Rapid NETosis Is an Effector Mechanism to Combat Ocular Herpes Infection.

Purpose: Neutrophils are known mediators of innate immunity, yet their effector function in herpesvirus infections remains poorly understood. Here, we elucidate the mechanistic action and pivotal role of neutrophil extracellular traps (NETs) during herpes simplex virus type 1 (HSV-1) ocular infection. Methods: Neutrophils were collected from mice for HSV-1 infection, fluorescence imaging, and immunoblotting assay. Tear samples from healthy subjects and patients with HSV-1 and mice were collected at L. V. Prasad Eye Institute, India, and at the University of Illinois, USA, respectively. For the in vivo study, C57BL/6 mice as well as diversity outbred mice were infected with HSV-1 (McKrae strain) followed by tear fluid collection at various time points (0-10 days). Samples were used for Flow cytometry, ELISA, and immunofluorescence assay. Human transcriptomic profile of keratitis dataset was used evaluate NETosis signaling pathways. We also performed neutrophil depletion studies. Results: Our data revealed a discernible temporal NET formation (NETosis) predominantly in the infected eye, across normal and diversity outbred murine models and human cases of HSV-1 infection. HSV-1 instigates swift NETosis governed by caspase-1 activation and myeloperoxidase secretion. Distinct accumulations of neutrophils, remaining unengaged in NET release in the contralateral eye post-infection, hinting at a proactive defensive posture in the uninfected eye. Moreover, neutrophil depletion accentuated ocular pathology, augmented viral load, and escalated disease scores, substantiating the protective effects of NETs in curtailing viral replication. Conclusions: Our report uncovers a previously unexplored mechanism of NETosis through pro-inflammatory cell death in response to ocular HSV-1 infection, and HPSE up-regulation, identifying new avenues for future studies.

Pathophysiology of reinfection by exogenous HSV-1 is driven by heparanase dysfunction.

Limited knowledge exists on exogenous DNA virus reinfections. Herpes simplex virus-1 (HSV-1), a prototype DNA virus, causes multiple human diseases including vision-threatening eye infections. While reinfection with an exogenous HSV-1 strain is considered plausible, little is known about the underlying mechanisms governing its pathophysiology in a host. Heparanase (HPSE), a host endoglycosidase, when up-regulated by HSV-1 infection dictates local inflammatory response by destabilizing tissue architecture. Here, we demonstrate that HSV-1 reinfection in mice causes notable pathophysiology in wild-type controls compared to the animals lacking HPSE. The endoglycosidase promotes infected cell survival and supports a pro-disease environment. In contrast, lack of HPSE strengthens intrinsic immunity by promoting cytokine expression, inducing necroptosis of infected cells, and decreasing leukocyte infiltration into the cornea. Collectively, we report that immunity from a recent prior infection fails to abolish disease manifestation during HSV-1 reinfection unless HPSE is rendered inactive.

Putative targeting by BX795 causes decrease in protein kinase C protein levels and inhibition of HSV1 infection.

Herpes simplex virus type-1 (HSV1) exploits cellular machinery for its own replicative advantage. Current treatment modalities against HSV1 cause toxicity and drug resistance issues. In the search for alternative forms of treatment, we have uncovered a small molecule, BX795, as a candidate drug with strong antiviral potential owing to its multitargeted mode of action. In this study, we show that in addition to a previously known mechanism of action, BX795 can directly interact with the proviral host factor protein kinase C (PKC) in silico. When administered to HSV1 or mock infected human corneal epithelial (HCE) cells, BX795 significantly reduces the protein level and perinuclear localization of proviral PKC-α and PKC-ζ isoforms. This activity closely mimics that of a known PKC inhibitor, Bisindolylmaleimide I (BIM I), which also inhibits viral replication. Taken together our studies demonstrate a previously unknown mechanism by which BX795 exerts its antiviral potential.

STAND alert! Prokaryotic immunity for recognition and defense against bacteriophages.

In a recent article, Gao et al. diversify our knowledge of prokaryotic innate immunity by characterizing a novel bacterial defense system that utilizes nucleotide-binding oligomerization-like receptors (NLRs) for recognizing phage proteins.

Postinfection Metabolic Reprogramming of the Murine Trigeminal Ganglion Limits Herpes Simplex Virus-1 Replication.

