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Project Group 5: Virulence Factors in Salmonella and Campylobacter

Head:
Roman Gerlach

Staff:

Dipl. Biol. Kathrin Blank
Medical laboratory assistant Christiane Schmidt
Dipl. Biol. Thorsten Wille

Subject

With more than 31,400 cases every year in Germany (2009) salmonelloses are one of the most frequent bacterial infectious diseases. The actual incidence is probably far higher as mild courses of the disease are not recorded. The serovars typhimurium and enteritidis are the Salmonella pathogens that occur in humans in almost 80% of all cases. Both serovars have a broad host spectrum, and livestock in particular constitutes a readily available reservoir for these Salmonella. Of the more than 2,500 serovars described of Salmonella enterica, a few are however highly adapted to humans. The serovars typhi, paratyphus A, B und C can cause severe systemic infections. The serovar typhi, the agent of Typhus abdominalis, almost only plays a role as a travel-associated disease with 65 notified cases in Germany (2009). These bacteria are endemic in many parts of South East Asia and in countries with inadequate hygiene standards. What is particularly worrying is the increase in multi-resistant isolates from these countries in recent years. Another trend, probably triggered by the targeted control of Salmonella typhi by means of vaccinations, is the rapid spread of NTS (nontyphoidal Salmonella) with severe systemic courses. Infections with NTS mainly involve the serovars typhimurium and enteritidis and constitute a major problem in Sub-Saharan Africa where there is a high level of fatalities amongst children and HIV-positive adults. Although the hygiene standards as well as the average immune status of the population in Germany are far higher, the spread of these pathogens constitutes a latent risk. As a consequence of the globalised movement of goods and individuals, these pathogens along with their undesirable properties (e.g. antibiotic resistance) can rapidly spread through the population.

Although Salmonella enterica is probably the best characterised gram-negative pathogen, many virulence mechanisms of this bacterium have not yet been fully understood. The focus of our work is on understanding the molecular mechanisms that cause pathogenicity. This work encompasses both the further characterisation of known virulence factors as well as the identification of new genes with a virulence function. Given the availability of more than 40 genome sequences of Salmonella enterica it is possible using bioinformatics methods to identify candidate genes and candidate regions and to further characterise them using a spectrum of methods consisting of molecular biology, biochemistry and biophysics. We pay special attention to (I) areas acquired in the genome with potential virulence function through Horizontal Gene Transfer (HGT, i.e. from other bacteria species) and (II) secretion systems and their substrates. Secretion systems play a decisive role for all pathogenic bacteria as they can influence other bacteria or host cells and enter into interactions with them.

A so-called Type I secretion system is encoded by the Salmonella Pathogenicity 4 (SPI-4) which is widespread within the species Salmonella. SPI-4 was identified as an important factor in the colonisation of cattle but is also expressed by bacteria in the blood of sepsis patients. We were able to demonstrate that the substrate of the Type I secretion system SiiE encoded by siiCDF is a giant, repetitive protein. SiiE can mediate the adhesion of bacteria specifically to polarised epithelial cells. The intestinal epithelium, which consists of polarised cells, is one of the most important barriers of the body to invasive bacteria like Salmonella. The way in which this system functions is unique from several angles: SiiE, the adhesive responsible for the mediation of adhesion is not permanently bound to the bacteria surface but is mainly to be found in the surrounding medium. Only when a signal is given of host cell contact can the protein be detected on the surface of the bacteria. A more in-depth understanding of this mechanism could help not only to provide more insight into the interaction between Salmonella and the intestinal epithelium and the foundations of Salmonella enteritis but also translate these findings into new therapy concepts. Firstly, the adhesion of SiiE to the host cell could be prevented (anti-adhesion strategy); secondly the mechanisms of host recognition of Salmonella could be inhibited. Both strategies would then lead to far less interaction between the pathogens and the intestinal epithelium, and prevent further spread and persistence.

3D reconstruction of confocal microscopic data shows Salmonella (green) with SiiE on the surface (red) of epithelial cells (A, blue). B: enlargement of the bacteria.Interaction of Salmonella typhimurium with polarised epithelial cells

3D reconstruction of confocal microscopic data shows Salmonella (green) with SiiE on the surface (red) of epithelial cells (A, blue). B: enlargement of the bacteria.

