Additive Effects of N-Acetylcysteine and [R₄W₄] Combination Treatment on Mycobacterium avium

Abstract

Mycobacterium avium is an opportunistic pathogen and a leading contributor to nontuberculous mycobacterial infections in immunocompromised individuals. However, treatment duration, antibiotic toxicity, and resistance present challenges in the management of mycobacterium infections, prompting the need for novel treatment. N-acetylcysteine (NAC) has demonstrated potent antimycobacterial activity, while antimicrobial peptides such as the cyclic [R₄W₄] have shown additive effects when combined with first-line antibiotics. This study aimed to investigate the mechanism and efficacy of NAC and [R₄W₄] combination therapy against M. avium. A membrane depolarization assay was used to evaluate the effects of NAC and [R₄W₄] on M. avium cell membrane integrity. Antimycobacterial activity was assessed by treating cultures with varying concentrations of NAC, [R₄W₄], a combination, or a sham treatment. The same regimens were applied to M. avium-infected THP-1-derived macrophages to assess intracellular efficacy. NAC and [R₄W₄] each disrupted the M. avium membrane potential, with enhanced effects in combination. The combination treatment significantly reduced M. avium survival in both the culture and infected macrophages compared with NAC alone and untreated controls. [R₄W₄] and NAC also demonstrated potent antibacterial activity, while the lowest MIC and the combination of [R₄W₄] and NAC displayed additive effects, indicating an improved bacterial inhibition compared to individual treatments. These findings demonstrate the additive activity of NAC and [R₄W₄] against M. avium in vitro and suggest that combining antioxidant compounds with antimicrobial peptides may represent a promising strategy for treating mycobacterial infections.

1. Introduction

Mycobacterium avium complex (MAC) is the most common group of nontuberculous mycobacteria (NTM) [1]. There has been a rapid increase in MAC infections associated with the increasing prevalence of immunocompromised patients globally [2]. Of the growing list of identified MAC species, the three most important human pathogenic species are M. avium, M. intracellulare, and M. chimaera. Additionally, M. avium is the most clinically significant human pathogenic species within MAC, representing approximately 80% of pulmonary NTM diseases [3,4]. These acid-fast slow-growing mycobacteria are found throughout the environment in soil, water sources, milk and food products, and animal reservoirs [5,6]. MAC are especially suited to endure in a range of environments due to their oligotrophicity, lending to their ability to survive in low carbon environments, as well as their biofilm production, which provides a scaffolding for surviving in water supplies [2].

Infection may be transmitted to humans through inhalation, ingestion, dermal contact, or medical devices [4]. Host immunity against M. avium involves both innate and adaptive immune responses. Like Mycobacterium tuberculosis (M. tb), M. avium is an intracellular pathogen, able to survive within macrophages by evading host machinery using defense mechanisms like inhibiting phagosome–lysosome fusion and inhibiting enzymes crucial for effective oxidative burst [4,7]. In a healthy individual, when activated macrophages housing M. avium are activated and shuttled to lymph nodes, the activated macrophages produce IL-12, which activates natural killer (NK) cells and T lymphocytes. NK cells release cytokines TNF-α, IFN-γ, and GM-CSF to upregulate macrophages. Th1 lymphocytes recognize antigenic presentation on major histocompatibility complex class II (MHC II), prompting the release of IFN-γ and IL-2 to further upregulate macrophage response. Ultimately, the macrophages isolate the pathogen within a granuloma, where they are subdued but able to continue to survive by evading the host immune response [2,7]. Immunocompromised patients who are unable to mount an effective immune response fail to upregulate macrophages enough to control infection. At-risk patients include those with severe acquired immunodeficiency syndrome (AIDS), severe combined immunodeficiency disease (SCID), and blood cell dyscrasias like leukemias and lymphomas, diabetics, and individuals receiving inflammatory modulators like corticosteroids, TNF-α inhibitors, and immunosuppressants [8,9,10,11,12].

M. avium causes three main infections: pulmonary MAC (MAC-PD), disseminated MAC (D-MAC), and MAC-associated lymphadenitis (MAC-L) [13]. MAC-PD presents as either fibrous–cavernous, which is associated with pre-existing lung disease such as chronic obstructive pulmonary disease (COPD), or nodular form, which has a predilection for non-smoking post-menopausal women [2]. D-MAC presents in severely immunocompromised patients and can be found in the lungs, liver, bowel, spleen, bone marrow, brain, blood, adrenal glands, urinary tract, and lymph nodes [14]. Buchacz et al. found that half of patients with severe acquired immunodeficiency syndrome (AIDS) had disseminated MAC infection prior to antiviral therapy [15]. MAC-L often presents as cervical lymphadenitis in immunocompromised children under the age of 5 [16,17].

