Mucosal Gene Expression in Response to SARS-CoV-2 Is Associated with Viral Load.

Little is known about the relationships between symptomatic early severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load and upper airway mucosal gene expression and immune response. To examine the association of symptomatic SARS-CoV-2 early viral load with upper airway mucosal gene expression, we profiled the host mucosal transcriptome from nasopharyngeal swab samples from 68 adults with symptomatic, mild-to-moderate coronavirus disease 19 (COVID-19). We measured SARS-CoV-2 viral load using reverse transcription-quantitative PCR (RT-qPCR). We then examined the association of SARS-CoV-2 viral load with upper airway mucosal immune response. We detected SARS-CoV-2 in all samples and recovered >80% of the genome from 95% of the samples from symptomatic COVID-19 adults. The respiratory virome was dominated by SARS-CoV-2, with limited codetection of other respiratory viruses, with the human Rhinovirus C being identified in 4 (6%) samples. This limited codetection of other respiratory viral pathogens may be due to the implementation of public health measures, like social distancing and masking practices. We observed a significant positive correlation between SARS-CoV-2 viral load and interferon signaling (OAS2, OAS3, IFIT1, UPS18, ISG15, ISG20, IFITM1, and OASL), chemokine signaling (CXCL10 and CXCL11), and adaptive immune system (IFITM1, CD300E, and SIGLEC1) genes in symptomatic, mild-to-moderate COVID-19 adults, when adjusting for age, sex, and race. Interestingly, the expression levels of most of these genes plateaued at a cycle threshold (CT) value of ~25. Overall, our data show that the early nasal mucosal immune response to SARS-CoV-2 infection is viral load dependent, potentially modifying COVID-19 outcomes. IMPORTANCE Several prior studies have shown that SARS-CoV-2 viral load can predict the likelihood of disease spread and severity. A higher detectable SARS-CoV-2 plasma viral load was associated with worse respiratory disease severity. However, the relationship between SARS-CoV-2 viral load, airway mucosal gene expression, and immune response remains elusive. We profiled the nasal mucosal transcriptome from nasal samples collected from adults infected with SARS-CoV-2 during spring 2020 with mild-to-moderate symptoms using a comprehensive metatranscriptomics method. We observed a positive correlation between SARS-CoV-2 viral load, interferon signaling, chemokine signaling, and adaptive immune system in adults with COVID-19. Our data suggest that early nasal mucosal immune response to SARS-CoV-2 infection was viral load dependent and may modify COVID-19 outcomes.

Children with eosinophilic esophagitis non-responsive to combination therapy have distinct esophageal transcriptomic and microbiome profile.

BACKGROUND: A combination of proton-pump inhibitors (PPI) and topical steroids (TS) is used to treat children with eosinophilic esophagitis (EoE). However, a subset of children do not respond to this combination therapy. We aimed to identify the esophageal transcriptional, cell composition, and microbial differences between the non-responders (EoE-PPI-TSnr; n = 7) and responders (EoE-PPI-TSr; n = 7) to the combination therapy for EoE and controls (n = 9) using metatranscriptomics. METHODS: Differential gene expression analysis was used to identify transcriptional differences, validated using the EoE diagnostic panel (EDP). Deconvolution analysis was performed to identify differences in their cell type composition. Microbiome analysis was conducted from esophageal biopsies RNAseq data, and microbial abundance was correlated with esophageal gene expression. RESULTS: In all, 3164 upregulated and 3154 downregulated genes distinguished EoE-PPI-TSnr from EoE-PPI-TSr. Eosinophilic inflammatory response, cytokine signaling, and collagen formation pathways were significantly upregulated in EoE-PPI-TSnr. There was a 56% overlap in dysregulated genes between EoE-PPI-TSnr and EDP, with a perfect agreement in the directionality of modulation. Eosinophils, dendritic cells (DCs), immature DCs, megakaryocytic-erythroid progenitors, and T helper type 1 cells were significantly higher in EoE-PPI-TSnr. There was no significant difference in microbiome diversity. The relative abundance of Fusobacterium sp. and Acinetobacter sp. notably differed in EoE-PPI-TSnr and correlated with the key pathways. CONCLUSION: Our results provide critical insights into the molecular, cellular, and microbial factors associated with the lack of response to PPI and TS combination therapy in children with EoE. This study advances our understanding of the pathobiology of EoE while guiding personalized treatment strategies.

