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

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-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 (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-Ery^S (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-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)%) (p < 0.001); and (iii) down- and up-regulation of fnbA/B and sarA, 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.

Keywords: MRSA; MSSA; SCCmec; argH; arginine; fnbA; fnbB; fumC; fumarate; malate; sarA; sdhA; sdhB; tricarboxylic acid cycle.

Figure 1

The urea and tricarboxylic acid (TCA)-cycles. Arginine is synthesized via the urea cycle. Carbamoyl phosphate reacts with ornithine to generate citrulline. Addition of aspartate to citrulline creates L-argininosuccinate. ATP is cleaved to AMP and pyrophosphate to drive this reaction forward. Arginine is cleaved off of L-argininosuccinate by the enzyme encoded by argH and can be used for protein synthesis. Hydrolysis of arginine generates ornithine and urea. Fumarate is the other product of the ArgH-catalyzed reaction and can be used in the TCA-cycle. Acetyl-CoA derived from pyruvate and other catabolic pathways enters the TCA-cycle. The acetyl group condenses with four-carbon oxaloacetate to produce citrate. Citrate rearranges to isocitrate, which is decarboxylated and forms NADH + H⁺ by transferring 2H⁺ + 2e⁻. 2-Oxoglutarate is decarboxylated and transfers 2H⁺ + 2e⁻ to form NADH + H⁺, while incorporating CoA to form succinyl-CoA. Succinate forms fumarate by transferring 2H⁺ + 2e⁻ resulting in FADH₂. Water is incorporated, and oxaloacetate is formed when 2H⁺ + 2e⁻ are transferred to form NADH + H⁺. The pathway marked in green highlights the proposed model for NADH reoxidation (Arnon-Buchanan cycle).

Figure 2

Effect of argininosuccinate lyase (argH), aconitate hydratase A (acnA), isocitrate dehydrogenase (icd), citrate synthase II (gltA), fumarate hydratase class II (fumC), succinate—CoA ligase (subunit beta) (sucC), succinate dehydrogenase (flavoprotein subunit) (sdhA), succinate dehydrogenase iron-sulfur protein (sdhB), and arginase (rocF) Tn mutants on the biofilm phenotype by UAS391-Ery^S. Flow biofilm images were captured after 17 h growth employing ZEN 2012 software (Zeiss) as 84 combined tile images consisting of one µm² horizontal tiles covering the entire microchannel.

Figure 3

Fluorescence microscopy observations of no flow biofilm matrix structure obtained from UAS391-Ery^S, argininosuccinate lyase (argH), fumarate hydratase class II (fumC), succinate dehydrogenase (flavoprotein subunit) (sdhA), succinate dehydrogenase iron-sulfur protein (sdhB), and citrate synthase II (gltA) Tn mutants. Since microscopy images of gltA::Tn, acnA::Tn, icd::Tn, sucC::Tn, and rocF::Tn, as well as corresponding complemented mutant biofilms were comparable to each other, the matrix formed by gltA::Tn serves as an example picture for all. The top row shows total cells stained with SYTO™ 9 and PI. The middle row shows PNAG stained with WGA Texas Red™-X Conjugate, and is combined with Bright-field imaging. The bottom row shows the protein component stained with FilmTracer™ SYPRO™ Ruby Biofilm Matrix.

Figure 4

Enzymatic treatment of preformed 24 (no flow) or 17 (flow) h-old biofilms of UAS391-Ery^S, ATCC^® 25923™ and urea or TCA-cycle Tn mutants with 100 µg/mL proteinase K (A,C) or 100 U/mL DNase I (B,D) under no flow (no flow) (A,B) and dynamic (flow) (C,D) conditions. Blanks refer to incubation in either culture medium with 10 mM Tris-HCl for proteinase K treatment or culture medium for DNase I treatment. Error bars represent 95% confidence intervals. ** refers to p < 0.001.