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10.1186/s11671-024-04107-4
Research
Polyvinylpyrrolidone capped silver nanoparticles enhance the autophagic clearance of Acinetobacter baumannii from human pulmonary cells
Sharma Saroj
Tiwari Vishvanath vishvanath@curaj.ac.in
vishvanathtiwari@hotmail.com

https://ror.org/056y7zx62 grid.462331.1 0000 0004 1764 745X Department of Biochemistry, Central University of Rajasthan, Ajmer, 305817 India
23 9 2024
23 9 2024
12 2024
19 1 15421 5 2024
9 9 2024
© The Author(s) 2024
2024
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Acinetobacter baumannii, an opportunistic pathogen has shown an upsurge in its multi-drug resistant isolates. OmpA of A. baumannii induces incomplete autophagy and apoptosis in host cells. Various therapeutic alternatives are under investigation against A. baumannii. Here, the major emphasis has been laid on comparing the efficacy of AgNP with different capping agents. OmpA targeted lead, Ivermectin capped AgNP (IVM-AgNP) has been compared with the antibacterial polyvinylpyrrolidone capped AgNP (PVP-AgNP) for their role in the modulations of host autophagy. Upregulation of p62 and LC3B confirmed by real-time PCR analysis indicated an increased autophagic flux upon the treatment with AgNPs. The elongation and closure of autophagic vacuoles was also supported by upregulated Atg genes (Atg4, Atg3, Atg5) in A. baumannii infected cells after treatment with AgNP. Autophagic flux increased on treatment with PVP-AgNP as suggested by the rise in mcherryLC3B fluorescence in A549 cells treated with PVP-AgNP as compared to the GFP-LC3B of IVM-AgNP. This suggests that PVP-AgNP treatment more effectively promotes the elongation and maturation stages of autophagy by increasing autophagic flux. These results indicate that capped AgNPs have the efficiency to revert the incomplete autophagy induced by A. baumannii back to normal autophagic levels.

Supplementary Information

The online version contains supplementary material available at 10.1186/s11671-024-04107-4.

Keywords

Acinetobacter baumannii
Autophagy
ESKAPE pathogens
Outer membrane protein A
Silver nanoparticles
Real-time PCR
Fluorescence microscopy
Autophagosome
Multi-drug resistance
Extreme drug resistance
http://dx.doi.org/10.13039/501100001411 Indian Council of Medical Research ICMR/AMR/Adhoc/291/2022-ECD-II Tiwari Vishvanath issue-copyright-statement© Springer Science+Business Media, LLC, part of Springer Nature 2024
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pmcIntroduction

In the past three decades, the opportunistic nosocomial pathogen Acinetobacter baumannii has been identified as one of the most bothersome pathogens in clinical settings [1]. Its ability to develop resistance to antibiotics has clinically raised its significance as a nosocomial pathogen [2, 3]. Antibiotic resistance is one of the major concerns related to the isolates of A. baumannii as multi-drug resistance (MDR), extreme-drug resistance (XDR), and pan-drug resistance (resistance to current antibiotics) have been reported globally [4, 5]. During the onset of infections in the host cells, two major mechanisms of the host defence are activated; autophagy and apoptosis [6]. Various studies have emphasized the role of OmpA in the modulations of host autophagy [7, 8]. It induces incomplete autophagy by inhibiting the fusion of autophagosomes and lysosomes and hence A. baumannii can persist inside the host cells [7]. Various approaches are being investigated in the elimination of the pathogen such as combinatorial usage of antibiotics, bacteriophage-based approaches, and the use of nanoparticle-based formulations [9–12]. Nanoparticles offer a more promising approach as an alternative to traditional therapeutics [13]. Since they also act as drug carriers, they modulate the pharmacokinetics, reduce toxicity, and help in the release of drugs at specific targets [14]. In one study, Raquel et.al. reported that the biogenic silver nanoparticles (AgNP) decreased the load of intracellular parasites and the proliferation rates in Toxoplasma gondii-infected HeLa cells [15]. They concluded that AgNP controls the infection via immunomodulation, induction of autophagy and apoptosis [15]. Biofabricated AgNP has also shown an antibacterial effect against Gram-positive bacteria Bacillus subtilis [16]. Lignin-capped nanoparticles also inhibited the growth of various MDR bacteria such as Pseudomonas aeruginosa, A. baumannii, Klebsiella pneumoniae, and Staphylococcus aureus [17]. The enhanced antibacterial activity of imipenem in conjugation with capped-AgNP was monitored against A. baumannii [18]. The antibacterial activity of polyvinylpyrrolidone (PVP)-capped AgNP has been shown on carbapenem-resistant strains of A. baumannii. Significant decrease in the viability of intracellular bacteria and adherence to A549 cells have been reported [19]. Studies have also shown higher efficacy of AgNP on Gram-negative bacteria via the utilization of different mechanisms of action like cell membrane leakage, inhibition of synthesis of cell wall, and generation of reactive oxygen species [20–22]. The mechanism of uptake of AgNP is via the endocytic pathway and the AgNP-containing endosomes mature to acidic late endosomes which later fuse with the autophagic pathway at lower non-cytotoxic doses. However, at cytotoxic concentrations, AgNP promotes apoptotic cell death [23–26]. AgNP also exhibits a very high efficacy in killing intracellular bacteria in S. aureus [27]. Hence this crosslink of pathways could be utilized in the study of modulations in autophagy induced by A. baumannii infections. A. baumannii is notoriously known to disrupt the fusion of autophagosomes and lysosomes to increase its chances of survival inside the host cells. It also disrupts the membrane of the lysosomes which release Cathepsin D upon the infection of A. baumannii [7, 28].

