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Infect Immun. Oct 2011; 79(10): 4010–4018.
PMCID: PMC3187240

The Burkholderia pseudomallei Δasd Mutant Exhibits Attenuated Intracellular Infectivity and Imparts Protection against Acute Inhalation Melioidosis in Mice [down-pointing small open triangle]

F. C. Fang, Editor

Abstract

Burkholderia pseudomallei, the cause of serious and life-threatening diseases in humans, is of national biodefense concern because of its potential use as a bioterrorism agent. This microbe is listed as a select agent by the CDC; therefore, development of vaccines is of significant importance. Here, we further investigated the growth characteristics of a recently created B. pseudomallei 1026b Δasd mutant in vitro, in a cell model, and in an animal model of infection. The mutant was typified by an inability to grow in the absence of exogenous diaminopimelate (DAP); upon single-copy complementation with a wild-type copy of the asd gene, growth was restored to wild-type levels. Further characterization of the B. pseudomallei Δasd mutant revealed a marked decrease in RAW264.7 murine macrophage cytotoxicity compared to the wild type and the complemented Δasd mutant. RAW264.7 cells infected by the Δasd mutant did not exhibit signs of cytopathology or multinucleated giant cell (MNGC) formation, which were observed in wild-type B. pseudomallei cell infections. The Δasd mutant was found to be avirulent in BALB/c mice, and mice vaccinated with the mutant were protected against acute inhalation melioidosis. Thus, the B. pseudomallei Δasd mutant may be a promising live attenuated vaccine strain and a biosafe strain for consideration of exclusion from the select agent list.

INTRODUCTION

Burkholderia pseudomallei, a Gram-negative saprophyte and facultative intracellular pathogen, is a common cause of environmentally acquired septicemia in Southeast Asia and northern Australia (7, 10, 44). It is the etiological agent of the disease melioidosis and is listed as a category B select agent by the U.S. Centers for Disease Control and Prevention. Bacterial select agent research is currently focused on basic research into virulence and pathogenesis to fulfill the five main points of the Public Health Security and Bioterrorism Preparedness and Response Act of 2002 (43). One of the goals is to develop and maintain medical countermeasures (such as drugs, vaccines, and other biological products, medical devices, and other supplies) against biological agents and toxins in case of a bioterrorism event. To combat potential foul play associated with intentional release of select agents, a focus on vaccine development for first responders, such as military and health service professionals, is of the utmost importance (26). Currently, there are no vaccines against B. pseudomallei, and treatment entails prolonged regimens of intravenous and orally administered antibiotic therapy (30).

In a recent publication, we described an engineered B. pseudomallei strain with a deletional mutation in the aspartate-β-semialdehyde dehydrogenase (asd) gene, which is auxotrophic for diaminopimelate (DAP) in rich medium and auxotrophic for DAP, lysine, methionine, and threonine in minimal medium (28); this is consistent with similar mutations in many other bacterial species (13, 1820). DAP is a diamino acid that cross-links to d-alanine in neighboring peptidoglycan strands, and the Δasd mutant exhibits the “pop-and-die” phenotype associated with an inability to synthesize DAP for cell wall biosynthesis. Previous works have created asd mutants in Salmonella enterica serovar Typhimurium (13) and Legionella pneumophila (17) and demonstrated a growth requirement for DAP. In addition, the S. Typhimurium Δasd strain has been extensively used in clinical studies with human subjects as a vaccine delivery strain (25, 40). The pathway for synthesizing DAP from aspartate is absent in mammals; therefore, no DAP is present in mammalian hosts, including humans (13, 36). The other amino acids (lysine, methionine, and threonine) made from aspartate via Asd are essential amino acids in humans, affording another possible level of nutrient limitation in vivo. Without a considerable exogenous concentration of DAP, the Δasd mutant is unable to cross-link its cell wall and cannot replicate. Even when supplied with high levels of DAP, intracellularly replicating L. pneumophila Δasd did not recover to wild-type levels of pathogenicity in either macrophage or protozoan infection models (17).

