MALDI-TOF MS analysis of Burkholderia pseudomallei and closely related species isolated from soils and water in Khon Kaen, Thailand.

Melioidosis is caused by Burkholderia pseudomallei (Bp) acquired from the environment. Conventional identification methods for environmental Bp are challenging due to the presence of closely related species. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is accurate for bacterial identification, but has been little used to identify Bp from environmental samples. This study aims to evaluate MALDI-TOF MS for the identification of Bp and closely related species isolated from environmental samples in Thailand using whole-genome sequencing (WGS) as the gold standard, including determining the best sample preparation method for this purpose. We identified Bp (n = 22), Burkholderia spp. (n = 28), and other bacterial species (n = 32) using WGS. MALDI-TOF analysis of all Bp isolates yielded results consistent with WGS. A decision-tree algorithm identified 16 important variable peaks, using the protein extraction method (PEM), demonstrating distinct MALDI-TOF profiles for the three categories (Bp, Burkholderia spp. and "other bacterial species"). Three biomarker peaks (4060, 5196, and 6553 Da) could discriminate Bp from other Burkholderia and closely related species with 100% sensitivity and specificity. Hence, the MALDI-TOF technique has shown its potential as a species discriminatory tool, providing results comparable to WGS for classification and surveillance of environmental Bp.

Modeling and computational characterization of a Xanthomonas sp. Hypothetical protein identifies a remote ortholog of Burkholderia lethal factor 1.

Burkholderia Lethal Factor 1 (BLF1) is a deamidase first characterized in Burkholderia pseudomallei. This enzyme inhibits cellular protein synthesis by deamidating a glutamine residue to a glutamic acid in its target protein, the eukaryotic translation initiation factor 4 A (eIF4A). In this work, we present the characterization of a hypothetical protein from Xanthomonas sp. Leaf131 as the first report of a BLF1 family ortholog outside of the Burkholderia genus. Although standard sequence similarity searches such as BLAST were not able to detect the homology between the Xanthomonas sp. Leaf131 hypothetical protein sequence and BLF1, our computed structure model for the Xanthomonas sp. hypothetical protein revealed structural similarities with an RMSD of 2.7 Å/164 Cα atoms and a TM-score of 0.72 when superposed. Structural comparisons of the Xanthomonas model structure against BLF1 and Escherichia coli cytotoxic necrotizing factor 1 (CNF1) revealed that the conserved signature LXGC motif and putative catalytic residues are structurally aligned thus signifying a level of functional or mechanistic similarity. Protein-protein docking analysis and molecular dynamics simulations also demonstrated that eIF4A could still be a possible target substrate for deamidation by XLF1 as it is for BLF1. We therefore propose that this Xanthomonas hypothetical protein be renamed as Xanthomonas Lethal Factor 1 (XLF1). Our work also provides further evidence of the utility of programs such as AlphaFold in bridging the computational function annotation transfer gap despite very low sequence identities of under 20%.Communicated by Ramaswamy H. Sarma.

Identification and characterization of two drug-like fragments that bind to the same cryptic binding pocket of Burkholderia pseudomallei DsbA.

Disulfide-bond-forming proteins (Dsbs) play a crucial role in the pathogenicity of many Gram-negative bacteria. Disulfide-bond-forming protein A (DsbA) catalyzes the formation of the disulfide bonds necessary for the activity and stability of multiple substrate proteins, including many virulence factors. Hence, DsbA is an attractive target for the development of new drugs to combat bacterial infections. Here, two fragments, bromophenoxy propanamide (1) and 4-methoxy-N-phenylbenzenesulfonamide (2), were identified that bind to DsbA from the pathogenic bacterium Burkholderia pseudomallei, the causative agent of melioidosis. The crystal structures of oxidized B. pseudomallei DsbA (termed BpsDsbA) co-crystallized with 1 or 2 show that both fragments bind to a hydrophobic pocket that is formed by a change in the side-chain orientation of Tyr110. This conformational change opens a `cryptic' pocket that is not evident in the apoprotein structure. This binding location was supported by 2D-NMR studies, which identified a chemical shift perturbation of the Tyr110 backbone amide resonance of more than 0.05 p.p.m. upon the addition of 2 mM fragment 1 and of more than 0.04 p.p.m. upon the addition of 1 mM fragment 2. Although binding was detected by both X-ray crystallography and NMR, the binding affinity (Kd) for both fragments was low (above 2 mM), suggesting weak interactions with BpsDsbA. This conclusion is also supported by the crystal structure models, which ascribe partial occupancy to the ligands in the cryptic binding pocket. Small fragments such as 1 and 2 are not expected to have a high energetic binding affinity due to their relatively small surface area and the few functional groups that are available for intermolecular interactions. However, their simplicity makes them ideal for functionalization and optimization. The identification of the binding sites of 1 and 2 to BpsDsbA could provide a starting point for the development of more potent novel antimicrobial compounds that target DsbA and bacterial virulence.

