Unraveling the mechanistic insights of sophorolipid-capped gold nanoparticle-induced cell death in Vibrio cholerae

ABSTRACT Cholera is one of the leading causes of mortality and morbidity worldwide. The treatment of cholera includes rehydration therapy, often combined with the administration of antibiotics (in severe cases). With the emergence and rapid spread of antimicrobial resistance, such microbial pathogens have become a compelling medical problem and a global threat. To combat the resistance to antibiotics, there is an urgent need for the discovery of novel and potent antimicrobial agents. The discovery of antimicrobial nanoparticles as an alternative to the existing antimicrobial regime has increased the hope for its use as one of the most promising tools against an array of microbial pathogens, including multidrug-resistant bacteria. Our recent report demonstrated that sophorolipid-capped gold nanoparticles (AuNPs-SL) exhibit potent antibacterial activity against Vibrio cholerae; however, the killing mechanism remained obscure. Metal nanoparticle-mediated killing of microbes occurs through non-specific mechanisms. To develop antimicrobial nanoparticles with better efficacy, knowledge of their specific mechanism of action is essential. In this work, we deduced the mechanistic insights of AuNPs-SL mediated cell death of V. cholerae. We observed that AuNPs-SL treatment evokes reactive oxygen species (ROS) formation. The surge in the ROS level triggers the overexpression of ROS-responsive genes, depolarization of the membrane, change in membrane permeability and leakage of intracellular proteins and DNA, depletion of ATP levels in the cell, DNA damage, and subsequent cell death. This study shows the possibility of use of AuNPs-SL as an alternative potential antimicrobial agent. IMPORTANCE Vibrio cholerae, a Gram-negative bacterium, is the causative agent of a fatal disease, “cholera.” Prevention of cholera outbreak is possible by eliminating the bacteria from the environment. However, antimicrobial resistance developed in microorganisms has posed a threat and challenges to its treatment. Application of nanoparticles is a useful and effective option for the elimination of such microorganisms. Metal-based nanopaticles exhibit microbial toxicity through non-specific mechanisms. To prevent resistance development and increase antibacterial efficiency, rational designing of nanoparticles is required. Thus, knowledge on the exact mechanism of action of nanoparticles is highly essential. In this study, we explore the possible mechanisms of antibacterial activity of AuNPs-SL against V. cholerae. We show that the interaction of AuNPs-SL with V. cholerae enhances ROS production and membrane depolarization, change in permeability, and leakage of intracellular content. This action leads to the depletion of cellular ATP level, DNA damage, and subsequent cell death.

cause cholera.Cholera is the second leading cause of mortality in children under 5 years of age and morbidity in adults (3).Out of the 200 serotypes of V. cholerae described, so far, only two, O1 and O139, are known to be the most virulent.
The treatment of cholera primarily includes rehydration therapy, often combined with the administration of antibiotics such as tetracycline, fluoroquinolones, and azithromycin for severe cases (4).In fact, oral rehydration was first introduced in the 1960s as a treatment against fluid loss and its replenishment that occur during acute diarrhea.It contains an iso-osmolar amount of glucose, electrolyte solution with base, and citrate, which is used to treat body water loss and metabolic acidosis.In decades following the first introduction of oral rehydration therapy, its preparation and administration have increased our capacity to control the outbreaks of diarrhea especially in developing countries and have largely remained effective.
In case of severe acute diarrhea, oral dose of antibiotic is administered as soon as the patient can tolerate oral medication.In most countries, doxycycline is recommended as the first line of treatment; however, azithromycin and ciprofloxacin are also given on a case-to-case basis.However, in the last few decades, extensive prescription and misuse of antibiotics enhanced the prevalence of antimicrobial resistance (AMR) cases, which poses a great threat to global health.In a recent study, it has been observed that V. cholerae may harbor as many as 40 different AMR encoding genes, making them capable of gaining resistance against as many as 22 antibiotics of nine different classes (5).Therefore, there is an urgent need to change the existing therapeutic strategies and to discover new antibiotics to combat AMR cases caused by V. cholerae.
Research outcomes in the past two decades have established nanoparticles as an alternative drug delivery as well as an antimicrobial agent to combat antibiotic-resist ant microorganisms.Recently, metallic nanoparticles have been reported to exhibit antimicrobial activity against AMR bacterial strains (6).For example, nickel and Ni(OH) 2 nanoparticles possess antimicrobial activities against multidrug-resistant (MDR) Klebsiella pneumoniae and Escherichia coli (7), copper nanoparticles against Micrococcus luteus, Staphylococcus aureus, E. coli, K. pneumoniae, Pseudomonas aeruginosa, and few fungal strains (8).The antimicrobial properties of silver nanoparticles against bacteria, fungi, and viruses are also well reported in the literature (9).Zinc oxide nanoparticles and gold nanoparticles (AuNPs) synthesized chemically are known to exhibit antibacterial activity against V. cholerae and are found to be efficient in lowering the bacterial burden (10).Zn is involved in immunocompetence and resistance of mucosa against infection and is a structural component of more than 200 enzymes.Silver nanoparticles are also potent against V. cholerae infection.Beside that, gold nanomaterials are considered to be one of the most suitable nanomaterials for biomedical applications, owing to their inherent biological inertness, well-established surface modification procedures, and facile and rapid preparation (11).
Biosurfactants produced by microbes also possess antimicrobial and antiadhesive activity and are amphiphilic in nature (12)(13)(14).Sophorolipid (SL) is a glycolipid biosur factant with potential antifungal, antibiofilm, and hyphal growth inhibition activity (15).It shows antibacterial activity against Gram-positive bacteria but does not exhibit potent activity against Gram-negative bacteria (16).It is biodegradable and eco-friendly and therefore can be utilized in the greener synthesis of gold nanoparticles.We have demonstrated that sophorolipid-capped gold nanoparticles (AuNPs-SL) exhibit potent antimicrobial activity against both Gram-negative and Gram-positive bacteria but with higher efficacy against Gram-negative bacteria (17).However, AuNPs (uncapped) and SL independently lack antimicrobial activity toward Gram-negative bacteria.Although it is known that AuNPs-SL exhibit antibacterial properties, its molecular mechanism is obscure.
In this study, we report the plausible molecular mechanism of AuNPs-SL mediated killing of V. cholerae.We observe that AuNPs-SL treatment evokes reactive oxygen species (ROS) production, causes DNA damage, alters membrane potential, and depletes ATP levels that ultimately cause apoptotic cell death in V. cholerae.

