Active Immunization with an Octa-Valent Staphylococcus aureus Antigen Mixture in Models of S. aureus Bacteremia and Skin Infection in Mice

Proteomic studies with different Staphylococcus aureus isolates have shown that the cell surface-exposed and secreted proteins IsaA, LytM, Nuc, the propeptide of Atl (pro-Atl) and four phenol-soluble modulins α (PSMα) are invariantly produced by this pathogen. Therefore the present study was aimed at investigating whether these proteins can be used for active immunization against S. aureus infection in mouse models of bacteremia and skin infection. To this end, recombinant His-tagged fusions of IsaA, LytM, Nuc and pro-Atl were isolated from Lactococcus lactis or Escherichia coli, while the PSMα1-4 peptides were chemically synthesized. Importantly, patients colonized by S. aureus showed significant immunoglobulin G (IgG) responses against all eight antigens. BALB/cBYJ mice were immunized subcutaneously with a mixture of the antigens at day one (5 μg each), and boosted twice (25 μg of each antigen) with 28 days interval. This resulted in high IgG responses against all antigens although the response against pro-Atl was around one log lower compared to the other antigens. Compared to placebo-immunized mice, immunization with the octa-valent antigen mixture did not reduce the S. aureus isolate P load in blood, lungs, spleen, liver, and kidneys in a bacteremia model in which the animals were challenged for 14 days with a primary load of 3 × 10⁵ CFU. Discomfort scores and animal survival rates over 14 days did not differ between immunized mice and placebo-immunized mice upon bacteremia with S. aureus USA300 (6 × 10⁵ CFU). In addition, this immunization did not reduce the S. aureus isolate P load in mice with skin infection. These results show that the target antigens are immunogenic in both humans and mice, but in the used animal models do not result in protection against S. aureus infection.     

The Tarenaya hassleriana Genome Provides Insight into Reproductive Trait and Genome Evolution of Crucifers

The Brassicaceae, including Arabidopsis thaliana and Brassica crops, is unmatched among plants in its wealth of genomic and functional molecular data and has long served as a model for understanding gene, genome, and trait evolution. However, genome information from a phylogenetic outgroup that is essential for inferring directionality of evolutionary change has been lacking. We therefore sequenced the genome of the spider flower (Tarenaya hassleriana) from the Brassicaceae sister family, the Cleomaceae. By comparative analysis of the two lineages, we show that genome evolution following ancient polyploidy and gene duplication events affect reproductively important traits. We found an ancient genome triplication in Tarenaya (Th-α) that is independent of the Brassicaceae-specific duplication (At-α) and nested Brassica (Br-α) triplication. To showcase the potential of sister lineage genome analysis, we investigated the state of floral developmental genes and show Brassica retains twice as many floral MADS (for MINICHROMOSOME MAINTENANCE1, AGAMOUS, DEFICIENS and SERUM RESPONSE FACTOR) genes as Tarenaya that likely contribute to morphological diversity in Brassica. We also performed synteny analysis of gene families that confer self-incompatibility in Brassicaceae and found that the critical SERINE RECEPTOR KINASE receptor gene is derived from a lineage-specific tandem duplication. The T. hassleriana genome will facilitate future research toward elucidating the evolutionary history of Brassicaceae genomes.     

The seirena B Class Floral Homeotic Mutant of California Poppy (Eschscholzia californica) Reveals a Function of the Enigmatic PI Motif in the Formation of Specific Multimeric MADS Domain Protein Complexes

The products of B class floral homeotic genes specify petal and stamen identity, and loss of B function results in homeotic conversions of petals into sepals and stamens into carpels. Here, we describe the molecular characterization of seirena-1 (sei-1), a mutant from the basal eudicot California poppy (Eschscholzia californica) that shows homeotic changes characteristic of floral homeotic B class mutants. SEI has been previously described as EScaGLO, one of four B class–related MADS box genes in California poppy. The C terminus of SEI, including the highly conserved PI motif, is truncated in sei-1 proteins. Nevertheless, like the wild-type SEI protein, the sei-1 mutant protein is able to bind CArG-boxes and can form homodimers, heterodimers, and several higher order complexes with other MADS domain proteins. However, unlike the wild type, the mutant protein is not able to mediate higher order complexes consisting of specific B, C, and putative E class related proteins likely involved in specifying stamen identity. Within the PI motif, five highly conserved N-terminal amino acids are specifically required for this interaction. Several families lack this short conserved sequence, including the Brassicaceae, and we propose an evolutionary scenario to explain these functional differences.     

Molecular evidence for the presence of the endosymbiontic bacteria Wolbachia in Cotesia populations (Hymenoptera: Braconidae)

Cotesia are larval endoparasitoids and are potential biocontrol agents for various insect pests. In the present study, we attempted to detect the allele-specific ftsZ gene of Wolbachia in Cotesia spp. obtained from different geographical locations. Samples of Cotesia plutellae originating from India (Bangalore and Ludhiana), Kenya, and South Africa and samples of C. glomerata from India (Solan) confirmed the presence of Wolbachia. However, C. plutellae samples from the Kullu region of India did not show infection with Wolbachia. ftsZ sequences showed a high degree of homology within the species (> 99%). The low genetic distance in the infected Cotesia populations indicated a single ancestral type, and distance to and from the outgroup suggested that populations are from the same species. Phylogenetic grouping of Cotesia species on the basis of geographic origin showed that the Wolbachia strain is closely related to strains that infect phylogenetically distant hosts.     

