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Glanders: off to the races with Burkholderia mallei

Gregory C. Whitlock , D. Mark Estes , Alfredo G. Torres
DOI: http://dx.doi.org/10.1111/j.1574-6968.2007.00949.x 115-122 First published online: 1 December 2007

Abstract

Burkholderia mallei, the etiologic agent of the disease known as glanders, is primarily a disease affecting horses and is transmitted to humans by direct contact with infected animals. The use of B. mallei as a biological weapon has been reported and currently, there is no vaccine available for either humans or animals. Despite the history and highly infective nature of B. mallei, as well as its potential use as a bio-weapon, B. mallei research to understand the pathogenesis and the host responses to infection remains limited. Therefore, this minireview will focus on current efforts to elucidate B. mallei virulence, the associated host immune responses elicited during infection and discuss the feasibility of vaccine development.

Keywords
  • Burkholderia
  • Burkholderia mallei
  • glanders
  • virulence factors
  • immune responses

History and clinical importance

Burkholderia mallei, the etiologic agent of glanders disease, is a Gram-negative, nonmotile, facultative intracellular bacterium. Most known members of the family Burkholderiaceae are resident in the soil; however, B. mallei is an obligate mammalian pathogen. Horses are highly susceptible to infection and are considered to be the natural reservoir for infection, although mules and donkeys are susceptible as well (Neubauer et al., 2005). Glanders maintains a lengthy history dating back to Aristotle in c. 350 bc when the disease was given the name ‘melis’. Identification of the etiologic agent B. mallei was described in 1882 by isolating an organism from the infected liver and spleen of a horse (Schadewaldt, 1975). Since its discovery, the pathogen has been classified as Loefflerella mallei, Pfeifferella mallei, Malleomyces mallei, Actinobacillus mallei, Corynebacterium mallei, Mycobacterium mallei, Pseudomonas mallei and Bacillus mallei. The current genus Burkholderia was a result of DNA–DNA homology values, cellular lipid and fatty acid composition and phenotypic characteristics, as well as 16S rRNA gene typing in 1992 (Yabuuchi et al., 1992). Members of this genus include Burkholderia pseudomallei, the causative agent of melioidosis (Dance, 1990), Burkholderia cepacia, an important pathogen in cystic fibrosis patients (Burns et al., 1996), and Burkholderia thailandensis, a relatively low-virulence bacterium (Glass et al., 2006). Clinically, B. mallei infected solipeds can present with either a chronic (horses) or an acute (mules and donkeys) form. Although eradication has been successful in the United States, glanders is endemic among domestic animals in Africa, Asia, the Middle East and Central and South America. The primary route of equine infection is most likely the consumption of feed or water contaminated with nasal discharges of infected animals, although a cutaneous form also exists, known as farcy. Chronically infected animals present a variety of signs and symptoms dependent on the route of infection including mucopurulent nasal discharge, lung lesions and nodules involving the liver and spleen. Acute infection results in high fever and emaciation, with ulceration of the nasal septum, accompanied by mucopurulent to hemorrhagic discharge. Pathological changes are limited in gut-associated lymphatic tissues, with the majority of pathology occurring in the lungs and airways (Schlater, 1992). Human infection with B. mallei has occurred rarely and sporadically among laboratory workers and those in direct contact with infected animals (Srinivasan et al., 2001). Person-to-person spread of B. mallei is extremely rare. The bacteria usually enter the body through the eyes, nose, mouth or breaks in the skin. In humans, glanders is characterized by initial onset of fever, rigors and malaise, culminating in a rapid onset of pneumonia, bacteremia, pustules and abscesses, leading to death in 7–10 days without antibiotic treatment. The course of infection is dependent on the route of exposure. Direct contact with the skin can lead to a localized cutaneous infection. Inhalation of aerosol or dust containing B. mallei can lead to septicemic, pulmonary or chronic infections of the muscle, liver and spleen. The disease has a 95% case fatality rate for untreated septicemia infections and a 50% case fatality rate in antibiotic-treated individuals (Mandell et al., 1995).

