An Efficient, Counter-Selection-Based Method for Prophage Curing in <em>Pseudomonas aeruginosa</em> Strains

Prophages are bacteriophages in the lysogenic state, where the viral genome is inserted within the bacterial chromosome. They contribute to strain genetic variability and can influence bacterial phenotypes. Prophages are highly abundant among the strains of the opportunistic pathogen Pseudomonas aeruginosa and were shown to confer specific traits that can promote strain pathogenicity. The main difficulty of studying those regions is the lack of a simple prophage-curing method for P. aeruginosa strains. In this study, we developed a novel, targeted-curing approach for prophages in P. aeruginosa. In the first step, we tagged the prophage for curing with an ampicillin resistance cassette (ampR) and further used this strain for the sacB counter-selection marker’s temporal insertion into the prophage region. The sucrose counter-selection resulted in different variants when the prophage-cured mutant is the sole variant that lost the ampR cassette. Next, we validated the targeted-curing with local PCR amplification and Whole Genome Sequencing. The application of the strategy resulted in high efficiency both for curing the Pf4 prophage of the laboratory wild-type (WT) strain PAO1 and for PR2 prophage from the clinical, hard to genetically manipulate, 39016 strain. We believe this method can support the research and growing interest in prophage biology in P. aeruginosa as well as additional Gram-negative bacteria

DNA fluorescent <i>in situ</i> hybridization confirms the presence of VZV genomes in nuclei of hESC-derived neurons quiescently infected with VZV.

<p>Neurons were infected with VZV-ORF66-GFP and <i>in situ</i> hybridization performed on isolated nuclei as described in the methods. (A) shows FISH of nuclei from productively and (B) from quiescently infected neurons. Almost all quiescently, infected FISH+ neurons contained only one puncta in their nuclei. (C) FISH for VZV genomes in quiescently infected neurons receiving the PI3K inhibitor LY as a reactivation stimulus. After LY treatment there was a slight decrease in the percentage of FISH+ nuclei in the preparations, but 25% of labeled nuclei contained 2 or more puncta, suggesting additional sites of VZV genomes had appeared. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004885#ppat.1004885.t001" target="_blank">Table 1</a> for quantification of the puncta. Scale Bar = 5 μm.</p

Reactivation of quiescent VZV in hESC-derived neurons induced by growth factor withdrawal.

<p>(A&C) hESC-derived neurons were incubated with low MOI VZV in the presence of ACV. Two weeks after exposure to virus and one week after removal of ACV, no GFP expression was detected in 50% of neuron-containing wells. At this time point, growth factors (GF) were withdrawn from the medium in wells that were GFP negative. By 4 days after GF withdrawal, massive loss of neurites was observed (B), eventually resulting in death of the cells in the wells by day 5 after treatment. However 30% of the initially GFP negative wells receiving GF-withdrawal treatment contained single and small foci of ORF66 protein-expressing neurons. (D). A and B are phase micrographs, C and D fluorescence micrographs of the same microscopic fields. Scale bar = 100μm.</p

RNA expression in productively and quiescently-infected hESC-derived neurons.

<p>A) The upper two histograms show the counts of reads from RNASeq analysis of quiescently-infected neurons and the lower two those of productively infected neurons aligned to the annotated vOKA genome. Note the difference in the Y-axis scale between the sets of histograms depicting productive and quiescently infected cultures. In the annotated genome at the bottom of the Fig, ORFs depicted in red (increased) and green (reduced) are those displaying statically significant differences in enrichment between quiescent and productively infected neurons. B) Fold-changes between the relative expression of transcripts of VZV ORFs between quiescent and productively infected neurons. The duplicated genes of the short repeats region of the genome are enriched in quiescently-infected neurons. ORFs for which significant differences were detected are denoted by asterisks.</p

Reactivation stimuli increase the number of VZV genomes and transcripts in quiescently-infected human neurons.

<p>Wells of quiescently infected neurons were induced to reactivate VZV using growth factor withdrawal (GF n = 2 for each time point) or treatment with PI3K inhibitor LY294002 (LY) 2, 4, or 7 weeks (n = 5 for each time point) after infection. DNA and RNA were extracted from the wells and VZV genomes (A) and transcripts (B) of ORF63 and ORF31 were quantified. Both treatments increased the levels of both viral genomes and transcripts to varying degrees at all time points tested, indicating at least a partial reactivation of VZV. In all experiments reactivating VZV with LY, changes in nucleic acid levels measured were statistically significant.</p

VZV DNA and transcripts are present in hESC-derived neurons productively or quiescently infected with VZV.

