Biology subjects

Blutt, S.

Publications and source records attributed to Blutt, S..

8 recordsLinked to original sources

bioRxiv2026

RSV & rhinovirus coinfection order dictates outcome via type III interferon-mediated interference in infant respiratory epithelium

Coinfection with respiratory syncytial virus (RSV) and rhinovirus (RV) is the most frequently detected coinfection in infants, yet mechanisms governing viral interference and disease outcome remain poorly understood. Although infection order is a key determinant of coinfection outcome involving RSV, influenza, rhinovirus, and SARS-CoV-2, its role in RSV/RV coinfections has not been defined. Using infant-derived human nasal organoids at air-liquid interface, we demonstrate that coinfection order fundamentally determines the outcome of RSV/RV interactions. An initial RSV infection almost completely inhibits secondary RV coinfection, whereas primary RV infection only partially impairs a subsequent RSV coinfection. These distinct outcomes are accompanied by divergent temporal antiviral and pro-inflammatory responses. Mechanistically, we identify type III interferons as a sufficient mediator of RSV-induced restriction of RV, establishing a host-driven mechanism of viral interference. In contrast, partial impairment of RSV following RV infection occurs through an interferon-independent mechanism. Despite marked differences, RSV/RV coinfections do not exacerbate epithelial injury or innate immune responses beyond that induced by RSV alone, indicating RSV is the dominant driver of pathology in coinfections. Collectively, our data establishes coinfection order as a critical determinant of RSV/RV outcome and reveals novel and mechanistic insight into critical viral-host interactions in the infant nasal epithelium.

microbiology↗

bioRxiv2026

D-alanine aminotransferase (Dat) promotes Staphylococcus aureus colonization fitness on human nasal respiratory epithelium

Nasal colonization by Staphylococcus aureus is an established risk factor for invasive infection, yet bacterial determinants promoting fitness on human nasal mucosa remain incompletely defined. To identify genes required for early colonization of human nasal respiratory epithelium, we colonized human nasal epithelial organoids differentiated at air-liquid interface (HNO-ALI) with a high-density transposon (Tn) library of the methicillin-resistant USA300 strain LAC. TnSeq analysis identified 165 genes that met our threshold for candidate colonization fitness factors. Among these, genes involved in D-alanine biosynthesis and use were enriched, including two encoding the enzymes that separately synthesize D-alanine in S. aureus: alanine racemase 1 (alr1) and D-alanine aminotransferase (dat). Disruption of dat reduced colonization fitness in competition with the parental strain by [≥] 1,000 fold across 4 different strains from clonal complexes 8, 5, and 30. In competition with the parental strain during HNO-ALI colonization, a dat::Tn mutant was 34-fold less fit than an alr1::Tn mutant. Genetic complementation with single-copy dat expressed from its native operon promoter restored parental colonization levels. Supplementation with exogenous D-alanine or L-alanine also rescued the dat::Tn colonization defect, whereas D-glutamate did not, consistent with Dat primarily producing D-alanine on nasal mucosa. Complementation with dat under control of a putative 5 intra-operon promoter substantially restored colonization but failed to support growth in chemically defined medium lacking L-alanine, suggesting a new layer of environment-specific regulation of dat transcription. Together, these findings demonstrate that Dat is a major source of D-alanine during colonization of human nasal mucosa and is required for S. aureus fitness in this environment. AUTHOR SUMMARYStaphylococcus aureus is the second leading cause of death due to bacterial infection globally, and nasal colonization is a major risk factor for invasive disease. Using a physiologically relevant, host-derived model of human nasal respiratory epithelium (HNO-ALI) and TnSeq, we identified 165 candidate genes contributing to S. aureus fitness during nasal mucosal colonization. We found that D-alanine aminotransferase (Dat) is the predominant source of D-alanine during nasal colonization, whereas alanine racemase (Alr1) predominates in rich medium, revealing an environment-specific hierarchy of D-alanine biosynthesis. Disruption of dat caused a > 1,000-fold defect in colonization in competition with the parental strain across multiple S. aureus clonal complexes, supporting a conserved role for dat in nasal colonization fitness. Additionally, we provide evidence that dat transcription from a previously cryptic promoter might be regulated by nasal mucosal conditions. Alr1 is proposed as an antimicrobial target in other bacterial pathogens; however, our data suggest that targeting Dat may be more effective for S. aureus nasal decolonization.

