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Antimicrob Agents Chemother. 2017 April; 61(4): e02545-16.
Published online 2017 March 24. Prepublished online 2017 January 17. doi:  10.1128/AAC.02545-16
PMCID: PMC5365713

Bacterium-Derived Cell-Penetrating Peptides Deliver Gentamicin To Kill Intracellular Pathogens

ABSTRACT

Commonly used antimicrobials show poor cellular uptake and often have limited access to intracellular targets, resulting in low antimicrobial activity against intracellular pathogens. An efficient delivery system to transport these drugs to the intracellular site of action is needed. Cell-penetrating peptides (CPPs) mediate the internalization of biologically active molecules into the cytoplasm. Here, we characterized two CPPs, α1H and α2H, derived from the Yersinia enterocolitica YopM effector protein. These CPPs, as well as Tat (trans-activator of transcription) from HIV-1, were used to deliver the antibiotic gentamicin to target intracellular bacteria. The YopM-derived CPPs penetrated different endothelial and epithelial cells to the same extent as Tat. CPPs were covalently conjugated to gentamicin, and CPP-gentamicin conjugates were used to target infected cells to kill multiple intracellular Gram-negative pathogenic bacteria, including Escherichia coli K1, Salmonella enterica serovar Typhimurium, and Shigella flexneri. Taken together, CPPs show great potential as delivery vehicles for antimicrobial agents and may contribute to the generation of new therapeutic tools to treat infectious diseases caused by intracellular pathogens.

KEYWORDS: antimicrobial drug delivery, cell-penetrating peptides, CPP-translocated antimicrobials, gentamicin, intracellular pathogenic bacteria

INTRODUCTION

Bacterial infections caused by intracellular pathogens are particularly difficult to eradicate, since these pathogens have adapted to an intracellular lifestyle to evade host immune responses (1). Examples of such pathogens are Salmonella and Shigella spp., which inject effector proteins via the type three secretion system (T3SS) to promote their engulfment into intestinal epithelial cells (2,4), and Escherichia coli K1, the leading cause of neonatal meningitis, which invades the endothelial cells that constitute the blood-brain barrier (5, 6). However, these bacteria have evolved quite distinct intracellular lifestyles. While Shigella organisms escape the phagocytic vacuole to reach the host cell cytosol, where they replicate (7, 8), Salmonella organisms live and propagate inside Salmonella-containing vacuoles (SCV) (2, 9). Similar to Salmonella, E. coli K1 resides in a membrane-bound vacuole (10, 11), where it prevents lysosomal fusion and degradation. In this way, the pathogens that reside in a protected niche constitute a reservoir for recurrence and reinfections (12, 13). Although several antibiotics, such as sulfonamides, tetracycline, chloramphenicol, ampicillin, and nalidixic acid, are used to treat intracellular pathogens, the choice of these antimicrobial agents has become limited due to the increasing rates of multidrug resistance (MDR) of clinical isolates (14,16). This development indicates that many antimicrobials will no longer be useful and presents a challenge for the development of effective substitute therapies and novel antibiotics. Furthermore, their ability to live and replicate inside host cells protects intracellular bacteria not only from the host immune response but also from the action of non-cell-permeable antibiotics (17). Indeed, a significant number of the most commonly used antibiotics, such as β-lactams and aminoglycosides, do not achieve therapeutic concentrations in intracellular infected cells due to their poor cell permeability (18). The development of novel strategies to enhance the uptake of bioactive antibiotics through the plasma membrane would improve therapies for infectious diseases that are currently difficult to treat.

Cell-penetrating peptides (CPPs) are small peptides that can autonomously translocate through the plasma membrane and mediate the transport of attached cargo molecules (19). CPPs are classified into three major classes according to their chemical and physical properties: cationic, amphipathic, and hydrophobic (20,23). Depending on several factors, such as secondary structure, the particular cargo, size, or the type of targeted cells (24), CPPs can employ different mechanisms to enter eukaryotic cells. These mechanisms include endocytosis followed by endosomal escape, which can be differentiated in different pathways, such as macropinocytosis, clathrin-mediated endocytosis, lipid raft-mediated endocytosis, or caveola-mediated endocytosis, or via direct membrane penetration, for example, by pore formation or micelle formation (21, 22, 25, 26). In addition, over the past few years, CPPs have been used to mediate the transport of several bioactive molecules of different natures, such as imaging agents, proteins and peptides, oligonucleotides, and nanoparticles, stressing their versatility as delivery agents into either mammalian, plant, or bacterial cells (23, 24, 27). Thus, due to their ability to translocate different types of active biomolecules across the plasma membrane, CPPs represent a most promising approach to delivery agents for non-cell-permeable antimicrobial molecules.

The aminoglycoside antibiotic gentamicin, which is used to treat acute life-threatening infections, has a poor ability to traverse eukaryotic cell membranes. Thus, despite the fact that gentamicin has a high bactericidal activity against extracellular bacteria, it fails to reach therapeutic levels in intracellular compartments, resulting in substantially reduced efficacy against intracellular bacterial infections. Therefore, in this study, gentamicin was chosen as a proof-of-principle compound for the development of a novel CPP-based delivery system. In particular, two CPPs derived from the Y. enterocolitica effector protein YopM were employed and their functional properties were characterized. We had previously shown that the Y. enterocolitica-derived YopM protein can autonomously translocate into eukaryotic cells independently of the T3SS (28,30). Autonomously cell-penetrating recombinant YopM is functional and downregulates the expression of proinflammatory cytokines (4, 34), suggesting a dual use of YopM as a delivery vehicle for cargo molecules and as a biological therapeutic for immunomodulation (31, 32). The translocation ability is conferred by the two α-helices at the N terminus, which act as a protein transduction domain (PTD), denoted 2αH. The 2αH domain is able to deliver heterologous cargo, e.g., green fluorescent protein (GFP) or YopE, into eukaryotic cells (28). Moreover, each of the two α-helices has protein transduction abilities, being recognized as two separate CPPs, namely, α1H and α2H. Thus far, our work with recombinant YopM has focused on the evaluation of its potential as a novel biologic therapeutic agent for the treatment of autoimmune diseases (33). Nevertheless, YopM protein transduction domains harbor great potential as delivery vehicles for other cargos. The use of bacterium-derived transport tools is not a completely new strategy, as several studies had described the use of bacterial toxins or other bacterium-derived effector proteins to target host cells. In this regard, nontoxic B subunits of bacterial toxins such as Shiga or anthrax toxins have been used to deliver diverse cargos and have been classified (depending on the attached components) as oncotoxins, mitotoxins, or immunotoxins (34, 35). Moreover, bacterial T3SS have already been engineered to deliver fusion proteins to their sites of action for vaccination and the treatment of inflammatory diseases, cancer, neurodegenerative diseases, intracellular infections, or chronic viral infections (36,38).

In this study, we characterized the α1H and α2H peptides of YopM as cell-penetrating peptides and investigated their ability to deliver cargo. We developed YopM-derived CPPs and the Tat peptide, derived from the HIV-1 trans-activator of transcription (39, 40), as a delivery system to transport gentamicin into eukaryotic cells. We show that these CPPs are able to increase the intracellular delivery and the efficacy of gentamicin in killing intracellular pathogenic bacteria. Thus, CPPs can be applied to engineer very promising antimicrobial tools to treat infectious diseases.