Herpes simplex virus type-1 (HSV-1) infections are known to alter the host metabolism for efficient propagation in vitro. However, in vivo metabolic perturbations upon prolonged HSV-1 infection remain poorly understood. We used high-resolution liquid chromatography coupled with mass spectrometry (LC-MS) and functional assays to determine the state of the trigeminal ganglion (TG) tissue metabolism upon prolonged corneal HSV-1 infection in a murine model. The metabolomics data indicated significant alterations in the host metabolic profile. After HSV-1 infection, the TG microenvironment assumed downregulation of central carbon metabolism and nucleotide synthesis pathways. We validated our observations using in vitro and ex vivo models through targeted inhibition of crucial metabolic polyamine pathways identified in our metabolomics screen. Our findings collectively suggested that HSV-1 infection altered the host metabolic product regulations that limit the energy and macromolecular precursors required for viral replication. IMPORTANCE The more severe ocular pathologies associated with HSV-1 infection are significant vision loss, ocular morbidity, and herpetic keratitis. The current clinical landscape lacks curative drugs and vaccines against HSV-1, a heavy burden associated with this neurotropic, ubiquitous pathogen. The virus is notoriously successful in establishing latency in the host TG, where it remains dormant with periodic reactivations in response to various stimuli like stress and immunosuppression. Metabolic perturbations in tissue microenvironment likely aid the virus in establishing its latent state along with subsequent reactivations yet remain poorly characterized. Here, we used mass spectrometry coupled with statistical data analysis to study the host metabolome in the TG during HSV-1 infection and identify metabolites that likely regulate infection.

OPTN (optineurin)-mediated selective autophagy prevents neurodegeneration due to herpesvirus infection.

Very little is known about the mechanisms that restrict neurotropic herpesviruses such as herpes simplex virus-1 (HSV-1) from infecting the central nervous system (CNS) and causing widespread death of neurons. Likewise, HSV-1 is thought to play a role in chronic neurodegeneration, yet a direct association has remained elusive. To address these issues, we recently showed that the selective macroautophagy/autophagy receptor OPTN (optineurin) specifically targets HSV-1 proteins VP16 and gB for degradation to prevent viral spread in the brain. OPTN deficiency alters host cytokine expression and tissue-specific immune signaling, and enhances necroptotic death of infected neurons. HSV-1-infected optn knockout mice show higher susceptibility to lethal CNS infection and the surviving animals demonstrate cognitive deficiency. Our research suggests that OPTN-mediated autophagy provides an intrinsic immune barrier against neurotropic viruses and protects the CNS from neurodegenerative stress.

Intrinsic Antiviral Activity of Optineurin Prevents Hyperproliferation of a Primary Herpes Simplex Virus Type 2 Infection.

Very little knowledge exists on virus-specific host cell intrinsic mechanisms that prevent hyperproliferation of primary HSV type 2 (HSV-2) genital infections. In this study, we provide evidence that the Nemo-related protein, optineurin (OPTN), plays a key role in restricting HSV-2 infection both in vitro and in vivo. Contrary to previous reports regarding the proviral role of OPTN during Sendai virus infection, we demonstrate that lack of OPTN in cells causes enhanced virus production. OPTN deficiency negatively affects the host autophagy response and results in a marked reduction of CCL5 induction. OPTN knockout (OPTN-/-) mice display exacerbated genital disease and dysregulated T cell frequencies in infected tissues and lymph nodes. A human transcriptomic profile dataset provides further credence that a strong positive correlation exists between CCL5 upregulation and OPTN expression during HSV-2 genital infection. Our findings underscore a previously unknown OPTN/CCL5 nexus that restricts hyperproliferative spread of primary HSV-2 infection, which may constitute an intrinsic host defense mechanism against herpesviruses in general.

Heparanase-Induced Activation of AKT Stabilizes ß-Catenin and Modulates Wnt/ß-Catenin Signaling during Herpes Simplex Virus 1 Infection.

