Browsing by Author "Yang, X. Frank"
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Item A Fur family protein BosR is a novel RNA-binding protein that controls rpoS RNA stability in the Lyme disease pathogen(Oxford University Press, 2024) Raghunandanan, Sajith; Priya, Raj; Alanazi, Fuad; Lybecker, Meghan C.; Schlax, Paula Jean; Yang, X. Frank; Microbiology and Immunology, School of Medicine2´-O-methylation (Nm) is one of the most abundant modifications found in both mRNAs and noncoding RNAs. It contributes to many biological processes, such as the normal functioning of tRNA, the protection of mRNA against degradation by the decapping and exoribonuclease (DXO) protein, and the biogenesis and specificity of rRNA. Recent advancements in single-molecule sequencing techniques for long read RNA sequencing data offered by Oxford Nanopore technologies have enabled the direct detection of RNA modifications from sequencing data. In this study, we propose a bio-computational framework, Nm-Nano, for predicting the presence of Nm sites in direct RNA sequencing data generated from two human cell lines. The Nm-Nano framework integrates two supervised machine learning (ML) models for predicting Nm sites: Extreme Gradient Boosting (XGBoost) and Random Forest (RF) with K-mer embedding. Evaluation on benchmark datasets from direct RNA sequecing of HeLa and HEK293 cell lines, demonstrates high accuracy (99% with XGBoost and 92% with RF) in identifying Nm sites. Deploying Nm-Nano on HeLa and HEK293 cell lines reveals genes that are frequently modified with Nm. In HeLa cell lines, 125 genes are identified as frequently Nm-modified, showing enrichment in 30 ontologies related to immune response and cellular processes. In HEK293 cell lines, 61 genes are identified as frequently Nm-modified, with enrichment in processes like glycolysis and protein localization. These findings underscore the diverse regulatory roles of Nm modifications in metabolic pathways, protein degradation, and cellular processes. The source code of Nm-Nano can be freely accessed at https://github.com/Janga-Lab/Nm-Nano.Item Acetyl-Phosphate Is Not a Global Regulatory Bridge between Virulence and Central Metabolism in Borrelia burgdorferi(Public Library of Science (PLoS), 2015) Richards, Crystal L.; Lawrence, Kevin A.; Su, Hua; Yang, Youyun; Yang, X. Frank; Dulebohn, Daniel P.; Gherardini, Frank C.; Department of Microbiology and Immunology, IU School of MedicineIn B. burgdorferi, the Rrp2-RpoN-RpoS signaling cascade is a distinctive system that coordinates the expression of virulence factors required for successful transition between its arthropod vector and mammalian hosts. Rrp2 (BB0763), an RpoN specific response regulator, is essential to activate this regulatory pathway. Previous investigations have attempted to identify the phosphate donor of Rrp2, including the cognate histidine kinase, Hk2 (BB0764), non-cognate histidine kinases such as Hk1, CheA1, and CheA2, and small molecular weight P-donors such as carbamoyl-phosphate and acetyl-phosphate (AcP). In a report by Xu et al., exogenous sodium acetate led to increased expression of RpoS and OspC and it was hypothesized this effect was due to increased levels of AcP via the enzyme AckA (BB0622). Genome analyses identified only one pathway that could generate AcP in B. burgdorferi: the acetate/mevalonate pathway that synthesizes the lipid, undecaprenyl phosphate (C55-P, lipid I), which is essential for cell wall biogenesis. To assess the role of AcP in Rrp2-dependent regulation of RpoS and OspC, we used a unique selection strategy to generate mutants that lacked ackA (bb0622: acetate to AcP) or pta (bb0589: AcP to acetyl-CoA). These mutants have an absolute requirement for mevalonate and demonstrate that ackA and pta are required for cell viability. When the ΔackA or Δpta mutant was exposed to conditions (i.e., increased temperature or cell density) that up-regulate the expression of RpoS and OspC, normal induction of those proteins was observed. In addition, adding 20mM acetate or 20mM benzoate to the growth media of B. burgdorferi strain B31 ΔackA induced the expression of RpoS and OspC. These data suggest that AcP (generated by AckA) is not directly involved in modulating the Rrp2-RpoN-RpoS regulatory pathway and that exogenous acetate or benzoate are triggering an acid stress response in B. burgdorferi.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.Item Borrelia burgdorferi elongation factor EF-Tu is an immunogenic protein during Lyme borreliosis(Nature, 2015-09) Carrasco, Sebastian E.; Yang, Youyun; Troxell, Bryan; Yang, Xiuli; Pal, Utpal; Yang, X. Frank; Department of Microbiology & Immunology, IU School of MedicineBorrelia burgdorferi, the etiological agent of Lyme disease, does not produce lipopolysaccharide but expresses a large number of lipoproteins on its cell surface. These outer membrane lipoproteins are highly immunogenic and have been used for serodiagnosis of Lyme disease. Recent studies have shown that highly conserved cytosolic proteins such as enolase and elongation factor Tu (EF-Tu) unexpectedly localized on the surface of bacteria including B. burgdorferi, and surface-localized enolase has