Biology subjects

Lewis, L. A.

Publications and source records attributed to Lewis, L. A..

5 recordsLinked to original sources

bioRxiv2025

The gonococcal vaccine candidate antigen NGO1701 is a N. gonorrhoeae periplasmic copper storage protein

The increasing worldwide trend of antibiotic-resistant Neisseria gonorrhoeae strains highlights the urgent need for new therapeutic strategies against this sexually transmitted pathogen, including a gonococcal vaccine. We previously designed a bioinformatics-based candidate selection pipeline (CASS) and identified potential novel gonococcal vaccine targets among hypothetical proteins expressed during natural human infection. One of these candidates, NGO1701, is a predicted periplasmic four-helix bundle protein with amino acid sequence homology to the copper storage protein 1 (Csp1) from Methylosinus trichosporium OB3b. In this study, we confirmed that purified NGO1701 binds 15 Cu(I) ions per monomer in vitro, supporting its function as Csp in N. gonorrhoeae. Using a ngo1701 deletion mutant generated in N. gonorrhoeae F62, we investigated its role in bacteria physiology. We showed that ablation of Csp was not limiting for bacterial growth and fitness in vitro, but the {Delta}csp strain became significantly more susceptible to copper mediated toxicity. This phenotype was rescued by csp gene complementation, indicating a role in protection against copper toxicity. Our results indicate that Csp participates in periplasmic copper homeostasis in N. gonorrhoeae, buffering excess copper to reduce toxicity and playing a putative role in copper delivery to important copper-enzymes. Csp does not appear to be involved in bacterial host cell interaction and activation in vitro, since no difference in the ability of N. gonorrhoeae to adhere/invade epithelial cells or induce IL-8 secretion was reported among wild type, csp deletion mutant and complemented strains. Furthermore, sera from mice immunized with NGO1701 failed to recognize {Delta}csp by dot blot and ELISA, and the seras ability to kill N. gonorrhoeae was abrogated against {Delta}csp. However, both functions were restored after gene complementation, supporting the relevance of Csp as a potential vaccine target. Allelic analysis of Neisseria species revealed that this gene is absent in N. meningitidis, thus making it a gonococcal-specific target. Author SummaryCopper is essential for bacterial metabolism but can be toxic in excess. Here, we identify NGO1701 as a copper storage protein (Csp) in Neisseria gonorrhoeae, capable of sequestering Cu(I) ions. Deletion of csp led to increased copper sensitivity, while overexpression restored resistance, suggesting a role in copper homeostasis. The {Delta}csp mutant also showed reduced growth in cobalt and manganese, likely due to metal interference by copper toxicity. Beyond detoxification, Csp may supply copper to essential cuproenzymes like cytochrome cbb3 oxidase and nitric oxide reductase, which support bacterial survival under host-imposed stress. Although Csp is not required for N. gonorrhoeae host cell interactions, it is a strong immune target. Immune recognition of N. gonorrhoeae {Delta}csp by anti- NGO1701 mouse sera was nearly abolished and the serum bactericidal activity was abrogated compared to N. gonorrhoeae F62 wild type bacteria, highlighting Csps potential as a target for therapeutic or vaccine strategies against N. gonorrhoeae.

immunology↗

bioRxiv2024

Spatial top-down proteomics for the functional characterization of human kidney

BackgroundThe Human Proteome Project has credibly detected nearly 93% of the roughly 20,000 proteins which are predicted by the human genome. However, the proteome is enigmatic, where alterations in amino acid sequences from polymorphisms and alternative splicing, errors in translation, and post-translational modifications result in a proteome depth estimated at several million unique proteoforms. Recently mass spectrometry has been demonstrated in several landmark efforts mapping the human proteoform landscape in bulk analyses. Herein, we developed an integrated workflow for characterizing proteoforms from human tissue in a spatially resolved manner by coupling laser capture microdissection, nanoliter-scale sample preparation, and mass spectrometry imaging. ResultsUsing healthy human kidney sections as the case study, we focused our analyses on the major functional tissue units including glomeruli, tubules, and medullary rays. After laser capture microdissection, these isolated functional tissue units were processed with microPOTS (microdroplet processing in one-pot for trace samples) for sensitive top-down proteomics measurement. This provided a quantitative database of 616 proteoforms that was further leveraged as a library for mass spectrometry imaging with near-cellular spatial resolution over the entire section. Notably, several mitochondrial proteoforms were found to be differentially abundant between glomeruli and convoluted tubules, and further spatial contextualization was provided by mass spectrometry imaging confirming unique differences identified by microPOTS, and further expanding the field-of-view for unique distributions such as enhanced abundance of a truncated form (1-74) of ubiquitin within cortical regions. ConclusionsWe developed an integrated workflow to directly identify proteoforms and reveal their spatial distributions. Where of the 20 differentially abundant proteoforms identified as discriminate between tubules and glomeruli by microPOTS, the vast majority of tubular proteoforms were of mitochondrial origin (8 of 10) where discriminate proteoforms in glomeruli were primarily hemoglobin subunits (9 of 10). These trends were also identified within ion images demonstrating spatially resolved characterization of proteoforms that has the potential to reshape discovery-based proteomics because the proteoforms are the ultimate effector of cellular functions. Applications of this technology have the potential to unravel etiology and pathophysiology of disease states, informing on biologically active proteoforms, which remodel the proteomic landscape in chronic and acute disorders.