RESULTS

Chemical and physical properties of YopM-derived peptides α1H and α2H.

The Yersinia effector protein YopM is a horseshoe-shaped protein that consists of two antiparallel α-helices at the N terminus and several leucine-rich repeats (LRR), whose numbers vary among the different Yersinia spp. and strains (41). We have previously identified the N-terminal domain of Y. enterocolitica YopM as the protein transduction domain (PTD) (28). This domain consists of the first 86 residues and comprises the two α-helices, 2αH, which mediate the autonomous penetration of the protein into eukaryotic cells (28) (Fig. 1A). In addition, each helix alone is sufficient to mediate protein translocation into the cell cytoplasm (28). Therefore, the first helix (α1H; corresponding to the YopM residues 34 to 51) and the second helix (α2H; corresponding to the YopM residues 53 to 73) of YopM each can be considered single cell-penetrating peptides (CPPs).

FIG 1
α1H and α2H peptide sequence analysis and modeling. (A) YopM consists of two N-terminal, antiparallel α-helices (green) and a variable number (13 to 22) of leucine-rich repeats (LRR) (orange). The YopM-derived CPPs are highlighted ...

To characterize the Y. enterocolitica YopM-derived peptides α1H and α2H, we tried to elucidate how the chemical and physical properties of the peptides could influence their interaction with the membrane by using several bioinformatics tools. The YopM region comprising residues 34 to 73 was subjected to a secondary structure prediction using PSIPRED software (42). A secondary α-helical structure was predicted for amino acids (aa) 36 to 48 and for aa 56 to 71, where the first and second helices are located, according to the crystal structure of the Y. pestis YopM elucidated by Evdokimov et al. (41) (Fig. 1B). The first 33 residues of the protein (YopM1–33) could not be resolved by crystal structure analysis, indicating that this part of the protein is disordered (41). However, residues 1 to 33 seem to stabilize the N-terminal domain of YopM when expressed recombinantly as the 2αH peptide (unpublished results).

With MCPEP software (43), a computational tool to predict peptide secondary structures by Monte Carlo simulation, we observed that α1H and α2H acquire α-helical conformations in both aqueous solutions and in a lipid bilayer environment. From the simulation of the two peptides on the membrane surface, the α1H peptide seems to lie on the membrane surface with four residues, which are distributed along the peptide sequence, coming into contact with the lipid bilayer (red residues and three-dimensional [3D] modeling) (Fig. 1C). In α2H, on the other hand, the four residues, which are in closer contact with the membrane, are all located in the second half of the peptide sequence (Fig. 1D). This suggests that α1H has a stronger contact with the membrane than does α2H. The amphipathic nature of both α-helices is shown in a helical wheel projection (Fig. 1E) using HeliQuest software.

CPPs are classified according to their physicochemical characteristics. Three major classes have been identified: cationic, amphipathic, and hydrophobic peptides (25). To further characterize the nature of the YopM-derived CPPs, we analyzed the hydrophobic/hydrophilic content of the peptide sequences (see Table S1 in the supplemental material). While α1H has a defined hydrophobic region, α2H shows interspersed hydrophobic patches along the sequence. Altogether, this analysis suggests that α1H belongs to the hydrophobic category of CPPs, whereas α2H would fit both amphipathic and hydrophobic classifications.

α1H and α2H can penetrate different eukaryotic cells.

The penetration abilities of the YopM-derived CPPs were investigated in different eukaryotic cell lines and compared to the performance of the Tat peptide. The fluorescein isothiocyanate (FITC)-labeled peptides were incubated in HeLa cells and human brain microvascular endothelial cells (HBMEC) at 37°C and analyzed by using flow cytometry in a quenched time-lapse uptake assay (46). In this assay, 0.2% trypan blue was added to completely quench the extracellular fluorescence and to measure only fluorescence arising from the intracellular FITC-labeled peptides. The intracellular fluorescence increased for all peptides in both cell lines with different kinetics. In HeLa cells, Tat-FITC showed progressive uptake over time, with a maximum intracellular fluorescence at 6 h (Fig. 2A). α1H- and α2H-FITC had similar uptake kinetics, with fluorescence intensity reaching a plateau after 2 h. However, α2H-FITC showed less efficient uptake, with only half of the intracellular fluorescence intensity seen for α1H-FITC. The uptake of the FITC-labeled peptides strongly increased in the first hour of incubation in HBMEC and remained constant over time, indicative of a fast uptake (Fig. 2B). The cellular uptake of CPP-FITC was also analyzed by using fluorescence microscopy, which revealed a homogeneous distribution of the fluorescence of FITC-labeled peptides in the cell cytosol (Fig. 2C). In HeLa cells and, to a lesser extent, in HBMEC, the three CPP-FITC conjugates appeared to be distributed in clusters inside the cells. This phenotype resembles vesicle entrapment and suggests endosomal compartmentalization (53, 54). In HBMEC, the CPP-FITC fluorescence intensity was very low due to a fast bleaching of the fluorophore, resulting in a low signal. Nevertheless, the intracellular distribution of the peptides was still visible. Overall, these data suggest that these YopM-derived peptides can translocate into the cytoplasm of endothelial and epithelial cell lines to the same extent as Tat, revealing a CPP translocation ability.

FIG 2
α1H, α2H, and Tat uptake and localization in eukaryotic cells. (A and B) Fluorescence-activated cell sorting (FACS) analysis of CPP-FITC uptake in HeLa cells (A) and HBMEC (B). CPP-FITC (750 nM) was incubated continuously with cells at ...

YopM-derived CPPs enter cells preferentially by endocytosis and partially by direct penetration.

It is known that CPP can reach the cellular cytosol via two major routes, endocytosis or direct penetration (22). To investigate which of these routes is preferentially employed by the YopM-derived CPPs rather than Tat, the uptake of the FITC-labeled CPPs was analyzed by using flow cytometry at 4°C, when energy- and temperature-dependent processes, such as endocytosis, are blocked (55). The uptake of Tat-FITC and α1H-FITC at 4°C was strongly reduced in both HeLa cells and HBMEC (Fig. 3A and andB)B) compared to uptake at 37°C (Fig. 2A and andB).B). This observation indicated that these peptides use endocytosis as a primary entry route in these cell lines. However, while in HeLa cells the maximum fluorescence intensity measured was 10 arbitrary units (AU), in HBMEC the intracellular fluorescence of the peptides was much higher (18 AU for α1H-FITC and 30 AU for Tat-FITC at 6 h). This indicates that in this cell line, even at 4°C, the peptides were still able to efficiently translocate into the cell. No significant differences were observed in the uptake of α2H-FITC at 4°C on both cell lines compared to the uptake of the peptide at 37°C. This suggests that α2H-FITC uptake is not strictly dependent on endocytosis.

FIG 3
CPP-FITC enters cells via endocytosis and partially via direct translocation. (A and B) FACS analysis of CPP-FITC uptake in HeLa (A) and HBMEC (B) cells. CPP-FITC (750 nM) was incubated continuously with cells at 4°C, and the fluorescence intensity ...

