Gene transfer into hepatocytes using asialoglycoprotein receptor mediated endocytosis of DNA complexed with an artificial tetra-antennary galactose ligand

We have constructed an artificial ligand for the hepatocyte-specific asialoglycoprotein receptor for the purpose of generating a synthetic delivery system for DNA. This ligand has a tetra-antennary structure, containing four terminal galactose residues on a branched carrier peptide. The carbohydrate residues of this glycopeptide were introduced by reductive coupling of lactose to the alpha- and epsilon-amino groups of the two N-terminal lysines on the carrier peptide. The C-terminus of the peptide, containing a cysteine separated from the branched N-terminus by a 10 amino acid spacer sequence, was used for conjugation to 3-(2-pyridyldithio)propionate-modified polylysine via disulfide bond formation. Complexes containing plasmid DNA bound to these galactose-polylysine conjugates have been used for asialoglycoprotein receptor-mediated transfer of a luciferase gene into human (HepG2) and murine (BNL CL.2) hepatocyte cell lines. Gene transfer was strongly promoted when amphipathic peptides with pH-controlled membrane-disruption activity, derived from the N-terminal sequence of influenza virus hemagglutinin HA-2, were also present in these DNA complexes. Thus, we have essentially borrowed the small functional domains of two large proteins, asialoglycoprotein and hemagglutinin, and assembled them into a supramolecular complex to generate an efficient gene-transfer system

Label-free quantitative proteomic analysis of the oral bacteria Fusobacterium nucleatum and Porphyromonas gingivalis to identify protein features relevant in biofilm formation

Background The opportunistic pathogens Fusobacterium nucleatum and Porphyromonas gingivalis are Gram-negative bacteria associated with oral biofilm and periodontal disease. This study investigated interactions between F. nucleatum and P. gingivalis proteomes with the objective to identify proteins relevant in biofilm formation. Methods We applied liquid chromatography-tandem mass spectrometry to determine the expressed proteome of F. nucleatum and P. gingivalis cells grown in biofilm or planktonic culture, and as mono- and dual-species models. The detected proteins were classified into functional categories and their label-free quantitative (LFQ) intensities statistically compared. Results The proteomic analyses detected 1,322 F. nucleatum and 966 P. gingivalis proteins, including abundant virulence factors. Using univariate statistics, we identified significant changes between biofilm and planktonic culture (p-value ≤0.05) in 0,4% F. nucleatum, 7% P. gingivalis, and 14% of all proteins in the dual-species model. For both species, proteins involved in vitamin B2 (riboflavin) metabolism had significantly increased levels in biofilm. In both mono- and dual-species biofilms, P. gingivalis increased the production of proteins for translation, oxidation-reduction, and amino acid metabolism compared to planktonic cultures. However, when we compared LFQ intensities between mono- and dual-species, over 90% of the significantly changed P. gingivalis proteins had their levels reduced in biofilm and planktonic settings of the dual-species model. Conclusions The findings suggest that P. gingivalis reduces the production of multiple proteins because of the F. nucleatum presence. The results highlight the complex interactions of bacteria contributing to oral biofilms, which need to be considered in the design of prevention strategies.publishedVersio

Repeated bronchoscopy in health and obstructive lung disease: is the airway microbiome stable?