In this study, various molecules used for capping silver nanoparticles with known activities against A. baumannii have been investigated for their interaction with OmpA of A. baumannii through an in-silico approach. Further, a previously screened lead molecule (Ivermectin; IVM) against OmpA of A. baumannii has been used to cap AgNP (data under review) and compared to a well-known antibacterial PVP-AgNP [19, 29]. IVM has been reported to mediate the induction of autophagy via the AKT/mTOR pathway and shows antibacterial activities against methicillin-resistant strains of S. aureus (MRSA), M. ulcerans, and Mycobacterium tuberculosis [30–33]. Therefore, an attempt has been made to understand the comparative efficacy of the IVM-capped AgNP and PVP-AgNP [19] on the modulations of autophagy-mediated clearance of A. baumannii. Modulations in autophagy-related genes at mRNA and protein levels has been studied using real-time PCR, western blotting, and microscopic methods. This would improve the current understanding of the role of AgNP as an alternative to current therapeutics used for the treatment of infections caused by A. baumannii.

Results

In-silico screening of capping agents for their binding with the OmpA protein of A. baumannii

HDOCK was used to check the interaction of various capping agents with OmpA of A. baumannii. The Phyre2 modelled OmpA of A. baumannii and various capping agents were uploaded to the HDOCK server. The docking scores with the terminal of binding for the capping agents with OmpA are enlisted in Table 1. Chitosan and PVP showed effective binding at the N-terminal of OmpA (with docking scores of − 251.7 and − 250.78, respectively) (Fig. 1). PVP binds to the surface exposed portion of OmpA whereas chitosan binds laterally to the β-barrel strands. Hence, PVP was selected to study the modulations in A. baumannii-mediated autophagy via its interaction with OmpA. Apart from that, IVM was previously screened and it is known to interact with OmpA and modulate the autophagy induced by A. baumannii (Data under review). Hence, both PVP and IVM were taken for further study. Table 1 Table representing the docking scores and binding terminal of different capping agents with OmpA of A. baumannii

S. no.	Name of the compound	Docking score	Binding terminal	
1	Polyethyleneglycol monoesterate	− 87.5	C-terminal	
2	Ethylenediamine tetraacetic acid (EDTA)	− 134.48	C-terminal	
3	Gallic acid	− 96.98	C-terminal	
4	Chitosan	− 251.7	N-terminal	
5	Citric acid	− 108.18	C-terminal	
6	Polyvinylpyrrolidone (PVP)	− 250.78	N-terminal	
7	Ivermectin	− 171.97	N-terminal	

Fig. 1 Docked structures of capping agents with OmpA of A. baumannii (brown represents OmpA and yellow represents the capping agents)

