University of Groningen Breaking walls: combined peptidic activities against Gram-negative human pathogens Li, Qian

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University of Groningen

Breaking walls: combined peptidic activities against Gram-negative human pathogens

Li, Qian

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Chapter

4

High synergistic

antimicrobial activity

against Gram-negative bacteria

of synthetic L-peptides

with vancomycin or nisin

Qian Li1, Manuel Montalban-Lopez1,2, Oscar P. Kuipers1

1Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, the Netherlands 2 Department of Microbiology, Faculty of Sciences, University of Granada, Spain

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Abstract

The development and dissemination of antibiotic-resistant bacterial pathogens is a global threat to public health. Novel compounds and/ or therapeutic strategies are required to face the challenge posed by, especially, Gram-negative pathogenic bacteria. The Gram-negative pathogens have developed high levels of resistance, often due to their poorly permeable or impermeable outer-membrane. The outer mem-brane avoids that very potent molecules against Gram-positive bac-teria can reach their target in Gram-negative microorganisms. Here, designed peptides (further termed GNPs), either alone or combined with vancomycin or nisin, were tested against selected Gram- negative pathogens demonstrating highly synergistic activity leading to dra-matically reduced minimal inhibitory concentration values (up to 32-fold). We observed that GNP-6 was very efficient alone and showed a modest synergism when combined with either vancomycin or nisin against the Gram-negative pathogens tested. In contrast, GNP-8 ex-hibited an astonishing synergism with vancomycin or nisin in spite of its poor activity when tested alone. Considering the size, charge and potency of the GNPs, GNP-6 and GNP-8 were selected for further tests. Overall, this approach constitutes a potent novel weapon against (drug- resistant) Gram-negative bacteria.

Key words:

Gram-negative bacteria, pathogens, outer-membrane-penetrating peptides, vancomycin, nisin, synergism

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CH APTER 4: I nt ro duc tio n

4 Introduction

With the alarming increase of antibiotic resistance, which has now reached a critical point, there is a major and serious worldwide threat to public health. Virtually no new broad- or narrow-spectrum antibi-otics have been developed for the last half century, while the existing drugs are rapidly becoming ineffective [1, 2]. Over the years, mankind became more and more dependent on antibiotics [1]. Unfortunately, the widespread use has undoubtedly increased the occurrence of bacterial antibiotic resistance [3, 4]. Already 700,000 deaths per year occur due to the spread of antimicrobial resistance (AMR), and this number is estimated to increase to 1,000,000 by the year 2050 [5]. Many of these infections are caused by Gram-negative pathogens. Therefore, search-ing new antibiotics or new therapeutic strategies against Gram-negative organisms is important and urgent.

Notably, the protective outer-membrane of Gram-negative bacteria functions as an efficient barrier to prevent several antimicrobials from reaching their targets at the cytoplasmic membrane and/or the cyto-plasm, which complicates the development of novel therapies against (multidrug-resistance (MDR)) Gram-negative pathogens [6, 7].

Vancomycin is a glycopeptide antibiotic used in therapeutics, and its mechanism to kill Gram-positive bacteria is binding the lipid II pen-tapeptide, thus blocking the synthesis of the cell wall [8, 9]. Nisin has been used in food industry as natural-preservative for decades due to its high activity against Gram-positive bacteria and the lack of toxicity in humans [10, 11]. Nisin has a dual mode of action which involves binding to lipid II, abducting it from the site of cell-wall synthesis and subsequently forming pores in the cytoplasmic membrane [12–15]. Vancomycin is one of the most effective and safe medicines, according

to the list of WHO [16]. Both vancomycin and nisin are highly effective against Gram-positive bacteria with minimal inhibitory concentrations (MICs) at nanomolar levels [17–20]. However, their activity against Gram-negative bacteria is much lower due to the fact that they do not easily reach the inner membrane. Therefore, we hypothesized that if we can find compounds that can perturb the Gram-negative outer membrane, this will enable nisin and vancomycin to enter the peri-plasm and reach the inner membrane, which contains their target lipid

