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Infection and Immunity, June 2005, p. 3740-3744, Vol. 73, No. 6
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.6.3740-3744.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Deletion of the Ferric Uptake Regulator Fur Impairs the In Vitro Growth and Virulence of Actinobacillus pleuropneumoniae

Ilse Jacobsen,1 Jörg Gerstenberger,1 Achim D. Gruber,2 Janine T. Bossé,3 Paul R. Langford,3 Isabel Hennig-Pauka,1,4 Jochen Meens,1 and Gerald-F. Gerlach1*

Department of Infectious Diseases, Institute for Microbiology,1 Department of Pathology,2 Clinic for Pigs and Small Ruminants, University of Veterinary Medicine Hannover Foundation, Hannover, Germany,4 Department of Paediatrics, Imperial College London, St. Mary's Campus, London W2 1PG, United Kingdom3

Received 14 December 2004/ Returned for modification 24 January 2005/ Accepted 14 February 2005


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ABSTRACT
 
In order to investigate the role of the ferric uptake regulator Fur in the porcine lung pathogen Actinobacillus pleuropneumoniae, we constructed an isogenic in-frame deletion mutant, A. pleuropneumoniae {Delta}fur. This mutant showed constitutive expression of transferrin-binding proteins, growth deficiencies in vitro, and reduced virulence in an aerosol infection model.


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TEXT
 
Iron is essential for most bacteria but of limited availability inside the host (21). Iron uptake systems developed to overcome this problem are often regulated by the ferric uptake regulator protein, Fur. Additionally, Fur has been shown to regulate virulence and virulence-associated factors (5, 21). Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, expresses two iron-regulated transferrin-binding proteins, TbpA and TbpB, which are essential for colonization of the host (3). Transcriptional regulation of the tbpB gene has been suggested to be mediated by Fur (13). Recently, the sequence of the fur gene of A. pleuropneumoniae 4074 has been published by Hsu et al. (17) (GenBank accession no. AF330229). In order to elucidate the role of Fur in A. pleuropneumoniae, an isogenic deletion mutant was constructed and analyzed in vitro and in vivo.

Construction and complementation of the isogenic deletion mutant A. pleuropneumoniae {Delta}fur. Primers oFU1a and oFU2 were used to amplify a 1,250-bp fragment containing the 447-bp fur gene of the A. pleuropneumoniae wild-type (wt) strain AP76. This fragment was cloned, and a 171-bp in-frame deletion (positions 157 to 327 of the fur open reading frame) was constructed (Table 1). By using the conjugative plasmid pFU600 (Table 1) in a single-step transconjugation system as described previously (23), this deletion was introduced by allelic replacement into the A. pleuropneumoniae wt, resulting in the mutant strain A. pleuropneumoniae {Delta}fur. The deletion was confirmed by PCR, pulsed-field gel electrophoresis, Southern blot analyses, and nucleotide sequencing (data not shown). In order to complement A. pleuropneumoniae {Delta}fur, the fur gene was amplified and cloned into the broad-host-range vector pLS88. The resulting plasmids pFU1310 and pFU1311 (Table 1), carrying the fur gene in either orientation, were transformed into A. pleuropneumoniae {Delta}fur by electroporation according to the protocol of Tung and Chow (29). The resulting transformants were confirmed by PCR analyses.


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  		TABLE 1. Strains, plasmids, and primers used in this study

Fur-mediated regulation of TbpB and ExbB. Fur-mediated regulation has been proposed for the small transferrin-binding protein TbpB (13), and it has been shown that the TbpB and ExbB protein-encoding genes are transcriptionally linked (28). Hence, we analyzed TbpB and ExbB expression under standard and iron-restricted conditions using Western blot analyses as described previously (11, 28).

In A. pleuropneumoniae {Delta}fur, TbpB and ExbB were constitutively expressed, whereas the A. pleuropneumoniae wt showed only low expression levels under standard culturing conditions and a clear upregulation upon iron restriction (Fig. 1). Complementation of A. pleuropneumoniae {Delta}fur with plasmid pFU1310 but not plasmid pFU1311 restored iron-dependent regulation of TbpB and ExbB expression (Fig. 1). The transcriptional start point of the exbBD-tbpBA transcript was determined using the 5'-RACE system for rapid amplification of cDNA ends, version 2.0 (Invitrogen, Groningen, The Netherlands) and was found to be an "A" 41 bp upstream of the tonB start codon within a putative Fur box (GATAATGATTTTCATTAAC, 94% identical with the consensus sequence [7]; boldfaced letter indicates transcriptional start point), as is typical for Fur-regulated promoters (30). Together the results of the genetic analyses and the expression studies strongly suggest that expression of the exbBD-tbpBA operon is regulated by Fur.



