ABSTRACT
Iron/heme acquisition systems are critical for microorganisms to acquire iron from the human host, where iron sources are limited due to the nutritional immune system and insolubility of the ferric form of iron. Prior work has shown that a variety of gallium compounds can interfere with bacterial iron acquisition. This study explored the intra- and extracellular antimicrobial activities of gallium protoporphyrin (GaPP), gallium mesoporphyrin (GaMP), and nanoparticles encapsulating GaPP or GaMP against the Gram-negative pathogens Pseudomonas aeruginosa and Acinetobacter baumannii, including clinical isolates. All P. aeruginosa and A. baumannii isolates were susceptible to GaPP and GaMP, with MICs ranging from 0.5 to ∼32 μg/ml in iron-depleted medium. Significant intra- and extracellular growth inhibition was observed against P. aeruginosa cultured in macrophages at a gallium concentration of 3.3 μg/ml (5 μM) of all Ga(III) compounds, including nanoparticles. Nanoparticle formulations showed prolonged activity against both P. aeruginosa and A. baumannii in previously infected macrophages. When the macrophages were loaded with the nanoparticles 3 days prior to infection, there was a 5-fold decrease in growth of P. aeruginosa in the presence of single emulsion F127 copolymer nanoparticles encapsulating GaMP (eFGaMP). In addition, all Ga(III) porphyrins and nanoparticles showed significant intracellular and antibiofilm activity against both pathogens, with the nanoparticles exhibiting intracellular activity for 3 days. Ga nanoparticles also increased the survival rate of Caenorhabditis elegans nematodes infected by P. aeruginosa and A. baumannii. Our results demonstrate that Ga nanoparticles have prolonged in vitro and in vivo activities against both P. aeruginosa and A. baumannii, including disruption of their biofilms.
INTRODUCTION
Extensively drug-resistant Gram-negative bacteria, such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae, are becoming a global health care threat (1). They show resistance to many commercially available antibiotics that target general metabolic pathways, including synthesis of proteins, RNA, DNA, and the cell wall (2, 3). Gram-negative bacteria are particularly prone to the development of antibiotic resistance because they are protected by an outer membrane from toxic compounds, possess a variety of efflux pumps, and exhibit a high transformation rate, allowing for rapid transmission of resistance genes from other bacteria. Drugs that target new, but conserved, bacterial therapeutic targets are needed to address this growing public health challenge.
Iron is an essential nutrient for nearly all living organisms. Although iron is one of the most abundant elements on earth, the bioavailability of free iron under physiological conditions is quite limited (4, 5). The ferric form of iron is insoluble and is sequestered by host proteins such as transferrin, lactoferrin, and ferritin (6, 7). These proteins serve to create an iron limited environment for invading bacteria that limits their ability to replicate and cause disease, thereby creating a system of “nutritional immunity” (8–10).
Both Gram-negative and Gram-positive pathogens have developed efficient systems to acquire iron from the iron-limited environment of the human host. Like many pathogens, P. aeruginosa and A. baumannii release siderophores, small high-affinity iron chelators, to capture free and/or protein-bound extracellular iron (Fe3+), which is then transported into the bacteria. Pyoverdin and pyochelin are the siderophores produced by P. aeruginosa, and acinetobactin and fimsbactin are the major siderophores released from A. baumannii (11).
Another major source of iron in vivo is heme and heme-containing proteins, including hemoglobin, which contains ∼70% of heme in the human host (12, 13). Thus, many pathogenic microbes, including P. aeruginosa and A. baumannii, possess efficient systems to acquire heme directly or through a hemophore released from the bacteria (8). Once internalized, the heme is degraded to release free iron by bacterial heme oxygenase or is used as a cofactor in bacterial heme-binding proteins, such as cytochromes and catalases.
Because of highly efficient heme acquisition systems of pathogens, various types of porphyrins, a mimic of heme or hemin, have been explored as antimicrobial agents. A number of studies have demonstrated that porphyrins complexed with a metal other than iron exhibit antimicrobial activity against Gram-negative and Gram-positive bacteria, as well as mycobacteria, by interfering with iron/heme acquisition systems (14–16). Gallium(III) has particularly been proven to have therapeutic potential as an iron-targeting metal due to its physical and chemical similarity to Fe(III). This allows Ga(III) to bind to iron utilizing proteins and participate in many intracellular processes (17–19). Incorporation of Ga(III) in place of iron disrupts the iron-dependent redox process because Ga(III) cannot be reduced to Ga(II) under physiological conditions. Like gallium salts, such as gallium nitrate, gallium chloride, and gallium citrate, it has been reported that gallium(III) meso- and protoporphyrin, a hemin mimetic, exhibit antimicrobial and antibiofilm activity against several bacteria, including P. aeruginosa (20–22), Neisseria gonorrhoeae (14), Haemophilus ducreyi (14), A. baumannii (22–24), Porphyromonas gingivalis (15), Staphylococcus aureus (25), and Mycobacterium abscessus (26).
We also have previously demonstrated that gallium(III) tetraphenylporphyrin (GaTP) and its nanoparticles are excellent growth inhibitors of mycobacteria and HIV-1 in vitro (27–29). Nanoparticles encapsulating GaTP exhibited prolonged growth-inhibiting activity against mycobacteria residing in macrophages. In the present study, the effects of Ga(III) mesoporphyrin (GaMP) and Ga(III) protoporphyrin (GaPP) and nanoparticles encapsulating GaMP or GaPP against P. aeruginosa and A. baumannii were studied under iron-free, iron-rich, and hemin-rich conditions. We also investigated the antibiofilm and intracellular and extracellular antibacterial activities of these Ga(III) porphyrins and nanoparticles against both P. aeruginosa and A. baumannii bacteria to assess their potential for further development as antimicrobial agents.
RESULTS
Cytotoxicity.The cytotoxicity of gallium nanoparticles, as well as that of gallium porphyrins (GaPP and GaMP), was assessed for up to 7 days in THP-1 macrophages (see Fig. S1 in the supplemental material). No toxicity, as determined by the resazurin assay, was found at up to 66 μg/ml (100 μM) gallium nanoparticles and gallium porphyrins at 7 days of incubation. However, 70 to 80% cytotoxicity was observed at 132 and 198 μg/ml GaPP, β-cyclodextrin and F127 block copolymer nanoparticles (FGaPP), and β-cyclodextrin nanoparticle encapsulating GaPP (CDGaPP) at day 7.
