# Polysaccharide synthesis operon modulates Rickettsia-endothelial cell interactions

## TL;DR

This study shows that a bacterial gene cluster in Rickettsia helps it avoid immune detection and survive in human cells, and disrupting this cluster could lead to effective vaccines.

## Contribution

The study identifies the polysaccharide synthesis operon (pso) as a key factor in Rickettsia immune evasion and vaccine potential.

## Key findings

- The pso gene cluster is essential for Rickettsia to evade immune detection and survive in endothelial cells.
- Disrupting pso reduces Rickettsia virulence and enhances protective immunity in a mouse model.
- O-antigen, produced by pso, is critical for immune evasion and pathogenesis.

## Abstract

Pathogenic Rickettsia species target vascular endothelial cells and cause systemic vasculitis. As obligate intracellular bacterial pathogens, Rickettsia must secure nutritional resources within the cytoplasm of endothelial cells while simultaneously subverting the innate immune defense system. With advances in rickettsial and host genetics, recent studies have identified novel molecular mechanisms involved in the complex interactions between Rickettsia and endothelial cells. However, it remains unclear how Rickettsia shields pathogen-derived immune stimulants, such as lipopolysaccharides (LPS) and peptidoglycan fragments, from immune recognition during intracellular replication. Prior work described two Rickettsia conorii variants with kkaebi transposon insertions in the polysaccharide synthesis operon (pso). Biochemical and immunological analyses revealed that pso is responsible for the biosynthesis of O-antigen (O-Ag) and the proper assembly of surface proteins. In the present work, we document that pso variant HK2 exhibits reduced capacities to adhere to and invade microvascular endothelial cells. Despite the low intracellular abundance, HK2 induced significantly higher levels of proinflammatory cytokines and chemokines, leading to premature cell death. Notably, HK2 exhibited defective intracellular survival in bone marrow-derived macrophages. This inability to dampen endothelial cell-mediated immune stimulation and resist macrophage-induced bactericidal activities resulted in the rapid elimination of viable Rickettsia in the mouse model of spotted fever. Further, when tested as a live-attenuated vaccine, HK2 elicited robust protective immunity against lethal spotted fever pathogenesis. Our work highlights the crucial role of pso in enabling Rickettsia to evade immune surveillance during intracellular replication within endothelial cells, ultimately delaying pathogen-induced programmed cell death and escaping immune defense mechanisms.

Rickettsia exhibits an obligate intracellular lifecycle, circulating between mammalian hosts and arthropod vectors. In human patients, pathogenic Rickettsia species target vascular endothelial cells and cause systemic vasculitis, leading to lethal outcomes without prompt antibiotic treatment. Strikingly, despite the ongoing reductive genome evolution, pathogenic Rickettsia species have evolved to evade endothelial immune detection mechanisms and exploit macrophage immune responses for their survival. Unfortunately, the factors enabling rickettsial intracellular survival are incompletely understood. Thus, additional insights into the underlying molecular mechanisms will assist in developing vaccines and therapeutics for rickettsioses. We carried out a series of tissue culture and animal infection studies in which we compared the virulence of Rickettsia conorii transposon insertional mutants expressing antigenically distinct O-Ag, a critical constituent of LPS. Our experimental results suggest that rickettsial O-Ag plays an important role during the intracellular lifecycle in endothelial cells and macrophages, contributing to immune evasion and systemic vasculitis. Further, we report that, without O-Ag, R. conorii exhibited significantly reduced virulence with excellent potency to elicit protective immunity against spotted fever pathogenesis. Our studies provide a strong foundation for immunological studies defining rickettsial innate immune evasion strategies and host immunity against rickettsioses.