The dissemination of pathogens through blood and their establishment within organs lead to severe clinical outcomes. However, the within-host dynamics that underly pathogen spread to and clearance from systemic organs remain largely uncharacterized. In animal models of infection, the observed pathogen population results from the combined contributions of bacterial replication, persistence, death, and dissemination, each of which can vary across organs. Quantifying the contribution of each these processes is required to interpret and understand experimental phenotypes. Here, we leveraged STAMPR, a new barcoding framework, to investigate the population dynamics of extraintestinal pathogenic E. coli, a common cause of bacteremia, during murine systemic infection. We show that while bacteria are largely cleared by most organs, organ-specific clearance failures are pervasive and result from dramatic expansions of clones representing less than 0.0001% of the inoculum. Clonal expansion underlies the variability in bacterial burden between animals, and stochastic dissemination of clones profoundly alters the pathogen population structure within organs. Despite variable pathogen expansion events, host bottlenecks are consistent yet highly sensitive to infection variables, including inoculum size and macrophage depletion. We adapted our barcoding methodology to facilitate multiplexed validation of bacterial fitness determinants identified with transposon mutagenesis and confirmed the importance of bacterial hexose metabolism and cell envelope homeostasis pathways for organ-specific pathogen survival. Collectively our findings provide a comprehensive map of the population biology that underlies bacterial systemic infection and a framework for barcode-based high-resolution mapping of infection dynamics.
Barcode and TIS read counts, in addition to all scripts required to analyze these data, are provided at github.com/hullahalli/stampr_rtisan. Sequencing reads have been deposited in SRA
- Matthew K Waldor
- Karthik Hullahalli
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: All animal experiments were conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and the Animal Welfare Act of the United States Department of Agriculture using protocols reviewed and approved by Brigham and Women's Hospital Committee on Animals (Institutional Animal Care and Use Committee protocol number 2016N000416 and Animal Welfare Assurance of Compliance number A4752-01)
- Bavesh D Kana, University of the Witwatersrand, South Africa
© 2021, Hullahalli & Waldor
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Members of the bacterial T6SS amidase effector (Tae) superfamily of toxins are delivered between competing bacteria to degrade cell wall peptidoglycan. Although Taes share a common substrate, they exhibit distinct antimicrobial potency across different competitor species. To investigate the molecular basis governing these differences, we quantitatively defined the functional determinants of Tae1 from Pseudomonas aeruginosa PAO1 using a combination of nuclear magnetic resonance (NMR) and a high-throughput in vivo genetic approach called deep mutational scanning (DMS). As expected, combined analyses confirmed the role of critical residues near the Tae1 catalytic center. Unexpectedly, DMS revealed substantial contributions to enzymatic activity from a much larger, ring-like functional hot spot extending around the entire circumference of the enzyme. Comparative DMS across distinct growth conditions highlighted how functional contribution of different surfaces is highly context-dependent, varying alongside composition of targeted cell walls. These observations suggest that Tae1 engages with the intact cell wall network through a more distributed three-dimensional interaction interface than previously appreciated, providing an explanation for observed differences in antimicrobial potency across divergent Gram-negative competitors. Further binding studies of several Tae1 variants with their cognate immunity protein demonstrate that requirements to maintain protection from Tae activity may be a significant constraint on the mutational landscape of tae1 toxicity in the wild. In total, our work reveals that Tae diversification has likely been shaped by multiple independent pressures to maintain interactions with binding partners that vary across bacterial species and conditions.
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