1. Introduction
Novel antimicrobial and engineering approaches are urgently needed to cope with antibiotic resistance. Antimicrobial peptides (AMPs), a class of naturally occurring small (generally less than 50 amino acids) and positively charged peptides [
1], have attracted attention in clinical research because of their broad-spectrum activity against a diverse group of microorganisms, including antibiotic-resistant pathogens [
2]. On the other hand, cytokines are a group of small immunomodulatory proteins that play a central role in host defense by orchestrating the antimicrobial functions and conferring greater protection against different infectious agents [
3]. One of the most studied cytokines is interferon-gamma (IFN-γ), which has proven to be a potent immunoprophylactic agent [
4,
5].
However, under physiological conditions, AMPs are subjected to proteolytic degradation and peptide inactivation by nonspecific interactions with anionic substances, which result in the low bioavailability and poor in vivo stability of these small molecules [
6]. Furthermore, most cytokines, including IFN-γ, have very short half-lives, so their immunological function is limited [
3]. Therefore, the accumulation of these proteins in naturally occurring bacterial inclusion bodies (IBs) and soluble self-assembling protein-only nanoparticles (PNPs) seems an appealing alternative to overcome these limitations. In 2017, Serna et al. described for the first time the use of PNPs as antibacterial agents [
7]. These soluble PNPs were obtained following a modular protein design based on the fusion of a cationic peptide to a C-terminal his-tagged scaffold protein [
8]. This modular configuration was presented as a transversal platform which has been replicated with several scaffold proteins [
9,
10]. The cationic α-helical GWH1 antimicrobial peptide [
11], once fused to the amino terminus of green fluorescent protein (GFP), promoted the oligomerization in PNPs of around 50 nm size which showed a wide bactericidal effect against different pathogenic bacteria in cell cultures [
7]. Due to the functional and structural versatility of this system, we wondered whether the fusion design between GWH1 and IFN-γ could also lead to the formation of these highly stable nanosized oligomers (PNPs). Besides, bacterial IBs, once considered as waste by-products derived from recombinant protein production, now provide a useful source of ready-to-use active protein. Inside these structures, therapeutic proteins are stored in native and native-like conformations and are released under physiological conditions [
12,
13,
14]. The benefits of this system lie in the protective effect against degradation and the sustained release of the protein, which in both cases can significantly increase their half-lives.
The aim of the present study is to characterize and to evaluate, in an in vivo mastitis mice model, the direct and non-direct antibacterial effects of two different protein designs (GWH1-GFP and GWH1-IFN-γ) assembled in two protein formats, namely, PNPs and IBs. Furthermore, we wanted to test the synergy of both activities, immunomodulation and bactericidal effect, in a single polypeptide with the potential to form PNPs. The results obtained here could throw some light on the development of new protein-based mastitis therapies but with transversal applicability in any clinical problem needing unconventional antimicrobial therapies.
4. Discussion
Novel therapies are urgently needed to tackle the reduced cure rates related to the development of bacterial resistance [
29]. One alternative includes the use of alpha-helical AMPs [
30]. However, large-scale production of synthetic small peptides is challenging [
31] and recombinant protein production in prokaryotic host is presented here as a promising alternative. Several experimental strategies have been explored to cope with the inherent toxicity of such peptides, when recombinantly produced, towards the prokaryotic host, including the fusion to partner proteins that seem to mask the antimicrobial activity [
32,
33,
34], as well as the accumulation in the insoluble cell fraction of AMPs fused to scaffold proteins [
35]. In any case, regardless of the mechanism of action of alpha-helical AMPs against microorganisms, the recombinant production of these peptides might be achieved by the neutralization of their net charge by fusion to solubility-enhancing proteins [
36,
37,
38] or by enhancing formation of IBs in producing cells by the fusion to aggregation-prone proteins [
39].
