Dual function for an effector immunity protein in pseudomonas aeruginosa biofilm biogenesis

The National Institute of Allergy and Infectious Diseases awarded Trustees of Dartmouth College $158,190 on December 20, 2025, under the Allergy and Infectious Diseases Research program (CFDA 93.855) to investigate dual functions of an effector immunity protein in Pseudomonas aeruginosa biofilm biogenesis.

The research tests the hypothesis that TSIT, a Type VI secretion system immunity protein in P. aeruginosa, functions as both a traditional T6SS immunity protein and as a transcription factor regulating biofilm formation. P. aeruginosa forms robust biofilms in the airways of people with cystic fibrosis and is a major cause of morbidity and mortality in this population. Preliminary data demonstrate that expression of TSIT alone restores biofilm production in a TSET operon mutant, indicating the immunity protein's importance in biofilm growth. The study will elucidate the mechanism underlying this phenotype and, because TSIT is conserved across gram-negative bacterial pathogens, will provide insights into biofilm regulation with potential clinical treatment applications.

The award runs through July 31, 2027, with place of performance in Hanover, New Hampshire. This is a Project Grant, classified as direct assistance funding to the recipient institution.

Abstract

Cystic fibrosis (CF) is characterized by the formation of thick mucus in the respiratory tract contributing to chronic infections of numerous pathogens, including commonly pseudomonas aeruginosa (PA). PA forms robust biofilms, aggregates of bacteria encased in a protective extracellular matrix, in the airways of people with CF (PWCF) and is a major cause of morbidity and mortality for PWCF. PA harbors a wide repertoire of competitive mechanisms that mediate host-microbe and microbe-microbe interactions. One such defensive mechanism is the type VI secretion system (T6SS) which is known to be expressed during respiratory infection in PWCF. The T6SS apparatus transports diverse effectors, one of which is tset. The tset operon consists of a paar/tip protein, chaperone proteins, the effector tset, and its cognate immunity protein tsit. Preliminary results found that deletion of the tset operon decreases PA biofilm growth in association with human CF bronchial epithelial cells and expression of tsit alone is sufficient to restore biofilm production in the tset operon mutant. Thus, the T6SS immunity protein tsit is important for PA biofilm growth, but the mechanism is unknown. Tsit has sequential and structural homology to a general lysr-type transcriptional regulator (LTTR). Lttrs are known to regulate diverse genes including those involved in virulence, metabolism, and quorum sensing. Homology to lttrs suggests a secondary, unconventional function for tsit, in addition to its role as a traditional T6SS immunity protein. This application aims to test the hypothesis that tsit has a secondary function as a transcription factor that regulates biofilm. As tsit is conserved across several gram-negative bacterial pathogens, this work will provide far reaching insights into biofilm regulation and thus clinical treatment. The applicant plans to pursue a career in academia in the area of host-pathogen interactions. Support for this application will enable her to hone skills in bioinformatics and biochemistry, acquire experience mentoring/teaching, experience with manuscript and grant preparation, and experience presenting to diverse audiences. This application will not only provide a broad range of laboratory techniques, but also provide transferrable skills for her long-term goal of becoming an academic researcher.