Under pathological conditions like herpes simplex virus 1 (HSV-1) infection, host-pathogen interactions lead to major reconstruction of the host protein network, which contributes to the dysregulation of signaling pathways and disease onset. Of note is the upregulation of a multifunctional host protein, heparanase (HPSE), following infection, which serves as a mediator in HSV-1 replication. In this study, we identify a novel function of HPSE and highlight it as a key regulator of ß-catenin signal transduction. The regulatory role of HPSE on the activation, nuclear translocation, and signal transduction of ß-catenin disrupts cellular homeostasis and establishes a pathogenic environment that promotes viral replication. Under normal physiological conditions, ß-catenin is bound to a group of proteins, referred to as the destruction complex, and targeted for ubiquitination and, ultimately, degradation. We show that virus-induced upregulation of HPSE leads to the activation of Akt and subsequent stabilization and activation of ß-catenin through (i) the release of ß-catenin from the destruction complex, and (ii) direct phosphorylation of ß-catenin at Ser552. This study also provides an in-depth characterization of the proviral role of ß-catenin signaling during HSV-1 replication using physiologically relevant cell lines and in vivo models of ocular infection. Furthermore, pharmacological inhibitors of this pathway generated a robust antiviral state against multiple laboratory and clinical strains of HSV-1. Collectively, our findings assign a novel regulatory role to HPSE as a driver of ß-catenin signaling in HSV-1 infection. IMPORTANCE Heparanase (HPSE) and ß-catenin have independently been implicated in regulating key pathophysiological processes, including neovascularization, angiogenesis, and inflammation; however, the relationship between the two proteins has remained elusive thus far. For that reason, characterizing this relationship is crucial and can lead to the development of novel therapeutics. For HSV-1 specifically, current antivirals are not able to abolish the virus from the host, leaving patients susceptible to episodes of viral reactivation. Identifying a host-based intervention can provide a better alternative with enhanced efficacy and sustained relief.

mTORC2 confers neuroprotection and potentiates immunity during virus infection.

Herpes simplex virus type-1 (HSV-1) causes ocular and orofacial infections. In rare cases, HSV-1 can cause encephalitis, which leads to permanent brain injuries, memory loss or even death. Host factors protect humans from viral infections by activating the immune response. However, factors that confer neuroprotection during viral encephalitis are poorly understood. Here we show that mammalian target of rapamycin complex 2 (mTORC2) is essential for the survival of experimental animals after ocular HSV-1 infection in vivo. We find the loss of mTORC2 causes systemic HSV-1 infection due to defective innate and adaptive immune responses, and increased ocular and neuronal cell death that turns lethal for the infected mice. Furthermore, we find that mTORC2 mediated cell survival channels through the inactivation of the proapoptotic factor FoxO3a. Our results demonstrate how mTORC2 potentiates host defenses against viral infections and implicate mTORC2 as a necessary factor for survival of the infected host.

Protease, Growth Factor, and Heparanase-Mediated Syndecan-1 Shedding Leads to Enhanced HSV-1 Egress.

Heparan sulfate (HS) and heparan sulfate proteoglycans (HSPGs) are considered important for the entry of many different viruses. Previously, we demonstrated that heparanase (HPSE), the host enzyme responsible for cleaving HS chains, is upregulated by herpes simplex virus-1 (HSV-1) infection. Higher levels of HPSE accelerate HS removal from the cell surface, facilitating viral release from infected cells. Here, we study the effects of overexpressing HPSE on viral entry, cell-to-cell fusion, plaque formation, and viral egress. We provide new information that higher levels of HPSE reduce syncytial plaque formation while promoting egress and extracellular release of the virions. We also found that transiently enhanced expression of HPSE did not affect HSV-1 entry into host cells or HSV-1-induced cell-to-cell fusion, suggesting that HPSE activation is tightly regulated and facilitates extracellular release of the maturing virions. We demonstrate that an HSPG-shedding agonist, PMA; a protease, thrombin; and a growth factor, EGF as well as bacterially produced recombinant heparinases resulted in enhanced HSV-1 release from HeLa and human corneal epithelial (HCE) cells. Our findings here underscore the significance of syndecan-1 functions in the HSV-1 lifecycle, provide evidence that the shedding of syndecan-1 ectodomain is another way HPSE works to facilitate HSV-1 release, and add new evidence on the significance of various HSPG shedding agonists in HSV-1 release from infected cells.

Heparan Sulfate Binding Cationic Peptides Restrict SARS-CoV-2 Entry.

A novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. While the world is striving for a treatment modality against SARS-CoV-2, our understanding about the virus entry mechanisms may help to design entry inhibitors, which may help to limit the virus spreading. Owing to the importance of cellular ACE2 and heparan sulfate in SARS-CoV-2 entry, we aimed to evaluate the efficacy of cationic G1 and G2 peptides in virus entry inhibition. In silico binding affinity studies revealed possible binding sites of G1 and G2 peptides on HS and ACE2, which are required for the spike-HS and spike-ACE2 interactions. Prophylactic treatment of G1 and G2 peptide was also proved to decrease the cell surface HS, an essential virus entry receptor. With these two mechanisms we confirm the possible use of cationic peptides to inhibit the entry of SARS-CoV-2.

