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.

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.

A Fur family protein BosR is a novel RNA-binding protein that controls rpoS RNA stability in the Lyme disease pathogen.

The σ54-σS sigma factor cascade plays a central role in regulating differential gene expression during the enzootic cycle of Borreliella burgdorferi, the Lyme disease pathogen. In this pathway, the primary transcription of rpoS (which encodes σS) is under the control of σ54 which is activated by a bacterial enhancer-binding protein (EBP), Rrp2. The σ54-dependent activation in B. burgdorferi has long been thought to be unique, requiring an additional factor, BosR, a homologue of classical Fur/PerR repressor/activator. However, how BosR is involved in this σ54-dependent activation remains unclear and perplexing. In this study, we demonstrate that BosR does not function as a regulator for rpoS transcriptional activation. Instead, it functions as a novel RNA-binding protein that governs the turnover rate of rpoS mRNA. We further show that BosR directly binds to the 5' untranslated region (UTR) of rpoS mRNA, and the binding region overlaps with a region required for rpoS mRNA degradation. Mutations within this 5'UTR region result in BosR-independent RpoS production. Collectively, these results uncover a novel role of Fur/PerR family regulators as RNA-binding proteins and redefine the paradigm of the σ54-σS pathway in B. burgdorferi.

BadR directly represses the expression of the glycerol utilization operon in the Lyme disease pathogen.

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.

The Rrp2-RpoN-RpoS pathway plays an important role in the blood-brain barrier transmigration of the Lyme disease pathogen.

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.

CD1d-dependent neuroinflammation impairs tissue repair and functional recovery following a spinal cord injury.

Tissue damage resulting from a spinal cord injury (SCI) is primarily driven by a robust neuroimmune/neuroinflammatory response. This intricate process is mainly governed by a multitude of cytokines and cell surface proteins in the central nervous system (CNS). However, the critical components of the neuroimmune/neuroinflammatory response during SCI are still not well-defined. In this study, we investigated the impact of CD1d, an MHC class I-like molecule mostly known for presenting lipid antigens to natural killer T (NKT) cells and regulating immune/inflammatory responses, on neuroimmune/neuroinflammatory responses induced by SCI. We observed an increased expression of CD1d on various cell types within the spinal cord, including microglia/macrophages, oligodendrocytes (ODCs), and endothelial cells (DCs), but not on neurons or astrocytes post-SCI. In comparison to wildtype (WT) mice, a T10 contusive SCI in CD1d knockout (CD1dKO or Cd1d -/- ) mice resulted in markedly reduced proinflammatory cytokine release, microglia/macrophage activation and proliferation. Following SCI, the levels of inflammatory cytokines and activation/proliferation of microglia/macrophages were dramatically reduced, while anti-inflammatory cytokines such as IL-4 and growth factors like VEGF were substantially increased in the spinal cord tissues of CD1dKO mice when compared to WT mice. In the post-acute phase of SCI (day 7 post-SCI), CD1dKO mice had a significantly higher frequency of tissue-repairing macrophages, but not other types of immune cells, in the injured spinal cord tissues compared to WT mice. Moreover, CD1d-deficiency protected spinal cord neuronal cells and tissue, promoting functional recovery after a SCI. However, the neuroinflammation in WT mouse spinal cords was independent of the canonical CD1d/NKT cell axis. Finally, treatment of injured mice with a CD1d-specific monoclonal antibody significantly enhanced neuroprotection and improved functional recovery. Therefore, CD1d promotes the proinflammatory response following a SCI and represents a potential therapeutic target for spinal cord repair. Significance Statement: The cell surface molecule, CD1d, is known to be recognized by cells of the immune system. To our knowledge, this is the first observation that the CD1d molecule significantly contributes to neuroinflammation following a spinal cord injury (SCI) in a manner independent of the CD1d/NKT cell axis. This is important, because this work reveals CD1d as a potential therapeutic target following an acute SCI for which there are currently no effective treatments.

A cross-sectional study on COVID19 mortality among people below 30 years of age in Tamilnadu-2020.

INTRODUCTION: The COVID19 pandemic has turned out to be one of the public health* burdens in 2020. The fear of deaths due to COVID19 has surmounted even in developed countries and hasn't spared young age. This study aims in assessing the mortality due to COVID19 among patients below 30years of age in TamilNadu. METHODS: The data was collected from a publicly available secondary data source(www.stopcorona.tn.gov.in)which is an official COVID19 state dashboard. Details of the young COVID19 deaths* under 30yrs of age, their gender, symptoms, Co-morbidities, date of symptoms, date of admission, and death were collected till October 2020. A total of 158 deaths were included in the analysis. Fischer exact test and Mann Whitney U test* were used and p-value <0.05 was considered significant. RESULTS: Among the 158 COVID19 deaths under 30 years of age, the median age affected was 25 years(IQR-7) and 70.3% (n-111) had at least one co-morbidity*. The median time interval between symptom onset and hospital admission was 3 days (IQR-3) and between admission and death was 4 days(IQR-7).There was a significant association of myocarditis, refractory seizures, Central nervous system involvement as the cause of death in the age group 0-15years, compared with 16-30years(p < 0.05). The majority of deaths occurred with a late presentation, also patients with higher age were admitted after 2 days of symptoms and the results were statistically significant(p < 0.05). CONCLUSION: Understanding the age-dependent risk gradient and their trend of this new virus at young age* is essential for public health planning and prevent future deaths, future research gateways.

