RESUMO
Mannheimia haemolytica causes respiratory disease in cattle. Amyloid proteins are a major component of biofilms; they aid in adhesion and confer resistance against several environmental insults. The amyloid protein curli is highly resistant to protease digestion and physical and chemical denaturation and binds Congo red (CR) dye. The purpose of this study was to characterize an approximately 50-kDa CR-binding amyloid-like protein (ALP) expressed by M. haemolytica. This protein resisted boiling and formic acid digestion and was recognized by a polyclonal anti-Escherichia coli curli serum, suggesting its relationship with amyloid proteins. Immunolabeling and transmission electron microscopy showed that antibodies bound long, thin fibers attached to the bacterial surface. Mass spectrometry analysis indicated that these fibers are M. haemolytica OmpP2-like proteins. The purified protein formed filaments in vitro, and antiserum against it reacted positively with biofilms. An in silico analysis of its amino acid sequence indicated it has auto-aggregation properties and eight amyloid peptides. Rabbit polyclonal antibodies generated against this ALP diminished the adhesion of ATCC 31612 and BA1 M. haemolytica strains to A549 human epithelial cells, indicating its participation in cell adhesion. ALP expressed by M. haemolytica may be important in its pathogenicity and ability to form biofilms.
Assuntos
Proteínas Amiloidogênicas/química , Proteínas da Membrana Bacteriana Externa/química , Biofilmes/crescimento & desenvolvimento , Mannheimia haemolytica/química , Células A549 , Sequência de Aminoácidos , Proteínas Amiloidogênicas/biossíntese , Proteínas Amiloidogênicas/genética , Proteínas Amiloidogênicas/isolamento & purificação , Animais , Aderência Bacteriana , Proteínas da Membrana Bacteriana Externa/biossíntese , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Bovinos , Vermelho Congo/química , Expressão Gênica , Humanos , Soros Imunes/química , Soros Imunes/isolamento & purificação , Mannheimia haemolytica/genética , Mannheimia haemolytica/isolamento & purificação , Mannheimia haemolytica/metabolismo , Modelos Moleculares , Peso Molecular , Pasteurelose Pneumônica , Ligação Proteica , Estrutura Secundária de Proteína , Coelhos , Alinhamento de Sequência , OvinosRESUMO
Gallibacterium anatis has the ability to hemagglutinate rabbit erythrocytes; however, no bacterial component has yet been associated with this function. In the present work, a protein of approximately 65 kDa with hemagglutinating activity for glutaraldehyde-fixed chicken erythrocytes was purified by ion interchange chromatography from G. anatis F149(T) secreted proteins. The protein was recognized by a rabbit polyclonal serum against a hemagglutinin from Avibacterium paragallinarum. The 65 kDa purified protein presented identity with a G. anatis filamentous hemagglutinin by mass spectrometric analysis. As well, the bacterial surface of G. anatis was labeled by immune gold assays using a polyclonal serum against the 65-kDa protein. A similar protein was recognized in four other G. anatis strains by immunoblots using the same antiserum. The protein binds sheep or pig biotinylated fibrinogen, suggesting an interaction with basement membrane eukaryotic cells components, and the protein is present in G. anatis biofilms. Overall, the results suggest that the 65 kDa hemagglutinin is a common antigen and a potential virulence factor in G. anatis.