1. Microbiology and Infectious Disease
  2. Plant Biology
Download icon

Host-associated microbe PCR (hamPCR) enables convenient measurement of both microbial load and community composition

  1. Derek S Lundberg  Is a corresponding author
  2. Pratchaya Pramoj Na Ayutthaya
  3. Annett Strauß
  4. Gautam Shirsekar
  5. Wen-Sui Lo
  6. Thomas Lahaye
  7. Detlef Weigel  Is a corresponding author
  1. Max Planck Institute for Developmental Biology, Germany
  2. University of Tübingen, Germany
Tools and Resources
  • Cited 1
  • Views 2,313
  • Annotations
Cite this article as: eLife 2021;10:e66186 doi: 10.7554/eLife.66186

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.

The following data sets were generated

Article and author information

Author details

  1. Derek S Lundberg

    Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, Germany
    For correspondence
    derek.lundberg@gmail.com
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7970-1595

  2. Pratchaya Pramoj Na Ayutthaya

    Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, Germany
    Competing interests
    No competing interests declared.

  3. Annett Strauß

    ZMBP-General Genetics, University of Tübingen, Tübingen, Germany
    Competing interests
    No competing interests declared.

  4. Gautam Shirsekar

    Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, Germany
    Competing interests
    No competing interests declared.

  5. Wen-Sui Lo

    Department of Evolutionary Biology, Max Planck Institute for Developmental Biology, Tübingen, Germany
    Competing interests
    No competing interests declared.

  6. Thomas Lahaye

    ZMBP-General Genetics, University of Tübingen, Tübingen, Germany
    Competing interests
    No competing interests declared.

  7. Detlef Weigel

    Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, Germany
    For correspondence
    weigel.elife@gmail.com
    Competing interests
    Detlef Weigel, Senior editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2114-7963

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

  1. Rebecca Bart, The Donald Danforth Plant Science Center, United States

Publication history

  1. Received: December 31, 2020
  2. Accepted: July 19, 2021
  3. Accepted Manuscript published: July 22, 2021 (version 1)
  4. 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

  • 2,313
    Page views
  • 376
    Downloads
  • 1
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Categories and tags

Research organisms

Further reading

    1. Cell Biology
    2. Microbiology and Infectious Disease

    Toxoplasma bradyzoites exhibit physiological plasticity of calcium and energy stores controlling motility and egress

    Yong Fu et al.
    Research Article

    Toxoplasma gondii has evolved different developmental stages for disseminating during acute infection (i.e. tachyzoites) and for establishing chronic infection (i.e. bradyzoites). Calcium ion (Ca2+) signaling tightly regulates the lytic cycle of tachyzoites by controlling microneme secretion and motility to drive egress and cell invasion. However, the roles of Ca2+ signaling pathways in bradyzoites remain largely unexplored. Here we show that Ca2+ responses are highly restricted in bradyzoites and that they fail to egress in response to agonists. Development of dual-reporter parasites revealed dampened Ca2+ responses and minimal microneme secretion by bradyzoites induced in vitro or harvested from infected mice and tested ex vivo. Ratiometric Ca2+ imaging demonstrated lower Ca2+ basal levels, reduced magnitude, and slower Ca2+ kinetics in bradyzoites compared with tachyzoites stimulated with agonists. Diminished responses in bradyzoites were associated with down-regulation of Ca2+-ATPases involved in intracellular Ca2+ storage in the endoplasmic reticulum (ER) and acidocalcisomes. Once liberated from cysts by trypsin digestion, bradyzoites incubated in glucose plus Ca2+ rapidly restored their intracellular Ca2+ and ATP stores leading to enhanced gliding. Collectively, our findings indicate that intracellular bradyzoites exhibit dampened Ca2+ signaling and lower energy levels that restrict egress, and yet upon release they rapidly respond to changes in the environment to regain motility.

    1. Microbiology and Infectious Disease

    The Lyme disease agent co-opts adiponectin receptor-mediated signaling in its arthropod vector

    Xiaotian Tang et al.
    Research Article Updated

    Adiponectin-mediated pathways contribute to mammalian homeostasis; however, little is known about adiponectin and adiponectin receptor signaling in arthropods. In this study, we demonstrate that Ixodes scapularis ticks have an adiponectin receptor-like protein (ISARL) but lack adiponectin, suggesting activation by alternative pathways. ISARL expression is significantly upregulated in the tick gut after Borrelia burgdorferi infection, suggesting that ISARL signaling may be co-opted by the Lyme disease agent. Consistent with this, RNA interference (RNAi)-mediated silencing of ISARL significantly reduced the B. burgdorferi burden in the tick. RNA-seq-based transcriptomics and RNAi assays demonstrate that ISARL-mediated phospholipid metabolism by phosphatidylserine synthase I is associated with B. burgdorferi survival. Furthermore, the tick complement C1q-like protein 3 interacts with ISARL, and B. burgdorferi facilitates this process. This study identifies a new tick metabolic pathway that is connected to the life cycle of the Lyme disease spirochete.

    1. Microbiology and Infectious Disease
    2. Physics of Living Systems

    Distinguishing different modes of growth using single-cell data

    Prathitha Kar et al.
    Research Article

    Collection of high-throughput data has become prevalent in biology. Large datasets allow the use of statistical constructs such as binning and linear regression to quantify relationships between variables and hypothesize underlying biological mechanisms based on it. We discuss several such examples in relation to single-cell data and cellular growth. In particular, we show instances where what appears to be ordinary use of these statistical methods leads to incorrect conclusions such as growth being non-exponential as opposed to exponential and vice versa. We propose that the data analysis and its interpretation should be done in the context of a generative model, if possible. In this way, the statistical methods can be validated either analytically or against synthetic data generated via the use of the model, leading to a consistent method for inferring biological mechanisms from data. On applying the validated methods of data analysis to infer cellular growth on our experimental data, we find the growth of length in E. coli to be non-exponential. Our analysis shows that in the later stages of the cell cycle the growth rate is faster than exponential.