Susanne Jacobsen

Comparative Evaluation of Low-Molecular-Mass Proteins from Mycobacterium tuberculosis Identifies Members of the ESAT-6 Family as Immunodominant T-Cell Antigens

ABSTRACT Culture filtrate from Mycobacterium tuberculosiscontains protective antigens of relevance for the generation of a new antituberculosis vaccine. We have identified two previously uncharacterized M. tuberculosis proteins (TB7.3 and TB10.4) from the highly active low-mass fraction of culture filtrate. The molecules were characterized, mapped in a two-dimensional electrophoresis reference map of short-term culture filtrate, and compared with another recently identified low-mass protein, CFP10 (F. X. Berthet, P. B. Rasmussen, I. Rosenkrands, P. Andersen, and B. Gicquel. Microbiology 144:3195–3203, 1998), and the well-described ESAT-6 antigen. Genetic analyses demonstrated that TB10.4 as well as CFP10 belongs to the ESAT-6 family of low-mass proteins, whereas TB7.3 is a low-molecular-mass protein outside this family. The proteins were expressed in Escherichia coli, and their immunogenicity was tested in cultures of peripheral blood mononuclear cells from human tuberculosis (TB) patients, Mycobacterium bovisBCG-vaccinated donors, and nonvaccinated donors. The two ESAT-6 family members, TB10.4 and CFP10, were very strongly recognized and induced gamma interferon release at the same level (CFP10) as or at an even higher level (TB10.4) than ESAT-6. The non-ESAT-6 family member, TB7.3, for comparison, was recognized at a much lower level. CFP10 was found to distinguish TB patients from BCG-vaccinated donors and is, together with ESAT-6, an interesting candidate for the diagnosis of TB. The striking immunodominance of antigens within the ESAT-6 family is discussed, and hypotheses are presented to explain this targeting of the immune response during TB infection.

Mapping and identification of Mycobacterium tuberculosis proteins by two-dimensional gel electrophoresis, microsequencing and immunodetection.

Mycobacterium tuberculosis is the infectious agent giving rise to human tuberculosis. The entire genome of M. tuberculosis, comprising approximately 4000 open reading frames, has been sequenced. The huge amount of information released from this project has facilitated proteome analysis of M. tuberculosis. Two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) was applied to fractions derived from M. tuberculosis culture filtrate, cell wall, and cytosol, resulting in the resolution of 376, 413, and 395 spots, respectively, in silver‐stained gels. By microsequencing and immunodetection, 38 culture filtrate proteins were identified and mapped, of which 12 were identified for the first time. In the same manner, 23 cell wall proteins and 19 cytosol proteins were identified and mapped, with 9 and 10, respectively, being novel proteins. One of the novel proteins was not predicted in the genome project, and for four of the identified proteins alternative start codons were suggested. Fourteen of the culture filtrate proteins were proposed to possess signal sequences. Seven of these proteins were microsequenced and the N‐terminal sequences obtained confirmed the prediction. The data presented here are an important complement to the genetic information, and the established 2‐D PAGE maps (also available at: www.ssi.dk/publichealth/tbimmun) provide a basis for comparative studies of protein expression.

Two antifungal thaumatin-like proteins from barley grain

Antifungal activity has been associated with 2 immunochemically distinct proteins, protein R and S (M r ∼23 kDa; pI 9‐10), which were isolated in pure form from barley grain. The proteins are homologous with thaumatin‐ and pathogenesis‐related proteins of the PR5 family. The proteins inhibit growth of i.a. Trichoderma viride and Candida albicans in microtiter plate assays and act synergistically with barley grain chitinase C. Like maize zeamatin, protein R and S but not chitinase C retarded fungal growth in synergism with nikkomycin Z, a nucleoside‐peptide inhibitor of fungal chitin synthesis. Although no inhibition of α‐amylases or serine proteases could be associated with protein R or S the results indicate that the homologous maize grain bifunctional inhibitor of insect α‐amylase and trypsin is very similar to or identical with maize zeamatin, which was proposed to have permeabilizing activity towards fungal membranes. Thus, in addition to the intensely sweet properties of thaumatin, multiple unrelated defense functions against insect and fungal pests can now be associated with the family of thaumatin‐homologous proteins.

