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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.
Four aerobic bacteria with bacteriolytic capabilities were isolated from the brackish water site Strait Uzynaral of Lake Balkhash in Kazakhstan. The morphology and physiology of the bacterial isolates have subsequently been analyzed. Using matrix assisted laser desorption ionization-time of flight mass spectrum and partial 16S rRNA gene sequence analyses, three of the isolates have been identified as Pseudomonas veronii and one as Paenibacillus apiarius. We determined the capability of both species to lyse pre-grown cells of the Gram-negative strains Pseudomonas putida SBUG 24 and Escherichia coli SBUG 13 as well as the Gram-positive strains Micrococcus luteus SBUG 16 and Arthrobacter citreus SBUG 321 on solid media. The bacteriolysis process was analyzed by creating growth curves and electron micrographs of co-cultures with the bacteriolytic isolates and the lysis sensitive strain Arthrobacter citreus SBUG 321 in nutrient-poor liquid media. One metabolite of Paenibacillus apiarius was isolated and structurally characterized by various chemical structure determination methods. It is a novel antibiotic substance.
The anaerobic bacterium Clostridioides difficile represents one of the most problematic pathogens, especially in hospitals. Dysbiosis has been proven to largely reduce colonization resistance against this intestinal pathogen. The beneficial effect of the microbiota is closely associated with the metabolic activity of intestinal microbes such as the ability to transform primary bile acids into secondary ones. However, the basis and the molecular action of bile acids (BAs) on the pathogen are not well understood. We stressed the pathogen with the four most abundant human bile acids: cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA) and lithocholic acid (LCA). Thin layer chromatography (TLC), confocal laser scanning microscopy (CLSM), and electron microscopy (EM) were employed to track the enrichment and destination of bile acids in the bacterial cell. TLC not only revealed a strong accumulation of LCA in C. difficile, but also indicated changes in the composition of membrane lipids in BA-treated cells. Furthermore, morphological changes induced by BAs were determined, most pronounced in the virtually complete loss of flagella in LCA-stressed cells and a flagella reduction after DCA and CDCA challenge. Quantification of both, protein and RNA of the main flagella component FliC proved the decrease in flagella to originate from a change in gene expression on transcriptional level. Notably, the loss of flagella provoked by LCA did not reduce adhesion ability of C. difficile to Caco-2 cells. Most remarkably, extracellular toxin A levels in the presence of BAs showed a similar pattern as flagella expression. That is, CA did not affect toxin expression, whereas lower secretion of toxin A was determined in cells stressed with LCA, DCA or CDCA. In summary, the various BAs were shown to differentially modify virulence determinants, such as flagella expression, host cell adhesion and toxin synthesis. Our results indicate differences of BAs in cellular localization and impact on membrane composition, which could be a reason of their diverse effects. This study is a starting point in the elucidation of the molecular mechanisms underlying the differences in BA action, which in turn can be vital regarding the outcome of a C. difficile infection.
Clostridioides difficile is an intestinal human pathogen that uses the opportunity of a depleted microbiota to cause an infection. It is known, that the composition of the intestinal bile acid cocktail has a great impact on the susceptibility toward a C. difficile infection. However, the specific response of growing C. difficile cells to diverse bile acids on the molecular level has not been described yet. In this study, we recorded proteome signatures of shock and long-term (LT) stress with the four main bile acids cholic acid
(CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), and lithocholic acid (LCA). A general overlapping response to all tested bile acids could be determined particularly in shock experiments which appears plausible in the light of their common steroid structure. However, during LT stress several proteins showed an altered abundance
in the presence of only a single or a few of the bile acids indicating the existence of specific adaptation mechanisms. Our results point at a differential induction of the groEL and dnaKJgrpE chaperone systems, both belonging to the class I heat shock genes. Additionally, central metabolic pathways involving butyrate fermentation and the reductive Stickland fermentation of leucine were effected, although CA caused a
proteome signature different from the other three bile acids. Furthermore, quantitative proteomics revealed a loss of flagellar proteins in LT stress with LCA. The absence of flagella could be substantiated by electron microscopy which also indicated less
flagellated cells in the presence of DCA and CDCA and no influence on flagella formation by CA. Our data break down the bile acid stress response of C. difficile into a general and a specific adaptation. The latter cannot simply be divided into a response to primary and secondary bile acids, but rather reflects a complex and variable adaptation process enabling C. difficile to survive and to cause an infection in the intestinal tract.
Clostridioides difficile is an intestinal human pathogen that uses the opportunity of a depleted microbiota to cause an infection. It is known, that the composition of the intestinal bile acid cocktail has a great impact on the susceptibility toward a C. difficile infection. However, the specific response of growing C. difficile cells to diverse bile acids on the molecular level has not been described yet. In this study, we recorded proteome signatures of shock and long-term (LT) stress with the four main bile acids cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), and lithocholic acid (LCA). A general overlapping response to all tested bile acids could be determined particularly in shock experiments which appears plausible in the light of their common steroid structure. However, during LT stress several proteins showed an altered abundance in the presence of only a single or a few of the bile acids indicating the existence of specific adaptation mechanisms. Our results point at a differential induction of the groEL and dnaKJgrpE chaperone systems, both belonging to the class I heat shock genes. Additionally, central metabolic pathways involving butyrate fermentation and the reductive Stickland fermentation of leucine were effected, although CA caused a proteome signature different from the other three bile acids. Furthermore, quantitative proteomics revealed a loss of flagellar proteins in LT stress with LCA. The absence of flagella could be substantiated by electron microscopy which also indicated less flagellated cells in the presence of DCA and CDCA and no influence on flagella formation by CA. Our data break down the bile acid stress response of C. difficile into a general and a specific adaptation. The latter cannot simply be divided into a response to primary and secondary bile acids, but rather reflects a complex and variable adaptation process enabling C. difficile to survive and to cause an infection in the intestinal tract.
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.
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.
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.
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.