title: The Role of Bacterial RNA as a Pathogen-Associated Molecular Pattern in Orientia tsutsugamushi Infection
title: Die Rolle Bakterieller RNA als Pathogen-Assoziiertes Molekulares Muster in Orientia tsutsugamushi-Infektionen
titleType: AlternativeTitle
Creators: name: Mehl, Jonas
Publisher: name: Philipps-Universität Marburg
Year: 2024
Subjects: vita-PAMPs
Toll-like receptors
innate immunity
scrub typhus
Orientia tsutsugamushi
Natural sciences &amp; mathematics
Naturwissenschaften
RNA recognition

description: Orientia tsutsugamushi (Ot) is a Gram-negative, obligate intracellular bacterium, annually infecting one million people with Scrub typhus. This vector-borne neglected disease is seasonally transmitted by larval mites in the Asia Pacific region. The BSL3 pathogen Ot induces high levels of pro-inflammatory cytokines, as shown in vitro as well as in murine models in vivo and in severe cases of Scrub typhus in humans. To date, it is unknown whether this inflammatory response is beneficial and protective, or rather represents an overshooting activation of innate host immunity. Of note, the pathogen-associated molecular patterns (PAMPs) inducing this inflammation, as well as the corresponding host receptors, have remained elusive. Earlier studies concluded the unknown PAMP must be a “heat-stable compound”, as both live and heat-killed (hk) Ot induced cytokines in vitro. 

Preliminary data had implied that transfected Ot RNA induced cytokine production in macrophages (MΦ), and that the murine Toll-like Receptors (TLRs) 7 and 13 might be differentially involved in sensing of live and dead (hk) Ot. Thus, the aims of this project were to elucidate if Ot RNA could be the heat-stable PAMP described in earlier studies, to investigate the possible role of bacterial RNA in live/dead-discrimination, and to identify the host receptors and RNA ligands involved in innate recognition of Ot.
 
To characterize the role of TLR13 in sensing of bacterial RNA ligands in a suitable cellular system, the receptor was knocked down in immortalized murine MΦ using CRISPR interference. Additionally, bone marrow-derived dendritic cells (BMDC) were generated from wildtype (wt) or TLR7-knockout mice to study the role of TLR7. In order to investigate the heat susceptibility of Ot immune stimulation and recognition via these RNA-specific receptors, cells were challenged with Ot exposed to temperatures from 37 to 70 °C. While live Ot induced cytokines via TLR7 in BMDC, Ot inactivated at medium temperatures, e.g. 56 °C, lost immunostimulatory capacity. These 56 °C hk Ot contained only residual, strongly degraded RNA, as determined by fluorometric quantification and Bioanalyzer electrophoresis. In contrast to these deadRNA- Ot, the RNA was conserved and only partially fragmented in bacteria heat-killed at higher temperatures, e.g. 70 °C. These deadRNA+ Ot strongly induced cytokines via TLR13 in MΦ. 

Next-generation RNA-sequencing revealed further differences in RNA composition between live and deadRNA+ Ot – both on the RNA species and on the single transcript level. Despite that, transfected whole RNA from both live and deadRNA+ Ot was sensed via TLR13. In in vitro infections however, TLR13 was only activated by deadRNA+ Ot but not by live Ot, suggesting that the TLR13 ligand is shielded in live Ot. Narrowing down possible TLR13 RNA ligands, transfection of size-fractionated Ot RNA revealed that TLR13 senses an Ot RNA &gt;200 nucleotides. The Ot 23S ribosomal RNA (rRNA) contains a known TLR13-recognition sequence, making it a likely TLR13-agonist. Thus, it was cloned, in vitro transcribed and upon transfection induced cytokines only in wt but not in TLR13 knockdown MΦ, identifying the Ot 23S rRNA as a potent TLR13 ligand. While transfected RNA from live Ot was not sensed via TLR7 in BMDC, live Ot infection induced cytokines via this receptor through a yet unknown RNA ligand. Additionally, different phagocytosis pathways of live and dead Ot might contribute to differential receptor activation, as cytokine induction by deadRNA+ Ot was more susceptible to macropinocytosis inhibitors than the cytokine response to live Ot. 

The human RNA-sensing TLR8 was discussed to be the functional equivalent of the murine TLR13, hence its role in Ot recognition was addressed next. Reporter cells overexpressing the human TLR8 sensed both live and deadRNA+ Ot. Additionally, cytokine induction by live or deadRNA+ Ot was abrogated upon treatment with a TLR8-specific inhibitor in human primary peripheral blood mononuclear cells. Thus, the human TLR8 combined the functions of its murine counterparts TLR7 and 13. 

In summary, this work unraveled RNA-dependent signaling pathways through which Ot activates innate immune responses. This study identifies RNA as the heat-stable Ot PAMP described in earlier reports. However, live and dead Ot were sensed by different RNA-sensing TLRs in mice (but not humans), potentially due to different RNA content, RNA availability or phagocytosis pathways. This argues for the involvement of different RNA ligands, rather than one heat-stable RNA that is sensed in both live and dead Ot.
As shown in the example of Ot, bacterial heat-killing at different temperatures lead to RNA degradation of a varying degree, which modified the capacity of the bacterium to activate the innate immunity via RNA-receptors. In contrast to the vitaPAMP-hypothesis, claiming that only live bacteria induce immune responses via RNA, this study showed that dead bacteria can also be sensed via RNA-sensing receptors - however only if they contain RNA of sufficient integrity and concentration.