We next analyzed the involvement of endocytosis in the uptake of these CPPs and, in particular, which endocytic pathway might be involved. For this, the intracellular uptake of CPP-FITC was determined in the presence of different inhibitors of endocytosis (Fig. 3C). The uptake of α1H-FITC was strongly reduced by methyl-β-cyclodextrin (MβCD) (56) and dynasore (57), a disruptor of lipid rafts and an inhibitor of dynamin- and clathrin-mediated endocytosis, respectively, and, to lesser extents, by nocodazole, an inhibitor of microtubule polymerization (58), and filipin, a disruptor of lipid rafts (59). These data suggest both that α1H-FITC enters cells preferentially by clathrin-dependent and lipid raft-dependent endocytosis and also that microtubule-dependent macropinocytosis is involved. The uptake of α2H-FITC was strongly reduced only in the presence of MβCD, while no effect was observed in the presence of filipin (Fig. 3C). While the first reagent removes cholesterol from the membrane, the second simply binds and masks cholesterol in the membrane (60), indicating only a minimal involvement for lipid raft-mediated endocytosis in α2H-FITC uptake. In addition, more than a 70% reduction of Tat-FITC uptake was observed with MβCD and dynasore, and an almost 50% reduction of uptake was observed in the presence of cytochalasin D (inhibitor of F-actin elongation) (61), filipin, and nocodazole (Fig. 3C). Even though these data are not statistically significant, there appears to be a trend. Amiloride, an inhibitor of macropinocytosis (19, 62), did not affect the uptake of any of the CPP-FITC.

To determine whether direct penetration was also involved in the translocation of these peptides, a membranolysis assay was performed (46). This assay is based on the measurement of the fluorescence emission of the cell-impermeable compound propidium iodide (PI) once it intercalates into DNA during an ongoing incubation of cells with FITC-labeled CPPs. PI accumulated in both HeLa cells and HBMEC upon incubation with CPP-FITC, indicating that CPP-FITC internalization induces a partial destabilization of the membrane integrity in these two cell lines (Fig. 3D and andE).E). However, the percentage of the overall PI uptake measured during CPP-FITC incubation was considerably lower than that for treatment with 0.2% Triton X-100, which was used as a positive control for membrane permeabilization, allowing PI diffusion. These results show that endocytosis seems to be the major entry mechanism for all CPPs. α2H-FITC prefers lipid raft-mediated endocytosis in HeLa cells, while Tat-FITC and α1H-FITC seemed to enter via endocytosis through multiple endocytic pathways. However, direct translocation might occur as a parallel entry mechanism for CPP in HeLa cells and HBMEC, as uptake at 4°C was not completely blocked.

Cellular localization of CPP-FITC conjugates with respect to intracellular localization of bacteria.

After we confirmed that the CPP-FITC conjugates reached endosomal compartments and finally the cytosol, we investigated the ability of CPP conjugates to target intracellular bacteria in infected cells. For this purpose, after infection of HBMEC with E. coli K1 and HeLa cells with Salmonella enterica serovar Typhimurium or Shigella flexneri, CPP-FITC conjugates were added directly to infected cells for 1 h. Since the CPP appear to initially enter the endosomal pathways via different mechanisms of endocytosis and, moreover, intracellular bacteria resist and survive in vacuoles (particularly E. coli, Salmonella, and, during early stages of infection, Shigella) (10, 63, 64), colocalization studies of the lysosome-associated membrane protein 1 (LAMP-1) with bacteria and CPP-FITC conjugates were performed. As already described by other groups, intracellular E. coli and Salmonella are mainly localized in LAMP-1-positive vacuoles (Fig. 4A and andB).B). Although the total intracellular amount differed between the single CPPs, Tat-, α1H-, and α2H-FITC conjugates appeared to be distributed in the cytosol and were partially present in LAMP-1-positive compartments (Fig. 4A and andB,B, yellow signal in merged images). Along with this, clear colocalization of CPP-FITC conjugates with LAMP-1-positive bacterium-containing vacuoles was observed (Fig. 4A and andB,B, white arrows). This indicates that after uptake into infected cells, CPPs also reached intracellular Salmonella and E. coli in vacuoles.

FIG 4
Immunofluorescence analysis of infected cells after treatment with CPP-gentamicin in LAMP1-positive compartments. (A) CLSM of HBMEC and infected HBMEC with E. coli K1 either left untreated or treated with the indicated CPP-FITC conjugates for 1 h. (B ...

In contrast, intracellular Shigella isolates are only partially localized in LAMP-1-positive vacuoles and, moreover, appear to be free in the cytosol (Fig. 4C). As described before (64), these bacteria likely have already escaped from vacuoles at this point of infection. However, colocalization of Tat-, α1H-, and α2H-FITC with Shigella could still be observed in the cytosol of infected cells but not in LAMP-1-positive compartments (Fig. 4C, white arrows). So far, it is unclear whether the CPP targeted the cytosolic bacteria from the cytoplasm and accumulated on their cell wall or whether the CPPs were carried along with Shigella during their escape from vacuoles. Both possibilities might be feasible, as the CPP could also be found in LAMP-1-positive endosomes in the absence of bacteria as well as distributed in the cytosol (Fig. 4C).

Although the exact mechanism of how Tat, α1H, and α2H target intracellular bacteria has to be evaluated in future studies, these CPPs appear to be useful to deliver an antibiotic such as gentamicin to sites of intracellular bacteria in infected host cells.

Conjugation of CPP-gentamicin does not alter the antibacterial activity of gentamicin.

The ability of CPPs to deliver attached cargo into cells makes them an attractive option as a drug delivery system. In this context, we used CPPs to deliver the aminoglycoside antibiotic gentamicin. Our conjugation strategy was based on the formation of a covalent bond between gentamicin and the peptides using the cross-linker succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC). SMCC allows conjugation between an amine group, provided by gentamicin, and a thiol group, provided by a cysteine present in the peptide sequence. For Tat the cysteine was added to the sequence at the N terminus and for α1H at the C terminus, while for α2H the cysteine of the original sequence was used. Gentamicin was first functionalized by conjugation with the linker via a stable amide bond. Subsequently, the addition of the CPP to the reaction mixture allowed the formation of CPP-gentamicin conjugates by formation of a thioether bond between the maleimide group of the linker and the thiol group of the Cys residue of the specific peptide (Fig. 5A).

FIG 5
CPP-gentamicin conjugates maintain gentamicin antibacterial activity and are not cytotoxic. (A) Schematic representation of CPP-gentamicin conjugation. Gentamicin and the cross-linker SMCC were mixed in PBS and incubated for 2 h at RT to form a stable ...

