Biology subjects

Checa, A.

Publications and source records attributed to Checa, A..

4 recordsLinked to original sources

bioRxiv2025

Pseudolysogeny-mediated evolutionary trade-offs favor phage therapy by limiting antibiotic resistance and virulence in Cutibacterium acnes

Phage therapy has mostly focused on strictly lytic phages, yet the ecological and evolutionary implications of pseudolysogeny remain poorly understood. Pseudolysogeny--where a phage genome persists in a non-integrated, latent state within the host--has been largely overlooked due to concerns about therapeutic efficacy. Here, we demonstrate that pseudolysogeny in Cutibacterium acnes confers superinfection resistance but imposes substantial fitness costs, including reduced biofilm formation, impaired interspecies competitiveness, and reversal of antibiotic resistance. Pseudolysogenic phages were also capable of killing starved C. acnes cells through direct lytic replication. No evidence of transduction of clindamycin resistance by these phages was detected. In a 3-month proof-of-concept study, topical application of a pseudolysogenic phage significantly reduced C. acnes abundance, acne lesions, and inflammation, with no adverse effects and persistence of viable phages post-treatment. Importantly, no phage-resistant C. acnes clones were detected during the in vivo study, likely due to the evolutionary trade-offs associated with pseudolysogeny that diminish the bacterial ecological fitness. These findings highlight pseudolysogeny as a mechanism that can be leveraged to enhance phage therapy outcomes while maintaining microbiome stability and limiting antibiotic resistance evolution.

microbiology↗

bioRxiv2024

Characterization of HLA-DR immunopeptidome of bronchoalveolar lavage cells in patients with newly diagnosed rheumatoid arthritis and healthy current-smoker controls

Evidence suggests that self-tolerance is breached in the lung prior to the clinical onset of rheumatoid arthritis (RA) in the joints. The human leukocyte antigen DR (HLA-DR) shared epitope (SE) represents the strongest genetic risk factor for sero-positive RA. However, to our knowledge, the HLA-DR immunopeptidome of the RA lung and its link to HLA-DR genotype has not been investigated to date. The objective of this study was to optimize the methods for characterizing the HLA-DR immunopeptidome of lung immune cells and apply it to newly diagnosed RA patients versus current-smoker healthy controls, as well as to investigate the connection with the HLA-DR genotype. The HLA-DR immunopeptidome method was improved to facilitate characterization from as few as 6 million bronchoalveolar lavage (BAL) cells per subject, consisting primarily of alveolar macrophages. This method was applied to newly diagnosed RA patients naive to treatment (n=9, LURA cohort), as well as healthy current-smoker controls (n=10, COSMIC cohort). For five of the RA patients, a 6-month follow-up after initiation of the standard-of-care treatment regime was also included. After isolation and purification, peptide samples were separated by nano-flow liquid chromatography coupled to an Orbitrap mass spectrometer equipped with ion mobility device (FAIMS). Mass spectra acquired in data dependent acquisition mode were then searched against a human proteome database. Subsequently, the identified peptides were deconvoluted to their predicted binding HLA-DR allele using MHCMotifDecon based on the sequenced genotype of the individual. An optimized sample preparation and analytic method enabled the detection of over 23,000 peptides from over 3,000 source proteins with between 1,000 and 5,000 peptides identified per sample. Notably, the application of FAIMS with three compensation voltages allowed for efficient transfer of 2+, 3+, and 4+ peptide ions while removing singly charged background ions. Hierarchical clustering revealed that the immunopeptidome was more driven by the HLA-DR genotype than by RA disease or sex. However, since the HLA-DR genotype is a strong risk factor for RA, these results are convoluted. When deconvoluting the peptides to their predicted binding allele, the HLA-DRB1 alleles *01:01, *04:01, *04:04, *04:05, *04:07, and *10:01 were consistently assigned more peptides than other alleles. Except for *04:07 these alleles belong to the SE risk factor alleles, providing a potential explanation between HLA-SE and RA pathogenesis. Native peptides from known citrullinated and non-modified RA autoantigens (such as -enolase and calreticulin) were detected and validated as binders in prediction algorithms. No significant differences were found between base line and follow-up (post-treatment) samples from RA patients. Taken together, this data characterizes the HLA-DR immunopeptidome in the lung of early RA in an unprecedented manner, which together with future immunogenicity studies will help our understanding of the connection between the lung and the pathogenesis of RA. Finally, more peptides predicted to bind to SE alleles and *04:07 compared to other alleles demands further study on the relative expression of HLA-DR alleles and presentation mechanisms to understand the implications for RA.

