Systematic mutagenesis of genes encoding predicted autotransported proteins of Burkholderia pseudomallei identifies factors mediating virulence in mice, net intracellular replication and a novel protein conferring serum resistance.

Burkholderia pseudomallei is the causative agent of the severe tropical disease melioidosis, which commonly presents as sepsis. The B. pseudomallei K96243 genome encodes eleven predicted autotransporters, a diverse family of secreted and outer membrane proteins often associated with virulence. In a systematic study of these autotransporters, we constructed insertion mutants in each gene predicted to encode an autotransporter and assessed them for three pathogenesis-associated phenotypes: virulence in the BALB/c intra-peritoneal mouse melioidosis model, net intracellular replication in J774.2 murine macrophage-like cells and survival in 45% (v/v) normal human serum. From the complete repertoire of eleven autotransporter mutants, we identified eight mutants which exhibited an increase in median lethal dose of 1 to 2-log10 compared to the isogenic parent strain (bcaA, boaA, boaB, bpaA, bpaC, bpaE, bpaF and bimA). Four mutants, all demonstrating attenuation for virulence, exhibited reduced net intracellular replication in J774.2 macrophage-like cells (bimA, boaB, bpaC and bpaE). A single mutant (bpaC) was identified that exhibited significantly reduced serum survival compared to wild-type. The bpaC mutant, which demonstrated attenuation for virulence and net intracellular replication, was sensitive to complement-mediated killing via the classical and/or lectin pathway. Serum resistance was rescued by in trans complementation. Subsequently, we expressed recombinant proteins of the passenger domain of four predicted autotransporters representing each of the phenotypic groups identified: those attenuated for virulence (BcaA), those attenuated for virulence and net intracellular replication (BpaE), the BpaC mutant with defects in virulence, net intracellular replication and serum resistance and those displaying wild-type phenotypes (BatA). Only BcaA and BpaE elicited a strong IFN-γ response in a restimulation assay using whole blood from seropositive donors and were recognised by seropositive human sera from the endemic area. To conclude, several predicted autotransporters contribute to B. pseudomallei virulence and BpaC may do so by conferring resistance against complement-mediated killing.

Comparative experimental subcutaneous glanders and melioidosis in the common marmoset (Callithrix jacchus).

Glanders and melioidosis are caused by two distinct Burkholderia species and have generally been considered to have similar disease progression. While both of these pathogens are HHS/CDC Tier 1 agents, natural infection with both these pathogens is primarily through skin inoculation. The common marmoset (Callithrix jacchus) was used to compare disease following experimental subcutaneous challenge. Acute, lethal disease was observed in marmosets following challenge with between 26 and 1.2 × 10(8) cfu Burkholderia pseudomallei within 22-85 h. The reproducibility and progression of the disease were assessed following a challenge of 1 × 10(2) cfu of B. pseudomallei. Melioidosis was characterised by high levels of bacteraemia, focal microgranuloma progressing to non-necrotic multifocal solid lesions in the livers and spleens and multi-organ failure. Lethal disease was observed in 93% of animals challenged with Burkholderia mallei, occurring between 5 and 10.6 days. Following challenge with 1 × 10(2) cfu of B. mallei, glanders was characterised with lymphatic spread of the bacteria and non-necrotic, multifocal solid lesions progressing to a multifocal lesion with severe necrosis and pneumonia. The experimental results confirmed that the disease pathology and presentation is strikingly different between the two pathogens. The marmoset provides a model of the human syndrome for both diseases facilitating the development of medical countermeasures.

Correction: Identification of a Predicted Trimeric Autotransporter Adhesin Required for Biofilm Formation of Burkholderia pseudomallei.

[This corrects the article DOI: 10.1371/journal.pone.0079461.].

Identification of a predicted trimeric autotransporter adhesin required for biofilm formation of Burkholderia pseudomallei.

