Melioidosis is a disease caused by infection with Burkholderia pseudomallei. The molecular basis for the pathogenicity of B. pseudomallei is poorly understood. However, recent work has identified the first toxin from this bacterium and shown that it inhibits host protein synthesis. Here, we review the illness that is potentially associated with biological warfare, the pathogen and its deadly molecular mechanism of action, as well as therapeutic developments that may follow.

Introduction

Melioidosis is a chronic infectious disease that is endemic in parts of Southeast Asia and Northern Australia, as well as sporadic in South and Central America, the Middle East and in some African countries. Burkholderia pseudomallei, the causative agent of melioidosis [1], is also named Whitmore's bacillus in reference to a British officer who discovered the bacteria in abscesses of dead opium addicts in Burma in 1911 [2]. The pathogen is a saprophytic bacterium that lives in moist soils and surface waters of the endemic regions. It has no requirement to pass through an animal to replicate and the pathogen infects humans by accident [3]. It is mostly transmitted by cutaneous contamination with the natural environment, e.g. infection of scratches and cuts resulting from agricultural work but also by ingestion and inhalation of contaminated particles or water. Similar to the Bubonic plague or Anthrax, the inhaled form is more virulent, causing a higher number of casualties during the wet seasons in which storms stimulate the formation of aerosols. Melioidosis is a daily drama for families living in the endemic areas as people, often working barefoot in rice fields, are frequently infected during their childhood. It was reported that 80% of 4-year-old children are seropositive for B. pseudomallei in endemic northeast Thailand [4].

Pathogenesis

Melioidosis is very difficult to diagnose as it mimics many symptoms common to tuberculosis, typhoid fever or malaria. These include acute pneumonia and septicaemia, hyperthermia, purulent coughing, sneezing and skin abscesses, organ abscesses, and further neurological lesions. Thus the disease has been nicknamed ‘The Great Mimicker’. In addition, B. pseudomallei colonies are able to switch between seven types of morphology on Ashdown's agar plates, each with distinct biological patterns associated with variable pathogenicity [5], complicating even furthermore the identification of the pathogen. There is no vaccine despite numerous trials [6]. The pathogen is naturally resistant to many antimicrobial agents and actively pumps some drugs out of the cell [7]. The typical treatment for melioidosis involves administering multiple antibiotics for up to 5 months. The pathogen can either induce an immediate acute form of the disease, in the first few weeks of incubation, which kills 20–50% of its victims even with medical treatment [8], or stay dormant in the host organism for decades [9] before activation by poorly defined mechanisms. Between 1965 and 1972, it has been estimated that 225000 US soldiers engaged in the Vietnam conflict were exposed to and became seropositive for B. pseudomallei. In particular, helicopter pilots were particularly susceptible through frequent exposure to contaminated soil aerosols created during take-off and landing manoeuvres [10]. The profile of melioidosis in the West was subsequently raised and the disease was nicknamed ‘The Vietnam Time Bomb’, although clearly many other conflicts have left soldiers exposed in endemic regions, for example, during the Sino-Japanese (1894–1945) and Indochina (1946–1979) Wars.

A potential biological warfare agent

In 2002, the classification of B. pseudomallei as a category B biological warfare agent [11] has since escalated research interests. The use of the pathogen by terrorists is also a concern [12] as it grows easily even in colder climates outside tropical regions. Furthermore, it survives at least a decade in distilled water [13] and would ensure a long-term contamination of the targeted population and environment. The pathogen exhibits a broad range of hosts, and has been shown to cause the disease in almost all animals including cattle, pigs, horses, dogs, cats, dolphins [14] and pigeons. Recently, it was reported that B. pseudomallei is also able to infect tomato plants, raising security concerns for bioterrorism as plants could easily be used as new hosts for growing the pathogen [15]. Burkholderia mallei, a closely related bacterium causes the disease Glanders in horses, donkeys and mules. B. mallei is thought to have been the first ever biological warfare agent to be used and was employed by German forces against Allies during World War I (1914–1918) to deliberately contaminate livestock and humans [16,17].

