Correction: Two stable variants of Burkholderia pseudomallei strain MSHR5848 express broadly divergent in vitro phenotypes associated with their virulence differences.

[This corrects the article DOI: 10.1371/journal.pone.0171363.].

Two stable variants of Burkholderia pseudomallei strain MSHR5848 express broadly divergent in vitro phenotypes associated with their virulence differences.

Burkholderia pseudomallei (Bp), the agent of melioidosis, causes disease ranging from acute and rapidly fatal to protracted and chronic. Bp is highly infectious by aerosol, can cause severe disease with nonspecific symptoms, and is naturally resistant to multiple antibiotics. However, no vaccine exists. Unlike many Bp strains, which exhibit random variability in traits such as colony morphology, Bp strain MSHR5848 exhibited two distinct and relatively stable colony morphologies on sheep blood agar plates: a smooth, glossy, pale yellow colony and a flat, rough, white colony. Passage of the two variants, designated "Smooth" and "Rough", under standard laboratory conditions produced cultures composed of > 99.9% of the single corresponding type; however, both could switch to the other type at different frequencies when incubated in certain nutritionally stringent or stressful growth conditions. These MSHR5848 derivatives were extensively characterized to identify variant-associated differences. Microscopic and colony morphology differences on six differential media were observed and only the Rough variant metabolized sugars in selective agar. Antimicrobial susceptibilities and lipopolysaccharide (LPS) features were characterized and phenotype microarray profiles revealed distinct metabolic and susceptibility disparities between the variants. Results using the phenotype microarray system narrowed the 1,920 substrates to a subset which differentiated the two variants. Smooth grew more rapidly in vitro than Rough, yet the latter exhibited a nearly 10-fold lower lethal dose for mice than Smooth. Finally, the Smooth variant was phagocytosed and replicated to a greater extent and was more cytotoxic than Rough in macrophages. In contrast, multiple locus sequence type (MLST) analysis, ribotyping, and whole genome sequence analysis demonstrated the variants' genetic conservation; only a single consistent genetic difference between the two was identified for further study. These distinct differences shown by two variants of a Bp strain will be leveraged to better understand the mechanism of Bp phenotypic variability and to possibly identify in vitro markers of infection.

The impact of inducing germination of Bacillus anthracis and Bacillus thuringiensis spores on potential secondary decontamination strategies.

AIMS: Decontamination and remediation of a site contaminated by the accidental or intentional release of fully virulent Bacillus anthracis spores are difficult, costly and potentially damaging to the environment. Development of novel decontamination strategies that have minimal environmental impacts remains a high priority. Although ungerminated spores are amongst the most resilient organisms known, once exposed to germinants, the germinating spores, in some cases, become susceptible to antimicrobial environments. We evaluated the concept that once germinated, B. anthracis spores would be less hazardous and significantly easier to remediate than ungerminated dormant spores. METHODS AND RESULTS: Through in vitro germination and sensitivity assays, we demonstrated that upon germination, B. anthracis Ames spores and Bacillus thuringiensis Al Hakam spores (serving as a surrogate for B. anthracis) become susceptible to environmental stressors. The majority of these germinated B. anthracis and B. thuringiensis spores were nonviable after exposure to a defined minimal germination-inducing solution for prolonged periods of time. Additionally, we examined the impact of potential secondary disinfectant strategies including bleach, hydrogen peroxide, formaldehyde and artificial UV-A, UV-B and UV-C radiation, employed after a 60-min germination-induction step. Each secondary disinfectant employs a unique mechanism of killing; as a result, germination-induction strategies are better suited for some secondary disinfectants than others. CONCLUSIONS: These results provide evidence that the deployment of an optimal combination strategy of germination-induction/secondary disinfection may be a promising aspect of wide-area decontamination following a B. anthracis contamination event. SIGNIFICANCE AND IMPACT OF THE STUDY: By inducing spores to germinate, our data confirm that the resulting cells exhibit sensitivities that can be leveraged when paired with certain decontamination measures. This increased susceptibility could be exploited to devise more efficient and safe decontamination measures and may obviate the need for more stringent methods that are currently in place.

D-cycloserine or similar physiochemical compounds may be uniquely suited for use in Bacillus anthracis spore decontamination strategies.

