Effect of TraN key residues involved in DNA binding on pIP501 transfer rates in Enterococcus faecalis.

Conjugation is a major mechanism that facilitates the exchange of antibiotic resistance genes among bacteria. The broad-host-range Inc18 plasmid pIP501 harbors 15 genes that encode for a type IV secretion system (T4SS). It is a membrane-spanning multiprotein complex formed between conjugating donor and recipient cells. The penultimate gene of the pIP501 operon encodes for the cytosolic monomeric protein TraN. This acts as a transcriptional regulator by binding upstream of the operon promotor, partially overlapping with the origin of transfer. Additionally, TraN regulates traN and traO expression by binding upstream of the PtraNO promoter. This study investigates the impact of nine TraN amino acids involved in binding to pIP501 DNA through site-directed mutagenesis by exchanging one to three residues by alanine. For three traN variants, complementation of the pIP501∆traN knockout resulted in an increase of the transfer rate by more than 1.5 orders of magnitude compared to complementation of the mutant with native traN. Microscale thermophoresis (MST) was used to assess the binding affinities of three TraN double-substituted variants and one triple-substituted variant to its cognate pIP501 double-stranded DNA. The MST data strongly correlated with the transfer rates obtained by biparental mating assays in Enterococcus faecalis. The TraN variants TraN_R23A-N24A-Q28A, TraN_H82A-R86A, and TraN_G100A-K101A not only exhibited significantly lower DNA binding affinities but also, upon complementation of the pIP501∆traN knockout, resulted in the highest pIP501 transfer rates. This confirms the important role of the TraN residues R23, N24, Q28, H82, R86, G100, and K101 in downregulating pIP501 transfer. Although TraN is not part of the mating pair formation complex, TraE, TraF, TraH, TraJ, TraK, and TraM were coeluted with TraN in a pull-down. Moreover, TraN homologs are present not only in Inc18 plasmids but also in RepA_N and Rep_3 family plasmids, which are frequently found in enterococci, streptococci, and staphylococci. This points to a widespread role of this repressor in conjugative plasmid transfer among Firmicutes.

Vibrio cholerae's ToxRS bile sensing system.

The seventh pandemic of the diarrheal cholera disease, which began in 1960, is caused by the Gram-negative bacterium Vibrio cholerae. Its environmental persistence provoking recurring sudden outbreaks is enabled by V. cholerae's rapid adaption to changing environments involving sensory proteins like ToxR and ToxS. Located at the inner membrane, ToxR and ToxS react to environmental stimuli like bile acid, thereby inducing survival strategies for example bile resistance and virulence regulation. The presented crystal structure of the sensory domains of ToxR and ToxS in combination with multiple bile acid interaction studies, reveals that a bile binding pocket of ToxS is only properly folded upon binding to ToxR. Our data proposes an interdependent functionality between ToxR transcriptional activity and ToxS sensory function. These findings support the previously suggested link between ToxRS and VtrAC-like co-component systems. Besides VtrAC, ToxRS is now the only experimentally determined structure within this recently defined superfamily, further emphasizing its significance. In-depth analysis of the ToxRS complex reveals its remarkable conservation across various Vibrio species, underlining the significance of conserved residues in the ToxS barrel and the more diverse ToxR sensory domain. Unravelling the intricate mechanisms governing ToxRS's environmental sensing capabilities, provides a promising tool for disruption of this vital interaction, ultimately inhibiting Vibrio's survival and virulence. Our findings hold far-reaching implications for all Vibrio strains that rely on the ToxRS system as a shared sensory cornerstone for adapting to their surroundings.