microbiology↗

bioRxiv2026

RSV Infects the Human Nasal Epithelium via the Basolateral Route with Distinct Subgroup Infectivity and Basal Cell Tropism

Respiratory syncytial virus (RSV) causes millions of lower respiratory tract infections (LRTIs) in young children, older adults, and immunocompromised populations every year. RSV infection initiates in the upper respiratory tract and can progress to the lower airways resulting in bronchiolitis, pneumonia, and even death. RSV primarily infects epithelial cells apically, but we hypothesized that basolateral exposure of the respiratory epithelium could provide an alternative mechanism of infection that contributes to LRTI development. Using a human nose organoid-air liquid interface (HNO-ALI) model, we performed apical and basolateral inoculations with contemporaneous RSV strains (RSV/A/ON and RSV/B/BA) representing the two RSV subgroups (A and B) in both adult and infant derived HNO-ALIs. Basolateral RSV exposure resulted in delayed viral replication and apical release compared to apical infection. A statistically significant difference in basolateral infection frequency was observed between RSV/B/BA and RSV/A/ON (81.3% versus 25%). Basolateral infection selectively targeted a rare basal cell population, while preserving epithelial integrity. Using undifferentiated HNO-ALIs, we determined for the first time that Krt23+ activated basal cells (ABCs) are uniquely susceptible to RSV infection, a finding we confirmed in fully differentiated HNO-ALIs. Together, our findings show that RSV can infect the respiratory epithelium from the basolateral side by initially targeting a rare subset of basal cells before spreading apically to ciliated cells. Moreover, RSV/B/BA may have an advantage over RSV/A/ON in utilizing the basolateral infection route. These findings highlight an alternative RSV infection pathway and could be a potential mechanism for RSV spread to the lower airways. ImportanceUnderstanding the pathogenesis of RSV is essential to understanding and preventing acute and long-term sequelae from infection. The canonical understanding of RSV infection is that the virus infects and is restricted to the apical ciliated cells upon inhalation or fomite exposure. We demonstrate that an alternative route of infection - the basolateral route, can be utilized by RSV to infect the apical ciliated cells of the respiratory epithelium. We also show for the first time a novel difference in infectivity between the two contemporaneous RSV strains (RSV/A/ON and RSV/B/BA). In addition, we describe a rare basal subset-the Krt23+ activated basal cells that are uniquely susceptible to RSV thus expanding the known cellular tropism of RSV. Infection of basal cells can impact airway differentiation, homeostasis, and remodeling. Overall, our findings expand on the pathogenesis of RSV and indicate there are alternative mechanisms of infection and cell populations that are susceptible to RSV.

