Direct α-Hydroxy Acid Loading onto a Bacterial Thiotemplate Assembly Line via a Multienzyme Gateway.

Various nonribosomal peptide synthetases (NRPSs) create structural and functional diversity by incorporating α-hydroxy acids into peptide backbones. Trigonic acid, an unusual cyclopropanol-substituted hydroxy acid, is the source of the molecular warhead of malleicyprol, a critical virulence factor of human and animal pathogens of the Burkholderia pseudomallei (BP) group. The process of selecting and loading this building block remained enigmatic as the NRPS module designated for this task is incomplete. Using a combination of bioinformatics, mutational analyses, targeted metabolomics, and in vitro biochemical assays, we show that two trans-acting enzymes are required to load this central building block onto the modular assembly line. An adenylation-thiolation didomain enzyme (BurJ) activates trigonic acid, followed by the translocation of the enzyme-bound α-hydroxy acid thioester by an FkbH-like protein with a mutated phosphatase domain (BurH). This specialized gateway is the first reported direct loading of an α-hydroxy acid onto a bona fide NRPS module in bacteria and expands the synthetic biology toolbox for the site-specific incorporation of non-canonical building blocks. Moreover, insight into the biochemical basis of virulence factor biosynthesis can provide a foundation for developing enzyme inhibitors as anti-virulence therapeutics against BP pathogen infections.

Crystal structure of betaine aldehyde dehydrogenase from Burkholderia pseudomallei.

Burkholderia pseudomallei infection causes melioidosis, which is often fatal if untreated. There is a need to develop new and more effective treatments for melioidosis. This study reports apo and cofactor-bound crystal structures of the potential drug target betaine aldehyde dehydrogenase (BADH) from B. pseudomallei. A structural comparison identified similarities to BADH from Pseudomonas aeruginosa which is inhibited by the drug disulfiram. This preliminary analysis could facilitate drug-repurposing studies for B. pseudomallei.

Protein-Ligand Dissociation Rate Constant from All-Atom Simulation.

Dissociation of a ligand isoniazid from a protein catalase was investigated using all-atom molecular dynamics (MD) simulations. Random acceleration MD (τ-RAMD) was used, in which a random artificial force applied to the ligand facilitates its dissociation. We have suggested a novel approach to extrapolate such obtained dissociation times to the zero-force limit assuming never before attempted universal exponential dependence of the bond strength on the applied force, allowing direct comparison with experimentally measured values. We have found that our calculated dissociation time was equal to 36.1 s with statistically significant values distributed in the interval of 0.2-72.0 s, which quantitatively matches the experimental value of 50 ± 8 s despite the extrapolation over 9 orders of magnitude in time.

Bacteriophage origin of some minimal ATP-dependent DNA ligases: a new structure from Burkholderia pseudomallei with striking similarity to Chlorella virus ligase.

DNA ligases, the enzymes responsible for joining breaks in the phosphodiester backbone of DNA during replication and repair, vary considerably in size and structure. The smallest members of this enzyme class carry out their functions with pared-down protein scaffolds comprising only the core catalytic domains. Here we use sequence similarity network analysis of minimal DNA ligases from all biological super kingdoms, to investigate their evolutionary origins, with a particular focus on bacterial variants. This revealed that bacterial Lig C sequences cluster more closely with Eukaryote and Archaeal ligases, while bacterial Lig E sequences cluster most closely with viral sequences. Further refinement of the latter group delineates a cohesive cluster of canonical Lig E sequences that possess a leader peptide, an exclusively bacteriophage group of T7 DNA ligase homologs and a group with high similarity to the Chlorella virus DNA ligase which includes both bacterial and viral enzymes. The structure and function of the bacterially-encoded Chlorella virus homologs were further investigated by recombinantly producing and characterizing, the ATP-dependent DNA ligase from Burkholderia pseudomallei as well as determining its crystal structure in complex with DNA. This revealed that the enzyme has similar activity characteristics to other ATP-dependent DNA ligases, and significant structural similarity to the eukaryotic virus Chlorella virus including the positioning and DNA contacts of the binding latch region. Analysis of the genomic context of the B. pseudomallei ATP-dependent DNA ligase indicates it is part of a lysogenic bacteriophage present in the B. pseudomallei chromosome representing one likely entry point for the horizontal acquisition of ATP-dependent DNA ligases by bacteria.

