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Gracias Fernandes da Costa Catalão, Maria João
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- Modulation of Cystatin F in Human Macrophages Impacts Cathepsin-Driven Killing of Multidrug-Resistant Mycobacterium tuberculosisPublication . Mandal, Manoj; Pires, David; Catalão, Maria João; Azevedo-Pereira, José Miguel; Anes, ElsaTuberculosis (TB) treatment relies primarily on 70-year-old drugs, and prophylaxis suffers from the lack of an effective vaccine. Among the 10 million people exhibiting disease symptoms yearly, 450,000 have multidrug or extensively drug-resistant (MDR or XDR) TB. A greater understanding of host and pathogen interactions will lead to new therapeutic interventions for TB eradication. One of the strategies will be to target the host for better immune bactericidal responses against the TB causative agent Mycobacterium tuberculosis (Mtb). Cathepsins are promising targets due to their manipulation of Mtb with consequences such as decreased proteolytic activity and improved pathogen survival in macrophages. We recently demonstrated that we could overcome this enzymatic blockade by manipulating protease inhibitors such as cystatins. Here, we investigate the role of cystatin F, an inhibitor that we showed previously to be strongly upregulated during Mtb infection. Our results indicate that the silencing of cystatin F using siRNA increase the proteolytic activity of cathepsins S, L, and B, significantly impacting pathogen intracellular killing in macrophages. Taken together, these indicate the targeting of cystatin F as a potential adjuvant therapy for TB, including MDR and XDR-TB.
- Characterization and functional analysis of the Gp1 accessory lysis protein in Mycobacterium smegmatis infection by the Mycobacteriophage Ms6Publication . Catalão, Maria João; Pimentel, Madalena Maria Vilela, 1961-; Pereira, J. Moniz, 1949-The majority of phages described to date (≈96%) is endowed with a tail and presents a double-stranded DNA (dsDNA) genome. Each infection cycle terminates with the strictly programmed and regulated lysis of the host brought about by two phage-encoded proteins, a murein-degrading enzyme, the endolysin, which is essential in achieving rapid hydrolysis of cell wall peptidoglycan, and a second membrane-embedded protein, the holin, which serves to release or activate the endolysin at a precisely defined time, thus bringing about an effective burst of the infected host. Targeting of endolysins to peptidoglycan can be achieved in two ways: through pore formation by canonical holins at a defined time or by continuous export of endolysins endowed with export signals during synthesis, assisted by the host Sec translocon. Mycobacteriophage Ms6 is a temperate bacteriophage that infects Mycobacterium smegmatis and possesses an unusual lytic cassette: in addition to the endolysin-holin lysis system encoded by genes lysA (gp2) and hol (gp4) respectively, the Ms6 lytic cassette comprises three accessory lysis proteins encoded by genes gp1, gp3 (lysB) and gp5 that are restricted to mycobacteriophages. Mycobacteria are Gram-positive bacteria that have evolved a complex cell wall, comprising a peptidoglycan-arabinogalactan polymer with covalently bound mycolic acids of considerable size, a variety of extractable lipids, and pore-forming proteins which provide an extraordinary efficient permeability barrier to noxious compounds and contribute to the high intrinsic resistance of mycobacteria to many drugs. To overcome the disadvantage that a complex cell wall may represents for a successful infective cycle, mycobacteriophages have evolved new lysis strategies by acquiring, through their evolution, genes that likely confer a substantial selective advantage over those without them by providing faster and more complete lysis. The present work underscores a new model for endolysin export in mycobacteriophage Ms6. Several lines of evidence indicate that gp1 encodes a secretion chaperone-like protein that binds the endolysin, assists the export to the extracytoplasmic environment independently of holin function and is required to accomplish an efficient lysis of M. smegmatis. Construction of different Ms6 derivatives deleted in different regions of the lysis operon demonstrated that the gene products of hol and gp5, although nonessential for phage viability, appear to play a role in controlling the timing of lysis. Remarkably, during M. smegmatis infection two endolysin forms (Lysin384 and Lysin241) are synthesized and both enzymes were shown to be essential for the normal timing, progression and completion of host cell lysis. In conclusion, this work highlights the role of the accessory lysis protein Gp1 and revealed alternative pathways for mycobacteria lysis, demonstrating that the presence of the mycobacterium-specific lysis factor Gp1, may confer a selective advantage not only for fitness under different environmental conditions but also as an alternative to lysis exclusively holin-dependent. Mycobacteriophages – Molecular chaperones – Holins – Endolysins - Lysis timing - Phage therapy - Mycobacteria
- Development of Chitosan Particles Loaded with siRNA for Cystatin C to Control Intracellular Drug-Resistant Mycobacterium tuberculosisPublication . Pires, David; Mandal, Manoj; Matos, Ana I.; Peres, Carina; Catalão, Maria João; Azevedo-Pereira, José M.; Satchi-Fainaro, Ronit; Florindo, Helena F; Anes, ElsaThe golden age of antibiotics for tuberculosis (TB) is marked by its success in the 1950s of the last century. However, TB is not under control, and the rise in antibiotic resistance worldwide is a major threat to global health care. Understanding the complex interactions between TB bacilli and their host can inform the rational design of better TB therapeutics, including vaccines, new antibiotics, and host-directed therapies. We recently demonstrated that the modulation of cystatin C in human macrophages via RNA silencing improved the anti-mycobacterial immune responses to Mycobacterium tuberculosis infection. Available in vitro transfection methods are not suitable for the clinical translation of host-cell RNA silencing. To overcome this limitation, we developed different RNA delivery systems (DSs) that target human macrophages. Human peripheral blood-derived macrophages and THP1 cells are difficult to transfect using available methods. In this work, a new potential nanomedicine based on chitosan (CS-DS) was efficiently developed to carry a siRNA-targeting cystatin C to the infected macrophage models. Consequently, an effective impact on the intracellular survival/replication of TB bacilli, including drug-resistant clinical strains, was observed. Altogether, these results suggest the potential use of CS-DS in adjunctive therapy for TB in combination or not with antibiotics.
- Liposomal Delivery of Saquinavir to Macrophages Overcomes Cathepsin Blockade by Mycobacterium tuberculosis and Helps Control the Phagosomal Replicative NichesPublication . Pires, David; Mandal, Manoj; Pinho, Jacinta O.; Catalão, Maria João; Almeida, António J.; Azevedo-Pereira, J. M.; Gaspar, Maria Manuela; Anes, ElsaMycobacterium tuberculosis is able to establish a chronic colonization of lung macrophages in a controlled replication manner, giving rise to a so-called latent infection. Conversely, when intracellular bacteria undergo actively uncontrolled replication rates, they provide the switch for the active infection called tuberculosis to occur. Our group found that the pathogen is able to manipulate the activity of endolysosomal enzymes, cathepsins, directly at the level of gene expression or indirectly by regulating their natural inhibitors, cystatins. To provide evidence for the crucial role of cathepsin manipulation for the success of tuberculosis bacilli in their intracellular survival, we used liposomal delivery of saquinavir. This protease inhibitor was previously found to be able to increase cathepsin proteolytic activity, overcoming the pathogen induced blockade. In this study, we demonstrate that incorporation in liposomes was able to increase the efficiency of saquinavir internalization in macrophages, reducing cytotoxicity at higher concentrations. Consequently, our results show a significant impact on the intracellular killing not only to reference and clinical strains susceptible to current antibiotic therapy but also to multidrug- and extensively drug-resistant (XDR) Mtb strains. Altogether, this indicates the manipulation of cathepsins as a fine-tuning strategy used by the pathogen to survive and replicate in host cells.