Deletion of the Ferric Uptake Regulator Fur Impairs the In Vitro Growth and Virulence of Actinobacillus pleuropneumoniae
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感染与免疫杂志 2005年第6期
Department of Infectious Diseases, Institute for Microbiology
Department of Pathology
Clinic for Pigs and Small Ruminants, University of Veterinary Medicine Hannover Foundation, Hannover, Germany
Department of Paediatrics, Imperial College London, St. Mary's Campus, London W2 1PG, United Kingdom
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 fur. This mutant showed constitutive expression of transferrin-binding proteins, growth deficiencies in vitro, and reduced virulence in an aerosol infection model.
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 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 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 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 fur by electroporation according to the protocol of Tung and Chow (29). The resulting transformants were confirmed by PCR analyses.
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 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 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.
Growth deficiencies of A. pleuropneumoniae fur. Actinobacillus pleuropneumoniae 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.
A. pleuropneumoniae 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 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 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 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 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 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.
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 fur was successful only from altered lung tissue (Table 2). This supports the hypothesis that A. pleuropneumoniae 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 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 fur cannot be excluded, although pigs infected with A. pleuropneumoniae fur did mount an immune response to ApxII similar to that of wt-infected pigs.
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.
REFERENCES
1. Reference deleted.
2. Anderson, C., A. A. Potter, and G. F. Gerlach. 1991. Isolation and molecular characterization of spontaneously occurring cytolysin-negative mutants of Actinobacillus pleuropneumoniae serotype 7. Infect. Immun. 59:4110-4116.
3. Baltes, N., I. Hennig-Pauka, and G. F. Gerlach. 2002. Both transferrin binding proteins are virulence factors in Actinobacillus pleuropneumoniae serotype 7 infection. FEMS Microbiol. Lett. 209:283-287.
4. Baltes, N., W. Tonpitak, G. F. Gerlach, I. Hennig-Pauka, A. Hoffmann-Moujahid, M. Ganter, and H. J. Rothkotter. 2001. Actinobacillus pleuropneumoniae iron transport and urease activity: effects on bacterial virulence and host immune response. Infect. Immun. 69:472-478.
5. Cooksley, C., P. J. Jenks, A. Green, A. Cockayne, R. P. Logan, and K. R. Hardie. 2003. NapA protects Helicobacter pylori from oxidative stress damage, and its production is influenced by the ferric uptake regulator. J. Med. Microbiol. 52:461-469.
6. Dehio, C., and M. Meyer. 1997. Maintenance of broad-host-range incompatibility group P and group Q plasmids and transposition of Tn5 in Bartonella henselae following conjugal plasmid transfer from Escherichia coli. J. Bacteriol. 179:538-540.
7. de Lorenzo., V, S. Wee, M. Herrero, and J. B. Neilands. 1987. Operator sequences of the aerobactin operon of plasmid ColV-K30 binding the ferric uptake regulation (fur) repressor. J. Bacteriol. 169:2624-2630.
8. Dubrac, S., and D. Touati. 2000. Fur positive regulation of iron superoxide dismutase in Escherichia coli: functional analysis of the sodB promoter. J. Bacteriol. 182:3802-3808.
9. Frey, J., J. T. Bosse, Y. F. Chang, J. M. Cullen, B. Fenwick, G. F. Gerlach, D. Gygi, F. Haesebrouck, T. J. Inzana, and R. Jansen. 1993. Actinobacillus pleuropneumoniae RTX-toxins: uniform designation of haemolysins, cytolysins, pleurotoxin and their genes. J. Gen. Microbiol. 139:1723-1728.
10. Furano, K., and A. A. Campagnari. 2003. Inactivation of the Moraxella catarrhalis 7169 ferric uptake regulator increases susceptibility to the bactericidal activity of normal human sera. Infect. Immun. 71:1843-1848.
