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

Abstract

In methicillin-sensitive <i>Staphylococcus aureus</i> (MSSA), the tricarboxylic acid (TCA) cycle is known to negatively regulate production of the major biofilm-matrix exopolysaccharide, PIA/PNAG. However, methicillin-resistant <i>S. aureus</i> (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-Ery^s), 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 (<i>argH</i>::Tn and <i>rocF</i>::Tn), and in the TCA-cycle encoded succinyl-CoA synthetase, succinate dehydrogenase, aconitase, isocitrate dehydrogenase, fumarate hydratase class II, and citrate synthase II (<i>sucC</i>::Tn, <i>sdhA/B</i>::Tn, <i>acnA</i>::Tn, <i>icd</i>::Tn, <i>fumC</i>::Tn and <i>gltA</i>::Tn). Biofilm formation was significantly decreased under no flow and flow conditions by <i>argH::</i>Tn, <i>fumC::</i>Tn<i>,</i> and <i>sdhA/B::</i>Tn (range OD₄₉₂ 0.374-0.667; integrated densities 2.065-4.875) compared to UAS391-Ery^S (OD₄₉₂ 0.814; integrated density 10.676) (<i>p</i> ≤ 0.008). Cellular and matrix stains, enzymatic treatment (Proteinase K, DNase I), and reverse-transcriptase PCR-based gene-expression analysis of fibronectin-binding proteins <i>(fnbA/B)</i> and the staphylococcal accessory regulator <i>(sarA)</i> on pre-formed UAS391-Ery^s 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-Ery^s (on average 22 and 25 (±4)%) (<i>p</i> < 0.001); and (iii) down- and up-regulation of <i>fnbA/B</i> and <i>sarA</i>, respectively, in Tn-mutants compared to UAS391-Ery^S (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.