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CH APTER 4: H ig h sy ner gi stic a nt imicr ob ia l ac tiv ity a ga in st G ra m-n ega tiv e b ac ter ia o f sy nt het ic L -p ep tides w ith va nco m ycin o r ni sin

4

II, resulting in substantive activity against Gram-negative pathogens. Synthetic peptides active against Gram-negative bacteria have been considered to combat infections caused by Gram-negative species [21]. However, their activity alone is usually insufficient. Remarkably,

in vitro synergism has been identified between combinations of

an-tibiotics or anan-tibiotics with other compounds, which can be used to target specific problematic pathogens [22–26]. Thus, the combination of these peptides that can alter the barrier function of the outer mem-brane combined with the potent effect of nisin or vancomycin might constitute a venue to effectively combat Gram-negative bacteria.

Here, nine peptides, which have been reported to exert modest ac-tivity against Gram-negative bacteria (GNPs) were selected from liter-ature to work as possible Gram-negative outer membrane- penetrating or -disturbing peptides (Table 1). Their efficacy, when combined with either vancomycin or nisin, is studied here.

1. Materials and methods

1.1. Materials

Nisin was purified to homogeneity by reversed-phase high perfor-mance liquid chromatography (RP-HPLC), operated with an Agilent 1260 series HPLC. Solvents used for RP-HPLC were solvent A (0.1 % trifluoracetic acid (TFA) in MilliQ water) and solvent B (0.1 % TFA in acetonitrile). The peptides were purified with a C12 column (4 µm proteo 90 Å column, 250 × 4.6 mm (Phenomenex)), as described pre-viously [35].

Vancomycin, sodium chloride (NaCl), Luria-Bertani (LB) and Mueller- Hinton broth (MHB) were purchased from Sigma-Aldrich (Oakville, Ontario, Canada). Sodium phosphate tablets were purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA). Synthesized

peptides were supplied by Pepscan (Lelystad, the Netherlands).

1.2. Bacterial strains and growth conditions

The bacteria used in this study are listed in Table 2. All the bacteria used were from lab collections and were obtained from the Belgian Co-ordinated Collections of Microorganisms (BCCM).

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CH APTER 4: M at er ia ls a nd m et ho ds

Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterobacter aerogenes were grown in

Luria-Bertani (LB) broth shaken (200 rpm) or on LB agar at 37 °C. All the strains were used to test the minimum inhibitory concentration (MIC) of nisin, vancomycin, synthesized peptides (GNPs) and com-binations thereof. All the strains were reactivated from a 20 % glycerol stock stored at −80 °C. They were subcultured at least 3 times before being tested in the experiments.

Table 1. List of GNPs used in this work.

Name Sequences Name in literature Reference Source GNP-1 GNNRPVYIPQPRPPHPRL Api1b [27] Honey bee GNP-2 VDKPPYLPRPRPPRRIYNR Oncocin [28] Milkweed bug GNP-3 VDKGSYLPRPTPPRPIYNRN-NH2 Pyrrhocoricin [29] Pyrrhocoris

apterus

GNP-4 RLLFRKIRRLKR EC5 [30] Predicted using Antimicrobial peptide database GNP-5 RIWVIWRR-NH2 Bac8c [31] Bovine GNP-6 RRLFRRILRWL-NH2 RW-BP100 [32] Synthetic AMP.

based on a cecro-pin A-melittin hybrid GNP-7 GIGKHVGKALKGLKGLLKGLGEC ADP-1 [33] Anuran

GNP-8 RIVQRIKKWLR-NH2 This work Human GNP-9 KRIVQRIKKWLR-NH2 KR-12-a2 [34] Human (LL-37

derived frag-ment)

Table 2. Strains used in this work.

