IU Indianapolis ScholarWorks :: Browsing by Author "Raghunandanan, Sajith"

Browsing by Author "Raghunandanan, Sajith"

Now showing 1 - 8 of 8

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 Medicine
2´-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.
A systemic approach to identify non-abundant immunogenic proteins in Lyme disease pathogens
(American Society for Microbiology, 2024) Yaş, Ozlem Buyuktanir; Coleman, Adam S.; Lipman, Rachel M.; Sharma, Kavita; Raghunandanan, Sajith; Alanazi, Fuad; Rana, Vipin S.; Kitsou, Chrysoula; Yang, Xiuli; Pal, Utpal; Microbiology and Immunology, School of Medicine
Borrelia burgdorferi, the pathogen of Lyme disease, differentially produces many outer surface proteins (Osp), some of which represent the most abundant membrane proteins, such as OspA, OspB, and OspC. In cultured bacteria, these proteins can account for a substantial fraction of the total cellular or membrane proteins, posing challenges to the identification and analysis of non-abundant proteins, which could serve as novel pathogen detection markers or as vaccine candidates. Herein, we introduced serial mutations to remove these abundant Osps and generated a B. burgdorferi mutant deficient in OspA, OspB, and OspC in an infectious 297-isolate background, designated as OspABC- mutant. Compared to parental isolate, the mutant did not reflect growth defects in the cultured medium but showed differential mRNA expression of representative tested genes, in addition to gross changes in cellular and membrane protein profiles. The analysis of differentially detectable protein contents of the OspABC- mutant, as compared to the wild type, by two-dimensional gel electrophoresis followed by liquid chromatography-mass spectrometry, identified several spirochete proteins that are dominated by proteins of unknown functions, as well as membrane transporters, chaperons, and metabolic enzymes. We produced recombinant forms of two of these represented proteins, BBA34 and BB0238, and showed that these proteins are detectable during spirochete infection in the tick-borne murine model of Lyme borreliosis and thus serve as potential antigenic markers of the infection. IMPORTANCE: The present manuscript employed a systemic approach to identify non-abundant proteins in cultured Borrelia burgdorferi that are otherwise masked or hidden due to the overwhelming presence of abundant Osps like OspA, OspB, and OspC. As these Osps are either absent or transiently expressed in mammals, we performed a proof-of-concept study in which their removal allowed the analysis of otherwise less abundant antigens in OspABC-deficient mutants and identified several immunogenic proteins, including BBA34 and BB0238. These antigens could serve as novel vaccine candidates and/or genetic markers of Lyme borreliosis, promoting new research in the clinical diagnosis and prevention of Lyme disease.
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 Medicine
Glycerol 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.
Dormant Mycobacterium tuberculosis converts isoniazid to the active drug in a Wayne’s model of dormancy
(Nature, 2018) Raghunandanan, Sajith; Jose, Leny; Kumar, Ramakrishnan Ajay; Dermatology, School of Medicine
Isoniazid (INH) is one among the four first-line drugs used in the treatment of tuberculosis. The bactericidal activity of INH is due to its ability to inhibit mycolic acid synthesis, which is an integral component of the mycobacterial cell wall. Non-replicating Mycobacterium tuberculosis (MTB) is phenotypically resistant to INH. The exact mechanism of this resistance is not clear, although the inability of dormant MTB to convert the pro-drug into an active form is thought to be one of the possible reasons. Employing targeted metabolomics approach, we show that dormant MTB can metabolize INH into its active INH-NAD+ adduct form. Further we show that the dormant bacteria have unaltered gene expression levels of katG and inhA (INH metabolizing enzymes). Transcript levels of drug efflux pump proteins which were low during dormancy did not increase in response to INH treatment. These findings point to an alternative mechanism for INH resistance in dormant MTB, which needs to be further elucidated.
MCP5, a methyl-accepting chemotaxis protein regulated by both the Hk1-Rrp1 and Rrp2-RpoN-RpoS pathways, is required for the immune evasion of Borrelia burgdorferi
(bioRxiv, 2024-06-10) Raghunandanan, Sajith; Zhang, Kai; Zhang, Yan; Sze, Ching Wooen; Priya, Raj; Luo, Yongliang; Lynch, Michael J.; Crane, Brian R.; Li, Chunhao; Yang, X. Frank; Microbiology and Immunology, School of Medicine
Borrelia (or Borreliella) burgdorferi, the causative agent of Lyme disease, is a motile and invasive zoonotic pathogen, adept at navigating between its arthropod vector and mammalian host. While motility and chemotaxis are well established as essential for its enzootic cycle, the function of methyl-accepting chemotaxis proteins (MCPs) in the infectious cycle of B. burgdorferi remains unclear. In this study, we demonstrate that MCP5, one of the most abundant MCPs in B. burgdorferi, is differentially expressed in response to environmental signals as well as at different stages of the pathogen’s enzootic cycle. Specifically, the expression of mcp5 is regulated by the Hk1-Rrp1 and Rrp2-RpoN-RpoS pathways, which are critical for the spirochete’s colonization of the tick vector and mammalian host, respectively. Infection experiments with an mcp5 mutant revealed that spirochetes lacking MCP5 could not establish infections in either C3H/HeN mice or Severe Combined Immunodeficiency (SCID) mice, which are defective in adaptive immunity, indicating the essential role of MCP5 in mammalian infection. However, the mcp5 mutant could establish infection and disseminate in NOD SCID Gamma (NSG) mice, which are deficient in both adaptive and most innate immune responses, suggesting a crucial role of MCP5 in evading host innate immunity. In the tick vector, the mcp5 mutants survived feeding but failed to transmit to mice, highlighting the importance of MCP5 in transmission. Our findings reveal that MCP5, regulated by the Rrp1 and Rrp2 pathways, is critical for the establishment of infection in mammalian hosts by evading host innate immunity and is important for the transmission of spirochetes from ticks to mammalian hosts, underscoring its potential as a target for intervention strategies.
