Fusobacterium nucleatum adhesin FadA binds vascular endothelial cadherin and alters endothelial integrity.

Fusobacterium nucleatum is a Gram-negative oral anaerobe, capable of systemic dissemination causing infections and abscesses, often in mixed-species, at different body sites. We have shown previously that F. nucleatum adheres to and invades host epithelial and endothelial cells via a novel FadA adhesin. In this study, vascular endothelial (VE)-cadherin, a member of the cadherin family and a cell-cell junction molecule, was identified as the endothelial receptor for FadA, required for F. nucleatum binding to the cells. FadA colocalized with VE-cadherin on endothelial cells, causing relocation of VE-cadherin away from the cell-cell junctions. As a result, the endothelial permeability was increased, allowing the bacteria to cross the endothelium through loosened junctions. This crossing mechanism may explain why the organism is able to disseminate systemically to colonize in different body sites and even overcome the placental and blood-brain barriers. Co-incubation of F. nucleatum and Escherichia coli enhanced penetration of the endothelial cells by the latter in the transwell assays, suggesting F. nucleatum may serve as an 'enabler' for other microorganisms to spread systemically. This may explain why F. nucleatum is often found in mixed infections. This study reveals a possible novel dissemination mechanism utilized by pathogens.

Polyphasic characterization and genetic relatedness of low-virulence and virulent Listeria monocytogenes isolates.

BACKGROUND: Currently, food regulatory authorities consider all Listeria monocytogenes isolates as equally virulent. However, an increasing number of studies demonstrate extensive variations in virulence and pathogenicity of L. monocytogenes strains. Up to now, there is no comprehensive overview of the population genetic structure of L. monocytogenes taking into account virulence level. We have previously demonstrated that different low-virulence strains exhibit the same mutations in virulence genes suggesting that they could have common evolutionary pathways. New low-virulence strains were identified and assigned to phenotypic and genotypic Groups using cluster analysis. Pulsed-field gel electrophoresis, virulence gene sequencing and multi-locus sequence typing analyses were performed to study the genetic relatedness and the population structure between the studied low-virulence isolates and virulent strains. RESULTS: These methods showed that low-virulence strains are widely distributed in the two major lineages, but some are also clustered according to their genetic mutations. These analyses showed that low-virulence strains initially grouped according to their lineage, then to their serotypes and after which, they lost their virulence suggesting a relatively recent emergence. CONCLUSIONS: Loss of virulence in lineage II strains was related to point mutation in a few virulence genes (prfA, inlA, inlB, plcA). These strains thus form a tightly clustered, monophyletic group with limited diversity. In contrast, low-virulence strains of lineage I were more dispersed among the virulence strains and the origin of their loss of virulence has not been identified yet, even if some strains exhibited different mutations in prfA or inlA.

Identification of oral bacterial DNA in synovial fluid of patients with arthritis with native and failed prosthetic joints.

OBJECTIVE: We examined the presence of bacterial DNA in synovial fluids of native or clinically aseptically failed prosthetic joints from patients having periodontal disease and arthritis to determine whether there is bacterial spread from the oral cavity to the joints. METHODS: A total of 36 subjects were enrolled in the study. Among these, 11 were diagnosed with rheumatoid arthritis (RA) and 25 were diagnosed with osteoarthritis (OA). Eight patients with OA and 1 patient with RA had failed prostheses. Synovial fluid was aspirated from the affected hip or knee joint. Pooled subgingival plaque samples were collected, followed by clinical periodontal examination. Bacterial DNA was extracted from the collected synovial fluid and dental plaque samples were followed by polymerase chain reactions and DNA sequence analysis of the 16S-23S rRNA genes. RESULTS: Of the 36 patients, bacterial DNA was detected in the synovial fluid samples from 5 patients (13.9%): 2 with RA (1 native and 1 failed prosthetic joints) and 3 with OA (1 native and 2 failed prosthetic joints). Of these 5 patients, 2 were diagnosed with periodontitis and had identical bacterial clones (Fusobacterium nucleatum and Serratia proteamaculans, respectively) detected in both the synovial fluid and the dental plaque samples. Fusobacterium nucleatum was the most prevalent, detected in 4 of the 5 positive samples. No cultures were done and no patients were treated with antibiotics or developed clinical infection. CONCLUSIONS: The present findings of bacterial DNA in the synovial fluid suggest the possibility of organisms translocating from the periodontal tissue to the synovium. We suggest that patients with arthritis or failed prosthetic joints be examined for the presence of periodontal diseases and be treated accordingly.