Solution Structure of the BPSL1445 Protein of Burkholderia pseudomallei Reveals the SYLF Domain Three-Dimensional Fold.

The SYLF domain is an evolutionary conserved protein domain with phosphatidylinositol binding ability, whose three-dimensional structure is unknown. Here, we present the solution structure and the dynamics characterization of the SYLF domain of the bacterial BPSL1445 protein. BPSL1445 is a seroreactive antigen and a diagnostic marker of Burkholderia pseudomallei, the etiological agent of melioidosis, a severe infectious disease in the tropics. The BPSL1445 SYLF domain (BPSL1445-SYLF) consists of a ß-barrel core, with two flexible loops protruding out of the barrel and three helices packing on its surface. Our structure allows for a more precise definition of the boundaries of the SYLF domain compared to the previously reported one and suggests common ancestry with bacterial EipA domains. We also demonstrate by phosphatidyl-inositol phosphate arrays and nuclear magnetic resonance titrations that BPSL1445-SYLF weakly interacts with phosphoinositides, thus supporting lipid binding abilities of this domain also in prokaryotes.

A multi-country study using MALDI-TOF mass spectrometry for rapid identification of Burkholderia pseudomallei.

BACKGROUND: Burkholderia pseudomallei is the bacterial causative agent of melioidosis, a difficult disease to diagnose clinically with high mortality if not appropriately treated. Definitive diagnosis requires isolation and identification of the organism. With the increased adoption of MALDI-TOF MS for the identification of bacteria, we established a method for rapid identification of B. pseudomallei using the Vitek MS, a system that does not currently have B. pseudomallei in its in-vitro diagnostic database. RESULTS: A routine direct spotting method was employed to create spectra and SuperSpectra. An initial B. pseudomallei SuperSpectrum was created at Shoklo Malaria Research Unit (SMRU) from 17 reference isolates (46 spectra). When tested, this initial SMRU SuperSpectrum was able to identify 98.2 % (54/55) of Asian isolates, but just 46.7 % (35/75) of Australian isolates. Using spectra (430) from different reference and clinical isolates, two additional SMRU SuperSpectra were created. Using the combination of all SMRU SuperSpectra with seven existing SuperSpectra from Townsville, Australia 119 (100 %) Asian isolates and 31 (100 %) Australian isolates were correctly identified. In addition, no misidentifications were obtained when using these 11 SuperSpectra when tested with 34 isolates of other bacteria including the closely related species Burkholderia thailandensis and Burkholderia cepacia. CONCLUSIONS: This study has established a method for identification of B. pseudomallei using Vitek MS, and highlights the impact of geographical differences between strains for identification using this technique.

Melioidosis patient serum-reactive synthetic tetrasaccharides bearing the predominant epitopes of Burkholderia pseudomallei and Burkholderia mallei O-antigens.

