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Lee, S. E.

Publications and source records attributed to Lee, S. E..

35 records · Page 2Linked to original sources

Single-cell RNA-seq reveals alterations in peripheral CX3CR1 and nonclassical monocytes in familial tauopathy

BackgroundEmerging evidence from mouse models is beginning to elucidate the brains immune response to tau pathology, but little is known about the nature of this response in humans. In addition, it remains unclear to what extent tau pathology and the local inflammatory response within the brain influence the broader immune system. MethodsTo address these questions, we performed single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) from carriers of pathogenic variants in MAPT, the gene encoding tau. ResultsAnalysis of [~]181,000 individual PBMC transcriptomes from MAPT pathogenic variant carriers (n = 8) and healthy non-carrier controls (n = 8) demonstrated striking differential expression in monocytes and natural killer (NK) cells. We observed a marked reduction in the expression of CX3CR1 - the gene encoding the fractalkine receptor that is known to modulate tau pathology in mouse models - in monocytes and NK cells. We also observed a significant reduction in the abundance of nonclassical monocytes and dysregulated expression of nonclassical monocyte marker genes, including FCGR3A. Finally, we identified reductions in TMEM176A and TMEM176B, genes thought to be involved in the inflammatory response in human microglia. We confirmed differential expression of select biologically relevant genes dysregulated in our scRNA-seq data using droplet digital PCR as an orthogonal technique for quantitative validation. ConclusionsOur results suggest that human peripheral immune cell expression and abundance are modulated by tau-associated pathophysiologic changes. CX3CR1 and nonclassical monocytes in particular will be a focus of future work exploring the role of these peripheral signals in additional tau-associated neurodegenerative diseases.

neuroscience↗

Sex differences in pain: Spinal cord injury in female and male mice elicits neuropathic pain symptoms

Spinal cord injury (SCI) in humans frequently causes intractable chronic pain. Females are susceptible to worsened pain compared to males, and females may show higher pain prevalence after SCI. Despite this difference in clinical prevalence of SCI pain, few preclinical studies have systematically studied in rodents sex differences in SCI-elicited pain-related behaviors. Here, we leverage data from a large cohort of mice to test whether contusion SCI consistently causes pain symptoms in mice, and to establish whether female (vs. male) mice display heightened hypersensitivity after SCI. Mechanical and heat sensory thresholds were assessed using the von Frey test and Hargreaves test, respectively. In an initial experiment, female mice receiving moderate 60 kDyn SCI or moderate-to-severe 75 kDyn SCI at T9 both exhibited mechanical and heat pain symptoms compared to sham controls. 75 kDyn SCI caused excess motor deficits that confounded defining pain sensitivity at acute times, so the moderate SCI force was used for subsequent experiments. Next, adult female and male C57BL6/J mice received sham surgery or T9 moderate contusion SCI. Comparing female to male mice after SCI, we reveal that mice of both sexes displayed mechanical and heat hypersensitivity compared to sham controls, from acute-to-chronic post-injury times. Females had amplified SCI-elicited hypersensitivity compared to males. Our data suggest that thoracic contusion SCI elicits consistent and persistent pain-associated symptoms, which are more intense in female vs. male mice. These results have important implications for uncovering sex-specific mechanisms and therapeutic targets to ameliorate neuropathic pain after SCI.

neuroscience↗

Anxiety-like behaviors in mice unmasked: Revealing sex differences in anxiety using a novel light-heat conflict test

