IAI FigSearch
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Other Versions of this Article:
IAI.00379-07v1
76/1/30    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pei, J.
Right arrow Articles by Ficht, T. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pei, J.
Right arrow Articles by Ficht, T. A.
Infection and Immunity, January 2008, p. 30-37, Vol. 76, No. 1
0019-9567/08/$08.00+0     doi:10.1128/IAI.00379-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Cytotoxicity in Macrophages Infected with Rough Brucella Mutants Is Type IV Secretion System Dependent{triangledown}

Jianwu Pei,1 Qingmin Wu,2 Melissa Kahl-McDonagh,1 and Thomas A. Ficht1*

Department of Veterinary Pathobiology, Texas A&M University and Texas Agricultural Experiment Station, College Station, Texas 77843-4467,1 Department of Preventive Veterinary Medicine, College of Veterinary Medicine, China Agricultural University, Beijing 100094, China2

Received 13 March 2007/ Returned for modification 19 April 2007/ Accepted 3 October 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Smooth Brucella spp. inhibit macrophage apoptosis, whereas rough Brucella mutants induce macrophage oncotic and necrotic cell death. However, the mechanisms and genes responsible for Brucella cytotoxicity have not been identified. In the current study, a random mutagenesis approach was used to create a mutant bank consisting of 11,354 mutants by mariner transposon mutagenesis using Brucella melitensis rough mutant 16M{Delta}manBA as the parental strain. Subsequent screening identified 56 mutants (0.49% of the mutant bank) that failed to cause macrophage cell death (release of 10% or less of the lactate dehydrogenase). The absence of cytotoxicity during infection with these mutants was independent of demonstrable defects in in vitro bacterial growth or uptake and survival in macrophages. Interrupted genes in 51 mutants were identified by DNA sequence analysis, and the mutations included interruptions in virB encoding the type IV secretion system (T4SS) (n = 36) and in vjbR encoding a LuxR-like regulatory element previously shown to be required for virB expression (n = 3), as well as additional mutations (n = 12), one of which also has predicted roles in virB expression. These results suggest that the T4SS is associated with Brucella cytotoxicity in macrophages. To verify this, deletion mutants were constructed in B. melitensis 16M by removing genes encoding phosphomannomutase/phosphomannoisomerase ({Delta}manBA) and the T4SS ({Delta}virB). As predicted, deletion of virB from 16M{Delta}manBA and 16M resulted in a complete loss of cytotoxicity in rough strains, as well as the low level cytotoxicity observed with smooth strains at extreme multiplicities of infection (>1,000). Taken together, these results demonstrate that Brucella cytotoxicity in macrophages is T4SS dependent.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Brucella spp., the causative agents of brucellosis, are facultative intracellular bacteria with a broad host range. These bacteria can survive and multiply in macrophages and trophoblasts, causing abortion in a variety of animals and undulant fever in humans. The economic losses caused by brucellosis in livestock industries can have dramatic consequences for agriculture and public health, especially in developing countries. Because of the public health concerns and the absence of safe and efficacious human vaccines, Brucella melitensis, B. abortus, and B. suis have been classified as category B agents on the CDC biodefense list (31).

To survive in hostile environments, intracellular bacteria have developed various strategies or virulence factors to evade elimination from the host. A number of bacteria, including Brucella, inhibit host cell apoptosis (13, 17, 19, 41, 43), which presumably enhances bacterial survival in the host. Inactivation of defense systems is another strategy used by bacteria to survive in their hosts (37). Cytotoxicity of Brucella in macrophages was originally described more than 40 years ago (14, 15). Our recent studies revealed that this cytotoxicity is macrophage specific and resembles oncosis and necrosis, not apoptosis (32, 33). The mechanism by which Brucella kills macrophages has been characterized as pore formation-mediated lysis that requires bacterial protein synthesis (33). However, the genes responsible for the cytotoxicity have not been identified.

In an effort to elucidate the molecular mechanisms and identify Brucella genes responsible for macrophage cytotoxicity, a mutant bank was created using the B. melitensis rough mutant 16M{Delta}manBA as the genetic background. The bank was screened with J774.A1 murine macrophages to identify mutants that failed to induce cytotoxic cell death. The results revealed that the type IV secretion system (T4SS) is essential for cytotoxic death of macrophages induced by Brucella infection.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial strains and media. The bacteria used in these experiments included B. melitensis 16M, 16M{Delta}manBA, 16M{Delta}virB2, and 16M{Delta}manBA{Delta}virB2, B. abortus S2308, S2308manBA::Tn5 (CA180) (2), S2308{Delta}virB2 (21), and S2308virB10::Tn5 (BA114) (20), and Escherichia coli β2155 with plasmid pSC189. Brucella strains were grown for 72 h at 37°C on tryptic soy agar (TSA) or in tryptic soy broth (Difco, Detroit, MI) unless stated otherwise. E. coli β2155 was grown on TSA containing 50 µg/ml diaminopimelic acid (DAP) and 100 µg/ml kanamycin (46).

Cell culture and reagents. Murine macrophage-like cell line J774.A1 and RAW 264.7 murine macrophages were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 0.1 mM nonessential amino acids (complete DMEM) as previously described (32). The cells were passaged every 3 to 5 days and discarded after passage 15. Mouse alveolar macrophage line MH-S (CRL-2019; ATCC) was maintained in RPMI 1640 with 10% (vol/vol) fetal bovine serum, sodium pyruvate, and 50 µM β-mercaptoethanol. CytoTox 96 nonradioactive cytotoxicity assay kits were purchased from Promega (Madison, WI). The autoinducer N-dodecanoyl-DL-homoserine lactone (C12-HSL) was obtained from Sigma.

