RtxA1-induced expression of the small GTPase Rac2 plays a key role in the pathogenicity of Vibrio vulnificus.

Infection with the human pathogen Vibrio vulnificus leads to the generation of reactive oxygen species (ROS) via NAD(P)H oxidase (Nox) in host cells. In the present study, we employed mutant V. vulnificus strains to identify an essential virulence factor responsible for this ROS generation. We found that repeats-in-toxin A1 (RtxA1) expressed by V. vulnificus acts via Nox1 to induce significant ROS generation in the intestine epithelial cells, which ultimately results in cell death. Furthermore, RtxA1 modulates the small GTPase Rac2, which is known to play an important role in the activation of Nox. When mice were infected by the oral method, in contrast with the wild-type bacteria, an RtxA1-deficient V. vulnificus mutant was unable to induce ROS generation within the intestine and failed to cause death. These findings strongly suggest that RtxA1-induced Rac2 expression is a critical step underlying the pathogenicity of V. vulnificus.

Inhibition of airway allergic disease by co-administration of flagellin with allergen.

Bacterial flagellin, which activates Toll-like receptor 5 and cytosolic pattern recognition receptor Ipaf, has a strong immunomodulatory activity. In the present study, we examined whether intranasal co-administration of flagellin with allergen could modulate established airway hyperresponsiveness and Th2 response using an ovalbumin (OVA)-sensitized mouse model. Balb/c mice sensitized with OVA were treated with OVA-flagellin (FlaB) mixture three times at 1-week intervals. Seven days after the final OVA-FlaB administration, the mice were challenged with OVA inhalation, and airway responses and OVA-specific immune responses were evaluated. The OVA-FlaB treatment significantly suppressed OVA-induced airway hyperresponsiveness, airway eosinophilic inflammation, and OVA-specific Th2 cytokine productions in splenocytes. These results indicate that flagellin co-administered with allergen can modulate airway inflammatory response through inhibition of Th2 responses, and flagellin can be considered as a component for allergen-specific immunotherapy.

The pyrH gene of Vibrio vulnificus is an essential in vivo survival factor.

We have suggested an important role of the pyrH gene during the infectious process of Vibrio vulnificus. Previously, we have identified 12 genes expressed preferentially during human infections by using in vivo-induced antigen technology. Among the in vivo-expressed genes, pyrH encodes UMP kinase catalyzing UMP phosphorylation. Introduction of a deletion mutation to the pyrH gene was lethal to V. vulnificus, and an insertional mutant showed a high frequency of curing. We constructed a site-directed mutant strain (R62H/D77N) on Arg-62 and Asp-77, both predicted to be involved in UMP binding, and characterized the R62H/D77N strain compared with the previously reported insertional mutant. We further investigated the essential role of the pyrH gene in the establishment of infection using the R62H/D77N strain. Cytotoxicity was decreased in the R62H/D77N strain, and the defect was restored by an in trans complementation. The intraperitoneal 50% lethal dose of the R62H/D77N strain increased by 26- and 238,000-fold in normal and iron-overloaded mice, respectively. The growth of the R62H/D77N strain in 50% HeLa cell lysate, 100% human ascitic fluid, and 50% human serum was significantly retarded compared to that of the isogenic wild-type strain. The R62H/D77N mutant also had a critical defect in the ability to survive and replicate even in iron-overloaded mice. These results demonstrate that pyrH is essential for the in vivo survival and growth of V. vulnificus and should be an attractive new target for the development of antibacterial drugs and replication-controllable live attenuated vaccines.

Identification of Actinobacillus actinomycetemcomitans using species-specific 16S rDNA primers.

The purpose of this study was to develop species-specific PCR primers for use in the identification and detection of Actinobacillus actinomycetemcomitans. These primers target variable regions of the 16S ribosomal RNA coding gene (rDNA). We assessed the specificity of the primers against 9 A. actinomycetemcomitans strains and 11 strains (3 species) of the Haemophilus genus. Primer sensitivity was determined by testing serial dilutions of the purified genomic DNAs of A. actinomycetemcomitans ATCC 33384T. Our obtained data revealed that we had obtained species-specific amplicons for all of the tested A. actinomycetemcomitans strains, and that none of these amplicons occurred in any of the other species. Our PCR protocol proved able to detect as little as 4 fg of A. actinomycetemcomitans chromosomal DNA. Our findings suggest that these PCR primers are incredibly sensitive, and should prove suitable for application in epidemiological studies, as well as the diagnosis and monitoring of periodontal pathogens after treatment for periodontitis.

Detection of Tannerella forsythia and/or Prevotella intermedia might be useful for microbial predictive markers for the outcome of initial periodontal treatment in Koreans.

A proportion of diseased sites in periodontal disease do not respond to the initial treatment, which might be due in part to the presence of specific microbial pathogens. The aim of this study was to clarify the value of microbial screening for predicting the outcome of periodontal treatment in Koreans using a polymerase chain reaction (PCR). This study enrolled 32 adults with periodontal disease. Microbial and clinical examinations were performed at the baseline and after the initial treatment (professional toothbrushing, scaling, and root planing). Subgingival plaque samples were taken from four sites in each subject (total 128 samples). PCR was used to detect the four putative pathogenic bacteria. There was an improvement in the average of each clinical measurement after the initial treatment. However, approximately half of the sites exhibiting bleeding upon probing (BOP) at the baseline still exhibited bleeding after treatment. There was a close association between the presence of BOP and the presence of Tannerella forsythia (formerly Bacteroides forsythus) and/or Prevotella intermedia. Furthermore, the sites harboring both T. forsythia and P. intermedia at the baseline had a poorer response to treatment than the sites where these two species were not detected. Therefore, microbial screening for T. forsythia and P. intermedia might be useful for predicting the treatment outcome in Koreans.

Pi30 DNA probe may be useful for the identification of Prevotella intermedia at the species or strain level.

Recently, we introduced a new method for the rapid screening of bacterial species-or subspecies-specific DNA probes, named the "inverted dot blot hybridization screening method." This method has subsequently been then applied to develop species-or strain-specific DNA probes for Prevotella intermedia and Prevotella nigrescens. In a previous study, the inverted dot blot hybridization data showed that a probe, Pi30, was specific for P. intermedia. In this study, the DNA probe Pi30 was evaluated by Southern blot analysis to determine if it could distinguish P. intermedia from P. nigrescens. The data showed that the probe Pi30 reacted with the genomic DNAs from the reference strains and clinical isolates of both P. intermedia and P. nigrescens, but the size of the signal bands was different. In addition, the probe Pi30 reacted with a 1.4 kbp fragment from the genomic DNAs digested with Pst I of the P. intermedia strains but not with any fragments of P. nigrescens strains. The result indicates that the probe Pi30 could be useful for the identification of P. intermedia by restriction fragment length polymorphism (RFLP) at the species or strain level.

A new method for rapid screening of bacterial species- or subspecies-specific DNA probes.

A simple assay for the rapid screening of bacterial species- or subspecies-specific DNA probes for the random cloning method is presented, involving the use of genomic DNAs as probes and recombinant plasmid DNAs containing genomic DNA digested with HindIII as targets. The optimal amount of target DNAs and the concentration of digoxigenin-labeled genomic DNA probes were 20 ng and 100 ng ml(-1) (or 10 ng and 200 ng ml(-1)), respectively. The method was applied to the development of Fusobacterium nucleatum subspecies-specific probes. Our results showed that four out of 96 probes were F. nucleatum subspecies-specific, which was confirmed by Southern blot analysis. Our results indicate that the new method can be used for the rapid screening of species- or subspecies-specific probes.