11. Gerlach, G. F., C. Anderson, A. A. Potter, S. Klashinsky, and P. J. Willson. 1992. Cloning and expression of a transferrin-binding protein from Actinobacillus pleuropneumoniae. Infect. Immun. 60:892-898.
12. Goethe, R., O. F. Gonzales, T. Lindner, and G. F. Gerlach. 2000. A novel strategy for protective Actinobacillus pleuropneumoniae subunit vaccines: detergent extraction of cultures induced by iron restriction. Vaccine 19:966-975.
13. Gonzalez, G. C., R. H. Yu, P. R. Rosteck, Jr., and A. B. Schryvers. 1995. Sequence, genetic analysis, and expression of Actinobacillus pleuropneumoniae transferrin receptor genes. Microbiology 141:2405-2416.
14. Hannan, P. C., B. S. Bhogal, and J. P. Fish. 1982. Tylosin tartrate and tiamutilin effects on experimental piglet pneumonia induced with pneumonic pig lung homogenate containing mycoplasmas, bacteria and viruses. Res. Vet. Sci. 33:76-88.
15. Harris, A. G., F. E. Hinds, A. G. Beckhouse, T. Kolesnikow, and S. L. Hazell. 2002. Resistance to hydrogen peroxide in Helicobacter pylori: role of catalase (KatA) and Fur, and functional analysis of a novel gene product designated 'KatA-associated protein', KapA (HP0874). Microbiology 148:3813-3825.
16. Horsburgh, M. J., E. Ingham, and S. J. Foster. 2001. In Staphylococcus aureus, fur is an interactive regulator with PerR, contributes to virulence, and is necessary for oxidative stress resistance through positive regulation of catalase and iron homeostasis. J. Bacteriol. 183:468-475.
17. Hsu, Y. M., N. Chin, C. F. Chang, and Y. F. Chang. 2003. Cloning and characterization of the Actinobacillus pleuropneumoniae fur gene and its role in regulation of ApxI and AfuABC expression. DNA Sequence 14:169-181.
18. Jacobsen, I., I. Hennig-Pauka, N. Baltes, M. Trost, and G. F. Gerlach. 2005. Enzymes involved in anaerobic respiration appear to play a role in Actinobacillus pleuropneumoniae virulence. Infect. Immun. 73:226-234.
19. Jacobsen, M. J., and J. P. Nielsen. 1995. Development and evaluation of a selective and indicative medium for isolation of Actinobacillus pleuropneumoniae from tonsils. Vet. Microbiol. 47:191-197.
20. Leiner, G., B. Franz, K. Strutzberg, and G. F. Gerlach. 1999. A novel enzyme-linked immunosorbent assay using the recombinant Actinobacillus pleuropneumoniae ApxII antigen for diagnosis of pleuropneumonia in pig herds. Clin. Diagn. Lab. Immunol. 6:630-632.
21. Litwin, C. M., and S. B. Calderwood. 1993. Role of iron in regulation of virulence genes. Clin. Microbiol. Rev. 6:137-149.
22. Loprasert, S., R. Sallabhan, W. Whangsuk, and S. Mongkolsuk. 2000. Characterization and mutagenesis of fur gene from Burkholderia pseudomallei. Gene 254:129-137.
23. Oswald, W., W. Tonpitak, G. Ohrt, and G. Gerlach. 1999. A single-step transconjugation system for the introduction of unmarked deletions into Actinobacillus pleuropneumoniae serotype 7 using a sucrose sensitivity marker. FEMS Microbiol. Lett. 179:153-160.
24. Raleigh, F. A., K. Lech, and R. Brent. 1989. Select topics from classical bacterial genetics, p. 1.4.1-1.4.14. In F. M. Ausubel et al. (ed.), Current protocols in molecular biology. Publishing Associates and Wiley Interscience, New York, N.Y.