Melioidosis and glanders, respectively caused by the Gram-negative bacteria Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm), are considered as urgent public health issues in developing countries and potential bioterrorism agents. Bp and Bm lipopolysaccharides (LPS) have been identified as attractive vaccine candidates for the development of prophylactic measures against melioidosis and glanders. Bp and Bm express structurally similar LPSs wherein the O-antigen (OAg) portion consists of a heteropolymer whose repeating unit is a disaccharide composed of d-glucose and 6-deoxy-l-talose residues, the latter being diversely acetylated and methylated. Herein we report the synthesis of two tetrasaccharides mimicking the main substitution epitopes of Bp and Bm LPS OAgs. The assembly of the tetrasaccharides was achieved using a sequential glycosylation strategy while relying on the late-stage epimerization of the inner rhamnose into a 6-deoxy-l-talose residue. We show that these synthetic compounds strongly react with culture-confirmed Thai melioidosis patient serum and closely mimic the antigenicity of native Bp OAg. Our results suggest that these tetrasaccharides could be suitable candidates for the development of vaccines and/or diagnostic tools against melioidosis and glanders.

Structural characterization of Class 2 OLD family nucleases supports a two-metal catalysis mechanism for cleavage.

Overcoming lysogenization defect (OLD) proteins constitute a family of uncharacterized nucleases present in bacteria, archaea, and some viruses. These enzymes contain an N-terminal ATPase domain and a C-terminal Toprim domain common amongst replication, recombination, and repair proteins. The in vivo activities of OLD proteins remain poorly understood and no definitive structural information exists. Here we identify and define two classes of OLD proteins based on differences in gene neighborhood and amino acid sequence conservation and present the crystal structures of the catalytic C-terminal regions from the Burkholderia pseudomallei and Xanthamonas campestris p.v. campestris Class 2 OLD proteins at 2.24 Å and 1.86 Å resolution respectively. The structures reveal a two-domain architecture containing a Toprim domain with altered architecture and a unique helical domain. Conserved side chains contributed by both domains coordinate two bound magnesium ions in the active site of B. pseudomallei OLD in a geometry that supports a two-metal catalysis mechanism for cleavage. The spatial organization of these domains additionally suggests a novel mode of DNA binding that is distinct from other Toprim containing proteins. Together, these findings define the fundamental structural properties of the OLD family catalytic core and the underlying mechanism controlling nuclease activity.

The structure of lipopolysaccharide transport protein B (LptB) from Burkholderia pseudomallei.

The thick outer membrane (OM) of Gram-negative bacteria performs an important protective role against hostile environments, supports cell integrity, and contributes to surface adhesion and in some cases also to virulence. A major component of the OM is lipopolysaccharide (LPS), a complex glycolipid attached to a core containing fatty-acyl chains. The assembly and transport of lipid A, the membrane anchor for LPS, to the OM begins when a heteromeric LptB2FG protein complex extracts lipid A from the outer leaflet of the inner membrane. This process requires energy, and upon hydrolysis of ATP one component of the heteromeric assembly, LptB, triggers a conformational change in LptFG in support of lipid A transport. A structure of LptB from the intracellular pathogen Burkholderia pseudomallei is reported here. LptB forms a dimer that displays a relatively fixed structure irrespective of whether it is in complex with LptFG or in isolation. Highly conserved sequence and structural features are discussed that allow LptB to fuel the transport of lipid A.

Biomarker discovery in the biofilm-forming process of Burkholderia pseudomallei by mass-spectrometry.

A serious human infectious disease called Melioidosis is a result of Burkholderia pseudomallei infection. Treatment for infected individuals is difficult due to a wide range of ineffective antibiotics including a high level of antibiotic tolerance which has been known to be caused by biofilm production. However, biofilm forming processes of this bacterium are not well documented despite multiple-methodologies being applied. In this study, we utilized a proteomics strategy called whole cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (whole cell MALDI-TOF MS) to discover a potential biomarker relating biofilm forming in B. pseudomallei. The results presented a novel specific type of enzyme amylo-alpha-1, 6-glucosidase, which was demonstrated by a higher level of gene expression during the biofilm development. Our results also suggested a list of candidate markers that might be involved in this scenario. Eventually, this knowledge may expand valuable data to the biofilm study that may increase effective treatments for people infected with B. pseudomallei and possibly other antibiotic tolerant bacteria.

Impact of nutritional stress on drug susceptibility and biofilm structures of Burkholderia pseudomallei and Burkholderia thailandensis grown in static and microfluidic systems.

