Faculty Bibliography

Trichuris nematodes reproduce within the microbiota-rich mammalian intestine and lay thousands of eggs daily, facilitating their sustained presence in the environment and hampering eradication efforts. Here, we show that bacterial byproducts facilitate the reproductive development of nematodes. First, we employed a pipeline using the well-characterized, free-living nematode C. elegans to identify microbial factors with conserved roles in nematode reproduction. A screen for E. coli mutants that impair C. elegans fertility identified genes in fatty acid biosynthesis and ethanolamine utilization pathways, including fabH and eutN. Additionally, Trichuris muris eggs displayed defective hatching in the presence of fabH- or eutN-deficient E. coli due to reduced arginine or elevated aldehydes, respectively. T. muris reared in gnotobiotic mice colonized with these E. coli mutants displayed morphological defects and failed to lay viable eggs. These findings indicate that microbial byproducts mediate evolutionarily conserved transkingdom interactions that impact the reproductive fitness of distantly related nematodes.

Gastrointestinal effects associated with Coronavirus Disease 2019 (COVID-19) are highly variable for reasons that are not understood. In this study, we used intestinal organoid-derived cultures differentiated from primary human specimens as a model to examine interindividual variability. Infection of intestinal organoids derived from different donors with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) resulted in orders of magnitude differences in virus replication in small intestinal and colonic organoid-derived monolayers. Susceptibility to infection correlated with angiotensin I converting enzyme 2 (ACE2) expression level and was independent of donor demographic or clinical features. ACE2 transcript levels in cell culture matched the amount of ACE2 in primary tissue, indicating that this feature of the intestinal epithelium is retained in the organoids. Longitudinal transcriptomics of organoid-derived monolayers identified a delayed yet robust interferon signature, the magnitude of which corresponded to the degree of SARS-CoV-2 infection. Interestingly, virus with the Omicron variant spike (S) protein infected the organoids with the highest infectivity, suggesting increased tropism of the virus for intestinal tissue. These results suggest that heterogeneity in SARS-CoV-2 replication in intestinal tissues results from differences in ACE2 levels, which may underlie variable patient outcomes.

The Th17 cell-lineage-defining cytokine IL-17A contributes to host defense and inflammatory disease by coordinating multicellular immune responses. The IL-17 receptor (IL-17RA) is expressed by diverse intestinal cell types, and therapies targeting IL-17A induce adverse intestinal events, suggesting additional tissue-specific functions. Here, we used multiple conditional deletion models to identify a role for IL-17A in secretory epithelial cell differentiation in the gut. Paneth, tuft, goblet, and enteroendocrine cell numbers were dependent on IL-17A-mediated induction of the transcription factor ATOH1 in Lgr5⁺ intestinal epithelial stem cells. Although dispensable at steady state, IL-17RA signaling in ATOH1⁺ cells was required to regenerate secretory cells following injury. Finally, IL-17A stimulation of human-derived intestinal organoids that were locked into a cystic immature state induced ATOH1 expression and rescued secretory cell differentiation. Our data suggest that the cross talk between immune cells and stem cells regulates secretory cell lineage commitment and the integrity of the mucosa.

In this issue of JEM, Fay et al. (2021. J. Exp. Med.https://doi.org/10.1084/jem.20211220) cohouse dirty pet store mice and rats with clean laboratory mice to gain insights into infection dynamics, discover new viruses, and identify relationships between viruses and the microbiome.

Extracellular vesicles of endosomal origin, exosomes, mediate intercellular communication by transporting substrates with a variety of functions related to tissue homeostasis and disease. Their diagnostic and therapeutic potential has been recognized for diseases such as cancer in which signaling defects are prominent. However, it is unclear to what extent exosomes and their cargo inform the progression of infectious diseases. We recently defined a subset of exosomes termed defensosomes that are mobilized during bacterial infection in a manner dependent on autophagy proteins. Through incorporating protein receptors on their surface, defensosomes mediated host defense by binding and inhibiting pore-forming toxins secreted by bacterial pathogens. Given this capacity to serve as decoys that interfere with surface protein interactions, we investigated the role of defensosomes during infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19. Consistent with a protective function, exosomes containing high levels of the viral receptor ACE2 in bronchioalveolar lavage fluid from critically ill COVID-19 patients was associated with reduced ICU and hospitalization times. We found ACE2+ exosomes were induced by SARS-CoV-2 infection and activation of viral sensors in cell culture, which required the autophagy protein ATG16L1, defining these as defensosomes. We further demonstrate that ACE2+ defensosomes directly bind and block viral entry. These findings suggest that defensosomes may contribute to the antiviral response against SARS-CoV-2 and expand our knowledge on the regulation and effects of extracellular vesicles during infection.

Epidemic RNA viruses seem to arise year after year leading to countless infections and devastating disease. SARS-CoV-2 is the most recent of these viruses, but there will undoubtedly be more to come. While effective SARS-CoV-2 vaccines are being deployed, one approach that is still missing is effective antivirals that can be used at the onset of infections and therefore prevent pandemics. Here, we screened FDA-approved compounds against SARS-CoV-2. We found that atovaquone, a pyrimidine biosynthesis inhibitor, is able to reduce SARS-CoV-2 infection in human lung cells. In addition, we found that berberine chloride, a plant-based compound used in holistic medicine, was able to inhibit SARS-CoV-2 infection in cells through direct interaction with the virion. Taken together, these studies highlight potential avenues of antiviral development to block emerging viruses. Such proactive approaches, conducted well before the next pandemic, will be essential to have drugs ready for when the next emerging virus hits.

