Biology subjects

Wolz, C.

Publications and source records attributed to Wolz, C..

10 recordsLinked to original sources

bioRxiv2026

Phagocytosis Events Shift the Activation Trajectory of Neutrophils towards Serial Eating of S. aureus

Neutrophil recruitment and activation are essential for the control of microbial infections, yet how these cells adapt functionally upon entry into inflamed tissues remains incompletely understood. In particular, how temporal cues and direct interaction with the pathogen differentially shape neutrophil heterogeneity and effector functions remains poorly defined. Here, we employed in vivo time-stamping of tissue entry to disentangle time-dependent versus pathogen interaction-specific drivers of neutrophil heterogeneity during S. aureus skin infection. We show that canonical aging and activation markers progressively accumulated with increasing time since arrival in the inflamed tissue. Strikingly, however, phagocytic activity at the single-cell level was uncoupled from tissue dwell time and instead strictly correlated with each neutrophil's history of prior uptake of S. aureus. Furthermore, pathogen engagement induced an immediate shift towards a maximally activated phenotype. Therefore, neutrophil functional heterogeneity in infected tissues is governed less by residence time and more by discrete pathogen encounters which act as a key instructive event for subsequently enhanced phagocytosis. This suggests that the balance of microenvironmental and pathogen-derived cues tailors neutrophil activation in order to optimize antimicrobial clearance.

immunology↗

bioRxiv2025

The phage {Phi}13-encoded transcriptional regulator Ltr controls phage assembly in Staphylococcus aureus

Temperate phages play a central role in evolution and pathogenicity of Staphylococcus aureus. Sa3int phages, in particular, contribute highly human-specific virulence factors that promote immune evasion and survival within the host. The reversible excision of these phages which occurs without phage production and bacterial lysis allows the simultaneous expression of phage virulence genes and the hlb gene where they usually integrate. However, the regulatory mechanisms controlling phage assembly and the cross-talk with host factors remain poorly understood. In this study, we analyzed the regulatory mechanism controlling late gene transcription in Sa3int phage {Phi}13. We identified a functional promoter, P23, located upstream of the late phage genes that control DNA processing and packaging, capsid assembly, bacterial lysis and immune evasion. SAOUHSC_02200, the gene located upstream of P23, encodes for a late transcriptional regulator (Ltr). Mutating the P23 TATA-box or the ltr gene abolished P23 activity and formation of mature intact phage particles, thus confirming the role of Ltr in regulating P23 activity. Four direct repeats upstream of the P23 transcriptional start site were identified as potential Ltr binding sites. RT-qPCR analysis confirmed that Ltr-dependent P23 activation is essential for expression of late genes and the subsequent propagation of {Phi}13. Furthermore, comparative analysis of P23 activity and ltr expression in different host strain backgrounds revealed strain-specific differences that appear to depend on the alternative sigma factor SigB and its downstream effector SpoVG. These findings establish Ltr as the major regulator of late gene expression in {Phi}13 and reveal bacterial host factors that control successful phage assembly, and bacterial lysis. ImportanceThe dynamic integration and excision of highly prevalent Sa3int phages in Staphylococcus aureus is considered a regulatory switch that enables bacterial adaptation to specific niches. These phages carry several human-specific virulence genes and integrate into the hlb virulence gene. It was assumed that they undergo a mechanism termed active lysogeny, which allows the phages to be excised reversibly without phage production or bacterial lysis. Here, we have identified a new phage-encoded late transcriptional regulator (Ltr) that controls the expression of all late phage genes and thus phage assembly and lysis. We found that Ltr activity is regulated by the alternative sigma factor B and its downstream effector SpoVG. Restriction of SpoVG, and consequently phage assembly, likely contributes to the maintenance of Sa3int phages, even under phage-inducing conditions. This may be relevant in certain infectious conditions where both the phage-encoded virulence genes and the gene that is usually interrupted by the phage are required for infectivity.

microbiology↗

bioRxiv2024

Systematic classification of phage receptor-binding proteins predicts surface glycopolymer structure in Staphylococcus pathogens

