# A type III secretion system is required for Bordetella atropi invasion of host cells in vivo

## TL;DR

Bordetella atropi uses a type III secretion system to invade host cells, forming structures that help it enter intestinal cells in nematodes.

## Contribution

The study identifies a T3SS as essential for B. atropi invasion and a new effector gene, deiA, involved in host cell invasion.

## Key findings

- A T3SS is required for B. atropi to invade host intestinal cells, forming membrane ruffles similar to those in Salmonella/Shigella.
- Deletion of the effector gene deiA reduces invasion events and infection rates in host nematodes.
- The virulence regulator bvgS is essential for T3SS-mediated invasion, suggesting it controls T3SS activity.

## Abstract

Bordetella atropi is an intracellular bacterial pathogen that infects the intestinal epithelia of the nematode host Oscheius tipulae. We previously showed that the bacteria use filamentation as a novel cell-to-cell spreading mechanism once inside the intestinal cell. However, how the bacteria invade the host cells and what factors contribute to B. atropi infection process remain unknown. In this study, we investigate the roles of type III (T3SS) and type VI secretion systems (T6SS) in B. atropi pathogenesis, which are employed by many bacterial pathogens, both extracellular and intracellular, to deliver effectors that manipulate host physiology to their advantage. We found that the two T6SSs encoded in B. atropi genome played no obvious roles in the invasion or intracellular spreading. In contrast, a T3SS was required for intestinal cell invasion. T3SS mutants showed loss of host cell protrusions from the apical surface that normally engulf invading wild type bacteria, as seen by both electron microscopy and confocal fluorescent microscopy. These protrusions bear morphological similarities to membrane ruffles triggered by the T3SS-mediated invasion seen in other pathogens such as Salmonella and Shigella spp. Additionally, we conducted dual transcriptomics and saw upregulation of T3SS in vivo, along with several putative effectors and the virulence regulator BvgS of the genus Bordetellae. We knocked out these effector candidates and found that deletion of one of these genes, deiA (decreased invasion protein A), leads to a reduction in the number of invasion events and overall percentage of infected animals in the population. In addition, deletion of the virulence regulator bvgS resulted in a complete loss of B. atropi invasion, suggesting it may regulate T3SS for host cell invasion.

The free-living nematodes, C. elegans and its related species, are useful models to study host-pathogen interactions in whole animal settings since they share many similarities in immune signaling pathways to mammals. We added a new pathogen representative of a major class, intracellular bacteria, to the list of pathogens that can be studied using these animals. The new pathogen, Bordetella atropi, belongs to the Bordetella genus that includes important human pathogens like B. pertussis, the causative agent of whooping cough. We showed that B. atropi requires a specialized machinery known as type III secretion system to manipulate host intestinal epithelial cells to form distinctive morphological structures like those observed during invasion of Salmonella or Shigella species. These structures wrap around and engulf the invading bacteria, eventually lead to the uptake of the bacteria into host cell cytoplasm, and thus result in successful invasion of host cells by the pathogen. We also identified a gene that we named deiA for decreased invasion protein A whose protein product contributes to the capacity of the pathogen to invade host cells. Our results established a system for studying intracellular bacterial pathogenesis in whole animals, potentially with high conservation at the mechanistic level to mammalian systems.