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Molecular and Structural Microbiology

MEMBERS

  • Alejandro Buschiazzo, PhD (Principal Investigator)
  • Joaquín Dalla Rizza (Technician)
  • Juan Andrés Imelio (PhD student)
  • Nicole Larrieux (Technician)
  • Cecilia Nieves (MSc student)
  • Marcos Nieves (PhD student)
  • Fabiana San Martin (PhD student)
  • Felipe Trajtenberg, PhD (Research Scientist)
  • Leticia Zarantonelli, PhD (Associate Research Scientist)
 Past members
  • Horacio Botti
  • Mathias Ferrari
  • Sofía Horjales
  • Frank Lehmann
  • Natalia Lisa (currently Honorary Research Associate)
  • Ariel Mechaly (currently Honorary Research Associate)
  • Natalia Morero
  • Natalia Ruétalo

 

Research

We want to understand at the molecular level how microorganisms carry on important biological processes, focusing on vital functions such as signal sensing and transduction, and the subsequent cell regulation that allows cells to adapt and maintain homeostasis. Our experimental approaches combine molecular microbiology with structural biology and biochemistry.

Structural Biology leads us also to protein engineering. One of our current challenges is to contribute to the development of vaccines against the infectious diseases caused by the microbial agents we are studying.

Current lines of research

I- Signaling and regulation in microorganisms

Bacterial two-component systems (TCSs) and different kinds of regulator proteins in bacteria constitute the main protein systems that we work on. The common theme is how cells use proteins to sense extra- and intra-cellular signals in order to regulate specific functions.

TCS

To understand the molecular means by which bacteria transduce signals, adapting to a changing environment, during the last few years we have been using a non-pathogenic model (Bacillus subtilis) focusing our efforts in elucidating the molecular mechanisms of signaling and regulation of lipid synthesis in Gram+ bacteria. Our main contribution concerns the structural studies of the TCS DesK/DesR. DesK is a trans-membrane histidine kinase that, together with its cognate response regulator DesR, regulates the membrane’s fluidity in response to cold shock in B. subtilis. Previous structural and biochemical work with the entire cytoplasmic region of DesK from B. subtilis (Albanesi et al., Proc Natl Acad Sci USA 2009, 106:16185-90; Trajtenberg et al., J Biol Chem 2010, 285:24892-903), allowed us to propose a mechanistic model that appears to be general for histidine kinase-mediated signal transduction. The crystal structure of full-length DesR in its activated state and of its receiver domain in the active and inactive configurations, were later determined in our lab, revealing the molecular details of the activation switch (Trajtenberg et al., mBio 2014, 5:e02105-14). More recently, we were able to visualize the complex of DesK and DesR in two distinct steps of the signaling cycle (Trajtenberg et al., eLife 2016, 5:e21422): the dephosphorylation (phosphatase) reaction, and the phosphoryl-transfer (phosphotransferase), the latter constituting the very first TCS complex to be crystallized in this state and solved at high resolution.

Fig_DesKFig_DesR

More recently, we have extended the study of TCS-mediated signaling & regulation in pathogenic bacteria. With this purpose we have launched molecular and structural studies of several TCSs in Leptospira spp., spirochetal bacteria that cause leptospirosis. This disease is the most widespread zoonosis in the world, reemerging as a major health problem. In Uruguay its prevalence as a veterinary issue is very significant. A collaborative partnership has been established with Albert Ko’s lab (Yale Univ) and Mathieu Picardeau’s (Institut Pasteur). We have progressed in the understanding of the  metabolic regulation of heme, which at difference with other Spirochetes, is critical for Leptospira survival. Controlled via a TCS, we have been able to solve the structure of the receiver domain of the response regulator HemR, understanding its role as a transcriptional activator and repressor of key genes in a heme regulon (Morero et al., Mol Microbiol 2014, 94:340-52). We are actively searching for the signal(s) sensed by HemK. We have more recently focused our attention also in the structural biology of other TCSs and phosphatases, pathogen-specific, that we have identified through a genetic approach knocking-out the genes and verifying virulence attenuation in the hamster model of acute disease (Adhikarla et al., Front Cell Infect Microbiol 2018, 8:45).