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  		FIG. 1. Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (top) and corresponding Western blots of whole-cell lysates from wt A. pleuropneumoniae (lanes 1 and 2), the uncomplemented isogenic deletion mutant A. pleuropneumoniae {Delta}fur (lanes 3 and 4), or A. pleuropneumoniae {Delta}fur complemented with plasmid pFU1310 (lanes 5 and 6) or pFU1311 (lanes 7 and 8) grown under standard (odd-numbered lanes) or iron-depleted (even-numbered lanes) conditions. Ten micrograms of protein was loaded per lane. The blots were developed with antibodies directed against TbpB (center) and ExbB (bottom), respectively. The closed arrowhead indicates the position of the TbpB protein; the open arrowhead indicates the position of the ExbB protein.

Growth deficiencies of A. pleuropneumoniae {Delta}fur. Actinobacillus pleuropneumoniae {Delta}fur showed significantly reduced growth (i) in nonagitated PPLO broth under an atmosphere with 5% CO2 (Fig. 2A; P < 0.01 by Student's t test), (ii) under anaerobic conditions (growth for 16 h in an anaerobic chamber) (Fig. 2B; P < 0.01 by Student's t test), (iii) on NAD (10 µg/ml)-supplemented PPLO agar plates (Fig. 2C), and (iv) on selective NAD-supplemented blood agar (Fig. 2D) containing crystal violet (1 µg/ml), lincomycin (2 µg/ml), bacitracin (100 µg/ml), or nystatin (50 µg/ml) (19), due to iron-dependent sensitivity to bacitracin (Fig. 2E and F). Complementation with fur in trans completely (plasmid pFU1310) or partially (plasmid pFU1311) restored growth on both solid media (Fig. 2C and D), thereby confirming that the absence of fur is responsible for these growth deficiencies. These deficiencies might be due to (i) increased iron uptake leading to toxic concentrations of iron in the cell, as described for other pathogens (10, 27), (ii) redundant energetic investment in the production of Fur-regulated proteins in the presence of sufficient iron, or (iii) downregulation of metabolic enzymes positively regulated by Fur.



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  		FIG. 2. Growth characteristics of A. pleuropneumoniae {Delta}fur in liquid (A, B) and on solid (C, D, E, F) medium. (A and B) Growth of wt A. pleuropneumoniae and its isogenic mutant A. pleuropneumoniae {Delta}fur in supplemented PPLO medium under a 5% CO2 atmosphere (A) and under anaerobic conditions (B). The asterisk indicates statistical significance (P < 0.01) as determined by Student's t test. Data in panels A and B were derived from five and three independent experiments, respectively. The central symbol in each box indicates the geometric mean, the hinges indicate the values in the middle half of the data, and the top and bottom symbols indicate the maximum and minimum values, respectively. (C and D) Growth of wt A. pleuropneumoniae and its isogenic mutant A. pleuropneumoniae {Delta}fur on supplemented PPLO agar (C) and on selective supplemented blood agar (D); pFU1310 and pFU1311 indicate the position of A. pleuropneumoniae {Delta}fur complemented in trans with the respective plasmid. (E and F) Growth of wt A. pleuropneumoniae and its isogenic mutant A. pleuropneumoniae {Delta}fur on supplemented blood agar containing bacitracin (100 µg/ml) alone (E) or bacitracin (100 µg/ml) and the iron chelator calcium trisodium pentetate (F).