Burkholderia pseudomallei is the causative agent of melioidosis and regarded as a bioterrorism threat. It can adapt to the nutrient-limited environment as the bacteria can survive in triple distilled water for 16 years. Moreover, B. pseudomallei exhibits intrinsic resistance to diverse groups of antibiotics in particular while growing in biofilms. Recently, nutrient-limited condition influenced both biofilm formation and ceftazidime (CAZ) tolerance of B. pseudomallei were found. However, there is no information about how nutrient-limitation together with antibiotics used in melioidosis treatment affects the structure of the biofilm produced by B. pseudomallei. Moreover, no comparative study to investigate the biofilm architectures of B. pseudomallei and the related B. thailandensis under different nutrient concentrations has been reported. Therefore, this study aims to provide new information on the effects of four antibiotics used in melioidosis treatment, viz. ceftazidime (CAZ), imipenem (IMI), meropenem (MEM) and doxycycline (DOX) on biofilm architecture of B. pseudomallei and B. thailandensis with different nutrient concentrations under static and flow conditions using confocal laser scanning microscopy. Impact of nutritional stress on drug susceptibility of B. pseudomallei and B. thailandensis grown planktonically or as biofilm was also evaluated. The findings of this study indicate that nutrient-limited environment enhanced survival of B. pseudomallei in biofilm after exposure to the tested antibiotics. The shedding planktonic B. pseudomallei and B. thailandensis were also found to have increased CAZ tolerance in nutrient-limited environment. However, killing activities of MEM and IMI were stronger than CAZ and DOX on B. pseudomallei and B. thailandensis both in planktonic cells and in 2-day old biofilm. In addition, MEM and IMI were able to inhibit B. pseudomallei and B. thailandensis biofilm formation to a larger extend compared to CAZ and DOX. Differences in biofilm architecture were observed for biofilms grown under static and flow conditions. Under static conditions, biofilms grown in full strength modified Vogel and Bonner's medium (MVBM) showed honeycomb-like architecture while a knitted-like structure was observed under limited nutrient condition (0.1×MVBM). Under flow conditions, biofilms grown in MVBM showed a multilayer structure while merely dispersed bacteria were found when grown in 0.1×MVBM. Altogether, this study provides more insight on the effect of four antibiotics against B. pseudomallei and B. thailandensis in biofilm under different nutrient and flow conditions. Since biofilm formation is believed to be involved in disease relapse, MEM and IMI may be better therapeutic options than CAZ for melioidosis treatment.

Comprehensive analysis of clinical Burkholderia pseudomallei isolates demonstrates conservation of unique lipid A structure and TLR4-dependent innate immune activation.

Burkholderia pseudomallei is an environmental bacterium that causes melioidosis, a major community-acquired infection in tropical regions. Melioidosis presents with a range of clinical symptoms, is often characterized by a robust inflammatory response, may relapse after treatment, and results in high mortality rates. Lipopolysaccharide (LPS) of B. pseudomallei is a potent immunostimulatory molecule comprised of lipid A, core, and O-polysaccharide (OPS) components. Four B. pseudomallei LPS types have been described based on SDS-PAGE patterns that represent the difference of OPS-type A, type B, type B2 and rough LPS. The majority of B. pseudomallei isolates are type A. We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) followed by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QqTOF MS) and gas chromatography to characterize the lipid A of B. pseudomallei within LPS type A isolates. We determined that B. pseudomallei lipid A is represented by penta- and tetra-acylated species modified with 4-amino-4-deoxy-arabinose (Ara4N). The MALDI-TOF profiles from 171 clinical B. pseudomallei isolates, including 68 paired primary and relapse isolates and 35 within-host isolates were similar. We did not observe lipid A structural changes when the bacteria were cultured in different growth conditions. Dose-dependent NF-κB activation in HEK cells expressing TLR4 was observed using multiple heat-killed B. pseudomallei isolates and corresponding purified LPS. We demonstrated that TLR4-dependent NF-κB activation induced by heat-killed bacteria or LPS prepared from OPS deficient mutant was significantly greater than those induced by wild type B. pseudomallei. These findings suggest that the structure of B. pseudomallei lipid A is highly conserved in a wide variety of clinical and environmental circumstances but that the presence of OPS may modulate LPS-driven innate immune responses in melioidosis.