25. Rossi-Campos, A., C. Anderson, G. F. Gerlach, S. Klashinsky, A. A. Potter, and P. J. Willson. 1992. Immunization of pigs against Actinobacillus pleuropneumoniae with two recombinant protein preparations. Vaccine 10:512-518.
26. Sheehan, B. J., J. T. Bosse, A. J. Beddek, A. N. Rycroft, J. S. Kroll, and P. R. Langford. 2003. Identification of Actinobacillus pleuropneumoniae genes important for survival during infection in its natural host. Infect. Immun. 71:3960-3970.
27. Staggs, T. M., J. D. Fetherston, and R. D. Perry. 1994. Pleiotropic effects of a Yersinia pestis fur mutation. J. Bacteriol. 176:7614-7624.
28. Tonpitak, W., S. Thiede, W. Oswald, N. Baltes, and G. F. Gerlach. 2000. Actinobacillus pleuropneumoniae iron transport: a set of exbBD genes is transcriptionally linked to the tbpB gene and required for utilization of transferrin-bound iron. Infect. Immun. 68:1164-1170.
29. Tung, W. L., and K. C. Chow. 1995. A modified medium for efficient electrotransformation of E. coli. Trends Genet. 11:128-129.
30. Vassinova, N., and D. Kozyrev. 2000. A method for direct cloning of fur-regulated genes: identification of seven new fur-regulated loci in Escherichia coli. Microbiology 146:3171-3182.
31. Willson, P. J., W. L. Albritton, L. Slaney, and J. K. Setlow. 1989. Characterization of a multiple antibiotic resistance plasmid from Haemophilus ducreyi. Antimicrob. Agents Chemother. 33:1627-1630.(Ilse Jacobsen, Jrg Gerste)
Department of Pathology
Clinic for Pigs and Small Ruminants, University of Veterinary Medicine Hannover Foundation, Hannover, Germany
Department of Paediatrics, Imperial College London, St. Mary's Campus, London W2 1PG, United Kingdom
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 fur. This mutant showed constitutive expression of transferrin-binding proteins, growth deficiencies in vitro, and reduced virulence in an aerosol infection model.
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 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 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 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 fur by electroporation according to the protocol of Tung and Chow (29). The resulting transformants were confirmed by PCR analyses.
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 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 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.
Growth deficiencies of A. pleuropneumoniae fur. Actinobacillus pleuropneumoniae 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.
A. pleuropneumoniae 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 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 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 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 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 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.
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 fur was successful only from altered lung tissue (Table 2). This supports the hypothesis that A. pleuropneumoniae 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 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 fur cannot be excluded, although pigs infected with A. pleuropneumoniae fur did mount an immune response to ApxII similar to that of wt-infected pigs.
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.
REFERENCES
1. Reference deleted.
2. Anderson, C., A. A. Potter, and G. F. Gerlach. 1991. Isolation and molecular characterization of spontaneously occurring cytolysin-negative mutants of Actinobacillus pleuropneumoniae serotype 7. Infect. Immun. 59:4110-4116.
3. Baltes, N., I. Hennig-Pauka, and G. F. Gerlach. 2002. Both transferrin binding proteins are virulence factors in Actinobacillus pleuropneumoniae serotype 7 infection. FEMS Microbiol. Lett. 209:283-287.
4. Baltes, N., W. Tonpitak, G. F. Gerlach, I. Hennig-Pauka, A. Hoffmann-Moujahid, M. Ganter, and H. J. Rothkotter. 2001. Actinobacillus pleuropneumoniae iron transport and urease activity: effects on bacterial virulence and host immune response. Infect. Immun. 69:472-478.
5. Cooksley, C., P. J. Jenks, A. Green, A. Cockayne, R. P. Logan, and K. R. Hardie. 2003. NapA protects Helicobacter pylori from oxidative stress damage, and its production is influenced by the ferric uptake regulator. J. Med. Microbiol. 52:461-469.
