Undergraduate Journal of Experimental Microbiology and Immunology
The Undergraduate Journal of Experimental Microbiology and Immunology (UJEMI) is dedicated to the publication of scientific articles authored by undergraduate students studying in fields related to microbiology and immunology. Founded in 2001, our suite of publications currently includes UJEMI, UJEMI+, UJEMI-Methods, and UJEMI-Perspectives.
Microgravity-Induced Taxonomic Shifts Enable Robust Random Forest Prediction of Microgravity Exposure in Mice
As human space exploration transitions toward longer-duration missions, understanding physiological adaptations to microgravity is crucial for maintaining crew health. While previous literature has primarily focused on the physical impacts of bone demineralization and skeletal unloading, utilizing the gut microbiome as a highly sensitive biosensor for systemic physiological shifts remains overlooked. This study aims to address this gap by identifying distinct microbial signatures that may predict a host's environmental exposure to microgravity. We hypothesized that microgravity exposure would produce specific microbial changes that could be used to train a Random Forest (RF) machine learning model to predict microgravity exposure in mice. To analyze if microgravity was producing the hypothesized effect on the microbiome, we utilized 16S rRNA sequencing data from the NASA Open Science Data Repository (dataset OSD-417) to analyze fecal samples from mice exposed to microgravity aboard the International Space Station (ISS), comparing them against ground control cohorts over 4.5-week and 9-week mission durations. Microbial community changes were assessed through alpha and beta diversity metrics, core microbiome analysis, differential abundance analysis, and indicator species analysis (ISA), which subsequently informed the training of the RF classification model to predict microgravity exposure. Our results demonstrated that while overall microbial richness and evenness remained relatively stable under microgravity conditions, some microbial taxa underwent significant restructuring. Prolonged microgravity exposure notably decreased the number of shared core microbiome taxa between flight and ground control mice, an effect that persisted even after the mice returned to Earth for recovery. Differential abundance analysis identified a trend towards enrichment of taxa under prolonged microgravity exposure. Furthermore, ISA identified specific taxa, such as Lachnoclostridium, Colidextribacter and Turicibacter, that were strongly associated with microgravity conditions. Leveraging these specific microbial shifts, the RF model successfully distinguished between microgravity-exposed and unexposed microbiomes with high accuracy. Ultimately, these findings indicate that the mice gut microbiome produces a robust predictive signal under microgravity.
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Probiotic Use Does Not Correlate with Overall Gut Microbiome Composition Across Medication Administration Routes in Multiple Sclerosis but Is Correlated with An Increased Abundance of Specific Anti-Inflammatory Taxa in Oral Medication Users
The gut microbiome has been increasingly recognized as an important factor in the pathogenesis of multiple sclerosis (MS). While MS medications and probiotic supplementation have been shown to independently modify gut microbial communities, their potential interaction remains unclear. In this study, we analyzed 16S rRNA gene sequencing data from the International Multiple Sclerosis Microbiome Study (iMSMS) to investigate whether probiotic use interacts with oral and non-oral medication routes to influence the gut microbiome in individuals with MS. Alpha and beta diversity analyses revealed no significant differences in microbial richness, evenness, or overall community composition across treatment groups. Similarly, no shifts toward microbiome profiles resembling those of untreated individuals with MS or healthy controls were observed, and predictive functional profiling showed no significant differences in pathway abundance. However, indicator species analysis found more indicator taxa present in probiotic-supplemented oral treatment groups compared to no probiotics and non-oral groups as well as identified three enriched taxa, Staphylococcus, Pediococcus, and Bacillus, potentially associated with probiotic use in oral treatment groups. Our findings suggest probiotic use does not significantly alter global gut microbiome composition but may be associated with subtle, taxa-specific changes in individuals receiving oral medications. Further investigation is needed to examine the possible functional and clinical implications of these localized microbial changes.
Alcohol Consumption and Cancer Status Do Not Shape Distinct Oral Microbial Communities Among Current Smokers
The prevalence of head and neck cancer (HNC) and pancreatic cancer is strongly associated with major lifestyle risk factors, particularly smoking and alcohol consumption. The oral microbiome is a dynamic ecosystem susceptible to alteration by tobacco and alcohol use, resulting in dysbiosis implicated in cancer progression. While the independent effects of smoking and alcohol on oral microbial composition and their contribution to such cancer risks have been characterized, the combined influence of alcohol consumption and smoking status on shaping unique microbial signatures and the subsequent association with HNC and pancreatic cancer remains unexplored. This study investigated the interaction between alcohol consumption levels among current smokers and oral microbial diversity, and assessed the association of the resulting microbial profiles with HNC and pancreatic cancer. We used a large-scale combined dataset, encompassing alcohol, smoking and cancer status information, with oral wash samples subjected to 16s rRNA gene sequencing. We conducted analyses on alpha and beta diversity, core microbiome, indicator species, differential abundance, and potential functional pathways to identify metabolic capacities associated with lifestyle and cancer variables. Contrary to our initial prediction that alcohol use and cancer would drive distinct taxonomic signatures, our findings suggested that alcohol use and cancer status (HNC or pancreatic cancer) did not significantly shape the oral microbiome composition among current smokers, lacking unique taxonomic signatures or indicator taxa. Microbial alpha and beta diversity did not differ significantly across alcohol consumption and cancer status groups. Differential abundance analysis revealed a general downregulation of microbial species, irrespective of cancer status or alcohol use. Functional analysis using PICRUSt2 revealed virtually no differences of metabolic function due to the consumption of alcohol. Overall, the present study suggests that while alcohol consumption and HNC or pancreatic cancer does not impact the taxonomic structure of the oral microbiome among current smokers, they may induce specific functional shifts relevant to cancer progression.
Iron Supplementation Does Not Significantly Alter Gut Microbiome Diversity or Function in 12-Month Old Anemic Infants, But Is Associated With Increased Hemoglobin and Ferritin Levels
Anemia, characterized by reduced red blood cells (RBCs) or hemoglobin, impairs oxygen transport and causes cognitive, physiological, and behavioral effects. Iron supplementation, including ferrous sulfate (FeSO₄) and multiple micronutrient powders (MNP), is a common treatment for anemia by replenishing iron stores and normalizing hemoglobin levels. However, despite being common treatments, their comparative effects on the gut microbiome remain unclear, and research on MNP's impact on hemoglobin and ferritin levels is limited. This study analyzed the effects of different iron supplementation types on gut microbiome composition in 12-month-old anemic infants and examined associations with hemoglobin and ferritin levels. We hypothesized iron supplementation would produce measurable shifts in the composition and functional potential of the infant gut microbiome. Using 16S rRNA sequencing, we assessed alpha and beta diversity, differential abundance, predictive functional analyses, and linear regression with hemoglobin and ferritin. No significant differences in gut microbiome composition or predicted function were found among 12-month-old anemic infants receiving different supplementation types. However, increased hemoglobin and ferritin levels were associated with greater microbial diversity, supporting the broader benefits of iron supplementation. Our study provides valuable insights into the effects of iron supplementation on the gut microbiome, and the role microbial differences play in hemoglobin and ferritin levels. These findings suggest neither FeSO₄ nor MNP disrupts gut microbiome composition or function, meaning both can be recommended for anemic infants without concern for gastrointestinal dysbiosis. Additionally, the positive association between higher hemoglobin and ferritin levels with microbial diversity further supports their use as beneficial treatments.
Intestinal and Diffuse Gastric Cancer Subtypes Exhibit Distinct Microbiome Profiles with Helicobacter Enriched in Male Diffuse Tumours
Gastric cancer (GC) remains one of the most commonly diagnosed cancers and a leading cause of cancer mortality globally. Clinical outcomes are heavily influenced by histological subtype, classified by the Lauren classification system as either intestinal or diffuse, and host factors such as biological sex. While the role of Helicobacter pylori as a key driver of carcinogenesis is well-documented, the role of the broader gastric microbiome and how biological sex modifies these interactions remains a critical knowledge gap. Here we investigate how gastric microbiome diversity, taxonomic composition, and predicted functional potential differ between the intestinal and diffuse subtypes, and whether biological sex modifies these patterns. Using a 16S rRNA gene sequencing dataset from a cohort of 45 GC patients (15 intestinal, 30 diffuse), we performed differential abundance and metabolic functional prediction analyses. We found that overall community composition, but not alpha diversity, differed significantly between subtypes. Moreover, within the diffuse subtype, female tumours exhibited significantly higher alpha diversity and a distinct microbial community compared to males. Taxonomic profiling revealed an enrichment of genera, including both Helicobacter and Lactobacillus in diffuse cases, with the males showing a significantly higher relative abundance of Helicobacter (p < 0.001). Predicted functional analysis further revealed increased metabolic enrichment in the diffuse subtype of male patients, including pathways related to lipid biosynthesis and overall virulence in H. pylori. These findings demonstrate that the gastric microbiome reflects histological heterogeneity and highlights the importance of biological sex in shaping gastric tumour-associated microbial patterns.
