Lack of a type-2 glycosyltransferase in the fish pathogen Flavobacterium psychrophilum determines pleiotropic changes and loss of virulence.

Flavobacterium psychrophilum is an important fish pathogen, responsible for Cold Water Disease, with a significant economic impact on salmonid farms worldwide. In spite of this, little is known about the bacterial physiology and pathogenesis mechanisms, maybe because it is difficult to manipulate, being considered a fastidious microorganism. Mutants obtained using a Tn4351 transposon were screened in order to identify those with alteration in colony morphology, colony spreading and extracellular proteolytic activity, amongst other phenotypes. A F. psychrophilum mutant lacking gliding motility showed interruption of the FP1638 locus that encodes a putative type-2 glycosyltransferase (from here on referred to as fpgA gene, Flavobacterium psychrophilum glycosyltransferase). Additionally, the mutant also showed a decrease in the extracellular proteolytic activity as a consequence of down regulation in the fpgA mutant background of the fpp2-fpp1 operon promoter, responsible for the major extracellular proteolytic activity of the bacterium. The protein glycosylation profile of the parental strain showed the presence of a 22 kDa glycosylated protein which is lost in the mutant. Complementation with the fpgA gene led to the recovery of the wild-type phenotype. LD50 experiments in the rainbow trout infection model show that the mutant was highly attenuated. The pleiotropic phenotype of the mutant demonstrated the importance of this glycosyltranferase in the physiology and virulence of the bacterium. Moreover, the fpgA mutant strain could be considered a good candidate for the design of an attenuated vaccine.

Expression of nitric oxide synthase (NOS) genes in channel catfish is highly regulated and time dependent after bacterial challenges.

Nitric oxide is well known for its roles in immune responses. As such, its synthesizing enzymes have been extensively studied from various species including some teleost fish species. However, the NOS genes have not been characterized in channel catfish (Ictalurus punctatus). In this study, we identified and characterized three NOS genes including one NOS1 and two NOS2 genes in channel catfish. Comparing with the NOS genes from other fish species, the catfish NOS genes are highly conserved in their structural features. Phylogenetic and syntenic analyses allowed determination of NOS1 and NOS2 genes of channel catfish and their orthology relationships. Syntenic analysis, as well as the phylogenetic analysis, indicated that the two NOS2 genes of catfish were lineage-specific duplication. The NOS genes were broadly expressed in most tested tissues, with NOS1 being expressed at the highest levels in the brain, NOS2b1 highly expressed in the skin and gill, and NOS2b2 lowly expressed in most of the tested tissues. The most striking findings of this study was that the expression of the NOS genes are highly regulated after bacterial infection, with time-dependent expression patterns that parallel the migration of macrophages. After Edwardsiella ictaluri challenge, dramatically different responses among the three NOS genes were observed. NOS1 was only significantly in the skin early after infection, while NOS2b1 was rapidly upregulated in gill, but more up-regulated in trunk kidney with the progression of the disease, suggesting such differences in gene expression may be reflective of the migration of macrophages among various tissues of the infected fish. In contrast to NOS1 and NOS2b1, NOS2b2 was normally expressed at very low levels, but it is induced in the brain and liver while significantly down-regulated in most other tissues.

Ornithobacterium rhinotracheale has neuraminidase activity causing desialylation of chicken and turkey serum and tracheal mucus glycoproteins.

Neuraminidases (sialidases) are virulence factors of several poultry pathogens. Ornithobacterium rhinotracheale is a well known poultry pathogen causing respiratory disease in chickens and turkeys all over the world. We investigated whether O. rhinotracheale has neuraminidase enzymatic activity (NEAC). We tested NEAC in 47 O. rhinotracheale strains isolated from turkeys and chickens in eight countries. All strains showed relatively strong NEAC and considerable levels of NEAC were detected also in "cell-free supernatants" of their pelleted cells. Zymography using neuraminidase-specific chromogenic substrate indicated that a protein with molecular mass of ~40kDa and isoelectric point (pI) of ~8.0 is a putative neuraminidase of O. rhinotracheale. Notably, the genome of the type strain of O. rhinotracheale, DSM 15997 contains a gene (Ornrh_1957) encoding a putative neuraminidase with such Mw (39.5 kDa) and pI (8.5). We sequenced a corresponding genomic region of 20 O. rhinotracheale strains and found five distinct types of the neuraminidase gene (termed nanO) sequences. Most diversified nanO sequence was found in two strains isolated from chickens in Hungary in 1995. Their nanO sequences differ from that of the type strain (LMG 9086(T)) in 27 nucleotides. O. rhinotracheale neuraminidase showed capacity to cleave sialic acid from chicken and turkey glycoproteins. It cleaved sialic acid from SAα(2-6)gal moiety of their serum proteins, including immunoglobulin G (IgG) and transferrin. O. rhinotracheale also desialylated chicken and turkey tracheal mucus glycoprotens with SAα(2-3)gal moieties. This study provides the first evidence that O. rhinotracheale has neuraminidase which can desialylate glycoproteins of its natural hosts.