Lipid transfer protein isolated from noni seeds displays antibacterial activity in vitro and improves survival in lethal sepsis induced by CLP in mice

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Research paper

Lipid transfer protein isolated from noni seeds displays antibacterial

activity

in vitro

and improves survival in lethal sepsis induced by CLP

in mice

Adson A. Souza

a

, Andrea S. Costa

a

, Dy

ely C.O. Campos

a

, Andressa H.M. Batista

b

,

Gleilton W.P. Sales

b

, N

adia A.P. Nogueira

b

, Keila M.M. Alves

c

,

Andrelina Noronha Coelho-de-Souza

d

, Herm

ogenes D. Oliveira

a,*

a_{Department of Biochemistry and Molecular Biology, Federal University of Cear}_{a, Campus do Pici Prof. Prisco Bezerra, 60440-900, Fortaleza, CE, Brazil} b_{Department of Clinical and Toxicological Analysis, Faculty of Pharmacy, Dentistry and Nursing, Federal University of Cear}_{a, 60431-372, Fortaleza, CE,}

Brazil

c

Center for Laboratory Veterinary Medicine of Fortaleza - LAFORVET, 60135-130, Fortaleza, CE, Brazil

d_{Laboratory of Experimental Physiology, Superior Institute of Biomedical Sciences, State University of Cear}_{a, 60714-903, Fortaleza, CE, Brazil}

a r t i c l e

i n f o

Article history:

Received 21 September 2017 Accepted 20 March 2018 Available online 22 March 2018

Keywords: Morinda citrifoliaL. Lipid transfer protein Antimicrobial activity Sepsis

a b s t r a c t

In the present study, we aimed to evaluate the antibacterial activity of a lipid transfer protein isolated fromMorinda citrifoliaL. seeds, namedMcLTP1, and to investigate its effect in the cecal ligation and

puncture (CLP) mouse sepsis model. Antimicrobial assays revealed that McLTP1 (12.5e800mg/mL)

signiﬁcantly reduced Staphylococcus aureus (ATCC 6538P and ATCC 14458) and Staphylococcus

epi-dermidis(ATCC 12228) planktonic growth, reaching maximal inhibition of approximately 50% and 98%,

respectively. Furthermore,McLTP1inhibited bioﬁlm formation of bothS. aureusstrains, achieving

per-centages ranging from 39.1% to 69.1% (200e800mg/mL) for ATCC 6538P and 34.4%e63% (12.5e800mg/

mL) for ATCC 14458. A synergistic interaction between McLTP1and oxacillin against S. aureus and

S. epidermidiswas also observed, as determined by fractional inhibitory concentration indices of 0.18 and

0.38, respectively.McLTP1showed no signiﬁcant inhibitory effect against Gram-negative bacteria. In the

in vivoexperiments, sepsis was lethal to 83% of the animals, 72 h after CLP. In contrast, 100% of the

animals treated withMcLTP1(8 mg/kg) before (intraperitoneal injection or oral dose) or after (oral dose)

CLP were still alive 3 days later. In addition, oral or intraperitoneal administration ofMcLTP1(8 mg/kg)

signiﬁcantly reduced the body weight loss, fever, leukocytosis, organ damage, and the level of inﬂ

am-matory serum cytokines induced by sepsis. In conclusion,McLTP1could be exploited for its antimicrobial

properties, and can be considered a potential therapeutic candidate for the management of clinical sepsis.

reserved.

1. Introduction

The emergence of microbial resistance is one of the greatest public health problems of this century. According to recent esti-mates, by 2050, this threat will generate 300 million premature deaths, costing the global economy approximately $100 trillion

[1,2]. Due to microbial resistance to drugs and the lack of new

an-tibiotics, an increase in infectious diseases’severity and the risk of

complications associated with bacterial infections has been

observed [2]. In the case of severe infections, which ultimately lead

to sepsis, this health state is characterized by systemic

Abbreviations:AMP, antimicrobial peptide; LTP, lipid transfer protein;McLTP1, LTP isolated fromMorinda citrifoliaL. seeds; FICI, fractional inhibitory concentration index; CLP, cecal ligation and puncture; IL, interleukin; TNF-a, tumor necrosis factor-a; MCP-1, monocyte chemotactic peptide 1; IFN-g, interferon-g; CBA, cyto-metric bead array; MIC, minimum inhibitory concentration; BHI, brain heart infu-sion; TSB, tryptic soy broth.