AuNPs-SL evokes ROS production in V. cholerae
The nanoparticle-mediated killing of bacteria is known to be associated with ROS production and alteration in membrane structure and function that subsequently lead to cell death (18).To investigate whether AuNPs-SL treatment against V. cholerae causes ROS production, we measured ROS levels using a fluorescent dye, 2,7-dichlorodihydro fluorescein diacetate (H 2 DCFDA).ROS measurement was done in a dose-dependent manner of AuNPs-SL, from 10 to 25 µg/mL, based on minimum inhibitory concentra tion (MIC) calculations of AuNPs-SL against V. cholerae (MIC: 25 µg/mL) (17).Synthe sis of AuNPs-SL was carried out according to the previously reported method (17).Fig. S1 shows the characterization of a representative batch of AuNPs-SL along with its transmission electron microscopy (TEM) image.With increasing concentrations of AuNPs-SL, it has been observed that the ROS levels also increase from 2.5-fold to 20.0-fold (Fig. 1A).To further validate the AuNPs-SL mediated ROS production, we used different ROS scavengers like N-acetyl-L-cysteine (NAC), tiron (Tr), ascorbate, thiourea (TU), and sodium pyruvate (SP) (19).Surprisingly, it has been found that NAC is highly efficient in scavenging AuNPs-SL mediated ROS levels, which is followed by Tr, ascorbate (all ~5-fold to 6-fold), TU, and SP (both ~3-fold) (Fig. 1B).Tr, SP, and TU scavenge -O 2 , H 2 O 2 , and • OH ROS species respectively.NAC replenishes the intracellular glutathione level or thiol-disulfide exchange to combat ROS-mediated redox imbalance of the cell (20,21).Nevertheless, the supplementation of these scavengers in the growth medium rescues the growth of V. cholerae treated with AuNPs-SL (Fig. 1C).Although ascorbate and SP exhibit a significant ROS scavenging effect, neither of the compounds were able to rescue the growth of the dying cells (Fig. 1B and C).Similar phenomena were also observed with cells treated with silver nanoparticles (22).The mitigation of ROS levels and growth restoration in the presence of ROS scavengers clearly indicates that AuNPs-SL treatment evokes ROS production in V. cholerae.

Dithiothreitol (DTT) restores the growth defect of AuNPs-SL treated cells
The restoration of growth in the presence of NAC make us stipulate the possibility of redox imbalance in the cell.DTT is a powerful reducing agent that has been shown to protect cells from oxidative stress and restore the cell viability (23).We also used DTT as a supplement to the AuNPs-SL treated cells and measured the ROS level.As a result, DTT treatment has been shown to reduce ROS levels in AuNPs-SL treated cells (Fig. S2A).To check whether DTT also restores growth, we performed spot assay in the presence of DTT.Likely DTT was found to restore the growth of AuNPs-SL treated cells (Fig. S2C).Being a strong reducing agent, we thought that DTT may alter the physical properties of nanoparticles and make them inactive.To rule out this possibility we checked the UV-spectrum of the nanoparticles after incubation with DTT.In DTT-incubated nanoparti cles, no significant changes in UV spectra were found (Fig. S2D), that provides another line of evidence that AuNPs-SL evokes ROS in V. cholerae.

