Biology subjects

Gutierrez, D.

Publications and source records attributed to Gutierrez, D..

7 recordsLinked to original sources

bioRxiv2025

Diversity, structure-function relationships and evolution of cell wall-binding domains of staphylococcal phage endolysins

Endolysins, encoded by phages, lyse bacterial hosts at the end of the replication cycle by degrading peptidoglycan. Consequently, they have evolved in response to host cell wall structures, leading to complex modular architectures, particularly in Gram-positive bacteria. These architectures feature diverse enzymatically active domains (EADs) and cell wall-binding domains (CBDs). This study investigates the structure-function relationships of CBDs in staphylococcal phage endolysins, exploring their evolutionary origins and the extent to which binding specificity can be predicted from sequence data. A set of 182 staphylococcal endolysin sequences was analyzed, revealing predominantly three-domain architectures, occasionally disrupted by species-specific mobile genetic elements. Most CBDs exhibited an SH3-like fold, classified into two major subfamilies: SH3b_P (including the well-characterized SH3_5 family) and SH3b_T. The composition of endolysin domains correlated with specific CBD families, suggesting co-evolution of CBDs and compatible EADs to ensure functional synergy. To assess binding properties, 24 CBDs were fused to eGFP and tested against a panel of staphylococci, revealing diverse specificity profiles. However, no clear correlation emerged between binding specificity, phylogenetic subgroups, or bacterial hosts. This suggests that minor structural modifications significantly impact function and that CBD specificity is not a major selective pressure in the staphylococcal bacteria-phage interface.

molecular biology↗

bioRxiv2024

Poly (ADP-ribose) polymerase-1 regulates HIV-1 replication in human CD4+ T cells

The cellular enzyme poly (ADP-ribose) polymerase-1 (PARP-1) regulates multiple processes that are potentially implicated in HIV-1 infection. However, the role of PARP-1 in HIV-1 infection remains controversial, with reports indicating or excluding that PARP-1 influence early steps of the HIV-1 life cycle. Most of these studies have been conducted with Vesicular Stomatitis virus Glycoprotein G (VSV-G)-pseudotyped, single-round infection HIV-1; limiting our understanding of the role of PARP-1 in HIV-1 replication. Therefore, we evaluated the effect of PARP-1 deficiency or inhibition in HIV-1 replication in human CD4+ T cells. Our data showed that PARP-1 knockout increased viral replication in SUP-T1 cells. Similarly, a PARP-1 inhibitor that targets PARP-1 DNA-binding activity enhanced HIV-1 replication. In contrast, inhibitors affecting the catalytic activity of the enzyme were inactive. In correspondence with the pharmacological studies, mutagenesis analysis indicated that the DNA-binding domain was required for the PARP-1 anti-HIV-1 activity, but the poly-ADP-ribosylation activity was dispensable. Our results also demonstrated that PARP-1 acts at the production phase of the viral life cycle since HIV-1 produced in cells lacking PARP-1 was more infectious than control viruses. The effect of PARP-1 on HIV-1 infectivity required Env, as PARP-1 deficiency or inhibition did not modify the infectivity of Env-deleted, VSV-G-pseudotyped HIV-1. Furthermore, virion-associated Env was more abundant in sucrose cushion-purified virions produced in cells lacking the enzyme. However, PARP-1 did not affect Env expression or processing in the producer cells. In summary, our data indicate that PARP-1 antagonism enhances HIV-1 infectivity and increases levels of virion-associated Env. ImportanceDifferent cellular processes counteract viral replication. A better understanding of these interfering mechanisms will enhance our ability to control viral infections. We have discovered a novel, antagonist effect of the cellular enzyme poly (ADP-ribose) polymerase-1 (PARP-1) in HIV-1 replication. Our data indicate that PARP-1 deficiency or inhibition augment HIV-1 infectivity in human CD4+ T cells, the main HIV-1 target cell in vivo. Analysis of the mechanism of action suggested that PARP-1 antagonism increases in the virus the amounts of the viral protein mediating viral entry to the target cells. These findings identify for the first time PARP-1 as a host factor that regulates HIV-1 infectivity, and could be relevant to better understand HIV-1 transmission and to facilitate vaccine development.

microbiology↗

bioRxiv2024

Molecular Myelin Dysfunction in the Most Common Inherited Peripheral Neuropathies - CMT1A and HNPP

