Secondary Logo

Journal Logo


Innate immune response to Burkholderia mallei

Saikh, Kamal U.; Mott, Tiffany M.

Author Information
Current Opinion in Infectious Diseases: June 2017 - Volume 30 - Issue 3 - p 297-302
doi: 10.1097/QCO.0000000000000362
  • Open



Burkholderia mallei are the etiological agent of a highly contagious, acute, or chronic, usually fatal disease of solipeds, known as glanders. This obligate mammalian, facultative intracellular pathogen is a Gram-negative, nonmotile, nonspore-forming bacillus which is widely regarded as a host-adapted deletion clone of Burkholderia pseudomallei, an environmental saprophytic pathogen that causes the disease melioidosis. Although horses, donkeys, and mules constitute the only known natural reservoirs for B. mallei, humans and other mammalian hosts [e.g., camels, nonhuman primates (NHPs), goats, dogs, cats, rabbits, hamsters, guinea pigs, and mice] are susceptible to infection and display similar disease progression and disease [1–7]. Glanders transmits amongst animals via respiratory secretions and exudates from skin lesions. In human infections, the primary modes of B. mallei transmission are via direct contact with damaged skin, invasion of mucous membranes, and deposition into the lung. Depending on the route of exposure, the disease course of glanders infection can range from acute to chronic and manifest in multiple forms, such as localized, pulmonary, disseminated, and septicemic. The clinical and pathological presentation of B. mallei infections bare a striking resemblance to B. pseudomallei infections, including their ability to remain quiescent and persist in the host following apparent clinical resolution [8]. Owing to the reasons above, in addition to their highly infectious nature as an aerosol, both pathogens are classified as Tier 1 select agents by the federal select agent program. Currently, no licensed vaccines are available for either disease, and medical therapeutic options are limited.

Both B. pseudomallei and B. mallei thrive intracellularly via modulation of host immune responses, which attributes to their resilience against current medical countermeasures. Despite the characterization of many B. pseudomallei virulence factors, its strategies for circumventing intracellular host defenses remain ill defined. Comparatively, even less is known for B. mallei. Limited understanding of these survival tactics poses a major challenge in the development of effective therapeutics. Thus, delineating the specific molecular mechanisms utilized by these pathogens to dysregulate host immune responses is paramount. The majority of research and review articles are focused on host immune responses to B. pseudomallei. This review will concentrate on recent advances in characterizing B. mallei-specific host immune responses, specifically innate immune responses. 

Box 1
Box 1:
no caption available


Although mechanisms can vary among Burkholderia spp., adhesion and invasion of host epithelial cells are vital steps during infection and appear to contribute to the overall virulence [9▪]. For successful infection of host cells, B. mallei depend on the strategic utilization of a multitude of virulence factors and mechanisms to manipulate many host processes and pathways. Recently, a combined computational and experimental approach was utilized to systemically assess nine B. mallei virulence factors and their interactions with host proteins to elucidate mechanisms of B. mallei pathogenicity [10▪▪]. Topological analyses of B. mallei–host protein–protein interactions suggest that B. mallei targets multifunctional intracellular host proteins, host proteins that interact with each other, and proteins with a large number of interacting partners. Host processes broadly influenced by these protein–protein interactions include the ubiquitination degradation system and focal adhesion pathways [10▪▪]. These results are consistent with the previous work that reported TssN protein interactions with the polyubiquitin-B protein and with the cullin-1a protein. These host proteins interact with tumor necrosis factor (TNF) receptor-associated factor 6 and IκB inhibitor-α, components central to Toll-like receptor (TLR) signaling [11]. These studies provide some insights into B. mallei pathogenesis, and on the proposed hypothesis that B. mallei modulate innate immune responses by interfering with host ubiquitination directly or in combination with other pathogen proteins.

A comprehensive assessment of murine macrophages infected with a diverse panel of Burkholderia spp. resulted in the uniform production of cytokines interleukin 1 β(IL-1β), tumor necrosis factorα (TNFα), and murine keratinocyte-derived protein chemokine, a murine homolog of human IL-8 [12]. Compared with B. pseudomallei-infected macrophages, B. mallei-infected macrophages secreted significantly higher levels of IL-6 and IL-10, which suggest these two pathogens differentially modulated host signaling cascades. Additionally, macrophages expressed lL-1β, IL-10, TNF receptor superfamily member 1B, and IL-36α mRNA, at significantly higher levels when infected with B. mallei compare with the other Burkholderia spp. [12], suggesting the existence of gene-based differences in the host inflammatory response that is unique to B. mallei.

