Peter R. Jungblut

Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida albicans

Neutrophils are the first line of defense at the site of an infection. They encounter and kill microbes intracellularly upon phagocytosis or extracellularly by degranulation of antimicrobial proteins and the release of Neutrophil Extracellular Traps (NETs). NETs were shown to ensnare and kill microbes. However, their complete protein composition and the antimicrobial mechanism are not well understood. Using a proteomic approach, we identified 24 NET-associated proteins. Quantitative analysis of these proteins and high resolution electron microscopy showed that NETs consist of modified nucleosomes and a stringent selection of other proteins. In contrast to previous results, we found several NET proteins that are cytoplasmic in unstimulated neutrophils. We demonstrated that of those proteins, the antimicrobial heterodimer calprotectin is released in NETs as the major antifungal component. Absence of calprotectin in NETs resulted in complete loss of antifungal activity in vitro. Analysis of three different Candida albicans in vivo infection models indicated that NET formation is a hitherto unrecognized route of calprotectin release. By comparing wild-type and calprotectin-deficient animals we found that calprotectin is crucial for the clearance of infection. Taken together, the present investigations confirmed the antifungal activity of calprotectin in vitro and, moreover, demonstrated that it contributes to effective host defense against C. albicans in vivo. We showed for the first time that a proportion of calprotectin is bound to NETs in vitro and in vivo.

Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42.

Bacillus amyloliquefaciens FZB42 is a Gram-positive, plant-associated bacterium, which stimulates plant growth and produces secondary metabolites that suppress soil-borne plant pathogens. Its 3,918-kb genome, containing an estimated 3,693 protein-coding sequences, lacks extended phage insertions, which occur ubiquitously in the closely related Bacillus subtilis 168 genome. The B. amyloliquefaciens FZB42 genome reveals an unexpected potential to produce secondary metabolites, including the polyketides bacillaene and difficidin. More than 8.5% of the genome is devoted to synthesizing antibiotics and siderophores by pathways not involving ribosomes. Besides five gene clusters, known from B. subtilis to mediate nonribosomal synthesis of secondary metabolites, we identified four giant gene clusters absent in B. subtilis 168. The pks2 gene cluster encodes the components to synthesize the macrolactin core skeleton.

Translocation of the Helicobacter pylori CagA protein in gastric epithelial cells by a type IV secretion apparatus

Helicobacter pylori is one of the most common bacterial pathogens, infecting about 50% of the world population. The presence of a pathogenicity island (PAI) in H. pylori has been associated with gastric disease. We present evidence that the H. pylori protein encoded by the cytotoxin‐associated gene A (cagA) is translocated and phosphorylated in infected epithelial cells. Two‐dimensional gel electrophoresis (2‐DE) of proteins isolated from infected AGS cells revealed H. pylori strain‐specific and time‐dependent tyrosine phosphorylation and dephosphorylation of several 125–135 kDa and 75–80 kDa proteins. Immunoblotting studies, matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS), cell fractionation and confocal microscopy demonstrated that one of the 125–135 kDa proteins represents the H. pylori CagA protein, which is translocated into the host cell membrane and the cytoplasm. Translocation of CagA was dependent on functional cagA gene and virulence (vir) genes of a type IV secretion apparatus composed of virB4, virB7, virB10, virB11 and virD4 encoded in the cag PAI of H. pylori. Our findings support the view that H. pylori actively translocates virulence determinants, including CagA, which could be involved in the development of a variety of gastric disease.

Comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains: towards functional genomics of microbial pathogens