The autotransporters are a large and diverse family of bacterial secreted and outer membrane proteins, which are present in many Gram-negative bacterial pathogens and play a role in numerous environmental and virulence-associated interactions. As part of a larger systematic study on the autotransporters of Burkholderia pseudomallei, the causative agent of the severe tropical disease melioidosis, we have constructed an insertion mutant in the bpss1439 gene encoding an unstudied predicted trimeric autotransporter adhesin. The bpss1439 mutant demonstrated a significant reduction in biofilm formation at 48 hours in comparison to its parent 10276 wild-type strain. This phenotype was complemented to wild-type levels by the introduction of a full-length copy of the bpss1439 gene in trans. Examination of the wild-type and bpss1439 mutant strains under biofilm-inducing conditions by microscopy after 48 hours confirmed that the bpss1439 mutant produced less biofilm compared to wild-type. Additionally, it was observed that this phenotype was due to low levels of bacterial adhesion to the abiotic surface as well as reduced microcolony formation. In a murine melioidosis model, the bpss1439 mutant strain demonstrated a moderate attenuation for virulence compared to the wild-type strain. This attenuation was abrogated by in trans complementation, suggesting that bpss1439 plays a subtle role in the pathogenesis of B. pseudomallei. Taken together, these studies indicate that BPSS1439 is a novel predicted autotransporter involved in biofilm formation of B. pseudomallei; hence, this factor was named BbfA, Burkholderia biofilm factor A.

Characterization of the Burkholderia pseudomallei K96243 capsular polysaccharide I coding region.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic to regions of Southeast Asia and Northern Australia. Both humans and a range of other animal species are susceptible to melioidosis, and the production of a group 3 polysaccharide capsule in B. pseudomallei is essential for virulence. B. pseudomallei capsular polysaccharide (CPS) I comprises unbranched manno-heptopyranose residues and is encoded by a 34.5-kb locus on chromosome 1. Despite the importance of this locus, the role of all of the genes within this region is unclear. We inactivated 18 of these genes and analyzed their phenotype using Western blotting and immunofluorescence staining. Furthermore, by combining this approach with bioinformatic analysis, we were able to develop a model for CPS I biosynthesis and export. We report that inactivating gmhA, wcbJ, and wcbN in B. pseudomallei K96243 retains the immunogenic integrity of the polysaccharide despite causing attenuation in the BALB/c murine infection model. Mice immunized with the B. pseudomallei K96243 mutants lacking a functional copy of either gmhA or wcbJ were afforded significant levels of protection against a wild-type B. pseudomallei K96243 challenge.

Development of an acute model of inhalational melioidosis in the common marmoset (Callithrix jacchus).

Studies of inhalational melioidosis were undertaken in the common marmoset (Callithrix jacchus). Following exposure to an inhaled challenge with aerosolized Burkholderia pseudomallei, lethal infection was observed in marmosets challenged with doses below 10 cfu; a precise LD(50) determination was not possible. The model was further characterized using a target challenge dose of approximately 10(2) cfu. A separate pathogenesis time-course experiment was also conducted. All animals succumbed, between 27 and 78 h postchallenge. The challenge dose received and the time to the humane endpoint (1 °C below normal body temperature postfever) were correlated. The first indicator of disease was an increased core body temperature (T(c) ), at 22 h postchallenge. This coincided with bacteraemia and bacterial dissemination. Overt clinical signs were first observed 3-5 h later. A sharp decrease (typically within 3-6 h) in the T(c) was observed prior to humanely culling the animals in the lethality study. Pathology was noted in the lung, liver and spleen. Disease progression in the common marmoset appears to be consistent with human infection in terms of bacterial spread, pathology and physiology. The common marmoset can therefore be considered a suitable animal model for further studies of inhalational melioidosis.

The cluster 1 type VI secretion system is a major virulence determinant in Burkholderia pseudomallei.

The Burkholderia pseudomallei K96243 genome encodes six type VI secretion systems (T6SSs), but little is known about the role of these systems in the biology of B. pseudomallei. In this study, we purified recombinant Hcp proteins from each T6SS and tested them as vaccine candidates in the BALB/c mouse model of melioidosis. Recombinant Hcp2 protected 80% of mice against a lethal challenge with K96243, while recombinant Hcp1, Hcp3, and Hcp6 protected 50% of mice against challenge. Hcp6 was the only Hcp constitutively produced by B. pseudomallei in vitro; however, it was not exported to the extracellular milieu. Hcp1, on the other hand, was produced and exported in vitro when the VirAG two-component regulatory system was overexpressed in trans. We also constructed six hcp deletion mutants (Δhcp1 through Δhcp6) and tested them for virulence in the Syrian hamster model of infection. The 50% lethal doses (LD(50)s) for the Δhcp2 through Δhcp6 mutants were indistinguishable from K96243 (<10 bacteria), but the LD(50) for the Δhcp1 mutant was >10(3) bacteria. The hcp1 deletion mutant also exhibited a growth defect in RAW 264.7 macrophages and was unable to form multinucleated giant cells in this cell line. Unlike K96243, the Δhcp1 mutant was only weakly cytotoxic to RAW 264.7 macrophages 18 h after infection. The results suggest that the cluster 1 T6SS is essential for virulence and plays an important role in the intracellular lifestyle of B. pseudomallei.

