A Novel 16S rRNA PCR-Restriction Fragment Length Polymorphism Assay to Accurately Distinguish Zoonotic Capnocytophaga canimorsus and C. cynodegmi.

The zoonotic bacteria Capnocytophaga canimorsus and C. cynodegmi, the predominant Capnocytophaga species in the canine oral biota, can cause human local wound infections or lethal sepsis, usually transmitted through dog bites. Molecular surveying of these Capnocytophaga species using conventional 16S rRNA-based PCR is not always accurate due to their high genetic homogeneity. In this study, we isolated Capnocytophaga spp. from the canine oral cavity and identified them using 16S rRNA and phylogenetic analysis. A novel 16S rRNA PCR-restriction fragment length polymorphism (RFLP) method was designed based on our isolates and validated using published C. canimorsus and C. cynodegmi 16S rRNA sequences. The results showed that 51% of dogs carried Capnocytophaga spp. Among these, C. cynodegmi (47/98, 48%) was the predominant isolated species along with one strain of C. canimorsus (1/98, 1%). Alignment analysis of 16S rRNA sequences revealed specific site nucleotide diversity in 23% (11/47) of the C. cynodegmi isolates, which were misidentified as C. canimorsus using previously reported species-specific PCR. Four RFLP types could be classified from all the isolated Capnocytophaga strains. The proposed method demonstrates superior resolution in distinguishing C. cynodegmi (with site-specific polymorphism) from C. canimorsus and especially in distinguishing C. canimorsus from other Capnocytophaga species. After in silico validation, this method was revealed to have an overall detection accuracy of 84%; notably, accuracy reached 100% in C. canimorsus strains isolated from human patients. Overall, the proposed method is a useful molecular tool for the epidemiological study of Capnocytophaga in small animals and for the rapid diagnosis of human C. canimorsus infections. IMPORTANCE With the increased number of small animal breeding populations, zoonotic infections associated with small animals need to be taken more seriously. Capnocytophaga canimorsus and C. cynodegmi are part of common biota in the mouths of small animals and can cause human infections through bites or scratches. In this study, C. cynodegmi with site-specific 16S rRNA sequence polymorphisms was erroneously identified as C. canimorsus during the investigation of canine Capnocytophaga by conventional PCR. Consequently, the prevalence of C. canimorsus is incorrectly overestimated in epidemiological studies in small animals. We designed a new 16S rRNA PCR-RFLP method to accurately distinguish zoonotic C. canimorsus from C. cynodegmi. After validation against published Capnocytophaga strains, this novel molecular method had high accuracy and could detect 100% of C. canimorsus-strain infections in humans. This novel method can be used for epidemiological studies and the diagnosis of human Capnocytophaga infection following exposure to small animals.

Caveolin-1 facilitates cyclooxygenase-2 protein degradation.

Cyclooxygenase-2 (COX-2) plays major roles in diverse physiological and pathological processes such as inflammation and tumorigenesis. Transcriptional control of COX-2 has been extensively investigated and characterized, but its post-translational control is less clear. Here, we report a novel mechanism by which COX-2 is degraded. Protein levels of caveolin-1 (Cav-1) and COX-2 showed an inverse relation in colon cancer cell lines. COX-2 proteins in lung and colon tissues were higher in Cav-1 null mice than in wild-type mice. RNAi knockdown of Cav-1 increased COX-2 protein level and decreased ubiquitinated COX-2 accumulation. In addition, deletion of the carboxy (C)-terminus of COX-2, which contains a unique 19-amino acid segment compared with COX-1, resulted in reduced Cav-1 binding and attenuated COX-2 degradation. COX-1 and green fluorescence protein containing the C-terminus of COX-2 resulted in enhanced degradation. Our findings suggest that Cav-1 binds COX-2 in endoplasmic reticulum (ER) and carries it for degradation via ER associated degradation. The C-terminal region of COX-2 is required for Cav-1 binding and degradation. These results indicate a novel function of Cav-1 in controlling COX-2 expression, which may regulate physiological functions and have tumor suppression effects.