Biology subjects

Rajmani, R. S.

Publications and source records attributed to Rajmani, R. S..

At least 19 recordsLinked to original sources

bioRxiv2026

Viral Protease Nsp5 Hijacks La Autoantigen to Orchestrate the Translation to Replication Transition in SARS-CoV-2

Viruses genetically encode proteins to regulate their life cycle. To translate this genetic information, most viruses exploit host translation machinery. Host dependency leads to competition between viral RNA and host mRNAs. Most viruses overcome it by inhibiting cap-dependent translation of host mRNAs and shifting its own translation into cap-independent mode. This switch is largely orchestrated by host RNA binding proteins (RBPs) by binding to viral 5UTRs. Here we demonstrate that human La autoantigen (La protein) specifically binds to GCAC sequence near the initiator AUG within the 5UTR of SARS-CoV-2 RNA promoting viral non-structural protein synthesis by cap independent translation, while suppressing replication early in infection. Interestingly, around 18-24 h post-infection, the viral protease Nsp5 cleaves La at a conserved LQ site, abrogating its 5'UTR interaction, which correlates with reduced polysome association and increased viral RNA availability for replication. MD simulation and docking study suggests that the cleavage disrupts La dimerization and truncates its C-terminal intrinsically disordered region, reducing RNA-binding affinity and conformational stability required for translation-competent ribonucleoprotein complexes. Results put forward a unique mechanism by which Nsp5-mediated proteolysis of La drives the temporal switch from translation to replication of SARS-CoV-2.

microbiology↗

bioRxiv2026

Lysine specific demethylase 1 (LSD1) regulates host alpha-ketoglutarate levels to modulate lipid peroxidation during Mycobacterium tuberculosis infection

Mycobacterium tuberculosis (Mtb) subverts host immune responses via modulation of host epigenome and metabolism. In this study, we underscore a role for the epigenetic modifier, Lysine Specific Demethylase 1 (LSD1), in regulating macrophage metabolism to support mycobacterial pathogenesis. In ex vivo and in vivo infection models, LSD1 inhibition reduced mycobacterial CFU alleviating lung pathology. Metabolomic analysis of Mtb infected, LSD1 deficient macrophages revealed increased levels of alpha-ketoglutarate (AKG), a crucial TCA cycle metabolite via regulating genes implicated in glutamine breakdown. Moreover, exogenous addition of AKG resulted in reduced oxidative stress and attenuated lipid peroxidation (LPO) with a consequent decrease in Mtb survival. Blocking glutamine breakdown in LSD1 deficient macrophages failed to reduce LPO and promoted Mtb intracellular survival, highlighting the role of LSD1-AKG axis in this immunomodulation. Dietary supplementation of AKG to Mtb infected mice improved lung pathology, limited Mtb dissemination and reduced the levels of oxidative Malondialdehyde adducts. Therefore, we highlight a host protective role of AKG during Mtb pathogenesis through suppression of lipid peroxidation and uncover an epigenetic-metabolic axis exploited by Mtb, thereby positing dietary supplementation of AKG as a potential therapeutic strategy against Tuberculosis.

immunology↗

bioRxiv2026

Eco-friendly approach to indenodiazepinones from o-formylynones and o-phenylenediamines: Novel pharmacophoric constructs and their anti-bacterial activity