In 1993, the WHO declared tuberculosis a global emergency on the basis that there are 8 million new cases per year. The complete genome of the strain H37Rv of the causative microorganism, Mycobacterium tuberculosis, comprising 3924 genes has been sequenced. We compared the proteomes of two non‐virulent vaccine strains of M. bovis BCG (Chicago and Copenhagen) with two virulent strains of M. tuberculosis (H37Rv and Erdman) to identify protein candidates of value for the development of vaccines, diagnostics and therapeutics. The mycobacterial strains were analysed by two‐dimensional electrophoresis (2‐DE) combining non‐equilibrium pH gradient electrophoresis (NEPHGE) with SDS–PAGE. Distinct and characteristic proteins were identified by mass spectrometry and introduced into a dynamic 2‐DE database (http://www.mpiib‐berlin.mpg.de/2D‐PAGE). Silver‐stained 2‐DE patterns of mycobacterial cell proteins or culture supernatants contained 1800 or 800 spots, respectively, from which 263 were identified. Of these, 54 belong to the culture supernatant. Sixteen and 25 proteins differing in intensity or position between M. tuberculosis H37Rv and Erdman, and H37Rv and M. bovis BCG Chicago, respectively, were identified and categorized into protein classes. It is to be hoped that the availability of the mycobacterial proteome will facilitate the design of novel measures for prevention and therapy of one of the great health threats, tuberculosis.

PROTEOMICS IN HUMAN DISEASE : CANCER, HEART AND INFECTIOUS DISEASES

In recent years, genomics has increased the understanding of many diseases. Proteomics is a rapidly growing research area that encompasses both genetic and environmental factors. The protein composition represents the functional status of a biological compartment. The five approaches presented here resulted in the detection of disease‐associated proteins. Calgranulin B was upregulated in colorectal cancer, and hepatoma‐derived aldose reductase‐like protein was reexpressed in a rat model during hepatocarcinogenesis. In these two investigations, attention was focused on one protein, obviously differing in amount, directly after two‐dimensional electrophoresis (2‐DE). Additional methods, such as enzyme activity measurements and immunohistochemistry, confirmed the disease association of the two candidates resulting from 2‐DE subtractive analysis. The following three investigations take advantage of the holistic potential of the 2‐DE approach. The comparison of 2‐DE patterns from dilated cardiomyopathy patients with those of controls revealed 25 statistically significant intensity differences, from which 12 were identified by amino acid analysis, Edman degradation or matrix‐assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS). A human myocardial 2‐DE database was constructed, containing 3300 protein spots and 150 identified protein species. The number of identified proteins was limited by the capacity of our group, rather than by the principle of feasibility. Another field where proteomics proves to be a valuable tool in identifying proteins of importance for diagnosis is proteome analysis of pathogenic microorganisms such as Borrelia burgdorferi (Lyme disease) and Toxoplasma gondii (toxoplasmosis). Sera from patients with early or late symptoms of Lyme borreliosis contained antibodies of various classes against about 80 antigens each, containing the already described antigens OspA, B and C, flagellin, p83/100, and p39. Similarly, antibody reactivity to seven different marker antigens of T. gondii allowed differentiation between acute and latent toxoplasmosis, an important diagnostic tool in both pregnancy and immunosuppressed patients.

Comparative proteome analysis of Helicobacter pylori

Helicobacter pylori, the causative agent of gastritis, ulcer and stomach carcinoma, infects approximately half of the worlds population. After sequencing the complete genome of two strains, 26695 and J99, we have approached the demanding task of investigating the functional part of the genetic information containing macromolecules, the proteome. The proteins of three strains of H. pylori, 26695 and J99, and a prominent strain used in animal models SS1, were separated by a high‐resolution two‐dimensional electrophoresis technique with a resolution power of 5000 protein spots. Up to 1800 protein species were separated from H. pylori which had been cultivated for 5 days on agar plates. Using matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) peptide mass fingerprinting we have identified 152 proteins, including nine known virulence factors and 28 antigens. The three strains investigated had only a few protein spots in common. We observe that proteins with an amino acid exchange resulting in a net change of only one charge are shifted in the two‐dimensional electrophoresis (2‐DE) pattern. The expression of 27 predicted conserved hypothetical open reading frames (ORFs) and six unknown ORFs were confirmed. The growth conditions of the bacteria were shown to have an effect on the presence of certain proteins. A preliminary immunoblotting study using human sera revealed that this approach is ideal for identifying proteins of diagnostic or therapeutic value. H. pylori 2‐DE patterns with their identified protein species were added to the dynamic 2D‐PAGE database (http://www.mpiib‐berlin.mpg.de/2D‐PAGE/). This basic knowledge of the proteome in the public domain will be an effective instrument for the identification of new virulence or pathogenic factors, and antigens of potentially diagnostic or curative value against H. pylori.