Live attenuated vaccines are particularly effective vaccines, because live bacteria may replicate modestly in the host, similar to situations encountered during an actual infection. In addition, live attenuated vaccines contain complex epitopes not found in subunit or heat-inactivated vaccines, and thus they stimulate parts of the immune system that could otherwise be neglected (e.g., a strong Th1 response) (12). Previous studies testing the efficacy of auxotrophic B. pseudomallei and Burkholderia mallei strains as live attenuated vaccines have resulted in various degrees of success (4, 42). In order to determine if the Δasd strain may be appropriate as a future vaccine candidate, we evaluated the growth and attenuation of the B. pseudomallei 1026b Δasd mutant in vitro and in cell culture. Animal studies were carried out to determine virulence levels and attenuation of the Δasd strain. Efficacy of the B. pseudomallei 1026b Δasd strain as a live attenuated vaccine against inhalation melioidosis was then ascertained in a BALB/c mouse model.

MATERIALS AND METHODS

Bacterial strains, media, and culture conditions.

All manipulations of B. pseudomallei were conducted in CDC/USDA-approved and -registered biosafety level 3 (BSL3) facilities at the University of Hawaii at Manoa and Colorado State University, and experiments with select agents were performed in accordance with the recommended BSL3 practices (32). Derivatives of Escherichia coli strains EPMax10B (Bio-Rad), E1345, E1354, E1869, and E1889 (Table 1) were routinely used for cloning or plasmid mobilization into B. pseudomallei as described previously (24, 28). Luria-Bertani (LB) medium (Difco) was used to culture E. coli strains. B. pseudomallei strains were cultured in LB or 1× M9 minimal medium supplemented with 20 mM glucose (MG). Antibiotics and nonantibiotic antibacterials in solid media were utilized as follows: for E. coli, glyphosate (GS) at 0.3% (wt/vol) and phosphinothricin (PPT) at 0.3% (wt/vol); for B. pseudomallei, GS at 0.3% (wt/vol) and PPT at 2.5% (wt/vol). Growth of E. coli Δasd strains and preparation of DAP were carried out as previously described (2). Selections for bar and gat genes in E. coli and B. pseudomallei strains were performed as previously described (28). B. pseudomallei Δasd::gat strains were grown on LB containing 200 μg/ml DAP or on MG containing 1 mM Lys, 1 mM Met, 1 mM Thr, and 200 μg/ml meso-DAP, as described previously (28).

Table 1.
Strains used in the study

Molecular methods and reagents.

Molecular methods, PCR conditions, and conjugation into select agents were conducted as described previously (2, 28, 35).

Engineering of B. pseudomallei ΔasdBp::gat-FRT.

B. pseudomallei ΔasdBp::gat- FRT was engineered as described previously (28); briefly, the allelic replacement vector pBAKA-ΔasdBp::FRT-gat was conjugally introduced into B. pseudomallei strain 1026b, and selection of the mutation was carried out on MG medium plus 200 μg/ml DAP, 0.3% GS, and 1 mM (each) of Lys, Met, and Thr (these 3 amino acids [3AA] are required for the specific Δasd mutation). Colonies were streaked on the same medium supplemented with 0.1% p-chlorophenylalanine (cPhe) to counterselect against pheS. GS-resistant mutants were purified once on LB plus DAP and patched again on MG plus 0.3% GS, 0.1% cPhe, and 1 mM 3AA with or without 200 μg/ml DAP to confirm the phenotype.

Construction of single-copy rfp-containing vectors.

The red fluorescent protein gene (rfp) was optimized for the codon preference of B. pseudomallei, and the constitutive B. pseudomallei rpsL promoter (PS12) was incorporated upstream of the gene (29). Constructed as previously described (29), rfp was cloned from pUC57-PS12-rfp into mini-Tn7-PCS12-bar to yield mini-Tn7-bar-rfp (Fig. 1). The mini-Tn7-PCS12-bar (28) construct was digested with PstI and SpeI and ligated to the rfp fragment obtained from pUC57-PS12-rfp digested with PstI and XbaI, producing mini-Tn7-bar-rfp. Next, the complementation and fluorescent tagging transposon was constructed by digesting mini-Tn7-bar-asdBp (28) with PstI and SpeI and ligating it to the rfp fragment from PstI- and XbaI-digested pUC57-PS12-rfp, yielding the single-copy complementation/fluorescence tagging vector mini-Tn7-bar-asdBp-rfp. In addition to the bar-based vector, the fluorescence tagging vector mini-Tn7-gat-rfp, based on gat and constructed as previously described (29), was also utilized.

Fig. 1.
Mini-Tn7-bar-rfp, single-copy tagging vector based on phosphinothricin resistance, harboring rfp driven by the PS12 promoter. After insertion aided by pTNS3-asdEc (29), the non-antibiotic resistance marker, which is flanked by identical FRTs, can be removed ...