We have conceptualised and executed an efficient, eco-friendly, one-flask, synthetic approach of general applicability to functionally enriched and potentially pharmacophoric tetracyclic indenodiazepinones through a base-mediated reaction cascade between a multifunctional o-formylynones and commercial o-phenylenediamines. The pertinence of this operationally simple, no-waste protocol, embodying many green and sustainable features, has been scoped with 26 variegated examples. This controlled one-pot assembly of novel tetracyclic indenodiazepinones with an unusual placement of an olefin through a reaction cascade involving Schiff base formation, intramolecular aza-Michael addition and a Mannich reaction between o-formylynones and o-phenylenediamines rapidly generates a diverse library poised for exploring their therapeutic potential and medicinal chemistry applications. The newly accessed indenodiazepinones exhibit promising antibacterial activities against many microorganisms and are non-cytotoxic even at higher concentrations. Interestingly, one of the compounds, 3v did not develop resistance in Salmonella Typhimurium (STM) upon repeated exposure to sub-lethal doses up to the 160th cycle, whereas resistance against clinically used antibacterial ciprofloxacin was attained at the 128th cycle. These preliminary findings open avenues towards the development of novel antimicrobial agents with enhanced durability against drug resistance and these efforts are disclosed in this report. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/697289v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1d1d9beorg.highwire.dtl.DTLVardef@2e8560org.highwire.dtl.DTLVardef@36742eorg.highwire.dtl.DTLVardef@105ae7c_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Efficient elimination of microbial infections in wounds using plasma activated water hydrogel

Surgical site infections are among the most common postoperative complications and remain a major global health concern, further intensified by increasing antimicrobial resistance. Plasma-activated water (PAW) has emerged as a novel antimicrobial strategy, though earlier studies suggested its activity depends on an acidic pH. To overcome this limitation, we previously developed a high-strength, neutral pH-buffered formulation (hs-PAbW) optimized for human clinical use, which preserved strong antimicrobial properties. In the present work, we created a hydrogel incorporating hs-PAbW at a bactericidal yet non-cytotoxic concentration (hs-PAbW-20) and evaluated its therapeutic performance in mouse wound models. In Balb/c mice, hs-PAbW-20 gel markedly reduced Pseudomonas aeruginosa and Staphylococcus aureus loads, demonstrating efficacy comparable to neomycin and soframycin. The gel also enhanced wound closure by regulating key genes involved in tissue repair. In a diabetic C57BL/6 model, hs-PAbW-20 gel similarly lowered P. aeruginosa burden and accelerated healing. Collectively, these results highlight hs-PAbW-20 gel as a promising next-generation wound therapy capable of addressing the shortcomings of traditional antibiotic-based treatments.

microbiology↗

bioRxiv2025

Infection with Mycobacterium tuberculosis orchestrates the PRMT5-dependent methylation of NCOA4 to govern host ferroptosis

Mycobacterium tuberculosis (Mtb) increases availability of free iron resulting in ferroptosis of host macrophages to facilitate its survival and dissemination. A critical factor for elevated levels of labile iron is the overt accumulation of the protein nuclear receptor coactivator 4 (NCOA4), that promotes autophagic degradation of ferritin in a process termed as ferritinophagy. Here, we identify a novel post-translational modification on NCOA4 that is essential for its interaction with ferritin in iron-replete condition of Mtb-infected cells. Specifically, protein arginine methyltransferase 5 (PRMT5), confers a symmetric-dimethylation on NCOA4 that promotes ferritinophagy-mediated ferroptosis. Using loss-of-function studies, we show that PRMT5 is required for lipid peroxidation, bacterial survival, and dissemination in Mtb-infected mice. Also, overexpression of a methylation-deficient mutant of NCOA4 phenocopy depletion of PRMT5 and reduced ferritinophagy in Mtb-infected cells. Furthermore, PRMT5-mediated methylation reduces the nuclear availability of NCOA4 and impairs its co-activatory role to nuclear receptors such as vitamin D3. Thus, our findings uncover the key interaction between NCOA4 and ferritin that regulates ferroptosis and mycobacterial survival during infection. Perturbation of this interaction results in reduced Mtb loads and alleviated disease pathology.

microbiology↗

bioRxiv2025

LuxS-mediated AI-2 signaling regulates chemotaxis, flagellar motility, and virulence genes in Salmonella Typhimurium and contributes to colonization in the murine gut

