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Science. Author manuscript; available in PMC May 31, 2012.
Published in final edited form as:
PMCID: PMC3364511
EMSID: UKMS48011

A Burkholderia pseudomallei Toxin Inhibits Helicase Activity of Translation Factor eIF4A

Abstract

The structure of BPSL1549, a protein of unknown function from Burkholderia pseudomallei reveals a similarity to E. coli cytotoxic necrotizing factor 1. We found that BPSL1549 acted as a potent cytotoxin against eukaryotic cells and was lethal when administered to mice. Expression levels of bpsl1549 correlate with conditions expected to promote or suppress pathogenicity. BPSL1549 promotes deamidation of Gln339 of the translation initiation factor eIF4A, abolishing its helicase activity and inhibiting translation. We propose to name BPSL1549 Burkholderia Lethal Factor 1 (BLF1).

The bacterium Burkholderia pseudomallei is the causative agent of melioidosis (1) and, in endemic areas including Southeast Asia, can be isolated from moist soil and stagnant water. B. pseudomallei infects many tissue, causing subclinical infections, acute septicaemia and sub-acute and chronic disease. The pathogen has the ability to remain latent in a host for decades (2) and infections in troops serving in endemic areas has resulted in the pathogen being referred to by the nickname, the “Vietnam Time Bomb” (3). There is no vaccine and the organism is multidrug resistant complicating treatment. The molecular mechanisms that underlie disease are largely unknown. To address this, a programme of structure determination on proteins of unknown function from B. pseudomallei has been initiated.

The structure of BPSL1549, a 23 kDa protein of unknown function, was determined at 1.04 Å resolution (Table S1, Fig. S1A). The fold consists of a sandwich of two curved mixed beta sheets decorated on the outside by alpha helices and loops (Fig 1A) and was similar to the catalytic domain of E. coli cytotoxic necrotizing factor 1 (CNF1-C) (4). Eleven strands of the BPSL1549 beta sandwich have identical sequence order and direction to those of CNF1-C, with an rmsd of 3.9 Å for 170 superposed α-carbon atoms (Fig. 1B). However, on the peripheries of the structures there are differences in both the extensions to the β-strands and the helices and loops, with only one helix in common.

Fig. 1
Structural analysis of BPSL1549

CNF1-C inactivates Rho GTPases by deamidation of a glutamine necessary for GTPase hydrolysis, thereby affecting actin cytoskeleton assembly (5). CNF1-C and BPSL1549 showed virtually no sequence identity, except at the active sites where the LSGC motif of CNF1-C was conserved in BPSL1549 (Fig. S1B,C). The cysteine of this motif, Cys866/Cys94 in CNF1-C and BPSL1549, respectively, is the crucial nucleophile in the CNF1-C catalytic triad responsible for deamidase activity (4). His881, the second component of the CNF1-C triad was present in BPSL1549 (as His106) and superposed well (Fig. 1C). In CNF1-C the third component of the triad is a main chain carbonyl, but in BPSL1549 the OG1 of Thr88 hydrogen bonds to the histidine. A structurally conserved tyrosine (Tyr164 in BPSL1549, Tyr962 in CNF1-C) hydrogen bonds to the main chain carbonyl of the catalytic cysteine (Fig. 1C).

The conservation of key catalytic residues with CNF1-C, suggested BPSL1549 was a glutamine deamidase. However, the molecular surface around the BPSL1549 active site cavity was broader and shallower than that of CNF1-C suggesting the deamidation target was different (Fig S2). Nevertheless, the structure implied that, like CNF1-C, BPSL1549 might be cytotoxic. Intraperitoneal injection of Balb/C mice with BPSL1549 was lethal by day 14 (Supplementary online material). BPSL1549 was also toxic to J774 macrophage cells within 3 days (Fig. 2A). In contrast, growth of 3T3 cells was insensitive to BPSL1549 unless the protein delivery reagent BioPORTER was included (Fig. S3A). The differential sensitivity between macrophages and fibroblasts may reflect uptake by active macropinocytosis in macrophages, although how the toxin accesses the cytoplasm is unclear.

