Biology subjects

Garrido, J. J.

Publications and source records attributed to Garrido, J. J..

12 recordsLinked to original sources

bioRxiv2026

A single Omicron mutation reshapes ORF3a-driven host-cell remodelling

SARS-CoV-2 ORF3a remodels host membranes, but the structural basis and metabolic consequences of this process remain unclear. Here, we combine complementary imaging approaches to define ORF3a function at nanometric scale, identifying underlying mechanisms, and determining how Omicron variant rewire this activity. ORF3a from the ancestral Wuhan strain disrupts Golgi cisternae, drives the formation of ORF3a dense vesicles, remodels mitochondrial architecture, and promotes lipid droplet expansion. Multi-omics analyses further reveal selective triacylglycerol accumulation linked to DGAT1 upregulation, which we validate pharmacologically through DGAT1 inhibition. In contrast, Omicron ORF3a variant, despite carrying only the Thr223Ile substitution within the {beta}7-{beta}8 loop at the bottom of the cytosolic domain, induced a dramatic phenotypic shift: ORF3a localizes to multivesicular bodies, preserves Golgi architecture, and fails to induce lipid accumulation. All together, these results identify ORF3a as a regulator of membrane organization and lipid homeostasis, showing how minimal sequence variation rewires host-cell remodelling. Graphical TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/742305v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@135032borg.highwire.dtl.DTLVardef@1633d4borg.highwire.dtl.DTLVardef@4d4115org.highwire.dtl.DTLVardef@1ebdf65_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2026

Homeostatic regulation of intrinsic neuronal excitability in visual thalamic relay cells induced by brief monocular deprivation

Homeostatic plasticity of intrinsic excitability (IE) in the visual system has been essentially shown at the cortical level but whether thalamic nuclei also express homeostatic plasticity of IE is unknown. We show here that 4 days of monocular deprivation (MD) at eye opening induces a homeostatic change in IE in dorsal lateral geniculate nucleus (dLGN) neurons. Neurons recorded in the dLGN region activated by the deprived eye are more excitable than neurons recorded in the dLGN region activated by the open eye. No significant changes were observed following 7 days of MD, however. Enhanced excitability in neurons from the deprived side after 4 days of MD was associated with a reduced Kv1-dependent LTP-IE, a smaller voltage ramp, and a reduced inter-spike interval, suggesting that Kv1 channels are down-regulated in deprived dLGN neurons. Furthermore, the ankyrin G signal of the axon initial segment was larger in deprived dLGN neurons compared with open ones, indicating that Nav1 channel number also undergoes homeostatic regulation, and Kv1.1 channel signals were lower in deprived neurons compared to open ones. In addition, electrical coupling was found to be strengthened in neurons displaying enhanced IE following either brief (4 days) or long (10 days) MD. These results suggest that homeostatic and Hebbian plasticity in the dLGN share common expression mechanisms involving the regulation of Kv1 channels, Nav1 channels and electrical coupling between relay neurons.

neuroscience↗

bioRxiv2026

Omicron-Enhanced Immunosuppressive Effects of SARS-CoV-2 ORF3a and ORF9b Accessory Proteins on Monocytic Inflammatory Response

This study investigates the poorly understood roles of SARS-CoV-2 accessory proteins using monocytic THP-1 cells expressing individual viral ORFs. ORF3a, ORF7b, and ORF9b were identified as major immunomodulators that suppress host inflammatory signaling. Specifically, cells expressing ORF3a or ORF9b exhibited reduced Toll-like receptor 4 (TLR4)-mediated production of key proinflammatory molecules--CCL2, CCL4, and IL-1{beta}--resulting in diminished immune cell recruitment. Importantly, Omicron-associated mutations in ORF3a (T223I) and ORF9b (P10S+{Delta}E27N28A29) amplified this immunosuppressive effect, leading to stronger transcriptomic suppression consistent with Omicrons reduced pathogenicity and clinical outcomes. These findings suggest that SARS-CoV-2 accessory proteins, particularly ORF3a and ORF9b, play pivotal roles in modulating monocytic immune responses. Enhanced suppression in Omicron variants highlights an evolutionary adaptation contributing to immune evasion and milder disease manifestations.

immunology↗

bioRxiv2026

Genome-scale TraDIS reveals dynamic and conserved fitness requirements of Salmonella Typhimurium across sequential host niches during porcine infection