Engineering of rfp-tagged B. pseudomallei strains and complemented mutants.

E1354 was utilized as the conjugal donor to introduce the single-copy vector mini-Tn7-bar-rfp into the B. pseudomallei Δasd mutant for fluorescent tagging, producing B. pseudomallei Δasd::gat-FRT/attTn7-bar-rfpasd/rfp). The mini-Tn7-bar-asdBp-rfp construct was introduced into the Δasd mutant for complementation and fluorescent tagging, yielding B. pseudomallei Δasd::gat-FRT/attTn7-bar-asdBp-rfpasd/complement/rfp). The mini-Tn7-gat-rfp construct was introduced into wild-type B. pseudomallei strain 1026b for fluorescent tagging of the wild type (wt), resulting in B. pseudomallei attTn7-gat-rfp (wt/rfp). These strains were obtained from a triparental mating experiment using the pTNS3-asdEc helper plasmid, and bacteria containing the integrated transposon were selected and screened via PCR as described previously (8, 24, 28). The mini-Tn7 system allows site-specific insertion of the transposon at a neutral site in the chromosome, downstream of any glmS gene, of which B. pseudomallei has three (8). In all cases the transposon had inserted at the highly favored glmS2 site. Fluorescence was verified by fixing the bacteria with fresh 1% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min followed by imaging with a Zeiss Axio Observer D.1 fluorescence microscope and the accompanying AxioVision release 4.7 software.

Construction of fluorescent strains, including selection for glyphosphate or phosphinothricin-resistant colonies, and transposon integration and screening were performed as previously described (8, 24, 28, 29). Sample preparation and fluorescent imaging were also carried out as previously described (29).

Growth analysis of the rfp-tagged B. pseudomallei Δasd mutant, complemented Δasd mutant, and wild-type strain.

Growth curve experiments were performed on the three RFP-labeled B. pseudomallei strains engineered above (wt/rfp, Δasd/rfp, and Δasd/complement/rfp). These strains were grown overnight at 37°C in LB medium, where the Δasd mutant was supplemented with 200 μg/ml of DAP. Overnight cultures were then washed twice with 1× M9 medium to remove trace amounts of DAP and resuspended in an equal volume of 1× M9 medium. Resuspended cultures were diluted 100-fold into fresh LB medium, without DAP, and shaken at 225 rpm at 37°C. At each time point, 300-μl aliquots were removed and diluted 2-fold in LB medium, and their optical densities were measured at 600 nm using an Eppendorf Biophotometer.

DAP dependency of B. pseudomallei Δasd/rfp.

A growth curve experiment was performed on the B. pseudomallei Δasd/rfp and B. pseudomallei wt/rfp strains. The strains were grown overnight and washed with 1× M9 and inoculated into LB media described above, supplemented with different concentrations of DAP (0, 50, 100, 200, and 500 μg/ml). At each indicated time point, 300-μl aliquots were removed and the optical densities at 600 nm were determined.

Intracellular replication assays.

Both murine macrophage RAW264.7 and human cervical carcinoma HeLa cell lines were grown in a 5% CO2 environment at 37°C in Dulbecco's modified Eagle's medium (DMEM) with 10% (vol/vol) fetal bovine serum (FBS). Gibco 100× antibiotic/antimycotic was added at a 1× working concentration (containing 100 U/ml of penicillin, 100 μg/ml of streptomycin, and 250 ng/ml of amphotericin B) to the cell culture medium during cell culture growth but was omitted during the infection assay. Intracellular replication assays were performed using a modified kanamycin protection assay as previously described (22). Briefly, cells (HeLa and RAW264.7 lines) were cultured in DMEM to confluence, scraped from cell culture flasks, and seeded at 1 × 105 cells per well into 24-well Corning CellBIND culture plates. To prepare cells for infection study, cells were allowed to attach overnight and were washed twice with 1× PBS in the morning.

