An essential virulence protein of Brucella abortus, VirB4, requires an intact nucleoside-triphosphate-binding domain

Masahisa Watarai1, Sou-ichi Makino1 and Toshikazu Shirahata1

Department of Veterinary Microbiology, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan1

Author for correspondence: Masahisa Watarai. Tel: +81 155 49 5387. Fax: +81 155 49 5402. e-mail: watarai{at}obihiro.ac.jp


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Brucella abortus is a facultative intracellular bacterium capable of surviving inside macrophages. The VirB complex, which is highly similar to conjugative DNA transfer apparatuses, is required for intracellular replication. A conserved NTP-binding domain in VirB4 suggests that one or both proteins couple energy by NTP hydrolysis to transport of putative effector molecule(s). Here it is shown that a mutant strain of B. abortus that contains an in-frame deletion in virB4 is unable to replicate in macrophages and survives in mice. Intracellular replication and virulence in mice are fully restored by expressing virB4 in trans, indicating that VirB4 is essential for intracellular replication and virulence in mice. An alteration within the NTP-binding region of VirB4 by site-directed mutagenesis abolished complementation of a virB4 mutant, demonstrating that an intact NTP-binding domain is critical for VirB4 function. Intracellular replication was inhibited in wild-type B. abortus after introducing a plasmid expressing a mutant VirB4 altered in the NTP-binding region. The dominant negative phenotype suggests that VirB4 either functions as a multimer or interacts with some other component(s) necessary for intracellular replication. Wild-type B. abortus-containing phagosomes lack the glycoprotein LAMP-1, which is an indicator of the normal endocytic pathway. Mutant strains were found in phagosomes that co-localized with LAMP-1, indicating that VirB4 containing the intact NTP-binding region is essential for evasion of fusion with lysosomes.

Keywords: type IV secretion, macrophage


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Brucella spp. are Gram-negative bacteria that cause abortion and infertility in numerous domestic and wild mammals, and a disease known as undulant fever in humans (Acha & Szylres, 1980 ). The bacterium is endemic in many underdeveloped countries and is responsible for large economic losses and chronic infections in human beings (Zavala et al., 1994 ). Brucella spp. are facultative intracellular pathogens that survive in a variety of cells, including macrophages, and their virulence and ability to cause chronic infections are thought to be due to their ability to avoid the killing mechanisms within macrophages (Baldwin & Winter, 1994 ; Sangari & Aguero, 1996 ). The molecular mechanisms of their virulence and infection are incompletely understood. Studies with HeLa cells have confirmed the observations that Brucella inhibits phagosome–lysosome fusion and transits through an intracellular compartment that resembles an autophagosome. Bacteria replicate in a different compartment, containing protein markers normally associated with the endoplasmic reticulum, as shown by confocal microscopy and immunogold electron microscopy (Comerci et al., 2001 ; Pizarro-Cerda et al., 1998a , b ).

Genetic loci encoding export mechanisms specializing in transferring a variety of multimolecular complexes across the bacterial membrane to the extracellular space or into other cells have been described. These complexes, named type IV secretion systems, have been reported in organisms such as Agrobacterium tumefaciens (virB genes) (Kuldau et al., 1990 ; Stachel & Nester, 1986 ), Bordetella pertussis (ptl genes) (Kotob et al., 1995 ; Weiss et al., 1993 ), Escherichia coli (tra genes) (Pohlman et al., 1994 ; Winans & Walker, 1985 ), Legionella pneumophila (dot/icm genes) (Segal et al., 1998 , Vogel et al., 1998 ) and Helicobacter pylori (cag genes) (Covacci et al., 1999 ). Recently, the virB operon of Brucella has been identified (O’Callaghan et al., 1999 ; Sieira et al., 2000 ). This operon comprises 13 ORFs, designated virB1 to virB11, orf12 and orf13, that share homology with components of other bacterial type IV secretion systems involved in the intracellular trafficking of pathogens. Polar mutations introduced in the first gene of the operon, virB1, abolish the ability of Brucella to replicate intracellularly, indicating that this system is essential for the intracellular lifestyle of this pathogen. Mice infected with polar and non-polar mutations in virB10 demonstrated that the virB operon is a major determinant of Brucella virulence (Sieira et al., 2000 ). Thus, Brucella abortus VirB proteins are thought to be constituent elements of the secretion apparatus, but their specific molecular functions are unknown.

