Linocin and OmpW Are Involved in Attachment of the Cystic Fibrosis-Associated Pathogen Burkholderia cepacia Complex to Lung Epithelial Cells and Protect Mice against Infection.

Members of the Burkholderia cepacia complex (Bcc) cause chronic opportunistic lung infections in people with cystic fibrosis (CF), resulting in a gradual lung function decline and, ultimately, patient death. The Bcc is a complex of 20 species and is rarely eradicated once a patient is colonized; therefore, vaccination may represent a better therapeutic option. We developed a new proteomics approach to identify bacterial proteins that are involved in the attachment of Bcc bacteria to lung epithelial cells. Fourteen proteins were reproducibly identified by two-dimensional gel electrophoresis from four Bcc strains representative of two Bcc species: Burkholderia cenocepacia, the most virulent, and B. multivorans, the most frequently acquired. Seven proteins were identified in both species, but only two were common to all four strains, linocin and OmpW. Both proteins were selected based on previously reported data on these proteins in other species. Escherichia coli strains expressing recombinant linocin and OmpW showed enhanced attachment (4.2- and 3.9-fold) to lung cells compared to the control, confirming that both proteins are involved in host cell attachment. Immunoproteomic analysis using serum from Bcc-colonized CF patients confirmed that both proteins elicit potent humoral responses in vivo Mice immunized with either recombinant linocin or OmpW were protected from B. cenocepacia and B. multivorans challenge. Both antigens induced potent antigen-specific antibody responses and stimulated strong cytokine responses. In conclusion, our approach identified adhesins that induced excellent protection against two Bcc species and are promising vaccine candidates for a multisubunit vaccine. Furthermore, this study highlights the potential of our proteomics approach to identify potent antigens against other difficult pathogens.

Immunoproteomic analysis of proteins expressed by two related pathogens, Burkholderia multivorans and Burkholderia cenocepacia, during human infection.

Burkholderia cepacia complex (Bcc) is an opportunistic bacterial pathogen that causes chronic infections in people with cystic fibrosis (CF). It is a highly antibiotic resistant organism and Bcc infections are rarely cleared from patients, once they are colonized. The two most clinically relevant species within Bcc are Burkholderia cenocepacia and Burkholderia multivorans. The virulence of these pathogens has not been fully elucidated and the virulence proteins expressed during human infection have not been identified to date. Furthermore, given its antibiotic resistance, prevention of infection with a prophylactic vaccine may represent a better alternative than eradication of an existing infection. We have compared the immunoproteome of two strains each from these two species of Bcc, with the aim of identifying immunogenic proteins which are common to both species. Fourteen immunoreactive proteins were exclusive to both B. cenocepacia strains, while 15 were exclusive to B. multivorans. A total of 15 proteins were immunogenic across both species. DNA-directed RNA polymerase, GroEL, 38kDa porin and elongation factor-Tu were immunoreactive proteins expressed by all four strains examined. Many proteins which were immunoreactive in both species, warrant further investigations in order to aid in the elucidation of the mechanisms of pathogenesis of this difficult organism. In addition, identification of some of these could also allow the development of protective vaccines which may prevent colonisation.

Gliotoxin effects on fungal growth: mechanisms and exploitation.

Although initially investigated for its antifungal properties, little is actually known about the effect of gliotoxin on Aspergillus fumigatus and other fungi. We have observed that exposure of A. fumigatus to exogenous gliotoxin (14 µg/ml), under gliotoxin-limited growth conditions, results in significant alteration of the expression of 27 proteins (up- and down-regulated >1.9-fold; p<0.05) including de novo expression of Cu, Zn superoxide dismutase, up-regulated allergen Asp f3 expression and down-regulated catalase and a peroxiredoxin levels. Significantly elevated glutathione GSH levels (p<0.05), along with concomitant resistance to diamide, were evident in A. fumigatus ΔgliT, lacking gliotoxin oxidoreductase, a gliotoxin self-protection gene. Saccharomyces cerevisiae deletents (Δsod1 and Δyap1) were hypersensitive to exogenous gliotoxin, while Δgsh1 was resistant. Significant gliotoxin-mediated (5 µg/ml) growth inhibition (p<0.001) of Aspergillus nidulans, Aspergillus terreus, Aspergillus niger, Cochliobolus heterostrophus and Neurospora crassa was also observed. Growth of Aspergillus flavus, Fusarium graminearum and Aspergillus oryzae was significantly inhibited (p<0.001) at gliotoxin (10 µg/ml), indicating differential gliotoxin sensitivity amongst fungi. Re-introduction of gliT into A. fumigatus ΔgliT, at a different locus (ctsD; AFUA_4G07040, an aspartic protease), with selection on gliotoxin, facilitated deletion of ctsD without use of additional antibiotic selection markers. Absence of ctsD expression was accompanied by restoration of gliT expression, and resistance to gliotoxin. Thus, we propose gliT/gliotoxin as a useful selection marker system for fungal transformation. Finally, we suggest incorporation of gliotoxin sensitivity assays into all future fungal functional genomic studies.

