Harald Gotten Wiker

The clinical course of tuberculosis (TB) infection, bacterial load and the morphology of lesions vary between pulmonary and extrapulmonary TB. Antigens expressed in abundance during infection could represent relevant antigens in the development of diagnostic tools, but little is known about the in vivo expression of various M. tuberculosis antigens in different clinical manifestations. The aim of this study was to study the differences in the presence of major secreted as well as somatic mycobacterial antigens in host tissues during advanced rapidly progressing and fatal pulmonary disease with mainly pneumonic infiltrates and high bacterial load, and to compare this to the presence of the same antigens in TB lymphadenitis cases, which is mainly chronic and self-limiting disease with organised granulomas and lower bacterial load. Human pulmonary (n = 3) and lymph node (n = 17) TB biopsies, and non-TB controls (n = 12) were studied. Ziehl-Neelsen stain, nested PCR 1S6110 and immunohistochemistry were performed. Major secreted (MPT32, MPT44, MPT46, MPT51, MPT53, MPT59, MPT63, and MPT64) and somatic mycobacterial antigens (Mce1A, Hsp65, and MPT57) were detected by using rabbit polyclonal antibodies. Plenty of bacilli were detectable with Ziehl-Neelsen stain in the lung biopsies while no bacilli were detected in the lymph node biopsies. All the cases were shown to be positive by PCR. Both secretory and somatic antigens were expressed in abundance in pulmonary infiltrates, while primarily somatic antigens were detected in the lymphadenitis cases. Of the secreted antigens, only MPT64 was consistently detected in both cases, indicating a preferential accumulation of this antigen within the inflammatory cells, even if the cells of the granuloma can efficiently restrict bacterial growth and clear away the secreted antigens. This study shows that major secreted mycobacterial antigens were found in high amounts in advanced pulmonary lesions without proper granuloma formation, while their level of staining was very low, or absent, in the lymph node TB lesions with organised granulomas and very low bacillary load, with one exception of MPT64, suggesting its role in the persistence of chronic infection. These findings have implication for development of new diagnostic tools.

Non-tuberculous mycobacteria (NTM) are different from Mycobacterium tuberculosis (MTB) both in their ubiquitous environmental distribution and in their reduced capacity to cause disease. While often neglected in favour of other infectious diseases, NTM may interfere with important aspects of TB control and management, namely the efficacy of new anti-tuberculosis (TB) vaccines; the immuno-diagnostic Tuberculin skin test (TST) and QuantiFERON TB Gold In Tube assay (QFTGIT); and immune biomarkers explored for their diagnostic and/or predictive potential. Our objective was therefore to explore host immune biomarkers in children who had NTM isolated from respiratory and/or gastric specimens. The present study was nested within a prospective cohort study of BCG-vaccinated neonates in Southern India. In this setting, immune biomarkers from peripheral blood were analyzed in 210 children aged RAB33A (pTGFβ1, IL-2 and IL-6 (all pRAB33A, TGFBR2 and IL-10 (all p<0.05) were differentially expressed in children with NTM present when compared to children that were culture negative for MTB and NTM (controls). Carriage of NTM may reduce the specificity of future diagnostic and predictive immune biomarkers relevant to TB management. Non-tuberculous mycobacteria (NTM) are a ubiquitous group of mycobacteria found in the environment. They are opportunistic pathogens causing human disease, especially in immunocompromised individuals. Differentiation between NTM infection and tuberculosis (TB) can be difficult. Data on incidence of NTM in TB endemic countries is limited due to resource intensive methods required for identification and a considerable workload due to other diseases. The present study was based on children investigated for TB and classified according to chest X-rays and mycobacterial culture reports. We explored host immune biomarkers which are potentially relevant to TB management, in children with confirmed NTM exposure. The findings from the present study suggest that NTM exposure modulates TB-relevant immune biomarkers in the host by eliciting some of the same immune responses as MTB infection. This is may be of importance when evaluating immunological correlates of protection in the setting of TB vaccine trials and potential TB diagnostic biomarkers.