The three bacterial strains used in this experiment were the same as used above for the complementation growth study. To investigate whether exogenous DAP allowed intracellular infection, two series of infection were carried out with the Δasd/rfp strain. One series was allowed to infect during the entire course of the study in the presence of DAP, and the other had DAP omitted from the medium after 1 h of infection (T = 1). During the first hour of infection, both series of the B. pseudomallei Δasd/rfp strain were supplemented with 200 μg/ml of DAP in the cell culture medium, so as not to bias the invasion ability during attachment and internalization. Assays with the wild-type and complemented Δasd/rfp mutant strains were carried out essentially as those with the Δasd/rfp mutant, except that no DAP was added. Briefly, B. pseudomallei strains were grown to a high cell density, washed twice with 1× PBS, and then diluted to ~1 × 106 CFU/ml. At time zero, 1 ml of DMEM containing diluted bacteria was added to the macrophage monolayers (multiplicity of infection [MOI], 10:1). After allowing the infection to progress for 1 h, the medium was removed and the monolayers were washed twice with 1× PBS to remove any unattached bacteria. Next, fresh DMEM with 700 μg/ml each of amikacin and kanamycin was added to the monolayers to kill any noninternalized bacteria and inhibit extracellular bacterial replication. During the assay, medium was removed from the wells at three time points (2, 6, and 24 h postinfection), and the infected cell monolayers were washed twice with 1× PBS and then lysed with 0.1% Triton X-100. Serial dilutions of the lysates were plated on Brucella agar (Difco) plus 4% (vol/vol) glycerol (BAG) medium at 37°C, as described previously (6, 14). BAG medium was supplemented with 200 μg/ml of DAP when enumerating B. pseudomallei Δasd/rfp colonies. Colonies were counted within 48 h. Experiments with both HeLa and RAW264.7 cell lines, in combination with all bacterial strains, were performed in triplicate, and the standard errors of the means (SEM) were calculated for each.

RAW264.7 macrophage cytotoxicity assay.

Macrophages were cultured as described above and seeded into a 96-well CellBIND plate at ~5 × 104 cells per well. A kanamycin protection and infection assay was carried out, as described above for the intracellular replication assay, with bacteria infected at an MOI of 10:1. At 2, 6, 12, and 24 h postinfection, the cellular supernatant was removed, and lactate dehydrogenase (LDH) levels were determined using the CytoTox 96 nonradioactive cytotoxicity assay (Promega). LDH levels of infected monolayers were compared and normalized to maximal LDH levels (after complete monolayer lysis using 0.1% Triton X-100) to determine the percent cytotoxicity. The cytotoxicity assay was carried out in triplicate, and the SEMs were calculated.

Light microscopy and time course of B. pseudomallei wt/rfp, Δasd/rfp, and Δasd/complement/rfp infection of RAW264.7 murine macrophages.

Light microscopy of infected cell monolayers was carried out as described previously (29), except for a few modifications. Glass coverslips were sterilized in 70% (vol/vol) ethanol and then treated for 4 h with 150 μg/ml poly-l-lysine in sterile double-distilled water (ddH2O). The glass coverslips were washed twice with ddH2O and allowed to air dry within a sterile petri dish overnight. In our experience, glass coverslips treated with poly-l-lysine provided the best surface for cell attachment and microscopic imaging. The 22- by 22-mm coverslips were placed at the bottoms of the wells in a 6-well Corning CellBIND plate prior to seeding. RAW264.7 macrophages were seeded at ~8 × 105 cells per well and allowed to attach overnight, and the infection was initiated by adding different bacterial strains at an MOI of 10:1. At 1 h postinfection, the coverslips were washed twice with 1× PBS, and then fresh DMEM containing 700 μg/ml of kanamycin was added to inhibit extracellular bacterial replication. At 2, 6, 12, and 24 h postinfection, the medium was removed and the cell monolayers were washed twice with 1× PBS and fixed with 1% paraformaldehyde for 30 min. After 30 min, the paraformaldehyde was removed and the coverslips were washed twice with 1× PBS. For safe removal of fixed samples from the BSL3 cabinet for imaging, this method must be initially tested by incubating fixed coverslips for 5 days in LB to confirm the absence of growth and viable bacteria. Coverslips were mounted with a slide-mounting buffer containing 50% glycerol in 1× PBS. Images were obtained as previously described (29).

Animal studies.