Structural and biophysical characterization of the Burkholderia pseudomallei IspF inhibitor L-tryptophan hydroxamate.

The enzyme 2-methylerythritol 2,4-cyclodiphosphate synthase, IspF, is essential for the biosynthesis of isoprenoids in most bacteria, some eukaryotic parasites, and the plastids of plant cells. The development of inhibitors that target IspF may lead to novel classes of anti-infective agents or herbicides. Enantiomers of tryptophan hydroxamate were synthesized and evaluated for binding to Burkholderia pseudomallei (Bp) IspF. The L-isomer possessed the highest potency, binding BpIspF with a KD of 36 µM and inhibited BpIspF activity 55% at 120 µM. The high-resolution crystal structure of the L-tryptophan hydroxamate (3)/BpIspF complex revealed a non-traditional mode of hydroxamate binding where the ligand interacts with the active site zinc ion through the primary amine. In addition, two hydrogen bonds are formed with active site groups, and the indole group is buried within the hydrophobic pocket composed of side chains from the 60 s/70 s loop. Along with the co-crystal structure, STD NMR studies suggest the methylene group and indole ring are potential positions for optimization to enhance binding potency.

Taurultams incorporating arylsulfonamide: First in vitro inhibition studies of α-, ß- and γ-class Carbonic Anhydrases from Vibrio cholerae and Burkholderia pseudomallei.

A new series of taurultambenzenesulfonamides 1-17 were prepared and considered for their inhibitory activity in vitro against the Carbonic Anhydrases from Vibrio cholerae (VchCA-α, VchCA-ß and VchCA-γ) and Burkholderia pseudomallei (BpsCA-ß and BpsCA-γ). Among the compounds tested, derivatives 4, 5, 7, 10, 12, and 16 resulted in highly effective VchCAα inhibitors (KI values spanning within the 6.1-9.6 nM range) and endowed with excellent Selectivity Indexes (SIs; KI VchCA-α/KI hCA II) all comprised between 0.04 and 0.09. Potent in vitro inhibitors for the BpsCA-γ were also identified (KIs of 18.9-19.5 nM). The results here reported may represent the blueprint for the future development of a new generation of CA-based antibiotics integrated with free of resistance mechanisms of action adopted from known drugs.

A study of inhibitors of d-glycero-ß-d-manno-heptose-1-phosphate adenylyltransferase from Burkholderia pseudomallei as a potential antibiotic target.

d-Glycero-ß-d-manno-heptose-1-phosphate adenylyltransferase from Burkholderia pseudomallei (BpHldC) is the fourth enzyme in the ADP-l-glycero-ß-d-manno-heptose biosynthesis pathway producing a lipopolysaccharide core. Therefore, BpHldC is an anti-melioidosis target. Three ChemBridge compounds purchased from ChemBridge Corporation (San Diego, CA) were found to have an effective inhibitory activity on BpHldC. Interestingly, ChemBridge 7929959 was the most effective compound due to the presence of the terminal benzyl group. The enzyme kinetic study revealed that most of them show mixed type inhibitory modes against ATP and ßG1P. The induced-fit docking indicated that the medium affinity of ChemBridge 7929959 is originated from its benzyl group occupying the substrate-binding pocket of BpHldC. The inhibitory role of terminal aromatic groups was proven with ChemBridge 7570508. Combined with the previous study, ChemBridge 7929959 is found to work as a dual inhibitor against both HldC and HddC. Therefore, three ChemBridge compounds can be developed as a potent anti-melioidosis agent with a novel inhibitory concept.

A triple-targeting inhibitory activity of Rose Bengal on polysaccharide biosynthesis of Burkholderia pseudomallei.

