Theoretical and quantitative aspects of iron acquisition by a bovine isolate of Pasteurella multocida A:3

Abstract

Current epidemiological data (VIDA UK) relating to bovine pneumonic pasteurellosis, a disease of welfare and economic significance worldwide, indicate that Pasteurella multocida serotype A has overtaken Mannheimia haemolytica as the leading cause of disease. Iron acquisition mechanisms in P. multocida, as with the majority of bacteria, are an essential element for survival and proliferation and a major virulence factor, although the detailed processes involved are not defined. A mathematical approach described here has constructed a reaction network representing iron acquisition from host transferrin, suggested target genes for quantitative RT-PCR (qRT-PCR) and provided data for initial theoretical modelling. Primers were designed for qRT-PCR study of 8 iron acquisition genes: fur, tbpA, tonB, exbB, exbD, fbpA, fbpB and fbpC and applied to the analysis of total RNA extracted from P. multocida A:3 grown in vitro in iron replete and iron restricted conditions in a 4 x 1L benchtop fermenter (B. Braun Biotech); 3 vessels iron restricted (αα-dipyridyl), 1 control. Growth rates were measured from hourly sequential samples by estimated and live viable counts. Results for qRT-PCR revealed differences in expression of iron restricted outer membrane protein (IROMP) genes in iron replete and iron restricted conditions. In replete conditions, all genes showed a similar transcription pattern with a steady increase from 0-4 hours, followed by a plateau from 4-8 hours. Under iron restriction, all genes with the exception of fur exhibited a faster initial phase of transcription from 0-2 hours followed by a drop at 2 hours and a subsequent recovery at 6-8 hours. The transcription pattern for fur showed an initial drop from 0-2 hours followed by a steady increase in expression tending to plateau between 6-8 hours. The differing transcription pattern of fur is expected due to its role as a transcriptional repressor of IROMP expression. Surprisingly, bacterial growth rate was not suppressed by iron restriction using 200 µM αα-dipyridyl, suggesting that IROMP expression under these conditions was sufficient to provide the necessary iron. The subsequent drop in transcription may represent a period wherein acquired iron is metabolised after which a recovery in transcription is triggered by the need to replenish intracellular iron levels. Results suggest that the initial period of growth from 0-3 hours should be studied in greater depth.