Sequence TTKF ↓ QE defines the site of proteolytic cleavage in Mhp683 protein, a novel glycosaminoglycan and cilium adhesin of Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae colonizes the ciliated respiratory epithelium of swine, disrupting mucociliary function and inducing chronic inflammation. P97 and P102 family members are major surface proteins of M. hyopneumoniae and play key roles in colonizing cilia via interactions with glycosaminoglycans and mucin. The p102 paralog, mhp683, and homologs in strains from different geographic origins encode a 135-kDa pre-protein (P135) that is cleaved into three fragments identified here as P45(683), P48(683), and P50(683). A peptide sequence (TTKF↓QE) was identified surrounding both cleavage sites in Mhp683. N-terminal sequences of P48(683) and P50(683), determined by Edman degradation and mass spectrometry, confirmed cleavage after the phenylalanine residue. A similar proteolytic cleavage site was identified by mass spectrometry in another paralog of the P97/P102 family. Trypsin digestion and surface biotinylation studies showed that P45(683), P48(683), and P50(683) reside on the M. hyopneumoniae cell surface. Binding assays of recombinant proteins F1(683)-F5(683), spanning Mhp683, showed saturable and dose-dependent binding to biotinylated heparin that was inhibited by unlabeled heparin, fucoidan, and mucin. F1(683)-F5(683) also bound porcine epithelial cilia, and antisera to F2(683) and F5(683) significantly inhibited cilium binding by M. hyopneumoniae cells. These data suggest that P45(683), P48(683), and P50(683) each display cilium- and proteoglycan-binding sites. Mhp683 is the first characterized glycosaminoglycan-binding member of the P102 family.     

Cell wall-linked cryptococcal phospholipase B1 is a source of secreted enzyme and a determinant of cell wall integrity

Phospholipase B (Plb1) is secreted by pathogenic fungi and is a proven virulence determinant in Cryptococcus neoformans. Cell-associated Plb1 is presumptively involved in fungal membrane biogenesis and remodelling. We have also identified it in cryptococcal cell walls. Motif scanning programs predict that Plb1 is attached to cryptococcal membranes via a glycosylphosphatidylinositol (GPI) anchor, which could regulate Plb1 export and secretion. A functional GPI anchor was identified in cell-associated Plb1 by (G)PI-specific phospholipase C (PLC)-induced release of Plb1 from strain H99 membrane rafts and inhibition of GPI anchor synthesis by YW3548, which prevented Plb1 secretion and transport to membranes and cell walls. Plb1 containing beta-1,6-linked glucan was released from H99 (wild-type strain) cell walls by beta-1,3 glucanase, consistent with covalent attachment of Plb1 via beta-1,6-linked glucans to beta-1,3-linked glucan in the central scaffold of the wall. Naturally secreted Plb1 also contained beta-1,6-linked glucan, confirming that it originated from the cell wall. Plb1 maintains cell wall integrity because a H99 deletion mutant, DeltaPLB1, exhibited a morphological defect and was more susceptible than H99 to cell wall disruption by SDS and Congo red. Growth of DeltaPLB1 was unaffected by caffeine, excluding an effect of Plb1 on cell wall biogenesis-related signaling pathways. Environmental (heat) stress caused Plb1 accumulation in cell walls, with loss from membranes and reduced secretion, further supporting the importance of Plb1 in cell wall integrity. This is the first demonstration that Plb1 contributes to fungal survival by maintaining cell wall integrity and that the cell wall is a source of secreted enzyme.     

