The connection between alginate regulation and anti-CRISPR: Q&A with Dr. Adair Borges

Issue 75 | May 15, 2020
15 min read
Capsid and Tail

The structure of an AmrZ tetramer bound to operator DNA. Source:

This week, Capsid & Tail’s volunteer editor Dr. Rohit Kongari talks to UCSF’s Dr. Adair Borges about the unexpected connection she’s uncovered between phage anti-CRISPRs and the Pseudomonas alginate pathway.

What’s New

James Doub (University of Maryland School of Medicine) and colleagues published their successful eradication of a 72-year-old man’s prosthetic joint infection with phage therapy, which was done alongside Adaptive Phage Therapeutics and the US Naval Medical Research Center. Of note, the phage appeared to cause unusual effects on the patient’s liver, pointing toward a need for clinical trials to optimize the dose of phage therapy and better understand its effects on the body.

BiotechPhage TherapyResearch paper

What happens to the bystanders of a phage-host interaction? Anushila Chatterjee (University of Colorado School of Medicine) and colleagues published a new preprint on how phage infection induces the type 7 secretion system in Enterococcus faecalis, which antagonizes neighboring cells.

Phage-host interactionsPreprintResearch paper

Shweta Karambelkar (Indian Institute of Science) and colleagues published a interesting report on how phage Mu uniquely protects its DNA from diverse host defense systems in Nucleic Acids Research. They characterized Mu’s “Mom” protein, which is responsible for DNA modification/protection, and has evolved to make a ‘giant chemical leap’ from acetylation to methylcarbamoylation activity.

Molecular BiologyPhage defense systemsResearch paper

Oliver Bezuidt (University of Pretoria, South Africa) and colleagues published a new paper in mSystems that sheds light on Antarctic phage-host interactions. Their metagenomic analysis picked up DISARM, BREX and CRISPR-Cas defense systems in Antarctic polar deserts, and these directly correlated with the metavirome of these communities. Overall it seems that phage-host interactions are active and important here.

MetagenomicsMetaviromicsPhage defense systemsResearch paper

Andrej Balaz (Geneton Ltd, Slovakia) and colleagues published a preprint describing PHERI, a phage host exploration tool that uses a machine learning algorithm to predict the host of a phage based on its sequence. Preprint | Application download | Github

Bioinformatics ToolResearch paperPreprint

Metagenomics is enabling discovery of millions of new viral sequences, but no tools previously existed to estimate their quality. Stephen Nayfach (DOE Joint Genome Institute) and colleagues created CheckV, an automated pipeline for assessing the quality of metagenome-assembled viral genomes. They validated the tool on public datasets and found that just 3.6% of viral sequences are high-quality or complete. Preprint | Tool.

PreprintResearch paperBioinformatics ToolMetagenomics

Jason Schrad (Michigan State University) and colleagues published a structural characterization in Cell (through cryoEM, cryoET, and SEM) of how giant virus infection is initiated. They also use proteomics to identify proteins released from capsids during infection. Their work suggests the molecular forces that trigger infection are conserved among disparate giant viruses.

Research paperStructural Biology

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Seeking phage data for undergraduate Bioanalytical Phage course

Maria Cambraia Guimaro, PhD, University of Maryland, Baltimore County

I am a Postdoctoral Teaching Associate working for STEM BUILD, an undergraduate success initiative investigating ways to enhance the diversity and success of STEM students. We offer an 8-week Summer Course-Based Undergraduate Research Experience during their sophomore year. Initially, we were planning to have the students performing molecular biology experiments and chemistry analytical experiments about phages or their environments. Usually, the students perform some analysis, write an abstract, and present a poster at a local undergraduate symposium.

As we will not be able to perform bench experiments on campus due to COVID-19, I am trying to get access to previously generated data related to phages (or to their original environment) to analyze with our students. I am seeking researchers willing to share any spare analytical data (chromatography, mass spectrometry, pH, samples moisture, cations, salt concentration, organic matter, microscopy images, etc) that can be used in our class. You would be included in any analysis or material produced by our students during this summer experience. 
Additionally, is anyone aware of any online bank or open database where researchers deposit analytical/chemical data from phages? The data could be also related to the environment where these phages were collected (soil, water, etc) or any other analysis related to phages.

If you can help, please contact me at [email protected].

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Special Issue: Viruses of Microbes 2020

Submissions are open to the special issue of the journal Viruses, “Viruses of Microbes 2020: The Latest Conquests on Viruses of Microbes”.

