RNA-basierte Kontrolle der Expression des Typ III Sekretionssystems von Yersinia pseudotuberculosis

Expression of Ysc-Yop type III secretion system requires a tight regulation, which is conferred by two RNA-binding proteins YopD and CsrA. The underlying study demonstrated that expression of the transcriptional master regulator lcrF of the Ysc-Yop T3SS is controlled in intertwined feedback loops by these RNA-binding proteins. CsrA is a global acting post-transcriptional regulator that coordinates metabolic and virulence gene expression. This study demonstrated that expression of csrA is precisely controlled in an autoregulatory feedback loop, indirectly on the transcriptional and directly on the post-transcriptional level. CsrA was found to directly bind to two sites in the 5'-UTR of its transcript, from which one site is located within the ribosomal binding site. By this mechanism, CsrA was shown to block translational initiation and restrict CsrA synthesis when a certain CsrA amount is reached. Under non-secretion conditions, the RNA-binding protein YopD was shown to bind to the csrA transcript thereby masking a CsrA binding site and preventing the direct autoinhibition of csrA. Analysis of the role of CsrA and YopD in the lcrF regulation demonstrated a direct interaction of both RNA-binding proteins with the yscW-lcrF transcript. The 5'-UTR of lcrF contains an RNA-thermometer structure, which prevents ribosomal access through a base-pairing mechanism. Furthermore, this study demonstrated, CsrA interacts with the ribosomal binding site of lcrF and modulates the opening state of the RNA thermometer. Both regulators modulate the stability of the lcrF transcript. CsrA stabilizes and YopD destabilizes the lcrF mRNA. This is caused by the YopD- and CsrA-dependent control of the RNA degradosome. Furthermore, new putative CsrA targets could be identified by an interactome analysis with FLAG-tagged CsrA protein. At body host temperature, CsrA specifically interacts with tRNAs and transcripts of tRNA processing proteins. In summary, this study revealed the complex interplay of YopD and CsrA in controlling the expression of the T3SS master regulator lcrF. Both regulators are in involved in the post-transcriptional control of lcrF expression under non-secretion conditions and confer an antagonistic regulation.Die Expression des Ysc-Yop Typ III Sekretionssystem setzt eine präzise Regulation voraus. Diese erfolgt durch die zwei RNA-bindenden Proteine YopD und CsrA. Die vorliegende Studie zeigte, dass die Expression des transkriptionellen Hauptregulators des Ysc-Yop T3SS lcrF in einer ineinander verschlungenen Rückkopplung durch diese RNA-bindenden Proteine kontrolliert wird. CsrA ist ein globaler posttranskriptioneller Regulator, der sowohl die Expression von metabolischen als auch Virulenzgenen koordiniert. Diese Studie zeigte, dass die Expression von csrA präzise in einer autoregulatorischen Rückkopplung kontrolliert wird - indirekt auf transkriptioneller und direkt auf posttranskriptioneller Ebene. Es konnte gezeigt werden, dass CsrA das eigene Transkript an zwei Stellen bindet, von der eine mit der ribosomalen Bindestelle überlappt. Durch diesen Mechanismus, blockt CsrA die Translation seines Transkripts und begrenzt die Synthese von weiteren CsrA Molekülen. Unter Nicht-Sekretionsbedingungen bindet das RNA-bindende Protein YopD das csrA-Transkript und maskiert somit eine der beiden CsrA Bindestelle. Dadurch wird die direkte Autoinhibition von csrA behindert. Die Analyse der Rolle von YopD und CsrA in der lcrF Regulation zeigte die direkte Interaktion beider RNA-Bindeproteine mit dem yscW-lcrF Transkript. Die 5'-UTR von lcrF enthält ein RNA-Thermometer, welches den ribosomalen Zugang durch einen Basenpaarungsmechanismus einschränkt. Desweiteren konnte in dieser Studie gezeigt werden, dass CsrA mit der ribosomalen Bindestelle von lcrF interagiert und den Öffnungsgrad des RNA-Thermometers moduliert. Die lcrF-RNA-Stabilität wird durch beide Regulatoren moduliert. CsrA stabilisiert und YopD destabilisiert das lcrF-Transkript. Dies wird durch die CsrA- und YopD-bedingte Kontrolle des RNA Degradosoms verursacht. Außerdem konnten neue mögliche CsrA-Ziel-Transkripte durch eine Interaktionsanalyse mit FLAG-markiertem CsrA-Protein aufgedeckt werden. Bei 37 °C interagiert CsrA spezifisch mit tRNAs sowie den Transkripten von tRNA-modifizierenden Proteinen. Zusammengefasst beleuchtete diese Studie das komplexe Zusammenspiel von YopD und CsrA in der Kontrolle des T3SS-Hauptregulators lcrF. Beide Regulatoren sind an der posttranskriptionellen Kontrolle der lcrF-Expression beteiligt, haben aber einen entgegengesetzten Effekt

