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Title: Pasteurella haemolytica leukotoxin and its interaction with target cells
Author: Saadati, Mojtaba
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1995
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A number of Pasteurella haemolytica isolates from cattle and sheep, including representatives of all serotypes and untypables, was examined for leukotoxin (LktA) production at the end of the log phase of growth in brain-heart infusion broth. The bacteria produced most toxin with very high and high aeration, at 37°C, in a low concentration of NaCl and at alkaline pH (pH8). There were marked differences in leukotoxic activity in culture supernate samples, as measured by chemiluminescence-inhibition assays with bovine and ovine neutrophils, even between strains of the same serotype. There was also some variation in the amount and mol. wt of the LktA protein produced by different strains, as judged by SDS-PAGE, immunoblotting and ELISA. Some strains produced normal amounts of LktA protein which had only low leukotoxic activity. Most strains (28/32) produced LktA of 105 kDa whereas four strains produced a higher mol. wt form of c.108 kDa, including two of the five serotype A2 strains examined. Thus, the P. haemolytica isolates showed considerable heterogeneity in terms of leukotoxin production, mol. wt and activity, even within a given serotype. Serial passage of P. haemolytica did not affect the production of leukotoxin (LktA). The mol. wt of LktA was more than 300 kDa as judged by ultrafiltration, due presumably to aggregation. Attempts to disaggregate LktA with SDS, zwittergent, EGTA and urea were not successful. Although P. haemolytica can produce LktA in the absence of calcium, for activity of LktA, calcium was found to be necessary. Active and non-activated rLktA was prepared after transformation of IktA and IktC genes into different strains of E. coli. The biological activities of active recombinant LktA were the same as that of native LktA, but the amount of leukotoxin obtained from E. coli was greater than that from P. haemolytica. Although previous reports had shown that LktA affected and killed only ruminant leukocytes, in this study it was found that LktA had different effects on ruminant and non-ruminant neutrophils. Leukotoxin killed ruminant neutrophils and bovine lymphoma (BL3) cells by cell swelling and lysis, but it had no effect on rabbit and guinea pig neutrophils and a mouse macrophage cell line (J774.2). Surprisingly, native and recombinant LktA partially inhibited the CL response of human neutrophils, although it did not kill the cells. This partial inhibition was due to leukotoxin and not to other components of bacteria such as LPS. Slightly different results were found by a cell tracking assay in which the neutrophils were incubated with active and non-activated recombinant LktA. The active toxin killed bovine neutrophils, but caused the migration of human neutrophils. However, the same concentration of non-activated rLktA had no effect either on CL response or movement of human neutrophils. The reason why LktA promoted movement of human neutrophils, but partially inhibited the CL response deserves further investigation as it suggests two opposing effects of LktA i.e. inhibition of chemiluminescence and production of cell movement. Although non-activated leukotoxin had no effect on CL response of different ruminant and non-ruminant target cells, it bound to these cells in a similar way to that of the active form of rLktA. The active and non-active LktA bound to target cells at 4°C, but to a lesser extent than at 37°C but no lysis of ruminant cells was observed at 4°C, indicating that binding and subsequent lysis can be dissociated and probably represent two separate events. Different monoclonal antibodies were used in an attempt to neutralise the toxicity of LktA, but none of them could completely neutralise its activity. A polyclonal antibody raised against rLktA completely neutralised the homologous LktA but only partially neutralised heterologous LktA from other straiiBof P. haemolytica. In contrast, bovine convalescent serum neutralised homologous and heterologous LktA. In addition to its leukotoxin activity, LktA also has haemolytic activity. This activity is weak and variable in different isolates of P. haemolytica. Nonactivated LktA had no haemolytic activity. Non-activated rLktA was activated in vitro in the presence of LktC and cytosolic activating factor (CAF). This activated toxin affect bovine neutrophils some and had effect on rabbit neutrophils, as judged by CL inhibition assay. However, lower LktA activity was found with the m-v/fro-activated toxin than with m-vivo-activated rLktA.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available