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Item Phylogenomics, Epigenomics, Virulome, and Mobilome of Gram-negative Bacteria Co-resistant to Carbapenems and Polymyxins: A One-Health Systematic Review and Meta-analyses(medRxiv, 2021-07-07) Ramaloko, Winnie Thabisa; Sekyere, John Osei; Microbiology and Immunology, School of MedicineGram-negative bacteria (GNB) continue to develop resistance against important antibiotics including last-resort ones such as carbapenems and polymyxins. An analysis of GNB with co-resistance to carbapenems and polymyxins from a One Health perspective is presented. Data of species name, country, source of isolation, resistance genes (ARGs), plasmid type, clones and mobile genetic elements (MGEs) were deduced from 129 articles from January 2016 to March 2021. Available genomes and plasmids were obtained from PATRIC and NCBI. Resistomes and methylomes were analysed using BAcWGSTdb and REBASE whilst Kaptive was used to predict capsule typing. Plasmids and other MEGs were identified using MGE Finder and ResFinder. Phylogenetic analyses were done using RAxML and annotated with MEGA 7. A total of 877 isolates, 32 genomes and 44 plasmid sequences were analysed. Most of these isolates were reported in Asian countries and were isolated from clinical, animal and environmental sources. Colistin resistance was mostly mediated by mgrB inactivation (37%; n = 322) and mcr-1 (36%; n = 312), while OXA-48/181 was the most reported carbapenemase. IncX and IncI were the most common plasmids hosting carbapenemases and mcr genes. The isolates were co-resistant to other antibiotics, with floR (chloramphenicol) and fosA3 (fosfomycin) being common; E. coli ST156 and K. pneumoniae ST258 strains were common globally. Virulence genes and capsular KL-types were also detected. Type I, II, III and IV restriction modification systems were detected, comprising various MTases and restriction enzymes. The escalation of highly resistant isolates drains the economy due to untreatable bacterial infections, which leads to increasing global mortality rates and healthcare costs.Item IFN-γ mediates Paneth cell death via suppression of mTOR(eLife Sciences, 2021-10-11) Araujo, Alessandra; Safronova, Alexandra; Burger, Elise; López-Yglesias, Américo; Giri, Shilpi; Camanzo, Ellie T.; Martin, Andrew T.; Grivennikov, Sergei; Yarovinsky, Felix; Microbiology and Immunology, School of MedicinePaneth cells constitutively produce antimicrobial peptides and growth factors that allow for intestinal homeostasis, host protection, and intestinal stem cell replication. Paneth cells rely heavily on the glycolytic metabolic program, which is in part controlled by the kinase complex Mechanistic target of rapamycin (mTORC1). Yet, little is known about mTOR importance in Paneth cell integrity under steady-state and inflammatory conditions. Our results demonstrate that IFN-γ, a crucial mediator of the intestinal inflammation, acts directly on murine Paneth cells to alter their mitochondrial integrity and membrane potential, resulting in an TORC1-dependent cell death mechanism distinct from canonical cell death pathways including apoptosis, necroptosis, and pyroptosis. These results were established with the purified cytokine and a physiologically relevant common Th1-inducing human parasite Toxoplasma gondii. Given the crucial role for IFN-γ, which is a cytokine frequently associated with the development of inflammatory bowel disease and compromised Paneth cell functions, the identified mechanisms underlying mTORC1-dependent Paneth cell death downstream of IFN-γ may provide promising novel approaches for treating intestinal inflammation.Item Gut community structure as a risk factor for infection in Klebsiella pneumoniae-colonized patients(American Society for Microbiology, 2024) Vornhagen, Jay; Rao, Krishna; Bachman, Michael A.; Microbiology and Immunology, School of MedicineThe primary risk factor for infection with members of the Klebsiella pneumoniae species complex is prior gut colonization, and infection is often caused by the colonizing strain. Despite the importance of the gut as a reservoir for infectious K. pneumoniae, little is known about the association between the gut microbiome and infection. To explore this relationship, we undertook a case-control