Secreted proteins of mycobacteria and their role during infection

Abstract

One-third of the world’s population is infected with the causative agent of tuberculosis, Mycobacterium tuberculosis. A fraction of infected individuals have an active form of the disease, while the remainder is able to control, but not eradicate the bacilli. The progression and outcome of the disease is dependent on precise interactions between the bacterium and its host, and the balance between the two organisms reflects thousands of years of co-evolution and adaptations. The host immune system recognises structural features of the bacilli, and immediately initiates responses upon pathogen entry. This does in turn lead to the development of highly specific immune responses that prime infected cells to become efficient mycobacterial killers. The bacterium on the other hand, produces molecules that modulate the responses in its own favour, and is therefore able to survive within the host cells for a prolonged time. In this thesis, our primary focus has been to investigate various aspects of the proteins produced by the bacterium. A broader knowledge in this field might contribute to the development of new vaccines, diagnostic markers, and drug targets, as well as providing increased understanding about the biology of this lethal pathogen. In paper I, we determined the best algorithm for predicting mycobacterial proteins that are secreted via the general secretory pathway, which is responsible for secretion of a majority of secreted proteins. Proteins processed by this system harbour an Nterminal signal peptide that is cleaved off as the protein is released on the exterior of the cell. The SignalP v3.0 hidden Markov model was found to be the best performing algorithm, and it identified the signal peptide of all proteins in a positive validation set, and did not generate any false positive results from a negative set. It also correctly identified the majority of the signal peptide cleavage sites. In paper II, we identified 30 proteins with predicted signal peptides that contrary to the prediction were observed to be uncleaved. These proteins were found to be enriched in the membrane fraction of the bacterium. A comparison of amino acid sequences showed these uncleaved signal peptides to be similar to peptides known to be cleaved, but with some variations in the amino acid composition, particularly in the -3 and -5 positions relative to the cleavage site. Some of the proteins had a transmembrane domain spanning the predicted cleavage site, and which might interfere with the signal peptide processing. A combination of features probably explains why most of these proteins are not processed by the signal peptidase. It is possible that these uncleaved signal peptides act as hydrophobic anchors for their respective proteins, and cause them to be retained in the membrane. In paper III, we studied a long term (up to 17 months) course of infection with two BCG strains in mice, and investigated the in vivo expression of secreted and somatic mycobacterial proteins. BCG Pasteur is phylogenetically more distant from the original BCG strain than BCG Russia. According to the general belief that serial passages of a laboratory strain causes successive attenuation, BCG Pasteur should be more attenuated. However, in our mouse model, BCG Pasteur was more persistent and induced greater pathology than BCG Russia. The increased virulence of BCG Pasteur might be related to a higher expression of proteins with antioxidant properties, which make the bacilli more able to resist killing mechanisms of the host cells. At 2 and 3 months after infection we investigated the expression of mycobacterial antigens by immunohistochemistry using 7 different polyclonal rabbit antibodies. The staining patterns revealed strong staining, particularly in macrophagedominant areas, but also in lymphocyte-dominant areas and in bronchial associated lymphoid tissues. In addition, we observed scattered cells with high antigen load in the normal lung parenchyma. This likely reflects the ability of the bacilli to migrate from established foci to more distant cells and remain unnoticed by the host’s danger signalling system preventing it to alert other immune cells of the spreading infection. At the time point investigated, the immunohistochemical analyses revealed great similarities in protein expression between the BCG strains. In paper IV, we investigated the M. tuberculosis-expression and localisation of secreted and somatic proteins in human lungs and lymph node tissues. We found a higher frequency of staining of the secreted proteins in loosely organised cell aggregates in the lungs, as compared to well-structured lymph node granulomas. In the latter, some major secreted proteins were only detected in the necrotic centres and not in the surrounding cells. Antibodies to the proteins of the antigen 85 complex gave generally low staining intensities, while anti-MPT64 produced strong staining of cells in all granulomas. Accumulation of proteins like MPT64 might eventually modulate immune responses to facilitate persistence of mycobacteria and pathogenesis of disease. Finally, staining with anti-Mce1A was found more frequently in organised granulomas, compared to regions without well-structured granulomas. This is in line with other studies that implicate a role for this mycobacterial protein in granuloma formation, and emphasises that both the host and the pathogen favour this structure.