AuNPs-SL alters the expression profile of ROS-responsive genes and the genes involved in stress responses
To mitigate the ROS, the living organisms possess their own line of defense and activate their ROS-responsive genes which act through different pathways.Therefore, the gene expression profile of selected genes has been checked in AuNPs-SL treatment by reverse transcription-quantitative PCR (RT-qPCR) (Fig. 2).A list of primer used for the study is provided in Table 1.The soxSR regulon is activated by ROS and regulates the expression of more than 40 genes.Superoxide radical upregulates the soxR, and the oxidized form of soxR acts as a transcription activator of sodA (24).sodA converts the superoxide anion (-O 2 ) to O 2 and H 2 O 2 that ultimately gets reduced to H 2 O by catalase (25).The expression of sodA was upregulated ~5-fold in AuNPs-SL treated cells (Fig. 2).sodA converts superoxide to H 2 O 2 , which activates the OxyR (a dual regulator activated in the presence of H 2 O 2 ), and OxyR further activates the genes involved in redox balance and metal homeostasis (26).In a study, it has been found that the V. cholerae ΔoxyR mutant is highly sensitive to ROS and has a growth defect even in the presence of rich growth media (27).An increase in intracellular H 2 O 2 level triggers the expression of catalase (gene regulated by OxyR) (26).The genes katG, grx (glutaredoxin) , fur, and dps (Codes for DNA-binding protein, Dps) are also regulated by OxyR (28) (29) (30).Around (~) 4-fold upregulation of oxyR and five to six fold upregulation of hydroxyl peroxidase detoxification gene (katE) was observed in AuNPs-SL treated V. cholerae (Fig. 2).We also checked the expression level of OxyR regulated genes like grx, dps, and fur.The gene grx acts as an antioxidant and plays an important role in iron-sulfur cluster formation (29) (30).We found a ~6-fold increase in the expression levels of grx (Fig. 2) in treated cells.The growth of Δdps strain is significantly hampered in oxidative stress as well as in starvation, acidic stress, and metal stress (31).Dps sequestrates iron, limits the Fenton reaction (in Fenton reaction, there is oxidation of Fe 2+ to Fe 3+ in the presence of H 2 O 2 to form hydroxyl free radical) and mitigates ROS levels (32) (33).The non-specific DNA binding of Dps protects DNA from ROS-mediated damage and the physical association of DNA with the toxic combination of Fe 2+ and H 2 O 2 .The global regulator of iron homeo stasis fur also protects cells from ROS by depleting free iron levels needed for Fenton reaction (34) (35).AuNPs-SL treatment increases the expression level of iron storage and dps and ferric uptake regulator (fur) by ~2.5-fold and ~2-fold, respectively (Fig. 2).ROS is known to cause damage to cellular DNA.DNA damage triggers the SOS response, and RecA upregulation is the hallmark of this process.We observed two fold upregulation of recA in the treated cells (Fig. 2).Beside that, in bacteria, the outer membrane acts as a selective barrier and limits the import of various toxic substances into the cell   (36).For example, the OmpU is a major porin, which is highly conserved among Vibrio species.It facilitates movement of uncharged small molecules from the outer membrane to periplasmic space as well as involved in the adhesion/colonization of the bacteria (37).It has also been reported that in iron depletion, the expression of omp is downregulated as they act as a receptor of the siderophore complex and heme-compound transporter in V. cholerae (38).To understand the role of porins in AuNPs-SL treatment, we checked the expression of ompU and found its downregulation (Fig. 2).Downregulation of ompU facilitates the selective entry of nanoparticles and may also control the free iron levels to slow down the Fenton reaction.Moreover, the downregulation of ompU is also an indicator of impaired cell evasion and biofilm formation (37).Biofilm formation requires cell-to-cell communication and quorum sensing.The luxO gene regulates the cell-to-cell communication and cell signaling (required in different physiological processes like motility, colony formation, and biofilm formation) (39).luxO is upregulated by seven fold (Fig. 2).Its upregulation depicts the possibility of lifestyle switching in V. cholerae upon AuNPs-SL treatment.For survival in stressful niches, V. cholerae employs remodeling of outer membrane assembly and activates secretion systems (which is required for the export of several proteins like chitin, cholera toxin, and protease) (40) (41), and activation of the general secretion pathway (gsp) genes is required for this.AuNPs-SL treatment upregulates the expression of gsp by three fold (Fig. 2).
In conclusion, the upregulation of ROS-responsive genes sodA, oxyR, katE, etc, iron storage protein dps, fur, SOS response gene recA, outer membrane gene ompU, and genes involved in quorum sensing (luxO) and secretion systems gsp indicates that AuNPs-SL treatment evokes ROS production, causes DNA damage, and alters cell-to-cell communication to counter its toxic effects.

AuNPs-SL treatment causes DNA damage and apoptosis in V. cholerae
ROS measurement and RT-qPCR data clearly demonstrate that AuNPs-SL treatment of V. cholerae evokes ROS generation.Induction of ROS damages DNA by creating lesions in bases, sugar, and DNA protein cross-links within the single-and double-strand bases of DNA (42).Overexpression of dps and recA (Fig. 2) (the DNA protecting and SOS response genes, respectively) further confirms the possibility of DNA damage in AuNPs-SL treated cells.To probe this, we performed terminal deoxynucleotidyl transferase-mediated dUTP-X nick end labeling (TUNEL) assay.In this assay, the fluorescence is measured from the free 3′-OH of the damaged DNA (synthesized from fluorescein-labeled dUTP by exogenously supplied terminal deoxynucleotidyl transferase).Thus, when cellular DNA is fragmented, the incorporation of labeled dUTP increases the fluorescence inside the cells.The AuNPs-SL treatment (at 25 µg/mL) increases the fluorescence intensity by 20-fold (Fig. 3A and B), which manifests severe DNA damage and fragmentation.Further, DNA fragmentation initiates the programmed cell death or apoptosis.Therefore, to probe the AuNPs-SL mediated apoptotic cell death, we performed apoptosis assay by using annexin V allophycocyanin conjugate.Annexin V has a higher affinity for phosphatidylserine, which becomes exposed to the outer leaflet in the cell membrane undergoing apoptosis.We observed a ~5-fold increase in the fluorescence intensity of annexin V in treated cells (25 µg/mL AuNPs-SL); however, the effect of a lower amount of AuNPs-SL (10 µg/mL) was not significant (Fig. 3C and D).Nevertheless, TUNEL assay and apoptosis assay exhibit apoptotic cell death of V. cholerae upon AuNPs-SL treatment.