The cooperative response through AI-2 allows Salmonella to coordinate and regulate its community behaviours crucial for its survival and infection. Understanding these mechanisms is essential for developing strategies to interfere with Salmonellas ability to cause disease. In this study, we report that Salmonella Typhimurium uses AI-2 signaling to modulate the expression of chemotaxis and motility-associated genes and the formation of flagella on the bacterial cell surface. The AI-2 acts as a chemoattractant, and the cascade mediates the chemotactic response of Salmonella. Such regulations assist in the attachment and invasion of Salmonella into intestinal epithelial cells both in vitro and in a murine model. Furthermore, we determined that HilD, known to regulate motility by induction of a chemoreceptor and SPI-1 in Salmonella, along with key SPI-1 genes, is underexpressed in the absence of LuxS/AI-2 signaling, which unravels a complex regulatory network by AI-2 in Salmonella. Beyond the initial phase, this signaling facilitates survival in the presence of polymyxin B and supports intracellular survival and persistence within epithelial cells by regulating the pmrD/AB system, thereby evading the antimicrobial peptide immune response. Additionally, our findings show that Salmonella activates its LuxS/AI-2 signalling cascade within the Caco-2 epithelial cells. Complimentary observation through transcriptomic profiling revealed that LuxS/AI-2 signalling governs the coordinated regulation of genes implicated in distinct phases of Salmonella pathogenesis. Lastly using a mouse model, we show that AI-2 signaling is critical for organ colonization and virulence of Salmonella, and inhibition of LuxS/AI-2 signaling in combination with antibiotics can be an alternative therapeutic approach. Thus, our study dissects an integrated regulatory mechanism by which AI-2 signaling mediates critical processes during multiple stages of Salmonella Typhimurium pathogenesis.

microbiology↗

bioRxiv2025

SIRT2 protects against Japanese encephalitis virus infection in mice

Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic RNA virus that causes Japanese encephalitis (JE) and poses a major threat to public health in Southeast Asia and the Western Pacific. Current strategies rely on prophylactic methods to prevent disease, as no effective antiviral therapy exists. Here, we report that SIRT2, an NAD+-dependent deacetylase enzyme, mediates antiviral activity against JEV infection in mice. Interestingly, our study reveals that SIRT2 is downregulated in JEV infection, SIRT2 genetic deficiency/small molecule inhibition increases viral yield in neuronal cells and mice brains thereby reducing the survival rate in the infected mice, whereas SIRT2 gene therapy to the JEV-infected mice by Adeno-associated virus vector reduced the JEV load in mice brains and improved the survival rate. SIRT2 deficiency activates inflammatory cytokines and chemokines response in the JEV-infected mice brains through activating NF-{kappa}B transcription factor. Mechanistically, SIRT2 deacetylates NF-{kappa}B to reduce the transcriptional factor activity of NF-{kappa}B that down-regulates the Beclin-1-mediated autophagy, which is needed for the JEV replication. Overall, the present findings establish SIRT2 as a potential regulator of JEV infection.

microbiology↗

bioRxiv2025

Development of a thermostable and broadly neutralizing pan-sarbecovirus vaccine candidate

