PE_PGRS proteins of Mycobacterium tuberculosis: A specialized molecular task force at the forefront of host–pathogen interaction

To the PE_PGRS protein subfamily belongs a group of surface-exposed mycobacterial antigens that in Mycobacterium tuberculosis (Mtb) H37Rv accounts to more than 65 genes, 51 of which are thought to express a functional protein. PE_PGRS proteins share a conserved structural architecture with three main domains: the N-terminal PE domain; the PGRS domain, that can vary in sequence and size and is characterized by the presence of multiple GGA-GGX amino acid repeats; the highly conserved sequence containing the GRPLI motif that links the PE and PGRS domains; the unique C-terminus end that can vary in size from few to up to ≈ 300 amino acids. pe_pgrs genes emerged in slow-growing mycobacteria and expanded and diversified in MTBC and few other pathogenic mycobacteria. Interestingly, despite sequence homology and apparent redundancy, PE_PGRS proteins seem to have evolved a peculiar function. In this review, we summarize the actual knowledge on this elusive protein family in terms of evolution, structure, and function, focusing on the role of PE_PGRS in TB pathogenesis. We provide an original hypothesis on the role of the PE domain and propose a structural model for the polymorphic PGRS domain that might explain how so similar proteins can have different physiological functions

Mapping and characterization of G-quadruplexes in Mycobacterium tuberculosis gene promoter regions

Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), one of the top 10 causes of death worldwide in 2015. The recent emergence of strains resistant to all current drugs urges the development of compounds with new mechanisms of action. G-quadruplexes are nucleic acids secondary structures that may form in G-rich regions to epigenetically regulate cellular functions. Here we implemented a computational tool to scan the presence of putative G-quadruplex forming sequences in the genome of Mycobacterium tuberculosis and analyse their association to transcription start sites. We found that the most stable G-quadruplexes were in the promoter region of genes belonging to definite functional categories. Actual G-quadruplex folding of four selected sequences was assessed by biophysical and biomolecular techniques: all molecules formed stable G-quadruplexes, which were further stabilized by two G-quadruplex ligands. These compounds inhibited Mycobacterium tuberculosis growth with minimal inhibitory concentrations in the low micromolar range. These data support formation of Mycobacterium tuberculosis G-quadruplexes in vivo and their potential regulation of gene transcription, and prompt the use of G4 ligands to develop original antitubercular agents

Improved protection in guinea pigs after vaccination with a recombinant BCG expressing MPT64 on its surface

Abstract The lack of an efficient vaccine against tuberculosis is still one of the major problems threatening global human health. In previous work we showed that expression of the protective antigen MPT64 on the surface of Mycobacterium bovis BCG, the only approved vaccine against tuberculosis, strongly improved its immunogenicity and protective potential in mice. In this work we demonstrate that the same recombinant strain is able to induce better protection than wild type BCG also in guinea pigs preventing Mycobacterium tuberculosis dissemination and lung pathology, making this strain a strong candidate for further testing

exploiting the mycobacterial cell wall to design improved vaccines against tuberculosis

The only vaccine available against tuberculosis (TB), the Bacille Calmette-Guerin (BCG), does not provide effective protection against the most common forms of adult TB and in recent years efforts have been made to develop a new and improved vaccine. Among the strategies implemented, the generation of a new live attenuated mycobacterial strain is seen as one of the most promising and feasible, for scientific, ethical and practical reasons. The new understanding of the biology of the tubercle bacilli and of host-pathogen interaction processes, coupled with the possibility to engineer BCG or M. tuberculosis, opened new avenues to design "intelligent" vaccines, capable of eliciting the immune response associated with protection while avoiding the induction of the host immune response associated with immunopathology. The complex and highly immunogenic mycobacterial cell wall can shape the general and antigen specific immune response elicited following immunization, and the possibility to exploit this knowledge may lead to the development of new vaccines that could help conquer this ancient human disease

Development of a repressible mycobacterial promoter system based on two transcriptional repressors

