The Path(ogen) Less Traveled

Before I was dropped off at college, I distinctly remember the ominous warning from my mother: be careful, don’t get meningitis! The disease is known for occurring in infants and college students, but I didn’t really know what it was…until this summer, now that I’m studying it. While there are several pathogens that are able to cause meningitis, Dr. Perfect’s lab studies Cryptococcus neoformans, or the fungal pathogen readily found in the environment causing cryptococcal meningitis. Though its environmental ubiquity seems alarming, Crypto. is actually an opportunistic infection, meaning it only causes disease when it is able to take advantage of weakened immune systems, such as those with HIV/AIDS, transplant recipients on immunosuppressants, or possibly even survivors of COVID-19. Even so, the prevalence of cryptococcal meningitis is significantly higher in poorer countries with less access to necessary treatments and supplies. Duke Hospital’s own survival rate for Crypto. infections is around 80%, while the global survival rate is only 50% (skewed largely by the populations lacking in adequate healthcare resources). Despite this frightening statistic, research on Crypto. is largely underfunded due to the fact that cryptococcal meningitis is a noncommunicable disease, or it cannot be spread person to person via a cough or a sneeze the way that the flu, COVID-19, or other common diseases can.

Luckily, Dr. Perfect’s lab is doing the hard work, spending every day working towards understanding Crypto. and trying to find new ways to target it through drugs and other methods to help prevent or cure future cryptococcal meningitis infections. This is where I come in (with my bench mentor/graduate student Julia, of course). One of the most interesting things about Crypto. is where it causes infection: the brain/spinal cord. A vast majority of other pathogens struggle significantly with the lack of nutrients and overall harsh environment of cerebral spinal fluid in the central nervous system, which is why Crypto. is such an intriguing fungus; where others fail at mere survival, Crypto. seems to thrive in this nutrient-deficient environment, hence its deadly survival rates. One project I am helping work with Julia to look at this summer is one particular set of genes that may be influencing this growth. These genes regulate nitrogen catabolism, or the identifying and processing of available nitrogen sources for use. Nitrogen catabolite repression (NCR) is the name for the regulation of these genes via certain regulators based on the nitrogen sources available (normal, easily metabolized sources results in a negative regulation — turning the genes off — and sources that require several steps to break down cause a positive regulation response — activating the genes encoding for certain necessary enzymes to break the nitrogenous compounds down –). Our first steps so far have been looking at two potential NCR genes, tagging them with a fluorescent tag (we are using mCherry, but for comparison it’s like GFP), and looking for any interactions between the proteins based on localization (if the proteins are co-localizing, we will see a certain fluorescent pattern showing them near each other). Knowing if these genes work together to regulate nitrogen catabolism is critical for moving forward in the overall investigation of identifying these regulation pathways in Crypto.

These last two weeks, we have been assembling all the necessary pieces to create the tagged strains of Crypto. I first had to design primers that would work in a PCR reaction to amplify each of the 5 segments needed for the tagged strain (the 5’ UTR, for attaching to a plasmid backbone, the NAT antibiotic resistance cassette, for incentive to integrate the plasmid into Crypto., the promoter + gene itself, the mCherry fluorescent tag, and the 3’ UTR region to attach to the other end of the plasmid backbone). The PCR results so far have been mostly successful, with two stubborn fragments still left to successfully amplify (let’s just say I’ve run a LOT of electrophoresis gels this week). Once we know that the PCR worked, we cut out the bands and extracted the DNA from them to be saved until all the pieces are ready. This week, we are finally going to do a Gibson assembly to put all the puzzle pieces together.

These two weeks so far have been a whirlwind of reading papers and learning techniques and working towards an exciting goal; I feel like a supersaturated sponge, but I can’t wait to keep learning more!

My first (of many) electrophoresis gels showing the successful PCR reactions for gene 1: URE3. Our 2nd fragment had the best band, meaning it worked the best (NAT cassette), and was extracted, but most of the other four fragments had to be run again in a new PCR reaction.

This was the gel electrophoresis showing the PCR reactions for gene 2: TAR1. The brighter bands were the successful amplifications, and the ones that were very faint or didn’t show up at all in the column had to be run again. Here, we were able to gel extract from 1 and 2, and some from 3 and 4 (This was a vast improvement from the first URE3 gel, which was one of the first PCRs and gels I ran in the lab).

 

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