Swapping of Periplasmic Domains between Brucella suis VirB8 and a pSB1
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《感染与免疫杂志》
Institut National de la Sante et de la Recherche Medicale U431, UFR Medecine, CS83021, Avenue Kennedy, 30908 Nmes Cedex 02, France
Department of Biology, McMaster University, 1280 Main St. West, Hamilton, Ontario LS8 4K1, Canada
ABSTRACT
A Brucella suis mutant with a nonpolar deletion in the virB8 gene was attenuated in a macrophage infection model. Complementation with the B. suis VirB8 protein expressed from the virB promoter restored virulence. Expression of TraJ, a VirB8 homologue from plasmid pSB102, did not restore virulence; however, virulence was partially restored by a chimeric protein containing the N terminus of the B. suis VirB8 protein and the C-terminal periplasmic domain of TraJ.
TEXT
Type IV secretion systems (T4SS) are used by gram-negative bacteria to transport macromolecules across the bacterial envelope into a recipient cell (4, 6). Transported substrates include nucleoprotein complexes, transported during bacterial conjugation or during transfer of transferred DNA from Agrobacterium tumefaciens, as well as protein virulence factors translocated into eukaryotic host cells by pathogens.
The T4SS is a complex multiprotein structure spanning the bacterial envelope. Most T4SS are composed of up to 11 individual protein building blocks plus, in many cases, a 12th "coupling protein," thought to bring the macromolecular substrates to the secretion system. Biochemical analysis of the A. tumefaciens VirB system, the T4SS paradigm, is beginning to show how the individual components assemble in the bacterial envelope and how the substrates are secreted (5). The VirB1 protein is a murein-digesting lytic transglycosylase that locally digests the peptidoglycan, allowing the assembly of the T4SS. The VirB3, VirB6, and VirB7 to VirB10 proteins are believed to form a channel-like structure spanning both bacterial membranes; the VirB4 and VirB11 proteins, with the VirD4 coupling protein, are ATPases associated with the cytoplasmic face of the inner membrane; and the VirB2 and VirB5 proteins form a pilus-like structure on the bacterial surface.
The VirB8 protein has been shown to play a key role in the assembly of the T4SS machinery. Using microscopic, biochemical, and genetic approaches to study function in A. tumefaciens, it was discovered that VirB8 acts as a nucleation center that is required to recruit VirB9 and VirB10 into clusters in the outer membrane (10) and to localize VirB proteins at the cell pole (8). The amino terminus of VirB8, encompassing the first 67 amino acids, contains a short cytoplasmic tail followed by a single hydrophobic transmembrane domain. The carboxy-terminal moiety of the protein of 172 amino acids is believed to be entirely periplasmic. Recently, the three-dimensional structures of the periplasmic domain of VirB8 from both Brucella suis and A. tumefaciens have been determined (1, 18). Using site-directed mutagenesis to change selected residues of the B. suis VirB8 protein, we have shown that changes that inhibit VirB8 dimerization or that inhibit the interactions with VirB4 or VirB10 also affect T4SS assembly and virulence (12). Here, we describe the construction and characterization of the B. suis strain with a nonpolar deletion of virB8 used in that study and show, for the first time, complementation with a chimeric protein containing the N terminus of the B. suis VirB8 protein and the C-terminal periplasmic domain of TraJ, a VirB8 homologue from plasmid pSB102.