Strains Characteristics Purpose References

Escherichia coli LMG 15862 beta lactamase Indicator strain Lab collection (BCCM)

Klebsiella pneumoniae

LMG 20218 beta lactamase Indicator strain Lab collection(BCCM)

Pseudomonas aeruginosa LMG 6395 Indicator strain Lab collection (BCCM)

Acinetobacter baumannii LMG 01041 Indicator strain Lab collection (BCCM)

Enterobacter aerogenes LMG 02094 Indicator strain Lab collection (BCCM)

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1.3. Determination of the minimum inhibitory concentration (MIC)

MIC tests were performed in triplicate by liquid growth inhibition mi-crodilution assays in sterile polypropylene microtiter plates according to Wiegand et al. [36]. Briefly, the indicator strains were first streaked on agar plates and then incubated overnight. 3~5 colonies were randomly picked and resuspended in saline solution (0.9 % NaCl (w/v)) until the OD625 of the bacterial suspension was ca. 0.08~0.13 (1~2 × 108 colo-ny forming units (CFU)/mL). The suspension was diluted in a ratio of 1:100 using Mueller-Hinton Broth (MHB) so the final inoculum was 5 × 105 CFU/mL. A growth control with no antimicrobial added and a sterility control were included for each strain in each plate. The antimicrobials were 2-fold serially diluted and 50 µl diluted bacterial suspension was added to each well to make the final volume 100 µl. The inoculum size in the wells was monitored plating an aliquot of the growth control. The microtiter plate and the count plates were in-cubated at 37 °C for 16~20 h without shaking. Growth inhibition was assessed measuring the OD600 using a microplate reader (Tecan infinite F200). The lowest concentration of the antimicrobials that inhibits visible growth of the indicator is identified as the MIC.

1.4. Synergy test and corresponding algorithm

We conducted standard chequerboard broth microdilution assays to test the synergistic effect of combined antimicrobials [37, 38]. Van-comycin or nisin (Drug X) was loaded two-fold serially diluted in the X-axis, while GNPs (Drug Y) were two fold serially diluted in the Y-axis (Figure 1). The initial concentration of both peptides was their individual MIC. 50 µl fresh bacterial suspension prepared as above was added to the wells to achieve 100 µl final volume per well.

To determine whether the combination is additive, synergistic or antagonistic, the fractional inhibitory concentration (FIC) indices [39] were calculated using the formula FICI = FICa + FICb = MICac/ MICa + MICbc/MICb [37, 39]. The MICa and MICb is the MIC of compound A or B alone, respectively. MICac is the MIC of com-pound A in combination with comcom-pound B and MICbc is the MIC of compound B when it was combined with compound A. The FIC corresponds to the MIC of a compound in combination with the

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CH APTER 4: R es ul ts

other compound divided by the MIC of the compound alone. FICa is FIC of compound A while FICb is FIC of compound B. The FICI is interpreted as follows: synergistic, FICI ≤ 0.5; additive, 0.5 < FICI ≤ 1; indifferent, 1 <FICI < 2; antagonistic, FICI > 2.

2. Results

2.1. Minimal inhibitory concentration (MICs) of antimicrobial compounds alone against Gram-negative pathogens

Activity tests of vancomycin, nisin and GNPs were performed against five clinically relevant Gram-negative pathogens. MICs values are listed in Table 3. As it can be seen, MICs of GNPs were very different from each other among peptides and pathogens. The MICs of GNPs varied from 0.5 µM (e.g. GNP-1 against E. coli) to more than 256 µM (e.g. GNP-4 against K. pneumoniae and E. aerogenes). GNP-5, GNP-6 and GNP-7 were the most interesting candidates, exerting activity against

Figure 1. Schematic picture of Synergy determination plate. A factorial dose matrix was used

to trial all mixtures of the two serially diluted single compounds. No antibiotics were added to the wells of growth control, while only medium was added in the wells of sterilization control.

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all the Gram-negative bacteria tested at low micromolar concentrations. GNP-4 showed the poorest activity amongst all the GNPs against the entire panel. Notably, GNP-1 demonstrated a potent activity against some species, e.g. 0.5 µM against E. coli, but failed to show any activity against P. aeruginosa or A. baumannii at the concentrations tested.