immunology↗

bioRxiv2024

Endurance exercise with reduced muscle glycogen content influences substrate utilization and attenuates acute mTORC1- and autophagic signaling in human type I and type II muscle fibers

BackgroundExercising with low muscle glycogen content can improve training adaptation, but the mechanisms underlying the muscular adaptation are still largely unknown. In this study, we measured substrate utilization and cell signaling in different muscle fiber types during exercise and investigated a possible link between these variables. MethodsFive subjects performed a single leg cycling exercise in the evening (day 1) with the purpose of reducing glycogen stores. The following morning (day 2), they performed two-legged cycling at [~]70% of VO2peak for 1h. Muscle biopsies were taken from both legs pre- and post-exercise for enzymatic analyses of glycogen, metabolite concentrations using LC-MS/MS-based quantification, and protein signaling using Western blot in pools of type I or type II fibers. ResultsGlycogen content was 60-65% lower for both fiber types (P<0.01) in the leg that exercised on day 1 (low leg) compared to the other leg with normal level of glycogen (normal leg) before the cycling exercise on day 2. Glycogen utilization during exercise was significantly less in both fiber types in the low compared to the normal leg (P<0.05). In the low leg, there was a 14- and 6-fold increase in long-chain fatty acids conjugated to carnitine in type I and type II fibers, respectively, post-exercise. This increase was 3-4 times larger than in the normal leg (P<0.05). Post-exercise, mTORSer2448 phosphorylation was increased in both fiber types in the normal leg (P<0.05) but remained unchanged in both fiber types in the low leg together with an increase in eEF2Thr56 phosphorylation in type I fibers (P<0.01). Exercise induced a reduction in the autophagy marker LC3B-II in both fiber types and legs, but the post-exercise level was higher in both fiber types in the low leg (P<0.05). Accordingly, the LC3B-II/I ratio decreased only in the normal leg (75% for type I and 87% for type II, P<0.01). ConclusionsStarting an endurance exercise session with low glycogen availability leads to profound changes in substrate utilization in both type I and type II fibers. This may reduce the mTORC1 signaling response, primarily in type I muscle fibers, and attenuate the normally observed reduction in autophagy.

physiology↗

bioRxiv2022

Endothelial-derived sphingolipids are required for vascular development and systemic lipid homeostasis

Serine palmitoyl transferase (SPT), the rate-limiting enzyme in the de novo synthesis of sphingolipids (SL), is needed for embryonic development, physiological homeostasis, and response to stress. The functions of de novo SL synthesis in vascular endothelial cells (EC), which line the entire circulatory system, are not well understood. Here we show that the EC de novo synthesis not only impacts vascular development but also maintains normal SL metabolic homeostasis in the circulatory system and peripheral organs. Mice with an endothelial-specific gene knockout of SPTLC1 (Sptlc1 ECKO), an essential subunit of the SPT complex, exhibited EC-intrinsic effects including reduced EC proliferation and tip/stalk cell differentiation, resulting in delayed retinal vascular development. In addition, Sptlc1 ECKO mice had reduced pathological retinal neovascularization in the oxygen-induced retinopathy model, suggesting that EC SL produced from the de novo pathway are needed for efficient VEGF signaling within the vascular system. Post-natal deletion of the EC Sptlc1also showed cell-extrinsic effects, including rapid reduction of several SL metabolites in plasma, red blood cells and peripheral organs (lung and liver) but not in the retina, part of the central nervous system (CNS). In the liver, EC de novo SL synthesis was required for acetaminophen-induced ceramide elevation and hepatotoxicity. These results suggest that EC-derived SL metabolites are in constant flux between the vasculature, circulatory elements, and parenchymal cells of non-CNS organs. Taken together, our data point to the central role of the endothelial SL biosynthesis in maintaining vascular development and neovascular proliferation, non-CNS tissue metabolic homeostasis and hepatocyte response to stress.

developmental biology↗