Wall teichoic acids (WTAs) are major surface polymers of staphylococcal pathogens and commensals, whose variable structure governs interaction with host receptors, immunoglobulins, and bacteriophages. The ribitol phosphate (RboP) WTA type contributes to virulence, for instance in Staphylococcus aureus, but we lack comprehensive knowledge of WTA types and cognate phages. We developed a computational pipeline to identify the receptor-binding proteins (RBPs) in 335 Staphylococcus phage genomes, yielding multiple distinct RBP clusters. Notably, many phages had two separate RBPs with in part different WTA preferences. RBP representatives differed in specificity for RboP WTA glycosylation types, recapitulating the specificity of the corresponding phage. Based on these results, we created a publicly available bioinformatic tool to predict phage host specificity based on RBP similarity. The RboP WTA specific {Phi}13-RBP also revealed that the presence of RboP WTA on non-aureus staphylococci is more common than previously thought. Our approach facilitates the characterization of opportunistic Staphylococcus pathogens according to WTA types, which has major implications for phage-mediated interspecies horizontal gene transfer and future phage therapies.

microbiology↗

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 capsular polysaccharide obstructs wall teichoic acid functions in Staphylococcus aureus

BackgroundThe cell envelope of Staphylococcus aureus contains two major secondary cell wall glycopolymers: capsular polysaccharide (CP) and wall teichoic acid (WTA). Both the CP and the WTA are attached to the cell wall and play distinct roles in S. aureus colonization, pathogenesis, and bacterial evasion of host immune defenses. ObjectiveWe aimed to investigate whether CP interferes with WTA-mediated properties. MethodsStrains with natural heterogeneous expression of CP, strains with homogeneous high CP expression and CP-deficient strains were compared to WTA deficient controls regarding WTA dependent phage binding, cell adhesion, IgG deposition, and virulence in vivo. ResultsWTA-mediated phage adsorption, specific antibody deposition and cell adhesion were negatively correlated with CP expression. WTA, but not CP, enhanced the bacterial burden in a mouse abscess model, while CP overexpression resulted in intermediate virulence in vivo. ConclusionsCP protects the bacteria from WTA-dependent opsonization and phage binding. This protection comes at the cost of diminished adhesion to host cells. The highly complex regulation and mostly heterogeneous expression of CP has probably evolved to ensure the survival and optimal physiological adaptation of the bacterial population as a whole.

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↗

bioRxiv2021

Alpha-hemolysin of Staphylococcus aureus impairs thrombus formation

Toxins are key virulence determinants of pathogens and can impair the function of host immune cells including platelets. Insights into pathogen toxin interference with platelets will be pivotal to improve treatment of patients with bacterial bloodstream infections. In this study, we deciphered the effects of Staphylococcus aureus toxins -hemolysin, LukAB, LukDE and LukSF on human platelets and compared the effects with the pore forming toxin pneumolysin of Streptococcus pneumoniae. In contrast to pneumolysin, -hemolysin initially activates platelets as indicated by CD62P and IIb{beta}3 integrin expression, but the resulting pores also induce alterations in the phenotype of platelets and induce apoptosis of platelets. The presence of small amounts of -hemolysin (0.2 {micro}g/mL) in whole blood abrogates thrombus formation indicating that in systemic infections with S. aureus the stability of formed thrombi is impaired. This might be of high clinical relevance for S. aureus induced endocarditis of the aortic valves. Stabilizing the thrombi by inhibiting -hemolysin induced impairment of platelets likely reduces the risk for septic (micro-)embolization. However, in contrast to pneumolysin, -hemolysin induced platelets damage could not be neutralized by intravenous immune globulins. In contrast to -hemolysin, S. aureus bi-component pore forming leukocidins LukAB, LukED and LukSF do not bind to platelets and had no significant effect on platelet activation and viability. Main point 1: -hemolysin forms pores in platelets, which first activate but then result in apoptosis and impairs thrombus formation and stability Main point 2: Polyvalent immunoglobulins do not neutralize the mode of action of the toxin

microbiology↗

bioRxiv2020

Inducible expression of (pp)pGpp synthetases in Staphylococcus aureus is associated with activation of stress response genes

