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Microorganisms 2018, 6(4), 113; https://doi.org/10.3390/microorganisms6040113

Enzymes Catalyzing the TCA- and Urea Cycle Influence the Matrix Composition of Biofilms Formed by Methicillin-Resistant Staphylococcus aureus USA300

1
Department of Medical Microbiology, Vaccine & Infectious Diseases Institute, University of Antwerp, 2610 Wilrijk, Belgium
2
Structural & Molecular Microbiology, VIB-VUB Center for Structural Biology, 1050 Brussels, Belgium
3
Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussels, Belgium
4
Viral Genetics, Vrije Universiteit Brussel, 1050 Brussels, Belgium
*
Author to whom correspondence should be addressed.
Received: 27 July 2018 / Revised: 17 October 2018 / Accepted: 25 October 2018 / Published: 29 October 2018
(This article belongs to the Special Issue Staphylococcus aureus Infection and Antimicrobial Resistance)
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Abstract

In methicillin-sensitive Staphylococcus aureus (MSSA), the tricarboxylic acid (TCA) cycle is known to negatively regulate production of the major biofilm-matrix exopolysaccharide, PIA/PNAG. However, methicillin-resistant S. aureus (MRSA) produce a primarily proteinaceous biofilm matrix, and contribution of the TCA-cycle therein remains unclear. Utilizing USA300-JE2 Tn-mutants (NARSA) in genes encoding TCA- and urea cycle enzymes for transduction into a prolific biofilm-forming USA300 strain (UAS391-Erys), we studied the contribution of the TCA- and urea cycle and of proteins, eDNA and PIA/PNAG, to the matrix. Genes targeted in the urea cycle encoded argininosuccinate lyase and arginase (argH::Tn and rocF::Tn), and in the TCA-cycle encoded succinyl-CoA synthetase, succinate dehydrogenase, aconitase, isocitrate dehydrogenase, fumarate hydratase class II, and citrate synthase II (sucC::Tn, sdhA/B::Tn, acnA::Tn, icd::Tn, fumC::Tn and gltA::Tn). Biofilm formation was significantly decreased under no flow and flow conditions by argH::Tn, fumC::Tn, and sdhA/B::Tn (range OD492 0.374−0.667; integrated densities 2.065−4.875) compared to UAS391-EryS (OD492 0.814; integrated density 10.676) (p ≤ 0.008). Cellular and matrix stains, enzymatic treatment (Proteinase K, DNase I), and reverse-transcriptase PCR-based gene-expression analysis of fibronectin-binding proteins (fnbA/B) and the staphylococcal accessory regulator (sarA) on pre-formed UAS391-Erys and Tn-mutant biofilms showed: (i) < 1% PIA/PNAG in the proteinaceous/eDNA matrix; (ii) increased proteins under no flow and flow in the matrix of Tn mutant biofilms (on average 50 and 51 (±11)%) compared to UAS391-Erys (on average 22 and 25 (±4)%) (p < 0.001); and (iii) down- and up-regulation of fnbA/B and sarA, respectively, in Tn-mutants compared to UAS391-EryS (0.62-, 0.57-, and 2.23-fold on average). In conclusion, we show that the biofilm matrix of MRSA-USA300 and the corresponding Tn mutants is PIA/PNAG-independent and are mainly composed of proteins and eDNA. The primary impact of TCA-cycle inactivation was on the protein component of the biofilm matrix of MRSA-USA300. View Full-Text
Keywords: MRSA; MSSA; SCCmec; argH; sdhB; sdhA; fumC; tricarboxylic acid cycle; fnbA; fnbB; sarA; arginine; fumarate; malate MRSA; MSSA; SCCmec; argH; sdhB; sdhA; fumC; tricarboxylic acid cycle; fnbA; fnbB; sarA; arginine; fumarate; malate
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De Backer, S.; Sabirova, J.; De Pauw, I.; De Greve, H.; Hernalsteens, J.-P.; Goossens, H.; Malhotra-Kumar, S. Enzymes Catalyzing the TCA- and Urea Cycle Influence the Matrix Composition of Biofilms Formed by Methicillin-Resistant Staphylococcus aureus USA300. Microorganisms 2018, 6, 113.

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