Internship at ACTA

Internships at ACTA

• Bacterial interaction in dual-species biofilms

  The group of Oral Microbiology of the dentistry faculty of the Free University and the University of Amsterdam is mostly interested in the roles of bacteria and bacteria interaction in dental infectious diseases including periodontitis and dental caries. The pathogens involved and their interactions with commensal bacteria are studied by molecular approaches.

  Recently, it was discovered that there are tens of thousands bacterial species present in the oral cavity. These bacteria are organized in a form of biofilm. It was known that strictly anaerobic bacteria Fusobacterium nucleatum and Porphyromonas gingivalis are pathogens for periodontitis, while facultative anaerobic bacteria Streptococcus mutans was a pathogen resulted in dental caries formation. However, not all bacterial species in the biofilm are pathogenic. Commensal bacteria refer to the bacterial species which are beneficial for the oral health. It is our interest to study the interaction between pathogen and commensal bacteria in an in vitro biofilm model. The factors which influence the interaction will be also studied.

  Our preliminary study already showed that cario-pathogen S. mutans displayed strain-dependent biofilm formation and antimicrobial resistance. In this project, the influence of commensal bacteria on biofilm formation on various S. mutans strains will be studied mainly by microscopic method. Fluorescent protein labeled bacteria, live/dead staining kit will be used to visualize the interactions in the biofilm.

  The goal of this project is

  1. Adapt the in vitro biofilm model for bacterial visualization
  2. Construct various fluorescent protein labeled bacteria.
  3. Visualize the bacterial interaction with microscopic method.

  Techniques

  Techniques that will be used in this project are:

  • PCR
  • cloning
  • electro-transformation
  • anaerobic culturing
  • microscopic examination
  • epifluorescent microscope
  • CSLM, etc.

  Contacts

  • Dongmei Deng
    Department of Preventive Dentistry
    Academic Centre for Dentistry Amsterdam (ACTA)
    Gustav Mahlerlaan 3004, 1081LA Amsterdam
    E-mail: d.deng@acta.nl
    Tel.: 020-598 06 75

• A potential biomarker for beneficial host-bacterial commensalism

  Skin-derived anti-leukoproteinase (SKALP), also known as elafin or ESI, is a potent elastase inhibitor for human leukocyte elastase and porcine pancreatic elastase. Several protective functions of SKALP have been suggested since the protein was identified from human epidermis. It might provide protection to the epithelium against the breakdown of extracellular matrix due to the trafficking of PMNs and mononuclear cells; it might involve in the protection of epidermal integrity; and it might involve in cell cycle regulation and be suppressed in carcinomas.

  Recently we observed in our study that the expression of SKALP in epidermis was greatly increased in an organotypic cell model after exposing to an oral commensal biofilm. The biofilm was formed under the condition that simulated healthy oral environment. We want to further investigate if the expression level of elafin in oral epithelial cells is linked to the pathogenicity of oral biofilms.

  The aims of this project are 1. to establish a simple model for studding the role of SKALP in a host-microbe interaction using monolayer oral epithelial cells; 2. to examine the differential expression of SKALP in oral epithelial cells under the challenge of pathogenic and commensal biofilms.

  Techniques:

  Cell culture; ELISA, Immunostaining, Microscopy, Real-time PCR, RNA extraction; Anaerobic bacterial culturing; Biofilm formation;

  Contacts:

  • Dongmei Deng
    d.deng@acta.nl

• Explore the role of oral bacteria in the chemoresistance of pancreatic cancer an internship

  Pancreatic cancer is a highly lethal disease with a 5-year survival rate below 8% and is projected to become the second leading cause of cancer-related deaths in 2020. The reasons for this poor prognosis include the detection of the tumor in the late disease stage and the poor response to chemotherapy. So far the most common chemotherapeutic protocols are i.e. FOLFIRINOX (a combination of 5-fluorouracil (5-FU), leucovorin, irinotecan and oxaliplatin) and gemcitabine/nab-paclitaxel. These protocols only resulted in modest survival benefits (< 1 year). Growing evidence suggests that microbes can influence the efficacy of anticancer treatments. Recent studies, including ours, demonstrate that bacteria are a component of pancreatic tumor microenvironment, and they may also play a critical role in mediating resistance to chemotheraphy. (REF1)
  
  The microbiota isolated from the pancreas tissue indicated that these microbes might come from oral cavity.  Remarkably, a most recent prospective study showed that the presence of two oral pathogens, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, was associated with an elevated risk of developing pancreatic cancer.(REF2) In addition, high levels of antibodies against Porphyromonas gingivalis were shown to double the risk of pancreatic cancer.(REF3) However, the definitive role for microbiota as either cause or reaction after tissue inflammation which is commonly associated to pancreatic cancer remains unclear. All these data support further studies aimed at unraveling the complex interaction between oral bacteria and pancreatic cancer.
  
  Since a recent study showed the role of bacteria in resistance to gemcitabine, the present project will focus on the potential effects of oral bacteria on the chemoresistance of pancreatic cells to the different chemotherapeutic drugs which are used in the standard treatment of pancreatic cancer patients.
  
  To this end, the specific goals of the project include 1) to understand the involvement of oral bacteria in the resistance of pancreatic cells to chemotherapies and 2) to explore the molecular mechanisms of this involvement. To achieve these goals, several oral bacterial species, including the two oral pathogen mentioned above will be coincubated with pancreatic cancer cells. The resistance of the cells to selected chemotherapy (eg, gemcitabine, nab-paclitaxel, oxaliplatin, irinotecan and 5-fluorouracil) will be evaluated with the Sulforhodamine-B (SRB) colorimetric assay. Various isogenic knockout mutants of oral bacteria and genetically modified pancreatic cells will be included in the assay to investigate the underlying mechanism.

  Techniques

  Techniques to be used in this project include:

  • Aerobic and anaerobic culturing of bacteria
  • Culturing of human cancer cell
  • SRB colorimetric assay for the evaluation of drug sensitivity/resistance, through the determination of the IC50 (eg, the drug concentration causing 50% inhibition of the cancer cell growth)
  • Microscopic examination of cellular features
  • Flow cytometry (for the analysis of celle cycle and apoptosis induction)

  Contacts

  • Dr. Ilaria Carnevale, i.carnevale@vumc.nl; 
  • Dr. Dongmei Deng, d.deng@acta.nl

  References

  1. Geller et al., Science 357, 1156-1160 (2017)
  2. Farrell JJ et al., Gut 61, 582-588 (2012)
  3. Michaud DS, Izard J. Cancer J 20, 203-206 (2014)