Iron supplementation rescues AuNPs-SL mediated growth defect in V. cholerae
In bacterial cells, iron uptake and storage are critically controlled and regulated by cellular physiology and homeostatic mechanism.To mitigate the elevated ROS levels caused by AuNPs-SL, dps (an iron storage protein) and fur (iron uptake regulator) genes have been upregulated (Fig. 2).They counter the free iron levels to inhibit free radical formation by Fenton reaction.This leads to the imbalance in the availability of iron for other physiological processes.Iron is an essential micronutrient for the growth and metabolism of microorganisms and works as a cofactor for various enzymes, viz, [Fe-S] cluster containing ferredoxins, heme-containing cytochromes, and fumarases (43).Therefore, the effect of external iron supplementation was checked in V. cholerae.Growth studies revealed that iron supplementation rescues the growth of AuNPs-SL treated cells (Fig. 4A).In addition to that, iron supplementation significantly decreased ROS production in the treated cells (Fig. 4B).Since the Fe-S clusters are the integral part of the electron transport chain (ETC) and their malfunctioning causes ROS generation (44), the supplementation of iron may repair the impaired Fe-S cluster assemblies in AuNPs-SL treatment that further decreases the ROS formation and the cells regained their growth and viability.Since increased ROS levels cause the overexpression of genes involved in iron-sulphur cluster formation, iron sequestration, and storage and iron homeostasis and protect the cell from ROS toxicity by depleting free iron required for Fenton reaction, we checked the expression of few selected genes, e.g., sod A, rec A, lux O, and oxy R in AuNPs-SL treated V. cholerae cells in the presence of Mohr's salt (ammonium iron(II) sulfate).The expression level of all the selected four genes was downregulated in Mohr's salt supplemented in comparison to the AuNPs-SL treated cells (Fig. S3).Mohr's salt, which is resistant to environmental oxidation, is more capable of Fe 2+ stabilization, prevention of Fenton reaction, and reduction of ROS stress and is capable of rescuing the cells from ROS toxicity.

AuNPs-SL treatment damages the cell membrane and causes membrane leakage in V. cholerae
The bactericidal activity of AuNPs-SL against V. cholerae suggests the interaction of the AuNPs-SL with the cell membrane.Therefore, we checked the architecture of the outer surface and cell membrane of V. cholerae by TEM.The TEM images showed the extensive damage to outer surface and ruptured membrane in the AuNPs-SL treated cells as compared to the untreated cells (Fig. 5Ai and Aii).When the cell membrane is damaged, there is a possibility of leakage of intracellular components such as proteins and DNA in the surrounding environment.Therefore, we measured the amount of protein and DNA in the extracellular media.Likely, in AuNPs-SL treated cells, we observed a concentration dependent increase in the release of protein and DNA content (Fig. 5B and C).Treatment with 25 and 50 µg/mL of AuNPs-SL resulted in two fold and four fold increase in the DNA leakage, respectively.To strengthen our findings, we also measured the protein content using β-lactamase plasmid in V. cholerae.The molecular weight of β-lactamase is approximately between 30 and 40 kDa (45).In the SDS -PAGE gel image, a distinct and obvious protein band is present below 35 kDa from total protein isolated from the treated plasmid cells which are very faint in untreated plasmid cells and completely missing in untreated and treated cells without plasmid.Agarose gel images (Fig. 5E) of equally loaded DNA (by volume 20 μL) clearly depict the higher DNA amount with smear in AuNPs-SL treated plasmid cells as compared to unreated plasmid cells.The smear throughout the lane of treated plasmid also indicates the DNA fragmentation.To further validate our findings, we performed nitrocefin-based chromogenic assay.Nitrocefin, a chromogenic cephalosporin, becomes red when β-lactamase hydrolyzes its amide link.It is frequently used for the detection of microbes producing beta-lactamase enzymes.Treated plasmid cells have a higher intensity of red color than untreated plasmid cells; however, no color change occurs in cells lacking plasmid (Fig. 5F).The intensity of red color for samples increases with increasing time.These experiments clearly demonstrate that AuNPs-SL treatment of V. cholerae leaks the intracellular contents.The membrane damage also causes osmotic shock to the cells (30) (46).To protect the cells from osmotic shock, the sodium and potassium ions play a very important role.Therefore, we measured the concentration of these ions using inductively coupled plasma mass spectrometry (ICP-MS) and observed that AuNPs-SL treatment led to an increase in K + and Na + (Table 2).TEM imaging, extracellular protein and DNA content measurement, and the increased level of K + and Na + show that AuNPs-SL severely damages the cells to exhibit apoptotic cell death.

