RESUMEN
Respiratory diseases in ruminants are a main cause of economic losses to farmers worldwide. Approximately 25% of ruminants experience at least one episode of respiratory disease during the first year of life. Mannheimia haemolytica is the main etiological bacterial agent in the ruminant respiratory disease complex. M. haemolytica can secrete several virulence factors, such as leukotoxin, lipopolysaccharide, and proteases, that can be targeted to treat infections. At present, little information has been reported on the secretion of M. haemolytica A2 proteases and their host protein targets. Here, we obtained evidence that M. haemolytica A2 proteases promote the degradation of hemoglobin, holo-lactoferrin, albumin, and fibrinogen. Additionally, we performed biochemical characterization for a specific 110 kDa Zn-dependent metalloprotease (110-Mh metalloprotease). This metalloprotease was purified through ion exchange chromatography and characterized using denaturing and chaotropic agents and through zymography assays. Furthermore, mass spectrometry identification and 3D modeling were performed. Then, antibodies against the 110 kDa-Mh metalloprotease were produced, which achieved great inhibition of proteolytic activity. Finally, the antibodies were used to perform immunohistochemical tests on postmortem lung samples from sheep with suggestive histology data of pneumonic mannheimiosis. Taken together, our results strongly suggest that the 110-Mh metalloprotease participates as a virulence mechanism that promotes damage to host tissues.
Asunto(s)
Mannheimia haemolytica , Pasteurelosis Neumónica , Enfermedades de las Ovejas , Bovinos , Ovinos , Animales , Pasteurelosis Neumónica/diagnóstico , Pasteurelosis Neumónica/microbiología , Metaloproteasas/metabolismo , Péptido Hidrolasas/metabolismo , Rumiantes , Colagenasas/metabolismo , Zinc/metabolismo , Enfermedades de las Ovejas/microbiologíaRESUMEN
Actinobacillus seminis is an autochthonous gram-negative bacterium that affects reproductive organs, causing epididymitis, low fertility, and occasional abortions in ovine and goats. The virulence factors and the pathogenicity mechanisms of A. seminis have not been clearly elucidated yet. In this work, biofilm production by A. seminis in in vitro assays is described and characterized. After 48-h incubation at 37 °C in trypticase soy broth, A. seminis formed biofilms containing an extracellular matrix comprised mainly of fibrillar material. Microaerophilia or the presence of calcium diminished biofilm formation in approximately 50% and 70%, respectively, but low iron concentrations increased it 40%. Through enzymatic digestion, it was found that proteins were the main component of these biofilms. Structural observations through scanning electron microscopy indicated the presence of a high amount of fibrillar material in which bacteria were immersed. Antibodies against different bacterial surface proteins, such as anti-biofilm matrix and anti-adhesin, diminished biofilm formation in 70% and 25%, respectively; whereas furanone C-30 and LED-209, compounds described as quorum-sensing inhibitors, completely inhibited biofilm formation. In conclusion, environmental conditions can influence strongly biofilm formation in A. seminis, and this could be an advantageous strategy that allows bacteria to persist inside a host.