Biology subjects

Lopez, C. A.

Publications and source records attributed to Lopez, C. A..

9 recordsLinked to original sources

bioRxiv2025

Incorporation of ChiA into AcMNPV occlusion bodies and its implications for oral infectivity

Baculoviruses are insect-specific viruses primarily employed in biological pest control and recombinant protein expression. As biopesticides, they offer numerous advantages over chemical pesticides and hold significant potential for application in integrated pest management (IPM). However, their slower action compared to conventional synthetic strategies has limited their widespread adoption. This study aimed to investigate the impact of incorporating a viral protein, AcChiA chitinase, which disrupts the gut peritrophic membrane of susceptible insects, into AcMNPV occlusion bodies in trans. This was achieved through the development of an insect cell line that facilitates the passive incorporation of this protein. The presence of ChiA was detected in dissolved occlusion bodies (OBs) preparations from infected transgenic lines, in a method-dependent manner, with the stronger signals observed in chimeric polyhedra. It is hypothesized that ChiA in OBs will increase peritrophic membrane permeability and facilitate primary infection. In bioassays, OBs produced in ChiA-expressing lines were associated with higher larval mortality than OBs produced in Sf9, consistent with a ChiA-mediated effect. Notably, the addition of ChiA proteins from stably transformed insect cell lines improved infection rates, particularly in recombinant baculoviruses, which exhibited a significant reduction in occlusion-derived virion (ODV) occlusion. This underscores the critical role of these proteins in the oral infection process. Consequently, it was possible to incorporate the ChiA protein from stably transformed insect cell lines into wild-type and recombinant baculovirus occlusion bodies, paving the way for potential applications in IPM with AcMNPV.

bioengineering↗

bioRxiv2024

A blueprint for biomolecular condensation driven by bacterial microcompartment encapsulation peptides

Bacterial microcompartments (BMC) are protein organelles with diverse metabolic capabilities. Their functional diversity is determined by an enzymatic core that is sequestered within a structurally conserved protein shell architecture. Segregation of protein cargo into the BMC is enabled by encapsulation peptides (EPs), which are short helical domains fused to core proteins through a disordered linker. Here, we investigate how EPs drive multicomponent cargo assembly into biomolecular condensates. In vitro experiments supported by molecular dynamics simulations demonstrate the importance of both conserved hydrophobic packing and electrostatic interactions in stabilizing trimeric EP bundles. Topological rearrangements of EP domains can drive programmable liquid-or gel-like partitioning in vitro and in vivo. This partitioning is found to be EP-specific, modular and can co-assemble at least three fluorescent reporters. In summary, we describe the molecular features necessary to drive biomolecular condensation using a widespread peptide tag. This work can serve as a blueprint for implementing EP biotechnology across diverse applications.

molecular biology↗

bioRxiv2024

Insertion and Anchoring of HIV-1 Fusion Peptide into Complex Membrane Mimicking Human T-cell

A fundamental understanding of how HIV-1 envelope (Env) protein facilitates fusion is still lacking. The HIV-1 fusion peptide, consisting of 15 to 22 residues, is the N-terminus of the gp41 subunit of the Env protein. Further, this peptide, a promising vaccine candidate, initiates viral entry into target cells by inserting and anchoring into human immune cells. The influence of membrane lipid reorganization and the conformational changes of the fusion peptide during the membrane insertion and anchoring processes, which can significantly affect HIV-1 cell entry, remains largely unexplored due to the limitations of experimental measurements. In this work, we investigate the insertion of the fusion peptide into an immune cell membrane mimic through multiscale molecular dynamics simulations. We mimic the native T-cell by constructing a 9-lipid asymmetric membrane, along with geometrical restraints accounting for insertion in the context of gp41. To account for the slow timescale of lipid mixing while enabling conformational changes, we implement a protocol to go back and forth between atomistic and coarse-grained simulations. Our study provides a molecular understanding of the interactions between the HIV-1 fusion peptide and the T-cell membrane, highlighting the importance of conformational flexibility of fusion peptides and local lipid reorganization in stabilizing the anchoring of gp41 into the targeted host membrane during the early events of HIV-1 cell entry. Importantly, we identify a motif within the fusion peptide critical for fusion that can be further manipulated in future immunological studies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/606381v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@d80eedorg.highwire.dtl.DTLVardef@bc2482org.highwire.dtl.DTLVardef@f715org.highwire.dtl.DTLVardef@15dba11_HPS_FORMAT_FIGEXP M_FIG Table of Content. C_FIG

