The RNA Chaperone Hfq Is Essential for Virulence and Modulates the Expression of Four Adhesins in Yersinia enterocolitica

In Enterobacteriaceae, the RNA chaperone Hfq mediates the interaction of small RNAs with target mRNAs, thereby modulating transcript stability and translation. This post-transcriptional control helps bacteria adapt quickly to changing environmental conditions. Our previous mutational analysis showed that Hfq is involved in metabolism and stress survival in the enteropathogen Yersinia enterocolitica. In this study we demonstrate that Hfq is essential for virulence in mice and influences production of surface pathogenicity factors, in particular lipopolysaccharide and adhesins mediating interaction with host tissue. Hfq inhibited the production of Ail, the Ail-like protein OmpX and the MyfA pilin post-transcriptionally. In contrast Hfq promoted production of two major autotransporter adhesins YadA and InvA. While protein secretion in vitro was not affected, hfq mutants exhibited decreased protein translocation by the type III secretion system into host cells, consistent with decreased production of YadA and InvA. The influence of Hfq on YadA resulted from a complex interplay of transcriptional, post-transcriptional and likely post-translational effects. Hfq regulated invA by modulating the expression of the transcriptional regulators rovA, phoP and ompR. Therefore, Hfq is a global coordinator of surface virulence determinants in Y. enterocolitica suggesting that it constitutes an attractive target for developing new antimicrobial strategies

Lah is a transmembrane protein and requires Spa10 for stable positioning of Woronin bodies at the septal pore of Aspergillus fumigatus

Woronin bodies are specialized, fungal-specific organelles that enable an immediate closure of septal pores after injury to protect hyphae from excessive cytoplasmic bleeding. In most Ascomycetes, Woronin bodies are tethered at the septal pore by so-called Lah proteins. Using the pathogenic mold Aspergillus fumigatus as a model organism, we show that the C-terminal 288 amino acids of Lah (LahC(288)) bind to the rim of the septal pore. LahC(288) essentially consists of a membrane spanning region and a putative extracellular domain, which are both required for the targeting to the septum. In an A. fumigatus rho4 deletion mutant that has a severe defect in septum formation, LahC(288) is recruited to spot-like structures in or at the lateral membrane. This suggests that LahC is recruited before Rho4 starts to govern the septation process. Accordingly, we found that in wild type hyphae Lah is bound before a cross-wall emerges and thus enables a tethering of Woronin bodies at the site of the newly formed septum. Finally, we identified Spa10, a member of a recently described family of septal poreassociated proteins, as a first protein that directly or indirectly interacts with LahC to allow a stable positioning of Woronin bodies at the mature septum

The RNA Chaperone Hfq Is Essential for Virulence and Modulates the Expression of Four Adhesins in Yersinia enterocolitica

In Enterobacteriaceae, the RNA chaperone Hfq mediates the interaction of small RNAs with target mRNAs, thereby modulating transcript stability and translation. This post-transcriptional control helps bacteria adapt quickly to changing environmental conditions. Our previous mutational analysis showed that Hfq is involved in metabolism and stress survival in the enteropathogen Yersinia enterocolitica. In this study we demonstrate that Hfq is essential for virulence in mice and influences production of surface pathogenicity factors, in particular lipopolysaccharide and adhesins mediating interaction with host tissue. Hfq inhibited the production of Ail, the Ail-like protein OmpX and the MyfA pilin post-transcriptionally. In contrast Hfq promoted production of two major autotransporter adhesins YadA and InvA. While protein secretion in vitro was not affected, hfq mutants exhibited decreased protein translocation by the type III secretion system into host cells, consistent with decreased production of YadA and InvA. The influence of Hfq on YadA resulted from a complex interplay of transcriptional, post-transcriptional and likely post-translational effects. Hfq regulated invA by modulating the expression of the transcriptional regulators rovA, phoP and ompR. Therefore, Hfq is a global coordinator of surface virulence determinants in Y. enterocolitica suggesting that it constitutes an attractive target for developing new antimicrobial strategies

