Flagellar glycosylation in Burkholderia pseudomallei and Burkholderia thailandensis

Glycosylation of proteins is known to impart novel physical properties and biological roles to proteins from both eukaryotes and prokaryotes. In this study, gel-based glycoproteomics were used to identify glycoproteins of the potential biothreat agent Burkholderia pseudomallei and the closely related but nonpathogenic B. thailandensis. Top-down and bottom-up mass spectrometry (MS) analyses identified that the flagellin proteins of both species were posttranslationally modified by novel glycans. Analysis of proteins from two strains of each species demonstrated that B. pseudomallei flagellin proteins were modified with a glycan with a mass of 291 Da, while B. thailandensis flagellin protein was modified with related glycans with a mass of 300 or 342 Da. Structural characterization of the B. thailandensis carbohydrate moiety suggests that it is an acetylated hexuronic acid. In addition, we have identified through mutagenesis a gene from the lipopolysaccharide (LPS) O-antigen biosynthetic cluster which is involved in flagellar glycosylation, and inactivation of this gene eliminates flagellar glycosylation and motility in B. pseudomallei. This is the first report to conclusively demonstrate the presence of a carbohydrate covalently linked to a protein in B. pseudomallei and B. thailandensis, and it suggests new avenues to explore in order to examine the marked differences in virulence between these two species.     

Knockout and pullout recombineering for naturally transformable Burkholderia thailandensis and Burkholderia pseudomallei

Phage λ-Red proteins are powerful tools for pulling and knocking out chromosomal fragments but have been limited to the γ-proteobacteria. Procedures are described here to easily knock out (KO) and pull out (PO) chromosomal DNA fragments from naturally transformable Burkholderia thailandensis and Burkholderia pseudomallei. This system takes advantage of published compliant counterselectable and selectable markers (sacB, pheS, gat and the arabinose-utilization operon) and λ-Red mutant proteins. pheS-gat (KO) or oriT-ColE1ori-gat-ori1600-rep (PO) PCR fragments are generated with flanking 40- to 45-bp homologies to targeted regions incorporated on PCR primers. One-step recombination is achieved by incubation of the PCR product with cells expressing λ-Red proteins and subsequent selection on glyphosate-containing medium. This procedure takes ~10 d and is advantageous over previously published protocols: (i) smaller PCR products reduce primer numbers and amplification steps, (ii) PO fragments suitable for downstream manipulation in Escherichia coli are obtained and (iii) chromosomal KO increases flexibility for downstream processing.     

Detection and differentiation of Burkholderia pseudomallei, Burkholderia mallei and Burkholderia thailandensis by multiplex PCR

Burkholderia pseudomallei, a Gram-negative bacterium that causes melioidosis may be differentiated from closely related species of Burkholderia mallei that causes glanders and non-pathogenic species of Burkholderia thailandensis by multiplex PCR. The multiplex PCR consists of primers that flank a 10-bp repetitive element in B. pseudomallei and B. mallei amplifying PCR fragment of varying sizes between 400-700 bp, a unique sequence in B. thailandensis amplifying a PCR fragment of 308 bp and the metalloprotease gene amplifying a PCR fragment of 245 bp in B. pseudomallei and B. thailandensis. The multiplex PCR not only can differentiate the three Burkholderia species but can also be used for epidemiological typing of B. pseudomallei and B. mallei strains.     

Targeted mutagenesis of Burkholderia thailandensis and Burkholderia pseudomallei through natural transformation of PCR fragments

Burkholderia pseudomallei is the causative agent of melioidosis, an overwhelming, rapidly fatal septic infection, and B. thailandensis is a closely related, less virulent species. Both organisms are naturally competent for DNA transformation, and this report describes a procedure exploiting this property for the rapid generation of marked deletion mutations by using PCR products. The method was employed to create 61 mutant strains. Several selectable elements were employed, including elements carrying loxP and FRT recombinase recognition sites to facilitate resistance marker excision. Chromosomal mutations could also be transferred readily between strains by transformation. The availability of simple procedures for creating defined chromosomal mutations and moving them between strains should facilitate genetic analysis of virulence and other traits of these two Burkholderia species.     

Differences in genomic macrorestriction patterns of arabinose-positive (Burkholderia thailandensis) and arabinose-negative Burkholderia pseudomallei.

