Two‐dimensional proteome reference maps for the human pathogenic filamentous fungus Aspergillus fumigatus

The filamentous fungus Aspergillus fumigatus has become the most important airborne fungal pathogen causing life‐threatening infections in immunosuppressed patients. We established a 2‐D reference map for A. fumigatus. Using MALDI‐TOF‐MS/MS, we identified 381 spots representing 334 proteins. Proteins involved in cellular metabolism, protein synthesis, transport processes and cell cycle were most abundant. Furthermore, we established a protocol for the isolation of mitochondria of A. fumigatus and developed a mitochondrial proteome reference map. 147 proteins represented by 234 spots were identified.     

Analysis of cotton (Gossypium hirsutum) root proteomes during a compatible interaction with the black root rot fungus Thielaviopsis basicola

A proteomic approach was used to uncover the inducible molecular defense mechanism of cotton root occurring during the compatible interaction with Thielaviopsis basicola. Microscopic observation of cotton root inoculated with a suspension of conidia showed that this necrotrophic hemibiotroph fungus interacts with the plant and completes its life cycle in our experimental system. 2‐DE analysis of root extracts taken after 1, 3, 5, and 7 days postinoculation and cluster analysis of the protein expression levels showed four major profiles (constant, upregulated, one slightly downregulated, and one dramatically downregulated). Spots significantly (p<0.05) upregulated were analyzed by LC‐MS/MS and identified using MASCOT MS/MS ion search software and associated databases. These proteins included defense and stress related proteins, such as pathogenesis‐related proteins and proteins likely to be involved in the oxidative burst, sugar, and nitrogen metabolism as well as amino acid and isoprenoid synthesis. While many of the identified proteins are common components of the defense response of most plants, a proteasome subunit and a protein reported to be induced only in cotton root following Meloidogyne incognita infection were also identified.     

Exo‐ and surface proteomes of the probiotic bacterium Lactobacillus acidophilus NCFM

Lactobacillus acidophilus NCFM is a well‐known probiotic bacterium extensively studied for its beneficial health effects. Exoproteome (proteins exported into culture medium) and surface proteome (proteins attached to S‐layer) of this probiotic were identified by using 2DE followed by MALDI TOF MS to find proteins potentially involved in bacteria–host interactions. The exo‐ and surface proteomes included 43 and 39 different proteins from 72 and 49 successfully identified spots, respectively. Twenty‐two proteins were shared between the two proteomes; both contained the major surface layer protein that participates in host interaction as well as several well‐known and putative moonlighting proteins. The exoproteome contained nine classically‐secreted (containing a signal sequence) and ten nonclassically‐secreted proteins, while the surface proteome contained four classically‐secreted and eight nonclassically secreted proteins. Identification of exo‐ and surface proteomes contributes describing potential protein‐mediated probiotic–host interactions.     

Optimization of a phenol extraction‐based protein preparation method amenable to downstream 2DE and MALDI‐MS based analysis of bacterial proteomes

2DE is one of the most efficient and widely used methods for resolving complex protein mixtures. For efficient analysis of complex samples, high‐resolution separation of proteins on 2D gel is essential, and for that purpose good sample preparation is crucial. In this study, we have improvized a method for preparing bacterial total cellular proteome, from a strategy applied earlier to recalcitrant plant tissues, which gave high‐quality resolution on 2DE. The method involving phenol extraction followed by methanol/ammonium acetate precipitation was first optimized for the chemolithotrophic proteobacteria Tetrathiobacter kashmirensis WT001 and Pseudaminobacter salicylatoxidans KCT001 that did not yield quality protein preps in conventional trichloroacetic acid/acetone precipitation method. Subsequently, to validate its general applicability, the method was evaluated against the trichloroacetic acid/acetone precipitation method for two other model bacteria, i.e. Escherichia coli DH5α and Mycobacterium smegmatis mc²6. Identification of at least four proteins each from the outer membrane, periplasm, and cytoplasm of T. kashmirensis by MALDI‐MS not only proved the efficiency of the method in extracting proteins from the different cellular compartments but also the amenability of the obtained protein spots toward MALDI‐MS based identification.     

