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Multidrug-resistant gram-negative pathogens such as Escherichia coli have become increasingly difficult to treat and therefore alternative treatment options are needed. Targeting virulence factors like biofilm formation could be one such option. Inhibition of biofilm-related structures like curli and cellulose formation in E. coli has been shown for different phenolic natural compounds like epigallocatechin gallate. This study demonstrates this effect for other structurally unrelated phenolics, namely octyl gallate, scutellarein and wedelolactone. To verify whether these structurally different compounds influence identical pathways of biofilm formation in E. coli a broad comparative RNA-sequencing approach was chosen with additional RT-qPCR to gain initial insights into the pathways affected at the transcriptomic level. Bioinformatical analysis of the RNA-Seq data was performed using DESeq2, BioCyc and KEGG Mapper. The comparative bioinformatics analysis on the pathways revealed that, irrespective of their structure, all compounds mainly influenced similar biological processes. These pathways included bacterial motility, chemotaxis, biofilm formation as well as metabolic processes like arginine biosynthesis and tricarboxylic acid cycle. Overall, this work provides the first insights into the potential mechanisms of action of novel phenolic biofilm inhibitors and highlights the complex regulatory processes of biofilm formation in E. coli.
Summary
Outer membrane extensions are common in many marine bacteria. However, the function of these surface enlargements or extracellular compartments is poorly understood. Using a combined approach of microscopy and subproteome analyses, we therefore examined Pseudoalteromonas distincta ANT/505, an Antarctic polysaccharide degrading gamma‐proteobacterium. P. distincta produced outer membrane vesicles (MV) and vesicle chains (VC) on polysaccharide and non‐polysaccharide carbon sources during the exponential and stationary growth phase. Surface structures of carbohydrate‐grown cells were equipped with increased levels of highly substrate‐specific proteins. At the same time, proteins encoded in all other polysaccharide degradation‐related genomic regions were also detected in MV and VC samples under all growth conditions, indicating a basal expression. In addition, two alkaline phosphatases were highly abundant under non‐limiting phosphate conditions. Surface structures may thus allow rapid sensing and fast responses in nutritionally deprived environments. It may also facilitate efficient carbohydrate processing and reduce loss of substrates and enzymes by diffusion as important adaptions to the aquatic ecosystem.
The iron-regulated surface determinant protein B (IsdB) of Staphylococcus aureus is involved in the acquisition of iron from hemoglobin. Moreover, IsdB elicits an adaptive immune response in mice and humans. Here, we show that IsdB also has impact on innate immunity. IsdB induces the release of proinflammatory cytokines, including IL-6 and IL-1β, in innate immune cells of humans and mice. In silico analysis and thermophoresis show that IsdB directly binds to TLR4 with high affinity. TLR4 sensing was essential for the IsdB-mediated production of IL-6, IL-1β, and other cytokines as it was abolished by blocking of TLR4-MyD88-IRAK1/4-NF-κB signaling. The release of IL-1β additionally required activation of the NLRP3 inflammasome. In human monocytes infected with live S. aureus, IsdB was necessary for maximal IL-1β release. Our studies identify S. aureus IsdB as a novel pathogen-associated molecular pattern that triggers innate immune defense mechanisms.
Abstract
Amphidiploid fungal Verticillium longisporum strains Vl43 and Vl32 colonize the plant host Brassica napus but differ in their ability to cause disease symptoms. These strains represent two V. longisporum lineages derived from different hybridization events of haploid parental Verticillium strains. Vl32 and Vl43 carry same‐sex mating‐type genes derived from both parental lineages. Vl32 and Vl43 similarly colonize and penetrate plant roots, but asymptomatic Vl32 proliferation in planta is lower than virulent Vl43. The highly conserved Vl43 and Vl32 genomes include less than 1% unique genes, and the karyotypes of 15 or 16 chromosomes display changed genetic synteny due to substantial genomic reshuffling. A 20 kb Vl43 lineage‐specific (LS) region apparently originating from the Verticillium dahliae‐related ancestor is specific for symptomatic Vl43 and encodes seven genes, including two putative transcription factors. Either partial or complete deletion of this LS region in Vl43 did not reduce virulence but led to induction of even more severe disease symptoms in rapeseed. This suggests that the LS insertion in the genome of symptomatic V. longisporum Vl43 mediates virulence‐reducing functions, limits damage on the host plant, and therefore tames Vl43 from being even more virulent.
The deep-sea tubeworm Riftia pachyptila lacks a digestive system but completely relies on bacterial endosymbionts for nutrition. Although the symbiont has been studied in detail on the molecular level, such analyses were unavailable for the animal host, because sequence information was lacking. To identify host-symbiont interaction mechanisms, we therefore sequenced the Riftia transcriptome, which served as a basis for comparative metaproteomic analyses of symbiont-containing versus symbiont-free tissues, both under energy-rich and energy-limited conditions. Our results suggest that metabolic interactions include nutrient allocation from symbiont to host by symbiont digestion and substrate transfer to the symbiont by abundant host proteins. We furthermore propose that Riftia maintains its symbiont by protecting the bacteria from oxidative damage while also exerting symbiont population control. Eukaryote-like symbiont proteins might facilitate intracellular symbiont persistence. Energy limitation apparently leads to reduced symbiont biomass and increased symbiont digestion. Our study provides unprecedented insights into host-microbe interactions that shape this highly efficient symbiosis.