BALB/c mice between 4 and 6 weeks of age were purchased from Jackson Laboratories (Bar Harbor, ME). Animals were housed in microisolator cages under pathogen-free conditions. The Institutional Animal Care and Use Committee at Colorado State University approved the animal experiments conducted for these studies. B. pseudomallei infections were done using intranasal (i.n.) inoculation (31). Animals were anesthetized with 100 mg of ketamine/kg of body weight plus 10 mg/kg xylazine. The desired challenge dose of B. pseudomallei was suspended in PBS, and 20 μl was delivered i.n., into alternating nostrils. For the challenge studies, groups of 5 mice were challenged with the wild-type or mutant strain. For the vaccination studies, mice (n = 10) were administered 1 × 107 CFU Δasd mutant B. pseudomallei intranasally and then boosted in the same manner 3 weeks later. Two weeks following the boost, mice were challenged intranasally with 4 × 103 CFU wild-type B. pseudomallei 1026b. For all B. pseudomallei challenge and survival studies, animals were monitored for disease symptoms twice daily and were euthanized according to predetermined humane end points. Lungs, liver, and spleen were removed and homogenized using a tissue stomacher (Teledyne Tekmar, Mason, OH), and homogenates were plated in serial dilutions to determine bacterial counts in the B. pseudomallei-challenged mice 75 days postinfection. Statistical differences in survival times were determined by Kaplan-Meier curves followed by the log-rank test (Prism5 software; GraphPad, La Jolla, CA).

RESULTS

Construction and growth analysis of the rfp-tagged B. pseudomallei Δasd, Δasd/complement, and wild-type strains.

In previous work (28), we developed two nonantibiotic markers, bar and gat, which are effective for the genetic manipulation of B. pseudomallei. In this study it became apparent that another marker besides gat was needed for fluorescent tagging of B. pseudomallei during infection studies. Therefore, we constructed a new non-antibiotic-based single-copy transposon vector (mini-Tn7-bar-rfp) (Fig. 1) for stable site-specific insertion of rfp genes without the need for plasmid maintenance. We used the gat select agent-compliant nonantibiotic marker to fluorescently tag wild-type bacteria as previously described (29). However, mini-Tn7-bar-rfp-based constructs were used to fluorescently tag the B. pseudomallei Δasd::gat-FRT strain, as well as to tag and complement this mutant strain.

The amino acid requirements of the B. pseudomallei Δasd/rfp mutant were previously demonstrated by showing that the mutant could not grow in the absence of methionine, threonine, and DAP on minimal medium plates (28). In this study, we wanted to show that this mutant is unable to grow in rich liquid medium in the absence of DAP. A growth curve experiment was initiated to allow the comparison of growth between B. pseudomallei wt/rfp, B. pseudomallei Δasd/rfp, and the B. pseudomallei Δasd/complement/rfp strains in a rich nutrient source (LB medium). The B. pseudomallei Δasd mutant displayed an inability to grow compared to the wild-type strain (Fig. 2A). This was expected, as in previously published work the B. pseudomallei 1026b Δasd mutant began to lyse after 6 h without DAP (28). When the B. pseudomallei Δasd/rfp strain was complemented using a transposon containing a single copy of the B. pseudomallei asd gene, the growth defect of the mutant was abolished and normal growth was restored. This indicated that the growth defect exhibited by the mutant was solely caused by deletion of the asd gene.

Fig. 2.
Growth curve experiments performed with B. pseudomallei strains. (A) B. pseudomallei strains were grown in the absence of DAP. The wild-type strain and the Δasd strain complemented with a single copy of the asd gene on a site-specific transposon ...

We next investigated the effects of different concentrations of DAP on growth of the Δasd/rfp mutant in rich medium (Fig. 2B). In the presence of DAP, however, all curves showed a significant growth lag. Compared to the wild type, the optical density at 600 nm eventually reached wild-type levels. This demonstrated that the Δasd mutant can grow well when DAP is added to the medium and not at all in the absence of DAP.

The B. pseudomallei Δasd mutant is highly attenuated in intracellular replication.

Assessment of the Δasd mutant attenuation in HeLa and RAW264.7 cell infection models was necessary before animal vaccination. In agreement with previous work (22), our experience suggested that an MOI of 10:1 would initiate an infection that would maximally affect the cell monolayer within 24 h. Internalization was very inefficient in HeLa cells, with only ~500 CFU out of ~1 × 106 CFU internalized by the monolayers (Fig. 3A). As shown in Fig. 3A, the B. pseudomallei Δasd/rfp strain was unable to replicate in HeLa cells. It was able to attach and become internalized as well as the wild type, as indicated by similar intracellular CFU obtained at 2 h postinfection. However, by 6 h postinfection (T = 6), the Δasd/rfp mutant alone (without DAP) was only able to replicate modestly. By 24 h postinfection, intracellular mutant bacteria were undetectable. However, when complemented with a single wild-type copy of the asd gene, the Δasd mutant strain behaved exactly as the wild type, reaching a maximum of ~1 × 106 CFU. Interestingly, the Δasd/rfp strain could infect HeLa cell monolayers when the growth medium was supplemented with 200 μg/ml of DAP and only slowed down the rate of decline in RAW 264.7 macrophages. This indicated that a sufficient amount of DAP was transported into HeLa cells.