Burkholderia mallei organisms are highly infectious as an aerosol, and infection requires few organisms, offering the potential for intentional release as a biological threat agent. The use of B. mallei as a biological warfare agent during the American Civil War, World Wars I and II and the Russian invasion of Afghanistan has been reported, and, as such, is now considered a select agent due to its potential use as a bio-weapon (Wheelis, 1998; Rotz et al., 2002; Horn, 2003). As a result of this potential threat, research involving B. mallei is confined to biosafety level 3 facilities in the United States and other countries worldwide. Despite the history and highly infective nature of B. mallei, as well as its potential use as a bio-weapon, B. mallei research to understand the pathogenesis and the host responses to infection remains limited. Therefore, this minireview will focus on current efforts to elucidate B. mallei virulence; the associated host immune responses elicited during infection; and discuss the feasibility of vaccine development.

Burkholderia mallei pathogenic determinants

The B. mallei strain ATCC 23344, isolated in 1944 from postmortem human cultures of blood and secretions, was sequenced to reveal two circular chromosomes comprising a total of c. 5.8 Mb and a G/C content of c. 69% (Nierman et al., 2004). A total of 5535 predicted protein-encoding ORFs have been identified in the genome. Genomic comparisons with the closely related species B. pseudomallei and B. thailandensis reveal significant homology, being c. 99% identical between conserved genes in B. pseudomallei, although B. mallei contains roughly 1.41 Mb less DNA than B. pseudomallei (Godoy et al., 2003; Nierman et al., 2004). Comparative analysis of B. mallei with virulence-associated genes found in pathogenic bacteria has revealed genes encoding an exopolysaccharide capsule, a lipopolysaccharide, type IV pili, as well as type III (plant- and animal-like) and type VI secretion systems (Fig. 1). Interestingly, most of the secretion systems and virulence-associated genes described so far are located in chromosome 2, while genes involved in metabolism, capsule production and lipopolysaccharide biosynthesis are found in chromosome 1.

1

Circular diagrams of chromosome 1 and chromosome 2 in Burkholderia mallei. Locations of selected genes encoding known virulence factors (black letters) and potential virulence-associated elements (gray letters) as displayed in PATHEMA (http://pathema.tigr.org/tigr-scripts/Burkholderia/PathemaHomePage.cgi). The size of the two chromosomes and the locus tag number for each one of the genes are also depicted.

Initial reports suggested that B. mallei lacked a capsule (Jennigs, 1963; Kovalev, 1971). However, subsequent studies have demonstrated that a polysaccharide capsule is present in B. mallei, and this extracellular structure is a virulence determinant (Popov et al., 1995, 2000; Fritz et al., 1999, 2000; DeShazer et al., 2001). Using subtractive hybridization, DeShazer et al. identified a polysaccharide gene cluster in B. mallei ATCC 23344 antigenically similar to B. pseudomallei. Further molecular characterization indicated wcbF as an essential gene for the production of capsular polysaccharide. Additionally, demonstration of a surface capsule on B. mallei ATCC 23344 by immunogold electron microscopy has been noted (Fritz et al., 2000; DeShazer et al., 2001). Challenge with the wild type and the capsule negative mutant showed a >105-fold difference in virulence in Syrian hamsters and a >103-fold difference in BALB/c mice when infected by the intraperitoneal and aerosol route respectively, demonstrating that the capsular polysaccharide is a key virulence factor (DeShazer et al., 2001). Similar studies with B. pseudomallei have identified genes responsible for the production of capsular polysaccharides demonstrating comparable virulence association (Reckseidler et al., 2001).