Growth inhibition of Gram-negative A. baumannii and P. aeruginosa by gallium(III) porphyrins under iron-depleted conditions.The susceptibilities of reference strains and clinical isolates of A. baumannii and P. aeruginosa to Ga(NO3)3 and Ga(III) porphyrins [GaPP, GaMP, and gallium(III) phthalocyanine chloride (GaPC)] were determined using the broth microdilution method in iron-depleted BM2 or RPMI 1640 medium containing 10% fetal bovine serum (FBS) (Table 1). GaPP and GaMP demonstrated similar growth inhibition, with MIC values of 4 to 8 μg/ml for all A. baumannii strains and 4 to 32 μg/ml for all P. aeruginosa strains. The exception was a clinical isolate, A. baumannii B1365-15, which showed low MIC values for both GaMP (0.25 μg/ml) and GaPP (0.5 μg/ml). However, GaPC was not as active as the other gallium porphyrins, showing MICs of >32 μg/ml against both P. aeruginosa and A. baumannii (Table 1). This could be due to the fact that GaPC possesses larger phthalic groups, which may limit binding to a hemophore or heme transporter to cross the bacterial outer membrane. The MICs of Ga(NO3)3 ranged from 0.5 to 128 μg/ml against all A. baumannii strains, whereas the MICs for P. aeruginosa strains ranged from 1 to 4 μg/ml. Thus, the susceptibility of A. baumannii to Ga(NO3)3 is more strain dependent, and resistance to Ga(NO3)3 is more common than for P. aeruginosa (22).
MICs in iron-limited BM2 medium
Interestingly, significant GaPP antimicrobial activity was observed at concentrations lower than the MIC of GaPP for P. aeruginosa strains, demonstrating 60 to 70% growth inhibitions at concentrations up to 32 ng/ml (Fig. 1 and S2) under iron-depleted conditions. A detailed mechanistic study is in progress to elucidate the mechanism that results in this increased antimicrobial activity of GaPP.
MICs of gallium nitrate and gallium protoporphyrin against P. aeruginosa PA103 and clinical isolate B1384 in iron-depleted BM2 medium. Data represent mean ± standard deviation of triplicates.
Reversal of growth inhibition of A. baumannii and P. aeruginosa by an exogenous iron source, ferric ammonium citrate.We tested the growth-inhibiting effects of Ga(NO3)3, GaPP, and GaMP in the presence of two different iron sources, ferric ammonium citrate (FAC) and hemin (Fig. 2 and 3). Interestingly, the addition of FAC (100 μg/ml) increased the growth rate of A. baumannii, resulting in an ∼100% increase in the bacterial population compared to the positive untreated control (no hemin, FAC, or gallium compound) at 24 h (Fig. 2A). However, the growth rate of P. aeruginosa was not affected significantly by excess FAC, showing only an ∼20% increase in the population, without reaching stationary phase at 24 h (Fig. 3A). This result suggests that A. baumannii may possess less efficient mechanisms for acquiring/utilizing extracellular free iron than P. aeruginosa to achieve maximum rates of growth; hence, it requires excess FAC.
Rescue effect of hemin and iron on A. baumannii growth inhibition by Ga(NO3)3 and Ga(III) porphyrins. Growth inhibition of A. baumannii ATCC 19606 by Ga(III) was reversed in the presence of 100 μg/ml ferric ammonium citrate (FAC) or 50 μg/ml hemin under iron-depleted conditions for 24 h. Data represent mean ± standard deviation of triplicates. (G) Simplified % growth compared to untreated positive control (no FAC, hemin, or gallium compound) at 24 h of incubation from growth curves in panels A and D. (H) Simplified % growth compared to untreated positive control (no FAC, hemin, or gallium compound) at 24 h of incubation from growth curves in panels B and E. (I) Simplified % growth compared to untreated positive control (no FAC, hemin, or gallium compound) at 24 h of incubation from growth curves in panels C and F. (+), no gallium compounds.
Rescue effect of hemin and iron on P. aeruginosa growth inhibition by Ga(NO3)3 and Ga(III) porphyrins. Growth inhibition of P. aeruginosa PA103 by Ga(III) was reversed in the presence of 100 μg/ml ferric ammonium citrate (FAC) or 50 μg/ml hemin under iron-depleted conditions. Data represent mean ± standard deviation of triplicates. (G) Simplified % growth compared to untreated positive control (no FAC, hemin, or gallium compound) at 24 h of incubation from growth curves in panels A and D. (H) Simplified % growth compared to untreated positive control (no FAC, hemin, or gallium compound) at 24 h of incubation from growth curves in panels B and E. (I) Simplified % growth compared to untreated positive control (no FAC, hemin, or gallium compound) at 24 h of incubation from growth curves in panels C and F. (+), no gallium compounds.
Growth inhibition of A. baumannii and P. aeruginosa by Ga(NO3)3 was reversed to a greater extent than with GaPP or GaMP by the presence of excess FAC (100 μg/ml). Interestingly, A. baumannii growth inhibited by Ga(NO3)3 at concentrations ranging from 1 to 32 μg/ml was fully reversed by the presence of excess FAC, while only 44% recovery was observed with P. aeruginosa at 32 μg/ml Ga(NO3)3 (Fig. 2G and 3G). This is consistent with the possibility that A. baumannii has a less efficient extracellular iron acquisition mechanism, as observed in the growth curve of FAC-treated A. baumannii strains. Growth inhibition of A. baumannii by GaPP (>8 μg/ml) or GaMP (>4 μg/ml) was not reversed by the presence of excess FAC (Fig. 2H and I). However, the presence of FAC recovered 40 and 27% of P. aeruginosa growth compared to conditions in the presence of GaPP or GaMP at 32 μg/ml, respectively. The reversal of growth inhibition of P. aeruginosa growth by FAC may be in agreement with less susceptibility to GaPP and GaMP, whose MICs are 2- and 4-fold higher than A. baumannii MICs. These data suggest that gallium porphyrin, a mimic of hemin, is more targeted to the process of heme utilization instead of a free iron-utilizing pathway.