Single-pot derivatisation strategy for enhanced gliotoxin detection by HPLC and MALDI-ToF mass spectrometry.

Gliotoxin is produced by non-ribosomal peptide synthesis and secreted from certain fungi, including Aspergillus fumigatus. It is an epipolythiodioxopiperazine that contains an intact disulphide bridge and is the focus of intense research as a consequence of its negative immunomodulatory properties. Gliotoxin detection is generally enabled by reversed-phase-high-performance liquid chromatography (RP-HPLC), with absorbance detection (220-280 nm), or liquid chromatography-mass spectrometry, yet detection is not readily achievable by matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-ToF MS). We have developed a single-pot derivatisation strategy which uses sodium borohydride-mediated reduction of gliotoxin followed by immediate alkylation of exposed thiols by 5'-iodoacetamidofluorescein to yield a stable product, diacetamidofluorescein-gliotoxin (GT-(AF)(2)), of molecular mass 1103.931 Da ((M+H)+). This product is readily detectable by RP-HPLC and exhibits a 6.8-fold increase in molar absorptivity compared with gliotoxin, which results in a higher sensitivity of detection (40 ng; 125 pmoL). GT-(AF)(2) also fluoresces (excitation/emission, 492:518 nm). Unlike free gliotoxin, the product (>800 fmol) is detectable by MALDI-ToF MS. Sporidesmin A can also be detected by RP-HPLC and MALDI-ToF MS (>530 fmol) using this strategy. We also demonstrate that the strategy facilitates detection of gliotoxin (mean ± SD = 3.55 ± 0.07 µg 100 µL(-1); n = 2) produced by A. fumigatus, without the requirement for organic extraction of culture supernatants and associated solvent removal. GT-(AF)(2) is also detectable (150 ng; 460 pmol) by thin-layer chromatography.

Recombinant expression and immunological characterisation of proteins derived from human metapneumovirus.

BACKGROUND: Human metapneumovirus (HMPV) has been shown to cause respiratory infection, accounting for approximately 7% of all such disease, and contributes to the development of asthma in humans. HMPV has a worldwide distribution with infectivity rates approaching 100%, and immunocompromised patients are particularly at risk from viral exposure. No anti-HMPV vaccine is available and diagnosis is primarily based on in-house molecular or serological tests, in part due to limited availability of recombinant HMPV antigens. OBJECTIVE: To generate a panel of HMPV-derived recombinant antigens, develop standardised ELISA systems for HMPV IgG detection and explore the nature of B cell memory against HMPV to underpin future vaccine studies. STUDY DESIGN: HMPV viral RNA was isolated from a clinical specimen and RT-PCR was conducted. The HMPV M and P genes were cloned and expressed in Escherichia coli. The HMPV N gene was cloned and expressed in insect cells using the baculovirus expression system. Each purified recombinant antigens was subsequently employed in HMPV-specific ELISA. RESULTS: High-level expression, and purification, of both HMPV matrix (M) (10 mg/g cells) and phosphoprotein (P) (3.82 mg/g cells) were achieved in an E. coli expression system. Recombinant HMPV (N) was successfully expressed in, and purified from the baculovirus expression system. Overall, a 99% HMPV IgG seroprevalence was observed (n = 96) using HMPV M-, N- and P-ELISA, respectively. The M antigen proved to be the most diagnostically useful with 99% of specimens tested exhibiting anti-M protein reactivity. A high correlation was observed between anti-M and N IgG reactivity (r = 0.96), with significant correlation also evident for anti-N and P IgG reactivity (r = 0.74). Lowest correlation was evident for anti-M and P IgG reactivity (r = 0.57). Finally, the first demonstration of HMPV-specific B cell memory (ranging 1-15 spot forming cells (SFC)/million cells) was achieved against M and P antigens in 40% of individuals tested. CONCLUSION: This work describes robust diagnostic systems for HMPV and new insight into antigen-specific B cell memory against HMPV.

The role of glutathione S-transferase GliG in gliotoxin biosynthesis in Aspergillus fumigatus.