CPP-gentamicin conjugates were checked for possible cytotoxic effects in HeLa cells and HBMEC by both lactate dehydrogenase (LDH) and bromodeoxyuridine (BrdU) assays. Each single component of the conjugation reaction (single CPPs, gentamicin, SMCC, and dimethyl sulfoxide [DMSO]) was tested separately. In HeLa cells, only Tat-gentamicin, α1H, and gentamicin showed a slight increase in the release of LDH compared to the respective controls (DMSO for Tat-gentamicin or phosphate-buffered saline [PBS] for the others), indicating a partial disruption of cell membrane (Fig. 5B). In HBMEC, treatment with α2H-gentamicin led to a significant increase in LDH release (10-fold) compared to cells treated with DMSO, indicating a slight cytotoxic effect of the compound. In addition, treatment with Tat or gentamicin alone showed a minimal increase in the LDH release compared to PBS treatment. However, in all cases, the increase in LDH release was not significantly higher than that of the untreated cells, indicating an overall noncytotoxic effect of the CPP-gentamicin conjugates or of the single compounds. In the BrdU proliferation assay, on the other hand, only unconjugated SMCC showed a significant decrease in both HeLa and HBMEC proliferation compared to untreated cells (Fig. 5C). In all other cases, CPP-gentamicin slightly reduced cell proliferation by about 20 to 30%, although this was not significant. The reduction of cell proliferation induced by CPP-gentamicin was similar to that induced by free gentamicin, which caused a 20% reduction in cell proliferation for both cell lines. In addition, unconjugated CPP and gentamicin together did not show a significant decrease in cell proliferation.

To ensure that the CPP-conjugated gentamicin maintained its antimicrobial activity, the bactericidal effect of CPP-gentamicin conjugates was tested in a functionality assay against different Gram-negative bacteria, such as E. coli K1 RS218, Salmonella enterica serovar Typhimurium, and Shigella flexneri. Incubation of Tat-gentamicin, α2H-gentamicin, and α1H-gentamicin in liquid cultures of E. coli K1, Salmonella, and Shigella inhibited bacterial growth with efficiencies comparable to that of unconjugated gentamicin, indicating that conjugated gentamicin retained its antimicrobial activity (Fig. 5D to toFF).

To exclude possible side effects caused by single components of the conjugation reaction, liquid cultures of E. coli K1, Salmonella, and Shigella were also incubated with 600 μg/ml of CPP or SMCC and 6% DMSO, and the OD600 was measured. While α1H and α2H did not show any effect on bacterial viability, Tat had a strong bactericidal effect against E. coli K1, causing a 10-fold decrease in the OD600 after 4 h compared to the control (Fig. 5D). Interestingly, Shigella growth was also partially (though not statistically significantly) affected by Tat (Fig. 5E), whereas no differences were observed for Salmonella (Fig. 5F). Similarly, SMCC and DMSO caused a significant reduction in E. coli K1 and Salmonella growth compared to the control, indicating that SMCC itself is not toxic for the bacterium (Fig. 5D and andF).F). A reduction of Shigella growth was observed in the presence of SMCC, whereas DMSO did not affect bacterial growth (Fig. 5E).

Finally, the susceptibility of the three pathogens to the CPP-gentamicin conjugates was determined by MIC assay. The MIC of CPP-gentamicin conjugates ranged between 1 and 2 μg/ml for all three bacteria, which is only about 2- to 4-fold higher than that for free gentamicin (0.5 μg/ml) (Table 1). The only exception is the effect of Tat-gentamicin on E. coli K1 RS218, which showed the same MIC as that for gentamicin. The same conjugates had a MIC of 8 μg/ml on a gentamicin-resistant strain, while a concentration of more than 32 μg/ml of a single CPP was needed to inhibit the bacterial growth.

TABLE 1
MIC of conjugated CPP and gentamicin on intracellular pathogens

CPP-mediated delivery of gentamicin increases antibiotic efficiency against intracellular pathogens.

As the CPP-gentamicin conjugates were shown to be effective as bactericidal compounds, we next investigated the ability of the CPP to mediate the delivery of functional gentamicin into infected eukaryotic cells and to target and kill intracellular bacteria. A modified gentamicin protection assay was developed to use CPP-gentamicin as an antibacterial for both extra- and intracellular bacteria. In this, CPP-gentamicin conjugates were directly applied to infected cells. HBMEC infected with E. coli K1 and treated with CPP-gentamicin showed a strong reduction in the recovered intracellular bacteria. Tat-gentamicin was the most effective, with a 986-fold reduction of bacteria compared to treatment with nonconjugated gentamicin, whereas treatment with α2H- and α1H-gentamicin caused a 50- and 14-fold reduction, respectively (Fig. 6A). Control treatment with SMCC, DMSO, and PBS did not affect the intracellular bacteria. Doxycycline, which as a cell-permeable antibiotic served as a positive control, killed all intracellular E. coli K1 RS218 organisms (Fig. 6A).

FIG 6
CPP-gentamicin efficiently reduced the amount of intracellular E. coli K1, Shigella, and Salmonella in infected cells. (A) HBMEC were infected with E. coli K1 RS218 before adding CPP-gentamicin (200 μg/ml gentamicin, 600 μg/ml CPP and ...

Similarly, a reduced amount of intracellular E. coli K1 was also observed in HBMEC treated with CPP-gentamicin compared to unconjugated gentamicin treatment by a fluorescence microscopy analysis (Fig. S1).

Treatment with unconjugated Tat, α2H, and α1H and gentamicin together caused a 5-, 8-, and 7-fold reduction, respectively, of intracellular E. coli K1 compared to free gentamicin, indicating, even though not significantly, that during the uptake process, CPPs cause transient membrane destabilization leading to gentamicin influx (Fig. 6B), consistent with the slight PI uptake during CPP-FITC penetration (Fig. 3D). While α1H and α2H did not induce any changes in intracellular bacterial numbers, Tat reduced intracellular bacterial loads 130-fold compared to PBS treatment but not significantly compared to gentamicin treatment (Fig. 6B).

Finally, the CPP-gentamicin antibacterial activity also proved to be effective against infection of HeLa cells with Salmonella and Shigella. In Shigella-infected cells, a significantly reduced number of intracellular bacteria was counted after treatment with Tat-gentamicin (1,950-fold), α2H-gentamicin (246-fold), or α1H-gentamicin (138-fold) compared to gentamicin treatment (Fig. 6C). In Salmonella-infected cells, the number of intracellular bacteria was reduced between 65- and 88-fold after incubation with CPP-gentamicin conjugates compared to gentamicin treatment (Fig. 6D). Single CPPs, SMCC, and DMSO had no effect on Shigella- and Salmonella-infected cells. In contrast, doxycycline did show a reduction of intracellular bacteria by approximately one log phase compared to gentamicin (Fig. 6C and andDD).

