Mycobacterium tuberculosis gene expression profiling within the context of protein networks.

As one of the world's most successful intracellular pathogens, Mycobacterium tuberculosis, the causative agent of human tuberculosis, is responsible for two to three million deaths annually. The pathogenicity of M. tuberculosis relies on its ability to survive and persist within host macrophage cells during infection. It is of central importance, therefore, to identify genes and pathways that are involved in the survival and persistence of M. tuberculosis within these cells. Utilizing genome-wide DNA arrays we have identified M. tuberculosis genes that are specifically induced during macrophage infection. To better understand the cellular context of these differentially expressed genes, we have also combined our array analyses with computational methods of protein network identification. Our combined approach reveals certain signatures of M. tuberculosis residing within macrophage cells, including the induction of genes involved in DNA damage repair, fatty acid degradation, iron metabolism, and cell wall metabolism

An improved strategy for selective and efficient enrichment of integral plasma membrane proteins of mycobacteria.

Mycobacterial plasma membrane proteins play essential roles in many cellular processes, yet their comprehensive proteomic profiling remains challenging. This is mainly due to obstacles related to their extraction and solubilization. To tackle this problem, we have developed a novel procedure to selectively enrich mycobacterial plasma membrane proteins based on alkaline sodium carbonate washing of crude membranes followed by Triton X-114 phase partitioning. The present study assesses the efficiency of this method by proteome analysis of plasma membrane proteins from Mycobacterium bovis BCG. Extracted proteins were separated in parallel by 1-D SDS-PAGE and 2-DE and then analyzed by LC-MS/MS and MALDI-MS/MS. Our study revealed 125 proteins, of which 54 contained 1-14 predicted transmembrane domains (TMD) including nine novel proteins. The 1-D SDS-PAGE-based proteome analysis identified 81 proteins, of which 49 (60.5%) harbored TMD. This approach also revealed many hydrophobic membrane-associated/periplasmic proteins lacking TMD, but only few soluble proteins. The identified proteins were characterized with regard to biological functions and physicochemical properties providing further evidence for the high efficiency of the prefractionation method described herein

Complementary analysis of the Mycobacterium tuberculosis proteome by two-dimensional electrophoresis and isotope-coded affinity tag technology.

Classical proteomics combined two-dimensional gel electrophoresis (2-DE) for the separation and quantification of proteins in a complex mixture with mass spectrometric identification of selected proteins. More recently, the combination of liquid chromatography (LC), stable isotope tagging, and tandem mass spectrometry (MS/MS) has emerged as an alternative quantitative proteomics technology. We have analyzed the proteome of Mycobacterium tuberculosis, a major human pathogen comprising about 4,000 genes, by (i) 2-DE and mass spectrometry (MS) and by (ii) the isotope-coded affinity tag (ICAT) reagent method and MS/MS. The data obtained by either technology were compared with respect to their selectivity for certain protein types and classes and with respect to the accuracy of quantification. Initial datasets of 60,000 peptide MS/MS spectra and 1,800 spots for the ICAT-LC/MS and 2-DE/MS methods, respectively, were reduced to 280 and 108 conclusively identified and quantified proteins, respectively. ICAT-LC/MS showed a clear bias for high M(r) proteins and was complemented by the 2-DE/MS method, which showed a preference for low M(r) proteins and also identified cysteine-free proteins that were transparent to the ICAT-LC/MS method. Relative quantification between two strains of the M. tuberculosis complex also revealed that the two technologies provide complementary quantitative information; whereas the ICAT-LC/MS method quantifies the sum of the protein species of one gene product, the 2-DE/MS method quantifies at the level of resolved protein species, including post-translationally modified and processed polypeptides. Our data indicate that different proteomic technologies applied to the same sample provide complementary types of information that contribute to a more complete understanding of the biological system studied

Natural Transmission of <em>Plasmodium berghei</em> Exacerbates Chronic Tuberculosis in an Experimental Co-Infection Model

<div><p>Human populations are rarely exposed to one pathogen alone. Particularly in high incidence regions such as sub-Saharan Africa, concurrent infections with more than one pathogen represent a widely underappreciated public health problem. Two of the world’s most notorious killers, malaria and tuberculosis, are co-endemic in impoverished populations in the tropics. However, interactions between both infections in a co-infected individual have not been studied in detail. Both pathogens have a major impact on the lung as the prime target organ for aerogenic <em>Mycobacterium tuberculosis</em> and the site for one of the main complications in severe malaria, malaria-associated acute respiratory distress syndrome (MA-ARDS). In order to study the ramifications caused by both infections within the same host we established an experimental mouse model of co-infection between <em>Mycobacterium tuberculosis</em> and <em>Plasmodium berghei</em> NK65, a recently described model for MA-ARDS. Our study provides evidence that malaria-induced immune responses impair host resistance to <em>Mycobacterium tuberculosis</em>. Using the natural routes of infection, we observed that co-infection exacerbated chronic tuberculosis while rendering mice less refractory to <em>Plasmodium.</em> Co-infected animals presented with enhanced inflammatory immune responses as reflected by exacerbated leukocyte infiltrates, tissue pathology and hypercytokinemia accompanied by altered T-cell responses. Our results - demonstrating striking changes in the immune regulation by co-infection with <em>Plasmodium</em> and <em>Mycobacterium</em> - are highly relevant for the medical management of both infections in humans.</p> </div

<i>Pb</i>NK65 co-infection alters T cell responses in <i>M. tuberculosis</i> infected mice.

