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

Abstract

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.

IgM cleavage by Streptococcus suis reduces IgM bound to the bacterial surface and is a novel complement evasion mechanism

“…Previous work showed that the deletion mutant 10∆ide Ssuis is attenuated in survival in porcine blood with high specific anti- S. suis IgM titers ex vivo [10]. To investigate if the attenuation is due to the inability of the mutant to cleave porcine IgM, blood survival assays with external complementation of the mutant with functional rIde Ssuis and non-functional rIde Ssuis _C195S were performed.…”

“…Previous research indicated, that the IgM protease Ide Ssuis of S. suis is involved in complement evasion [10]. However, as conclusions were based on the comparison of the wt to the mutant 10∆ide Ssuis only, it remained to be shown that the identified phenotypes depend on the lack of the IgM cleavage activity.…”

“…Previous experiments with the specific classical complement pathway inhibitor EGTA MgCl 2 and unencapsulated S. suis showed that C3 deposition on S. suis depends on the classical complement pathway [10]. The importance of the classical complement pathway in immunity to streptococcal infection was demonstrated by Brown et al who identified it as the dominant pathway required for innate immunity to Streptococcus pneumoniae in mice [31].…”

“…The possession of an IgM protease can be pivotal for pathogens early in infection and in young animals when IgM titers are highest. Previous studies of our group showed, that expression of Ide Ssuis reduced IgM-mediated C3 deposition on the bacterial surface and has a positive effect on survival of S. suis in porcine blood in the presence of specific IgM titers [10]. The aim of this study was to investigate if Ide Ssuis is a cysteine protease and to specifically test if the IgM cleavage activity of Ide Ssuis is involved in complement evasion and virulence.…”

“…Virulence factors involved specifically in complement evasion on the other hand are scarce. Proven or potential candidates are the polysaccharide capsule [8], factor H binding proteins FhB and Fhbp [9] and lastly the IgM protease Ide Ssuis [10]. Ide Ssuis , expressed universally among S. suis serotypes, is a highly specific protease, with porcine IgM as its sole substrate [11].…”