Isolation of Total RNA from Pseudomonas aeruginosa within Biofilms for Measuring Gene Expression

Summary

This protocol presents a method to isolate RNA from Pseudomonas aeruginosa biofilms grown in chamber slides for high throughput sequencing.

Abstract

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that causes infections in the airways of cystic fibrosis (CF) patients. P. aeruginosa is known for its ability to form biofilms that are protected by a matrix of exopolysaccharides. This matrix allows the microorganisms to be more resilient to external factors, including antibiotic treatment. One of the most common methods of biofilm growth for research is in microtiter plates or chambered slides. The advantage of these systems is that they allow for the testing of multiple growth conditions, but their disadvantage is that they produce limited amounts of biofilm for RNA extraction. The purpose of this article is to provide a detailed, step by step protocol on how to extract total RNA from small amounts of biofilm of sufficient quality and quantity for high throughput sequencing. This protocol allows for the study of gene expression within these biofilm systems.

Introduction

Most chronic bacterial infections, such as pulmonary infections in cystic fibrosis (CF) patients and prosthesis related infections, are characterized by the growth of organisms within biofilms. Biofilms¹ are communities of bacteria encased in a matrix composed primarily of polysaccharides². Bacteria within biofilms can be slow growing, metabolically dormant, and in anaerobic, hypoxic conditions. Biofilms are more resistant to antibiotics due to factors such as decreased antibiotic penetration, increased expression of drug efflux pumps, and decreased cell division³. For these and other reasons, they are of great research interest.

In order to accurately study persistent infections such as chronic Pseudomonas aeruginosa infections in CF patients, the growth conditions seen with biofilm formation need to be accurately reflected in vitro. A common, high throughput method is to grow them in chamber slides or microtiter plates and monitor biofilm formation by confocal microscopy⁴. It is known that a key regulator in the transition from a planktonic, or free-floating, to biofilm bacterial lifestyle is the secondary messenger, cyclic-di-GMP⁵. Increased cyclic-di-GMP levels increase the expression of specific genes that promote biofilm growth. Small non-coding regulatory RNAs and quorum sensing also play important roles in regulation of biofilm formation⁵. Measuring biofilm gene expression by sequencing extracted bacterial RNA can be challenging. P. aeruginosa, for example, produces three exopolysaccharides (Psl, Pel and alginate), which are produced in significant amounts in biofilms^6,7. These polysaccharides can interfere with RNA extraction and purification leading to impure preparations containing low levels of bacterial mRNA⁸. Commercially available RNA extraction kits are able to produce high quality RNA from planktonic bacterial cultures but may not work as well with biofilm cultures^9,10,11. There are a few commercial RNA extraction kits that claim to work for biofilms, one of which we use with this method.

In this manuscript, we describe the procedures for growing P. aeruginosa biofilms in chamber slides and extracting bacterial mRNA for high throughput sequencing^12,13. Utilizing clinical isolates collected from sputum samples from CF patients, we demonstrate that these methods can be used for isolates with varying growth characteristics. In comparison to prior publications, this protocol is described in detail to enable better success in studying bacterial biofilm gene expression^11,14,15,16.

Protocol

The Research Ethics Board (REB) is required for the collection and processing of sputum samples from human subjects. This study was approved by the Hospital for Sick Children (REB#1000019444). Research Ethics Board (REB) is required to collect and store sputum samples from human subjects. These studies were approved by the Hospital for Sick Children REB#1000058579. 1. Biofilm formation Grow Pseudomonas aeruginosa isolates obtained from the sputum samples of CF patients use…

Representative Results

The general overview of the method is shown in Figure 1. We previously used 8-well chamber slides to grow P. aeruginosa biofilms and expose them to antibiotics before then examining them via confocal microscopy at different time points12,13. This method can be used to extract total RNA directly from biofilms grown in this system in order to study gene expression changes post treatment. This protocol has been optimized for <e…

Discussion

Total RNA is successfully extracted from 17 different bacterial biofilm samples in triplicate, yielding a total of 51 samples. The forty-nine RNA libraries are pooled and successfully sequenced. Overall, this validates our quality criteria with a 96 % success rate even though more than half the samples are considered to be low abundance and of sub-optimal quality^34,35,36,37.

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Materials

Agilent 2100 Bioanalyzer	Agilent	G2939BA	Automated electrophoresis of biomolecules
Agilent RNA 6000 pico kit	Agilent	5067-1513	High sensitivity RNA electrophoresis chip to generate a RIN
DNA/RNA Lysis Buffer	Zymo Research	D7001-1-50	A guanidinium thiocyanate and N-Lauroylsarcosine-based lysis buffer sold as part of a nucleic acid purification kit
DNA/RNA Prep Buffer	Zymo Research	D7010-2-10	A guanidine HCl and ethanol buffer used for purification of DNA and RNA
DNA/RNA Shield	Zymo Research	R1100-50	DNA and RNA preservation/protection reagent
DNA/RNA Wash Buffer	Zymo Research	D7010-3-6	A salt and ethanol buffer used for purification of DNA and RNA
DNBSEQ G-400RS	MGI	G-400RS	High throughput sequencer
MGIEasy RNA Directional Library Prep Set	MGI	1000006386	Generate libraries for MGI high-throughput sequencing platforms from total RNA.
Mini-Beadbeater-96	BioSpec	1001	A high energy, high throughput cell disrupter
NEBNext rRNA Depletion Kit (bacteria)	New England Biolabs	E7850X	Efficient and specific depletion of bacterial rRNA (5S, 16S, 23S)
Nunc Lab-Tek II chamber slide system	Thermo Fisher Scientific	154534	8-well chamber slide with removable wells
Qubit Fluorometer	Thermo Fisher Scientific	Q33238	Fluorometer for DNA, RNA and proteins
Qubit RNA HS Assay Kit	Thermo Fisher Scientific	Q32852	High sensitivity fluorometric assay to measure RNA concentration
Spin-Away Filters	Zymo Research	C1006-50-F	Silica-based spin column primarily used to bind or remove genomic DNA
Sterile inoculation loops, 1 uL	Sarstedt	86.1567.050	Sterile, disposable inoculation loops for manipulation of microorganisms
ZR BashingBead Lysis tubes	Zymo Research	S6003-50	2 mL tubes containing 0.1 and 0.5 mm bead lysis matrix for homogenizing biological samples
Zymo Spin IIICG Columns	Zymo Research	C1006-50-G	Silica-based spin column for purification of DNA and RNA
Zymo-Spin III-HRC Filters	Zymo Research	C1058-50	Remove inhibitors such as polyphenolic compounds, humic/fulvic acids, tannins, melanin, etc.
Zymobiomics DNA/RNA Miniprep kit	Zymo Research	R2002	DNA and RNA dual extraction kit
Zymobiomics HRC Prep solution	Zymo Research	D4300-7-30	To be used with Zymo-Spin III-HRC Filters to remove PCR inhibitors