Dirk Bumann

Robust Salmonella metabolism limits possibilities for new antimicrobials

New antibiotics are urgently needed to control infectious diseases. Metabolic enzymes could represent attractive targets for such antibiotics, but in vivo target validation is largely lacking. Here we have obtained in vivo information about over 700 Salmonella enterica enzymes from network analysis of mutant phenotypes, genome comparisons and Salmonella proteomes from infected mice. Over 400 of these enzymes are non-essential for Salmonella virulence, reflecting extensive metabolic redundancies and access to surprisingly diverse host nutrients. The essential enzymes identified were almost exclusively associated with a small subgroup of pathways, enabling us to perform a nearly exhaustive screen. Sixty-four enzymes identified as essential in Salmonella are conserved in other important human pathogens, but almost all belong to metabolic pathways that are inhibited by current antibiotics or that have previously been considered for antimicrobial development. Our comprehensive in vivo analysis thus suggests a shortage of new metabolic targets for broad-spectrum antibiotics, and draws attention to some previously known but unexploited targets.

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.

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.

Caspase-11 activation requires lysis of pathogen-containing vacuoles by IFN-induced GTPases

Lipopolysaccharide from Gram-negative bacteria is sensed in the host cell cytoplasm by a non-canonical inflammasome pathway that ultimately results in caspase-11 activation and cell death. In mouse macrophages, activation of this pathway requires the production of type-I interferons, indicating that interferon-induced genes have a critical role in initiating this pathway. Here we report that a cluster of small interferon-inducible GTPases, the so-called guanylate-binding proteins, is required for the full activity of the non-canonical caspase-11 inflammasome during infections with vacuolar Gram-negative bacteria. We show that guanylate-binding proteins are recruited to intracellular bacterial pathogens and are necessary to induce the lysis of the pathogen-containing vacuole. Lysis of the vacuole releases bacteria into the cytosol, thus allowing the detection of their lipopolysaccharide by a yet unknown lipopolysaccharide sensor. Moreover, recognition of the lysed vacuole by the danger sensor galectin-8 initiates the uptake of bacteria into autophagosomes, which results in a reduction of caspase-11 activation. These results indicate that host-mediated lysis of pathogen-containing vacuoles is an essential immune function and is necessary for efficient recognition of pathogens by inflammasome complexes in the cytosol.

Identification of Surface Proteins of Helicobacter pylori by Selective Biotinylation, Affinity Purification, and Two-dimensional Gel Electrophoresis

Helicobacter pylori is a widespread human pathogen that can cause gastric ulcers and cancer. To identify surface proteins that may play a role in pathogen-host interactions and represent potential targets for the control of this infection, we selectively biotinylated intact H. pylori with the hydrophilic reagent sulfosuccinimidyl-6-(biotinamido)-hexanoate and purified the labeled proteins by membrane isolation, solubilization, and affinity chromatography. After separation of 82 biotinylated proteins on two-dimensional gels, 18 were identified with comparison to proteome data and peptide mass fingerprinting. Among the identified proteins, 9 have previously been shown to be surface-exposed, 7 are associated with virulence, and 11 are highly immunogenic in infected patients. In conclusion, this generally applicable combined proteome approach facilitates the rapid identification of promising targets for the control of H. pylori and might be applicable to numerous other human pathogens although larger biotinylation reagents might be required in some cases to prevent permeation of porin channels in the outer membrane.

Microbial quest for food in vivo: ‘Nutritional virulence’ as an emerging paradigm

Microbial access to host nutrients is a fundamental aspect of infectious diseases. Pathogens face complex dynamic nutritional host microenvironments that change with increasing inflammation and local hypoxia. Since the host can actively limit microbial access to nutrient supply, pathogens have evolved various metabolic adaptations to successfully exploit available host nutrients for proliferation. Recent studies have unraveled an emerging paradigm that we propose to designate as ‘nutritional virulence’. This paradigm is based on specific virulence mechanisms that target major host biosynthetic and degradation pathways (proteasomes, autophagy and lysosomes) or nutrient‐rich sources, such as glutathione, to enhance host supply of limiting nutrients, such as cysteine. Although Cys is the most limiting cellular amino acid, it is a metabolically favourable source of carbon and energy for various pathogens that are auxotrophic for Cys but utilize idiosyncratic nutritional virulence strategies to generate a gratuitous supply of host Cys. Therefore, proliferation of some intracellular pathogens is restricted by a host nutritional rheostat regulated by certain limiting amino acids, and pathogens have evolved idiosyncratic strategies to short circuit the host nutritional rheostat. Deciphering mechanisms of microbial ‘nutritional virulence’ and metabolism in vivo will facilitate identification of novel microbialand host targets for treatment and prevention of infectious diseases. Host–pathogen synchronization of amino acid auxotrophy indicates that this nutritional synchronization has been a major driving force in the evolution of many intracellular bacterial pathogens.