This issue is associated with the 2021 meeting from the International Society of Viruses of Microbes (ISVM) to be held in Guimarães, Portugal in 2021, and intends to be a platform for the latest advances on research of microbial viruses, from ecology and evolution, to virus structure and function, and from virus-host interaction to biotechnology applications and phage therapy.

ISVM members receive a 10% discount and a limited number of fee waivers is available. Please contact guest editors in advance by email about planned submissions. Guest editors: Tessa Quax, Marianne De Paepe, Karin Holmfeldt.

Special IssueViruses of Microbes

The connection between alginate regulation and anti-CRISPR: Q&A with Dr. Adair Borges

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Postdoctoral Researcher

Bioinformatics, Biotechnology, Molecular Biology, Phage isolation, Phage Therapy, Phage-host interactions, Teaching, Phage Genomics, RNA Sequencing

I am a Phage Biologist at heart with a strong technical background in Biochemistry, Bioinformatics, Phage Genomics, Microbiology and Molecular Biology. I worked on the role of phage spanins in host lysis for my PhD under the guidance of Dr. Ryland Young at Texas A&M University. I am currently working on understanding the host-phage interactions between Staphylococcus aureus and phage K towards use in phage therapy applications.

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Graduate Student
Bondy-Denomy lab, UCSF

I am interested in the host-pathogen interactions of bacteria and their phages. In graduate school, I studied how phages use anti-CRISPRs to protect themselves from the bacterial CRISPR-Cas immune system.

This week, Dr. Rohit Kongari brings us a Q&A with Dr. Adair Borges from the lab of Dr. Joe Bondy-Denomy at the University of California, San Francisco. The Q&A is focused on their story, recently published in Nature Microbiology, on how bacterial alginate regulators and their phage homologs repress CRISPR-Cas immunity.

Rohit: Howdy Adair, thanks for doing this interview for Capsid & Tail. Can you give us a quick background about yourself, how your interest in phages started, and eventually how you got to Joe’s lab at UCSF?

Adair: I am originally from Billings, Montana, but I went to University of Pittsburgh for my undergrad majoring in Microbiology. Being interested in research, I worked in the lab of Dr. Jon Boyle, who studied the eukaryotic parasite Toxoplasma gondii. I became part of an amazing undergrad research community at Pitt, of which Dr. Graham Hatfull was a key part. I got introduced to phages through a few classes taught by Dr. Hatfull and other researchers. That’s where I first learned about basic phage biology, CRISPR, and phage genomics, and got super interested in them. From there, I moved to UCSF for grad school. While I was not sure what I was going to do, I knew I liked evolution, host-pathogen interactions, and microbiology. At the end of my first year there, Joe Bondy-Denomy, my eventual PI, was an incoming Sandler Fellow starting his lab on phages and host-pathogen interactions. A late rotation in his lab turned out to be a great experience and the science was interesting. I liked working with Joe too, so I ended up joining his lab.

How and when did you get involved in the alginate regulation project? What was the path to finding the connection between host alginate regulation and CRISPR-Cas immunity?

Adair: It was one of the first projects started in Joe’s lab, initiated by the second author on the paper and the then-research technician in the lab, Bardo Castro. Over a year and a half, he worked on screening for regulators of CRISPR-Cas in Pseudomonas aeruginosa (PA). Once he left for grad school, I took over the project and started putting pieces of a story together from the mutants he had isolated. Starting with just one transposon hit in the sensory kinase kinB, which plugs into the host alginate regulation pathway, it took us a long time to figure out exactly which core set of genes involved in the pathway were responsible for regulating CRISPR. I spent most of my time understanding the relevance in terms of Pseudomonas physiology and host-phage interactions. One of the biggest things that threw us off was the regulation being linked to surface sensing. Bardo was able to show that a deletion mutant of a transcriptional regulator in this pathway, AmrZ, showed elevated CRISPR activity in a plaquing assay but I could never detect any differences in the expression or activity of CRISPR related proteins. Initially confused by these results, we later realized what we had overlooked in our experimental setup. The samples for our qPCR/protein level assays were being collected from liquid phase culture whereas the plaque assay was a read-off from growth on a solid surface. Once I started looking into phage resistance in liquid cultures and protein levels from growth on a top-agar overlay, it became clear to us that regulation of CRISPR by AmrZ was coupled to host growth on a surface. The storyline presented in the paper is pretty close to how we kept digging deeper using different assays to find whether the regulation was at the
transcriptional or translational level.

I can only imagine how time-consuming all those assays and analyses were. So how common are alginate regulation systems in other bacterial species, and what is their relevance to host physiology?