biophysics↗

bioRxiv2023

Predicting Permeation of Compounds across the Outer Membrane of P. aeruginosa Using Molecular Descriptors: Advantages and Limitations

The ability of Gram-negative pathogens to adapt and protect themselves against antibiotics is a growing threat to public health. The low permeability of the outer membrane (OM) in combination with effective multidrug efflux pumps, constitute the two main antibiotic resistance mechanisms. Though much efforts have been devoted to discover new antibiotics that can bypass these defense mechanisms, no new antibiotic classes have been introduced into clinics in the last 35 years. Models that identify specific descriptors of molecular properties and predict the likelihood that a given compound is capable of successfully permeate the OM and inhibit bacterial growth while avoiding efflux could facilitate the discovery of novel classes of antibiotics. Here we evaluate 174 molecular descriptors of 1260 antimicrobial compounds and study their correlations with antibacterial activity in Gram-negative Pseudomonas aeruginosa. While part of these descriptors are computed using traditional approaches based on the physicochemical properties intrinsic to the compounds, ensemble docking and all-atom molecular dynamics (MD) simulations are used to derive additional bacterium-specific mechanistic properties. Descriptors of compound permeation across the OM were calculated using all-atom MD simulations of the compounds in different subregions of the OM model. Descriptors of interactions with efflux pumps were calculated from ensemble docking of compounds targeting specific binding pockets of MexB, the major efflux transporter of P. aeruginosa. Using these descriptors and the measured antibacterial inhibitory concentrations of compounds, we design and implement a statistical protocol to identify a subset of the molecular properties that are predictive of whether a given compound is a strong or weak permeator across the Gram-negative OM. Our results indicate that 88.4% of the compounds that show measurable antibacterial activity, follow very consistent rules of permeation, which highlight the critical role that the interaction between the compound and the OM have at predicting permeation. The remaining 11.6% of the compounds, although less predictive, are characterized by distinctive structural markers that can be used to minimize classification errors. An implementation of the permeation rules and the structural markers uncovered in our study is shown, and it demonstrates the accuracy of our approach in a set of previously unseen compounds. Taken together, our analysis sheds new light on the key molecular properties that drug candidates should have in order to be effective at OM permeation/inhibition of P. aeruginosa, and opens the gate to similar data-driven studies in other Gram-negative pathogens.