Host-Adaptation of Burkholderia pseudomallei Alters Metabolism and Virulence: a Global Proteome Analysis.

Little is known about the evolution, adaptation and pathogenesis of Burkholderia pseudomallei within host during acute melioidosis infection. Melioidosis is a potential life threatening disease contracted through inhalation, ingestion, inoculation or direct entry of the organism into the blood stream via wounds or skin abrasions from contaminated soil and water. Environmental B. pseudomallei strain (Bp MARAN ), isolated during a melioidosis outbreak in Pahang, Malaysia was injected intra-peritoneally into a mouse and passaged strain was recovered from spleen (Bpmouse-adapted). A gel-based comparative proteomics profiling approach was used, to map and identify differentially expressed proteins (fold-change ≥ 2; p-value ≤ 0.05) between the strains. A total of 730 and 685 spots were visualised in the Bp MARAN and Bpmouse-adapted strains, respectively. Of the 730 spots (Bp MARAN as reference gel), 87 spots were differentially regulated (44 up- and 43 down-regulated). The identified proteins were classified as proteins related to metabolism, stress response, virulence, signal transduction, or adhesion. In comparison, it was found that those proteins related to adhesins, virulence factors and stress- response were up-regulated and could possibly explain the adaptation of the bacteria in the host. Investigating the differentially expressed proteins may provide better perspective of bacterial factors which aid survivability of B. pseudomallei in host.

Structural diversity of Burkholderia pseudomallei lipopolysaccharides affects innate immune signaling.

Burkholderia pseudomallei (Bp) causes the disease melioidosis. The main cause of mortality in this disease is septic shock triggered by the host responding to lipopolysaccharide (LPS) components of the Gram-negative outer membrane. Bp LPS is thought to be a weak inducer of the host immune system. LPS from several strains of Bp were purified and their ability to induce the inflammatory mediators TNF-α and iNOS in murine macrophages at low concentrations was investigated. Innate and adaptive immunity qPCR arrays were used to profile expression patterns of 84 gene targets in response to the different LPS types. Additional qPCR validation confirmed large differences in macrophage response. LPS from a high-virulence serotype B strain 576a and a virulent rough central nervous system tropic strain MSHR435 greatly induced the innate immune response indicating that the immunopathogenesis of these strains is different than in infections with strains similar to the prototype strain 1026b. The accumulation of autophagic vesicles was also increased in macrophages challenged with highly immunogenic Bp LPS. Gene induction and concomitant cytokine secretion profiles of human PBMCs in response to the various LPS were also investigated. MALDI-TOF/TOF was used to probe the lipid A portions of the LPS, indicating substantial structural differences that likely play a role in host response to LPS. These findings add to the evolving knowledge of host-response to bacterial LPS, which can be used to better understand septic shock in melioidosis patients and in the rational design of vaccines.

Expression and crystallographic studies of D-glycero-ß-D-manno-heptose-1-phosphate adenylyltransferase from Burkholderia pseudomallei.

The Gram-negative bacterium Burkholderia pseudomallei is the causative agent of melioidosis. D-glycero-ß-D-manno-Heptose-1-phosphate adenylyltransferase (HldC) is the fourth enzyme of the ADP-L-glycero-ß-D-manno-heptose biosynthesis pathway, which produces an essential carbohydrate comprising the inner core of lipopolysaccharide. Therefore, HldC is a potential target of antibiotics against melioidosis. In this study, HldC from B. pseudomallei has been cloned, expressed, purified and crystallized. Synchrotron X-ray data from a selenomethionine-substituted HldC crystal were also collected to 2.8 Šresolution. The crystal belonged to the primitive triclinic space group P1, with unit-cell parameters a = 74.0, b = 74.0, c = 74.9 Å, α = 108.4, ß = 108.4, γ = 108.0°. Eight protomers are present in the unit cell and three out of five selenomethionines were found in each protomer using the PHENIX software suite. A full structural determination is in progress to elucidate the structure-function relationship of the protein.