Construction and characterization of a mutant with nonpolar deletion of VirB8 in B. suis 1330. A plasmid with a nonpolar deletion of virB8 was created by inverse PCR (Fig. 1) using pIN33 as a template. Bacterial strains, plasmids, and PCR primers are described in Tables 1 and 2. To construct pIN33, a 3.1-kb XmaI-NheI DNA fragment containing virB6 to virB9 from B. suis 1330 was cloned into pBluescript SK(–) linearized with XmaI and SpeI. Primers VirBdel8F, located at the 5' end of virB9, and VirBdel8R, located at the 3' end of virB7, were designed to create an in-frame deletion of virB8 after inverse PCR and religation of the phosphorylated amplification product. Sequencing of the resulting pIN2 plasmid showed that the deletion of virB8 was in frame and that no errors were introduced by the PCR. An XmaI-XbaI restriction fragment containing the whole insert was cloned into the suicide plasmid pSDM3005, which carries the sacB and a kanamycin resistance gene (19). The resulting pIN3 was electroporated into B. suis 1330, and plasmid integration by homologous recombination was selected by kanamycin resistance. Excision events were selected by plating on trypticase soy agar supplemented with 5% sucrose. Sucrose-resistant colonies were tested for kanamycin sensitivity and then by PCR and Southern blotting to confirm the virB8 deletion and the absence of plasmid sequences (data not shown). The DNA sequence of the 2.4-kb XmaI-XbaI fragment surrounding the virB8 deletion amplified from BS1008 was determined to confirm that no additional mutation had been introduced.
The deletion is nonpolar and does not affect regulation of virB expression. We have previously shown that VirB proteins are expressed at a very low level by B. suis in rich or minimal medium at neutral pH, whereas transcription of the B. suis virB operon is strongly induced in acid minimal medium, conditions thought to mimic the environment encountered in the phagosome (2, 13). We used Western blotting with antibodies raised against recombinant B. suis VirB5, VirB8, VirB9, VirB10, and VirB12 to compare expression of these proteins by wild-type B. suis and the BS1008 mutant cultured in virB operon-inducing medium at either pH 7.0 or pH 4.5 as described previously (3, 14). Low-level expression of the five VirB proteins was detected in wild-type B. suis after incubation at neutral pH, and very strong induction was observed in acid minimal medium (Fig. 2). As expected, VirB8 was not detected in BS1008 grown under any conditions. VirB5, VirB9, VirB10, and VirB12 were expressed in BS1008, showing that the deletion was truly nonpolar and that the regulation of the operon in response to acid pH was not affected.
The BS1008 mutant is attenuated in macrophages. J774 murine macrophage-like cells were infected with wild-type B. suis or BS1008 in a standard gentamicin protection assay as described previously (11), and the intracellular survival and multiplication of the bacteria were followed at different times after the beginning of the infection. At early time points (2 and 6 h postinfection), no differences were seen between the two strains. However, while the wild-type strain multiplied 1,000-fold over 48 h, the BS1008 mutant had the expected attenuated phenotype, being unable to multiply, confirming that the presence of VirB8 is essential for B. suis virulence (Fig. 3B and C).
Homologous complementation of BS1008. To complement the virB8 deletion in BS1008, we constructed a plasmid allowing expression of B. suis virB8 from the virB promoter. Plasmid pIN9 was obtained from pBBR1-MCS (9) by insertion of a 330-bp BamHI-ClaI fragment encompassing the tac promoter, the multiple cloning site, and the FLAG peptide from pFLAG-CTC (Sigma), allowing constitutive expression of FLAG-tagged proteins in Brucella (no lacI). Next, the tac promoter was replaced by a 1.1-kb PCR-amplified fragment encompassing the B. suis virB promoter. The resulting plasmid, pIN34, contains a unique NdeI restriction site providing an initiation codon downstream of the virB1 Shine-Dalgarno sequence and a unique BglII restriction site upstream of the sequence coding the FLAG peptide. The sequence coding the B. suis virB8 gene product was finally amplified by PCR, using primers containing NdeI and BglII sites. Two versions were constructed, with the downstream primer designed either to introduce a stop codon before the FLAG tag-encoding sequence allowing expression of wild-type VirB8 (pIN38) or to create a FLAG-tagged VirB8 protein (pIN39). These plasmids were introduced into BS1008 by electroporation and their ability to complement the virulence defect assessed. Complementation of BS1008 with pIN38 restored its ability to multiply within macrophages (Fig. 3B and C). Multiplication was slightly reduced during the first 24 h compared to that of the wild type but reached wild-type levels by 48 h postinfection. These slight differences may be due to the fact that expression of VirB8 from pIN38 (10 to 20 copies) was much higher than in the wild-type strain (Fig. 2). The high levels of expression of VirB8 did not, however, affect the levels of the other VirB proteins in the cells. Expression of the FLAG-tagged VirB8 (pIN39) complemented far less efficiently than that of the wild-type protein. Interestingly, expression of the FLAG-tagged VirB8 protein also reduced the virulence of wild-type B. suis, suggesting that the FLAG-tagged protein interferes with T4SS assembly or function (data not shown).