Conjugation and mutagenesis. Conjugation was conducted as previously described (46). Briefly, B. melitensis 16M{Delta}manBA was grown on solid medium for 72 h at 37°C. E. coli β2155 containing pSC189 was grown on solid medium supplemented with 50 µg/ml DAP for 24 h at 37°C. Bacteria were harvested in 5 ml of peptone saline (1% [wt/vol] Bacto peptone [Difco] and 0.5% [wt/vol] NaCl) containing DAP. Equal volumes of the two bacterial suspensions were mixed to obtain a donor-to-recipient ratio of approximately 1:100 and centrifuged at 13,400 x g for 2 min, and then the pellets were resuspended in 100 µl of peptone saline supplemented with DAP. Conjugation mixtures containing B. melitensis 16M{Delta}manBA and E. coli β2155 bearing pSC189 were spotted onto 24-mm-diameter nitrocellulose filters on the surface of a TSA plate containing DAP. After 2 h of incubation at 37°C, bacteria on the filter surfaces were resuspended in 1.0 ml of peptone saline. Serial dilutions of the conjugation mixtures were prepared and plated onto TSA plates supplemented with kanamycin (100 µg/ml) to evaluate the transformation efficiency of Brucella. The remaining bacterial conjugation mixture was stored in peptone saline containing 20% (vol/vol) glycerol at –80°C.

Selection of noncytotoxic mutants. Based on the efficiency of conjugation, the frozen mixtures were diluted and plated onto TSA plates supplemented with 100 µg/ml of kanamycin. Single colonies were transferred from the TSA plates to 96-well microplates containing tryptic soy broth supplemented with 100 µg/ml kanamycin and incubated for 48 h at 37°C. Ten microliters of the 48-h cultures of each mutant was used to inoculate J774.A1 cells cultured in 96-well plates at a multiplicity of infection (MOI) of approximately 1,000. The plates were centrifuged for 5 min at 200 x g to synchronize uptake and then incubated at 37°C for 20 min. An equal volume of complete DMEM containing 100 µg/ml of gentamicin was added to each well, and incubation was continued at 37°C for 48 h. The mutants lacking cytotoxicity were identified using phase-contrast microscopy, and the results were confirmed using the lactate dehydrogenase (LDH) release assay.

Sequence analysis and identification of interrupted loci. Genomic DNA from selected mutants was isolated as previously described and digested with HaeIII or RsaI (46). The digested DNA was self-ligated and amplified by PCR using the following mariner-specific primers: forward primer 5'-CAACACTCAACCCTATCTCG-3' and reverse primer 5'-CACTCAACCCTATCTCGGTC-3'. PCR products were purified from agarose gels using a QIAquick gel purification kit (Qiagen) and were sequenced using the reverse primer at the Gene Technologies Laboratory, Texas A&M University. The sequences were blasted against the B. melitensis genome database to identify the disrupted genes using MacVector (Accelrys, Inc).

Intracellular survival assay. Monolayers of macrophages cultured in 24-well plates were infected with B. melitensis and selected mutants at an MOI of 100, and bacterial survival was determined as previously described (32).

Macrophage cytotoxicity assay. LDH release into cell culture supernatants was detected using the CytoTox 96 nonradioactive cytotoxicity assay as previously described (32). Cell death was expressed as the percentage of LDH release, which was calculated using the following formula: percentage of LDH release = 100 x (test LDH release – spontaneous release)/(maximum release – spontaneous release). The maximum release was determined following dissolution of cell monolayers using 1% (vol/vol) Triton X-100.

Autoinducer C12-HSL treatment of Brucella-infected cells. Macrophages cultured in 24-well plates were infected with 16M{Delta}manBA or CA180 at an MOI of 50 or with 16M or S2308 at an MOI of 1,000 as described above. The infected cells were treated with fresh media containing various concentrations of C12-HSL at 1 h postinfection (p.i.). Cytotoxicity was determined at 24 h p.i. using the LDH release assay.

Statistical analysis. Statistical significance was determined using Student's t test; a P value of <0.05 was considered significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of mutants with minimal cytotoxicity in macrophages. Our previous studies have shown that cytotoxicity of rough Brucella strains requires bacterial protein synthesis (33). To identify the responsible genes, a mutant bank consisting of 11,354 mutants was generated via conjugation between E. coli β2155 bearing pSC189 and B. melitensis 16M{Delta}manBA (46). Since 16M{Delta}manBA is cytotoxic to murine macrophages, mutants that do not induce macrophage cell death were expected to have their cytotoxicity-related genes interrupted. In the first round of screening, macrophage cytotoxicity was determined by light microscopy observation as previously reported (32). Macrophages infected with noncytotoxic mutants at an MOI of ~1,000 were easily differentiated from macrophages infected with cytotoxic mutants (Fig. 1). After one round of screening, 350 mutants that did not exhibit cytotoxicity in macrophages were identified. To eliminate mutants that failed to induce cytotoxic cell death as a result of poor growth alone, mutants exhibiting poor in vitro growth based on absorbance values for the microtiter dish cultures were eliminated from consideration. The cytotoxicity of the remaining mutants was confirmed using the LDH release assay, in which 56 mutants (0.49% of the mutant bank) exhibited minimum LDH release (10% or less) in at least two separate assays (Table 1).