biophysics↗

bioRxiv2022

Zika virus replicates in the vagina of mice with intact interferon signaling

Zika virus (ZIKV) is unusual among flaviviruses in its ability to spread between humans through sexual contact, as well as by mosquitoes. Sexual transmission has the potential to change the epidemiology and geographic range of ZIKV compared to mosquito-borne transmission and potentially could produce distinct clinical manifestations, so it is important to understand the host mechanisms that control susceptibility to sexually transmitted ZIKV. ZIKV replicates poorly in wild-type mice following subcutaneous inoculation, so most ZIKV pathogenesis studies use mice lacking IFN-{beta} signaling (e.g. Ifnar1-/-). However, we found that wild-type mice support ZIKV replication following intravaginal infection, although the infection remained localized to the lower female reproductive tract. Vaginal replication was not a unique property of ZIKV, as other flaviviruses that generally are restricted in wild-type mice also were able to replicate in the vagina. Vaginal ZIKV infection required a high-progesterone state (pregnancy or pre-treatment with depot medroxyprogesterone acetate (DMPA)), identifying a key role for hormonal status in susceptibility to vaginal infection. Progesterone-mediated susceptibility did not appear to result from a compromised epithelial barrier, blunted antiviral gene induction, or changes in vaginal leukocyte populations, leaving open the mechanism by which progesterone confers susceptibility to vaginal ZIKV infection. Progesterone treatment is a key component of mouse vaginal infection models for herpes simplex virus and Chlamydia, but the mechanisms by which DMPA increases susceptibility to those pathogens also remain poorly defined. Understanding how progesterone mediates susceptibility to ZIKV vaginal infection may provide insights into host mechanisms influencing susceptibility to diverse sexually transmitted pathogens. IMPORTANCEZika virus (ZIKV) is transmitted by mosquitoes, similarly to other flaviviruses. However, ZIKV is unusual in its ability also to spread through sexual transmission. We found that ZIKV was able to replicate in the vaginas of wild-type mice, even though these mice do not support ZIKV replication by other routes, suggesting that the vagina is particularly susceptible to ZIKV infection. Vaginal susceptibility was dependent on a high progesterone state, which is a common feature of mouse vaginal infection models for other pathogens, through mechanisms that have remained poorly defined. Understanding how progesterone mediates susceptibility to ZIKV vaginal infection may provide insights into host mechanisms that influence susceptibility to diverse sexually transmitted pathogens.

microbiology↗

bioRxiv2021

Zika virus-specific IgM elicited during pregnancy exhibits ultrapotent neutralization

Congenital Zika virus (ZIKV) infection results in neurodevelopmental deficits in up to 14% of infants born to ZIKV-infected mothers. Neutralizing antibodies are a critical component of protective immunity. Here, we demonstrate that plasma IgM responses contribute to ZIKV immunity in pregnancy, mediating neutralization up to three months post symptoms. From a ZIKV-infected pregnant woman, we established a B cell line secreting a pentameric ZIKV-specific IgM (DH1017.IgM) that exhibited ultrapotent ZIKV neutralization dependent on the IgM isotype. DH1017.IgM targets a novel envelope dimer epitope within Domain II. The arrangement of this epitope on the virion is compatible with concurrent engagement of all ten antigen-binding sites of DH1017.IgM, a solution not achievable by IgG antibodies. DH1017.IgM protected against lethal ZIKV challenge in mice. Our findings identify a unique role of antibodies of the IgM isotype in protection against ZIKV and posit DH1017.IgM as a safe and effective candidate immunoprophylactic, particularly during pregnancy. Key pointsO_LIPlasma IgM contributes to early ZIKV neutralization during pregnancy C_LIO_LIUltrapotent neutralization by pentameric DH1017.IgM mAb depends on isotype C_LIO_LIDH1017.IgM can engage all binding sites concurrently through different angles of approach C_LIO_LIDH1017.IgM protects mice against lethal ZIKV challenge C_LI

immunology↗

bioRxiv2020

Multiscale Characterization of Complex Binding Interactions of Cellulolytic Enzymes Highlights Limitations of Classical Approaches