Reversal of growth inhibition of A. baumannii and P. aeruginosa by exogenous hemin.The effect of exogenous hemin as an iron source on the growth inhibition of both pathogens was also investigated. Hemin exhibited a different pattern of rescue than FAC on the growth of both bacteria. As seen in Fig. 2D and 3D, A. baumannii had long lag and exponential phases compared to the untreated control, whereas there was no significant difference in the growth of P. aeruginosa when exposed to hemin (50 μg/ml). Hemin reversed the GaPP- and GaMP-mediated growth inhibition of A. baumannii better than did FAC, showing 45 to 90% growth recovery compared to the untreated controls (Fig. 2H and I). Hemin and FAC showed similar recovery patterns for GaPP- and GaMP-mediated growth inhibition of P. aeruginosa (Fig. 3H and I). These results support the hypothesis that GaPP and GaMP are competitive inhibitors of heme uptake and/or utilization. In comparison to FAC, hemin was less effective in reversing growth of both pathogens inhibited by Ga(NO3)3 (Fig. 2D, 2G, 3D, and 3G). It was observed that there was a loss of the recovery effect of hemin at concentrations higher than 8 μg/ml Ga(NO3)3 against P. aeruginosa and 35% recovery at 32 μg/ml Ga(NO3)3 against A. baumannii, suggesting that A. baumannii may have lower efficacy systems in extracting iron from hemin. As a result, observations from growth curve and rescue experiments imply that A. baumannii has less effective iron and heme acquisition systems than does P. aeruginosa.
Extracellular and intracellular antimicrobial activities of gallium porphyrins and nanoparticles against P. aeruginosa and A. baumannii.To study extracellular and intracellular growth-inhibiting activities of Ga(III) porphyrins, we designed and conducted two different experiments. First, GaPP or GaMP nanoparticles were prepared by encapsulation to investigate their duration of activity. β-Cyclodextrin (CDGaPP) and F127 block copolymer (FGaPP) nanoparticles containing GaPP were formulated using high-pressure homogenization. A single emulsion method was used to formulate F127 copolymer nanoparticles encapsulating GaMP (eFGaMP). The nanoparticles were characterized by dynamic light scattering. The size of the FGaPP was 674 ± 27 nm, with a polydispersity index (PDI) of 0.39 ± 0.003, and the ζ potential was −16.4 ± 0.65. The size of CDGaPP was 427 ± 29.5 nm, with a PDI of 0.43 ± 0.03, and the ζ potential was −20.9 ± 0.5. The size of eFGaMP was 300 ± 20 nm, the PDI is 0.4, and the ζ potential was +7 ± 0.5. The Ga loading efficiency in all nanoparticles was ∼70%.
Prior to studies of extra- and intracellular antimicrobial activity in macrophages, the antimicrobial activity of these nanoparticle suspensions was determined using a microdilution method. Table 2 shows the lowest inhibitory concentration against P. aeruginosa and A. baumannii in iron-depleted BM2 medium. Ga(III) porphyrin-loaded nanoparticles were as effective as the free drugs against both pathogens, exhibiting MICs of 4 to 8 μg/ml.
MICs of gallium nanoparticles against P. aeruginosa and A. baumannii
THP-1 macrophages in RPMI 1640 medium supplemented with 10% FBS were infected at a multiplicity of infection (MOI) of 1 with either P. aeruginosa or A. baumannii, followed by treatment with Ga(III) porphyrins or nanoparticles for 3 days to determine their extracellular and intracellular inhibitory activities (Fig. 4). We observed that internal and external P. aeruginosa-infected macrophages were more susceptible to gallium porphyrins and nanoparticles than A. baumannii-infected macrophages. Interestingly, when A. baumannii-infected macrophages were treated with 3.3 to ∼13.2 μg/ml Ga(III) compounds, no inhibition was observed against either extracellular or intracellular bacteria at days 1 and 3 postinfection. This resulted in death of the macrophages (Fig. S3). In contrast, treatment of the THP-1 macrophages infected with P. aeruginosa with 3.3 μg/ml (5 μM) of the Ga(III) compounds resulted in >99.9% inhibition of both intracellular and extracellular growth (Fig. 4C and D and S4), indicating a high therapeutic potential of GaPP and GaMP against P. aeruginosa.
Extra- and intracellular antimicrobial activities of gallium porphyrins and nanoparticles against A. baumannii ATCC 19606 (Ab) and P. aeruginosa PA103 (Pa). THP-1 macrophages were infected at an MOI of 1 for 1 h, followed by treatment with Ga(III) compounds (66 μg/ml [100 μM] for A. baumannii, 3.3 μg/ml [5 μM] for P. aeruginosa) for 3 days. ns, not significant. Multiple comparisons were performed using ANOVA, and Student’s t test was used to compare two groups (GraphPad Prism 6.0). ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05 compared to positive control.
Although A. baumannii is more susceptible to GaPP and GaMP than is P. aeruginosa (Table 1), a 20-fold increase in the concentration of Ga(III) inhibitors was required to exert antimicrobial activity against intracellular and extracellular A. baumannii in cocultured macrophages (Fig. 4A and B). At a concentration of 66 μg/ml (100 μM), Ga(III) inhibitors showed significant inhibitory activity against both intracellular and extracellular A. baumannii. GaMP and its nanoparticles (eFGaMP) showed the best inhibitory activities among the Ga(III) inhibitors tested. GaMP reduced extracellular and intracellular growth of A. baumannii by 2.5- and 2-fold compared to GaPP, respectively (Fig. 4A and B). Although FGaPP and CDGaPP nanoparticles were active in reducing external and internal A. baumannii by about 60- and 2-fold compared to their nontreated controls, respectively, after 3 days of incubation, they did not exert enhanced antimicrobial activity compared to free GaPP against the bacilli. However, enhanced antimicrobial activity against external and internal A. baumannii was observed for eFGaMP compared to its respective GaMP and GaPP nanoparticles, suggesting that these nanoparticles exert improved activity based on properties such as size and surface potential and/or sustained drug release (27). Here, the smaller size and better penetrating positive ζ potential of eFGaMP may have contributed to the enhancement of long-acting antimicrobial efficacy.