Charcot-Marie-Tooth Disease Type 1A (CMT1A) and Hereditary Neuropathy with Liability to Pressure Palsies (HNPP) are the most common inherited peripheral neuropathies and arise from copy number variation of the Peripheral Myelin Protein 22 gene (PMP22). While secondary axon degeneration has been proposed as a primary driver of pathogenesis, our prior work demonstrated neuromuscular deficits in CMT1A mice in the absence of overt axonal loss, prompting investigation into primary myelin dysfunction. Here, we reveal that altered PMP22 dosage profoundly disrupts molecular architecture at critical myelin subdomains, Schmidt-Lanterman incisures (SLIs) and Nodes of Ranvier. Using high-resolution confocal imaging of teased peripheral nerve fibers from CMT1A and HNPP model mice, we identified disorganization of adherens junctions, mislocalization of Connexin29, and altered distribution of nodal ion channels in CMT1A and HNPP, with several defects more pronounced in CMT1A, aligning with clinical severity. Notably, Kv1.2 and Caspr mislocalization along the internode and Nav nodal widening suggest disruption of axoglial domains essential for saltatory conduction. Together, these phenotypes support a model in which PMP22 governs myelin architecture, likely through adherens junction regulation, with its dysregulation predicted to impair metabolic support and axonal ion homeostasis, thereby compromising the structural and functional integrity of myelin and contributing directly to disease pathogenesis. These findings shift the pathogenic paradigm for CMT1A and HNPP from axonal degeneration to primary myelin failure and highlight actionable molecular targets for therapeutic intervention. This study offers mechanistic insight into CMT1A and HNPP and provides a conceptual framework with broad relevance to other dysmyelinating disorders. Main PointsO_LIPMP22 copy number variation disrupts myelin architecture at SLIs and Nodes of Ranvier. C_LIO_LIAdherens junction and axoglial domain defects are more severe in CMT1A than HNPP. C_LIO_LIFindings support primary myelin dysfunction as a key driver of pathogenesis. C_LI

neuroscience↗

bioRxiv2024

The new SH3_T domain increases the structural and functional variability among SH3b-like CBDs from staphylococcal phage endolysins

Endolysins, proteins encoded by phages to lyse their hosts and release their progeny, have evolved to adapt to the structural features of each host. The endolysins from Staphylococcus-infecting phages typically feature complex architectures with two enzymatically active domains (EADs) and one cell wall-binding domain (CBD) belonging to the bacterial SH3 (SH3b) superfamily. This study focuses on three SH3b-like CBDs from exemplary staphylococcal phage endolysins (LysRODI, LysC1C, and LysIPLA5) that were structurally and functionally characterized. While RODI_CBD and C1C_CBD were assigned to the well-known SH3_5 family, a new family, SH3b_T, was identified using the CBD from LysIPLA5 as a model. GFP-fused CBDs were created to assess their differential binding to a collection of staphylococcal strains. IPLA5_CBD showed enhanced binding to Staphylococcus epidermidis, while RODI_CBD and C1C_CBD exhibited distinct binding profiles, with RODI_CBD targeting Staphylococcus aureus specifically and C1C_CBD displaying broad binding. Sequence comparisons suggested that a few differences in key amino acids could be responsible for the latter binding difference. The CBDs modulated the activity spectrum of synthetic EAD-CBD combinations in accordance with the previous binding profiles, but in a manner that was also dependent on the EAD present in the fusion protein. These results serve as a context for the diversity and versatility of SH3b domains in staphylococcal endolysins, providing insights on how (i) the CBDs from this superfamily have diverged to adapt to diverse bacterial ligands in spite of sharing a common fold; and (ii) the evolution of specificity relies on the EAD-CBD combination rather than solely the CBD. IMPORTANCEClinical management of bacterial infections is nowadays compromised by the rise in antimicrobial resistance. The development of new antimicrobial therapies with diverse modes of action is therefore of pivotal importance to complement the current standard of care. Phage endolysins are a new class of antibacterial agents based on rapid peptidoglycan degradation. The natural reservoir of phage endolysins offers a practically infinite diversity. This works reveals a broadly spread but still unknown phage endolysin domain targeting staphylococci while providing structural-functional insights that are paramount to understand the evolution of endolysins and how they can be applied as an antimicrobial.

molecular biology↗

bioRxiv2023

Connectome architecture shapes large-scale cortical reorganization in schizophrenia: a worldwide ENIGMA study

