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
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ABSTRACT |
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Keywords: type IV secretion, macrophage
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INTRODUCTION |
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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 (OCallaghan 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.
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METHODS |
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Construction of in-frame deletion mutant of virB4.
pMAW14 (virB4) was constructed by cloning two PCR fragments into SalI/SacI-cleaved pSR47s (Andrews et al., 1998
). Fragment 1 was a 1640 bp SalIBamHI 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 BamHISacI 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 (virB4) was introduced into DH5
(
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 EcoRIBamHI 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; 13871389) 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 EcoRIBamHI 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 EcoRIBamHI fragment from pMAW17 into EcoRI/BamHI-cleaved pBBR1MCS-2.
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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.
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RESULTS |
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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 phagosomelysosome 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 (
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 (
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.
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DISCUSSION |
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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
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 phagosomelysosome 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.
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ACKNOWLEDGEMENTS |
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Received 27 July 2001;
revised 29 November 2001;
accepted 23 January 2002.