Association between Oral Microbiome and Esophageal Diseases: A State-of-the-Art Review.

BACKGROUND: Esophageal conditions result in significant morbidity and mortality worldwide. There is growing enthusiasm for discerning the role of microbiome in esophageal diseases. Conceivably, the focus has been on examining the role of local microbiome in esophageal diseases although this is somewhat limited by the invasive approach required to sample the esophageal tissue. Given the ease of sampling the oral cavity combined with the advances in genomic techniques, there is immense interest in discovering the role of the oral microbiome in esophageal conditions. SUMMARY: In this review, we aim to discuss the current evidence highlighting the association between the oral microbiome and esophageal diseases. In particular, we have focused on summarizing the alterations in oral microbiome associated with malignant, premalignant, and benign esophageal cancers, inflammatory and infectious conditions, and esophageal dysmotility diseases. Identifying alterations in the oral microbiome is a key to advancing our understanding of the etiopathogenesis and progression of esophageal diseases, promoting novel diagnostics, and laying the foundation for personalized treatment approaches. KEY MESSAGES: Further studies are needed to unravel the mechanisms by which the oral microbiome influences the development and progression of esophageal diseases, as well as to investigate whether alterations in the oral microbiome can impact the natural history of various esophageal diseases.

SARS-CoV-2 infection and viral load are associated with the upper respiratory tract microbiome.

BACKGROUND: Little is known about the relationships between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the respiratory virus responsible for the ongoing coronavirus disease 2019 (COVID-19) pandemic, and the upper respiratory tract (URT) microbiome. OBJECTIVE: We sought to compare the URT microbiome between SARS-CoV-2-infected and -uninfected adults and to examine the association of SARS-CoV-2 viral load with the URT microbiome during COVID-19. METHODS: We characterized the URT microbiome using 16S ribosomal RNA sequencing in 59 adults (38 with confirmed, symptomatic, mild to moderate COVID-19 and 21 asymptomatic, uninfected controls). In those with COVID-19, we measured SARS-CoV-2 viral load using quantitative reverse transcription PCR. We then examined the association of SARS-CoV-2 infection status and its viral load with the ⍺-diversity, ß-diversity, and abundance of bacterial taxa of the URT microbiome. Our main models were all adjusted for age and sex. RESULTS: The observed species index was significantly higher in SARS-CoV-2-infected than in -uninfected adults (ß linear regression coefficient = 7.53; 95% CI, 0.17-14.89; P = .045). In differential abundance testing, 9 amplicon sequence variants were significantly different in both of our comparisons, with Peptoniphilus lacrimalis, Campylobacter hominis, Prevotella 9 copri, and an Anaerococcus unclassified amplicon sequence variant being more abundant in those with SARS-CoV-2 infection and in those with high viral load during COVID-19, whereas Corynebacterium unclassified, Staphylococcus haemolyticus, Prevotella disiens, and 2 Corynebacterium_1 unclassified amplicon sequence variants were more abundant in those without SARS-CoV-2 infection and in those with low viral load during COVID-19. CONCLUSIONS: Our findings suggest complex associations between SARS-CoV-2 and the URT microbiome in adults. Future studies are needed to examine how these viral-bacterial interactions can impact the clinical progression, severity, and recovery of COVID-19.

Persistent Gut Microbial Dysbiosis in Children with Acute Lymphoblastic Leukemia (ALL) During Chemotherapy.