shown to contribute to the enzootic cycle of B. burgdorferi. In this study, we studied the immunogenicity, surface localization, and function of B. burgdorferi EF-Tu. We found that EF-Tu is highly immunogenic in mice, and EF-Tu antibodies were readily detected in Lyme disease patients. On the other hand, active immunization studies showed that EF-Tu antibodies did not protect mice from infection when challenged with B. burgdorferi via either needle inoculation or tick bites. Borrelial mouse-tick cycle studies showed that EF-Tu antibodies also did not block B. burgdorferi migration and survival in ticks. Consistent with these findings, we found that EF-Tu primarily localizes in the protoplasmic cylinder of spirochetes and is not on the surface of B. burgdorferi. Taken together, our studies suggest that B. burgdorferi EF-Tu is not surfaced exposed, but it is highly immunogenic and is a potential serodiagnostic marker for Lyme borreliosis.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 Corrigendum: Role of HK2 in the Enzootic Cycle of Borrelia burgdorferi(Frontiers Media, 2021-03-31) Liu, Qiang; Xu, Haijun; Zhang, Yan; Yang, Jing; Du, Jimei; Zhou, Yan; Yang, X. Frank; Lou, Yongliang; Microbiology and Immunology, School of MedicineItem Developing In Vitro and In Vivo Models for Lyme Neuroborreliosis (LNB)(2023-08) Alanazi, Fuad Fahad; Yang, X. Frank; Bauer, Margaret E.; Yu, Andy Q.; Relich, Ryan F.; Nass, Richard M.Lyme neuroborreliosis (LNB) is a neurologic disorder caused by infection with Borrelia burgdorferi, resulting in inflammation in the central and peripheral nervous systems. LNB remains poorly understood due to the lack of a suitable experimental model. The non-human primate model for LNB presents significant impediments, such as high costs, specialized training, ethical considerations, and low infection frequency. Finding alternative models is imperative to advance LNB research. This study aims to develop alternative in vitro and in vivo models for LNB. First, we developed an in vitro transwell assay to identify the factors required for the blood-brain barrier (BBB) transmigration of B. burgdorferi. Second, we established a middle-aged mouse model for studying neuroinflammation associated with LNB. Last, we further characterized a Caenorhabditis elegans (C. elegans) model to study B. burgdorferi-associated neuron damage. With these models, we discovered that the Rrp2-RpoN-RpoS pathway in B. burgdorferi is essential for B. burgdorferi to cross the BBB and that the outer surface protein C (OspC) controlled by this pathway plays a vital role in crossing the BBB. We found that B. burgdorferi is detectable in the brains of middle-aged mice but not in younger mice and triggers host immune response, resulting in elevated levels of cytokines such as TNF-alpha, IFN-γ, and IL-9 and reduction in microglia in the infected mice. Lastly, we demonstrated that C. elegans can feed on B. burgdorferi, which may result in neurodegeneration. This provides a powerful tool for screening pathogen and host factors involved in neuroborreliosis. Overall, the in vitro and in vivo models developed in this study will significantly advance LNB research, which may lead to the development of new treatments and improved patient outcomes.Item DhhP, a Cyclic di-AMP Phosphodiesterase of Borrelia burgdorferi, Is Essential for Cell Growth and Virulence(ASM, 2014-05) Ye, Meiping; Zhang, Jun-Jie; Fang, Xin; Lawlis, Gavin B.; Troxell, Bryan; Zhou, Yan; Gomelsky, Mark; Lou, Yongliang; Yang, X. Frank; Department of Microbiology and Immunology, IU School of MedicineCyclic di-AMP (c-di-AMP) is a recently discovered second messenger in bacteria. Most of work on c-di-AMP signaling has been done in Gram-positive bacteria, firmicutes, and actinobacteria, where c-di-AMP signaling pathways affect potassium transport, cell wall structure, and antibiotic resistance. Little is known about c-di-AMP signaling in other bacteria. Borrelia burgdorferi, the causative agent of Lyme disease, is a spirochete that has a Gram-negative dual membrane. In this study, we demonstrated that B. burgdorferi BB0619, a DHH-DHHA1 domain protein (herein designated DhhP), functions as c-di-AMP phosphodiesterase. Recombinant DhhP hydrolyzed c-di-AMP to pApA in a Mn2+- or Mg2+-dependent manner. In contrast to c-di-AMP phosphodiesterases reported thus far, DhhP appears to be essential for B. burgdorferi growth both in vitro and in the mammalian host. Inactivation of the chromosomal dhhP gene could be achieved only in the presence of a plasmid-encoded inducible dhhP gene. The conditional dhhP mutant had a dramatic increase in intracellular c-di-AMP level in comparison to the isogenic wild-type strain. Unlike what has been observed in Gram-positive bacteria, elevated cellular c-di-AMP in B. burgdorferi did not result in an increased resistance to β-lactamase antibiotics, suggesting that c-di-AMP's functions in spirochetes differ from those in Gram-positive bacteria. In addition, the dhhP mutant was defective in induction of the σS factor, RpoS, and the RpoS-dependent outer membrane virulence factor OspC, which uncovers an important role of c-di-AMP in B. burgdorferi virulence.Item Differential