Objectives
Biofilm removal is the decisive factor for the control of peri-implantitis. Cold atmospheric pressure plasma (CAP) can become an effective aid due to its ability to destroy and to inactivate bacterial biofilm residues. This study evaluated the cleaning efficiency of CAP, and air-polishing with glycine (APG) or erythritol (APE) containing powders alone or in combination with CAP (APG + CAP, APE + CAP) on sandblasted/acid etched, and anodised titanium implant surface.
Materials and methods
On respective titanium discs, a 7-day ex vivo human biofilm was grown. Afterwards, the samples were treated with CAP, APG, APE, APG + CAP, and APE + CAP. Sterile and untreated biofilm discs were used for verification. Directly after treatment and after 5 days of incubation in medium at 37 °C, samples were prepared for examination by fluorescence microscopy. The relative biofilm fluorescence was measured for quantitative analyses.
Results
Air-polishing with or without CAP removed biofilms effectively. The combination of air-polishing with CAP showed the best cleaning results compared to single treatments, even on day 5. Immediately after treatment, APE + CAP showed insignificant higher cleansing efficiency than APG + CAP.
Conclusions
CAP supports mechanical cleansing and disinfection to remove and inactivate microbial biofilm on implant surfaces significantly. Here, the type of the powder was not important. The highest cleansing results were obtained on sandblasted/etched surfaces.
Clinical relevance.
Microbial residuals impede wound healing and re-osseointegration after peri-implantitis treatment. Air-polishing treatment removes biofilms very effectively, but not completely. In combination with CAP, microbial free surfaces can be achieved. The tested treatment regime offers an advantage during treatment of peri-implantitis.
Bacterial kidney disease (BKD) is a chronic bacterial disease affecting both wild and farmed salmonids. The causative agent for BKD is the Gram-positive fish pathogen Renibacterium salmoninarum. As treatment and prevention of BKD have proven to be difficult, it is important to know and identify the key bacterial proteins that interact with the host. We used subcellular fractionation to report semi-quantitative data for the cytosolic, membrane, extracellular, and membrane vesicle (MV) proteome of R. salmoninarum. These data can aid as a backbone for more targeted experiments regarding the development of new drugs for the treatment of BKD. Further analysis was focused on the MV proteome, where both major immunosuppressive proteins P57/Msa and P22 and proteins involved in bacterial adhesion were found in high abundance. Interestingly, the P22 protein was relatively enriched only in the extracellular and MV fraction, implicating that MVs may play a role in host–pathogen interaction. Compared to the other subcellular fractions, the MVs were also relatively enriched in lipoproteins and all four cell wall hydrolases belonging to the New Lipoprotein C/Protein of 60 kDa (NlpC/P60) family were detected, suggesting an involvement in the formation of the MVs.
We analyzed the proteomic response of the Gram-negative fish pathogen A. salmonicida to iron limitation, an elevated incubation temperature, and the antibiotic florfenicol. Proteins from different subcellular fractions (cytosol, inner membrane, outer membrane, extracellular and outer membrane vesicles) were enriched and analyzed. We identified several iron-regulated proteins that were not reported in the literature for A. salmonicida before. We could also show that hemolysin, an oxidative-stress-resistance chaperone, a putative hemin receptor, an M36 peptidase, and an uncharacterized protein were significantly higher in abundance not only under iron limitation but also with an elevated incubation temperature. This may indicate that these proteins involved in the infection process of A. salmonicida are induced by both factors. The analysis of the outer membrane vesicles (OMVs) with and without applied stresses revealed significant differences in the proteomes. OMVs were smaller and contained more cytoplasmic proteins after antibiotic treatment. After cultivation with low iron availability, several iron-regulated proteins were found in the OMVs, indicating that A. salmonicida OMVs potentially have a function in iron acquisition, as reported for other bacteria. The presence of iron-regulated transporters further indicates that OMVs obtained from ‘stressed’ bacteria might be suitable vaccine candidates that induce a protective anti-virulence immune response.
Proteomic Adaptation of Clostridioides difficile to Treatment with the Antimicrobial Peptide Nisin
(2021)
Background
Peri-implantitis therapy is a major problem in implantology. Because of challenging rough implant surface and implant geometry, microorganisms can hide and survive in implant microstructures and impede debridement. We developed a new water jet (WJ) device and a new cold atmospheric pressure plasma (CAP) device to overcome these problems and investigated aspects of efficacy in vitro and safety with the aim to create the prerequisites for a clinical pilot study with these medical devices.
Methods
We compared the efficiency of a single treatment with a WJ or curette and cotton swab (CC) without or with adjunctive use of CAP (WJ + CAP, CC + CAP) to remove biofilm in vitro from rough titanium discs. Treatment efficacy was evaluated by measuring turbidity up to 72 h for bacterial re-growth or spreading of osteoblast-like cells (MG-63) after 5 days with scanning electron microscopy. With respect to application safety, the WJ and CAP instruments were examined according to basic regulations for medical devices.
Results
After 96 h of incubation all WJ and CC treated disks were turbid but 67% of WJ + CAP and 46% CC + CAP treated specimens were still clear. The increase in turbidity after WJ treatment was delayed by about 20 h compared to CC treatment. In combination with CAP the cell coverage significantly increased to 82% (WJ + CAP) or 72% (CC + CAP), compared to single treatment 11% (WJ) or 10% (CC).
Conclusion
The newly developed water jet device effectively removes biofilm from rough titanium surfaces in vitro and, in combination with the new CAP device, biologically acceptable surfaces allow osteoblasts to grow. WJ in combination with CAP leads to cleaner surfaces than the usage of curette and cotton swabs with or without subsequent plasma treatment. Our next step will be a clinical pilot study with these new devices to assess the clinical healing process.