Prophylactic or therapeutic antibiotic use along with chemotherapy treatment potentially has a long-standing adverse effect on the resident gut microbiota. We have established a case-control cohort of 32 pediatric and adolescent acute lymphoblastic leukemia (ALL) patients and 25 healthy siblings (sibling controls) to assess the effect of chemotherapy as well as antibiotic prophylaxis on the gut microbiota. We observe that the microbiota diversity and richness of the ALL group is significantly lower than that of the control group at diagnosis and during chemotherapy. The microbiota diversity is even lower in antibiotics-exposed ALL patients. Although the gut microbial diversity tends to stabilize after 1-year post-chemotherapy, their abundances were altered because of chemotherapy and prophylactic antibiotic treatments. Specifically, the abundances of mucolytic gram-positive anaerobic bacteria, including Ruminococcus gnavus and Ruminococcus torques, tended to increase during the chemotherapy regimen and continued to be elevated 1 year beyond the initiation of chemotherapy. This dysbiosis may contribute to the development of gastrointestinal complications in ALL children following chemotherapy. These findings set the stage to further understand the role of the gut microbiome dynamics in ALL patients and their potential role in alleviating some of the adverse side effects of chemotherapy and antibiotics use in immunocompromised children.

Gastrointestinal microbial populations can distinguish pediatric and adolescent Acute Lymphoblastic Leukemia (ALL) at the time of disease diagnosis.

BACKGROUND: An estimated 15,000 children and adolescents under the age of 19 years are diagnosed with leukemia, lymphoma and other tumors in the USA every year. All children and adolescent acute leukemia patients will undergo chemotherapy as part of their treatment regimen. Fortunately, survival rates for most pediatric cancers have improved at a remarkable pace over the past three decades, and the overall survival rate is greater than 90 % today. However, significant differences in survival rate have been found in different age groups (94 % in 1-9.99 years, 82 % in ≥10 years and 76 % in ≥15 years). ALL accounts for about three out of four cases of childhood leukemia. Intensive chemotherapy treatment coupled with prophylactic or therapeutic antibiotic use could potentially have a long-term effect on the resident gastrointestinal (GI) microbiome. The composition of GI microbiome and its changes upon chemotherapy in pediatric and adolescent leukemia patients is poorly understood. In this study, using 16S rRNA marker gene sequences we profile the GI microbial communities of pediatric and adolescent acute leukemia patients before and after chemotherapy treatment and compare with the microbiota of their healthy siblings. RESULTS: Our study cohort consisted of 51 participants, made up of matched pediatric and adolescent patients with ALL and a healthy sibling. We elucidated and compared the GI microbiota profiles of patients and their healthy sibling controls via analysis of 16S rRNA gene sequencing data. We assessed the GI microbiota composition in pediatric and adolescent patients with ALL during the course of chemotherapy by comparing stool samples taken before chemotherapy with stool samples collected at varying time points during the chemotherapeutic treatment. The microbiota profiles of both patients and control sibling groups are dominated by members of Bacteroides, Prevotella, and Faecalibacterium. At the genus level, both groups share many taxa in common, but the microbiota diversity of the patient group is significantly lower than that of the control group. It was possible to distinguish between the patient and control groups based on their microbiota profiles. The top taxa include Anaerostipes, Coprococcus, Roseburia, and Ruminococcus2 with relatively higher abundance in the control group. The observed microbiota changes are likely the result of several factors including a direct influence of therapeutic compounds on the gut flora and an indirect effect of chemotherapy on the immune system, which, in turn, affects the microbiota. CONCLUSIONS: This study provides significant information on GI microbiota populations in immunocompromised children and opens up the potential for developing novel diagnostics based on stool tests and therapies to improve the dysbiotic condition of the microbiota at the time of diagnosis and in the earliest stages of chemotherapy.

Mining host-pathogen protein interactions to characterize Burkholderia mallei infectivity mechanisms.