Sugar nucleotidyltransferases (SNTs) participate in various biosynthesis pathways constructing polysaccharides in Gram-negative bacteria. In this study, a triple-targeting inhibitory activity of Rose Bengal against SNTs such as d-glycero-α-d-manno-heptose-1-phosphate guanylyltransferase (HddC), d-glycero-ß-d-manno-heptose-1-phosphate adenylyltransferase (HldC), and 3-deoxy-d-manno-oct-2-ulosonic acid cytidylyltransferase (KdsB) from Burkholderia pseudomallei is provided. Rose Bengal effectively suppresses the nucleotidyltransferase activity of the three SNTs, and its IC50 values are 10.42, 0.76, and 5.31 µM, respectively. Interestingly, Rose Bengal inhibits the three enzymes regardless of their primary, secondary, tertiary, and quaternary structural differences. The experimental results indicate that Rose Bengal possesses the plasticity to shape its conformation suitable to interact with the three SNTs. As HddC functions in the formation of capsular polysaccharides and HldC and KdsB produce building blocks to constitute the inner core of lipopolysaccharide, Rose Bengal is a potential candidate to design antibiotics in a new category. In particular, it can be developed as a specific antimelioidosis agent. As the mortality rate of the infected people caused by B. pseudomallei is quite high, there is an urgent need for specific antimelioidosis agents. Therefore, a further study is being carried out with derivatives of Rose Bengal.

Inhibition of d-glycero-ß-d-manno-heptose 1-phosphate adenylyltransferase from Burkholderia pseudomallei by epigallocatechin gallate and myricetin.

Flavonoids play beneficial roles in various human diseases. In this study, a flavonoid library was employed to probe inhibitors of d-glycero-ß-d-manno-heptose-1-phosphate adenylyltransferase from Burkholderia pseudomallei (BpHldC) and two flavonoids, epigallocatechin gallate (EGCG) and myricetin, have been discovered. BpHldC is one of the essential enzymes in the ADP-l-glycero-ß-d-manno-heptose biosynthesis pathway constructing lipopolysaccharide of B. pseudomallei. Enzyme kinetics study showed that two flavonoids work through different mechanisms to block the catalytic activity of BpHldC. Among them, a docking study of EGCG was performed and the binding mode could explain its competitive inhibitory mode for both ATP and ßG1P. Analyses with EGCG homologs could reveal the important functional moieties, too. This study is the first example of uncovering the inhibitory activity of flavonoids against the ADP-l-glycero-ß-d-manno-heptose biosynthesis pathway and especially targeting HldC. Since there are no therapeutic agents and vaccines available against melioidosis, EGCG and myricetin can be used as templates to develop antibiotics over B. pseudomallei.

Functional redundancy of Burkholderia pseudomallei phospholipase C enzymes and their role in virulence.

Phospholipase C (PLC) enzymes are key virulence factors in several pathogenic bacteria. Burkholderia pseudomallei, the causative agent of melioidosis, possesses at least three plc genes (plc1, plc2 and plc3). We found that in culture medium plc1 gene expression increased with increasing pH, whilst expression of the plc3 gene was pH (4.5 to 9.0) independent. Expression of the plc2 gene was not detected in culture medium. All three plc genes were expressed during macrophage infection by B. pseudomallei K96243. Comparing B. pseudomallei wild-type with plc mutants revealed that plc2, plc12 or plc123 mutants showed reduced intracellular survival in macrophages and reduced plaque formation in HeLa cells. However, plc1 or plc3 mutants showed no significant differences in plaque formation compared to wild-type bacteria. These findings suggest that Plc2, but not Plc1 or Plc3 are required for infection of host cells. In Galleria mellonella, plc1, plc2 or plc3 mutants were not attenuated compared to the wild-type strain, but multiple plc mutants showed reduced virulence. These findings indicate functional redundancy of the B. pseudomallei phospholipases in virulence.

Detecting the nature and solving the crystal structure of a contaminant protein from an opportunistic pathogen.