Two virB proteins, VirB4 and VirB11, contain the putative NTP-binding site (Sieira et al., 2000 ) first described by Walker et al. (1982) . Nucleotide-binding proteins might have several roles in the secretion process, including providing energy for transport or signalling the opening of a gate or channel by a kinase activity. In this study, we examined one of these putative nucleotide-binding proteins, VirB4, to determine its importance in intracellular replication and the role that the putative NTP-binding site might play in the Brucella virulence.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and media.
All Bru. abortus derivatives are from strain 544, which is a smooth virulent Bru. abortus biovar 1 strain (Table 1). Bru. abortus strains were maintained as frozen glycerol stocks and were cultured on Brucella broth (BBL) or Brucella broth containing 1·5% agar.


View this table:
[in this window]
[in a new window]
 
Table 1. Bacterial strains and plasmids used in this study

 
Initial isolation of replication-proficient plasmids was from E. coli strain DH5{alpha}. For the propagation of suicide plasmids requiring R6K {pi} protein, E. coli strain DH5{alpha} ({lambda}pir) (Kolter et al., 1978 ) was used. Kanamycin was used at 40 µg ml-1.

Construction of in-frame deletion mutant of virB4.
pMAW14 ({Delta}virB4) was constructed by cloning two PCR fragments into SalI/SacI-cleaved pSR47s (Andrews et al., 1998 ). Fragment 1 was a 1640 bp SalI–BamHI fragment spanning a site located 1620 nt upstream of the 5' end of virB4 to 20 nt downstream from the 5' end and was amplified by PCR using primers 5'-GTCGACCAAGTCAACAGGTACAATCTG-3' (SalI site underlined) and 5'-GGATCCGCCCATTATGATTCTCTTTTG-3' (BamHI site underlined) (nucleotide positions 690 and 2290 in GenBank accession no. AF226278, respectively; Sieira et al., 2000 ). Fragment 2 was a 1617 bp BamHI–SacI fragment spanning the region starting 6 nt upstream of the 3' end of virB4 to a position 1611 nt downstream from the 3' end and was amplified using primers 5'-GGATCCAGGTGACACTATGAAGAAGAT-3' (BamHI site underlined) and 5'-GAGCTCGCGGCTGAAGATTGCACTCGA-3' (SacI site underlined) (nucleotide positions 4785 and 6385 in AF226278, respectively; Sieira et al., 2000 ).

pMAW14 ({Delta}virB4) was introduced into DH5{alpha} ({lambda}pir), and subsequently the plasmid was transferred into Bru. abortus 544 by using electroporation (Gene Pulser; Bio-Rad). Cells were spread onto Brucella agar containing 5% sucrose and incubated at 37 °C to isolate recombinants in which the entire plasmid integrated into the appropriate chromosomal site. The resulting sucrose-resistant colonies were tested for kanamycin sensitivity which indicates loss of the suicide vector. The kanamycin-sensitive colonies thus selected were analysed to confirm that in-frame deletion had occurred in the virB4 gene by PCR amplification.

pMAW15 (virB4+) was constructed by cloning a PCR fragment into EcoRI/BamHI-cleaved pBBR1MCS-2 (Kovach et al., 1995 ). The 2786 bp EcoRI–BamHI PCR fragment spanned a site located 270 nt upstream of the 5' end of virB4 to a position 20 nt downstream from the 3' end (Sieira et al., 2000 ) and was amplified using the primers 5'-GAATTCAGCCATGTTGTTTGGGGTTCC-3' (EcoRI site underlined) and 5'-GGATCCAGAATTATCTTCTTCATAGTG-3' (BamHI site underlined).

Construction of a point mutation in the NTP-binding domain of VirB4.
A point mutation was made in the coding sequence (AAA; 1387–1389) for the invariant Lys463 residue (K463) within the Walker-type NTP-binding motif of VirB4 (Fig. 1). pMAW16 was constructed by cloning a 2786 bp EcoRI–BamHI fragment from pMAW15 into EcoRI/BamHI-cleaved pKF19k for site-directed mutagenesis (TAKARA). The coding sequence for Lys463 (AAA) was mutated to encode Arg (AGA) using the site-directed mutagenesis system Mutan-Express Km (TAKARA) and mutagenic oligonucleotide 5'-CAGGACAGTTCTACCAGCGCC-3' which includes the codon that alters Lys463 to Arg (K463R). The point mutation within this region of virB4 was confirmed by DNA sequence analysis and the resultant plasmid was designated pMAW17. pMAW18 (virB4 K463R) was constructed by cloning a 2786 bp EcoRI–BamHI fragment from pMAW17 into EcoRI/BamHI-cleaved pBBR1MCS-2.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 1. Genetic map of the virB4 flanking region and alteration of the putative nucleoside-binding region of VirB4. (a) Construction of an in-frame deletion mutation is shown for {Delta}virB4. Nucleotides are numbered as described previously by Sieira et al. (2000) . (b) The consensus Walker box A region is shown with the corresponding region from VirB4 (aa 457–464). The K463R alteration made by site-directed mutagenesis is underlined.