Objective Little is known concerning the stability of the lower airway microbiome. We have compared the microbiota identified by repeated bronchoscopy in healthy subjects and patients with ostructive lung diseaseases (OLD). Methods 21 healthy controls and 41 patients with OLD completed two bronchoscopies. In addition to negative controls (NCS) and oral wash (OW) samples, we gathered protected bronchoalveolar lavage in two fractions (PBAL1 and PBAL2) and protected specimen brushes (PSB). After DNA extraction, we amplified the V3V4 region of the 16S rRNA gene, and performed paired-end sequencing (Illumina MiSeq). Initial bioinformatic processing was carried out in the QIIME-2 pipeline, identifying amplicon sequence variants (ASVs) with the DADA2 algorithm. Potentially contaminating ASVs were identified and removed using the decontam package in R and the sequenced NCS. Results A final table of 551 ASVs consisted of 19 × 106 sequences. Alpha diversity was lower in the second exam for OW samples, and borderline lower for PBAL1, with larger differences in subjects not having received intercurrent antibiotics. Permutational tests of beta diversity indicated that within-individual changes were significantly lower than between-individual changes. A non-parametric trend test showed that differences in composition between the two exams (beta diversity) were largest in the PSBs, and that these differences followed a pattern of PSB > PBAL2 > PBAL1 > OW. Time between procedures was not associated with increased diversity. Conclusion The airways microbiota varied between examinations. However, there is compositional microbiota stability within a person, beyond that of chance, supporting the notion of a transient airways microbiota with a possibly more stable individual core microbiome.publishedVersio

Label-free quantitative proteomic analysis of the oral bacteria Fusobacterium nucleatum and Porphyromonas gingivalis to identify protein features relevant in biofilm formation

Background The opportunistic pathogens Fusobacterium nucleatum and Porphyromonas gingivalis are Gram-negative bacteria associated with oral biofilm and periodontal disease. This study investigated interactions between F. nucleatum and P. gingivalis proteomes with the objective to identify proteins relevant in biofilm formation. Methods We applied liquid chromatography-tandem mass spectrometry to determine the expressed proteome of F. nucleatum and P. gingivalis cells grown in biofilm or planktonic culture, and as mono- and dual-species models. The detected proteins were classified into functional categories and their label-free quantitative (LFQ) intensities statistically compared. Results The proteomic analyses detected 1,322 F. nucleatum and 966 P. gingivalis proteins, including abundant virulence factors. Using univariate statistics, we identified significant changes between biofilm and planktonic culture (p-value ≤0.05) in 0,4% F. nucleatum, 7% P. gingivalis, and 14% of all proteins in the dual-species model. For both species, proteins involved in vitamin B2 (riboflavin) metabolism had significantly increased levels in biofilm. In both mono- and dual-species biofilms, P. gingivalis increased the production of proteins for translation, oxidation-reduction, and amino acid metabolism compared to planktonic cultures. However, when we compared LFQ intensities between mono- and dual-species, over 90% of the significantly changed P. gingivalis proteins had their levels reduced in biofilm and planktonic settings of the dual-species model. Conclusions The findings suggest that P. gingivalis reduces the production of multiple proteins because of the F. nucleatum presence. The results highlight the complex interactions of bacteria contributing to oral biofilms, which need to be considered in the design of prevention strategies

Consistent biofilm formation by Streptococcus pyogenes emm 1 isolated from patients with necrotizing soft tissue infections

Objectives: Biofilm formation has been demonstrated in muscle and soft tissue samples from patients with necrotizing soft tissue infection (NSTI) caused by Streptococcus pyogenes, but the clinical importance of this observation is not clear. Although M-protein has been shown to be important for in vitro biofilm formation in S. pyogenes, the evidence for an association between emm type and biofilm forming capacity is conflicting. Here we characterize the biofilm forming capacity in a collection of S. pyogenes isolates causing NSTI, and relate this to emm type of the isolates and clinical characteristics of the patients. Methods: Bacterial isolates and clinical data were obtained from NSTI patients enrolled in a multicenter prospective observational study. Biofilm forming capacity was determined using a microtiter plate assay. Results: Among 57 cases, the three most frequently encountered emm types were emm1 (n = 22), emm3 (n = 13), and emm28 (n = 7). The distribution of biofilm forming capacity in emm1 was qualitatively (narrow-ranged normal distribution) and quantitatively (21/22 isolates in the intermediate range) different from other emm types (wide ranged, multimodal distribution with 5/35 isolates in the same range as emm1). There were no significant associations between biofilm forming capacity and clinical characteristics of the patients. Conclusions: The biofilm forming capacity of emm1 isolates was uniform and differed significantly from other emm types. The impact of biofilm formation in NSTI caused by S. pyogenes on clinical outcomes remains uncertain