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Carvalho, L. P.

Publications and source records attributed to Carvalho, L. P..

4 recordsLinked to original sources

Mycobacterium tuberculosis overcomes phosphate starvation by extensively remodelling its lipidome with phosphorus-free lipids

Tuberculosis (TB) is the biggest cause of death from infectious disease worldwide [1-4]. The causative agent, Mycobacterium tuberculosis (Mtb), possesses a complex cell envelope that determines many of the key physiologic and virulence properties that facilitate infection, which comprises multiple classes of unique lipids [5-7]. The macrophage phagosome is a key reservoir of infection in pulmonary TB and multiple studies have shown that inorganic phosphate (Pi) is limiting in this environment [8-11]. The ability of Mtb to sense and respond to phosphate starvation is required for virulence in animal models and replication in human macrophages in vitro [12-14]. Here, we show that during Pi restriction the Mtb lipidome is markedly remodelled such that phospholipids are replaced with multiple classes of phosphorus-free lipids, some of which have not been documented before. Further, we discover that Mtb can metabolise phospholipid polar heads derived from host pulmonary surfactant as an alternative phosphate source, which sustains cell division for several generations during Pi restriction. These dual manipulations of phospholipid metabolism provide Mtb with an escape from phosphate restriction specific to the infection of alveolar macrophages, one of the earliest steps in establishing pulmonary tuberculosis. The changes in envelope lipidome remodelling, akin to that observed in some marine and terrestrial bacteria [15-20] suggests that standard Mtb culture conditions that use media with high concentrations of Pi do not reflect the physiologic environment during infection, thereby potentially undermining vaccine and drug development for tuberculosis. Moreover, the distinct Mtb phosphate-free lipids and the metabolic pathways that generate them could provide new antibiotic targets.

microbiology↗

Nitrogen utilisation by the metabolic generalist pathogen Mycobacterium tuberculosis

Bacterial metabolism is fundamental to pathogenesis and has a dominant effect on bacterial killing by antibiotics. Here we explore how Mycobacterium tuberculosis utilises amino acids as nitrogen sources, using a combination of bacterial physiology and stable isotope tracing coupled to liquid chromatography mass spectrometry metabolomics methods. Our results define core properties of the nitrogen metabolic network from M. tuberculosis, such as: (i) the lack of homeostatic control of amino acid pool sizes; (ii) similar rates of utilisation of different amino acids as sole nitrogen sources; (iii) improved nitrogen utilisation from amino acids compared to ammonium; and (iv) co-metabolism of nitrogen sources. Finally, we discover that alanine dehydrogenase, is involved in ammonium assimilation in M. tuberculosis, in addition to its essential role in alanine utilisation. This study represents the first in-depth analysis of nitrogen source utilisation by metabolic generatlist M. tuberculosis and reveals a flexible metabolic network with characteristics that are likely product of evolution in the human host.

microbiology↗

Antibiotic resistance evasion is explained by rare mutation frequency and not by lack of compensatory mechanisms

Drug resistant infections represent one of the most challenging medical problems of our time. D-cycloserine is an antibiotic used for decades without appearance and dissemination of antibiotic resistant strains, making it an ideal model compound to understand what drives resistance evasion. We investigated why Mycobacterium tuberculosis fails to become resistant to D-cycloserine. To address this question we employed a combination of bacterial genetics, genomics, biochemistry and fitness analysis in vitro, in macrophages and in mice. Altogether, our results suggest that the ultra-low mutation frequency associated with D-cycloserine resistance is the dominant factor delaying the appearance of clinical resistance to this antibiotic in bacteria infecting humans, and not lack of potential compensatory mechanisms.\n\nOne Sentence SummaryWe show that the lack of D-cycloserine resistance in Mycobacterium tuberculosis is due its ultra-low mutation frequency rather than lack of compensatory mechanisms.

microbiology↗

Discovery of a novel stereospecific β-hydroxyacyl-CoA lyase/thioesterase shared by three metabolic pathways in Mycobacterium tuberculosis

The vast number of poorly characterised enzymes in Mycobacterium tuberculosis (Mtb) is one of the key barriers precluding a better understanding of the biology that underpins pathogenesis. Here, we investigated the Mtb orphan enzyme Rv2498c to delineate its physiological role. Our results from in vitro enzymatic assays, phylogenetic analysis, X-ray crystallography and in vivo Mtb experiments, de-orphan Rv2498c as a multi-functional {beta}-hydroxyacyl-CoA lyase/thioesterase ({beta}-HAClyase/thioesterase) that participates in three different metabolic pathways: L-leucine catabolism, itaconate dissimilation, and glyoxylate shunt. Moreover, the deletion of the rv2498c gene from the Mtb genome resulted in attenuation in the mouse model compared to infection with the parent strain. To the best of our knowledge, this is the first report of an (R)-3-hydroxyl-3-methylglutaryl-CoA for leucine catabolism and an itaconate-specific resistance mechanism in Mtb.

biochemistry↗