Anxiety and chronic pain afflict hundreds of millions worldwide. Both anxiety and pain are more prevalent in females compared to males. Unfortunately, robust sex differences in human anxiety are not recapitulated in rodent tests, and results from rodent pain studies frequently fail to translate clinically. Therefore, there is a need to develop tests that reflect the differential salience of anxiety or pain-related stimuli between the sexes. Accordingly, here we introduce the Thermal Increments Dark-Light (TIDAL) conflict test. The TIDAL test places an anxiety-relevant stimulus (dark vs. illuminated chamber) in conflict with a heat-related stimulus (incrementally heated vs. isothermic chamber); mice freely explore the dark-heating and illuminated-isothermic chambers. Here, we aim to determine whether the TIDAL conflict test reveals in mice underappreciated sex differences in anxiety and/or heat sensitivity. We establish in four distinct experiments that females on the TIDAL conflict test persist substantially longer on the dark-heated plate, suggesting that females as compared to males exhibit elevated anxiety-like behavior. Mice more strongly prefer the dark plate on the TIDAL conflict test compared to control thermal place preference with both chambers illuminated. We also reveal that an anxiety-relieving drug, paroxetine, reduces mouse preference for the heating dark plate, supporting the validity of the TIDAL test. Therefore, our new TIDAL conflict test reliably unmasks the relative salience of anxiety (vs. heat sensitivity): mice that are female exhibit robust anxiety-like behaviors not consistently observed in classical tests. Future studies should incorporate TIDAL and other conflict tests to better understand rodent behavior and to identify mechanisms underlying anxiety and pain.

neuroscience↗

Radiogenomics of C9orf72 expansion carriers reveals global transposable element de-repression and enables prediction of thalamic atrophy and clinical impairment

Hexanucleotide repeat expansion (HRE) within C9orf72 is the most common genetic cause of frontotemporal dementia (FTD). Thalamic atrophy occurs in both sporadic and familial FTD but is thought to distinctly affect HRE carriers. Separately, emerging evidence suggests widespread de-repression of transposable elements (TEs) in the brain in several neurodegenerative diseases, including C9orf72 HRE-mediated FTD (C9-FTD). Whether TE activation can be measured in peripheral blood and how the reduction in peripheral C9orf72 expression observed in HRE carriers relates to atrophy and clinical impairment remain unknown. We used the FreeSurfer pipeline and its extensions to assess the effects of C9orf72 HRE and clinical diagnosis (n = 78) on atrophy of thalamic nuclei. We also generated a novel, whole-blood RNA-seq dataset to determine the relationships between peripheral C9orf72 expression, TE activation, thalamic atrophy, and clinical severity (n = 114). We confirmed global thalamic atrophy and reduced C9orf72 expression in HRE carriers. Moreover, we identified disproportionate atrophy of the right mediodorsal lateral nucleus in HRE carriers and showed that C9orf72 expression associated with clinical severity, independent of thalamic atrophy. Strikingly, we found global peripheral activation of TEs, including the human endogenous LINE-1 element, L1HS. L1HS levels were associated with atrophy of multiple pulvinar nuclei, a thalamic region implicated in C9-FTD. Integration of peripheral transcriptomic and neuroimaging data from HRE carriers revealed atrophy of specific thalamic nuclei; demonstrated that C9orf72 levels relate to clinical severity; and identified marked de-repression of TEs, including L1HS, which predicted atrophy of FTD-relevant thalamic nuclei. Significance StatementPathogenic repeat expansion in C9orf72 is the most frequent genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis (C9-FTD/ALS). The clinical, neuroimaging, and pathological features of C9-FTD/ALS are well-characterized, whereas the intersections of transcriptomic dysregulation and brain structure remain largely unexplored. Herein, we utilized a novel radiogenomic approach to examine the relationship between peripheral blood transcriptomics and thalamic atrophy, a neuroimaging feature disproportionately impacted in C9-FTD/ALS. We confirmed reduction of C9orf72 in blood and found broad dysregulation of transposable elements--genetic elements typically repressed in the human genome--in symptomatic C9orf72 expansion carriers, which associated with atrophy of thalamic nuclei relevant to FTD. C9orf72 expression was also associated with clinical severity, suggesting that peripheral C9orf72 levels capture disease-relevant information.

