Genetic Analysis and Serological Detection of Novel O-Antigen Gene Clusters of Plesiomonas shigelloides

Abstract

Plesiomonas shigelloides, a member of the family Vibrionaceae, is a gram-negative, rod-shaped, facultative anaerobic bacterium with flagella. P. shigelloides has been isolated from such sources as freshwater, surface water, and many wild and domestic animals. P. shigelloides contains 102 Oantigens and 51 H-antigens. The diversity of O-antigen gene clusters is relatively poorly understood. In addition to O1 and O17 reported by other laboratories, and the 12 O serogroups (O2, O10, O12, O23, O25, O26, O32, O33, O34, O66, O75, and O76) reported previously by us, in the present study, nine new P. shigelloides serogroups (O8, O17, O18, O37, O38, O39, O44, O45, and O61) were sequenced and annotated. The genes for the O-antigens of these nine groups are clustered together in the chromosome between rep and aqpZ. Only O38 possesses the wzm and wzt genes for the synthesis and translocation of O-antigens via the ATP-binding cassette (ABC) transporter pathway; the other eight use the Wzx/Wzy pathway. Phylogenetic analysis using wzx and wzy showed that both genes are diversified. Among the nine new P. shigelloides serogroups, eight use wzx/wzy genes as targets. In addition, we developed an O-antigen-specific PCR assay to detect these nine distinct serogroups with no cross reactions among them.

Keywords: O-antigen gene clusters; PCR assay; Plesiomonas shigelloides; serogroups.

Fig. 1

The O-antigen gene clusters of nine P. shigelloides serogroups O8, O17, O18, O37, O38, O39, O44, O45 and O61.

Fig. 2. Proposed biosynthesis pathways for sugars in the nine P. shigelloides serogroups clusters.

*Indicates the genes that were found outside the OPS clusters. Putative pathways are denoted by a broken line. MnaA, UDP-Nacetylglucosamine- 2-epimerase; Gne, UDP-N-acetylglucosamine-4-epimerase; Gna, UDP-GalNAcA synthetase; WbgZ, C-5 epimerase; FnlA*, 4,6-dehydratase, 3- and 5-epimerase; QnlA, dTDP-4-dehydrorhamnose reductase; QnlB, C-2 epimerase; GalU*, UTP-glucose-1-phosphate uridylyltransferase; GalE, UDP-glucose-4-epimerase; RmlA, glucose-1-phosphate thymidylyltransferase; RmlB, dTDP-D-glucose 4,6-dehydratase; RmlC, dTDP-4-keto-6-deoxy-Dglucose3,5-epimerase; RmlD, dTDP-6-deoxy-L-mannose-dehydrogenase. ManA*, phosphomannose isomerase; ManB*, phosphomannomutase; ManC*, mannose-1-phosphate guanylyltransferase; Gmd, GDP-mannose 4,6-dehydratase; Fcl, GDP-L-fucose synthetase. DManNAc, 2-acetamido-2-deoxy-D-mannose; D-GalNAc, 2-acetamido-2-deoxy-D-galactose; D-GalNAcA, 2-Acetamido-2- deoxy-D-galacturonic acid; L-AltNAcA,2-Acetamido-2-deoxy-L-altruronic acid; D- FucNAc4N, 2-Acetamido-4-amino-2, 4- dideoxy-D-fucose; L-RhaNAc, 2-acetamido-2,6-dideoxy-L-mannose (N-acetyl-L-rhamnosamine); D-GlcNAc, 2-acetamido- 2-deoxy-D-glucose; L-QuiNAc, 2-Acetamido-2-deoxy-L-quinovose (2-acetamido-2,6-dideoxy-L-glucose); D-Glc, Dglucose; D-Gla, D-galactose; L-Rha, L-rhamnose (6-deoxy-L-mannose); L-Fuc, L-fucose; D-Man, D-mannose.

Fig. 3

Comparison of the O-antigen gene clusters of P. shigelloides O39 and O44.

Fig. 4. Phylogenetic analysis.

An unrooted phylogenetic tree constructed by the neighbor-joining method based on the wzx (A) and wzy (B) genes is shown. Bootstrap values were based on 1000 replications and only values greater than 50% are shown.

Fig. 5. PCR detection and sensitivity.

A. Line 1: Layout of the nine single-plex PCR with specific primers for each serotype. Line 2: Negative control. Line 3: Specificity of the PCR for P. shigelloides O61 without cross reaction with the other eight serogroups. Line 4: Mock sample with two serotypes of P. shigelloides 37 and O61. Line 5: Mock sample with three serotypes of P. shigelloides O37, O38 and O61. B. Sensitivity of detection with the genomic DNA of P. shigelloides O61. M, DL 500 bp Marker; Lane 1: 100 ng; Lane 2: 10 ng; Lane 3: 1 ng; Lane 4: 0.1 ng; Lane 5: 0.01 ng; Lane 6: 1 pg; Lane 7: 0.1 pg; Lane 8: 0.01 pg; and Lane 9: negative control.