*Corresponding author.

E-mail address:hermogenes@ufc.br(H.D. Oliveira).

Contents lists available atScienceDirect

Biochimie

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b i o c h i

https://doi.org/10.1016/j.biochi.2018.03.011

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inﬂammatory response syndrome (SIRS), affecting the patient's

entire body and resulting in high morbidity and mortality [3e6].

Unfortunately, there has been a drop in the number of new

antibiotics introduced to the market in recent decades [7]. The

challenges of ﬁnding new antimicrobial agents with alternative

bacterial targets, lowerﬁnancial returns compared to those of other

disease therapies, and heavier regulatory guidelines are factors

involved in this decline [7,8]. Because of this crisis of antibiotic

development and the absence of drugs approved for the treatment of sepsis, research into novel approaches and alternative sources of

bioactive compounds has been greatly stimulated [6,8].

Plant antimicrobial peptides (AMPs) have emerged as promising

molecules with therapeutic potential in human health [9]. They are

small (usually 2e9 kDa), basic, and cysteine-rich molecules, with

disulﬁde bridges in their internal structures that generate highly

stable globular conformations [9,10]. According to sequence

ho-mologies, pattern distributions of disulﬁde bridges, and

three-dimensional structures, AMPs are divided into nine classes: thio-nins, defensins, shepherdins, cyclotides, snakins, heveins, knottins,

Ib-AMPs, and non-speciﬁc lipid transfer proteins (nsLTPs) [9e11].

Although several studies have shown that AMPs present broad

spectrumin vitroactivities against bacteria and fungi [9e11], these

activities have not been explored extensively throughin vivo

ex-periments (e.g., in strategies involving systemic infectious diseases, such as sepsis).

Recently, an antimicrobial peptide belonging to the lipid

trans-fer protein class, namedMcLTP1, was isolated fromMorinda citrifolia

L. (noni) seeds (UniProtKB accession number: C0HJH5). This LTP was shown to be thermostable and resistant to pepsin, trypsin, and chymotrypsin digestion; it was capable of displaying potent

anal-gesic and anti-inﬂammatory activities when orally administered to

mice [12].

The aim of this study was to evaluate thein vitroandin vivo

antibacterial properties ofMcLTP1, with the latter involving both

prophylactic and therapeutic interventions in a mouse sepsis

model. Because the actual efﬁcacy of McLTP1 in vivo may be

different from thatin vitrodue to interactions with components of

the human/animal body, performing bothin vitroandin vivoassays

provided a more complete understanding of the peptide’s activity.

The outcomes will demonstrate that these molecules have poten-tial to address burgeoning antimicrobial resistance issues.

2. Materials and methods

2.1. Plant material and puriﬁcation of McLTP1

M. citrifoliaL. var.citrifoliaseeds were obtained from EMBRAPA

Agroindustria Tropical, Fortaleza, Ce, Brazil. After extraction from

defatted noni seedﬂour,McLTP1was puriﬁed using a combination

of trichloroacetic acid precipitation and size exclusion

chroma-tography, as previously published by Campos et al. [12]. Samples

obtained from gel ﬁltration and containingMcLTP1 were

lyophi-lized and stored at 20_{C for further use in biological assays. The}

purity of McLTP1 was assessed by sodium dodecyl

sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with a 15%

poly-acrylamide gel under non-reducing conditions [13]. The proteins

were visualized with Coomassie Brilliant Blue R-250 staining.

McLTP1samples used in biological assays were prepared based on

soluble total protein concentrations estimated by the method of Bradford [14].