Synthesis and characterization of nanoparticles

We have capped AgNP with IVM to check its activity against A. baumannii infections in comparison to the PVP-AgNP. Dynamic Light Scattering (DLS) analysis indicated that the hydrodynamic size of the synthesised AgNP was 136.2 nm and 256.4 nm for PVP-AgNP and IVM-AgNP, respectively (Fig. 2A, B). The PVP-AgNP were further characterised using Fourier Transform Infrared Spectroscopy (FTIR) showing a sharp peak at 3434, 2922, and 2862 cm−1 (Fig. 2C). The FTIR peak of IVM- AgNP (Fig. 2D) is somewhat like the AgNP but more intense peak at 2920, 1383, and 1018 cm−1 with some minor peaks at 744, 576 cm−1 were observed. Synthesis of IVM-AgNP was further confirmed using UV–visible spectra (Fig. 2E), and maximum absorbance was observed around 420 nm. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analysis of the IVM-AgNP showed homogenous distribution of the nanoparticles (Fig. 2F, G). Scanning electron microscopy analysis of the IVM-AgNP showed that AgNP are oblate spheroid-shaped (Fig. 2H). Further, to evaluate the size of the capped AgNPs, SEM analysis of both the synthesised AgNPs was performed, and the result showed that the size of both (IVM-AgNP and PVP-AgNP) nanoparticles was below 50 nm (Supplementary Fig. S1).Fig. 2 DLS spectra representing the size of the synthesized A PVP-AgNP B IVM-AgNP. FTIR spectra showing % transmittance at different wavenumber (cm−1) of the C PVP-AgNP D IVM-AgNP. E UV–visible spectra of the IVM-AgNP F TEM G SEM analysis of the IVM-AgNP showing homogenous distribution of the nanoparticles H SEM analysis showing the shape of the IVM-AgNP

Capped-AgNP do not affect the viability of pulmonary cells

The percentage cell viability of A549 cells was analysed in the presence of the synthesized AgNP. PVP-AgNP has already been reported to be non-cytotoxic to A549 cells [19]. Analysis of the cytotoxic effect of the AgNP was compared with the untreated control. MTT assay showed that AgNP at the concentration of 16 µg/ml was non-cytotoxic to A549 cells. This fulfils our criteria of studying the modulations in autophagy of the host cells upon the infection of A. baumannii (Supplementary Fig. S2A) in the presence of AgNP.

Decreased EtBr accumulation shows OmpA inhibiting activity of capped-AgNP

To confirm the OmpA-inhibiting activity of the capped nanoparticles, an EtBr accumulation assay was performed in the presence of each AgNP at a concentration 16 µg/ml. A decline was observed in the accumulation of EtBr in A. baumannii treated with each AgNP as compared to the untreated control. This shows inhibition of the non-specific porin OmpA by AgNP (Supplementary Fig. S2B) mainly by the capped IVM and PVP molecule on AgNPs.

A. baumannii internalization in A549 cells and uptake of AgNP

In the present study, the aim is to target the persistence of pathogenic A. baumannii in the autophagic vacuoles via capped AgNPs. To check the intake of AgNP, we have capped AgNP with FITC (green fluorescence) and analysed using a multimode imaging system. Figure 3A represents the uptake/interaction of fluorescent nanoparticles by A. baumannii. The cells were stained with DAPI (blue, binds to DNA) to observe the localization of fluorescently labelled nanoparticles. It was observed that the location of DAPI and FITC are at a similar position that shows FITC-tagged AgNPs binds to the bacterial cell. Apart from that, the engulfment of A. baumannii in A549 cells was also monitored. For this, A. baumannii was tagged with FITC and A549 cells were infected. Figure 3B represents the uptake of the bacteria by A549 cells showing that at 6-h infection, A. baumannii is internalized in A549 cells. Similarly, to target the bacteria via the autophagic process, it is also important that FITC-tagged AgNPs enter the A549 cells. Hence, we have also monitored the uptake of FITC-tagged AgNPs by A549 cells, and results showed that FITC fluorescence had been scattered around the nucleus in the cytosol of A549 cells (Fig. 3C). Altogether, the results confirm the interaction of FITC tagged AgNP with the membrane of A. baumannii as well as the uptake of the AgNP by A549 cells.Fig. 3 A Fluorescence microscopy analysis to observe the interaction of FITC tagged IVM-AgNP by A. baumannii; image represents the DAPI stained cells (blue), localization of FITC tagged IVM-AgNP in cells (green) and the DAPI-FITC merge image at 60 × magnification. B Represents the internalization of FITC labelled A. baumannii by A549 cells after infection for 6 h at 100:1 MOI (20 × magnification). Similarly, C Represents the uptake of FITC-tagged IVM-AgNP by A549 cells at 20 × magnification