Non-typhoidal salmonellosis remains a major global cause of foodborne gastrointestinal disease, with pigs representing an important reservoir of Salmonella enterica serovar Typhimurium. The emergence of host-adapted, multidrug-resistant lineages has further reinforced the need to understand the genetic basis of bacterial persistence and pathogenicity within physiologically relevant hosts. Here, we applied transposon-directed insertion sequencing (TraDIS) to systematically define the genetic requirements of a highly host-adapted, multidrug-resistant S. Typhimurium DT104 isolate across sequential host niches during porcine infection. A high-density transposon mutant library ([~]1.2 million mutants) was subjected to in vivo selection in the ileal mucosa and mesenteric lymph nodes, as well as ex vivo infection of primary porcine neutrophils. Across all conditions, we identified 1,813 conditionally essential genes, revealing strong niche-specific fitness signatures and a progressive increase in selective stringency along the infection route. Ileal colonization was primarily driven by determinants of invasion, motility and lipopolysaccharide biosynthesis. In contrast, survival within neutrophils depended on resistance to antimicrobial stresses and extensive metabolic rewiring, whereas persistence in lymph nodes required a broader functional repertoire integrating virulence, motility, envelope remodelling and metabolic adaptation, including bacterial microcompartment-associated pathways. Despite this marked heterogeneity, we identified a conserved porcine host-conditioned essential genome, comprising core invasion functions, RNA metabolism and genome maintenance pathways, and central metabolism. Notably, the outer membrane lipid asymmetry system Mla and the twin-arginine translocation (Tat) pathway emerged as conserved bottlenecks for in vivo fitness across all host-associated environments. Together, these findings establish a hierarchical model of S. Typhimurium adaptation during porcine infection and provide a systems-level view of tissue-specific and conserved genetic requirements underpinning persistence in a major zoonotic reservoir.

microbiology↗

bioRxiv2025

Single-Cell Dissection of Immunometabolic Rewiring in the Porcine Ileum during Salmonella Typhimurium Infection

Salmonella Typhimurium is a major zoonotic pathogen, with pigs acting as important subclinical carriers. To explore the specific intestinal immune response at the cellular level, we used Single-cell RNA sequencing (scRNA-seq), enabling detailed analysis of immune cell types and gene expression profiles during infection. In addition to enterocytes, our results revealed the presence of diverse immune populations, including monocytes/macrophages, dendritic cells, innate lymphoid cells (ILCs), thirteen T cell subtypes and five B cell populations were identified, revealing pronounced infection-driven alterations in cellular composition and transcriptional states. Among T cells, naive and follicular CD4+/CD8+ {beta} T cells and NK T cells were expanded, whereas effector CD8+ T cells and CD2- and SELLhi {gamma}{delta} T cells were decreased. B-cell populations shifted toward activated and cycling states, with decreased antibody-secreting, resting, and transitioning cells. Dendritic cells and monocyte/macrophage populations were expanded, and group 3 ILCs and enterocytes were markedly reduced. Pathway analyses revealed robust cell type-specific immunometabolic remodeling, including enhanced protein-folding and stress-adaptive pathways in T and B cells, heightened inflammatory, interferon, and cytokine signaling in myeloid populations, and coordinated metabolic and immune adjustments in epithelial cells, highlighting the complexity of host responses to Salmonella infection. This study provides the first scRNA-seq landscape of the porcine ileum during S. Typhimurium infection, offering insight into host immune cell dynamics and immunometabolic responses.

genomics↗

bioRxiv2025

SARS-CoV-2 ORF7a Drives Mitochondrial Dysfunction via PDK4 Activation and Complex I Inhibition

SARS-CoV-2 reprograms host metabolism to promote viral replication and evade immune responses. While infection is known to impair mitochondrial function and enhance glycolysis, the role of viral accessory proteins in these alterations remains unclear. Here, we investigate the metabolic impact of the accessory protein ORF7a. Lentiviral expression of ORF7a in human lung epithelial (A549) and monocytic (THP1) cells, coupled with integrated transcriptomic, proteomic, and metabolomic analyses, revealed profound dysregulation of glucose and lipid metabolism. ORF7a impaired oxidative phosphorylation, reducing basal and maximal respiration, inducing mitochondrial depolarization, and increasing reactive oxygen species. Mechanistically, ORF7a upregulated pyruvate dehydrogenase kinase 4 (PDK4), promoting pyruvate dehydrogenase (PDH) complex phosphorylation and suppressing pyruvate oxidation. However, pharmacological PDK4 inhibition did not restore respiration. High-resolution respirometry in frozen samples revealed impaired complex I function, while Blue Native-PAGE demonstrated defective respiratory supercomplex assembly. By linking enzymatic inhibition with structural destabilization, our study uncovers a functional vulnerability of the mitochondrial respiratory chain to viral manipulation. These findings establish ORF7a as a key modulator of host metabolic reprogramming and highlight mitochondrial pathways as potential therapeutic targets in COVID-19.