Fig. 3.
Infection of HeLa and RAW264.7 cells by B. pseudomallei and the Δasd strain. HeLa (A) and RAW264.7 (B) cell monolayers were infected at an MOI of 10:1. The complemented Δasd strain showed no decrease in its ability to invade and replicate ...

B. pseudomallei was internalized efficiently and replicated much more significantly within RAW264.7 cells (Fig. 3B). The B. pseudomallei Δasd/rfp strain was not internalized efficiently or was killed more efficiently by macrophages than the wild type, as indicated by the 1-log difference in CFU at 2 h postinfection. The Δasd mutant could not sustain a wild-type level of replication even in the presence of 200 μg/ml of DAP, unlike HeLa cell infection. By the end of the assay (24 h), the Δasd/complement/rfp and the wt/rfp strains replicated to a similar level (~1 × 106 CFU) within the RAW264.7 macrophage monolayer. This indicated that even a single copy of the asdBp gene can restore the mutant's abilities to grow within cells.

Cytotoxicity and light/fluorescence microscopy time course of the B. pseudomallei 1026b Δasd mutant infection of RAW264.7 murine macrophages.

Although the Δasd mutant does not replicate to high cell numbers like the wild-type, it was of interest whether or not the Δasd mutant damages the cell monolayers comparably to the wild-type. LDH assays of all strains infecting RAW264.7 monolayers at 2 and 6 h postinfection revealed little differences in cytotoxicity compared to the noninfected control (Fig. 4A), while LDH levels of the wild type and complement began to rise at 12 h postinfection. The wild-type-infected and complemented Δasd mutant-infected monolayers had surpassed the maximum cytotoxicity at 24 h (determined by lysing the initially seeded macrophages), reaching ~100% (Fig. 4A). Independently of DAP, the Δasd mutant did not damage the monolayers to the same level as the wild type and was still comparable to the noninfected control. LDH levels of the noninfected macrophage monolayer rose at 24 h, indicating the spontaneous lysis of macrophages at high confluence, a usual occurrence where rapid division leads to low nutrient availability, macrophage death, and LDH release.

Fig. 4.
(A) Cytotoxicity of B. pseudomallei strains to the RAW264.7 murine macrophage cell line. RAW264.7 cells were infected with the Δasd mutant (in the presence or absence of DAP), the complemented mutant strain, and wild-type strain. Between 2 h and ...

Intracellular replication and host cell cytotoxicity were then placed in a visual context by tracking the rfp-tagged bacteria via fluorescence microscopy. Visible in representative images from 24 h postinfection was a pervasive red fluorescence indicative of high numbers of intracellular bacteria (Fig. 4B). The majority of macrophages were joined together in MNGCs. Upon closer inspection (Fig. 5), the MNGCs were observed teaming with bacteria in both the wt/rfp- and Δasd/complement/rfp-infected monolayers. The bacteria-containing protrusions were clearly visible, extending from the surfaces of the macrophages (Fig. 5). The Δasd mutant-infected monolayers neither contained high numbers of replicating bacteria nor did they show any sign of MNGC formation in the monolayer at 24 h, and in fact they appeared as healthy as the noninfected controls (Fig. 4B). Although not unexpected, the data reaffirm that the mutant was internalized by the macrophages but was unable to produce cytopathology (or MNGCs), with or without DAP, consistent with the wild-type infection.

Fig. 5.
Intracellular replication of B. pseudomallei. RAW264.7 murine macrophage monolayers were visualized using a combination of differential interference contrast and red fluorescence microscopy 24 h postinfection with the B. pseudomallei Δasd/complement ...

Attenuation, vaccination, and acute protection of the Δasd mutant in mice.

