Dual pathogenicity island transfer by piggybacking lateral transduction

Lateral transduction (LT) is the process by which temperate phages mobilize large sections of bacterial genomes. Despite its importance, LT has only been observed during prophage induction. Here, we report that superantigen-carrying staphylococcal pathogenicity islands (SaPIs) employ a related but more versatile and complex mechanism of gene transfer to drive chromosomal hypermobility while self-transferring with additional virulence genes from the host. We found that after phage infection or prophage induction, activated SaPIs form concatamers in the bacterial chromosome by switching between parallel genomic tracks in replication bubbles. This dynamic life cycle enables SaPIbov1 to piggyback its LT of staphylococcal pathogenicity island vSaα, which encodes an array of genes involved in host-pathogen interactions, allowing both islands to be mobilized intact and transferred in a single infective particle. Our findings highlight previously unknown roles of pathogenicity islands in bacterial virulence and show that their evolutionary impact extends beyond the genes they carry

Genome hypermobility by lateral transduction

Genetic transduction is a major evolutionary force that underlies bacterial adaptation. Here we report that the temperate bacteriophages of Staphylococcus aureus engage in a distinct form of transduction we term lateral transduction. Staphylococcal prophages do not follow the previously described excision-replication-packaging pathway but instead excise late in their lytic program. Here, DNA packaging initiates in situ from integrated prophages, and large metameric spans including several hundred kilobases of the S. aureus genome are packaged in phage heads at very high frequency. In situ replication before DNA packaging creates multiple prophage genomes so that lateral-transducing particles form during normal phage maturation, transforming parts of the S. aureus chromosome into hypermobile regions of gene transfer

Lipid metabolic perturbation is an early-onset phenotype in adult spinster mutants: a Drosophila model for lysosomal storage disorders

Intracellular accumulation of lipids and swollen dysfunctional lysosomes are linked to several neurodegenerative diseases, including lysosomal storage disorders (LSD). Detailed characterization of lipid metabolic changes in relation to the onset and progression of neurodegeneration is currently missing. We systematically analyzed lipid perturbations in spinster (spin) mutants, a Drosophila model of LSD-like neurodegeneration. Our results highlight an imbalance in brain ceramide and sphingosine in the early stages of neurodegeneration, preceding the accumulation of endomembranous structures, manifestation of altered behavior, and buildup of lipofuscin. Manipulating levels of ceramidase and altering these lipids in spin mutants allowed us to conclude that ceramide homeostasis is the driving force in disease progression and is integral to spin function in the adult nervous system. We identified 29 novel physical interaction partners of Spin and focused on the lipid carrier protein, Lipophorin (Lpp). A subset of Lpp and Spin colocalize in the brain and within organs specialized for lipid metabolism (fat bodies and oenocytes). Reduced Lpp protein was observed in spin mutant tissues. Finally, increased levels of lipid metabolites produced by oenocytes in spin mutants allude to a functional interaction between Spin and Lpp, underscoring the systemic nature of lipid perturbation in LSD

Phenomic analysis of chronic granulomatous disease reveals more severe integumentary infections in X-Linked compared with autosomal recessive chronic granulomatous disease

BACKGROUND : Chronic granulomatous disease (CGD) is an inborn error of immunity (IEI), characterised by recurrent bacterial and fungal infections. It is inherited either in an Xlinked (XL) or autosomal recessive (AR) mode. Phenome refers to the entire set of phenotypes expressed, and its study allows us to generate new knowledge of the disease. The objective of the study is to reveal the phenomic differences between XL and AR-CGD by using Human Phenotype Ontology (HPO) terms. METHODS : We collected data on 117 patients with genetically diagnosed CGD from Asia and Africa referred to the Asian Primary Immunodeficiency Network (APID network). Only 90 patients with sufficient clinical information were included for phenomic analysis. We used HPO terms to describe all phenotypes manifested in the patients. RESULTS : XL-CGD patients had a lower age of onset, referral, clinical diagnosis, and genetic diagnosis compared with AR-CGD patients. The integument and central nervous system were more frequently affected in XL-CGD patients. Regarding HPO terms, perianal abscess, cutaneous abscess, and elevated hepatic transaminase were correlated with XL-CGD. A higher percentage of XL-CGD patients presented with BCGitis/BCGosis as their first manifestation. Among our CGD patients, lung was the most frequently infected organ, with gastrointestinal system and skin ranking second and third, respectively. Aspergillus species, Mycobacterium bovis, and Mycobacteirum tuberculosis were the most frequent pathogens to be found. CONCLUSION : Phenomic analysis confirmed that XL-CGD patients have more recurrent and aggressive infections compared with AR-CGD patients. Various phenotypic differences listed out can be used as clinical handles to distinguish XL or AR-CGD based on clinical features.The Society for Relief of Disabled Children and Jeffrey Modell Foundation.https://www.frontiersin.org/journals/immunologydm2022Paediatrics and Child Healt

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

PATHOGENICITY ISLAND-MEDIATED PROPHAGE INDUCTION

Ph.DDOCTOR OF PHILOSOPHY (SOM

Genetic transduction by phages and chromosomal islands: the new and noncanonical

No abstract available