study comparing the gut community structure of K. pneumoniae-colonized intensive care and hematology/oncology patients. Cases were K. pneumoniae-colonized patients infected by their colonizing strain (N = 83). Controls were K. pneumoniae-colonized patients who remained asymptomatic (N = 149). First, we characterized the gut community structure of K. pneumoniae-colonized patients agnostic to case status. Next, we determined that gut community data is useful for classifying cases and controls using machine learning models and that the gut community structure differed between cases and controls. K. pneumoniae relative abundance, a known risk factor for infection, had the greatest feature importance, but other gut microbes were also informative. Finally, we show that integration of gut community structure with bacterial genotype data enhanced the ability of machine learning models to discriminate cases and controls. Interestingly, inclusion of patient clinical variables failed to improve the ability of machine learning models to discriminate cases and controls. This study demonstrates that including gut community data with K. pneumoniae-derived biomarkers improves our ability to classify infection in K. pneumoniae-colonized patients. IMPORTANCE: Colonization is generally the first step in pathogenesis for bacteria with pathogenic potential. This step provides a unique window for intervention since a given potential pathogen has yet to cause damage to its host. Moreover, intervention during the colonization stage may help alleviate the burden of therapy failure as antimicrobial resistance rises. Yet, to understand the therapeutic potential of interventions that target colonization, we must first understand the biology of colonization and if biomarkers at the colonization stage can be used to stratify infection risk. The bacterial genus Klebsiella includes many species with varying degrees of pathogenic potential. Members of the K. pneumoniae species complex have the highest pathogenic potential. Patients colonized in their gut by these bacteria are at higher risk of subsequent infection with their colonizing strain. However, we do not understand if other members of the gut microbiota can be used as a biomarker to predict infection risk. In this study, we show that the gut microbiota differs between colonized patients who develop an infection versus those who do not. Additionally, we show that integrating gut microbiota data with bacterial factors improves the ability to classify infections. Surprisingly, patient clinical factors were not useful for classifying infections alone or when added to microbiota-based models. This indicates that the bacterial genotype and the microbial community in which it exists may determine the progression to infection. As we continue to explore colonization as an intervention point to prevent infections in individuals colonized by potential pathogens, we must develop effective means for predicting and stratifying infection risk.Item TL1A priming induces a multi-cytokine Th9 cell phenotype that promotes robust allergic inflammation in murine models of asthma(Elsevier, 2024) Niese, Michelle L.; Pajulas, Abigail L.; Rostron, Cameron R.; Cheung, Cherry C. L.; Krishnan, Maya S.; Zhang, Jilu; Cannon, Anthony M.; Kaplan, Mark H.; Microbiology and Immunology, School of MedicineMulti-cytokine-producing Th9 cells secrete IL-9 and type 2 cytokines and mediate mouse and human allergic inflammation. However, the cytokines that promote a multi-cytokine secreting phenotype have not been defined. Tumor necrosis factor superfamily member TL1A signals through its receptor DR3 to increase IL-9. Here we demonstrate that TL1A increases expression of IL-9 and IL-13 co-expressing cells in murine Th9 cell cultures, inducing a multi-cytokine phenotype. Mechanistically, this is linked to histone modifications allowing for increased accessibility at the Il9 and Il13 loci. We further show that TL1A alters the transcription factor network underlying expression of IL-9 and IL-13 in Th9 cells and increases binding of transcription factors to Il9 and Il13 loci. TL1A-priming enhances the pathogenicity of Th9 cells in murine models of allergic airway disease through the increased expression of IL-9 and IL-13. Lastly, in both chronic