The Laboratory of Molecular and Structural Microbiology (LMSM) seeks to understand how bacteria sense signals from their environment and internal milieu, to thereafter respond and adapt. Pathogenic bacteria are a particular interest, focused on the Spirochete genus Leptospira.
Signaling is mediated by proteins, which change their 3D structures in a signal-dependent way, structures that are often stabilized by phosphorylation. Phosphoryl-transfer along such signal transduction pathways is thus a key enzymatic reaction, the mechanistic features of which our lab wants to uncover at the molecular level.
Our lab uses X-ray crystallography, to image the 3D structures of target proteins, such as sensory histidine kinases and response regulators, alone and in complex. By combining these high-resolution images with other sources of information —especially issued from biochemistry, genetics and microbiology experiments— we wish to understand the function and then contribute to the development of applications, such as vaccines against microbial diseases.
The Leptospira genus comprises many species, at least 10 of them cause a serious disease: leptospirosis. This zoonosis (that is, a disease that transmits from animals to humans), affects the reproductive capacity of cattle in Uruguay. It also causes an acute disease in humans, sometimes deadly, for which there are still no effective vaccines. Signaling systems in Leptospira shall uncover virulence and pathogenesis mechanisms, still poorly understood in these spirochetes. Key virulence proteins will be attractive targets to develop intervention strategies, and effectively control the zoonosis, with anticipated veterinarian and public health applications.
Signaling and regulation in microorganisms
We are particularly interested in two component systems (TCS) in bacteria. TCSs are central in mediating signaling and regulation, almost ubiquitous in prokaryotes and archaea, they are also present in fungi and plants. Other regulatory proteins, such as one component systems, are also being studied. The general question behind this line of research is: how do cells use these sensory and regulatory proteins to detect extra- and intra-cellular signals, and then regulate specific functions? To answer this question, saprophytic bacteria (such as Bacillus subtilis and Leptospira biflexa) are studied, as well as pathogenic ones (Leptospira interrogans, L. borpetersenii, L. noguchii, Enterococcus faecium, Mycobacterium tuberculosis). In these models, several key biological processes are studied, such as the regulation of lipid synthesis (Albanesi et al., Proc Natl Acad Sci USA 2009, 106: 16185, Trajtenberg et al., J Biol Chem 2010, 285: 24892, Trajtenberg et al. al., mBio 2014, 5: e02105; Trajtenberg et al., eLife 2016, 5: e21422; Imelio et al., Bio-protocol 2017, 7: e2510; Lara et al., 2018 submitted), heme metabolism (Morero et al., Mol Microbiol 2014 , 94: 340), or virulence in pathogenicity (Adhikarla et al., Front Cell Infect Microbiol 2018, 8:45).
Molecular and structural biology of Leptospira
Different species of Leptospira cause leptospirosis. This zoonosis is the most widespread one around the world, reemerging as an important problem in human and animal health. In Uruguay, it is a significant issue, provoking cattle abortions and reproductive failure, and transmitting to humans causing acute disease. Our lab wants to elucidate the molecular mechanisms that determine and/or regulate virulence and pathogenicity in Leptospira. There are currently two main projects to do this: the study of the motility apparatus and systematic efforts to isolate and type local Leptospira strains in Uruguay.
i- The motility apparatus of Leptospira. Active translational motility of leptospira (swimming) is central for the virulence of the pathogenic species that cause leptospirosis. The essential organelle used for swimming is the flagellum. Our lab is currently studying the detailed molecular architecture of the flagellar appendage. The filament of these spirochetes’ flagella is confined within the periplasm, a unique feature shared by all Spirochetes, including bacteria like Treponema pallidum (the agent of syphilis) or Borrelia burgdorferi (Lyme disease). In collaboration with the Ko and Sindelar labs in Yale University, as well as the Picardeau lab at the Institut Pasteur (Paris), we have shown that the flagellar filament from Leptospira is much more complex compared to better-known bacterial models used so far as a paradigm of swimming motility in bacteria. As an example, filaments from Salmonella and other Enterobacteria are built as homopolymers of a single protein species (flagellin), whereas Leptospira comprise at least eight different proteins polymerized into the periplasmic filament assembly (Wunder et al., Mol Microbiol 2016, 101: 457; San Martin et al., Acta Crystallogr F 2017, 73:123; Wunder et al., Front Cell Infect Microbiol 2018, 8:130). We have recently crystallized two new proteins from both pathogenic and saprophytic Leptospira spp. (San Martín et al., Acta Crystallogr F 2017, 73:123),and solved their 3D structures (unpublished results), revealing novel protein folds. We are now moving forward towards their physiological interactions and interaction partners, enabling for the flagellar filament role in spirochete motility.
ii- isolation and typing of autochthonous Leptospira spp. strains. This line is being developed in the context of a multicentric collaborative project, aims to isolating native strains from Leptospira spp. from biological samples obtained from infected cattle and other animal reservoirs. These isolates are typed by complementary techniques: classic serologic methods and novel molecular approaches, ultimately achieving greater sensitivity and specificity (Zarantonelli et al., PLoS Negl Trop Dis 2018, 12:e0006694). This effort has led to the creation of a biobank of strains of Leptospira, which up to now was not available in Uruguay, reporting the identity of the serovars that circulate in natural infections. This biobank will be useful in the formulation of more efficacious vaccines, in the improvement of diagnostic methods, as well as in further investigations of leptospirosis in Uruguay.