Dissociation of DNA damage sensing by endoglycosidase HPSE.

Balance between cell proliferation and elimination is critical in handling threats both exogenous and of internal dysfunction. Recent work has implicated a conserved but poorly understood endoglycosidase heparanase (HPSE) in the restriction of innate defense responses, yet biochemical mediators of these key functions remained unclear. Here, an unbiased immunopurification proteomics strategy is employed to identify and rank uncharacterized interactions between HPSE and mediators of canonical signaling pathways linking cell cycle and stress responses. We demonstrate with models of genotoxic stress including herpes simplex virus infection and chemotherapeutic treatment that HPSE dampens innate responses to double-stranded DNA breakage by interfering with signal transduction between initial sensors and downstream mediators. Given the long-standing recognition of HPSE in driving late-stage inflammatory disease exemplified by tissue destruction and cancer metastasis, modulation of this protein with control over the DNA damage response imparts a unique strategy in the development of unconventional multivalent therapy.

Entry receptor bias in evolutionarily distant HSV-1 clinical strains drives divergent ocular and nervous system pathologies.

PURPOSE: Herpes simplex virus-1 (HSV-1) infection leads to varying pathologies including the development of ocular lesions, stromal keratitis and encephalitis. While the role for host immunity in disease progression is well understood, the contribution of genetic variances in generating preferential viral entry receptor usage and resulting immunopathogenesis in humans are not known. METHODS: Ocular cultures were obtained from patients presenting distinct pathologies of herpes simplex keratitis (HSK). Next-generation sequencing and subsequent analysis characterized genetic variances among the strains and estimated evolutionary divergence. Murine model of ocular infection was used to assess phenotypic contributions of strain variances on damage to the ocular surface and propagation of innate immunity. Flow cytometry of eye tissue identified differential recruitment of immune cell populations, cytokine array probed for programming of local immune response in the draining lymph node and histology was used to assess inflammation of the trigeminal ganglion (TG). Ex-vivo corneal cultures and in-vitro studies elucidated the role of genetic variances in altering host-pathogen interactions, leading to divergent host responses. RESULTS: Phylogenetic analysis of the clinical isolates suggests evolutionary divergence among currently circulating HSV-1 strains. Mutations causing alterations in functional host interactions were identified, particularly in viral entry glycoproteins which generated a receptor bias to herpesvirus entry mediator, an immune modulator involved in immunopathogenic diseases like HSK, leading to exacerbated ocular surface pathologies and heightened viral burden in the TG and brainstem. CONCLUSIONS: Our data suggests receptor bias resulting from genetic variances in clinical strains may dictate disease severity and treatment outcome.

Disruption of innate defense responses by endoglycosidase HPSE promotes cell survival.

The drive to withstand environmental stresses and defend against invasion is a universal trait extant in all forms of life. While numerous canonical signaling cascades have been characterized in detail, it remains unclear how these pathways interface to generate coordinated responses to diverse stimuli. To dissect these connections, we followed heparanase (HPSE), a protein best known for its endoglycosidic activity at the extracellular matrix but recently recognized to drive various forms of late-stage disease through unknown mechanisms. Using herpes simplex virus-1 (HSV-1) infection as a model cellular perturbation, we demonstrate that HPSE acts beyond its established enzymatic role to restrict multiple forms of cell-intrinsic defense and facilitate host cell reprogramming by the invading pathogen. We reveal that cells devoid of HPSE are innately resistant to infection and counteract viral takeover through multiple amplified defense mechanisms. With a unique grasp of the fundamental processes of transcriptional regulation and cell death, HPSE represents a potent cellular intersection with broad therapeutic potential.

Dysregulation of Cell Signaling by SARS-CoV-2.

Pathogens usurp host pathways to generate a permissive environment for their propagation. The current spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection presents the urgent need to understand the complex pathogen-host interplay for effective control of the virus. SARS-CoV-2 reorganizes the host cytoskeleton for efficient cell entry and controls host transcriptional processes to support viral protein translation. The virus also dysregulates innate cellular defenses using various structural and nonstructural proteins. This results in substantial but delayed hyperinflammation alongside a weakened interferon (IFN) response. We provide an overview of SARS-CoV-2 and its uniquely aggressive life cycle and discuss the interactions of various viral proteins with host signaling pathways. We also address the functional changes in SARS-CoV-2 proteins, relative to SARS-CoV. Our comprehensive assessment of host signaling in SARS-CoV-2 pathogenesis provides some complex yet important strategic clues for the development of novel therapeutics against this rapidly emerging worldwide crisis.