Heterologous complementation with chimeric VirB8 proteins. The VirB proteins encoded by the virB operon of Brucella have been shown to display only limited similarity with proteins encoded by several previously described T4SS (11). Recently, a broad-host-range plasmid isolated from a microbial population residing in the rhizosphere of alfalfa, pSB102, has been shown to contain a tra operon encoding 12 proteins with high levels of similarity (30 to 75% identity, 53 to 88% similarity) to the VirB proteins from Brucella (16). In preliminary experiments, we introduced this plasmid into a B. suis strain with polar insertion in virB2 by conjugation but detected no complementation (data not shown). BS1008 is an ideal tool to study the function of VirB8 and its homologues from other bacteria, as it carries a nonpolar deletion in VirB. To determine whether the Brucella VirB8 protein could be replaced by TraJ, its homolog from pSB102 that shares more than 50% identity with VirB8, the traJ gene from pSB102 was amplified by PCR and cloned into pIN34 to give pIN54 (expressing TraJ without a FLAG tag), which was introduced into BS1008. The resulting strain (BS1008 pIN54) was still attenuated in J774 cells, suggesting that the pSB102 TraJ protein could not replace VirB8 (Fig. 3A). A more detailed comparison of the sequences of the two proteins shows that the cytoplasmic and transmembrane domains (residues 1 to 77) of TraJ display only 39% identity with the corresponding part of VirB8, whereas the periplasmic domain displays more than 63% identity with VirB8 (Fig. 3A). To assess whether the protein fragments complement, we constructed two chimeric genes composed of the 5' parts of traJ or virB8 ligated to the 3' part of the other gene and cloned them into pIN34 to give pIN56 (TraJ-VirB8) and pIN57 (VirB8-TraJ). These two plasmids were introduced into BS1008 and the resulting strains assayed for virulence. At 24 h postinfection, the levels of bacteria expressing both of the chimeras were similar to that of BS1008 (Fig. 3B); however, at 48 h postinfection, the strain complemented with the VirB8-TraJ chimera had regained some ability to survive and multiply intracellularly, reaching levels almost 100 times higher than that of BS1008 (Fig. 3C). The complementation was still partial, however, with bacterial levels 100 times lower than that of BS1008 complemented with wild-type VirB8.
There have been very few examples of heterologous complementation between components of T4SS. This is probably due to their complex architecture and the multiple protein-protein interactions required for their assembly. Components of the Bartonella Trw system have high levels of similarity with those of the Trw system encoded by, and required for, the conjugative transfer of IncW plasmid R388. The Bartonella TrwD (VirB11 family, 79% identity) and TrwH (VirB7 family, 59% identity) proteins from these T4SS fully (TrwD) or partially (TrwH) complemented the conjugation deficiency of the corresponding R388 mutants (17). Hppner et al. (7) found that the B. suis VirB1 and pKM101 TraL proteins could partially complement the T4SS assembly and function of an Agrobacterium virB1 mutant. The complementation was dependent on the lytic transglycosylase active site of the protein. Similarly, Schmidt-Eisenlohr et al. (15) found that TraC, the VirB5 homologue from plasmid pKM101, could partially complement the pilus assembly defect seen in an A. tumefaciens virB5 mutant. B. suis VirB4 fully complemented a virB4 gene defect in A. tumefaciens (21). Here, we give the first example of heterologous complementation of a virulence defect in a mammalian pathogen. Interestingly, we find that only the chimeric protein with the periplasmic domain of TraJ is functional. To date, VirB8 has been found to interact only with itself or other proteins through the periplasmic domain (20); this suggests that the 63% similarity between TraJ and VirB8 is sufficient to maintain these interactions. The N-terminal cytoplasmic and transmembrane regions of TraJ and VirB8 show only 39% similarity. The lack of complementation by the chimeric protein with the TraJ N terminus shows how important this region is for VirB8 function. This suggests that the constraints for insertion of the N terminus into the membrane or its interaction with unknown Brucella proteins during T4SS biogenesis is too strong to allow it to be replaced. Alternatively, the N terminus may be involved in specific interactions with a transported substrate.
