptomic (via RNA-seq) and proteomic analyses and identified several gene clusters with differential expression in the natural mutant, including the DosR regulon and the molybdenum cofactor biosynthesis genes, both of which were found in lower abundance in the natural mutant. We also demonstrated lesser virulence of the natural mutant in the guinea pig animal model. Overall, our findings suggest that the ON-A natural mutant is less fit to cause disease, but nevertheless has the potential to cause extended transmission in at-risk populations.</p></div

Differentially expressed genes from the DevR regulon.

<p>Differentially expressed genes from the DevR regulon.</p

Differentially expressed genes in the molybdenum cofactor biosynthesis loci 1.

<p>Organization of genes involved in MoCo biosynthesis in red. Bars represent gene expression fold change between ON-A WT in relation to ON-A NM. *Genes with statistically significant values (p-value <0.01).</p

Survival analysis of guinea pigs infected with ON-A NM and ON-A WT.

<p>Thirty five guinea pigs per strain were infected with ON-A NM (Closed squares) or ON-A WT (closed circles). Log-rank test indicates the two strains differ in their ability to cause rapid disease (p-value <0.05).</p

Lung pathology and CFUs of infected guinea pigs.

<p>(A) Lung pathology scores of lung of guinea pigs infected with ON-A WT (black bars) and ON-A NM (grey bars. (B) CFUs in the lung of guinea pigs infected with ON-A WT (black circles) and ON-A NM (red squares). Each point is represented by 5 animals per strain. The 90 day post infection point includes animals euthanized at days 78 (n = 2) and 108 (n = 3) for the ON-A NM group and days 63 (n = 1), 73 (n = 1), 82 (n = 1) and 96 (n = 2) for the ON-A WT group. * Statistically significant difference (p-value < 0.05).</p

Comparison of Mean Days to Death of various treatment groups of guinea pigs.

<p>Mean Days to Death (MDD) was determined by dividing the summed total number of surviving days of each treatment group with the number of animals/group. Results are expressed as means±SEM of eight animals/group. Note: MDD for BCG/Ad groups may have been underestimated as 40–60% of animals in these treatment groups were still surviving at the time study termination (74 weeks). **p≤0.01, *p≤0.05 compared to saline control or between BCG and BCG/Ad i.n.</p

Kaplan-Meier curves of percent survival of <i>M.tb</i>-infected guinea pigs over the course of 74 weeks post-challenge.

<p>Groups of guinea pigs (eight animals per group) were treated as depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005856#pone-0005856-g001" target="_blank">Fig. 1B</a> and their survival rate was determined on a weekly basis up to 74 weeks when the entire study was terminated. p≤0.01 BCG, BCG/Ad i.n and BCG/Ad i.m compared to saline control; p≤0.05 BCG vs. BCG/Ad i.n; p≤0.1 BCG vs. BCG/Ad i.m.</p

Experimental regimens used for both short-term and long-term guinea pig studies.

<p>Experimental regimens used for both short-term and long-term guinea pig studies.</p

Dynamics and comparison of cytokine levels in mouse lung homogenates.

<p>A. Comparison of pro-inflammatory cytokines levels throughout the infection with pairs of clinical and laboratory clonal <i>Mtb</i> strains (t-test, *p<0.05). Limit of detection (LD) for IFN-γ: 0.5pg/mL, TNF-α: 0.9 pg/mL, and IL-6: 1.4 pg/mL.) Pair comparison between mice infected B). IL-10 (LD: 16.8 pg/mL) and IL-2 (LD: 0.1 pg/mL) levels in mice infected with laboratory and clinical <i>Mtb</i> pairs. Bars represent the mean values of cytokine concentration for five mice and the error bars represent the standard deviation.</p

AhpC leves from soluble cellular fractions of <i>Mtb</i> cultures.

<p>Western blot using anti-AhpC_<i>Mtb</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166807#pone.0166807.ref017" target="_blank">17</a>] from <b>c</b>ulture supernatants and cytosolic fraction of the four strains used in the study grown in GAS media (1 and 2 represent the two culture replicates of each strain used). CFP of H37Rv from BEI was used as a positive control. *indicates the lane for the ladder.</p