Zoonotic spillover of sarbecoviruses to humans resulted in theSARS-CoV-1 outbreak in 2003 and the current COVID-19 pandemic caused by SARS-CoV-2. In both cases, the viral spike protein (S) is the principal target of neutralising antibodies that prevent infection. Within spike, the immunodominant receptor-binding domain (RBD) is the primary target of neutralising antibodies in COVID-19 convalescent sera and vaccine recipients. We have constructed stabilized RBD derivatives of different sarbecoviruses: SARS-CoV-1 (Clade 1a), WIV-1 (Clade 1a), RaTG13 (Clade 1b), RmYN02 (Clade 2) and BtKY72 (Clade 3). Stabilization enhanced yield by an 3-23-fold. The RBD derivatives were conformationally intact as assayed by binding to multiple broadly neutralizing antibodies. The stabilized RBDs show significant enhancement in apparent Tm, exhibit resistance to a 2-hour incubation at temperatures up to 60 in PBS in contrast to corresponding WT RBDs, and show prolonged stability of over 15 days at 37 after lyophilization. In mice immunizations, both stabilization and trimerization significantly enhanced elicited neutralization titers by [~]100 fold. The stabilized RBD cocktail elicited high neutralizing titers against both homologous and heterologous pseudoviruses. The immunogenicity of the vaccine formulation was assessed in both naive and SARS-CoV-2 pre-immunized mice, revealing an absence of immune imprinting, thus indicating its suitability for use in future sarbecovirus-origin epidemics or pandemics. Author summaryThe COVID-19 pandemic was caused by the sarbecovirus SARS-CoV-2. Phylogenetically, sarbecoviruses are divided into four clades: Clade 1a, Clade 1b, Clade 2 and Clade 3 and within these clades, are many other sarbecovirus strains with pandemic or epidemic potential. It is therefore important to develop a broadly protective, pan-sarbecovirus vaccine formulation that can be cheaply and rapidly produced. While mRNA vaccine formulations are efficacious, they have stringent low temperature storage requirements and there is limited manufacturing expertise in low and middle income countries for this modality. Neutralizing antibodies are important for protection and in the case of SARS-CoV-2 are primarily directed against the Receptor Binding Domain (RBD) of the surface spike protein. In this study, we have designed and developed an adjuvanted, protein subunit, pan sarbecovirus vaccine formulation using stabilized RBD derivatives from diverse sarbecoviruses as immunogens. We demonstrate that the stabilized RBD derivatives have considerably enhanced yield and thermal stability relative to corresponding WT proteins and that the formulation remains stable for up to two weeks at 37{degrees}C. The formulation was found to be highly immunogenic in both naive and pre-immunized mice, eliciting neutralizing titers well above the known protective threshold, indicating its suitability for use in future sarbecovirus-origin epidemics or pandemics.

immunology↗

bioRxiv2025

Activin A mediated KAT8 expression induces ferroptosis during Mycobacterium tuberculosis infection

Activin A, a secretory glycoprotein, has been shown to be upregulated in TB patients, and its levels are correlated with disease severity. Here, we identify a functional role for activin A and the downstream SMAD2/3 signalling in Mtb pathogenesis and dissemination. Molecular assays, including ChIP and loss-of-function analysis, demonstrated that Activin A regulates the expression of lysine acetyltransferase (KAT) 8, which in turn regulates HO-1 levels and Mtb-induced ferroptosis. Mechanistically, we identify KAT8-mediated acetylation of NRF2 during Mtb infection, leading to enhanced nuclear availability of the transcription factor and increased expression of HO-1. Finally, utilizing an in vivo mouse model of TB, we show that the pharmacological inhibition of activin A receptor and KAT8 restricts Mtb burden, dissemination and ameliorates TB pathology. Thus, we report a novel role for activin A in regulating NRF2 localisation and outline its potential consequences during TB.

immunology↗

bioRxiv2025

Bioenergetic reprogramming of macrophages reduces drug tolerance in Mycobacterium tuberculosis

Eradication of Mycobacterium tuberculosis (Mtb) requires strategies targeting bacteria inside the host. Mtb exhibits heterogeneity in redox metabolism inside macrophages to evade killing by anti-TB drugs. If and how macrophage physiology correlates with bacterial redox heterogeneity and drug tolerance remains unclear. Using a fluorescent reporter of mycobacterial redox potential, flow sorting, and RNA sequencing of infected macrophages, we characterized transcriptional and metabolic responses of macrophages harboring redox-diverse Mtb populations. We found that macrophages with suppressed glycolysis and elevated oxidative phosphorylation (OXPHOS) correlated with Mtb populations exhibiting reductive stress and drug tolerance. Conversely, macrophages with elevated glycolysis and suppressed OXPHOS displayed higher mitochondrial reactive oxygen species through reverse electron transport, resulting in oxidative stress in Mtb and enhancing drug efficacy. Computational and genetic approaches identified Nrf2 as a key regulator of macrophage bioenergetics driving redox heterogeneity and drug tolerance in Mtb. Redirecting macrophage metabolism from OXPHOS to glycolysis using an FDA- approved antiemetic drug, meclizine, subverted redox heterogeneity and diminished drug tolerance in macrophages and mice. The pharmacological profile of meclizine (Cmax and AUClast) indicated no adverse interactions with first-line anti-TB drugs in mice. Our data demonstrate the feasibility of reprogramming macrophage metabolism to reduce drug tolerance in Mtb infection.