and memory-recall models of allergic airway disease, blockade of TL1A signaling decreases the multi-cytokine Th9 cell population and attenuates the allergic phenotype. Taken together, these data demonstrate that TL1A promotes the development of multi-cytokine Th9 cells that drive allergic airway diseases and that targeting pathogenic T helper cell-promoting cytokines could be an effective approach for modifying disease.Item Recruiting transgender men in the Southeastern United States for genital microbiome research: Lessons learned(Public Library of Science, 2024-08-12) Van Gerwen, Olivia T.; Sherman, Z. Alex; Kay, Emma Sophia; Wall, Jay; Lewis, Joy; Eastlund, Isaac; Graves, Keonte J.; Richter, Saralyn; Pontius, Angela; Aaron, Kristal J.; Siwakoti, Krishmita; Rogers, Ben; Toh, Evelyn; Elnaggar, Jacob H.; Taylor, Christopher M.; Van Wagoner, Nicholas J.; Muzny, Christina A.; Microbiology and Immunology, School of MedicineBackground: Transgender men (TGM) are underrepresented in genital microbiome research. Our prospective study in Birmingham, AL investigated genital microbiota changes over time in TGM initiating testosterone, including the development of incident bacterial vaginosis (iBV). Here, we present lessons learned from recruitment challenges encountered during the conduct of this study. Methods: Inclusion criteria were assigned female sex at birth, TGM or non-binary identity, age ≥18 years, interested in injectable testosterone but willing to wait 7 days after enrollment before starting, and engaged with a testosterone-prescribing provider. Exclusion criteria were recent antibiotic use, HIV/STI infection, current vaginal infection, pregnancy, or past 6 months testosterone use. Recruitment initiatives included community advertisements via flyers, social media posts, and referrals from local gender health clinics. Results: Between February 2022 and October 2023, 61 individuals contacted the study, 17 (27.9%) completed an in-person screening visit, and 10 (58.8%) of those screened were enrolled. The primary reasons for individuals failing study screening were having limited access to testosterone-prescribing providers, already being on testosterone, being unwilling to wait 7 days to initiate testosterone therapy, or desiring the use of topical testosterone. Engagement of non-White TGM was also minimal. Conclusion: Despite robust study inquiry by TGM, screening and enrollment challenges were faced including engagement by TGM not yet in care and specific study eligibility criteria. Excitement among TGM for research representation should be leveraged in future work by engaging transgender community stakeholders at the inception of study development, particularly regarding feasibility of study inclusion and exclusion criteria, as well as recruitment of TGM of color. These results also highlight the need for more clinical resources for prescribing gender-affirming hormone therapy, especially in the Southeastern US.Item Characterization of Vaginal Microbial Community Dynamics in the Pathogenesis of Incident Bacterial Vaginosis, a Pilot Study(Wolters Kluwer, 2023) Elnaggar, Jacob H.; Lammons, John W.; Taylor, Christopher M.; Toh, Evelyn; Ardizzone, Caleb M.; Dong, Amy; Aaron, Kristal J.; Luo, Meng; Tamhane, Ashutosh; Lefkowitz, Elliot J.; Quayle, Alison J.; Nelson, David E.; Muzny, Christina A.; Microbiology and Immunology, School of MedicineBackground: Despite more than 60 years of research, the etiology of bacterial vaginosis (BV) remains controversial. In this pilot study, we used shotgun metagenomic sequencing to characterize vaginal microbial community changes before the development of incident BV (iBV). Methods: A cohort of African American women with a baseline healthy vaginal microbiome (no Amsel criteria, Nugent score 0-3 with no Gardnerella vaginalis morphotypes) were followed for 90 days with daily self-collected vaginal specimens for iBV (≥2 consecutive days of a Nugent score of 7-10). Shotgun metagenomic sequencing was performed on select vaginal specimens from 4 women, every other day for 12 days before iBV diagnosis. Sequencing data were analyzed through Kraken2 and bioBakery 3 workflows, and specimens were classified into community state types. Quantitative polymerase chain reaction was performed to compare the correlation