Another surface structure in B. mallei is the lipopolysaccharide, which is predicted to possess a lipid component (Lipid A), a core region and cell wall antigens (O antigens). Analysis of lipopolysaccharide profiles of B. mallei strains has helped aid the identification of the gene cluster responsible for O polysaccharide biosynthesis in B. mallei ATCC 23344 (Burtnick et al., 2002). Sequence analysis revealed a 19.9-kb contiguous sequence containing the entire B. mallei O polysaccharide biosynthetic gene cluster. Sequence alignment of the B. pseudomallei and B. mallei O polysaccharide biosynthetic regions revealed 99% identity at the nucleotide level, with nomenclature consistent with that of B. pseudomallei. Resistance to the bactericidal effects of normal human serum (NHS) has been demonstrated for both B. pseudomallei and B. mallei ATCC 23344, while those strains lacking intact O polysaccharide moieties (NCTC 120 and ATCC 15310) were completely killed following incubation with 30% NHS, implicating lipopolysaccharide as a potential virulence determinant (DeShazer et al., 1998; Burtnick et al., 2002). Furthermore, B. mallei lipopolysaccharide has been shown to activate hTLR4 complexes on HEK 293-hTLR4/MD2-CD14-expressing cells as well as stimulate cytokine and chemokine responses by antigen-presenting cells (Brett et al., 2007).

It is well known that the interaction of bacteria with host cell membranes is a prerequisite step for the infectious process to occur. The adherent capability of Burkholderia species has received little attention, with the exception of B. pseudomallei, B. thailandensis and B. cepacia (Chiu et al., 2001; Brown et al., 2002; Kespichayawattana et al., 2004). Investigations of temperature regulation on adherence to eukaryotic cells in vitro using the clinical isolate B. pseudomallei 08 revealed increased adherence when grown at 30 vs. 37 °C (Brown et al., 2002). Additionally, adherence demonstrated a statistically significant increase when experiments were conducted with stationary-phase cultures as opposed to those grown to the log phase (Brown et al., 2002). Conversely, temperature failed to modulate the direct adherence of B. pseudomallei K96243 to eukaryotic cells (Boddey et al., 2006). Burkholderia pseudomallei K96243 contains a type IV pilin protein, PilA, which contributes to adherence to cultured respiratory cell lines, implicating this pili as a potential mediator in the pathogenic process (Essex-Lopresti et al., 2005). The ability to colonize the eukaryotic monolayer may involve multiple cellular interactions, depending on the strain: bacterium–cell interactions (adherence) and bacterium–bacterium interactions (microcolonies). To the authors’ knowledge, no adherence or microcolony studies exist in the literature for B. mallei, although a type IV pilin protein has been identified, showing no protective characteristics following active or passive immunization (Fernandes et al., 2007). The lack of protection offered by type IV pili immunization may, in part, be due to a lack of pili production during infection. Although antipilin antibody titers were detected in immunized animals, it is possible that these antibodies were the result of preformed pili in the inoculating dose.

An additional feature common to B. mallei, B. pseudomallei and B. cepacia is the localization of infection to the lungs and respiratory airways. Aerogenic exposure of B. mallei to BALB/c mice includes localization of the pathogen in the upper and lower sections of the respiratory tract and transportation of bacteria within alveolar macrophages to regional lymph nodes, suggestive of intracellular survival (Lever et al., 2003). Additionally, certain species of Burkholderia are capable of intracellular invasion in both phagocytic and nonphagocytic cell lines, such as RAW 264.7, J774.2, J774.1, A549, HeLa and Vero cells (Jones et al., 1996; Harley et al., 1998; Pilatz et al., 2006; Ribot & Ulrich, 2006). Investigations into the intracellular characteristics of B. mallei have predominantly focused on the type III secretion system (TTSS) as a possible mechanism responsible for the invasion, escape from intracellular compartments and survival in host cells. Burkholderia mallei carries two chromosomal regions encoding for TTSSs: one with similarity to animal-like TTSS and a second TTSS similar to that found in plant pathogens (Fig. 1). The B. mallei ATCC 23344 animal-like TTSS encodes 30 ORFs, and it is genetically similar to that of B. pseudomallei (Hueck, 1998). These B. mallei TTSS genes are clustered in a distinct region of chromosome 2, sharing homology with both Salmonella inv/spa/prg and Shigella ipa/mxi/spa TTSSs (Table 1). Correlations of the B. mallei TTSS and pathogenicity have been established. A functional animal-like TTSS is required for full pathogenicity of B. mallei ATCC 23344 in the BALB/c mouse and Syrian hamster models of infection (Ulrich & DeShazer, 2004). Mutations in the B. mallei bsaQ and bsaZ genes (homologues of Salmonella invA and spaS genes) demonstrated decreased virulence as evident by survival, clinical signs and bacterial burden of the lungs, liver and spleen (Ulrich & DeShazer, 2004). Although B. mallei TTSS mutants display attenuated virulence, the protection offered by these mutants has been shown to be suboptimal, suggesting that vaccine candidates may exist within the TTSS, e.g. the TTSS effector molecules or the TTSS needle cap protein (G.C. Whitlock & A.G. Torres, unpublished data). The requirement of a competent TTSS for intracellular survival of B. mallei within phagocytic cell lines has been documented; however, the experimental approach adopted was limited to the contribution of a functional TTSS apparatus by disruption of protein delivery (Ribot & Ulrich, 2006). Therefore, identification of putative effector molecules is essential to decipher the molecular mechanisms associated with the B. mallei TTSS. In silico analysis reveals a second gene cluster encoding a plant-like TTSS in B. mallei, which displays 99% sequence identity to B. pseudomallei, and in a lesser degree to plant TTSS analogs (Rainbow et al., 2002). Identification of a plant host for B. mallei has not been determined, although the presence of an apparently intact plant-like TTSS within the B. mallei genome (Table 2) suggests that a possible contribution of this secretion system to bacterial virulence may exist.

