Pseudomonas aeruginosa
is a Gram-negative bacterium which can cause serious infections among immune-depressed people including cystic fibrosis patients where it can colonize the lungs causing chronic ...infections. Iron is essential for
P. aeruginosa
and can be provided via three sources under aerobic conditions: its own siderophores pyochelin (PCH) and pyoverdine (PVD), xenosiderophores, or heme, respectively. Pyoverdine is the high affinity siderophore and its synthesis and uptake involve more than 30 genes organized in different operons. Its synthesis and uptake are triggered by iron scarcity via the Fur regulator and involves two extra cytoplasmic sigma factors (ECF), PvdS for the biosynthesis of PVD and FpvI for the uptake via the TonB-dependent FpvA outer membrane transporter and other periplasmic and inner membrane proteins. It appeared recently that the regulation of PVD biosynthesis and uptake involves other regulators, including other ECF factors, and LysR regulators. This is the case especially for the genes coding for periplasmic and inner membrane proteins involved in the reduction of Fe
3+
to Fe
2+
and the transport of ferrous iron to the cytoplasm that appears to represent a crucial step in the uptake process.
is an opportunistic pathogen, which causes chronic infections, especially in cystic fibrosis (CF) patients where it colonizes the lungs via the build-up of biofilms. Tobramycin, an aminoglycoside, is ...often used to treat
infections in CF patients. Tobramycin at sub-minimal inhibitory concentrations enhances both biofilm biomass and thickness
; however, the mechanism(s) involved are still unknown. Herein, we show that tobramycin increases the expression and activity of SigX, an extracytoplasmic sigma factor known to be involved in the biosynthesis of membrane lipids and membrane fluidity homeostasis. The biofilm enhancement by tobramycin is not observed in a
mutant, and the
mutant displays increased membrane stiffness. Remarkably, the addition of polysorbate 80 increases membrane fluidity of
-mutant cells in biofilm, restoring the tobramycin-enhanced biofilm formation. Our results suggest the involvement of membrane fluidity homeostasis in biofilm development upon tobramycin exposure.IMPORTANCEPrevious studies have shown that sub-lethal concentrations of tobramycin led to an increase biofilm formation in the case of infections with the opportunistic pathogen
. We show that the mechanism involved in this phenotype relies on the cell envelope stress response, triggered by the extracytoplasmic sigma factor SigX. This phenotype was abolished in a
-mutant strain. Remarkably, we show that increasing the membrane fluidity of the mutant strain is sufficient to restore the effect of tobramycin. Altogether, our data suggest the involvement of membrane fluidity homeostasis in biofilm development upon tobramycin exposure.
Chronic hepatitis B virus (HBV) infection is a leading cause of hepatocellular carcinoma (HCC), but its role in the transformation process remains unclear. HBV encodes a small protein, known as HBx, ...which is required for infection and has been implicated in hepatocarcinogenesis. Here we show that HBx induces lagging chromosomes during mitosis, which in turn leads to formation of aberrant mitotic spindles and multinucleated cells. These effects require the binding of HBx to UV‐damaged DNA binding protein 1 (DDB1), a protein involved in DNA repair and cell cycle regulation, and are unexpectedly attributable to HBx interfering with S‐phase progression and not directly with mitotic events. HBx also affects S‐phase and induces lagging chromosomes when expressed from its natural viral context and, consequently, exhibits deleterious activities in dividing, but not quiescent, hepatoma cells. Conclusion: In addition to its reported role in promoting HBV replication, the binding of HBx to DDB1 may induce genetic instability in regenerating hepatocytes and thereby contribute to HCC development, thus making this HBV–host protein interaction an attractive target for new therapeutic intervention. (HEPATOLOGY 2008.)
Physiological signals have shown to be reliable indicators of stress in laboratory studies, yet large-scale ambulatory validation is lacking. We present a large-scale cross-sectional study for ...ambulatory stress detection, consisting of 1002 subjects, containing subjects' demographics, baseline psychological information, and five consecutive days of free-living physiological and contextual measurements, collected through wearable devices and smartphones. This dataset represents a healthy population, showing associations between wearable physiological signals and self-reported daily-life stress. Using a data-driven approach, we identified digital phenotypes characterized by self-reported poor health indicators and high depression, anxiety and stress scores that are associated with blunted physiological responses to stress. These results emphasize the need for large-scale collections of multi-sensor data, to build personalized stress models for precision medicine.
SigX, one of the 19 extra-cytoplasmic function sigma factors of P. aeruginosa, was only known to be involved in transcription of the gene encoding the major outer membrane protein OprF. We conducted ...a comparative transcriptomic study between the wildtype H103 strain and its sigX mutant PAOSX, which revealed a total of 307 differentially expressed genes that differed by more than 2 fold. Most dysregulated genes belonged to six functional classes, including the "chaperones and heat shock proteins", "antibiotic resistance and susceptibility", "energy metabolism", "protein secretion/export apparatus", and "secreted factors", and "motility and attachment" classes. In this latter class, the large majority of the affected genes were down-regulated in the sigX mutant. In agreement with the array data, the sigX mutant was shown to demonstrate substantially reduced motility, attachment to biotic and abiotic surfaces, and biofilm formation. In addition, virulence towards the nematode Caenorhabditis elegans was reduced in the sigX mutant, suggesting that SigX is involved in virulence-related phenotypes.