Decreased fluorescence of internalized AgNP in A. baumannii infected A549 cells

Fluorescence microscopy analysis of FITC-tagged AgNP in uninfected and infected A549 cells was performed to check intracellular concentration of AgNPs. Figure 4 represents the modulations in the fluorescence of the internalized AgNP in both conditions. In both the infected IVM-AgNP and PVP-AgNP treated A549 cells (Fig. 4B, D), a decrease in the internal fluorescence intensity of the cells was observed as compared to the uninfected AgNP-treated cells (Fig. 4A, C). The data is represented graphically in Fig. 4E. This might be due to the rise in the autophagic flux that result into better clearance of AgNPs via autophagosomes in the presence of A. baumannii.Fig. 4 A Fluorescence microscopy analysis to observe the modulations in the fluorescence of the internalized AgNP. A549 cells were treated with FITC tagged AgNP with or without infection of A. baumannii where blue fluorescence corresponds to DAPI and green to FITC. A Uninfected A549 cells treated with FITC tagged PVP-AgNP B A. baumannii infected A549 cells treated with FITC tagged PVP-AgNP. Similarly, C Uninfected A549 cells treated with FITC tagged IVM-AgNP D A. baumannii infected A549 cells treated with FITC tagged IVM-AgNP. E Corrected total cell fluorescence was measured using Image J to quantify the changes in the internal fluorescence of A549 cells. Images represent best out of the biological replicates at 20 × magnification. All Statistical analysis was performed using One way ANOVA; p < 0.05 and error bars indicate mean with SD

Treatment with AgNPs modulates autophagy in A. baumannii-infected A549 cells

A549 cell line (pulmonary epithelial cells) has been selected to study the modulations in autophagy-related genes due to the ability of A. baumannii in the establishment of lung infections. This analysis was performed with the help of real-time PCR for 2-, 4-, and 6-h duration of infection at mRNA levels. For study at mRNA level, different genes facilitating different stages of autophagy; initiation (p62, Beclin-1, and CALCOCO2), elongation and maturation of autophagic vacuoles (Atg3, Atg4B, Atg5, Atg16L1, Atg7, LC3A, LC3B, and LC3C) have been analysed. Relative expression of p62 was highly upregulated in both IVM-AgNP and PVP-AgNP, where PVP-AgNP showed more enhanced upregulation (Fig. 5). This upregulation of p62 indicates the recognition and targeting of the pathogen to the autophagic machinery as it is one the major cargo receptors. The levels of CALCOCO2 were restored in PVP-AgNP treated samples compared to that of the uninfected control. All result together showed that A. baumannii is majorly eliminated via the p62-mediated mechanism, where CALCOCO2 has a lesser or no role in the autophagic clearance of the bacteria (Fig. 5). Similar results were observed in the case of Beclin-1. Levels of Atg5 were significantly downregulated in the IVM-AgNP and PVP-AgNP as compared to the control; this indicates that AgNP is less favouring to the elongation phase of the autophagy. A similar trend was observed in the case of Atg7. IVM-AgNP showed an increase in the levels of ATG16L1 whereas PVP-AgNP showed an unusual downregulation in the levels of ATG16L1. The levels of Atg4B were unaffected in IVM-AgNP in comparison to the control but were highly upregulated in PVP-AgNP. Similar results were observed in the case of Atg3 (Fig. 5). LC3B is a biomarker of autophagy, whose levels are upregulated in AgNP-treated samples as compared to the control (Fig. 5). This indicates the increase in the autophagic flux as high levels of LC3B show a rise in the induction of autophagy as well as increase in the turnover of LC3B from LC3A. This is again supported by the low levels of mRNA of LC3A in AgNP-treated samples. LC3C is crucial for the recruitment of cargo which is internalised via the plasma membrane to the autophagosome initiation sites [34]. We also analysed the effect of only IVM and PVP in modulating LC3B and p62 in A549 cells using real-time PCR (represented in Supplementary Fig. S3). The relative gene expression analysis for the various autophagy-related genes for 2 and 4 h is shown in Supplementary Figs. S4 and S5.Fig. 5 Real-time PCR data representing the relative expression analysis at 100:1MOI for different autophagy-related genes for 6 h of infection in A549 cells for control, IVM-AgNP treated A. baumannii infected A549 cells, and with PVP-AgNP treated A. baumannii infected A549 cells. All the experiments were performed in triplicates and normalized to 18s rRNA. Statistical analysis was performed with One-way ANOVA where p-value is < 0.05 and error bars represent the mean with SD