II- Leptospira motility

This line is focused on studying the molecular architecture of the flagellar filament of Leptospira spp. These flagella are  periplasmic (a unique feautre in Spirochetes) and are essential for normal translational motility, which is absolutely needed for virulence in pathogenic species. The molecular bases that link the flagellum to the cell body enabling  the swimming motion, are yet to be discovered. Our evidence show that the leptospiral filament is structurally more complex in its protein constitution than flagella from better known bacterial models (in which the filament is built from a single protein species: flagellin, such as in Salmonella). In this way we have contributed to discovering protein FcpA, a novel filament component (Wunder et al. Mol Microbiol 2016, 101:457-70) key for normal flagellar structure and function. More recently, we have crystallized FcpA from pathogenic and saprophytic Leptospira species (San Martin et al., Acta Crystallogr F 2017, 73:123-129), and determined its crystal structure (unpublished results). Currently we are making progress in uncovering its physiologic interactors to decipher its role(s) in the flagellar assembly.

lepto

III- Isolation and typing of autochthonous Leptospira serovars 

This line, developed in the context of a collaborative multicentric project, pursues the aim of isolating autochthonous strains of Leptospira bacteria   from biologic samples obtained from bovine cattle, suspect of infection. Such isolates are being typed with complementary techniques: serologic and novel molecular approaches, with higher sensitivity/specificity (Zarantonelli et al., 2018 manuscript under revision). This effort has led to the creation of a biobank of Leptospira strains, which up to now was not available in the public domain in Uruguay, informing the identity of circulating serovars. Such a biobank shall be instrumental in the formulation of vaccines with higher efficacy.

IV- Structural virology

Started as a collaboration with Dr Otto Pritsch (Inst Pasteur de Montevideo & Medical School UdelaR), we have focused on the structural aspects of the capsid from bovine leukemia virus (BLV). We have solved the crystallographic structure of the native, non-engineered capsid of BLV, providing with detailed information about key residues involved in the self-assembly of this supramolecular structure (Obal et al. Science 2015). We are currently interested in uncovering the interactions of BLV capsid with host cell proteins so that we understand the mechanisms used by the virus to regulate assembly and uncoating along its life cycle.

capsid3

V- Collaborative work

  1. Dr Hugo Gramajo (Instituto de Biologia Molecular y Celular IBR, Rosario, Argentina) and his team, aimed at elucidating the crystal structures of transcription factors from Mycobacterium tuberculosis, involved in the regulation of lipid metabolism.
  2. Drs Mathieu Picardeau (Institut Pasteur, Paris, France) and Albert Ko (Yale University, New Haven, USA), working in in Leptospira motility and molecular mechanisms of pathogenesis.

EDUCATION-COURSES

  • Organization of several courses and workshops :

    • Course “Macromolecular Crystallography: introduction and applications” – 2010 (Institut Pasteur de Montevideo)
    • School “Macromolecular Crystallography School – From data processing to structure refinement and beyond”. Course co-organized with CCP4 (UK), with annual periodicity since 2013, foundational year when the workshop took place at the Institut Pasteur de Montevideo. Every other year (2014, 2016) the course was held at the Instituto de Fisica de Sao Carlos (Univ de Sao Paulo, Sao Carlos, Brazil), alternating with the Inst Pasteur de Montevideo venue (in 2015 and 2017).
    • Hands-on workshop: “Isolation of Leptospira spp. strains from field cases of bovine leptospirosis”. 2014 (Institut Pasteur de Montevideo, INIA (estacion La Estanzuela); Universidad de la Republica, Instituto de Higiene, Fac de Medicina; and DILAVE (Min de Ganaderia, Agricutlura y Pesca). With participation of invited professors from Massey University (New Zealand).
    • Workshop “Modern Approaches in Drug Discovery for Neglected Infectious Diseases”. 2014 Institut Pasteur de Montevideo.
    • Hands-on workshop: “Integrative methods in Structural Biology to enhance high impact research in health and disease” 2016 Institut Pasteur de Montevideo, co-organized with the University of Oxford and the European Structural Biology network Instruct. 

    Training and supervision of students :

