Luiz Pedro Carvalho: Projects

Functional genomics and mycobacterial enzymology

Genomics and proteomics continue to deliver a wealth of information about genes and their putative proteins, but experimental functional validation remains a great challenge. For instance, hundreds of genes from Mycobacterium tuberculosis (Mtb) are predicted to encode enzymes that are involved in biosynthesis and degradation of cofactors, amino acids, carbohydrates and lipids, but very few of these have had their function revealed and validated. In addition, several proteins encoded in the mycobacterial genome do not share any obvious homology with proteins seen in other organisms. Approximately 35 per cent of these orphan proteins are predicted to be essential and therefore could become potential targets for drug discovery, if their function was known.

We employ modern, high-resolution mass spectrometry-based metabolomic methods such as activity-based metabolic profiling and global metabolomic profiling to uncover substrates and products of orphan enzymes, therefore annotating their catalytic function and metabolic role. In the case of Rv1692, our results indicate that contrary to its initial annotation as a nucleotide phosphatase it is a glycerol-phosphate phosphatase. In addition, global metabolomic profiling of a clean genetic knockout indicate for the first time its role in lipid polar head catabolism, a pathway that was not mapped or demonstrated to exist in M. tuberculosis before (see Figure 1). We are currently applying these methods to other mycobacterial orphan enzymes in an attempt to map new enzymatic activities and uncharted metabolic pathways.

In addition to finding new enzymatic functions and metabolic pathways, we are studying how metabolite-driven control of enzymatic activity and metabolic pathways takes place, at both protein and whole cell levels. Allosteric regulation by small molecule ligands is a highly-efficient, evolutionary conserved regulatory mechanism that has been poorly investigated in M. tuberculosis and represents another untapped source for novel antimicrobial drug discovery.

 

Selected publications

Pisco, JP; de Chiara, C; Pacholarz, KJ; Garza-Garcia, A; Ogrodowicz, RW; Walker, PA; Barran, PE; Smerdon, SJ and de Carvalho, LPS (2017) Uncoupling conformational states from activity in an allosteric enzymeNature Communications 8, 203

Ganapathy, U; Marrero, J; Calhoun, S; Eoh, H; de Carvalho, LPS; Rhee, K and Ehrt, S (2015) Two enzymes with redundant fructose bisphosphatase activity sustain gluconeogenesis and virulence in Mycobacterium tuberculosisNature Communications 6, 7912

Larrouy-Maumus G, Biswas T, Hunt DM, Kelly G, Tsodikov OV and de Carvalho LP. (2013) Discovery of a glycerol 3-phosphate phosphatase reveals glycerophospholipid polar head recycling in Mycobacterium tuberculosisPNAS 110(28): 11320-11325

Antibiotic research and systems pharmacology

Understanding the mechanisms of action and resistance to antibiotics and promising lead compounds is key to their rational improvement and to the development of strategies and molecules that can potentially bypass resistance, shorten treatment and be less toxic. We apply modern metabolomic, proteomic, transcriptomic and chemical biological methods to define both pharmacodynamics and (intrabacterial) pharmacokinetics of antibiotics and antimycobacterial agents. Knowledge gained by these studies will rationally guide further improvements to the drugs. One of the drugs we are interested in studying is the antibiotic D-cycloserine (DCS), a second-line drug used as cornerstone for the treatment of multidrug-resistant tuberculosis.

Our studies to date indicate that: (1) DCS inhibits both Alanine racemase and D-Ala:D-Ala ligase in vitro, as expected; however, unlike has been observed in other species (2) DCS is a slow-onset inhibitor of M. tuberculosis D-Ala:D-Ala ligase; (3) metabolomics and labelling experiments revealed that DCS successfully decreases the levels of D-Ala:D-Ala in M. tuberculosis without a full blockage of the synthesis of D-Ala, indicating poor inhibition of alanine racemase; and (4) that in M. tuberculosis, recovery of the pool size of D-Ala:D-Ala following DCS treatment is delayed, indicating for the first a post-antibiotic effect for DCS, consistent with time-dependent inhibition of D-Ala:D-Ala ligase. Taken together, these results indicate that DCS kills M. tuberculosis by inhibiting D-Ala:D-Ala ligase, not alanine racemase (see Figure 2). These results are therefore of great importance for the efficient design/development of novel inhibitors of the D-Ala branch of peptidoglycan biosynthesis in M. tuberculosis.