The changes at the protein level were monitored with the help of western blotting of LC3B and p62 (Fig. 6). The turnover of LC3B from LC3A was found to be downregulated in IVM-AgNP and PVP-AgNP both as compared to the control (Fig. 6A). Now this is in contradiction to the mRNA levels of both genes. One possible explanation for this could be due to the sequestering of the cargo bacteria to the late autophagic vesicles; that is towards the completion of autophagy. Higher levels of mRNA for LC3B signifies the need for an increase in the autophagic flux which is required for the bacterial clearance as well as the processing of the AgNP by host cells. Similarly, the changes in the levels of p62 also support the targeting of the cargo to the final stages of autophagic degradation (Fig. 6B). As both LC3B and p62 are accumulated during the inhibition of autophagy, but the treatment with IVM-AgNP and PVP-AgNP properly regulates the completion of autophagy, hence the reduction in the levels of both was observed.Fig. 6 Western blot representing the analysis of the ratio of A LC3B/LC3A and B p62 for control, IVM-AgNP treated A. baumannii infected A549 cells, and PVP-AgNP treated A. baumannii infected A549 cells for 6 h. All the data was normalized to GAPDH. The figure represents the modulations in the turnover of LC3B from LC3A indicating the changed autophagic flux and similarly the recruitment of cargo (p62) for A. baumannii clearance. The full image of the western blot has been represented in the Supplementary Fig. S6

Fluorescence microscopy analysis showed AgNPs mediate enhancement of the autophagic flux

Analysis of different samples under fluorescence microscopy was performed after infecting the mcherry-GFP-LC3 transfected A549 cells with A. baumannii at 100:1 MOI. The results showed decrease in the GFP and an increase in mcherry fluorescence in the cells that were infected with PVP-AgNP treated A. baumannii (Fig. 7C) as compared to the uninfected control (Fig. 7A). The increase in the GFP is a measure of the recruitment of cargo to the autophagic vacuoles. The increase in mcherry fluorescence indicates the completion stages of autophagy, as it marks the recruitment of the cargo to the late autophagolysosmes, which is supported by decrease in the green fluorescence. This could be seen in Fig. 7C, where the samples were treated with PVP-AgNP and GFP was highly downregulated and mcherry fluorescence was increased. This indicates that PVP-AgNPs has higher efficiency to promote the completion of autophagy as compared to IVM-AgNPs.Fig. 7 Fluorescence microscopy analysis of green (GFP) and red fluorescence (mcherry) in A549 cells transfected with mcherryGFP-LC3 which were infected with A. baumannii (100:1 MOI) for A Control B IVM-AgNP treated A. baumannii infected A549 cells, and C PVP-AgNP treated A. baumannii infected A549 cells for 6 h in A549 cells D Corrected Total Cell Fluorescence (Green and Red fluorescence from GFP and mcherry, respectively) quantified using ImageJ software for analyzing the changes in fluorescence during different treatment conditions. Images represent best out of the biological replicates at 20 × magnification. All Statistical analysis was performed using 2way ANOVA; p < 0.05 and error bars indicate mean with SD