Burkholderia pathogenicity relies on protein virulence factors to control and promote bacterial internalization, survival, and replication within eukaryotic host cells. We recently used yeast two-hybrid (Y2H) screening to identify a small set of novel Burkholderia proteins that were shown to attenuate disease progression in an aerosol infection animal model using the virulent Burkholderia mallei ATCC 23344 strain. Here, we performed an extended analysis of primarily nine B. mallei virulence factors and their interactions with human proteins to map out how the bacteria can influence and alter host processes and pathways. Specifically, we employed topological analyses to assess the connectivity patterns of targeted host proteins, identify modules of pathogen-interacting host proteins linked to processes promoting infectivity, and evaluate the effect of crosstalk among the identified host protein modules. Overall, our analysis showed that the targeted host proteins generally had a large number of interacting partners and interacted with other host proteins that were also targeted by B. mallei proteins. We also introduced a novel Host-Pathogen Interaction Alignment (HPIA) algorithm and used it to explore similarities between host-pathogen interactions of B. mallei, Yersinia pestis, and Salmonella enterica. We inferred putative roles of B. mallei proteins based on the roles of their aligned Y. pestis and S. enterica partners and showed that up to 73% of the predicted roles matched existing annotations. A key insight into Burkholderia pathogenicity derived from these analyses of Y2H host-pathogen interactions is the identification of eukaryotic-specific targeted cellular mechanisms, including the ubiquitination degradation system and the use of the focal adhesion pathway as a fulcrum for transmitting mechanical forces and regulatory signals. This provides the mechanisms to modulate and adapt the host-cell environment for the successful establishment of host infections and intracellular spread.

A second-generation protein-protein interaction network of Helicobacter pylori.

Helicobacter pylori infections cause gastric ulcers and play a major role in the development of gastric cancer. In 2001, the first protein interactome was published for this species, revealing over 1500 binary protein interactions resulting from 261 yeast two-hybrid screens. Here we roughly double the number of previously published interactions using an ORFeome-based, proteome-wide yeast two-hybrid screening strategy. We identified a total of 1515 protein-protein interactions, of which 1461 are new. The integration of all the interactions reported in H. pylori results in 3004 unique interactions that connect about 70% of its proteome. Excluding interactions of promiscuous proteins we derived from our new data a core network consisting of 908 interactions. We compared our data set to several other bacterial interactomes and experimentally benchmarked the conservation of interactions using 365 protein pairs (interologs) of E. coli of which one third turned out to be conserved in both species.

Using host-pathogen protein interactions to identify and characterize Francisella tularensis virulence factors.

BACKGROUND: Francisella tularensis is a select bio-threat agent and one of the most virulent intracellular pathogens known, requiring just a few organisms to establish an infection. Although several virulence factors are known, we lack an understanding of virulence factors that act through host-pathogen protein interactions to promote infection. To address these issues in the highly infectious F. tularensis subsp. tularensis Schu S4 strain, we deployed a combined in silico, in vitro, and in vivo analysis to identify virulence factors and their interactions with host proteins to characterize bacterial infection mechanisms. RESULTS: We initially used comparative genomics and literature to identify and select a set of 49 putative and known virulence factors for analysis. Each protein was then subjected to proteome-scale yeast two-hybrid (Y2H) screens with human and murine cDNA libraries to identify potential host-pathogen protein-protein interactions. Based on the bacterial protein interaction profile with both hosts, we selected seven novel putative virulence factors for mutant construction and animal validation experiments. We were able to create five transposon insertion mutants and used them in an intranasal BALB/c mouse challenge model to establish 50 % lethal dose estimates. Three of these, ΔFTT0482c, ΔFTT1538c, and ΔFTT1597, showed attenuation in lethality and can thus be considered novel F. tularensis virulence factors. The analysis of the accompanying Y2H data identified intracellular protein trafficking between the early endosome to the late endosome as an important component in virulence attenuation for these virulence factors. Furthermore, we also used the Y2H data to investigate host protein binding of two known virulence factors, showing that direct protein binding was a component in the modulation of the inflammatory response via activation of mitogen-activated protein kinases and in the oxidative stress response. CONCLUSIONS: Direct interactions with specific host proteins and the ability to influence interactions among host proteins are important components for F. tularensis to avoid host-cell defense mechanisms and successfully establish an infection. Although direct host-pathogen protein-protein binding is only one aspect of Francisella virulence, it is a critical component in directly manipulating and interfering with cellular processes in the host cell.

Novel Burkholderia mallei virulence factors linked to specific host-pathogen protein interactions.