of read counts with bacterial abundance. Results: Common BV-associated bacteria such as G. vaginalis , Prevotella bivia , and Fannyhessea vaginae were increasingly identified in the participants before iBV. Linear modeling indicated significant increases in G. vaginalis and F . vaginae relative abundance before iBV, whereas the relative abundance of Lactobacillus species declined over time. The Lactobacillus species decline correlated with the presence of Lactobacillus phages. We observed enrichment in bacterial adhesion factor genes on days before iBV. There were also significant correlations between bacterial read counts and abundances measured by quantitative polymerase chain reaction. Conclusions: This pilot study characterizes vaginal community dynamics before iBV and identifies key bacterial taxa and mechanisms potentially involved in the pathogenesis of iBV.Item Borrelia burgdorferi Secretes c-di-AMP as an Extracellular Pathogen-Associated Molecular Pattern to Elicit Type I Interferon Responses in Mammalian Hosts(bioRxiv, 2024-08-20) Priya, Raj; Ye, Meiping; Raghunanadanan, Sajith; Liu, Qiang; Li, Wei; Lou, Yongliang; Sintim, Herman O.; Yang, X. Frank; Microbiology and Immunology, School of MedicineBorrelia burgdorferi (B. burgdorferi), an extracellular spirochetal pathogen, elicits a type-I interferon (IFN-I) response that contributes to the pathology of Lyme disease, including the development and severity of Lyme arthritis. However, the specific Pathogen-Associated Molecular Patterns (PAMPs) of B. burgdorferi responsible for triggering the IFN-I response are not well understood. Previous studies have identified an unknown, nuclease-resistant component in B. burgdorferi culture supernatants that significantly stimulates the IFN-I response, but its identity remains unknown. In this study, we reveal that B. burgdorferi secretes cyclic-di-adenosine monophosphate (c-di-AMP) as a key extracellular PAMP, inducing the host IFN-I response in macrophages. Using genetically manipulated B. burgdorferi strains, we demonstrate a requirement of c-di-AMP for stimulating IFN-I response by macrophages ex vivo. Additionally, infecting mice with B. burgdorferi alongside exogenous c-di-AMP resulted in a markedly increased IFN-I response in mouse tissues. Furthermore, inactivation or inhibition of the host STING signaling pathway significantly reduced the IFN-I response, indicating that c-di-AMP-induced IFN-I production is STING-dependent. Our findings identify c-di-AMP as a crucial PAMP secreted by B. burgdorferi to elicit the host IFN-I response via activation of STING signaling pathway, suggesting that targeting c-di-AMP production could represent a novel therapeutic strategy against Lyme arthritis.Item Targeting Nrf2 with 3 H-1,2-dithiole-3-thione to moderate OXPHOS-driven oxidative stress attenuates IL-17A-induced psoriasis(Elsevier, 2023-03) Liu, Chuan-Teng; Yen, Jui-Hung Jimmy; Brown, Dennis A.; Song, Ying-Chyi; Chu, Mei-Yun; Hung, Yu-Hsiang; Tang, Yi-Huan; Wu, Po-Yuan; Yen, Hung-Rong; Microbiology and Immunology, School of MedicinePsoriasis, a chronic autoimmune disease characterized by the hyperproliferation of keratinocytes in the epidermis and parakeratosis, significantly impacts quality of life. Interleukin (IL)− 17A dominates the pathogenesis of psoriasis and facilitates reactive oxygen species (ROS) accumulation, which exacerbates local psoriatic lesions. Biologic treatment provides remarkable clinical efficacy, but its high cost and unignorable side effects limit its applications. 3 H-1,2-Dithiole-3-thione (D3T) possesses compelling antioxidative capacities against several diseases through the nuclear factor erythroid 2-related factor 2 (Nrf2) cascade. Hence, we aimed to evaluate the effect and mechanism of D3T in psoriasis. We found that D3T attenuates skin thickening and scaling by inhibiting IL-17A-secreting γδT cells in imiquimod (IMQ)-induced psoriatic mice. Interleukin-17A markedly enhanced IL-6 and IL-8 expression, lipid peroxidation, the contents of nitric oxide and hydrogen peroxide, oxidative phosphorylation and the MAPK/NF-κB pathways in keratinocytes. IL-17A also inhibited the Nrf2-NQO1-HO-1 axis and the activities of superoxide dismutase and glutathione peroxidase. D3T significantly reversed these parameters