Max Van Belkum

Mentor: Dr. Mansour Mohamadzadeh

College of Veterinary Medicine

Major

Nutritional Science and Applied Physiology and Kinesiology

Minor

N/A

Research Interests

  • Human Microbiome
  • Immunology
  • Nutritional Science

Academic Awards

  • Florida Bright Futures
  • Potter-Chafin Award (2017)
  • University Scholars Program (2017-2018)

Organizations

  • Association of Future Physician-Scientists
  • American Medical Student Association (AMSA)

Volunteer

  • Aces in Motion Tennis Coach
  • Covenant Hospice
  • Shands Clinical Volunteer

Hobbies and Interests

  • Tennis
  • Ping-Pong
  • Super Smash Bros.
  • Going to the Gym

Research Description

Illuminating Probiotic Propionibacterium Freudenreichii UF1 to Understand Interaction with the Infant Microbiome
Formula has long been considered a viable alternative to breast milk as a means of providing nourishment to infants. However, according to a meta-analysis of over 9000 abstracts, formula fed infants are at a greater relative risk of a plethora of diseases, including but not limited to: gastroenteritis, severe lower respiratory tract infections, asthma, obesity, type 1 and 2 diabetes, childhood leukemia, sudden infant death syndrome (SIDS), and necrotizing enterocolitis . However, encouraging breast-feeding has proven challenging, as only 49% of infants were breastfed for 6 months in 2011 as recommended. Alternatively, our lab looks to augment infant formula in order to improve the health of those not able to be breastfed. P. UF1 is the bacterium subspecies of Propionibacterium freudenreichii that my lab isolated from the fecal samples of premature infants that have been fed human breast milk but not in premature infants that had been formula fed. Since then, the purpose of the lab has largely been to elucidate the function of the bacterium. Mice are used as test subjects, with the end goal of using P. UF1 for human therapy. This bacterium has been shown (in accordance with our unpublished data) to have probiotic effects in multiple scenarios, including producing beneficial short chain fatty acids like propionate, eliciting a decrease in inflammatory cytokines, sustaining regulatory T cells (Treg cells), and markedly mitigating necrotizing enterocolitis (NEC) in neonatal mice that were subjected to NEC. If the results are promising enough, there is a possibility that the lab will work towards creating a probiotic formula with P. UF1 for premature infants that that will be combined with human breast milk. My proposed research project is to find a way to get this specific bacterium to stably fluoresce in order to understand its function in vivo. The challenge is that traditional fluorescent proteins require oxygen to illuminate, while P. UF1 is an anaerobic bacterium. Recent studies have shown flavin mononucleotide (FMN)-based fluorescent proteins (FbFP) including PpFbFP (from Pseudomonas putida) can be used in a variety of anaerobes, and they illuminate under both aerobic and anaerobic conditions. Until now, there is no report regarding the use of fluorescence genes in anaerobic Propionibacteria. Therefore, developing a stable system for labeling P. UF1 is of great importance to understand how this bacterium is interacting with the host immune system. Briefly, the PpFbFP gene was codon-optimized and cloned into P. UF1-E. coli shutter vector pYMZ, generating plasmid pYMZ-P4-PpFbFP which express His6-tagged PpFbFP gene under control of P4 promoter. Following transformation into P. UF1, plasmid-harboring P. UF1 was identified, and expression of PpFbFP was confirmed by qRT-PCR and Western blot. The labeled bacteria had a maximal excitation wavelength of 495 nm at an excitation wavelength of 450 nm (Figure available upon request) and flow cytometry demonstrated that the bacterium has fluorescence (Figure available upon request). Microscopy observation indicated that P. UF1 were successfully labeled but only 5-10% of them had bright fluorescence (Figure available upon request), possibly due to the plasmid instability. To generate a stably inherited PpFbFP-labeled P. UF1 mutant, homologous recombination strategy will be used to insert a single copy of P4-His6-PpFbFP gene fragment into the chromosome of P. UF1 (Fig. 1d). We hypothesize that if the proper location of the chromosome for the gene PpFbFp is used, then we will be able to observe stable fluorescence of P. UF1 bacteria, thus further allowing us to elucidate its function in vivo and potentially improve the health of human infants. The proposed timeline for the project is as follows: For the rest of spring 2017, I will be actively reading literature on the PpFbFp gene, construction of the plasmid for gene insertion, and P. UF1 electroporation. If ahead of schedule, I may begin experimenting on gene insertion. By the summer of 2017, I will have begun experimentation. By September, I hope to have preliminary results that will spur further experimentation, either into other ways to get the bacteria to fluoresce or using the newfound fluorescence to elucidate its function in vivo. The first stipend will be used to fund this.