A proteomic evaluation of Pyrenophora tritici‐repentis,causal agent of tan spot of wheat,reveals major differences between virulent and avirulent isolates

Pyrenophora tritici‐repentis causes tan spot, an important foliar disease of wheat. The fungus produces multiple host‐specific toxins, including Ptr ToxB, a chlorosis‐inducing protein encoded by the ToxB gene. A homolog of ToxB is also found in avirulent isolates of the fungus. In order to improve understanding of the role of this homolog and evaluate the general pathogenic ability of P. tritici‐repentis, we compared the proteomes of avirulent race 4 and virulent race 5 isolates of the pathogen. Western blotting analysis revealed the presence of Ptr ToxB in spore germination and culture fluids of race 5 but not race 4. A comprehensive proteome‐level comparison by 2‐DE indicated 133 differentially abundant proteins in the secretome (29 proteins) and mycelium (104 proteins) of races 4 and 5, of which 63 were identified by MS/MS. A number of the proteins found to be up‐regulated in race 5 have been implicated in microbial virulence in other pathosystems, and included the secreted enzymes α‐mannosidase and exo‐β‐1,3‐glucanase, heat‐shock and BiP proteins, and various metabolic enzymes. These proteome‐level differences suggest a reduced general pathogenic ability in race 4 of P. tritici‐repentis, irrespective of toxin production. Such differences may reflect an adaptation to a saprophytic habit.     

A proteomics approach to study synergistic and antagonistic interactions of the fungal–bacterial consortium Fusarium oxysporum wild‐type MSA 35

Fusarium oxysporum is an important plant pathogen that causes severe damage of many economically important crop species. Various microorganisms have been shown to inhibit this soil‐borne plant pathogen, including non‐pathogenic F. oxysporum strains. In this study, F. oxysporum wild‐type (WT) MSA 35, a biocontrol multispecies consortium that consists of a fungus and numerous rhizobacteria mainly belonging to γ‐proteobacteria, was analyzed by two complementary metaproteomic approaches (2‐DE combined with MALDI‐Tof/Tof MS and 1‐D PAGE combined with LC‐ESI‐MS/MS) to identify fungal or bacterial factors potentially involved in antagonistic or synergistic interactions between the consortium members. Moreover, the proteome profiles of F. oxysporum WT MSA 35 and its cured counter‐part CU MSA 35 (WT treated with antibiotics) were compared with unravel the bacterial impact on consortium functioning. Our study presents the first proteome mapping of an antagonistic F. oxysporum strain and proposes candidate proteins that might play an important role for the biocontrol activity and the close interrelationship between the fungus and its bacterial partners.     

Proteome reference map for the plant growth‐promoting bacterium Pseudomonas putida UW4

A proteome reference map containing 326 2‐D gel spots representing 275 different proteins was constructed for the plant growth‐promoting bacterium Pseudomonas putida UW4. Protein identifications were obtained using Q‐TOF MS/MS spectra matching to homologous proteins from other Pseudomonas strains and confirmed by PMF analysis. This data set is accessible at http://world‐2dpage.expasy.org/repository/ and will aid in further characterization of Pseudomonas strains and interactions of plant growth‐promoting bacterium with the plant rhizosphere environment.     

Development of polymorphic markers for the root pathogen Thielaviopsis basicola using ISSR‐PCR

Thielaviopsis basicola is a soil‐borne fungal pathogen affecting many important agricultural crops. Little is known regarding the population biology or origin of this pathogen. Polymorphic markers developed for Ceratocystis fimbriata, a species complex phylogenetically closely related to T. basicola, were tested and found not to be useful for T. basicola. In this study 14 primer pairs, seven of which resulted in the amplification of single polymorphic fragments in T. basicola were developed. These primers will enable further studies on this economically important pathogen, and will result in an enhanced understanding of its population structure in different parts of the world.     

A two‐dimensional proteome map of the aflatoxigenic fungus Aspergillus flavus

The filamentous fungus Aspergillus flavus is an opportunistic soil‐borne pathogen that produces aflatoxins, the most potent naturally occurring carcinogenic compounds known. This work represents the first gel‐based profiling analysis of A. flavus proteome and establishes a 2D proteome map. Using 2DE and MALDI‐TOF‐MS/MS, we identified 538 mycelial proteins of the aflatoxigenic strain NRRL 3357, the majority of which were functionally annotated as related to various cellular metabolic and biosynthetic processes. Additionally, a few enzymes from the aflatoxin synthesis pathway were also identified.     

