Biology subjects

Moraes, T.

Publications and source records attributed to Moraes, T..

7 recordsLinked to original sources

bioRxiv2026

Human breast milk extracellular vesicles from mothers with asthma differentially modulate the release of inflammatory cytokines by primary human airway smooth muscle cells in a recipient-cell specific manner

Breastfeeding provides health benefits in childhood, reducing the frequency of gastrointestinal and respiratory infections. Breastmilk (BM) is a rich source of bioactive molecules including extracellular vesicles (EVs), which exert immunomodulatory signalling in recipient cells, with cargo that is affected by maternal characteristics. Here we investigated the biophysical characteristics of BM-EVs from mothers with (asthmatic BM-EVs) or without asthma (control BM-EVs) and their effect on the release of cytokines from primary human hTERT-immortalized airway smooth muscle cells (hASMs) from asthmatic or non-asthmatic (control) donors. BM-EVs were isolated using size exclusion chromatography (N=5/group), characterized biophysically and by EV-specific protein markers. In addition, BM-EV were co-cultured (48h) with primary hASM cells from both non-asthmatic (control) and asthmatic donors to determine the effect on cytokine release. All participants were Caucasian and the BM was collected 12-15 weeks postpartum. BM-EVs showed the presence of intact and small-EVs ([~]100 nm). Asthmatic BM-EVs appeared to have a smaller average EV size (135.6 nm) vs. controls (148.3 nm, p=0.0613), but [~]5-fold higher concentration of both total (p=0.0014) and small EVs (p=0.0016). The expression of EV subtype protein expression was reduced in asthmatic BM-EVs vs. control BM-EVs: CD63 by 86% (p=0.0224), flotillin-1 by 40% (p=0.0196), CD9 by 24% (p=0.0646) and HSP70 by 69% (p=0.0873). Asthmatic BM-EVs co-cultured with hASMs from control donors decreased pro-inflammatory cytokine release: MCP-1 by 55% (p=0.0286), IL-6 by 45% (p=0.0801) and IL-2 by 32% (p=0.0970) vs. control-BM-EVs. Conversely, asthmatic BM-EVs co-cultured with hASMs from asthmatic donors increased secretion of anti-inflammatory cytokine IL-10 by 32% (p=0.0660), and IL-1Ra by 75% (p=0.0875), and pro-inflammatory IL-2 by 57% (p=0.0688) vs. control-BM-EVs. Internalization of control and asthmatic BM-EVs was confirmed by labelled EV uptake experiments. No detrimental effects on cell viability with BM-EV treatment were observed. In summary, asthmatic BM-EVs are smaller and enriched in BM, and exert differential effects on cytokine release in a BM-donor and recipient-cell specific manner. Given that BM can enter infant airways, the immunomodulatory effects of BM-EVs on hASMs warrants further investigation to delineate the under underlying mechanisms.

cell biology↗

bioRxiv2025

Rationally designed minimized TbpB confers broad protection against meningococcal infection

Transferrin binding protein B (TbpB), an iron acquisition protein, has long been recognized as a promising vaccine candidate targeting the pathogenic Neisseria species, including Neisseria meningitidis, the cause of meningococcal disease, and Neisseria gonorrhoeae, the cause of gonorrhea. A challenge to the development of this protein as a vaccine immunogen is the extent of antigenic variability it exhibits, which complicates the selection of a single variant to elicit a broadly cross-protective immune response. We have utilized structure-informed antigen engineering to develop a minimized version of TbpB consisting of the proteins carboxy-terminal lobe with its variable surface loops removed. Here, we reveal the effectiveness of this "loopless C-lobe" as an independent immunogen, with structural characterization and stability studies to demonstrate its integrity, and murine immunization and challenge studies that establish its ability to elicit robust protective efficacy by using N. meningitidis invasive infection and nasopharyngeal colonization models. The breadth of protection provided, as measured by both in vitro analysis and cross-protection mouse challenge studies, indicate that a single loopless C-lobe elicits a broadly cross-protective immune response against the diverse panel of meningococcal strains tested, and that the cross-reactivity is superior to that offered by the intact TbpB or the native C-lobe. Together, this study demonstrates the utility of structure-informed antigen engineering towards the development of broadly efficacious protein-based vaccines. ImportanceSurface-exposed proteins on bacterial pathogens are enticing candidate vaccine targets, however their exposure to the immune system frequently leads to high levels of antigenic variation, a factor that complicates the development of broadly protective vaccines. Here, we undertake an antigen engineering approach to develop a minimized version of a surface lipoprotein, transferrin binding protein B, where variable regions of the protein have been removed to focus the immune response to conserved regions of this antigen. We combine structural studies and mouse infection models of Neisseria meningitidis, the cause of meningococcal disease, and Neisseria gonorrhoeae, the causative agent of gonorrhea, to reveal that our strategic minimizing of the protein immunogen focuses the immune response to extend the resulting breadth of cross-reactivity and cross-protection.

