Project

Detection and characterization of the virulence-associated markers of the zoonotic pathogen Streptococcus suis by Real-Time PCR, organoids and CRISPR technology.

Background:

Streptococcus suis (SS) infections can give rise to systemic infections such as meningitis in pigs and humans. SS is often found in swine tonsils as part of the normal porcine microbiota. The capacity of SS to cause infection is difficult to predict since SS strains can display high, medium or low virulence. So far, only two main virulence associated markers, Extracellular Factor (EF) and Muramidase-Released Protein (MRP) have been proposed to determine the potential virulence of SS isolates. However, the function of MRP and EF has not been fully elucidated and, due to their high genetic variability, their detection is often misled in SS strains isolated from diseased pigs.

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In a collection of 1700 clinical and non-clinical SS isolates from different geographical origins, 54 genes were highly virulence-associated in the clinical strains and we consider these "candidate" virulence markers. 

In this project the student will work on setting up a multiplex Real-Time PCR (multiplex RT-PCR) for 6 selected virulence markers, to detect potentially virulent SS strains in tonsils swabs of piglets. In addition, the function of the selected genes will be characterized by the generation of knockout mutants using a CRISPR/Cas9 system that we have operational in the lab. Phenotypic assays, which include the growth of KO mutants under infection-mimicking conditions and the use of swine cell lines and tonsillar organoids, can also be part of the thesis research.

Objectives:

A reliable detection method for the identification of virulent SS strains is missing. The development of a fast diagnostic method to detect highly virulent strains in pig tonsil swabs could effectively help to prevent SS infections. Moreover the characterization of the function of the selected virulence-associate makers could help to elucidate their role in virulence.

Methodology:

Microbiology:

o   Lab media preparation

o   Growth, storage and handling of bacterial pathogens. Bacterial enumeration by Colony Forming Unit (CFU) counting

o   Growth of SS in infection-mimicking conditions such as porcine serum or blood (established in our lab)

 Molecular biology:

o   Design of primers/ fluorescent probes that anneal to conserved sregion of candidate virulence marker genes

o   Set up of multiplex Real Time PCR for high-throughput detection (Bio-Rad CFX96 - 5-channel)

o   Extraction of bacterial genomic DNA from tonsil swabs with bead-beading method (established in our lab)

o   Generation KO mutant with CRISPR/Cas9 system (established in our lab)

Host-microbe interactions:

o   Use of tonsillar organoids (established in our lab) to investigate adhesion/ invasion to and translocation across tonsillar cells by WT and KO SS strains.

Requirements:

o   Students should follow an MSc program in biology, biotechnology or a similar study with background in molecular biology and some basic microbiological lab practices.

o   Interest in infectious (zoonotic) diseases

o   Motivated, enthusiastic and focused

o   Some specific experimental procedures may require a student to work longer days

 

Due to the nature of this research project, two students could collaborate during the research.

Contact information:

Postdoc Dr Laura Ferrando laura.ferrando@wur.nl and PhD candidate alex.gussak@wur.nl