Tightly regulated gene expression systems represent invaluable tools for studying gene function and for the validation of drug targets in bacteria. While several regulated bacterial promoters have been characterized, few of them have been successfully used in mycobacteria. In this article we describe the development of a novel repressible promoter system effective in both fast- and slow-growing mycobacteria based on two chromosomally encoded repressors, dependent on tetracycline (TetR) and pristinamycin (Pip), respectively. This uniqueness results in high versatility and stringency. Using this method we were able to obtain an ftsZ conditional mutant in Mycobacterium smegmatis and a fadD32 conditional mutant in Mycobacterium tuberculosis, confirming their essentiality for bacterial growth in vitro. This repressible promoter system could also be exploited to regulate gene expression during M. tuberculosis intracellular growt

Functional Dissection of the PE Domain Responsible for Translocation of PE_PGRS33 across the Mycobacterial Cell Wall

PE are peculiar exported mycobacterial proteins over-represented in pathogenic mycobacterial species. They are characterized by an N-terminal domain of about 110 amino acids (PE domain) which has been demonstrated to be responsible for their export and localization. In this paper, we characterize the PE domain of PE_PGRS33 (PERv1818c), one of the best characterized PE proteins. We constructed several mutated proteins in which portions of the PE domain were deleted or subjected to defined mutations. These proteins were expressed in different mycobacterial species and their localization was characterized. We confirmed that the PE domain is essential for PE_PGRS33 surface localization, and demonstrated that a PE domain lacking its first 30 amino acids loses its function. However, single amino acid substitutions in two regions extremely well conserved within the N-terminal domain of all PE proteins had some effect on the stability of PE_PGRS33, but not on its localization. Using Mycobacterium marinum we could show that the type VII secretion system ESX-5 is essential for PE_PGRS33 export. Moreover, in M. marinum, but not in Mycobacterium bovis BCG and in Mycobacterium tuberculosis, the PE domain of PE_PGRS33 is processed and secreted into the culture medium when expressed in the absence of the PGRS domain. Finally, using chimeric proteins in which different portions of the PERv1818c domain were fused to the N-terminus of the green fluorescent protein, we could hypothesize that the first 30 amino acids of the PE domain contain a sequence that allows protein translocation

Essentiality of mmpL3 and impact of its silencing on Mycobacterium tuberculosis gene expression

MmpL3 is an inner membrane transporter of Mycobacterium tuberculosis responsible for the export of trehalose momomycolate, a precursor of the mycobacterial outer membrane component trehalose dimycolate (TDM), as well as mycolic acids bound to arabinogalactan. MmpL3 represents an emerging target for tuberculosis therapy. In this paper, we describe the construction and characterization of an mmpL3 knockdown strain of M. tuberculosis. Downregulation of mmpL3 led to a stop in bacterial division and rapid cell death, preceded by the accumulation of TDM precursors. MmpL3 was also shown to be essential for growth in monocyte-derived human macrophages. Using RNA-seq we also found that MmpL3 depletion caused up-regulation of 47 genes and down-regulation of 23 genes (at least 3-fold change and false discovery rate <= 1%). Several genes related to osmoprotection and metal homeostasis were induced, while several genes related to energy production and mycolic acids biosynthesis were repressed suggesting that inability to synthesize a correct outer membrane leads to changes in cellular permeability and a metabolic downshift

Development of a repressible mycobacterial promoter system based on two transcriptional repressors

Tightly regulated gene expression systems represent invaluable tools for studying gene function and for the validation of drug targets in bacteria. While several regulated bacterial promoters have been characterized, few of them have been successfully used in mycobacteria. In this article we describe the development of a novel repressible promoter system effective in both fast- and slow-growing mycobacteria based on two chromosomally encoded repressors, dependent on tetracycline (TetR) and pristinamycin (Pip), respectively. This uniqueness results in high versatility and stringency. Using this method we were able to obtain an ftsZ conditional mutant in Mycobacterium smegmatis and a fadD32 conditional mutant in Mycobacterium tuberculosis, confirming their essentiality for bacterial growth in vitro. This repressible promoter system could also be exploited to regulate gene expression during M. tuberculosis intracellular growth