2.2. Reagents and culture media

Gentamicin and oxacillin were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). The antimicrobial agents were diluted

according to the Clinical and Laboratory Standards Institute (CLSI)

guidelines [15]. The culture media, Brain Heart Infusion broth (BHI)

and Tryptic soy broth (TSB), were obtained from Himedia®

(Mumbai, Maharashtra, India). Crystal violet, which was used to

stain bacterial bioﬁlms, was purchased from Laborclin LTDA

(Pin-hais, PR, Brazil). Other solvents and chemicals were analytical grade and obtained from local suppliers.

2.3. Antibacterial activity

2.3.1. Bacterial strains

Three Gram-negative bacteria strains (Escherichia coli ATCC

10536,Pseudomonas aeruginosaATCC 902, andKlebsiella

pneumo-niaeATCC 10031) and three Gram-positive strains (Staphylococcus

epidermidisATCC 12228,Staphylococcus aureus ATCC 6538P, and

ATCC 14458) were tested for their susceptibility to McLTP1. To

prepare bacterial cell suspensions for antimicrobial activity assays,

all strains were inoculated in BHI liquid medium at 37_{C for 24 h. A}

saline inoculum suspension of each culture was then prepared to a

ﬁnal concentration of 106CFU/mL.

2.3.2. Antibacterial susceptibility test

The antibacterial susceptibility test was performed in triplicate in 96-well culture plates in BHI medium, based on the

micro-dilution broth method described previously [15].McLTP1was

dis-solved in sterile distilled water,ﬁltered using 0.22

m

m membranes,

and tested at 12.5e800

m

g/mL. Gentamicin and oxacillin (positive

controls) were also prepared in sterile distilled water and two-fold

serially diluted from 0.048 to 50

m

g/mL. Minimum inhibitory

con-centrations (MICs) were assessed visually after an incubation

period of 24 h (37_{C) and were recorded as the lowest sample}

concentration at which there was no growth. To assess cell growth, the absorbance was measured at 620 nm (A620), using a microplate reader (Bio Tek, Winooski, VT, USA). Controls without antibiotics or

McLTP1were also measured. BHI broth containing only the

anti-microbial drugs at the concentrations tested was used as a blank. The planktonic culture turbidity measured in the absence of drugs was considered 100% growth, and was used as a reference to

determine the percent growth inhibition of theMcLTP1-treated

cultures.

2.3.3. Effect of McLTP1on staphylococcal bioﬁlm formation

The effect ofMcLTP1 on S. aureusbioﬁlm formation was

per-formed in triplicate using polystyrene ﬂat-bottomed microtiter

plates, as previously reported [16].S.aureusinocula (strains ATCC

14458 and ATCC 6538P) were cultured in TSB supplemented with

1% (w/v) glucose to aﬁnal concentration of 106CFU/mL and

incu-bated withMcLTP1(12.5e800

m

g/mL) at 37C for 24 h. After the

incubation period, the plate wells were washed with sterile saline (0.85% NaCl) to remove unattached cells, while the adherent cells

wereﬁxed for 15 min with 99% methanol. Then, the adherent

bio-ﬁlms were stained with 2% (w/v) crystal violet solution for 15 min.

The excess crystal violet was removed by washing with ultrapure water, and the plate was air-dried. Finally, the crystal violet was dissolved by adding acetic acid 33% (v/v) with shaking in an orbital

shaker for 15 min. Bioﬁlm formation was determined by measuring

the absorbance at 570 nm (A570) using a microplate reader. The

percent inhibition was calculated, taking theA570of non-treated

controls as 100% [17].

2.3.4. Combination effect of antibiotics with McLTP1

Chequerboard titration was performed using McLTP1

(100e400

m

g/mL) and the standard drugs, gentamicin and oxacillin,

at sub-MIC concentrations (MIC/16eMIC/2).S.aureus(ATCC 14458

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determine the fractional inhibitory concentration index (FICI),

ac-cording to a previously published method [18]. The inoculum size

and the culture conditions were the same as those used for the

antibacterial susceptibility test. FICI values0.5 were considered

synergistic (SYN), 0.5<FICI4.0 indifferent (IND), and >4.0,

antagonistic (ANT) [19].