We first tested the Δasd mutant for attenuation in vivo. The 50% lethal dose (LD50) of B. pseudomallei 1026b in BALB/c mice has been determined to be approximately 900 CFU via the inhalation route (14). An i.n. dose of 4,500 CFU has been experimentally determined to produce 100% mortality in BALB/c mice after 3 days (21, 31). Intranasal inoculation mimics inhalation melioidosis and produces a characteristic acute pneumonic infection to which BALB/c mice succumb within a few days. Five BALB/c mice were challenged i.n. with 4,500 CFU of B. pseudomallei 1026b, and another five BALB/c mice were challenged i.n. with 1 × 107 CFU (5 logs × LD50). Survival of the mice was then monitored. After 3 days, mice challenged with wild-type B. pseudomallei had all been euthanized due to progressive infection (Fig. 6A). In contrast, mice infected with asd mutant B. pseudomallei showed no outward signs of infection and were observed for 75 days postchallenge, and all remained healthy during this period (Fig. 6A). Thus, the Δasd mutant was highly attenuated compared to the wild-type strain of B. pseudomallei. To assess possible bacterial persistence in vivo, mice challenged with mutant B. pseudomallei were euthanized on day 75, and the lungs, livers, and spleens were homogenized, diluted, and plated on LB agar. Bacteria were not detected in any organ, based on assays with limits of detection of approximately 50 CFU/organ, indicating the mutant bacteria did not persist in organs typically infected during the chronic phase of infection with virulent B. pseudomallei. The numbers of mice used in these studies were judged to be adequate (31) to ensure that the mutant bacterium was avirulent in immunocompetent mice.

Fig. 6.
(A) The B. pseudomallei 1026b Δasd mutant is avirulent in mice. Mice (n = 5 animals per group) were challenged i.n. with either 4,500 CFU B. pseudomallei 1026b (wild type) or 1 × 107 CFU B. pseudomallei 1026b Δasd mutant, and survival ...

We then considered if the Δasd mutant could be used as a vaccine against inhalation melioidosis in BALB/c mice. Numerous publications support the fact that single vaccinations with attenuated live B. pseudomallei vaccines are generally unable to protect mice from developing chronic melioidosis (4, 15, 41). Therefore, we investigated whether an i.n. prime-boost vaccination strategy could extend protection against development of chronic melioidosis. The i.n. route of infection and vaccination emulates aerosol exposure/vaccination, and typically vaccination at the route of pathogen entry generally leads to more effective disease prevention (3). Ten BALB/c mice were primed with an i.n. vaccination of 1 × 107 CFU of the B. pseudomallei 1026b Δasd mutant. Three weeks later, the same mice were boosted with another i.n. vaccination of 1 × 107 CFU of the Δasd mutant. The time period between the initial exposure and the boost would presumably allow for an adaptive cellular and humoral immune response to occur. Two weeks postboost, the mice were challenged with 4 × 103 CFU of wild-type strain 1026b, and survival was compared to unvaccinated mice challenged with the same amount of the wild type. The boost was administered 2 weeks before the infection to further enhance the immune response, presumably allowing time for enhanced adaptive immunity (33, 38). The data showed that vaccinated mice survived significantly longer than the unvaccinated control mice (Fig. 6B). While the prime-boost strategy used in this study protected mice from acute infection, it failed to protect mice from development of chronic B. pseudomallei infection, as nearly all of the vaccinated and challenged mice developed infection of organs at secondary sites, particularly the spleen (data not shown).

DISCUSSION

The essentiality of the asd gene in E. coli has been known for some time, but its requirement for growth and infectivity of select agent species has not been thoroughly investigated. This study evaluated the growth and pathogenicity of the B. pseudomallei Δasd mutant produced in the previous work (28) and its potential for use as a live attenuated vaccine. By performing growth experiments, it was found that without DAP the mutant is unable to replicate and complementation, with a single copy of wild-type asdBp, is sufficient for in vitro and intracellular replication compared to the wild-type bacterium in both HeLa and RAW264.7 cells. These studies demonstrated that by adding DAP, the Δasd strain can be easily propagated within a laboratory setting and, by complementing the asd gene, a markerless balanced lethal system could be used for various B. pseudomallei studies (37). It is important to note that while adding DAP during asd mutant infection can reestablish wild-type growth levels in some cell lines (i.e., HeLa cells), it is not homologous to replication of the mutant after single-copy complementation, where high levels of replication are seen within both cell lines. This may have important implications in future subcutaneous vaccine experiments due to the epithelial nature of the HeLa cell line.