Pathogenicity of B. pseudomallei

B. pseudomallei is a Gram-negative, aerobic, motile and capsulated bacterium. Outside its natural habitat in the moist soils of melioidosis-endemic regions, it lives as an intracellular invasive pathogen in the cytoplasm of infected animal host cells [1,18]. It was suggested that it spreads from cell to cell by inducing actin polymerization which in turn would cause cell fusion and formation of multinucleated giant cells [19]. Recently, it was also reported that it multiplies as an intercellular micro-organism in the xylem vessels of tomato plants [15]. The sequence of the B. pseudomallei genome was completed in 2004 [20]. It revealed that the two circular chromosomes have distinct evolutionary origins, harbouring genes encoding various virulence and survival factors that were acquired from other micro-organisms. The large chromosome (4.07 Mb) encodes many of the core functions associated with central metabolism and cell growth. The small chromosome (3.17 Mb) encodes accessory functions associated with virulence, adaptation and survival in different environments. The pathogen secretes many exotoxins including various enzymes implicated in the necrosis of tissues (proteases, lipases, lecithinases, catalases and haemolysins). Activity of other pathogenic factors depends on bacterial effectors that increase the virulence of the bacterium in the host cells and allow its survival. Recently, the bacterial effector CHBP (Cif homologue from B. pseudomallei) was shown to be a potent inhibitor of the eukaryotic ubiquitination pathway, which deamidates Gln40 in ubiquitin and ubiquitin-like protein NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8), resulting in cell cycle arrest and actin stress fibre formation [21]. In addition, the pathogen produces several types of secretion systems that are thought to mediate the transport and secretion of bacterial effectors into the host cells. They play an important role in the virulence and the intracellular lifestyle of B. pseudomallei [22,23]. Cellular superoxide is used by infected hosts to kill a broad range of intracellular pathogens. A superoxide dismutase C enzyme was recently isolated from B. pseudomallei and was shown to be involved in the intracellular survival of the bacterium [24]. However, bacterial pathogens usually produce lethal toxin(s) which have yet to be characterized in B. pseudomallei.

Identification of the first lethal toxin from B. pseudomallei

In 2007, proteome analysis of B. pseudomallei and the non-pathogenic related Burkholderia thailandensis strain highlighted the expression of 14 hypothetical proteins of unknown function in the pathogenic bacterium [25]. This study prompted an investigation of some of these hypothetical proteins using X-ray crystallography, which resulted in the determination of the structure of one protein, BPSL1549, which on subsequent investigation was found to be the first toxin from B. pseudomallei [26]. Consequently, BPSL1549 was renamed BLF1 (Burkholderia lethal factor 1). Recombinant BLF1 is toxic and kills mice challenged by the intraperitoneal route, as well as cultures of macrophages. Moreover, a partial in-frame deletion of the gene encoding BLF1 in B. pseudomallei makes it 100-fold less potent at killing mice [26]. The amino-acid sequence of BLF1 does not share homology with other known factors, but the crystal structure revealed that BLF1 is structurally related to the C-terminal domain of the CNF1 (cytotoxic necrotizing factor 1) produced by some pathogenic strains of Escherichia coli. However, BLF1 does not affect deamidation of the GTPase protein Rho and cytoskeleton assembly as reported for CNF1 [27]. In contrast, BLF1 specifically deamidates Gln339 of eIF4A (eukaryotic initiation factor 4A). This modification inactivates the RNA helicase activity required for melting mRNA secondary structures during initiation of translation, but does not alter the ATPase activity of eIF4A. In turn, this leads to extensive inhibition of protein synthesis in human cells (Figure 1). Accordingly, other eIF4A mutations that uncouple ATPase and/or RNA helicase activities are known to inhibit translation [29]. There are three eIF4A isoforms: isoforms I and II are involved in translation. In contrast, eIF4A isoform III is a component of the exon junction complex involved in NMD (nonsense-mediated mRNA decay) and is not likely to be a substrate of BLF1 since the residue structurally equivalent to Gln339 in eIF4A isoforms I and II is proline in eIF4A isoform III.

The toxin BLF1 inhibits translation by deamination of initiation factor eIF4A

Figure 1
The toxin BLF1 inhibits translation by deamination of initiation factor eIF4A

Cartoon representing a human cell infected with the invasive intracellular form of the Burkholderia pseudomallei bacterium (Bpm). The pathogen produces numerous virulence factors including the BLF1 toxin depicted by grey triangles. In the nucleus of the host cell, transcription of genes led to co-transcriptional processing and nuclear export of mature mRNAs that are translated into proteins in the cytoplasm. During translation, mRNAs are first circularized by a ribonucleoprotein complex of initiation which interacts simultaneously with the CAP (represented by a black circle) and the polyA tail of the mRNA. It is composed, among other proteins, of the eIF4F factor, a trimeric complex containing the eIF4A, eIF4E and eIF4G subunits, which can associate with the PolyA binding protein (PABP). Secondary structures of mRNAs have to be resolved by the RNA helicase activity of the eIF4A factor for progression into the active elongation mode of protein synthesis. Deamidation of Gln339 of eIF4A to glutamic acid by the toxin BLF1 abolishes its RNA helicase activity and sequesters mRNA and ribosomal subunits in stalled initiation complexes. In vitro, one molecule of BLF1 is able to inactivate ~700 molecules eIF4A per minute, a turnover number similar in range to the cleavage of N-glycosidic bonds at A4324 of 28S RNA by the potent translation poison ricin [28]. Death of cells is likely to occur when protein synthesis stops once all active ribosomes have been inactivated.