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The discovery that the bacterial defense mechanism, CRISPR-Cas9, can be reprogrammed as a gene editing tool has revolutionized the field of gene editing. CRISPR-Cas9 can introduce a ...double-strand break at a specific targeted site within the genome. Subsequent intracellular repair mechanisms repair the double strand break that can either lead to gene knock-out (via the non-homologous end-joining pathway) or specific gene correction in the presence of a DNA template via homology-directed repair. With the latter, pathological mutations can be cut out and repaired. Advances are being made to utilize CRISPR-Cas9 in patients by incorporating its components into non-viral delivery vehicles that will protect them from premature degradation and deliver them to the targeted tissues. Herein, CRISPR-Cas9 can be delivered in the form of three different cargos: plasmid DNA, RNA or a ribonucleoprotein complex (RNP). We and others have recently shown that Cas9 RNP can be efficiently formulated in lipid-nanoparticles (LNP) leading to functional delivery in vitro. In this study, we compared LNP encapsulating the mRNA Cas9, sgRNA and HDR template against LNP containing Cas9-RNP and HDR template. Former showed smaller particle sizes, better protection against degrading enzymes and higher gene editing efficiencies on both reporter HEK293T cells and HEPA 1–6 cells in in vitro assays. Both formulations were additionally tested in female Ai9 mice on biodistribution and gene editing efficiency after systemic administration. LNP delivering mRNA Cas9 were retained mainly in the liver, with LNP delivering Cas9-RNPs additionally found in the spleen and lungs. Finally, gene editing in mice could only be concluded for LNP delivering mRNA Cas9 and sgRNA. These LNPs resulted in 60 % gene knock-out in hepatocytes. Delivery of mRNA Cas9 as cargo format was thereby concluded to surpass Cas9-RNP for application of CRISPR-Cas9 for gene editing in vitro and in vivo.
Previous studies indicated that female sex might be a modifier in Stargardt disease, which is an ABCA4-associated retinopathy.
To investigate whether women are overrepresented among individuals with ...ABCA4-associated retinopathy who are carrying at least 1 mild allele or carrying nonmild alleles.
Literature data, data from 2 European centers, and a new study. Data from a Radboudumc database and from the Rotterdam Eye Hospital were used for exploratory hypothesis testing.
Studies investigating the sex ratio in individuals with ABCA4-AR and data from centers that collected ABCA4 variant and sex data. The literature search was performed on February 1, 2023; data from the centers were from before 2023.
Random-effects meta-analyses were conducted to test whether the proportions of women among individuals with ABCA4-associated retinopathy with mild and nonmild variants differed from 0.5, including subgroup analyses for mild alleles. Sensitivity analyses were performed excluding data with possibly incomplete variant identification. χ2 Tests were conducted to compare the proportions of women in adult-onset autosomal non-ABCA4-associated retinopathy and adult-onset ABCA4-associated retinopathy and to investigate if women with suspected ABCA4-associated retinopathy are more likely to obtain a genetic diagnosis. Data analyses were performed from March to October 2023.
Proportion of women per ABCA4-associated retinopathy group. The exploratory testing included sex ratio comparisons for individuals with ABCA4-associated retinopathy vs those with other autosomal retinopathies and for individuals with ABCA4-associated retinopathy who underwent genetic testing vs those who did not.
Women were significantly overrepresented in the mild variant group (proportion, 0.59; 95% CI, 0.56-0.62; P < .001) but not in the nonmild variant group (proportion, 0.50; 95% CI, 0.46-0.54; P = .89). Sensitivity analyses confirmed these results. Subgroup analyses on mild variants showed differences in the proportions of women. Furthermore, in the Radboudumc database, the proportion of adult women among individuals with ABCA4-associated retinopathy (652/1154 = 0.56) was 0.10 (95% CI, 0.05-0.15) higher than among individuals with other retinopathies (280/602 = 0.47).
This meta-analysis supports the likelihood that sex is a modifier in developing ABCA4-associated retinopathy for individuals with a mild ABCA4 allele. This finding may be relevant for prognosis predictions and recurrence risks for individuals with ABCA4-associated retinopathy. Future studies should further investigate whether the overrepresentation of women is caused by differences in the disease mechanism, by differences in health care-seeking behavior, or by health care discrimination between women and men with ABCA4-AR.