Cholera is a contagious diarrheal disease that leads to about 20,000 to 140,000 yearly deaths. It is caused by a bacterium called Vibrio cholerae, which can survive in harsh conditions and many environments. It often contaminates water, where it lives in an energy-conserving mode. But when humans consume Vibrio cholerae-contaminated water or food, the bacterium can sense its new environment and switch into a high-energy consuming state, causing fever, diarrhea, and vomiting. Vibrio cholerae recognizes bile acid in the human stomach, which signals that the bacterium has reached ideal conditions for causing disease. So far, it has been unclear, how exactly the bacterium detects bile acid. Understanding how these bacteria sense bile acid, could help scientists develop new ways to prevent cholera outbreaks or treat infections. Gubensäk et al. analysed two proteins from the Vibrio cholerae bacterium, called ToxR and ToxS, which are located below the bacteria's protective membrane. More detailed analyses showed that the two proteins bind together, forming a bile-binding pocket. When correctly assembled, this bile-sensing machine detects bile concentrations in the body, allowing the bacterium to adapt to the local conditions. Using crystal structures, a series of interaction studies, and modeling software, Gubensäk et al. detailed step-by-step how the two proteins sense bile acid and help the bacteria adapt and thrive in the human body. The results confirm the results of previous studies that implicated ToxR and ToxS in bile sensing and provide new details about the process. Scientists may use this information to develop new ways to interfere with the bacteria's bile-sensing and gut adaptation processes. They may also use the information to screen for existing drugs that block bile sensing and then test as cholera treatments or prevention strategies in clinical trials. New cholera treatment or prevention approaches that don't rely on antibiotics may help public health officials respond to growing numbers of cholera outbreaks and to prevent the spread of antibiotic-resistant bacteria.

Small Things Matter: The 11.6-kDa TraB Protein is Crucial for Antibiotic Resistance Transfer Among Enterococci.

Conjugative transfer is the most important means for spreading antibiotic resistance genes. It is used by Gram-positive and Gram-negative bacteria, and archaea as well. Conjugative transfer is mediated by molecular membrane-spanning nanomachines, so called Type 4 Secretion Systems (T4SS). The T4SS of the broad-host-range inc18-plasmid pIP501 is organized in a single operon encoding 15 putative transfer proteins. pIP501 was originally isolated from a clinical Streptococcus agalactiae strain but is mainly found in Enterococci. In this study, we demonstrate that the small transmembrane protein TraB is essential for pIP501 transfer. Complementation of a markerless pIP501∆traB knockout by traB lacking its secretion signal sequence did not fully restore conjugative transfer. Pull-downs with Strep-tagged TraB demonstrated interactions of TraB with the putative mating pair formation proteins, TraF, TraH, TraK, TraM, and with the lytic transglycosylase TraG. As TraB is the only putative mating pair formation complex protein containing a secretion signal sequence, we speculate on its role as T4SS recruitment factor. Moreover, structural features of TraB and TraB orthologs are presented, making an essential role of TraB-like proteins in antibiotic resistance transfer among Firmicutes likely.

The periplasmic domains of Vibriocholerae ToxR and ToxS are forming a strong heterodimeric complex independent on the redox state of ToxR cysteines.

The transmembrane protein ToxR plays a key role in the virulence expression system of Vibrio cholerae. The activity of ToxR is dependent on its periplasmic sensor domain (ToxRp) and on the inner membrane protein ToxS. Herein, we present the Nuclear Magnetic Resonance NMR solution structure of the sensory ToxRp containing an intramolecular disulfide bond. The presented structural and dynamic experiments with reduced and oxidized ToxRp propose an explanation for the increased proteolytic sensitivity of reduced ToxR. Additionally, for the first time, we could identify the formation of a strong heterodimer complex between the periplasmic domains of ToxR and ToxS in solution. NMR interaction studies reveal that binding of ToxS is not dependent on the redox state of ToxR cysteines, and formed complexes are structurally similar. By monitoring the proteolytic cleavage of ToxRp with NMR, we additionally provide a direct evidence of ToxS protective function. Taken together our results suggest that ToxR activity is regulated by its stability which is, on the one hand, dependent on the redox states of its cysteines, influencing the stability of its fold, and on the other hand, on its interaction with ToxS, which binds independent on the cysteines and acts as a protection against proteases.

Differentiation of hMSC and hPDLSC induced by PGE2 or BMP-7 in 3D models.