Next, the duration of activity of the nanoparticles in macrophages was investigated. Here, the bacterial load to infect THP-1 macrophages was increased (MOI, 20) to achieve higher intracellular infection, and the intracellular activity of the Ga(III) inhibitors (66 μg/ml) was determined against both pathogens residing in macrophages in RPMI 1640 medium supplemented with 10% FBS (Fig. 5 and S5). After initial infection, the macrophages were washed and treated with gentamicin to remove extracellular bacteria in order to investigate the inhibitory effect of Ga(III) only on bacteria initially residing in macrophages. As shown in Fig. 5, all inhibitors showed excellent growth-inhibiting activity, resulting in killing more than 99.9% of P. aeruginosa PA103 within macrophages at day 1 and 3 postinfection. In contrast, A. baumannii residing in macrophages were more resistant to Ga(III) than was P. aeruginosa, but significant growth inhibition was observed up to day 2 postinfection. Compared to the positive control, 3- and 1-fold reductions of A. baumannii growth by GaPP were observed, while GaMP showed >200- and 4-fold growth inhibition within macrophages at day 1 and day 2, respectively (Fig. 5). Similarly, F127 polymer nanoparticles encapsulating GaMP (eFGaMP) demonstrated better antimicrobial activity, resulting in >250- and 4.5-fold growth inhibition compared to the positive control, 70- and 3.5-fold reduction compared to FGaPP, and 23- and 2-fold reduction compared to CDGaPP at days 1 and 2 postinfection, respectively. These observations suggest that A. baumannii is more susceptible to GaMP and eFGaMP, and excellent growth inhibition was achieved by all Ga(III) porphyrins and nanoparticles against P. aeruginosa (Fig. 5). This result was also confirmed by visual inspection of wells of the plate (Fig. S5). Turbidity from the wells containing macrophages and A. baumannii was observed at day 2, indicating that the bacillus escaped from the macrophages and was growing in RPMI 1640 medium. However, no turbidity was found from the wells infected with P. aeruginosa at days 1 and 3 (Fig. S5 and S6).
Long-acting intracellular antimicrobial activities of gallium porphyrins and nanoparticles within THP-1 macrophages infected with A. baumannii ATCC 19606 (A and B) or P. aeruginosa PA103 (C and D). THP-1 macrophages were infected at an MOI of 20 for 1 h, followed by treatment with the Ga(III) compounds (66 μg/ml) for 24 h (day 1 postinfection). After washing, the macrophages were incubated for 1 (A. baumannii, day 2) or 2 (P. aeruginosa, day 3) days. Multiple comparisons were performed using ANOVA, and Student’s t test was used to compare two groups (GraphPad Prism 6.0). ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05 compared to positive control. ns, not significant. Data represent the mean ± standard deviation of triplicates.
Potential of Ga(III) porphyrins and their nanoparticles loaded into macrophages to prevent infection.We examined to what extent uninfected macrophages that had been treated with FGaPP, CDGaPP, and eFGaMP were protected from subsequent bacterial challenge over time. THP-1 macrophages were incubated with Ga(III) inhibitors for 24 h (day 0), following which the extracellular Ga(III) compound was removed and the macrophages were infected at days 0, 1, 3, and 5 posttreatment. Overall, the Ga(III) inhibitors (66 μg/ml) showed prolonged inhibitory activity against P. aeruginosa infection up to 3 days, whereas an inhibitory effect was not observed against A. baumannii at Ga(III) concentrations up to 198 μg/ml. This may be due to the inability to achieve an adequate concentration of Ga(III) inside the macrophage in RPMI 1640 medium (with 10% FBS) for A. baumannii.
The antimicrobial effect of all Ga(III) inhibitors present in macrophages was slowly lost over time against P. aeruginosa, as noted by the waning inhibitory effect at days 3 and 5 post-drug loading (Fig. 6). These Ga(III) inhibitors displayed different time-dependent antimicrobial activity. GaPP and FGaPP lost activity at day 3, while β-cyclodextrin nanoparticle encapsulating GaPP (CDGaPP) showed significant inhibitory activity on the same day, suggesting more prolonged drug retention and slow drug release from CDGaPP and rapid export of free GaPP from macrophages. In contrast, GaMP demonstrated more prolonged activity, resulting in 1.6-fold higher growth reduction in P. aeruginosa within macrophages compared to the positive control at day 3 (Fig. 6C). The best activity was seen with nanoparticles encapsulating GaMP (eFGaMP), which exhibited 2.7- and 4.4-fold higher reduction in P. aeruginosa than GaMP and the positive control, respectively.
Prolonged intracellular antimicrobial activity of Ga(III) within macrophages against P. aeruginosa. THP-1 macrophages were treated with Ga(III) compounds (66 μg/ml) for 24 h (day 0). After washing, the pretreated macrophages were incubated for designated days until infection with P. aeruginosa PA103 at an MOI of 20 for 3 h. Multiple-comparison testing was performed using ANOVA, and Student’s t test was used to compare two groups (GraphPad Prism 6.0). ****, P < 0.0001; ***, P < 0.001, **, P < 0.01; *, P < 0.05 compared to positive control. ns, not significant. Data represent the mean ± standard deviation of triplicates.
In contrast to the results with P. aeruginosa, none of the gallium porphyrins and nanoparticles (66 μg/ml) showed any inhibitory activity against A. baumannii within macrophages at day 0 or day 3 (Fig. S7). Pretreatment of macrophages with concentrations up to 198 μg/ml of the Ga(III) inhibitors failed to reduce viable A. baumannii cells within macrophages, which is consistent with the result described above with posttreatment of A. baumannii-infected macrophages.
Activities of GaPP and nanoparticles against biofilms of A. baumannii and P. aeruginosa.Biofilms are responsible for tolerance and resistance to antibiotics, thereby increasing the importance of developing novel antibiofilm therapies. To determine the effect of GaPP and its nanoparticles (FGaPP and CDGaPP) against biofilms of both bacterial species, biofilms grown on plasma-coated wells were treated with 5× the MIC (40 μg/ml) in iron-depleted medium (30). The tetracycline antibiotic minocycline was used as a positive control for antibiofilm activity for comparison with gallium compounds. At a concentration of 40 μg/ml, all GaPP and their nanoparticles exhibited significant reduction of viable bacterial biofilms, showing >400- and >45-fold decreases in A. baumannii and P. aeruginosa biofilms, respectively, compared to the positive control (Fig. 7).
