A tale of two proteins: relationship between HilD & FliZ in SPI1 regulation

Non-typhoid Salmonella are major pathogens that cause gastroenteritis and enteric fever. Salmonella invades the host epithelium using the Type Three Secretion System (T3SS) encoded on Salmonella Pathogenicity Island 1 (SPI1). SPI1 T3SS gene expression is tightly regulated by HilA, a transcriptional activator encoded on SPI1. The expression of hilA is regulated by three homologous proteins, HilD, HilC, and RtsA, which self-activate their own expression and each other’s expression, forming a complex feed-forward loop. HilD, HilC, and RtsA work independently and collectively to regulate hilA expression. To better understand the protein interactions that regulate SPI1 expression, we have characterized the nature of the interaction between HilD, HilC, and RtsA. Using biochemical and genetic experiments, we show that HilD, HilC, and RtsA form homodimers and heterodimers in solution. We have identified a possible N-terminal dimerization domain and shown that alanine substitution of this region results in decreased dimerization of HilD and HilC and negatively affects hilA expression. In Salmonella, regulation of SPI1 machinery represents one of the most complex signal integration systems required for successful invasion of the host epithelium. Numerous environmental signals that control SPI1 expression are integrated at the level of HilD. FliZ, encoded in the flagellar regulon, is a positive regulator of SPI1 that acts solely through HilD to control the expression of hilA. To better understand the complex network of signal integration at the level of HilD, we characterized the nature of the relationship between HilD and FliZ. Using genetic studies, we show that FliZ does not facilitate HilD dimerization, but controls HilD protein activity by regulating the DNA binding ability of HilD. Two-hybrid systems and co-immunoprecipitation (co-IP) studies show that HilD and FliZ do not interact directly at the protein level. To identify the intermediate regulator between HilD and FliZ, we performed three alternative and complementary experiments to screen the Salmonella genome. RNA-Seq analysis was conducted in ΔfliZ, wildtype, and pFliZ backgrounds to identify genes transcriptionally regulated by FliZ. Random transposon mutagenesis screen was performed to isolate mutants that did not respond to FliZ-mediated activation of SPI1. Additionally, we demonstrate that hilA expression can be activated by FliZ in E. coli, suggesting that the intermediate regulator is shared between E. coli and Salmonella. Therefore, a systematic genome-wide screen of non-essential genes was conducted using the E. coli Keio collection. In summary, we were unable to identify the intermediate protein between HilD and FliZ using these screening approaches, and the precise mechanism by which FliZ regulates HilD remains unknown. Further investigation into the role of FliZ in HilD regulation will provide deeper insight into the signal integration mechanisms controlling SPI expression at the level of HilD protein and the coordination of complex signal transduction pathways involved in Salmonella invasion.

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

• Narm, Koh Eun

Contributors dc:contributor

• Slauch, James M.
• Slauch, James M
• Vanderpool, Carin K
• Cronan, John E
• Metcalf, William W.

• Salmonella
• Spi1
• Fliz
• Salmonella Pathogenesis

Statement dc:rights

• Copyright 2025 Koh Eun Narm

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/130191

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F130191