Influenza A Hemagglutinin and Neuraminidase Phylogenetic Groups Are Associated with Minimal Effects on the Cloacal Microbiome of Infected Wild Mallard Ducks
Influenza A viruses (IAV) are highly contagious respiratory pathogens responsible for several global pandemics. Typically classified into subtypes by its envelope proteins hemagglutinin (HA) and neuraminidase (NA), individual IAV HA and NA subtypes are often aggregated into two larger groups per protein based on phylogeny, used to predict the pathogenicity of a particular virus. Importantly, mallard ducks are major IAV reservoirs, contributing to zoonotic transmission to humans. Research has observed IAV-induced changes in cloacal microbiome composition within mallards; however, the effects of viral subtypes remain largely uncharacterized. This research seeks to characterize these HA and NA phylogenetic subgroup effects, evaluate how they differ from one another, and predict phylogenetic subgroups of infections. 16S rRNA sequencing data from cloacal microbiomes of uninfected and IAV-infected mallards were used to explore this. Alpha- and beta-diversity, differential abundance, indicator taxa, and predicted functional potential analyses supported previous findings of IAV-induced changes to cloacal microbiome composition and function respectively, but showed no significant differences among either HA or NA phylogenetic subgroups. Consequently, RandomForest predictive models–trained to predict HA or NA subgroup of infection using samples’ genera abundances–exhibited weak predictive power. Altogether, these findings suggested that the effects of IAV infection on the cloacal microbiomes of mallards were independent of HA and NA phylogenetic subgroups. With a persisting lack of understanding of different IAV HA and NA proteins’ effects on host microbiomes, this research highlights the importance of further examining IAV viral proteins and their effects on host microbiomes.
Gut Microbiome and Blood Metabolic Biomarkers Are Not Strong Predictors of Opioid Use Disorder
Opioid use disorder (OUD) is a chronic condition that can be characterized by an overwhelming desire to use opioids, heightened opioid tolerance, and withdrawal symptoms when opioid use is stopped. Given the relapsing nature of the condition, early intervention is crucial to enable timely treatment. Recent studies suggest that OUD involves dynamic interactions between the brain, immune system, and gut, resulting in significant alterations of the gut microbiome and changes in systemic biomarkers such as cytokines, chemokines, and other metabolic markers. However, the integration of combining gut microbiome data with blood-based biomarker data and their potential as diagnostic markers remain unclear. To address this gap, we compared alpha and beta diversity between OUD-positive and OUD-negative patients and found no significant differences in gut microbiome composition. We then conducted a differential abundance analysis using DESeq2 to identify gut microbial taxa that differed significantly between the two groups and combined the top 20 taxa with significant biomarkers associated with OUD to develop a predictive model using the Random Forest machine learning algorithm for identifying OUD status. We identified 203 taxa that were differentially abundant and 6 out of 21 statistically significant biomarkers that were associated with OUD. However, both individual or combined gut microbial and biomarker profiles showed no predictive power for OUD status. Our results indicate that gut microbiome and blood metabolic biomarker data, either individually or in combination, are insufficient for reliably predicting OUD status and therefore lack utility as a reliable clinical diagnostic tool.
Low Dietary Vitamin C Intake Is Associated with Pro-Inflammatory Taxa and Oxidative Stress Related Functional Pathways in Parkinson’s Disease with No Correlation to Age of Onset
Vitamin C, a dietary antioxidant, has been extensively studied for its neuroprotective role in the development of Parkinson’s disease (PD). PD and antioxidants have also been found to exhibit contrasting influences on gut microbiome composition. Despite this, there is limited research investigating the potential role of the gut microbiome as a mediator between dietary vitamin C intake and PD age of onset. To address this gap, we examined the association between dietary vitamin C intake, gut microbiome composition, and PD age of onset using 16S rRNA sequencing data from Cirstea et al. The gut microbiome composition of PD patients (n = 197) was analyzed in relation to vitamin C intake levels, but no significant differences in gut microbiome diversity (alpha and beta) were found. Four microbial genera were identified as distinct gut microbial signatures linked with vitamin C intake (indicator taxa & differential abundance analysis). Namely, lower intake correlated with pro-inflammatory Escherichia-Shigella (P = 0.048, Stat = 0.714), and high intake with three taxa: Victivallis (P = 0.008, Stat = 0.568), an unclassified genus belonging to the Muribaculaceae family (P = 0.005, Stat = 0.573), and UCG-005 of the Oscillospiraceae family (P adjusted = 0.040, ANCOMBC2). Predicted functional analysis displayed an association between high vitamin C intake and the underrepresentation of 15 metabolic pathways related to vitamin C metabolism and oxidative stress (P < 0.05, LinDA). This distinct metabolic profile suggests that high vitamin C intake may regulate oxidative stress by modulating the gut microbiome, potentially influencing PD progression. Nevertheless, taxa associated with vitamin C intake levels showed no predictive power for PD age of onset (adjusted R2 = 0.068). Overall, our findings highlight dietary vitamin C’s association with shifts in the PD microbiota composition and functional potential. Yet, this variation does not appear to be reflected in the age of onset. Therefore, while vitamin C intake may be associated with microbiome differences, our findings do not support vitamin C alone as a key factor in PD prevention.
Sex-specific Differences in Oral Microbiome Composition and Predicted Functional Pathways Emerge at High Degrees of COVID-19 Severity
Sex-specific differences in the oral microbiome may contribute to variability in Coronavirus disease 2019 (COVID-19) severity, yet their influence on oral microbial composition and predicted function remains unknown. To address this gap, this study investigated whether distinct sex-specific patterns exist in microbial taxonomic and functional diversity across COVID-19 severity levels in a Mexican saliva cohort. We hypothesized that the taxa driving dysbiosis would differ by sex due to host genetic variances and predicted that the overall severity of dysbiosis would be higher in males compared to females of the same severity level. Furthermore, we hypothesized that males with severe infection would exhibit stronger enrichment of inflammatory pathways, such as lipopolysaccharide (LPS) biosynthesis and antimicrobial resistance genes. Microbial composition and diversity were characterized through secondary analysis of 16S ribosomal RNA (rRNA) sequencing data using alpha and beta diversity analyses, indicator species analysis, and differential abundance testing, while predicted functional pathways were inferred using PICRUSt2. Alpha diversity showed no sex associated differences at any severity levels, whereas beta diversity indicated that sex contributed to variance only in severe cohorts. Indicator Species Analysis (ISA) identified distinct mortality signatures, with deceased males associated with Acinetobacter and deceased females characterized by Veillonella and Isobaculum. Differential abundance testing revealed sex-specific dysbiosis increased with disease severity, with males exhibiting greater enrichment in non-commensal genera in severe and deceased cohorts. The integrated analysis highlighted distinct patterns of normalized counts between males and females across severity levels, where males exhibited high loads of opportunistic taxa while females showed depletion as severity increased. Functional inference of MetaCyc pathways indicated that COVID-19 severity was the primary driver of predicted functional variation, whereas sex effects were limited and emerged primarily in Severe positive and Deceased strata. Male-enriched pathways included steroid metabolism, amino acid degradation, and cell envelope-related functions, suggesting heightened microbial functional shifts in severe disease, partially supporting the heightened functional shifts in severe disease. Overall, these findings suggest sex influences oral microbiome composition and predicted functional pathways mainly in severe and fatal cases of COVID-19 infection, especially in males.