Deletion of the Bacillus anthracis capB homologue in Francisella tularensis subspecies tularensis generates an attenuated strain that protects mice against virulent tularaemia.

As there is currently no licensed vaccine against Francisella tularensis, the causative agent of tularaemia, the bacterium is an agent of concern as a potential bioweapon. Although F. tularensis has a low infectious dose and high associated mortality, it possesses few classical virulence factors. An analysis of the F. tularensis subspecies tularensis genome sequence has revealed the presence of a region containing genes with low sequence homology to part of the capBCADE operon of Bacillus anthracis. We have generated an isogenic capB mutant of F. tularensis subspecies tularensis SchuS4 and shown it to be attenuated. Furthermore, using BALB/c mice, we have demonstrated that this capB strain affords protection against significant homologous challenge with the wild-type strain. These data have important implications for the development of a defined and efficacious tularaemia vaccine.

Bioavailability and efficacy of levofloxacin against Francisella tularensis in the common marmoset (Callithrix jacchus).

Pharmacokinetic and efficacy studies with levofloxacin were performed in the common marmoset (Callithrix jacchus) model of inhalational tularemia. Plasma levofloxacin pharmacokinetics were determined in six animals in separate single-dose and multidose studies. Plasma drug concentrations were analyzed using liquid chromatography-tandem mass spectrometry-electrospray ionization. On day 7 of a twice-daily dosing regimen of 40 mg/kg, the levofloxacin half-life, maximum concentration, and area under the curve in marmoset plasma were 2.3 h, 20.9 microg/ml, and 81.4 microg/liter/h, respectively. An efficacy study was undertaken using eight treated and two untreated control animals. Marmosets were challenged with a mean of 1.5 x 10(2) CFU of Francisella tularensis by the airborne route. Treated animals were administered 16.5 mg/kg levofloxacin by mouth twice daily, based on the pharmacokinetic parameters, beginning 24 h after challenge. Control animals had a raised core body temperature by 57 h postchallenge and died from infection by day 5. All of the other animals survived, remained afebrile, and lacked overt clinical signs. No bacteria were recovered from the organs of these animals at postmortem after culling at day 24 postchallenge. In conclusion, postexposure prophylaxis with orally administered levofloxacin was efficacious against acute inhalational tularemia in the common marmoset. The marmoset appears to be an appropriate animal model for the evaluation of postexposure therapies.

Characterization of lethal inhalational infection with Francisella tularensis in the common marmoset (Callithrix jacchus).

The intracellular Gram-negative pathogen Francisella tularensis is the causative agent of tularaemia and is prevalent in many countries in the northern hemisphere. To determine whether the common marmoset (Callithrix jacchus) would be a suitable non-human primate model of inhalational tularaemia, a pathophysiology study was undertaken. Ten animals were challenged with approximately 10(2) c.f.u. F. tularensis strain SCHU S4 (F. tularensis subsp. tularensis). To look for trends in the infection, pairs of animals were sacrificed at 24 h intervals between 0 and 96 h post-challenge and blood and organs were assessed for bacteriology, pathology and haematological and immunological parameters. The first indication of infection was a raised core temperature at 3 days post-challenge. This coincided with a number of other factors: a rapid increase in the number of bacteria isolated from all organs, more pronounced gross pathology and histopathology, and an increase in the immunological response. As the disease progressed, higher bacterial and cytokine levels were detected. More extensive pathology was observed, with multifocal lesions seen in the lungs, liver and spleen. Disease progression in the common marmoset appears to be consistent with human clinical and pathological features of tularaemia, indicating that this may be a suitable animal model for the investigation of novel medical interventions such as vaccines or therapeutics.