Figure 1
The toxin BLF1 inhibits translation by deamination of initiation factor eIF4A

Cartoon representing a human cell infected with the invasive intracellular form of the Burkholderia pseudomallei bacterium (Bpm). The pathogen produces numerous virulence factors including the BLF1 toxin depicted by grey triangles. In the nucleus of the host cell, transcription of genes led to co-transcriptional processing and nuclear export of mature mRNAs that are translated into proteins in the cytoplasm. During translation, mRNAs are first circularized by a ribonucleoprotein complex of initiation which interacts simultaneously with the CAP (represented by a black circle) and the polyA tail of the mRNA. It is composed, among other proteins, of the eIF4F factor, a trimeric complex containing the eIF4A, eIF4E and eIF4G subunits, which can associate with the PolyA binding protein (PABP). Secondary structures of mRNAs have to be resolved by the RNA helicase activity of the eIF4A factor for progression into the active elongation mode of protein synthesis. Deamidation of Gln339 of eIF4A to glutamic acid by the toxin BLF1 abolishes its RNA helicase activity and sequesters mRNA and ribosomal subunits in stalled initiation complexes. In vitro, one molecule of BLF1 is able to inactivate ~700 molecules eIF4A per minute, a turnover number similar in range to the cleavage of N-glycosidic bonds at A4324 of 28S RNA by the potent translation poison ricin [28]. Death of cells is likely to occur when protein synthesis stops once all active ribosomes have been inactivated.

Potential therapeutic applications related to the discovery of the basis for melioidosis

The characterization of the lethal toxin BLF1 and its deadly molecular mechanism of action in human cells pave the way for the identification of molecules which might prevent the modification of eIF4A by BLF1. Such molecules may be useful in limiting the cellular damage that results from a Burkholderia infection and be useful as part of a combined therapy with antibiotics. Furthermore, inactivation of the toxin BLF1 is also of interest as it may allow the production of a vaccine for the first time. Indeed, a C94S mutation in BLF1 inhibits its activity as a glutamine deamidase [26] and may be a useful starting point for production of a vaccine. Although this mutation still retains the ability to bind eIF4A and inhibits it at high concentrations, further engineering of BLF1 may be required before a suitable candidate for vaccination is identified.

BLF1 also offers alternative applications in medicine. Several inhibitors of protein synthesis have been described as anticancer agents including inhibitors of eIF4A [30]. As a potent eIF4A inhibitor, BLF1 might prove useful as a potential anticancer agent. The product of BLF1-modified eIF4A, bearing a substitution of Gln339 to Glu339, is itself a potent translation inhibitor. Thus both BLF1 and eIF4A-Glu339 are potential new molecular tools for targeted chemotherapy of tumours. However, eIF4A-Glu339 may display advantages in therapeutic use because it would be more stable in patients as, unlike BLF1, it is less likely to trigger an immune response. Clearly, the efficient delivery of BLF1 or eIF4A-Glu339 to tumours would be an important prerequisite for their therapeutic use.

Summary

Melioidosis is a bacterial infectious disease caused by B. pseudomallei, a saprophyte that lives in moist soils and stagnant waters of endemic regions. The pathogen accidentally infects large numbers of people across the world, and could potentially be utilized as a biological warfare agent because of its long persistence in animals and the environment. In recent work, the first ever toxin was identified from B. pseudomallei [26]. The toxin, named BLF1, is a potent inhibitor of cellular protein synthesis which deamidates Glu339 in the translation initiation factor eIF4A. This disrupts the RNA helicase activity of eIF4A which is required to unwind secondary structure in mRNA and allow easy access for the ribosome. The characterization of BLF1 and its deadly mechanism of action bring new therapeutic hopes to scientists. However, the agent of melioidosis is well packed with an arsenal of virulence factors, and it is plausible that actions aimed at silencing BLF1 activity may trigger the bacterium to induce other lethal uncharacterized virulence factors.

RNA UK 2012: An Independent Meeting held at The Burnside Hotel, Bowness-on-Windermere, Cumbria, U.K., 20–22 January 2012. Organized and Edited by Raymond O'Keefe and Mark Ashe (Manchester, U.K.).

Abbreviations

     
  • BLF1

    Burkholderia lethal factor 1

  •  
  • CNF1

    cytotoxic necrotizing factor 1

  •  
  • eIF4A

    eukaryotic initiation factor 4A

Funding

S.A.W. acknowledges support from the Biotechnology and Biological Sciences Research Council and the Wellcome Trust.

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