Biological relevance of colony morphology and phenotypic switching by Burkholderia pseudomallei

Melioidosis is a notoriously protracted illness and is difficult to cure. We hypothesize that the causative organism, Burkholderia pseudomallei, undergoes a process of adaptation involving altered expression of surface determinants which facilitates persistence in vivo and that this is reflected by changes in colony morphology. A colony morphotyping scheme and typing algorithm were developed using clinical B. pseudomallei isolates. Morphotypes were divided into seven types (denoted I to VII). Type I gave rise to other morphotypes (most commonly type II or III) by a process of switching in response to environmental stress, including starvation, iron limitation, and growth at 42 degrees C. Switching was associated with complex shifts in phenotype, one of which (type I to type II) was associated with a marked increase in production of factors putatively associated with in vivo concealment. Isogenic types II and III, derived from type I, were examined using several experimental models. Switching between isogenic morphotypes occurred in a mouse model, where type II appeared to become adapted for persistence in a low-virulence state. Isogenic type II demonstrated a significant increase in intracellular replication fitness compared with parental type I after uptake by epithelial cells in vitro. Isogenic type III demonstrated a higher replication fitness following uptake by macrophages in vitro, which was associated with a switch to type II. Mixed B. pseudomallei morphologies were common in individual clinical specimens and were significantly more frequent in samples of blood, pus, and respiratory secretions than in urine and surface swabs. These findings have major implications for therapeutics and vaccine development.     

Molecular analysis of T and B cell repertoires in mice immunized with Opisthorchis viverrini antigens.

B10 mice were immunized with an Opisthorchis viverrini somatic extract and then their responses were analyzed. The antigenic fractions of the extract were separated by SDS-polyacrylamide gel electrophoresis, electroblotted to nitrocellulose membranes and solubilized for use in lymphocyte culture. Antibody specificity was also visualized by immunoblotting using immunized mouse sera. The Mr of the main immunogenic fractions for T cells ranged from 28 to 46 kDa, whereas those recognized by antibodies were 45, 52, 56, 59, 65, 69, 75 and 81 kDa. The results indicate a striking difference in the antigenic recognition pattern of T and B cells which may be important for selecting antigen molecules for immunological studies of this trematode infection in man.     

Molecular identification and phylogenetic analysis of nuclear rDNA sequences among three opisthorchid liver fluke species (Opisthorchiidae: Trematoda)

In this study, we describe the development of a fast and accurate molecular identification system for human-associated liver fluke species (Opisthorchis viverrini, Opisthorchis felineus, and Clonorchis sinensis) using the PCR-RFLP analysis of the 18S-ITS1-5.8S nuclear ribosomal DNA region. Based on sequence variation in the target rDNA region, we selected three species-specific restriction enzymes within the ITS1 regions, generating different restriction profiles among the species: MunI for O. viverrini, NheI for O. felineus, and XhoI for C. sinensis, respectively. Each restriction enzyme generated different-sized fragments specific to the species examined, but no intraspecific polymorphism or cross-reaction between the species was detected in their restriction pattern. These results indicate that PCR-linked restriction analysis of the ITS1 region allows for the rapid and reliable molecular identification among these opisthorchid taxa. In addition, phylogenetic analysis of rDNA sequences using different methods (MP, ML, NJ, and Bayesian inference) displayed O. viverrini and O. felineus as a sister group, but this relationship was not strongly supported. The failure of recovering a robust phylogeny may be due to the relatively small number of synapomorphic characters shared among the species, yielding weak phylogenetic signal. Alternatively, rapid speciation within a very short period time could be another explanation for the relatively poorly resolved relationships among these species. Our data are insufficient for discriminating between sudden cladogenesis and other potential causes of poor resolution. Further information from independent loci might help resolve this phylogeny.     

Comparative in vivo and in vitro analyses of putative virulence factors of Burkholderia pseudomallei using lipopolysaccharide, capsule and flagellin mutants

Burkholderia pseudomallei is a gram-negative bacillus that is the causative agent of melioidosis. We evaluated host–pathogen interaction at different levels using three separate B. pseudomallei mutants generated by insertional inactivation. One of these mutants is defective in the production of the polysaccharide side chains associated with lipopolysaccharide; one does not produce the capsular polysaccharide with the structure -3)-2- O -acetyl-6-deoxy-β- d - manno -heptopyranose-(1-; and the third mutant does not produce flagellin. We compared the in vivo virulence in BALB/c mice, the in vitro fate of intracellular survival inside human polymorphonuclear cells (PMNs) and macrophages (Mφs) and the susceptibility to killing by 30% normal human serum, reactive nitrogen and oxygen intermediates and antimicrobial peptides with that of their wild-type counterpart. The lipopolysaccharide and capsule mutants demonstrated a marked reduction in virulence for BALB/c mice, but the flagellin mutant was only slightly less virulent than the parent strain. The results from the BALB/c mice experiments correlated with survival in Mφs. The lipopolysaccharide and capsule mutants were also more susceptible to killing by antimicrobial agents. All bacteria were equally susceptible to killing by PMNs. Altogether, the data suggest that lipopolysaccharide and capsule and, to a much lesser extent, flagella, are most likely associated with the virulence of this bacterium and highlight the importance of intracellular killing by PMNs and Mφs in disease pathogenesis.