Antimicrobial Peptides and Cell-Penetrating Peptides for Treating Intracellular Bacterial Infections

Bacterial infections caused by intracellular pathogens are difficult to control. Conventional antibiotic therapies are often ineffective, as high doses are needed to increase the number of antibiotics that will cross the host cell membrane to act on the intracellular bacterium. Moreover, higher doses of antibiotics may lead to elevated severe toxic effects against host cells. In this context, antimicrobial peptides (AMPs) and cell-penetrating peptides (CPPs) have shown great potential to treat such infections by acting directly on the intracellular pathogenic bacterium or performing the delivery of cargos with antibacterial activities. Therefore, in this mini-review, we cover the main AMPs and CPPs described to date, aiming at intracellular bacterial infection treatment. Moreover, we discuss some of the proposed mechanisms of action for these peptide classes and their conjugation with other antimicrobials.


INTRODUCTION
Over the years, bacteria have been adapting to survive, become resistant to drugs, and make the host's phagocytes their home (Cornejo et al., 2017). Intracellular bacteria have received special attention since they are extremely challenging to detect and treat (Mitchell et al., 2016). Once internalized, they can remain inactive but persist, causing recurrent infections and chronic illnesses (Monack et al., 2004). Many of the treatments currently used for internalized bacteria are ineffective at therapeutical doses (Monack et al., 2004). For example, conventional antibiotics used in treatments against extracellular bacteria have reduced permeability in intracellular bacterial infections (Proctor et al., 2006;Lehar et al., 2015).
Although antibiotics can kill the internalized bacteria, these microorganisms have shown different resistance profiles, thus often rendering the treatment ineffective (Proctor et al., 2006;Lehar et al., 2015). In this current scenario, the search for effective alternative ways of killing intracellular bacteria is imperative. Promising alternatives are antimicrobial peptides (AMPs) and cell-penetrating peptides (CPPs) for direct antibacterial activities or for the delivery of antibacterial cargos and peptide nucleic acids (PNAs) within the host cell (Splith and Neundorf, 2011;Le et al., 2017).
Antimicrobial peptides (AMPs) are commonly found in nature, also called natural antibiotics (Felıćio et al., 2017). These molecules play a critical role in the first line of defense against pathogens, being expressed constitutively or induced by different types of cells in response to infectious or inflammatory stimuli (Kumar et al., 2018;Yount et al., 2019).
Based on their activities and structural profiles, new and promising active molecules have been designed; among them, we can cite AMPs with intracellular antibacterial activity (Creṕin et al., 2020). In general, these peptides are constituted of five to 50 amino acid residues and manage to cross the host's first barrier, the plasma membrane (Felıćio et al., 2017). Once internalized by endocytosis or macropinocytosis (Figure 1), they can present direct actions in vacuoles or cytosol, where the bacteria are found. Moreover, they can interfere with some internal signaling pathways (e.g., maturation of IL-1b, and increased levels of TNF-a, IL-1b, and IL-10 to eliminate intracellular bacteria) . By interfering with the intracellular pathways, some specific mediators are increased, including those responsible for chemotactic activities, stimulation of the oxidative metabolism in phagocytes, increasing protein production in the acute inflammation phase and stimulating CD4 + and CD8 + cells (Trinchieri, 1997;O'Shea et al., 2002).
Cell-penetrating peptides (CPPs) are also considered intracellular antibacterial agents as they can penetrate bacterial cell membranes without seriously compromising their integrity. CPPs are small peptides that can autonomously translocate across plasma membranes and mediate transport of cargo molecules (Gomarasca et al., 2017). Charge molecules that can be coupled to CPPs include biological products, such as proteins, oligonucleotides, nanoparticles, and small molecule drugs (Hoffmann et al., 2018). The conjugation of CPP-charges can occur by a covalent and non-covalent bond (Hoffmann et al., 2018). The main feature of the plasma membrane is to protect the internal content of the cells. Therefore, the plasma membrane is a semipermeable barrier and essential for cell survival. The transport of compounds through the membrane occurs freely with small molecules or specific channels in larger molecules (Gomarasca et al., 2017;Ruseska and Zimmer, 2020). CPPs have a high permeability rate, cross the membrane of different cell types, present low cytotoxicity and do not activate the host's immune response (Ruseska and Zimmer, 2020).
These peptides can either exert a direct antibacterial mechanism or perform the delivery of bioactive molecules, including other antimicrobial agents and PNAs (Reissmann, 2014). PNAs are synthetic DNA derivatives applied in hybridization-based microbial diagnostics. They are able to reduce the proliferation of various bacteria due to the antisense FIGURE 1 | Schematic representation of the mechanism involved in bacteria, AMPs, and CPPs internalization into macrophages. Free-floating bacteria can invade the host cell by zipper or trigger mechanisms. Once inside the host cell, intracellular bacteria can occupy both the cytosol and vacuoles. At their therapeutic dose, conventional antibiotics usually present low permeability, rendering the intracellular bacterial infection treatment ineffective. By contrast, increasing antibiotics' concentration can favor their permeabilization across the host cell membrane to treat the infection. However, as a consequence, it can also trigger cell toxicity. AMPs and CPPs have been used as alternative therapies. These peptide classes can translocate through the host cell plasma membrane via endocytosis, phagocytosis and macropinocytosis. In some cases, CPPs can also form transient toroidal pores or interact with membrane receptors. Once inside the cell, AMPs usually trigger direct antibacterial activities, whereas CPPs have been commonly used in conjunction with PNA and cargos aiming at essential bacterial genes silencing or direct antibacterial activities, respectively. targeting of essential genes (Barkowsky et al., 2019). PNAs coupled to CPPs are able to support translocation over the cell membrane in bacteria ( Figure 1) and, thus, increase antimicrobial efficiency (Barkowsky et al., 2019). The CPPs' internalization mechanisms depend on two factors, including the target cell and the load coupled to the CPPs (when used in association with cargos) (Ruseska and Zimmer, 2020). In this mini-review, we cover recent findings on the usage of AMPs and CPPs as promising intracellular antibacterial candidates, which act by interfering with cell-penetrating properties, and their suggested mechanism of action and conjugation with other antimicrobial agents. Obligatory or facultative intracellular lifestyles are characteristic of bacterial evolution, enabling these microorganisms' growth and replication in different niches (Otten et al., 2018). When bacteria start the cell internalization process, some of them can be detected by the host defense mechanisms, activating macrophage recruitment. However, bacteria that evade this process can also internalize macrophages, thus being protected from the host's immune response and antibiotic action (Maurin and Raoult, 2001;Canton and Kima, 2012). Thus, intracellular bacteria reside directly in the mammalian cell-host cytoplasm or vacuoles, manipulating endocytic or secretory pathways to recruit necessary supplements to guarantee their replication (Loeuillet et al., 2006;Canton and Kima, 2012;Yeh et al., 2020). Different systems can facilitate the entry of intracellular bacteria in professional phagocytes, including type III secretion system (T3SS) and type IV secretion system (T4SS) (Coburn et al., 2007). These systems can translate specific proteins (effector proteins) to mediate pathways of the pathogen's internalization. The purpose of bacterial effector proteins is to facilitate the entry of intracellular bacteria into the host cell, providing survival and replication in the cytoplasm or intracellular organelle (Ham et al., 2011;Yeh et al., 2020).