We are currently investigating additional molecular and cellular mechanisms for DCS action in M. tuberculosis and how this antibiotic has evaded clinical resistance thus far.

Selected publications

Larrouy-Maumus, G; Marino, LB; Madduri, AVR; Ragan, TJ; Hunt, DM; Bassano, L; Gutierrez, MG; Moody, DB; Pavan, FR and de Carvalho, LPS (2016) Cell-envelope remodeling as a determinant of phenotypic antibacterial tolerance in Mycobacterium tuberculosisACS Infectious Diseases 2, 352-36

Prosser, GA; Rodenburg, A; Khoury, H; de Chiara, C; Howell, S; Snijders, AP and de Carvalho, LPS (2016) Glutamate racemase is the primary target of β-chloro-ᴅ-alanine in Mycobacterium tuberculosisAntimicrobial Agents and Chemotherapy 60, 6091-6099

Prosser GA and de Carvalho LP. (2013) Metabolomics reveal D-Alanine:D-Alanine ligase as the target of D-cycloserine in Mycobacterium tuberculosisACS Med Chem Letters 4(12):1233-1237

Host-pathogen metabolism

Metabolomic studies allow the discovery and simultaneous visualisation of entire biochemical pathways directly. The information gathered by combination of metabolomics and stable isotope tracing is not accessible by genomic, transcriptomic or proteomic means and is, by definition, the representation of all regulatory cascades, and therefore the closest possible readout of phenotype. Using these methodologies we intend to evaluate regulatory, kinetic and topological aspects of M. tuberculosis metabolic networks on a systems biology level. In a second step, we would like to define which nodes of M. tuberculosis metabolic network are 'connected' to the macrophage metabolic network, in an attempt of understanding the metabolic basis for infection.

In a series of studies carried out in collaboration with Olivier Neyrolles group in France, we were able to assign the substrate specificity and metabolic role of two mycobacterial transporters (see Figure). Their biochemical function was then used to understand their role during infection. These studies represent the first applications of high resolution mass spectrometry-based metabolomic profiling and stable isotope tracing to assign function to orphan transporters. We are now interested in expanding these studies to other important orphan transporters in a focused effort to map unknown nutrient acquisition pathways in M. tuberculosis.

In addition to this work we are engaged in a series of studies aimed a mapping the macrophage metabolic network. Macrophages are immune cells that are both responsible for killing M. tuberculosis and when subverted, one of the niches where it can reside during active and latent infection. In addition, several other human pathogens such as HIV can infect macrophages. Finally, macrophages play a key role in cancer progression and understanding their metabolic requirements might assist on the development of novel antitumor compounds and strategies.

Selected publications

Gouzy A, Larrouy-Maumus G, Bottai D, Levillain F, Dumas A, Wallach JB, Brandi I, de Chastellier C, Wu TD, Poincloux R, Brosch R, Gerquin-Kern JL, Schnappinger D, de Carvalho LP, Poquet Y, and Neyrolles O. (2014) Mycobacterium tuberculosis exploits asparagine to assimilate nitrogen and resist acid stress during infectionPLoS Pathogens 10(2):e1003928

Gouzy A, Larrouy-Maumus G, Wu TD, Moreau F, Peixoto A, Lugo-Villarino G, Levillain F, Lepourry L, Guerquin-Kern JL, de Carvalho LP, Poquet Y, and Neyrolles O. (2013) Aspartate is required for Mycobacterium tuberculosis nitrogen assimilation and host colonization.  Nat Chem Bio 9(11): 674-676

Goldstone D, Ennis-Adenrian V, Hedden J, Groom HCT, Rice G, Christodoulou E, Walker P, Kelly G, Haire LF, Yap MW, de Carvalho LP, Stoye J, Crow YJ, Taylor I and Webb M. (2011) HIV-1 restriction factor SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase.  Nature 480(7377): 379-382

 

Luiz Pedro Carvalho

Luiz Pedro Carvalho

luiz.carvalho@crick.ac.uk
+44 (0)20 379 62300

  • Qualifications and history
  • 2000-1 MSc Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
  • 2001-6 PhD Albert Einstein College of Medicine, New York, USA
  • 2006-11 Post doctoral fellow Cornell University, New York, USA
  • 2011-2015 Group Leader at Medical Research Council National Institute for Medical Research, London, UK
  • 2015 Group Leader, the Francis Crick Institute, London, UK