Role of Hsp100/Clp Protease Complexes in Controlling the Regulation of Motility in Bacillus subtilis

The Hsp100/Clp protease complexes of Bacillus subtilis ClpXP and ClpCP are involved in the control of many interconnected developmental and stress response regulatory networks, including competence, redox stress response, and motility. Here we analyzed the role of regulatory proteolysis by ClpXP and ClpCP in motility development. We have demonstrated that ClpXP acts on the regulation of motility by controlling the levels of the oxidative and heat stress regulator Spx. We obtained evidence that upon oxidative stress Spx not only induces the thiol stress response, but also transiently represses the transcription of flagellar genes. Furthermore, we observed that in addition to the known impact of ClpCP via the ComK/FlgM-dependent pathway, ClpCP also affects flagellar gene expression via modulating the activity and levels of the global regulator DegU-P. This adds another layer to the intricate involvement of Clp mediated regulatory proteolysis in different gene expression programs, which may allow to integrate and coordinate different signals for a better-adjusted response to the changing environment of B. subtilis cells.DFG/Tu106/6DFG/Tu106/

Neutrophil-derived reactive agents induce a transient SpeB negative phenotype in Streptococcus pyogenes

Background Streptococcus pyogenes (group A streptococci; GAS) is the main causative pathogen of monomicrobial necrotizing soft tissue infections (NSTIs). To resist immuno-clearance, GAS adapt their genetic information and/or phenotype to the surrounding environment. Hyper-virulent streptococcal pyrogenic exotoxin B (SpeB) negative variants caused by covRS mutations are enriched during infection. A key driving force for this process is the bacterial Sda1 DNase. Methods Bacterial infiltration, immune cell influx, tissue necrosis and inflammation in patient´s biopsies were determined using immunohistochemistry. SpeB secretion and activity by GAS post infections or challenges with reactive agents were determined via Western blot or casein agar and proteolytic activity assays, respectively. Proteome of GAS single colonies and neutrophil secretome were profiled, using mass spectrometry. Results Here, we identify another strategy resulting in SpeB-negative variants, namely reversible abrogation of SpeB secretion triggered by neutrophil effector molecules. Analysis of NSTI patient tissue biopsies revealed that tissue inflammation, neutrophil influx, and degranulation positively correlate with increasing frequency of SpeB-negative GAS clones. Using single colony proteomics, we show that GAS isolated directly from tissue express but do not secrete SpeB. Once the tissue pressure is lifted, GAS regain SpeB secreting function. Neutrophils were identified as the main immune cells responsible for the observed phenotype. Subsequent analyses identified hydrogen peroxide and hypochlorous acid as reactive agents driving this phenotypic GAS adaptation to the tissue environment. SpeB-negative GAS show improved survival within neutrophils and induce increased degranulation. Conclusions Our findings provide new information about GAS fitness and heterogeneity in the soft tissue milieu and provide new potential targets for therapeutic intervention in NSTIs.publishedVersio

Prothrombotic and Proinflammatory Activities of the β-Hemolytic Group B Streptococcal Pigment

A prominent feature of severe streptococcal infections is the profound inflammatory response that contributes to systemic toxicity. In sepsis the dysregulated host response involves both immunological and nonimmunological pathways. Here, we report a fatal case of an immunocompetent healthy female presenting with toxic shock and purpura fulminans caused by group B streptococcus (GBS; serotype III, CC19). The strain (LUMC16) was pigmented and hyperhemolytic. Stimulation of human primary cells with hyperhemolytic LUMC16 and STSS/NF-HH strains and pigment toxin resulted in a release of proinflammatory mediators, including tumor necrosis factor, interleukin (IL)-1β, and IL-6. In addition, LUMC16 induced blood clotting and showed factor XII activity on its surface, which was linked to the presence of the pigment. The expression of pigment was not linked to a mutation within the CovR/S region. In conclusion, our study shows that the hemolytic lipid toxin contributes to the ability of GBS to cause systemic hyperinflammation and interferes with the coagulation system

A bacterial secreted translocator hijacks riboregulators to control type III secretion in response to host cell contact.

