One Step Forward with Dry Surface Biofilm (DSB) of Staphylococcus aureus: TMT-Based Quantitative Proteomic Analysis Reveals Proteomic Shifts between DSB and Hydrated Biofilm

Abstract

The Gram-positive bacterium Staphylococcus aureus is responsible for serious acute and chronic infections worldwide and is well-known for its biofilm formation ability. Recent findings of biofilms on dry hospital surfaces emphasise the failures in current cleaning practices and disinfection and the difficulty in removing these dry surface biofilms (DSBs). Many aspects of the formation of complex DSB biology on environmental surfaces in healthcare settings remains limited. In the present study, we aimed to determine how the protein component varied between DSBs and traditional hydrated biofilm. To do this, biofilms were grown in tryptic soy broth (TSB) on removable polycarbonate coupons in the CDC biofilm reactor over 12 days. Hydrated biofilm (50% TSB for 48 h, the media was then changed every 48 h with 20% TSB, at 37 °C with 130 rpm). DSB biofilm was produced in 5% TSB for 48 h at 35 °C followed by extended periods of dehydration (48, 66, 42 and 66 h at room temperature) interspersed with 6 h of 5% TSB at 35 °C. Then, we constructed a comprehensive reference map of 12-day DSB and 12-day hydrated biofilm associated proteins of S. aureus using a high-throughput tandem mass tag (TMT)-based mass spectrometry. Further pathway analysis of significantly differentially expressed identified proteins revealed that proteins significantly upregulated in 12-day DSB include PTS glucose transporter subunit IIBC (PtaA), UDP-N-acetylmuramate-L-alanine ligase (MurC) and UDP-N-acetylenolpyruvoylglucosamine (MurB) compared to 12-day hydrated biofilm. These three proteins are all linked with peptidoglycan biosynthesis pathway and are responsible for cell-wall formation and thicker EPS matrix deposition. Increased cell-wall formation may contribute to the persistence of DSB on dry surfaces. In contrast, proteins associated with energy metabolisms such as phosphoribosyl transferase (PyrR), glucosamine--fructose-6-phosphate aminotransferase (GlmS), galactose-6-phosphate isomerase (LacA), and argininosuccinate synthase (ArgG) were significantly upregulated whereas ribosomal and ABC transporters were significantly downregulated in the 12-day hydrated biofilm compared to DSB. However, validation by qPCR analysis showed that the levels of gene expression identified were only partially in line with our TMT-MS quantitation analysis. For the first time, a TMT-based proteomics study with DSB has shed novel insights and provided a basis for the identification and study of significant pathways vital for biofilm biology in this reference microorganism.

Staphylococcus aureus cell wall phenotypic changes associated with biofilm age and water stress: a key contributing factor for biocide resistance

“…While in DSB, UDP-Nacetylenolpyruvylglucosamine reductase (MurB) and UDP-N-acetylmuramic acid: L-alanine ligase (MurC) were upregulated. murB catalyses the second committed step in peptidoglycan biosynthesis converting enolpyruvyl UDP-acetylglucosamine to UDP-N-acetylmuramic acid to which MurC adds a L-alanine 31 . The increased peptidoglycan production in biofilm cells results in increased cell wall thickness which was confirmed using TEM.…”

Repurposing of the antimalarial agent tafenoquine to combat MRSA

“…Using R software, differentially expressed proteins (DEPs) were considered as a P value of < 0.05 and |fold change| of ≥1.5. Gene Ontology (GO), InterPro (IPR), and subcellular were analyzed using GSEA (gsea-3.0) enrichment ( 39 ). All the raw data and the corresponding txt files have been deposited in ProteomeXchange via the iProX partner repository ( https://www.iprox.cn//page/project.html?id=IPX0006384000 ) with a data set identifier of IPX0006384000.…”

How biofilm changes our understanding of cleaning and disinfection

“…It is likely that DSB provides a mean for pathogen survival (including desiccation sensitive one) in a dry state in the environment, and act as a pathogen reservoir [ 26 , 128 ]. In S. aureus , proteome analysis between DSB and hydrated biofilms showed differences in the up-regulation in DSB of proteins involved in peptidoglycan biosynthesis pathway related to cell-wall formation and thicker EPS matrix deposition, which was hypothesised to contribute to DSB persistence on dry surfaces [ 129 ]. Biofilms are likely to play a role in bacterial persistence, even Gram-negative ones, in dry environments.…”