Abiotic Soil Conditions Are the Driving Predictors of Organic Matter Removal in British Columbia Forests
Soil quality, encompassing biotic and abiotic properties, is a key contributor to forest health. Wood harvesting can impact soil quality by altering nutrient availability, pH, moisture, and the soil microbiome. However, it is unclear whether wood harvesting affects abiotic and biotic soil properties similarly across different sites. Identifying robust indicators of wood harvesting can help develop improved forest recovery monitoring and management strategies. Machine learning (ML) models are beneficial for monitoring and identifying indicators of forest recovery, but presently no model has been developed using soil quality to assess forest perturbation and recovery following wood harvesting. To address these gaps, this study developed a Random Forest (RF) model using abiotic soil properties and microbial taxa to predict the level of organic matter (OM) removal in BC forests and identify which soil properties and taxa are the most important predictors. We used soil samples collected from 6 BC Long-Term Soil Productivity (LTSP) program sites, with data collected for 16S ribosomal RNA sequences, as well as the abiotic properties. These samples were taken from sites subjected to different severities of OM removal that correspond to common wood harvesting methods, along with unharvested reference sites. Genera associated with OM removal were identified and used with abiotic soil properties to develop RF models that predicted OM removal with 78–89% accuracy. Abiotic factors, specifically carbon content and pH, were more important for predicting OM removal than microbial genera. Amongst microbial genera, Actinophytocola, Inquilinus, and Puia were most important for predicting OM removal.
Hypertension and Depression Comorbidities Are Associated with Altered Gut Microbial Evenness and Taxa Abundance in Individuals Aged 60 and Older
Hypertension and depression affect substantial proportions of the population, especially older individuals. They correlate with significant changes in the gut microbiome, implicating microbiome composition as a possible therapeutic target. However, little exploration has been done on gut microbiome diversity and composition between hypertension and depression comorbidities. It is unknown how demographic variables such as age and sex affect microbiome diversity, particularly in the context of hypertension and depression comorbidities. We aimed to characterize the compositional changes in the gut microbiome between hypertension-only, depression-only and hypertension and depression comorbidities when stratified by sex and age. Using a 16S rRNA amplicon sequencing dataset, we conducted alpha diversity, beta diversity, taxonomic abundance, differential abundance and functional pathways analysis to determine the differences between the three health conditions, as well as sample stratification by sex and by age. Patients 60 years of age and over showed significant differences in their gut microbiome between conditions. An increase in evenness compared to control, which were patients without hypertension or depression, was found in the hypertension and hypertension and depression comorbidities, leading to the conclusion that a decrease in beneficial taxa, such as Faecalibacterium, occurred in healthy controls, along with an increase in rarer taxa. However, the enriched taxa in hypertension, depression and hypertension and depression cohorts showed both similar and opposing differential abundance to what was previously published. Studies using clinical diagnosis with larger sample sizes would benefit further exploration of microbial changes that occur in older patients with hypertension, depression and hypertension and depression comorbidity.
High-LET Radiation Disrupts Gut Microbiome Function in a Dose-Dependent Manner with Unaided Recovery Limited to Low Dose Exposure
In the last few decades there has been a surge in interest in space travel, not just from government-backed research institutions like NASA, but also from private business owners looking to expand their ventures into commercial space flight. However, this brings up the concern of radiation exposure outside the confines of the atmosphere and what risks it may pose to our gut microbiota and, by association, our health. Previous studies with mouse models have shown that exposure to space-type radiation causes decreased diversity and relative abundance of beneficial taxa (as well as increased abundance of pathogenic taxa) but did not explore the temporal dynamics of these changes. Our study investigates the temporal changes in the gut microbiome after radiation exposure to determine what dose presents the limit to unaided recovery, what specific taxa maintain the strongest foothold soon after exposure, and what key metabolic functions are lost or gained. We utilized a NASA-collected 16S rRNA dataset, obtained from radiation-exposed mice at 10 days and 30 days post-treatment and compared diversity metrics, differentially abundant taxa, and metabolic functional potential between the two timepoints. We found that unaided recovery of alpha diversity to pre-exposure levels was only observed up to a dose 0.1 Gy, while beta diversity displayed a nonlinear response to absorbed radiation dose. Unusually, core microbiome analysis revealed that 0.1 Gy and 1 Gy had the most similarities in genera. At 0.1 Gy, the Lachnoclostridium and Lachnospiraceae FCS020 were able to recover from 10 to 30 days, while at 1 Gy, Bifidobacterium, Clostridium sensu stricto 1, and Dubosiella exhibited decreased relative abundance from 10 to 30 days. At 0.1 Gy, pathways related to purine degradation were found to have increased abundance at 30 days. In contrast, we observed widespread decreases across many metabolic pathways at 1 Gy, suggesting a broader disruption of microbial metabolism over adaptive response. The observed dose dependent changes in differential abundance of important taxa, coupled to dose dependent functional shifts identify potential target taxa for medical restoration post radiation exposure and metabolites and vitamins for supplement development.
Gut Microbiome Metabolic Pathways Diverge Between Fish Orders in Actinopterygians
Actinopterygians, ray-finned fishes, represent the most diverse and extensively studied fish class, comprising over 30,000 species. The gut microbiome of Actinopterygians plays a critical role in host behaviour and immune function, driving increasing research interest in this area. The Minich et al. dataset comprises samples from multiple fish orders in diverse environments. For this study, four orders–Perciformes, Blenniiformes, Centrarchiformes, and Labriformes–in rocky reef environments within Actinopterygians were selected from Minich et al. dataset for comparative analysis, as no previous research has focused specifically on these orders. The V4 region of the 16S rRNA gene was utilized to address this research gap. Analyses included Alpha and Beta Diversity, Indicator Species, Core Microbiome, ANCOM-BC, and Functional Pathway analyses to assess compositional and functional variation of gut microbiome between the four orders. Results indicated minimal differences in Alpha and Beta Diversity among four fish orders. Indicator species were identified in all four orders except Perciformes. Core Microbiome Analysis revealed numerous order-specific genera; however, ANCOM-BC detected no significant differences in relative abundance. Functional pathway analysis demonstrated notable metabolic trends between Blenniiformes and Perciformes as well as Centrarchiformes and Perciformes, with limited divergence observed between Labriformes and Perciformes. These findings suggest that compositions of gut microbiome between selected four orders could be similar yet may display significant differences in gut microbiome metabolic pathways.
Chronic Wasting Disease Pathology States in White-Tailed Deer Differentially Affect the Gut Microbiome in a Sex-Dependent Manner
Chronic wasting disease is a fatal, contagious, neurodegenerative prion disease that affects both captive and free-ranging Cervid populations in North America, Scandinavia, and South Korea. Previous studies have shown links between chronic wasting disease status and the microbiome, as well as links between other prion diseases and microbiome shifts. However, there is a gap in knowledge for how the microbiome is affected during different stages of chronic wasting disease progression. Here, we expand this research by analysing the microbiome of white-tailed deer at different pathological stages of the disease: negative, lymph node positive, and brain and lymph node positive. We used a previously published 16S rRNA dataset to determine diversity metrics, core microbiome, indicator taxa, and differential abundance. Our results showed no significant difference in either sex for alpha and beta diversity. In contrast to this, we found a significant shift in core microbiome composition for male deer across pathology states, as well as a significant change in differential abundance for both sexes across pathology states. We found shifts in differential abundance in bacterial phyla associated with carbohydrate fermentation, short-chain fatty acid production, and energy metabolism. Our findings suggest that the microbiome shifts as disease progresses in terms of its composition and abundance, but not in terms of diversity metrics, and that these shifts are sex-dependent. Our differential abundance findings suggest that chronic wasting disease pathology states cause shifts related to core metabolic guilds rather than individual taxa.
Overall Gut Microbiome Community Composition Is Conserved Across Adult Multiple Sclerosis Onset Ages, with Modest Taxa-Level Differences
Multiple sclerosis (MS) can present at different ages in adults, leading to severe and progressive disability. As such, understanding the risk factors for MS onset may support earlier risk stratification and monitoring. The gut microbiome, trillions of intestinal microorganisms that shape host metabolism and immune tone has been repeatedly linked to MS risk and clinical course. Yet, whether the gut microbiome is linked to onset age and disease severity remains unclear. In this study, we thus aimed to explore the association between gut microbiome composition, age of MS onset, and disease severity. Using 16S rRNA profiles from the International MS Microbiome Study (iMSMS), we analyzed patients with MS and stratified them into early (18–29 years old), mid (30–49 years old), and late (≥50 years old) onset groups. We then assessed alpha (Shannon, Faith’s phylogenetic diversity) and beta diversity (Bray-Curtis), then used indicator species analysis and DESeq2 to identify onset-linked taxa and shifts in abundance patterns. Finally, we tested whether these microbiome features relate to disease burden, measured by Multiple Sclerosis Severity Scores (MSSS and relapse counts. We trained a random-forest model to compare the predictive contribution of host clinical variables and microbial features for classifying onset groups. Overall, we found no difference in alpha and beta diversity across onset groups. Moreover, core microbiome analysis showed that MS onset age groups shared most of their core amplicon sequence variants, while only a small subset of genera differed between groups. Overall community composition (Bray-Curtis dissimilarities) explained a modest variation in MSSS and showed no association with relapse count. Together, these results suggest that while microbiome diversity is broadly conserved across MS onset age groups, taxa-specific differences exist, but their association with severity was small and relapse count showed no association.
