An Infrared Nanospectroscopy Study of X. vesicatoria Xcv Bv5–4a Cell Envelope Alterations Produced by P. fluorescens SF4c tailocins.

MARICRUZ FERNÁNDEZ; SONIA FISCHER; VIVIANA LÓPEZ RAMÍREZ; RAUL O. FREITAS; FRANCISCO C. B. MAIA; GUSTAVO M. MORALES

Encuentro; 30th edition of LNLS Annual Users? Meeting (RAU); 2020

Brazilian Center for Research in Energy and Materials (CNPEM)

The continuing global spread of antibiotic-resistant bacterial pathogens requires the development of alternative therapeutic strategies and bacteriocins are envisioned as an alternative [1]. Bacteriocins are peptides or proteins produced by bacteria that exhibit narrow or broader activity spectra. Pseudomonas fluorescens SF4c produces tailocins, a phage-tail-like bacteriocins with antimicrobial activity against Xanthomonas vesicatoria Xcv Bv5-4a, a phytopathogenic strains responsible of bacterial-spot disease in tomato. We demonstrated, that tailocins adhere to the cell envelope of strain Xcv Bv5-4a and cause the cellular death [2]. Although it has been proposed that the action mechanism involve the pore formation in the membrane, direct structural evidence of the damage at the molecular level is missing. Here we report studies using a high resolution microscopy (AFM) in combination with a spectroscopic technique (IR scattering Scanning NearField Optical Microscopy, IR s-SNOM) to obtain information of surface topography simultaneously with opto-vibrational information in the near field overcoming the Abbe's diffraction limit of spatial resolution. Besides the morphological differences observed between individuals X. vesicatoria Xcv Bv5-4a cells untreated (control) and treated with tailocins of P. fluorescens SF4c; the principal component analysis (PCA) computed on the set of all acquired spectra allows to differentiate between the treated and control cells. The region from 1800 to 900 cm-1 assigned mainly to vibratory modes related to membrane phospholipids, ν C=O of lipids (1755 cm-1 ) and νas P=O of phosphate groups (1240 cm-1 ) are modified, and can be associated to alterations in the structure of the cell envelope of Xcv Bv5-4a cells when are treated with tailocins. The results demonstrate that IR s-SNOM facilities available at the LNLS enable to study the e biochemical nature to single bacterium level at a nanoscale, without any special sample preparation.