microbiology↗

bioRxiv2024

Hydrogen sulfide (H2S) coordinates redox balance, carbon metabolism, and mitochondrial bioenergetics to suppress SARS-CoV-2 infection.

Viruses exploit host metabolism for replication and immune-regulation. Understanding how SARS-CoV-2 alters the host metabolism may lead to treatments for COVID-19. We report that a ubiquitous gaseous molecule, hydrogen sulfide (H2S), regulates redox, metabolism, and mitochondrial bioenergetics to control SARS-CoV-2. Virus replication is associated with down-regulation of the H2S-producing enzymes cystathionine-{beta}-synthase (CBS), cystathionine-{gamma}-lyase (CTH), and 3-mercaptopyruvate sulfurtransferase (3-MST), resulting in diminished endogenous H2S levels. Inhibition of CTH resulted in SARS-CoV-2 proliferation. A slow-releasing H2S donor, GYY4137, diminished virus replication by inducing Nrf2/Keap1 pathway, restoring redox balance and mitochondrial bioenergetics. Treatment of SARS-CoV-2-infected animals with GYY4137 suppressed viral replication, ameliorated respiratory pathology, and restored antioxidant gene expression. Notably, whole-body plethysmography showed improved pulmonary function variables, including pulmonary obstruction and end-expiratory pause upon GYY4137 treatment in vivo. Data extend our understanding of H2S-mediated regulation of viral-infections, and open new avenues for investigating the pathogenic mechanisms and therapeutic opportunities for coronavirus-associated disorders.

microbiology↗

bioRxiv2024

The resilience of Salmonella to bile stress is impaired due to the reduced efflux pump activity mediated by the antioxidant enzyme YqhD

Bile salts play a critical role in modulating the host gut. They have antimicrobial properties wherein they disrupt the bacterial membrane and produce reactive oxygen species, causing DNA damage. Bile-resistant pathogen like Salmonella regulates their metabolic activity to counteract the effects of bile. This study explores the role of YqhD, a putative alcohol dehydrogenase, in Salmonellas bile salt susceptibility. We observed increased survival of yqhD mutant in the in vitro studies in LB media with bile, liver cell line HepG2 and C57BL/6 mice on treatment with 8% sodium cholate in the intestine. Bile salts are produced for the digestion of fat. Replacing the chow diet with a high-fat diet (HFD) in mice increased organ burden in C57BL/6 mice of the yqhD mutant. Mutation of yqhD on bile salt exposure leads to increased reactive oxygen species and modulation of antioxidant genes in the bacteria. The oxidative stress of the yqhD mutant is indispensable for improved survival when exposed to bile salt. The addition of antioxidant glutathione in vitro reduced the enhanced growth of the yqhD mutant. Similarly, in the gp91-/- phox mice, there was a reduced organ burden of yqhD mutant on exposure to HFD compared to chow-fed mice. Furthermore, the yqhD mutant increases AcrAB efflux pump activity regulated by RamA/R regulon. TeaserRedox gene yqhD regulates bile salt susceptibility by modulating Salmonellas ROS and efflux pump activity. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=185 SRC="FIGDIR/small/625033v1_figa1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@14e30f0org.highwire.dtl.DTLVardef@6f14a0org.highwire.dtl.DTLVardef@b3291dorg.highwire.dtl.DTLVardef@503988_HPS_FORMAT_FIGEXP M_FIG A. STM {Delta}yqhD has less organ burden in the C57BL/6 mice than STM WT, and there was a similar organ burden in gp91phox-/-. On HFD exposure, the organ burden of STM {Delta}yqhD became similar to STM WT in C57BL/6 mice, and there was reduced organ of STM {Delta}yqhD burden when gp91phox-/- were exposed to HFD. B. On bile salt exposure, YqhD in Salmonella reduces ROS in bacteria and expression of RamA, decreasing the AcrAB efflux pump activity and reducing survival. C_FIG