6. Dehio, C., and M. Meyer. 1997. Maintenance of broad-host-range incompatibility group P and group Q plasmids and transposition of Tn5 in Bartonella henselae following conjugal plasmid transfer from Escherichia coli. J. Bacteriol. 179:538-540.
7. de Lorenzo., V, S. Wee, M. Herrero, and J. B. Neilands. 1987. Operator sequences of the aerobactin operon of plasmid ColV-K30 binding the ferric uptake regulation (fur) repressor. J. Bacteriol. 169:2624-2630.
8. Dubrac, S., and D. Touati. 2000. Fur positive regulation of iron superoxide dismutase in Escherichia coli: functional analysis of the sodB promoter. J. Bacteriol. 182:3802-3808.
9. Frey, J., J. T. Bosse, Y. F. Chang, J. M. Cullen, B. Fenwick, G. F. Gerlach, D. Gygi, F. Haesebrouck, T. J. Inzana, and R. Jansen. 1993. Actinobacillus pleuropneumoniae RTX-toxins: uniform designation of haemolysins, cytolysins, pleurotoxin and their genes. J. Gen. Microbiol. 139:1723-1728.
10. Furano, K., and A. A. Campagnari. 2003. Inactivation of the Moraxella catarrhalis 7169 ferric uptake regulator increases susceptibility to the bactericidal activity of normal human sera. Infect. Immun. 71:1843-1848.
11. Gerlach, G. F., C. Anderson, A. A. Potter, S. Klashinsky, and P. J. Willson. 1992. Cloning and expression of a transferrin-binding protein from Actinobacillus pleuropneumoniae. Infect. Immun. 60:892-898.
12. Goethe, R., O. F. Gonzales, T. Lindner, and G. F. Gerlach. 2000. A novel strategy for protective Actinobacillus pleuropneumoniae subunit vaccines: detergent extraction of cultures induced by iron restriction. Vaccine 19:966-975.
13. Gonzalez, G. C., R. H. Yu, P. R. Rosteck, Jr., and A. B. Schryvers. 1995. Sequence, genetic analysis, and expression of Actinobacillus pleuropneumoniae transferrin receptor genes. Microbiology 141:2405-2416.
14. Hannan, P. C., B. S. Bhogal, and J. P. Fish. 1982. Tylosin tartrate and tiamutilin effects on experimental piglet pneumonia induced with pneumonic pig lung homogenate containing mycoplasmas, bacteria and viruses. Res. Vet. Sci. 33:76-88.
15. Harris, A. G., F. E. Hinds, A. G. Beckhouse, T. Kolesnikow, and S. L. Hazell. 2002. Resistance to hydrogen peroxide in Helicobacter pylori: role of catalase (KatA) and Fur, and functional analysis of a novel gene product designated 'KatA-associated protein', KapA (HP0874). Microbiology 148:3813-3825.
16. Horsburgh, M. J., E. Ingham, and S. J. Foster. 2001. In Staphylococcus aureus, fur is an interactive regulator with PerR, contributes to virulence, and is necessary for oxidative stress resistance through positive regulation of catalase and iron homeostasis. J. Bacteriol. 183:468-475.
17. Hsu, Y. M., N. Chin, C. F. Chang, and Y. F. Chang. 2003. Cloning and characterization of the Actinobacillus pleuropneumoniae fur gene and its role in regulation of ApxI and AfuABC expression. DNA Sequence 14:169-181.
18. Jacobsen, I., I. Hennig-Pauka, N. Baltes, M. Trost, and G. F. Gerlach. 2005. Enzymes involved in anaerobic respiration appear to play a role in Actinobacillus pleuropneumoniae virulence. Infect. Immun. 73:226-234.
19. Jacobsen, M. J., and J. P. Nielsen. 1995. Development and evaluation of a selective and indicative medium for isolation of Actinobacillus pleuropneumoniae from tonsils. Vet. Microbiol. 47:191-197.
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