Multiple Sclerosis Severity and Depression Contribute to Differences Fueled by Rare Taxa in the Gut Microbiome of Patients
Multiple Sclerosis (MS) is a chronic demyelinating immune disease with rising global prevalence, and depression represents one of its most common comorbidities. Both MS and depression have been independently associated with gut microbiome perturbations, however the extent to which MS disease severity and depressive status jointly modulate microbial diversity and taxonomic structure remains unresolved. Using a paired household 16S rRNA dataset, we performed an integrative analysis of alpha and beta diversity, core microbiome membership, and differential abundance across six experimental groups differentiated by MS severity (RRMS, PMS) and depression presence. Alpha diversity analyses indicated that richness differed significantly in PMS patients with or without depression, while Beta diversity analysis confirmed this and revealed additional significant comparisons between several MS and depression groups. In contrast, taxonomic composition remained comparatively stable across groups and abundance only differed exclusively in the PMS group and in two species, Varibaculum cambriense and Blautia fusiformis. The divergence between distinct diversity effects and minimal taxonomic shift implies that rare taxa abundance may disproportionately contribute to observed diversity patterns.
Fibre Intake and Exercise May Promote a Healthier Shift in the Gut Microbiome of Individuals with Cardiovascular Disease
Cardiovascular diseases (CVD) consist of a variety of diseases affecting the arteries, veins, and/or heart and are currently one of the leading causes of death worldwide. There are several risk factors for these diseases, including several of the lifestyle changes that may occur with westernization of a culture. Specifically, this shift is often associated with decreased fibre intake and a more sedentary lifestyle. While other studies have reported on the effects of fibre intake and exercise separately, the combined effect of these lifestyle changes on the gut microbiome and how it may influence cardiometabolic status has not yet been fully understood. We hypothesized that fibre and exercise would contribute to a healthier and more diverse microbiome, offering a protective effect against CVD. To test this, we used a 16S rRNA dataset from a population newly undergoing westernization to compare alpha and beta diversity metrics, differential abundance analysis, predictive functional analyses, and network co-occurrences. Although no significant difference in microbial diversity metrics, we identified changes within specific taxa and functional pathways. Within the abnormal cardiometabolic groups, fibre and exercise appeared to offer a rescuing effect by increasing the abundance of several beneficial bacteria and suppressing harmful pathways. Finally, network co-occurrence analyses showed increased network complexity within the dysfunctional cardiometabolic group, as well as a beneficial community associated with physical activity. Overall, it appears that adequate fibre and exercise may offer protection against CVD through healthier microbiome composition and networks, supporting the hypothesis. However, these changes are not yet detectable at the level of alpha and beta diversity metrics, although may become detectable as westernization progresses.
Analysis of Representative Thermodesulfobacteriota Taxa in Wetland Sediments Reveals a Prevalence of Geobacteraceae in Low Conductivity Rather Than the Predicted Desulfobulbaceae Enrichment in High Conductivity
Wetland ecosystems rely on complex microbial communities to maintain soil homeostasis through long-distance electron transport (LDET). Thermodesulfobacteriota is one of the most important soil microbes that facilitate LDET; however, it remains poorly understood how different soil conditions, such as conductivity, influence how these bacteria thrive in wetland environments. This study evaluated the impact of soil conductivity using a 16S rRNA dataset of 55 Northeastern American wetland samples. It was observed that low-conductivity environments, defined by a below 0.3 mS/cm threshold, exhibit significantly higher phylogenic richness and community homogeneity compared to high-conductivity sites. While alpha diversity remained stable, taxonomic profiling indicated a significant increase in the relative abundance of Geobacter in low conductivity environments (p < 0.05). The Spearman’s rank correlation identified a strong negative relationship between conductivity and Geobacter abundance (rho = -0.499), whereas Desulfobulbaceae abundance remained consistent across samples. These results demonstrate that Geobacter abundance is significantly higher in low-conductivity wetland sediments, identifying soil conductivity as an environmental factor associated with the distribution of conductive microbial guilds. These findings highlight environmental conductivity as a primary factor shaping the distribution of conductive microbial guilds in freshwater aquatic systems.
Fermented and Fresh Vegetable Consumption Results in Distinct Taxonomic Composition Shifts in the Gut Microbiome of Women
The diversity of the human gut microbiota is integral to gastrointestinal health and is largely influenced by diet. Probiotics are living bacteria that can provide health benefits when consumed. Fermentation of food encourages the growth of microbes with potential probiotic properties. Previous research studied the relationship between the human gut microbiome and consumption of different diets–specifically fresh versus fermented vegetables–with a focus on taxonomic composition rather than functional profiles. The analysis also focused on non-sex-specific microbiome responses and had poor characterization of the durability and long-term effects of microbial changes from fermented vegetables on the gut. We compared the consumption of fermented vegetables to fresh vegetables to see if they enhance gut microbiota composition and function to promote long-term gut health in women over time. We employed beta diversity analysis with linear mixed-effects of alpha and beta diversity modeling, differential abundance analysis, and functional analysis to investigate the longitudinal outcomes and functional metabolic pathways of fresh versus fermented vegetable consumption on the gut microbiomes of individuals with and without constipation. Our findings show that although cross-sectional analyses revealed no significant difference in gut microbiome beta diversity between the two diets, both diets induced immediate and sustained longitudinal shifts in microbial composition. Longitudinal analysis of alpha diversity revealed consumption of fermented vegetables led to more sustained shifts in microbial composition compared to fresh vegetables. Differential abundance analysis supports the finding that consumption of fermented vegetables created stronger and broader changes in microbial taxa, having both predicted beneficial and non-beneficial host effects. Compared to fresh vegetable consumption, fermented vegetable consumption also enriched specific functional pathways with variable host benefits. These findings can assist the development of dietary strategies that benefit the long-term gut microbial function and health in women.
Disease-Modifying Therapies in Progressive Multiple Sclerosis Show Limited Impact on the Gut Microbiome
Multiple sclerosis (MS) is an autoimmune disease that targets the myelin in the central nervous system, leading to severe neurodegeneration. Although there is no cure, disease-modifying therapies (DMTs) are used to slow disease progression and manage symptoms. In studies of MS and of the relapsing-remitting subtype (RRMS), DMTs have been shown to alter the gut microbiome. Previous research identifies the microbiome as a potential driving factor for MS disease progression, therefore, understanding the impacts of DMTs on this system is crucial for effective MS treatment. However, the impacts of DMTs on the primary progressive MS (PPMS) and secondary progressive MS (SPMS) subtypes remain underexplored. This study investigates the potential effects of DMTs on the gut microbiome of PPMS and SPMS patients, grouped as progressive MS (PMS) patients. DMTs were grouped by mechanism into two categories: immunomodulators and T/B cell therapies. Using stool samples from treated PMS patients, untreated PMS patients, and genetically unrelated healthy household controls, we performed microbial diversity, differential abundance, and potential functional pathway. Ultimately, our research found no significant differences in gut microbial diversity or indicator taxa between PMS treatment status and across treatment types. We discovered few commensal species unique to treatment groups and identified consistent depletion of anti-inflammatory taxa in samples from DMT-treated patients. Moreover, our research suggests that immunomodulators may alter metabolic, stress response, and drug resistance-related functional pathways. Overall, more research is necessary to elucidate the relationship between DMTs and the gut microbiome in PMS.
Lactose Intolerance Had No Significant Effect on the Diversity, Composition, and Function of the Human Gut Microbiome in Patients with Multiple Sclerosis
As a neurodegenerative, autoimmune disorder, multiple sclerosis (MS) has been linked to the gut-brain axis through the gut microbiota, which is critical for immune system development. Shifts in microbiome composition related to disease and diet have been associated with differential health outcomes; however, their combined effects remain largely unknown. In this study, we investigated whether lactose intolerance (LI), a common dietary restriction, differentially affects the gut microbiome of individuals with MS compared to healthy controls. Using 16S amplicon sequencing data obtained from Zhou et al. (2022), we analyzed gut microbiome diversity, composition, and function across four groups: Healthy No Dietary Restrictions (HNDR) compared to Healthy Lactose Intolerant (HLI), and MS No Dietary Restrictions (MSNDR) compared to MS Lactose Intolerant (MSLI). The results for alpha diversity metrics (Observed, Shannon index) were not significant. Beta diversity metrics (Bray-Curtis) were also insignificant between MSNDR and MSLI, although a significant difference was found between HNDR and HLI. Taxonomic shifts between no dietary restriction (NDR) and LI individuals in both the healthy and MS groups were observed to be minimal, further supported by the overlapping genera found from the core microbiome analysis and the lack of significance in differentially abundant taxa. Functional analyses revealed that there were no significant metabolic pathways pertaining to any taxa. The results suggest that diversity, composition, and function of the human gut microbiome in patients with MS are largely unaffected by lactose intolerance.