Projects

(I) Mechanisms of host cell recognition of Salmonella:

  • We would like to obtain greater understanding of the processes that play a role in the recognition of host cells, particularly epithelial cells. These findings could furnish at least a partial explanation of the differences observed in the host tropism of various Salmonella serovars.
  • Establishment and development of methods which facilitate the identification of transient protein-protein interactions: FRET (Fluorescence Resonance Energy Transfer), PCAs (Protein Complementation Assays) and in vivo cross-linking and identification of the interaction partners using mass spectroscopy
    Studies on the modulation of virulence functions as a response to host cells and host cell contact as well as defence functions of the epithelia.

(II) Identification and characterisation of virulence factors:

  • For the purposes of understanding the pathogenesis of Salmonella infections, it is necessary to characterise the underlying virulence mechanisms in the context of the host. Establishment and development of fine-tuned infection models on the basis of cell cultures to quantify adhesion, invasion and intracellular survival
  • Identification of in vitro conditions for the expression of virulence factors and loci using various reporter systems (GFP, luciferases)
  • Use of biochemical, biophysical and microscopic methods to determine the sub-cellular localisation of virulence-associated proteins

Co-operation partners

  • Prof. Dr. rer nat. Michael Hensel, Lehrstuhl für Mikrobiologie Universität Osnabrück
  • Prof. Dr. rer physiol. Markus Schnare, Institut für Immunologie, Philipps-Universität Marburg
  • Dr. med. Jonathan Jantsch, Mikrobiologisches Institut, Universitätsklinikum Erlangen
  • PD Dr. Lothar Jänsch und Dr. Tobias Reinl, AG Zelluläre Proteomforschung, HZI Braunschweig
  • Prof. Dr. Susanne Häußler, AG Chronische Pseudomonas-Infektionen, HZI Braunschweig

Situations vacant

Information about situations vacant can be accessed on Stellenausschreibungen des Robert Koch-Instituts.

For projects within the framework of work placements, Bachelor, Master or Diploma studies, we are always looking for committed, highly motivated students. For these and other enquiries please contact the group leader.

Date: 30.06.2011

Publications

  • Rabsch W, Trüpschuch S, Windhorst D, Gerlach RG (2011): Typing phages and and prophages of Salmonella.
    In: Steffen Porwollik (eds), Salmonella: From Genome to Function. Hethersett UK: Caister Academic Press.

  • Ott L, Holler M, Gerlach RG, Hensel M, Rheinlaender J, Schaffer TE, Burkovski A (2010): Corynebacterium diphtheriae invasion-associated protein (DIP1281) is involved in cell surface organization, adhesion and internalization in epithelial cells.
    BMC Microbiol. 10 (1): 2. more

  • Gerlach RG, Jäckel D, Hölzer SU, Hensel M (2009): Rapid oligonucleotide-based recombineering of the chromosome of Salmonella enterica.
    Appl. Environ. Microbiol. 75 (6): 1575-1580. more

  • Gerlach RG, Claudio N, Rohde M, Jäckel D, Wagner C, Hensel M (2008): Cooperation of Salmonella pathogenicity islands 1 and 4 is required to breach epithelial barriers.
    Cell. Microbiol. 10 (11): 2364-2376. more

  • Jantsch J, Chakravortty D, Turza N, Prechtel AT, Buchholz B, Gerlach RG et al. (2008): Hypoxia and hypoxia inducible factor-1α modulate lipopolysaccharide-induced dendritic cell activation and function.
    J. Immunol. 180 (7): 4679-4705. more

  • Gerlach RG, Jäckel D, Stecher B, Wagner C, Lupas A, Hardt WD, Hensel M (2007): Salmonella Pathogenicity Island 4 Encodes A Giant Non-fimbrial Adhesin and The Cognate Type I Secretion System.
    Cellular Microbiology Jul;9 (7): 1834-50.

  • Gerlach RG, Hensel M (2007): Protein Secretion Systems and Adhesins: The Molecular Armory of Gram-negative Pathogens.
    International Journal of Medical Microbiology Oct;297 (6): 401-15.

  • Gerlach RG, Hensel M (2007): Salmonella Pathogenicity Islands In Host Specificity, Host Pathogen-interactions and Antibiotics Resistance of Salmonella enterica.
    Berliner und Münchener Tierärztliche Wochenschrift 120 (7/8): 317-27.

  • Gerlach RG, Hölzer SU, Jäckel D, Hensel M (2007): Rapid Engineering of Bacterial Reporter Gene Fusions Using Red Recombination.
    Applied and Environmental Microbiology Jul;73 (13): 4234-42.

  • Gerlach RG, Jäckel D, Geymeier N, Hensel M (2007): Salmonella Pathogenicity Island 4-mediated Adhesion Is Co-regulated With Invasion Genes.
    Salmonella enterica. Infection and Immunit Oct;75 (10): 4697-709.