View this table:
1

Burkholderia mallei Type III secretion (animal-like) system

Protein or gene nameSimilarity to Burkholderia speciesSimilarity to other speciesPredicted function and characteristics
BicPBicP (B. pseudomallei)Tir (Shewanella baltica)Type III secretion chaperone
BopABopA (B. pseudomallei)IcsB (S. dysenteriae)Putative type III secreted intercellular spread protein
BMAA1522BURPS1106A_A2066 (B. pseudomallei)Unknown
BopEBopE (B. pseudomallei)SopE (S. enterica)Type III secreted G-nucleotide exchange factor
BapCBapC (B. pseudomallei)Putative transglycosylase (Ralstonia eutropha)Putative exported and invasion protein
BapBBapB protein (Burkholderia thailandensis)ACP (Thermotoga maritima)Acyl carrier protein of unknown function
BapAISS (Ostreococcus tauri)Putative protein disulfide-isomerase
BprDBipD (B. pseudomallei)IpaD (S. dysenteriae)Type III secretion needle tip protein (IpaD/SipD/SspD family)
BprABprA (B. pseudomallei) and HNS (B. vietnamiensis)Putative HNS-like regulatory protein
BipCBipC (B. pseudomallei)IpaC (S. flexneri)Type III secretion effector (IpaC/SipC family)
BipBBipB protein (B. pseudomallei)SipB (S. enterica)Type III secreted cell invasion protein
BicABicA (B. pseudomallei)SicA (Salmonella enterica)Type III secretion low calcium response chaperone (LcrH/SycD family)
BsaZBsaZ (B. pseudomallei)SpaS (S. typhimurium)Type III secretion apparatus protein (YscU/HrpY family)
BsaYBsaY (B. pseudomallei)YsaT (Y. enterocolitica)Type III secretion apparatus protein (SpaR/YscT/HrcT family)
BsaXBsaX (B. pseudomallei)YsaS (Y. enterocolitica)Type III secretion apparatus protein
BsaWBsaW (B. pseudomallei)YsaR (Y. enterocolitica)Type III secretion apparatus protein (YscR/HrcR family)
BsaVBsaV (B. pseudomallei)SpaO (S. enterica)Type III secretion system apparatus protein (YscQ/HrcQ family)
BsaUBsaU (B. pseudomallei)Putative surface presentation of antigens protein
BsaTBsaT (B. pseudomallei)Putative surface presentation of antigens protein
BsaSBsaS (B. pseudomallei)SpaL (S. enterica)Type III secretion system ATPase
BsaRBsaR (B. thailandensis)SpaK (S. enterica)Type III secretion system protein
BsaQBsaQ (B. thailandensis)YsaV (Y. enterocolitica)Type III secretion system apparatus protein
BsaPBsaP (B. pseudomallei)MxiC (S. flexneri)Type III secretion regulator (YopN/LcrE/InvE/MxiC family)
BsaOBsaO (B. pseudomallei)InvG (S. enterica)Type III secretion outer membrane pore (YscC/HrcC family)
BsaNBsaN (B. pseudomallei)InvF (S. enterica)Type III secretion system transcriptional regulator (AraC family)
BMAA1546BURPS1106A_A2090 (B. pseudomallei)Conserved hypothetical protein of unknown function
BsaMBsaM (B. pseudomallei)PrgH (S. enterica)Type III secretion system needle protein (PrgH/EprH family)
BsaLBsaM (B. pseudomallei)EprI (E. coli O157:H7)Type III secretion system needle protein
BsaKBsaM (B. pseudomallei)PrgJ (S. typhimurium)Needle complex minor subunit
BsaJBsaM (B. pseudomallei)EprK (E. coli O157:H7)Type III secretion apparatus lipoprotein (YscJ/HrcJ family)
OrgAOrgA (B. pseudomallei)OrgA (S. typhimurium)Oxygen-regulated type III secretion system protein (OrgA/MxiK family)
BMAA1552Bpse17_02004987 (B. pseudomallei)OrgB (S. typhimurium)Oxygen-regulated type III secretion apparatus protein (HrpE/YscL family)
View this table:
2