The current clinical practice guidelines recommended by the American Thoracic Society (ATS), European Respiratory Society (ERS), European Society of Clinical Microbiology and Infectious Diseases (ESCMID), and Infectious Disease Society of America (IDSA) suggest a once daily regimen of azithromycin, rifampicin or rifabutin, and ethambutol for fibrocavitary or severe nodular MAC-PD and thrice-weekly for non-severe nodular MAC-PD [18]. For severe fibrocavitary cases, the addition of an aminoglycoside injection has been added to the daily regimen [18]. This treatment course has a history of low success rates (61.4%), difficult long-term maintenance, drug–drug reactions, and has yielded macrolide resistance which poses a population health risk as macrolide-resistant MAC-PD has poor treatment success [18,19]. There is a current need to develop novel therapies and adjunctive therapies to improve the treatment success rate, reduce treatment duration, and limit antimicrobial resistance in the treatment of M. avium.

Antimicrobial peptides (AMPs) are emerging as novel treatment options for multidrug-resistant bacteria. [R₄W₄] is an antimicrobial amphipathic cyclic peptide containing four arginine (R) and four tryptophan (W) residues. As an amphiphilic peptide, it can adhere to the cell wall due to an electrostatic attraction with negatively charged bacterial members through ionic bonding, and subsequently perturbs the cell wall through a hydrophobic interaction with cell wall lipids [20]. Previous studies have demonstrated the efficacy of cyclic [R₄W₄] as an adjunctive antimicrobial therapy against various human pathogens including methicillin-resistant Staphylococcus aureus, Escherichia coli, and Klebsiella pneumonia, Pseudomonas aeruginosa [21,22,23,24]. [R₄W₄] has also been demonstrated to be efficacious against acid-fast bacterium such as M. tuberculosis and M. avium, though its mechanistic effects have yet to be elucidated [23,24].

Studies have demonstrated that N-acetylcysteine (NAC) functions as a cysteine prodrug and precursor for glutathione synthesis [25]. NAC has been used to treat cysteine/GSH deficiencies in various conditions, including AIDS, cystic fibrosis, COPD, diabetes, and more [26]. We have previously demonstrated that NAC induces the acidification of phagosomes and reduces the burden of M. tb in granulomas [27]. Given the ability of NAC to reduce M. tb survivability by mediating host evasion mechanisms, we hypothesize that a combination of NAC and cyclic [R₄W₄] may offer a potent novel treatment method against M. avium. In this study, we aim to evaluate the direct mechanistic and antimycobacterial effects that NAC and [R₄W₄] may possess against M. avium. We also aim to determine whether [R₄W₄] has additive effects when added alongside NAC.

2. Results

2.1. Membrane Depolarization Potential by [R₄W₄], NAC, and Their Combination

We first assessed if [R₄W₄] and NAC have direct effects on the M. avium cell membrane. The membrane depolarization potential for [R₄W₄] at concentrations of 2 µg/mL, 4 µg/mL, and 8 µg/mL was measured with membrane potential-sensitive fluorescence dye 3,3″-dipropylthiadicarbocyanine iodine (DisC₃(5)). Additionally, membrane depolarization was assessed for NAC 20 mM and a combination treatment (NAC 20 mM + [R₄W₄] 8 µg/mL) using 3 µg/mL of Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) as the positive control and 20 µg/mL of rifampicin (RIF) as the negative control. Untreated samples (buffer only) served as the baseline control.

DisC₃(5) is a fluorescent dye that, when incubated, binds to the bacterial cell membrane in a polarized state [28]. Upon treatment with membrane-disrupting agents, intracellular contents are released, leading to membrane depolarization and a loss of membrane integrity. This depolarization causes the dye to be expelled from the membrane, resulting in an increase in fluorescence, which can be quantitatively measured.

The [R₄W₄] treatment showed an increase in fluorescence, with a statistically significant increase with the addition of 8 µg/mL of [R₄W₄] (p = 0.0006) (Figure 1). NAC 20 mM also produced a significant increase in fluorescence (p < 0.0001) similar to the positive control CCCP (p < 0.0001). The combination treatment resulted in a significant increase in fluorescence compared to the untreated baseline (p < 0.0001), with over a 2-fold increase in fluorescence compared with [R₄W₄] 8 µg/mL (p < 0.0001) and 20 mM NAC (p < 0.0001) singular treatment (Figure 1). In contrast, RIF treatment led to minimal and statistically nonsignificant changes in fluorescence (Figure 1).

These findings suggest that both [R₄W₄] and NAC have cell membrane depolarizing and disrupting effects, with enhanced effects when used in combination.