AIMS: As observed in the aftermath of the anthrax attacks of 2001, decontamination and remediation of a site contaminated by the accidental or intentional release of Bacillus anthracis spores is difficult, costly and potentially damaging to the environment. The identification of novel strategies that neutralize the threat of spores while minimizing environmental damage remains a high priority. We investigated the efficacy of d-cycloserine (DCS), an antibiotic and inhibitor of the spore-associated enzyme (alanine racemase) responsible for converting l-alanine to d-alanine, as a spore germination enhancer and antimicrobial agent. METHODS AND RESULTS: We characterized the impact of DCS exposure on both germinating spores and vegetative cells of fully virulent B. anthracis by evaluating spore germination kinetics, determining the minimum inhibitory concentrations (MICs) required to affect growth of the bacteria and performing macrophage viability assays. DCS enhanced germination induced by l-alanine and also efficiently killed the newly germinated spores. Furthermore, DCS proved nontoxic to macrophages at concentrations that provided protection from the killing effects of spores. Similar tests were conducted with Bacillus thuringiensis (subspecies kurstaki and Al Hakam) to determine its potential as a possible surrogate for B. anthracis field trials. Bacillus thuringiensis spores responded in a similar manner to B. anthracis spores when exposed to DCS. CONCLUSIONS: These results further support that DCS augments the germination response of spores in the presence of l-alanine but also reveal that DCS is bactericidal towards germinating spores. SIGNIFICANCE AND IMPACT OF THE STUDY: DCS (or similar compounds) may be uniquely suited for use as part of decontamination strategies by augmenting the induction of spore germination and then rendering the germinated spores nonviable.

Characterization of a multi-component anthrax vaccine designed to target the initial stages of infection as well as toxaemia.

Current vaccine approaches to combat anthrax are effective; however, they target only a single protein [the protective antigen (PA) toxin component] that is produced after spore germination. PA production is subsequently increased during later vegetative cell proliferation. Accordingly, several aspects of the vaccine strategy could be improved. The inclusion of spore-specific antigens with PA could potentially induce protection to initial stages of the disease. Moreover, adding other epitopes to the current vaccine strategy will decrease the likelihood of encountering a strain of Bacillus anthracis (emerging or engineered) that is refractory to the vaccine. Adding recombinant spore-surface antigens (e.g. BclA, ExsFA/BxpB and p5303) to PA has been shown to augment protection afforded by the latter using a challenge model employing immunosuppressed mice challenged with spores derived from the attenuated Sterne strain of B. anthracis. This report demonstrated similar augmentation utilizing guinea pigs or mice challenged with spores of the fully virulent Ames strain or a non-toxigenic but encapsulated ΔAmes strain of B. anthracis, respectively. Additionally, it was shown that immune interference did not occur if optimal amounts of antigen were administered. By administering the toxin and spore-based immunogens simultaneously, a significant adjuvant effect was also observed in some cases. Thus, these data further support the inclusion of recombinant spore antigens in next-generation anthrax vaccine strategies.

Humanized theta-defensins (retrocyclins) enhance macrophage performance and protect mice from experimental anthrax infections.

Retrocyclins are humanized versions of the -defensin peptides expressed by the leukocytes of several nonhuman primates. Previous studies, performed in serum-free media, determined that retrocyclins 1 (RC1) and RC2 could prevent successful germination of Bacillus anthracis spores, kill vegetative B. anthracis cells, and inactivate anthrax lethal factor. We now report that retrocyclins are extensively bound by components of native mouse, human, and fetal calf sera, that heat-inactivated sera show greatly enhanced retrocyclin binding, and that native and (especially) heat-inactivated sera greatly reduce the direct activities of retrocyclins against spores and vegetative cells of B. anthracis. Nevertheless, we also found that retrocyclins protected mice challenged in vivo by subcutaneous, intraperitoneal, or intranasal instillation of B. anthracis spores. Retrocyclin 1 bound extensively to B. anthracis spores and enhanced their phagocytosis and killing by murine RAW264.7 cells. Based on the assumption that spore-bound RC1 enters phagosomes by "piggyback phagocytosis," model calculations showed that the intraphagosomal concentration of RC1 would greatly exceed its extracellular concentration. Murine alveolar macrophages took up fluorescently labeled retrocyclin, suggesting that macrophages may also acquire extracellular RC1 directly. Overall, these data demonstrate that retrocyclins are effective in vivo against experimental murine anthrax infections and suggest that enhanced macrophage function contributes to this property.

Processing, assembly and localization of a Bacillus anthracis spore protein.