The unintentional crystallization of contaminant proteins in the place of target recombinant proteins is sporadically reported, despite the availability of stringent expression/purification protocols and of software for the detection of contaminants. Typically, the contaminant protein originates from the expression organism (for example Escherichia coli), but in rare circumstances contaminants from different sources have been reported. Here, a case of contamination from a Serratia bacterial strain that occurred while attempting to crystallize an unrelated protein from Burkholderia pseudomallei (overexpressed in E. coli) is presented. The contamination led to the unintended crystallization and structure analysis of a cyanase hydratase from a bacterial strain of the Serratia genus, an opportunistic enterobacterium that grows under conditions similar to those of E. coli and that is found in a variety of habitats, including the laboratory environment. In this context, the procedures that were adopted to identify the contaminant based on crystallographic data only are presented and the crystal structure of Serrata spp. cyanase hydratase is briefly discussed.

Benzylaminoethylureido-Tailed Benzenesulfonamides Show Potent Inhibitory Activity against Bacterial Carbonic Anhydrases.

A series of benzylaminoethylureido-tailed benzenesulfonamides was analyzed for their inhibition potential against bacterial carbonic anhydrases (CAs) such as VhCA α, ß, and γ from Vibrio cholerae, and BpsCA ß and γ-CAs from Burkholderia pseudomallei. Growing drug resistance against antibiotics demands alternative targets and mechanisms of action. As CA is essential for the survival of bacteria, such enzymes have the potential for developing new antibiotics. Most of the compounds presented excellent inhibition potential against VhCA γ compared to α and ß, with Ki values in the range of 82.5-191.4 nM. Several sulfonamides exhibited excellent inhibition against BpsCA ß with Ki values in the range of 394-742.8 nM. Recently it has been demonstrated that sufonamide CA inhibitors are effective against vancomycin-resistant enterococci. These data show that CA inhibition of pathogenic bacteria may lead to a new class of antibiotics.

NMR fragment screening reveals a novel small molecule binding site near the catalytic surface of the disulfide-dithiol oxidoreductase enzyme DsbA from Burkholderia pseudomallei.

The presence of suitable cavities or pockets on protein structures is a general criterion for a therapeutic target protein to be classified as 'druggable'. Many disease-related proteins that function solely through protein-protein interactions lack such pockets, making development of inhibitors by traditional small-molecule structure-based design methods much more challenging. The 22 kDa bacterial thiol oxidoreductase enzyme, DsbA, from the gram-negative bacterium Burkholderia pseudomallei (BpsDsbA) is an example of one such target. The crystal structure of oxidized BpsDsbA lacks well-defined surface pockets. BpsDsbA is required for the correct folding of numerous virulence factors in B. pseudomallei, and genetic deletion of dsbA significantly attenuates B. pseudomallei virulence in murine infection models. Therefore, BpsDsbA is potentially an attractive drug target. Herein we report the identification of a small molecule binding site adjacent to the catalytic site of oxidized BpsDsbA. 1HN CPMG relaxation dispersion NMR measurements suggest that the binding site is formed transiently through protein dynamics. Using fragment-based screening, we identified a small molecule that binds at this site with an estimated affinity of KD ~ 500 µM. This fragment inhibits BpsDsbA enzymatic activity in vitro. The binding mode of this molecule has been characterized by NMR data-driven docking using HADDOCK. These data provide a starting point towards the design of more potent small molecule inhibitors of BpsDsbA.

Functional analysis of BPSS2242 reveals its detoxification role in Burkholderia pseudomallei under salt stress.

A bpss2242 gene, encoding a putative short-chain dehydrogenase/oxidoreductase (SDR) in Burkholderia pseudomallei, was identified and its expression was up-regulated by ten-fold when B. pseudomallei was cultured under high salt concentration. Previous study suggested that BPSS2242 plays important roles in adaptation to salt stress and pathogenesis; however, its biological functions are still unknown. Herein, we report the biochemical properties and functional characterization of BPSS2242 from B. pseudomallei. BPSS2242 exhibited NADPH-dependent reductase activity toward diacetyl and methylglyoxal, toxic electrophilic dicarbonyls. The conserved catalytic triad was identified and found to play critical roles in catalysis and cofactor binding. Tyr162 and Lys166 are involved in NADPH binding and mutation of Lys166 causes a conformational change, altering protein structure. Overexpression of BPSS2242 in Escherichia coli increased bacterial survival upon exposure to diacetyl and methylglyoxal. Importantly, the viability of B. pseudomallei encountered dicarbonyl toxicity was enhanced when cultured under high salt concentration as a result of BPSS2242 overexpression. This is the first study demonstrating that BPSS2242 is responsible for detoxification of toxic metabolites, constituting a protective system against reactive carbonyl compounds in B. pseudomallei..

