Biology subjects

Korn, N.

Publications and source records attributed to Korn, N..

4 recordsLinked to original sources

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↗

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↗

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↗