Understanding small RNA-mediated post-transcriptional regulation of Salmonella pathogenicity island 1 activator HilA in Salmonella typhimurium

Salmonella Pathogenicity Island 1 (SPI1) encodes a Type-3 secretion system (T3SS) essential for Salmonella invasion of intestinal epithelial cells. Many environmental and regulatory signals control SPI1 gene expression, but in most cases, the molecular mechanisms remain unclear. Many regulatory signals control SPI1 at a post-transcriptional level, and we have identified a number of small RNAs (sRNAs) that control the SPI1 regulatory circuit. The transcriptional regulator HilA activates the expression of the genes encoding the SPI1 T3SS structural and primary effector proteins. The hilA mRNA 5' untranslated region (UTR) is ~350 nucleotides in length and binds the RNA chaperone Hfq, suggesting it is a likely target for sRNA-mediated regulation. I compared two high-throughput approaches for sRNA target identification. I developed a modified MS2-Affinity Purification and Sequencing (MAPS) protocol and employed rGRIL-Seq (reverse Global sRNA Target Identification by Ligation and Sequencing). These methods used hilA 5' UTR tagging to capture cognate sRNA regulators. Comparative analysis revealed distinct advantages and limitation of each approach for dissecting target-focused sRNA discovery. rGRIL-Seq analysis revealed the potential sponge function of hilA 5’ UTR, identifing an sRNA-mediated SPI-1 post-transcriptional regulatory network composed of four sRNAs: Stnc2090, Stnc290, Stnc540 and Stnc3630, the results also defined sRNA InvR binding site within the hilA ribosome binding site region. Further genetic and molecular characterization showed that InvR directly base-pairs with hilA mRNA to prevent translation initiation, functioning to counteract HilA overactivation by HilD. This work revealed the structure of sRNA regulatory networks controlling SPI-1. Expression of master regulators such as HilD is extensively targeted by multiple sRNAs, while downstream effectors like HilA receive minimal direct post-transcriptional regulation. This pattern likely reflects evolutionary optimization for regulatory efficiency. The limited direct hilA regulators may result from evolutionary pressure against inappropriate activation of this essential virulence factor. These results advance our understanding of post-transcriptional regulatory network in SPI-1 virulence gene expression and demonstrate the sophisticated molecular strategies employed by Salmonella to integrate environmental signals for precise virulence gene-expression control.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Microbiology

Grantor

University of Illinois Urbana-Champaign

Year dc:date

2025

Author dc:creator

• Hou, Yutong

Contributors dc:contributor

• Vanderpool, Carin K.
• Slauch, James M.
• Whitaker, Rachel
• Mera, Paola

• Salmonella Pathogenicity Island
• Salmonella
• sRNA
• Post-transcriptional regulation
• HilA
• Gene regulation
• Virulence

Statement dc:rights

• Copyright 2025 Yutong Hou

Language dc:language

en

Handle dc:identifier

https://hdl.handle.net/2142/132740

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/132740