 
Cell culture.
Bone-marrow-derived macrophages from female BALB/c mice were prepared as described by Watarai et al. (2001) . After culturing in L-cell conditioned medium, the macrophages were replated for use by lifting cells in PBS on ice for 5 to 10 min, harvesting by centrifugation and resuspending in RPMI 1640 containing 10% fetal bovine serum. The mouse macrophage-like cell line J774 was maintained in RPMI 1640 containing 10% fetal bovine serum. The macrophages were seeded (2x105–3x105 per well) in 24-well tissue culture plates for all assays.

Determination of efficiency of intracellular growth of bacteria.
Bacteria were deposited onto macrophages at an m.o.i. of 20 by centrifugation at 150 g for 5 min at room temperature and were then incubated at 37 °C in 5% CO2 for 1 h. The macrophages were then washed once with medium and incubated with 30 µg gentamicin ml-1. At different time points, cells were washed and lysed with 1% Triton X-100 and the number of bacteria was counted in plates of a suitable dilution.

Detection of bacteria in macrophages.
Mouse bone-marrow-derived macrophages were infected with Bru. abortus as described above. Infected monolayers were washed extensively with fresh medium to remove non-cell-associated Bru. abortus. Infected cells were fixed in periodate/lysine/paraformaldehyde (PLP) (McLean & Nakane, 1974 ) containing 5% sucrose for 30 min at 37 °C, washed three times in PBS and permeabilized in 0·1% Triton X-100 for 30 min at room temperature. Samples were then washed three times in blocking buffer (2%, v/v, goat serum in PBS) for 5 min and stained with anti-Bru. abortus rabbit serum (diluted 1:1000) in blocking buffer for 1 h at 37 °C. To visualize antibodies, samples were washed three times in blocking buffer, incubated with FITC-conjugated goat anti-rabbit IgG (Zymed) diluted 1:500 in blocking buffer for 1 h at 37 °C and visualized in mounting medium (90% glycerol containing 1 mg phenylenediamine ml-1 in PBS, pH 9·0).

LAMP-1 staining.
Infected macrophages were fixed in PLP-sucrose for 1 h at 37 °C. Samples were washed three times in PBS and wells were successively incubated three times for 5 min in blocking buffer (2% goat serum in PBS) at room temperature. All antibody-probing steps were carried out for 1 h at 37 °C in a humidified incubator. After blocking, samples were stained with anti-Bru. abortus polyclonal rabbit serum diluted 1:1000 in blocking buffer to identify extracellular bacteria. Samples were washed three times for 5 min with blocking buffer, stained with Cascade-Blue-conjugated goat anti-rabbit IgG diluted 1:500 in blocking buffer and incubated as above. Samples were washed three times in PBS for 5 min and then permeabilized in -20 °C methanol for 10 s. After incubating three times for 5 min with blocking buffer, samples were stained with anti-LAMP-1 rat mAb ID4B (obtained from Developmental Studies Hybridoma Bank of the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA, and the Department of Biology, University of Iowa, Iowa City, IA, USA) diluted 1:100 in blocking buffer (Swanson & Isberg, 1996 ). After washing three times for 5 min in blocking buffer, samples were stained simultaneously with TRITC-conjugated goat anti-rat IgG (Molecular Probes). Samples were placed in mounting medium and visualized by fluorescence microscopy.

Fluorescence microscopy.
The specimens were analysed using an Olympus IX70 inverted phase microscope and the images were collected using a cooled CCD camera (CoolSNAP; Roper Scientific) and processed using Openlab software (Improvision) on a Power Macintosh G4 computer.