Characterization of homologous sphingosine-1-phosphate lyase isoforms in the bacterial pathogen Burkholderia pseudomallei.

Sphingolipids (SLs) are ubiquitous elements in eukaryotic membranes and are also found in some bacterial and viral species. As well as playing an integral structural role, SLs also act as potent signaling molecules involved in numerous cellular pathways and have been linked to many human diseases. A central SL signaling molecule is sphingosine-1-phosphate (S1P), whose breakdown is catalyzed by S1P lyase (S1PL), a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the cleavage of S1P to (2E)-hexadecenal (2E-HEX) and phosphoethanolamine. Here, we show that the pathogenic bacterium, Burkholderia pseudomallei K96243, encodes two homologous proteins (S1PL2021 and S1PL2025) that display moderate sequence identity to known eukaryotic and prokaryotic S1PLs. Using an established MS-based methodology, we show that recombinant S1PL2021 is catalytically active. We also used recombinant human fatty aldehyde dehydrogenase to develop a spectrophotometric enzyme-coupled assay to detect 2E-HEX formation and measure the kinetic constants of the two B. pseudomallei S1PL isoforms. Furthermore, we determined the X-ray crystal structure of the PLP-bound form of S1PL2021 at 2.1 Å resolution revealing that the enzyme displays a conserved structural fold and active site architecture comparable with known S1PLs. The combined data suggest that B. pseudomallei has the potential to degrade host SLs in a S1PL-dependent manner.

In vivo Distribution and Clearance of Purified Capsular Polysaccharide from Burkholderia pseudomallei in a Murine Model.

Burkholderia pseudomallei is the causative agent of melioidosis, a severe infection prominent in northern Australia and Southeast Asia. The "gold standard" for melioidosis diagnosis is bacterial isolation, which takes several days to complete. The resulting delay in diagnosis leads to delayed treatments, which could result in death. In an attempt to develop better methods for early diagnosis of melioidosis, B. pseudomallei capsular polysaccharide (CPS) was identified as an important diagnostic biomarker. A rapid lateral flow immunoassay utilizing CPS-specific monoclonal antibody was developed and tested in endemic regions worldwide. However, the in vivo fate and clearance of CPS has never been thoroughly investigated. Here, we injected mice with purified CPS intravenously and determined CPS concentrations in serum, urine, and major organs at various intervals. The results indicate that CPS is predominantly eliminated through urine and no CPS accumulation occurs in the major organs. Immunoblot analysis demonstrated that intact CPS was excreted through urine. To understand how a large molecule like CPS was eliminated without degradation, a 3-dimenational structure of CPS was modeled. The predicted CPS structure has a rod-like shape with a small diameter that could allow it to flow through the glomerulus of the kidney. CPS clearance was determined using exponential decay models and the corrected Akaike Information Criterion. The results show that CPS has a relatively short serum half-life of 2.9 to 4.4 hours. Therefore, the presence of CPS in the serum and/or urine suggests active melioidosis infection and provides a marker to monitor treatment of melioidosis.

Identification of Candidate Host Cell Factors Required for Actin-Based Motility of Burkholderia pseudomallei.

Intracellular actin-based motility of the melioidosis pathogen Burkholderia pseudomallei requires the bacterial factor BimA. Located at one pole of the bacterium, BimA recruits and polymerizes cellular actin to promote bacterial motility within and between cells. Here, we describe an affinity approach coupled with mass spectrometry to identify cellular proteins recruited to BimA-expressing bacteria under conditions that promote actin polymerization. We identified a group of cellular proteins that are recruited to the B. pseudomallei surface in a BimA-dependent manner, a subset of which were independently validated with specific antisera including the ubiquitous scaffold protein Ras GTPase-activating-like protein (IQGAP1). IQGAP1 integrates several key cellular signaling pathways including those involved in actin dynamics and has been shown to be involved in the adhesion of attaching and effacing Escherichia coli to infected cells and invasion of host cells by Salmonella enterica serovar Typhimurium. Although a direct interaction between BimA and IQGAP1 could not be detected using either conventional pulldown or yeast two hybrid techniques, confocal microscopy revealed that IQGAP1 is recruited to B. pseudomallei actin tails in infected cells, and siRNA-mediated knockdown highlighted a role for this protein in controlling the length and actin density of B. pseudomallei actin tails.