2.2. Synergism of GNPs and vancomycin/nisin

The synergism between GNPs and either vancomycin or nisin was measured using chequerboard broth microdilution assays. All the GNPs were combined with vancomycin or nisin and initially tested against E. coli in the initial screening. As it is shown in Table 4, except GNP-8, all the peptides showed an additive effect with vancomycin against E. coli (the FICI is interpreted as follows: synergistic, FICI ≤ 0.5; additive, 0.5 < FICI ≤ 1). Nisin displayed an additive effect with GNP-2 and GNP-3 while synergistic with the others against E. coli.

The FICI of nisin in combination with GNP-4 against E. coli was 0.375 which means the combination of 3 µM of nisin and 8 µM of GNP-4 was sufficient to completely inhibit the growth of E. coli. How-ever, 3 µM or 8 µM are still rather high concentrations for in vivo tests. What is more, the MICs of GNP-4 against K. pneumoniae, P. aeruginosa,

A. baumannii and E. aerogenes are even higher than the MIC against E. coli. So, if we consider a similar trend, the final concentration of

GNP-4 in combination with nisin will probably be even higher than

Table 3. MIC value (µM) of GNPs, vancomycin and nisin alone against 5 Gram-negative pathogens.

E. coli

LMG15862 K. pneumoniae LMG20218 P. aeruginosaLMG 6395 A. baumannii LMG01041 E. aerogenesLMG 02094

GNP-1 0.5 5 >64 >64 8 GNP-2 4 8 >64 16 >64 GNP-3 3 16 32 8 >32 GNP-4 64 >256 >128 >128 >256 GNP-5 3 6 3 2 5 GNP-6 2 4 3 2 8 GNP-7 6 12 13 2 8 GNP-8 12 32 16 >64 128 GNP-9 4 64 16 16 32 Vancomycin 64 128 128 32 192 Nisin 12 48 36 6 32

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Table 4. Combined activity of GNPs and vancomycin or nisin against 5 Gram-negative

patho-gens.

Pathogens Antibiotic GNP MICa

(µM) MICb (µM) MICac(µM) MICbc (µM) FICI

E. coli LMG15862 Vancomycin GNP-1 64 0.5 32 0.125 0.75 GNP-2 64 4 32 2 1 GNP-3 64 3 32 1.5 1 GNP-4 64 64 16 16 0.75 GNP-5 64 3 32 0.75 0.75 GNP-6 64 2 16 1 0.75 GNP-7 64 6 16 3 0.75 GNP-8 64 12 4 1.5 0.188 GNP-9 64 4 32 0.25 0.563 Nisin GNP-1 12 0.5 1.5 0.13 0.375 GNP-2 12 4 1.5 2 0.625 GNP-3 12 3 3 1.5 0.75 GNP-4 12 64 3 8 0.375 GNP-5 12 3 0.75 0.75 0.313 GNP-6 12 2 1.5 0.5 0.375 GNP-7 12 6 1.5 1.5 0.375 GNP-8 12 12 1.5 1.5 0.25 GNP-9 12 4 1.5 1 0.375 K. pneumoniae LMG20218 Vancomycin GNP-1 128 5 64 0.625 0.625 GNP-5 128 6 64 3 1 GNP-6 128 4 32 1 0.5 GNP-7 128 12 64 3 0.75 GNP-8 128 32 4 2 0.094 GNP-9 128 64 8 8 0.188 Nisin GNP-1 48 5 6 1.25 0.375 GNP-5 48 6 6 1.5 0.375 GNP-6 48 4 3 0.5 0.188 GNP-7 48 12 3 0.75 0.125 GNP-8 48 32 0.75 1 0.047 GNP-9 48 64 0.75 4 0.078 P. aeruginosa LMG6395 Vancomycin GNP-1 128 >64 128 32 <1.5 GNP-5 128 3 128 1.5 1.5 GNP-6 128 3 32 1.5 0.75 GNP-7 128 13 128 6.5 1.5 GNP-8 128 16 16 2 0.25 GNP-9 128 16 64 8 1 Nisin GNP-1 36 >64 4.5 64 <1.13 GNP-5 36 3 2.25 0.75 0.313 GNP-6 36 3 4.5 0.75 0.375 GNP-7 36 13 4.5 3.25 0.375 GNP-8 36 16 4.5 1 0.188 GNP-9 36 16 4.5 1 0.188 CH APTER 4: R es ul ts

