Investigation of the role of TamAB in Salmonella pathogenesis and outer membrane homeostasis

Salmonella survive and replicate in macrophages, which normally kill bacteria by exposing them to various harsh conditions and antimicrobial effectors, many of which target the bacterial cell envelope. The PhoPQ two-component system responds to the phagosome environment and induces factors that protect the outer membrane, allowing adaptation and growth in the macrophage. To find the role of TamAB in Salmonella pathogenesis, we show that PhoPQ induces the transcription of the tamAB operon both in vitro and in macrophages. The TamA protein is structurally similar to BamA, an essential protein in the BAM complex that assembles Beta-barrel proteins in the outer membrane, while TamB is an AsmA-family protein implicated in lipid transport between the inner and outer membranes. We show that the BAM machinery is stressed in vitro under low Mg2+ and low pH conditions that mimic the phagosome. Mutations affecting BAM function have been previously shown to confer significant virulence defects. Although loss of tamAB alone confers no virulence defect, a tamAB deletion confers a synthetic phenotype in bam mutant backgrounds in animals and macrophages, and it also does so in vitro upon treatment with vancomycin or sodium dodecyl sulfate. Recently, it has been shown that TamB, YhdP and YdbH, three members of AsmA-like proteins, are involved in phospholipid transport to the outer membrane in Escherichia coli. Mutations affecting YhdP also confer synthetic phenotypes with bam mutations in the animal, but this interaction is not evident in vitro. Thus, in the harsh phagocytic environment of the macrophage, the outer membrane BAM machinery is compromised, and the TamAB system, and perhaps other PhoPQ-regulated factors, are induced to compensate. It is most likely that TamAB and other systems assist the BAM complex indirectly by affecting outer membrane properties. We took global approaches to detect any other involved genes/pathways that work with or are involved with this phenomenon. LC/MS analysis of various tam and bam mutations showed RcsF, YhdV, PqiC, OmpX, Blc, and BamE significantly decreased in abundance in the outer membrane in the tamAB background compared to the control groups. RNA sequencing suggests that the expression of these genes is not affected by a tamAB deletion. Western blot analysis showed that BamE and PqiC have similar abundance in the membrane when expressed from the pWKS30 plasmid. Furthermore, RNA sequencing showed 217 genes with a significantly different expression pattern in the tamAB background than controls. A qPCR showed that cadB transcription increased in the tamAB background. Suppressor analysis of spontaneous mutants in bamB tamAB background revealed that galE deletion suppressed the vancomycin sensitivity. Additionally, the removal of mlaA and pldA rescued the sensitivity to vancomycin. It is known that galE deletion changes the outer membrane architecture by shortening the LPS sugar, and mlaA and pldA deletions increase the phospholipids in the outer leaflets.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Microbiology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2023

Author dc:creator

• Ramezanifard, Rouhallah

Contributors dc:contributor

• Slauch, James M
• Imlay, James A
• Cronan, John E
• Kehl-Fie, Thomas E

• Tamab
• Salmonella
• Outer Membrane

Statement dc:rights

• Copyright 2023 Rouhallah Ramezanifard

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/122225

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F122225