Our lab works in collaboration with Dr. Hugo Gramajo (Institute of Molecular and Cellular Biology, IBR, Rosario, Argentina) and his team, to elucidate crystallographic structures and mechanisms of action of Mycobacterium tuberculosis transcription factors (one component regulator systems), playing essential roles in regulating fatty acid metabolism in this pathogen.
We also collaborate with Dr. Mathieu Picardeau (Institut Pasteur, Paris, France) and Dr. Albert Ko (Yale University, New Haven, USA) on motility of Leptospira and pathogenesis mechanisms of these bacteria at the molecular level.
In addition, our lab integrates a multicentric consortium in Uruguay, working together with teams from the Veterinary Laboratories Division (“Miguel C Rubino” DILAVE) of the Ministry of Livestock, Agriculture and Fishery (Alejandra Suanes, Rodolfo Rivero), National Institute of Agricultural Research (Franklin Riet), Hygiene Institute at the Medical School (Felipe Schelotto), addressing problems of isolation, typing, diagnostics and genomics of Leptospira.
Theoretical-practical workshop: “Isolation of Leptospira spp. strains from field cases of bovine leptospirosis” (2014) Institut Pasteur de Montevideo, INIA (La Estanzuela); Universidad de la República, Hygiene Institute, School of Medicine; and DILAVE (Ministry of Livestock, Agriculture and Fishery), with invited professor from Massey University (New Zealand).
Theoretical-practical workshop: “Integrative methods in Structural Biology to enhance high impact research in health and disease” (2016) Institut Pasteur de Montevideo, co-organized by Oxford University and Instruct, the European Network of Structural Biology.
2017-2019 – “Structural and functional studies of the endoflagella of Leptospira: an essential component of the pathogenicity of spirochetes”– Uruguayan National Agency for Research and Innovation (ANII), Clemente Estable Fund #FCE_3_2016_1_126797 (Uruguay). Principal Investigator F Trajtenberg.
2017-2018 – “Bank of native strains from zoonotic diseases that affect Uruguayan cattle for R&D of Uruguayan industry” Ministry of Industry, Energy and Mining (Ministerio de Industria, Energía y Minería MIEM), Fondo Industrial Dirección Nacional de Industrias, # 2016-8-2-002671 (Uruguay). Role: collaborator group.
2018-2021 – “Molecular mechanism of bacterial signalling: directionality from the signal to the response”. Uruguayan National Agency for Research and Innovation (ANII), Clemente Estable Fund #FCE_1_2017_1_136291 (Uruguay). Principal Investigator A Buschiazzo. Partners: Dr Sergio Pantano (Lab of Biomolecular Simulations, IP Montevideo) and Dr Marcelo Martí (Biological Chemistry Dept & INQUIMAE, Universidad de Buenos Aires, Argentina).
2017-2021 – International Joint Unit “Integrative Microbiology of Zoonotic Agents”. Institut Pasteur de Montevideo (Uruguay) and Institut Pasteur (Francia). Role: Principal Investigator. Co-PI: Dr M. Picardeau (Institut Pasteur, France).
2015-2018 – “Creation and characterization of a bank Leptospira spp. strains isolated from bovine cases of leptospirosis in Uruguay”. Uruguayan National Agency for Research and Innovation (ANII), Programa Alianzas # ALI_1_2014_1_4982 (Uruguay). Principal Investigator: A Buschiazzo. Partners: Prof F Schelotto (Medical School, Universidad de la República, Uruguay), Vet. Alejandra Suanes (Ministry of Agriculture, DILAVE) and Vet Franklin Riet (Uruguayan National Agency for Research in Agriculture, INIA).
2019-2020 – Capacity building funds for the “Centro de Biología Estructural del Mercosur – CeBEM”. Ministry of Education and Culture (MEC), Dirección para el Desarrollo de la Investigación y el Conocimiento (D2C2), Uruguay. Co-PIs A Buschiazzo and R Radi (CEINBIO, Medical School, Udelar), heads of the two Uruguayan CeBEM nodes (www.cebem-lat.org).