Antifungal activity of chitin-binding PR-4 type proteins from barley grain and stressed leaf

Antifungal activity in vitro has been associated with barley leaf and grain proteins which are homologous with pathogenesis related proteins of type 4 (PR‐4) from tobacco and tomato and with C terminal domains of potato win and Hevea hevein precursor proteins. One protein (pI ∼9.3, M r ∼13.7 kDa) from barley grain and two very similar proteins from leaves infected with Erysiphe graminis were isolated by chitin affinity chromatography, but none of the proteins showed chitinase activity in vitro. The leaf proteins were increased several fold in response to either Erysiphe infection or NiCl2 infiltration and accumulated extracellularly. The three barley proteins were found to inhibit growth of Trichoderma harzianum in microtiter plate assays using ∼‐10 μg/ml concentrations and in lower concentrations in a synergistic way when mixed either with barley chitinase C (a PR‐3 type protein) or with barley protein R (a PR‐5 type protein). Structurally similar proteins were detected in wheat, rye and oats grain extracts.

Implications of high-temperature events and water deficits on protein profiles in wheat (Triticum aestivum L. cv. Vinjett) grain.

Increased climatic variability is resulting in an increase of both the frequency and the magnitude of extreme climate events. Therefore, cereals may be exposed to more than one stress event in the growing season, which may ultimately affect crop yield and quality. Here, effects are reported of interaction of water deficits and/or a high‐temperature event (32°C) during vegetative growth (terminal spikelet) with either of these stress events applied during generative growth (anthesis) in wheat. Influence of combinations of stress on protein fractions (albumins, globulins, gliadins and glutenins) in grains and stress‐induced changes on the albumin and gliadin proteomes were investigated by 2‐DE and MS. The synthesis of individual protein fractions was shown to be affected by both the type and time of the applied stresses. Identified drought or high‐temperature‐responsive proteins included proteins involved in primary metabolism, storage and stress response such as late embryogenesis abundant proteins, peroxiredoxins and α‐amylase/trypsin inhibitors. Several proteins, e.g. heat shock protein and 14‐3‐3 protein changed in abundance only under multiple high temperatures.

Purification and characterization of three chitinases and one β-1,3-glucanase accumulating in the medium of cell suspension cultures of barley (Hordeum vulgare L.)

Abstract Three basic chitinases and one basic β-1,3-glucanase were secreted into the medium when embryogenic cell suspensions of barley ( Hordeum vulgare L.) cv. ‘Igri’ were cultured as undifferentiated aggregates in the presence of 2,4-D. The enzymes were purified by affinity and ion exchanged chromatography. Two of the chitinases were identified as the previously described endochitinases T and C from barley grain. The third and novel chitinase, designated K, was the major basic chitinase in the medium constituting 4% of the soluble protein. Chitinase K was found to be a 33-kDa endochitinase with pI at 8.7. Further analysis showed that this enzyme is also expressed in barley grain. The amino acid composition and five partial amino acid sequences covering 93 residues of chitinase K were determined. A high similarity was found between chitinase K and barley chitinase T and C as well as basic chitinases from barley aleurone and barley, bean and potato leaves. The purified β-1,3-glucanase with a molecular weight (MW) of 32 kDa and pI ≥ 9.8 constituted 1% of the soluble protein in the medium. Based on similar MW, pI and amino acid composition as well as identical N-terminal sequence (24 residues) this enzyme was identified as the previously described β-1,3-endoglucanase GII from germinating barley grain. The results suggest that secretion of chitinase and β-1,3-glucanase from the embryogenic cell suspensions of barley corresponds to accumulation of the same enzymes in barley grain.