High‐accuracy and high‐throughput proteomic methods have completely changed the way we can identify and characterize proteins. MS‐based proteomics can now provide a unique supplement to genomic data and add a new level of information to the interpretation of genomic sequences. Proteomics‐driven genome annotation has become especially relevant in microbiology where genomes are sequenced on a daily basis and limitations of an in silico driven annotation process are well recognized. In this review paper, we outline different strategies on how one can design a proteogenomic experiment, for example on genome‐sequenced (synonymous proteogenomics) versus unsequenced organisms (ortho‐proteogenomics) or with the aid of other “omic” data such as RNA‐seq. We touch upon many challenges that are encountered during a typical proteogenomic study, mostly concerning bioinformatics methods and downstream data analysis, but also related to creation and use of sequence databases. A large list of proteogenomic case studies of different microorganisms is provided to illustrate the mapping of MS/MS‐derived peptide spectra to genomic DNA sequences. These investigations have led to accurate determination of translational initiation sites, pointed out eventual read‐throughs or programmed frameshifts, detected signal peptide processing or other protein maturation events, removed questionable annotation assignments, and provided evidence for predicted hypothetical proteins.

Tuberculosis, the disease caused by Mycobacterium tuberculosis, remains a relevant public health issue. This is due mostly to the coepidemiology with HIV/AIDS, the appearance of multidrug‐resistant strains globally, and failure of BCG (bacillus Calmette–Guerin) vaccination to confer complete protection. This bacterium was one of the first to have its genome sequenced, yet over a decade after the release of the genomic information, the characterization of its phylogenetic tree and of different strain variants inside this species revealed that much is still needed to be done for a full understanding of the M. tuberculosis genome and proteome. Current methods using LC‐MS/MS and hybrid high‐resolution mass spectrometers can identify 2400–2800 proteins of the 4000 predicted genes in M. tuberculosis. In this article, we review relevant details of this bacterium's pathology and immunology, describing articles where proteomics helped the community to tackle some of the organism biology, from understanding strain diversity, cellular structure composition, immunogenicity, and host–pathogen interactions. Finally, we will discuss the challenges yet to be fulfilled in order to better characterize M. tuberculosis by proteomics.

Background: The potential causes for variation in virulence between distinct M. tuberculosis strains are still not fully known. However, differences in protein expression are probably an important factor. In this study we used a label-free quantitative proteomic approach to estimate differences in protein abundance between two closely related M. tuberculosis strains; the virulent H37Rv strain and its attenuated counterpart H37Ra. Results: We were able to identify more than 1700 proteins from both strains. As expected, the majority of the identified proteins had similar relative abundance in the two strains. However, 29 membrane-associated proteins were observed with a 5 or more fold difference in their relative abundance in one strain compared to the other. Of note, 19 membrane- and lipo-proteins had higher abundance in H37Rv, while another 10 proteins had a higher abundance in H37Ra. Interestingly, the possible protein-export membrane protein SecF (Rv2586c), and three ABC-transporter proteins (Rv0933, Rv1273c and Rv1819c) were among the more abundant proteins in M. tuberculosis H37Rv. Conclusion: Our data suggests that the bacterial secretion system and the transmembrane transport system may be important determinants of the ability of distinct M. tuberculosis strains to cause disease.

Background: Membrane- and membrane-associated proteins are important for the pathogenicity of bacteria. We have analysed the content of these proteins in virulentMycobacterium tuberculosisH37Rv using Triton X-114 detergent-phase separation for extraction of lipophilic proteins, followed by their identification with high resolution mass spectrometry.Results: In total, 1417 different proteins were identified.In silicoanalysis of the identified proteins revealed that 248 proteins had at least one predicted trans-membrane region. Also, 64 of the identified proteins were predicted lipoproteins, and 54 proteins were predicted as outer membrane proteins. Three-hundred-and-ninety-five of the observed proteins, including 91 integral membrane proteins were described for the first time. Comparison of abundance levels of the identified proteins was performed using the exponentially modified protein abundance index (emPAI) which takes into account the number of the observable peptides to the number of experimentally observed peptide ions for a given protein. The outcome showed that among the membrane-and membrane-associated proteins several proteins are present with high relative abundance. Further, a close examination of the lipoprotein LpqG (Rv3623) which is only detected in the membrane fractions ofM. tuberculosisbut not inM. bovis, revealed that the homologous gene inM. bovislack the signal peptide and lipobox motif, suggesting impaired export to the membrane.Conclusions: Altogether, we have identified a substantial proportion of membrane- and membrane-associated proteins ofM. tuberculosisH37Rv, compared the relative abundance of the identified proteins and also revealed subtle differences between the different members of theM. tuberculosiscomplex.

To investigate how the risk of active tuberculosis disease is influenced by time since original infection and to determine whether the risk of reactivation of tuberculosis increases or decreases with age.