Crystal Structure of a Tetrameric Type II ß-Carbonic Anhydrase from the Pathogenic Bacterium Burkholderia pseudomallei.

Carbonic anhydrase (CA) is a zinc enzyme that catalyzes the reversible conversion of carbon dioxide to bicarbonate and proton. Currently, CA inhibitors are widely used as antiglaucoma, anticancer, and anti-obesity drugs and for the treatment of neurological disorders. Recently, the potential use of CA inhibitors to fight infections caused by protozoa, fungi, and bacteria has emerged as a new research line. In this article, the X-ray crystal structure of ß-CA from Burkholderia pseudomallei was reported. The X-ray crystal structure of this new enzyme was solved at 2.7 Å resolution, revealing a tetrameric type II ß-CA with a "closed" active site in which the zinc is tetrahedrally coordinated to Cys46, Asp48, His102, and Cys105. B. pseudomallei is known to encode at least two CAs, a ß-CA, and a γ-CA. These proteins, playing a pivotal role in its life cycle and pathogenicity, offer a novel therapeutic opportunity to obtain antibiotics with a different mechanism of action. Furthermore, the new structure can provide a clear view of the ß-CA mechanism of action and the possibility to find selective inhibitors for this class of CAs.

Burkholderia ubonensis Meropenem Resistance: Insights into Distinct Properties of Class A ß-Lactamases in Burkholderia cepacia Complex and Burkholderia pseudomallei Complex Bacteria.

Burkholderia pseudomallei, the founding member of the B. pseudomallei complex (Bpc), is a biothreat agent and causes melioidosis, a disease whose treatment mainly relies on ceftazidime and meropenem. The concern is that B. pseudomallei could enhance its drug resistance repertoire by the acquisition of DNA from resistant near-neighbor species. Burkholderia ubonensis, a member of the B. cepacia complex (Bcc), is commonly coisolated from environments where B. pseudomallei is present. Unlike B. pseudomallei, in which significant primary carbapenem resistance is rare, it is not uncommon in B. ubonensis, but the underlying mechanisms are unknown. We established that carbapenem resistance in B. ubonensis is due to an inducible class A PenB ß-lactamase, as has been shown for other Bcc bacteria. Inducibility is not sufficient for high-level resistance but also requires other determinants, such as a PenB that is more robust than that present in susceptible isolates, as well as other resistance factors. Curiously and diagnostic for the two complexes, both Bpc and Bcc bacteria contain distinct annotated PenA class A ß-lactamases. However, the protein from Bcc bacteria is missing its essential active-site serine and, therefore, is not a ß-lactamase. Regulated expression of a transcriptional penB'-lacZ (ß-galactosidase) fusion in the B. pseudomallei surrogate B. thailandensis confirms that although Bpc bacteria lack an inducible ß-lactamase, they contain the components required for responding to aberrant peptidoglycan synthesis resulting from ß-lactam challenge. Understanding the diversity of antimicrobial resistance in Burkholderia species is informative about how the challenges arising from potential resistance transfer between them can be met.IMPORTANCEBurkholderia pseudomallei causes melioidosis, a tropical disease that is highly fatal if not properly treated. Our data show that, in contrast to B. pseudomallei, B. ubonensis ß-lactam resistance is fundamentally different because intrinsic resistance is mediated by an inducible class A ß-lactamase. This includes resistance to carbapenems. Our work demonstrates that studies with near-neighbor species are informative about the diversity of antimicrobial resistance in Burkholderia and can also provide clues about the potential of resistance transfer between bacteria inhabiting the same environment. Knowledge about potential adverse challenges resulting from the horizontal transfer of resistance genes between members of the two complexes enables the design of effective countermeasures.

Virulence of the emerging pathogen, Burkholderia pseudomallei, depends upon the O-linked oligosaccharyltransferase, PglL.