Gastric Cancer and Parkinson’s Disease Drive Distinct Changes to the Microbiome
Gastric cancer (GC) and Parkinson’s disease (PD) are age-associated diseases that have been linked to gut microbiome dysbiosis. Despite the significance of the gut microbiome in the onset of these two diseases, no study to date has compared them with each other to identify unique or shared effects on the microbiome. In this study, we analyzed 16S rRNA amplicon sequencing data derived from gastric mucosal samples for GC and stool samples for PD to indirectly assess potential shared microbial profiles. Here, we examined alpha diversity differences between GC and PD groups and their relative controls, revealing significant Shannon diversity changes associated with GC, but not with PD. Further, there were no shared taxa between the core microbiome of GC and PD. Indicator species analysis identified several genera that showed strong associations with GC, whereas no genera were significantly associated with PD, indicating distinct microbial signatures between the two disease states. In our differential abundance analysis, we found distinct, non-overlapping patterns of gut microbial dysbiosis in PD and GC, with PD showing relatively limited taxonomic shifts. In contrast, GC was associated with more extensive alterations in microbial composition, characterized by a greater number of significantly changed taxa and larger fold changes. Additionally, through our functional profiling, we observed significantly enriched metabolic signatures that were only revealed in the GC group. Overall, our findings suggest that GC and PD drive divergent changes to the microbiome, with GC exhibiting more severe dysbiosis than PD. These data show that each condition is characterized by a highly specific and non-overlapping microbial profile. Our work in this area suggests the need for a specialized and standardized dataset directly comparing the microbiome of GC and PD patients for further investigation.
Alcohol Intake Does Not Have a Strong Impact on the Gut Microbiome's Diversity, Core Microbiome and Metabolic Function Compared to Inflammatory Bowel Disease
Gut microbiome dysbiosis has been strongly associated with major changes to the gastrointestinal (GI) environment by various physiological conditions, with inflammatory bowel disease (IBD) and more recently, chronic alcohol intake noted as significant contributing factors. The disruption of the gut microbiota balance ultimately results in the activation of specific bacterial pathways associated with inflammation in GI tract (GIT) tissues. Previous literature has investigated the effects of both IBD and high alcohol consumption independently in affecting the gut microbiome. However, limited research has concomitantly studied whether these two conditions induce similar patterns and metabolic pathways of gut microbiota. Our study aimed to examine if low and high alcohol intake patients exhibit significant microbiome compositional and functional differences comparable to IBD’s dysbiosis patterns between Crohn’s Disease and Ulcerative Colitis, including metabolic pathways. We analysed 16S rRNA sequencing datasets from alcohol and IBD groups independently using QIIME2 and R for alpha, beta diversity and core taxa analyses, and PICRUSt2 was used for functional pathway analysis. Our findings show that the alcohol-intake patient groups did not exhibit significant differences in diversity, core microbiome, or metabolic functions. Therefore, chronic alcohol intake minimally disrupts microbiome diversity, core microbiome, and metabolic functions compared to IBD, and no shared functional pathways are identified between alcohol and IBD patient groups.
Farm-living Exposure Is Associated with Minor Differences in Gut Microbiome Functional Potential but not With Taxonomic Composition and Diversity in Parkinson’s Disease Patients
Parkinson’s disease (PD) is the fastest-growing neurodegenerative disorder worldwide, with increasing evidence indicating that environmental exposures, such as farm-living, are linked to its onset and development. PD is also characterized by a taxonomically and functionally distinct gut microbiome. Notably, major shifts in the microbiome have similarly been observed in individuals with farm-living exposure, raising the question of whether this environmental factor may shape the gut microbiome in the context of PD. In this study, we investigated whether the gut microbiome of PD patients with a history of farm-living differs from that of patients without such exposure in terms of diversity, composition, and functional potential. We used 16S rRNA data from fecal samples from 197 PD patients collected by Cirstea et al. to conduct our analyses. Alpha and beta diversity showed no difference based on farm-living status among PD patients. No taxonomic differences were detected, with no differentially abundant or indicator taxa identified. Predictive functional analysis revealed a significant reduction in several KEGG Orthology terms in PD patients with farm-living exposure compared to those without. Although no large-scale functional trends were observed, several terms involved in metabolic and genetic processes were less abundant in patients with farm-living history. Finally, gut microbial genera of PD patients with farm-living exposure were found to have marginal predictive capacity for early vs. late age of PD onset. Overall, these results suggest that farm-living is not associated with any taxonomic differences in the gut microbiome of PD patients, but may be linked to minor alterations in functional potential. These findings provide a foundation for further research investigating whether environmental exposures, like farm-living, influence the gut microbiome, and how such alterations may influence the development or pathogenesis of neurodegenerative diseases like PD.
Catch Method Is a Primary, Confounding Factor in Fish Microbiome Composition Across Four Body Sites
The composition of the fish microbiome plays key roles in immune response and host protection. Investigations into factors modulating the fish microbiome have key implications in understanding the vertebrate microbiome given the large number of fish species. Previous studies have identified strong effects of fish species and habitat on various fish body site microbiomes. However, the contribution of fish morphology to taxonomic and functional diversity of the microbiome has not been extensively characterized. In studies on terrestrial animals, effects of morphological variables on the microbiome have revealed strong effects, with the species-area relationship shown to correlate increased microbiome diversity with increased organism size. In this study, we aimed to determine if morphological variables describing fish species located in Southern California correlate with changes in microbiome taxonomy across body sites. Here we investigate the microbiome of four different body sites, gill, skin, midgut, and hindgut, using 16S rRNA sequencing collected from 101 fish species off the coast of Southern California. Initial analysis of alpha and beta diversity revealed the primary driver of microbiome diversity differences to be, not a morphological variable, but the method by which fish were caught. Subsequent core microbiome, differential abundance, and indicator taxa analysis revealed that the effects of catch method are body site-specific and drive differences in microbial composition at the ASV level. These findings reveal a novel confounding variable in fish microbiome studies, potentially informing updated best practices and study designs for fish microbiome sampling. Future directions include establishing causality of the method of catch either through direct impacts on microbial composition of collected samples or biasing the taxonomic profile of sampled fish.
Motor Impairment in Parkinson’s Disease Exhibits Sex-Specific Differences in Gut Microbiome Composition but Not Functionality
Parkinson’s disease (PD) is a common neurodegenerative disease characterized by chronic and progressive motor and cognitive symptoms. Currently, no cure for PD exists and risk factors that mediate susceptibility and progression are elusive. Investigation into the factors that lead to PD neuropathogenesis may provide therapeutic avenues for not only relieving motor and cognitive symptoms in patients, but also preventing their onset. Further, identification of a reliable biomarker for diagnosis may support intervention and monitoring of PD progression. In this study, we examined whether progressive motor and cognitive impairment in PD were associated with changes in gut microbiome diversity, taxa composition and function. Using sequenced 16S rRNA V4 regions of the gut microbiome of PD patients, we performed sex-controlled alpha and beta diversity correlations across cognitive and motor impairment PD severity metrics to uncover microbiome alterations associated with PD progression. We then analyzed compositional and taxa-level differences across sex-controlled severity metric groupings and determined whether identified microbiome alterations were functional in PD pathogenesis. Our findings show that motor impairment in PD correlated with increased gut microbiome diversity in males but not females. Conversely, cognitive impairment in PD did not correlate with altered gut microbiome composition in either sex. Further, we demonstrate that motor impairment corresponded with more core microbiome alterations than cognitive impairment in PD patients, and a sex-specific differential abundance of microbial taxa between moderate versus mild motor impaired PD patients. That said, observed compositional and taxa-level differences in motor impaired PD patients did not translate to functional changes with respect to predicted microbial pathways. Our work provides insight into possible biomarkers of motor impairment progression in PD patients and future directions for exploring microbiome alterations and their relationship with PD severity.