INTRACELLULAR INFECTIONS CAUSED
Intracellular bacteria can invade different cell types, including neutrophils, keratinocytes, and intestinal epithelial cells (Ham et al., 2011;Abu-Humaidan et al., 2018). The strategies used by intracellular bacteria to invade non-phagocytic host cells include the zipper and trigger mechanisms ( Figure 1) (Ham et al., 2011). Bacteria that use the zipper mechanism express proteins on their membrane surfaces, which bind to the host cell's plasma membrane receptors. The binding of surface proteins (bacteria and host cells) promotes pathogen-host cell interaction and triggers signaling cascades that reorganize the actin cytoskeleton, helping the bacteria to be internalized (Cossart and Sansonetti, 2004;Ham et al., 2011). By contrast, the trigger mechanism is characterized by the type III secretion system (Veiga and Cossart, 2006). The bacterium secretes effector proteins into the cytoplasm to modulate the actin cytoskeleton, polymerizing the actin, thus forming a pseudopod and allowing the bacteria's entry into the cytoplasm (Veiga and Cossart, 2006;ÓCroínıń and Backert, 2012).
Another problem is that bacterial infections caused by intracellular pathogens are incredibly difficult to eradicate (Cossart and Sansonetti, 2004), as the antibiotics' concentration internalized in host cells is lower than their minimum inhibitory concentration (MIC) (Harish and Menezes, 2015). Such disparities in antibiotic concentration could lead to further drug resistance. The antimicrobials currently used to treat intracellular pathogens are sulfonamides, quinolones, tetracyclines, and beta-lactams (Harish and Menezes, 2015;Bongers et al., 2019). It is suggested that sulfonamides are captured by host cells by active mechanisms and have a bacteriostatic effect on the intracellular pathogen. Quinolones enter and accumulate in subcellular compartments. Beta-lactams enters the cell by passive diffusion, interfering with the intracellular bacteria peptidoglycan synthesis. Finally, tetracyclines enter the host cells through organic cations active transport and interfere with bacterial protein synthesis (Bongers et al., 2019). Nevertheless, the recurrent choice of these antibiotics has become limited in clinical cases with resistant bacterial strains (Harish and Menezes, 2015).
The increase in resistance to multiple drugs reinforces the urgent need to develop new intracellularly active antibacterial agents capable of overcoming the low cellular permeability of the antimicrobials currently used (Weissman et al., 2016).