The modification of the C terminus of VirB8 by addition of a FLAG epitope leads to a protein that not only has a reduced ability to complement the virB8 deletion but also has a dominant negative effect on the virulence of the wild-type strain. This suggests that the protein could still retain part of its functionality and be able to initiate assembly of T4SS that are not functional. Another possible explanation is suggested by the decreased amount of VirB8 we have detected by Western blotting, using either anti-VirB8 or anti-FLAG antibodies, of several samples of bacteria cultured in minimal medium at acidic pH compared to those cultured at neutral pH (not shown). This result could reflect the fact that FLAG peptide, when present at the C-terminal end of the protein, disrupts protein-protein interactions and/or targets VirB8 dimers to proteolysis, thus explaining the decreased virulence of the strain expressing the VirB8 protein with a FLAG compared to that expressing the protein without the FLAG. A more detailed biochemical analysis of this will be possible using the Agrobacterium surrogate system that we have recently described (3).
ACKNOWLEDGMENTS
We thank Jacques Godfroid for raising the anti-VirB12 serum, Susanne Schneiker for plasmid pSB102, and Annette Vergunst for pSDML3005.
This work was supported by INSERM, the European Community (QLK2-CT-2001-01200), Universite de Montpellier 1 (BQR), La Region Languedoc Roussillon, and La Ville de Nmes. Christian Baron was supported by the Canadian Institutes of Health Research (CIHR; grant MOP-64300).
FOOTNOTES
Corresponding author. Mailing address: Institut National de la Sante et de la Recherche Medicale U431, UFR Medecine, CS83021, Avenue Kennedy, 30908 Nmes Cedex 02, France. Phone: 00 33 4 66 02 81 46. Fax: 00 33 4 66 02 81 48. E-mail: david.ocallaghan@univ-montp1.fr.
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Department of Biology, McMaster University, 1280 Main St. West, Hamilton, Ontario LS8 4K1, Canada
ABSTRACT
A Brucella suis mutant with a nonpolar deletion in the virB8 gene was attenuated in a macrophage infection model. Complementation with the B. suis VirB8 protein expressed from the virB promoter restored virulence. Expression of TraJ, a VirB8 homologue from plasmid pSB102, did not restore virulence; however, virulence was partially restored by a chimeric protein containing the N terminus of the B. suis VirB8 protein and the C-terminal periplasmic domain of TraJ.
TEXT
Type IV secretion systems (T4SS) are used by gram-negative bacteria to transport macromolecules across the bacterial envelope into a recipient cell (4, 6). Transported substrates include nucleoprotein complexes, transported during bacterial conjugation or during transfer of transferred DNA from Agrobacterium tumefaciens, as well as protein virulence factors translocated into eukaryotic host cells by pathogens.
The T4SS is a complex multiprotein structure spanning the bacterial envelope. Most T4SS are composed of up to 11 individual protein building blocks plus, in many cases, a 12th "coupling protein," thought to bring the macromolecular substrates to the secretion system. Biochemical analysis of the A. tumefaciens VirB system, the T4SS paradigm, is beginning to show how the individual components assemble in the bacterial envelope and how the substrates are secreted (5). The VirB1 protein is a murein-digesting lytic transglycosylase that locally digests the peptidoglycan, allowing the assembly of the T4SS. The VirB3, VirB6, and VirB7 to VirB10 proteins are believed to form a channel-like structure spanning both bacterial membranes; the VirB4 and VirB11 proteins, with the VirD4 coupling protein, are ATPases associated with the cytoplasmic face of the inner membrane; and the VirB2 and VirB5 proteins form a pilus-like structure on the bacterial surface.