The Novel Monoclonal IgG₁-Antibody AB90-E8 as a Diagnostic Tool to Rapidly Distinguish <i>Aspergillus fumigatus</i> from Other Human Pathogenic <i>Aspergillus</i> Species

In most cases, invasive aspergillosis (IA) is caused by A. fumigatus, though infections with other Aspergillus spp. with lower susceptibilities to amphotericin B (AmB) gain ground. A. terreus, for instance, is the second leading cause of IA in humans and of serious concern because of its high propensity to disseminate and its in vitro and in vivo resistance to AmB. An early differentiation between A. fumigatus and non-A. fumigatus infections could swiftly recognize a potentially ineffective treatment with AmB and lead to the lifesaving change to a more appropriate drug regime in high-risk patients. In this study, we present the characteristics of the monoclonal IgG1-antibody AB90-E8 that specifically recognizes a surface antigen of A. fumigatus and the closely related, but not human pathogenic A. fischeri. We show immunostainings on fresh frozen sections as well as on incipient mycelium picked from agar plates with tweezers or by using the expeditious tape mount technique. All three methods have a time advantage over the common procedures currently used in the routine diagnosis of IA and outline the potential of AB90-E8 as a rapid diagnostic tool

Functional Orthodontic Treatment of Mandibular Condyle Fractures in Children and Adolescent Patients: An MRI Follow-Up

Background: The purpose of this study was to retrospectively evaluate and follow up a conservative treatment approach with functional orthodontic appliances for the management of mandibular condyle fractures in children and adolescent patients. Methods: Between 2020 and 2022, the treatment records of patients with mandibular condyle fractures receiving a functional orthodontic treatment (FOT) were evaluated. In addition to the clinical and functional findings, magnetic resonance images of the mandibular condyles and surrounding structures were assessed. Results: Out of 61 patients, 8 met the inclusion criteria. The follow-up examination records showed no functional limitations. In 75% of cases, mild midline deviations persisted (mean 1.1 mm) without significant alterations to the occlusal relationships. Magnetic resonance imaging (MRI) showed the remodeling of the condyles and the restitution of the ramus heights, even in dislocated and displaced fractures. In three cases, a partial displacement of the articular disc was observed at the follow-up. No differences in the remodeling patterns were noted depending on age, sex, or fracture location. Conclusions: A FOT led to favorable functional and morphologic outcomes, supporting the concept of a conservative functional approach in children and adolescent patients. Functional adjunctive therapy should be considered in the conservative treatment of mandibular condyle fractures in growing patients

The RNA chaperone Hfq impacts growth, metabolism and production of virulence factors in Yersinia enterocolitica.

To adapt to changes in environmental conditions, bacteria regulate their gene expression at the transcriptional but also at the post-transcriptional level, e.g. by small RNAs (sRNAs) which modulate mRNA stability and translation. The conserved RNA chaperone Hfq mediates the interaction of many sRNAs with their target mRNAs, thereby playing a global role in fine-tuning protein production. In this study, we investigated the significance of Hfq for the enteropathogen Yersina enterocolitica serotype O:8. Hfq facilitated optimal growth in complex and minimal media. Our comparative protein analysis of parental and hfq-negative strains suggested that Hfq promotes lipid metabolism and transport, cell redox homeostasis, mRNA translation and ATP synthesis, and negatively affects carbon and nitrogen metabolism, transport of siderophore and peptides and tRNA synthesis. Accordingly, biochemical tests indicated that Hfq represses ornithine decarboxylase activity, indole production and utilization of glucose, mannitol, inositol and 1,2-propanediol. Moreover, Hfq repressed production of the siderophore yersiniabactin and its outer membrane receptor FyuA. In contrast, hfq mutants exhibited reduced urease production. Finally, strains lacking hfq were more susceptible to acidic pH and oxidative stress. Unlike previous reports in other Gram-negative bacteria, Hfq was dispensable for type III secretion encoded by the virulence plasmid. Using a chromosomally encoded FLAG-tagged Hfq, we observed increased production of Hfq-FLAG in late exponential and stationary phases. Overall, Hfq has a profound effect on metabolism, resistance to stress and modulates the production of two virulence factors in Y. enterocolitica, namely urease and yersiniabactin

Influence of <i>hfq</i> on indole production and ornithine decarboxylase activity.