microbiology↗

bioRxiv2024

Salmonella effector SseL induces PD-L1 up-regulation and T cell inactivation via beta-catenin signalling axis

The upregulation of PD-L1 by various pathogens is a recognized strategy to evade the adaptive immune response. Salmonella infection also upregulates PD-L1 levels causing culling of the activated T-cell; however, the underlying mechanism behind this upregulation is not known. Our findings indicate that the upregulation of PD-L1 is through Salmonella pathogenicity island 2 (SPI-2) encoded effectors since PFA-fixed STM WT and STM{Delta}ssaV (which is unable to secrete effector proteins) did not alter PD-L1 levels. We have further investigated the role of the SPI-2 effector SseL (a deubiquitinase known to affect the NF-B pathway) in PD-L1 upregulation. Our study identifies SPI-2 effector SseL to be crucial for upregulating PD-L1 in vitro as well as in vivo murine models. The increase in PD-L1 levels induced by STM WT facilitates colonization in secondary infection sites in C57BL/6 mice, including the liver and spleen, while the STM{Delta}sseL strain exhibits significant colonization defects. Notably, despite the reduced colonization capacity of STM{Delta}sseL, infected mice exhibit earlier mortality associated with heightened inflammation. We further elucidated the molecular mechanism behind PD-L1 upregulation and observed that bacterial effector SseL helps in the stabilization of {beta}-catenin inside the cell. {beta}-catenin thus translocates into the nucleus and directly regulates the transcriptional levels of PD-L1, which is abrogated upon using {beta}-catenin/TCF inhibitor FH535. Collectively, our study elucidates the mechanism by which Salmonella mediates immune suppression through PD-L1 upregulation. O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/620790v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@12ba486org.highwire.dtl.DTLVardef@3e8b40org.highwire.dtl.DTLVardef@28546corg.highwire.dtl.DTLVardef@1bea897_HPS_FORMAT_FIGEXP M_FIG Abstract figure: Schematic representation of SseL mediated PDL1 upregulation and further affecting the T cell proliferation C_FIG

immunology↗

bioRxiv2024

Odoribacter splanchnicus mitigates Salmonella-induced gut inflammation and its associated pathogenesis via its secreted bacteriocin