Infected macrophages further assessed for changes in their host-signaling dynamics showed increased phosphorylation of adenosine monophosphate-activated protein kinase; regulators of nuclear factor-kappa B signaling pathway (e.g., IκB inhibitor-α, Glycogen synthase kinase (GSK)3β, Src, and STAT1) and mitogen-activated protein kinases (e.g., p38, Extracellular-signal regulated kinase 1/2, and c-Myc) [13▪]. The degrees in which target host proteins or processes are modulated correlated to the differences in pathogenicity observed amongst Burkholderia species. In infected macrophages, B. mallei were a stronger inducer of Inducible nitric oxide synthase expression and interferon-gamma (IFNβ) production compared with B. pseudomallei. Based on these data, in addition to current knowledge of signaling transduction, a representitive network of signaling pathways and axes was constructed to illustrate the activation of signaling cascades in response to Burkholderia spp infection [13▪]. Based on canonical pathways downstream of TLR4, induction of phosphorylated forms of adenosine monophosphate-activated protein kinase-α1, GSK3β, and Src play key roles in regulating the inflammatory response of Burkholderia spp. infections.

Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria, and a potent stimulator of host innate immune responses. Structure–activity relationship studies of TLR4 agonist suggest the biological activity of LPS correlates with the composition of its lipid A moiety [14]. Evaluation of B. mallei LPS showed the acylation of lipid A had a greater effect on its biological activity than their length [15▪▪]. Thus, overall differential macrophage activation may be related to B. mallei LPS, which is similar to the B. pseudomallei LPS and bares a penta-acylated lipid A with 4-amino-4-deoxyarabinose in almost half of its molecules, and appears to be a weaker macrophage activator as compared with enterobacterial LPS. Consistent with this, a significant reduction in mRNA expression or secretion of IL-6, TNFα, and IL-1β is exhibited when stimulated with purified B. mallei LPS compared with E. coli LPS-treated macrophages. Compared with E. coli-infected macrophages, B. mallei-infected macrophages also produce reduced levels of both IFN-dependent genes and mediators (IFNβ and Nitric oxide) and cytokines [TNFα, IL-6, IL-10, Granalocyte-macrophage colony stimulating factor (GM-CSF), and regulated on activation normal T cell expressed and secreted (RANTES)].

B. mallei must overcome a gamut of antibacterial mechanisms and products (e.g., Adenosine monophosphates and reactive oxygen and nitrogen species) critical to innate immunity to establish persistent infection. B. mallei Frederick Memorial Hospital (FMH) isolates collected from mice spleens 60 days postinfection showed attenuated abilities to replicate and induce cytotoxicity in macrophage assays [16]. One B. mallei isolate displayed a change in its LPS phenotype, from smooth to rough, resulting from the loss of its O-polysaccharide (OPS) during the course infection [16]. These phenotypic changes were conceived to stem from the infection shifting from an acute to a chronic or subclinical form, which is less prone to stimulate host immune responses. Earlier studies highlighted that genetic and phenotypic characteristics potentially associated with persistence of both B. pseudomallei and B. mallei[17,18]. Further studies, including sequencing the OPS biosynthetic gene cluster of this B. mallei FMH strain may provide insight into the genetic basis for the loss of OPS. Intriguingly, OPS modification and loss is a hallmark of chronic Pseudomonas aeruginosa infection [19].


Highlighting the susceptibility of B. mallei to cell-mediated immune responses, previous studies compared the survival rates of infected BALB/c and IFNγ knockout mice. BALB/c mice survived more than 37 days longer than IFNγ knockout mice and showed significantly lower levels of bacterial colonization, which illustrates the importance of IFNγ-mediated immunity for control of infection [20]. Macrophages and human pulmonary alveolar type II cells contribute to innate immunity by secreting inflammatory cytokines during B. mallei infection [21]. When exposed to heat-killed B. mallei, primary Peripheral blood Mononuclear Cells (PBMCs) from NHPs and humans elicit the strong production of IFNγ, TNFα, IL-6, and IL-1β [22▪▪]. Cytokine responses varied among the NHPs, in which the African green monkey appears to be most responsive, compared with Rhesus or Cynomologus species, suggesting the inflammatory responses vary within mammalian species [22▪▪]. Similar results were observed with aerosol exposure of B. mallei FMH 23344 strain to NHPs conducted at USAMRIID, where most of the African green monkeys died but all Rhesus or Cynomologous species survived (Personal communication). The immune-signaling mechanism for the strong cellular response demonstrated that myeloid differentiation primary response protein 88 (MyD88)-mediated signaling contributes to proinflammatory cytokine responses [22▪▪]. These results were consistent with earlier reports which showed that MyD88−/− mice were highly susceptible to pulmonary challenges with B. mallei and had significantly short survival time, increased bacterial burdens, and severe organ pathology compared with wild-type mice [23]. Recruitment of inflammatory monocytes and Dendritic Cells to the lungs and local production of IL-12, followed by Natural killer cell cell production of IFNγ, are the key cellular responses required for early protection from B. mallei infection.