<p>(A and B) The concentration of indole present in culture supernatants was determined after growth in LB at 27°C. (A) Bacteria were grown for four hours at 27°C. Data represent mean and standard deviation of at least three independent experiments each performed with triplicate independent cultures. (B) Complementation analysis. Bacterial cultures were grown for 16 h at 27°C, since strains carrying plasmids were delayed in their indole production. Because of the variability of indole concentration produced by parental strains carrying plasmids (between 0.07 and 1.5 mM in four independent experiments), results were expressed relative to the indole produced by the parental strain JB580v carrying the control vector(which was set at 100%). Data represent mean and standard deviation of four independent experiments each performed with at least triplicate independent cultures. Significance was calculated with Student‘s unpaired <i>t</i>-test (*<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001). (C) Ornithine decarboxylase activity detected using the API-20E strip. All wells are positive (negative wells remain yellow), but wells inoculated with <i>hfq</i>-negative strains turn red, whereas those inoculated with parental strains are more orange.</p

Pesticin sensitivity assay^a.

<p>^a , all strains were tested in duplicate at least twice and a representative experiment is shown; ^b, size of growth inhibition obtained with undiluted pesticin preparate; ^c, MID: minimum inhibitory dilution factor for pesticin preparate to inhibit bacterial growth.</p

Role of <i>hfq</i> in production of yersiniabactin and its receptor FyuA.

<p>(A) Immunodetection of FyuA in strains grown for 24 h in LB supplemented with DIP (LBD). Loading was as follows: 1, WA fyuA; 2, WA-314; 3, SOR4; 4, JB580v; 5, SOR17; 6, WA-314(pACYC184ts); 7, WA-314(pAhfq); 8, SOR4(pACYC184ts); and 9, SOR4(pAhfq). Upper panel shows the immunoblot. The relative signal for each band compared to wild type (which was set to 100%) is indicated. Bottom panel shows part of Coomassie blue-stained gel used as loading control. (B) Reporter assay measuring yersiniabactin production. Following growth for 24 h in LBD at 37°C, bacterial culture supernatants were harvested. They were applied to a reporter strain which expresses luciferase in response to yersiniabactin. Luciferase activity was determined after incubation of the reporter strain for 24 h at 37°C. Results are the mean and standard deviation of duplicate cultures each assessed in triplicate. Significance was calculated with Student‘s unpaired <i>t</i>-test (**<i>P</i><0.01; ***<i>P</i><0.001). Similar results were obtained in three independent experiments.</p

Growth of <i>Y</i>. <i>enterocolitica</i> strains in BHI (A, B) and LB (C,D).

<p>(A and B) Bacteria were grown in BHI at 27°C (A) and 37°C (B): parental strains WA-314 (black diamonds) and JB580v (black squares), <i>hfq</i>-negative strains SOR3 (white diamonds), SOR4 (white triangles) and SOR17 (white squares). (C and D) Growth in LB of complemented strains at 27°C (C) and 37°C (D): JB580v(pACYC184) (black squares and straight line, parental strain harbouring the control plasmid), JB580v(pAhfq) (white square and dotted line, parental strain with complementing plasmid), SOR17(pACYC184) (black triangle and black line, <i>hfq</i>-negative strain with control plasmid), and SOR17(pAhfq) (white triangle and dotted line, complemented <i>hfq</i> strain). Full complementation of the growth defect of strains SOR3 and SOR4 was also observed after introduction of plasmid pAhfq (data not shown). Results are the mean and standard deviation (error bars) of two cultures and are representative of at least two independent experiments.</p