Faculty Bibliography

Soil-transmitted intestinal worms known as helminths colonize over 1.5 billion people worldwide. Although helminth colonization has been associated with altered composition of the gut microbiota, such as increases in Clostridia, individual species have not been isolated and characterized. Here, we isolate and sequence the genome of 13 Clostridia from the Orang Asli, an indigenous population in Malaysia with a high prevalence of helminth infections. Metagenomic analysis of 650 fecal samples from urban and rural Malaysians confirm the prevalence of species corresponding to these isolates and reveal a specific association between Peptostreptococcaceae family members and helminth colonization. Remarkably, Peptostreptococcaceae isolated from the Orang Asli display superior capacity to promote the life cycle of whipworm species, including hatching of eggs from Trichuris muris and Trichuris trichiura. These findings support a model in which helminths select for gut colonization of microbes that support their life cycle.

In recent years, the contribution of exosomes to immunity, inflammation and host-pathogen interaction have been appreciated. Exosomes are small secreted extracellular vesicles from endosomal origin that contain a myriad of cellular molecules (protein, nucleic acids), including surface receptors. We have reported a pathogen-induced and macroautophagy/autophagy-dependent class of exosomes coined as "defensosomes", which protect the host from membrane-targeting toxins. In a recent study, we found that defensosomes decorated with ACE2, the SARS-CoV-2 cellular receptor, are produced in the lungs of patients with COVID-19, and that increased concentration of ACE2-loaded defensosomes is associated with decreased hospitalization length. Mechanistically, SARS-CoV-2 induces the production of ACE2-coated defensosomes, a process requiring the autophagy machinery, which in turn binds and neutralizes the virus. We propose that defensosomes represent a new form of autophagy-mediated innate immunity that contributes to the host's armamentarium against pathogens.

Although microbial populations in the gut microbiome are associated with COVID-19 severity, a causal impact on 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. We first demonstrate SARS-CoV-2 infection induces gut microbiome dysbiosis in mice, which correlated with alterations to Paneth cells and goblet cells, and markers of barrier permeability. Samples collected from 96 COVID-19 patients at two different clinical sites also revealed substantial gut microbiome dysbiosis, including blooms of opportunistic pathogenic bacterial genera known to include antimicrobial-resistant species. Analysis of blood culture results testing for secondary microbial bloodstream infections with paired microbiome data indicates that bacteria may 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.

Polymorphisms in the Apolipoprotein L1 (APOL1) gene are common in ancestrally African populations, and associate with kidney injury and cardiovascular disease. These risk variants (RV) provide an advantage in resisting Trypanosoma brucei, the causal agent of African trypanosomiasis, and are largely absent from non-African genomes. Clinical associations between the APOL1 high risk genotype (HRG) and disease are stronger in those with comorbid infectious or immune disease. To understand the interaction between cytokine exposure and APOL1 cytotoxicity, we established human umbilical vein endothelial cell (HUVEC) cultures representing each APOL1 genotype. Untreated HUVECs were compared to IFNÉ£-exposed; and APOL1 expression, mitochondrial function, lysosome integrity, and autophagic flux were measured. IFNÉ£ increased median APOL1 expression across all genotypes 22.1 (8.3 to 29.8) fold (p=0.02). Compared to zero risk variant-carrying HUVECs (0RV), HUVECs carrying 2 risk variant copies (2RV) showed both depressed baseline and maximum mitochondrial oxygen consumption (p<0.01), and impaired mitochondrial networking on MitoTracker assays. These cells also demonstrated a contracted lysosomal compartment, and an accumulation of autophagosomes suggesting a defect in autophagic flux. Upon blocking autophagy with non-selective lysosome inhibitor, hydroxychloroquine, autophagosome accumulation between 0RV HUVECs and untreated 2RV HUVECs was similar, implicating lysosomal dysfunction in the HRG-associated autophagy defect. Compared to 0RV and 2RV HUVECs, HUVECs carrying 1 risk variant copy (1RV) demonstrated intermediate mitochondrial respiration and autophagic flux phenotypes, which were exacerbated with IFNÉ£ exposure. Taken together, our data reveal that IFNÉ£ induces APOL1 expression, and that each additional RV associates with mitochondrial dysfunction and autophagy inhibition. IFNÉ£ amplifies this phenotype even in 1RV HUVECs, representing the first description of APOL1 pathobiology in variant heterozygous cell cultures.

Loss of Paneth cells and their antimicrobial granules compromises the intestinal epithelial barrier and is associated with Crohn's disease, a major type of inflammatory bowel disease^1-7. Non-classical lymphoid cells, broadly referred to as intraepithelial lymphocytes (IELs), intercalate the intestinal epithelium^8,9. This anatomical position has implicated them as first-line defenders in resistance to infections, but their role in inflammatory disease pathogenesis requires clarification. The identification of mediators that coordinate crosstalk between specific IEL and epithelial subsets could provide insight into intestinal barrier mechanisms in health and disease. Here we show that the subset of IELs that express γ and δ T cell receptor subunits (γδ IELs) promotes the viability of Paneth cells deficient in the Crohn's disease susceptibility gene ATG16L1. Using an ex vivo lymphocyte-epithelium co-culture system, we identified apoptosis inhibitor 5 (API5) as a Paneth cell-protective factor secreted by γδ IELs. In the Atg16l1-mutant mouse model, viral infection induced a loss of Paneth cells and enhanced susceptibility to intestinal injury by inhibiting the secretion of API5 from γδ IELs. Therapeutic administration of recombinant API5 protected Paneth cells in vivo in mice and ex vivo in human organoids with the ATG16L1 risk allele. Thus, we identify API5 as a protective γδ IEL effector that masks genetic susceptibility to Paneth cell death.