Evaluation of Soluble Ag43α as a Competitive Inhibitor: Evidence for Kinetic Delay and Structural Reinforcement in Escherichia coli Biofilms
Escherichia coli biofilms are a significant source of persistent and chronic infections, with the Antigen 43 (Ag43) protein often helping to mediate their formation through surface self-association, promoting autoaggregation. This study looked to examine whether soluble Ag43α, the passenger domain and functional subunit of Ag43, could function as a competitive inhibitor to block these surface interactions, thereby preventing autoaggregation and biofilm formation. Recombinant Ag43α was expressed in E. coli BL21(DE3) and purified via nickel affinity chromatography. Structural integrity was validated through a limited proteolysis assay, which revealed a trypsin-resistant core, consistent with a folded β-helix. The functional effects of soluble Ag43α were assessed with autoaggregation kinetics and crystal violet staining assays in both fimbriae-deficient (ΔfimA) and fimbriated E. coli backgrounds. Results confirmed that Ag43 expression could drive rapid autoaggregation relative to an empty vector control. While soluble Ag43α did not abolish this phenotype, higher concentrations (100 µg/mL) created a modest and reproducible delay in optical density decline, a proxy for sedimentation. In static biofilm assays, soluble Ag43α did not inhibit biofilm formation in fimbriae-deficient strains. Unexpectedly, in fimbriated strains, Ag43α at low doses (20 µg/mL) significantly enhanced biofilm formation, roughly doubling the intensity in crystal violet staining, while high doses (100 µg/mL) returned biomass to baseline levels. This could possibly be explained by low-dose Ag43α serving as a molecular scaffold in the matrix rather than as a blocker, but at high doses, it ends up saturating binding sites. Furthermore, disruption assays on pre-established biofilms showed no significant reductions across all conditions, indicating that mature Ag43 matrices are resistant to soluble protein interference. These findings ultimately suggest that Ag43 matrices are formed with high avidity interactions, but may be delayed in their formation by the introduction of soluble Ag43α.
Cutting Glutamate Decarboxylase Loose: Autotransporter-Based Secretion to Improve GABA Yield
Gamma-aminobutyric acid is an important metabolite used in neuroscience research. It serves as the primary inhibitory neurotransmitter in the brain, regulating various neurological processes. It is also utilized widely in industrial applications, including food and nutraceutical production. Engineered microbial biosynthesis has emerged as a sustainable alternative to chemical synthesis of this metabolite, with bacterial autotransporter-based surface display platforms enabling extracellular conversion of glutamate to gamma-aminobutyric acid by glutamate decarboxylase. Autotransporters are a promising secretion strategy because they allow proteins to fold after translocation across the outer membrane, making them suitable for transporting large enzymes to the outer surface of bacteria, where they can be exposed to extracellular proteases or other activating processes. Previous work has shown that the Bordetella resistance to killing A protein autotransporter can display glutamate decarboxylase on the cell surface, but limited enzymatic activity suggests constraints in functionality. In this study, we aimed to improve gamma-aminobutyric acid production by engineering an autotransporter system containing a protease cleavage site to enable the release of glutamate decarboxylase into the extracellular environment. The recombinant construct was expressed in bacterial strains containing or lacking protease activity to compare surface-bound and secreted forms of the enzyme. Protein expression, localization, and cleavage were assessed using Western blot analysis of whole cell lysates, cytoplasmic and membrane fractions, and concentrated extracellular proteins. The recombinant protein was detected only at low intracellular levels in OmpT-positive cells, and neither surface display nor extracellular secretion of glutamate decarboxylase was observed, likely due to a deletion within the construct’s N-terminus signal peptide that impaired Sec-dependent translocation across the inner membrane. Although functional conversion of glutamate to gamma-aminobutyric acid was not evaluated, this study establishes a framework for evaluating enzyme localization and extracellular secretion in autotransporter systems and supports future efforts to optimize microbial production of gamma-aminobutyric acid.
Engineering Surface Display of E3 Nanobody Using the BrkA Autotransporter in Escherichia coli
More than a century after its first identification, Alzheimer’s disease (AD) remains a neurodegenerative disorder with no cure. The accumulation of extracellular amyloid-beta (Aβ) plaques is a central pathological feature of AD, and impaired clearance contributes to disease progression. Previous in vitro studies have shown that physically coupling Aβ-targeting antibodies to Aβ-degrading enzymes, such as neprilysin (NEP), enhances the efficiency and specificity of amyloid cleavage by localizing enzymatic activity to the substrate. These findings motivate the development of co-expression-based clearance strategies in which targeting and degradation components function in close proximity. As an initial proof-of-concept toward a targeted co-expression clearance system, this study establishes a bacterial surface-display model to validate presentation of an Aβ-targeting nanobody in a controlled and accessible manner. Specifically, Escherichia coli was engineered to surface display the Aβ-binding nanobody E3 using the BrkA type V autotransporter system from Bordetella pertussis. A recombinant BrkA-based expression construct (pJMMS) was generated via Gibson Assembly® by fusing E3 to the 5′ end of the brkA passenger domain within the pENS backbone and transformed into E. coli UT5600. Colony PCR and nanopore sequencing confirmed correct insertion of the E3 sequence, and surface localization was verified using a trypsin accessibility assay. Together, these results establish a modular proof-of-concept platform for nanobody-guided targeting that can be extended to future co-expression of Aβ-degrading enzymes. This work provides a foundational engineering step toward targeted amyloid clearance systems, including future applications in engineered microglia.
Utilizing the BrkA Autotransporter System for Heavy Metal-binding with Surface Display of 6xHis on Escherichia coli
Heavy metal contamination in aquatic environments is a global concern, disrupting marine ecosystems and posing severe risks to human health. Consequently, bioremediation strategies to sequester heavy metals using microbial constructs have gained increasing interest. One approach involves genetically engineered microorganisms to surface display metal-binding peptides. In this study, we investigated if Escherichia coli can express the BrkA autotransporter protein to surface display a hexahistidine (6xHis) tag for metal sequestering. Two constructs, UT5600_pENS and UT5600_pPALMC1, were generated, each encoding BrkA with a 6xHis tag in differing locations. The expression of 6xHis on the cell surface was validated through a trypsin accessibility assay, SDS-PAGE, and western blot analysis. A nickel bead-binding assay and zinc strip concentration test were then developed to assess metal-binding capacity. However, although previous research has indicated the ability of 6xHis to sequester cadmium ions (Cd²⁺), our constructs did not appear to bind to nickel resin or zinc ions (Zn²⁺). To explore this outcome, the zeta potential of nickel resin was measured and revealed a slight negative charge, suggesting that there could be weak electrostatic repulsion between the resin and the negatively charged cell surface. Additionally, AlphaFold was used to predict the three-dimensional (3D) structures of the BrkA-6xHis constructs encoded by pENS and pPALMC1. Structural visualizations and western blot analysis suggested that repositioning of the 6xHis insert could improve its accessibility, however further research is required to optimize the metal-binding ability of histidine based autotransporter constructs.
Deletion of DsbA Does Not Improve Translocation of Cysteine-Rich Recombinant BrkA-EC10 in the BrkA Secretion Pathway
BrkA is an autotransporter native to Bordetella pertussis that can be utilized to display proteins of interest in its passenger domain on the surface of Escherichia coli. However, cysteine-rich recombinant passengers such as EC10 may introduce disulfide bonding, which may obstruct translocation through the β-barrel and reduce surface expression. Prior research on autotransporter secretion implicated presence of periplasmic DsbA chaperone and its role in introducing disulfide bonds as a possible explanation for reduced secretion efficiency of the passenger. Though the periplasmic transit process remains a contested area, this study aims to elucidate the role of DsbA, a protein native to E. coli, in the inhibition of BrkA-EC10 translocation in order to optimize the BrkA autodisplay system for cysteine-rich proteins. To compare and evaluate expression of BrkA and BrkA-EC10 in wild-type BW25113 K-12 E. coli (WT) and JW3832 K-12 E. coli (ΔdsbA), as well as any possible pleiotropic effects, we employed a growth curve assay, a trypsin accessibility assay, and a Western blot. Ultimately, our results indicated that ΔdsbA did not impair or enhance the translocation of the BrkA-EC10, failing to restore expression to levels observed for BrkA in our WT strain. Additionally, we found BrkA-EC10 expressed in the ΔdsbA strain exhibited reduced cell viability. These findings suggest there may be more factors at play for the translocation of BrkA-EC10 beyond DsbA and disulfide bonding.