2.2. Combined Antimicrobial Effects of the Combination of [R₄W₄] and NAC Against M. avium Compared to That of [R₄W₄] Alone

In this experiment, we measured the additive antimicrobial effects of the combination of [R₄W₄] and NAC at different concentrations compared to [R₄W₄] alone against M. avium at three post-treatment time points to evaluate whether the addition of NAC to [R₄W₄] can produce an additive effect. Figure 2 depicts the colony-forming unit (CFU) counts of M. avium measured at 3 h (Figure 2A), 4 days (Figure 2B), and 8 days (Figure 2C) after treatment with various concentrations of [R₄W₄], NAC, or their combinations. The statistical analysis demonstrated that, at 3 h post-treatment, with [R₄W₄] at 8 µg/mL, all the tested concentrations of NAC (5 mM, 10 mM, and 20 mM) and the combination of [R₄W₄] and NAC showed a significant reduction in M. avium colonies compared to the untreated control. This reduction reflected bacteriostatic activity, as colony counts remained above the initial inoculum. Furthermore, the combination treatments R+N (4,10), R+N (2,20), and R+N (8,20) demonstrated a significant reduction in M. avium colonies compared to respective [R₄W₄] alone (p < 0.05), which demonstrated a significantly enhanced efficacy over the individual treatments alone, suggesting an additive antimicrobial effect of [R₄W₄] and NAC (Figure 2A). However, [R₄W₄] at 4 µg/mL exhibited no significant reduction in M. avium colonies at 3 h post-treatment.

At day 4 post-treatment, all the treatment categories, except [R₄W₄] at 2 µg/mL, showed a significant reduction in M. avium colonies compared to the untreated control. However, as illustrated in Figure 2B, [R₄W₄] exerted a dose-dependent antibacterial efficacy that was not observed in NAC. The combination treatments R+N (8,20) and R+N (2,20) produced robust reductions in CFUs compared to either [R₄W₄] or NAC alone; however, they could not reach the statistically significant level (Figure 2B), further supporting the additive effect of [R₄W₄] and NAC. The combination R+N (8,5) did not produce any further increase in additive effect, which is similar to both R (8) and N (5).

On the other hand, at day 8 post-treatment, all the concentrations of [R₄W₄] alone, NAC alone, and the combinations exhibited a significant reduction in M. avium colonies. As represented in Figure 2C, both [R₄W₄] and NAC exhibited a dose-dependent antimicrobial activity. Interestingly, NAC at 10 mM and 20 mM and the combinations, particularly R+N (4,10), R+N (8,20), and R+N (2,20), resulted in CFU counts approaching the zero colonies of M. avium (Figure 2C), showing a sustained and enhanced antibacterial activity for higher concentrations of NAC and the combination of [R₄W₄] and NAC with three treatments. Moreover, the combination treatment R+N (2,20) showed a significant reduction in M. avium colonies compared to the respective [R₄W₄] (Figure 2C). Overall, the findings suggest that the combination of [R₄W₄] and NAC exhibits a more potent antimicrobial effect against M. avium than either [R₄W₄] or NAC alone, supporting an additive effect.

2.3. Additive Antimicrobial Effect of the [R₄W₄] and NAC Combination Against M. avium Within THP-1 Macrophages Compared to Either [R₄W₄] or NAC Alone

We evaluated the additive antimicrobial activity of [R₄W₄] in combination with NAC to determine whether the addition of NAC enhances the reduction in M. avium within THP-1 macrophages compared to a treatment with either [R₄W₄] or NAC alone. Figure 3 shows the CFU counts of M. avium within THP-1 macrophages at 3 h (A), 4 days (B), and 8 days (C) following treatment with different concentrations of [R₄W₄], NAC, or their combinations.

At the 3 h time point, NAC 10 mM (p < 0.05) and 20 mM (p < 0.0001) produced a significant reduction in intracellular M. avium relative to the untreated controls (Figure 3A). Furthermore, the combination treatment R+N (4,10) (p < 0.05), R+N (8,20) (p < 0.0001), and R+N (2,20) (p < 0.0001) significantly diminished CFU counts compared to the untreated control. Moreover, R+N (8,20) and R+N (2,20) demonstrated a significant reduction in CFU count compared to the respective [R₄W₄] alone (p < 0.0001), which indicates an additive antimicrobial effect of [R₄W₄] and NAC. In contrast, [R₄W₄] could not decrease CFUs at 3 h when administered alone (Figure 3A).

By day 4, all treatment groups except for [R₄W₄] at 2 µg/mL and 8 µg/mL showed a significant reduction in CFUs compared with untreated M. avium in macrophages (Figure 3B). As shown in Figure 3B, NAC treatment alone exhibited a dose-dependent decrease in CFU counts. Moreover, all the combinational treatments exhibited a significant reduction in M. avium colonies compared to the respective [R₄W₄] alone (p < 0.001), which suggests the continued additive antimicrobial effect of [R₄W₄] and NAC against M. avium. However, only the combinational treatment R+N (2,5) significantly decreased CFUs compared to the respective NAC alone (p < 0.01).