(A and B) Antibiofilm activity of GaPP and its nanoparticles against A. baumannii ATCC 19606 (A) and P. aeruginosa PA103 (B). Biofilms were grown on plasma-coated wells and treated with inhibitor for 24 h in BM2 medium. Data represent the mean ± standard deviation of triplicates. (+), bacterial growth in the absence of inhibitors. Multiple-comparison testing was performed using ANOVA, and Student’s t test was used to compare two groups (GraphPad Prism 6.0).
Long-acting antibiofilm activities of GaMP and its nanoparticle eFGaMP against A. baumannii and P. aeruginosa.To determine whether GaMP and eFGaMP exhibited prolonged antibiofilm activity against both pathogens, the biofilms formed in human plasma were treated with GaMP or eFGaMP at a concentration of 150 μg/ml, the required concentration to effectively disrupt biofilm, for 24 h, following which the external drug was removed and the remaining undisrupted biofilm was further incubated. At day 3 following Ga(III) loading, GaMP and its nanoparticles showed more than 1,200 times enhanced biofilm disruption of both pathogens compared to the positive control. It was also observed that nanoparticle-encapsulated GaMP revealed 3.5-fold higher antibiofilm activity than did free GaMP, suggesting prolonged release of GaMP from the nanoparticles. However, there was no significant difference in disruption of biofilm growth of either pathogen between GaMP and eFGaMP at day 6 postloading (Fig. 8).
(A and B) Prolonged antibiofilm activities of GaMP and its nanoparticles against A. baumannii ATCC 19606 (A) and P. aeruginosa PA103 (B). Biofilms were grown on plasma-coated wells and treated with Ga(III) inhibitor for 24 h in BM2 medium. After washing free of external drugs, the biofilms were further incubated for 3 and 6 days. Data represent the mean ± standard deviation of triplicates. (+), bacterial growth in the absence of gallium inhibitors. Multiple-comparison testing was performed using ANOVA, and Student’s t test was used to compare two groups (GraphPad Prism 6.0). ****, P < 0.0001 compared to (+).
Antimicrobial activity of Ga(III) nanoparticles against infected Caenorhabditis elegans.C. elegans wild-type strain N2 worms were infected with P. aeruginosa or A. baumannii at an optical density at 600 nm (OD600) of 0.1 in slow-killing (SK) medium. The antimicrobial activity of the Ga(III) nanoparticle (eFGaMP) was assessed by determining the survival rate. P. aeruginosa- or A. baumannii-infected worms that were untreated did not survive at 17 h of incubation. However, at 17 h, 38%, 82%, and 76% of P. aeruginosa-infected worms and 18%, 55%, and 100% of A. baumannii-infected worms remained alive in the presence of 50, 100, and 200 μg/ml eFGaMP, respectively (Fig. 9).
Antimicrobial activity against infected C. elegans. Synchronized L4∼young adult C. elegans were infected with pathogens in slow-killing medium (SK) containing gallium nanoparticles (eFGaMP). A number of survived C. elegans were counted at 17 h of incubation at 23°C. Student’s t test was used to compare two groups.**, P < 0.01; *, P < 0.05 compared to positive control.
DISCUSSION
Bacteria develop resistance to antibiotics by destroying, modifying, or removing antibiotics or altering their targets. Recently, the World Health Organization (WHO) listed P. aeruginosa and A. baumannii as “priority pathogens” that are a threat to human health, emphasizing the impact of Gram-negative bacteria in developing multidrug resistance (MDR) to antibiotics and urging the development of new antimicrobial drugs (31). In search of a new strategy to fight these emerging MDR bacteria, disruption of iron/heme acquisition and utilization by gallium-based compounds have been explored by several groups (19). Ga (III)-based compounds, in the form of salts or complexed with other compounds, have shown broad-spectrum antimicrobial activity against mycobacteria and Gram-negative and Gram-positive bacteria (18, 32). Ga nitrate, Ga citrate, and Ga maltolate have shown significant inhibition of growth and biofilm formation against many bacteria in vitro (33–35).
Because of the abundance of heme as an iron source in vivo, the ability of gallium nitrate to exert sustained growth inhibition may be more limited against organisms able to acquire iron from heme-containing compounds. In the present study, we found that growth of A. baumannii had a longer lag phase and enhanced exponential phase than did P. aeruginosa in the presence of hemin. Our results suggest that iron may play a role in the transition from lag phase into exponential phase for A. baumannii.
In contrast to results with A. baumannii, with P. aeruginosa, a longer lag phase was not observed in the presence of excess hemin (50 μg/ml), nor did hemin promote bacterial growth. However, in a recent report, P. aeruginosa growth was promoted in the presence of hemin at concentrations up to 16 μg/ml (20), but higher concentrations may be toxic. These data suggest that P. aeruginosa has more active heme uptake machinery for iron accumulation during lag phase. Paradoxically, the efficient heme uptake during lag phase could lead to heme accumulation and increase heme toxicity.
The growth curves in the presence of hemin and Ga(NO3)3 suggest that growth rescue by hemin is strain and Ga(NO3)3 concentration dependent. As shown in Fig. 2D and G, the addition of hemin (50 μg/ml) restored 30% of the A. baumannii growth at 24 h inhibited by 32 μg/ml Ga(NO3)3, indicating that A. baumannii can efficiently obtain iron by degrading internalized heme in the presence of Ga(NO3)3. In contrast, hemin did not show the ability to significantly reverse Ga(NO3)3 inhibition of P. aeruginosa growth compared to rescue of A. baumannii growth (Fig. 3D and G). Although hemin rescued 40% and 30% of the growth inhibition of P. aeruginosa at 2 and 4 μg/ml Ga(NO3)3, respectively, no rescue was observed in P. aeruginosa growth inhibited by Ga(NO3)3 at ≥8 μg/ml. This suggests that P. aeruginosa has a limited ability to obtain iron from heme compared to A. baumannii in the presence of Ga(NO3)3.