While schizophrenia is considered a prototypical network disorder characterized by widespread brain-morphological alterations, it still remains unclear whether distributed structural alterations robustly reflect underlying network layout. Here, we tested whether large-scale structural alterations in schizophrenia relate to normative structural and functional connectome architecture, and systematically evaluated robustness and generalizability of these network-level alterations. Leveraging anatomical MRI scans from 2,439 adults with schizophrenia and 2,867 healthy controls from 26 ENIGMA sites and normative data from the Human Connectome Project (n=207), we evaluated structural alterations of schizophrenia against two network susceptibility models: i) hub vulnerability, which examines associations between regional network centrality and magnitude of disease-related alterations; ii) epicenter mapping, which identify regions whose typical connectivity profile most closely resembles the disease-related morphological alterations. To assess generalizability and specificity, we contextualized the influence of site, disease stages, and individual clinical factors and compared network associations of schizophrenia with that found in affective disorders. Schizophrenia-related structural alterations co-localized with interconnected functional and structural hubs and harbored temporo-paralimbic and frontal epicenters. Findings were robust across sites and related to individual symptom profiles. We observed localized unique epicenters for first-episode psychosis and early stages, and transmodal epicenters that were shared across first-episode to chronic stages. Moreover, transdiagnostic comparisons revealed overlapping epicenters in schizophrenia and bipolar, but not major depressive disorder, yielding insights in pathophysiological continuity within the schizophrenia-bipolar-spectrum. In sum, cortical alterations over the course of schizophrenia robustly follow brain network architecture, emphasizing marked hub susceptibility and temporo-frontal epicenters at both the level of the group and the individual. Subtle variations of epicenters across disease stages suggest interacting pathological processes, while associations with patient-specific symptoms support additional inter-individual variability of hub vulnerability and epicenters in schizophrenia. Our work contributes to recognizing potentially common pathways to better understand macroscale structural alterations, and inter-individual variability in schizophrenia.

neuroscience↗

bioRxiv2022

A bioluminescence-based ex vivo burn wound model for real-time assessment of novel antibacterial compounds

The silent pandemic of antibiotic resistance is thriving, prompting the urgent need for the development of new antibacterial drugs. However, within the preclinical pipeline, in vitro screening conditions can differ significantly from the final in vivo settings. To bridge the gap between in vitro and in vivo assays, we developed a pig skin-based bioluminescent ex vivo burn wound infection model, enabling real-time assessment of antibacterials in a longitudinal, non-destructive manner. We provide a proof-of-concept for A. baumannii NCTC13423, a multidrug-resistant clinical isolate, which was equipped with the luxCDABE operon as a reporter using a Tn7-based tagging system. This bioluminescence model provided a linear correlation between the number of bacteria and a broad dynamic range (104 to 109 CFU). This longitudinal model was subsequently validated using a fast-acting enzybiotic as an antibacterial. Since this model combines a realistic, clinically relevant yet strictly controlled environment with real-time measurement of bacterial burden, we put forward this ex vivo model as a valuable tool to assess the preclinical potential of novel antibacterial compounds. Summary statementHere, we demonstrate the potential of a bioluminescence-based ex vivo model for the longitudinal assessment of antibacterials. Moreover, we also provide a proof-of-concept with an engineered lysin.

molecular biology↗

bioRxiv2022

Development of genome-driven, lifestyle-informed primers for identification of the cereal infecting pathogens Xanthomonas translucens pathovars undulosa and translucens.

Bacterial leaf streak, blight and black chaff caused by Xanthomonas translucens pathovars are major diseases affecting small grains. Xanthomonas translucens pv. translucens and X. translucens pv. undulosa are seedborne pathogens that cause similar symptoms on barley, but only X. translucens pv. undulosa causes bacterial leaf streak of wheat. Recent outbreaks of X. translucens have been a concern for wheat and barley growers in the Northern Great Plains and Upper Midwest; however, there are limited diagnostic tools for pathovar differentiation. We developed a multiplex PCR based on whole-genome differences to distinguish X. translucens pv. translucens and X. translucens pv. undulosa. We validated the primers across different Xanthomonas and non-Xanthomonas strains. To our knowledge, these are the first multiplex PCR to distinguish X. translucens pv. translucens and X. translucens pv. undulosa. These molecular tools will support disease management strategies enabling detection and pathovar incidence analysis of X. translucens.

microbiology↗