Figure 1
View larger version (58K):
[in this window]
[in a new window]

  		FIG. 1. Noncytotoxic rough mutant infection in murine macrophages. J774.A1 macrophages were infected with mutants from the rough mutant bank at an MOI of ~1,000. The infected cells were observed using light microscopy at 24 h p.i. (A) Cytotoxic mutant-infected cells. (B) Noncytotoxic mutant (20G1)-infected cells. (C) Uninfected cells.

 

View this table:
[in this window]
[in a new window]

  		TABLE 1. Interrupted genes of noncytotoxic mutants that are persistent in macrophages

 
Since a reduction in cytotoxicity may also result from defects in uptake and/or intracellular survival in macrophages, each of the mutants was characterized by using a gentamicin protection assay. The results revealed that the uptake of the mutants was not affected compared with that of the parental rough strain and remained 10-fold higher than the uptake of smooth organisms (Fig. 2). However, in contrast to the parental strain, which was gradually cleared from the macrophages as a result of cytotoxicity (32), the numbers of the mutants increased over the first 24 h (100%) (P < 0.001 compared with uptake), and the mutants persisted at that level for the duration of the experiment (48 h) (Fig. 2 and Table 1).


Figure 2
View larger version (19K):
[in this window]
[in a new window]

  		FIG. 2. Noncytotoxic mutants replicate in murine macrophages. J774.A1 macrophages were infected with 16M, 16M{Delta}manBA, or noncytotoxic double mutants at an MOI of 100. Bacterial uptake and survival were determined using the gentamicin protection assay. The results for 19 representative noncytotoxic double mutants are shown. The symbols indicate the mean values from three independent experiments. An asterisk indicates that the P value is <0.001 for a comparison with uptake.

 
T4SS is required for cytotoxicity of rough B. melitensis strains. To identify interrupted genes in mutants lacking cytotoxicity, an inverse PCR was used to amplify DNA products for sequencing as previously described (20, 46). The results of sequencing identified the genes interrupted in 51 mutants; the genes in 5 mutants remained unidentified (Table 1). Of the mutants identified, 36 had Himar1 insertions in the virB locus encoding the T4SS, 3 had Himar1 insertions in the vjbR locus encoding a LuxR-like regulatory element necessary for T4SS synthesis, and 12 had Himar1 interruptions in 12 other genes, 2 of which (BMEII1093 and BMEII0653-4) have a known relationship with a bacterial type III secretion system and cytotoxicity in other species (23, 47) (Table 1). These results suggest that there is a critical relationship between the expression of the T4SS and the cytotoxicity observed during B. melitensis infection in macrophages. Table 1 shows the distribution of transposon insertions in the T4SS operon. virB4 had the highest frequency of insertion, while no interruptions were detected in virB3, virB7, and virB12. The lack of detection of interruptions in virB3 and virB7 may have resulted from the small size of these genes. The failure to detect interruptions in virB12 was presumed to be due to the failure of the protein to contribute to the T4SS function (30, 39).

To verify that the phenotype observed was the result of loss of the gene function identified, a manBA and virB2 double-gene-knockout mutant and a virB2 single-gene-knockout mutant were constructed as previously described (21). As predicted, the 16M{Delta}manBA mutant was cytotoxic to macrophages, while the 16M{Delta}manBA{Delta}virB2 mutant (rough) was noncytotoxic even at an MOI as high as 2,000 (Fig. 3A). The cytotoxicity observed with smooth strain 16M at an elevated MOI may have been the result of the presence of rough organisms that arose spontaneously in Brucella cultures or from low-level cytotoxic activity with smooth strains (Fig. 3A). Although it was not possible to distinguish between the two possibilities, deletion of virB2 completely eliminated the cytotoxicity observed with the smooth organism (Fig. 3A). To determine whether elimination of cytotoxicity is species specific, the cytotoxicities of B. abortus S2308, S2308{Delta}virB2, S2308virB10::Tn5, and S2308manBA::Tn5 were evaluated using the LDH release assay (Fig. 3B). Interruption of virB10 or knockout of virB2 eliminated the cytotoxicity of S2308 when an extreme MOI was used in the infection. These results confirmed the essential role of T4SS for Brucella cytotoxicity (Fig. 3).


Figure 3
View larger version (20K):
[in this window]
[in a new window]

  		FIG. 3. Brucella cytotoxicity in macrophage is T4SS dependent. (A) J774.A1 macrophages were infected with B. melitensis 16M or 16M-derived mutants at various MOI. (B) J774.A1 macrophages were infected with B. abortus S2308 or S2308-derived mutants at various MOI. Macrophage cytotoxicity was determined at 24 h p.i. using the LDH release assay. The data are the means ± standard deviations of a representative experiment that was repeated twice with similar results.

 
To determine whether the observed virB-dependent cytotoxicity is cell type specific, several different cell lines were infected with the mutants. The results revealed that 16M{Delta}manBA is cytotoxic to RAW 264.7 murine macrophages (Fig. 4A), MH-S mouse alveolar macrophages, (Fig. 4B), and THP-1 human monocytes (data not shown) but is not cytotoxic to HeLa cells (data not shown). These results are consistent with previously published results obtained using B. abortus rough mutants (32). Again, 16M{Delta}manBA{Delta}virB2 was not cytotoxic to all the cell types tested (Fig. 4A and 4B and data not shown). Taken together, the results show that T4SS is essential for Brucella-induced macrophage cytotoxicity.