Discussion and conclusion

Apart from the serious threat of the multi-drug resistant nature of A. baumannii, clearance of the intracellular bacteria is a serious concern. According to the available literature, the survival of the pathogen inside the host cells is related to its ability to modify the basic host defence responses or delay the process of the clearance of the bacteria [6, 7]. AgNPs have been reported to have a variety of properties including their antibacterial nature against various microbes [35, 36]. Different capping agents have been used to enhance the efficacy and stability of nanoparticles. In addition to that, capping agents such as EDTA, Chitosan, Citric acid, Gallic acid, Poly glutamic acid, and PVP show antibacterial activity [18, 19, 29, 37]. The main aim of this study was to target the modulation in host autophagy caused by the infections of A. baumannii. Hence, the capping agents were docked with OmpA. OmpA is a surface-exposed protein with eight antiparallel β-barrel strands at the N-terminal embedded in the outer membrane and the periplasmic globular C-terminal [38]. The docking score and the site of binding indicated that PVP was best interacting with OmpA among the selected leads. Apart from that, capping the nanoparticles with PVP increases their stability by making them tolerant to the changes in the pH and ionic composition of the environment, and they are less toxic to the host cells [39–41]. They have also been reported to show better antibacterial activity inside the in-vivo system [42]. In addition to that, the selected lead molecule IVM also exhibits high efficacy of antibacterial activity against multi-drug resistant and extremely drug-resistant strains of Mycobacterium [31] and is also clinically approved. IVM is also known for its antibacterial activity against Staphylococcus aureus [33]. Furthermore, IVM is also known to enhance autophagy via the utilization of the AKT/mTOR pathway [30]. This is in correlation to the area of interest as A. baumannii-induced autophagy has also been reported to follow the mTOR pathway [8]. Moreover, the mechanism of uptake of the AgNP is mediated via the endosomes and has been attributed to the enhancement of autophagy in the host cells as it crosslinks with the autophagic pathway [43]. The exploitation of both IVM- and PVP-AgNP-mediated autophagic modulations in A. baumannii infected cells could improve the chances of elimination of intracellular bacterial infections. Decreased fluorescence of the internalized IVM-AgNP and PVP-AgNP upon infection of A. baumannii as compared to the uninfected AgNP-treated A549 cells (control) hints towards enhanced autophagic flux in the cells. This possibility could be analyzed by quantifying the internalization of AgNP and comparing the size and AgNP dose-dependent modulations of autophagy in the infected host cells. Hence the modulations in autophagy of the host have been tested in the presence of IVM-AgNP and already known antibacterial PVP-AgNP. In the initiation stages of autophagy (p62, Beclin-1, and CALCOCO2) PVP-AgNP proved to be a better treatment than IVM-AgNP. This could be justified by the increase in the levels of p62, and CALCOCO2 and Beclin-1 were reverted to normal uninfected control. Atg genes, which facilitate the elongation of autophagic vacuoles are downregulated by PVP-AgNP and promote the closure of the autophagosome and the recycling of the LC3 for new cycles of cargo targeting. In conclusion, the levels of LC3B/LC3A and p62 in western blot indicate the completion of the autophagic process on the treatment with AgNP which is furthermore supported by the confocal microscopy data.

The decreased green and upregulated red fluorescence due to the GFP and mcherry, respectively in the PVP-AgNP treated samples mark the completion of autophagy. PVP-AgNP treatment resulted in the recruitment of more cargo receptors at the initiation phase of autophagy and promoted the genes responsible for the enclosure of the autophagic vacuoles. Similarly, IVM-AgNP also showed a likewise trend but PVP-AgNP showed better enhancement of autophagic clearance. There could be two possible explanations for the mechanism of action of AgNP. Firstly, the enhanced levels of autophagy in the presence of AgNP could be due to the individual role of AgNP in the induction of autophagy via crosslinking of the endocytic and autophagic pathways. Secondly, due to the antibacterial activity of PVP-AgNP, a lot of debris accumulates in the host cells which is caused by the damaged membrane and generation of reactive oxygen species in the pathogen [20–22]. Hence PVP-AgNP promotes the enhanced clearance of A. baumannii through autophagy as compared to the IVM-AgNP. Another reason for the enhanced autophagic clearance of the A. baumannii by PVP-AgNP is due to the smaller size of PVP-AgNP than IVM-AgNP, as the rate of autophagic flux is also monitored by the size of nanoparticles. The diverse mechanisms of action of nanoparticles against drug-resistant strains of A. baumannii, increase the probability of eliminating the intracellular bacterial infections in the host. More exploration of the intracellular bacteria and its targeting by autophagy will provide further insight into the development of therapeutics in future.

Materials and methods

In-silico binding of nanoparticle capping agents with OmpA of A. baumannii

OmpA was modelled by submitting the protein sequence (AXV53527.1) to the Phyre2 server (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) [44]. The model was refined and validated using GalaxyWeb and PSVS, respectively [45, 46]. The modelled structure of OmpA and various capping agents such as gallic acid, EDTA, chitosan, PVP, citric acid, PGA, and PEG, along with IVM were submitted to the HDOCK server (HDOCK Server (hust.edu.cn)). HDOCK uses a hybrid algorithm of template-based and template-free docking for the prediction of interaction between the receptor and ligand [47]. It predicts docking scores, confidence scores, and ligand RMSD (Å) for the docked complexes. The molecules were further shortlisted based on docking scores and the terminal of binding to OmpA.

Bacterial culture

A Wild-type strain of A. baumannii (AB5075) was grown in Muller Hinton Broth (MHB, Himedia). The culture was inoculated from a single colony and grown in an incubator shaker at 37 °C according to the published protocol [19]. Centrifugation and 1xPBS wash (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4) were given to the culture before coinfection studies.

Culture of A549 cell line

The human pulmonary cell line, A549 was cultured according to the published protocol [19]. The passage of the cells was performed every 2–3 days and the cells were seeded in 35 mm dish or 6 well plates for the infection studies.

