PREreview of Bioorthogonal labeling of chitin in pathogenic Candida species reveals biochemical mechanisms of hyphal growth and homeostasis

based on: https://www.biorxiv.org/content/10.1101/2024.08.27.609898v1.full

The major goal of this manuscript is to create a less-perturbative method for visualizing chitin in C. albicans, a human pathogen. The central premise builds on the (famous) work of Bertozzi that used a “click” compatible GlcNacs in S. cerevisiae expressing the promiscuous C. albicans Hxk1, which is needed to phosphorylated the modified GlcNac building blocks. This manuscript brings the concept full circle by using Hxk1 in its native system to create a minimally perturbative GlcNac incorporation scheme. The demonstration is convincing and I image this reagent will find use in the fungal and pathogenesis communities. I have a few questions about the work that could help me understand the potential use cases

• In the lead up to Fig 3, much is made about the media shift to avoid the GlcNac building blocks being metabolized and incorporated into anything but chitin. The data in Fig 3 definitely speak to the positive aspects of incorporation into chitin, but I’m unsure whether there is additional data that can speak to how much goes to other sources beyond chitin or if a media switch to rely on GlcNac with these reagents could teach us anything new about metabolism. 

• I understand many of the downsides vs. Calcofluor white for perturbation. But the performance of the fluorescent labeled chitin in their scheme could be more contrasted with the previous “standard” in a more complex imaging setup then what is shown here. Duration of growth, timelapse, etc. 

• The conclusions around: "two-pronged strategy that the pathogen uses to sense the presence of bacteria and transform from a non-pathogenic state into the pathogenic hyphae form. " - are a bit overstated given the biochemical complexity of how they are triggering the transition in this specific paper. However, this does not detract from the value of the reagents they create to answer this exact question with more carefully controlled experiments. 

• I wonder how these reagents would do in a mammalian infection setting. I’m thinking particularly in imaging applications where we want to see the chitin of fungal pathogens in human tissues. Could start in cell culture, of course.

In addition I have a few minor quibbles:

• the pedant in me views homology as qualitative, not something that is quantified in percentages - perhaps you mean sequence identity or similarity? regardless the ambiguity in that quantification should be addressed

• Sloppy wording around ". This indicates that Hxk1 is essential and likely required for GlcNAc incorporation into the fungal cell wall." - essential for labeled GlcNac not growth, right? Can't tell from the microscopy because -GFP looks the same as dead.

• Hxk1, Nag1, and Dac1 - confused why all three gene knockouts are necessary - or does this just speak to use of a pre-existing strain with all three deleted? 

James Fraser

Competing interests

The author declares that they have no competing interests.