Fig. 2
Toxic effects of BPSL1549

On the basis of the abolition of the biological activity of CNF1 when Cys866 is mutated to serine, a BPSL1549C94S mutant was created. In mice BPSL1549C94S was non-toxic, whereas in J774 cells some toxicity (p < 0.05) was observed, but only at the highest toxin concentration tested (Fig 2A). Thermal stability studies (Fig. S3B,C) together with the BPSL1549C94S structure (Table S1) confirmed that this point mutation does not disrupt the structure and that C94 is required for BPSL1549 activity.

To investigate a role for bpsl1549 in bacterial virulence, an in-frame deletion mutant of bpsl1549 in B. pseudomallei was constructed. Whilst we cannot exclude the influence of polarity effects in the Δbpsl1549 strain, compared to the wild type strain the mutant was significantly attenuated when mice were challenged by the i.p. route (Fig. 2B). The median lethal dose of B. pseudomallei Δbpsl1549 was 1.26 × 105 colony forming units (CFU), 100 times higher than that for the wild type B. pseudomallei strain K96243, consistent with a role for bpsl1549 in virulence. Moreover, bpsl1549 expression was highly upregulated in response to multiple virulence cues (Fig. S4).

An affinity column was used to purify BPSL1549 binding-partners from human cell extract. The major protein purified was translation initiation factor eIF4A which was confirmed by immunoblotting (Fig. 3A). Interaction with endogenous eIF4A was verified by coimmunoprecipitation with FLAG-BPSL1549. FLAG-BPSL1549C94S precipitated more eIF4A than FLAG-BPSL1549, suggesting that release of the substrate from the toxin requires completion of deamidation (Fig. 3B).

Fig. 3
BPSL1549 modifies eIF4A and blocks translation

The BPSL1549:eIF4A interaction suggested BPSL1549 might inhibit translation and its expression in human cells led to 90% or greater reduction in gene expression for two reporters. Reduced translation inhibition by BPSL1549C94S may result from binding eIF4A (Fig. 3B,C). The reporter mRNA levels were unaffected, consistent with BPSL1549 blocking translation (Fig. 3C, right panel). BPSL1549 reduced endogenous protein synthesis monitored by 35S-Met/Cys incorporation at levels similar to that seen with the eIF4A inhibitor hippuristanol (6), whereas BPSL1549C94S did not (Fig. 3D). The ongoing translation in the presence of BPSL1549 may arise from eIF4A-independent mRNAs. To assess the impact of BPSL1549 on translation initiation we monitored the abundance of ribosomal subunits, monosomes and polysomes in cell extracts (Fig. 3E). BPSL1549 enhanced the size of the 80S peak and reduced the size of the polysome peaks with polyA binding protein (PABP) being absent in this region of the gradient. This suggested translation initiation was stalled, mirroring the situation seen with the eIF4A specific inhibitor pateamine A (7).

A translational block can trigger cytoplasmic stress granule formation either by an eIF2α phosphorylation-dependent or independent pathways induced by cellular stresses or by inactivation of eIF4A, 4G, 4B,4H or PABP respectively (8). Sodium arsenate treatment led to both stress granule formation and eIF2α phosphorylation (Fig. S5). BPSL1549 expression also caused stress granule formation, but not eIF2α phosphorylation, consistent with activation of the eIF2α phosphorylation-independent pathway. BPSL1549C94S failed to trigger stress granule formation efficiently, consistent with its reduced ability to inhibit translation. .

To identify the modification in eIF4A we used MS to analyse FLAG-eIF4AI purified from human cells expressing BPSL1549 (Fig. S6). MS revealed deamidation of Gln339 to Glu (Fig. 4A). Gln339 is in the C-terminal domain of eIF4A and is the first glutamine of a conserved Gln-Gln pair lying between motifs V and VI (9). This loop is adjacent to the cleft between the enzyme’s two domains and lies between the ATP and RNA binding sites (Fig S7) suggesting modification might inhibit eIF4A ATPase or helicase activities. In vitro, BPSL1549 deamidates eIF4A Gln339 with a turnover number of ~700 per minute, similar to the rate of depurination catalysed by ricin (10) (Fig. S8, S9). Consistent with Gln339 being the target residue, BPSL1549 binds less efficiently to eIF4AIQ339E, the product of the deamidation reaction (Fig. S10). Two isoforms of eIF4A (I and II) have a glutamine at position 339, whereas eIF4AIII, an exon junction complex subunit, has proline at this position and is unlikely to be a BPSL1549 substrate.