The speciation of the proteome

IntroductionIn proteomics a paradox situation developed in the last years. At one side it is basic knowledge that proteins are post-translationally modified and occur in different isoforms. At the other side the protein expression concept disclaims post-translational modifications by connecting protein names directly with function.DiscussionOptimal proteome coverage is today reached by bottom-up liquid chromatography/mass spectrometry. But quantification at the peptide level in shotgun or bottom-up approaches by liquid chromatography and mass spectrometry is completely ignoring that a special peptide may exist in an unmodified form and in several-fold modified forms. The acceptance of the protein species concept is a basic prerequisite for meaningful quantitative analyses in functional proteomics. In discovery approaches only top-down analyses, separating the protein species before digestion, identification and quantification by two-dimensional gel electrophoresis or protein liquid chromatography, allow the correlation between changes of a biological situation and function.ConclusionTo obtain biological relevant information kinetics and systems biology have to be performed at the protein species level, which is the major challenge in proteomics today.

Proteome Analysis of Secreted Proteins of the Gastric Pathogen Helicobacter pylori

ABSTRACT Secreted proteins (the secretome) of the human pathogen Helicobacter pylori may mediate important pathogen-host interactions, but such proteins are technically difficult to analyze. Here, we report on a comprehensive secretome analysis that uses protein-free culture conditions to minimize autolysis, an efficient recovery method for extracellular proteins, and two-dimensional gel electrophoresis followed by peptide mass fingerprinting for protein resolution and identification. Twenty-six of the 33 separated secreted proteins were identified. Among them were six putative oxidoreductases that may be involved in the modification of protein-disulfide bonds, three flagellar proteins, three defined fragments of the vacuolating toxin VacA, the serine protease HtrA, and eight proteins of unknown function. A cleavage site for the amino-terminal passenger domain of VacA between amino acids 991 and 992 was determined by collision-induced dissociation mass spectrometry. Several of the secreted proteins are interesting targets for antimicrobial chemotherapy and vaccine development.

Finding one's way in proteomics: a protein species nomenclature

Our knowledge of proteins has greatly improved in recent years, driven by new technologies in the fields of molecular biology and proteome research. It has become clear that from a single gene not only one single gene product but many different ones - termed protein species - are generated, all of which may be associated with different functions. Nonetheless, an unambiguous nomenclature for describing individual protein species is still lacking. With the present paper we therefore propose a systematic nomenclature for the comprehensive description of protein species. The protein species nomenclature is flexible and adaptable to every level of knowledge and of experimental data in accordance with the exact chemical composition of individual protein species. As a minimum description the entry name (gene name + species according to the UniProt knowledgebase) can be used, if no analytical data about the target protein species are available.

Immunoproteomics of Helicobacter pylori infection and relation to gastric disease

The Gram negative bacterium Helicobacter pylori is a human pathogen which infects the gastric mucosa and causes an inflammatory process leading to gastritis, ulceration and cancer. A systematic, proteome based approach was chosen to detect candidate antigens of H. pylori for diagnosis, therapy and vaccine development and to investigate potential associations between specific immune responses and manifestations of disease. Sera from patients with active H. pylori infection (n = 24), a control group with unrelated gastric disorders (n = 12) and from patients with gastric cancer (n = 6) were collected and analyzed for the reactivity against proteins of the strain HP 26695 separated by two‐dimensional electrophoresis. Overall, 310 antigenic protein species were recognized by H. pylori positive sera representing about 17% of all spots separated. Out of the 32 antigens most frequently recognized by H. pylori positive sera, nine were newly identified and 23 were confirmed from other studies. Three newly identified antigens which belong to the 150 most abundant protein species of H. pylori, were specifically recognized by H. pylori positive sera: the predicted coding region HP0231, serine protease HtrA (HP1019) and Cag3 (HP0522). Other antigens were recognized differently by sera from gastritis and ulcer patients, which may identify them as candidate indicators for clinical manifestations. The data from these immunoproteomic analyses are added to our public database (http://www.mpiib‐berlin.mpg.de/2D‐PAGE). This platform enables one to compile many protein profiles and to integrate data from other studies, an approach which will greatly assist the search for more immunogenic proteins for diagnostic assays and vaccine design.