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RNA-based control of Listeria adaptation to stress and virulence

Research summary:

Over the past few years, our group has been interested in the study of surface proteins of the bacterium Listeria monocytogenes. Ingestion of this bacterial pathogen in contaminated food results in serious pathologies such as meningoencephalitis and abortion. The properties of this pathogen that hinder its control in food include the growth at low temperature (4ºC) and its ability to cope with stress conditions such as high osmolarity and low pH. One of our aims is to analyze the protein content associated with the cell wall when L. monocytogenes adapts to grow at low temperature (4ºC) and to investigate its role in the adaptive response to cold.

In addition, RNA sequencing data (RNASeq) at 4ºC show changes in the expression of a defined group of small non-coding RNA (sRNAs) with regulatory function. Here, we intend to elucidate regulatory molecular mechanisms responsible for the adaptive response to cold. In particular, to identify new transcriptional regulators and regulatory RNAs we are analyzing gene expression patterns during the cold 'acclimatization' phase (Fig. 1). Due to the strong effects of temperature on RNA structure and function and given that the majority of sRNAs are expressed both at host temperature (37ºC) and 4ºC we consider that the same regulatory RNA could function differently at the two temperatures, a concept not yet explored in bacterial pathogens.

Finally, we would like to decipher how RNA-based regulatory networks control changes in the surface proteins profile during the temperature transition and their connection to virulence. Some important functions for infection are also expressed by the pathogen at high levels during growth at 4ºC. Understanding the biology of L. monocytogenes in the cold with the participation of regulatory RNAs could unveil novel targets for contamination control in refrigerated food and extrapolate the results to other pathogens with similar capabilities for growing at low temperatures.

Image

Figure 1: Gene expression analysis during L. monocytogenes adaptation to cold. Left panel. Lab procedure to the explore molecular changes in Listeria adaptation to refrigeration temperatures. Exponentially-growing bacteria at optimal temperature are subjected to a sudden temperature downshift and subsequently collected along the acclimation and cold-adapted phases. Right panel. Hierarchical clustering analysis of Listeria gene expression patterns at the different time points after temperature downshift.

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* For external calls please dial 34 91196 followed by the extension number
Last nameNameLaboratoryExt.*e-mailProfessional category
Pucciarelli MorroneMaría Graciela1084497mg.pucciarelli(at)uam.esProfesor Titular Universidad, GA

Relevant publications:

  • An alternative penicillin-binding protein involved in Salmonella relapses following ceftriaxone therpy. Castanheira, S., López-Escarpa, D., Pucciarelli, MG, Cestero, JJ, Baquero, F. García-del Portillo, F. EBioMedicine (2020) 55: 102771. doi: 10.1016/ j.ebiom. 2020.102771.
  • Two distinct penicillin binding proteins promote cell division in different Salmonella lifestyles. Castanheira S, Cestero JJ, García-Del Portillo F. *, Pucciarelli MG.* Microbial Cell (2018) 5(3):165-168. doi: 10.15698/mic2018.03.622.
  • Bley-Folly B, Ortega AD* , Hubmann G, Bonsing-Vedelaar S, Wijma HJ, van der Meulen P, Milias-Argeitis A, Heinemann M† (2018) Assessment of the interaction between the flux-signaling metabolite fructose-1,6-bisphosphate and the bacterial transcription factors CggR and Cra. Mol Microbiol. 109:278-290. doi: 10.1111/mmi.14008.
  • Pucciarelli MG, García-del Portillo F. Within-host envelope remodelling and its impact in bacterial recognition. Current Issues Molecular Biology (2018) 25:43-60. doi: 10.21775/cimb.025.043. Epub 2017 Sep 6.
  • Litsios A*, Ortega ÁD*, Wit EC, Heinemann M†. (2018) Metabolic-flux dependent regulation of microbial physiology. Current Opinion Microbiology. 42:71-78. doi: 10.1016/j.mib.2017.10.029.
  • Castanheira S, Cestero JJ, Rico-Pérez G, García P, Cava F, Ayala JA, Pucciarelli MG*, García-Del Portillo F.* A specialized peptidoglycan synthase promotes Salmonella cell division inside host cells. mBIO (2017) 8(6) doi: 10.1128/mBio.01685-17.
  • Impens F, Rolhion N, Radoshevich L, Bécavin C, Duval M, Mellin J, García-del Portillo F, Pucciarelli MG, Williams AH, Cossart P. N-terminomics identifies Prli42 as a membrane miniprotein conserved in Firmicutes and critical for stressosome activation in Listeria monocytogenes. Nature Microbiology (2017) 2:17005. doi: 10.1038/nmicrobiol.2017.5.
  • García-del Portillo F, Pucciarelli MG. RNA-Seq unveils new attributes of the heterogeneous Salmonella-host cell communication. RNA Biology (2017) 14:429-435. doi: 10.1080/ 15476286.2016.1276148.
  • Pucciarelli MG, Rodríguez L, García-Del Portillo F. A disulfide bond in the membrane protein IgaA is essential for repression of the RcsCDB system. Frontiers Microbiology (2017) 8:2605. doi: 10.3389/fmicb.2017.02605. eCollection 2017.
  • Pucciarelli MG*, García-Del Portillo F. Salmonella lifestyles and their impact on host-to-host transmission. Microbiology Spectrum (2017) 5(4). doi: 10.1128/microbiolspec.MTBP-0009-2016.
  • Pinheriro J, Reis O, Vieira A, Moura IM, Zanolli Moreno L, Carvalho F, Pucciarelli MG, García del-PortilloF, Sousa S, Cabanes D. Listeria monocytogenes encodes a functional ESX-1 secretion system whose expression is detrimental to in vivo infection. Virulence (2017) 18;8(6):993-1004. doi: 10.1080/21505594.2016.1244589.
  • Radzikowski JL, Vedelaar S, Siegel D, Ortega AD, Schmidt A, Heinemann M†. (2016) Bacterial persistence is an active sigma(S) stress response to metabolic flux limitation. Mol Sys Biol 12(9): 882. doi: 10.15252/msb.20166998.
  • Quereda JJ, García-del Portillo F, Pucciarelli MG*. Listeria monocytogenes remodels the cell surface in the blood-stage. Environmental Microbiology Reports (2016), doi: 10.1111/1758-2229.12416.
  • Rico-Pérez G, Pezza A, Pucciarelli MG, de Pedro MA, Soncini FC,García-Del Portillo F. A novel peptidoglycan D,L-endopeptidase induced by Salmonella inside eukaryotic cells contributes to virulence. Molecular Microbiology (2016) 99:546-56. doi: 10.1111/mmi.13248.
  • Ortega AD, Quereda JJ, Pucciarelli MG, García-del Portillo F. Bacterial non-coding RNAs in the interaction of intracellular pathogens with eukaryotic cells. Frontiers in Cellular and Infection Microbiology (2014), 4:162. doi: 10.3389/fcimb.2014.00162.
  • Quereda JJ, Ortega AD, Pucciarelli MG*, García-del Portillo F*. The Listeria small RNA Rli27 regulates a cell wall protein inside eukaryotic cells by targeting a long 5’-UTR variant. PLoS Genetics (2014),10(10): e1004765. doi: 10.1371/journal.pgen.1004765.
  • Quereda JJ, Pucciarelli MG*. Deletion of the membrane protein Lmo0412 increases the virulence of Listeria monocytogenes. Microbes Infection (2014), Aug;16(8):623-32.