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8 µM thus discouraging any in vivo test. The positive effect on E. coli led us to test the effect of only the most potent combinations on other Gram-negative bacteria, therefore discarding GNP-2, GNP-3, and GNP-4 (Table 4).

Notably, the combination of GNP-8 with either vancomycin or nisin displayed an astonishing synergism against all the five Gram- negative pathogens. The effective concentration of both compounds was reduced more than 16-fold in the combination in some cases. We observed that GNP-5, GNP-6 and GNP-7 were very efficient when tested alone and showed a modest synergism with either vancomycin

Pathogens Antibiotic GNP MICa

(µM) MICb (µM) MICac(µM) MICbc (µM) FICI

A. baumannii LMG01041 Vancomycin GNP-1 32 >64 16 16 <0.75 GNP-5 32 2 16 0.5 0.75 GNP-6 32 2 8 0.5 0.5 GNP-7 32 2 16 0.25 0.625 GNP-8 32 >64 2 1 <0.078 GNP-9 32 16 4 4 0.375 Nisin GNP-1 6 >64 1.5 64 <1.5 GNP-5 6 2 0.75 0.5 0.375 GNP-6 6 2 0.75 0.5 0.375 GNP-7 6 2 0.75 0.5 0.375 GNP-8 6 >64 0.19 4 <0.094 GNP-9 6 16 0.19 1 0.094 E. aerogenes LMG02094 Vancomycin GNP-1 192 8 192 8 2 GNP-5 192 5 192 5 2 GNP-6 192 8 64 2 0.583 GNP-7 192 8 192 4 1.5 GNP-8 192 128 12 8 0.125 GNP-9 192 32 48 4 0.375 Nisin GNP-1 32 8 4 4 0.625 GNP-5 32 5 4 1.25 0.375 GNP-6 32 8 2 1 0.188 GNP-7 32 8 4 1 0.25 GNP-8 32 128 4 8 0.188 GNP-9 32 32 2 8 0.313

Green: the lowest FICI for the specific Gram-negative pathogen and antibiotic in this table; Red: FICI < 0.1.

Note: MICa is the MIC of vancomycin or nisin alone; MICb corresponds to the MIC of GNPs when used alone; MICac is the MIC of vancomycin or nisin in combination with the GNPs at the MICbc concentration. MICbc is the MIC of GNPs when used with the MICac concentration of vancomycin or nisin.

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CH APTER 4: Di sc us sio n

or nisin against the Gram-negative pathogens tested. Among them, GNP-6 was more synergistic with either vancomycin or nisin than GNP-5. On the contrary, GNP-8 and GNP-9 showed a very strong synergy with either vancomycin or nisin (in some cases, FICI < 0.1) and a modest anti-Gram-negative effect alone. Interestingly, GNP-8 exerted stronger synergy with either vancomycin or nisin than GNP-9, which has a higher net charge. Our data prove that the combination of selected GNPs with vancomycin or nisin drastically boosts the activity of these compounds against Gram-negative pathogens.