B. pseudomallei demonstrate an ability to escape autophagosomes in host phagocyte in vitro as well as in murine models and human cases of melioidosis, thus avoiding immune responses [24]. The recurring illness of melioidosis patients in endemic areas can potentially be because of relapse or reinfection. Bacteria can become quiescent and subclinical to avoid host immune mechanisms of clearance. An earlier report indicated that nonfunctional mutations in BPSS0180, a type VI cluster-associated gene capable of inducing autophagy in both phagocytic and nonphagocytic mammalian cells, resulted in significant colocalization of B. pseudomallei with autophagy marker light chain3 and impaired intracellular survival [25]. A recent report suggests that B. pseudomallei evade autophagy [26]. Consistent with these earlier reports, recent results from our laboratory also suggest that lack of autophagy correlate with intracellular persistence of bacteria with aerosol exposure not only of B. pseudomallei but also B. mallei in spleens of BALB/c and C57/BL6 mice with chronic infection (Alam et al. 2016; manuscript submitted). Memisevic et al.[10▪▪] suggests that multiple B. mallei virulence factors such as BMAA1865, BMAA0728 (TssN), and BMAA0553 influence critical host processes related to modulation of host ubiquitination, phagosome escape, interference with host cycloskeleton rearrangement, and focal adhesion and a means to modulate and adapt the host cell environment to advance infection. Further studies may shed light on whether any of these B. mallei proteins are directly or indirectly linked in the evasion of host autophagy processes.


Antibiotic resistance associated with Burkholderia infection is on the rise [27]. Even with optimal antibiotic treatment, the mortality from acute severe melioidosis is high (30–50% in Thailand, 19% Australia) and mortality rates can be as high as 40% for cases of glanders [28–30]. Recently, Waag [31] reported that mice experimentally exposed to B. mallei suggest that although antibiotics can be efficacious after prolonged interval between exposure and treatment, but only if the animals were previously vaccinated. Thus, it is likely that both vaccination against B. mallei and postexposure therapeutic approaches would be required for complete protection against B. mallei exposure.


Primary cellular responses by analyses of IL-1β and other inflammatory cytokine responses by comparison with E. coli LPS, African green monkeys appears to be most responsive to B. mallei or B pseudomallei than Cynomolgus or Rhesus [22▪▪]. Characterization of the immune-signaling mechanism for cellular inflammatory response revealed that MyD88-mediated signaling contributed to the B. mallei and B. pseudomallei induced proinflammatory responses. Notably, B. mallei, B. pseudomallei, or purified LPS from these pathogens induced MyD88-mediated reporter activity was inhibited and inflammatory cytokine production was attenuated by a MyD88 inhibitor [22▪▪]. In the scenario of dysregulating inflammatory responses with established B. mallei infection that often leads to septicemia and immune pathogenesis, thus MyD88-targeted therapeutic intervention may be a potential strategy for therapy.


For complete protection against Burkholderia pathogens, previous vaccine efforts focused on inducing both cellular and humoral immune responses [32]. Possible candidates include whole-cell killed, subunit glycoconjugate, and live attenuated vaccines, as recently reviewed by Aschenbroich et al.[33]. These vaccines showed limited efficacy that resulted in partial protection and bacterial dissemination in murine models of infection. Live attenuated recombinant Salmonella expressing B. mallei LPS O antigen showed protection in a murine infection model of B. thilandensis, a surrogate for biothreat Burkholderia spp., and suggest a promising platform for vaccine development [34].

Recently, two live attenuated B. mallei strains consisting of mutations in ubiquitination and phagosomal escape (ΔtssN) or iron transport (ΔtonB) show protection against lethal challenges in models of murine glanders [35▪,36▪▪]. Analysis of the immune responses observed in vaccination-challenge studies was performed to understand how these mutants modulate immune responses. BALB/c mice surviving exposure to aerosolized ΔtssN showed elevated expression of proinflammatory cytokines and chemokines: IL-1α, IL-1β, IL-2, IL-4, IL-10, IL-12, MIG, macrophage inhibitory protein-1α, and TNFα, and Vascular endothelial groth factor [35▪]. This modulation of host responses showed ΔtssN capable of inducing prolonged innate immunity despite its high degree of attenuation. Mice immunization with ΔtssN demonstrated 67% survival rates at 21 days postwild-type challenge [35▪]. Authors suggested the partial protection afforded by ΔtssN immunization was mainly driven by innate immunity as BALB/c mice failed to show increased expression of proinflammatory cytokines and chemokines after ΔtssN prime and boost regimens.