The VirB8 protein has been shown to play a key role in the assembly of the T4SS machinery. Using microscopic, biochemical, and genetic approaches to study function in A. tumefaciens, it was discovered that VirB8 acts as a nucleation center that is required to recruit VirB9 and VirB10 into clusters in the outer membrane (10) and to localize VirB proteins at the cell pole (8). The amino terminus of VirB8, encompassing the first 67 amino acids, contains a short cytoplasmic tail followed by a single hydrophobic transmembrane domain. The carboxy-terminal moiety of the protein of 172 amino acids is believed to be entirely periplasmic. Recently, the three-dimensional structures of the periplasmic domain of VirB8 from both Brucella suis and A. tumefaciens have been determined (1, 18). Using site-directed mutagenesis to change selected residues of the B. suis VirB8 protein, we have shown that changes that inhibit VirB8 dimerization or that inhibit the interactions with VirB4 or VirB10 also affect T4SS assembly and virulence (12). Here, we describe the construction and characterization of the B. suis strain with a nonpolar deletion of virB8 used in that study and show, for the first time, complementation with a chimeric protein containing the N terminus of the B. suis VirB8 protein and the C-terminal periplasmic domain of TraJ, a VirB8 homologue from plasmid pSB102.
Construction and characterization of a mutant with nonpolar deletion of VirB8 in B. suis 1330. A plasmid with a nonpolar deletion of virB8 was created by inverse PCR (Fig. 1) using pIN33 as a template. Bacterial strains, plasmids, and PCR primers are described in Tables 1 and 2. To construct pIN33, a 3.1-kb XmaI-NheI DNA fragment containing virB6 to virB9 from B. suis 1330 was cloned into pBluescript SK(–) linearized with XmaI and SpeI. Primers VirBdel8F, located at the 5' end of virB9, and VirBdel8R, located at the 3' end of virB7, were designed to create an in-frame deletion of virB8 after inverse PCR and religation of the phosphorylated amplification product. Sequencing of the resulting pIN2 plasmid showed that the deletion of virB8 was in frame and that no errors were introduced by the PCR. An XmaI-XbaI restriction fragment containing the whole insert was cloned into the suicide plasmid pSDM3005, which carries the sacB and a kanamycin resistance gene (19). The resulting pIN3 was electroporated into B. suis 1330, and plasmid integration by homologous recombination was selected by kanamycin resistance. Excision events were selected by plating on trypticase soy agar supplemented with 5% sucrose. Sucrose-resistant colonies were tested for kanamycin sensitivity and then by PCR and Southern blotting to confirm the virB8 deletion and the absence of plasmid sequences (data not shown). The DNA sequence of the 2.4-kb XmaI-XbaI fragment surrounding the virB8 deletion amplified from BS1008 was determined to confirm that no additional mutation had been introduced.
The deletion is nonpolar and does not affect regulation of virB expression. We have previously shown that VirB proteins are expressed at a very low level by B. suis in rich or minimal medium at neutral pH, whereas transcription of the B. suis virB operon is strongly induced in acid minimal medium, conditions thought to mimic the environment encountered in the phagosome (2, 13). We used Western blotting with antibodies raised against recombinant B. suis VirB5, VirB8, VirB9, VirB10, and VirB12 to compare expression of these proteins by wild-type B. suis and the BS1008 mutant cultured in virB operon-inducing medium at either pH 7.0 or pH 4.5 as described previously (3, 14). Low-level expression of the five VirB proteins was detected in wild-type B. suis after incubation at neutral pH, and very strong induction was observed in acid minimal medium (Fig. 2). As expected, VirB8 was not detected in BS1008 grown under any conditions. VirB5, VirB9, VirB10, and VirB12 were expressed in BS1008, showing that the deletion was truly nonpolar and that the regulation of the operon in response to acid pH was not affected.
The BS1008 mutant is attenuated in macrophages. J774 murine macrophage-like cells were infected with wild-type B. suis or BS1008 in a standard gentamicin protection assay as described previously (11), and the intracellular survival and multiplication of the bacteria were followed at different times after the beginning of the infection. At early time points (2 and 6 h postinfection), no differences were seen between the two strains. However, while the wild-type strain multiplied 1,000-fold over 48 h, the BS1008 mutant had the expected attenuated phenotype, being unable to multiply, confirming that the presence of VirB8 is essential for B. suis virulence (Fig. 3B and C).