immunology↗

bioRxiv2023

Metabolic and mitochondria alterations induced by SARS-CoV-2 accessory proteins ORF3a, ORF9b, ORF9c and ORF10

Antiviral signaling, immune response and cell metabolism in human body are dysregulated by SARS-CoV-2, the causative agent of the COVID-19. Here, we show that SARS-CoV-2 accessory proteins ORF3a, ORF9b, ORF9c and ORF10 induce a significant mitochondrial and metabolic reprogramming in A549 lung epithelial cells. While all four ORFs caused mitochondrial fragmentation and altered mitochondrial function, only ORF3a and ORF9c induced a marked structural alteration in mitochondrial cristae. ORF9b, ORF9c and ORF10 induced largely overlapping transcriptomes. In contrast, ORF3a induced a distinct transcriptome, including the downregulation of numerous genes for proteins with critical mitochondrial functions and morphology. Genome-Scale Metabolic Models predicted common and private metabolic flux reprogramming, notably a depressed amino acid metabolism, and an enhanced metabolism of specific lipids distinctly induced by ORF3a. These findings reveal metabolic dependencies and vulnerabilities prompted by SARS-CoV-2 accessory proteins that may be exploited to identify new targets for intervention. One-Sentence SummaryMitochondria and metabolic alterations induced by SARS- CoV-2 accessory proteins ORF3a, ORF9b, ORF9c, ORF10 in pulmonary cells unravel new targets of intervention.

immunology↗

bioRxiv2023

SARS-CoV-2 accessory proteins involvement in inflammatory and profibrotic processes through IL11 signaling

SARS-CoV-2, the cause of the COVID19 pandemic, possesses eleven accessory proteins encoded in its genome. Their roles during infection are still not completely understood. Transcriptomic analysis revealed that both WNT5A and IL11 were significantly up-regulated in A549 cells expressing individual accessory proteins ORF6, ORF8, ORF9b or ORF9c from SARS-CoV-2 (Wuhan-Hu-1 isolate). IL11 signaling-related genes were also differentially expressed. Bioinformatics analysis disclosed that both WNT5A and IL11 were involved in pulmonary fibrosis idiopathic disease. Functional assays confirmed their association with profibrotic cell responses. Subsequently, data comparison with lung cell lines infected with SARS-CoV-2 or lung biopsies from patients with COVID19 evidenced altered gene expression that matched those obtained in this study. Our results show ORF6, ORF8, ORF9b and ORF9c involvement in inflammatory and profibrotic responses. Thus, these accessory proteins could be targeted by new therapies against COVID19 disease. Research topic(s)Viral diseases, COVID19 insights

immunology↗

bioRxiv2023

Rescue of normal excitability in LGI1-deficient epileptic neurons

Leucine-rich Glioma Inactivated 1 (LGI1) is a glycoprotein secreted by neurons, the deletion of which leads to Autosomal Dominant Lateral Temporal Lobe Epilepsy. Recently, we showed that LGI1 deficiency in a mouse model (KO-Lgi1) decreased Kv1.1 channel density at the axon initial segment (AIS) and at presynaptic terminals, thus enhancing both intrinsic excitability and glutamate release. However, the precise conditions for rescuing normal excitability in KO-Lgi1 neurons have still not been reported. Here we show that the selective expression of LGI1 in KO-Lgi1 neurons with the use of single-cell electroporation reduces intrinsic excitability, and restores both the Kv1.1 mediated D-type current and Kv1.1 immunostaining at the AIS. In addition, we show that the homeostatic shortening of the AIS length observed in KO-Lgi1 neurons is prevented in neurons electroporated with the Lgi1 gene. Furthermore, we reveal a spatial gradient of both intrinsic excitability and Kv1.1 immunostaining that is centred on the electroporated neuron. We conclude that expression of LGI1 restores normal excitability through the expression of functional Kv1 channels at the AIS.