Rescue of Ga(NO3)3 inhibition of both pathogens was also observed with the addition of ferric ammonium citrate (FAC) (Fig. 2A and 3A), in which the recovery is strain dependent. A. baumannii growth was fully restored at Ga(NO3)3 concentrations ranging from 1 to 32 μg/ml by the addition of FAC (100 μg/ml). In contrast, P. aeruginosa showed Ga(NO3)3 concentration-dependent rescue, with 44% recovery by FAC at 32 μg/ml Ga(NO3)3. Altogether, A. baumannii may have more active iron/heme acquisition systems to obtain extracellular iron sources under iron-depleted conditions when in the presence of Ga(NO3)3.
The recovery of bacterial growth with FAC is lower than with hemin when A. baumannii growth was inhibited by Ga porphyrins (Fig. 2). FAC (100 μg/ml) lost its rescue effect at 8 and 4 μg/ml GaPP and GaMP, respectively, whereas hemin (50 μg/ml) rescued A. baumannii growth at 32 μg/ml GaMP and GaPP. This result suggests that gallium porphyrins may target hemoproteins involved in heme uptake pathway other than iron-binding proteins, and gallium was also not released from porphyrins by heme oxygenase, as has been suggested for iron (36, 37). Thus, targeting heme-dependent biological processes is promising for the development of antimicrobial agents for A. baumannii. In contrast, hemin and FAC demonstrated similar recovery efficacy against P. aeruginosa growth inhibited by GaPP and GaMP (Fig. 3).
Upon invasion of pathogens, the human immune system plays important roles in bacterial clearance. Monocytes and macrophages are among the first immune cells to respond to invading pathogens by phagocytizing them and recruiting other immune cells to sites of infection. If they survive, pathogens phagocytized by macrophages face more challenges for obtaining iron than do extracellular organisms. Thus, they might be more sensitive to gallium if adequate intracellular levels are achieved. Multifunctional nanoparticles that target monocytes and macrophages with high selectivity, targeted-drug delivery, and clinical use have shown efficacy in inhibiting intracellular pathogens (38). Thus, F127 polymer and β-cyclodextrin nanoparticles encapsulating Ga(III) porphyrins were formulated, and their in vitro efficacies were assessed against both Gram-negative pathogens present inside/outside THP-1 macrophages.
The populations of external and internal A. baumannii bacteria in cocultured THP-1 cells were considerably higher than those observed with P. aeruginosa after 3 day of incubation with 3.3 μg/ml gallium inhibitors (Fig. 4, S3, and S4). It should be noted that there were no extracellular bacteria initially present in the cultures; following initial incubation with the bacteria, the remaining extracellular bacteria were washed extensively to avoid bacterial killing of THP-1 cells. Remarkably, regardless of the Ga(III) compounds used, intracellular and extracellular P. aeruginosa growth was substantially reduced at a Ga(III) concentration of 3.3 μg/ml, killing more than 99.9% compared to the positive control. In contrast, a concentration of 66 μg/ml Ga(III) compounds was required to exhibit significant inhibition of A. baumannii in RPMI 1640 medium and THP-1 cells (Fig. 4). This difference was also observed from the experiment set where the bacterial load was increased (MOI, 20) to infect THP-1 cells and higher Ga(III) concentrations were loaded to the infected cells to investigate the intracellular antimicrobial activity (Fig. 5 and S5). These results suggest that intracellular A. baumannii is more resistant and can escape from the THP-1 cell better than can P. aeruginosa. Although GaPP and GaMP have similar MIC values against both pathogen in iron-depleted broth (Table 1), their intra- and extracellular activities were not dependent on the respective MICs. However, our results indicate that all gallium compounds can penetrate THP-1 cells and inhibit bacterial growth, and nanoparticles are loaded into THP-1 cells.
Our previous works demonstrated that macrophage-targeted Ga(III) nanoparticles have a prolonged antimicrobial effect against Mycobacterium abscessus, Mycobacterium avium, Mycobacterium tuberculosis, and Mycobacterium smegmatis within macrophages because of sustained Ga(III) release over time (27–29, 39). In the current work, we found that THP-1 cells preloaded with GaMP, CDGaPP, or eFGaMP exhibited resistance to P. aeruginosa infection, but only for 3 days, losing all activity at day 5 postloading. GaPP and FGaPP lost activity even at day 3. Promisingly, gallium nanoparticles (eFGaMP) showed 5 and 3 times better antimicrobial activity than did the positive control and the respective GaMP activity at day 3 postloading against P. aeruginosa, confirming the sustained release of GaMP from these nanoparticles for up to 3 days (Fig. 6). To the contrary, at concentrations ranging from 66 to 198 μg/ml, all of the gallium compounds preloaded into macrophages, except eFGaMP, failed to show any prolonged antimicrobial activity against A. baumannii (Fig. S7). A significant inhibitory effect was only observed at 198 μg/ml eFGaMP at day 0. Our results suggest that P. aeruginosa bacteria residing in macrophages are more susceptible to Ga(III) porphyrins than are A. baumannii bacteria, possibly indicating a better ability of A. baumannii in macrophages to access intracellular iron even in the presence of Ga(III) porphyrins.
In the present study, we found that intracellular and extracellular activities of gallium porphyrins in RPMI 1640 medium supplemented with 10% FBS are different from those observed, as determined by MICs and growth curve studies in the presence of FAC and hemin under iron-depleted conditions. GaMP and GaPP demonstrated potential antimicrobial activity in the presence of FAC, while they failed to show relevant activity against A. baumannii in cocultured macrophages in RPMI 1640 medium supplemented with 10% FBS. This low susceptibility of A. baumannii in RPMI 1640 medium supplemented with 10% FBS may be due to albumin that binds gallium porphyrins. Ga(III) and indium(III) phthalocyanines strongly bind to bovine serum albumin (BSA) (40). Hemin also interacts with BSA, resulting in the formation of a stacking noncovalent complex (41). Human albumin is known to transport several ligands, including hemin, fatty acids, and bilirubin, by forming stable complexes (42). Taken together, our data imply that gallium porphyrin binds and forms a complex with albumin present in FBS. P. aeruginosa, but not A. baumannii, may have the ability to extract heme or Ga porphyrin from the albumin complex. In support of this possibility, higher GaPP and GaMP MICs (>128 μg/ml) were observed against A. baumannii in RPMI 1640 medium containing 10% FBS compared to those in iron-depleted medium, while P. aeruginosa showed MICs of 1 μg/ml for both GaMP and GaPP.