Burkholderia mallei is an infectious intracellular pathogen whose virulence and resistance to antibiotics makes it a potential bioterrorism agent. Given its genetic origin as a commensal soil organism, it is equipped with an extensive and varied set of adapted mechanisms to cope with and modulate host-cell environments. One essential virulence mechanism constitutes the specialized secretion systems that are designed to penetrate host-cell membranes and insert pathogen proteins directly into the host cell's cytosol. However, the secretion systems' proteins and, in particular, their host targets are largely uncharacterized. Here, we used a combined in silico, in vitro, and in vivo approach to identify B. mallei proteins required for pathogenicity. We used bioinformatics tools, including orthology detection and ab initio predictions of secretion system proteins, as well as published experimental Burkholderia data to initially select a small number of proteins as putative virulence factors. We then used yeast two-hybrid assays against normalized whole human and whole murine proteome libraries to detect and identify interactions among each of these bacterial proteins and host proteins. Analysis of such interactions provided both verification of known virulence factors and identification of three new putative virulence proteins. We successfully created insertion mutants for each of these three proteins using the virulent B. mallei ATCC 23344 strain. We exposed BALB/c mice to mutant strains and the wild-type strain in an aerosol challenge model using lethal B. mallei doses. In each set of experiments, mice exposed to mutant strains survived for the 21-day duration of the experiment, whereas mice exposed to the wild-type strain rapidly died. Given their in vivo role in pathogenicity, and based on the yeast two-hybrid interaction data, these results point to the importance of these pathogen proteins in modulating host ubiquitination pathways, phagosomal escape, and actin-cytoskeleton rearrangement processes.

The protein interaction network of bacteriophage lambda with its host, Escherichia coli.

Although most of the 73 open reading frames (ORFs) in bacteriophage λ have been investigated intensively, the function of many genes in host-phage interactions remains poorly understood. Using yeast two-hybrid screens of all lambda ORFs for interactions with its host Escherichia coli, we determined a raw data set of 631 host-phage interactions resulting in a set of 62 high-confidence interactions after multiple rounds of retesting. These links suggest novel regulatory interactions between the E. coli transcriptional network and lambda proteins. Targeted host proteins and genes required for lambda infection are enriched among highly connected proteins, suggesting that bacteriophages resemble interaction patterns of human viruses. Lambda tail proteins interact with both bacterial fimbrial proteins and E. coli proteins homologous to other phage proteins. Lambda appears to dramatically differ from other phages, such as T7, because of its unusually large number of modified and processed proteins, which reduces the number of host-virus interactions detectable by yeast two-hybrid screens.

Mucosal Microbiome Disruption in Acute Laryngeal Injury Following Intubation.

The objective of this study was to characterize mucosal microbial shifts in patients with acute laryngeal injury (ALgI) after intubation. This cross-sectional study included 20 patients with ALgI who underwent early endoscopic intervention with tissue culture, 20 patients with idiopathic subglottic stenosis (iSGS) who underwent tissue culture during the routine endoscopic intervention, and 3 control patients who underwent mucosal swab culture. 70% of the ALgI patients had a positive culture compared to 5% of the iSGS patients and none of the controls. The most identified microbes isolated from ALgI patients included Staphylococcus species in 30% and Streptococcus species in 25%. The high rate of pathologic bacterial infiltration into postintubation laryngeal wounds supports efforts to reduce bacterial colonization of endotracheal tubes and highlights the role of culture-directed antibiotic therapy as a part of early intervention to improve outcomes for patients with ALgI.

From TgO/GABA-AT, GABA, and T-263 Mutant to Conception of Toxoplasma.

