Thimerosal and Animal Brains: New Data for Assessing Human Ethylmercury Risk

Background: To better understand effects of iron restriction on Actinobacillus pleuropneumoniae and to identify new potential vaccine targets, we conducted transcript profiling studies using a DNA microarray containing all 2025 ORFs of the genome of A. pleuropneumoniae serotype 5b strain L20. This is the first study involving the use of microarray technology to monitor the transcriptome of A. pleuropneumoniae grown under iron restriction. Results: Upon comparing growth of this pathogen in iron-sufficient versus iron-depleted medium, 210 genes were identified as being differentially expressed. Some genes (92) were identified as being up-regulated; many have confirmed or putative roles in iron acquisition, such as the genes coding for two TonB energy-transducing proteins and the hemoglobin receptor HgbA. Transcript profiling also led to identification of some new iron acquisition systems of A. pleuropneumoniae. Genes coding for a possible Yfe system (yfeABCD), implicated in the acquisition of chelated iron, were detected, as well as genes coding for a putative enterobactin-type siderophore receptor system. ORFs for homologs of the HmbR system of Neisseria meningitidis involved in iron acquisition from hemoglobin were significantly up-regulated. Down-regulated genes included many that encode proteins containing Fe-S clusters or that use heme as a cofactor. Supplementation of the culture medium with exogenous iron re-established the expression level of these genes. Conclusion: We have used transcriptional profiling to generate a list of genes showing differential expression during iron restriction. This strategy enabled us to gain a better understanding of the metabolic changes occurring in response to this stress. Many new potential iron acquisition systems were identified, and further studies will have to be conducted to establish their role during iron restriction. Published: 13 March 2007 BMC Genomics 2007, 8:72 doi:10.1186/1471-2164-8-72 Received: 9 June 2006 Accepted: 13 March 2007 This article is available from: http://www.biomedcentral.com/1471-2164/8/72 © 2007 Deslandes et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Page 1 of 20 (page number not for citation purposes) BMC Genomics 2007, 8:72 http://www.biomedcentral.com/1471-2164/8/72 Background Actinobacillus pleuropneumoniae, etiological agent of porcine pleuropneumonia, causes great commercial losses to the swine industry worldwide [1]. Transmission of this highly contagious disease that affects pigs of all ages occurs mostly by aerosol and close contact with infected animals [2]. During 24 to 48 hours of the acute phase of the disease, formation of extensive and fibrinohemorrhagic lung lesions is often fatal. Animals that survive the disease may become asymptomatic carriers of the bacteria, developing localized and necrotizing lesions associated with pleuritis [3]. Based on differences of capsular polysaccharides, fifteen serotypes have been identified: serotypes 1 to 12 and 15 belong to biotype 1, which is NAD-dependent; serotypes 13 and 14 are classified in biotype 2, which is NAD-independent [4]. In North America, serotypes 1, 5 and 7 are prevalent, while serotypes 2 and 9 are most commonly found in Europe. Despite many years of research, the total complement of bacterial components that are involved in infection by A. pleuropneumoniae has yet to be identified. Several virulence factors have been proposed: capsular polysaccharides, lipopolysaccharides (LPS), Apx toxins and various iron acquisitions systems [2]. However, the overall contribution of each component to the infection process remains unclear. Although less virulent, an acapsular mutant was still serum-resistant, showed higher adhesion to piglet tracheal frozen sections and could still be re-isolated from lungs of infected animals [5]. LPS apparently plays a role in adhesion in vivo, as these molecules show in vitro adhesion to many biological components [4]. The Apx toxins contribute to development of lesions typically associated with the disease [6] and mutants missing Apx toxins are avirulent in pigs and mice [7]. However, different A. pleuropneumoniae serotypes secrete different sets of Apx toxins, and the relative contribution of the four different Apx toxins (ApxI to IV) is still not clear. Low availability of iron in the host represents a major stress for bacterial pathogens and is considered a signal that leads to significant changes in cell processes. Iron atoms are often linked with sulphur in Fe-S clusters in the catalytic core of enzymes involved in diverse functions such as respiration, ATP generation, and DNA replication and repair, which might account for this phenomenon. Iron is an essential element for almost every living organism. However in the host, molecules such as transferrin, lactoferrin, haptoglobin and hemoglobin in extra-cellular fluids bind free iron and iron-containing molecules very tightly [8]. While bacteria generally need free iron concentrations of about 10-7 M, its concentration may be 10-24 M in the mammalian host [9]. To counteract the effect of these iron-withholding