Comparative genomics reveals high biological diversity and specific adaptations in the industrially and medically important fungal genus Aspergillus.

BACKGROUND: The fungal genus Aspergillus is of critical importance to humankind. Species include those with industrial applications, important pathogens of humans, animals and crops, a source of potent carcinogenic contaminants of food, and an important genetic model. The genome sequences of eight aspergilli have already been explored to investigate aspects of fungal biology, raising questions about evolution and specialization within this genus. RESULTS: We have generated genome sequences for ten novel, highly diverse Aspergillus species and compared these in detail to sister and more distant genera. Comparative studies of key aspects of fungal biology, including primary and secondary metabolism, stress response, biomass degradation, and signal transduction, revealed both conservation and diversity among the species. Observed genomic differences were validated with experimental studies. This revealed several highlights, such as the potential for sex in asexual species, organic acid production genes being a key feature of black aspergilli, alternative approaches for degrading plant biomass, and indications for the genetic basis of stress response. A genome-wide phylogenetic analysis demonstrated in detail the relationship of the newly genome sequenced species with other aspergilli. CONCLUSIONS: Many aspects of biological differences between fungal species cannot be explained by current knowledge obtained from genome sequences. The comparative genomics and experimental study, presented here, allows for the first time a genus-wide view of the biological diversity of the aspergilli and in many, but not all, cases linked genome differences to phenotype. Insights gained could be exploited for biotechnological and medical applications of fungi.

Common versus noble Bacillus subtilis differentially responds to air and argon gas plasma.

The applications of low-temperature plasma are not only confined to decontamination and sterilization but are also found in the medical field in terms of wound and skin treatment. For the improvement of already established and also for new plasma techniques, in-depth knowledge on the interactions between plasma and microorganism is essential. In an initial study, the interaction between growing Bacillus subtilis and argon plasma was investigated by using a growth chamber system suitable for low-temperature gas plasma treatment of bacteria in liquid medium. In this follow-up investigation, a second kind of plasma treatment-namely air plasma-was applied. With combined proteomic and transcriptomic analyses, we were able to investigate the plasma-specific stress response of B. subtilis toward not only argon but also air plasma. Besides an overlap of cellular responses due to both argon and air plasma treatment (DNA damage and oxidative stress), a variety of gas-dependent cellular responses such as growth retardation and morphological changes were observed. Only argon plasma treatments lead to a phosphate starvation response whereas air plasma induced the tryptophan operon implying damage by photooxidation. Biological findings were supported by the detection of reactive plasma species by optical emission spectroscopy and Fourier transformed infrared spectroscopy measurements.

Characterization of the global impact of low temperature gas plasma on vegetative microorganisms.

Plasma medicine and also decontamination of bacteria with physical plasmas is a promising new field of life science with huge interest especially for medical applications. Despite numerous successful applications of low temperature gas plasmas in medicine and decontamination, the fundamental nature of the interactions between plasma and microorganisms is to a large extent unknown. A detailed knowledge of these interactions is essential for the development of new as well as for the enhancement of established plasma-treatment procedures. In the present work we introduce for the first time a growth chamber system suitable for low temperature gas plasma treatment of bacteria in liquid medium. We have coupled the use of this apparatus to a combined proteomic and transcriptomic analyses to investigate the specific stress response of Bacillus subtilis 168 cells to treatment with argon plasma. The treatment with three different discharge voltages revealed not only effects on growth, but also clear evidence of cellular stress responses. B. subtilis suffered severe cell wall stress, which was made visible also by electron microscopy, DNA damages and oxidative stress as a result of exposure to plasma. These biological findings were supported by the detection of reactive plasma species by OES measurements.

Human immune proteome in experimental colonization with Staphylococcus aureus.

More than 20% of adults are persistently colonized with Staphylococcus aureus. When hospitalized, these carriers have increased risks of infection with their own strains. However, a recent study demonstrated a lower incidence of bacteremia-related death among carriers than among noncarriers, raising the question whether the adaptive immune system plays a protective role. In fact, S. aureus carriers mount a highly specific neutralizing antibody response against superantigens of their colonizing strains. We now used 2-dimensional immunoblotting to investigate the profiles of antibodies from healthy individuals against S. aureus extracellular proteins. Moreover, we tested whether symptom-free experimental colonization of these individuals with an S. aureus strain of low virulence, 8325-4, is sufficient to induce an antibody response. Sera obtained before and 4 weeks after colonization were screened for immunoglobulin G (IgG) antibody binding to extracellular staphylococcal proteins. At baseline, most volunteers harbored IgG directed against conserved virulence factors, including alpha-hemolysin (Hla), beta-hemolysin (Hlb), phospholipase C (Plc), staphylococcal serine protease (SspA), and cysteine protease (SspB). However, the variability of spot patterns and intensities was striking and could be important in case of infection. Experimental nasal colonization with S. aureus 8325-4 did not elicit new antibodies or boost the humoral response. Thus, the high antibody prevalence in humans is likely not induced by short-term nasal colonization, and presumably minor infections are required to trigger anti-S. aureus antibody responses.

Proteomic analysis of Legionella-containing phagosomes isolated from Dictyostelium.