AuNPs-SL causes membrane depolarization in V. cholerae
Severe membrane damage and intracellular leakage in AuNPs-SL treatment may alter the other physical features of the membrane.It is evident that both ROS production and membrane damage cause membrane depolarization (47).To investigate whether AuNPs-SL also depolarizes the membrane in V. cholerae, we measured the membrane potential in the varying concentrations of AuNPs-SL using two different dyes, DiBAC 4 and DiOC 2 .DiBAC 4 is an anionic lipophilic bis-oxonol dye.When the membrane depolarizes, as the membrane potential shifts from negative to positive, the concentration of the dye entering the cells increases (i.e., the higher the membrane depolarization, the higher the oxonol fluorescence intensity).In our experimental setup, when the V. cholerae cells were treated with 10 and 25 µg/mL of AuNPs-SL, membrane depolarization increased by 13% and 16%, respectively (Fig. 6A and B).Also with DiOC 2 , there is a concentrationdependent change in the membrane potential as indicated by the intensity of DiOC 2 (Fig. 6C and D); the change in fluorescence intensity of DiOC 2 is directly proportional to the loss of membrane potential of the cells (48).ROS formation is linked with membrane depolarization (47).To demonstrate this link, we used DTT supplementation because DTT rescues the cells from ROS and also helps in decreasing membrane depolarization (49).Surprisingly, it has been observed that DTT rescues the AuNPs-SL treated cells, but it is not able to restore the membrane depolarization (Fig. S2B).

AuNPs-SL decreases ATP production in V. cholerae
ETC components possess Fe-S clusters and are located in the membrane (50).ROS formation, growth rescue in iron supplementation, depolarized membrane, and altered membrane architecture manifest the impairment of electron transport chain.The aforementioned impairment of ETC in AuNPs-SL treated V. cholerae prompted us to investigate whether AuNPs-SL treatment causes energy crisis in the cell or not.We measured the intracellular ATP level using a luciferase based ATP bioluminescence assay and observed a ~50% reduction in ATP levels in AuNPs-SL treated cells.However, no dose-dependent decrease in ATP levels was observed in the treatment range (10 and 25 µg/mL) of AuNPs-SL (Fig. 6E).(53).AuNPs are used in drug delivery and photothermal, therapeutic, radiosensitizing, and gene transfection (54).In our previous work, we demonstrated the antibacterial, antibiofilm, and growth inhibitory effect on non-dividing cells of AuNPs-SL (17).AuNPs-SL were also effective against V. cholerae grown in virulent conditions, and IC 50 was found to be at AuNPs-SL-50; 90% of inhibition was observed at AuNPs-SL-100 and complete eradication at AuNPs-SL-200 (Fig. S4A).The current work aims to uncover the mechanistic insights of AuNPs-SL mediated cell death of V. cholerae.Like antibiotic stress (55), we observed that AuNPs-SL treatment preliminarily causes ROS formation (Fig. 1).The concentration-dependent increase of ROS levels in the AuNPs-SL nanoparticle treatment (Fig. 1A), the growth restoration, and decrease in ROS levels in the presence of ROS scavengers (NAC, ascorbate, tiron, SP, and TU) (Fig. 1B and C) demonstrate that AuNPs-SL evokes ROS in V. cholerae.Induction of ROS leads to the cascade of events in the cells.Primarily, it induces ROSresponsive genes to counter the ROS levels, e.g., oxyR, soxR, katE, sodA, fur, and dps (56).In this study, we observed the upregulation of the selected genes (sodA, oxyR, katE, dps, fur, and grx) involved in the intracellular ROS mitigation (Fig. 2).Prior studies suggest that ROS production causes DNA damage, membrane damage, impaired Fe-S cluster biogenesis, lowered ATP production, etc. (57) (58) (59).RecA is the hallmark of SOS response (60).The upregulation of recA in treated cells probed the possibility of DNA damage and subsequent apoptotic cell death in the treated cells that has been further exhibited by TUNEL assay (Fig. 3A and B) and apoptosis assay (Fig. 3C and D).
Rescue of AuNPs-SL treated V. cholerae by iron supplementation shows the possibility of impaired Fe-S biogenesis, which further slows down the ETC and subsequent leakage of electrons (Fig. 4).The leakage of electrons from ETC could be the major reason for ROS generation.Impairment of ETC also manifests energy crisis in the cell (61).ROS causes various physiological assaults to the cell, Therefore, we were curious to know the process of entry of AuNPs-SL in to the cells and their effects.The bacterial membrane is selective in nature; thus, downregulation of porin ompU indicates the resistance of the AuNPs-SL into the cells.To understand the nanoparticle and membrane interaction, the TEM imaging was done.TEM images showed that AuNPs-SL cause physical damage to the membrane (Fig. 5Aii) by interacting with the outer membrane of V. cholerae.Physical damage of membrane led to uncontrolled entry of nanoparticles inside the cell.Damage to the outer surface cause osmotic imbalances in the cell, which lead to the leakage of intracellular content like DNA and proteins (Fig. 5).The upregulation of luxO and gsp (Fig. 2) further depicts that the nanoparticles manipulate the cell-to-cell communica tion and signaling to combat stress by switching the lifestyle of bacteria.The relation between nanoparticle treatment and iron homeostasis perturbation is a matter of future investigation to gain insights into the metabolic rewiring of V. cholerae due to AuNPs-SL treatment.The molecular mechanism of AuNPs-SL mediated killing of V. cholerae is summarized in the model (Fig. 7).The cell death model presented in Fig. 7 depicts the interaction of the AuNPs-SL with the cell surface, membrane damage, their entry in to the cell, and subsequent cascade of events.Membrane structure alteration and damage has various consequences like the entry of more AuNPs-SL, osmotic imbalance, and intracellular content leakage.The increased titer of nanoparticles affects various physiological processes, e.g., ROS generation by impairing Fe-S biogenesis.ROS induces the cascade of events such as DNA damage and apoptotic cell death.The damaged membrane induces the intracellular leakage of proteins and DNA and alters osmotic balance.Damaged membrane also affects ETC, causes membrane depolarization, and ultimately lowers the energy state of cells, which is the lifeline of every living organism (Fig. 6).Few studies using metallic nanoparticles have employed silver, zinc, or selenium nanoparticles as antimicrobials against V. cholerae (62).Chatterjee et al. used gold nanoparticles of different shapes and sizes for the eradication of biofilm of Vibrio cholerae in an in vivo animal model.They found inhibition of biofilm and compromised production and structure of cholera toxin, and reduction of fluid accumulation in infected mice treated with nanoparticles (63).Similarly, we speculate that AuNPs-SL might inhibit the expression and production of several vital proteins having crucial roles in survival of V. cholerae.
Greener AuNPs-SL have the ability to reduce cell viability at all stages of the V. cholerae life cycle, including active (planktonic) and latent (biofilm and non-dividing) cells (previous findings) and virulent condition (this study).Therefore, AuNPs-SL can be used as an alternative therapy at any stage/form to combat V. cholerae infection.These nanoparticles interact with the cell membrane, create ROS surge, upregulate genes.deplete ATP, cause DNA damage, alter membrane potential, and initiate loss of cellular content, all of which influence Vibrio's survival.However, the in vivo study is still remaining to establish it as a formulated drug.
This study provides a new, inexpensive alternate method to reduce the infectious dose of pathogen contaminated water and potential of an alternative in vivo therapy against cholera infection.Further studies are being carried out to find out the toxicity (both acute and long term) and carcinogenicity and clearance of the nanoparticle in the animal model.Besides that, the efficacy of the AuNPs-SL is being tested for lowering of toxin production and mutagenicity.Further efficacy of these nanoparticles could be tested against MDR strain of V. cholerae.Further, we are also interested to understand the cross-talk of iron homeostasis and metabolic rewiring of bacteria in nanoparticle treatment.This study will further strengthen its use as a combinatorial antibiotic therapy.