Utility of a Lateral Flow Immunoassay (LFI) to Detect Burkholderia pseudomallei in Soil Samples.

BACKGROUND: Culture is the gold standard for the detection of environmental B. pseudomallei. In general, soil specimens are cultured in enrichment broth for 2 days, and then the culture broth is streaked on an agar plate and incubated further for 7 days. However, identifying B. pseudomallei on the agar plates among other soil microbes requires expertise and experience. Here, we evaluate a lateral flow immunoassay (LFI) developed to detect B. pseudomallei capsular polysaccharide (CPS) in clinical samples as a tool to detect B. pseudomallei in environmental samples. METHODOLOGY/PRINCIPAL FINDINGS: First, we determined the limit of detection (LOD) of LFI for enrichment broth of the soil specimens. Soil specimens (10 grams/specimen) culture negative for B. pseudomallei were spiked with B. pseudomallei ranging from 10 to 105 CFU, and incubated in 10 ml of enrichment broth in air at 40°C. Then, on day 2, 4 and 7 of incubation, 50 µL of the upper layer of the broth were tested on the LFI, and colony counts to determine quantity of B. pseudomallei in the broth were performed. We found that all five soil specimens inoculated at 10 CFU were negative by LFI on day 2, but four of those five specimens were LFI positive on day 7. The LOD of the LFI was estimated to be roughly 3.8x106 CFU/ml, and culture broth on day 7 was selected as the optimal sample for LFI testing. Second, we evaluated the utility of the LFI by testing 105 soil samples from Northeast Thailand. All samples were also tested by standard culture and quantitative PCR (qPCR) targeting orf2. Of 105 soil samples, 35 (33%) were LFI positive, 25 (24%) were culture positive for B. pseudomallei, and 79 (75%) were qPCR positive. Of 11 LFI positive but standard culture negative specimens, six were confirmed by having the enrichment broth on day 7 culture positive for B. pseudomallei, and an additional three by qPCR. The LFI had 97% (30/31) sensitivity to detect soil specimens culture positive for B. pseudomallei. CONCLUSIONS/SIGNIFICANCE: The LFI can be used to detect B. pseudomallei in soil samples, and to select which samples should be sent to reference laboratories or proceed further for bacterial isolation and confirmation. This could considerably decrease laboratory workload and assist the development of a risk map for melioidosis in resource-limited settings.

Flexible vs Rigid Epitope Conformations for Diagnostic- and Vaccine-Oriented Applications: Novel Insights from the Burkholderia pseudomallei BPSL2765 Pal3 Epitope.

Peptides seldom retain stable conformations if separated from their native protein structure. In an immunological context, this potentially affects the development of selective peptide-based bioprobes and, from a vaccine perspective, poses inherent limits in the elicitation of cross-reactive antibodies by candidate epitopes. Here, a 1,4-disubstituted-1,2,3-triazole-mediated stapling strategy was used to stabilize the native α-helical fold of the Pal3 peptidic epitope from the protein antigen PalBp (BPSL2765) from Burkholderia pseudomallei, the etiological agent of melioidosis. Whereas Pal3 shows no propensity to fold outside its native protein context, the engineered peptide (Pal3H) forms a stable α-helix, as assessed by MD, NMR, and CD structural analyses. Importantly, Pal3H shows an enhanced ability to discriminate between melioidosis patient subclasses in immune sera reactivity tests, demonstrating the potential of the stapled peptide for diagnostic purposes. With regard to antibody elicitation and related bactericidal activities, the linear peptide is shown to elicit a higher response. On these bases, we critically discuss the implications of epitope structure engineering for diagnostic- and vaccine-oriented applications.