Biology subjects

Kafer, L. A.

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

3 recordsLinked to original sources

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

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

Nasal microbionts differentially colonize and elicit cytokines in human nasal epithelial organoids

Nasal colonization by Staphylococcus aureus or Streptococcus pneumoniae is associated with an increased risk of infection by these pathobionts, whereas nasal colonization by Dolosigranulum species is associated with health. Human nasal epithelial organoids (HNOs) physiologically recapitulate human nasal respiratory epithelium with a robust mucociliary blanket. We reproducibly monocolonized HNOs with these three bacteria for up to 48 hours with varying kinetics across species. HNOs tolerated bacterial monocolonization with localization of bacteria to the mucus layer and with minimal cytotoxicity compared to uncolonized HNOs. Human nasal epithelium exhibited both species-specific and general cytokine responses, without induction of type I interferons, consistent with colonization rather than infection. Only live S. aureus colonization robustly induced IL-1 family cytokines, suggestive of inflammasome signaling. D. pigrum and live S. aureus decreased CXCL10, whereas S. pneumoniae increased CXCL11, chemokines involved in antimicrobial responses to both viruses and bacteria. Overall, HNOs are a compelling model system to reveal host-microbe dynamics at the human nasal mucosa. IMPORTANCEHuman nasal microbiota often includes highly pathogenic members, many of which are antimicrobial resistance threats, e.g., methicillin-resistant Staphylococcus aureus and antibiotic-resistant Streptococcus pneumoniae. Preventing colonization by nasal pathobionts decreases infections and transmission. In contrast, nasal microbiome studies identify candidate beneficial bacteria that might resist pathobiont colonization, e.g., Dolosigranulum pigrum. Discovering how these microbionts colonize the human nasal passages and means to reduce pathobiont colonization is limited by previous models. This creates an urgent need for human-based models that exemplify bacterial nasal colonization. We addressed this need by developing human nasal epithelial organoids (HNOs) as a new model system of bacterial nasal colonization. HNOs accurately represent the mucosal surface of the human nasal passages enabling exploration of bacterial-epithelial interactions, which is crucial since the epithelium instigates the initial innate immune response to bacteria. Here, we identified differential epithelial cytokine responses to these three bacteria setting the stage for future research.

microbiology↗