Growth conditions and cell viability
Gram-negative V. cholerae EL Tor N16961 strain was used in this study.In all the experiments, unless mentioned otherwise, growth was done at 37°C in Luria broth (LB) at 200 rpm in an orbital incubator shaker.For experiments, the nanoparticle treatment was given at an optical density at 600 nm (OD 600 ) of 0.2.To perform growth assays, Bioscreen C growth curve machine was used.In the growth assays, log phase cells were diluted 1,000 times, and 100 µL of diluted culture was added to the honeycomb plate with the indicated supplements, and volume was made up with sterile LB to 200 µL.OD 600 was measured at an interval of 1 hour, and a graph was plotted.For spot assay, an LB agar plate with different concentrations of AuNPs-SL (25 µg/mL), Mohr's salt (1.5 mM), and a combination of the two was prepared.V. cholerae cells at log phase were diluted to a ratio of 1:10 2 , 1: 10 4 , and 1:10 6 , and 10 µL of each was spotted incubated at 37°C overnight.The images of plates were taken and representative images are shown.

Nanoparticle synthesis
Synthesis of AuNPs-SL was done as mentioned earlier (17).Briefly, the synthesis of AuNPs-SL involves the addition of 40 µL of sophorolipid (100 mg/mL) in 10 mL of a chloroauric solution with a concentration of 400 µg/mL (pH 5.5 ± 0.2) with a few drops of freshly prepared sodium borohydride solution (NaBH 4 , 100 mM).

Measurement of ROS
ROS was measured using ROS-sensitive fluorescent probe H 2 DCFDA (Sigma Aldrich).Once the dye enters the cell, it senses the ROS and gets oxidized to form the fluorescent product, 2′,7′-dichlorofluorescein (64).Concentration of AuNPs-SL was taken in the range of 10-25 µg/mL based on our previous finding of antimicrobial activity of AuNPs-SL against V. cholerae.Cells were grown until OD 600 of 0.2 and were treated with different concentrations of AuNPs-SL.After treatment, the cells were spun down and washed thrice with phosphate buffered saline (PBS) and stained with H 2 DCFDA (10 µM) for 1 hour at 37°C.After incubation, the fluorescence of 20,000 cells was measured using a flow cytometer (Accuri C 6 ) in the FL1 channel.The normalized mean fluorescence intensity (MFI) of three independent experiments was plotted with ±SD.The ROS was also measured by supplementing ROS quenchers.The cells were treated with AuNPs-SL-25 and supplemented with 10 mM of respective quenchers.