Legionella pneumophila, the agent of Legionnaires' disease, replicates intracellularly within specialized phagosomes of human macrophages and amoebae. In this study, we have developed a protocol for the isolation of Legionella-containing phagosomes from Dictyostelium discoideum. Cell fractionation, two-dimensional gel electrophoresis and MALDI-TOF MS combined with genomic data identified 157 phagosome host proteins. In addition to proteins with an evident role in phagosome maturation, we identified proteins for which a function remains to be elucidated. Possible interactions of coronin with cytosolic NADPH oxidase components and protein kinase C inhibitors which together may lead to an inhibition of phagosomal superoxide generation are discussed. Comparative proteomics of phagosomes containing highly virulent L. pneumophila Corby versus less virulent L. hackeliae revealed distinctive protein expression patterns, e.g., an abundance of RhoGDI in L. hackeliae degrading phagosomes versus little RhoGDI in L. pneumophila Corby replicative phagosomes. We present a kinetic dissection of phagosome maturation including the complex alterations of the phagosome protein composition. A reference flow chart suggests so far unrecognized consequences of infection for host cell physiology, actin degradation on phagosomes, and a putative cysteine proteinase inhibitor interference with lysosomal enzyme sorting and activation processes.

Proteomic analysis of the bacterial pathogen Bartonella henselae and identification of immunogenic proteins for serodiagnosis.

Bartonella henselae is a slow growing, fastidious and facultative intracellular pathogen causing cat scratch disease and vasculoproliferative disorders. To date, knowledge about the pathogenicity of this human pathogenic bacterium is limited and, additionally, serodiagnosis still needs further improvement. Here, we investigated the proteome of B. henselae using 2-D SDS-PAGE and MALDI-TOF-MS. We provide a comprehensive 2-D proteome reference map of the whole cell lysate of B. henselae with 431 identified protein spots representing 191 different proteins of which 16 were formerly assigned as hypothetical proteins. To unravel immunoreactive antigens, we applied 2-D SDS-PAGE and subsequent immunoblotting using 33 sera of patients suffering from B. henselae infections. The analysis revealed 79 immunoreactive proteins of which 71 were identified. Setting a threshold of 20% seroreactivity, 11 proteins turned out to be immunodominant antigens potentially useful for an improved Bartonella-specific serodiagnosis. Therefore, we provide for the first time (i) a comprehensive 2-D proteome map of B. henselae for further proteome-based studies focussed on the pathogenicity of B. henselae and (ii) an integrated view into the humoral immune responses targeted against this newly emerged human pathogenic bacterium.

Proteomic analysis of antioxidant strategies of Staphylococcus aureus: diverse responses to different oxidants.

The high resolution 2-D protein gel electrophoresis technique combined with MALDI-TOF MS and a recently developed fluorescence-based thiol modification assay were used to investigate the cellular response of Staphylococcus aureus to oxidative stress. Addition of hydrogen peroxide, diamide, and the superoxide generating agent paraquat to exponentially growing cells revealed complex changes in the protein expression pattern. In particular, proteins involved in detoxification, repair systems, and intermediary metabolism were found to be up-regulated. Interestingly, there is only a small overlap of proteins induced by all these stressors. Exposure to hydrogen peroxide mediated a significant increase of DNA repair enzymes, whereas treatment with diamide affected proteins involved in protein repair and degradation. The activity of proteins under oxidative stress conditions can be modulated by oxidation of thiol groups. In growing cells, protein thiols were found to be mainly present in the reduced state. Diamide mediated a strong increase of reversibly oxidized thiols in a variety of metabolic enzymes. By contrast, hydrogen peroxide resulted in the reversible oxidation especially of proteins with active site cysteines. Moreover, high levels of hydrogen peroxide influenced the pI of three proteins containing cysteines within their active sites (GapA1, AhpC, and HchA) indicating the generation of sulfinic or sulfonic acid by irreversible oxidation of thiols.

Proteomic characterization of the whole secretome of Legionella pneumophila and functional analysis of outer membrane vesicles.

Secretion of effector molecules is one of the major mechanisms by which the intracellular human pathogen Legionella pneumophila interacts with host cells during infection. Specific secretion machineries which are responsible for the subfraction of secreted proteins (soluble supernatant proteins [SSPs]) and the production of bacterial outer membrane vesicles (OMVs) both contribute to the protein composition of the extracellular milieu of this lung pathogen. Here we present comprehensive proteome reference maps for both SSPs and OMVs. Protein identification and assignment analyses revealed a total of 181 supernatant proteins, 107 of which were specific to the SSP fraction and 33 of which were specific to OMVs. A functional classification showed that a large proportion of the identified OMV proteins are involved in the pathogenesis of Legionnaires' disease. Zymography and enzyme assays demonstrated that the SSP and OMV fractions possess proteolytic and lipolytic enzyme activities which may contribute to the destruction of the alveolar lining during infection. Furthermore, it was shown that OMVs do not kill host cells but specifically modulate their cytokine response. Binding of immunofluorescently stained OMVs to alveolar epithelial cells, as visualized by confocal laser scanning microscopy, suggested that there is delivery of a large and complex group of proteins and lipids in the infected tissue in association with OMVs. On the basis of these new findings, we discuss the relevance of protein sorting and compartmentalization of virulence factors, as well as environmental aspects of the vesicle-mediated secretion.