Introduction: Traveler's diarrhea is the most common travel related illness and is often encountered in the outpatient setting. Most pathogens that cause traveler's diarrhea are poorly characterized by conventional stool testing such as stool culture and ova and parasite exam. Multiplex polymerase chainreaction based gastrointestinal pathogen panels (GI PCR) offer advantages in sensitivity and turnaround time, however their impact on clinical management is unclear. Our aim was to evaluate the clinical impact of GI PCR testing in outpatients presenting with traveler's diarrhea.
Method(s): We performed a retrospective study comparing outpatients presenting with acute gastrointestinal symptoms at an academic medical center who received stool testing with GI PCR from September 2015 to April 2019, to patients who presented with similar symptoms from February 2009 to April 2012, when GI PCR was unavailable, and received stool culture and ova and parasite exam ("conventional testing"). Patients who received GI PCR were matched by age and sex to patients who received conventional testing. We recorded pathogens isolated, demographic data, history of travel within 30 days of presentation, presenting symptoms and rates of antibiotic therapy.
Result(s): We identified 1,018 outpatients who received GI PCR or conventional stool testing. (n5509 each). There were 70 patients in the conventional cohort and 78 in the GI PCR cohort, respectively, with traveler's diarrhea. In patients who received GI PCR, we observed higher diagnostic yield in patients with traveler's diarrhea who received GI PCR, compared to patients without a history of recent travel (54 vs. 39%, p50.01, Table 1). There was no difference in diagnostic yield in patients who received conventional stool testing (9 vs 10%, p50.74). Patients with travelers' diarrhea were more likely to have pathogenic species of Escherichia coli (E. coli) identified on testing (45 vs. 22%, p, 0.01, Table 2), particularly Enteroaggregative E. coli (23 vs. 6%, p, 0.01). Patients with traveler's diarrhea were more likely to receive antibiotics whether they received GI PCR (38 vs. 27%, p50.04) or conventional testing (35 vs. 25%, p50.06).
Conclusion(s): GI PCR was associated with a higher diagnostic yield in patients presenting with travelers' diarrhea. Patients with travelers' diarrhea were significantly more likely to have pathogenic E. coli species identified on PCR testing, with up to 45% of patients testing positive

Introduction: Multiplex gastrointestinal pathogen panels (GI PCR) for the detection of enteric infection offer advantages in turnaround time and sensitivity compared to conventional modalities of stool testing. Previous research on GI PCR has focused almost exclusively on hospitalized patients, however, most cases of gastroenteritis are managed in the outpatient setting. Our aim was to compare GI PCR to conventional stool testing in outpatients with gastroenteritis and evaluate the impact on clinical decision-making and management.
Method(s): We performed a retrospective study of outpatients presenting with acute gastrointestinal symptoms at an academicmedical center from September 2015 to April 2019 who received stool testing with GI PCR, and from February 2009 to April 2012 who received stool culture and ova and parasite exam ("conventional testing"). Patients who received GI PCR were matched by age and sex to patients who received conventional testing. We recorded pathogens isolated, demographic data, presenting symptoms, risk factors, antibiotic therapy, and downstream associated healthcare utilization including abdominal imaging, endoscopy, emergency room visits, hospitalizations, and abdominal surgeries. Univariate and multivariate linear regression was used to estimate the effect of testing on empiric antibiotic therapy.
Result(s): We identified 1,018 outpatients who received GI PCR or conventional stool testing. (n = 509 each). A pathogen was isolated in 208 (41%) patients with GI PCR and 49 (10%, P< 0.01, Table 1) with conventional culture. There were no significant differences in overall rates of antibiotic therapy or average duration of therapy, but GI PCR was associated with less empiric therapy (47% vs 70%, P < 0.01), and higher rates of narrowing (13 vs 4%) and discontinuation (3 vs 0%, P < 0.01) of empiric antibiotics after the initial visit, with a 1.6-fold lower likelihood (95% CI 1.04-2.57, P= 0.04) of initiating empiric antibiotics on multivariable analysis. There were no significant differences in other outcomes.
Conclusion(s): GI PCR testing detected more pathogens compared to conventional stool testing, without a significant increase in antibiotics prescribed. PCR testing was associated with changes in prescribing patterns for antibiotics, with a shift towards less empiric therapy

The microbial populations in the gut microbiome have recently been associated with COVID-19 disease severity. However, a causal impact of the gut microbiome on COVID-19 patient health has not been established. Here we provide evidence that gut microbiome dysbiosis is associated with translocation of bacteria into the blood during COVID-19, causing life-threatening secondary infections. Antibiotics and other treatments during COVID-19 can potentially confound microbiome associations. We therefore first demonstrate that the gut microbiome is directly affected by SARS-CoV-2 infection in a dose-dependent manner in a mouse model, causally linking viral infection and gut microbiome dysbiosis. Comparison with stool samples collected from 97 COVID-19 patients at two different clinical sites also revealed substantial gut microbiome dysbiosis, paralleling our observations in the animal model. Specifically, we observed blooms of opportunistic pathogenic bacterial genera known to include antimicrobial-resistant species in hospitalized COVID-19 patients. Analysis of blood culture results testing for secondary microbial bloodstream infections with paired microbiome data obtained from these patients suggest that bacteria translocate from the gut into the systemic circulation of COVID-19 patients. These results are consistent with a direct role for gut microbiome dysbiosis in enabling dangerous secondary infections during COVID 19.