Burkholderia mallei Type III secretion (plant-like) system

Protein or gene nameSimilarity to Burkholderia speciesSimilarity to other speciesPredicted function and characteristics
BMAA1617BPSS1607 (B. pseudomallei)HrpB1 (Xanthomonas oryzae pv. oryzicola)Type III secretion system protein
BMAA1618Bpse17_02005433 (B. pseudomallei)RimL (Anabaena variabilis)Putative acetyltransferase
BMAA1619BURPS305_2288 (B. pseudomallei)Hypothetical protein of unknown function
BMAA1620BURPS1106A_A2187 (B. pseudomallei)Hypothetical protein of unknown function
HrpBHrpB (B. pseudomallei)HrpB (Ralstonia solanacearum)Type III secretion transcriptional regulator (AraC/XylS family)
BMAA1622BPSS1611 (B. pseudomallei) and HpaB (B. cenocepacia)Type III secretion chaperone
BMAA1623BURPS1710b_A0670 (B. pseudomallei)Hypothetical protein of unknown function
BMAA1624Bpse110_02005542 (B. pseudomallei) and BTH_II0759 (B. thailandensis)Putative type III secretion protein
BMAA1625PseudogeneAuthentic frameshift of a type III secretion inner membrane protein
BMAA1626BpseP_03005479 (B. pseudomallei) and BTH_II0756 (B. thailandensis)Putative type III secretion protein
SctS (EscS)Hrp (B. pseudomallei)HrcS (Xanthomonas oryzae)Type III secretion inner membrane protein (HrpO family)
SctR (EscR)SctR (B. pseudomallei) and YscR (B. cepacia)Type III secretion apparatus protein (YscR/HrcR family)
SctQSctQ (B. pseudomallei) and SPOA (B. thailandensis)Type III secretion inner membrane protein (YscQ/HrcQ family)
SctV (EscV)HrcV (B. pseudomallei) and EscV (B. dolosa)Type III secretion inner membrane protein (HrcV family)
BMAA1631BpseP_03005485 (B. pseudomallei)HrcU protein (Xanthomonas oryzae)Type III secretion inner membrane protein (YscU/HrpY family)
BMAA1632BPSS1622 (B. pseudomallei)HrpB1 (Xanthomonas oryzae)Type III secretion protein (HrpB1/HrpK family)
BMAA1633HrpB2 (B. pseudomallei)HrpB2 (Xanthomonas oryzae)Type III secretion protein
BMAA1634PseudogeneAuthentic frameshift of a type III secretion inner membrane protein
BMAA1635HrpB4 (B. pseudomallei)HrpB4 (Xanthomonas campestris)Type III secretion protein
SctLSctL (B. pseudomallei)HrpF (Ralstonia solanacearum)Type III secretion inner membrane protein (HrpE/YscL family)
SctNSctN (B. pseudomallei)HrpB6 (Xanthomonas oryzae)Type III secretion system ATPase
BMAA_BmtmRNA3sRNA
SctTSctT (B. pseudomallei)HrcT (Acidovorax avenae)Type III secretion inner membrane protein (SpaR/YscT/HrcT family)