2.4. Effect of McLTP1on lethal sepsis induced by cecal ligation and

puncture (CLP) in mice

2.4.1. Animals

In this study, male Swiss albino mice (45 days old; 35 g) were used. The animals were housed in polypropylene cages under

standard environmental conditions (24±1_{C, humidity 45}

e65%,

and 12 h light/dark cycle) and received food and waterad libitum.

All animals were treated in compliance with the ethical standards established by the National Guidelines for the Use of Experimental Animals of Brazil and by the Directive 2010/63/EU of the European Parliament and of the Council of the European Union. The experi-mental protocols were approved by the Committee for the Ethical

Use of Animals of the Federal University of Ceara (CEUA-UFC no.

108/2016).

2.4.2. Sepsis model

The experimental induction of sepsis was performed using the

CLP model, as outlined previously [20]. In brief, before the surgery,

all mice were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) solution. Under aseptic conditions, a laparotomy was performed with a median longitudinal incision of approximately 1.5 cm, allowing full expo-sure of the cecum. A non-obstructive ligation with cotton thread 4-0 was placed in the region immediately below the ileocecal valve, followed by a single perforation with an 18-gauge needle. After perforation, to allow leakage of a small amount of fecal content, the cecum was softly compressed and then returned to the peritoneal cavity. Sham-operated mice were submitted to the same surgical procedure, without ligation and perforation of the cecum.

Subse-quently, the abdomen was closed in two layers. Forﬂuid

replace-ment, isotonic saline (37_{C, 4% of body weight) was administered}

subcutaneously, immediately after the surgical procedure. Then, all animals were returned to their cages, with free access to food and water, and were observed over 72 h.

2.4.3. Experimental design

The mice were randomized intoﬁve groups (n¼8 per group). 1)

Sham operated, 2) sepsis induction but receiving only sterile saline (0.15 M NaCl), 3) sepsis induction and receiving a single

intraperi-toneal dose ofMcLTP1 (8 mg/kg) 1 h before surgery (prophylactic

effect of intraperitonealMcLTP1), 4) sepsis induction and receiving

a single oral dose ofMcLTP1 (8 mg/kg) 2 h before surgery

(pro-phylactic effect of oral McLTP1), and 5) sepsis induction and

receiving oral McLTP1 (8 mg/kg) immediately after surgery, and

then every 6 h over a 72-h period (therapeutic effect of oral

McLTP1). The survival, body weights, and rectal temperatures of the

animals were monitored every 12 h for 72 h after the surgery.

2.4.4. Total and differential blood cell counts

Blood was collected aseptically under anesthesia from the retro-orbital plexus at time intervals of 24 and 48 h. The blood was assayed with an automatic hematology analyzer BC-2800 (Shenz-hen Mindray Bio-Medical Electronics Co., S(Shenz-henz(Shenz-hen, Guangdong, China) to determine the white blood cell, granulocyte, and lymphocyte counts.

2.4.5. Histopathologic analysis

Seventy-two hours after CLP, all animals were euthanized with an overdose of anesthetic (300 mg/kg ketamine and 30 mg/kg xylazine). Tissue samples of the liver and superior lobe of the right lung were removed for histopathologic analysis. The organs were

ﬁxed in phosphate-buffered saline (PBS) containing 4%

para-formaldehyde, and were then embedded in parafﬁn. A microtome

was used to produce sections that were mounted on slides,

deparafﬁnized, and then stained with hematoxylin and eosin. The

following parameters were evaluated in the stained tissue sections:

vascular congestion, hemorrhage, and cellular inﬁltration,

accord-ing to the followaccord-ing scores: 0, absent; 1, weak; 2, moderate; and 3,

strong, as described by Zhou et al. [21]. Histological assessment was

performed randomly and blindly under a light microscope (Nikon E200).