RT-qPCR
Genes were selected on the basis of their role in intracellular ROS scavenging (estab lished by published findings).To check the expression profile of the selected genes in the presence of AuNPs-SL, RT-qPCR was performed.At higher concentrations of AuNPs-SL, isolated RNA shears/degrades; therefore, a lower concentration of AuNPs-SL (10 µg/mL) was used for the study.RNA extraction was done using the Trizol reagent method (65), and cleanup was done using Qiagen Kit.The RNA was checked for its quality and quantified using Nano-Drop spectrophotometer.One-step SYBER Green Master Mix reaction mixture (Invitrogen) was used to perform the qPCR reaction in a Fast Real-time PCR system (Applied Biosystem) with 200 ng of RNA per reaction.The data of triplicate experiments of biological duplicates are plotted with SD.

TUNEL assay
TUNEL assay was performed to check nanoparticle-mediated DNA fragmentation in V. cholerae using a kit.Briefly, cells were grown with AuNPs-SL (25 µg/mL) and nalidixic acid (5 µg/mL) for 3 hours.After treatment, the cells were harvested and washed with PBS (pH 7.2), fixed with 2% formaldehyde (15 minutes on ice), and treated post-fixation with 70% ethanol.The fixed cells were permeabilized with permeabilization buffer (0.1% Triton X-100 and 0.1% sodium citrate), while being kept on ice for 2 minutes.After washing, the cells were resuspended in 50 µL of the solution containing enzyme (terminal deoxynu cleotidyl transferase) and reaction mixture in the ratio of 1:9.After 1 hour of incubation at 37°C, the cells were washed again and resuspended in PBS.The flow cytometer data of 20,000 cells were acquired using an FL1 laser of Accuri C 6 flow cytometer and the mean fluorescence intensity of three independent experiments was plotted with SD.

Apoptosis assay
To check the nanoparticle-induced apoptosis in V. cholerae, we performed an annexin affinity assay.In brief, the cells were treated with AuNPs-SL and were washed twice with PBS buffer.Staining was done with annexin V allophycocyanin conjugate (5 µg/mL) for 20 minutes.After staining, the cells were washed with PBS to remove the unbound stain and were suspended in PBS.The fluorescence intensity of 20,000 cells was measured using the FL4 filter of the Accuri C 6 flow cytometer.The MFI values of three independent experiments were plotted with SD.

Transmission electron microscopy
To check the alterations in the ultrastructure of V. cholerae membrane in AuNPs-SL treatment, TEM was performed by following protocol (66) with slight modifications.In brief, both treated (AuNPs-SL 25 µg/mL for 3 hours) and untreated cells were pelleted and washed with PBS twice.Fixation was done with modified Karnovsky's fixative containing 2% (vol/vol) glutaraldehyde and 2% (vol/vol) paraformaldehyde in 0.1-M sodium cacodylate buffer (pH 7.2) at 4°C for 2 hours (67).Fixation was followed by washing with PBS and post-fixation treatment with 0.2-M sodium cacodylate buffer and osmium tetroxide [200 µL of 2% (wt/vol)] at 4°C for 90 minutes.The sample was then washed thrice with 0.1-M sodium cacodylate buffer and resuspended in the same buffer containing 2% agarose.A thin section was cut using a microtome followed by its gradual dehydration with acetone solution (once at 30%, 50%, 70%, and 90% and twice at 100%).Finally, the sections were examined under TEM in JEOL-2100.The representative images have been shown.

Measurement of leaked cellular contents (protein and DNA)
To determine the leakage of cellular contents (protein and DNA), the cells were treated with different concentrations of nanoparticles (AuNPs-SL, 25 and 50 µg/mL) followed by a Bradford assay and nanodrop readings to quantify the protein and DNA contents of the samples, respectively.For measurement of protein, 1 mL of cell sample (treated and untreated) was withdrawn after 2-hour incubation of AuNPs-SL treatment.The cells were pelleted, and the supernatant was used for protein quantification by the Bradford method.For measurement of DNA, untreated and treated cells were harvested, and DNA was extracted from the supernatant as per manufacturer instruction (ZR Fungal/Bac terial DNA Kit catalog no.D6005).DNA quantification was done by Nanodrop 1,000 spectrophotometer (Nanodrop Technologies Inc., USA).For agarose gel analysis, DNA was isolated manually from the supernatant from untreated and treated (AuNPs-SL-25) by precipitating the supernatant with chilled isopropanol followed by washing with ethanol.Isolated DNA was quantified and electrophoresed on 1% agarose gel.Isolation of the total protein was carried out after the completion of the treatment time.In a typical procedure, samples were spun down, and the supernatant was collected for further study.An equal volume of solution, i.e., a mixture of methanol and chloroform (4:1), was added to the cell soup with proper mixing and was kept at 4°C for 5 minutes.It was then centrifuged at 7,000 rpm for 10 minutes at 4°C.The upper layer was carefully discarded without disturbing the ring/interface.An equal volume of methanol was added, vortexed gently, and centrifuged at 10,000 rpm for 10 minutes at 4°C.The pellet was washed again and dried to remove residue from the solvent.It was resuspended into Tris-NaCl buffer and quantified using Bradford assay.The samples were analyzed on a 12% SDS-PAGE gel.β-Lactamase assay was carried out using nitrocefin reagent.Nitrocefin is a β-lactam that changes color when a β-lactamase breaks it down.Fifty microliters of untreated and treated cell supernatant was mixed with the 2-µL nitrocefin solution (5 mM) and incubated in the dark to observe the color change.