mechanisms of the host, bacteria have evolved different iron acquisition systems, often relying on the secretion of siderophores, small (<1000 Da) molecules with high affinity for iron, or on surface receptors specific for iron-containing host proteins [10]. Studies in our laboratory have led to the identification, expression and characterization of the A. pleuropneumoniae hydroxamate siderophore receptor FhuA [11,12] and a hemoglobin binding receptor HgbA [13]. A. pleuropneumoniae also possesses a transferrin receptor complex composed of two outer membrane (OM) proteins: a 100 kDa TbpA may form a transmembrane channel enabling transport of iron across the OM; a 60 kDa lipoprotein TbpB acts as an auxillary molecule [2,14,15]. Energization of these OM transporters relies on the transduction of the proton motive force from the cytoplasmic membrane (CM) by the TonB-ExbB-ExbD complex [16] that is anchored in the CM and spans the periplasm. In A. pleuropneumoniae, two different TonB systems have been identified: genes tonB1-exbB1-exbD1 are transcriptionally linked to the tbpA-tbpB genes [17]; and a second system with genes tonB-exbB2-exbD2 was also identified [18]. Transport of iron across the CM is apparently accomplished by the AfuABC ABC transporter [19]. It has also been shown that A. pleuropneumoniae can use exogenous siderophores and may be able to secrete an iron chelator in response to iron stress [20]. The ferric uptake regulator Fur protein has been identified in many pathogenic bacteria, including A. pleuropneumoniae [21]. Using Fe2+ as a cofactor, the Fur protein can interact with a specific sequence termed the Fur box in the promoter region of genes implicated in iron acquisition processes, thereby repressing transcription. When iron becomes scarce, the Fur protein loses its cofactor and becomes inactive. The fact that transcription of some genes seems to be under positive control of active Fur protein [22,23] was recently explained by the discovery of RyhB, a small non-coding RNA which belongs to the Fur regulon [24]. When transcribed, the RyhB RNA down-regulates the mRNA level of those genes that seemed to be positively regulated by Fur. To better understand the mechanisms used by A. pleuropneumoniae that address iron restriction and to gain insights into strategies used by this pathogen under conditions mimicking the in vivo environment, we evaluated gene expression profiles of A. pleuropneumoniae grown under iron restriction. Our study identified 210 differentially expressed genes, of which 92 are up-regulated. Within the latter set, components of previously unrecognized iron acquisition systems were identified: a putative enterochelin-like siderophore receptor, a potential Yfe system for the acquisition of chelated iron, a putative hemoglobin acquisition system homologous to the N. meningitidis HmbR system, and a putative Fe2+-specific porin system. Page 2 of 20 (page number not for citation purposes) BMC Genomics 2007, 8:72 http://www.biomedcentral.com/1471-2164/8/72 Results and Discussion Microarray analysis of mRNA levels during growth of A. pleuropneumoniae under iron-restricted conditions To assess the response of A. pleuropneumoniae to iron restriction, the reference strain S4074 was grown in BHI broth containing 50 μg/ml EDDHA, a concentration sufficient to cause iron restriction [11]. This strain was chosen because it is the strain that has been the most studied over time, but also because major problems were encountered with RNA extraction from strain L20. Preliminary CGH studies conducted in our lab showed that 95% of the genes of the A. pleuropneumoniae 5b L20 genome are conserved between both strains. Growth curves established the optimum growth phase for RNA extraction (data not shown). At 50 μg/ml of EDDHA, bacterial growth is almost completely inhibited within an hour of addition. By adding the iron chelator at an optical density of 0.1, iron-restricted cultures and iron-rich cultures were harvested concurrently at an optical density of 0.3. Under these growth conditions, we identified 210 differentially expressed genes, with an estimated false discovery rate (FDR) of 3.22%: 118 were down-regulated (Table 1) and 92 were up-regulated (Table 2). In order to confirm that these variations were not caused by the chelator, control experiments where iron was supplemented to the restricted medium were conducted. Exogenous iron, in the form of FeCl3, was added to a final concentration of 50 μg/ml to the iron-depleted medium. Growth curves indicated that this concentration of FeCl3 was sufficient to promote growth at a similar level as in the BHI broth. Under these conditions, the expression pattern was highly similar to that seen in BHI broth: we identified only 30 differentially expressed genes, out of 2025, with an estimated FDR of 2.5%, 26 of which were up-regulated, while only 4 were down-regulated (data not-shown). Only 12 genes significantly differentially expressed in the iron-supplemented medium were identified as such in the irondepleted versus BHI broth experiment, but with reversed levels of variation. Gene lldD (ap2032), which was up-regulated in the iron-depleted medium, was