The stringent response is characterized by the synthesis of the messenger molecules pppGpp, ppGpp or pGpp (here collectively designated (pp)pGpp). The phenotypic consequences resulting from (pp)pGpp accumulation vary among species and can be mediated by different underlying mechanisms. Most genome-wide analyses have been performed under stress conditions, which often mask the immediate effects of (pp)pGpp-mediated regulatory circuits. In Staphylococcus aureus, (pp)pGpp can be synthesized via the RelA-SpoT-homolog (RSHSau) upon amino acid limitation or via one of the two small (pp)pGpp synthetases RelP or RelQ, upon cell wall stress. We used RNA-Seq to compare the global effects in response to transcriptional induction of the synthetase domain of RSH (RSH-Syn), RelP or RelQ without the need to apply additional stress conditions. Enzyme expression resulted in changes in the nucleotide pool similar to induction of the stringent response via the tRNA synthetase inhibitor mupirocin: a reduction in the GTP pool, an increase in the ATP pool and synthesis of pppGpp, ppGpp and pGpp. Induction of all three enzymes resulted in similar changes in the transcriptome. However, RelQ was less active than RSH-Syn and RelP, indicating strong restriction of its (pp)pGpp-synthesis activity in vivo. Genes involved in the SOS response, iron storage (e.g. ftnA, dps), oxidative stress response (e.g., katA, sodA) and the the psm1-4 and psm{beta}1-2 operons coding for cytotoxic, phenole soluble modulins (PSMs) were highly upregulated upon (pp)pGpp synthesis. Analyses of the ftnA, dps and psm genes in different regulatory mutants revealed that their (pp)pGpp-dependent regulation can occur independent of the regulators PerR, Fur, SarA or CodY. Moreover, psm expression is uncoupled from expression of the quorum sensing system Agr, the main known psm activator. The expression of central genes of the oxidative stress response protects the bacteria from anticipated ROS stress derived from PSMs or exogenous sources. Thus, we identified a new link between the stringent response and oxidative stress in S. aureus that is likely crucial for survival upon phagocytosis. SignificanceMost bacteria make use of the second messenger (pp)pGpp to reprogram bacterial metabolism under nutrient-limiting conditions. In the human pathogen Staphylococcus aureus, (pp)pGpp plays an important role in virulence, phagosomal escape and antibiotic tolerance. Here, we analyzed the immediate consequences of (pp)pGpp synthesis upon transcriptional induction of the (pp)pGpp-producing enzymes RSH, RelP or RelQ. (pp)pGpp synthesis provokes immediate changes in the nucleotide pool and severely impacts the expression of hundreds of genes. A main consequence of (pp)pGpp synthesis in S. aureus is the induction of ROS-inducing toxic phenol-soluble modulins (PSMs) and simultaneous expression of the detoxifying system to protect the producer. This mechanism is likely of special advantage for the pathogen after phagocytosis.

microbiology↗

bioRxiv2019

The 5’-NAD cap of RNAIII modulates toxin production in Staphylococcus aureus isolates

1Nicotinamide adenosine dinucleotide (NAD) has been found to be covalently attached to the 5-ends of specific RNAs in many different organisms, but the physiological consequences of this modification are largely unknown. Here we report the occurrence of several NAD-RNAs in the opportunistic human pathogen Staphylococcus aureus. Most prominently, RNAIII, a central quorum-sensing regulator of this bacteriums physiology, was found to be 5-NAD-capped to a significant extent. NAD incorporation efficiency into RNAIII was found to depend in vivo on the -1 position of the P3 promoter. Reduction of RNAIIIs NAD content led to a decreased expression of alpha- and delta-toxins, resulting in reduced cytotoxicity of the modified strains. These effects to not seem to be due to changes in RNAIIIs secondary structure upon NAD attachment, as indicated by largely unaltered patterns in in vitro chemical probing experiments. Our study represents a large step towards establishing a biological function of the 5-NAD cap, which for RNAIII in S. aureus is to modulate the expression of virulence factors.\n\n2 ImportanceNumerous organisms, including bacteria, are endowed with a 5-NAD cap in specific RNAs. While the presence of the 5-NAD cap modulates the stability of the modified RNA species, a significant biological function and phenotype have not been assigned so far. Here, we show the presence of a 5-NAD cap in RNAIII from S. aureus, a dual-function regulatory RNA involved in quorum-sensing processes and regulation of virulence factor expression. We also demonstrate that altering the natural NAD modification ratio of RNAIII leads to a decrease in exotoxin production, thereby modulating bacteriums virulence. Our work unveils a new layer of regulation of RNAIII and the agr system that might be linked to the redox state of the NAD molecule in the cell.

microbiology↗

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↗