Regenerative therapies of pathogenic tissue defects are gaining increasing importance in periodontology. Among others, the osteogenic effect of BMP-7 seems to play a major role in the development of teeth and alveolar bone. Human periodontal ligament stem cells (hPDLSC), as well as human mesenchymal stem cells (hMSC), show the ability to differentiate into various types of tissues. Regarding prostaglandin E2, many studies have confirmed that it is involved in the inflammation associated to periodontitis stimulating osteoclasts, which ultimately leads to resorption of tooth supporting bone. Herein, we aimed to investigate how PGE2 influences regenerative processes. The influence of PGE2 and BMP-7 on the osteogenic differentiation of hMSC and hPDLSC was determined in a 3D cell culture model using qRT-PCR, immunocytochemistry and REM. BMP-7 enhanced the expression of osteogenic markers in hMSC and lowered it in hPDLSC-TERT. BMP-7 had a lower osteogenic effect on hPDLSC-hTERT than on hMSC, while PGE2 decreases the osteogenic differentiation in both cell types, thus, inhibiting anabolic processes. Both cell types presented good proliferation and adhesion onto the scaffolds. The well-developed structural morphology and the support of osteogenic differentiation suggest that the scaffolds are potential candidate materials for bone regeneration. The positivity for Cap in hPDLSC and more in hMSC immunostaining samples indicates the initiation of neocementogenesis as part of periodontal regeneration. In conclusion, BMP7, in particular combined with MSC, seems to have a favourable application also in periodontal regeneration. Our results show that inflammation plays an important role in periodontal regeneration. PGE2 is a key mediator, which stimulates bone resorption also via a mechanism involving the inhibition of osteogenic differentiation of MSC as well as PDLSC. Therefore, regenerative approaches should always be conducted in combination with anti-inflammatory measures oriented to control inflammation.

Clinical association between chronic periodontitis and the leukocyte extravasation inhibitors developmental endothelial locus-1 and pentraxin-3.

This clinical study aimed to determine whether periodontal disease is associated with expression of developmental endothelial locus-1 (Del-1) and pentraxin-3 (PTX-3), endogenous inhibitors of leukocyte extravasation in humans. Expression of DEL1, PTX3, interleukin-17A (IL17A), and lymphocyte function-associated antigen-1 (LFA1) was determined, using RT-PCR and melting curve analysis, in biopsies of gingival tissues from 95 patients: 42 with moderate periodontitis; 40 with severe periodontitis; and 13 healthy controls. Relative expression of DEL1 and PTX3 was statistically significantly weaker in patients with periodontitis than in the control subjects. On the contrary, both IL17A and LFA1 showed statistically significant stronger expression in patients with periodontitis than in healthy controls. Correlation analysis, performed using Spearman's test, showed that expression of DEL1 was statistically significantly linked to periodontitis (ρ = -0.103) and to age (ρ = -0.134), but not to the gender of the patient, and that expression of PTX3 was significantly correlated with periodontitis (ρ = -0.354). Expression of neutrophil extravasation inhibitors DEL1 and PTX3 show significant, but weak, association with the clinical manifestation of chronic periodontitis.

Sox2 is a potent inhibitor of osteogenic and adipogenic differentiation in human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) are a promising target for cell-based bone regeneration. However, their application for clinical use is limited because hMSCs lose their ability for cell division and differentiation during longer in vitro cultivation. The osteogenic differentiation is regulated through a complex network of molecular signal transduction pathways where the canonical Wnt pathway plays an important role. Sox2, a known key factor for maintenance of cellular pluripotency in stem cells, is supposed to influence the Wnt pathway in osteoblasts. In this study, we overexpressed Sox2 in immortalized hMSCs by lentiviral gene transfer. Sox2 overexpression significantly reduced the osteogenic and adipogenic differentiation potentials. This effect was abolished by knockdown of Sox2 overexpression. In addition, Oct4 and Nanog, other key transcription factors for pluripotency, are strongly upregulated when Sox2 is overexpressed. Furthermore, Dkk1, a target gene of the Sox2-Oct4 heterodimer and a Wnt antagonist, is downregulated. Sox2 overexpression causes higher expression levels of ß-catenin, the central transcription factor of the canonical Wnt pathway. These results suggest that Sox2 keeps hMSCs in an undifferentiated state by influencing the canonical Wnt pathway. Regulated expression of Sox2 may be a promising tool to cultivate hMSCs in sufficient quantities for cell and gene therapy applications.