At day 8, every concentration of [R₄W₄] alone, NAC alone, and the combinations produced a significant reduction in CFUs compared to the untreated control (Figure 3B). At this time point, three treatments of [R₄W₄] alone demonstrated a dose-dependent reduction in CFU counts that was missing at the 3 h and 4 days post-treatment time points. Similarly, NAC alone continued to show a dose-dependent decrease in M. avium colonies. Furthermore, all the combinational treatments except R+N (8,5) produced a significant reduction in CFU counts compared to the respective [R₄W₄] alone (p < 0.01), indicating a continued additive antibacterial effect against M. avium. Notably, the combination treatments R+N (8,20) and R+N (2,20) almost cleared off all the bacteria from macrophages, which suggests a complete recovery from M. avium infection. Moreover, R+N (4,10) and R+N (8,5) significantly reduced the CFU counts relative to the respective NAC alone (p < 0.05).

Overall, the findings suggest that both [R₄W₄] and NAC exert dose-dependent intracellular antimicrobial activity against M. avium, particularly at the 8 days post-treatment time point, and that their combination produced a more significant bacterial reduction than either [R₄W₄] or NAC alone. Together, these results support an additive antibacterial effect of [R₄W₄] and NAC that significantly reduced the burden of M. avium colonies.

2.4. Minimum Inhibitory Concentration (MIC) of [R₄W₄] and NAC Against M. avium

The antibacterial activities of [R₄W₄] and NAC were assessed individually to determine their minimum inhibitory concentrations (MICs). As shown in

Table 1. Presentation of mean percentage of inhibition and MICs of different concentrations of [R₄W₄] and NAC.

Treatment	Mean % Inhibition ± SD	MIC
R4W4 1	76.51 ± 33.12	6 µg/mL
R4W4 2	55.06 ± 18.92
R4W4 4	82.28 ± 45.33
R4W4 6	158.18 ± 11.45
R4W4 8	145.05 ± 13.14
NAC 2.5	89.97 ± 25.97	5 mM
NAC 5	136.68 ± 22.86
NAC 10	94.97 ± 7.95
NAC 15	140.44 ± 3.00
NAC 20	176.00 ± 6.16

, both compounds exhibited an inhibition of M. avium growth.

[R₄W₄] showed a gradual increase in inhibitory activity with increasing concentrations, reaching its maximum inhibition at 6 µg/mL, beyond which no significant additional inhibition was observed. This concentration was therefore considered the MIC of [R₄W₄].

Similarly, NAC displayed an increasing trend of inhibition across the tested concentrations from 2.5 to 20 mM, with a complete inhibition observed at 5 mM, which was identified as its MIC.

These results indicate that both [R₄W₄] and NAC possess notable antibacterial activity against M. avium when used individually, and the MIC for [R₄W₄] was 6 µg/mL and for NAC was 5 mM.

2.5. Checkerboard Synergy Analysis of [R₄W₄] and NAC

The checkerboard microdilution assay was performed to evaluate the interaction between [R₄W₄] and NAC against M. avium. The calculated fractional inhibitory concentration (FIC) indices are presented in

Table 2. FIC index of [R₄W₄] and NAC combination treatments determined by checkerboard assay.

Treatment	FIC Index
R+N (2,5)	1.33
R+N (2,20)	4.33
R+N (4,10)	2.67
R+N (8,20)	5.33
R+N (8,5)	2.33

.

As shown in Table 2, the combination R+N (2,5) exhibited an FIC index of 1.33, suggesting an additive or indifferent interaction. Similarly, combinations R+N (4,10) (FIC = 2.67) and R+N (8,5) (FIC = 2.33) also fell within the additive or indifferent range, indicating that the compounds acted independently at these concentrations.

In contrast, higher-concentration combinations such as R+N (2,20) (FIC = 4.33) and R+N (8,20) (FIC = 5.33) showed no additive or indifferent interactions; however, in our M. avium survival assays using CFUs, we observed an additive effect with these two combination treatments (Figure 2 and Figure 3).

Overall, the results indicate that [R₄W₄] and NAC did not exhibit a strong synergy against M. avium under the tested conditions. The additive or indifferent effects observed at combinations R+N (2,5), R+N (4,10), and R+N (8,5) suggest an additive antimycobacterial action.