(*, corresponding author; †, equal contribution) 

Doctoral theses:

  • Gadea Rico-Pérez. “Metabolismo del peptidoglicano de Salmonella en el interior de células eucariotas” Department of Molecular Biology, Facultad de Ciencias, UAM. 15 de octubre 2015. Apto Cum Laude. Thesis supervisor: M. Graciela Pucciarelli and Francisco García-del Portillo.
  • Lorena Domínguez Acuña. “Análisis de funciones de Salmonella enterica que responden al estado de no proliferación en el interior de fibroblastos”. Department of Molecular Biology, Facultad de Ciencias, UAM. 17 de octubre 2012. Apto Cum Laude. Thesis supervisor: M. Graciela Pucciarelli and Francisco García-del Portillo.
  • Enrique Calvo Alcocer. “Aplicación de técnicas proteómicas al estudio de nuevos factores de virulencia en Listeria”. Facultad de Ciencias, UAM. 14 de enero 2010. Award to the best Thesis in Molecular Biology, ERA-NET Pathogenomics Network. Apto Cum Laude. Thesis supervisor: M. Graciela Pucciarelli and Francisco García-del Portillo.

Patents:

  • García-del Portillo, F., Rico-Pérez, G., Ramón-Marquès, E, San Félix, A.R., Velázquez, S., De La Puente, S., García, E., De Castro, S., Pucciarelli, M.G.
    Title:  Peptides containing D-Alanine (D-Ala) or related amino alcohols.
    Ref:  P201931007 (year 2019).
    Entities: CSIC-UAM
  • García-del Portillo, F., Castanheira, S., Cestero, J.J., López, D. Pucciarelli, M.G.
    Title:  Inhibitors of intracellular bacterial growth and persistence as antibiotics.
    Ref:  EP20382026 (year 2020).
    Entities: CSIC-UAM

Research projects and grants:

  • Regulación por RNA en la adaptación de Listeria al frío. IP: M. Graciela Pucciarelli and Álvaro D. Ortega. Programa Nacional de Biotecnología, Ministerio de Ciencia, Innovación y Universidades (ref. PGC2018-096364-B-I00).  Desde 01-01-2019 hasta 31-12-2021.
  • Análisis molecular de proteínas de superficie de Listeria y su aplicabilidad en higiene alimentaria. IP: M. Graciela Pucciarelli. Programa Nacional de Biotecnología, Ministerio de Economía y Competitividad (ref. BIO2014-55238-R). Desde 01-01-2015 hasta 30-06-2018.
  • Regulación de las proteínas de superficie LPXTG de Listeria monocytogenes. IP: M. Graciela Pucciarelli. Programa Nacional de Biotecnología, Ministerio de Ciencia e Innovación (ref. BIO2010- 18962). Desde 01-01-2011 hasta 31-12-2013.
  • Post-genómica funcional en Listeria: papel en virulencia de nuevas proteínas de superficie. IP: M. Graciela Pucciarelli. Programa Nacional de Biotecnología, Ministerio de Educación y Ciencia (ref. BIO2006- 14230). Desde 01-10-2006 hasta 30-09-2009.
  • Training network to understand and exploit mechanisms of sensory perception in bacteria (PATHSENSE). European Commission. Marie Sklodowska-Curie Training Networks (ITN). Call: H2020-MSCA-ITN-2016. Ref.: MSCA-ITN-ETN-721456. Starting date: 01/04/2017.
  • Red de control de Salmonelosis en Iberoamérica (SALMOIBER). Entidad: CYTED, Programa Iberoamericano de Ciencia y Tecnología para el Desarrollo. Ref: 215RT0493. Duración: 2015- 2018. I.P.: Francisco García del Portillo, CNB-CSIC. 8 grupos.

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