Adair: Alginate is an exopolysaccharide that is actually not very common in the bacterial world. It is made only by brown seaweed, Pseudomonas and Azotobacter. Alginate production is not only quite specific to Pseudomonas, but also an important part of its virulence, antibiotic resistance, and overall pathogenicity. Overproduction of alginate is strongly selected for in the Pseudomonas strains from clinical Cystic Fibrosis isolates. The mucoid growth phenotype we observe in many pathogenic strains is in fact due to overproduction of alginate. Some of the Pseudomonas genes involved in the alginate biosynthesis pathway are host bacterial transcription regulators involved in other processes as well. For example, AlgU which is a transcriptional regulator involved in one of the initial stages of the alginate pathway also acts as sigma E, a bacterial sigma factor with other important functions.

Your paper discusses the presence of homologs of such regulators in phages and mobile genetic elements (MGEs). How unique is that, and is there any common feature about those phages or genetic elements that carry these homologs?

Adair: We looked specifically for homologs of AmrZ and mostly found them in Pseudomonas phages and plasmids. The 15 Mobile Genetic Elements (MGEs) mentioned in the paper that have an AmrZ homolog are highly diverse and include temperate phages, lytic phages, and plasmids. There is no clear evidence for alginate regulator homologs being enriched in any class of these elements preferentially and it is still an open question as to why these elements carry these regulators. We explored it in the context of CRISPR immunity where it makes sense for phages to carry such repressors. Since our lab has an anti-CRISPR focus, we think of these repressors as early or upstream anti-CRISPRs that inhibit CRISPR activity by repressing the transcription and biogenesis of new CRISPR-Cas complexes. But given how pleiotropic these regulators are, it is highly possible that there are multiple other effects on the phage life cycle in addition to repressing CRISPR.

So AmrZ is a surface-dependent CRISPR-Cas regulator. Are there other examples of CRISPR regulation or interference being dependent on growth conditions?

Adair: Bacterial physiological state is definitely important for CRISPR. It had already been shown in both Pseudomonas and Serratia that quorum sensing (QS) plays an important role in activating CRISPR. Dr. Bonnie Bassler’s lab showed that Pseudomonas activates CRISPR upon reaching high cell density (where the risk of phage infection is high) through QS, and Dr. Peter Fineran’s lab demonstrated that multiple different CRISPR systems in Serratia were activated in a coordinated fashion, similarly through QS. These two groups have also shown that temperature and metabolic status, respectively, also impact CRISPR. However, our findings are the first instance where CRISPR-Cas is being turned off through sensing the physical environment, the signal being growth on a surface. It makes sense because the risk of phage infection is significantly lesser on a surface due to limited phage dispersal compared to liquid culture. Dr. George O’Toole’s lab has a lot of great work showing that PA has also been shown to beis hypersensitive to CRISPR-Cas self-targeting during surface association, so repressing CRISPR-Cas likely counters the risk of autoimmunity as well.

Apart from the identification of a new CRISPR-Cas regulation mechanism, what do you think is the most interesting takeaway from your work, for this story in particular?

Adair: I think it once again highlights how ingenious phages are at manipulating their hosts. In this case, Pseudomonas phages and mobile genetic elements using AmrZ homologs are now able to regulate CRISPR-Cas. Essentially, a mechanism employed by the host to regulate CRISPR-Cas and limit self-toxicity is being exploited by these genetic parasites for their own benefit. In our analysis, we identified a few strains with multiple prophages, that each carried their own copy of AmrZ, in addition to the host copy. So, there is probably a profound impact on the transcriptional landscape of the cell in those strains with potential individual contributions to the overall effect. It is incredible to think that AmrZ has evolved into a “guns for hire” role in this arms race between bacteria and their predators, being used by both sides.

This interview has been lightly edited for clarity.

Adair (@AdairLBorges) recently graduated from the Bondy-Denomy lab at UCSF and is starting a new postdoctoral position as a Miller Fellow in Dr. Jill Banfield’s lab at UC Berkeley soon.

Want to learn more about anti-CRISPRs?

If you are interested in more new anti-CRISPR work from the Bondy-Denomy lab, check out Caroline Mahendra’s work showing that broad-spectrum anti-CRISPRs on plasmids facilitate horizontal gene transfer, and Dr. Beatriz Osuna’s paper on a bi-functional anti-CRISPR that induces Cas9 degradation!

Rohit Kongari helped us produce this week’s article by helping us source and write the What’s New and Jobs sections. Thanks Rohit!!

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