neuroscience↗

Amplification of Olfactory Signals by Anoctamin 9 is Essential for Mammalian Olfaction: a Risk Factor for the Covid-19-associated Anosmia

Sensing smells of foods, prey, or predators determines animal survival. Olfactory sensory neurons in the olfactory epithelium (OE) detect odorants, where cAMP and Ca2+ play a significant role in transducing odorant inputs to electrical activity. Here we show Anoctamin 9, a cation channel activated by cAMP/PKA pathway, is expressed in the OE and amplifies olfactory signals. Ano9- deficient mice had reduced olfactory behavioral sensitivity, electro-olfactogram signals, and neural activity in the olfactory bulb. In line with the difference in olfaction between birds and other vertebrates, chick ANO9 failed to respond to odorants, whereas chick CNGA2, a major transduction channel, showed greater responses to cAMP. Importantly, single-cell transcriptome data from Covid-19 patients revealed that Ano9 transcripts were markedly suppressed among genes in the olfactory signal pathway. The signal amplification by ANO9 is essential for mammalian olfactory transduction, whose downregulation may be a risk factor for the olfactory dysfunction in Covid-19 patients.

cell biology↗

Low diversity and instability of the sinus microbiota over time in adults with cystic fibrosis

BackgroundChronic rhinosinusitis (CRS) is a common, yet underreported and understudied manifestation of upper respiratory disease in people with cystic fibrosis (CF). There are currently no standard of care guidelines for the management of CF CRS, but treatment of upper airway disease may ameliorate lower airway disease. We sought to inform future treatment guidelines by determining whether changes to sinus microbial community diversity and specific taxa known to cause CF lung disease are associated with increased respiratory disease and inflammation. MethodsWe performed 16S rRNA gene sequencing, supplemented with cytokine analyses, microscopy, and bacterial culturing, on samples from the sinuses of 27 adults with CF CRS at the University of Pittsburghs CF Sinus Clinic. At each study visit, participants underwent endoscopic paranasal sinus sampling and clinical evaluation. We identified key drivers of microbial community composition and evaluated relationships between diversity and taxa with disease outcomes and inflammation. FindingsSinus community diversity was low and the composition was unstable, with many participants exhibiting alternating dominance between Pseudomonas aeruginosa and Staphylococci over time. Despite a tendency for dominance by these two taxa, communities were highly individualized and shifted composition during exacerbation of sinus disease symptoms. Exacerbations were also associated with communities dominated by Staphylococcus spp. Reduced microbial community diversity was linked to worse sinus disease and the inflammatory status of the sinuses (including increased IL-1{beta}). Increased IL-1{beta} was also linked to worse sinus endoscopic appearance, and other cytokines were linked to microbial community dynamics. InterpretationTo our knowledge, this is the largest longitudinal study of microbial communities and cytokine secretion in CF CRS. Our work revealed previously unknown instability of sinus microbial communities and a link between inflammation, lack of microbial community diversity, and worse sinus disease. FundingCystic Fibrosis Foundation and US National Institutes of Health. Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSA search of the PubMed database on October 11, 2021 with the terms [cystic fibrosis sinus microbiome] yielded 16 results, and we have identified seven primary research articles on the CF CRS microbiome (including re-analyses of existing datasets). Most are cross-sectional cohort analyses, along with one prior longitudinal study of four adults at the University of Auckland, New Zealand. Together, these prior studies reveal similarities between CF CRS and CF sputum microbiomes, including low community diversity associated with sinus disease, the presence of common CF-associated microbes in the sinuses, and prevalence of sinus communities dominated by P. aeruginosa or Staphylococcus aureus. High levels of IL-1{beta} are linked to the presence of nasal polyps in CF CRS, and polymorphisms in the IL-1 receptor antagonist gene are associated with risk of CRS outside of the context of CF. Two prior studies of this cohort have been performed by our laboratory. One describes clinical indicators of CF sinus disease and the other links sinus infection biogeography to P. aeruginosa evolutionary genomics. Added value of this studyOur study is the first to examine longitudinal relationships between the host immune response (through cytokine profiling) and microbiota dynamics in CF CRS, including linking elevated IL-1{beta} to worse sinus disease through reduced sinus microbial community diversity. The longitudinal nature of our study also allowed us to uncover striking temporal instability of microbial communities in approximately half of our cohorts sinus microbial communities over two years, including switching between communities dominated by P. aeruginosa and Staphylococcus spp. This instability could hinder attempts to link the relative abundance of taxa to clinical outcomes of interest in cross-sectional studies (e.g., markers of disease progression). We also identified patterns of synergy and antagonism between specific taxa, and impacts of the host immune response in the sinuses on community composition. Implications of all the available evidenceTogether with prior CF CRS microbiome studies, our study underscores similarities between sinus and lower respiratory tract microbial community structure in CF, and we show how community structure tracks with inflammation and several disease measures. This work strongly suggests that clinical management of CRS could be leveraged to improve overall respiratory health in CF. Our work implicates elevated IL-1{beta} in reduced microbiota diversity and worse sinus disease in CF CRS, suggesting applications for existing therapies targeting IL-1{beta}. Finally, the widespread use of highly effective CFTR modulator therapy has led to less frequent availability of spontaneous expectorated sputum for microbiological surveillance of lung infections. A better understanding of CF sinus microbiology could provide a much-needed alternative site for monitoring respiratory infection status by important CF pathogens.