Biofilms have substantial influences on the pathogenesis of human infections by reducing antimicrobial susceptibility and enhancing virulence (43, 44). Recently, the Ga(III) porphyrins GaPP and GaMP have been described as potential antibiofilm therapies against P. aeruginosa and A. baumannii (21, 24). Here, the effects of and Ga(III) porphyrins and nanoparticles containing them on growth inhibition of biofilms with these two pathogens was studied. Our results are consistent with those of these previous studies, showing significant antibiofilm activity of both GaPP and GaMP (Fig. 7 and 8). Nanoparticles encapsulating GaPP (FGaPP and CDGaPP) exhibited antimicrobial effects against both biofilms at a concentration of 40 μg/ml (5× the MIC) for both pathogens, while no significant difference was found between nanoparticles and free GaPP. Considering the slow release of GaMP from nanoparticles (eFGaMP), higher concentrations of GaMP and nanoparticles (150 μg/ml) were used to evaluate long-acting antibiofilm activity (Fig. 8). Despite substantial antibiofilm effects against both pathogens at day 3 posttreatment, both GaPP and eFGaMP lost activity at day 6 posttreatment. This may be due to reduced drug release from the nanoparticles. In addition, pathogenic biofilms are protected by an extracellular matrix, by which the penetration of the nanoparticles may have been decreased, resulting in a loss of antibiofilm activity of nanoparticles at day 6 (Fig. 8). Antibiofilm activity was observed with eFGaMP nanoparticles up to day 3, demonstrating the potential of Ga(III) porphyrins as antibiofilm agents.
eFGaMP was also able to penetrate C. elegans and protect C. elegans from lethal infection with P. aeruginosa or A. baumannii. At 17 h, 38%, 82%, and 76% of P. aeruginosa-infected worms receiving 50, 100, and 200 μg/ml eFGaMP, respectively, survived, as did 18%, 55%, and 100% of A. baumannii-infected worms, respectively (Fig. 9). In the absence of treatment, 100% of the worms infected with either bacterial pathogen died. Thus, eFGaMP was able to penetrate C. elegans and kill the respective bacteria to extend the survival time of C. elegans.
In conclusion, long-acting FGaPP and eFGaMP nanoparticles were synthesized, and their intracellular and extracellular antimicrobial activities were tested against P. aeruginosa and A. baumannii strains, including clinical isolates. All P. aeruginosa and A. baumannii strains were susceptible to GaPP, GaMP, and nanoparticles, with MICs ranging from 0.5 to ∼32 μg/ml in iron-depleted medium. Significant intracellular and extracellular growth inhibition was observed against both P. aeruginosa (3.3 μg/ml) and A. baumannii (66 μg/ml) in infected macrophages with all Ga(III) inhibitors, including nanoparticles. Prolonged activity of nanoparticles was observed in previously infected (P. aeruginosa and A. baumannii) macrophages. Similarly, nanoparticle-treated macrophages showed prolonged activity, where they were infected at day 3 posttreatment, resulting in about 5- and 1-fold higher P. aeruginosa growth inhibition with eFGaMP and CDGaPP, respectively, than with the positive control. In addition, all gallium complexes showed antibiofilm activity, and nanoparticles showed prolonged antibiofilm activity up to 3 days against both pathogens. Gallium nanoparticles are also able to reduce bacterial infection in C. elegans and extend its survival time compared to the positive control. Our results also suggest that P. aeruginosa residing in macrophages is more susceptible to Ga(III) porphyrins than is A. baumannii. Thus, interfering and/or limiting bacterial access to host iron sources by targeting bacterial heme uptake pathways with Ga(III) metalloporphyrin derivatives is a potential antimicrobial and antibiofilm strategy for the future. In addition, more selective and macrophage-targeted gallium nanoparticles could increase the therapeutic index of such an approach.
MATERIALS AND METHODS
Materials, reagents, and strains.Human monocytic THP-1 cells, P. aeruginosa PA103 (ATCC 29260), and A. baumannii (ATCC 19606), were purchased from the American Type Culture Collection (ATCC) and used as the reference strains. Clinical isolates of P. aeruginosa and A. baumannii were obtained from the Clinical Pathology/Microbiology Laboratory at Nebraska Medicine, Omaha, NE. Gallium(III) protoporphyrin IX chloride (GaPP) and gallium(III) mesoporphyrin chloride (GaMP) were purchased from Frontier Scientific (Logan, UT). β-Cyclodextrin and gallium(III) phthalocyanine chloride (GaPC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). RPMI 1640 medium was purchased from Invitrogen (Hyclone). Difco tryptic soy agar (TSA) and broth (TSB) were purchased from BD (Sparks, MD). Gallium nitrate was obtained from Acros Organics (NJ, USA).
Cell culture.THP-1 cells were differentiated into THP-1 macrophages in the presence of phorbol 12-myristate 13-acetate (PMA; 100 nM) in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 50 μg/ml gentamicin at 37°C for 24 h in a humidified 5% CO2 incubator. After differentiation, all THP-1 macrophage experiments were performed in RPMI 1640 medium supplemented with 10% FBS.
Preparation of nanoparticles.F127 block copolymer (Aldrich) was used to prepare nanoparticles encapsulating gallium protoporphyrin (FGaPP), and β-cyclodextrin was used to prepare CDGaPP nanoparticles using a homogenization (sonication) method. GaPP (20 mg, 1% [wt/vol]) and polymer (2 mg, 0.1% [wt/vol]) were mixed and stirred in 2 ml of 10 mM HEPES for 24 h at room temperature. The mixture was sonicated (20% amplitude, model 500 Sonic Dismembrator; Fisher Scientific) on ice 5 times for 30 s, with a 1-min pause on ice between cycles. The nanoparticles were then stored at 4°C until needed. Gallium nanoparticles encapsulating gallium mesoporphyrin (eFGaMP) were prepared from F127 block copolymer using a single-emulsion technique. Ten milligrams of GaMP was dissolved in 1 ml of CH2Cl2 and added dropwise to 10 ml of 1% poly(vinyl alcohol) (PVA) with stirring. The mixture was sonicated over ice for 30 s 5 times with a 1-min rest on ice in between cycles. It was then stirred overnight at room temperature, rotavapored to remove CH2Cl2, and stored at 4°C until it was used. The nanoparticles were characterized by dynamic light scattering (Zetasizer Nano Series Nano-ZS system; Malvern Instruments, Inc., Westborough, MA USA). A standard curve was used to determine the concentration of Ga porphyrin entrapped in nanoparticles by measuring the absorbance at 400 nm in methanol (Agilent UV-Vis spectrophotometer).