3. Discussion

Antimicrobial peptides are emerging compounds of interest in the treatment of multidrug-resistant bacteria due to their fast killing, low toxicity, biodiversity, and low molecular weight [29]. [R₄W₄] is an amphipathic cyclic peptide shown to possess direct antimycobacterial effects [23,24]. [R₄W₄] has also been shown to provide additive effects in reducing M. avium survival when added alongside rifampicin and azithromycin [24]. Additionally, the addition of [R₄W₄], added in conjunction with isoniazid or pyrazinamide, reduced the bacterial burden in M. tb-infected peripheral blood mononuclear cells from healthy patients in vitro [23]. N-acetylcysteine (NAC) is another compound of interest in the treatment of mycobacterium infections. NAC has been shown to exhibit potent antimycobacterial activity against M. tb [30]. Furthermore, NAC provided synergistic effects to isoniazid and rifampicin for in vitro granuloma models against M. tb [27]. However, there has yet to be an exploration of the mechanistic effects of NAC and [R₄W₄] combination treatment during an M. avium infection.

Previous studies have demonstrated that the peptide [R₄W₄] functions as a cell wall disruptor in both Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), and Gram-negative species, including Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa [21,22,23,24]. In contrast, M. avium is an acid-fast bacterium with a complex cell wall architecture composed of peptidoglycan, arabinogalactan, and mycolic acids, which significantly differs from the structural composition of Gram-positive and Gram-negative bacterial cell walls [31]. Mycolic acids have been implicated in limiting antibiotic permeability, thereby contributing to intrinsic resistance mechanisms [32]. Notably, no prior studies have evaluated whether [R₄W₄] exerts cell wall-disruptive effects on mycobacterial species. To investigate this, we conducted a membrane depolarization assay to determine whether [R₄W₄] induces a disruption of the cell wall and membrane integrity in M. avium. This assay uses a fluorescent probe, DisC₃(5), that quenches and binds onto the cell membrane when the bacterial cell is polarized [28]. Cell membrane-disrupting agents alter the membrane potential leading to the depolarization and decline of membrane integrity, allowing the dye to release from the cell membrane in which the fluorescent intensity can be measured [33]. The addition of lower concentrations of [R₄W₄] in a M. avium-DisC₃(5) suspension resulted in an increase in fluorescence, with a significant increase in fluorescence with 8 µg/mL [R₄W₄] (Figure 1). Consistent with previous reports on MRSA and Klebsiella, [R₄W₄] demonstrates similar cell membrane-disrupting effects on the mycobacterial cell membrane [21,22].

We then also assessed if NAC would have any effect on the cell membrane. While several studies demonstrated that NAC has direct effects on mycobacterial growth and can augment host immunity against mycobacteria, limited data exists on the direct mechanistic antimycobacterial effects of NAC [27,30]. We observed a significant increase in fluorescence with the addition of NAC 20 mM similar to our positive control. This increase was compounded when adding NAC and R₄W₄ in combination. Even though CCCP served as positive control, it induced a lower fluorescence than the combination treatment. CCCP primarily works by collapsing membrane potential rather than being a maximal depolarizer. In contrast, NAC alongside R₄W₄ has an increased membrane depolarization, which disrupts membrane integrity, increasing membrane permeability. As such, it leads to more fluorescence dye release, increasing the recorded value much higher than that of CCCP. This finding is the first to suggest that R₄W₄ and NAC have cell wall- and membrane-disrupting effects on M. avium. Further studies are needed to corroborate these findings.

The direct effects of NAC and [R₄W₄] on M. avium survival in culture were assessed along with a combination treatment using a time kill assay (Figure 2A, Appendix A). In this assay, single and combination treatments were provided for each culture. The first set of cultures received treatment and terminated 3 h post-inoculation (3 h). A second set of cultures was treated immediately and treated again 3 days post-inoculation, before terminating 4 days post-inoculation (4 days). A third set of cultures was treated immediately, 4 days and 6 days post-inoculation, and terminated 8 days post-infection. This assay allows us to determine the kinetics of [R₄W₄] and NAC treatment and the effects of dosing frequency. The immediate effects of treatment are assessed 3 h post-infection, the effects of treatment on sustained infection are assessed 4 days post-infection, and the effects of chronic infection are assessed at 8 days post-infection. Furthermore, we observed the effects of a single dose (3 h), double dose (4 days), and triple dose (8 days).

