7th joint conference "Joint Microbiology & Infection Conference 2024" of the DGHM and VAAM

Prof. Dr.

Chair

Regulation of the CTXϕ phage life cycle by a quorum-sensing controlled small RNA in Vibrio cholerae

RNA biology

Vibrio cholerae is a major human pathogen that causes the diarrheal disease cholera. A crucial aspect in the pathogenesis of the disease is cell-cell communication, a.k.a quorum sensing (QS), which coordinates community behaviours such as biofilm formation and toxin production. The cholera toxin is encoded by the filamentous bacteriophage CTXϕ which is irreversibly integrated into the host chromosome. Lysogenic induction of CTXϕ is well studied at the transcriptional level and depends on the host-encoded SOS response regulator LexA and the phage-encoded repressor RstR. However, post-transcriptional regulation of CTXϕ has not been studied and identifying potential post-transcriptional regulators of CTXϕ is challenging as it is only poorly expressed under standard laboratory conditions. To address this question, we performed transcriptomic analysis of V. cholerae under virulence-inducing conditions, resulting in the identification of dozens of differentially expressed sRNAs, including the well-characterized TarB and CarZ sRNAs, as well as several uncharacterized sRNAs. To investigate the global RNA-RNA network under virulence conditions, we used RIL-seq (RNA-interaction-by-ligation-and-sequencing). Our analysis captured 1,395 RNA-RNA chimeras interacting with Hfq, including a novel virulence-related sRNA that we named CisR (CTX inhibiting small RNA). We discovered that CisR base-pairs with and inhibits the expression of the cep mRNA, encoding a core structural element of the CTXϕ phage, and that the CisR sRNA is processed by RNAse E from the 3&quot;UTR of the prtV mRNA, which encodes an M6 metalloprotease that is relevant for invasion of the mammalian host. Further, we found that two global regulators activate cisR transcription: the quorum-sensing regulator HapR in a cell density dependent manner, and the cyclic AMP receptor protein (CRP) in response to carbon starvation. Taken together, our data indicate that the core-genome encoded CisR sRNA controls the production of the horizontally acquired virulence gene that has a crucial role in the life cycle of the CTXϕ phage.

Oral Presentation

RIL-seq analysis identifies the sRNA CrfA as an RNA sponge rewiring carbon starvation in Caulobacter

Caulobacter crescentus is an environmental bacterium living in nutrient-poor habitats. Adaptation of Caulobacter to changing physiological conditions has been extensively studied at the transcriptional level, but little is known about post-transcriptional control. In bacteria, small regulatory RNAs (sRNAs) contribute significantly to this layer of regulation by base-pairing mRNA targets, often with the help of RNA-binding proteins (RBPs), of which Hfq is the only one known to interact with sRNAs in Caulobacter.To study the global Hfq-dependent RNA interactome in this species, we performed RIL-seq (RNA interaction by ligation and sequencing). Among the more than 1,000 new RNA-RNA interactions recovered in our analysis, we detected an abundant RNA pair involving the two sRNAs CrfA and R0014. CrfA has been previously identified in the Caulobacter response to carbon starvation, however, we now show that instead of acting directly on mRNA targets, CrfA functions as an RNA sponge, sequestering and inactivating the sRNA R0014. In turn, R0014 is a classical sRNA that base-pairs and represses its various target mRNAs encoding outer membrane receptors and components of the central metabolism.Our study uncovers CrfA as an important non-coding regulator involved in rewiring the transcriptome of Caulobacter under low nutrient conditions, and to prime the bacterium for future outgrowth.

Aspergillus fumigatus-infected neutrophils release extracellular RNAs bearing the capacity for host-pathogen cross-kingdom communication

Fungal infections were spotlighted by the WHO in 2022 as being an important medical challenge of the 21st century, causing up to 3.75 million deaths among 1 billion infections annually and case-fatality rates of up to 90% in some populations. Immunocompromised individuals are especially susceptible to fungal infections, which are caused by opportunistic pathogens like Aspergillus fumigatus. Due to shortcomings in treatment and early diagnosis as well as an increased emergence of antifungal resistance, new diagnostic and therapeutic strategies are essential. RNA therapeutics are a potentially beneficial approach that might emerge from the expanding field of extracellular RNAs (exRNA), where a variety of RNA species were demonstrated to mediate cross-kingdom communication in fungal infection contexts of plants, insects, and mammalian immune cells. These exRNAs can be released in association with RNA binding proteins (RBPs) or extracellular vesicles (EVs), of which the latter derived from A. fumigatus-infected polymorphonuclear leukocytes (PMNs), were shown to bear antifungal capacity. To uncover a possible involvement of exRNAs in antifungal defense, we investigated RNA isolates from large and small EV fractions alongside small RNA-sequencing. Utilizing capillary electrophoresis, the extracellular environment was revealed to be enriched for 20-200 nt small RNAs (sRNA), which according to sRNA-sequencing displayed high abundances of miRNAs. Surprisingly, these miRNAs were enriched for specific, conserved candidates with known antimicrobial properties and potentially play a gene modulatory role in the fungus in cooperation with fungal or human RNA interference (RNAi) effectors. Subsequent investigations focused on the characterization of the identified miRNAs, involvement of key RNAi components, and definition of their role in A. fumigatus defense. We hope that these findings will contribute to the foundation of the exRNAs as a mediator of host-pathogen interactions and benefit development of new RNA-based diagnostics and therapeutics against this important human pathogen in the future.

Raum 13

RNA Biology

Oral Session

Oral Sessions

Oral Session
RNA Biology

Appointment

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Topic

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Oral SessionRNA Biology

Appointment

Chair

Topic

Programme

Oral Session
RNA Biology