Project in Chile

Intestinal colonization of different strains of Listeria monocytogenes and interaction with the gut microbiota

University of Michigan investigator(s) and unit

Cristina Bares, PhD, Assistant Professor of Social Work

International colleague(s) name and unit

Paola Navarrete, PhD, Assistant Professor, Laboratory of Microbiology and Probiotics, INTA, University of Chile.

Purpose

To study the interaction of different strains of Listeria monocytogenes and the zebrafish (Danio rerio) model

Rationale

Listeria monocytogenes is a Gram positive foodborne bacterium that can infect a wide range of animals, including humans. The bacterium can produce septicemia, meningitis and chorioamnionitis which are associated with a high mortality. The severity of L. monocytogenes infection depends on the virulence of the strains and the immune status of the host. Immune-competent individuals that ingest L. monocytogenes contaminated food can have gastroenteritis that resolves in few days. However, immunocompromised individuals, infants, older people, and pregnant woman have a high risk of infection and have more severe symptoms.

The mechanisms involved in pathogenicity and virulence of this pathogen have been intensively studied. Some proteins and genes have been identified and related with the virulence of the pathogen. Some of these genes are involved with the adhesion and invasion of the pathogen into the host cells. However, to date, little is known about the intestinal phase of this pathogen, such as its replication in the gut or its relation with the gut microbiota.

A recent publication in a murine model showed that the gut microbiota markedly reduces L. monocytogenes colonization of the gut lumen and prevents systemic dissemination (Becattini et al., 2017). This study was performed with one pathogenic strain of L. monocytogenes (10403s). In our lab, we have a collection of L. monocytogenes isolates with different origin and phenotypic traits. In this project, we want to explore the intestinal behavior, such as growth or colonization, of these different strains of L. monocytogenes and evaluate if this behavior can be modulated by some early bacterial component of the gut microbiota of infant such as Lactobacillus, Bifidobacterium or Clostridium.

In this project we will use zebrafish. Compared to murine models, zebrafish have several advantages: they i) are easy to maintain, ii) develop rapidly (at 3 days post-fertilization (dpf) their mouth and digestive tract is open, so they can be colonized by different microorganisms), iii) in the early stages of development, larvae are transparent, which allows observing in vivo the colonization process, iv) the availability of transgenic lines expressing fluorescent proteins in different lineages of immune cells, allows the in vivo monitoring of physiological and pathophysiological processes, v) embryos and larvae are small enough to fit into 96-well plates, so it is possible to perform experiments with many individuals (high-throughput), vi) zebrafish's genome has been completely sequenced, vii) methodologies to obtain germ-free fish or gnotobiotic have been described (and have already been standardized and implemented in our laboratory), viii) the digestive tract share extensive homology with mammalian and it includes the accessory organs such as the liver, gallbladder and pancreas. Additionally, the gastrointestinal tract also has a next-distal functional differentiation. The initial morphogenesis is completed at 3 days post fertilization (dpf), stage in which they can colonize the digestive tract with microorganisms (Bates et al., 2006; Rawls et al., 2004).

Study design and methods

We will evaluate the effect of Lactobacillus, Bifidobacterium or Clostridium in the in vivo colonization of the zebrafish gut of different strains of Listeria monocytogenes. Colonization assays will be conducted according to methodology standardized in our lab (Caruffo et al, 2015 and 2016.). Colonization ability will be assessed from day 3 until day 9 dpf. These experiments will be independently performed 3 times.

Anticipated activities on project

The trainee will participate in the cultivation, DNA extraction, PCR amplification of different L. monocytogenes isolates as well as Lactobacillus, Bifidobacterium or Clostridium. and colonization studies in zebrafish larvae.

Techniques/methods trainee(s) should become familiar with in advance

  1. Handling of bacterial cultures (aerobic and anaerobic with anaerobic jar)
  2. Gram staining
  3. DNA extraction from bacteria
  4. PCR

Suggested readings

  • Bates, J.M., et al. (2006). Dev Biol 297(2): 374-386.
  • Bates, J.M., et al. (2007). Cell Host Microbe 2(6): 371-382.
  • Becattini, S. et al. (2017) J. Experimental Med. 214(7): 1973-1989.
  • Caruffo M, et al. (2015). Front Microbiol. 7;6:1093. doi: 10.3389/fmicb.2015.01093.
  • Caruffo M, et al. (2016). Front Cell Infect Microbiol. http://dx.doi.org/10.3389/fcimb.2016.00127
  • Rawls, J.F., et al. (2006). Cell 127(2): 423-433.