Numerous Gram-negative pathogens use a Type III Secretion System (T3SS) to promote virulence by injecting effector proteins into targeted host cells, which subvert host cell processes. Expression of T3SS and the effectors is triggered upon host cell contact, but the underlying mechanism is poorly understood. Here, we report a novel strategy of Yersinia pseudotuberculosis in which this pathogen uses a secreted T3SS translocator protein (YopD) to control global RNA regulators. Secretion of the YopD translocator upon host cell contact increases the ratio of post-transcriptional regulator CsrA to its antagonistic small RNAs CsrB and CsrC and reduces the degradosome components PNPase and RNase E levels. This substantially elevates the amount of the common transcriptional activator (LcrF) of T3SS/Yop effector genes and triggers the synthesis of associated virulence-relevant traits. The observed hijacking of global riboregulators allows the pathogen to coordinate virulence factor expression and also readjusts its physiological response upon host cell contact

Broad Spectrum Antibiotic Xanthocillin X Effectively Kills Acinetobacter baumannii via Dysregulation of Heme Biosynthesis

Isonitrile natural products exhibit promising antibacterial activities. However, their mechanism of action (MoA) remains largely unknown. Based on the nanomolar potency of xanthocillin X (Xan) against diverse difficult-to-treat Gram-negative bacteria, including the critical priority pathogen Acinetobacter baumannii, we performed in-depth studies to decipher its MoA. While neither metal binding nor cellular protein targets were detected as relevant for Xan’s antibiotic effects, sequencing of resistant strains revealed a conserved mutation in the heme biosynthesis enzyme porphobilinogen synthase (PbgS). This mutation caused impaired enzymatic efficiency indicative of reduced heme production. This discovery led to the validation of an untapped mechanism, by which direct heme sequestration of Xan prevents its binding into cognate enzyme pockets resulting in uncontrolled cofactor biosynthesis, accumulation of porphyrins, and corresponding stress with deleterious effects for bacterial viability. Thus, Xan represents a promising antibiotic displaying activity even against multidrug resistant strains, while exhibiting low toxicity to human cells.U.S. Department of Veterans Affair

Molecular profiling of tissue biopsies reveals unique signatures associated with streptococcal necrotizing soft tissue infections

Necrotizing soft tissue infections (NSTIs) are devastating infections caused by either a single pathogen, predominantly Streptococcus pyogenes, or by multiple bacterial species. A better understanding of the pathogenic mechanisms underlying these different NSTI types could facilitate faster diagnostic and more effective therapeutic strategies. Here, we integrate microbial community profiling with host and pathogen(s) transcriptional analysis in patient biopsies to dissect the pathophysiology of streptococcal and polymicrobial NSTIs. We observe that the pathogenicity of polymicrobial communities is mediated by synergistic interactions between community members, fueling a cycle of bacterial colonization and inflammatory tissue destruction. In S. pyogenes NSTIs, expression of specialized virulence factors underlies infection pathophysiology. Furthermore, we identify a strong interferon-related response specific to S. pyogenes NSTIs that could be exploited as a potential diagnostic biomarker. Our study provides insights into the pathophysiology of mono- and polymicrobial NSTIs and highlights the potential of host-derived signatures for microbial diagnosis of NSTIs

Discovery of the first small-molecule CsrA-RNA interaction inhibitors using biophysical screening technologies.

CsrA is a global post-transcriptional regulator protein affecting mRNA translation and/or stability. Widespread among bacteria, it is essential for their full virulence and thus represents a promising anti-infective drug target. Therefore, we aimed at the discovery of CsrA-RNA interaction inhibitors. Results & methodology: We followed two strategies: a screening of small molecules (A) and an RNA ligand-based approach (B). Using surface plasmon resonance-based binding and fluorescence polarization-based competition assays, (A) yielded seven small-molecule inhibitors, among them MM14 (IC50 of 4 µM). (B) resulted in RNA-based inhibitor GGARNA (IC50 of 113 µM)