In the absence of DAP, the mutant was unable to replicate in either the HeLa or RAW 264.7 cell infection models. Cytotoxicity data showed that the mutant did not cause increased death or distress to the macrophages, indicating that the mutant is unable to replicate within or cause significant damage to host cell macrophages via endotoxin or exoenzyme release. The link between cytotoxicity and inflammation has been known for some time and can be partially attributed to free radical release during cellular damage both in vitro and in vivo (27). Although inflammatory modulators were not measured in the cytotoxicity assay, a tentative hypothesis would place the corresponding inflammatory modulator levels in the same trend as LDH. By tagging the B. pseudomallei strains with RFP and tracking them in vitro during intracellular replication, we were able to confirm the intracellular location of the mutant and further demonstrate the utility of non antibiotic selectable markers in pathogenesis research.

B. pseudomallei Δasd mutants should be considered biosafe strains suitable for laboratory use and exclusion from the USDA/CDC select agent lists. First, the Δasd mutant was constructed by deleting several hundred bases in the middle of the asd gene (28), producing a stable mutant unable to revert. Additionally, as DAP is not present within mammals, there is no source of exogenous DAP, affording another level of safety for this strain. On the other hand, it can be seen that the B. pseudomallei Δasd mutant invades host cells although, like the B. pseudomallei purM mutant (31), the Δasd mutant is unable to replicate in the host, and bacterial persistence cannot occur. These data, together with the use of Δasd mutants of other species as vaccine delivery strains in humans (13, 41), provide strong evidence supporting the removal of this strain from select agent lists, as was previously done for the 1026b ΔpurM strain Bp82 (31).

Previous vaccination studies utilizing live attenuated strains and a single vaccination were unable to prevent death from chronic infection (1, 4, 9, 34, 39). However, we had reason to believe that the Δasd mutant would be more effective than previous live attenuated strains. It has been shown that a more protective immune response can be achieved by increasing short-term vaccine persistence, which we attempted with the booster vaccine (5, 23, 45). Unfortunately, while vaccination with the Δasd mutant did indeed protect against acute melioidosis, the vaccine failed to protect against chronic melioidosis. This failure might have been because the Δasd mutant vaccine was unable to persist long enough or disseminate and proliferate enough, even after the boost, to induce systemic protection. The route of vaccination can be important because of increased protection at the site of challenge (e.g., mucosal surfaces); however, this may not generate systemic protection (3). This is a possible reason for why mice were protected from the initial lung infection but eventually succumbed to systemic infection at secondary sites. Even so, protection from acute pneumonic melioidosis may provide a vital increase in survival time that could allow for the administration of antibiotic therapeutics.

Future investigations should be carried out to address whether short-term persistence, proliferation, and dissemination of the Δasd mutant, achieved by adding DAP to the vaccine, would provide systemic protection against chronic melioidosis. Within-host persistence of the Δasd mutant would then be contingent on the amount of DAP administered with the vaccine. Addition of DAP to mutant-infected HeLa cells, an epithelial cell line, did allow some intracellular replication; therefore, it may be highly beneficial to incorporate a subcutaneous vaccine containing DAP. Longer exposure to the cutaneous and subcutaneous dendritic cells could prolong T-cell activation at draining lymph nodes (33) and create the powerful cell-mediated immune response hypothetically necessary for sterile immunity (16). Other means of producing systemic dissemination and, perhaps, protection would be to incorporate an intramuscular or subcutaneous vaccination along with the inhaled vaccination. This two-pronged approach may give rise to longer protection from chronic or latent infection, which is proving more difficult to combat than acute melioidosis. Seemingly, the greatest prospect for an effective vaccine against melioidosis is a live attenuated strain. In conclusion, this initial work suggests the utility of the B. pseudomallei Δasd mutant as a live attenuated vaccine against acute melioidosis and further justifies its potential removal from the select agent list.

ACKNOWLEDGMENTS

This project was supported by award number AI065359 from the National Institute of Allergy and Infectious Diseases and by Center of Biomedical Research Excellence grant P20RR018727 from the National Center for Research Resources of the National Institutes of Health. S.W.D. and H.P.S. were supported by NIH NIAID grant AI065357.

The content of this report is solely the responsibility of the authors and does not necessarily represent the official view of the funding agencies.

Footnotes

[down-pointing small open triangle]Published ahead of print on 1 August 2011.

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