Virginia Research Day 2021

Studying Streptococcus suis via Multiple Sequencing Approaches

S. Tristan Stoyanof , Kevin Lahmers, and Clayton Caswell Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Blacksburg, VA

Introduction

Sequencing Methods

Streptococcus suis poses one of the greatest economic threats to the pork industry and is a growing health concern for those working in close proximity to the infected pigs. This Gram- positive pathogen is capable of spreading to nearly every organ and tissue within the host, partially due to the bacterium’s ability to cross tight junction barriers (i.e. the blood-brain barrier). On a genetic level, little is known as to how S. suis is able to do so. To combat this, we will establish not only what genes are essential within S. suis , but also which genes are conditionally important. To do so, transposon sequencing (Tn-seq) will be employed. Tn-seq utilizes the mariner transposon and it’s insertional affinity for T-A dinucleotides, a pattern that occurs ~128,000 times within the S. suis genome. Each individual bacterial cell will receive only one copy of the transposon, which will insert itself in one of the many potential T-A sites. This, carried out on the magnitude of 1x10 8 – 1x10 9 bacterial cells per experiment, provides an extremely high throughput method of generating, and later screening mutants. Alongside these Tn-seq experiments, mapping of the differential gene expression will be carried out via RNA sequencing. S. suis is capable of rapid changes in gene expression, which is largely dependent on the environment in which it resides. By simulating some of the many biologically relevant conditions, we can effectively get a snapshot of what genes are differentially regulated in response. Many of the growth/stress conditions used represent some of the varied niches S. suis has been shown to infect. These include simulated pig mucin, pig cerebral spinal fluid, pig serum, as well as a biofilm. While both projects are separate from one another, combined they begin to lay the genomic foundations for how this bacterium survives as such a successful pathogen within both humans and pigs.

Determining differential expression of genes (RNA Sequencing)

Determining essentiality or conditional importance of genes (Transposon Sequencing)

Vector transformed into S. suis ATCC 700794

Growth Condition X

THY (Control)

Limited Nutrients

Many Nutrients

No Antimicrobials

GGCTC TA GACC CCGAG AT CTGG

Low pH

Antimicrobials

1

Neutral pH

Gene regulation happens rapidly. Expression levels peak after ~20 minutes, before settling down as the bacteria begins to cope mRNA levels directly equate to gene expression, but their protein products aren’t always directly affected

Once per cell x 10 9 cells

1 2 3 Established Baseline mRNA Levels

3

Le Breton Y, Belew AT, McIver KS. Protocols for Tn-seq Analyses in the Group A Streptococcus. Methods Mol Biol. 2020 PMID: 32430812

More mRNA

Less mRNA

Same mRNA

GGCTC CCGAG

TA GACC AT CTGG

Krmit

truncated or non- functional product

Isolate total RNA. Deplete rRNA.

Isolate total RNA. Deplete rRNA.

Biologically Relevant Growth Conditions

rRNA comprises ~75% of the total RNA. It’s depletion will allow for better mRNA sequencing depth

Sequence, align to genome

Sequence, align to genome

Library added to THY (control) and chosen experimental condition(s)

• Cerebral spinal fluid • Whole blood • Serum • Nasal mucous • Biofilms

Cultures are allowed to grow for 24 hours

Comparisons can now be made between the control samples and any experimental condition

Genomic DNA extracted

GGCTC CCGAG

TA GACC AT CTGG

Krmit

Experimental Design

Conclusion

Both RNA-seq and Tn-seq pose as powerful starting points for understanding how a bacterial organism interacts with, and responds to, an environment. In the case of a pathogenic species, this dual sequencing approach also has the potential to rapidly identify potential therapeutic targets. This approach also has the capacity to be applied to a wide variety of both Gram- positive and -negative bacterial species with ease.

Now wherever an insertion mutation introduced a fitness disadvantage, those mutants won’t be present in the output, or will be present at a much lower rate. The opposite is also true.

The transposon, along with a portion of the host genome, is liberated and barcoded

Unique barcode

Krmit

Future Directions

Identifies growth condition

Aligns to S. suis ATCC700764 genome

Insertional Frequency = <1

Insertional Frequency = 0

Insertional Frequency = >1

Both halves of this project are currently being sequenced at Virginia Tech’s Genomic Sequencing Center. Once the conditional importance of the genes, as well as their differential expression, is elucidated, isogenic deletions will be made. Genes that fit this description will be either 2-fold over/under- expressed (RNA-Seq) or be missing from specific growth conditions (Tn-seq). There also exists the unique possibility of interplay between these two experiments. If a gene is differentially regulated (RNA-Seq) in a growth conditions, and no insertional mutants are detected in the same condition (Tn-Seq), that gene will represent an extremely attractive target for further study.

Acknowledgments

• Thank you to the Virginia Tech Genomics Sequencing center for handling the sequencing of both the RNA-seq and Tn-seq projects • Dr. Kevin McIver for the donation of the pKRMT vector and usage protocol • Dr. Kevin Lahmers for processing the raw data for ease of study

Le Breton Y, Belew AT, McIver KS. Protocols for Tn-seq Analyses in the Group A Streptococcus. Methods Mol Biol. 2020 PMID: 32430812

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