Untreated controls treated with a carrier vehicle demonstrated a 100-fold increase after 8 days post-infection. All treatments demonstrated inhibitory effects compared to the untreated control. When evaluating the effect of [R₄W₄], a small but observable dose-dependent response was observed with 8 µg/mL. Multiple doses of [R₄W₄] at 8 µg/mL appeared to induce a bacteriostatic effect, as CFU numbers were maintained at similar levels at the beginning of the experiment. Conversely, NAC treatment appeared to induce a bactericidal effect, with a 10-fold decrease in CFUs with two doses of NAC 20 mM at 4 days post-infection, and completely undetectable cultures with three doses of NAC 10 mM and NAC 20 mM at 8 days post-infection. When assessing the combination treatment at 3 h post-infection, while the combination treatments had significantly lower M. avium CFU counts than [R₄W₄], there were no significant differences between the NAC treatment and combination treatment, indicating that the immediate effects of the combination treatment can largely be attributed to the action of NAC. This is further evidenced by the combination treatment with [R₄W₄] at 8 µg/mL and NAC at 5 mM. The combination did not exhibit an additive effect, whereas the antibacterial effect was comparable to that of [R₄W₄] or NAC alone at their respective concentrations. This finding suggests that [R₄W₄] acts as a bacteriostatic agent, whereas NAC acts as bactericidal agent, however, in higher concentrations. An important note is that, at the 3 h time point, [R₄W₄] and NAC exhibited bacteriostatic activity, as colony counts remained above the initial inoculum, rather than demonstrating bactericidal effects. Additive effects can be observed with higher doses of [R₄W₄] and NAC with longer incubation times at 4 and 8 days (Figure 2B,C).

We then examined the effects of singular and NAC and [R₄W₄] combination treatments on intracellular M. avium survival in THP-1 cell-derived macrophages. NAC treatment has been previously demonstrated to significantly reduce M. avium burden in the A549 human lung cell line and MH-S macrophage cell line 3 and 5 days post-infection, with a dose-dependent response observed in A549-infected cells [30]. Similarly, NAC treatment in THP-1 cells in this study has been observed to significantly reduce M. avium survival, with a dose-dependent response observed 3 h, 4 days, and 8 days post-infection. [R₄W₄], however, only demonstrated a dose-dependent response at 8 days post-infection (Figure 3C).

The delay in efficacy of [R₄W₄] could potentially be attributed to the limited capacity of [R₄W₄] to passively transverse the host cell membrane. While the tryptophan residues may increase the affinity for the host cell membrane, the charged arginine residues may decrease [R₄W₄] solubility [34]. The entry of [R₄W₄] into the host cell potentially requires more active processes such as phagocytosis or pinocytosis, requiring more incubation time, though more studies are needed to confirm this hypothesis [35]. NAC can exert a rapid effect on the host antioxidant systems important for bacterial clearance and autophagy [36]. This would be consistent with the immediate effects of NAC 3 h post-infection compared to [R₄W₄] (Figure 3A). We previously tested liposomal formulations of [R₄W₄] in M. avium-infected THP-1 macrophages and found that liposomal [R₄W₄] can significantly reduce M. avium burden 4 days post-infection with 2 µg/mL and 4 µg/mL, indicating that liposomes may enhance the intracellular delivery of [R₄W₄] [37]. In this study, a combination treatment with NAC and [R₄W₄] significantly reduced M. avium survival at 4 and 8 days post-initial treatment in a dose-dependent response compared to untreated controls and NAC singular treatment (Figure 3B,C). Furthermore, combinations with higher concentrations (8 µg/mL [R₄W₄] + 20 mM NAC) resulted in undetectable levels of M. avium, indicating that the combination treatment augments the efficacy with increased dosing.

While prior experiments from our lab [23,24,27] have evaluated the effects of [R₄W₄] and NAC in combination with first-line antimycobacterials, the goal and aim of this study was to elucidate the additive and mechanistic effects of [R₄W₄] and NAC themselves.

We also measured the minimum inhibitory concentrations of [R₄W₄] and NAC (Table 1). The MIC results demonstrated that both R₄W₄ and NAC possess concentration-dependent antibacterial activity. R₄W₄ inhibited M. avium growth with a minimum inhibitory concentration of approximately 6 µg/mL, while NAC showed a complete inhibition at 5 mM. These findings suggest that both compounds are effective against M. avium, which is compatible with our CFU results (Figure 2 and Figure 3). We measured bacterial absorbance after 4 days of incubation, since M. avium is a slow growing bacterium and this incubation period will allow us to see visible changes in OD. Furthermore, when we attempted to measure the OD at 3 h post-incubation, we did not observe any changes in the absorbance. Additionally, we wanted to choose a time point that is consistent with our CFU study protocol.

The checkerboard assay was also conducted to evaluate the potential interaction between R₄W₄ and NAC. The majority of combinations, specifically R+N (2,5), R+N (4,10), and R+N (8,5), exhibited additive or indifferent interactions (FIC index between 1 and 4), indicating that the combination treatments maintain their individual activities without significantly enhancing or diminishing each other’s effects. Such additive behavior suggests that the compounds act through distinct pathways that do not interfere with one another. Therefore, more experiments are warranted in future studies to find out the individual specific mechanisms of [R₄W₄] and NAC. However, the R+N (8,20) and R+N (2,20) combinations showed an antagonistic effect (FIC > 4). These findings are in contrast to our CFU results, which indicate that these two combinations showed a significant inhibitory effect against M. avium (Figure 2 and Figure 3). M. avium survival assays using CFUs were performed many times, and our study findings were always consistent. Overall, the findings suggest that both R₄W₄ and NAC independently exhibit a strong antimycobacterial potential, and their combination results in an additive effect rather than synergy.

