Biology subjects

Salzer, A.

Publications and source records attributed to Salzer, A..

4 recordsLinked to original sources

bioRxiv2024

(p)ppGpp-mediated GTP homeostasis ensures the survival and antibiotic tolerance of Staphylococcus aureus during starvation by preserving the proton motive force

Upon nutrient limitation bacteria enter a nongrowing state, which allow bacterial survival and antibiotic tolerance. The mechanisms whether and how the messenger molecule (p)ppGpp contributes to the transition in Firmicutes is debated. Here we show for Staphylococcus aureus that (p)ppGpp-dependent restriction of the GTP pool is essential for the culturability of starved cells and for antibiotic tolerance. Elevated GTP levels in a starving (p)ppGpp-deficient mutant lead to a division-incompetent, dormant state characterized by reduced metabolic activity and alterations in membrane function and architecture. GTP level control of nucleotide sensitive promoters result in transcriptional downregulation of gene of the TCA cycle and electron transport chain. Increasing transcription of qoxABCD, a terminal oxidase of the respiratory chain, through mutation of the transcriptional start site partially restored the culturability of the (p)ppGpp-deficient mutant. Furthermore, we showed that the maintenance of proton motive force under nutritional stress contributes to antibiotic tolerance, supporting the idea of applying (p)ppGpp or PMF inhibitors to combat antibiotic-tolerant bacteria.

microbiology↗

bioRxiv2023

The biocide triclosan induces (p)ppGpp dependent antibiotic tolerance and alters SarA dependent biofilm structures in Staphylococcus aureus

The biocide triclosan is used extensively in both household and hospital settings. The chronic exposure to the biocide occurring in individuals that use triclosan-containing products results in low levels of triclosan present in the human body that has been linked to induction of antibiotic tolerance and altered biofilm formation. Here we aimed to unravel the molecular mechanisms involved in triclosan induced antibiotic tolerance and biofilm formation in Staphylococcus aureus. Triclosan treatment prior to planktonic exposure to bactericidal antibiotics resulted in 1,000 fold higher viable cell counts compared to non-pretreated cultures. Triclosan pretreatment also protected S. aureus biofilms against otherwise lethal doses of antibiotics as shown by live/dead cell staining and viable cell counting. Triclosan mediated antibiotic tolerance in planktonic and biofilm cultures required an active stringent response because a pppGpp0 strain was not protected from antibiotic killing. Incubation of S. aureus with triclosan also altered biofilm structure due to SarA-mediated overproduction of the polysaccharide intercellular adhesin (PIA) in the biofilm matrix. Thus, physiologically relevant concentrations of triclosan can trigger (p)ppGpp dependent antibiotic tolerance as well as SarA dependent biofilm formation. ImportanceThe prevalent bacterium Staphylococcus aureus infects skin lesions and indwelling devices, and this can cause sepsis with 33% mortality. Intrinsic to this is the formation of co-ordinated communities (biofilms) protected by a polysaccharide coat. S. aureus is increasingly difficult to eradicate due to its antibiotic resistance. Protection against Methicillin Resistant S. aureus (MRSA) includes pre-hospital admission washing with products containing biocides. The biocide triclosan is the predominant antibacterial compound in sewage in Ontario due to its use in household and hospital settings. Levels of triclosan accumulate with exposure in humans. The significance of our research is in identifying the mechanisms triggered by exposure of S. aureus to physiological levels of triclosan that go on to raise the tolerance of S. aureus to antibiotics and promote the formation of biofilms. This understanding will inform future criteria used to determine effective antimicrobial treatments.

microbiology↗

bioRxiv2019

The BIR2/BIR3-interacting Phospholipase D gamma 1 negatively regulates immunity in Arabidopsis