Recruitment of Host Proteins to the Coxiella Burnetii Vacuole in the Absence of the Sterol Reductase CBU1206(2020-08) Ratnayake, Rochelle Chashmi; Gilk, Stacey; Yang, X. Frank; Tran, Tuan M.; Sullivan, William J., Jr.Q fever is a heavily underdiagnosed and underreported infection caused by the obligate intracellular pathogen Coxiella burnetii. Following entry into the host cell, Coxiella replicates in the acidic phagolysosome-like parasitophorous vacuole termed the Coxiella Containing Vacuole (CCV). The CCV is a large and highly fusogenic compartment that actively fuses with the host endocytic pathway during maturation of the phagolysosome. Evidence suggests that the development of the CCV is sensitive to increasing cholesterol levels and leads to CCV acidification and bacterial death. Therefore, we hypothesize that CCV cholesterol concentration is carefully modulated through the Coxiella encoded sterol reductases (CBU1206 and CBU1158). A ∆CBU1206 mutant of Coxiella is hypersensitive to cholesterol and displays growth defects in intracellular replication and CCV development. Following fusion with the host endocytic pathway, the Coxiella NMII Phase II (WT) CCVs readily acquire host proteins such as LAMP1, CD63, Rab7, ORP1L, RILP, and LC3. These heterotypic events with the host endosomal cascade are presumed to provide selected subsets of endocytosed cargo and membrane. Therefore, I investigated whether ΔCBU1206 CCV heterotypic fusion events are defective due to altered lipid content on the CCV membrane. I observed increased accumulation of sterols on the ΔCBU1206 CCV membrane. Similar to WT, the mutant readily fuses host lysosomes and readily acquires the host glycoprotein LAMP1 but displays reduced localization of CD63 (LAMP3). Additionally, reduced localization of the late endosomal markers Rab7, ORP1L, and RILP was observed suggesting that late endosome fusion maybe defective in ΔCBU1206. Further, reduced localization of LC3 was also observed suggesting that the mutant may also be defective in fusing with autophagosomes. Finally, the mutant possesses a functional Type 4 Secretion System that secretes a moderate amount of effector proteins relative to WT. Considering the vast array of functions accomplished by the effectors secreted, the moderate effector secretion by the mutant could influence the endocytic pathway fusion processes as well as CCV development. Collectively, this body of work suggests that the lack of sterol reductase CBU1206 in Coxiella results in defective heterotypic fusion events of the CCV membrane that could alter pathogenesis and CCV expansion.Item The EbpA-RpoN Regulatory Pathway of the Pathogen Leptospira interrogans Is Essential for Survival in the Environment(American Society for Microbiology, 2017-01-17) Hu, Wei-Lin; Pappas, Christopher J.; Zhang, Jun-Jie; Yang, You-Yun; Yan, Jie; Picardeau, Mathieu; Yang, X. Frank; Microbiology and Immunology, School of MedicineLeptospira interrogans is the agent of leptospirosis, a reemerging zoonotic disease. It is transmitted to humans through environmental surface waters contaminated by the urine of mammals chronically infected by pathogenic strains able to survive in water for long periods. Little is known about the regulatory pathways underlying environmental sensing and host adaptation of L. interrogans during its enzootic cycle. This study identifies the EbpA-RpoN regulatory pathway in L. interrogans In this pathway, EbpA, a σ54 activator and putative prokaryotic enhancer-binding protein (EBP), and the alternative sigma factor RpoN (σ54) control expression of at least three genes, encoding AmtB (an ammonium transport protein) and two proteins of unknown function. Electrophoresis mobility shift assay demonstrated that recombinant RpoN and EbpA bind to the promoter region and upstream of these three identified genes, respectively. Genetic disruption of ebpA in L. interrogans serovar Manilae virtually abolished expression of the three genes, including amtB in two independent ebpA mutants. Complementation of the ebpA mutant restored expression of these genes. Intraperitoneal inoculation of gerbils with the ebpA mutant did not affect mortality. However, the ebpA mutant had decreased cell length in vitro and had a significantly lowered cell density at stationary phase when grown with l-alanine as the sole nitrogen source. Furthermore, the ebpA mutant has dramatically reduced long-term survival ability in water. Together, these studies identify a regulatory pathway, the EbpA-RpoN pathway, that plays an important role in the zoonotic cycle of L. interrogans IMPORTANCE: Leptospirosis is a reemerging disease with global importance. However, our understanding of gene regulation of the spirochetal pathogen Leptospira interrogans is still in its infancy, largely due to the lack of robust tools for genetic manipulation of this spirochete. Little is known about how the pathogen achieves its long-term survival in the aquatic environment. By utilizing bioinformatic, genetic, and biochemical methods, we discovered a regulatory pathway in L. interrogans, the EbpA-RpoN pathway, and demonstrated that this pathway plays an important role in environmental survival of this pathogen.