Preparing A549 cells and infection with bacterial culture

The cells were detached with trypsin–EDTA (0.05%) after washing two times with 1xPBS. Single cell suspension was diluted in DMEM and cells were centrifuged at 1000 rpm for 5 min. The dissolved pellet was used to seed cells at a cell density 0.3 × 106 in 6 well plates [19]. The culture for Wild-type AB5075 was grown till an OD600 of 0.4 and then treated with IVM-AgNP and PVP-AgNP at a concentration of 16µg/ml for two hours. This culture was used to infect the cells at 100:1 (Multiplicity of Infection) MOI for 2, 4, and 6 h. The pulmonary A549 cells (at 90% confluency) were incubated in only DMEM (without FBS and antibiotic) after PBS wash. AB5075 was added to these cells at 100:1 MOI and further, the cells were incubated in a 5% CO2 incubator at 37 °C for 6 h after infection.

Synthesis and characterization of AgNP

AgNPs were synthesized using the previous lab protocol where 0.01 M AgNO3, 5 ml (Fluka 34294) was mixed with 2-methoxyethanol, 10 ml (Sigma 284467). This mixture was stirred for 2 h on the magnetic stirrer. 0.2 g Polyvinylpyrrolidone (Aldrich 234257) was added as a stabilizer and stirred for 30 min. For capping the AgNP with Ivermectin, 280 µl of 5 mM Ivermectin (dissolved in 25 mM NaOH) was further added to the silver nanoparticles with stirring for 10 min [29]. For PVP-AgNPs, PVP was further added with stirring for 10min. Synthesized AgNP were pelleted down with centrifugation for 4 cycles of 30 min at 12000 g. The pellet was washed with 100% ethanol and then air dried. Characterization of the nanoparticles was performed using UV–vis spectroscopy (Multimode reader, BioTek), Fourier transform infrared spectroscopy (FT-IR Spectrum 2; Perkin Elmer), Scanning electron microscopy (Nova Nano FE-SEM 450), and Transmission electron microscopy (Tecnai G2 20 S-TWIN).

Preparation for studying uptake of AgNP by A. baumannii and A549 cells

A549 cells were grown on coverslips in 35mm dishes and then both A549 cells and A. baumannii were treated with FITC tagged IVM-AgNP at 16 µg/ml concentration for 6 h. A549 cells were washed with PBS thrice, fixed in 4% formaldehyde, and then stained with DAPI (1 µg/ml; Himedia TC229) for 10 min. The coverslips were mounted on the slides with the help of an Antifade mountant with DAPI (Invitrogen) and were then analyzed by fluorescence microscopy (BioTek). Similarly, for analysing the effects of A. baumannii infection on the internalization of AgNP, the FITC tagged AgNP treated infected A549 cells (at 100:1 MOI for 6 h) and then the cells were analyzed under the fluorescence microscope (BioTeK).

FITC labelling of A. baumannii

2ml of overnight grown culture of AB5075 was pelleted down at 5000 rpm for 10 min. The pellet was dissolved in 0.1M NaHCO3, incubated in 10 µg/ml FITC in the dark at room temperature for 30 min. After that, the suspension was again pelleted and washed three times with HBSS to remove any unbound dye. The FITC-labelled bacteria was finally dissolved in DMEM and used to infect A549 cells at MOI of 100:1 for 6 h.

Cytotoxicity analysis

For cytotoxicity analysis of A549 cells in the presence of IVM-AgNP and PVP-AgNP, A549 cells were seeded in 96 well plates and treated with varying doses of compounds. The cytotoxic effects of different compounds on A549 cells were analyzed using the previous lab protocol [19].

EtBr accumulation assay

The bacterial culture was grown till OD600 of 0.6 and then pelleted down at 15000 rpm for 5 min. Two PBS washes were given to the pellet and diluted such that the OD600 remained 0.4. To this diluted culture 0.4% of glucose was added as an energy source. This culture was added to 96 well plates and given treatment with IVM-AgNP and PVP-AgNP at 16 µg/ml. Finally, 0.5 µg/ml EtBr was added to the wells and the readings were taken at intervals of 2 min for 1 h using a multimode reader (BioTek). The excitation and emission wavelengths were 530 nm and 600 nm, respectively [49].