Cellulolytic microorganisms, like Trichoderma reesei or Clostridium thermocellum, frequently have non-catalytic carbohydrate-binding modules (CBMs) associated with secreted or cell surface bound multidomain carbohydrate-active enzymes (CAZymes) like cellulases. Mostly type-A family CBMs are known to promote cellulose deconstruction by increasing the substrate-bound concentration of cognate cellulase catalytic domains. However, due to the interfacial nature of cellulose hydrolysis and the structural heterogeneity of cellulose, it has been challenging to fully understand the role of CBMs on cellulase activity using classical protein-ligand binding assays. Here, we report a single-molecule CAZyme assay for an industrially relevant processive cellulase Cel7A (from T. reesei) to reveal how subtle CBM1 binding differences can drastically impact cellulase motility/velocity and commitment to initial processive motion for deconstruction of two well-studied crystalline cellulose allomorphs (namely cellulose I and III). We take a multifaceted approach to characterize the complex binding interactions of all major type-A family representative CBMs including CBM1, using an optical-tweezers based single-molecule CBM-cellulose bond rupture assay to complement several classical bulk ensemble protein-ligand binding characterization methods. While our work provides a basis for the cautious use of Langmuir-type adsorption models to characterize classical protein-ligand binding assay data, we highlight the critical limitations of using such overly simplistic models to gain a truly molecular-level understanding of interfacial protein binding interactions at heterogeneous solid-liquid interfaces. Finally, molecular dynamics simulations provided a theoretical basis for the complex binding behavior seen for CBM1 towards two distinct cellulose allomorphs reconciling experimental findings from multiscale analytical methods. Significance StatementMultimodal biomolecular binding interactions involving carbohydrate polymers (e.g., cellulose, starch, chitin, glycosaminoglycans) are fundamental molecular processes relevant to the recognition, biosynthesis, and degradation of all major terrestrial and aquatic biomass. Protein-carbohydrate binding interactions are also critical to industrial biotechnology operations such as enzymatically-catalyzed bioconversion of starch and lignocellulose into biochemicals like ethanol. However, despite the ubiquitous importance of such interfacial processes, we have a poor molecular-level understanding of protein-polysaccharide binding interactions. Here, we provide a comprehensive experimental and theoretical analysis of bulk ensemble versus single-molecule binding interactions of enzyme motors and associated non-catalytic binding domains with cellulosic polysaccharides to highlight the critical limitations of applying classical biochemical assay techniques alone to understanding protein adsorption or biological activity at solid-liquid interfaces.

biochemistry↗

bioRxiv2019

Oligomeric state of the ZIKV E protein defines protective immune responses.

The current leading Zika vaccine candidates in clinical testing are based on live or killed virus platforms, which have safety issues, especially in pregnant women. Zika subunit vaccines, however, have shown poor performance in preclinical studies. We hypothesized that Zika Envelope (E) protein subunit vaccines have performed poorly because the antigens tested have been recombinant E monomers that do not display critical quaternary structure epitopes present on Zika E protein homodimers that cover the surface of the virus. To test this hypothesis, we engineered and produced stable recombinant E protein homodimers. Unlike the E monomer, the dimer was recognized by strongly neutralizing monoclonal antibodies isolated from Zika-immune individuals. In a mouse model of vaccination, the dimeric antigen stimulated strongly neutralizing antibodies that targeted epitopes that were similar to epitopes recognized by human antibodies following natural Zika virus infection. In contrast, the monomer antigen stimulated lower levels of neutralizing antibodies directed to simple epitopes on domain III of E protein. In a mouse model of ZIKV challenge, only E dimer antigen stimulated protective antibodies, not the monomer. These results highlight the importance of mimicking the highly structured flavivirus surface when designing subunit vaccines. The flavivirus field has a long history of using E monomers as vaccine antigens with limited success. These results are applicable to developing second generation subunit vaccines against Zika as well as other medically important flaviviruses such as dengue and yellow fever viruses.

microbiology↗

bioRxiv2019

Unsupervised Machine Learning for Analysis of Coexisting Lipid Phases and Domain Growth in Biological Membranes

Phase separation in mixed lipid systems has been extensively studied both experimentally and theoretically because of its biological importance. A detailed description of such complex systems undoubtedly requires novel mathematical frameworks that are capable to decompose and categorize the evolution of thousands if not millions of lipids involved in the phenomenon. The interpretation and analysis of Molecular Dynamics (MD) simulations representing temporal and spatial changes in such systems is still a challenging task. Here, we present a new unsupervised machine learning approach based on Nonnegative Matrix Factorization, called NMFk, that successfully extracts physically meaningful features from neighborhood profiles derived from coarse-grained MD simulations of ternary lipid mixture. Our results demonstrate that leveraging NMFk can (a) determine the role of different lipid molecules in phase separation, (b) characterize the formation of nano-domains of lipids, (c) determine the timescales of interest and (d) extract physically meaningful features that uniquely describe the phase separation with broad implications.

bioinformatics↗