Toxoplasma gondii causes morbidity, mortality, and disseminates widely via cat sexual stages. Here, we find T. gondii ornithine aminotransferase (OAT) is conserved across phyla. We solve TgO/GABA-AT structures with bound inactivators at 1.55 Å and identify an inactivator selective for TgO/GABA-AT over human OAT and GABA-AT. However, abrogating TgO/GABA-AT genetically does not diminish replication, virulence, cyst-formation, or eliminate cat's oocyst shedding. Increased sporozoite/merozoite TgO/GABA-AT expression led to our study of a mutagenized clone with oocyst formation blocked, arresting after forming male and female gametes, with "Rosetta stone"-like mutations in genes expressed in merozoites. Mutations are similar to those in organisms from plants to mammals, causing defects in conception and zygote formation, affecting merozoite capacitation, pH/ionicity/sodium-GABA concentrations, drawing attention to cyclic AMP/PKA, and genes enhancing energy or substrate formation in TgO/GABA-AT-related-pathways. These candidates potentially influence merozoite's capacity to make gametes that fuse to become zygotes, thereby contaminating environments and causing disease.

Studying protein complexes by the yeast two-hybrid system.

Protein complexes are typically analyzed by affinity purification and subsequent mass spectrometric analysis. However, in most cases the structure and topology of the complexes remains elusive from such studies. Here we investigate how the yeast two-hybrid system can be used to analyze direct interactions among proteins in a complex. First we tested all pairwise interactions among the seven proteins of Escherichia coli DNA polymerase III as well as an uncharacterized complex that includes MntR and PerR. Four and seven interactions were identified in these two complexes, respectively. In addition, we review Y2H data for three other complexes of known structure which serve as "gold-standards", namely Varicella Zoster Virus (VZV) ribonucleotide reductase (RNR), the yeast proteasome, and bacteriophage lambda. Finally, we review an Y2H analysis of the human spliceosome which may serve as an example for a dynamic mega-complex.

Proton Pump Inhibitors Modulate Gene Expression Profile in Esophageal Mucosa and Microbiome.

OBJECTIVE: Proton pump inhibitors (PPIs) are commonly used to manage children with upper gastrointestinal symptoms and without a formal diagnosis. We investigated the effect of PPIs on esophageal mucosal transcriptome and active microbiota in children with normal esophagi. Furthermore, we examined whether the differences in host esophageal mucosal gene expression were driven by an underlying esophageal epithelial cell type composition. METHODS: Using metatranscriptomics, the host transcriptional and active microbial profiles were captured from 17 esophageal biopsy samples (PPI naïve [PPI-], n = 7; PPI exposed [PPI+], n = 10) collected from children without any endoscopic and histologic abnormalities in their esophagus (normal esophagus). Deconvolution computational analysis was performed with xCell to assess if the observed epithelial gene expression changes were related to the cell type composition in the esophageal samples. RESULTS: The median (IQR) age of our cohort was 14 years (12-16) with female (63%) preponderance. Both groups were similar in terms of their demographics and clinical features. Compared with PPI-, the PPI+ had upregulation of 27 genes including the MUC genes. The cell type composition was similar between the PPI- and PPI+ groups. Prevotella sp and Streptococcus sp were abundant in PPI+ group. CONCLUSIONS: In children with normal esophagus, PPI exposure can be associated with upregulation of esophageal mucosal homeostasis and epithelial cell function genes in a cell-type independent manner, and an altered esophageal microbiome. Additional studies are warranted to validate our findings and to investigate the causal effect of PPIs on the normal esophageal epithelium and microbial communities.

Upper respiratory tract microbiota dynamics following COVID-19 in adults.

To date, little is known about the effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the coronavirus disease 2019 (COVID-19) pandemic, on the upper respiratory tract (URT) microbiota over time. To fill this knowledge gap, we used 16S ribosomal RNA gene sequencing to characterize the URT microbiota in 48 adults, including (1) 24 participants with mild-to-moderate COVID-19 who had serial mid-turbinate swabs collected up to 21 days after enrolment and (2) 24 asymptomatic, uninfected controls who had mid-turbinate swabs collected at enrolment only. To compare the URT microbiota between groups in a comprehensive manner, different types of statistical analyses that are frequently employed in microbial ecology were used, including ⍺-diversity, ß-diversity and differential abundance analyses. Final statistical models included age, sex and the presence of at least one comorbidity as covariates. The median age of all participants was 34.00 (interquartile range=28.75-46.50) years. In comparison to samples from controls, those from participants with COVID-19 had a lower observed species index at day 21 (linear regression coefficient=-13.30; 95 % CI=-21.72 to -4.88; q=0.02). In addition, the Jaccard index was significantly different between samples from participants with COVID-19 and those from controls at all study time points (PERMANOVA q<0.05 for all comparisons). The abundance of three amplicon sequence variants (ASVs) (one Corynebacterium ASV, Frederiksenia canicola, and one Lactobacillus ASV) were decreased in samples from participants with COVID-19 at all seven study time points, whereas the abundance of one ASV (from the family Neisseriaceae) was increased in samples from participants with COVID-19 at five (71.43 %) of the seven study time points. Our results suggest that mild-to-moderate COVID-19 can lead to alterations of the URT microbiota that persist for several weeks after the initial infection.