immunology↗

bioRxiv2024

Phylogeny-based selection of representative variants with Navargator: Proof of principle using humoral cross-reactivity data from two immunization studies

BackgroundThe accessibility to the immune system of bacterial surface proteins makes them attractive targets for subunit vaccines. However, this same property also means they tend to exhibit high sequence variability. Achieving broad cross-protection usually necessitates that antigens from multiple isolates are included, but the choice of sequence variant is a non-trivial problem. Visual inspection of phylogenetic trees is the norm, but this is subjective and can be greatly influenced by the choice of viewing software. This has real-world implications, as groups have shown that the selection of non-optimal antigens likely led to lower cross-protection in the commercially available vaccines against Neisseria meningitidis serogroup B. Aim / MethodsTo address this problem, we have developed Navargator, bioinformatics software that takes a phylogenetic tree as input and identifies the variants that are the most similar to the greatest number of other sequences. The underlying premise is that cross-reactivity will be correlated with phylogenetic distances extracted from the tree; this was validated by several rodent immunization studies with the proteins transferrin-binding protein B and factor H binding protein from N. meningitidis and N. gonorrhoeae, measuring antibody-based cross-reactivity between an antigen panel using a custom high-throughput ELISA. ResultsNavargator has been made freely available both as an online tool and as source code for local installation. We implemented several different clustering methods, with exact algorithms for smaller datasets, and heuristics suitable for large trees of thousands of sequences. Our immunization studies have shown that this approach is sound, and that cross-reactivity is predicted well by phylogenetic distances in a sigmoidal manner. ConclusionsThe complexity of vaccine development rises sharply with each additional antigen included, so using the minimal number required is an important consideration. Navargator attempts to facilitate this in a systematic and generalizable manner. The user can run the analysis by selecting their desired number of representatives, or they can provide any form of cross-reactivity data and have the program identify a minimum reactivity threshold via correlation with the phylogenetic tree. The program will then identify the smallest number of representatives required to satisfy this threshold.

bioinformatics↗

bioRxiv2023

Investigating the importance of surface exposed loops in the gonococcal HpuB transporter for hemoglobin binding and utilization

Neisseria gonorrhoeae is the etiological agent of the sexually-transmitted infection gonorrhea and a global health challenge since no protective immunity results from infection and far fewer treatment options are available with increasing antimicrobial resistance. With no efficacious vaccines, researchers are exploring new targets for vaccine development and innovative therapeutics. The outer membrane TonB-dependent transporters (TdTs) produced by N. gonorrhoeae are considered promising antigen targets as they are highly conserved and play crucial roles in overcoming nutritional immunity. One of these TdTs, the hemoglobin transport system comprised of HpuA and HpuB, allows N. gonorrhoeae to acquire iron from hemoglobin (hHb). In the current study, mutations in the hpuB gene were generated to better understand the structure-function relationships in HpuB. This study is one of the first to demonstrate that N. gonorrhoeae can bind to and utilize hemoglobin produced by animals other than humans. This study also determined that when HpuA is absent, mutations targeting extracellular loop 7 of HpuB led to defective hHb binding and utilization. However, when the lipoprotein HpuA is present, these loop 7 mutants recovered their ability to bind hHB, although their growth phenotype remained significantly impaired. Interestingly, loop 7 contains putative heme binding motifs and a hypothetical -helical region. Taken together, these results highlight the importance of loop 7 in the functionality of HpuB in binding hHb, and extracting and internalizing iron.

microbiology↗

bioRxiv2021

Reconstitution of surface lipoprotein translocation reveals Slam as an outer membrane translocon in Gram-negative bacteria