    • 3 undergraduate students

    • 3 MSc students 

    • 4 PhD students

    • 3 postdoctoral fellows

    • Training of >10 students and interns from Uruguay and abroad

GRANTS

  1. Typing and diagnosis of Leptospira spp. using molecular approaches: towards the design of recombinant vaccines”. Uruguayan National Agency for Research and Innovation ANII, Fondo Sectorial Innovagro #FSA_1_2013_1_12557 (Uruguay), 2014-2017. Role: Principal Investigator. Partners: Vet Alejandra Suanes (Min of Agricultures, DILAVE) and Dr M Picardeau (Biology of Spirochetes Unit, IPasteur)
  2. Molecular mechanism of bacterial signaling: directionality from the signal to the response”. Uruguayan National Agency for Research and Innovation ANII, Fondo Clemente Estable #FCE_1_2017_1_136291 (Uruguay), 2018-2021. Role: Principal Investigator. Partners: Dr Sergio Pantano (Lab of Biomolecular Simulations, Inst Pasteur Montevideo) and Dr Marcelo Martí (Biological Chemistry Dept & INQUIMAE, Univ de Buenos Aires, Argentina).
  3. Creation and characterization of a bank of Leptospira spp. strains isolated from bovine cases of leptospirosis in Uruguay”. Uruguayan National Agency for Research and Innovation ANII, Program Alianzas # ALI_1_2014_1_4982 (Uruguay). Granted 2015-2018. Role: Principal Investigator. Partners: Prof F Schelotto (Medical School, Univ de la Republica, Uruguay), Vet Alejandra Suanes (Min of Agricultures, DILAVE) and Vet F Riet (Uruguayan National Agency for Research in Agriculture INIA).
  4. Integrative Microbiology of Zoonotic Agents”. Institut Pasteur de Montevideo (Uruguay) and Institut Pasteur (Francia), International Joint Unit – 2016-2021. Role: Principal Investigator. Co-PI: Dr M Picardeau (Institut Pasteur, France).
  5. Consolidation of the “Centro de Biologia Estructural del Mercosur – CeBEM”. Ministry of Education and Culture (MEC), Uruguay. Direccion de Innovación, Ciencia y Tecnología para el Desarrollo (DICYT). 2014-2018. Role: general coordinator; co-managed by A Buschiazzo and R Radi (heads of the 2 Uruguayan CeBEM nodes http://www.cebem-lat.org).

PUBLICATIONS

Publications last 5 years

Adhikarla H, Wunder EA Jr., Mechaly AE, Mehta S, Wang Z, Santos L, Bisht V, Diggle P, Murray G, Adler B, Lopez F, Townsend JP, Groisman E, Picardeau M, Buschiazzo A¶, Ko AI¶. Lvr, a signaling system that controls global gene regulation and virulence in pathogenic Leptospira. Front Cell Infect Microbiol. 2018 Feb 23;8:45. doi: 10.3389/fcimb.2018.00045. ¶ [Corresponding authors] 

San Martin F, Mechaly AE, Larrieux N, Wunder EA Jr, Ko AI, Picardeau M, Trajtenberg F, Buschiazzo A. Crystallization of FcpA from Leptospira, a novel flagellar protein that is essential for pathogenesis. Acta Crystallogr F Struct Biol Commun. 2017 Mar 1;73(Pt 3):123-129. doi: 10.1107/S2053230X17002096. 

Mechaly AE, Soto Diaz S, Sassoon N, Buschiazzo A, Betton JM, Alzari PM. Structural coupling between autokinase and phosphotransferase reactions in a bacterial histidine kinase. Structure. 2017 Jun 6;25(6):939-944.e3. doi: 10.1016/j.str.2017.04.011. 

Trajtenberg FImelio JA, Machado MR, Larrieux N, Marti MA, Obal G, Mechaly AEBuschiazzo A. Regulation of signaling directionality revealed by 3D snapshots of a kinase:regulator complex in action. Elife. 2016 Dec 12;5:e21422. doi: 10.7554/eLife.21422.

Morán-Barrio J*, Lisa MN*Larrieux N, Drusin SI, Viale AM, Moreno DM, Buschiazzo A¶, Vila AJ¶. Crystal structure of the metallo-β-lactamase GOB in the periplasmic dizinc form reveals an unusual metal site. Antimicrob Agents Chemother. 2016 Sep 23;60(10):6013-22. doi: 10.1128/AAC.01067-16. * [These authors contributed equally to this work]  ¶ [Corresponding authors]

Wunder EA, Figueira CP, Benaroudj N, Hu B, Tong BA, Trajtenberg F, Liu J, Reis MG, Charon NW, Buschiazzo A, Picardeau M, Ko AI. A novel flagellar sheath protein, FcpA, determines filament coiling, translational motility and virulence for the Leptospira spirochete. Mol Microbiol. 2016 Aug;101(3):457-70. doi: 10.1111/mmi.13403.