Homologous complementation of BS1008. To complement the virB8 deletion in BS1008, we constructed a plasmid allowing expression of B. suis virB8 from the virB promoter. Plasmid pIN9 was obtained from pBBR1-MCS (9) by insertion of a 330-bp BamHI-ClaI fragment encompassing the tac promoter, the multiple cloning site, and the FLAG peptide from pFLAG-CTC (Sigma), allowing constitutive expression of FLAG-tagged proteins in Brucella (no lacI). Next, the tac promoter was replaced by a 1.1-kb PCR-amplified fragment encompassing the B. suis virB promoter. The resulting plasmid, pIN34, contains a unique NdeI restriction site providing an initiation codon downstream of the virB1 Shine-Dalgarno sequence and a unique BglII restriction site upstream of the sequence coding the FLAG peptide. The sequence coding the B. suis virB8 gene product was finally amplified by PCR, using primers containing NdeI and BglII sites. Two versions were constructed, with the downstream primer designed either to introduce a stop codon before the FLAG tag-encoding sequence allowing expression of wild-type VirB8 (pIN38) or to create a FLAG-tagged VirB8 protein (pIN39). These plasmids were introduced into BS1008 by electroporation and their ability to complement the virulence defect assessed. Complementation of BS1008 with pIN38 restored its ability to multiply within macrophages (Fig. 3B and C). Multiplication was slightly reduced during the first 24 h compared to that of the wild type but reached wild-type levels by 48 h postinfection. These slight differences may be due to the fact that expression of VirB8 from pIN38 (10 to 20 copies) was much higher than in the wild-type strain (Fig. 2). The high levels of expression of VirB8 did not, however, affect the levels of the other VirB proteins in the cells. Expression of the FLAG-tagged VirB8 (pIN39) complemented far less efficiently than that of the wild-type protein. Interestingly, expression of the FLAG-tagged VirB8 protein also reduced the virulence of wild-type B. suis, suggesting that the FLAG-tagged protein interferes with T4SS assembly or function (data not shown).
Heterologous complementation with chimeric VirB8 proteins. The VirB proteins encoded by the virB operon of Brucella have been shown to display only limited similarity with proteins encoded by several previously described T4SS (11). Recently, a broad-host-range plasmid isolated from a microbial population residing in the rhizosphere of alfalfa, pSB102, has been shown to contain a tra operon encoding 12 proteins with high levels of similarity (30 to 75% identity, 53 to 88% similarity) to the VirB proteins from Brucella (16). In preliminary experiments, we introduced this plasmid into a B. suis strain with polar insertion in virB2 by conjugation but detected no complementation (data not shown). BS1008 is an ideal tool to study the function of VirB8 and its homologues from other bacteria, as it carries a nonpolar deletion in VirB. To determine whether the Brucella VirB8 protein could be replaced by TraJ, its homolog from pSB102 that shares more than 50% identity with VirB8, the traJ gene from pSB102 was amplified by PCR and cloned into pIN34 to give pIN54 (expressing TraJ without a FLAG tag), which was introduced into BS1008. The resulting strain (BS1008 pIN54) was still attenuated in J774 cells, suggesting that the pSB102 TraJ protein could not replace VirB8 (Fig. 3A). A more detailed comparison of the sequences of the two proteins shows that the cytoplasmic and transmembrane domains (residues 1 to 77) of TraJ display only 39% identity with the corresponding part of VirB8, whereas the periplasmic domain displays more than 63% identity with VirB8 (Fig. 3A). To assess whether the protein fragments complement, we constructed two chimeric genes composed of the 5' parts of traJ or virB8 ligated to the 3' part of the other gene and cloned them into pIN34 to give pIN56 (TraJ-VirB8) and pIN57 (VirB8-TraJ). These two plasmids were introduced into BS1008 and the resulting strains assayed for virulence. At 24 h postinfection, the levels of bacteria expressing both of the chimeras were similar to that of BS1008 (Fig. 3B); however, at 48 h postinfection, the strain complemented with the VirB8-TraJ chimera had regained some ability to survive and multiply intracellularly, reaching levels almost 100 times higher than that of BS1008 (Fig. 3C). The complementation was still partial, however, with bacterial levels 100 times lower than that of BS1008 complemented with wild-type VirB8.