Proteomic characterization of seeds from yellow lupin (Lupinus luteus L.)

Yellow lupin (Lupinus luteus L.) is a legume crop containing a large amount of protein in its seeds. In this study, we constructed a seed‐protein catalog to provide a foundation for further study of the seeds. A total of 736 proteins were identified in 341 2DE spots by nano‐LC‐MS/MS. Eight storage proteins were found as multiple spots in the 2DE gels. The 736 proteins correspond to 152 unique proteins as shown by UniRef50 clustering. Sixty‐seven of the 152 proteins were associated with KEGG‐defined pathways. Of the remaining proteins, 57 were classified according to a GO term. The functions of the remaining 28 proteins have yet to be determined. This is the first yellow lupin seed–protein catalog, and it contains considerably more data than previously reported for white lupin (L. albus L.).     

Quantitative analysis of cellular proteome alterations in human influenza A virus‐infected mammalian cell lines

Over the last years virus–host cell interactions were investigated in numerous studies. Viral strategies for evasion of innate immune response, inhibition of cellular protein synthesis and permission of viral RNA and protein production were disclosed. With quantitative proteome technology, comprehensive studies concerning the impact of viruses on the cellular machinery of their host cells at protein level are possible. Therefore, 2‐D DIGE and nanoHPLC‐nanoESI‐MS/MS analysis were used to qualitatively and quantitatively determine the dynamic cellular proteome responses of two mammalian cell lines to human influenza A virus infection. A cell line used for vaccine production (MDCK) was compared with a human lung carcinoma cell line (A549) as a reference model. Analyzing 2‐D gels of the proteomes of uninfected and influenza‐infected host cells, 16 quantitatively altered protein spots (at least ±1.7‐fold change in relative abundance, p<0.001) were identified for both cell lines. Most significant changes were found for keratins, major components of the cytoskeleton system, and for Mx proteins, interferon‐induced key components of the host cell defense. Time series analysis of infection processes allowed the identification of further proteins that are described to be involved in protein synthesis, signal transduction and apoptosis events. Most likely, these proteins are required for supporting functions during influenza viral life cycle or host cell stress response. Quantitative proteome‐wide profiling of virus infection can provide insights into complexity and dynamics of virus–host cell interactions and may accelerate antiviral research and support optimization of vaccine manufacturing processes.     

Identifying secreted proteins of Marssonina brunnea by degenerate PCR

Marssonina brunnea is an important fungal pathogen of the Populus genus. To further our understanding of the pathogenesis of M. brunnea, we initiated a proteome‐level study of the fungal secretome. Using de novo peptide sequencing by MS/MS, we obtained peptide sequences for 32 protein spots. Four proteins were identified by sequence homology to conserved proteins in public databases using MS‐driven BLAST. To identify additional protein spots, we combined a degenerate PCR method, based on the Consensus–DEgenerate Hybrid Oligonucleotide Primer (CODEHOP) method, and a rapid amplification of cDNA ends method to clone the full‐length cDNA fragments encoding the proteins identified in the gel. Using this method, we cloned the full‐length cDNA fragments encoding 11 M. brunnea‐specific proteins. This method provides an efficient approach to identification of species‐specific proteins of non‐sequenced organisms. Furthermore, we analyzed the expression patterns of these genes during infection. We found that most of the identified secreted proteins could be induced in artificial medium after hyphae entered poplar apoplast spaces. We propose that for the host‐specialized M. brunnea, the elongation of hyphae has evolved closely with the secretion of apoplastic proteins.     

Inhibition of photosynthesis and modification of the wheat leaf proteome by Ptr ToxB: A host‐specific toxin from the fungal pathogen Pyrenophora tritici‐repentis

Tan spot, caused by Pyrenophora tritici‐repentis, is an important foliar disease of wheat. The fungus produces the host‐specific, chlorosis‐inducing toxin Ptr ToxB. To better understand toxin action, we examined the effects of Ptr ToxB on sensitive wheat. Photosynthesis, as measured by infrared gas analysis, declined significantly within 12 h of toxin treatment, prior to the development of chlorosis at 48–72 h. Analysis by 2‐DE revealed a total of 102 protein spots with significantly altered intensities 12–36 h after toxin treatment, of which 66 were more abundant and 36 were less abundant than in the buffer‐treated control. The identities of 47 of these spots were established by MS/MS, and included proteins involved in the light reactions of photosynthesis, the Calvin cycle, and the stress/defense response. Based on the declines in photosynthesis and the identities of the differentially abundant proteins, we hypothesize that Ptr ToxB causes a rapid disruption in the photosynthetic processes of sensitive wheat, leading to the generation of ROS and oxidative stress. Although the photoprotective and repair mechanisms of the host appear to initially still be functional, they are probably overwhelmed by the continued production of ROS, leading to chlorophyll photooxidation and the development of chlorosis.     