microbiology↗

SprayNPray: user-friendly taxonomic profiling of genome and metagenome contigs

Shotgun sequencing of cultured microbial isolates/individual eukaryotes (whole-genome sequencing) and microbial communities (metagenomics) has become commonplace in biology. Very often, sequenced samples encompass organisms spanning multiple domains of life, necessitating increasingly elaborate software for accurate taxonomic classification of assembled sequences. While many software tools for taxonomic classification exist, SprayNPray offers a quick and user-friendly, semiautomated approach, allowing users to separate contigs by taxonomy (and other metrics) of interest. Easy installation, usage, and intuitive output, which is amenable to visual inspection and/or further computational parsing, will reduce barriers for biologists beginning to analyze genomes and metagenomes. This approach can be used for broad-level overviews, preliminary analyses, or as a supplement to other taxonomic classification or binning software. SprayNPray profiles contigs using multiple metrics, including closest homologs from a user-specified reference database, gene density, read coverage, GC content, tetranucleotide frequency, and codon-usage bias. The output from this software is designed to allow users to spot-check metagenome-assembled genomes, identify, and remove contigs from putative contaminants in isolate assemblies, identify bacteria in eukaryotic assemblies (and vice-versa), and identify possible horizontal gene transfer events.

bioinformatics↗

Therapeutic base and prime editing of COL7A1 mutations in recessive dystrophic epidermolysis bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin fragility disorder caused by loss-of-function mutations in the COL7A1 gene, which encodes type VII collagen (C7), a protein that functions in skin adherence. From 36 Korean RDEB patients, we identified a total of 69 pathogenic mutations (40 variants without recurrence), including point mutations (72.5%) and insertion/deletion mutations (27.5%). We used base and prime editing to correct mutations in fibroblasts from two patients (Pat1, who carried a c.3631C>T mutation in one allele, and Pat2, who carried a c.2005C>T mutation in one allele). We applied adenine base editors (ABEs) to correct the pathogenic mutation or to bypass a premature stop codon in Pat1-derived primary fibroblasts. To expand the targeting scope, we also utilized prime editors (PEs) to correct the mutations in Pat1- and Pat2-derived fibroblasts. Ultimately, we found that both ABE- and PE-mediated correction of COL7A1 mutations restored full-length C7 expression, reversed the impaired adhesion and proliferation exhibited by the patient-derived fibroblasts, and, following transfer of edited patient-derived fibroblasts into the skin of immunodeficient mice, led to C7 deposition within the dermal-epidermal junction. These results suggest that base and prime editing could be feasible strategies for ex vivo gene editing to treat RDEB.

