Rothia from the human nose inhibit Moraxella catarrhalis colonization with a secreted peptidoglycan endopeptidase

Reed M. Stubbendieck^1,2, Eishika Dissanayake³, Peter M. Burnham¹, Susan E. Zelasko^1,4, Mia I. Temkin¹, Sydney S. Wisdorf³, Rose F. Vrtis³, James E. Gern^3,5, Cameron R. Currie^1,6,7,8

¹Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706 ²Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK 74078 ³Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, WI 53792 ⁴Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, WI 53706 ⁵Department of Medicine, University of Wisconsin School of Medicine and Public Health, Madison, WI 53792 ⁶Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53706 ⁷Laboratory of Genetics, University of Wisconsin-Madison, Madison, WI 53706 ⁸David Braley Centre for Antibiotic Discovery, Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada

Introduction

This repository contains the code necessary to replicate the results and figures of our study on interactions between the pathobiont Moraxella catarrhalis and Rothia.

Datasets

The code in the Rmd document and the following code snippets expect the raw and derived datasets to be in the ./rawData/ and ./derivedData/ directories, respectively.

Raw datasets

The raw amplicon sequencing reads can be found in the Short Read Archive under BioProject accession PRJNA866994. The genome sequences of the Rothia generated for this study can be found under BioProject accession PRJNA867425. All other raw datasets can be found from FigShare: here.

Derived datasets

All derived datasets can be found from FigShare: here.

Prerequisites

Software

• antiSMASH
• anvi'o
• BiG-SCAPE
• Core Species Tree
• Mothur
• Prokka
• PyParanoid

R Packages

• Biostrings
• circlize
• cowplot
• ggmsa
• ggnewscale
• ggpubr
• ggtree
• phyloseq
• RColorBrewer
• readr
• reshape2
• rstatix
• seqmagick
• tidyverse
• vegan

Code

For all code, the thread_num needs to be changed before it can be run. All paths below assume that code is being run from the base project directory.

Processing reads with fastp

python3 ./scripts/python/run_fastp.py ./rawData/amplicon_reads/ ./derivedData/amplicon_reads/processed_reads/ thread_num

Mothur

conda activate mothur
mothur ./scripts/mothur/mothur_batch_file.txt
conda deactivate

Genome Annotation

conda activate prokka
python3 ./scripts/python/genome_annotation.py ./rawData/genomes/combined_genomes/ ./derivedData/annotations/combined_genomes/ ./rawData/genomes/rothia_genome_table.csv thread_num
conda deactivate

PyParanoid

conda activate pyparanoid
python3 ./scripts/python/run_pyparanoid_BuildGroups.py ./derivedData/annotations/combined_genomes/ ./derivedData/pyparanoid_out_combined/ ./rawData/genomes/combined_strainlist.txt RothiaDB thread_num
python3 scripts/python/parse_homolog_faa.py ./derivedData/pyparanoid_out_combined/RothiaDB/homolog.faa ./derivedData/pyparanoid_out_combined/RothiaDB/rothia_homolog_groups.tsv
conda deactivate

Core Species Tree

mkdir ./derivedData/phylogenetic_trees/rothia_tree/
cd ./derivedData/phylogenetic_trees/rothia_tree/
perl path/to/core_species_tree/core_species_tree.pl path/to/rawData/rothia_genomes/*.fna

Used FigTree to convert the ASTRAL-II core-genome phylogeny (./derivedData/rothia_tree/astral.species.tre) using propotional branch lengths based on the root (./derivedData/rothia_tree/astral.species_proportional.tre).

anvi'o

conda activate anvio-7
python3 ./scripts/python/run_anvio.py ./rawData/genomes/combined_genomes/ ./derivedData/anvio_out/ Rothia thread_num
anvi-import-misc-data -p ./derivedData/anvio_out/anvio_pangenome/Rothia-PAN.db -t layers ./rawData/genomes/rothia_genome_table.tsv
anvi-compute-genome-similarity --external-genomes ./derivedData/anvio_out/external_genomes --program pyANI --output-dir ./derivedData/anvio_out/ANI/ --num-threads thread_num --pan-db derivedData/anvio_out/anvio_pangenome/Rothia-PAN.db
anvi-display-pan -g ./derivedData/anvio_out/Rothia-GENOMES.db -p ./derivedData/anvio_out/anvio_pangenome/Rothia-PAN.db --server-only -P 8080
anvi-script-add-default-collection -p ./derivedData/anvio_out/anvio_pangenome/Rothia-PAN.db
anvi-summarize -g ./derivedData/anvio_out/Rothia-GENOMES.db -p ./derivedData/anvio_out/anvio_pangenome/Rothia-PAN.db -C DEFAULT
conda deactivate

antiSMASH

conda activate antismash
python3 ./scripts/python/run_antismash.py ./rawData/genomes/combined_genomes/ ./derivedData/rothia_antismash_output/ thread_num
conda deactivate

BiG-SCAPE

conda activate bigscape
 python3 /home/stubbendieck/BiG-SCAPE/bigscape.py --pfam_dir /home/stubbendieck/databases/ -c thread_num --mibig --include_singletons --mix --hybrids-off --verbose -i ./derivedData/rothia_antismash_output/gbks/ -o ./derivedData/rothia_bigscape/
 conda deactivate

Proteomics Output

python3 ./scripts/python/proteomics_output_convert.py ./rawData/proteomics/ ./derivedData/rothia_proteomics_output.tsv

ymls

antismash
anvio-7
bigscape
mothur
prokka
pyparanoid