Aim: We sought to characterize the contribution of the O-OTase, PglL, to virulence in two Burkholderia spp. by comparing isogenic mutants in Burkholderia pseudomallei with the related species, Burkholderia thailandensis. Materials & methods: We utilized an array of in vitro assays in addition to Galleria mellonella and murine in vivo models to assess virulence of the mutant and wild-type strains in each Burkholderia species. Results: We found that pglL contributes to biofilm and twitching motility in both species. PglL uniquely affected morphology; cell invasion; intracellular motility; plaque formation and intergenus competition in B. pseudomallei. This mutant was attenuated in the murine model, and extended survival in a vaccine-challenge experiment. Conclusion: Our data support a broad role for pglL in bacterial fitness and virulence, particularly in B. pseudomallei.

A new bivalent fluorescent fusion protein for differential Cu(II) and Zn(II) ion detection in aqueous solution.

Simple and easy to engineer metal-sensing molecules that are capable of differentiating metal ions and producing metal-specific signals are highly desirable. Metal ions affect the thermal stability of proteins by increasing or decreasing their resistance to unfolding. This work illustrates a new strategy for designing bivalent fluorescent fusion proteins capable of differentiating metal ions in solution through their distinct effects on a protein's thermal stability. A new dual purpose metal sensor was developed consisting of biotin protein ligase (BirA) from B. pseudomallei (Bp) fused to green fluorescent protein (GFP). When coupled with differential scanning fluorimetry of GFP-tagged proteins (DSF-GTP) for signal-transduction detection, Bp BirA-GFP yields distinct protein unfolding signatures with Zn(II) and Cu(II) ions in aqueous solutions. The limit of detection of the system is ∼1 µM for both metal species. The system can be used in a variety of high-throughput assay formats including for the screening of metal-binding proteins and chelators. Bp BirA-GFP has also the additional benefit of being useful in Cu(II) ion field-testing applications through simple visual observation of a temperature-dependent loss of fluorescence. Bp BirA-GFP is the first example of a 2protein-based dual purpose Cu(II) and Zn(II) ion sensor compatible with two different yet complementary signal-transduction detection systems.

Functional and structural analysis of trehalose-6-phosphate phosphatase from Burkholderia pseudomallei: Insights into the catalytic mechanism.

We report the functional and structural characterization of trehalose-6-phosphate phosphatase (TPP), from the Gram-negative bacterium B. pseudomallei that causes melioidosis, a severe infectious disease endemic in Southeast Asia and Northern Australia. TPP is a key enzyme in the trehalose biosynthesis pathway, which plays an important role in bacterial stress responses. Due to the absence of this biosynthetic pathway in mammals, TPP has drawn attention as a potential drug target, to combat antibiotic resistance. In this context, we present a detailed biochemical analysis of purified recombinant TPP, reporting its specific high catalytic activity toward the trehalose-6-phosphate substrate, and an absolute requirement for its Mg2+ cofactor. Furthermore, we present the crystal structure of TPP solved at 1.74 Å, revealing the canonical haloacid dehalogenase (HAD) superfamily fold and conserved substrate binding pocket, from which insights into the catalytic mechanism may be deduced. Our data represent a starting point for the rational design of antibacterial drugs.

Antibacterial activity of 2-amino-4-hydroxypyrimidine-5-carboxylates and binding to Burkholderia pseudomallei 2-C-methyl-d-erythritol-2,4-cyclodiphosphate synthase.

Enzymes in the methylerythritol phosphate pathway make attractive targets for antibacterial activity due to their importance in isoprenoid biosynthesis and the absence of the pathway in mammals. The fifth enzyme in the pathway, 2-C-methyl-d-erythritol-2,4-cyclodiphosphate synthase (IspF), contains a catalytically important zinc ion in the active site. A series of de novo designed compounds containing a zinc binding group was synthesized and evaluated for antibacterial activity and interaction with IspF from Burkholderia pseudomallei, the causative agent of Whitmore's disease. The series demonstrated antibacterial activity as well as protein stabilization in fluorescence-based thermal shift assays. Finally, the binding of one compound to Burkholderia pseudomallei IspF was evaluated through group epitope mapping by saturation transfer difference NMR.