Figure 4
View larger version (20K):
[in this window]
[in a new window]

  		FIG. 4. T4SS-dependent cytotoxicity of Brucella is macrophage specific. RAW 264.7 murine macrophages (A) and MH-S murine alveolar macrophages (B) were infected with 16M, 16M{Delta}virB2, 16M{Delta}manBA, or 16M{Delta}manBA{Delta}virB2 at various MOI. Macrophage cytotoxicity was determined at 24 h p.i. using the LDH release assay. The data are the means ± standard deviations of a representative experiment that was repeated twice with similar results.

 
C12-HSL treatment inhibits Brucella cytotoxicity. The quorum-sensing signal molecule C12-HSL has been identified in B. melitensis, and addition of this pheromone to a culture of B. melitensis or B. suis down-regulated virB transcription (11, 40). Recent studies have revealed that this effect is mediated by the transcriptional regulator VjbR (LuxR homolog) (11). Since interruptions in vjbR have been shown to prevent cytotoxicity, it is reasonable to assume that quorum sensing may play a role in regulating this activity. To determine whether the quorum-sensing pheromone has an effect on cytotoxicity induced by Brucella infection, J774.A1 macrophages were infected with Brucella and treated with various concentrations of C12-HSL. The LDH release levels detected at 24 h p.i. confirmed that C12-HSL inhibited Brucella cytotoxicity in a dose-dependent manner (Fig. 5). C12-HSL is not bactericidal or bacteriostatic to Brucella, since the survival and replication of Brucella were not affected by C12-HSL treatment at the highest concentration used in this study (data not shown).


Figure 5
View larger version (21K):
[in this window]
[in a new window]

  		FIG. 5. Autoinducer C12-HSL inhibits Brucella cytotoxicity in macrophages. J774.A1 macrophages were infected with 16M or S2308 at an MOI of 1,000 or with 16M{Delta}manBA or S2308manBA::Tn5 at an MOI of 50. C12-HSL was added to culture media at various concentrations. LDH release was detected at 24 h p.i. The data are the means ± standard deviations of a representative experiment that was repeated twice with similar results.

 
Double-knockout mutant 16M{Delta}manBA{Delta}virB2 persists in macrophages. To confirm the results obtained during the screening of the Himar1 transposon-derived mutants (Fig. 2), the uptake and survival of the double-knockout mutant 16M{Delta}manBA{Delta}virB2 in macrophages were evaluated using the gentamicin protection assay, and the results were compared with the results for single-gene-deletion mutants constructed using the same approach (Fig. 6). Consistent with previous results, the uptake of 16M{Delta}manBA{Delta}virB2 was not affected by the loss of the T4SS function, and the levels remained elevated throughout the course of the experiment. The 16M{Delta}manBA mutant was taken up at similar levels, consistent with the rough phenotype, while the {Delta}virB mutant was taken up at greatly reduced levels due to its smooth phenotype. However, in contrast to the sustained intracellular survival of the 16M{Delta}manBA{Delta}virB2 mutant, the values for the 16M{Delta}virB2 and 16M{Delta}manBA mutants decreased over time. It seems reasonable to speculate that the combined effects of the two mutations altered the intracellular trafficking and persistence of the organism.


Figure 6
View larger version (19K):
[in this window]
[in a new window]

  		FIG. 6. Double-gene-knockout mutant 16M{Delta}manBA{Delta}virB2 persists in murine macrophages. J774.A1 macrophages were infected with 16M, 16M{Delta}virB2, 16M{Delta}manBA, or 16M{Delta}manBA{Delta}virB2 at an MOI of 100. Bacterial uptake and survival in the macrophages were determined by the gentamicin protection assay. The data are the means ± standard deviations of a representative experiment that was repeated twice with similar results.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The cytotoxicity in macrophages induced by rough Brucella mutants, including B. melitensis, B. abortus, and B. suis rough mutants, was first reported more than 40 years ago (14, 15). Our recent studies indicated that the cytotoxicity reflects oncotic and necrotic cell death that requires bacterial protein synthesis and direct contact with macrophages (32, 33). In the current report, we demonstrate for the first time that the T4SS is critical for Brucella cytotoxicity in macrophages.

Mariner-based transposon mutagenesis has been demonstrated to be an efficient approach for bacterial mutation studies (1, 9, 38, 46). In the current study, a 2-h conjugation was used and no siblings were identified in the mutant library, which was consistent with previous reports (46). DNA sequencing of interrupted genes and identification of the insertion sites revealed that each of the 51 noncytotoxic mutants examined is unique (data not shown). Although 36 of the 51 mutants have transposon insertions in genes encoding the T4SS, it has been demonstrated that the virB operon is not a hot spot for Himar1 insertion (46). As discussed below, the failure to identify insertions in the virB12 locus argues against such an insertion bias.