A community effort towards a knowledge-base and mathematical model of the human pathogen Salmonella Typhimurium LT2.

BackgroundMetabolic reconstructions (MRs) are common denominators in systems biology and represent biochemical, genetic, and genomic (BiGG) knowledge-bases for target organisms by capturing currently available information in a consistent, structured manner. Salmonella enterica subspecies I serovar Typhimurium is a human pathogen, causes various diseases and its increasing antibiotic resistance poses a public health problem.ResultsHere, we describe a community-driven effort, in which more than 20 experts in S. Typhimurium biology and systems biology collaborated to reconcile and expand the S. Typhimurium BiGG knowledge-base. The consensus MR was obtained starting from two independently developed MRs for S. Typhimurium. Key results of this reconstruction jamboree include i) development and implementation of a community-based workflow for MR annotation and reconciliation; ii) incorporation of thermodynamic information; and iii) use of the consensus MR to identify potential multi-target drug therapy approaches.ConclusionTaken together, with the growing number of parallel MRs a structured, community-driven approach will be necessary to maximize quality while increasing adoption of MRs in experimental design and interpretation.

Safety and immunogenicity of live recombinant Salmonella enterica serovar Typhi Ty21a expressing urease A and B from Helicobacter pylori in human volunteers

Helicobacter pylori urease was expressed in the common live typhoid vaccine Ty21a yielding Ty21a(pDB1). Nine volunteers received Ty21a(pDB1) and three control volunteers received Ty21a. No serious adverse effects were observed in any of the volunteers. Ten out of 12 volunteers developed humoral immune responses to the Salmonella carrier as detected by antigen-specific antibody-secreting cells but only two volunteers seroconverted. A total of five volunteers showed responses in one or two out of three assays for cellular responses to the carrier (proliferation, IFN-gamma-secretion, IFN-gamma-ELISPOT). Three of the volunteers that had received Ty21a(pDB1) showed a weak but significant T-cell response to Helicobacter urease, while no volunteer had detectable humoral responses to urease. Ty21a(pDB1) is a suitable prototype to optimize Salmonella-based vaccination for efficient cellular responses that could mediate protective immunity against Helicobacter.

Rapidly maturing red fluorescent protein variants with strongly enhanced brightness in bacteria

A rapidly maturing variant of the red fluorescent protein DsRed was optimized for bacterial expression by random mutagenesis. The brightest variant contains six mutations, two of which (S4T and a silent mutation in codon 2) explain most of the fluorescence enhancement. The novel variants are expressed at 9–60‐fold higher levels in Escherichia coli compared to DsRed.T3, but are not superior fluorophores on a per molecule basis. In contrast to previously available DsRed variants, DsRed.T3_S4T is sufficiently bright to monitor Salmonella gene expression in infected animals using flow cytometry. However, no fluorescence enhancement was observed in Leishmania or HeLa cells, indicating that these novel variants are specifically useful for bacteria.

Correlation of T cell response and bacterial clearance in human volunteers challenged with Helicobacter pylori revealed by randomised controlled vaccination with Ty21a-based Salmonella vaccines

Background: Helicobacter pylori remains a global health hazard, and vaccination would be ideal for its control. Natural infection appears not to induce protective immunity. Thus, the feasibility of a vaccine for humans is doubtful. Methods: In two prospective, randomised, double-blind, controlled studies (Paul Ehrlich Institute application nos 0802/02 and 1097/01), live vaccines against H pylori were tested in human volunteers seronegative for, and without evidence of, active H pylori infection. Volunteers (n = 58) were immunised orally with Salmonella enterica serovar Typhi Ty21a expressing H pylori urease or HP0231, or solely with Ty21a, and then challenged with 2×105 cagPAI− H pylori. Adverse events, infection, humoral, cellular and mucosal immune response were monitored. Gastric biopsies were taken before and after vaccination, and postchallenge. Infection was terminated with antibiotics. Results: Vaccines were well tolerated. Challenge infection induced transient, mild to moderate dyspeptic symptoms, and histological and transcriptional changes in the mucosa known from chronic infection. Vaccines did not show satisfactory protection. However, 13 of 58 volunteers, 8 vaccinees and 5 controls, became breath test negative and either cleared H pylori (5/13) completely or reduced the H pylori burden (8/13). H pylori-specific T helper cells were detected in 9 of these 13 (69%), but only in 6 of 45 (13%) breath test-positive volunteers (p = 0.0002; Fisher exact test). T cells were either vaccine induced or pre-existing, depending on the volunteer. Conclusion: Challenge infection offers a controlled model for vaccine testing. Importantly, it revealed evidence for T cell-mediated immunity against H pylori infection in humans.