neuroscience↗

bioRxiv2022

Antiviral immune responses, cellular metabolism and adhesion are differentially modulated by SARS-CoV-2 ORF7a or ORF7b

SARS-CoV-2, the causative agent of the present COVID-19 pandemic, possesses eleven accessory proteins encoded in its genome, and some have been implicated in facilitating infection and pathogenesis through their interaction with cellular components. Among these proteins, accessory protein ORF7a and ORF7b functions are poorly understood. In this study, A549 cells were transduced to express ORF7a and ORF7b, respectively, to explore more in depth the role of each accessory protein in the pathological manifestation leading to COVID-19. Bioinformatic analysis and integration of transcriptome results identified defined canonical pathways and functional groupings revealing that after expression of ORF7a or ORF7b, the lung cells are potentially altered to create conditions more favorable for SARS-CoV-2, by inhibiting the IFN-I response, increasing proinflammatory cytokines release, and altering cell metabolic activity and adhesion. Based on these results, it is reasonable to suggest that ORF7a and ORF7b could be targeted by new therapies or used as future biomarkers during this pandemic.

immunology↗

bioRxiv2021

Humoral immune responses against seasonal coronaviruses predict efficiency of SARS-CoV-2 spike targeting, FcγR activation, and corresponding COVID-19 disease severity

Despite SARS-CoV-2 being a "novel" coronavirus, several studies suggest that detection of anti-spike IgG early in infection may be attributable to the amplification of humoral memory responses against seasonal hCoVs in severe COVID-19 patients. In this study, we examined this concept by characterizing anti-spike IgG from a cohort of non-hospitalized convalescent individuals with a spectrum of COVID-19 severity. We observed that anti-spike IgG levels positively correlated with disease severity, higher IgG cross-reactivity against betacoronaviruses (SARS-CoV-1 and OC43), and higher levels of proinflammatory Fc gamma receptor 2a and 3a (Fc{gamma}R2a & Fc{gamma}R3a) activation. In examining the levels of IgG targeting betacoronavirus conserved and immunodominant epitopes versus disease severity, we observed a positive correlation with the levels of IgG targeting the conserved S2FP region, and an inverse correlation with two conserved epitopes around the heptad repeat (HR) 2 region. In comparing the levels of IgG targeting non-conserved epitopes, we observed that only one of three non-conserved immunodominant epitopes correlated with disease severity. Notably, the levels of IgG targeting the receptor binding domain (RBD) were inversely correlated with severity. Importantly, targeting of the RBD and HR2 regions have both been shown to mediate SARS-CoV-2 neutralization. These findings show that, aside from antibody (Ab) targeting of the RBD region, humoral memory responses against seasonal betacoronaviruses are potentially an important factor in dictating COVID-19 severity, with anti-HR2-dominant Ab profiles representing protective memory responses, while an anti-S2FP dominant Ab profiles indicate deleterious recall responses. Though these profiles are masked in whole antigen profiling, these analyses suggest that distinct Ab memory responses are detectable with epitope targeting analysis. These findings have important implications for predicting severity of SARS-CoV-2 infections (primary and reinfections), and may predict vaccine efficacy in subpopulations with different dominant antibody epitope profiles.

microbiology↗

bioRxiv2021

Homeostatic regulation of axonal Kv1.1 channels accounts for both synaptic and intrinsic modifications in CA3 circuit

Homeostatic plasticity of intrinsic excitability goes hand-in-hand with homeostatic plasticity of synaptic transmission. However, the mechanisms linking the two forms of homeostatic regulation have not been identified so far. Using electrophysiological, imaging and immunohistochemical techniques, we show here that blockade of excitatory synaptic receptors for 2-3 days induces an up-regulation of synaptic strength at CA3-CA3 connexions and intrinsic excitability of CA3 pyramidal neurons. Activity-deprived connexions were found to express a high release probability, an insensitivity to dendrotoxin, and a lack of depolarization-induced presynaptic facilitation, indicating a loss of presynaptic Kv1.1 function. The down-regulation of Kv1.1 channels in activity-deprived neurons was confirmed by their broader action potentials measured in the axon that were insensitive to dendrotoxin. We conclude that regulation of axonal Kv1.1 channel constitutes a unique mechanism linking intrinsic excitability and synaptic strength that accounts for the functional synergy existing between homeostatic regulation of intrinsic excitability and synaptic transmission.

neuroscience↗