Plants have evolved effective strategies to defend themselves against pathogen invasion. Starting from the plasma membrane with the recognition of microbe-associated molecular patterns (MAMPs) via pattern recognition receptors, internal cellular signaling pathways are induced to ultimately fend off the attack. Phospholipase D (PLD) hydrolyzes membrane phospholipids to produce phosphatidic acid (PA), which has been proposed to play a second messenger role in immunity. The Arabidopsis PLD family consists of 12 members and for some a specific function in resistance towards a subset of pathogens has been shown. We demonstrate here that Arabidopsis PLD{gamma}1, but not its close homologs PLD{gamma}2 and PLD{gamma}3, is specifically involved in plant immunity. Genetic inactivation of PLD{gamma}1 resulted in increased resistance towards the virulent bacterium Pseudomonas syringae pv. tomato DC3000 and the necrotrophic fungus Botrytis cinerea. As pld{gamma}1 mutant plants responded with elevated levels of reactive oxygen species to MAMP-treatment, a negative regulatory function for this PLD isoform is proposed. Importantly, PA levels in pld{gamma}1 mutants were not affected compared to stressed wild-type plants, suggesting that alterations in PA levels are unlikely the cause for the enhanced immunity in the pld{gamma}1 line. Instead, the plasma-membrane-attached PLD{gamma}1 protein colocalized and associated with the receptor-like kinases BIR2 and BIR3, which are known negative regulators of pattern-triggered immunity. Moreover, complex formation of PLD{gamma}1 and BIR2 was further promoted upon MAMP-treatment. Hence, we propose that PLD{gamma}1 acts as a negative regulator of plant immune responses in complex with immunity-related proteins BIR2 and BIR3.\n\nOne-sentence summaryA phospholipase D is a novel negative regulator of plant immunity and forms complexes with regulatory receptor-like kinases.

plant biology↗

bioRxiv2019

Revisiting the regulation of the capsular polysaccharide biosynthesis gene cluster in Staphylococcus aureus

In Staphylococcus aureus, the capsular polysaccharide (CP) protects against phagocytosis, but also hinders adherence to endothelial cells and matrix proteins. Its biosynthesis is tightly controlled resulting in a heterogeneous phenotype within a population and CP being mainly detectable in non-growing cells. Capsular biosynthesis genes are encoded by a conserved capA-P operon whose expression is driven by an upstream promoter element (Pcap) in front of capA. The organization of Pcap is poorly understood, as is the interplay of different regulators that influence the early-Off/late-Heterogeneous cap transcription pattern. Here, we demonstrate that Pcap contains a main SigB-dependent promoter. The SigB consensus motif overlaps with a previously described inverted repeat that is crucial for cap expression. The essentiality of the inverted repeat is derived from this region acting as a SigB binding site rather than as an operator site for the proposed cap activators RbsR and MsaB. Furthermore, Pcap contains an extensive upstream region harboring a weak SigA-dependent promoter and binding sites for the cap repressors SaeR, CodY and Rot. We show that heterogeneous CP synthesis is determined by the combination of SigB activity and repressor binding to the upstream region. The direct SigB dependency and the upstream repressors are also sufficient to explain the temporal gene expression pattern at the transcriptional level. However, CP synthesis remains growth phase-dependent even when capA transcription is rendered constitutive, suggesting additional post-transcriptional regulatory circuits. Thus, the interference of multiple repressors with SigB-dependent promoter activity as well as post-transcriptional mechanisms ensure the appropriate regulation of CP synthesis.\n\nImportanceThe majority of bacterial pathogens produce an array of polysaccharides on their surface which are important virulence factors and thus serve as attractive vaccine candidates. However, the synthesis and assembly of these structures is highly variable and tightly regulated at various levels. In the human pathogen Staphylococcus aureus, the synthesis of the capsular polysaccharide (CP) is dependent on a complex regulatory network which ensures that CP is produced only in a fraction of stationary phase cells. Here, we determined main regulators that drive the peculiar CP expression pattern. We found that the interplay of the transcriptional repressors Sae, CodY and Rot with the alternative Sigma factor B is responsible for early-Off/late-Heterogeneous expression at the transcriptional level. The data also implicates post-transcriptional mechanisms that may act to avoid conflict in precursor usage by machineries involved in either synthesis of CP or other glycopolymers in growing bacterial cells.

microbiology↗