microbiology↗

bioRxiv2026

Streptococcus pneumoniae colonization modulates human nasal epithelial responses to respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is a major cause of morbidity and mortality in infants globally. Specific nasal bacterial genera are differentially associated with RSV severity in infants: Haemophilus and Streptococcus with more severe disease and Dolosigranulum with healthy controls or milder outcomes. We hypothesized these differential bacterial effects begin at the epithelial level. Therefore, we established human nasal epithelial organoids differentiated at air-liquid interface (HNO-ALI) as a model system to assess effects of individual nasal microbionts on the epithelial response to subsequent RSV infection and of RSV on those microbionts. Infant-derived HNO-ALI were monocolonized with either Streptococcus pneumoniae, nontypeable Haemophilus influenzae, or Dolosigranulum pigrum one day before viral infection. RSV reduced colonizing S. pneumoniae and D. pigrum levels without affecting H. influenzae. S. pneumoniae precolonization uniquely reduced RSV levels during infection. S. pneumoniae precolonization also modulated the epithelial transcriptional response to RSV infection more so than H. influenzae or D. pigrum, with a pronounced effect on genes involved in immune response, cell cycle, stress, and growth signaling. Gene set enrichment analysis showed S. pneumoniae precolonization blunted RSV-induced increase in inflammatory and immune responses, consistent with S. pneumoniae also modulating RSV-induced cytokine production. Furthermore, S. pneumoniae precolonization blocked RSV-mediated dysregulation of cell-cycle genes, consistent with preventing arrest. Bacterial rescue of cell-cycle progression is a potential mechanism for reduced infectious virion production, since cell-cycle arrest enhances RSV replication. HNO-ALI facilitated elucidation of bacterial-viral-epithelial interplay at a frequent site of viral infection, directly linking nasal bacterial colonization to RSV infection dynamics. IMPORTANCEMost RSV-related hospitalizations occur in healthy young children, yet predictors of RSV severity in this population are limited. The nasal passages are a major microbiota site exposed to frequent respiratory viral infections. Specific nasal bacterial genera/species correlate with RSV clinical outcomes; however, the biological basis of these associations is poorly defined. Here, we established human nasal epithelial organoids differentiated at air-liquid interface (HNO-ALI) as a tractable experimental system for defining the interplay between bacteria, virus, and epithelial cells in the human nasal passages, enabling mechanistic insight into how the nasal microbiota contribute to RSV disease severity. Using this approach, we found that precolonization with each of the common nasal bacteria S. pneumoniae, H. influenzae, or D. pigrum differentially affected the nasal epithelial response to RSV infection. This work highlights that the nasal epithelium actively integrates microbial and viral signals and that variation in bacterial colonization can shape viral infection trajectories.

microbiology↗

bioRxiv2024

Advancements in Human Norovirus Cultivation in Human Intestinal Enteroids

Human noroviruses (HuNoVs) are a significant cause of epidemic and sporadic acute gastroenteritis worldwide. The lack of a reproducible culture system hindered the study of HuNoV replication and pathogenesis for almost a half-century. This barrier was overcome with our successful cultivation of multiple HuNoV strains in human intestinal enteroids (HIEs), which has significantly advanced HuNoV research. We optimized culture media conditions and generated genetically-modified HIE cultures to enhance HuNoV replication in HIEs. Building upon these achievements, we now present new insights to this culture system, which involve testing different media, unique HIE lines, and additional virus strains. HuNoV infectivity was evaluated and compared in new HIE models, including HIEs generated from different intestinal segments of individual adult organ donors, HIEs from human intestinal organoids produced from directed differentiation of human embryonic stem cells into intestinal organoids that were transplanted and matured in mice before making enteroids (H9tHIEs), genetically-engineered (J4FUT2 knock-in [KI], J2STAT1 knock-out [KO]) HIEs, as well as HIEs derived from a patient with common variable immunodeficiency (CVID) and from infants. Our findings reveal that small intestinal HIEs, but not colonoids, from adults, H9tHIEs, HIEs from a CVID patient, and HIEs from infants support HuNoV replication with segment and strain-specific differences in viral infection. J4FUT2-KI HIEs exhibit the highest susceptibility to HuNoV infection, allowing the cultivation of a broader range of GI and GII HuNoV strains than previously reported. Overall, these results contribute to a deeper understanding of HuNoVs and highlight the transformative potential of HIE cultures in HuNoV research. ImportanceHuNoVs cause global diarrheal illness and chronic infections in immunocompromised patients. This manuscript reports approaches for cultivating HuNoVs in secretor positive human intestinal enteroids (HIEs). HuNoV infectivity was compared in new HIE models, including ones from i) different intestinal segments of single donors, ii) human embryonic stem cell-derived organoids transplanted into mice, iii) genetically-modified lines, and iv) a patient with chronic variable immunodeficiency disease. HIEs from small intestine, but not colon, support HuNoV replication with donor, segment and strain-specific variations. Unexpectedly, HIEs from one donor are resistant to GII.3 infection. The genetically-modified J4FUT2-KI HIEs enable cultivation of a broad range of GI and GII genotypes. New insights into strain-specific differences in HuNoV replication in HIEs support this platform for advancing understanding of HuNoV biology and developing potential therapeutics.

microbiology↗

bioRxiv2023

Functional Genomics of Gastrointestinal Escherichia coli Isolated from Patients with Cancer and Diarrhea