MR1 Tetramer-Based Artificial APCs Expand MAIT Cells from Human Peripheral Blood That Effectively Kill Glioblastoma Cells.

Immunotherapy for cancer treatment requires the activation of cytotoxic effector lymphocytes. Mucosal-associated invariant T (MAIT) cells are innate T cells that recognize the MHC class I-like molecule MR1. MAIT cells play an important role in the immune response against microbial infections and can directly kill tumor cells. Although MAIT cells can be expanded ex vivo, this method is time-consuming, expensive, and requires allogenic feeder layers. To overcome the limitations of conventional dendritic cell-based vaccines and ex vivo expansion of human T cells, an artificial APC (aAPC) approach to expand antitumor effector cells has several advantages. In this study, we explored an efficient in vitro method to amplify MR1-specific MAIT cells from human peripheral blood using aAPCs made by coating cell-sized latex beads with an Ag-loaded MR1 tetramer complex and anti-CD28 Ab. We further elucidated the cytotoxic potential of such expanded MAIT cells against three human glioblastoma multiforme (GBM) cell lines to explore their potential use as a novel immunotherapeutic tool, as the mostly lethal GBM poorly responds to conventional chemotherapy. When aAPCs were compared with the standard allogenic feeder layer-based approach for MAIT cell expansion, they were significantly more effective. Our results indicate that the aAPC-expanded MAIT cells remained functional, retained their original phenotype, secreted proinflammatory cytokines, and showed cytotoxicity against the GBM cell lines. Hence, MAIT cells have the potential to be a novel tool in immunotherapy approaches for the treatment of human GBM.

MR1 overexpression correlates with poor clinical prognosis in glioma patients.

BACKGROUND: Glioblastoma is the most common adult primary brain tumor with near-universal fatality. Major histocompatibility complex (MHC) class I molecules are important mediators of CD8 activation and can be downregulated by cancer cells to escape immune surveillance. MR1 is a nonclassical MHC-I-like molecule responsible for the activation of a subset of T cells. Although high levels of MR1 expression should enhance cancer cell recognition, various tumors demonstrate MR1 overexpression with unknown implications. Here, we study the role of MR1 in glioma. METHODS: Using multi-omics data from the Cancer Genome Atlas (TCGA), we studied MR1 expression patterns and its impact on survival for various solid tumors. In glioma specifically, we validated MR1 expression by histology, elucidate transcriptomic profiles of MR1 high versus low gliomas. To understand MR1 expression, we analyzed the methylation status of the MR1 gene and MR1 gene-related transcription factor (TF) expression. RESULTS: MR1 is overexpressed in all grades of glioma and many other solid cancers. However, only in glioma, MR1 overexpression correlated with poor overall survival and demonstrated global dysregulation of many immune-related genes in an MR1-dependent manner. MR1 overexpression correlated with decreased MR1 gene methylation and upregulation of predicted MR1 promoter binding TFs, implying MR1 gene methylation might regulate MR1 expression in glioma. CONCLUSIONS: Our in silico analysis shows that MR1 expression is a predictor of clinical outcome in glioma patients and is potentially regulated at the epigenetic level, resulting in immune-related genes dysregulation. These findings need to be validated using independent in vitro and in vivo functional studies.

Brain astrocytes and microglia express functional MR1 molecules that present microbial antigens to mucosal-associated invariant T (MAIT) cells.

It is unknown whether brain astrocytes and microglia have the capacity to present microbial antigens via the innate immune MR1/MAIT cell axis. We have detected MAIT cells in the normal mouse brain and found that both astrocytes and microglia are MR1+. When we stimulated brain astrocytes and microglia with E. coli, and then co-cultured them with MAIT cells, MR1 surface expression was upregulated and MAIT cells were activated in an antigen-dependent manner. Considering the association of MAIT cells with inflammatory conditions, including those in the CNS, the MR1/MAIT cell axis could be a novel therapeutic target in neuroinflammatory disorders.

Development of nsP2 protease based cell free high throughput screening assay for evaluation of inhibitors against emerging Chikungunya virus.

