Isolation of Pasteurella haemolytica leukotoxin mutants

• Published in: Infection and Immunity Vol. 63; no. 3; pp. 1027 - 1032
• Main Authors: MONJULA CHIDAMBARAM, SHARMA, B, PETRAS, S. F, REESE, C. P, FROSHAUER, S, WEINSTOCK, G. M
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01-03-1995
• Subjects: analytical methods; antigene; antigenos; antigens; Bacterial Toxins - genetics; Bacteriology; Biological and medical sciences; biological differences; Blotting, Southern; Blotting, Western; crecimiento; croissance; diferencias biologicas; difference biologique; Electrophoresis, Gel, Pulsed-Field; Exotoxins - genetics; Fundamental and applied biological sciences. Psychology; genetica; Genetics; genetique; growth; Mannheimia haemolytica - genetics; Mannheimia haemolytica - growth & development; Microbial Sensitivity Tests; Microbiology; Mutagenesis; mutant; mutantes; mutants; Nitrosoguanidines; Pasteurella haemolytica; pathogenicity; poder patogeno; pouvoir pathogene; Restriction Mapping; Selection, Genetic; technique analytique; tecnicas analiticas; toxinas; toxine; toxins; Pasteurellaceae; Leukotoxin; Mutagenesis; Restriction map; Bacteria; Isolation; Mutation; Pasteurella haemolytica
• ISSN: 0019-9567; 1098-5522
• DOI: 10.1128/IAI.63.3.1027-1032.1995

Two mutants of Pasteurella haemolytica A1 that do not produce leukotoxin were isolated. Following mutagenesis, colonies were screened with antiserum by a filter assay for absence of the secreted leukotoxin. The two mutants both appeared to produce normal amounts of other antigens, as judged by reactivity with polyclonal serum from an animal with pasteurellosis, and were not altered in beta-hemolytic activity as seen on blood agar plates. There was no evidence of either cell-associated or secreted leukotoxin protein when Western blots (immunoblots) were carried out with the polyclonal serum or with a monoclonal antibody directed against the leukotoxin. Southern blots revealed that both mutants show the wild-type restriction pattern at the leukotoxin locus, although the strain with the lktA2 mutation showed differences in other regions of the chromosome on analysis by pulsed-field gel electrophoresis. The strain with the lktA2 mutation grew more slowly than did the wild-type strain, while the strain with the lktA1 mutation was indistinguishable from the wild-type strain in its growth properties. The strain with the lktA1 mutation should be valuable in determining the role of the leukotoxin in virulence as well as in identifying other virulence factors of P. haemolytica.