Agrobacterium tumefaciens-Mediated Genetic Transformation of the Ect-endomycorrhizal Fungus Terfezia boudieri.

Mycorrhizal desert truffles such as Terfezia boudieri, Tirmania nivea, and Terfezia claveryi, form mycorrhizal associations with plants of the Cistaceae family. These valued truffles are still collected from the wild and not cultivated under intensive farming due to the lack of basic knowledge about their biology at all levels. Recently, several genomes of desert truffles have been decoded, enabling researchers to attempt genetic manipulations to enable cultivation. To execute such manipulations, the development of molecular tools for genes transformation into truffles is needed. We developed an Agrobacterium tumefaciens-mediated genetic transformation system in T. boudieri. This system was optimized for the developmental stage of the mycelia explants, bacterial optical density, infection and co-cultivation durations, and concentrations of the selection antibiotics. The pFPL-Rh plasmid harboring hph gene conferring hygromycin resistance as a selection marker and the red fluorescent protein gene were used as visual reporters. The optimal conditions were incubation with 200 µM of acetosyringone, attaining a bacterial optical density of 0.3 OD600; transfer time of 45 min; and co-cultivation for 3 days. This is the first report on a transformation system for T. boudieri, and the proposed protocol can be adapted for the transformation of other important desert truffles as well as ectomycorrhizal species.

Bacterial Pigment Prodigiosin Demonstrates a Unique Antiherpesvirus Activity That Is Mediated through Inhibition of Prosurvival Signal Transducers.

Herpes simplex virus (HSV) is among the most prevalent viral infections worldwide and remains incurable. While nucleoside analogs are used to relieve symptoms of infection, they suffer from having serious adverse effects and are unable to abolish the virus from the host. Here, we demonstrate a unique antiviral effect of prodigiosin (PG), a natural secondary metabolite produced by Serratia marcescens, on HSV infection. We show that PG naturally exerts antiviral activity against HSV-1 and HSV-2 infections. PG treatment resulted in robust inhibition of viral replication in vitro and ex vivo in cultured porcine corneas. Additionally, PG protected against HSV-1 infection and disease progression in a murine model of ocular infection. In our quest to determine the molecular mechanisms of its antiviral activity, we show that PG specifically inhibits NF-κB and Akt signaling pathways and promotes accelerated cell death in HSV-infected cells. Our findings reveal novel antiviral properties of PG, suggesting its high potential as an alternative treatment for herpetic diseases. They also provide new information on antiviral effects of HSV-bacterial metabolite interactions.IMPORTANCE In this article, we provide a new role for a commonly found bacterial pigment in controlling herpes simplex virus infection, for which diverse and multimodal antiviral agents are needed to prevent drug resistance. Serratia marcescens is a red pigment (prodigiosin)-producing Gram-negative bacillus that is naturally found in soil and water. It is associated with many kinds of human infections, including wound and eye infections, and meningitis. Taking cues from previous studies on prodigiosin, including possible proapoptotic anticancer properties, we investigated how it might affect HSV infection. Interestingly, we found that it is a potent virucidal compound that disrupts host signaling pathways needed for HSV growth and survival. The mode of antiviral action suggests potentially broad activity against enveloped viruses. Our results also indicate that interactions with commensal bacteria may inhibit HSV infection, underscoring the importance of studying these microbial metabolites and their implications for viral pathogenesis and treatment.

Environmental and socioeconomic effects of mosquito control in Europe using the biocide Bacillus thuringiensis subsp. israelensis (Bti).