Virulence in mice.
Virulence was determined by quantifying the survival of the strains in the spleen after 10 days. Six-week-old female BALB/c mice were injected intraperitoneally with approximately 104 c.f.u. brucellae in 0·1 ml saline. Groups of five mice were injected with each strain. At 10 days post-infection the mice were sacrificed by decapitation and their spleens were removed, weighed and homogenized in saline. Tissue homogenates were serially diluted with PBS and plated on Brucella agar to count the number of c.f.u. in each spleen.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Effect of alteration of the putative NTP-binding region of VirB4 on intracellular replication
To investigate the role of the virB4 gene in intracellular replication in macrophages, an in-frame deletion mutation was constructed in the virB4 gene. This deletion mutation was designated {Delta}virB4 and the mutant was designated Ba598 (Fig. 1a). As survival and multiplication in macrophages is an important virulence attribute of Brucella, we examined the intracellular replication of Brucella strains in two mouse macrophage models, using macrophage-like cell line J774 and bone-marrow-derived macrophages. J774 or mouse bone-marrow-derived macrophages were infected for various periods at an m.o.i. of 20. The results show that Ba598 ({Delta}virB4) failed to replicate in both J774 and bone-marrow-derived macrophages (Figs 2 and 3). The mutation was complemented in trans by introducing plasmid pMAW15, which carries a wild-type copy of virB4 (Ba603). Introduction of the complementing plasmid into Ba598 ({Delta}virB4) restored replication to 544 (wild-type) levels (Fig. 2).



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 2. Intracellular replication of Bru. abortus strains in J774 cells and mouse bone-marrow-derived macrophages. J774 cells (a) and mouse bone-marrow-derived macrophages (b) were infected with 544 (wild-type, {square}), Ba598 ({Delta}virB4, {lozenge}), Ba603 (virB4+, {circ}) or Ba616 (virB4 K463R, {triangleup}) for the indicated time. Data points and error bars represent the mean c.f.u. of triplicate samples from a typical experiment (performed at least four times) and their standard deviation.

 


View larger version (98K):
[in this window]
[in a new window]
 
Fig. 3. Immunofluorescence micrograph of intracellular replicated Bru. abortus. Mouse bone-marrow-derived macrophages were infected with 544 (wild-type; a, c) or Ba598 ({Delta}virB4; b, d) for 48 h, as described in Methods. (a, b) FITC-labelled intracellular bacteria; (c, d) phase-contrast images.

 
To determine whether the putative NTP-binding region of VirB4 plays a role in intracellular replication, we used site-directed mutagenesis to specifically alter virB4 such that the encoded Lys463 located in the Walker box A region of the protein is changed to an Arg residue (Fig. 1b). We introduced a plasmid, pMAW18, containing full-length virB4 with that specific mutation into Ba598 ({Delta}virB4) and the transformant was designated Ba616 (virB4 K463R). Ba616 failed to restore replication to 544 (wild-type) levels (Fig. 2). These results implied that the putative NTP-binding region of VirB4 is necessary for intracellular replication of Brucella.

The VirB proteins have been proposed to interact with other VirB proteins to form a multicomponent membrane apparatus which can mediate export of putative substrate(s) (Christie, 2001 ). If VirB4 constitutes one of the transporter subunits, or if the protein functions as a multimer, then a merodiploid expressing both the wild-type and mutant allele may differ phenotypically from 544 (wild-type). To test this possibility, we introduced pMAW18 (virB4 K463R) into the wild-type strain (Ba615) and then examined the ability of Ba615 to replicate in macrophages. Bone-marrow-derived macrophages were infected for 48 h at an m.o.i. of 20. Introducing a control plasmid containing either no virB4 or a wild-type copy of virB4 did not affect intracellular replication (5·1x105±0·5x105 or 5·4x105±0·8x105 c.f.u., respectively). In contrast, introducing a plasmid containing the mutated virB4 into 544 (wild-type) resulted in greatly reduced intracellular replication (7·6x104±0·7x104 c.f.u.). These data indicated that the virB4 K463R mutant exhibits a dominant negative phenotype.

The putative NTP-binding region of VirB4 is required for inhibition of phagosome–lysosome fusion by Bru. abortus early in infection
Phagosomes containing virulent Bru. abortus are reluctant to fuse with lysosomes, whereas dead Bru. abortus phagosomes co-localize with endocytic compartments in the early stage of infection in macrophages (Arenas et al., 2000 ). To test the ability of Bru. abortus to target properly within bone-marrow-derived macrophages early in infection, interaction of the mutants with the endocytic pathway was quantified by immunofluorescence localization of LAMP-1, a membrane protein of late endosomes and lysosomes (Chen et al., 1988 ; Harter & Mellman, 1992 ).

