Fatty acid kinase A is an important determinant of biofilm formation in Staphylococcus aureus USA300

BMC Genomics

Research Article

S. Malhotra-Kumar¹  S. De Backer²  A. Turlej-Rogacka²  B. B. Xavier²  H. Goossens²  P. Moons²  J. S. Sabirova³  H. De Greve⁴  J-P Hernalsteens⁵ 

[1] Department of Medical Microbiology, Campus Drie Eiken, University of Antwerp, S6, Universiteitsplein 1, B-2610, Wilrijk, Belgium;Laboratory of Medical Microbiology, Vaccine and Infectious Disease Institute, University of Antwerp, Antwerp, Belgium;Laboratory of Medical Microbiology, Vaccine and Infectious Disease Institute, University of Antwerp, Antwerp, Belgium;Viral Genetics Research Group, Vrije Universiteit Brussel, Brussels, Belgium;Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium;Structural and Molecular Microbiology, Structural Biology Research Center, VIB, Brussels, Belgium;Viral Genetics Research Group, Vrije Universiteit Brussel, Brussels, Belgium;

关键词: vfrB;    fakA;    MRSA;    S. aureus;    Transposon mutagenesis;    Whole genome sequencing;    SNPs;    USA300-UAS391;    USA300_UAS391;    USA300;    UAS391;   

DOI  :  10.1186/s12864-015-1956-8

 received in 2015-05-22, accepted in 2015-09-25,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA)-USA300 is notorious for its ability to cause community- and healthcare-acquired infections, which are even more difficult to treat when associated with a biofilm phenotype. We aimed to characterize the genetic determinants of biofilm formation in a USA300 skin abscess isolate (UAS391) that formed prolific biofilms.MethodsUSA300 S. aureus strains, TCH1516 and FPR3757, were found to be closely related based on whole genome mapping (Argus™ Optical Mapping System, Opgen Inc, Gaithersburg, USA) to UAS391 (96.3-99.1 % similarity, P=0.0151), however differed markedly in biofilm formation (P=0.0001) on a dynamic assay (BioFlux 200, Fluxion Biosciences, USA). Comparison of whole genome sequences of these strains identified differences in a total of 18 genes. Corresponding Tn (bursa aurealis-bearing) knockout mutants in these target genes were obtained from a publicly available mutant library of the same clonal lineage (USA300-JE2) and were characterized phenotypically for biofilm formation. Tn mutants showing significant differences in biofilm formationwere utilized for transduction into a plasmid-cured erythromycin-sensitive derivative of UAS391 and for complementation experiments. All strains were tested on the dynamic assay, and 17h-biofilms were stained (SYTO9, Life Technologies) and fluorescence intensity quantified by microscopy (Zeiss, ImageJ). Gene expression levels in Tn and transduced mutants were studied by quantitative reverse transcriptase PCR (StepOnePlusTM, Applied Biosystems®).ResultsComparison of the sequenced genomes of TCH1516, FPR3757 and UAS391 yielded a limited number of variant genes (n=18) that were hypothesized to account for the observed difference in biofilm-forming capacity. Screening of Tn mutants disrupted in these target genes identified one mutant (NE229) bearing a transposon insertion in SAUSA300_1119 (fakA), which exhibited increased biofilm formation similar to UAS391 (P=0.9320). Transduction experiments confirmed that fakA::Tn corresponded to 1.9- to 4.6-fold increase in biofilm formation depending on the USA300 strain background (P≤0.0007), while complementation of the TCH1516 wild-type fakA allele in UAS391 resulted in a 4.3-fold reduction in biofilm formation (P<0.0001).ConclusionsThis sequential approach, consisting of strain typing, genome comparison and functional genomics, identified fakA, a recently described fatty acid kinase in S. aureus that is essential for phospholipid synthesis and also impacts the transcription of numerous virulence factors, as a negative regulator of biofilm formation in S. aureus USA300.

【 授权许可】

CC BY   
© Sabirova et al. 2015