Effects of quantitative and qualitative differences in volatiles from host‐ and non‐host‐infested maize on the attraction of the larval parasitoid Cotesia kariyai

Cotesia kariyai Watanabe (Hymenoptera: Braconidae) is a specialist larval parasitoid of Mythimna separata Walker (Lepidoptera: Noctuidae). Cotesia kariyai wasps use herbivore‐induced plant volatiles (HIPVs) to locate hosts. However, complex natural habitats are full of volatiles released by both herbivorous host‐ and non‐host‐infested plants at various levels of intensity. Therefore, the presence of non‐hosts may affect parasitoid decisions while foraging. Here, the host‐finding efficiency of naive C. kariyai from HIPVs influenced by host‐ and non‐host‐infested maize [Zea mays L. (Poaceae)] plants was investigated with a four‐arm olfactometer. Ostrinia furnacalis Guenée (Lepidoptera: Crambidae) was selected as a non‐host species. One unit (1 U) of host‐ or non‐host‐infested plant was prepared by infesting a potted plant with five host or seven non‐host larvae. In two‐choice bioassays, host‐infested plants fed upon by different numbers of larvae, and various units of host‐ and non‐host‐infested plants (infestation units; 1 U, 2 U, and 3 U) were arranged to examine the effects of differences in volatile quantity and quality on the olfactory responses of C. kariyai with the assumption that volatile quantity and quality changes with differences in numbers of insects and plants. Cotesia kariyai was found to perceive quantitative differences in volatiles from host‐infested plants, preferring larger quantities of volatiles from larger numbers of larvae or plants. Also, the parasitoids discriminated between healthy plants, host‐infested plants, and non‐host‐infested plants by recognising volatiles released from those plants. Cotesia kariyai showed a reduced preference for host‐induced volatiles, when larger numbers of non‐host‐infested plants were present. Therefore, quantitative and qualitative differences in volatiles from host‐ and non‐host‐infested plants appear to affect the decision of C. kariyai during host‐habitat searching in multiple tritrophic systems.     

Comparative two‐dimensional polyacrylamide gel electrophoresis of the salivary proteome of children with autism spectrum disorder

In the last decades, prevalence of autism spectrum disorder (ASD) has been on the rise. However, clear aetiology is still elusive and improvements in early diagnosis are needed. To uncover possible biomarkers present in ASD, we used two‐dimensional polyacrylamide gel electrophoresis and nanoliquid chromatography‐tandem mass spectrometry (nanoLC‐MS/MS), to compare salivary proteome profiling of children with ASD and controls. A total of 889 spots were compared and only those spots with a fold change ≥1.7 and a P‐value <0.05 or a fold change of ≥3.0 between ASD cases and controls were analysed by nanoLC‐MS/MS. Alpha‐amylase, CREB‐binding protein, p532, Transferrin, Zn alpha2 glycoprotein, Zymogen granule protein 16, cystatin D and plasminogen were down‐regulated in ASD. Increased expression of proto‐oncogene Frequently rearranged in advanced T‐cell lymphomas 1 (FRAT1), Kinesin family member 14, Integrin alpha6 subunit, growth hormone regulated TBC protein 1, parotid secretory protein, Prolactin‐inducible protein precursor, Mucin‐16, Ca binding protein migration inhibitory factor‐related protein 14 (MRP14) was observed in individuals with ASD. Many of the identified proteins have previously been linked to ASD or were proposed as risk factors of ASD at the genetic level. Some others are involved in pathological pathways implicated in ASD causality such as oxidative stress, lipid and cholesterol metabolism, immune system disturbances and inflammation. These data could contribute to protein signatures for ASD presence, risk and subtypes, and advance understanding of ASD cause as well as provide novel treatment targets for ASD.     

