Two putative zinc metalloproteases contribute to the virulence of strains that cause avian necrotic enteritis.

Two putative zinc metalloproteases encoded by have been implicated in the pathogenesis of necrotic enteritis, an economically significant poultry disease that is caused by this anaerobic bacterium. These proteases have ~64% amino acid identity and are encoded by the and genes. We screened 83 isolates by PCR for the presence of these genes. The first gene, , is chromosomally located and was present in all screened strains of , regardless of their origin and virulence. The second gene, , is plasmid-borne and was only found in isolates derived from chickens with necrotic enteritis. We describe the generation of insertionally inactivated mutants of both and in a virulent isolate. For each mutant, a significant (< 0.001) reduction in virulence was observed in a chicken necrotic enteritis disease model. Examples of each mutant strain were characterized by whole genome sequencing, which showed that there were a few off-site mutations with the potential to affect the virulence of these strains. To confirm the importance of these genes, independently derived and mutants were constructed in different virulent isolates and shown to have reduced virulence in the experimental disease induction model. A double mutant also was generated and shown to have significantly reduced virulence, to the same extent as the respective single mutants. Our results provide evidence that both putative zinc metalloproteases play an important role in disease pathogenesis.