Comparative microbial analysis of paired amniotic fluid and cord blood from pregnancies complicated by preterm birth and early-onset neonatal sepsis.

BACKGROUND: 16S rRNA-based genomic analyses have revolutionized our understanding of infectious diseases. Many cases which were recognized as "idiopathic" are now known to have an infectious etiology. Here, we present a proof-of-concept study to examine the microbial link between intra-amniotic infection (IAI) and early-onset neonatal sepsis (EONS). RESULTS: Using culture independent methods, we analyzed paired amniotic fluid (AF) and cord blood (CB) samples from 36 singleton pregnancies complicated by preterm birth (PTB), IAI, and/or EONS. PTB cases were grouped as 1) Group 1- neonatal blood culture-positive EONS (n=6). 2) Group 2- neonatal blood culture-negative presumed EONS with positive IAI (n=16). 3) Group 3- neonatal blood culture-negative presumed EONS with no IAI (n=7); 4) Group 4- no EONS or IAI (n=7). In addition, samples from term healthy deliveries (n=8) served as technical controls. A total of 31 species (15 non-redundant) were identified in AF, of which only 1/3 were cultivated. Significantly fewer microorganisms were detected in CB, with a total of 18 species (7 non-redundant) identified, of which only 2 (Escherichia coli, Streptococcus agalactiae) were cultivated. Of those, Bergeyella, Fusobacterium nucleatum, and Sneathia sanguinegens had not been detected in EONS before. The novel species identified in AF by PCR include Peptoniphilus harei and Lachnospiraceae sp. The majority (72%) of CB species were also detected in the matching AF, with E. coli and F. nucleatum as the most prevalent. The 16S rRNA sequences of paired AF and CB were 99.9-100% identical, while no identical sequences were found between different pregnancies. CONCLUSIONS: Previously unrecognized, uncultivated or difficult-to-cultivate species are implicated in EONS. Microbial species in paired AF and CB likely share the same infectious origin. Given its prevalence in EONS, F. nucleatum should be placed on the same importance scale as E. coli.

Signal peptide of FadA adhesin from Fusobacterium nucleatum plays a novel structural role by modulating the filament's length and width.

FadA, a novel adhesin of periodontal pathogen Fusobacterium nucleatum is composed of two forms, pre-FadA and mature FadA (mFadA), constituting the functional FadA complex (FadAc). By electron microscopy, we observed that mFadA formed uniformly long and thin filaments, while FadAc formed heterogeneous filaments of varying lengths and widths, as well as "knots". Mutants in signal peptide or in the non-alpha-helical loop retaining heterogeneous structures had binding activity while those forming aggregates or long filaments lost activity. These observations suggest short filaments and knots may be the active forms of FadA. This is the first demonstration that a signal peptide is required for the assembly of a bacterial adhesin.

Significance of pretreatment analysis of hepatitis C virus genotype 1b hypervariable region 1 sequences to predict antiviral outcome.

The heterogeneity of hypervariable region 1 (HVR1), located at the amino terminus of the E2 envelope, may be involved in resistance to alpha interferon (IFN-alpha) treatment. We investigated whether peculiar HVR1 domain profiles before treatment were associated with the maintenance of sensitivity or the appearance of resistance to treatment. Fifteen patients infected with hepatitis C virus genotype 1b and treated with IFN with or without ribavirin were selected. Ten responded to treatment (groups R1 and R2) and five did not (group NR). The amino acid sequences of 150 naturally occurring HVR1 variants present in the serum before therapy were compared in relation to treatment outcome. HVR1 variants from the NR group contained a constant nonantigenic amino acid segment that was not found in HVR1 variants from the R groups.