3. Discussion

The widespread distribution and development of multidrug resistance among bacterial pathogens have become a serious public health crisis in the world. Better management of use of existing drugs and finding novel sources for new drugs are extremely urgent. In the last years, a renewed interest in conventional antibiotics for new applications as well as bacteriocins for clinical use has emerged as a plausible additional solution to combat (MDR) Gram-negative bacteria [25, 40–45]. In this work we attempt to improve the activity of the well-known antibiotic vancomycin and to expand the inhibitory effect of the antimicrobial nisin against Gram-negative bacteria. To do so, combination tests between modestly active peptides against Gram-negative species and known anti-Gram-positive antimicrobial drugs were performed. We selected a range of natural antimicrobial peptides and evaluated their intrinsic activity. In addition, as reported before, compounds with different targets on bacterial cells can display very good synergistic effects against pathogens, thereby reducing their individual toxicity [22, 25]. This strategy, if successful, could decrease the dose needed, while improving the sensitivity of pathogens to the antimicrobials. This can on one hand slow down the massive use of drugs and on the other hand delay antibiotic resistance [25].

In this work, several specific GNPs were selected to be synthesized including proline-rich compounds (GNP-1, GNP-2 and GNP-3), arginine-rich peptides (GNP-6 and GNP-9), or more random peptides (GNP-7). They were either from eukaryote origin (insects, amphibians

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and mammals) or designed according to antimicrobial databases. GNP-8 is a derivative of GNP-9 with a reduced charge, which was de-signed to overcome the renal clearance or toxicity that might be caused by highly cationic peptides [46]. The design of GNP-8 compromised the activity against Klebsiella, Acinetobacter and Enterobacter, it was similar against Pseudomonas and even improved against E. coli when compared to GNP-9 (Table 3). This suggests that the mechanism of action against these strains is not based only on the net charge but involves additional mechanisms. When combining these GNPs with ei-ther vancomycin or nisin, the concentration of the compounds needed to inhibit the growth of Gram-negative pathogens could be lowered by more than 30-fold in some cases. Moreover, the actual concentration needed for both compounds can be even less than 1 µM of each at the same time. This concentration range is quite attractive for further

in vivo characterization. Remarkably, the intrinsic activity of the GNPs

was not correlated with a better synergy with nisin or vancomycin. Thus, we could observe that GNP-8, with a modest activity against

the selected bacterial strains, achieved the best synergistic effect with either antimicrobial although the specific underlying mechanism is as yet unknown.

The target of vancomycin and nisin in bacteria is lipid II at the cyto-plasmic membrane [12]. Thus, a hypothesis to explain this synergism is that GNPs first work on the outer membrane of Gram- negative patho-gens and then enable vancomycin or nisin to reach the inner membrane (peptidoglycan) and inhibit the synthesis of the cell wall (Figure 2). It cannot be excluded that some of the peptides also reach the inner membrane and act synergistically also at that location. Previous works with sublethal damage of the outer membrane of Gram-negative bacte-ria show that nisin and vancomycin can readily inhibit Gram-negative bacteria [47–49]. We speculate that the higher the GNP-induced outer membrane damage the more accessible lipid II becomes. Therefore, GNPs that target the membrane rather than those having intracellular targets will have a more potent synergistic effect even if their intrinsic activity is lower, although this needs further experimental evidence. Our work proves that anti-Gram-positive antimicrobials and mem-brane disrupting peptides display strong synergistic activity against selected Gram-negative bacteria. A mechanism of gateway activity of

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CH APTER 4: A ck no w le dg em en ts

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membrane disrupting peptides is proposed. However, the full potential of the combinations of vancomycin/nisin and GNPs still needs to be further confirmed and realized after pharmacokinetic-pharmacody-namic studies. Considering the size, charge and potency of the GNPs, GNP-6 and GNP-8 were selected for further tests.

Acknowledgements

Qian Li was supported by the Chinese Scholarship Council (NO 201306770012).

Manuel Montalban-Lopez was supported by a grant of EU FW7 project SynPeptide.

Figure 2. Schematic overview of the hypothetical synergism of vancomycin/nisin and GNPs.

Left: vancomycin or nisin alone cannot penetrate the outer-membrane due to their size. Right: in the presence of GNPs, a (possibly transient) gap will be formed on the OM and vancomycin or nisin can reach the inner membrane.

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