Fig. 4
BPSL1549 inhibits eIF4AI RNA helicase activity

Expression of eIF4AI Q339E in vivo reduced expression of two reporters, indicating Q339E is dominant negative, whereas control mutation Q340E had no effect (Fig. 4B). Q339E had no effect on eIF4A1 ATPase activity or RNA and ATP binding (Fig. S11). However, Q339E reduced RNA helicase activity (Fig. 4C). eIF4AI mutations that uncouple the ATPase and helicase activities are known to act as inhibitors of translation (11). At molar ratios of 1:500 BPSL1549:eIF4A there was 47% inhibition of helicase activity indicative of enzymatic modification by BPSL1549. Whereas BPSL1549C94S only caused 15% inhibition of helicase activity at a molar ratio of 1:10 BPSL1549C94S:eIF4A, probably caused by BPSL1549C94S binding to eIF4A (Fig S12). Based on the closed conformation structure of eIF4AIII (12), Gln339 in eIF4AI would lie close to Asp337 and Arg368 and the 3′ adenine ribose hydroxyl of ATP (Fig. S13). Thus, deamidation of Gln339 probably disrupts interaction between these residues, influencing the functions of Asp337. In the Vasa RNA helicase, the structural equivalent of eIF4AI Asp337 is Asp534 and a D534A mutation uncouples ATP hydrolysis and RNA binding from the helicase activity (13).

We examined the impact of BPSL1549 on formation of the cap binding complex eIF4F. BPSL1549 had no impact on the levels of eIF4E, 4G1 or PABP bound, but increased the amount of eIF4A recovered (Fig. 4D), suggesting eIF4A was more stably bound within eIF4F. Endogenous eIF4A was readily exchanged with recombinant eIF4A within eiF4F when cell extracts were pre-incubated with the control splicing factor SC35 (Fig. 4E lanes 6,7) but in cell extracts pre-incubated with BPSL1549, recombinant eIF4A did not efficiently displace endogenous eiF4A from eIF4F. Thus recycling of eIF4A, required for efficient translation (11),(14) would be impaired.

Compared to CNF1-C a major difference with BPSL1549 is the lack of the receptor binding and translocation domains, essential for cytoplasmic delivery of CNF1-C. However, the intracytoplasmic lifestyle of B. pseudomallei removes the need for BPSL1549 to cross the eukaryotic cell membrane, though the absence of an obvious BPSL1549 signal sequence leaves its mechanism of secretion uncertain. The demonstration that BPSL1549 is a potent inhibitor of translation suggests that it is an important weapon in the armoury of B. pseudomallei and we suggest renaming it Burkholderia Lethal Factor 1 (BLF1). The identification of structurally unrelated papain-like glutamine deamidases CHBP in B. pseudomallei (15) and PMT in Pasteurella multocida (16) suggests this type of chemistry may be more generally employed by pathogenic bacteria than is currently recognised.

Supplementary Material

Acknowledgements

We thank S. Morley for providing translation factor antibodies, C.Hellen for the eIF4B vector, A. Goldman and M. Ashe for helpful suggestions, F. Salguero (VLA) and C. Taylor (Dstl) for histopathology analysis and Defence Science Organisation Laboratories, Singapore for human sera. SW and MJD acknowledge BBSRC support. RM, SN, NMM, MFR and DWR acknowledge Ministry of Science, Technology & Innovation, Government of Malaysia grant [07-05-16-MGI-GMB08] support and the British Council PMI-2 Initiative. AC thanks CONACYT for scholarship funding. MS-T acknowledges Ministry of Defence (UK) support. MJD acknowledges EPSRC support. RT acknowledges Wellcome Trust support [grant WT085162AIA]. We acknowledge ESRF and Diamond Synchrotron for beamtime and thank C. Mueller-Dieckmann and R. Flaig for assistance with stations ID29 and I04. Atomic coordinates and structure factors have been deposited in the Protein Databank (PDB) under accession codes XXXX and XXXX.

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