biochemistry↗

bioRxiv2021

Comparative analysis of host-associated variation in Phytophthora cactorum

Phytophthora cactorum is often described as a generalist pathogen, with isolates causing disease in a range of plant species. It is the causative agent of two diseases in the cultivated strawberry, crown rot (CR; causing whole plant collapse) and leather rot (LR; affecting the fruit). In the cultivated apple, P. cactorum causes girdling bark rots on the scion (collar rot) and rootstock (crown rot), as well as necrosis of the fine root system (root rot) and fruit rots. We investigated evidence for host specialisation within P. cactorum through comparative genomic analysis of 18 isolates. Whole genome phylogenetic analysis provided genomic support for discrete lineages within P. cactorum, with well supported non-recombing clades for strawberry CR and apple infecting isolates specialised to strawberry crowns and apple tissue. Isolates of strawberry CR are genetically similar globally, while there is more diversity in apple-infecting isolates. We sought to identify the genetic basis of host specialisation, demonstrating gain and loss of effector complements within the P. cactorum phylogeny, representing putative determinants of host boundaries. Transcriptomic analysis highlighted that those effectors found to be specific to a single host or expanded in the strawberry lineage are amongst those most highly expressed during infection of strawberry and give a wider insight into the key effectors active during strawberry infection. Many effectors that had homologs in other Phytophthoras that have been characterised as avirulence genes were present but not expressed in our tested isolate. Our results highlight several RxLR-containing effectors that warrant further investigation to determine whether they are indeed virulence factors and host-specificity determinants for strawberry and apple. Furthermore, additional work is required to determine whether these effectors are suitable targets to focus attention on for future resistance breeding efforts.

microbiology↗

bioRxiv2018

A novel sialylation site on Neisseria gonorrhoeae lipooligosaccharide links heptose II lactose expression with pathogenicity

Sialylation of lacto-W-neotetraose (LNnT) extending from heptose I (HepI) of gonococcal lipooligosaccharide (LOS) contributes to pathogenesis. Previously, gonococcal LOS sialyltransterase (Lst) was shown to sialylate LOS in Triton X-100 extracts of strain 15253, which expresses lactose from both HepI and HepII, the minimal structure required for mAb 2C7 binding. Ongoing work has shown that growth of 15253 in cytidine monophospho-W-acetylneuraminic acid (CMP-Neu5Ac)-containing media enables binding to CD33/Siglec-3, a cell surface receptor that binds sialic acid, suggesting that lactose termini on LOS of intact gonococci can be sialylated. Neu5Ac was detected on LOSs of strains 15253 and a MS11 mutant with only lactose from HepI and HepII by mass spectrometry; deleting HepII lactose rendered Neu5Ac undetectable. Resistance of HepII lactose Neu5Ac to desialylation by 2-3-specific neuraminidase suggested an 2-6-linkage. Although not associated with increased factor H binding, HepII lactose sialylation inhibited complement C3 deposition on gonococci. 15253 mutants that lacked Lst or HepII lactose were significantly attenuated in mice, confirming the importance of HepII Neu5Ac in virulence. All 75 minimally passaged clinical isolates from Nanjing, China, expressed HepII lactose, evidenced by reactivity with mAb 2C7; mAb 2C7 was bactericidal against the first 62 (of 75) isolates that had been collected sequentially and were sialylated before testing. mAb 2C7 effectively attenuated 15253 vaginal colonization in mice. In conclusion, this novel sialylation site could explain the ubiquity of gonococcal HepII lactose in vivo. Our findings reiterate the candidacy of the 2C7 epitope as a vaccine antigen and mAb 2C7 as an immunotherapeutic antibody.

microbiology↗

bioRxiv2018

Assessing Combinability of Phylogenomic Data using Bayes Factors

With the rapid reduction in sequencing costs of high-throughput genomic data, it has become commonplace to use hundreds of genes/sites to infer phylogeny of any study system. While sampling large number of genes has given us a tremendous opportunity to uncover previously unknown relationships and improve phylogenetic resolution, it also presents us with new challenges when the phylogenetic signal is confused by differences in the evolutionary histories of sampled genes. Given the addition of accurate marginal likelihood estimation methods into popular Bayesian software programs, it is natural to consider using the Bayes Factor (BF) to compare different partition models in which genes within any given partition subset share both tree topology and edge lengths. We explore using marginal likelihood to assess data subset combinability when data subsets have varying levels of phylogenetic discordance due to deep coalescence events among genes (simulated within a species tree), and compare the results with our recently-described phylogenetic informational dissonance index (D) estimated for each data set. BF effectively detects phylogenetic incongruence, and provides a way to assess the statistical significance of D values. We discuss methods for calibrating BFs, and use calibrated BFs to assess data combinability using an empirical data set comprising 56 plastid genes from green algae order Volvocales.

evolutionary biology↗