Foodborne pathogens pose a major global health hazard, worsened by drug-resistant strains. Therefore, designing novel therapeutic strategies is crucial. Here, we identified the protective role of gut-commensal Odoribacter splanchnicus (OS) against Salmonella in mice by promoting colonization resistance, preserving gut barrier integrity, and preventing acute inflammation and biofilm formation. In vitro, both OS and their culture supernatant inhibited Salmonella biofilm formation, intracellular proliferation in human intestinal cells, and virulence gene expression. Further, our results depicted that OSs protective role acts over a broad spectrum as it confers protection against Gram-positive, Listeria monocytogenes and Gram-negative, Salmonella Typhimurium. Notably, OS conferred protection even when administered post-infection in mice, highlighting its therapeutic potential. Using several biochemical and proteomics approaches, we characterized key OS-secreted molecules that limit intracellular Salmonella and Listeria replication in human intestinal epithelial cells by regulating key virulence effectors and flagella. Collectively, our study establishes OS as the broad-spectrum protective agent against Salmonella and Listeria infections, with promising therapeutic potential. Author SummaryFoodborne pathogens such as Salmonella and Listeria continue to threaten global health, especially with the rise of antibiotic resistance. In this study, we identify the gut commensal bacterium Odoribacter splanchnicus (OS) as a potent protectant against these pathogens. Using a mouse model, we show that OS enhances colonization resistance, maintains gut barrier integrity, and prevents both inflammation and biofilm formation during infection. Remarkably, OS also limits pathogen proliferation and virulence gene expression in human intestinal cells. These protective effects are mediated by both live OS and its secreted molecules within culture supernatant. Importantly, OS administration post-infection also conferred protection, indicating strong therapeutic potential. Biochemical and proteomic analyses revealed OS-secreted factors that suppress key virulence and motility pathways in pathogens. Our findings establish Odoribacter splanchnicus as a promising broad-spectrum biotherapeutic agent against major foodborne infections. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/609322v4_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@7ea42corg.highwire.dtl.DTLVardef@4c8074org.highwire.dtl.DTLVardef@855350org.highwire.dtl.DTLVardef@120decc_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Salmonella Typhimurium employ spermidine to exert protection against ROS-mediated cytotoxicity and rewires host polyamine metabolism to ameliorate its survival in macrophages

Salmonella infection involves a cascade of attacks and defence measures. After breaching the intestinal epithelial barrier, Salmonella is phagocytosed by the macrophages, inside which, the bacteria face multiple stresses and, consequently, employ appropriate countermeasures. We show that, in Salmonella, the polyamine spermidine activates a stress response mechanism by regulating critical antioxidant genes. Salmonella Typhimurium mutants for spermidine transport and synthesis cannot mount an antioxidative response, resulting in high intracellular ROS levels. These mutants are also compromised in their ability to be phagocytosed by macrophages. Furthermore, it regulates a novel enzyme in Salmonella, Glutathionyl-spermidine synthetase (GspSA), which is known to prevent the oxidation of proteins in E.coli. Moreover, the spermidine mutants and the GspSA mutant show significantly reduced survival in the presence of hydrogen peroxide in vitro, and lesser organ burden in the mouse model of Salmonella infection. Conversely, in macrophages isolated from gp91phox-/- mice, we observed a rescue in the attenuated fold proliferation previously observed upon infection. Interestingly, Salmonella upregulates polyamine biosynthesis in the host through its effectors from SPI-1 and SPI-2, which also solves the mystery of the attenuated proliferation observed in spermidine transport mutants. Thus, inhibition of this pathway in the host abrogates the proliferation of Salmonella Typhimurium in macrophages. From a therapeutic perspective, inhibiting host polyamine biosynthesis using an FDA-approved chemopreventive drug, D,L--difluoromethylornithine (DFMO), reduces Salmonella colonization and tissue damage in the mouse model of infection, while enhancing the survival of infected mice. Therefore, our work provides a mechanistic insight into the critical role of spermidine in stress resistance of Salmonella. It also reveals a strategy of the bacteria in modulating host metabolism to promote their intracellular survival and shows the potential of DFMO to curb Salmonella infection.

microbiology↗

bioRxiv2023

A host AAA-ATPase exhibits bacteriolytic activity for clearance of microbial infection

An array of host cytosol guarding factors impede bacterial proliferation and preserve cellular sterility. Amongst them, proteasomal degradation of ubiquitinated pathogens has emerged as a critical mechanism for ensuring cytosolic sanctity. We wondered how proteasomes, with their small size and inability to extract membrane-bound proteins, can eradicate pathogens. Here, we unveil a unique strategy, wherein VCP/p97, a host AAA-ATPase, eliminates pathogens by exerting mechanical force that physically unfolds and pulls out ubiquitinated proteins from bacterial membrane. Combining a single-molecule approach along with molecular dynamic simulation and in-vitro reconstitution, we demonstrate that protein extraction by p97 causes extensive membrane lysis and release of cytosolic contents from phylogenetically diverse microbes. Additionally, in an in-vivo mouse sepsis model, this segregase-dependent bactericidal effect of p97 abrogated microbial proliferation in host tissues. Overall, we discovered a distinct innate antimicrobial function of p97, that protects the host against lethal bacterial infections. One Sentence SummaryA host AAA-ATPase exhibits bacteriolytic activity.