Genotoxicity biomonitoring of sewage in two municipal wastewater treatment plants using the Tradescantia pallida var. purpurea bioassay

The genotoxicity of untreated and treated sewage from two municipal wastewater treatment plants (WTP BN and WTP SJN) in the municipality of Porto Alegre, in the southern Brazilian state of Rio Grande do Sul, was evaluated over a one-year period using the Tradescantia pallida var. purpurea (Trad-MCN) bioassay. Inflorescences of T. pallida var. purpurea were exposed to sewage samples in February (summer), April (autumn), July (winter) and October (spring) 2009, and the micronuclei (MCN) frequencies were estimated in each period. The high genotoxicity of untreated sewage from WTP BN in February and April was not observed in treated sewage, indicating the efficiency of treatment at this WTP. However, untreated and treated sewage samples from WTP SJN had high MCN frequencies, except in October, when rainfall may have been responsible for reducing these frequencies at both WTPs. Physicochemical analyses of sewage from both WTPs indicated elevated concentrations of organic matter that were higher at WTP SJN than at WTP BN. Chromium was detected in untreated and treated sewage from WTP SJN, but not in treated sewage from WTP BN. Lead was found in all untreated sewage samples from WTP SJN, but only in the summer and autumn at WTP BN. These results indicate that the short-term Trad-MCN genotoxicity assay may be useful for regular monitoring of municipal WTPs.     

Occurrence, genes and expression of the W/Se-containing class II benzoyl-coenzyme A reductases in anaerobic bacteria

Benzoyl-coenzyme A (CoA) reductases (BCRs) are key enzymes in the anaerobic degradation of aromatic compounds and catalyse the reductive dearomatization of benzoyl-CoA to cyclohexa-1,5-dienoyl-1-carboxyl-CoA. Class I BCRs are ATP-dependent FeS enzymes, whereas class II BCRs are supposed to be ATP-independent and contain W, FeS clusters, and most probably selenocysteine. The active site components of a putative eight subunit class II BCR, BamBCDEFGHI, were recently characterized in Geobacter metallireducens. In this organism bamB was identified as structural gene for the W-containing active site subunit; bamF was predicted to code for a selenocysteine containing electron transfer subunit. In this work the occurrence and expression of BCRs in a number of anaerobic, aromatic compound degrading model microorganisms was investigated with a focus on the BamB and BamF components. Benzoate-induced class II BCR in vitro activities were determined in the soluble protein fraction in all obligately anaerobic bacteria tested. Where applicable, the results were in agreement with Western blot analysis using BamB targeting antibodies. By establishing a specific bamB targeting PCR assay, bamB homologues were identified in all tested obligately anaerobic bacteria with the capacity to degrade aromatic compounds; a number of bamB sequences from Gram-negative/positive sulfate-reducing bacteria were newly sequenced. In several organisms at least two bamB paralogues per genome were identified; however, in nearly all cases only one of them was transcribed during growth on an aromatic substrate. These benzoate-induced bamB genes are proposed to code for the active site subunit of class II BCRs; the major part of them group into a phylogenetic subcluster within the bamB homologues. Results from in silico analysis suggested that all class II BCRs contain selenocysteine in the BamF, and in many cases also in the BamE subunit. The results obtained indicate that the distribution of the two classes of BCRs in anaerobic bacteria appears to be strictly ruled by the available free energy from the oxidation of the aromatic carbon source rather than by phylogenetic relationships.     

More than 100,000 detectable peptide species elute in single shotgun proteomics runs but the majority is inaccessible to data-dependent LC-MS/MS

Shotgun proteomics entails the identification of as many peptides as possible from complex mixtures. Here we investigate how many peptides are detectable by high resolution MS in standard LC runs of cell lysate and how many of them are accessible to data-dependent MS/MS. Isotope clusters were determined by MaxQuant and stringently filtered for charge states and retention times typical of peptides. This resulted in more than 100,000 likely peptide features, of which only about 16% had been targeted for MS/MS. Three instrumental attributes determine the proportion of additional peptides that can be identified: sequencing speed, sensitivity, and precursor ion isolation. In our data, an MS/MS scan rate of 25/s would be necessary to target all peptide features, but this drops to less than 17/s for reasonably abundant peptides. Sensitivity is a greater challenge, with many peptide features requiring long MS/MS injection times (>250 ms). The greatest limitation, however, is the generally low proportion of the target peptide ion intensity in the MS/MS selection window (the "precursor ion fraction" or PIF). Median PIF is only 0.14, making the peptides difficult to identify by standard MS/MS methods. Our results aid in developing strategies to further increase coverage in shotgun proteomics.