In the present work, we have designed GWH1-containing recombinant proteins, with the idea of building bifunctional molecules where each individual part of the recombinant fusion design could maintain its original function, and at the same time, act as a building block for protein self-assembling [
8,
10]. In that sense, it has been demonstrated that GFP, toxins and pro-apoptotic proteins are able to act as scaffold partners for protein self-assembling while retaining their biological activity [
9,
40]. Protein production was affected by the expression of these AMP carrying proteins. The addition of GWH1 provoked a drastic decrease in protein yields in comparison to the non-AMP-containing counterparts, see
Supplementary Table S1. However, proteins were produced in enough amounts to allow an effective purification process. Still, in the tested conditions, GWH1-IFN-γ arrangement was unable to promote the formation of soluble PNPs (
Figure 1D), suggesting the inability of the mouse IFN-γ to act as a scaffold domain for protein oligomerization. Mature mouse IFN-γ forms noncovalently linked homodimers of 20–25 kDa [
41]. The formation of dimers in both, IFN-γ and GWH1-IFN-γ could explain the bigger sizes observed by DLS and the bigger size of GWH1-IFN-γ over IFN-γ due to the presence of the GWH1 peptide. Moreover, the N-terminal addition of the GWH1 antimicrobial peptide has somehow diminished or truncated the mouse IFN-γ functionality as observed when comparing the activity of the IFN-γ and GWH1-IFN-γ proteins (
Figure 3B). This fact is not surprising, since there is evidence that the N-terminus region of mouse IFN-γ plays an important role in receptor binding. Therefore, the failure to bind prevents internalization and later events that result in the induction of the immune response [
42,
43].
Surprisingly, antimicrobial performance of GWH1-containing proteins in different hosts, showed an inhibitory activity against
E. coli, whereas
S. aureus was more resistant to the treatment (
Figure 2A,B). The different membrane composition may influence the mechanism of action of α-helical AMPs, especially for Gram-negative and Gram-positive bacteria [
44]. In this sense, the GWH1 constructs produced in this work, not only have displayed a preferred antimicrobial activity against the Gram-negative
E. coli, but, most importantly, the antimicrobial performance in this microorganism was enhanced when the GWH1-fusion proteins formed nanoparticles (
Figure 2A). The main mechanism of action of AMPs is membrane permeabilization and structural disruption. To achieve the antimicrobial effect, a minimal AMP concentration is required in the target surface. Such characteristic is described as the threshold concentration [
45] and is experimentally expressed as the peptide-to-lipid ratio (P/L). The propensity of GWH1-GFP to self-assemble in multimeric complexes could enhance the proximity of effective monomeric units on cell surface, diminishing the local peptide concentration required to reach higher P/L values. As the P/L ratio increases, the peptides start to insert and traverse the membrane. AMP monomeric forms require higher concentrations to achieve threshold concentrations. This was demonstrated in the case of the monomeric GWH1-GFP, which lacked a dose-dependent antimicrobial activity, only showing antimicrobial effect at the highest concentration, suggesting the need to reach a critical concentration in order to display its activity (
Figure 2D). A similar result was observed with the unassembled GWH1-IFN-γ (
Figure 2A). The presented evidence validates the multimeric format as a more effective arrangement than the monomeric format, when it comes to antimicrobial activity.
