Actinobacillus pleuropneumoniae, is a Gram-negative, facultative anaerobic coccobacillus of the Pasteurellaceae family . It is the causative agent of porcine pleuropneumonia. This highly infectious disease causes impaired animal welfare and serious economic losses in the swine industry, world-wide. The infection can lead to both peracute disease with rapid death and chronic infection resulting in asymptomatic carriers . Based on differences in capsular polysaccharides, 15 serotypes have been recognized . The serotypes differ greatly in both virulence potential, immunogenicity and in geographical distribution [4–8]. Due to differences in immunogenicity, vaccines raised against one serotype do not provide protection from infection by other serotypes .
A number of virulence factors have been described for A. pleuropneumoniae [2, 9–11]. Serotype variations in virulence potential seem to be primarily governed by the amount of capsule and the combination of RTX toxins, denoted apxI, apxII, and apxIII, produced by the individual serotypes [12, 13]. The most virulent combination, apxI and apxII, is produced by serotypes 1, 5, 9, and 11. ApxII and apxIII are found in the medium virulent serotypes 2, 3, 4, 6, 8, and 15. The remaining serotypes produce one toxin: apxII by serotypes 7, 12, and 13 and apxI by serotypes 10, and 14 . Serotypes 7 and 12 are also considered to be of medium virulence, while serotypes 10, 13 and 14 are only rarely isolated from disease [4, 14]. Still, observations of variation in pathogenic potential, even among serotypes and strains expressing the same apx toxins, indicate that other virulence determinants must be contributing to the observed differences in pathogenesis [2, 15–17]. Serotype 3 is generally believed to be less virulent than the remaining types [4, 18], although some serotype 3 strains showed no difference in pathogenicity when compared to other apxII/apxIII producing serotypes [7, 17].
An important virulence factor for bacteria is the ability to survive and grow in an iron-limited environment . Iron is involved in metabolic pathways, respiration, oxygen transport, DNA synthesis and synthesis of metabolites [19, 20] and is critical to the invading microorganisms for establishing infection. As part of the innate defense, the mammalian host keeps the levels of intracellular free iron to around 10-18M which is insufficient to allow bacterial growth . The low level of free iron in the host is maintained by high affinity proteins such as transferrin, lactoferrin, haem, haemoglobin (Hb), and ferritin .
Like other pathogens, A. pleuropneumoniae has adapted a number of strategies for scavenging host iron. The bacterium is able to use porcine transferrin as well as haem proteins as sole sources of iron. A. pleuropneumoniae genes known to be involved in iron uptake are the porcine transferrin specific outer membrane (OM) proteins, tbpA and tbpB, the co-transcribed tonB-exbB - exbD complex [21–23], and the second tonB system, designated exbB2 - exbD2 - tonB2 . Solely responsible for the Hb uptake in A. pleuropneumoniae is the haemoglobin binding protein, hgbA . The presence of a periplasmic binding protein-dependent transport system, homologuous to yfeABCD of Yersinia pestis has been documented in A. pleuropneumoniae and other Pasteurellaceae species [26–29]. A gene cluster sharing homology with the HmbR Hb receptor from N. meningitides has recently been identified by microarray analysis . In A. pleuropneumoniae, the putative Actinobacillus ferric uptake operon, afuABC, and the siderophore ferrichrome uptake, fhu, receptor are not regulated by iron-levels [30–33].
In many bacteria the intracellular iron level and utilization is controlled by the balance between the regulatory protein, fur (ferric-uptake regulator protein), and ryhB [20, 34]. Fur is a global gene regulator involved in numerous functions of the cell, such as respiration, glycolysis, purine metabolism, and redox-stress resistance . It represses transcription upon interaction with its co-repressor Fe2+, and causes de-repression in the absence of Fe2+ . The fur regulated ryhB, a small non-coding RNA (sRNA), acts by repressing iron-using proteins under iron-restricted conditions . Whether ryhB or other sRNAs are involved in the regulatory response of Pasteurellaceae remains to be demonstrated. Recently, however, homologues of the global sRNA regulator, Hfq, a key factor in regulations by sRNAs in bacteria, has also been identified in A. pleuropneumoniae, P. multocida, and H. influenza . Among the many Hfq-dependent regulators is ryhB .
Microarray analysis of gene regulation under iron restriction have been studied in A. pleuropneumoniae, Pasteurella multocida, Haemophilus influenza, Mannheimia haemolytica, and Haemophilus parasuis, respectively [26–29, 36]. Presently, however, the knowledge of intra-species variation in response to iron deprivation is limited for the Pasteurellaceae family. Only few comparative transcriptional profiling studies have been performed in this group and none for A. pleuropneumoniae . We used an in vitro model system to compare the response of moderate (serotypes 2, 3, 6, 7), and highly virulent strains (serotypes 1 and 5) of A. pleuropneumoniae to the iron restricted conditions found in the porcine host. The primary aims were: 1) to identify any variations in the transcriptional response among the serotypes which might contribute to the explanation of the observed differences in virulence, 2) to identify a set of genes defining the core modulon of A. pleuropneumoniae in response to iron limitation, 3) to develop a valid method for transcriptional comparison of multiple serotypes.