Induction, purification and characterization of barley leaf chitinase

Abstract Chitinase activity in susceptible and resistant primary leaves of barley ( Hordeum vulgare L.) was found to be induced fivefold and threefold, respectively, 7 days after inoculation with the powdery mildew fungus ( Erysiphe graminis f.sp. hordei ). A minor proportion of the total chitinase activity from control and infected leaves was detected in intracellular washing fluids from the leaf material. Six chitin binding proteins from barley leaves were characterized after purification by affinity and cation exchange chromatography. One of the purified proteins is an endochitinase with an apparent molecular weight of 30 kDa and a pI of 9.1. A partial amino acid sequence obtained after tryptic digestion of the protein shows high similarity to amino acid predicted from endochitinase cDNA clones from bean leaves, potato leaves and barley aleurone cells, as well as to an endochitinase from barley endosperm.

The extracellular proteome of Bifidobacterium animalis subsp. lactis BB-12 reveals proteins with putative roles in probiotic effects

Probiotics are live microorganisms that exert health‐promoting effects on the human host, as demonstrated for numerous strains of the genus Bifidobacterium. To unravel the proteins involved in the interactions between the host and the extensively used and well‐studied probiotic strain Bifidobacterium animalis subsp. lactis BB‐12, proteins secreted by the bacterium, i.e. belonging to the extracellular proteome present in the culture medium, were identified by 2‐DE coupled with MALDI‐TOF MS. Among the 74 distinct proteins identified, 31 are predicted to carry out their physiological role either outside the cell or on its surface. These proteins include solute‐binding proteins for oligosaccharides, amino acids and manganese, cell wall‐metabolizing proteins, and 18 proteins that have been described to interact with human host epithelial cells or extracellular matrix proteins. The potential functions include binding of plasminogen, formation of fimbriae, adhesion to collagen, attachment to mucin and intestinal cells as well as induction of immunomodulative response. These findings suggest a role of the proteins in colonization of the gastrointestinal tract, adhesion to host tissues, or immunomodulation of the host immune system. The identification of proteins predicted to be involved in such interactions can pave the way towards well targeted studies of the protein‐mediated contacts between bacteria and the host, with the goal to enhance the understanding of the mode of action of probiotic bacteria.

Improved tolerance to drought stress after anthesis due to priming before anthesis in wheat (Triticum aestivum L.) var. Vinjett

Summary Physiological and proteome insights were determined into the alleviating effects of pre-anthesis drought priming on drought stress during post-anthesis, which will be of importance for future drought-tolerance studies in cereals.

Proteome reference map of Lactobacillus acidophilus NCFM and quantitative proteomics towards understanding the prebiotic action of lactitol

Lactobacillus acidophilus NCFM is a probiotic bacterium adapted to survive in the gastrointestinal tract and with potential health benefits to the host. Lactitol is a synthetic sugar alcohol used as a sugar replacement in low calorie foods and selectively stimulating growth of L. acidophilus NCFM. In the present study the whole‐cell extract proteome of L. acidophilus NCFM grown on glucose until late exponential phase was resolved by 2‐DE (pH 3–7). A total of 275 unique proteins assigned to various physiological processes were identified from 650 spots. Differential 2‐DE (DIGE) (pH 4–7) of L. acidophilus NCFM grown on glucose and lactitol, revealed 68 spots with modified relative intensity. Thirty‐two unique proteins were identified in 41 of these spots changing 1.6–12.7‐fold in relative abundance by adaptation of L. acidophilus NCFM to growth on lactitol. These proteins included β‐galactosidase small subunit, galactokinase, galactose‐1‐phosphate uridylyltransferase and UDP‐glucose‐4‐epimerase, which all are potentially involved in lactitol metabolism. This first comprehensive proteome analysis of L. acidophilus NCFM provides insights into protein abundance changes elicited by the prebiotic lactitol.