Dataset Information

Proteome changes as consequence of altered c-di-GMP metabolism in BCG

ABSTRACT: Pellicles, a type of biofilm, have gathered a renewed interest in the field of tuberculosis as a structure that mimics some characteristics occurring during M. tuberculosis infection, such as antibiotic recalcitrance and chronicity of infection. In other bacteria, it has been well documented that the second messenger c-di-GMP modulates the transition from planktonic cells to biofilm formation. In this work, we used two shotgun proteomics approaches, a label-free one to identify proteins present at two weeks of pellicle formation, and isobaric labeling to assess the differences in the proteome occurring among pellicles formed by three strains of M. bovis BCG: the parental strain (WT), a previously reported knock out in BCG1419c (∆PDE) gene and a new knock out in BCG1416c (∆DGC) gene, in order to unravel the role played by genes involved in the metabolism c-di-GMP during pellicle mode of growth. Our findings strongly suggest that in M. bovis BCG, c-di-GMP may positively correlate with enhanced tolerance to nitrosative stress and negatively to cell wall permeability, therefore suggesting hypothesis relevant for adaption to tolerance to several antibiotics.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Mycobacterium Bovis Bcg Str. Pasteur 1173p2

TISSUE(S): Prokaryotic Cell

DISEASE(S): Tuberculosis

SUBMITTER: Gustavo Diaz

LAB HEAD: Nicole Kruh-Garcia

PROVIDER: PXD005894 | Pride | 2018-03-21

REPOSITORIES: Pride

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Publications

Immune response elicited by two rBCG strains devoid of genes involved in c-di-GMP metabolism affect protection versus challenge with M. tuberculosis strains of different virulence.

Segura-Cerda Cristian Alfredo CA Aceves-Sánchez Michel de Jesús MJ Marquina-Castillo Brenda B Mata-Espinoza Dulce D Barrios-Payán Jorge J Vega-Domínguez Perla Jazmín PJ Pedroza-Roldán César C Bravo-Madrigal Jorge J Vallejo-Cardona Alba Adriana AA Hernández-Pando Rogelio R Flores-Valdez Mario Alberto MA

Vaccine 20180315 16

Pellicles, a type of biofilm, have gathered a renewed interest in the field of tuberculosis as a structure that mimics some characteristics occurring during M. tuberculosis infection, such as antibiotic recalcitrance and chronicity of infection, and as a source of antigens for humoral response in infected guinea pigs. In other bacteria, it has been well documented that the second messenger c-di-GMP modulates the transition from planktonic cells to biofilm formation. In this work, we used the liv ...[more]

PMID: 29550192

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Project description:Shewanella oneidensis is an important model organism for bioremediation studies because of its diverse respiratory capabilities. In recent years, biofilm development of S. oneidensis has been extensively studied because it is essential to reduce solid metals. As a special form of biofilm, however, pellicles are largely overlooked. The goal of this work was to understand requirements of S. oneidensis pellicle formation and the molecular basis of pellicle formation. We demonstrated that successful pellicle formation and survival was likely to require the threshold level of cell density and higher concentration of oxygen. Proteinase K and EDTA were potent pellicle disrupter. DNA microarray experiments were used to study the gene expression profile of young air–liquid interface pellicle relative to planktonic cells, which indicated that the air–liquid interface pellicle was more metabolically active than the planktonic cells. Most notably, consistently up-regulation of iron or heme uptake and transportation proteins was observed in the S. oneidensis MR-1 pellicle. However, neither the hmuT nor hugA heme transport mutant was defective in pellicle formation. An examination of the influence of several metal cations on the anti-pellicle activity of EDTA showed that Ca (II), Mn(II), Cu(II), and Zn(II) fully protected S. oneidensis MR-1 pellicle against EDTA treatment while additional of iron enabled the initiation of pellicle formation but maturation was significantly impaired. Collectively, iron was less important than other metals with respect to pellicle formation in S. oneidensis. A fresh colony grown overnight on a LB plate was used to inoculate 50 ml LB and incubated in a shaker (200 rpm) to an OD600 of 0.8 at the room temperature. This culture was then diluted 500-fold with fresh LB, resulting in the starting cultures. Aliquots of 30ml starting cultures were transferred to 50-ml Pyrex beakers and allowed to develop pellicles at the room temperature. When a complete but thin (young pellicle) at the interface were formed (about 30h hours), planktonic culture and pellicle were separated and applied to centrifugation at 8000 rpm for 3 min at room temperature. 3 parallel starting cultures were used and 3 samlpes of pellicle cells or planktonic cells were collected at 30h. RNA from the pellicle cells was fluorescently labeled with Cy3, and that from the planktonic was labeled with Cy5.

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