2.4.6. Quantiﬁcation of cytokines by the cytometric bead array (CBA) technique

Blood samples from mice in group #2 (pretreatment with saline

1 h before CLP; n¼8), group #3 (intraperitoneal pretreatment with

McLTP11 h before CLP; n¼8), and group #5 (oral post-treatment

with McLTP1 immediately after CLP; n¼8) were collected from

the retro-orbital plexus 12 h after sepsis induction. Thereafter,

levels of tumor necrosis factor-

a

(TNF-

a

), interferon-

g

(IFN-

g

),

interleukin (IL)-6, IL-10, IL-12 p70, and monocyte chemotactic protein 1 (MCP-1) were measured from the serum samples by CBA

technique. Cytokine quantiﬁcation was performed using a mouse

inﬂammatory cytokine kit (Becton Dickinson Biosciences, San Jose,

CA, EUA), following the manufacturer’s protocol. Data were

analyzed using FCAP Array software (BD Biosciences).

2.5. Statistical analysis

Experimental data are expressed as means±standard

de-viations. Signiﬁcant differences among means were evaluated in

GraphPad Prism®

5 software (GraphPad Prism, Inc., San Diego, CA, USA) using one-way analysis of variance (ANOVA), followed by

Tukey’s post hoc tests. In all comparisons, signiﬁcant differences

were considered atp<0.05.

3. Results

3.1. Antibacterial activity of McLTP1

3.1.1. Effect on planktonic cells and bioﬁlm formation

McLTP1had a signiﬁcant inﬂuence on the planktonic growth of

the threeStaphylococcusstrains tested (Fig. 1).McLTP1could inhibit

~50% of the planktonic growth (p<_{0.05) of}S.aureusstrains, ATCC

6538P and ATCC 14458, at 12.5 and 200

m

g/mL, respectively. The

observed effect did not occur in a concentration-dependent manner for both strains. In contrast to that observed for the

S. aureus strains, McLTP1 inhibited S. epidermidis (ATCC 12228)

planktonic growth by ~20% at a concentration range of

12.5e200

m

g/mL (p<0.05), reaching a maximal inhibition of 98% at

800

m

g/mL. Despite the bacterial antiplanktonic activity displayed

by the antimicrobial peptide, it was not possible to calculate MIC,

because 800

m

g/mL McLTP1 (i.e., the maximum concentration

evaluated) was not able to inhibit the bacterial growth of the tested strains completely. Unlike the effects observed against the

Gram-positive bacteria,McLTP1did not inhibit the growth of the

Gram-negative bacterial strains at any tested concentration (data not shown).

The antibioﬁlm effects ofMcLTP1 at increasing concentrations

(12.5e800

m

g/mL) onS.aureus(ATCC 14458 and ATCC 6538P) are

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mL), McLTP1 could diminish bioﬁlm formation of both strains,

achieving inhibition of 39.1e69.1% for ATCC 6538P and ~63% for

ATCC 14458. At concentrations below 200

m

g/mL McLTP1, only

ATCC 14458 was susceptible, demonstrating a signiﬁcant

reduc-tion (p<0.05) in bioﬁlm formation activity, ranging from 34.4% (at

12.5

m

g/mL) to 55.6% (at 100

m

g/mL).

3.1.2. Effect of combination of antibiotics with McLTP1

Synergistic interactions were observed betweenMcLTP1and

oxacillin for the three bacterial strains, with indexes (FICI) ranging

from 0.18 to 0.38, as shown inTable 1. No synergism was observed

against the three bacterial strains with the cotreatment ofMcLTP1

and gentamicin at any concentration tested.

Fig. 1.Effect ofMcLTP1on planktonic growth ofStaphylococcusspp.S. aureus(ATCC 14458 and ATCC 6538P) andS. epidermidis(ATCC 12228). Absorbances (620 nm) were measured after 24 h at 37_{C in the absence or in the presence of}_Mc_{LTP1. *}_p_<_0.05

compared to control group (One-way ANOVA followed by Tukey's multiple com-parison test).

Fig. 2.Antibiofilm formation activity ofMcLTP1againstStaphylococcus aureusstrains. Data are expressed as the means of biofilm production percentage (±SD) of tripli-cates. *Indicates significant difference between untreated and treated bacteria cells (p<0.05; ANOVA Tukey’s post hoc test).