A genomic search of B. mallei ORFs assigned to conserved components of the highly regulated type VI secretion systems (T6SSs) revealed four putative T6SSs located on chromosome 2 and one truncated on chromosome 1 (BMA2826–2833) (Schell et al., 2007). Three of these T6SS gene clusters (clusters 1–3) possess >80% sequence conservation with B. pseudomallei and B. thailandensis, while cluster 4 (BMAA1897–1915) is exclusive to pathogenic B. mallei and B. pseudomallei. Cluster 1 (BMAA0729–0744) appears to be transcriptionally regulated by the VirAG two-component system and AraC-type regulator BMAA1517. A hemolysin-coregulated protein (Hcp) family member encoded in cluster 1 is secreted in vitro by this T6SS and displays immunogenic characteristics in mice, horses and humans (Schell et al., 2007). In addition, overexpression of VirAG or BMAA1517 results in enhanced secretion of the T6SS encoded protein Hcp1. Syrian hamsters injected intraperitoneal with B. mallei T6SS mutants demonstrated 100% survival compared with B. mallei ATCC 23344 wild-type challenged animals up to 14 days (Schell et al., 2007). Further, immunoglobulin recognition of Hcp1 in multiple infected hosts demonstrates the ability of Hcp1 expression in vivo, lending to its potential use as a candidate vaccine.

