Portrait of a pathogen: the Mycobacterium tuberculosis proteome in vivo

Abstract

Background: Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a facultative intracellular pathogen that can persist within the host. The bacteria are thought to be in a state of reduced replication and metabolism as part of the chronic lung infection. Many in vitro studies have dissected the hypothesized environment within the infected lung, defining the bacterial response to pH, starvation and hypoxia. While these experiments have afforded great insight, the picture remains incomplete. The only way to study the combined effects of these environmental factors and the mycobacterial response is to study the bacterial response in vivo.

Methodology/principal findings: We used the guinea pig model of tuberculosis to examine the bacterial proteome during the early and chronic stages of disease. Lungs were harvested thirty and ninety days after aerosol challenge with Mtb, and analyzed by liquid chromatography-mass spectrometry. To date, in vivo proteomics of the tubercle bacillus has not been described and this work has generated the first large-scale shotgun proteomic data set, comprising over 500 unique protein identifications. Cell wall and cell wall processes, and intermediary metabolism and respiration were the two major functional classes of proteins represented in the infected lung. These classes of proteins displayed the greatest heterogeneity indicating important biological processes for establishment of a productive bacterial infection and its persistence. Proteins necessary for adaptation throughout infection, such as nitrate/nitrite reduction were found at both time points. The PE-PPE protein class, while not well characterized, represented the third most abundant category and showed the most consistent expression during the infection.

Conclusions/significance: Cumulatively, the results of this work may provide the basis for rational drug design - identifying numerous Mtb proteins, from essential kinases to products involved in metal regulation and cell wall remodeling, all present throughout the course of infection.

Figure 1. Representative photomicrographs of A) day 30, B) day 90 post-infection guinea pig lungs, and C) uninfected guinea pig lungs.

Guinea pigs were aerosolized with a low dose infection of virulent Mtb (H37Rv). Tissues were stained (H&E) to demonstrate differences in granulomas based on size (primary granuloma, marked with the letter P) and the absence (secondary granuloma, marked with the letter S) of a necrotic core. Bars are 200 µm.

Figure 2. The percent of novel protein identifications taper after sequential injections of MS.

Illustration showing the number of proteins identified during 10 replicates (x-axis). The circles signify 50 ng injections of the same WCL digest peptide mixture (standard deviation = 13.1). Squares represent the additive effect of protein identifications after each replicate after combining the Sequest and Mascot result files in Scaffold (left y-axis). The triangles depict the percentage (right y-axis) of unique protein identifications gained per additional replicate.

Figure 3. Venn diagram depicting the breakdown of proteins identified at each time-point in this study.

While a similar amount of proteins were identified at each of the two infection time points, the overlap is only 28% and 27% of the total identification in the 30 and 90-day samples, respectively.

Figure 4

A) Comparison of functional categories. The 30-day (blue), 90-day (red) and total identifications (green) were broken down by functional categories. Categories codes are assigned by TubercuList (http://genolist.pasteur.fr/TubercuList/help/function-codes.html): 0 = virulence, detoxification & adaptation; 1 = lipid metabolism; 2 = information pathways; 3 = cell wall & cell processes; 5 = insertion sequences & phages; 6 = PE/PPE; 7 = intermediary metabolism & respiration; 8 = unknown; 9 = regulatory proteins; 10 = conserved hypotheticals and 16 = conserved hypotheticals with an ortholog in M. bovis. B) Venn diagram showing the overlap between the 30-day and 90-day infection in category 3 and C) category 7.

Figure 5. Changes over the course of infection of representative pathways from categories 3 & 7.

Each bar in the graph corresponds to the normalized spectral count for each protein within the same category. Each color signifies the infection time-point: blue = 30-day, red = 90-day and green = proteins found at both time-points, of which the area below the black line is the 30-day count and above equals the 90-day count. To the right of each category is the percent breakdown of the total spectral counts in the 30 versus 90-day samples.