Rutin as Deoxyribonuclease I Inhibitor

DNase I inhibitory potential of water extract of nine Hypericum species (H. umbellatum, H. barbatum, H. rumeliacum, H. rochelii, H. perforatum, H. tetrapterum, H. olympicum, H. hirsutum, H. linarioides) and the most important Hypericum secondary metabolites (hypericin, hyperforin, quercetin, and rutin) was investigated. All examined Hypericum extracts inhibited DNase I with IC₅₀ below 800 μg/ml, whereby H. perforatum was the most potent (IC₅₀=391.26±68.40 μg/ml). Among the investigated Hypericum secondary metabolites, rutin inhibited bovine pancreatic DNase I in a non‐competitive manner with IC₅₀ value of 108.90±9.73 μm . DNase I inhibitory ability of rutin was further confirmed on DNase I in rat liver homogenate (IC₅₀=137.17±16.65 μm ). Due to the involvement of DNase I in apoptotic processes the results of this study indicate the importance of frequent rutin and H. perforatum consumption in daily human nutrition. Rutin is a dietary component that can contribute to male infertility prevention by showing dual mechanism of sperm DNA protection, DNase I inhibition and antioxidant activity.     

Determination of Transgene Copy Number in Stably Transfected Mammalian Cells by PCR-Capillary Electrophoresis Assay

The quantitative determination of transgene copy number in stably transfected mammalian cells has been traditionally estimated by Southern blot analysis. Recently, other methods have become available for appraisal of gene copy number, such as real-time PCR. Herein we describe a new method based on a fluorescently labeled PCR, followed by capillary electrophoresis. We amplified our target gene (prothrombin) and the internal control originating from genomic DNA (18S rRNA) in the same PCR tube and calculated the mean peak height ratio of the target:control gene for every cell clone sample. With this approach we identified stably transfected cell clones bearing the same transgene copy number. The results of our assay were confirmed by real-time PCR. Our method proves to be fast, low-cost, and reproducible compared with traditionally used methods. This assay can be used as a rapid screening tool for the determination of gene copy number in gene expression experiments.     

Proteogenomic mapping of Mycoplasma hyopneumoniae virulent strain 232

Background

Mycoplasma hyopneumoniae causes respiratory disease in swine and contributes to the porcine respiratory disease complex, a major disease problem in the swine industry. The M. hyopneumoniae strain 232 genome is one of the smallest and best annotated microbial genomes, containing only 728 annotated genes and 691 known proteins. Standard protein databases for mass spectrometry only allow for the identification of known and predicted proteins, which if incorrect can limit our understanding of the biological processes at work. Proteogenomic mapping is a methodology which allows the entire 6-frame genome translation of an organism to be used as a mass spectrometry database to help identify unknown proteins as well as correct and confirm existing annotations. This methodology will be employed to perform an in-depth analysis of the M. hyopneumoniae proteome.

Results

Proteomic analysis indicates 483 of 691 (70%) known M. hyopneumoniae strain 232 proteins are expressed under the culture conditions given in this study. Furthermore, 171 of 328 (52%) hypothetical proteins have been confirmed. Proteogenomic mapping resulted in the identification of previously unannotated genes gatC and rpmF and 5-prime extensions to genes mhp063, mhp073, and mhp451, all conserved and annotated in other M. hyopneumoniae strains and Mycoplasma species. Gene prediction with Prodigal, a prokaryotic gene predicting program, completely supports the new genomic coordinates calculated using proteogenomic mapping.

Conclusions

Proteogenomic mapping showed that the protein coding genes of the M. hyopneumoniae strain 232 identified in this study are well annotated. Only 1.8% of mapped peptides did not correspond to genes defined by the current genome annotation. This study also illustrates how proteogenomic mapping can be an important tool to help confirm, correct and append known gene models when using a genome sequence as search space for peptide mass spectra. Using a gene prediction program which scans for a wide variety of promoters can help ensure genes are accurately predicted or not missed completely. Furthermore, protein extraction using differential detergent fractionation effectively increases the number of membrane and cytoplasmic proteins identifiable my mass spectrometry.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-576) contains supplementary material, which is available to authorized users.     