Meyer PA, Socias S, Key J, Ransey E, Tjon EC, Buschiazzo A, et al. Data publication with the structural biology data grid supports live analysis. Nat Commun. 2016 Mar 7;7:10882. doi: 10.1038/ncomms10882

Fouts DE, Matthias MA, Adhikarla H, Adler B, Amorim-Santos L, Berg DE, Bulach D, Buschiazzo A, Chang YF, Galloway RL, Haake DA, Haft DH, Hartskeerl R, Ko AI, Levett PN, Matsunaga J, Mechaly AE, Monk JM, Nascimento AL, Nelson KE, Palsson B, Peacock SJ, Picardeau M, Ricaldi JN, Thaipandungpanit J, Wunder EA Jr, Yang XF, Zhang JJ, Vinetz JM. What Makes a Bacterial Species Pathogenic?:Comparative Genomic Analysis of the Genus Leptospira. PLoS Negl Trop Dis. 2016 Feb 18;10(2):e0004403.

East A, Mechaly AE, Huysmans GH, Bernarde C, Tello-Manigne D, Nadeau N, Pugsley AP, Buschiazzo A, Alzari PM, Bond PJ, Francetic O. Structural Basis of Pullulanase Membrane Binding and Secretion Revealed by X-Ray Crystallography, Molecular Dynamics and Biochemical Analysis. Structure. 2016 Jan 5;24(1):92-104.

Saita E, Abriata LA, Tsai YT, Trajtenberg F, Lemmin T, Buschiazzo A, Dal Peraro M, de Mendoza D, Albanesi D. A coiled coil switch mediates cold sensing by the thermosensory protein DesK. Mol Microbiol. 2015 Oct;98(2):258-71.

Obal G*, Trajtenberg F*, Carrión F, Tomé L, Larrieux N, Zhang X, Pritsch O¶, Buschiazzo A¶. Conformational plasticity of a native retroviral capsid revealed by x-ray crystallography. Science. 2015 Jul 3;349::95-8. *[These authors contributed equally to this work]  ¶[Corresponding authors]

Methot SP, Litzler LC, Trajtenberg F, Zahn A, Robert F, Pelletier J, Buschiazzo A, Magor BG, Di Noia JM. Consecutive interactions with HSP90 and eEF1A underlie a functional maturation and storage pathway of AID in the cytoplasm. J Exp Med. 2015 Apr 6;212(4):581-96.

Trajtenberg F, Albanesi D, Ruétalo N, Botti H, Mechaly AE, Nieves M, Aguilar PS, Cybulski L, Larrieux N, Mendoza D, Buschiazzo A. Allosteric activation of bacterial response regulators: the role of the cognate histidine kinase beyond phosphorylation. (2014) mBio 5:e02105-14. 

Morero NRBotti H, Nitta KR, Carrión F, Obal G, Picardeau M, Buschiazzo A. HemR is an OmpR/PhoB-like response regulator from Leptospira, which simultaneously effects transcriptional activation and repression of key haem metabolism genes. Mol Microbiol. 2014 Oct;94:340-52.

Trajtenberg F*, Altabe S*, Larrieux N, Ficarra F, de Mendoza D, Buschiazzo A, Schujman GE. Structural insights into bacterial resistance to cerulenin. FEBS J. 2014 May;281(10):2324-38. *[These authors contributed equally to this work] 

Martinez A, Peluffo G, Petruk AA, Hugo M, Piñeyro D, Demicheli V, Moreno DM, Lima A, Batthyány C, Durán R, Robello C, Martí MA, Larrieux N, Buschiazzo A, Trujillo M, Radi R, Piacenza L. Structural and molecular basis of the peroxynitrite-mediated nitration and inactivation of Trypanosoma cruzi iron-superoxide dismutases (Fe-SODs) A and B: disparate susceptibilities due to the repair of Tyr35 radical by Cys83 in Fe-SODB through intramolecular electron transfer. J Biol Chem. 2014 May 2;289(18):12760-78.

Correa A*, Trajtenberg F*, Obal G, Pritsch O, Dighiero G, Oppezzo P, Buschiazzo A. Structure of a human IgA1 Fab fragment at 1.55 Å resolution: potential effect of the constant domains on antigen-affinity modulation. Acta Crystallogr D Biol Crystallogr. 2013 Mar;69(Pt 3):388-97. *[These authors contributed equally to this work] 

Albanesi D, Reh G, Guerin ME, Schaeffer F, Debarbouille M, Buschiazzo A, Schujman GE, de Mendoza D, Alzari PM. Structural basis for feed-forward transcriptional regulation of membrane lipid homeostasis in Staphylococcus aureus. PLoS Pathog. 2013 Jan;9(1):e1003108.  

CONTACT

Alejandro Buschiazzo (alebus@pasteur.edu.uy)