Real-time PCR analysis of human autophagy genes

For total RNA isolation from the infected A549 cells, TRIzol™ Reagent (Invitrogen) was used according to the manufacturing instructions and the concentration of RNA was estimated using nanodrop (Jenway Genova Nano). cDNA was synthesized using 1 µg RNA with the help of a Verso cDNA synthesis Kit (ThermoScientific). CFX96 Touch Real-Time PCR (Bio-Rad) was used to perform quantitative PCR using the SYBR green-based real-time PCR mix (Applied Biosystem real-time PCR mix). The list of primers used has been provided in the supplementary Table 1 along with their annealing temperatures (°C). The reaction conditions were set according to the instructions of the manufacturer (Supplementary Tables 1 and 3 represent the reaction mixture and reaction conditions for the real-time PCR used in this study). Relative expression analysis of the human autophagy genes was analyzed using 2–∆∆Ct method.

Western blotting analysis

Infected A549 cells were lysed using ice-cold RIPA lysis buffer on ice (150 mM NaCl, 5 mM EDTA pH 7.4, 50 mM Tris–Cl pH 6.8, 1% NP-40, 0.5% Sodium-deoxycholate, 0.1% SDS, and 1 mM PMSF). After adding RIPA lysis buffer, the cells were scraped and collected in 1.5 ml centrifuge tube. The tubes were vortexed for 30 s for 8–10 times, then the samples were incubated on ice for an hour, and finally, the protein was collected in the supernatant by centrifuging the lysate at 12000 g at 4 °C. The concentration of different protein samples was estimated using the Bradford assay. SDS-PAGE (12%) was performed for protein samples and transferred onto 0.2 µm PVDF membrane (Bio-Rad). Blocking was performed at room temperature using 5% skimmed milk for 1 h. Overnight incubation of the membrane was done with anti-rabbit LC3B (1:1000 dilution) (Cell Signaling Technologies), and GAPDH (1:1000 dilution) (Novus) primary antibody on a rocker shaker at 4 °C. The next day, the PVDF membrane was washed thrice with 1 × TBST (2 mM Tris: 150 mM NaCl, 0.01% w/v Tween-20) for 10 min each. Further incubation of PVDF was done with anti-mouse IgG HRP conjugated secondary antibody (1:2000 dilution) (R & D system) for 2 h. The protein expression was detected using ECL (WESTAR ANTARES, Cyanagen) on the LI-COR system. Densitometry analysis was performed using ImageJ software according to the protocol [48].

Transfection and fluorescence microscopy

For transfection of A549 cells with the fluorescent plasmid (Addgene; mcherryGFPLC3 Cat. 110060), 35 mm dishes were used for seeding of the cells on coverslips till they reached a confluency of 80%. Transfection of A549 cells was performed using Lipofectamine™ 3000 Transfection Reagent (Invitrogen). 2500 ng plasmid DNA of mcherryGFPLC3 was used in 5 µl of P3000™ Reagent and 3.75 µl of Lipofectamine™ 3000 Reagent was used for the transfection of plasmid DNA in A549 cells. For visualization studies, the A. baumannii infected A549 cells were fixed with 4% formaldehyde and stained with DAPI (1 µg/ml) (Himedia). The fixed cells were mounted on slides using an Antifade mountant containing DAPI (Invitrogen). The fixed slides were analyzed under a fluorescence microscope (BioTek). The fluorescence intensities were quantified using ImageJ software as corrected total cell fluorescence (CTCF).

Statistical analysis

GraphPad Prism and MS Excel were used to analyze all the data statistically.

Supplementary Information

Additional file1 (PDF 2030 KB)

Acknowledgements

Vishvanath Tiwari would also like to thank the ICMR (ICMR/AMR/Adhoc/291/2022-ECD-II). SS wants to thank the Central University of Rajasthan for providing Ph.D. fellowship.

Author contributions

Conceived and designed: V.T.; Data curation: S.S., V.T.; Analyze the data: V.T.; Wrote the manuscript: S.S., and V.T.; Proofread of the final version: V.T. All authors have read and agreed to the published version of the manuscript.

Funding

Vishvanath Tiwari would also like to thank the ICMR (ICMR/AMR/Adhoc/291/2022-ECD-II).

Data availability

All the data are available in the manuscript and its supplementary file (Figs. S1–S6 and Tables S1–S3).

Declarations

Conflict of interest

The authors declare no competing interests.

Informed consent

All the data are available in the manuscript, and all the authors agree to publish it. No third-party data is used; hence, this section is not applicable to the current manuscript.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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