BALB/c mice immunized with ΔtonB provided up to 100% survival at 21 days postwild-type challenge [36▪▪]. Compared with controls, immunized mice expressed moderated inflammatory cytokine/chemokine profiles with significant reductions reported in IL-6, GM-CSF, monocyte chemoattractant protein-1, and RANTES [36▪▪]. Authors correlated these results with reduced immune-mediated tissue damage observed in immunized mice. In cross-protection studies, ΔtonB-immunized mice challenged with B. pseudomallei K96243 demonstrated 75% survival 36 days postinfection [36▪▪]. Although these studies displayed protection and resulted in wild-type clearance, ΔtonB immunization was noted to result in persistence infection of the live attenuated mutant in the spleens of surviving mice. Despite persistence, the B. mallei tonB mutant shows potential as a candidate for further vaccine development and optimization.


B. mallei target intracellular host immune-signaling pathways for intracellular survival. Recent studies provide some understanding of pathogen–host protein interactions, dysregulation of macrophage activation, and immune evasion by B. mallei. Still, considerable gaps exist regarding the understanding of specific B. mallei protein(s) and signaling pathways that likely contribute to intracellular survival and evasion of host immune effector mechanisms. More focused research in delineating the molecular basis for host inability or dysregulation of the host immune effector mechanism manipulated by this pathogen is needed. This may limit persistent infection, and likely provide direction toward developing medical countermeasures.



Financial support and sponsorship



Army: Opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the U.S. Army.

Conflicts of interest

There are no conflicts of interest.


Papers of particular interest, published within the annual period of review, have been highlighted as:

▪ of special interest

▪▪ of outstanding interest


1. Loeffler F. The etiology of glanders [in German]. Arb Kaiserl Gesundh 1886; 1:141–198.
2. Kovalev GK. Glanders [review]. Zh Mikrobiol Epidemiol Immunobiol 1971; 48:63–70.
3. Wilkinson L. Glanders: medicine and veterinary medicine in common pursuit of a contagious disease. Med Hist 1981; 25:363–384.
4. Miller WR, Pannell L, Cravitz L, et al. Studies on certain biological characteristics of malleomyces mallei and malleomyces pseudomallei: II. Virulence and infectivity for animals. J Bacteriol 1948; 55:127–135.
5. Fritz DL, Vogel P, Brown DR, et al. Mouse model of sublethal and lethal intraperitoneal glanders (Burkholderia mallei). Vet Pathol 2000; 37:626–636.
6. Fritz DL, Vogel P, Brown DR, Waag DM. The hamster model of intraperitoneal Burkholderia mallei (glanders). Vet Pathol 1999; 36:276–291.
7. Neubauer H, Meyer H, Finke E. Human glanders. Int Rev Armed Forces Med Serv 1997; 70:258–265.
8. Dvorak GD, Spickler AR. Glanders. J Am Vet Med Assoc 2008; 233:570–577.
9▪. David J, Bell RE, Clark GC. Mechanisms of disease: host-pathogen interactions between Burkholderia species and lung epithelial cells. Front Cell Infect Microbiol 2015; 5:80.
10▪▪. Memisevic V, Zavaljevski N, Rajagopala SV, et al. Mining host-pathogen protein interactions to characterize Burkholderia mallei infectivity mechanisms. PLoS comput biol 2015; 11:e1004088.
11. Memisevic V, Zavaljevski N, Pieper R, et al. Novel Burkholderia mallei virulence factors linked to specific host-pathogen protein interactions. Mol Cell Proteomics 2013; 12:3036–3051.
12. Chiang CY, Ulrich RL, Ulrich MP, et al. Characterization of the murine macrophage response to infection with virulent and avirulent Burkholderia species. BMC Microbiol 2015; 15:259.
13▪. Chiang CY, Uzoma I, Lane DJ, et al. A reverse-phase protein microarray-based screen identifies host signaling dynamics upon Burkholderia spp. infection. Front Microbiol 2015; 6: article 683.
14. Alderson MR, McGowan P, Baldridge JR, Probst P. TLR4 agonists as immunomodulatory agents. J Endotoxin Res 2006; 12:313–319.
15▪▪. Korneev KV, Arbatsky NP, Molinaro A, et al. Structural relationship of the lipid A acyl groups to activation of murine toll-like receptor 4 by lipopolysaccharides from pathogenic strains of Burkholderia mallei, Acinetobacter baumannii, and Pseudomonas aeruginosa. Front Immunol 2015; 6:595.
16. Bernhards RC, Cote CK, Amemiya K, et al. Characterization of in vitro phenotypes of Burkholderia pseudomallei and Burkholderia mallei strains potentially associated with persistent infection in mice. Arch Microbiol 2016; 1–25.
17. Hayden HS, Lim R, Brittnacher MJ, et al. Evolution of Burkholderia pseudomallei in recurrent melioidosis. PLoS One 2012; 7:e36507.
18. Price EP, Sarovich DS, Webb JR, et al. Accurate and rapid identification of the Burkholderia pseudomallei near-neighbour, Burkholderia ubonensis, using real-time PCR. PLoS One 2013; 8:e71647.
19. Smith EE, Buckley DG, Wu Z, et al. Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients. Proc Natl Acad Sci U S A 2006; 103:8487–8492.
20. Rowland CA, Lertmemongkolchai G, Bancroft A, et al. Critical role of type 1 cytokines in controlling initial infection with Burkholderia mallei. Infect Immun 2006; 74:5333–5340.
21. Lu R, Popov V, Patel J, Eaves-Pyles T. Burkholderia mallei and Burkholderia pseudomallei stimulate differential inflammatory responses from human alveolar type II cells (ATII) and macrophages. Front Cell Infect Microbiol 2012; 2:165.
22▪▪. Alam S, Amemiya K, Bernhards RC, et al. Characterization of cellular immune response and innate immune signaling in human and nonhuman primate primary mononuclear cells exposed to Burkholderia mallei. Microb Pathog 2015; 78:20–28.
23. Goodyear A, Troyer R, Bielefeldt-Ohmann H, Dow S. MyD88-dependent recruitment of monocytes and dendritic cells required for protection from pulmonary Burkholderia mallei infection. Infect Immun 2012; 80:110–120.
24. Allwood EM, Devenish RJ, Prescott M, et al. Strategies for intracellular survival of Burkholderia pseudomallei. Front Microbiol 2011; 2:170.
25. Singh AP, Lai SC, Nandi T, et al. Evolutionary analysis of Burkholderia pseudomallei identifies putative novel virulence genes, including a microbial regulator of host cell autophagy. J Bacteriol 2013; 195:5487–5498.
26. Devenish RJ, Lai SC. Autophagy and burkholderia. Immunol Cell Biol 2015; 93:18–24.
27. Rhodes KA, Schweizer HP. Antibiotic resistance in Burkholderia species. Drug Resist Updat 2016; 28:82–90.
28. Van Zandt KE, Greer MT, Gelhaus HC. Glanders: an overview of infection in humans. Orphanet J Rare Dis 2013; 8:131.
29. White NJ. Melioidosis. Lancet 2003; 361:1715–1722.
30. Currie BJ, Fisher DA, Howard DM, et al. The epidemiology of melioidosis in Australia and Papua New Guinea. Acta Tropica 2000; 74:121–127.
31. Waag DM. Efficacy of postexposure therapy against glanders in mice. Antimicrob Agents Chemother 2015; 59:2236–2241.
32. Hatcher CL, Muruato LA, Torres AG. Recent advances in Burkholderia mallei and B. pseudomallei research. Curr Trop Med Rep 2015; 2:62–69.
33. Aschenbroich SA, Lafontaine ER, Hogan RJ. Melioidosis and glanders modulation of the innate immune system: barriers to current and future vaccine approaches. Expert Rev Vaccines 2016; 15:1163–1181.
34. Moustafa DA, Scarff JM, Garcia PP, et al. Recombinant Salmonella expressing Burkholderia mallei LPS O antigen provides protection in a murine model of melioidosis and glanders. PLoS One 2015; 10:e0132032.
35▪. Bozue JA, Chaudhury S, Amemiya K, et al. Phenotypic characterization of a novel virulence-factor deletion strain of Burkholderia mallei that provides partial protection against inhalational glanders in mice. Front Cell Infect Microbiol 2016; 6:21.
36▪▪. Mott TM, Vijayakumar S, Sbrana E, et al. Characterization of the Burkholderia mallei tonB vaccine development. PLoS Negl Trop Dis 2015; 9:e0003863.

Burkholderia mallei; cellular immunity; immune signaling; Innate Immune response; vaccine

Copyright © 2017 The Author(s). Published by Wolters Kluwer Health, Inc.