down-regulated in the iron-supplemented medium. Conversely, 11 genes that were down-regulated in the iron-depleted medium were up-regulated in the iron-supplemented medium. This indicates that the results obtained in the irondepleted versus BHI broth experiment can be attributed to iron restriction, and not to another effect of the chelator. Validation of microarray results by qRT-PCR Seventeen genes, representing a wide range of log2 ratio values, were selected for transcript level analysis using qRT-PCR. Seven genes were overexpressed during iron restriction (tonB1, hgbA, omp64, fetB2, apxIC, PM0741, NMB1668); eight genes were repressed (nrfA, nrfC, nfrE, ompW, dcuB2, dmsA, torA, ccmC); two genes were not affected (pedD, ap1465). We also investigated the transcript level of the exbB1, exbD1 and tbpA genes, all known to be transcriptionally linked to tonB1 [17] and previously used as positive controls to assess iron restriction [12]. However, they were not present on the AppChip1 as this region of the genome was in one of the few unsequenced areas when the microarrrays were designed. In all cases, genes that had been identified as upor down-regulated with the microarrays were confirmed by the qRT-PCR experiments. The exbB1, exbD1 and tbpA genes were also up-regulated. Genes not affected showed low level of variation during qRT-PCR analysis, and show good correlation with other results (Fig. 1). Overall, there was good correlation between the log2 ratios measured by microarray and log2 ratios from qRT-PCR data (R2 = 0.87). The log2 ratios observed with qRT-PCR were usually superior to those observed with the microarray. This outcome has been observed before [25,26] and probably reflects the detection limit of microarrays as well as the complex normalization methods that are used prior to the analysis. Genes expressed differentially under iron restriction To evaluate the effect of iron restriction on the porcine pathogen A. pleuropneumoniae, we performed microarray hybridization experiments. Given that iron plays a vital role in metabolic pathways through its presence in the structure of numerous enzymes [27] and its implication in the regulation of genes associated with virulence [28], we recorded important changes in the transcriptome of the bacteria under iron-restricted conditions. A total of 210 genes showed differential expression and the functional classification of these genes provides a significant overview of changes occurring in the bacteria. Numerous microarray studies have investigated effects of iron restriction in many different pathogens, including E. coli [29], H. pylori [30], H. parasuis [31], N. gonorrhoeae [25], N. meningitidis [32], as well as Pasteurella multocida [33], a well known animal pathogen closely related to A. pleuropneumoniae. Many genes that were identified as being ironregulated in the P. multocida study were homologs of some genes that were also identified in our study (Table 3), thus emphasizing the importance of their regulation during iron restriction. A common feature in all these studies is the high induction of genes related to iron acquisition as the products of these genes are essential for survival of the bacteria. (i) Down-regulated genes Down-regulated genes (Fig. 2) mostly belong to the functional class termed "Energy Metabolism"; 42 of the 118 repressed genes (35%) belong to this group, and they are amongst the most highly repressed. Almost all these genes encode proteins with Fe-S clusters, that use heme molecules as cofactors, or that are activated by Fe2+ or other divalent cations. These include genes coding for the different subunits of formate dehydrogenase (bisC, hybA, fdhE), Page 3 of 20 (page number not for citation purposes) BMC Genomics 2007, 8:72 http://www.biomedcentral.com/1471-2164/8/72 Table 1: A. pleuropneumoniae genes which are down-regulated during iron restriction Gene ID Gene Description Fold Change Hypothetical/Unclassified/Unknown ap0497 engA putative GTP binding protein -2.27 ap0491 glnE Unknown -1.98 ap1365 srmB uncharacterized conserved protein -1.85 ap1538 traC conserved hypothetical protein -1.72 ap0677 nfnB putative nitroreductase, FMN-dependent -1.70 ap1779 mscL conserved hypothetical protein -1.69 ap0802 dxr conserved hypothetical protein, distant homolog of PhoU -1.58 ap0787 cdsA putative transcriptional regulator -1.54 ap0685 mlc protein of unknown function -1.53 ap1297+ sspA predicted iron-dependent peroxidase -1.53 ap0973 abgB possible metal dependent peptidase, unclassified -1.48 ap1405 nth possible sodium/sulphate transporter -1.41 ap1725 mviN uncharacterized membrane protein, putative virulence factor -1.38 ap0622 aroC flp operon protein C -1.28 ap0989 fstX conserved hypothetical protein -1.27 Biosynthesis of cofactors ap0684 bioD1 probable dethiobiotin synthetase -3.49 ap1624 menA 1,4-dihydroxy-2-naphthoateoctaphenyltransferase -1.57 ap1131 hemC porphobilinogen deaminase -1.47 ap0447 hemA glutamyl-tRNA reductase -1.40 ap1080 hemN oxygen-independent corproporphyrinogen III oxydase -1.40 ap2005 menB naphthoate synthase -1.39 ap1684 ispH hydroxymethylbutenyl pyrophosphate reductase -1.37 ap2023 4-hydroxybenzoate synthetase -1.31