bioengineering↗

Visualization of reactive astrocytes in living brain of Alzheimer's disease patient

An early appearance of reactive astrocytes is a hallmark of Alzheimers disease (AD)1,2, providing a substrate for early diagnostic neuroimaging targets. However, there is no clinically validated neuroimaging probe to visualize the reactive astrogliosis in the human brain in vivo. Here, we report that PET/CT imaging with 11C-acetate and 18F-fluorodeoxyglucose (18F-FDG) functionally visualizes the reactive astrocyte-mediated neuronal hypometabolism in the brains with neuroinflammation and AD. We demonstrate that reactive astrocytes excessively absorb acetate through elevated monocarboxylate transporter-1 (MCT1), leading to aberrant GABA synthesis and release which suppresses neuronal glucose uptake through decreased glucose transporter-3 (GLUT3) in both animal and human brains. We propose the non-invasive functional PET/CT imaging for astrocytic acetate-hypermetabolism and neuronal glucose-hypometabolism as an advanced diagnostic strategy for early stages of AD.

neuroscience↗

Evolution and adaptation of Pseudomonas aeruginosa in the paranasal sinuses of people with cystic fibrosis

People with the genetic disorder cystic fibrosis (CF) harbor lifelong respiratory infections, with morbidity and mortality frequently linked to chronic lung infections dominated by the opportunistically pathogenic bacterium Pseudomonas aeruginosa. During chronic CF lung infections, a single clone of P. aeruginosa can persist for decades and dominate end-stage CF lung disease due to its propensity to adaptively evolve to the respiratory environment, a process termed "pathoadaptation". Chronic rhinosinusitis (CRS), chronic inflammation and infection of the sinonasal space, is highly prevalent in CF and the sinuses may serve as the first site in the respiratory tract to become colonized by bacteria that then proceed to seed lung infections. We identified three evolutionary genetic routes by which P. aeruginosa evolves in the sinuses of people with CF, including through the evolution of mutator lineages and proliferative insertion sequences and culminating in early genomic signatures of host-restriction. Our findings raise the question of whether a significant portion of the pathoadaptive phenotypes previously thought to have evolved in response to selective pressures in the CF lungs may have first arisen in the sinuses and underscore the link between sinonasal and lung disease in CF. Graphical abstract and highlightsO_LIPseudomonas aeruginosa undergoes adaptive evolution in the sinuses of people with CF C_LIO_LIOver time, pathoadapted strains display early signatures of genome degradation consistent with recent host restriction C_LIO_LIMutations previously thought to occur in CF lungs may have first evolved in sinuses C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/359844v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@13b36aborg.highwire.dtl.DTLVardef@8230e1org.highwire.dtl.DTLVardef@1570c3eorg.highwire.dtl.DTLVardef@1e4e530_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

Plant NLR targets P-type ATPase for executing plasma membrane depolarization leading to calcium influx and cell death

Hypersensitive response (HR) is a robust immune response mediated by plant nucleotide-binding and leucine-rich repeat receptor (NLR). However, the early molecular event linking NLR to cell death is obscure. Here we demonstrate that NLR targets plasma membrane H+-ATPases (PMA) generating electrochemical potential across the membrane. CCA309, an autoactive N-terminal domain of pepper coiled-coil NLR (CNL), associates with PMAs and its autoactivity is affected by silencing or overexpression of PMA. CCA309-induced extracellular alkalization accompanied with membrane depolarization is followed by calcium influx and cell death. CCA309 interacts with C-terminal regulatory domain of PMA and 14-3-3 negatively affects CCA309-induced cell death. Moreover, pharmacological experiments with fusicoccin, an irreversible PMA activator, confirmed that CC- and CNL-mediated cell death occurred through inhibiting PMA. We propose PMAs as the primary target of plasma membrane-associated CNL to disrupt electrochemical homeostasis leading to HR cell death.