Exploring the Therapeutic Potential for Subtilisin Carlsberg Surface Expression Using the BrkA Autotransporter System
Celiac disease is an autoimmune disorder characterized by damage to the small intestinal epithelium resulting from an immunogenic response to gluten. Patients with the disease may experience symptoms like diarrhea, fatigue, and nutrient malabsorption, highlighting that a strict gluten-free diet remains the only effective treatment. Subtilisin Carlsberg (SubC), a protease derived from Bacillus licheniformis, is capable of gluten degradation via its mature domain. A type V autotransporter system known as Bordetella resistance to killing A (BrkA) allows for surface expression of heterologous proteins. However, previous attempts to express the homologous subtilisin E in Escherichia coli using the BrkA type V autotransporter system were unsuccessful, likely due to the misfolding of the mature domain. In subtilisin-family proteases, proper folding is mediated by an N-terminal pro domain acting as a chaperone. Here, we propose the BrkA-mediated surface expression of SubC in E. coli, a common gut commensal, using a trans-complementation system. The pro (pCARP) and mature (pCARM) domains will be expressed on separate plasmids in the OmpT-deficient E. coli UT5600 strain. We hypothesize that the trans-complementation of these domains will enable proper folding and proteolytic activity of the mature domain. In our project, we successfully assembled the pCARM plasmid and confirmed the surface expression using a trypsin accessibility assay. However, assembly of the pCARP plasmid remained incomplete, preventing functional validation of the dual-plasmid system. This study explores pro-domain-assisted mature domain folding in-trans as a strategy for functional enzyme display, laying the groundwork for future therapeutic approaches for celiac disease. Future work should focus on completing the pCARP assembly and evaluating proteolytic activity under pH conditions representative of the small intestine to evaluate in vivo feasibility. This system could enable localized gluten degradation in the gut, improving health outcomes and quality of life for individuals with celiac disease.
Developing a Model for Antigen 43 Translocation Across Escherichia coli Inner and Outer Membranes
Antigen 43 (Ag43) is an autotransporter encoded by the metastable flu gene that mediates autoaggregation and flocculation in Escherichia coli. As a member of the type V secretion system, Ag43 is translocated to the outer membrane via the SecYEG translocon and the Bam complex. During this process, cleavage is expected to occur first between the signal peptide and passenger domain by SecYEG, followed by a second cleavage event between the passenger and autochaperon domains by an as-yet unidentified factor. These sequential processing events are predicted to produce three distinct protein fragments; In this study, we investigated Ag43 expression and time-dependent cleavage within E. coli BL21, DH5a and BW25113 strains, utilizing an arabinose inducible plasmid encoding Ag43. Within western blotting analysis, we show that the expected banding pattern for the current cleavage model was not observed, and propose revised models for Ag43 outer membrane translocation and processing in E. coli.
The BrkA Autochaperone Domain May Not Be Necessary for Promoting Correct Folding and Function of β-lactamase Enzyme
The Type V autotransporter system, exemplified by Bordetella resistance to killing (BrkA) from Bordetella pertussis, has been used as a platform for the surface display of heterologous proteins. While many autotransporters contain an internal autochaperone domain to facilitate folding of the passenger domain, its necessity for correct folding and function of heterologous passenger domains, such as β-lactamase is not yet studied. In this study, we investigated the role of BrkA autochaperone domain in the stability of ꞵ-lactamase passenger in Escherichia coli strain UT5600 using two engineered constructs: pBLacIn containing the autochaperone domain, and pBLacSwitch which lacks the autochaperone domain. Initial analysis of native BrkA revealed a cleaved passenger domain showing resistance to trypsin concentrations up to 15 μg/mL. Growth assays in the presence of Penicillin G were conducted in order to assess the functionality of ꞵ-lactamase without an autochaperone domain. There was no significant difference in growth between pBLacIn and pBLacSwitch, while the BrkA control consistently reached a lower peak OD600. Western blot analysis of pBLacIn and pBLacSwitch treated with 15 μg/mL showed an absence of detectable protein expression with and without trypsin, possibly due to their dimerization. These findings suggest that the BrkA autochaperone domain may not be necessary for promoting correct folding and function of ꞵ-lactamase passenger protein, however plasmid dimerization may be impacting these results.
Developing a Model to Investigate Escherichia coli Autoaggregation Inhibition by Distinct Phylogenetic Variants of Antigen 43
Autoaggregation by Escherichia coli promotes the formation of persistent biofilms that can cause chronic bacterial infections. Antigen 43 is a type V autotransporter that drives autoaggregation through self-association. Antigen 43 variants are grouped into six phylogenetic classes and display diverse capacities to form homotypic and heterotypic interactions during autoaggregation. Previous work proposed that soluble antigen 43 of the C1 class has therapeutic potential by competitive inhibition of antigen 43-mediated autoaggregation. Compared to variants of the C1 class, surface-displayed variants of the C4 class engage in broader heterotypic interactions with other antigen 43 classes. On this basis, we hypothesized that soluble C4 antigen 43 would display comparable inhibition of autoaggregation to soluble C1 antigen 43 but have greater therapeutic potential. To investigate this, we constructed a plasmid which expresses the passenger domain from the C4 class of antigen 43 by Gibson Assembly. We induced protein production from the plasmid using isopropyl β-D-1-thiogalactopyranoside and purified it by nickel resin chromatography. We then assessed autoaggregation in a BW25113 pBAD-Ag43 model at pH 7.4 to determine the capacity of the soluble passenger domain of antigen 43 of the C4 class to inhibit antigen 43-mediated autoaggregation and compared it to the C1 class. Technical replicates show that neither of the proteins significantly inhibit E. coli autoaggregation at the concentrations tested. This study provides a model through which E. coli autoaggregation by antigen 43 C1, C4, and other phylogenetic classes can be studied.
Utilizing the BrkA Autotransporter System to Surface Express LinB on Escherichia coli UT5600 for β-hexachlorocyclohexane Bioremediation
β-hexachlorocyclohexane (β-HCH) is a toxic byproduct of the organochlorine pesticide industry that poses severe risks to human and animal health, inducing developmental, hepatic, and neurological effects. A potential bioremediation strategy lies in bacteria from the genus Sphingobium that express LinB, a haloalkane dehalogenase that hydrolytically cleaves chlorine groups from HCH isomers, funneling them into degradation pathways and significantly reducing recalcitrance. Our study makes use of the Bordetella-resistant-to-killing autotransporter (BrkA) type Va secretion system from Bordetella pertussis, which can be harnessed to surface display foreign proteins in Gram-negative bacteria such as Escherichia coli. The use of the BrkA autotransporter to surface express LinB on the outer membrane of E. coli is explored as a potential future bioremediation strategy to break down β-HCH. To this end, surface localization of LinB could allow these E. coli cells to be introduced into contaminated environments, where extracellularly exposed LinB may directly interact with and degrade β-HCH. We designed a recombinant plasmid, pITAS, by inserting the linB gene into the BrkA passenger domain of the pOCS plasmid backbone, a vector expressing a 6x-Histidine (6x-His) tag for visualization on western blot and containing an OmpT cut site for future secretion experiments. The engineered construct was introduced into E. coli UT5600 cells, where LinB was confirmed to be displayed on the cell surface. This work establishes a foundation for future bioremediation strategies by employing the BrkA autotransporter for LinB surface expression to target β-HCH contamination.
Evaluating the Potential of BrkA Autotransporter to Co-Display a Functional LipA/LipB Heterodimeric Enzyme in Escherichia coli UT5600
High concentrations of lipids can obstruct sewer systems and form persistent oil layers. Microbial bioremediation presents a viable solution to mitigate lipid pollution. This study investigates whether the BrkA autotransporter can co-display functional heterodimeric Burkholderia cepacia lipase (LipA) and foldase (LipB) on Escherichia coli UT5600. Surface localization of LipA and LipB was confirmed using trypsin accessibility assays and flow cytometry, indicating lower levels of LipA relative to LipB when individually expressed. Functional analysis was conducted using a para-nitrophenyl palmitate assay and revealed modest lipase activity within co-expressing strains, although activity remained below that of a commercial lipase. To enable unique detection of LipA and LipB within the co-expression system, a Myc-tagged LipB plasmid was generated for use alongside His-tagged LipA. Together, these findings demonstrate the feasibility of BrkA-mediated co-display of functional heterodimeric LipA and LipB. While further optimization is necessary to improve lipase efficiency, these findings provide a foundation for the development of microbial systems for enzymatic lipid bioremediation.