The T4SS is a membrane-associated complex that has been identified in a variety of bacterial species and has multiple functions (4). One of the main functions is to translocate virulence factors into host cells. For example, Helicobacter pylori secretes the virulence factor CagA through its T4SS into host cells, thereby inducing NF-{kappa}B translocation and proinflammatory responses (5). Several effectors translocated by the Dot/Icm secretion system in Legionella have been identified (6, 25, 42). The T4SS identified in Brucella is encoded by 11 genes (virB1 through virB11) and is required for Brucella trafficking to replication niches and intracellular survival in host cells (7, 10, 12, 30). A possible twelfth component located downstream of the T4SS structural genes is expressed, but it is not required for bacterial virulence (39). Surprisingly, our current results show that the elimination of T4SS expression in rough mutants resulted in enhanced replication and persistence, suggesting that the T4SS is not required for rough mutant intracellular trafficking and survival in macrophages. Previous studies, including studies in our labs (unpublished data), revealed that rough Brucella strains invade macrophages by a different pathway than smooth strains, which influences both the intracellular trafficking and the ultimate fate of rough organisms (34, 36). The T4SS may play a different role in rough Brucella intracellular trafficking and survival in macrophages, which is under further investigation.

Our current study demonstrated for the first time that the T4SS is critical for Brucella cytotoxicity in macrophages. Although the factors translocated by the T4SS of Brucella have not been identified, our previous studies showed that the cytotoxicity requires bacterial protein synthesis and bacterial uptake, not just preexisting components of the bacteria (33). These results suggested that pores are formed in the host cell either as a result of a translocated "cytotoxin" or directly as a result of T4SS function. As such, a closer examination of the identities of the genes that contribute to the loss of cytotoxic function may be revealing. Because pore formation by cytotoxin insertion in the cellular membrane is cell type specific (44), the observation that Brucella cytotoxicity is macrophage specific (32) supports the cytotoxin-translocating hypothesis. It is plausible that the cytotoxin can insert pores into macrophage membranes and induce cell death but cannot do this in HeLa cell or BHK cell membranes. Since the putative cytotoxin has not been identified, it is impossible to explore the interaction of the toxin with its cell receptor. The fact that rough Brucella mutants are more cytotoxic than smooth strains (32) (Fig. 3) suggests that rough mutants secrete more toxin molecules into the host cell. The mechanisms are unclear, but it is plausible that O antigen expressed on the bacterial cell surface may impair T4SS function in smooth Brucella strains. This phenomenon has been reported for the type III secretion systems of Shigella and Yersinia (29, 45). In Shigella, shortening of the lipopolysaccharide molecule by O-antigen glucosylation enhanced the function of the type III secretion system. A minimal needle length is required for efficient functioning of the Yersinia enterocolitica type III secretion system. In smooth Brucella strains, the T4SS apparatus may be shielded by O antigen, and therefore the cytotoxin may not be easily secreted. In contrast, on the surface of rough Brucella strains, the T4SS is exposed by a shortening or lack of O antigen. Enhanced injection of toxin into host cells causes increased cell death.

It is not surprising that interruptions were not detected in virB12, since the product of this gene is not required for Brucella virulence (39), and it is therefore plausible that VirB12 is not required for T4SS assembly or a function pertaining to cytotoxicity. The failure to obtain mutants with interruptions in virB3 or virB7 may be due to the small sizes of these genes (350 and 173 bp, respectively), although the size of virB2 is similar and multiple insertions in this locus were identified (Table 1). Alternative explanations include nonsaturating mutagenesis of the Brucella genome and the absence of a contribution of these structural proteins to cytotoxicity.

Although cytotoxic activity has been described, the Brucella "cytotoxin" remains to be identified. Of the 22 identified genes related to the cytotoxicity of Brucella in macrophages, 9 are in the virB locus. Another gene encodes the regulatory component VjbR (LuxR homolog) that is necessary for virB expression in Brucella (11) (J. Weeks and T. A. Ficht, unpublished data). The remaining genes have no known or obvious direct link with T4SS activity in Brucella, but at least two of the interruptions are associated with virulence in other systems. One interruption occurs in the glycerol-3-phosphate regulon repressor (Table 1) that has been shown to regulate uptake of the autoinducer AI-2, which is important in bacterial cell-to-cell communication and in regulating the activity of LuxR (47). Another transposon interruption occurs just upstream of the gene encoding diguanylate cyclase/phosphodiesterase. Several members of the diguanylate cyclase/phosphodiesterase family of proteins in Pseudomonas aeruginosa have been shown to be associated with virulence and cytotoxicity mediated by the type III secretion system (23). Additionally, it is presumed that the other genes identified have an indirect role in cytotoxicity due to changes in the metabolic or energetic state of the bacteria, which may alter or prevent assembly of the T4SS. Although these activities are highly suggestive, additional experiments are necessary to confirm the contribution to T4SS activity and the potential links with cytotoxicity.

There are parallels between the activities associated with the Brucella T4SS and the activities associated with the dot/icm-encoded T4SS in Legionella pneumophila, although these systems are representatives of two different classes of T4SS. Similar to the evidence for Brucella described above, Legionella has been shown to form pores in host cells that may be important for bacterial egress and subsequent reinfection (3, 8, 18, 22, 26, 48). In addition, Brucella has been shown to induce necrotic and oncotic cell death in macrophages (32, 33). Cytotoxicity induced by L. pneumophila requires not only T4SS but also flagellin (28, 35). Recent evidence indicates that flagellin contamination of the macrophage cytosol through the T4SS triggers cell death (27). A flagellum has been described for Brucella (11, 16, 24), and it is associated with virulence, but the function of this structure is unclear at this time. In the current study, noncytotoxic mutants with interruptions within the flagellin gene were not observed and were presumed not to function in this context. However, we cannot rule out the possibility that Brucella flagellin is involved in macrophage cytotoxicity and that such mutants were stochastically missed. Direct determination of cytotoxicity in flagellin gene knockout mutants offers the best alternative.