ANTIMICROBIAL PEPTIDES
Antimicrobial peptides (AMPs) are multifunctional molecules that have been largely isolated from various organisms, including vertebrate and invertebrate animals, plants, and bacteria (Porto et al., 2017). AMPs can be useful in eliminating bacteria, parasites, fungi, and viruses (Yan and Hancock, 2001). In addition, immunomodulatory and anticancer properties have also been reported (Hancock and Sahl, 2006). Therefore, the range of physicochemical properties and mechanisms of action attributed to AMPs make these molecules promising antibacterial drug candidates, which are more capable of acting on different biosynthetic pathways than conventional antibiotics reported as working against extracellular and intracellular bacteria (Gottschalk et al., 2015;Silva and Gomes, 2017).
Insects have essential AMPs from their organism's innate system, a rich source to be prospected (Li et al., 2018). The DLP4 peptide, isolated from the hemolymph of Hermetia illucens, showed potent antibacterial activity against Grampositive and negative. In an assay using MRSA ATCC43300 in intracellular form with RAW 264.7 cells; DLP4 peptides and their analog DLP2 significantly reduced bacterial growth in vitro (DLP2 -1.89 -log and DLP4 -1.34 -log) ( Table 1) (Li et al., 2018).
Rational design is a modern approach used to improve existing peptide structures for a desired biological activity. The peptide WR12 (RWWRWWRRWWRR) is a de novo designed short synthetic peptide, composed exclusively of arginine and tryptophan amino acid residues. D-IK8 (irikirik) is a short synthetic peptide composed only of eight D-amino acids ). These peptides were tested for their intracellular antibacterial activity on human keratinocytes infected by MRSA and methicillin-sensitive S. aureus (MSSA) . The D-IK8 peptide reduced the intracellular bacterial load of MRSA and MSSA by 96%, at a concentration of 16 µM, whereas at this same concentration, the peptide WR12 reduced MRSA and MSSA loads by 40% (Table 1) .
Many AMPs have remarkable activity against bacteria, but the minority of AMPs manage to reach intracellular bacteria via nonlytic mechanisms to the host cell (Splith and Neundorf, 2011;Yang et al., 2020). An alternative is the CPPs, which can present intracellular antimicrobial activity or can be coupled to cargos with activity against intracellular bacteria (Splith and Neundorf, 2011).

CELL-PENETRATING PEPTIDES
Frankel and Pabo were the first researchers to discover a peptide capable of translocating to the nucleus of cells, known as the TAT peptide (transactivating transcription protein), encoded by human immunodeficiency virus type 1 (HIV-1) (Frankel and Pabo, 1988). Later, consecutive discoveries were made and, currently, more than 1,700 CPPs have been registered in an online database (http://crdd.osdd.net/raghava/cppsite/).
The mechanisms of CPP's penetration into cells can occur by more than one pathway. In general, the internalization of CPPs is closely related to the molecule conjugated to it (cargo), its concentration and the target cell type (Koren and Torchilin, 2012). Authors suggest that the mechanisms may include endocytosis followed by endosomal escape, which can be differentiated in many ways, including macropinocytosis, clathrin-mediated endocytosis, lipid raft-mediated endocytosis or caveola-mediated endocytosis, or via direct membrane penetration, for example, by transient toroidal pores or micelles formation (Figure 1) (Richard et al., 2003;Madani et al., 2011;Wang et al., 2014;Rizzuti et al., 2015).