Chikungunya virus has emerged as one of the most important global arboviral threats over the last decade. Inspite of large scale morbidity, with long lasting polyarthralgia, so far no licensed vaccine or antiviral is available. CHIKV nsP2 protease is crucial for processing of viral nonstructural polypeptide precursor to release enzymes required for viral replication, thus making it a promising drug target. In this study, high cell density cultivation (HCDC) of Escherichia coli in batch process was carried out to produce rCHIKV nsP2pro in a cost-effective manner. The purified nsP2pro and fluorogenic peptide substrate have been adapted for fluorescence resonance energy transfer (FRET) based high throughput screening (HTS) assay with Z' value and CV of 0.67 ± 0.054 and <10% respectively. We used this cell free HTS system to screen panel of metal ions and its conjugate which revealed zinc acetate as a potential candidate, which was further found to inhibit CHIKV in Vero cells. Scale-up process has not been previously reported for any of the arboviral nonstructural enzymes. The successful scale-up method for viral protease together with a HTS assay could lead to the development of industrial level large-scale screening platform for identification of protease inhibitors against emerging and re-emerging viruses.

TLR3 mediated innate immune response in mice brain following infection with Chikungunya virus.

Chikungunya virus (CHIKV) has received global attention due to the series of large-scale outbreaks in different parts of the world. Many unusual clinical severities including neurological complications and death were reported in recent outbreaks. The mechanism underlying the host immune response to CHIKV in the brain is poorly characterized. In this study, the neuropathogenesis of CHIKV with E1:A226V mutation was elucidated in 1 week old BALB/c mice. The virus was found to replicate in mice brain with peak titer of 10(4) on 6th day post infection. Immunohistochemical analysis revealed preferential virus localization in neuronal cells of cerebellum. The expression profiling of TLR, antiviral genes and cytokines in mice brain revealed significant up regulation of TLR3, TRAF-6, TICAM-1, MCP-1, CXCL-10, IL-6, IL-4, ISG-15, MX-2, IFN-ß, OAS-3 genes that ultimately resulted in virus clearance from brain by day 9-10 suggesting activation of innate immune pathway. Further the effect of poly I: C (Polyinosinic: Polycytidylic acid), a TLR-3 agonist and potent IFN inducer on CHIKV neuropathogenesis was studied. Pretreatment of mice with Poly I: C caused reduction of CHIKV titer in brain and offered 100% protection of animals. The protection was mediated by an increased induction of TLR3, IFN-ß and antiviral genes in mice brain. Our result demonstrates that pre immune stimulation of animals by Poly I: C is effective inhibitor of CHIKV replication and might be a promising prevention agent against this virus.

Cloning, expression and evaluation of diagnostic potential of recombinant capsid protein based IgM ELISA for chikungunya virus.

The resurgence of chikungunya virus in the form of unprecedented explosive epidemic with unusual clinical severity after a gap of 32 years is a point of major public health concern. Definitive diagnosis is critical in differentiating the disease, especially in dengue endemic areas. The immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) is widely used for diagnosis of chikungunya infection. However IgM ELISA based on whole virus antigen is associated with biohazard risk. The present study describes the development and evaluation of recombinant capsid protein based indirect IgM antibody capture micro plate enzyme linked immunosorbent assay (ELISA) for rapid and accurate diagnosis of chikungunya infection. The gene coding for capsid protein was cloned in frame with GST tag in pET41a+ vector and expressed in E. coli followed by purification with affinity chromatography. The comparative evaluation of in-house chikungunya IgM ELISA vis-a-vis commercially available SD ELISA kit with 90 chikungunya suspected acute phase human patient serum samples revealed 97% accordance. The overall sensitivity and specificity of the reported capsid protein based IgM ELISA was 100% and 95% respectively with 96% PPV and 100% NPV. These findings clearly demonstrated the usefulness of the recombinant capsid protein based CHIKV IgM ELISA for reliable clinical diagnosis of CHIKV infection in human patient.

Immunogenicity of Escherichia coli expressed envelope 2 protein of Chikungunya virus.

Chikungunya fever, a re-emerging infection, is an arthropod-borne viral disease prevalent in different parts of the world, particularly Africa and South East Asia. Chikungunya virus envelope 2 protein is involved in binding to host receptors and it contains specific epitopes that elicit virus neutralizing antibodies. A highly immunogenic, recombinant Chikungunya virus envelope 2 protein was produced by bioreactor in Escherichia coli for development of a suitable diagnostic and vaccine candidate. This protein was refolded and further purified to achieve biologically active protein. The biological function of refolded and purified recombinant envelope 2 protein of Chikungunya virus was confirmed by its ability to generate envelope 2 specific antibodies with high titers in animal models. These findings suggest that recombinant envelope 2 protein of Chikungunya virus in combination with compatible adjuvant is highly immunogenic. Thus, recombinant envelope 2 protein can be a potential diagnostic reagent and vaccine candidate against Chikungunya virus infection.