Molecular phylogeny of Neotropical bioluminescent beetles (Coleoptera: Elateroidea) in southern and central Brazil

Bioluminescence in beetles is found mainly in the Elateroidea superfamily (Elateridae, Lampyridae and Phengodidae). The Neotropical region accounts for the richest diversity of bioluminescent species in the world with about 500 described species, most occurring in the Amazon, Atlantic rainforest and Cerrado (savanna) ecosystems in Brazil. The origin and evolution of bioluminescence, as well as the taxonomic status of several Neotropical taxa in these families remains unclear. In order to contribute to a better understanding of the phylogeny and evolution of bioluminescent Elateroidea we sequenced and analyzed sequences of mitochondrial NADH2 and the nuclear 28S genes and of the cloned luciferase sequences of Brazilian species belonging to the following genera: (Lampyridae) Macrolampis, Photuris, Amydetes, Bicellonycha, Aspisoma, Lucidota, Cratomorphus; (Elateridae) Conoderus, Pyrophorus, Hapsodrilus, Pyrearinus, Fulgeochlizus; and (Phengodidae) Pseudophengodes, Phrixothrix, Euryopa and Brasilocerus. Our study supports a closer phylogenetic relationship between Elateridae and Phengodidae as other molecular studies, in contrast with previous morphologic and molecular studies that clustered Lampyridae/Phengodidae. Molecular data also supported division of the Phengodinae subfamily into the tribes Phengodini and Mastinocerini. The position of the genus Amydetes supports the status of the Amydetinae as a subfamily. The genus Euryopa is included in the Mastinocerini tribe within the Phengodinae/Phengodidae. Copyright © 2013 John Wiley & Sons, Ltd.     

Insights into the structural nature of the transition state in the Kir channel gating pathway

In a previous study we identified an extensive gating network within the inwardly rectifying Kir1.1 (ROMK) channel by combining systematic scanning mutagenesis and functional analysis with structural models of the channel in the closed, pre-open and open states. This extensive network appeared to stabilize the open and pre-open states, but the network fragmented upon channel closure. In this study we have analyzed the gating kinetics of different mutations within key parts of this gating network. These results suggest that the structure of the transition state (TS), which connects the pre-open and closed states of the channel, more closely resembles the structure of the pre-open state. Furthermore, the G-loop, which occurs at the center of this extensive gating network, appears to become unstructured in the TS because mutations within this region have a ‘catalytic’ effect upon the channel gating kinetics.     

Mycoplasma hyopneumoniae Surface proteins Mhp385 and Mhp384 bind host cilia and glycosaminoglycans and are endoproteolytically processed by proteases that recognize different cleavage motifs

P97 and P102 paralogues occur as endoproteolytic cleavage fragments on the surface of Mycoplasma hyopneumoniae that bind glycosaminoglycans, plasminogen, and fibronectin and perform essential roles in colonization of ciliated epithelia. We show that the P102 paralogue Mhp384 is efficiently cleaved at an S/T-X-F↓X-D/E-like site, creating P60(384) and P50(384). The P97 paralogue Mhp385 is inefficiently cleaved, with tryptic peptides from a 115 kDa protein (P115(385)) and 88 kDa (P88(385)) and 27 kDa (P27(385)) cleavage fragments identified by LC-MS/MS. This is the first time a preprotein belonging to the P97 and P102 paralogue families has been identified by mass spectrometry. The semitryptic peptide (752)IQFELEPISLNV(763) denotes the C-terminus of P88(385) and defines the novel cleavage site (761)L-N-V↓A-V-S(766) in Mhp385. P115(385), P88(385), P27(385), P60(384), and P50(384) were shown to reside extracellularly, though it is unknown how the fragments remain attached to the cell surface. Heparin- and cilium-binding sites were identified within P60(384), P50(384), and P88(385). No primary function was attributed to P27(385); however, this molecule contains four tandem R1 repeats with similarity to porcine collagen type VI (α3 chain). P97 and P102 paralogue families are adhesins targeted by several proteases with different cleavage efficiencies, and this process generates combinatorial complexity on the surface of M. hyopneumoniae.