Idiopathic subglottic stenosis arises at the epithelial interface of host and pathogen.

Background: Idiopathic subglottic stenosis (iSGS) is a rare fibrotic disease of the proximal airway affecting adult Caucasian women nearly exclusively. Life-threatening ventilatory obstruction occurs secondary to pernicious subglottic mucosal scar. Disease rarity and wide geographic patient distribution has previously limited substantive mechanistic investigation into iSGS pathogenesis. Result: By harnessing pathogenic mucosa from an international iSGS patient cohort and single-cell RNA sequencing, we unbiasedly characterize the cell subsets in the proximal airway scar and detail their molecular phenotypes. Results show that the airway epithelium in iSGS patients is depleted of basal progenitor cells, and the residual epithelial cells acquire a mesenchymal phenotype. Observed displacement of bacteria beneath the lamina propria provides functional support for the molecular evidence of epithelial dysfunction. Matched tissue microbiomes support displacement of the native microbiome into the lamina propria of iSGS patients rather than disrupted bacterial community structure. However, animal models confirm that bacteria are necessary for pathologic proximal airway fibrosis and suggest an equally essential role for host adaptive immunity. Human samples from iSGS airway scar demonstrate adaptive immune activation in response to the proximal airway microbiome of both matched iSGS patients and healthy controls. Clinical outcome data from iSGS patients suggests surgical extirpation of airway scar and reconstitution with unaffected tracheal mucosa halts the progressive fibrosis. Conclusion: Our data support an iSGS disease model where epithelial alterations facilitate microbiome displacement, dysregulated immune activation, and localized fibrosis. These results refine our understanding of iSGS and implicate shared pathogenic mechanisms with distal airway fibrotic diseases.

Species-specific transcriptomic changes upon respiratory syncytial virus infection in cotton rats.

The cotton rat (Sigmodon) is the gold standard pre-clinical small animal model for respiratory viral pathogens, especially for respiratory syncytial virus (RSV). However, without a reference genome or a published transcriptome, studies requiring gene expression analysis in cotton rats are severely limited. The aims of this study were to generate a comprehensive transcriptome from multiple tissues of two species of cotton rats that are commonly used as animal models (Sigmodon fulviventer and Sigmodon hispidus), and to compare and contrast gene expression changes and immune responses to RSV infection between the two species. Transcriptomes were assembled from lung, spleen, kidney, heart, and intestines for each species with a contig N50 > 1600. Annotation of contigs generated nearly 120,000 gene annotations for each species. The transcriptomes of S. fulviventer and S. hispidus were then used to assess immune response to RSV infection. We identified 238 unique genes that are significantly differentially expressed, including several genes implicated in RSV infection (e.g., Mx2, I27L2, LY6E, Viperin, Keratin 6A, ISG15, CXCL10, CXCL11, IRF9) as well as novel genes that have not previously described in RSV research (LG3BP, SYWC, ABEC1, IIGP1, CREB1). This study presents two comprehensive transcriptome references as resources for future gene expression analysis studies in the cotton rat model, as well as provides gene sequences for mechanistic characterization of molecular pathways. Overall, our results provide generalizable insights into the effect of host genetics on host-virus interactions, as well as identify new host therapeutic targets for RSV treatment and prevention.