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In vitro (Cappiello et al., 2016) Insect defensin-like peptide DLP4  (Brezden et al., 2016) internalization of conjugated CPPs could occur through energydependent macropinocytosis and clathrin-mediated endocytosis pathways (Li et al., 2018). CPPs have also been used to carry PNAs, aiming at silencing bacteria's essential genes (Bai et al., 2012). For a PNA to enter the cell, it is necessary to attach it to a CPP (Bai et al., 2012). For instance, the essential RNA polymerase a-subunit (encoded by the rpoA gene) in the intracellular pathogen Listeria monocytogenes has been a target for PNA-CPP therapies. Abushahba et al. evaluated five different CPPs [antennapedia, TAT, (RXR) 4XB, and (RFR) 4XB] coupled with a PNA targeting the gene cited above (Abushahba et al., 2016). Listeria monocytogenes was internalized in J774A.1 cells and treated with the PNA-CPP candidates at 2, 4, and 8 mM. Among the tested PNA-CPPs, PRXR showed a higher reduction in the bacterial load (Log 10 1.78), at a 2 mM (Table 1). However, the cell permeabilization mechanism of action triggered by this CPP is still unknown (Abushahba et al., 2016). Interestingly, studies have reported that encapsulated intracellular bacteria (e.g., S. Typhimurium LT2) are commonly more difficult to treat with PNA-CPP therapies (Cornejo et al., 2017). As an alternative, the electroporation-based delivery of CPP-PNA conjugates has been proposed, which has led to increased bioavailability, as this strategy does not involve the conjugate endocytosis (Cornejo et al., 2017).
Another applicability of CPPs includes the transport of antibiotics into the host cell, as these antimicrobials can be useful against intracellular bacteria, but have little permeability. To evaluate whether gentamicin (GM) would target intracellular bacteria, studies have proposed combining GM with CPPs (Gomarasca et al., 2017). CPPs, including a1H (KSK TEYYNAWAVWERNAPC), a2H (GNGEQREM AVSR LRDCLDRQA), and TAT peptide have been used in this regard (Gomarasca et al., 2017). In a study by Gomarasca et al (Trinchieri, 1997)., human brain microvascular cells (HBMEC) were infected with E. coli K1 and treated with 600 µg mL -1 of the three gentamicin-CPP conjugates, including a1H-gentamicin; a2H-gentamicin and Tat-gentamicin (Gomarasca et al., 2017). The best result was observed by treating the cells with Tatgentamicin, which led to a 6-log reduction in bacterial load ( Table 1) (Gomarasca et al., 2017). Transmission electron microscopy studies indicate that eukaryotic cells' surface integrity was maintained. Additionally, it was observed that the intracellular bacteria remain in endosomal compartments or vacuoles after treatment with Tat-gentamicin, ultimately losing their cell-wall integrity (Gomarasca et al., 2017). Similarly, the development of a cleavable kanamycin-CPP conjugate has been reported (Brezden et al., 2016). This conjugate (P14KanS) containing the disulfide linkage will be broken to release kanamycin when cleaved in the intracellular reduction environment. P14KanS presents high cell penetration potential and significant antibacterial potential against Mycobacterium tuberculosis and Salmonella, both in vitro and in vivo ( Table 1) (Brezden et al., 2016).

CONCLUSION
Intracellular bacterial infections pose an additional challenge compared to extracellular infections, as the antibacterial agent of choice has to cross the host's plasma membrane without harming the cell to eliminate the intracellular pathogen. High doses of conventional antibiotics are usually needed to successfully combat intracellular infections, considering their low cell-penetrating potential. As a consequence of these higher doses, host cell viability is often compromised. As alternative therapies, here we summarize recent findings on the usage of AMPs and CPPs for treating intracellular bacterial infections. As described, these classes of antimicrobials have shown the ability to reach the intracellular bacteria inside the host cell either to exert direct antibacterial activities or deliver cargo molecules with antibacterial potential. Moreover, many studies have reported the non-toxic effects of these peptides on the host cells, rendering them attractive drug candidates for countering intracellular infections.

AUTHOR CONTRIBUTIONS
DB and MC contributed equally. MC and OF corrected the manuscript. All authors contributed to the article and approved the submitted version.