Although disruption of the T4SS of Brucella prevents pore formation and cytotoxicity, additional work is required to determine the exact role of cytotoxicity in the survival and virulence of Brucella. In our recent studies, plaque formation was evaluated following 16M{Delta}manBA and 16M{Delta}manBA{Delta}virB2 infection, and the results revealed that, like Legionella egress, Brucella egress from infected cells is essential for cell-to-cell spread and continued replication of the organism (data not shown). The fact that this activity is more prominent in rough mutants of B. abortus and B. melitensis may explain why rough mutants arise spontaneously both in culture and during infection (J. E. Turse and T. A. Ficht, unpublished results). The appearance of rough mutants may be expected to enhance the cytotoxic function and egress from the cells.


   ACKNOWLEDGMENTS
 
This work was supported by grant R01 AI48496 to T.A.F. from NIH and by grant 1U54AI057156 from the Western Regional Center for Excellence.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Veterinary Pathobiology, Texas A&M University, College Station, TX 77843-4467. Phone: (979) 845-4118. Fax: (979) 862-1088. E-mail: tficht{at}cvm.tamu.edu Back

{triangledown} Published ahead of print on 15 October 2007. Back

Editor: V. J. DiRita


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Akerley, B. J., E. J. Rubin, A. Camilli, D. J. Lampe, H. M. Robertson, and J. J. Mekalanos. 1998. Systematic identification of essential genes by in vitro mariner mutagenesis. Proc. Natl. Acad. Sci. USA 95:8927-8932.[Abstract/Free Full Text]
2. Allen, C. A., L. G. Adams, and T. A. Ficht. 1998. Transposon-derived Brucella abortus rough mutants are attenuated and exhibit reduced intracellular survival. Infect. Immun. 66:1008-1016.[Abstract/Free Full Text]
3. Alli, O. A., L. Y. Gao, L. L. Pedersen, S. Zink, M. Radulic, M. Doric, and Y. Abu Kwaik. 2000. Temporal pore formation-mediated egress from macrophages and alveolar epithelial cells by Legionella pneumophila. Infect. Immun. 68:6431-6440.[Abstract/Free Full Text]
4. Backert, S., and T. F. Meyer. 2006. Type IV secretion systems and their effectors in bacterial pathogenesis. Curr. Opin. Microbiol. 9:207-217.[CrossRef][Medline]
5. Brandt, S., T. Kwok, R. Hartig, W. Konig, and S. Backert. 2005. NF-{kappa}B activation and potentiation of proinflammatory responses by the Helicobacter pylori CagA protein. Proc. Natl. Acad. Sci. USA 102:9300-9305.[Abstract/Free Full Text]
6. Bruggemann, H., C. Cazalet, and C. Buchrieser. 2006. Adaptation of Legionella pneumophila to the host environment: role of protein secretion, effectors and eukaryotic-like proteins. Curr. Opin. Microbiol. 9:86-94.[CrossRef][Medline]
7. Celli, J., C. de Chastellier, D. M. Franchini, J. Pizarro-Cerda, E. Moreno, and J. P. Gorvel. 2003. Brucella evades macrophage killing via VirB-dependent sustained interactions with the endoplasmic reticulum. J. Exp. Med. 198:545-556.[Abstract/Free Full Text]
8. Chen, J., K. S. de Felipe, M. Clarke, H. Lu, O. R. Anderson, G. Segal, and H. A. Shuman. 2004. Legionella effectors that promote nonlytic release from protozoa. Science 303:1358-1361.[Abstract/Free Full Text]
9. Chiang, S. L., and E. J. Rubin. 2002. Construction of a mariner-based transposon for epitope-tagging and genomic targeting. Gene 296:179-185.[CrossRef][Medline]
10. Comerci, D. J., M. J. Martinez-Lorenzo, R. Sieira, J. P. Gorvel, and R. A. Ugalde. 2001. Essential role of the VirB machinery in the maturation of the Brucella abortus-containing vacuole. Cell. Microbiol. 3:159-168.[CrossRef][Medline]
11. Delrue, R. M., C. Deschamps, S. Leonard, C. Nijskens, I. Danese, J. M. Schaus, S. Bonnot, J. Ferooz, A. Tibor, X. De Bolle, and J. J. Letesson. 2005. A quorum-sensing regulator controls expression of both the type IV secretion system and the flagellar apparatus of Brucella melitensis. Cell. Microbiol. 7:1151-1161.[CrossRef][Medline]
12. Delrue, R. M., M. Martinez-Lorenzo, P. Lestrate, I. Danese, V. Bielarz, P. Mertens, X. De Bolle, A. Tibor, J. P. Gorvel, and J. J. Letesson. 2001. Identification of Brucella spp. genes involved in intracellular trafficking. Cell. Microbiol. 3:487-497.[CrossRef][Medline]
13. Fernandez-Prada, C. M., E. B. Zelazowska, M. Nikolich, T. L. Hadfield, R. M. Roop II, G. L. Robertson, and D. L. Hoover. 2003. Interactions between Brucella melitensis and human phagocytes: bacterial surface O-polysaccharide inhibits phagocytosis, bacterial killing, and subsequent host cell apoptosis. Infect. Immun. 71:2110-2119.[Abstract/Free Full Text]