As expected, most phagosomes containing 544 (wild-type) did not co-localize with the late endosomal and lysosomal marker: 14·3±3·2% of the wild-type phagosomes were LAMP-1-positive (Fig. 4). In contrast, phagosomes containing Ba598 ({Delta}virB4) with severe intracellular growth defects were frequently stained brightly by an antibody specific for LAMP-1 (74·6±5·3% LAMP-1-positive) (Fig. 4). Introduction of the complementing plasmid into Ba598 ({Delta}virB4) restored evasion of the endocytic pathway to 544 (wild-type) levels (17·0±2·9% LAMP-1-positive). To determine whether the putative NTP-binding region of VirB4 plays a role in intracellular trafficking, interaction of Ba616 (virB4 K463R) with the endocytic pathway was quantified. Similar results were obtained for the virB4 mutant: 76·1±2·9% of the Ba616 (virB4 K463R) cells resided in a LAMP-1-positive compartment. Introducing a control plasmid containing either no virB4 or a wild-type copy of virB4 did not affect LAMP-1 staining (12·5±2·7 or 13·5±2·1 %, respectively). In contrast, introducing a plasmid containing the mutated virB4 into 544 (wild-type) resulted in 73·9±2·3% LAMP-1-positive phagosomes.



View larger version (101K):
[in this window]
[in a new window]
 
Fig. 4. Co-localization of virB4 mutant with late endosomal and lysosomal marker LAMP-1 in mouse bone-marrow-derived macrophages by immunofluorescence microscopy. Macrophages were infected with 544 (wild-type; a, c) or Ba598 ({Delta}virB4; b, d) for 1 h, fixed and stained for LAMP-1 co-localization (a, b) and intracellular bacteria (c, d).

 
The putative NTP-binding region of VirB4 is essential for virulence in mice
Groups of five mice were injected intraperitoneally with 104 c.f.u. of Bru. abortus 544 (wild-type), Ba598 ({Delta}virB4), Ba603 (virB4+) or Ba616 (virB4 K463R). At 10 days post-inoculation, mice were sacrificed and their spleens were weighed and examined for Brucella proliferation. The number of viable bacteria recovered from the spleens of mice injected with 544 (wild-type) and Ba603 (virB4+) were 3·8x107±1·4x107 and 8·6x107±0·7x106 c.f.u. per spleen, respectively. On the other hand, no viable bacteria were recovered from mice injected with Ba598 ({Delta}virB4) and Ba616 (virB4 K463R), based on counting the number of c.f.u. in each spleen. The weights of the spleens of mice injected with Ba598 ({Delta}virB4) (58·3±5·2 mg) and Ba616 (virB4 K463R) (57·2±4·9 mg) were markedly lower than those of mice injected with 544 (wild-type) (271·0±18 mg) and Ba603 (virB4+) (216·6±11 mg). These results indicated that the putative NTP-binding region is involved directly or indirectly in the splenomegaly typically associated with infection by wild-type Bru. abortus.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study, we have examined the importance of VirB4 for Bru. abortus intracellular replication and the functional significance of NTP binding or hydrolysis by this protein. VirB4 is a component of the VirB secretion apparatus that has a high sequence similarity to members of the type IV secretion system (Sieira et al., 2000 ). In A. tumefaciens, a type IV secretion system participates in delivering oncogenic T-DNA from the bacterium to the plant cell, whereas in Bor. pertussis it participates in the secretion of pertussis toxin (Christie, 2001 ; Christie & Vogel, 2000 ). Recently, O’Callaghan et al. (1999) and Sieira et al. (2000) have described the presence of a virB region in Brucella suis and Bru. abortus which is involved in intracellular growth in macrophages. We found that VirB4 is a critical component of the VirB secretion apparatus, as an in-frame deletion mutant of virB4 exhibited greatly impaired intracellular replication in the macrophage-like cell line J774 and mouse bone-marrow-derived macrophages. The fact that this mutation could be complemented by introducing a plasmid containing a wild-type copy of virB4 confirms that this defect was due to a mutation in virB4 rather than to a polar effect of the mutation.

