Obligate intracellular bacteria like Chlamydia trachomatis undergo a complex developmental cycle between infectious non-replicative (EBs) and non-infectious replicative (RBs) forms. EBs shortly after entering a host cell transform to RBs, a crucial process in infection, initiating chlamydial replication. As Chlamydia fail to replicate outside the host cell it is currently unknown how the replicative part of the developmental cycle is initiated. Here we show in a cell-free approach in axenic media that uptake of glutamine by the bacteria is crucial for peptidoglycan synthesis which has a role in Chlamydia replication. The increased requirement for glutamine in infected cells is satisfied by reprogramming the glutamine metabolism in a c-Myc-dependent manner. Glutamine is effectively taken up by the glutamine transporter SLC1A5 and metabolized via glutaminase. Interference with this metabolic reprogramming limited growth of Chlamydia. Intriguingly, Chlamydia failed to produce progeny in SLC1A5 knockout organoids and mice. Thus, we report on the central role of glutamine for the development of an obligate intracellular pathogenic bacterium and the reprogramming of host glutamine metabolism, which may provide a basis for innovative anti-infective strategies.
Transfer RNAs (tRNAs) are among the most heavily modified RNA species. Posttranscriptional tRNA modifications (ptRMs) play fundamental roles in modulating tRNA structure and function, and are being increasingly linked to human physiology and disease. Detection of ptRMs is often challenging, expensive and laborious. Restriction Fragment Length Polymorphism (RFLP) analyses study the patterns of DNA cleavage after restriction enzyme treatment; and have been used for the qualitative detection of modified bases on messenger RNAs. It is known that some ptRMs induce specific and reproducible base 'mutations' when tRNAs are reverse transcribed. For example, inosine, which derives from the deamination of adenosine, is detected as a guanosine when an inosine-containing tRNA is reverse transcribed, PCR-amplified and sequenced. ptRMdependent base changes on RT-PCR amplicons generated as a consequence of the reverse transcription reaction might create or abolish endonuclease restriction sites. The suitability of RFLP for the detection and/or quantification of ptRMs has not been studied thus far. Here we show that different ptRMs can be detected at specific sites of different tRNA types by RFLP. For the examples studied we show that this approach is able to reliably estimate the modification status of the sample, a feature that can be useful in the study of the regulatory role of tRNA modifications on gene expression.3
Inosine at the "wobble" position (I34) is one of the few essential posttranscriptional modifications in tRNAs (tRNAs). It results from the deamination of adenosine and occurs in bacteria on tRNA and in eukarya on six or seven additional tRNA substrates. Because inosine is structurally a guanosine analogue, reverse transcriptases recognize it as a guanosine. Most methods used to examine the presence of inosine rely on this phenomenon and detect the modified base as a change in the DNA sequence that results from the reverse transcription reaction. These methods, however, cannot always be applied to tRNAs because reverse transcription can be compromised by the presence of other posttranscriptional modifications. Here we present SL-ID (splinted ligation-based inosine detection), a reverse transcription-free method for detecting inosine based on an I34-dependent specific cleavage of tRNAs by endonuclease V, followed by a splinted ligation and polyacrylamide gel electrophoresis analysis. We show that the method can detect I34 on different tRNA substrates and can be applied to total RNA derived from different species, cell types, and tissues. Here we apply the method to solve previous controversies regarding the modification status of mammalian tRNA.
3 1 process in infection, initiating chlamydial replication. As Chlamydia fail to replicate 3 2 outside the host cell it is currently unknown how the transition from EBs to RBs is 3 3 initiated. Here we show in a cell-free approach in axenic media that uptake of glutamine 3 4 by the bacteria is critical to initiate EB-RB transition. These bacteria utilize glutamine to 3 5 synthesize cell wall peptidoglycan which has recently been detected in the septa of 3 6 replicating intracellular Chlamydia. The increased requirement for glutamine in infected 3 7 cells is achieved by reprogramming the glutamine metabolism in a c-Myc-dependent 3 8 manner. Glutamine was effectively taken up by the glutamine transporter SLC1A5 and 3 9 metabolized via glutaminase. Interference with this metabolic reprogramming limited 4 0 growth of Chlamydia. Intriguingly, Chlamydia failed to produce progeny in SLC1A5 4 1 knockout mice. Thus, we report on the central role of glutamine for the development of 4 2 an obligate intracellular pathogenic bacterium and the reprogramming of host glutamine 4 3
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.