We reported previously two biochemically and antigenically distinct biotypes of Burkholderia pseudomallei. These two distinct biotypes could be distinguished by their ability to assimilate L-arabinose. Some B. pseudomallei isolated from soil samples could utilize this substrate (Ara+), whereas the other soil isolates and all clinical isolates could not (Ara-). Only the Ara isolates were virulent in animals and reacted with monoclonal antibody directed at the surface envelope, most likely the exopolysaccharide component. In the present study, pulsed-field gel electrophoresis was employed for karyotyping of these previously identified B. pseudomallei strains. We demonstrate here that the DNA macrorestriction pattern allows the differentiation between B. pseudomallei, which can assimilate L-arabinose, and the proposed B. thailandensis, which cannot do so. Bacterial strains from 80 melioidosis patients and 33 soil samples were examined by genomic DNA digestion with NcoI. Two major reproducible restriction patterns were observed. All clinical (Ara-) isolates and 9 Ara- soil isolates exhibited macrorestriction pattern I (MPI), while 24 soil isolates (Ara+) from central and northeastern Thailand displayed macrorestriction pattern II (MPII). The study here demonstrated pulsed-field gel electrophoresis to be a useful tool in epidemiological investigation possibly distinguishing virulent B. pseudomallei from avirulent B. thailandensis or even identifying closely related species of Burkholderia.     

Development of a multiplex PCR assay for rapid identification of Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia mallei and Burkholderia cepacia complex

We have developed a multiplex PCR assay for rapid identification and differentiation of cultures for Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia mallei and Burkholderia cepacia complex. The assay is valuable for use in clinical and veterinary laboratories, and in a deployable laboratory during outbreaks.     

The wbiA locus is required for the 2-O-acetylation of lipopolysaccharides expressed by Burkholderia pseudomallei and Burkholderia thailandensis

Burkholderia pseudomallei and Burkholderia thailandensis express similar O-antigens (O-PS II) in which their 6-deoxy-alpha-L-talopyranosyl (L-6dTalp) residues are variably substituted with O-acetyl groups at the O-2 or O-4 positions. In previous studies we demonstrated that the protective monoclonal antibody, Pp-PS-W, reacted with O-PS II expressed by wild-type B. pseudomallei strains but not by a B. pseudomallei wbiA null mutant. In the present study we demonstrate that WbiA activity is required for the acetylation of the L-6dTalp residues at the O-2 position and that structural modification of O-PS II molecules at this site is critical for recognition by Pp-PS-W.     

Human single-chain Fv antibodies against Burkholderia mallei and Burkholderia pseudomallei

Much effort has been devoted to the development of mouse monoclonal antibodies that react specifically with Burkholderia mallei and Burkholderia pseudomallei for diagnostic and/or therapeutic purposes. Our present study focused on the screening of a phage-displayed nonimmune human single-chain Fv (scFv) antibody library against heat-killed B. mallei and B. pseudomallei for the generation of human scFv antibodies specific to the two pathogenic species of bacteria. Using two different panning procedures, we obtained seven different scFv phage antibodies that interacted with the heat-killed whole bacterial cells of B. mallei and B. pseudomallei. Our results demonstrate that panning of a human scFv antibody library against heat-killed whole bacterial cells may provide a valuable strategy for developing human monoclonal antibodies against the highly pathogenic bacteria.     

Actin-binding proteins from Burkholderia mallei and Burkholderia thailandensis can functionally compensate for the actin-based motility defect of a Burkholderia pseudomallei bimA mutant

Recently we identified a bacterial factor (BimA) required for actin-based motility of Burkholderia pseudomallei. Here we report that Burkholderia mallei and Burkholderia thailandensis are capable of actin-based motility in J774.2 cells and that BimA homologs of these bacteria can restore the actin-based motility defect of a B. pseudomallei bimA mutant. While the BimA homologs differ in their amino-terminal sequence, they interact directly with actin in vitro and vary in their ability to bind Arp3.     

Plasmacytoid dendritic cell bactericidal activity against Burkholderia pseudomallei

Melioidosis sepsis, caused by Burkholderia pseudomallei, is associated with high mortality due to an overwhelming inflammatory response. Plasmacytoid dendritic cells (pDC) are potent producers of type I interferons (IFN). This study investigated whether pDC and type I IFN play a role during the early stages of B. pseudomallei infection. Human and murine pDC internalised and killed B. pseudomallei as efficiently as murine conventional DC (cDC). pDC derived from B. pseudomallei-susceptible (BALB/c) mice demonstrated poor intracellular killing and increased IFN-alpha compared to pDC derived from B. pseudomallei-resistant (C57BL/6) mice. This is the first evidence of pDC bactericidal activity against B. pseudomallei infection.     

Burkholderia multivorans acts as an antagonist against the growth of Burkholderia pseudomallei in soil

In this study, it was demonstrated, by using agar diffusion tests and a Transwell system, that Burkholderia multivorans NKI379 has an antagonistic effect against the growth of B. pseudomallei. Bacterial representatives were isolated from agricultural crop soil and mixed to construct a partial bacterial community structure that was based on the results of reproducible patterns following PCR-denaturing gradient gel electrophoresis analysis of total soil chromosomes. The antagonistic effect of B. multivorans on B. pseudomallei was observed in this imitate community. In a field study of agricultural crop soil, the presence of B. pseudomallei was inversely related to the presence of the antagonistic strains B. multivorans or B. cenocepacia. B. multivorans NKI379 can survive in a broader range of pH, temperatures and salt concentrations than B. pseudomallei, suggesting that B. multivorans can adapt to extreme environmental changes and therefore predominates over B. pseudomallei in natural environments.     