MIC.A 2-fold serial microdilution method was used to determine the MIC in BM2 medium (45). Iron-depleted BM2 medium was prepared from potassium phosphate dibasic (6.97 g/liter), potassium phosphate monobasic (2.99 g/liter), ammonium sulfate (0.92 g/liter), magnesium sulfate·7H2O (0.24 g/liter), succinic acid (4.02 g/liter), and Casamino Acids (1g/liter), which was adjusted to pH 7.0 by adding 10 N NaOH. P. aeruginosa and A. baumannii were cultured in BM2 until they reached an OD625 of 0.4 (6.3 × 108 and 8.5 × 108 CFU/ml, respectively). Each well of the 96-well plate was inoculated with 5 × 105 CFU/ml and incubated in BM2 medium at 37°C for 24 h. The OD600 was measured to determine the lowest concentration of the compound that inhibited bacterial growth, using a BioTek Synergy H1 hybrid reader.
Cell viability assay.Cytotoxicity of gallium compounds was expressed as the percentage of the treated cells that remained viable relative to the negative control (nontreated cells). Cytotoxicity was measured using a resazurin reduction assay, as described previously (46). Differentiated THP-1 macrophages (3 × 105 cells/well in a tissue culture 48-well plate) were exposed to Ga(NO3)3, GaPP, GaMP, CDGaPP, FGaPP, and eFGaMP (33, 66, and 198 μg/ml) for 24 h (day 0) in RPMI 1640 medium supplemented with 10% FBS. After washing the cells with phosphate-buffered saline (PBS) three times, the treated cells were incubated for defined days, followed by determination of cell viability. The cytotoxicity was determined by measuring the fluorescence intensity of resorufin (the reduced form of resazurin) at excitation/emission (Ex/Em) of 560/590 nm (BioTek Synergy H1 hybrid reader).
Internal and external bacterial killing assay in cocultured THP-1 macrophages.Differentiated THP-1 macrophages (0.75 × 106 cells/well in a tissue culture 24-well plate) were incubated with P. aeruginosa PA103 or A. baumannii ATCC 19606 at an MOI of 1 or 20 for 1 h in RPMI 1640 medium supplemented with 10% FBS. The infected THP-1 cells were washed with PBS and incubated in 50 μg/ml gentamicin for 1 h to remove extracellular bacteria. After washing with PBS, the infected cells were treated with gallium porphyrins or gallium nanoparticles for 3 days before lysis to assess internal and external P. aeruginosa or A. baumannii content. After collecting medium to perform a colony counting assay (CFU), cells were washed with PBS several times and lysed with 0.05% SDS, and a CFU assay was performed to determine the number of internalized bacteria. Internal bacteria were normalized by dividing the number of CFU by the amount of whole protein of THP-1 cells. The whole-protein concentration was determined using the bicinchoninic acid (BCA) assay (47, 48).
For drug preloading experiments, THP-1 macrophages (0.75 × 106 cells/well) were treated with Ga(III) porphyrins or their respective nanoparticles for 24 h, followed by washing with PBS to remove external Ga(III) compounds. The preloaded macrophages were incubated with either pathogen at defined days following drug loading. Following incubation with the bacteria at an MOI of 1 for 3 h, the infected macrophages were incubated for an additional 1 h before lysis. CFU number and normalization were determined as described above.
Biofilm formation assay.The microtiter plate assay for biofilm formation was performed according to a reported method with modifications (49). Briefly, biofilms of P. aeruginosa PA103 and A. baumannii ATCC 19606 were formed in 96-well tissue culture plates, which were precoated with 20% human plasma (Sigma) in 0.05 M carbonate buffer (pH 9.4) at 4°C for 24 h. Overnight, the culture grown in LB medium supplemented with 3% NaCl, 0.5% Casamino Acids, and 0.5% glucose was diluted to an OD600 of 0.05 and used to form biofilms on the precoated wells. The plate was incubated at 37° C for 24 h and washed with PBS buffer, following which BM2 medium containing a gallium porphyrin or nanoparticles was added to each well in triplicate. After 24 h of incubation at 37°C, the biofilm was scraped into PBS buffer (100 μl), serially diluted, and plated on TSA plates to enumerate the CFU.
Caenorhabditis elegans killing assay.Wild-type N2 C. elegans was purchased from the Caenorhabditis Genetics Center at the University of Minnesota and cultured as described previously (50, 51). Synchronized L4 stage to young-adult worms grown on nematode growth medium (NGM) agar plates with Escherichia coli OP50 were washed with sterilized M9 buffer and resuspended in slow-killing (SK) medium. Twenty to ∼30 C. elegans worms were transferred to 96-well plates containing Ga(III) compounds and infected with P. aeruginosa PA103 (OD600, 0.1) or A. baumannii ATCC 19606 (OD600, 0.1) for 17 to ∼24 h at 25°C. The worms were considered to be dead if they did not move, even after gentle shaking of the plate.
Statistical analysis.All results are presented as the mean ± standard deviation of the mean. For multiple-comparison testing, a one- or two-way analysis of variance (ANOVA) (Tukey test, with GraphPad Prism 6.0) was performed to determine significant differences between means for bacteria residing in cells or for planktonic bacteria. Student’s t test was performed to compare two groups. Statistical significance was evaluated at P values of <0.05, 0.01, 0.001, and 0.0001.
ACKNOWLEDGMENTS
This work was supported in part by a VA Merit Review award to B.E.B.
We declare no competing financial interests.
FOOTNOTES
- Received 18 December 2018.
- Returned for modification 21 January 2019.
- Accepted 6 February 2019.
- Accepted manuscript posted online 19 February 2019.
Supplemental material for this article may be found at https://doi.org/10.1128/AAC.02643-18.
- Copyright © 2019 American Society for Microbiology.
