Alex Poret

And finally, an incredibly-loud thank you to all my coauthors, especially @contaminatedsci.bsky.social , Greg Priebe, and Matt Schaefers, who mentored me throughout this endeavor. I began this project as a college freshman, and now, years later, I’m ecstatic to see it out in the world!

In our paper, we further comment on the coexistence of different O-antigen phenotypes over a decade, speculate on the clonality of B. dolosa transmission, and more! Please let us know any questions, criticisms, and comments, or just want to speculate about reversions.

Also see related theory by labmate Paul Torrilo,  who also speculates reversion may be masking evolution across human hosts {10.1101/2023.09.14.557751}

Our work shows that repeated, de novo mutation in ordinary genes is a viable strategy to traverse changing environments, and suggests pathogen evolution may be oversimplified without frequent sequencing to resolve reversions.

Through in vitro and murine in vivo models, we find a tradeoff between tissue localization -- O-antigen-absent isolates survive better in immune cells at the expense of lung cells. We speculate spatial localization may be driving our observed phenotypic switching.

This creates a fascinating story, where B. dolosa repeatedly disrupts O-antigen expression in early infection and restores it in chronic, decades-long infection across patients via de novo mutation alone.

To our surprise and initial confusion, the new patients were initially infected with an O-antigen-expressing strain. However, new de novo mutations disrupting the O-antigen-synthesis pathway quickly emerged within each patient, mimicking the outbreak ancestor!

Two new recently infected patients allowed us to address this question. We sequenced 931 B. dolosa isolates from the first 38 months of their infections and the lung autopsy of the chronically-infected (~10 years) suspected source.

This stop codon independently reverted in 9/14 studied patients across years of infection, leading to a new hypothesis: does O-antigen absence facilitate transmission at the expense of chronic infection?

Back in 2011, @contaminatedsci.bsky.social and Michel et al. followed a Boston-area outbreak and discovered that the founding B. dolosa strain contained a premature stop codon that abrogated its O-antigen, a lipopolysaccharide sugar chain that mediates immune recognition. {10.1038/ng.997}

Here, we trace how an ordinary, non-hypermutable pathway is toggled on-and-off throughout an outbreak of Burkholderia dolosa, a rare pathogen that infects people with cystic fibrosis.

De novo mutations mediate phenotypic switching in an opportunistic human lung pathogen Bacteria evolving within human hosts encounter selective tradeoffs that render mutations adaptive in one context and deleterious in another. Here, we report that the cystic fibrosis-associated pathogen Burkholderia dolosa overcomes in-human selective tradeoffs by acquiring successive point mutations that alternate phenotypes. We sequenced the whole genomes of 931 respiratory isolates from two recently infected patients and an epidemiologically-linked, chronically-infected patient. These isolates are contextualized using 112 historical genomes from the same outbreak strain. Within both newly infected patients, diverse parallel mutations that disrupt O-antigen expression quickly arose, comprising 29% and 63% of their B. dolosa communities by 3 years. The selection for loss of O-antigen starkly contrasts with our previous observation of parallel O-antigen-restoring mutations after many years of chronic infection in the historical outbreak. Experimental characterization revealed that O-antigen loss increases uptake in immune cells while decreasing competitiveness in the mouse lung. We propose that the balance of these pressures, and thus whether O-antigen expression is advantageous, depends on tissue localization and infection duration. These results suggest that mutation-driven alternation during infection may be more frequent than appreciated and is underestimated without dense temporal sampling. ### Competing Interest Statement P.C.B. is a consultant to or holds equity in 10X Genomics, General Automation Lab Technologies/Isolation Bio, Celsius Therapeutics, Next Gen Diagnostics, Cache DNA, Concerto Biosciences, Stately, Ramona Optics, Bifrost Biosystems, and Amber Bio. His laboratory has received research funding from Calico Life Sciences, Merck, and Genentech for unrelated work.

How do bacteria navigate environmental variation in human hosts? 

 In our newest preprint, we find bacteria can use “permanent” solutions for temporary problems — mutation-mediated phenotypic switching! 

www.biorxiv.org/content/10.1...

De novo mutations mediate phenotypic switching in an opportunistic human lung pathogen https://www.biorxiv.org/content/10.1101/2024.02.06.579193v1 Bacteria evolving within human hosts encounter selective tradeoffs that render mutations adaptive in

De novo mutations mediate phenotypic switching in an opportunistic human lung pathogen https://www.biorxiv.org/content/10.1101/2024.02.06.579193v1