All Bacillus spores are encased in macromolecular shells. One of these is a proteinacious shell called the coat that, in Bacillus subtilis, provides critical protective functions. The Bacillus anthracis spore is the infectious particle for the disease anthrax. Therefore, the coat is of particular interest because it may provide essential protective functions required for the appearance of anthrax. Here, we analyse a protein component of the spore outer layers that was previously designated BxpA. Our data indicate that a significant amount of BxpA is located below the spore coat and associated with the cortex. By SDS-PAGE, BxpA migrates as a 9 kDa species when extracted from Sterne strain spores, and as 11 and 14 kDa species from Ames strain spores, even though it has predicted masses of 27 and 29 kDa, respectively, in these two strains. We investigated the possibility that BxpA is subject to post-translational processing as previously suggested. In B. subtilis, a subset of coat proteins is proteolysed or cross-linked by the spore proteins YabG or Tgl, respectively. To investigate the possibility that similar processing occurs in B. anthracis, we generated mutations in the yabG or tgl genes in the Sterne and Ames strains and analysed the consequences for BxpA assembly by SDS-PAGE. We found that in a tgl mutant of B. anthracis, the apparent mass of BxpA increased. This is consistent with the possibility that Tgl directs the cross-linking of BxpA into a form that normally does not enter the gel. Unexpectedly, the apparent mass of BxpA also increased in a yabG mutant, suggesting a relatively complex role for proteolysis in spore protein maturation in B. anthracis. These data reveal a previously unobserved event in spore protein maturation in B. anthracis. We speculate that proteolysis and cross-linking are ubiquitous spore assembly mechanisms throughout the genus Bacillus.

Effects of altering the germination potential of Bacillus anthracis spores by exogenous means in a mouse model.

Inhalational anthrax is the most severe form of anthrax. It has been shown in small-animal and non-human primate models that relatively large pools of ungerminated Bacillus anthracis spores can remain within the alveolar spaces for days to weeks post-inhalation or until transported to areas more favourable for germination and bacillary outgrowth. In this study, spores of the Ames strain that were exposed to germination-inducing media prior to intranasal delivery were significantly less infectious than spores delivered in either water or germination-inhibitory medium. The effect of manipulating the germination potential of these spores within the lungs of infected mice by exogenous germination-altering media was examined. The data suggested that neither inducing germination nor inhibiting germination of spores within the lungs protected mice from the ensuing infection. Germination-altering strategies could, instead, significantly increase the severity of disease in a mouse model of inhalational anthrax when implemented in vivo. It was shown that germination-altering strategies, in this study, were not beneficial to the infected host and are impractical as in vivo countermeasures.

Analysis of a novel spore antigen in Bacillus anthracis that contributes to spore opsonization.

The significance of Bacillus anthracis as an agent of bioterrorism has been well established. An understanding of both the pathogenesis and the host response is required to elucidate approaches to more rapidly detect and effectively prevent or treat anthrax. Current vaccine strategies are focused primarily on production of antibodies against the protective antigen components of the anthrax toxins, which are secreted by the bacilli. A better understanding of the dynamic morphology of the dormant and germinating spore and its interaction with the host immune system could be important in developing an optimally efficacious anthrax vaccine. A spore-associated protein was identified that was specific to the Bacillus cereus group of bacteria and referred to as spore opsonization-associated antigen A (SoaA). Immuno-electron microscopy localized this protein to the area of the cortex beneath the coat of the dormant spore. Although our data suggested that SoaA was found below the coat layers of the ungerminated spore, SoaA was involved in the interaction of spores with macrophages shortly after infection. To investigate further the specific properties of the SoaA protein, the soaA gene was inactivated in the B. anthracis Ames strain. The SoaA protein in the Ames strain of B. anthracis increased the phagocytic uptake of the spores in the presence of anti-spore antibodies. Unlike the wild-type strain, the mutant soaA : : Kan strain was not readily opsonized by anti-spore antibodies. While the mutant spores retained characteristic resistance properties in vitro and virulence in vivo, the soaA : : Kan mutant strain was significantly less suited for survival in vivo when competed against the wild-type Ames strain.

Fully virulent Bacillus anthracis does not require the immunodominant protein BclA for pathogenesis.

The BclA protein is the immunodominant epitope on the surface of Bacillus anthracis spores; however, its roles in pathogenesis are unclear. We constructed a BclA deletion mutant (bclA) of the fully virulent Ames strain. This derivative retained full virulence in several small-animal models of infection despite the bclA deletion.

Expression of sialylated or paragloboside-like lipooligosaccharides are not required for pustule formation by Haemophilus ducreyi in human volunteers.

The lipooligosaccharide (LOS) of Haemophilus ducreyi, the etiologic agent of chancroid, chemically and immunologically resembles human glycosphingolipid antigens. To test whether LOS that contains paragloboside-like structures was required for pustule formation, an isogenic mutant (35000HP-RSM2) was constructed in losB, which encodes D-glycero-D-manno-heptosyltransferase. 35000HP-RSM2 produces a truncated LOS whose major glycoform terminates in a single glucose attached to a heptose trisaccharide core and 2-keto-3-deoxyoctulosonic acid. Five human subjects were inoculated with 35000HP and 35000HP-RSM2 in a dose-response trial. For estimated delivered doses (EDDs) of >/=25 CFU, the pustule formation rates were 80% for 35000HP and 58% for 35000HP-RSM2. Preliminary data indicated that a previously described Tn916 losB mutant made a minor glycoform that does not require DD-heptose to form the terminal N-acetyllactosamine. If 35000HP-RSM2 made this glycoform, then 35000HP-RSM2 could theoretically make a sialylated glycoform. To test whether sialylated LOS was required for pustule formation, a second trial comparing an isogenic sialyltransferase mutant (35000HP-RSM203) to 35000HP was performed in five additional subjects. For EDDs of >/=25 CFU, the pustule formation rates were 30% for both 35000HP and 35000HP-RSM203. The histopathology and recovery rates of H. ducreyi from surface cultures and biopsies obtained from mutant and parent sites in both trials were similar. These results indicate that neither the expression of a major glycoform resembling paragloboside nor sialylated LOS is required for pustule formation by H. ducreyi in humans.