BrkA Autotransporter Mediates Surface Display but Inefficient Extracellular Release of LinB in Escherichia coli
β-Hexachlorocyclohexane (β-HCH) is a persistent environmental pollutant that resists degradation and accumulates in the ecosystem, posing risks to human health. Haloalkane dehalogenases such as LinB from Sphingobium species offer potential for bioremediation, but their intracellular localization limits access to extracellular substrates. To address this, we investigated the BrkA autotransporter system of Bordetella pertussis for surface display and potential protease-mediated secretion of LinB in Escherichia coli. The previously developed pITAS construct encodes a BrkA-LinB fusion protein containing an engineered OmpT cleavage site and an N-terminal 6xHis-tag, and was expressed in both E. coli UT5600 (OmpT-) and UT2300 (OmpT+) strains. Subcellular fractionation determined that BrkA-LinB localized predominantly to the outer membrane in both E. coli strains, with bands at ~170-180 kDa corresponding to the full-length protein. Trypsin accessibility assay showed loss of those bands upon treatment, further confirming surface display. In the OmpT+ strain, no His-tagged BrkA-LinB cleavage products were detected in the concentrated supernatant. In contrast, the positive control pOCS produced a ~65 kDa band in the supernatant, consistent with OmpT-mediated cleavage. These findings indicate that while BrkA supports surface display of LinB, OmpT-mediated cleavage and extracellular release were not observed in the pITAS construct. Given that surface display was confirmed in pITAS but cleavage products were only detected in pOCS, this suggests that insertion of LinB into the BrkA passenger domain may be affecting the accessibility of the engineered cleavage site.
Assessing BrkA-E3 Surface Conformation Using Trypsin Sensitivity Assays
BrkA is a type Vα autotransporter-based display systems from Bordetella pertussis whose passenger region can be engineered for recombinant protein display on the bacterial surface, where displayed proteins become extracellularly accessible for detection and ligand binding. E3 is an amyloid-β-targeting nanobody directed against a key pathological peptide in Alzheimer’s disease, and in this study we examined whether pJMMS, a plasmid encoding a BrkA-E3 fusion, retains a BrkA-like surface-accessibility pattern in Escherichia coli UT5600. Because anti-Aβ nanobodies may be useful in future surface-display-based screening and targeting strategies, it is important to determine whether E3 incorporation disrupts BrkA processing or accessibility. AlphaFold modelling suggested that the engineered fusion retained a largely BrkA-like architecture, although these models were used only to assess structural plausibility rather than as direct evidence of export or surface display. Anti-His Western blotting showed that pJMMS exhibited a trypsin-sensitive lower-molecular weight band similar to that of the positive BrkA control. Growth-curve analysis further suggested that expression of BrkA-E3 did not substantially impair bacterial growth relative to pENS. Together, these findings support the conclusion that E3 incorporation is compatible with BrkA processing and formation of a surface accessible BrkA-derived species.
Expression of Autotransporter BrkA Fails to Activate CpxAR Envelope Stress Response and Confer Heat Tolerance in Escherichia coli
Bordetella resistance to killing (BrkA) is a Type V autotransporter found in the outer membrane of Bordetella pertussis, the bacterium responsible for whooping cough. Previous work by Garcia et al. suggested that expression of BrkA in Escherichia coli may confer heat tolerance via a DegP-dependent mechanism. DegP is a temperature-dependent serine protease responsible for degrading misfolded periplasmic proteins; its expression is upregulated by the CpxAR two-component envelope stress response system under conditions of envelope stress. In this study, we investigated the effect of BrkA expression on envelope stress by measuring CpxAR activation using a luminescence (lux) reporter assay and assessed cell survival using optical density-based heat tolerance assays at 37℃ and 42℃. The luminescence assay demonstrated that BrkA expression did not activate the CpxAR response system. Furthermore, OD600 measurements revealed no enhanced heat tolerance in BrkA-expressing E. coli. Overall, our findings show that BrkA does not activate the CpxAR response system, which indicates that BrkA does not induce detectable envelope stress. This study provides insight into how BrkA is efficiently maintained in the membrane of B. pertussis.
Engineering of a Chimeric Hbp-BrkA Autotransporter for Surface Display in Escherichia coli
Autotransporters are a class of auto-display proteins used extensively in bacterial surface display systems, where a passenger domain is translocated to the cell exterior via a β-barrel domain anchored in the outer membrane. The hemoglobin-binding protease (Hbp) β-barrel has been established as a favourable translocator for chimeric autotransporter systems due to its high efficiency of outer membrane integration; however, its capacity to translocate the BrkA passenger domain from Bordetella pertussis has not been previously determined. In this study, we investigated whether the Hbp β-barrel could functionally replace the native BrkA β-barrel to support the surface display of the BrkA passenger domain in Escherichia coli BL21(DE3) by engineering and evaluating the chimeric plasmid pHAD (plasmid Hbp AutoDisplay). Sequence verification revealed a frameshift mutation within the Hbp β-barrel C-terminal region. Despite this, cells harbouring pHAD showed a significant increase in growth rate, and reduced but detectable levels of BrkA passenger domain in the membrane fraction. These results are suggestive of membrane association and partial surface exposure of the BrkA passenger domain mediated by the Hbp β-barrel, while highlighting the need for construct correction and optimized detection conditions to fully assess the surface-display capacity of this chimeric system.
Engineering the BrkA Autotransporter System to Surface Display an Anti-HER2 Nanobody in Escherichia coli
Breast cancer is the most frequently diagnosed cancer in women, and amplification of HER2 (human epidermal growth factor receptor 2), a tumour-associated antigen, defines an aggressive subtype while also providing a potential molecular marker for targeted therapies. This study investigates whether the Bordetella pertussis BrkA autotransporter system, which tolerates foreign protein insertion in its passenger domain, can be used to surface-display the HER2-specific nanobody C7b in Escherichia coli cells for targeted drug delivery and imaging applications. A recombinant BrkA-C7b plasmid (pBNJ) was constructed by inserting a C7b gene block into the 6x Histidine-tagged BrkA passenger domain within the pENS plasmid and expressed in UT5600 E. coli cells. Trypsin accessibility assays with western blotting confirmed the BrkA-C7b product to be surface accessible, however, trypsin susceptibility assays with western blotting indicated incomplete folding or misfolding of the construct, thereby suggesting a system that requires further optimization. These findings demonstrate surface display of C7b via the BrkA autotransporter, thus establishing a foundation for potential immunotherapies and tumour imaging strategies through further study of the BrkA-C7b anti-HER2 nanobody display system.
DegP Knockout in the JW0157 E. coli Strain Reveals No Improvement of Cysteine-Rich Recombinant BrkA-EC10 Surface Expression
The prevalence of global environmental and health-related issues stemming from historically high levels of heavy metal pollution in our natural surroundings has emphasized the need for more robust microbial biotechnology-based methods to remove these pollutants. As a result, bioremediation of heavy metal contaminants in environmental ecosystems using engineered microbial strains has become a significant area of academic interest. Recent studies have explored the use of the Type V autotransporter BrkA to display EC10, a phytochelator domain, on the surface of Escherichia coli as a viable strategy for microbially driven bioremediation. However, their findings reported reduced secretion of the BrkA–EC10 recombinant protein which was attributed to disulfide bond formation within the cysteine-rich EC10 domain. Subsequent experiments aimed to minimize disulfide bond formation using β-mercaptoethanol (βME) treatment and ∆dsbA strains. However, neither approaches improved BrkA–EC10 surface expression. The secretion pathway of BrkA-EC10 remains poorly understood, and no effective chemical or biological strategies have been identified to enhance BrkA–EC10 secretion in E. coli. Investigation of other periplasmic proteins interacting with BrkA-EC10 has identified the DegP protease as a potential contributor to BrkA-EC10 degradation. We hypothesize that E. coli cells lacking DegP exhibit enhanced cell surface expression of BrkA-EC10. Trypsin accessibility assay and Western blot analysis showed no significant improvement of BrkA-EC10 surface expression in ∆degP background E. coli strain JW0157. The liquid growth assay did show that the ∆degP background and intracellular expression of BrkA-EC10 does not significantly reduce cell viability or growth rate. However, as expected, the Cu2+ colorimetric assay shows that intracellularly expressed BrkA-EC10 does not cause a significant increase in Cu2+ heavy metal ion sequestration. These findings suggest that the removal of DegP protease alone is insufficient to significantly improve BrkA-EC10 surface expression and secretion, and that effective heavy metal ion sequestration may require high-efficiency surface expression of the BrkA-EC10 protein as a strategy for improved microbial bioremediation.