We describe the epidemiology and clinical characteristics of 29 patients with cancer and diarrhea in whom Enteroaggregative Escherichia coli (EAEC) was initially identified by GI BioFire panel multiplex. E. coli strains were successfully isolated from fecal cultures in 14 of 29 patients. Six of the 14 strains were identified as EAEC and 8 belonged to other diverse E. coli groups of unknown pathogenesis. We investigated these strains by their adherence to human intestinal organoids, cytotoxic responses, antibiotic resistance profile, full sequencing of their genomes, and annotation of their functional virulome. Interestingly, we discovered novel and enhanced adherence and aggregative patterns for several diarrheagenic pathotypes that were not previously seen when co-cultured with immortalized cell lines. EAEC isolates displayed exceptional adherence and aggregation to human colonoids compared not only to diverse GI E. coli, but also compared to prototype strains of other diarrheagenic E. coli. Some of the diverse E. coli strains that could not be classified as a conventional pathotype also showed an enhanced aggregative and cytotoxic response. Notably, we found a high carriage rate of antibiotic resistance genes in both EAEC strains and diverse GI E. coli isolates and observed a positive correlation between adherence to colonoids and the number of metal acquisition genes carried in both EAEC and the diverse E. coli strains. This work indicates that E. coli from cancer patients constitute strains of remarkable pathotypic and genomic divergence, including strains of unknown disease etiology with unique virulomes. Future studies will allow for the opportunity to re-define E. coli pathotypes with greater diagnostic accuracy and into more clinically relevant groupings.

microbiology↗

bioRxiv2021

The human nose organoid respiratory virus model: an ex-vivo human challenge model to study RSV and SARS-CoV-2 pathogenesis and evaluate therapeutics

There is an unmet need for pre-clinical models to understand the pathogenesis of human respiratory viruses; and predict responsiveness to immunotherapies. Airway organoids can serve as an ex-vivo human airway model to study respiratory viral pathogenesis; however, they rely on invasive techniques to obtain patient samples. Here, we report a non-invasive technique to generate human nose organoids (HNOs) as an alternate to biopsy derived organoids. We made air liquid interface (ALI) cultures from HNOs and assessed infection with two major human respiratory viruses, respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Infected HNO-ALI cultures recapitulate aspects of RSV and SARS-CoV-2 infection, including viral shedding, ciliary damage, innate immune responses, and mucus hyper-secretion. Next, we evaluated the feasibility of the HNO-ALI respiratory virus model system to test the efficacy of palivizumab to prevent RSV infection. Palivizumab was administered in the basolateral compartment (circulation) while viral infection occurred in the apical ciliated cells (airways), simulating the events in infants. In our model, palivizumab effectively prevented RSV infection in a concentration dependent manner. Thus, the HNO-ALI model can serve as an alternate to lung organoids to study respiratory viruses and testing therapeutics.

microbiology↗

bioRxiv2020

Obesity phenotypes are preserved in intestinal stem cell enteroids from morbidly obese patients

Obesity and obesity-related comorbidities are significant health care challenges. Bariatric surgery (BS) is the most effective therapy for treating obesity and type 2 diabetes. A barrier in the development of therapeutic alternatives is incomplete mechanistic understanding of the benefits of BS and the lack of human intestinal models that recapitulate the pathophysiology of obesity. Using adult intestinal stem cell-derived enteroid cultures established from healthy lean subjects and morbidly obese patients, including post-BS cases, four phenotypes correlating patient BMI and intestinal glucose absorption were identified suggesting that enteroids retain patient phenotype heterogeneity associated with healthy and diseased state. In a sub-population of obese patients, increased dietary glucose absorption and gluconeogenesis was due to significantly higher expression of intestinal carbohydrate transporters (SGLT1, GLUT2 and GLUT5) and gluconeogenic enzymes (PEPCK1 and G6Pase) compared to enteroids from lean subjects that demonstrated low glucose absorption and lacked gluconeogenesis. Enteroids established from successful BS cases exhibited low glucose absorption similar to that observed in lean subjects. These data show that human enteroids preserve the patient phenotype in long-term cultures and represent a reliable preclinical model to study the heterogeneity of the obesity mechanisms, which is necessary to determine the efficacy of therapeutic interventions.

physiology↗