microbiology↗

bioRxiv2023

Enhanced protective efficacy of a novel, thermostable, RBD-S2 fusion immunogen against SARS-CoV-2 and its variants

With the rapid emergence of variants of concern (VOC), the efficacy of currently licensed vaccines has reduced drastically. VOC mutations largely occur in the S1 subunit of Spike. The S2 subunit of SARS-CoV-2 is conserved and thus more likely to elicit broadly protective immune responses. However, the contribution of the S2 subunit in improving the overall efficacy of vaccines remains unclear. Therefore, we designed, characterized, and evaluated the immunogenicity and protective potential of a stabilized SARS-CoV-2 Receptor Binding Domain (RBD) fused to a stabilized S2. Designed immunogens were expressed as soluble proteins with approximately fivefold higher purified yield than the Spike ectodomain and formulated along with Squalene-in-water emulsion (SWE) adjuvant. S2 immunization failed to elicit a neutralizing immune response but significantly reduced lung viral titers in mice challenged with the heterologous Beta variant. In hamsters, SWE-formulated RS2 showed enhanced immunogenicity and efficacy relative to corresponding RBD and Spike formulations. Despite being based on the ancestral Wuhan strain of SARS-CoV-2, RS2 exhibited broad neutralization, including against Omicron variants (BA.1, BA.5 and BF.7), as well as the clade 1a WIV-1 and SARS-CoV-1 strains. RS2 sera also showed enhanced competition with both S2 directed and RBD Class 4 directed broadly neutralizing antibodies, relative to RBD and Spike elicited sera. When lyophilized, RS2 retained antigenicity and immunogenicity even after incubation at 37 {degrees}C for a month. The data collectively suggest that the RS2 immunogen is a promising modality to combat SARS-CoV-2 variants.

biophysics↗

bioRxiv2022

SIRT1 and SIRT3 mediated immuno-metabolic switch govern Salmonella survival within infected macrophages both in vitro and in vivo.

Sirtuins are the major players in host immuno-metabolic regulation. However, the role of sirtuins in the modulation of the immune metabolism pertaining to Salmonellosis is largely unknown. Here, our investigation focussed on the role of two important sirtuins, SIRT1 and SIRT3, shedding light on their impact on intracellular Salmonellas metabolic switch and pathogenesis establishment. Our study indicated the ability of the live Salmonella Typhimurium to differentially regulate the levels of SIRT1 and SIRT3 for maintaining the high glycolytic metabolism and low fatty acid metabolism in Salmonella. Perturbing SIRT1 or SIRT3 through knockdown or inhibition, resulted in a remarkable shift in the host metabolism to low fatty acid oxidation and high glycolysis. This switch led to decreased proliferation of Salmonella in the macrophages. Further, Salmonella-induced higher levels of SIRT1 and SIRT3 led to a skewed polarization state of the macrophages from a pro-inflammatory M1 state toward an immunosuppressive M2 making it more conducive for the intracellular life of Salmonella. Alongside, governing immunological functions by modulating p65 NF-{kappa}B acetylation, SIRT1, and SIRT3 also skew Salmonella-induced host metabolic switch by regulating the acetylation status of HIF-1 and PDHA1. Interestingly, though knock-down of SIRT1/3 attenuated Salmonella proliferation in macrophages, in in vivo mice-model of infection, inhibition or knockdown of SIRT1/3 led to more dissemination and higher organ burden which can be attributed to enhanced ROS and IL-6 production. Our study hence reports for the first time that Salmonella modulates SIRT1/3 levels to maintain its own metabolism for successful pathogenesis.

microbiology↗