Antigenic relatedness between Burkholderia pseudomallei and Burkholderia mallei

Burkholderia pseudomallei and Burkholderia mallei are causative agents of distinct diseases, namely, melioidosis and glanders, respectively. The two species are very closely related, based on DNA-DNA homology, base sequence of the 16S rRNA, and phenotypic characteristics. Based on the use of polyclonal antisera, B. pseudomallei and B. mallei are also found to be antigenically closely related to one another. We previously reported the production of monoclonal antibodies (MAbs) against B. pseudomallei antigens; one group was specific for the 200-kDa exopolysaccharide present on the surface of all B. pseudomallei isolates, and the other was specific for the lipopolysaccharide (LPS) structure present on more than 95% of the B. pseudomallei tested. In the present study, we showed that the MAbs against 200-kDa antigen of B. pseudomallei cross-reacted with a component present also in some B. mallei isolates (3/6), but the positive immunoblot reaction was noted below the 200-kDa position. On the other hand, none of the six B. mallei isolates reacted with the MAb specific for B. pseudomallei LPS. It was of interest to observe that only the 3 exopolysaccharide-positive B. mallei isolates reacted with a commercial MAb against B. mallei LPS. The data presented suggest that B. mallei can be classified antigenically into two types based on their reactivities with different MAbs, i.e., the presence or absence of exopolysaccharide and the types of lipopolysaccharide. The heterogeneity of the LPS from these two closely related organisms is most likely related to the differences in its O-polysaccharide side chain.     

Burkholderia thailandensis outer membrane vesicles exert antimicrobial activity against drug-resistant and competitor microbial species

Gram-negative bacteria secrete outer membrane vesicles (OMVs) that play critical roles in intraspecies, interspecies, and bacteria-environment interactions. Some OMVs, such as those produced by Pseudomonas aeruginosa, have previously been shown to possess antimicrobial activity against competitor species. In the current study, we demonstrate that OMVs from Burkholderia thailandensis inhibit the growth of drug-sensitive and drug-resistant bacteria and fungi. We show that a number of antimicrobial compounds, including peptidoglycan hydrolases, 4-hydroxy-3-methyl-2-(2-non-enyl)-quinoline (HMNQ) and long-chain rhamnolipid are present in or tightly associate with B. thailandensis OMVs. Furthermore, we demonstrate that HMNQ and rhamnolipid possess antimicrobial and antibiofilm properties against methicillin-resistant Staphylococcus aureus (MRSA). These findings indicate that B. thailandensis secretes antimicrobial OMVs that may impart a survival advantage by eliminating competition. In addition, bacterial OMVs may represent an untapped resource of novel therapeutics effective against bio-film-forming and multidrug-resistant organisms.     

Burkholderia uboniae sp. nov., L-arabinose-assimilating but different from Burkholderia thailandensis and Burkholderia vietnamiensis

A polar multitrichous gram-negative motile rod, EY 3383, originally identified as Burkholderia thailandensis, revealed a DNA-DNA reassociation rate of 36.7%, under stringent conditions, with the type strain of B. thailandensis, despite the 16S rDNA homology value between two type strains being as high as 97.9%. The strain was clearly differentiated from the type strain of B. thailandensis by physiological, bio-chemical, and nutritional characteristics, without significant difference in cellular fatty acid and lipid composition. Based on the results of 16S rDNA sequence analysis, DNA-DNA hybridization and phenotypic characterization, Burkholderia uboniae sp. nov. is herein proposed. The type strain is NCTC 13147=EY 3383, isolated on 8 December 1989 from surface soil along the roadside in Ubon Ratchathani, Thailand. Major respiratory quinone is ubiquinone-8(Q8). G+C content of DNA is 69.71%.     

Immunobiological properties of Burkholderia pseudomallei and Burkholderia mallei capsular substances

The biopolymer composition, immunotropic and immunogenic properties of the fractions of B. pseudomallei and B. mallei were under study. The first two capsular fractions of these agents were found to be similar in their biopolymer composition that was indicative of their close relations. At the same time the causative agents of glanders proved to have decreased content of high molecular glycoproteids and LPS fragments. In the causative agents of melioidosis, capsular fractions K3 and K4 were characterized by the domination of proteins with a molecular weight of 42-25 kD. Fraction K4 in B. pseudomallei and fraction K1 in B. mallei had pronounced immunosuppressing properties ensuring the protection of encapsulated microbial cells in the body. The biopolymers forming fractions K1, K2, K3 in B. pseudomallei and fraction K2 in B. mallei were characterized by immunomodulating properties.