Gliotoxin, a redox-active metabolite, is produced by the opportunistic fungal pathogen Aspergillus fumigatus, and its biosynthesis is directed by the gli gene cluster. Knowledge of the biosynthetic pathway to gliotoxin, which contains a disulfide bridge of unknown origin, is limited, although L-Phe and L-Ser are known biosynthetic precursors. Deletion of gliG from the gli cluster, herein functionally confirmed as a glutathione S-transferase, results in abrogation of gliotoxin biosynthesis and accumulation of 6-benzyl-6-hydroxy-1-methoxy-3-methylenepiperazine-2,5-dione. This putative shunt metabolite from the gliotoxin biosynthetic pathway contains an intriguing hydroxyl group at C-6, consistent with a gliotoxin biosynthetic pathway involving thiolation via addition of the glutathione thiol group to a reactive acyl imine intermediate. Complementation of gliG restored gliotoxin production and, unlike gliT, gliG was found not to be involved in fungal self-protection against gliotoxin.

Self-protection against gliotoxin--a component of the gliotoxin biosynthetic cluster, GliT, completely protects Aspergillus fumigatus against exogenous gliotoxin.

Gliotoxin, and other related molecules, are encoded by multi-gene clusters and biosynthesized by fungi using non-ribosomal biosynthetic mechanisms. Almost universally described in terms of its toxicity towards mammalian cells, gliotoxin has come to be considered as a component of the virulence arsenal of Aspergillus fumigatus. Here we show that deletion of a single gene, gliT, in the gliotoxin biosynthetic cluster of two A. fumigatus strains, rendered the organism highly sensitive to exogenous gliotoxin and completely disrupted gliotoxin secretion. Addition of glutathione to both A. fumigatus Delta gliT strains relieved gliotoxin inhibition. Moreover, expression of gliT appears to be independently regulated compared to all other cluster components and is up-regulated by exogenous gliotoxin presence, at both the transcript and protein level. Upon gliotoxin exposure, gliT is also expressed in A. fumigatus Delta gliZ, which cannot express any other genes in the gliotoxin biosynthetic cluster, indicating that gliT is primarily responsible for protecting this strain against exogenous gliotoxin. GliT exhibits a gliotoxin reductase activity up to 9 microM gliotoxin and appears to prevent irreversible depletion of intracellular glutathione stores by reduction of the oxidized form of gliotoxin. Cross-species resistance to exogenous gliotoxin is acquired by A. nidulans and Saccharomyces cerevisiae, respectively, when transformed with gliT. We hypothesise that the primary role of gliotoxin may be as an antioxidant and that in addition to GliT functionality, gliotoxin secretion may be a component of an auto-protective mechanism, deployed by A. fumigatus to protect itself against this potent biomolecule.

Proteomic studies in biomedically and industrially relevant fungi.

Historically, the proteomic investigation of filamentous fungi has been restrained by difficulties associated with efficient protein extraction and the lack of extensive fungal genome sequence databases. The advent of robust protein extraction and separation technologies, combined with protein mass spectrometry and emerging genome sequence data, is leading to the emergence of extensive new knowledge on the nature of these organisms. In this review, we discuss some recent technological advances and their role in exploring the proteome of Aspergillus spp., along with other biotechnologically relevant fungi.

Analysis of major intracellular proteins of Aspergillus fumigatus by MALDI mass spectrometry: identification and characterisation of an elongation factor 1B protein with glutathione transferase activity.

Aspergillus fumigatus is a recognised human pathogen, especially in immunocompromised individuals. The availability of the annotated A. fumigatus genome sequence will significantly accelerate our understanding of this organism. However, limited information is available with respect to the A. fumigatus proteome. Here, both a direct proteomic approach (2D-PAGE and MALDI-MS) and a sub-proteomic strategy involving initial glutathione affinity chromatography have been deployed to identify 54 proteins from A. fumigatus primarily involved in energy metabolism and protein biosynthesis. Furthermore, two novel eukaryotic elongation factor proteins (eEF1Bgamma), termed ElfA and B have been identified and phylogenetically confirmed to belong to the eEF1Bgamma class of GST-like proteins. One of these proteins (ElfA) has been purified to homogeneity, identified as a monomeric enzyme (molecular mass=20 kDa; pI=5.9 and 6.5), and found to exhibit glutathione transferase activity specific activities (mean+/-standard deviation, n=3) of 3.13+/-0.27 and 3.43+/-1.0 micromol/min/mg, using CDNB and ethacrynic acid, respectively. Overall, these data highlight the importance of new approaches to dissect the proteome of, and elucidate novel functions within, A. fumigatus.