Production of recombinant Chikungunya virus envelope 2 protein in Escherichia coli.

Chikungunya, a mosquito-borne viral disease caused by Chikungunya virus (CHIKV), has drawn substantial attention after its reemergence causing massive outbreaks in tropical regions of Asia and Africa. The recombinant envelope 2 (rE2) protein of CHIKV is a potential diagnostic as well as vaccine candidate. Development of cost-effective cultivation media and appropriate culture conditions are generally favorable for large-scale production of recombinant proteins in Escherichia coli. The effects of medium composition and cultivation conditions on the production of recombinant Chikungunya virus E2 (rCHIKV E2) protein were investigated in shake flask culture as well as batch cultivation of Escherichia coli. Further, the fed-batch process was also carried out for high cell density cultivation of E. coli expressing rE2 protein. Expression of rCHIKV E2 protein in E. coli was induced with 1 mM isopropyl-beta-thiogalactoside (IPTG) at ~23 g dry cell weight (DCW) per liter of culture and yielded an insoluble protein aggregating to form inclusion bodies. The final DCW after fed-batch cultivation was ~35 g/l. The inclusion bodies were isolated, solubilized in 8 M urea and purified through affinity chromatography to give a final product yield of ~190 mg/l. The reactivity of purified E2 protein was confirmed by Western blotting and enzyme-linked immunosorbent assay. These results show that rE2 protein of CHIKV may be used as a diagnostic reagent or for further prophylactic studies. This approach of producing rE2 protein in E. coli with high yield may also offer a promising method for production of other viral recombinant proteins.

Differential regulation of TLR mediated innate immune response of mouse neuronal cells following infection with novel ECSA genotype of Chikungunya virus with and without E1:A226V mutation.

Chikungunya virus (CHIKV) has received global attention due to the series of large-scale outbreaks in different parts of the world including Africa, Indian Ocean Islands, India and South-East Asia. The appearance of many unusual severe manifestations including neurological disorders was reported in post resurgence epidemics with implication of novel East Central South African (ECSA) genotype with E1:A226V mutation. The molecular mechanism of CHIKV neuropathogenesis is not yet understood and very little is known about the host-pathogen interactions. In the present study replication kinetics and innate immune response of ECSA genotype of CHIKV with and without A226V mutation were determined in mouse neuroblastoma cell line (N2a). The 226V mutant strain was more replication competent in N2a cells with a peak titer of 10(8)PFU/ml compared to 10(6)PFU/ml for A226 virus. Besides, the 226V mutant virus showed relatively less induction of antiviral genes i.e. IFN-ß, OAS-3, MX-2, ISG-15 and Toll like receptors 3 and 7 as compared to non mutant strain (A226). Further pretreatment of N2a cells either with Poly I: C, IFN-ß or TNF-α resulted in inhibition of CHIKV replication hence confirming the role of TLR mediated innate immune response in CHIKV pathogenesis. Differential regulation of TLRs and associated down stream antiviral genes might have attributed for increased pathogenesis of the 226V mutant novel ECSA genotype of CHIKV during the recent epidemics.

Yeast expressed recombinant Hemagglutinin protein of novel H1N1 elicits neutralising antibodies in rabbits and mice.

Currently available vaccines for the pandemic Influenza A (H1N1) 2009 produced in chicken eggs have serious impediments viz limited availability, risk of allergic reactions and the possible selection of sub-populations differing from the naturally occurring virus, whereas the cell culture derived vaccines are time consuming and may not meet the demands of rapid global vaccination required to combat the present/future pandemic. Hemagglutinin (HA) based subunit vaccine for H1N1 requires the HA protein in glycosylated form, which is impossible with the commonly used bacterial expression platform. Additionally, bacterial derived protein requires extensive purification and refolding steps for vaccine applications. For these reasons an alternative heterologous system for rapid, easy and economical production of Hemagglutinin protein in its glycosylated form is required. The HA gene of novel H1N1 A/California/04/2009 was engineered for expression in Pichia pastoris as a soluble secreted protein. The full length HA- synthetic gene having α-secretory tag was integrated into P. pastoris genome through homologous recombination. The resultant Pichia clones having multiple copy integrants of the transgene expressed full length HA protein in the culture supernatant. The Recombinant yeast derived H1N1 HA protein elicited neutralising antibodies both in mice and rabbits. The sera from immunised animals also exhibited Hemagglutination Inhibition (HI) activity. Considering the safety, reliability and also economic potential of Pichia expression platform, our preliminary data indicates the feasibility of using this system as an alternative for large-scale production of recombinant influenza HA protein in the face of influenza pandemic threat.