A. pleuropneumoniae {Delta}fur shows reduced virulence in an aerosol infection model. We previously showed that an A. pleuropneumoniae fur transposon-insertion mutant was highly attenuated in competition assays in vitro and in vivo (26). To investigate the effects of the fur deletion in vivo, we challenged clinically healthy pigs 7 to 9 weeks of age from an A. pleuropneumoniae-free herd by using a previously described aerosol infection model (4) with either A. pleuropneumoniae {Delta}fur (8 animals) or the wt A. pleuropneumoniae parental strain (12 animals) (Table 2). Animals challenged with the parental strain showed a significantly higher clinical score (Table 2; P = 0.015 by Student's t test), determined as described previously (18), and a significantly higher lung lesion score, determined by the method of Hannan et al. (14) (Table 2; P = 0.017 by the Wilcoxon test) than pigs challenged with A. pleuropneumoniae {Delta}fur. Further, all animals in both groups had developed antibodies against the ApxIIA toxin (20) as well as against surface-associated proteins (12) (Table 2). For histopathology, lung tissues were immersion-fixed in formalin and embedded in paraffin, and 5-µm thin sections were stained with hematoxylin and eosin. Lung tissue on day 7 after infection with wt A. pleuropneumoniae revealed acute pleuritis, bronchitis, thrombosis, vasculitis, and multifocal coagulative and liquefaction necroses lined by active immune cells (Fig. 3A). Lung lesions in pigs infected with A. pleuropneumoniae {Delta}fur differed insofar as the area of active immune defense was broader and included fibroblasts and collagen fibers, and cell debris was less prominent (Fig. 3C). This difference was even more distinct on day 21 postinfection. The histological differences observed, especially the presence of fibroblasts and collagen in the demarcation wall of lesions caused by A. pleuropneumoniae {Delta}fur as early as day 7 (Fig. 3C) postinfection and the absence of the prominent layer of decayed immune cells on day 21 (Fig. 3D), suggest that A. pleuropneumoniae {Delta}fur was more susceptible to the host immune response than the A. pleuropneumoniae wt and therefore was eliminated faster, with less destruction of immune cells.


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  		TABLE 2. Virulence of A. pleuropneumoniae AP76 and A. pleuropneumoniae {Delta}fur in an aerosol infection model



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  		FIG. 3. Histopathology of lung lesions revealed by a hematoxylin-and-eosin stain. Animals were infected with the A. pleuropneumoniae wt (A, B) or its isogenic mutant A. pleuropneumoniae {Delta}fur (C, D) and were sacrificed on day 7 (A, C) or 21 (B, D) postinfection. L, normal lung tissue, with some alveolar edema; F, demarcation by fibroblasts and collagen fibers; D, cell debris consisting mainly of decayed neutrophils and macrophages; N, center of necrosis. Histopathology was performed for four animals (two lesions each) per group; results similar to those shown here were found for each sample within the respective group.

The A. pleuropneumoniae wt could be reisolated in large numbers from surface swabs of lymph nodes and from intact and altered lung tissue of all animals, as well as from tonsils for 9 of 12 animals in the respective challenge group. In contrast, reisolation of A. pleuropneumoniae {Delta}fur was successful only from altered lung tissue (Table 2). This supports the hypothesis that A. pleuropneumoniae {Delta}fur is more susceptible to the host immune response and is cleared faster from healthy lung tissue than from the centers of necrotic areas, where it is shielded from the active immune response to a certain extent by necrotic tissue and fibrous demarcation.

The lack of persistence of A. pleuropneumoniae {Delta}fur on the respiratory epithelium might be mediated by the continuous expression of TbpB, since TbpB is highly immunogenic (25). An additional possible cause for the inability of fur mutants to persist is a deregulation of iron uptake, resulting in toxic levels of intracellular iron and reduced growth (16). Furthermore, in other bacteria, Fur was found to upregulate factors that might influence survival inside the host, such as superoxide dismutase (8, 22) and catalase (15). Finally, Hsu et al. (17) demonstrated that expression of ApxI, one of the RTX toxins that are major virulence factors of A. pleuropneumoniae (9), is positively regulated by Fur under high calcium conditions. Since the impact of Fur on the expression of ApxII and ApxIV, the only toxins present in A. pleuropneumoniae serotype 7, is unknown, a decreased toxin production as an additional cause for attenuation of A. pleuropneumoniae {Delta}fur cannot be excluded, although pigs infected with A. pleuropneumoniae {Delta}fur did mount an immune response to ApxII similar to that of wt-infected pigs.


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ACKNOWLEDGMENTS
 
This work was supported by Sonderforschungsbereich 587 (project A4) of the Deutsche Forschungsgemeinschaft (DFG), Bonn, Germany. I.J. is a fellow of the graduate college 745 (project A1) of the DFG. Vector sequencing was supported by a grant given by the Bundesministerium fuer Bildung und Forschung (BMBF) within the BioProfile program. P.R.L. and J.T.B. were supported by the Wellcome Trust and BBSRC.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institut für Mikrobiologie, Zentrum für Infektionsmedizin, Stiftung Tierärztliche Hochschule Hannover, Bischofsholer Damm 15, 30173 Hannover, Germany. Phone: 49-511-856 7598. Fax: 49-511-856 7697. E-mail: gfgerlach{at}gmx.de. Back

Editor: F. C. Fang


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Infection and Immunity, June 2005, p. 3740-3744, Vol. 73, No. 6
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.6.3740-3744.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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