<p>C57BL/6 mice were infected by aerosol with <i>M. tuberculosis</i> H37Rv (100 CFU/lung) and challenged with <i>Pb</i>NK65 sporozoites by mosquito bite 40 days later. Control mice were infected with <i>M. tuberculosis</i> or <i>Pb</i>NK65 alone, respectively. A) 12 days upon <i>Pb</i>NK65 infection, lungs, spleens and livers were analyzed for the presence of CD44 positive CD4 and CD8 effector T cells by flow cytometry. B) Whole lung and spleen lysates and purified liver lymphocytes were re-stimulated <i>in vitro</i> with PMA/Iono (50 ng/ml, respectively) and analyzed by flow cytometry for the presence of IL-2, TNF-α, IL-10 or IFN-γ producing CD4 and CD8 T cells. Results are shown as means ± SD (n = 3–5). Data from one out of two independent experiments are shown. Statistical analysis was performed by ANOVA (*p<0.05; **p<0.01; *** p<0.001).</p

Malaria co-infection increases inflammatory tissue responses in <i>M. tuberculosis</i> infected lungs.

<p>C57BL/6 mice were aerosol infected with <i>M. tuberculosis</i> H37Rv (100 CFU/lung) and 40 days later challenged with <i>Pb</i>NK65 sporozoites by mosquito bite. Control mice were infected with <i>M. tuberculosis</i> or <i>Pb</i>NK65 alone, respectively. A–C) Representative H&E stains of lungs 13 days after co-infection. Note, that <i>Pb</i>NK65 co-infection exacerbated tissue pathology compared to lungs of mice infected with <i>M. tuberculosis</i> alone (v  =  vessel, b  =  bronchus; asterisks indicate hemozoin loaded cells; arrow: neutrophils; arrowhead: monocytes). D) Histopathological scores from co-infected, <i>Pb</i>NK65 or <i>M. tuberculosis</i> infected lungs are shown. Pathology was most severe in co-infected animals, with the total score being significantly increased compared to <i>M. tuberculosis</i> infected lungs (n = 4 for co-infected and <i>Pb</i>NK65 infected; n = 5 for <i>M. tuberculosis</i> infected). E) Lung weights 12 days after co-infection. F–G) Lung leukocytes were analyzed for surface expression of CD11b and GR-1. Results are shown as means ± SD (n = 3–5). Results from one representative experiment out of two independent ones are shown. Statistical analysis was performed by ANOVA (*p<0.05; **p<0.01).</p

<i>Pb</i>NK65 associated liver damage is reduced in co-infected mice.

<p>C57BL/6 mice were aerosol infected with <i>M. tuberculosis</i> H37Rv (100 CFU/lung) and 40 days later challenged with <i>Pb</i>NK65 sporozoites by mosquito bite. Control mice were infected with <i>M. tuberculosis</i> or <i>Pb</i>NK65 alone, respectively. Representative H&E stains of liver sections 13 days after co-infection. Note, that <i>Pb</i>NK65 infection caused periportal inflammation (B; arrows) and tissue necrosis (B; arrowhead) which was reduced in livers of co-infected animals (A).</p

Comparative proteome analysis of culture supernatant proteins from virulent Mycobacterium tuberculosis H37Rv and attenuated M-bovis BCG Copenhagen

A comprehensive analysis of culture supernatant (CSN) proteins of Mycobacterium tuberculosis H37Rv was accomplished by combination of two-dimensional electrophoresis (2-DE), mass spectrometry, and N-terminal sequencing by Edman degradation. Analytical 2-DE gels resolved approximately 1250 protein spots from CSN of M. tuberculosis H37Rv, 381 of which were identified by mass spectrometry and/or Edman degradation. This study revealed 137 different proteins, 42 of which had previously been described as secreted. Comparative proteome analysis of CSN from virulent M. tuberculosis H37Rv and attenuated Mycobacterium bovis BCG Copenhagen identified 39 M. tuberculosis-specific spots containing 27 different proteins, representing candidate antigens for novel vaccines and diagnostics in tuberculosis. These included five proteins encoded by open reading frames absent from M. bovis BCG, e.g., early secretory antigen target (Esat6), as well as 22 novel differential proteins, such as acetyl-CoA C-acetyltransferase (Rv0243) and two putative Esat6-like proteins (Rv1198, Rv1793)

A new in vivo model to test anti-tuberculosis drugs using fluorescence imaging.

OBJECTIVES: The current method for testing new drugs against tuberculosis in vivo is the enumeration of bacteria in organs by cfu assay. Owing to the slow growth rate of Mycobacterium tuberculosis (Mtb), these assays can take months to complete. Our aim was to develop a more efficient, fluorescence-based imaging assay to test new antibiotics in a mouse model using Mtb reporter strains. METHODS: A commercial IVIS Kinetic® system and a custom-built laser scanning system with fluorescence molecular tomography (FMT) capability were used to detect fluorescent Mtb in living mice and lungs ex vivo. The resulting images were analysed and the fluorescence was correlated with data from cfu assays. RESULTS: We have shown that fluorescent Mtb can be visualized in the lungs of living mice at a detection limit of ∼8 × 10⁷ cfu/lung, whilst in lungs ex vivo a detection limit of ∼2 × 10⁵ cfu/lung was found. These numbers were comparable between the two imaging systems. Ex vivo lung fluorescence correlated to numbers of bacteria in tissue, and the effect of treatment of mice with the antibiotic moxifloxacin could be visualized and quantified after only 9 days through fluorescence measurements, and was confirmed by cfu assays. CONCLUSIONS: We have developed a new and efficient method for anti-tuberculosis drug testing in vivo, based on fluorescent Mtb reporter strains. Using this method instead of, or together with, cfu assays will reduce the time required to assess the preclinical efficacy of new drugs in animal models and enhance the progress of these candidates into clinical trials against human tuberculosis