In addition to the CPP-gentamicin antibacterial activity assay, we also performed electron microscopy analysis of infected cells after treatment with the different conjugates to document lysis of intracellular bacteria on the ultrastructural level. For this purpose, after infection of HBMEC and HeLa cells with bacteria for 1.5 h, CPP-gentamicin conjugates were added for an additional 1.5 h, and samples were subsequently prepared for electron microscopy. Transmission electron microscopy (TEM) revealed that after infection and incubation with CPP-gentamicin, the cells were morphologically similar to uninfected and untreated control cells (Fig. 7). To better illustrate the integrity of the eukaryotic cells, lower-magnification images of uninfected HBMEC and HeLa cells are shown (Fig. 7A and andC).C). In the absence of CPP-gentamicin, most of the intracellular Salmonella, Shigella, and E. coli K1 RS218 organisms reside in mainly intact endosomal compartments or vacuoles and showed an intact cell wall and regular cell morphology (Fig. 7A and andC).C). However, after additional incubation with α1H-, α2H-, or Tat-gentamicin, the majority of intracellular bacteria were clearly found in various states of lysis and digestion (Fig. 7B and andD).D). In most cases, Salmonella, Shigella, and E. coli K1 RS218 completely lost their cell wall integrity, and if they were present at all, the morphology of the bacteria was highly disturbed (Fig. 7B and andD),D), indicating the intracellular bactericidal activity of gentamicin after delivery by CPPs. Interestingly, after incubation with Tat-gentamicin, we observed in close proximity to the bacteria electron-dense fuzzy material, independent of the type of bacteria or cell (Fig. 7B and andD).D). Similar observations were made for extracellular bacteria, which were incubated with Tat-gentamicin (Fig. S2), indicating that this fuzzy material resembles detached components of the bacterial cell wall and might be a consequence of the membrane-destabilizing bactericidal activity of Tat as reported previously (65).

FIG 7
Electron microscopic analysis of infected cells after treatment with CPP-gentamicin. (A) HeLa cells and HeLa cells infected with Salmonella enterica serovar Typhimurium and Shigella flexneri before adding CPP-gentamicin. (B) HeLa cells infected with ...

Taken together, our results show that CPP-gentamicin conjugates target and efficiently kill both extracellular and intracellular pathogenic bacteria, such as E. coli K1 RS218, Salmonella enterica serovar Typhimurium, and Shigella flexneri. Thus, for combating bacterial infections involving intracellular pathogens, using CPP to engineer membrane-permeable antimicrobials might offer novel therapeutic options.

DISCUSSION

Cell-penetrating peptides are able to deliver active biological molecules into eukaryotic cells. While there are many examples of viral proteins that contain CPPs, only a few bacterial CPPs have been characterized (66). We previously identified a PTD in the Y. enterocolitica YopM effector protein (28). The PTD is localized at the N-terminal double α-helical domain of the protein, named 2αH, yet each single helix has protein transduction capacity. Here, we characterized the two YopM-derived PTDs, α1H and α2H, as cell-penetrating peptides and showed that they can deliver bioactive molecules through the eukaryotic cell plasma membrane.

The two peptides were predicted to assume an α-helical secondary conformation, in agreement with the Y. pestis YopM crystal structure (41). Simulations of membrane interactions indicated that α1H displays a stronger interaction with the lipid bilayer, while only a few residues of α2H condensed at the C-terminal side of the peptide interact with the membrane (Fig. 1). Hydrophobicity analyses revealed that while α1H has a main hydrophobic pattern with only a few basic residues, α2H possesses several hydrophobic residues interspersed with basic and acidic residues (see Table S1 in the supplemental material). Thus, considering the general classification of CPPs as cationic, amphipathic, or hydrophobic (27), we suggest that α1H belongs to the hydrophobic class, whereas α2H is a primary amphipathic peptide. The well-characterized Tat peptide belongs to the cationic peptide subgroup (40), and indeed, Tat contains more than 60% basic, positively charged residues.

In HBMEC, Tat, α1H, and α2H rapidly accumulated inside cells and were localized to the cytosol (Fig. 2B and andC).C). In addition, the reduced but still prominent uptake of the peptides at 4°C and the slight intracellular accumulation of PI in HBMEC suggested a major involvement of direct penetration over endocytosis (Fig. 3B and andE).E). In HeLa cells, the CPPs were distributed homogeneously in the cytosol with partial endosomal compartmentalization. This is consistent with our observations using endocytosis inhibitors in HeLa cells (Fig. 3C). While the uptake of α2H occurs mainly by lipid raft-mediated endocytosis, for both Tat and α1H, multiple endocytic pathways, such as macropinocytosis and clathrin-dependent and lipid raft-dependent endocytosis, could be involved in the cellular entry mechanism.

It is known that CPPs can utilize different entry routes simultaneously and that their uptake depends on many factors, such as the delivered cargo, physicochemical characteristics of the peptides, and, no less important, the particular experimental conditions (67). For Tat, several endocytic routes have been proposed, such as macropinocytosis (68), clathrin-mediated endocytosis (69), and caveola-mediated endocytosis (70). Further, it has been demonstrated that when conjugated to a fluorophore, Tat prefers clathrin-mediated endocytosis (69), which is in accordance with our findings. Interestingly, filipin, which binds cholesterol, failed to reduce α2H uptake. As cholesterol is important for endosomal vesicle formation, and as it was found that depletion of cholesterol inhibits the transport of endosomes to the Golgi apparatus (71), the inhibition of α2H uptake in the presence of MβCD might result from defective formation and trafficking of endocytic vesicles. Regardless of the mechanism, at least a fraction of the endocytosed peptides escaped from endosomes and was distributed into the cytosol (Fig. 2C).

However, as we have shown, the uptake at 4°C of both α1H and α2H in HeLa cells was strongly reduced but not completely inhibited, and PI was internalized in these cells during incubation with the two CPPs (Fig. 3A and andD).D). These data suggest that α1H and α2H employ direct translocation as an alternative or even parallel uptake mechanism to enter HeLa cells. This is in accordance with our previous findings for full-length YopM which primarily enters cells by endocytosis but also employs direct membrane penetration as an entry mechanism (28, 29). In contrast, Tat uptake was completely inhibited at 4°C in HeLa cells, suggesting that direct translocation of Tat can be excluded in this cell line.

More importantly, we could show that Tat, α1H, and α2H were able to target intracellular Salmonella and E. coli in LAMP-1-positive vacuoles and also Shigella in the cytosol (Fig. 4A to toC).C). Details of how the CPPs reach the intracellular bacteria are not known yet, but it is likely that after endocytosis and subsequent endosomal trafficking, CPP-containing vesicles fuse with bacterium-containing vacuoles. In contrast, penetration of bacterium-containing vacuoles by cytosolic CPPs appears to be unlikely, as the membrane leaflets of vacuoles are inverted with respect to the plasma membrane. Additionally, it is still elusive how the CPPs are able to target cytosolic Shigella. Either the CPPs were carried along with Shigella during their escape from vacuoles or the CPPs can target the cytosolic bacteria from the cytoplasm and accumulate on their surface due to electrostatic interactions of the positively charged peptides with negatively charged components of the bacterial cell wall. In any case, all peptides entered infected host cells and targeted intracellular bacteria, underlining the feasibility of delivering impermeable antimicrobial agents at the site of infections.

