Host-associated microbe PCR (hamPCR) enables convenient measurement of both microbial load and community composition
Abstract
The ratio of microbial population size relative to the amount of host tissue, or 'microbial load', is a fundamental metric of colonization and infection, but it cannot be directly deduced from microbial amplicon data such as 16S rRNA gene counts. Because existing methods to determine load, such as serial dilution plating, quantitative PCR, and whole metagenome sequencing, add substantial cost and/or experimental burden, they are only rarely paired with amplicon sequencing. We introduce host-associated microbe PCR (hamPCR), a robust strategy to both quantify microbial load and describe interkingdom microbial community composition in a single amplicon library. We demonstrate its accuracy across multiple study systems, including nematodes and major crops, and further present a cost-saving technique to reduce host overrepresentation in the library prior to sequencing. Because hamPCR provides an accessible experimental solution to the well-known limitations and statistical challenges of compositional data, it has far-reaching potential in culture-independent microbiology.
Data availability
All data in this manuscript have been deposited in the European Nucleotide Archive (ENA) under the project number PRJEB38287. At https://www.ebi.ac.uk/ena.
Article and author information
Author details
Funding
Deutsche Forschungsgemeinschaft (SPP 2125 DECRyPT)
- Derek S Lundberg
- Pratchaya Pramoj Na Ayutthaya
- Detlef Weigel
Human Frontiers Science Program Long-term Fellowship (LT000565/2015-L)
- Derek S Lundberg
Max Planck Society
- Derek S Lundberg
- Pratchaya Pramoj Na Ayutthaya
- Gautam Shirsekar
- Wen-Sui Lo
- Detlef Weigel
Cluster of Excellence EXC2124 Controlling Microbes to Fight Infection (390838134)
- Derek S Lundberg
- Pratchaya Pramoj Na Ayutthaya
- Gautam Shirsekar
- Wen-Sui Lo
- Detlef Weigel
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Rebecca Bart, The Donald Danforth Plant Science Center, United States
Version history
- Received: December 31, 2020
- Accepted: July 19, 2021
- Accepted Manuscript published: July 22, 2021 (version 1)
- Version of Record published: August 25, 2021 (version 2)
Copyright
© 2021, Lundberg et al.
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.
Metrics
-
- 4,547
- Page views
-
- 560
- Downloads
-
- 14
- Citations
Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Microbiology and Infectious Disease
- Plant Biology
Purinergic signaling activated by extracellular nucleotides and their derivative nucleosides trigger sophisticated signaling networks. The outcome of these pathways determine the capacity of the organism to survive under challenging conditions. Both extracellular ATP (eATP) and Adenosine (eAdo) act as primary messengers in mammals, essential for immunosuppressive responses. Despite the clear role of eATP as a plant damage-associated molecular pattern, the function of its nucleoside, eAdo, and of the eAdo/eATP balance in plant stress response remain to be fully elucidated. This is particularly relevant in the context of plant-microbe interaction, where the intruder manipulates the extracellular matrix. Here, we identify Ado as a main molecule secreted by the vascular fungus Fusarium oxysporum. We show that eAdo modulates the plant's susceptibility to fungal colonization by altering the eATP-mediated apoplastic pH homeostasis, an essential physiological player during the infection of this pathogen. Our work indicates that plant pathogens actively imbalance the apoplastic eAdo/eATP levels as a virulence mechanism.
-
- Microbiology and Infectious Disease
SARS-CoV-2 spike protein plays a key role in mediating viral entry and inducing host immune responses. It can adopt either an open or closed conformation based on the position of its receptor-binding domain (RBD). It is yet unclear what cause these conformational changes or how they influence the spike's functions. Here we show that Lys417 in the RBD plays dual roles in the spike's structure: it stabilizes the closed conformation of the trimeric spike by mediating inter-spike-subunit interactions; it also directly interacts with ACE2 receptor. Hence, a K417V mutation has opposing effects on the spike's function: it opens up the spike for better ACE2 binding while weakening the RBD's direct binding to ACE2. The net outcomes of this mutation are to allow the spike to bind ACE2 with higher probability, mediate viral entry more efficiently, but become more exposed to neutralizing antibodies. Given that residue 417 has been a viral mutational hotspot, SARS-CoV-2 may have been evolving to strike a balance between infection potency and immune evasion, contributing to its pandemic spread.