We have also demonstrated the importance of the putative NTP-binding motif of virB4 by specifically altering the region of virB4 that encodes a Walker box A motif. To date, two other organisms, A. tumefaciens and Bor. pertussis, have been shown to have a functionally important NTP-binding domain. The intact NTP-binding domain of VirB4 is essential for A. tumefaciens virulence (Berger & Christie, 1993 ) and the NTP-binding domain of PtlC, a member of a set of proteins necessary for the secretion of pertussis toxin from Bor. pertussis, is essential for transport of pertussis toxin across bacterial membranes (Cook et al., 1999 ). We have shown that alteration of Lys463 to Arg in Bru. abortus VirB4 protein had a marked effect on intracellular replication in macrophages and virulence in mice. As this mutation is in the putative NTP-binding region of VirB4, our results suggest that this region is critical for VirB4 function and support the idea that NTP binding is also an important aspect of VirB4 function. Three families of putative ATPases are associated with type IV transfer systems: (1) the TraG family of coupling proteins, (2) homologues of A. tumefaciens VirB4 protein, and (3) homologues of the RP4 TrbB and of A. tumefaciens VirB11 proteins (Christie, 2001 ). These proteins are ubiquitous among the type IV systems and are sometimes present in two or more copies. Further studies have provided evidence for transmembrane topology, self-association and a structural contribution to channel formation that is independent of VirB4 ATPase activity. Based on these properties, this family of ATPases might transduce information, possibly in the form of ATP-induced conformational changes, across the cytoplasmic membrane to extracytoplasmic subunits (Dang et al., 1999 ). Additional work needs to be done to determine the function of VirB4, since introduction of a plasmid containing an NTP-binding domain mutation into a wild-type strain of Bru. abortus resulted in drastically reduced intracellular replication. Our finding of a dominant negative phenotype for this mutation suggests that the altered protein may interfere with the action of the wild-type protein. Our study also indicates that VirB4 might interact with another component of the putative secretion system, possibly another molecule of VirB4, another VirB protein or unidentified substrate(s). These findings provide important information towards understanding the mechanism underpinning VirB function.

The mechanism of virulence of Brucella spp. is not yet fully understood. Brucella spp. infect their hosts through mucosae and wounds and initially enter into professional phagocytes where they survive and reproduce (Liautard et al., 1996 ). It is known that intracellular pathogens have developed a series of strategies to survive inside cells (Sinai & Joiner, 1997 ). Alteration of the normal process of phagosome maturation has been described for several micro-organisms, such as Mycobacterium, Legionella and Chlamydia (Sinai & Joiner, 1997 ). The intracellular survival of Brucella spp. has been documented for several cell types. Bru. abortus shows a different intracellular trafficking pattern between professional and non-professional phagocytes. Multiple observations have been reported that Bru. abortus is incorporated into phagosomes and remains in membrane-bound compartments until the host cell dies (Comerci et al., 2001 ; Pizarro-Cerda et al., 1998a , b ). In non-professional phagocytes, Brucella is located in structures that resemble the endoplasmic reticulum (Pizarro-Cerda et al., 1998a , b ). Other evidence has indicated that Brucella is transported through the autophagic pathway before accumulating in the endoplasmic reticulum (Comerci et al., 2001 ; Pizarro-Cerda et al., 1998a , b ). Macrophages are particularly important for the survival and spreading of Brucella during infection (Liautard et al., 1996 ) and these autophagosomes are not observed in macrophages. Arenas et al. (2000) monitored the intracellular transport of Bru. abortus in macrophages and observed the kinetics of the fusion of phagosomes with preformed lysosomes labelled with colloidal gold particles by electron microscopy. In that study, phagosomes containing live Bru. abortus delayed fusion with lysosomes and newly endocytosed material was not incorporated into these phagosomes (Arenas et al., 2000 ). In this study, we tested the ability of a wild-type strain, a {Delta}virB4 mutant and a mutant of the putative NTP-binding motif of virB4 to target properly within bone-marrow-derived macrophages early in infection. Bacterial phagosomes were scored for acquisition of the lysosomal glycoprotein LAMP-1, an abundant transmembrane protein found predominantly in late endosomes and lysosomes (Chen et al., 1988 ; Harter & Mellman, 1992 ). Our results, together with previous evidence, indicate that Bru. abortus prevents phagosome–lysosome fusion after uptake by macrophages.

This report shows that Bru. abortus VirB4 plays a critical role in intracellular growth and virulence in mice. Moreover, VirB4 may be a nucleotide-binding protein that interacts with other members of the VirB secretion apparatus to facilitate transport of unidentified substrate(s) across the bacterial membrane. Future investigations directed toward the clarification of the nature of the effector molecules may shed light on the molecular mechanism underlying the infection process of Bru. abortus.