In this regard, many antibiotics have poor cell permeability and hence fail to target intracellular pathogens. To overcome this disadvantage, the permeability for gentamicin has been enhanced by encapsulating the drug into pH-sensitive liposomes (12, 72). Nevertheless, liposomes possess low stability, low drug entrapment, difficult particle size control, and short half-lives (73). Here, we adopted a different strategy based on the use of CPPs as delivery vehicles. Many organic molecules, such as the anticancer drug doxorubicin (74) or the antifungal drug natamycin (75), have been successfully delivered by CPPs and their therapeutic efficacy has been increased. Recently, Abushahba et al. (76) used CPPs to deliver peptide nucleic acids (PNAs) targeting bacterial genes, such as RNA polymerase, to kill Listeria monocytogenes in vitro, or in cell culture and in C. elegans infection models. However, to our knowledge, an antibiotic has never been conjugated to and delivered by CPPs. Thus, Tat and the YopM-derived CPPs were used as a new delivery strategy for gentamicin. The chemical conjugation of Tat, α1H, and α2H peptides with gentamicin was mediated by the cross-linker SMCC (Fig. 5A), as it has already been used successfully to conjugate CPP with the organic molecule doxorubicin (74). The CPP-gentamicin conjugates showed strong antibacterial activity, inhibiting the growth of three exemplary Gram-negative pathogenic bacteria (E. coli K1 RS218, S. enterica serovar Typhimurium, and S. flexneri) in liquid culture to the same extent as free gentamicin (Fig. 5D to toF).F). Consistent with this, CPP-gentamicin showed a strong susceptibility to the pathogenic bacteria, with a MIC of only 1 to 2 μg/ml (and even 0.5 μg/ml for Tat-gentamicin on E. coli) compared to 0.5 μg/ml of unconjugated gentamicin (Table 1), whereas the single peptides did not show significant antibacterial activity, as their MIC in all cases measured >32 μg/ml. Even though such low concentrations of CPP-gentamicin conjugates are enough to inhibit bacterial growth, in this study we used a concentration of 200 μg/ml of gentamicin, both conjugated or unconjugated to CPPs, which is often used in standard gentamicin protection assays (50). A high concentration of antibiotic in this case allowed the complete killing of bacteria remaining extracellularly to allow for the detailed investigation of only intracellular ones.

In addition, CPP-gentamicin conjugates did not show significant cytotoxic effects on eukaryotic cells, as shown by both LDH and BrdU assays (Fig. 5B and andC).C). Although the low cytotoxicity of the conjugates seemed promising, several limitations, such as poor serum stability and considerable immunogenicity, which have been reported for CPPs (77, 78), might limit their in vivo applications. These potential drawbacks of CPP-antimicrobial conjugates need to be investigated in future studies, and if necessary, the conjugates need to be optimized to ensure their usability for therapeutic applications.

Importantly, CPP-gentamicin conjugates also showed antimicrobial activity in infected cells, drastically reducing the number of intracellular E. coli K1 RS218, Shigella, or Salmonella organisms in infected HBMEC or HeLa cells (Fig. 6A to toD).D). Moreover, lysis and destruction of intracellular bacteria after addition of CPP-gentamicin conjugates to infected cells was confirmed on the ultrastructural level by electron microscopy (Fig. 7). We used the cell-permeable doxycycline to compare its antimicrobial activity against intracellular bacteria with that of CPP-gentamicin. Tetracyclines are broad-spectrum antibiotics that exhibit activity against a wide range of Gram-positive and Gram-negative bacteria, including the enteric bacteria Salmonella and Shigella, and have been shown to diffuse in eukaryotic cells (79,81). For all tested bacteria, doxycycline showed an increased antibacterial activity compared to CPP-gentamicin (Fig. 6A to toDD).

The amount of recovered E. coli K1 RS218 from HBMEC-infected cells treated with unconjugated Tat was reduced compared to α1H or α2H treatment (Fig. 6B). Here, Tat might exert an antimicrobial activity before being degraded by cell-released proteases. Previous reports have identified Tat (47,58) as an antimicrobial peptide with broad-spectrum antibacterial activity against many Gram-positive and Gram-negative bacteria, including S. aureus, E. coli, and S. Typhimurium (65, 82). We also observed a marked bactericidal effect of Tat against E. coli K1 RS218 and partly against Shigella but not against Salmonella in functionality assays (Fig. 5D to toF).F). This bactericidal effect of Tat might also explain the massive cell wall destruction of bacteria observed by electron microscopy for samples that have been incubated with Tat-gentamicin (Fig. 7 and Fig. S2). Nevertheless, the antibacterial activity of Tat alone was not comparable to that of gentamicin, and α1H and α2H did not impair bacterial growth.

Incubation of unconjugated gentamicin and CPPs in E. coli K1-infected cells caused a 5- to 8-fold reduction in the number of intracellular bacteria compared to free gentamicin (Fig. 6B). As the uptake of CPPs leads to intracellular accumulation of PI in HBMEC, we cannot exclude completely that during the uptake process of CPPs some free gentamicin might diffuse into the infected cells, causing the observed bacterial killing. Overall, these results suggest that the antibacterial activity of CPP-gentamicin conjugates is dependent upon their effective intracellular penetration.

In summary, we developed and characterized two novel CPPs that are derived from the bacterial effector protein YopM, α1H and α2H. We showed that the YopM-derived CPPs, as well as the Tat peptide, might be used as vehicles to increase the cell permeability to and intracellular delivery of antimicrobial agents such as antibiotics (e.g., gentamicin). This delivery system can be adapted and modified further to enhance the uptake of other antimicrobials and thus improve the treatment of infectious diseases.

MATERIALS AND METHODS

Computational analyses.

The 3D structure of YopM derived from Y. enterocolitica was modeled based on the crystal structure of Y. pestis YopM (PDB entry 1JL5) (41) using PyMOL. Secondary structure predictions of the sequence of the YopM34-73 fragment were obtained via the web server PSIPRED (42). The secondary structure propensity in membrane or water environments, as well as the membrane simulation, was obtained for the peptide sequences via the MCPEP web server (43). The helical wheel projection was obtained by using the online tool HeliQuest (44).

Peptides and chemical reagents.

The sequences of the α1H and α2H peptides were obtained from the bacterial effector protein YopM, derived from Y. enterocolitica O:8 JB580v (pYV8081). All peptides were synthesized commercially by GenScript (Piscataway, NJ, USA). For conjugation purposes an N-terminal Cys residue was added to the Tat sequence and a C-terminal Cys to the α1H peptide sequence (indicated in boldface below). For α2H, the natural Cys residue was used. The lyophilized peptides Cys-Tat (47,57) (shorten Tat) (CYGRKKRRQRR), α1H (KSKTEYYNAWAVWERNAPC), and α2H (GNGEQREMAVSRLRDCLDRQA) were resuspended in water to a stock concentration of 10 mg/ml. Fluorescein isothiocyanate (FITC) was coupled to peptide N termini by GenScript. Lyophilized FITC-labeled peptides were resuspended in water at a stock concentration of 10 mg/ml, except α1H-FITC, which was resuspended in dimethylformamide (DMF) at 5 mg/ml. All chemicals were purchased from Sigma-Aldrich GmbH (Munich, Germany) unless otherwise stated.

CPP-gentamicin conjugation.