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 Coronavirus Disease 2019 (COVID-19). Consistent with a protective function, exosomes containing high levels of the viral receptor ACE2 in bronchoalveolar lavage fluid (BALF) from critically ill COVID-19 patients was associated with reduced intensive care unit (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.

In Malaysia, soil-transmitted helminth (STH) infections still persist among indigenous communities. In the past, local studies have focused mostly on epidemiologic aspects of STH infections with a scarcity of information on the efficacy of deworming treatment. The present study consisted of 2 phases: a cross-sectional phase on current epidemiological status and risk factors of STH infections and a longitudinal study over 6 weeks on triple dose albendazole efficacy against STH infections. A total of 253 participants were recruited at baseline and a pre-tested questionnaire was administered to obtain information on socio-demographics, environmental and behavioural risk factors. Stool samples were evaluated using a modified Kato-Katz technique. Cure rate (CR) and egg reduction rate (ERR) were assessed at 3 weeks following a 3-day course of 400mg albendazole treatment and infection status were observed again at 6 weeks. Baseline positivity of trichuriasis, ascariasis and hookworm infections were 56.1%, 11.9% and 20.2%, respectively. Multivariate analysis showed age below 18 years old (P = 0.004), without latrine in house (P = 0.042) and indiscriminate defecation (P = 0.032) were associated with STH infections. In the longitudinal study (N = 89), CR for trichuriasis was 64.6%, while CR of 100% was observed for both ascariasis and hookworm. ERR was above 90% for all three STH species. A rapid increased of Trichuris trichiura egg output was observed at 6 weeks. In conclusion, STH infections are highly prevalent among indigenous communities. Children and teenagers, poor sanitation and hygiene behaviour were determinants for STH infections. Triple dose albendazole is found to be efficacious against Ascaris lumbricoides and hookworm infections but has moderate curative effect with high ERR against T. trichiura. Although triple dose albendazole regimen has logistic challenges and may not be a routine option, consideration of this treatment regime may still be necessary in selective communities to reduce high intensity of T. trichiura infection.

Background/UNASSIGNED:The epidemiology of nosocomial bloodstream infections (NBSIs) in patients with coronavirus disease 2019 (COVID-19) is poorly understood, due in part to substantial disease heterogeneity resulting from multiple potential pathogens. Methods/UNASSIGNED:We identified risk factors for NBSIs and examined the association between NBSIs and mortality in a retrospective cohort of patients hospitalized with COVID-19 in 2 New York City hospitals during the height of the pandemic. We adjusted for the potential effects of factors likely to confound that association, including age, race, illness severity upon admission, and underlying health status. Results/UNASSIGNED:infections did not have an identifiable source and were not associated with common risk factors for infection by these organisms. Conclusions/UNASSIGNED:Pathogen species and mortality exhibited temporal differences. Early recognition of risk factors among COVID-19 patients could potentially decrease NBSI-associated mortality through early COVID-19 and antimicrobial treatment.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common causes of hospital-acquired pneumonia. To better manage patients with MRSA pneumonia, we require a greater understanding of the host-pathogen interactions during infection. MRSA research focuses on highly virulent and cytotoxic strains, which demonstrate robust phenotypes in animal models of infection. However, nosocomial infections are often caused by hospital-acquired MRSA (HA-MRSA) isolates that exhibit low cytotoxicity and few or no phenotypes in mice, thereby confounding mechanistic studies of pathogenesis. Consequently, virulence pathways utilized by HA-MRSA in nosocomial pneumonia are largely unknown. Here, we report that conditioning mice with broad-spectrum antibiotics lowers the barrier to pneumonia, thereby transforming otherwise avirulent HA-MRSA isolates into lethal pathogens. HA-MRSA isolates are avirulent in gnotobiotic mice, mimicking results in conventional animals. Thus, the observed enhanced susceptibility to infection in antibiotic-treated mice is not due to depletion of the microbiota. More generally, we found that antibiotic conditioning leads to increased susceptibility to infection by diverse antimicrobial-resistant (AMR) pathogens of low virulence. Treatment with antibiotics leads to dehydration and malnutrition, suggesting a potential role for these clinically relevant and reducible hospital complications in susceptibility to pathogens. In sum, the model described here mitigates the impact of low virulence in immunocompetent mice, providing a convenient model to gain fundamental insight into the pathogenesis of nosocomial pathogens. IMPORTANCE Antimicrobial-resistant (AMR) pathogens are responsible for over 2.8 million infections and over 35,000 deaths per year in the United States. To study these microbes, animal models that are susceptible to these pathogens are required. However, many of these pathogens exhibit low virulence in conventional mice, which has negatively impacted mechanistic studies. Here, we show that mice treated with antibiotics in their drinking water become exquisitely susceptible to low-virulence AMR pathogens. Surprisingly, the increased susceptibility was independent of the impact of antibiotics on the microbiome and seems to be due to an unintended consequence of antibiotic treatment: weight loss due to dehydration and caloric restriction. Unlike other models used to sensitize mice to low-virulence pathogens, our model does not reduce phagocyte numbers. Thus, here, we describe an immunocompetent mouse model to facilitate the identification of novel targets and accelerate the development of preventives and therapeutics to combat infections by AMR pathogens.