In conclusion, the findings of this study provide evidence for NAC and [R₄W₄] treatment as a potential strategy for clearing M. avium infections. Additional studies assessing optimal dose ranges for combination treatment are warranted. Moreover, the efficacy of NAC + [R₄W₄] against frontline M. avium drugs will have to be tested in a head-to-head comparison. Furthermore, in vivo efficacy studies and randomized controlled clinical trials are required to fully ascertain the efficacy and safety of NAC and [R₄W₄] treatment as a treatment modality against mycobacterial infections. Our laboratory has initiated in vivo studies using mouse models with M. avium infection with glutathione as a treatment, and we plan to extend this approach to evaluate [R₄W₄] and NAC combination treatment in this system. We also plan to assess the mechanistic effects of [R₄W₄] and NAC alone and the combination treatments through ATP Production Assays and other experiments.

4. Materials and Methods

4.1. Bacterial and Reagent Processing and Preparation

Mycobacterium avium was cultured in 7H9 media medium (Hi Media, Santa Maria, CA, USA) supplemented with albumin dextrose complex (ADC) (GEMINI, New York City, NY, USA) and maintained at 37 °C until reaching the logarithmic growth phase, determined by an optical density of 0.5 to 0.8 at A600. M. avium cultures were washed with phosphate-buffered saline (PBS) (Sigma, St. Louis, MO, USA), followed by mechanical disruption of bacterial clumps through vortexing with 3 mm sterile glass beads at 3 min intervals for processing. The resulting single cell M. avium suspension was filtered using a 5 µm syringe filter to eliminate any remaining bacterial aggregations. The processed single cell M. avium suspension was serially diluted and plated on 7H11 agar (Hi Media, Santa Maria, CA, USA) to enumerate bacterial numbers present in the processed stock. Aliquots of processed bacterial stocks were frozen and stored in a cryogenic freezer at −80 °C until use in the experimental trial. Stock solutions of NAC and [R₄W₄] were dissolved in nanopure water and sterilized through 0.22 m filtration.

4.2. Bacterial Cell Culture, Antibiotic Treatment, and CFU Counts

To assess direct antimycobacterial effects of singular or combination NAC+ [R₄W₄] treatment, processed M. avium bacterial cultures (2 × 10⁵ cells/well) were cultivated in 96-well tissue culture plates (Corning, Corning, NY, USA) containing 7H9 media and supplemented with various concentrations of [R₄W₄] treatment (2 µg/mL, 4 µg/mL, and 8 µg/mL), NAC single treatment (5 mM, 10 mM, and 20 mM), combination NAC+ [R₄W₄] treatment (5 mM, 10 mM, and 20 mM NAC with 2 µg/mL, 4 µg/mL, and 8 µg/mL [R₄W₄], respectively), or sham treatment (nanopure water) as a control. Treatments were applied immediately after infection and 3 days and 6 days post-infection. Treatment concentrations were selected from previous efficacy studies [24,27,30]. Each treatment category was cultured in triplicate. Treated M. avium cultures were incubated at 37 °C, with 5% CO₂. Incubation was terminated at 3 h, 4 days, and 8 days post-treatment (Appendix A). At termination, cell culture suspensions were collected, serially diluted, plated onto 7H11 agar in duplicate, and incubated for 2 weeks at 37 °C until visible colony formation. Colony-forming units (CFUs) were enumerated to assess M. avium viability post-treatment.

4.3. THP-1 Cell Preparation, Differentiation, and Infection

THP-1 cells were cultured in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) with 10% FBS and incubated at 37 °C with 5% CO₂. THP-1 cells were enumerated using a hemocytometer and trypan blue staining. THP-1 cultures grown to 2 × 10⁵ cells were harvested for infection studies. A 96-well tissue culture plate was coated with poly-L-lysine before loading THP-1 cells for cell adhesion. To induce macrophage differentiation, THP-1 cells were treated with 10 ng/mL of phorbol 12-myristate 13-acetate (PMA) and incubated at 37 °C with 5% CO₂ overnight. After overnight incubation, the 96-well plate was inspected under a microscope to visualize the formation of a differentiated cell monolayer to confirm macrophage differentiation. The supernatant was subsequently removed, and cells were washed with 1X PBS three times before reloading wells with RPMI with 10% FBS. Processed M. avium was inoculated to each well at a 1:1 MOI (M. avium to THP-1 cells) and incubated at 37 °C with 5% CO₂ for 1 h to allow for phagocytosis. After the incubation period, the supernatant was discarded, and nonphagocytosed bacteria were removed by washing with 1X PBS solution three times. Fresh RPMI with 10% FBS was added to each well.