Membrane depolarization assay
To perform membrane depolarization assay, the untreated and AuNPs-SL treated were grown for 3 hours.After incubation, the cells were centrifuged at 4,000 rpm for 10 minutes, and the cell pellet was washed with PBS.The washed cells were resuspended in 10 µM DiBAC 4 (1-mM DMSO) and incubated for 30 minutes at 37°C in the dark.The cells were pelleted and washed with PBS to remove the extra stain.Finally, the cells were suspended in PBS and the fluorescence intensity of 20,000 cells was measured using an FL1 laser of Accuri C 6 flow cytometer.
Loss of membrane potential was also analyzed by using dye 3,3′-diethyloxacarbocya nine iodide (DiOC 2 , BacLight bacterial membrane potential kit, and Molecular Probes/ Invitrogen), a membrane potential sensitive dye.For this, the cells were stained with 2.5 µM of dye for 20 minutes at 37°C in the dark.Thereafter, the cells were washed with PBS to remove the extra stain, and flow cytometer data were acquired for 10,000 cells using an FL1 laser of Accuri C 6 flow cytometer.

ATP measurement
Relative ATP estimation was done using ATP Bioluminescence Assay Kit CLS II (Roche) following the manufacturer's recommended protocol.Briefly, the cells were grown as ROS measurement protocol.Cells were harvested at 4,000 rpm for 10 minutes and washed with chilled PBS.To extract ATP, the cells were incubated in ATP extraction buffer (100-mM Tris-HCl, pH 7.75, and 4-mM EDTA, pH 8.0) at 100°C for 2 minutes, followed by separation of cell-free supernatant by centrifugation for 5 minutes at 1,000 g. ifty microliters each of the sample supernatant and luciferase reagents was added to wells in black 96-well microplate with gentle mixing, and the luminescence was measured using a luminometer.
The relative light units (RLUs) were recorded and normalized per milligram of protein.The normalized RLU's of three independent experiments were plotted with SD.

Statistical analysis
The data from three independent experiments were calculated as mean ± standard deviation.Most of fluorescent data are represented as normalized or fold change in MFI which is calculated by dividing treated sample to the untreated one.Either paired or unpaired t-test analysis (mentioned in the graph) was performed using GraphPad Prism version 8 software for analysis.

FIG 2
FIG 2 RT-qPCR in AuNPs-SL nanoparticle treatment.Fold change of selected genes in the presence of AuNPs-SL 25 µg/mL as compared to untreated samples.The data of biological duplicates have been plotted with ±standard deviation.

FIG 4
FIG 4 Iron supplementation rescues the growth and decreases ROS levels.The growth curve in the presence and absence of AuNPs-SL-25 µg/mL and iron supplement (Mohr's salt) (A).Fold change in MFI of H 2 DCFDA shows that iron decreases ROS levels (B).Spot assay in the presence of AuNPs-SL-25 µg/mL and Mohr's salt.**P value < 0.01 calculated through paired t-test (C).

FIG 5
FIG 5 AuNPs-SL cause membrane damage and leakage of intracellular contents.TEM images of V. cholerae (A) in the absence (Ai) and presence (Aii) of AuNPs-SL.Absorbance at 595 showing the leakage of protein (B).Nanodrop readings of DNA concentration at varying concentrations of AuNPs-SL (25 and 50 µg/mL) (C) (n = 3 ± SD).The data were analyzed using an unpaired t-test **P< 0.01 and ****P < 0.0001.SDS-PAGE gel image of total protein isolated from untreated and treated cells without and with β-lactamase plasmid (D).Agarose gel electrophoresis of untreated and treated cells containing β-lactamase plasmid (E).β-lactamase activity of the above-mentioned cells and conditioning at different time intervals (F).

FIG 7
FIG 7 Schematic representation of mechanistic insight of AuNPs-SL mediated killing of V. cholerae.AuNPs-SL interacts with the membrane to cause membrane damage and accumulates in the cell.Accumulation of nanoparticles causes ROS formation which has various physiological consequences like oxidative damage to DNA (which further lead to DNA fragmentation and apoptosis), activation of ROS-responsive genes, and impaired iron homeostasis.Membrane damage and ROS together lead to osmotic imbalances, leakage of intracellular content (protein and DNA), alteration in proton motive force, and membrane potential which ultimately depletes intracellular ATP content.

TABLE 1
List of primers used in the RT-qPCR a