14. Freeman, B. A., D. J. Kross, and R. Circo. 1961. Host-parasite relationships in brucellosis. II. Destruction of macrophage cultures by Brucella of different virulence. J. Infect. Dis. 108:333-338.[Medline]
15. Freeman, B. A., and B. H. Rumack. 1964. Cytopathogenic effect of Brucella spheroplasts on monocytes in tissue culture. J. Bacteriol. 88:1310-1315.[Abstract/Free Full Text]
16. Fretin, D., A. Fauconnier, S. Kohler, S. Halling, S. Leonard, C. Nijskens, J. Ferooz, P. Lestrate, R. M. Delrue, I. Danese, J. Vandenhaute, A. Tibor, X. DeBolle, and J. J. Letesson. 2005. The sheathed flagellum of Brucella melitensis is involved in persistence in a murine model of infection. Cell. Microbiol. 7:687-698.[CrossRef][Medline]
17. Galdiero, E., C. Romano Carratelli, M. Vitiello, I. Nuzzo, E. Del Vecchio, C. Bentivoglio, G. Perillo, and F. Galdiero. 2000. HSP and apoptosis in leukocytes from infected or vaccinated animals by Brucella abortus. New Microbiol. 23:271.[Medline]
18. Gao, L. Y., and Y. A. Kwaik. 2000. The mechanism of killing and exiting the protozoan host Acanthamoeba polyphaga by Legionella pneumophila. Environ. Microbiol. 2:79-90.[CrossRef][Medline]
19. Gross, A., A. Terraza, S. Ouahrani-Bettache, J. P. Liautard, and J. Dornand. 2000. In vitro Brucella suis infection prevents the programmed cell death of human monocytic cells. Infect. Immun. 68:342-351.[Abstract/Free Full Text]
20. Hong, P. C., R. M. Tsolis, and T. A. Ficht. 2000. Identification of genes required for chronic persistence of Brucella abortus in mice. Infect. Immun. 68:4102-4107.[Abstract/Free Full Text]
21. Kahl-McDonagh, M. M., and T. A. Ficht. 2006. Evaluation of protection afforded by Brucella abortus and Brucella melitensis unmarked deletion mutants exhibiting different rates of clearance in BALB/c mice. Infect. Immun. 74:4048-4057.[Abstract/Free Full Text]
22. Kirby, J. E., J. P. Vogel, H. L. Andrews, and R. R. Isberg. 1998. Evidence for pore-forming ability by Legionella pneumophila. Mol. Microbiol. 27:323-336.[CrossRef][Medline]
23. Kulasakara, H., V. Lee, A. Brencic, N. Liberati, J. Urbach, S. Miyata, D. G. Lee, A. N. Neely, M. Hyodo, Y. Hayakawa, F. M. Ausubel, and S. Lory. 2006. Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3'-5')-cyclic-GMP in virulence. Proc. Natl. Acad. Sci. USA 103:2839-2844.[Abstract/Free Full Text]
24. Letesson, J. J., P. Lestrate, R. M. Delrue, I. Danese, F. Bellefontaine, D. Fretin, B. Taminiau, A. Tibor, A. Dricot, C. Deschamps, V. Haine, S. Leonard, T. Laurent, P. Mertens, J. Vandenhaute, and X. De Bolle. 2002. Fun stories about Brucella: the "furtive nasty bug." Vet. Microbiol. 90:317-328.[CrossRef][Medline]
25. Luo, Z. Q., and R. R. Isberg. 2004. Multiple substrates of the Legionella pneumophila Dot/Icm system identified by interbacterial protein transfer. Proc. Natl. Acad. Sci. USA 101:841-846.[Abstract/Free Full Text]
26. Molmeret, M., O. A. Alli, M. Radulic, M. Susa, M. Doric, and Y. A. Kwaik. 2002. The C-terminus of IcmT is essential for pore formation and for intracellular trafficking of Legionella pneumophila within Acanthamoeba polyphaga. Mol. Microbiol. 43:1139-1150.[CrossRef][Medline]
27. Molofsky, A. B., B. G. Byrne, N. N. Whitfield, C. A. Madigan, E. T. Fuse, K. Tateda, and M. S. Swanson. 2006. Cytosolic recognition of flagellin by mouse macrophages restricts Legionella pneumophila infection. J. Exp. Med. 203:1093-1104.[Abstract/Free Full Text]
28. Molofsky, A. B., L. M. Shetron-Rama, and M. S. Swanson. 2005. Components of the Legionella pneumophila flagellar regulon contribute to multiple virulence traits, including lysosome avoidance and macrophage death. Infect. Immun. 73:5720-5734.[Abstract/Free Full Text]
29. Mota, L. J., L. Journet, I. Sorg, C. Agrain, and G. R. Cornelis. 2005. Bacterial injectisomes: needle length does matter. Science 307:1278.[Abstract/Free Full Text]
30. O'Callaghan, D., C. Cazevieille, A. Allardet-Servent, M. L. Boschiroli, G. Bourg, V. Foulongne, P. Frutos, Y. Kulakov, and M. Ramuz. 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.[CrossRef][Medline]
31. Pappas, G., P. Panagopoulou, L. Christou, and N. Akritidis. 2006. Brucella as a biological weapon. Cell. Mol. Life Sci. 63:2229-2236.[CrossRef][Medline]
32. Pei, J., and T. A. Ficht. 2004. Brucella abortus rough mutants are cytopathic for macrophages in culture. Infect. Immun. 72:440-450.[Abstract/Free Full Text]