   ACKNOWLEDGEMENTS
 
We thank M. E. Kovach for providing pBBR1MCS-2 and Y. Isayama for helpful suggestions. This work was supported, in part, by a grant from Grants-in-Aid for Scientific Research (12575029 and 13770129), Japan Society for the Promotion of Science.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Acha, P. & Szylres, B. (1980). Zoonoses and Communicable Diseases Common to Man and Animals. Washington, DC: Pan American Health Organization.

Andrews, H. L., Vogel, J. P. & Isberg, R. R. (1998). Identification of linked Legionella pneumophila genes essential for intracellular growth and evasion of the endocytic pathway. Infect Immun 66, 950-958.[Abstract/Free Full Text]

Arenas, G. N., Staskevich, A. S., Aballay, A. & Mayorga, L. S. (2000). Intracellular trafficking of Brucella abortus in J774 macrophages. Infect Immun 68, 4255-4263.[Abstract/Free Full Text]

Baldwin, C. L. & Winter, A. J. (1994). Macrophages and Brucella. Immunol Ser 60, 363-380.[Medline]

Berger, B. R. & Christie, P. J. (1993). The Agrobacterium tumefaciens virB4 gene product is an essential virulence protein requiring an intact nucleoside triphosphate-binding domain. J Bacteriol 175, 1723-1734.[Abstract]

Chen, J. W., Cha, Y., Yuksel, K. U., Gracy, R. W. & August, J. T. (1988). Isolation and sequencing of a cDNA clone encoding lysosomal membrane glycoprotein mouse LAMP-1. J Biol Chem 263, 8754-8758.[Abstract/Free Full Text]

Christie, P. J. (2001). Type IV secretion: intercellular transfer of macromolecules by systems ancestrally related to conjugation machines. Mol Microbiol 40, 294-305.[Medline]

Christie, P. J. & Vogel, J. P. (2000). Bacterial type IV secretion: conjugation systems adapted to deliver effector molecules to host cells. Trends Microbiol 8, 354-360.[Medline]

Comerci, D. J., Martinez-Lorenzo, M. J., Sieira, R., Gorvel, J. & Ugalde, R. A. (2001). Essential role of the VirB machinery in the maturation of the Brucella abortus-containing vacuole. Cell Microbiol 3, 159-168.[Medline]

Cook, D. M., Farizo, K. M. & Burns, D. L. (1999). Identification and characterization of PtlC, an essential component of the pertussis toxin secretion system. Infect Immun 67, 754-759.[Abstract/Free Full Text]

Covacci, A., Telford, J. L., Del Giudice, G., Parsonnet, J. & Rappuoli, R. (1999). Helicobacter pylori virulence and genetic geography. Science 284, 1328-1333.[Abstract/Free Full Text]

Dang, T. A., Zhou, X. R., Graf, B. & Christie, P. J. (1999). Dimerization of the Agrobacterium tumefaciens VirB4 ATPase and the effect of ATP-binding cassette mutations on the assembly and function of the T-DNA transporter. Mol Microbiol 32, 1239-1253.[Medline]

Harter, C. & Mellman, I. (1992). Transport of the lysosomal membrane glycoprotein lgp120 (lgp-A) to lysosomes does not require appearance on the plasma membrane. J Cell Biol 177, 311-325.

Kolter, R., Inuzuka, M. & Helinski, D. R. (1978). Trans-complementation-dependent replication of a low molecular weight origin fragment from plasmid R6K. Cell 15, 1199-1208.[Medline]

Kotob, S. I., Hausman, S. Z. & Burns, D. L. (1995). Localization of the promoter for the ptl genes of Bordetella pertussis, which encode proteins essential for secretion of pertussis toxin. Infect Immun 63, 3227-3230.[Abstract]

Kovach, M. E., Elzer, P. H., Hill, D. S., Robertson, G. T., Farris, M. A., Roop, R. M.II & Peterson, K. M. (1995). Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166, 175-176.[Medline]

Kuldau, G. A., De Vos, G., Owen, J., McCaffrey, G. & Zambryski, P. (1990). The virB operon of Agrobacterium tumefaciens pTiC58 encodes 11 open reading frames. Mol Gen Genet 221, 256-266.[Medline]

Liautard, J. P., Gross, A., Dornand, J. & Kohler, S. (1996). Interactions between professional phagocytes and Brucella spp. Microbiologia 12, 197-206.[Medline]

