338. The LiaR-Regulon of Daptomycin-Resistant Enterococcus faecium
Session: Poster Abstract Session: Emerging Resistance - Epidemiology and Mechanisms
Thursday, October 5, 2017
Room: Poster Hall CD
Background: The three component regulatory system LiaFSR is a major mediator of the cell envelope response to daptomycin (DAP). LiaR is the response regulator of the system and controls the transcription of genes associated with the DAP resistance (DAP-R) phenotype. Previous structural data in E. faecium indicate that dimerization of LiaR is important to increase the affinity for the target DNA region, a specific sequence of ca. 45 nt, and activate the stress response. Indeed, substitutions in LiaR associated with DAP-R increase the oligomeric state of LiaR. However, the genes regulated by E. faecium LiaR are unknown.

Methods: In order to identify genes regulated by LiaR in E. faecium, we performed comparative RNASeq experiments using a DAP-R E. faecium strain (R497) and a liaR deletion mutant derivative (R497ΔliaR). Total RNA was isolated in early exponential phase of growth and rRNA depleted to obtain cDNA libraries. Sequencing was performed on an Illumina platform. Differential gene expression in R497 vs R497ΔliaR was calculated by DeSeq2 (p-adjusted value ˂ 0.05). We used quantitative real time PCR (qRT-PCR) to validate the differential expression observed in the RNASeq analysis. Additionally, putative LiaR DNA binding-site sequences were identified in silico by the TFBS Perl module, using a consensus probabilistic weight matrix.

Results: We found a total of 70 genes differentially expressed between R497 vs R497ΔliaR; 41 were significantly under-expressed in R497ΔliaR. Inside this group, in silico analyses revealed a putative LiaR-DNA binding sequence upstream of 12 gene clusters. The highest regulated clusters were liaFSR and liaXYZ (the main target of LiaR in E. faecalis), a result confirmed by qRT-PCR assays. Five other genes were identified and included xpaC, EF0026, EF1533, EF1066, EF1503 (E. faecalis V583 nomenclature), predicted to encode an amino acid metabolism enzyme, two hypothetical proteins, an acetyltransferase and fructose-1,6-bisphosphatase, respectively.

Conclusion: Our experimental strategy identified genes that are regulated by LiaR in E. faecium. Dissection of the function of these genes would provide important insights into the enterococal cell envelope stress response and open new targets for therapeutic intervention.

Diana Panesso, PhD1, Mauricio Latorre, PhD2,3, Sandra Rincon, MSc1, William Miller, MD4, Lorena Diaz, PhD1, Jinnethe Reyes, PhD5, Rafael Rios, MSc1, Javier Ardila, MSc1, Lina Paola Carvajal, BSc1 and Cesar Arias, MD, PhD, FIDSA6, (1)Molecular Genetics and Antimicrobial Resistance Unit - International Center for Microbial Genomics, Universidad El Bosque, Bogota, Colombia, (2)INTA, Universidad de Chile, Santiago, Chile, (3)Universidad de O´Higgins, Rancagua, Chile, (4)Department of Internal Medicine, Division of Infectious Diseases, UTHealth McGovern Medical School, Houston, TX, (5)Universidad El Bosque, Bogota, Colombia, (6)Microbiology and Molecular Genetics, University of Texas McGovern Medical School, Houston, TX

Disclosures:

D. Panesso, None

M. Latorre, None

S. Rincon, None

W. Miller, None

L. Diaz, None

J. Reyes, None

R. Rios, None

J. Ardila, None

L. P. Carvajal, None

C. Arias, None

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