Sortase inhibitor phenyl vinyl sulfone inhibits Renibacterium salmoninarum adherence and invasion of host cells

Abstract

Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmonid fishes, is a Gram-positive diplococcobacillus belonging to the family Micrococcaceae. Analysis of the genome sequence of the bacterium demonstrated the presence of a sortase homolog (srtD), a gene specifying an enzyme found in Gram-positive bacteria and required for covalent anchoring of cell surface proteins. Interference of sortase activity is being examined as a target for therapeutic prevention of infection by several pathogenic Gram-positive bacterial species. In silico analysis identified 8 open reading frames containing sortase recognition motifs, suggesting these proteins are translocated to the bacterial cell wall. The sortase and potential sortase substrate genes are transcribed in R. salmoninarum, suggesting they encode functional proteins. Treatment of R. salmoninarum with phenyl vinyl sulfone (PVS) significantly reduced bacterial adherence to Chinook salmon fibronectin. In addition, the ability of the PVS-treated bacteria to adhere to Chinook salmon embryo cells in vitro was dramatically reduced compared to that of untreated bacteria. More importantly, PVS-treated bacteria were unable to invade and replicate within CHSE-214 cells (demonstrated by an intracellular growth assay and by light microscopy). When treated with PVS, R. salmoninarum was not cytopathic to CHSE-214 cells, whereas untreated bacteria produced cytopathology within a few days. These findings clearly show that PVS, a small molecule drug and a known sortase inhibitor, can interfere with the ability of R. salmoninarum to adhere and colonize fish cells, with a corresponding decrease in virulence.KEY WORDS: Renibacterium salmoninarum · Bacterial kidney disease · Anti-virulence chemotherapy · Adherence · Invasion · Host cell sortase inhibitor · Phenyl vinyl sulfone Resale or republication not permitted without written consent of the publisherDis Aquat Org 78: [115][116][117][118][119][120][121][122][123][124][125][126][127] 2007 1994, Kehoe 1994). MSCRAMs have specific C-terminal anchoring signals or cell wall sorting (CWS)-motifs that are recognized by a membrane transpeptidase called sortase. Sortase catalyzes covalent anchoring of these proteins to the peptidoglycan surface during cell wall synthesis (reviewed in Navarre & Schneewind 1999, Ton-That et al. 2004, Marraffini et al. 2006). Elimination of sortase activity and the consequent disruption of surface protein anchoring correlates with a dramatic decrease in pathogenicity of Gram-positive bacteria in animal infections (Mazmanian et al. 2000, Bolken et al. 2001, Bierne et al. 2002, Garandeau et al. 2002, Jonsson et al. 2002, Weiss et al. 2004. Sortase is also involved in the assembly of cell surface pili on Gram-positive bacteria, which often aid attachment to host cells (Ton-That & Schneewind 2003). Collectively, these and other findings suggest that sortases are promising targets for new antibacterial drugs (Schneewind et al. 1993, Frankel et al. 2004, Ton-That et al. 2004, Weis...

Genome Sequence of the Fish Pathogen Renibacterium salmoninarum Suggests Reductive Evolution away from an Environmental Arthrobacter Ancestor

“…strain FB24, respectively. Using the tripartite pattern search method of Boekhorst et al (5), as well as tools such as Pfam, SignalP, TMpred, and ProDom, we identified seven ORFs in the R. salmoninarum ATCC 33209 genome specifying potential sortase substrates containing the LAxTG motif (RSal 33209_2105, RSal33209_3407, RSal33209_2525, RSal33209_ 3273, RSal33209_0619, RSal33209_1326, and RSal33209_ 0402), some of which were predicted to be cell surface proteinases or adhesins (62). Two of the seven potential SrtD substrates identified through bioinformatics analysis are annotated as products of pseudogenes (RSal33209_0619 and RSal 33209_1326), and the current search may have failed to identify additional complete sortase substrates.…”

“…Treatment of R. salmoninarum with sortase inhibitors decreases the adherence of R. salmoninarum to fibronectin and fish cells in vitro, suggesting that these proteins have a role in attachment (62). Recent drug discovery efforts have focused on sortases as a possible therapeutic target, and it is possible that such efforts will be important in preventing R. salmoninarum infection (62). While R. salmoninarum SrtD is the only apparent active sortase, the presence of a degenerate srtA gene and at least one degenerate SrtA substrate with homologs in Arthrobacter may represent additional examples of specific genome decay in a pathway not needed for the intracellular lifestyle of R. salmoninarum.…”

“…Three of the seven sortase cleavage motif-encoding ORFs are also present in Arthrobacter, and two of these ORFs appear to be pseudogenes in R. salmoninarum. Treatment of R. salmoninarum with sortase inhibitors decreases the adherence of R. salmoninarum to fibronectin and fish cells in vitro, suggesting that these proteins have a role in attachment (62). Recent drug discovery efforts have focused on sortases as a possible therapeutic target, and it is possible that such efforts will be important in preventing R. salmoninarum infection (62).…”

“…This pathogen can also be transmitted horizontally from infected fish sharing a water supply (1,40). Prophylaxis or prevention of R. salmoninarum infection is challenging, due in part to the ability of this bacterium to survive phagocytosis and possibly replicate within macrophages (2,33,62,73). For Pacific salmon, there are no efficacious vaccines, and antibiotic therapy never fully eliminates the pathogen (15,55).…”

Comparative Pathogenomics of Bacteria Causing Infectious Diseases in Fish

“…Infected fish may take months to show signs of disease. bacterial kidney disease is one of the most difficult bacterial diseases of fish to treat [108], mainly due to its ability to evade phagocytosis and invade and survive in host cells [109, 110]. R. salmoninarum is very slow growing, and it is extremely difficult to apply genetic manipulation techniques to study its gene functions.…”