McLean, I. W. & Nakane, P. K. (1974). Periodate-lysine-paraformaldehyde fixative: a new fixative for immunoelectron microscopy. J Histochem Cytochem 22, 1077-1083.[Medline]

O’Callaghan, D., Cazevieille, C., Allardet-Servent, A., Boschiroli, M. L., Bourg, G., Foulongne, V., Frutos, P., Kulakov, Y. & Ramuz, M. (1999). A homologue of the Agrobacterium tumefaciens VirB and Bordetella pertussis Ptl type IV secretion systems is essential for intracellular survival of Brucella suis. Mol Microbiol 33, 1210-1220.[Medline]

Pizarro-Cerda, J., Meresse, S., Parton, R. G., van der Goot, G., Sola-Landa, A., Lopez-Goni, I., Moreno, E. & Gorvel, J. P. (1998a). Brucella abortus transits through the autophagic pathway and replicates in the endoplasmic reticulum of nonprofessional phagocytes. Infect Immun 66, 5711-5724.[Abstract/Free Full Text]

Pizarro-Cerda, J., Moreno, E., Sanguedolce, V., Mege, J. L. & Gorvel, J. P. (1998b). Virulent Brucella abortus prevents lysosome fusion and is distributed within autophagosome-like compartments. Infect Immun 66, 2387-2392.[Abstract/Free Full Text]

Pohlman, R. F., Genetti, H. D. & Winans, S. C. (1994). Common ancestry between IncN conjugal transfer genes and macromolecular export systems of plant and animal pathogens. Mol Microbiol 14, 655-668.[Medline]

Sangari, F. J. & Aguero, J. (1996). Molecular basis of Brucella pathogenicity: an update. Microbiologia 12, 207-218.[Medline]

Segal, G., Purcell, M. & Shuman, H. A. (1998). Host cell killing and bacterial conjugation require overlapping sets of genes within a 22-kb region of the Legionella pneumophila genome. Proc Natl Acad Sci U S A 95, 1669-1674.[Abstract/Free Full Text]

Sieira, R., Comerci, D. J., Sanchez, D. O. & Ugalde, R. A. (2000). A homologue of an operon required for DNA transfer in Agrobacterium tumefaciens is required in Brucella abortus for virulence and intracellular multiplication. J Bacteriol 182, 4849-4855.[Abstract/Free Full Text]

Sinai, A. P. & Joiner, K. A. (1997). Safe haven: the cell biology of nonfusogenic pathogen vacuoles. Annu Rev Microbiol 51, 415-462.[Medline]

Stachel, S. E. & Nester, E. W. (1986). The genetic and transcriptional organization of the vir region of the A6 Ti plasmid of Agrobacterium tumefaciens. EMBO J 5, 1445-1454.[Abstract]

Swanson, M. S. & Isberg, R. R. (1996). Identification of Legionella pneumophila mutants that have aberrant intracellular fates. Infect Immun 64, 2585-2594.[Abstract]

Vogel, J. P., Andrews, H. L., Wong, S. K. & Isberg, R. R. (1998). Conjugative transfer by the virulence system of Legionella pneumophila. Science 279, 873-876.[Abstract/Free Full Text]

Walker, M. E., Saraste, M., Runswick, M. J. & Gay, N. J. (1982). Distantly related sequences in the alpha and beta subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J 1, 945-951.[Medline]

Watarai, M., Andrews, H. L. & Isberg, R. R. (2001). Formation of a fibrous structure on the surface of Legionella pneumophila associated with exposure of DotH and DotO proteins after intracellular growth. Mol Microbiol 39, 313-329.[Medline]

Weiss, A. A., Johnson, F. D. & Burns, D. L. (1993). Molecular characterization of an operon required for pertussis toxin secretion. Proc Natl Acad Sci U S A 90, 2970-2974.[Abstract]

Winans, S. C. & Walker, G. C. (1985). Conjugal transfer system of the IncN plasmid pKM101. J Bacteriol 161, 402-410.[Medline]

Woodcock, D. M., Crowther, P. J., Doherty, J., Jefferson, S., DeCruz, E., Noyer-Weidner, M., Smith, S. S., Michael, M. Z. & Graham, M. W. (1989). Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants. Nucleic Acids Res 17, 3469-3478.[Abstract]

Zavala, I., Nava, A., Guerra, J. & Quiros, C. (1994). Brucellosis. Infect Dis Clin N Am 8, 225-241.[Medline]

Received 27 July 2001; revised 29 November 2001; accepted 23 January 2002.