There have been very few examples of heterologous complementation between components of T4SS. This is probably due to their complex architecture and the multiple protein-protein interactions required for their assembly. Components of the Bartonella Trw system have high levels of similarity with those of the Trw system encoded by, and required for, the conjugative transfer of IncW plasmid R388. The Bartonella TrwD (VirB11 family, 79% identity) and TrwH (VirB7 family, 59% identity) proteins from these T4SS fully (TrwD) or partially (TrwH) complemented the conjugation deficiency of the corresponding R388 mutants (17). Hppner et al. (7) found that the B. suis VirB1 and pKM101 TraL proteins could partially complement the T4SS assembly and function of an Agrobacterium virB1 mutant. The complementation was dependent on the lytic transglycosylase active site of the protein. Similarly, Schmidt-Eisenlohr et al. (15) found that TraC, the VirB5 homologue from plasmid pKM101, could partially complement the pilus assembly defect seen in an A. tumefaciens virB5 mutant. B. suis VirB4 fully complemented a virB4 gene defect in A. tumefaciens (21). Here, we give the first example of heterologous complementation of a virulence defect in a mammalian pathogen. Interestingly, we find that only the chimeric protein with the periplasmic domain of TraJ is functional. To date, VirB8 has been found to interact only with itself or other proteins through the periplasmic domain (20); this suggests that the 63% similarity between TraJ and VirB8 is sufficient to maintain these interactions. The N-terminal cytoplasmic and transmembrane regions of TraJ and VirB8 show only 39% similarity. The lack of complementation by the chimeric protein with the TraJ N terminus shows how important this region is for VirB8 function. This suggests that the constraints for insertion of the N terminus into the membrane or its interaction with unknown Brucella proteins during T4SS biogenesis is too strong to allow it to be replaced. Alternatively, the N terminus may be involved in specific interactions with a transported substrate.
The modification of the C terminus of VirB8 by addition of a FLAG epitope leads to a protein that not only has a reduced ability to complement the virB8 deletion but also has a dominant negative effect on the virulence of the wild-type strain. This suggests that the protein could still retain part of its functionality and be able to initiate assembly of T4SS that are not functional. Another possible explanation is suggested by the decreased amount of VirB8 we have detected by Western blotting, using either anti-VirB8 or anti-FLAG antibodies, of several samples of bacteria cultured in minimal medium at acidic pH compared to those cultured at neutral pH (not shown). This result could reflect the fact that FLAG peptide, when present at the C-terminal end of the protein, disrupts protein-protein interactions and/or targets VirB8 dimers to proteolysis, thus explaining the decreased virulence of the strain expressing the VirB8 protein with a FLAG compared to that expressing the protein without the FLAG. A more detailed biochemical analysis of this will be possible using the Agrobacterium surrogate system that we have recently described (3).
ACKNOWLEDGMENTS
We thank Jacques Godfroid for raising the anti-VirB12 serum, Susanne Schneiker for plasmid pSB102, and Annette Vergunst for pSDML3005.
This work was supported by INSERM, the European Community (QLK2-CT-2001-01200), Universite de Montpellier 1 (BQR), La Region Languedoc Roussillon, and La Ville de Nmes. Christian Baron was supported by the Canadian Institutes of Health Research (CIHR; grant MOP-64300).
FOOTNOTES
Corresponding author. Mailing address: Institut National de la Sante et de la Recherche Medicale U431, UFR Medecine, CS83021, Avenue Kennedy, 30908 Nmes Cedex 02, France. Phone: 00 33 4 66 02 81 46. Fax: 00 33 4 66 02 81 48. E-mail: david.ocallaghan@univ-montp1.fr.
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