plant biology↗

A three-dimensional Air-Liquid Interface Culture Model for the Study of Epstein-Barr virus Infection in the Nasopharynx

Epstein-Barr virus (EBV) infection is ubiquitous in humans and is associated with the cancer, nasopharyngeal carcinoma. EBV replicates in the differentiated layers of stratified keratinocytes but whether the other cell types of the airway epithelium are susceptible to EBV is unknown. Here, we demonstrate with primary nasopharyngeal cells grown at the air-liquid interface that the pseudostratified epithelium can be susceptible to EBV infection and we report that susceptible cell types with distinct EBV transcription profiles can be identified by single-cell RNA-sequencing. Although EBV infection in the nasopharynx has evaded detection in asymptomatic carriers, these findings demonstrate that EBV latent and lytic infection can occur in the cells of the nasopharyngeal epithelium.

microbiology↗

CRISPR/Cas9-mediated gene editing induces neurological recovery in an A53T-SNCA overexpression rat model of Parkinson's disease

To date, no publicly available disease-modifying therapy for Parkinsons disease has been developed. This can be partly attributed to the absence of techniques for in vivo deletion of the SNCA gene (encoding -synuclein), which is one of the key players in Parkinsons disease pathology. In particular, A53T-mutated SNCA (A53T-SNCA) is one of the most studied familial pathologic mutations in Parkinsons disease. Here we utilized a recently discovered genome editing technique, CRISPR/Cas9, to delete A53T-SNCA in vitro and in vivo. Among various CRISPR/Cas9 systems, SaCas9-KKH with a single guide RNA (sgRNA) targeting A53T-SNCA was packaged into adeno-associated virus. Adeno-associated virus carrying SaCas9-KKH significantly reduced A53T-SNCA levels in A53T-SNCA-overexpressed HEK293T cells, without off-target effects on wild-type SNCA. Furthermore, we tested the techniques in vivo therapeutic potential in a viral A53T-SNCA overexpression rat model of Parkinsons disease. Gene deletion of A53T-SNCA significantly prevented the overexpression of -synuclein, dopaminergic neurodegeneration, and parkinsonian motor symptoms, whereas a negative control without sgRNA did not. Our findings propose CRISPR/Cas9 system as a potential therapeutic tool for A53T-SNCA familial Parkinsons disease.

genetics↗

Undulating changes in human plasma proteome across lifespan are linked to disease

Aging is the predominant risk factor for numerous chronic diseases that limit healthspan. Mechanisms of aging are thus increasingly recognized as therapeutic targets. Blood from young mice reverses aspects of aging and disease across multiple tissues, pointing to the intriguing possibility that age-related molecular changes in blood can provide novel insight into disease biology. We measured 2,925 plasma proteins from 4,331 young adults to nonagenarians and developed a novel bioinformatics approach which uncovered profound non-linear alterations in the human plasma proteome with age. Waves of changes in the proteome in the fourth, seventh, and eighth decades of life reflected distinct biological pathways, and revealed differential associations with the genome and proteome of age-related diseases and phenotypic traits. This new approach to the study of aging led to the identification of unexpected signatures and pathways of aging and disease and offers potential pathways for aging interventions.

systems biology↗

The murine transcriptome reveals global aging nodes with organ-specific phase and amplitude

