| 1. |
|
|
| 2. |
- Klionsky, Daniel J., et al.
(författare)
-
Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
-
Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
-
Forskningsöversikt (refereegranskat)abstract
- In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
|
|
| 3. |
- Loh, Edmund, 1981-, et al.
(författare)
-
A trans-acting riboswitch controls expression of the virulence regulator PrfA in Listeria monocytogenes
- 2009
-
Ingår i: Cell. - : Elsevier BV. - 0092-8674 .- 1097-4172. ; 139:4, s. 770-779
-
Tidskriftsartikel (refereegranskat)abstract
- Riboswitches are RNA elements acting in cis, controlling expression of their downstream genes through a metabolite-induced alteration of their secondary structure. Here, we demonstrate that two S-adenosylmethionine (SAM) riboswitches, SreA and SreB, can also function in trans and act as noncoding RNAs in Listeria monocytogenes. SreA and SreB control expression of the virulence regulator PrfA by binding to the 5´-untranslated region of its mRNA. Absence of the SAM riboswitches SreA and SreB increases the level of PrfA and virulence gene expression in L. monocytogenes. Thus, the impact of the SAM riboswitches on PrfA expression highlights a link between bacterial virulence and nutrient availability. Together, our results uncover an unexpected role for riboswitches and a distinct class of regulatory noncoding RNAs in bacteria.
|
|
| 4. |
- Mandin, Pierre, et al.
(författare)
-
VirR, a response regulator critical for Listeria monocytogenes virulence.
- 2005
-
Ingår i: Mol Microbiol. - 0950-382X. ; 57:5, s. 1367-80
-
Tidskriftsartikel (refereegranskat)abstract
- Signature-tagged mutagenesis (STM) was used to identify new genes involved in the virulence of the Gram-positive intracellular pathogen Listeria monocytogenes. One of the mutants isolated by this technique had the transposon inserted in virR, a gene encoding a putative response regulator of a two-component system. Deletion of virR severely decreased virulence in mice as well as invasion in cell-culture experiments. Using a transcriptomic approach, we identified 12 genes regulated by VirR, including the dlt-operon, previously reported to be important for L. monocytogenes virulence. However, a strain lacking dltA, was not as impaired in virulence as the DeltavirR strain, suggesting a role in virulence for other members of the vir regulon. Another VirR-regulated gene is homologous to mprF, which encodes a protein that modifies membrane phosphatidyl glycerol with l-lysine and that is involved in resistance to human defensins in Staphylococcus aureus. VirR thus appears to control virulence by a global regulation of surface components modifications. These modifications may affect interactions with host cells, including components of the innate immune system. Surprisingly, although controlling the same set of genes as VirR, the putative cognate histidine kinase of VirR, VirS, encoded by a gene located three genes downstream of virR, was shown not to be essential for virulence. By monitoring the activity of VirR with a GFP reporter construct, we showed that VirR can be activated independently of VirS, for example through a mechanism involving variations in the level of intracellular acetyl phosphate. In silico analysis of the VirR-regulated promoters revealed a VirR DNA-binding consensus site and specific interaction between purified VirR protein and this consensus sequence was demonstrated by gel mobility shift assays. This study identifies a second key virulence regulon in L. monocytogenes, after the prfA regulon.
|
|
| 5. |
- Quereda, Juan J., et al.
(författare)
-
Role in virulence of phospholipases, listeriolysin O and listeriolysin S from epidemic Listeria monocytogenes using the chicken embryo infection model
- 2018
-
Ingår i: Veterinary research (Print). - : BioMed Central. - 0928-4249 .- 1297-9716. ; 49
-
Tidskriftsartikel (refereegranskat)abstract
- Most human listeriosis outbreaks are caused by Listeria monocytogenes evolutionary lineage I strains which possess four exotoxins: a phosphatidylinositol-specific phospholipase C (PlcA), a broad-range phospholipase C (PlcB), listeriolysin O (LLO) and listeriolysin S (LLS). The simultaneous contribution of these molecules to virulence has never been explored. Here, the importance of these four exotoxins of an epidemic lineage I L. monocytogenes strain (F2365) in virulence was assessed in chicken embryos infected in the allantoic cavity. We show that LLS does not play a role in virulence while LLO is required to infect and kill chicken embryos both in wild type transcriptional regulator of virulence PrfA -(PrfAWT) and constitutively active PrfA (PrfA*) backgrounds. We demonstrate that PlcA, a toxin previously considered as a minor virulence factor, played a major role in virulence in a PrfA* background. Interestingly, GFP transcriptional fusions show that the plcA promoter is less active than the hly promoter in vitro, explaining why the contribution of PlcA to virulence could be observed more importantly in a PrfA* background. Together, our results suggest that PlcA might play a more important role in the infectious lifecycle of L. monocytogenes than previously thought, explaining why all the strains of L. monocytogenes have conserved an intact copy of plcA in their genomes.
|
|
| 6. |
- Toledo-Arana, Alejandro, et al.
(författare)
-
The Listeria transcriptional landscape from saprophytism to virulence
- 2009
-
Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 459:7249, s. 950-956
-
Tidskriftsartikel (refereegranskat)abstract
- The bacterium Listeria monocytogenes is ubiquitous in the environment and can lead to severe food-borne infections. It has recently emerged as a multifaceted model in pathogenesis. However, how this bacterium switches from a saprophyte to a pathogen is largely unknown. Here, using tiling arrays and RNAs from wild-type and mutant bacteria grown in vitro, ex vivo and in vivo, we have analysed the transcription of its entire genome. We provide the complete Listeria operon map and have uncovered far more diverse types of RNAs than expected: in addition to 50 small RNAs (<500 nucleotides), at least two of which are involved in virulence in mice, we have identified antisense RNAs covering several open-reading frames and long overlapping 5' and 3' untranslated regions. We discovered that riboswitches can act as terminators for upstream genes. When Listeria reaches the host intestinal lumen, an extensive transcriptional reshaping occurs with a SigB-mediated activation of virulence genes. In contrast, in the blood, PrfA controls transcription of virulence genes. Remarkably, several non-coding RNAs absent in the non-pathogenic species Listeria innocua exhibit the same expression patterns as the virulence genes. Together, our data unravel successive and coordinated global transcriptional changes during infection and point to previously unknown regulatory mechanisms in bacteria.
|
|
