# Inc/GFP chimera protein-based interactomics reveals host cellular interactions of Cps0558, a novel Chlamydia psittaci inclusion protein

## TL;DR

This study identifies a new Chlamydia psittaci inclusion protein and its host interaction partners using a novel chimera-based method.

## Contribution

A novel Inc/GFP chimera protein-based interactomics approach is introduced for studying Chlamydia–host interactions.

## Key findings

- Cps0558 is a novel C. psittaci Inc protein that interacts with ACAD11, a lipid metabolism-related host protein.
- IncA interacts with host proteins involved in ubiquitylation, suggesting a role in modulating this system.
- The chimera-based method confirms interactions in the native infection context, supporting physiological relevance.

## Abstract

The obligate intracellular Gram-negative bacterium Chlamydia psittaci, a zoonotic pathogen transmissible between birds and humans, has played a pioneering role in research on its membrane-bound replicative niche termed the inclusion. Inclusion membrane proteins (Inc proteins) are crucial for Chlamydia–host interactions and were first identified in C. psittaci. This study investigates putative C. psittaci Inc proteins by a combination of in silico analyses, immunofluorescence and, strikingly, a new Inc/GFP chimera protein-based interactomics approach to identify host cellular interaction partners. Here, we report a novel C. psittaci Inc protein, Cps0558, along with respective host cellular interaction partners, in particular ACAD11, which is involved in lipid metabolism. We confirm their physical interaction in the native infection context, supporting the physiological relevance of our chimera-based screen. Furthermore, new interaction partners for the known Inc protein IncA are identified, revealing a potential role of IncA as modulator of the host ubiquitylation system. These results provide further insights into the biology of C. psittaci and present a novel tool for studying Inc proteins under conditions closely resembling their natural tertiary structure.

This study investigates putative Chlamydia psittaci Inc proteins by a combination of in silico analyses, immunofluorescence, and a new Inc/GFP chimera protein-based interactomics approach to identify host cellular interaction partners.