Positive feedback regulation between RpoS and BosR in the Lyme disease pathogen
(bioRxiv, 2024-09-15) Raghunandanan, Sajith; Priya, Raj; Lin, Gaofeng; Alanazi, Fuad; Zoss, Andrew; Warren, Elise; Yang, X. Frank; Microbiology and Immunology, School of Medicine
In Borrelia burgdorferi, the Lyme disease pathogen, differential gene expression is primarily controlled by the alternative sigma factor RpoS (σS). Understanding how RpoS levels are regulated is crucial for elucidating how B. burgdorferi is maintained throughout its enzootic cycle. Our recent studies have shown that a homolog of Fur/PerR repressor/activator, BosR, functions as an RNA-binding protein that controls the rpoS mRNA stability. However, the mechanisms of regulation of BosR, particularly in response to host signals and environmental cues, remain largely unclear. In this study, we revealed a positive feedback loop between RpoS and BosR, where RpoS post-transcriptionally regulates BosR levels. Specifically, mutation or deletion of rpoS significantly reduced BosR levels, while artificial induction of rpoS resulted in a dose-dependent increase in BosR levels. Notably, RpoS does not affect bosR mRNA levels but instead modulates the turnover rate of the BosR protein. Furthermore, we demonstrated that environmental cues do not directly influence bosR expression but instead induce rpoS transcription and RpoS production, thereby enhancing BosR protein levels. This discovery adds a new layer of complexity to the RpoN-RpoS pathway and suggests the need to re-evaluate the factors and signals previously believed to regulate RpoS levels through BosR.
The Rrp2-RpoN-RpoS pathway plays an important role in the blood-brain barrier transmigration of the Lyme disease pathogen
(American Society for Microbiology, 2023) Alanazi, Fuad; Raghunandanan, Sajith; Priya, Raj; Yang, X. Frank; Microbiology and Immunology, School of Medicine
Lyme disease, caused by Borrelia (or Borreliella) burgdorferi, is a complex multisystemic disorder that includes Lyme neuroborreliosis resulting from the invasion of both the central and peripheral nervous systems. However, factors that enable the pathogen to cross the blood-brain barrier (BBB) and invade the central nervous system (CNS) are still not well understood. The objective of this study was to identify the B. burgdorferi factors required for BBB transmigration. We utilized a transwell BBB model based on human brain-microvascular endothelial cells and focused on investigating the Rrp2-RpoN-RpoS pathway, a central regulatory pathway that is essential for mammalian infection by B. burgdorferi. Our results demonstrated that the Rrp2-RpoN-RpoS pathway is crucial for BBB transmigration. Furthermore, we identified OspC, a major surface lipoprotein controlled by the Rrp2-RpoN-RpoS pathway, as a significant contributor to BBB transmigration. Constitutive production of OspC in a mutant defective in the Rrp2-RpoN-RpoS pathway did not rescue the impairment in BBB transmigration, indicating that this pathway controls additional factors for this process. Two other major surface lipoproteins controlled by this pathway, DbpA/B and BBK32, appeared to be dispensable for BBB transmigration. In addition, both the surface lipoprotein OspA and the Rrp1 pathway, which are required B. burgdorferi colonization in the tick vector, were found not required for BBB transmigration. Collectively, our findings using in vitro transwell assays uncover another potential role of the Rrp2-RpoN-RpoS pathway in BBB transmigration of B. burgdorferi and invasion into the CNS.
YebC regulates variable surface antigen VlsE expression and is required for host immune evasion in Borrelia burgdorferi
(Public Library of Science, 2020-10-13) Zhang, Yan; Chen, Tong; Raghunandanan, Sajith; Xiang, Xuwu; Yang, Jing; Liu, Qiang; Edmondson, Diane G.; Norris, Steven J.; Yang, X. Frank; Lou, Yongliang; Microbiology and Immunology, School of Medicine
Borrelia burgdorferi, the Lyme disease pathogen causes persistent infection by evading the host immune response. Differential expression of the surface-exposed lipoprotein VlsE that undergoes antigenic variation is a key immune evasion strategy employed by B. burgdorferi. Most studies focused on the mechanism of VlsE antigen variation, but little is known about VlsE regulation and factor(s) that regulates differential vlsE expression. In this study, we investigated BB0025, a putative YebC family transcriptional regulator (and hence designated BB0025 as YebC of B. burgdorferi herein). We constructed yebC mutant and complemented strain in an infectious strain of B. burgdorferi. The yebC mutant could infect immunocompromised SCID mice but not immunocompetent mice, suggesting that YebC plays an important role in evading host adaptive immunity. RNA-seq analyses identified vlsE as one of the genes whose expression was most affected by YebC. Quantitative RT-PCR and Western blot analyses confirmed that vlsE expression was dependent on YebC. In vitro, YebC and VlsE were co-regulated in response to growth temperature. In mice, both yebC and vlsE were inversely expressed with ospC in response to the host adaptive immune response. Furthermore, EMSA proved that YebC directly binds to the vlsE promoter, suggesting a direct transcriptional control. These data demonstrate that YebC is a new regulator that modulates expression of vlsE and other genes important for spirochetal infection and immune evasion in the mammalian host.

Browsing by Author "Raghunandanan, Sajith"

Results Per Page

Sort Options