CPP and gentamicin were covalently conjugated using the heterobifunctional cross-linker succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC; Thermo Fisher Scientific, Braunschweig, Germany). CPP-gentamicin conjugates were prepared by mixing gentamicin with SMCC dissolved in DMSO. PBS was used as conjugation buffer. After 2 h of incubation at room temperature (RT), CPPs were added and additionally incubated for 2 h at RT. For CPP-gentamicin functionality assays, 50 μg/ml gentamicin (104 μM) was conjugated to 150 μg/ml CPP (90 μM Tat, 62 μM α2H, and 64 μM α1H) and 150 μg/ml SMCC (448 μM). For all other experiments, 600 μg/ml of CPP (361 μM Tat, 248 μM α2H, and 259 μM α1H) was conjugated to 200 μg/ml of gentamicin (418 μM) and 600 μg/ml of SMCC (1.79 mM). When free SMCC was used as a control, the N-hydroxysuccinimide ester group of SMCC was inactivated by incubation with 5 mM or 1.5 mM arginine and glutamic acid solution (pH 8.0) in PBS for 2 h at RT. Subsequently, 5 mM or 1.5 mM cysteine (pH 8.0) in PBS was added and incubated for an additional 2 h at RT to quench the maleimide group. The CPP-gentamicin conjugates or inactivated SMCC were stored at 4°C and used within 2 days.

Cell lines and culture.

HeLa (ATCC CCL-2) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 1 g/liter glucose, 10% fetal bovine serum (FBS), 1% nonessential amino acids (NEAA) (PAA Laboratories GmbH, Cölbe, Germany), 100 U/ml penicillin, and 100 μg/ml streptomycin. Human brain microvascular endothelial cells (HBMEC) were cultured in RPMI 1640 medium with 10% FBS, 10% Nu-serum (BD Bioscience, Heidelberg, Germany), 1% NEAA, 1 mM sodium pyruvate, 1% minimal Eagle's medium vitamins, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were grown at 37°C in a 5% CO2 atmosphere and subcultured up to 80% confluence before passaging. Before each experiment, the medium was removed and exchanged with fresh medium without antibiotics.

Bacterial strains.

E. coli K1 RS218, a clinical isolate from a newborn with meningitis (45), was grown in LB broth. Salmonella enterica serovar Typhimurium strain ATCC 14026 was grown in MacConkey broth purple (AppliChem, Darmstadt, Germany). Shigella flexneri strain DSM 4782 was grown in brain heart infusion (BHI) (BD Bioscience, Heidelberg, Germany). For plating, 1.5% agar (AppliChem, Darmstadt, Germany) was added.

Quenched time-lapse uptake assay and membranolysis monitoring.

The kinetics of CPP-FITC peptide uptake were measured by performing quenched time-lapse uptake assays (46). To measure peptide uptake rates, trypan blue was added to quench extracellular fluorescence. HeLa cells and HBMEC were grown to confluence, trypsinized, and resuspended in PBS prior to incubation with 750 nM CPP-FITC peptides at 37°C or 4°C. During the ongoing incubation, samples were taken at each time point, 0.2% trypan blue was added, and samples were directly analyzed by flow cytometry using a BD FACSCalibur flow cytometer (BD Biosciences, Heidelberg, Germany). To monitor potential deleterious effects of CPP-FITC on membrane integrity, 1 μg/ml propidium iodide (PI) was added during coincubation with CPP-FITC, and the fluorescence of intracellular PI was recorded by flow cytometry as a measure of membranolysis (47). As a positive control for disruption of membrane integrity, 0.2% Triton X-100 (AppliChem, Darmstadt, Germany) was added before the measurements. Viable cells were gated according to the side and forward scatter.

Endocytosis inhibitors.

To determine the involvement of endocytosis in CPP uptake, HeLa cells were pretreated with 3 mM amiloride, 2.5 μM cytochalasin D, 80 μM dynasore (Enzo Life Science, Antwerp, Belgium), 5 μg/ml filipin, 5 mM methyl-β-cyclodextrin (MβCD), or 20 μM nocodazole for 1 h at 37°C before adding 750 nM CPP-FITC. DMSO was used as a solvent control. After 6 h of incubation, samples were mixed with 0.2% trypan blue and the intracellular fluorescence of the FITC-labeled peptides was measured using flow cytometry.

Fluorescence microscopy.

To analyze CPP-FITC uptake and intracellular distribution, HeLa cells and HBMEC were cultured on 12-mm glass coverslips in 24-well plates and incubated with 1 μM CPP-FITC. Cells were washed twice with 0.05% Tween 20 for 20 min with gentle agitation to remove bound extracellular peptides. Cells were then fixed with 4% paraformaldehyde (PFA) and quenched with 0.2% glycine. Actin and DNA were counterstained with phalloidin-tetramethyl rhodamine isothiocyanate (TRITC) and with Draq5 (BioStatus, Shepshed, United Kingdom), respectively. Subsequently, immunofluorescence was evaluated with a BZ-9000 BIOREVO fluorescence microscope (Keyence, Osaka, Japan), adjusting the exposure settings to the negative controls.

To detect intracellular bacteria, HBMEC infected with E. coli K1 RS218 were treated with CPP-gentamicin, 200 μg/ml gentamicin, or 600 μg/ml SMCC or were left untreated for 90 min. Cells were then extensively washed and prepared as described above. Cellular and bacterial DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI; AppliChem, Darmstadt, Germany). The samples were analyzed with a BZ-9000 BIOREVO fluorescence microscope.

CLSM.

For confocal laser scanning microscopy (CLSM), all cells were grown in 8-well chamber slides (ibidi, Planegg/Martinsried, Germany). HBMEC were infected with E. coli K1 for 1.5 h at a multiplicity of infection (MOI) of 100:1 (bacteria to eukaryotic cells). Accordingly, HeLa cells were infected with Salmonella enterica serovar Typhimurium at an MOI of 100 for 1.5 h, and infection of HeLa cells with Shigella flexneri was carried out at an MOI of 10 for 1.5 h. After extensive washing, infected cells were incubated in fresh cell culture medium containing 100 μg/ml gentamicin for 18 h. After bacterial infections, CPP-FITC conjugates (10 μg/ml) were added directly to infected cells for 1 h. After washing with PBS, fixation with 4% paraformaldehyde, quenching with 0.2 M glycine (pH 7.2), permeabilization in 0.25% Triton X-100, and blocking with 5% bovine serum albumin (Sigma-Aldrich, Munich, Germany), cells were stained with anti-LAMP1 antibody (D2D11; Cell Signaling Technology, Denver, CO) and Cy3-conjugated goat anti-rabbit IgG (Sigma-Aldrich, Munich, Germany). Nuclei were counterstained with Draq5 (Cell Signaling Technology, Denver, CO). The preparations were mounted in fluorescence mounting medium (Dako/Biozol, Eching, Germany) and analyzed with a confocal laser-scanning microscope (LSM 510 META microscope equipped with a Plan-Apochromat 63×/1.4-numeric-aperture oil immersion objective; Carl Zeiss, Oberkochen, Germany).

CPP-gentamicin functionality assay.