in IL-17A-treated keratinocytes. ML‐385, a Nrf2 neutralizer, failed to improve D3T-induced anti-inflammatory and antioxidative effects in IL-17A-treated keratinocytes. We conclude that targeting Nrf2 with D3T to diminish oxidative and inflammatory damage in keratinocytes may attenuate psoriasis.Item Adiponectin pathway activation dampens inflammation and enhances alveolar macrophage fungal killing via LC3-associated phagocytosis(bioRxiv, 2024-08-24) Goli, Sri Harshini; Lim, Joo-Yeon; Basaran-Akgul, Nese; Templeton, Steven P.; Microbiology and Immunology, School of MedicineAlthough innate immunity is critical for antifungal host defense against the human opportunistic fungal pathogen Aspergillus fumigatus, potentially damaging inflammation must be controlled. Adiponectin (APN) is an adipokine produced mainly in adipose tissue that exerts anti-inflammatory effects in adipose-distal tissues such as the lung. We observed 100% mortality and increased fungal burden and inflammation in neutropenic mice with invasive aspergillosis (IA) that lack APN or the APN receptors AdipoR1 or AdipoR2. Alveolar macrophages (AMs), early immune sentinels that detect and respond to lung infection, express both receptors, and APN−/− AMs exhibited an inflammatory/M1 phenotype that was associated with decreased fungal killing. Pharmacological stimulation of AMs with AdipoR agonist AdipoRon partially rescued deficient killing in APN−/− AMs that was dependent on both receptors. Finally, APN-enhanced fungal killing was associated with increased activation of the non-canonical LC3 pathway of autophagy. Thus, our study identifies a novel role for APN in LC3-mediated killing of A. fumigatus.Item BadR directly represses the expression of the glycerol utilization operon in the Lyme disease pathogen(American Society for Microbiology, 2024) Zhang, Jun-Jie; Raghunandanan, Sajith; Wang, Qian; Priya, Raj; Alanazi, Fuad; Lou, Yongliang; Yang, X. Frank; Microbiology and Immunology, School of MedicineGlycerol utilization as a carbohydrate source by Borreliella burgdorferi, the Lyme disease spirochete, is critical for its successful colonization and persistence in the tick vector. The expression of the glpFKD (glp) operon, which encodes proteins for glycerol uptake/utilization, must be tightly regulated during the enzootic cycle of B. burgdorferi. Previous studies have established that the second messenger cyclic di-GMP (c-di-GMP) is required for the activation of glp expression, while an alternative sigma factor RpoS acts as a negative regulator for glp expression. In the present study, we report identification of a cis element within the 5´ untranslated region of glp that exerts negative regulation of glp expression. Further genetic screen of known and predicted DNA-binding proteins encoded in the genome of B. burgdorferi uncovered that overexpressing Borrelia host adaptation regulator (BadR), a known global regulator, dramatically reduced glp expression. Similarly, the badR mutant significantly increased glp expression. Subsequent electrophoretic mobility shift assay analyses demonstrated that BadR directly binds to this cis element, thereby repressing glp independent of RpoS-mediated repression. The efficiency of BadR binding was further assessed in the presence of c-di-GMP and various carbohydrates. This finding highlights multi-layered positive and negative regulatory mechanisms employed by B. burgdorferi to synchronize glp expression throughout its enzootic cycle.IMPORTANCEBorreliella burgdorferi, the Lyme disease pathogen, must modulate its gene expression differentially to adapt successfully to its two disparate hosts. Previous studies have demonstrated that the glycerol uptake and utilization operon, glpFKD, plays a crucial role in spirochetal survival within ticks. However, the glpFKD expression must be repressed when B. burgdorferi transitions to the mammalian host. In this study, we identified a specific cis element responsible for the repression of glpFKD. We further pinpointed Borrelia host adaptation regulator as the direct binding protein to this cis element, thereby repressing glpFKD expression. This discovery paves the way for a deeper exploration of how zoonotic pathogens sense distinct hosts and switch their carbon source utilization during transmission.