Haemophilus ducreyi produces a novel sialyltransferase. Identification of the sialyltransferase gene and construction of mutants deficient in the production of the sialic acid-containing glycoform of the lipooligosaccharide.

Haemophilus ducreyi, the cause of the sexually transmitted disease chancroid produces a lipooligosaccharide (LOS) containing a terminal sialyl N-acetyllactosamine trisaccharide. Previously, we reported the identification and characterization of the N-acetylneuraminic acid cytidylsynthetase gene (neuA). Forty-nine base pairs downstream of the synthetase gene is an open reading frame (ORF) encoding a protein with a predicted molecular weight of 34,646. This protein has weak homology to the polysialyltransferase of Escherichia coli K92. Downstream of this ORF is the gene encoding the H. ducreyi homologue of the Salmonella typhimurium rmlB gene. Mutations were constructed in the neuA gene and the gene encoding the second ORF by insertion of an Omega kanamycin cassette, and isogenic strains were constructed. LOS was isolated from each strain and characterized by SDS-polyacrylamide gel electrophoresis, carbohydrate, and mass spectrometric analysis. LOS isolated from strains containing a mutation in neuA or in the second ORF, designated lst, lacked the sialic acid-containing glycoform. Complementation studies were performed. The neuA gene and the lst gene were each cloned into the shuttle vector pLS88 after polymerase chain reaction amplification. Complementation of the mutation in the lst gene was observed, but we were unable to complement the neuA mutation. Since it is possible that transcription of the neuA gene and the lst gene were coupled, we constructed a nonpolar mutation in the neuA gene. In this construct, the neuA mutation was complemented, suggesting transcriptional coupling of the neuA gene and the lst gene. Sialyltransferase activity was detected by incorporation of 14C-labeled NeuAc from CMP-NeuAc into trichloroacetic acid-precipitable material when the lst gene was overexpressed in the nonpolar neuA mutant. We conclude that the lst gene encodes the H. ducreyi sialyltransferase. Since the lst gene product has little, if any, structural relationship to other sialyltransferases, this protein represents a new class of sialyltransferase.

Facile construction of mutations in Haemophilus ducreyi using lacZ as a counter-selectable marker.

Haemophilus ducreyi is a Gram-negative bacterium which is the causative agent of chancroid, an ulcerative sexually transmitted disease. In order to understand the pathogenesis of H. ducreyi disease, studies designed to identify potential virulence determinants and construct mutants deficient in the elaboration of these determinants have been undertaken in several laboratories. At the present time, construction of isogenic mutants is accomplished by electroporation of linearized DNA containing insertionally inactivated H. ducreyi genes followed by selection for the resistance marker encoded on the inactivated gene. In our experience, certain mutants are difficult to construct using this procedure. In the construction of strains containing lacZ as a reporter gene, we observed that the growth of lacZ expressing H. ducreyi was inhibited in the presence of X-gal. We have exploited this observation to develop a new strategy for the construction of isogenic H. ducreyi mutants.

Interaction of Legionella pneumophila with Acanthamoeba castellanii: uptake by coiling phagocytosis and inhibition of phagosome-lysosome fusion.

Legionella pneumophila is a facultative intracellular parasite able to survive within both human monocytes and amoebae. We have demonstrated that processing of L. pneumophila by the free-living amoeba Acanthamoeba castellanii shows many similarities to the processing of L. pneumophila by monocytes. These similarities include uptake of L. pneumophila by coiling phagocytosis and the subsequent confinement of L. pneumophila in a ribosome-studded phagosome. In addition, as in monocytes, inhibition of lysosomal fusion with phagosomes containing L. pneumophila was detected in amoebae. With all clinical isolates, inhibition of phagosomes-lysosome fusion correlated with virulence. However, with one of the environmental isolates tested, no significant difference in phagosome-lysosome fusion was observed between the virulent and avirulent forms. These results indicate that the avirulent form of this isolate differed from the virulent form in some other respect critical to intracellular survival. Therefore, intracellular multiplication of L. pneumophila within A. castellanii may not be solely dependent upon the inhibition of lysosomal fusion.