Bacillus thuringiensis subsp. israelensis (Bti) has been used in mosquito control programs to reduce nuisance in Europe for decades and is generally considered an environmentally-safe, effective and target-specific biocide. However, the use of Bti is not uncontroversial. Target mosquitoes and affected midges represent an important food source for many aquatic and terrestrial predators and reduction of their populations is likely to result in food-web effects at higher trophic levels. In the context of global biodiversity loss, this appears particularly critical since treated wetlands are often representing conservation areas. In this review, we address the current large-scale use of Bti for mosquito nuisance control in Europe, provide a description of its regulation followed by an overview of the available evidence on the parameters that are essential to evaluate Bti use in mosquito control. Bti accumulation and toxin persistence could result in a chronic expose of mosquito populations ultimately affecting their susceptibility, although observed increase in resistance to Bti in mosquito populations is low due to the four toxins involved. A careful independent monitoring of mosquito susceptibility, using sensitive bioassays, is mandatory to detect resistance development timely. Direct Bti effects were documented for non-target chironomids and other invertebrate groups and are discussed for amphibians. Field studies revealed contrasting results on possible impacts on chironomid abundances. Indirect, food-web effects were rarely studied in the environment. Depending on study design and duration, Bti effects on higher trophic levels were demonstrated or not. Further long-term field studies are needed, especially with observations of bird declines in Bti-treated wetland areas. Socio-economic relevance of mosquito control requires considering nuisance, vector-borne diseases and environmental effects jointly. Existing studies indicate that a majority of the population is concerned regarding potential environmental effects of Bti mosquito control and that they are willing to pay for alternative, more environment-friendly techniques.

Bacterial microbiota of Aedes aegypti mosquito larvae is altered by intoxication with Bacillus thuringiensis israelensis.

BACKGROUND: Insect microbiota is a dynamic microbial community that can actively participate in defense against pathogens. Bacillus thuringiensis (Bt) is a natural entomopathogen widely used as a bioinsecticide for pest control. Although Bt's mode of action has been extensively studied, whether the presence of microbiota is mandatory for Bt to effectively kill the insect is still under debate. An association between a higher tolerance and a modified microbiota was already evidenced but a critical point remained to be solved: is the modified microbiota a cause or a consequence of a higher tolerance to Bt? METHODS: In this study we focused on the mosquito species Aedes aegypti, as no work has been performed on Diptera on this topic to date, and on B. thuringiensis israelensis (Bti), which is used worldwide for mosquito control. To avoid using antibiotics to cure bacterial microbiota, mosquito larvae were exposed to an hourly increasing dose of Bti during 25 hours to separate the most susceptible larvae dying quickly from more tolerant individuals, with longer survival. RESULTS: Denaturing gradient gel electrophoresis (DGGE) fingerprinting revealed that mosquito larval bacterial microbiota was strongly affected by Bti infection after only a few hours of exposure. Bacterial microbiota from the most tolerant larvae showed the lowest diversity but the highest inter-individual differences. The proportion of Bti in the host tissue was reduced in the most tolerant larvae as compared to the most susceptible ones, suggesting an active control of Bti infection by the host. CONCLUSIONS: Here we show that a modified microbiota is associated with a higher tolerance of mosquitoes to Bti, but that it is rather a consequence of Bti infection than the cause of the higher tolerance. This study paves the way to future investigations aiming at unraveling the role of host immunity, inter-species bacterial competition and kinetics of host colonization by Bti that could be at the basis of the phenotype observed in this study.

Mechanistic approach for fabrication of gold nanoparticles by Nitzschia diatom and their antibacterial activity.

The problem of chemically synthesized nanoproducts motivated scientific community to explore ecofriendly methods of nanosynthesis. Diatoms belong to a group of aquatic, unicellular, photosynthetic microalgae have been scarcely investigated as a source of reducing and capping agents for nanosynthesis of pesticides and antibiotics. The present study reports a novel ecofriendly method for the fabrication of bioactive gold nanoparticles using locally isolated Nitzschia diatoms. The diatom-fabricated gold nanoparticles show characteristic ruby red colored with sharp absorbance peak at 529 nm. Electron microscopy confirmed irregular shape of gold nanoparticles, with average size of 43 nm and zeta potential of -16.8 mV. The effects of gold nanoparticles on diatom viability were investigated using light and electron microscopy. The mechanistic approach to shed light on how diatoms reacted after exposure to gold metal salt revealed that exposure to gold chloride triggers elevated levels of catalase and peroxidase (12.76 and 14.43 unit/mg protein, respectively) to relieve reactive oxygen species (ROS) stress induced by gold salt exposure. Investigation studies on mechanisms behind Nitzschia-mediated gold nanoparticles fabrication outlined the role of diatom proteins, polysaccharides in reduction, and stabilization of nanoparticles as confirmed by FT-IR analysis. Bioactivity of gold nanoparticles was accessed by coupling them with antibiotics (penicillin and streptomycin), which increased their antibacterial activity compared to individual nanoparticles and antibiotics (Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus). Overall, the present novel phyco-nanotechnological approach is a promising tool to be used as sustainable strategy in green nanotechnology as well as to reduce use of antibiotics in microbial control.