Aging is the single greatest cause of disease and death worldwide, and so understanding the associated processes could vastly improve quality of life. While the field has identified major categories of aging damage such as altered intercellular communication, loss of proteostasis, and eroded mitochondrial function1, these deleterious processes interact with extraordinary complexity within and between organs. Yet, a comprehensive analysis of aging dynamics organism-wide is lacking. Here we performed RNA-sequencing of 17 organs and plasma proteomics at 10 ages across the mouse lifespan. We uncover previously unknown linear and non-linear expression shifts during aging, which cluster in strikingly consistent trajectory groups with coherent biological functions, including extracellular matrix regulation, unfolded protein binding, mitochondrial function, and inflammatory and immune response. Remarkably, these gene sets are expressed similarly across tissues, differing merely in age of onset and amplitude. Especially pronounced is widespread immune cell activation, detectable first in white adipose depots in middle age. Single-cell RNA-sequencing confirms the accumulation of adipose T and B cells, including immunoglobulin J-expressing plasma cells, which also accrue concurrently across diverse organs. Finally, we show how expression shifts in distinct tissues are highly correlated with corresponding protein levels in plasma, thus potentially contributing to aging of the systemic circulation. Together, these data demonstrate a similar yet asynchronous inter- and intra-organ progression of aging, thereby providing a foundation to track systemic sources of declining health at old age.

genomics↗

A stealth adhesion factor contributes to Vibrio vulnificus pathogenicity: Flp pili play roles in host invasion, survival in the blood stream and resistance to complement activation

The tad operons encode the machinery required for adhesive Flp (fimbrial low-molecular-weight protein) pili biogenesis. Vibrio vulnificus, an opportunistic pathogen, harbors three distinct tad loci. Among them, only tad1 locus was highly upregulated in in vivo growing bacteria compared to in vitro culture condition. To understand the pathogenic roles of the three tad loci during infection, we constructed single, double and triple tad loci deletion mutants. Interestingly, only the{Delta} tad123 triple mutant cells exhibited significantly decreased lethality in mice. Ultrastructural observations revealed short, thin filamentous projections disappeared on the{Delta} tad123 mutant cells. Since the pilin was paradoxically non-immunogenic, a V5 tag was fused to Flp to visualize the pilin protein by using immunogold EM and immunofluorescence microscopy. The{Delta} tad123 mutant cells showed attenuated host cell adhesion, delayed RtxA1 exotoxin secretion and subsequently impaired translocation across the intestinal epithelium compared to wild type, which could be partially complemented with each wild type operon. The{Delta} tad123 mutant was susceptible to complement-mediated bacteriolysis, predominantly via the alternative pathway, suggesting stealth hiding role of the Tad pili. Taken together, all three tad loci cooperate to confer successful invasion of V. vulnificus into deeper tissue and evasion from host defense mechanisms, ultimately resulting in septicemia.\n\nAuthor SummaryTo understand the roles of the three Tad operons in the pathogenesis of V. vulnificus infection, we constructed mutant strain with single, double and triple Tad loci deletions. Employing a variety of mouse infection models coupled with molecular genetic analyses, we demonstrate here that all three Tad operons are required for V. vulnificus pathogenicity as the cryptic pili contribute to host cell and tissue invasion, survival in the blood, and resistance to complement activation.

microbiology↗

Multiple reference genome sequences of hot pepper reveal the massive evolution of plant disease resistance genes by retroduplication

Transposable elements (TEs) provide major evolutionary forces leading to new genome structure and species diversification. However, the role of TEs in the expansion of disease resistance gene families has been unexplored in plants. Here, we report high-quality de novo genomes for two peppers (Capsicum baccatum and C. chinense) and an improved reference genome (C. annuum). Dynamic genome rearrangements involving translocations among chromosome 3, 5 and 9 were detected in comparison between C. baccatum and the two other peppers. The amplification of athila LTR-retrotransposons, members of the gypsy superfamily, led to genome expansion in C. baccatum. In-depth genome-wide comparison of genes and repeats unveiled that the copy numbers of NLRs were greatly increased by LTR-retrotransposon-mediated retroduplication. Moreover, retroduplicated NLRs exhibited great abundance across the angiosperms, with most cases lineage-specific and thus recent events. Our study revealed that retroduplication has played key roles in the emergence of new disease-resistance genes in plants.

plant biology↗