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Fig. 3.Survival rates of animals with sepsis subjected toMcLTP1treatment. The animals were pretreated (i.p. and p.o.) or post-treated (p.o.) withMcLTP1(8 mg/kg), and survival assessed over 72 h. Data are expressed as the percentage survival of the animals (mean±SD for each group, n¼8). #Signiﬁcantly different (p<0.05; ANOVA Tukey’s post hoc test) from CLP group. Note: since similar survival rates were observed among sham operated group andMcLTP1pre and post-treated groups, the lines overlap.

Table 2

Antipyretic activity ofMcLTP1on lethal sepsis induced by CLP in mice. Treatment Dose (mg/kg) Rectal temperature (_C)

Normal After CLP

12 h 24 h 36 h 48 h 72 h

Vehicle _ 34.1±0.5 39.4±0.3 39.2±0.2 40.2±0.5 38.8±0.6 38.2±0.8

McLTP1pre-ip 8 mg/kg e 35.1±0.2# 34.1±0.6# 35.1±0.7# 34.0±0.4# 35.0±0.2#

McLTP1pre-p.o. 8 mg/kg e 37.8±0.4# 36.1±0.1# 35.5±0.1# 35.6±0.3# 35.5±0.6#

McLTP1post-p.o. 8 mg/kg e 34.1±0.1# 33.7±0.5# 34.1±0.7# 34.0±0.6# 34.0±0.1# Data are expressed as mean±S.D (n¼8/per group). ANOVA Tukey’s post hoc test (#p<0.05vs. vehicle).

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3.2. Effect of McLTP1on lethal sepsis induced by CLP in mice

3.2.1. Effects on survival, body weight, and rectal temperature As expected, the sham-operated group had a survival rate of 100% during the observation period. In contrast, the vehicle group

had a survival rate reduction of 50% within theﬁrst 24 h, decreasing

to 17% after 72 h. Both pretreatment (oral and intraperitoneal) and

post-treatment withMcLTP1(8 mg/kg) promoted a signiﬁcant level

of protection, with 100% of the mice surviving 72 h after CLP (Fig. 3).

Before the surgery, all animals weighed 35±0.1 g. In the

sham-operated group, the mice maintained their weight throughout the

experiment. However, a signiﬁcant weight loss was observed in the

vehicle group, with the weights of these mice reaching 26±0.1 g

72 h post-CLP. After three days, the groups pretreated orally or

intraperitoneally and post-treated orally with McLTP1 (8 mg/kg)

weighed 36±0.1, 33±0.1, and 34±0.1 g, respectively, which was

similar to that observed in the sham group (34±0.1 g).

Table 2shows the antipyretic effect mediated byMcLTP1 treat-ments in the lethal sepsis induced by CLP. The mice of the vehicle

group developed fever within theﬁrst 12 h after surgery. Fever was

also observed in the mice of the group orally pretreated with

McLTP1 by 12 h; however, it normalized afterward (72 h,

35.5±0.6_{C). Both} _Mc_LTP1 _{(8 mg/kg) pretreatment}

(intraperito-neal) and post-treatment (oral) presented a potent antipyretic ac-tivity, normalizing the body temperature of the mice even 12 h after sepsis induction.

3.2.2. Hematological alterations

Fig. 4shows the hematological parameters of the mice with sepsis induced by the CLP method. The vehicle group presented

with leukocytosis in theﬁrst 24 h after induction of sepsis, and this

condition persisted over the next 48 h of the experiment. On the Fig. 5.Effect ofMcLTP1on lung and liver organ damage in septic animals. (A) Representative images from the different experimental groups are presented (magniﬁcation400). (B) Histopathologic injury scores were graded from 0 (normal) to 3 (severe), according to the tissues damage of 8 animals per group. * and # indicate signiﬁcant differences compared with the sham and vehicle (saline) group, respectively (p<0.05; ANOVA Tukey’s post hoc test).

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contrary, the prophylactic and therapeutic treatments withMcLTP1

(8 mg/kg) signiﬁcantly reduced leukocytosis compared to that of

the vehicle control throughout this time.