4.4. THP-1 Macrophage Treatment and Infection Termination

To assess intracellular M. avium survival post-NAC or NAC+ [R₄W₄] treatment, various treatments were administered to wells containing M. avium-infected macrophages, including [R₄W₄] single treatment (2 µg/mL, 4 µg/mL, and 8 µg/mL), NAC single treatment (5 mM, 10 mM, or 20 mM), NAC+ [R₄W₄] combination treatment, or sham treatment (1X PBS) for control. Treatment categories were cultured in triplicate. Treatments were applied immediately after infection and 3 days and 6 days post-infection in triplicate using either sham treatment control or varying concentrations of singular or combination NAC and [R₄W₄] treatments. Treated M. avium-infected macrophages were incubated at 37 °C with 5% CO₂ until termination at 3 h, 4 days, and 8 days post-treatment. To terminate infection, the supernatant was removed from the well and loaded with ice cold MilliQ water to lyse cells and liberate phagocytosed M. avium. To enumerate cell growth post-treatment, cell lysates were serially diluted, plated onto 7H11 agar in duplicate, and incubated at 37 °C for 2 weeks until visible colony growth.

4.5. Membrane Depolarization Assay

The effects of treatments on M. avium cell membrane potential were assessed as previously described [33,38]. M. avium was cultured in Middlebrook 7H9 medium and grown to mid-logarithmic phase (OD₆₀₀ = 0.6). The bacterial culture was then centrifuged, washed, and resuspended in a buffer containing 5 mM HEPES and 5 mM dextrose (pH 7.2–7.4), followed by dilution to an OD₆₀₀ of 0.3. The fluorescent dye 3,3′-Dipropylthiadicarbocyanine iodide (DiSC₃(5)) was added to the cell suspension at a final concentration of 3 µM and incubated in the dark for 45 min to allow for dye quenching. Following incubation, the cell–dye mixture was aliquoted into a clear 96-well microplate. Potassium chloride (KCl) was added to stabilize the culture. Wells were then treated in triplicate with varying concentrations of either [R₄W₄] or N-acetylcysteine (NAC). Untreated cultures served as baseline measurement. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) at 3 µg/mL served as a positive control, while rifampicin was used as a negative control. Fluorescence measurements were taken over time using an excitation wavelength of 622 nm and an emission wavelength of 670 nm.

4.6. Determination of MIC Against M. avium and Additive Effects of the Combination Treatment

To determine the minimum inhibitory concentration of the treatments against M. avium, processed M. avium bacterial cultures (OD600 = 0.3) were added to 96-well tissue culture plates (Corning, NY, USA) containing 7H9 media (100 µL, 2 × 10⁵ cells/well). The wells were then supplemented with varying concentrations of [R₄W₄] treatment (1 µg/mL, 2 µg/mL, 4 µg/mL, 6 µg/mL, 8 µg/mL), NAC treatment (2.5 mM, 5 mM, 10 mM, 15 mM, 20 mM), or sham treatment (PBS) as control. Treatments were applied immediately after the infection and after 3 days. The cultures were incubated at 37 °C, with 5% CO₂. OD600 of the plates was measured using a Spectramax M2e microplate reader (Molecular Devices, San Jose, CA, USA) before (A1) and after (A2) incubation for each treatment category. The following formula was used to calculate the percent inhibition [39]:

$${\displaystyle \frac{OD600 Control-\left(OD600 T2-OD600 T1\right)}{OD600 Control}}\times 100\mathrm{\%}$$

where OD600 Control is the difference in absorbance of PBS control wells post- and pre-incubation for each time period and OD600 A1 and A2 are absorbances measured before and after incubation for each time period.

The calculated MIC was used to calculate the checkerboard assay as fractional inhibitory concentration (FIC) using the following formula [40,41]:

$$FIC={\displaystyle \frac{MIC A incombination}{MIC A alone}}+{\displaystyle \frac{MIC B incombination}{MIC B alone}}$$

where MIC A and B in combination are the MICs of the combination treatments and MIC A and B alone are the MICs of each respective compound separately. The FIC index was interpreted as follows:

≤0.5 = Synergistic effect;

>0.5–4 = Additive effect or indifferent;

>4 = Antagonistic effect.

4.7. Statistical Analysis

GraphPad Prism Software (Version 10.5.0) was utilized for statistical analysis. ANOVA with Bonferroni correction was employed to compare treatment categories. The data were presented as the mean ± standard error of the mean. Asterisks denoting statistically significant p-values were placed between comparison groups. Calculated p-values of <0.05 (*), <0.005 (**), <0.0005 (***), and <0.00001 (****) were all considered statistically significant.