At a human/livestock/wildlife interface, Escherichia coli populations were used to assess the risk of bacterial and antibiotic resistance dissemination between hosts. We used phenotypic and genotypic ...characterization techniques to describe the structure and the level of antibiotic resistance of E. coli commensal populations and the resistant Enterobacteriaceae carriage of sympatric African buffalo (Syncerus caffer caffer) and cattle populations characterized by their contact patterns in the southern part of Hwange ecosystem in Zimbabwe. Our results (i) confirmed our assumption that buffalo and cattle share similar phylogroup profiles, dominated by B1 (44.5%) and E (29.0%) phylogroups, with some variability in A phylogroup presence (from 1.9 to 12%); (ii) identified a significant gradient of antibiotic resistance from isolated buffalo to buffalo in contact with cattle and cattle populations expressed as the Murray score among Enterobacteriaceae (0.146, 0.258, and 0.340, respectively) and as the presence of tetracycline-, trimethoprim-, and amoxicillin-resistant subdominant E. coli strains (0, 5.7, and 38%, respectively); (iii) evidenced the dissemination of tetracycline, trimethoprim, and amoxicillin resistance genes (tet, dfrA, and blaTEM-1) in 26 isolated subdominant E. coli strains between nearby buffalo and cattle populations, that led us (iv) to hypothesize the role of the human/animal interface in the dissemination of genetic material from human to cattle and toward wildlife. The study of antibiotic resistance dissemination in multihost systems and at anthropized/natural interface is necessary to better understand and mitigate its multiple threats. These results also contribute to attempts aiming at using E. coli as a tool for the identification of pathogen transmission pathway in multihost systems.
Pf4 is a filamentous bacteriophage integrated as a prophage into the genome of Pseudomonas aeruginosa PAO1. Pf4 virions can be produced without killing P. aeruginosa. However, cell lysis can occur ...during superinfection when Pf virions successfully infect a host lysogenized by a Pf superinfective variant. We have previously shown that infection of P. aeruginosa PAO1 with a superinfective Pf4 variant abolished twitching motility and altered biofilm architecture. More precisely, most of the cells embedded into the biofilm were showing a filamentous morphology, suggesting the activation of the cell envelope stress response involving both AlgU and SigX extracytoplasmic function sigma factors. Here, we show that Pf4 variant infection results in a drastic dysregulation of 3,360 genes representing about 58% of P. aeruginosa genome; of these, 70% of the virulence factors encoding genes show a dysregulation. Accordingly, Pf4 variant infection (termed Pf4*) causes
reduction of P. aeruginosa virulence and decreased production of
-acyl-homoserine lactones and 2-alkyl-4-quinolones quorum-sensing molecules and related virulence factors, such as pyocyanin, elastase, and pyoverdine. In addition, the expression of genes involved in metabolism, including energy generation and iron homeostasis, was affected, suggesting further relationships between virulence and central metabolism. Altogether, these data show that Pf4 phage variant infection results in complex network dysregulation, leading to reducing acute virulence in P. aeruginosa. This study contributes to the comprehension of the bacterial response to filamentous phage infection.
Filamentous bacteriophages can become superinfective and infect P. aeruginosa, even though they are inserted in the genome as lysogens. Despite this productive infection, growth of the host is only mildly affected, allowing the study of the interaction between the phage and the host, which is not possible in the case of lytic phages killing rapidly their host. Here, we demonstrate by transcriptome and phenotypic analysis that the infection by a superinfective filamentous phage variant causes a massive disruption in gene expression, including those coding for virulence factors and metabolic pathways.
Abstract
Purpose:
Anal cancer is increasing in HIV+ men who have sex with men (MSM). Treatment options for its precursor, high-grade anal intraepithelial neoplasia (HGAIN), are suboptimal. In this ...phase I to II dose-finding study, we assessed the safety and efficacy of the human papillomavirus type 16 (HPV16) synthetic long peptide vaccine (SLP-HPV-01) in HIV+ MSM with HPV16-positive HGAIN.
Patients and Methods:
Four dosage schedules (1-5-10; 5-10-20; 10-20-40; and 40-40-40-40 μg) of SLP-HPV-01 were administered intradermally with a 3-week interval in 10 patients per dose level (DL). In each dose group, 5 patients also received 1 μg/kg pegylated IFNα-2b subcutaneously. Primary endpoints were safety and regression of HGAIN at 3, 6, and 12 months.
Results:
Eighty-one of 134 screened patients (60%) had HPV16-negative HGAIN lesions, leaving 53 eligible patients. Thirteen patients were excluded, leaving 40 men. The vaccine was well tolerated. One patient developed a generalized rash. The highest dosage level induced the strongest immune responses. There was no indication for stronger reactivity in the IFNα groups. Up to 18 months of follow-up, 8/38 intention-to-treat patients had a complete clinical and histologic response and one had a partial response (in total 9/38, 23.7%). At the highest dosage level, the clinical response was 4/10 (40%). Stronger immune responses were detected among clinical responders.
Conclusions:
The highest DL is safe, immunogenic, and associated with clinical responses to HPV16-induced lesions. However, as the majority of HGAIN is caused by the other HPV types, further studies should aim at pan-HPV vaccination to prevent or treat HGAIN.
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