33. Pei, J., J. E. Turse, Q. Wu, and T. A. Ficht. 2006. Brucella abortus rough mutants induce macrophage oncosis that requires bacterial protein synthesis and direct interaction with the macrophage. Infect. Immun. 74:2667-2675.[Abstract/Free Full Text]
34. Porte, F., A. Naroeni, S. Ouahrani-Bettache, and J. P. Liautard. 2003. Role of the Brucella suis lipopolysaccharide O antigen in phagosomal genesis and in inhibition of phagosome-lysosome fusion in murine macrophages. Infect. Immun. 71:1481-1490.[Abstract/Free Full Text]
35. Ren, T., D. S. Zamboni, C. R. Roy, W. F. Dietrich, and R. E. Vance. 2006. Flagellin-deficient Legionella mutants evade caspase-1- and Naip5-mediated macrophage immunity. PLoS Pathog. 2:e18.[CrossRef][Medline]
36. Rittig, M. G., A. Kaufmann, A. Robins, B. Shaw, H. Sprenger, D. Gemsa, V. Foulongne, B. Rouot, and J. Dornand. 2003. Smooth and rough lipopolysaccharide phenotypes of Brucella induce different intracellular trafficking and cytokine/chemokine release in human monocytes. J. Leukoc. Biol. 74:1045-1055.[Abstract/Free Full Text]
37. Rosenberger, C. M., and B. B. Finlay. 2003. Phagocyte sabotage: disruption of macrophage signalling by bacterial pathogens. Nat. Rev. Mol. Cell Biol. 4:385-396.[CrossRef][Medline]
38. Rubin, E. J., B. J. Akerley, V. N. Novik, D. J. Lampe, R. N. Husson, and J. J. Mekalanos. 1999. In vivo transposition of mariner-based elements in enteric bacteria and mycobacteria. Proc. Natl. Acad. Sci. USA 96:1645-1650.[Abstract/Free Full Text]
39. Sun, Y. H., H. G. Rolan, A. B. den Hartigh, D. Sondervan, and R. M. Tsolis. 2005. Brucella abortus VirB12 is expressed during infection but is not an essential component of the type IV secretion system. Infect. Immun. 73:6048-6054.[Abstract/Free Full Text]
40. Taminiau, B., M. Daykin, S. Swift, M. L. Boschiroli, A. Tibor, P. Lestrate, X. De Bolle, D. O'Callaghan, P. Williams, and J. J. Letesson. 2002. Identification of a quorum-sensing signal molecule in the facultative intracellular pathogen Brucella melitensis. Infect. Immun. 70:3004-3011.[Abstract/Free Full Text]
41. Tolomeo, M., P. D. Carlo, V. Abbadessa, L. Titone, S. Miceli, E. Barbusca, G. Cannizzo, S. Mancuso, S. Arista, and F. Scarlata. 2003. Monocyte and lymphocyte apoptosis resistance in acute and chronic brucellosis and its possible implications in clinical management. Clin. Infect. Dis. 36:1533-1538.[CrossRef][Medline]
42. VanRheenen, S. M., Z. Q. Luo, T. O'Connor, and R. R. Isberg. 2006. Members of a Legionella pneumophila family of proteins with ExoU (phospholipase A) active sites are translocated to target cells. Infect. Immun. 74:3597-3606.[Abstract/Free Full Text]
43. Wang, M., N. Qureshi, N. Soeurt, and G. Splitter. 2001. High levels of nitric oxide production decrease early but increase late survival of Brucella abortus in macrophages. Microb. Pathog. 31:221-230.[CrossRef][Medline]
44. Welch, R. A. 1991. Pore-forming cytolysins of gram-negative bacteria. Mol. Microbiol. 5:521-528.[Medline]
45. West, N. P., P. Sansonetti, J. Mounier, R. M. Exley, C. Parsot, S. Guadagnini, M. C. Prevost, A. Prochnicka-Chalufour, M. Delepierre, M. Tanguy, and C. M. Tang. 2005. Optimization of virulence functions through glucosylation of Shigella LPS. Science 307:1313-1317.[Abstract/Free Full Text]
46. Wu, Q., J. Pei, C. Turse, and T. A. Ficht. 2006. Mariner mutagenesis of Brucella melitensis reveals genes with previously uncharacterized roles in virulence and survival. BMC Microbiol. 6:102.[CrossRef][Medline]
47. Xavier, K. B., and B. L. Bassler. 2005. Interference with AI-2-mediated bacterial cell-cell communication. Nature 437:750-753.[CrossRef][Medline]
48. Zink, S. D., L. Pedersen, N. P. Cianciotto, and Y. Abu-Kwaik. 2002. The Dot/Icm type IV secretion system of Legionella pneumophila is essential for the induction of apoptosis in human macrophages. Infect. Immun. 70:1657-1663.[Abstract/Free Full Text]

Infection and Immunity, January 2008, p. 30-37, Vol. 76, No. 1
0019-9567/08/$08.00+0     doi:10.1128/IAI.00379-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Other Versions of this Article:
IAI.00379-07v1
76/1/30    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pei, J.
Right arrow Articles by Ficht, T. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pei, J.
Right arrow Articles by Ficht, T. A.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
J. Bacteriol. J. Virol. Eukaryot. Cell
Microbiol. Mol. Biol. Rev. Clin. Vaccine Immunol. All ASM Journals