CPP-gentamicin functionality assays were performed to assess the antibacterial activity of the conjugated gentamicin. The protocol was modified according to Jones et al. (48). Briefly, overnight cultures of E. coli K1, Salmonella, and Shigella were diluted 1:100 in fresh medium before adding CPP-gentamicin conjugates. The OD600 was measured before the addition of CPP-gentamicin (0 h) and after 4 h of incubation. As controls, the single components of the conjugation reactions (50 μg/ml gentamicin; 150 μg/ml unconjugated Tat, α1H, α2H, or inactivated SMCC; and 6% DMSO) were added separately to the bacterial cultures and the OD600 was measured as described above.

Measurements of the MIC (MIC assay).

The MIC of CPP-gentamicin on bacterial growth was determined according to guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST).

The MIC assay was performed in sterile 96-well microtiter plates with a final volume of 100 μl/well of Mueller-Hinton broth (MHB) medium. CPP-gentamicin conjugates were prepared as described for CPP-gentamicin assay. For the MIC assay, free gentamicin and CPP-gentamicin conjugates were serially diluted from 64 μg/ml to 0.5 μg/ml in 50 μl of MHB medium. Unconjugated CPP dilutions ranged from 192 μg/ml to 2 μg/ml in 50 μl of MHB medium.

Blood agar plates containing single colonies of each tested bacterium (E. coli K1, S. flexneri, and S. Typhimurium) were prepared 24 h before the MIC assay. Single colonies were inoculated in 0.5 ml of 0.048 M BaCl2 (1.17%, wt/vol, BaCl2 · 2H2O) and 99.5 ml of 0.18 M H2SO4 (1%, vol/vol) with constant stirring until they measured 0.5 ± 0.3 McFarland standard absorbance. The bacteria inoculum (50 μl; corresponding to 1 × 105 CFU) was added to each well containing serial dilutions of gentamicin, CPP-gentamicin, and unconjugated CPP, so that the final concentration ranged from 32 μg/ml to 0.25 μg/ml for the former and from 96 μg/ml to 1 μg/ml for the latter. The inhibition of growth was visually observed after 24 h of incubation at 37°C. Sensitive bacteria grow with <4 μg/ml gentamicin or conjugates, whereas resistant bacteria grow with >8 μg/ml gentamicin or conjugates (EUCAST 2012 clinical breakpoints, http://www.eucast.org/clinical_breakpoints).

CPP-gentamicin assay.

For CPP-gentamicin assay, HBMEC were infected as described before (49). Cells were infected with 20 μl of an overnight culture of E. coli K1 for 1.5 h. Salmonella and Shigella infection of HeLa cells was modified from protocols of references 50 and 51, respectively. Static cultures of Salmonella and Shigella were grown overnight. After overnight growth, 3.3 × 106 CFU/ml of Salmonella or Shigella at an MOI of 100 was added to the cells, the cells were centrifuged at 700 × g for 10 min to allow bacterial contact with the cells, and they were incubated for 1.5 h to allow for bacterial invasion.

CPP-gentamicin assays were performed to determine the ability of CPP-gentamicin conjugates to kill extra- and intracellular bacteria simultaneously. After bacterial infections, CPP-gentamicin conjugates were added directly to infected cells for 1.5 h. Alternatively, a mixture of unconjugated CPP and gentamicin (CPP + gentamicin), at the same concentration as that used in the conjugation reaction, was briefly premixed before adding to the cells. As controls, nonconjugated CPPs, free gentamicin, inactivated SMCC, DMSO, and Dulbecco's PBS (DPBS) were added at the same concentrations as those used in the conjugation reaction. In addition, 200 μg/ml doxycycline was used as a positive control. After incubation, cells were washed with PBS and lysed with 0.2% Triton-X (AppliChem, Darmstadt, Germany). The cell lysates containing the intracellular bacteria were then serially diluted in PBS and plated on agar plates for counting. The level of viable intracellular bacteria was calculated as the percentage of intracellular bacteria recovered after free gentamicin treatment.

Electron microscopy.

Infection of HBMEC and HeLa cells with bacteria and subsequent incubation with CPP-gentamicin was performed exactly as described for the CPP-gentamicin assay. After infection and incubation with CPP-gentamicin, cells were initially fixed in 2% glutaraldehyde in DPBS, pH 7.4, postfixed in 1% OsO4 (osmium tetroxide) in DPBS, dehydrated in inclusive “en bloc contrast” with uranyl acetate, and embedded in epon. Ultrathin sections (70 nm) of the samples were cut (UC6 ultramicrotome; Leica, Vienna, Austria) and subsequently counterstained with uranyl acetate and lead. The sample was analyzed at 80 kV on an FEI-Tecnai 12 electron microscope (FEI, Eindhoven, Netherlands). Photographs of selected areas were documented with a Veleta side-mounted transmission electron microscopy (TEM) camera (Emsis, Münster, Germany).

LDH assay.

The potential cytotoxicity of CPP-gentamicin conjugates was evaluated by measuring lactate dehydrogenase (LDH) release. The amount of LDH released upon the disruption of membrane integrity is proportional to the number of damaged cells (52). HeLa cells and HBMEC were incubated with CPP-gentamicin, unconjugated CPPs, SMCC, or gentamicin at the same concentration as that used in the conjugation reaction, 6% DMSO, or PBS for 2 h. The amount of LDH released in the supernatant or total LDH was measured by using the CytoTox 96 nonradioactive cytotoxicity assay kit (Promega GmbH, Mannheim, Germany) according to the manufacturer's instructions. The final result is presented as a percentage of LDH released in the supernatant versus total LDH upon cell lysis.

BdrU proliferation assay.

The potential cytotoxicity of CPP-gentamicin conjugates was evaluated by determining the number of proliferative cells in a BrdU assay. HeLa cells and HBMEC were incubated with CPP-gentamicin, unconjugated CPPs, SMCC, or gentamicin at the same concentration as that used in the conjugation reaction mixture for 1.5 h. After incubation, the medium was removed and the cells were incubated with new medium for 72 h to allow cell recovery. After allowing time for recovery, the cells were cultured for 3 h with labeling medium containing BrdU, which was incorporated into the newly synthesized DNA of proliferating cells. The amount of BrdU incorporated during DNA synthesis and cell division was detected according to the cell proliferation enzyme-linked immunosorbent assay BrdU colorimetric kit (Roche Diagnostic GmbH, Mannheim, Germany) by following the manufacturer's instructions. Each sample was analyzed in triplicate. The final result is presented as a percentage of cell proliferation compared to untreated cells.

Supplementary Material

Supplemental material:

ACKNOWLEDGMENTS

We thank the Institute of Hygiene, University of Münster, for kindly providing the Salmonella and Shigella strains and V. Humberg for assistance with CPP-gentamicin functionality experiments.

This work is part of the doctoral thesis of M.G.

This work was supported partially by grants of the Deutsche Forschungsgemeinschaft (Fellowship of the Graduate School of the Cells-in-Motion Cluster of Excellence [EXC 1003-CiM], University of Münster, Germany, to M.G. and the PP of PA689/13-1 [BP500061]) and by a personal fellowship to M.G. from the Medical Faculty Münster. T.F.C.M. has been funded by the National Center for Scientific and Technological Development, CNPq no. 204996/2014-7, from the Brazilian Government.

Footnotes

Supplemental material for this article may be found at https://doi.org/10.1128/AAC.02545-16.

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