Host immune responses to B. mallei infections

Currently, there is no available animal or human vaccine against B. mallei infection. Development of an effective vaccine will require an animal model capable of predicting immune components necessary to induce immunity while exhibiting pathogenesis seen in human disease. Among potential rodent model species for vaccine development, guinea-pigs and hamsters are the most susceptible to B. mallei infection. Mice are relatively intermediate in susceptibility on par with humans and are thus an ideal model for development of vaccine targets and immune functional studies. Mouse strains vary in the relative levels of susceptibility, with BALB/c mice ranking among the more susceptible strains and C57BL6 as the more resistant by both intraperitoneal and aerosol methods of infection (Fritz et al., 2000; Lever et al., 2003). Immune correlates of protection are not fully developed for B. mallei although its capacity to replicate inside the host cell suggests that type 1-mediated cellular immune responses will be linked to a protective response. Recent vaccine studies utilizing various nonviable B. mallei strains (encapsulated or not) have demonstrated that a mixed type 1/type 2 response is not protective. These studies evaluated both antigen-specific isotype (IgG1-type 2 vs. IgG2a-type 1) and cytokine responses to recall stimulation as a readout system (Amemiya et al., 2002). Following intraperitoneal inoculation of B. mallei ATCC 23344, BALB/c mice demonstrate a strong IFN-γ response, accompanied by IL-18, IL-12, IL-27 and IL-6 expression within 24 h after infection (Rowland et al., 2006). Stimulation of Toll-like receptor 9 (TLR9) with a CpG-containing oligodeoxynucleotide exhibited elevated IL-12 and IFN-γ levels following B. mallei aerosol challenge (Waag et al., 2006). Additionally, BALB/c mice pretreated intraperitoneal with CpG motifs showed increased survival and lower levels of B. mallei in the lungs and spleens (Waag et al., 2006). Aerogenic exposure to B. mallei auxotrophs shows enhanced production of whole bacillus-specific IgG2a over IgG1, demonstrating the potential in protection against aerosol challenge, in addition to studies that have used heat-killed or irradiated bacilli and the type 1 inductive cytokine IL-12 (Ulrich et al., 2005; Amemiya et al., 2006). IL-12, in combination with nonviable B. mallei, has been shown to be superior in inducing protection relative to killed bacteria alone. Much of the enhancement is thought to occur via increased production of IFN-γ and activation of macrophages although the cellular source of IFN-γ [conventional αβ T cells, natural killer (NK) cells, NK T cells or γδ T cells] is not well defined for B. mallei. In naive mice, IFN-γ production has been shown to be essential for protection from B. pseudomallei infection in the first 24 h (Santanirand et al., 1999; Haque et al., 2006). The studies of B. pseudomallei and T cell-mediated immunity suggest that IFN-γ-expressing cells should be the targets for an effective vaccination strategy. Burkholderia mallei heat-killed vaccinated BALB/c mice, depleted of TNF-α or IFN-γ, show 100% mortality following intraperitoneal challenge with B. mallei ATCC 23344 (G.C. Whitlock, R.A. Lukaszewski, B.M. Judy, S. Paessler, A.G. Torres & D.M. Estes, unpublished data). The effector role for IFN-γ in mediating protection against B. mallei may include both immunoregulatory and nonregulatory functions. Regardless, the requirement of IFN-γ, as demonstrated by administration of neutralizing antibody before infection, indicates that stimulation of IFN-γ response is a desirable goal for a B. mallei vaccine. Moreover, BALB/c mice with an established B. mallei chronic infection rapidly lose the ability to control the growth of the bacillus upon neutralization of TNF-α (G.C. Whitlock, R.A. Lukaszewski, B.M. Judy, S. Paessler, A.G. Torres & D.M. Estes, unpublished data), demonstrating a role for sustained TNF-α production in the maintenance of host survival throughout the course of B. mallei infection. Following depletion of B220+ cells in heat-killed vaccinated BALB/c mice, survival rates decrease as much as 100% relative to nondepleted controls when challenged intraperitoneal. Additionally, relatively resistant C57BL/6 mice deficient in mature B-cells (µMT) succumb to similar mortality rates (G.C. Whitlock, R.A. Lukaszewski, B.M. Judy, S. Paessler, A.G. Torres & D.M. Estes, unpublished data). Both antibody responses (IgG2a/IgG1 ratios) and passive immunotherapy with monoclonal antibodies to B. mallei lipopolysaccharide have demonstrated a role for antibody in protection to infection (Trevino et al., 2006). BALB/c mice injected intraperitoneal with anti-B. mallei monoclonal antibodies 18 h before whole-body aerosol challenge with 20 lethal doses 50% (LD50; 1.9 × 104 CFU) of B. mallei ATCC 23344 demonstrated 100% survival (Trevino et al., 2006). In contrast, administration of anti-B. mallei monoclonal antibodies 18 h postchallenge was ineffective in providing protection (Trevino et al., 2006). Although protection was achieved in the above study, experiments were limited to 14 days postchallenge. The bacterial burden of harvested spleens demonstrated significant numbers of B. mallei indicating a lack of sterilizing immunity, suggestive of an antibody-induced reduction of infecting organisms, perhaps below the lethal threshold. Clearance and/or limitation of available pathogen in the initial phase of infection would presumably limit the number of internalized organisms by host cells, thus circumventing the immune-evasive characteristics of an intracellular pathogen. In addition to humoral immunity, a specific cell-mediated immunity will most likely prove advantageous in the development of an efficacious vaccine regimen.

Future challenges: the B. mallei vaccine

Burkholderia infections are difficult to treat with antibiotics and as indicated before, no vaccine exists. Aerogenic vaccination with a B. mallei auxotroph promoted a Th1-like Ig response, suggesting that live-attenuated strains might serve as promising vaccine candidates (Ulrich et al., 2005). Further, B. mallei is susceptible to cell-mediated immune responses, promoting expression of type 1 cytokines, which suggests that the development of an effective vaccine should target the production of IFN-γ (Rowland et al., 2006). The use of these and other approaches relies on a reasonable understanding of the protective innate and adaptive host response, which, at present, is relatively unresolved for B. mallei and also in part for B. pseudomallei.

Acknowledgements

The authors thank Scott T. Moen for critically reading the manuscript. Burkholderia research performed in the authors’ labs is supported in part by the UTMB John Sealy Memorial Endowment Fund for Biomedical Research (to A.G.T.), by NIH grant U54 AI057156 (to D.M.E.), and by NIH/NIAID, Task Order 19 (Part-C) Contract No: N01-AI-30065. G.C.W. was funded by a fellowship from the UTMB Sealy Center for Vaccine Development.

Footnotes

  • Editor: Ian Henderson

References

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