<p>Neurons were infected with high or low MOI VZV-ORF66-GFP in the presence of acyclovir, and ACV removed after 6 days incubation. (A) DNA and RNA were extracted from the GFP negative wells 2, 4 or 7 weeks after infection. Levels of VZV genomes and transcripts for ORF63 and ORF31 (gB) were quantified using Taqman probes and digital qPCR and normalized to GAPDH. (B) Transcripts levels detected from both ORFs in quiescently and productively-infected neurons, showing much higher levels in productively-infected neurons.</p

Reactivation of VZV in quiescently infected hESC-derived neurons by PI3K inhibition at 34°C results in a productive, spreading infection.

<p>(A-F) A microscopic field showing neurons expressing GFP after reactivation at 34°C for 2 (A,B), 6 (C,D) and 14 (E,F) days after initiation of treatment. GFP expression is first observed in individual neurons, and spreads over time in the same initial foci of expression. (G-H) Another experiment showing the results of LY-induced reactivation at 34°C. The area depicted by the box in G is presented at higher magnification in H, showing the diffuse filling of neurons with GFP at 34<b>°</b>C. (I-N) In another reactivation experiment at 34°C, a focus of GFP expression (I&L) initially spreads over 3 days (J&M), but then contracts over a period of 4 days (K&N). Scale bars = 100μm.</p

Percentage of neuronal nuclei containing fluorescent puncta following DNA FISH to detect VZV genomes.

<p>Percentage of neuronal nuclei containing fluorescent puncta following DNA FISH to detect VZV genomes.</p

Quiescent infection and reactivation of VZV in hESC-derived neurons without the use of ACV.

<p>hESC-derived neurons were infected via their axons in compartmentalized microfluidic chambers as detailed in the methods. A) Quantification of VZV genomes and transcripts from ORF63 and ORF31 were quantified by digital qPCR from nuclei acids extracted from the cell-body compartment two weeks after infection. B) & C) Reactivation of VZV in axonally-infected neurons. (B) Reactivation of VZV in hESC-derived neurons by LY 2 weeks after quiescent axonal infection. Neurons infected as in A were treated for 4 days with LY at 37°C and DNA and RNA extracted. qPCR revealed an increase in VZV genomes and transcripts from ORF62 and ORF31. No GFP-positive neurons were observed under these reactivation conditions. (C) Reactivation of neurons axonally-infected by VZV at 34°C. A photomicrograph of a microfluidic chamber where the cell body compartment (CB) containing the somata of axonally infected neurons was treated for 4 days with LY at 34 degrees is shown. A cluster of neurons (upper box) expressing ORF66GFP as a result of the treatment (higher magnification image in upper inset). The axons in the axonal compartment (Ax) are coated by the GFP-fluorescent debris (lower box, higher magnification image in the lower inset) used to infect the axons. Ch = microfluidic channels connecting the cell body and axonal compartments. Scale Bars = 100μm.</p

Neuronal IRE-1 coordinates an organism-wide cold stress response by regulating fat metabolism

Cold affects many aspects of biology, medicine, agriculture, and industry. Here, we identify a conserved endoplasmic reticulum (ER) stress response, distinct from the canonical unfolded protein response, that maintains lipid homeostasis during extreme cold. We establish that the ER stress sensor IRE-1 is critical for resistance to extreme cold and activated by cold temperature. Specifically, neuronal IRE-1 signals through JNK-1 and neuropeptide signaling to regulate lipid composition within the animal. This cold-response pathway can be bypassed by dietary supplementation with unsaturated fatty acids. Altogether, our findings define an ER-centric conserved organism-wide cold stress response, consisting of molecular neuronal sensors, effectors, and signaling moieties, which control adaptation to cold conditions in the organism. Better understanding of the molecular basis of this stress response is crucial for the optimal use of cold conditions on live organisms and manipulation of lipid saturation homeostasis, which is perturbed in human pathologies.Ministry of Education (MOE)Ministry of Health (MOH)National Medical Research Council (NMRC)Published versionThis work was supported by funds from the Israel Science Foundation (ISF grant no. 689/19 to S.H.K.), the traveling fellowship from The Company of Biologists (JCSTF18118 to R.D.), the Singapore Ministry of Education Academic Research Fund Tier 3 (MOE-MOET32020-0001 to G.T.), the Ministry of Health, Singapore, National Medical Research Council Open Fund Individual Research Grant (MOH-000566 to G.T.), and the S Research Scholarship to J.H.K. (predoctoral fellowship)