Surface lipoproteins (SLPs) are peripherally attached to the outer leaflet of the outer membrane in many Gram-negative bacteria, playing significant roles in nutrient acquisition and immune evasion in the host. While the factors that are involved in the synthesis and delivery of SLPs in the inner membrane are well characterized, the molecular machineries required for the movement of SLPs to the surface are still not fully elucidated. In this study, we investigated the translocation of a surface lipoprotein TbpB through a Slam1-dependent pathway. Using purified components, we developed an in vitro translocation assay where unfolded TbpB is transported through Slam1 containing proteoliposomes, confirming Slam1 as an outer membrane translocon. While looking to identify factors to increase translocation efficiency, we discovered the periplasmic chaperone Skp interacted with TbpB in the periplasm of Escherichia coli. The presence of Skp was found to increase the translocation efficiency of TbpB in the reconstituted translocation assays. A knockout of Skp in Neisseria meningitidis revealed that Skp is essential for functional translocation of TbpB to the bacterial surface. Taken together, we propose a pathway for surface destined lipoproteins, where Skp acts as a holdase for Slam-mediated TbpB translocation across the outer membrane.

microbiology↗

bioRxiv2019

Transferrin binding protein B and Transferrin binding protein A2 expand the transferrin recognition range of Histophilus somni

The bacterial bipartite transferrin receptor is an iron acquisition system that is required for survival by several key human and animal pathogens. It consists of the TonB-dependent transporter Transferrin binding protein A (TbpA) and the surface lipoprotein Transferrin binding protein B (TbpB). Curiously, the Tbps are only found in host specific pathogens, and are themselves host specific, meaning that they will bind to the transferrin of their host species, but not to those of other animal species. While this phenomenon has long been established, neither the steps in the evolutionary process that led to this exquisite adaptation for the host, nor the steps that could alter it, are known. We sought to gain insight into these processes by studying Tbp specificity in Histophilus somni, a major pathogen of cattle. A past study showed that whole cells of H. somni specifically bind bovine transferrin, but not transferrin from sheep and goats, two bovids whose transferrins share 93% amino acid sequence identity with bovine transferrin. To our surprise, we found that H. somni can use sheep and goat transferrins as iron sources for growth, and that HsTbpB, but not HsTbpA, has detectable affinity for sheep and goat transferrins. Furthermore, a third transferrin binding protein, HsTbpA2, also showed affinity for sheep and goat transferrins. Our results show that H. somni TbpB and TbpA2 act to broaden the host transferrin recognition range of H. somni.\n\nImportanceHost restricted pathogens infect a single host species or a narrow range of host species. Histophilus somni, a pathogen that incurs severe economic losses for the cattle industry, infects cattle, sheep, and goats, but not other mammals. The transferrin binding proteins, TbpA and TbpB, are thought to be a key iron acquisition system in H. somni, however, surprisingly, they were also shown to be cattle transferrin-specific. In our study we find that H. somni TbpB, and another little-studied Tbp, TbpA2, bind sheep and goat transferrins as well as bovine transferrin. Our results suggest that TbpA2 may have allowed for host range expansion, and provide a mechanism for how host specificity in Tbp containing pathogens can be altered.

microbiology↗

bioRxiv2018

Super-resolution Molecular Map of Basal Foot Reveals Novel Cilium in Airway Multiciliated Cells

Motile cilia are beating machines that play a critical role in airway defense. During airway cell differentiation, hundreds of motile cilia are templated from basal bodies that extend a basal foot, an appendage that links motile cilia together to ensure beating coordination. This assembly has thus far escaped structural analysis because its size is below the resolution limit. Here, we determine the molecular architecture and identify basal foot proteins using a super-resolution-driven approach. Quantitative super-resolution image analysis shows that the basal foot is organized in three main regions linked by elongated coiled-coil proteins. FIB-SEM tomography and comparative super-resolution mapping of basal feet reveal that, among hundreds of motile cilia of an airway cell, a hybrid cilium with features of primary and motile cilia is harbored. The hybrid cilium is conserved in mammalian multiciliated cells and originates from parental centrioles. We further demonstrate that this novel cilium is a signalling centre whose cellular position is dependent on flow.

cell biology↗