Fitness consequences of nest infiltration by the mutualist‐exploiter Megalomyrmex adamsae

1. Fungus‐growing ants are obligate mutualists. Their nutrient‐rich fungus garden provides a valuable food store that sustains the ant hosts, but can also attract social parasites. 2. The ‘guest ant' Megalomyrmex adamsae Longino parasitises the fungus‐growing Trachymyrmex zeteki Weber queen just after nest founding. The parasitic queen infiltrates the incipient nest, builds a cavity in the fungal garden, and lays eggs that develop into workers and reproductive males and females. 3. This study compared young parasitised and non‐parasitised laboratory colonies by measuring garden growth and biomass, and the number of host workers and reproductives. Host queen survival and parasite colony growth were also monitored. 4. Parasitised Trachymyrmex colonies had reduced host worker and alate numbers, as well as lower garden biomass, compared with non‐parasitised control colonies, confirming that M. adamsae is a xenobiotic social parasite. Host queen survival was not significantly different between parasitised and control colonies. 5. This is the first study that experimentally infects host colonies with a xenobiotic social parasite to measure fitness cost to the host. The natural history of M. adamsae and the fungus‐growing ant mutualism are evaluated in the context of three general predictions of (Bronstein, Ecology Letters, 4 , 277–287, 2001a) regarding the cost of mutualism exploiters.     

A comparative study on the heparin‐binding proteomes of Toxoplasma gondii and Plasmodium falciparum

Toxoplasma gondii is an obligatory intracellular apicomplexan parasite which exploits host cell surface components in cell invasion and intracellular parasitization. Sulfated glycans such as heparin and heparan sulfate have been reported to inhibit cell invasion by T. gondii and other apicomplexan parasites such as Plasmodium falciparum. The aim of this study was to investigate the heparin‐binding proteome of T. gondii. The parasite‐derived components were affinity‐purified on the heparin moiety followed by MS fingerprinting of the proteins. The heparin‐binding proteins of T. gondii and P. falciparum were compared based on functionality and affinity to heparin. Among the proteins identified, the invasion‐related parasite ligands derived from tachyzoite/merozoite surface and the secretory organelles were prominent. However, the profiles of the proteins were different in terms of affinity to heparin. In T. gondii, the proteins with highest affinity to heparin were the intracellular components with functions of parasite development contrasted to that of P. falciparum, of which the rhoptry‐derived proteins were prominently identified. The profiling of the heparin‐binding proteins of the two apicomplexan parasites not only explained the mechanism of heparin‐mediated host cell invasion inhibition, but also, to a certain extent, revealed that the action of heparin on the parasite extended after endocytosis.     

Two‐dimensional proteome reference map for the radiation‐resistant bacterium Deinococcus geothermalis

2‐DE reference maps for Deinococcus geothermalis cytosolic and cell envelope proteomes were constructed. In total, 403 spots were identified as 299 different proteins. Unique in the proteomes were four subunits of V‐type ATPase and Deinococcus specific proteins constituting one‐fourth of cell envelope proteome. The cytoplasmic proteome included enzymes of the central carbon metabolism, chaperones, enzymes of protein and DNA repair, and oxidative stress. A total of 34 abundant proteins with unknown function may relate to the extreme stress tolerance of D. geothermalis.     

Protein reference mapping of dihydrofolate reductase‐deficient CHO DG44 cell lines using 2‐dimensional electrophoresis

Chinese hamster ovary (CHO) cells are the major mammalian host for producing various therapeutic proteins. Among CHO cells, the dihydrofolate reductase‐deficient CHO DG44 cell line has been used as a popular mammalian host because of the availability of a well‐characterized genetic selection and amplification system. However, this cell line has not been studied at the proteome level. Here, the first detailed proteome analysis of the CHO DG44 cell line is described. A protein reference map of the CHO DG44 cell line was established by analyzing whole cellular proteins using 2‐DE with various immobilized pH gradients (pHs 3–10, 5–8, and 3–6) in the first dimension and a 12% acrylamide gel in the second dimension. The map is composed of over 1400 silver‐stained protein spots. Among them, 179 protein spots, which represent proteins associated with various biological processes and cellular compartments, were identified based on MALDI‐TOF‐MS and MS/MS. This proteome database should be valuable for better understanding of CHO cell physiology and protein expression patterns which may lead to efficient therapeutic protein production.