In order to explore the influence of the multiple display configuration of AMPs in the in vivo settings, we analyzed the performance of the engineered protein nanoparticles in a mouse mastitis model. Infectious mastitis is one of the most relevant diseases in dairy cattle, and antibiotic usage is the leading strategy for its treatment and prevention, since this disease is the costliest for dairy producers [
46]. Despite the high antimicrobial activity showed by the multimeric format of GWH1-GFP, in vitro assays do not always correlate with in vivo efficacies inside the mammary gland [
47,
48]. Various aspects are influenced by the complex milk environment [
25], and, therefore, a proof of concept in vivo approach is necessary. To date, different studies have used mouse models to assess the effect of antimicrobials [
48,
49,
50,
51]. However, most of these studies were dedicated to the Gram-positive pathogen
S. aureus [
25] and only few of them characterized the efficacy of antimicrobial agents on other relevant mastitis-causing pathogens such as
E. coli [
52,
53]. To the best of our knowledge, this is the first study in which the antimicrobial capacity of AMP-containing nanoparticles has been determined in an in vivo mouse mastitis model of
E. coli infection. Based on the in vitro assays for GWH1-GFP PNPs, a single dose of 60 µmol/L, i.e., ten times the MIC measured in vitro for GWH1-GFP in
E. coli (
Figure 2A), was selected for the intramammary administration after the bacterial challenge. As a result, the GWH1-GFP PNPs further reduced by 1-Log the bacterial burden in glands when compared with the non-AMP-containing counterpart, GFP (
Figure 6A). This difference can be explained by the presence of the GWH1 antimicrobial peptide, which validates the functionality of this format in the milk environment and its efficacy as a promising anti-mastitis candidate. However, in
S. aureus challenged animals, GWH1-GFP PNPs reduced bacterial load at the same level as control GFP (UPs) (
Figure 7). In accordance with this, antimicrobial activity of GWHI-GFP PNPs in in vitro experiments against
S. aureus was lower than that observed against
E. coli (
Figure 2A). As previously mentioned, the differences in the structure and physical properties of the bacterial membrane might account for the dissimilar activity of the same AMP in Gram-negative and Gram-positive bacteria [
54]. On the other hand, in
E. coli, GWH1-IFN-γUPs were not as effective as GWH1-GFP PNPs (
Figure 6A), which supports the idea that the multimeric format is of utmost significance for reducing the local concentration of AMP necessary to achieve a deleterious membrane disruption, and, consequently, to increase the efficacy of the compound.
The IB format provides a protective environment against short-time degradation and a sustainable release of recombinant protein that may improve the effect over time [
55,
56,
57]. However, as it has been described before [
12], the amount of protein that can be released from IBs can vary depending on the IB-forming protein and the used experimental conditions during protein production process [
58]. All this, associated with the fact that IFN-γ IBs have previously demonstrate activity, even at higher level than the soluble counterpart [
12], urged us to test the efficacy of this format in an in vivo approach. The antimicrobial efficacy for GWH1-GFP IBs was partially reduced, showing a more disperse pattern which was not significant when compared to GFP IBs. This observation could be in accordance with the studied characteristics of GWH1-GFP IBs. The low availability of protein due to the poor release (
Figure 1C) associated with the fact that part of the released protein can be found in a monomeric state (
Figure 1E), which has been demonstrated here whereby bactericidal activity is reduced, may explain the low efficacy of this format in terms of antimicrobial activity. GWH1-IFN-γ and IFN-γ IBs slightly reduced the bacterial burden, being this latter statistically significant when compared to GFP IBs. This behavior could be associated with the increased release showed by both protein designs from IBs, especially for IFN-γ IBs (
Figure 1C). Additionally, it is well known that IFN-γ has a very short half-life and therefore its immunological function in the mammary gland is limited [
3]. The protective behavior provided by this oligomeric format may enhance its stability and merits further research. In fact, previous results showed that the activity of IFN- γ is preserved in protein released from IBs after a 96 h incubation at 37 °C [
12]. The cytokine embedded in IBs could be less sensitive towards proteolytic degradation, which could then result in a better immunological performance inside the mammary gland. In fact, in the in vivo assays, the activity of the proteins containing IFN-γ displayed a higher activity when administered in IB format.
Altogether, the results presented in this work show a better antimicrobial performance of the multimeric AMP format against the monomeric configuration, and, most importantly, provided significant data for considering the AMP-containing nanoparticles as a promising alternative for treatment of mastitis. However, it should be noted that efficacy studies in mice should only be considered as an intermediate proxy to predict efficacy in dairy cows [
51]. Overall, the versatility of the nanoparticle self-assembling format provides a valuable tool for testing a diversity of AMP with different scaffold proteins that could generate possible synergies or provide antimicrobial activity against other pathogens of interest.