3.2.3. Histological analysis

The severity of histopathologic changes and lesion scores are

shown inFig. 5. CLP induced lung and liver histopathological

al-terations, including congestion, inﬂammatory cell inﬁltration,

ne-crosis, and degeneration. All treatments with McLTP1 (8 mg/kg)

markedly attenuated these pathological changes by both routes (intraperitoneal and oral), especially in the pretreated mice, which showed tissue structures similar to those observed in the sham-operated group.

3.2.4. Production of cytokines

Cytokine levels were increased in the septic animals

(CLPþvehicle), as observed with IL-6, IL-10, MCP-1, and TNF-

a

levels (Fig. 6). In contrast, both pretreatment and post-treatment

withMcLTP1(intraperitoneal and oral, respectively) could reduce

(p<0.05) systemic production of all the aforementioned cytokines.

IFN-

g

and IL-12 p70 were detected at low levels (<10 pg/mL), and

there were no signiﬁcant differences amongMcLTP1treated groups

and the vehicle group.

4. Discussion

AMPs have been described as components of plant innate im-mune mechanisms involved in the responses to pathogen attacks

[10]. Due to their antimicrobial abilities, AMPs have been proposed

as promising tools in human health and agribusiness applications

[9]. Some classes of the AMP family, such as LTP members, have

been the focus of antimicrobial studiesin vitro, particularly against

phytopathogenic microorganisms. However, their effects on human

pathogens andin vivoapplications have remained unclear.

Pharmacological studies have shown that McLTP1 displays

antinociceptive and anti-inﬂammatory activities by intraperitoneal

or oral routes, demonstrating its potential use as a therapeutic

agent [12]. Despite this peptide belonging to the AMP family and

the potential biological effect observed forMcLTP1, its antibacterial

activity has not been assessed until now. Therefore, we explored

the properties ofMcLTP1 on bacterial species of clinical interest

in vitro, and we investigated its protective effect on mice with sepsis

induced by CLP, via prophylactic and therapeutic treatments.

In our experiments, we found thatMcLTP1inhibited the growth

of Gram-positive microorganisms (Staphylococcusspp.), but did not

show antimicrobial effects on E. coli, P. aeruginosa, and

K. pneumoniae. These results are similarly to those of a study on an

LTP isolated fromPhaseolus mungoseeds, which could inhibit the

growth ofS. aureusbut was not able to inhibit the Gram-negative

Salmonella typhimuriumused in that study [22]. Most plant LTPs

show speciﬁcity against a particular range of microorganisms and

exhibit varied efﬁcacy. Differences in the lipid composition of their

cell membranes are believed to be involved in the selective toxicity

of plant LTPs against pathogens [23]. Beyond this, the literature

shows that the outer cell wall of Gram-negative bacteria can confer low permeability to antibacterial molecules, diminishing their susceptibility to some antimicrobial agents compared to that of

Gram-positive microorganisms [24].

The action mechanism of the antimicrobial effects ofMcLTP1has

not been investigated in this work. However, based on the mode of action described for the antimicrobial activity of other LTPs, it is reasonable to assume that this peptide could act as a per-meabilizing membrane agent. The membrane pore-forming ca-pacity of LTPs occurs through electrostatic interactions between cationic amino acid residues present in the peptide structure and

membrane lipids, with no direct relationship to their ability to bind

and transfer lipids [10,11,23]. Reductions in the antimicrobial

ac-tivity of plant LTPs in the presence of high ionic-strength solutions, and a weaker effect observed for LTP isoforms with a smaller number of basic amino acid residues, support the mechanism of

action involving membrane targeting [23].

In addition to displaying bacterial antiplanktonic activity,

McLTP1was able to diminishS.aureusbioﬁlm formation.S.aureusis

a versatile and highly virulent pathogen involved in clinical

in-fections. It is commonly associated with strains producing bioﬁlms,

which are difﬁcult to treat, leading to bacteremia and metastatic

infections that are strongly associated with high morbidity and

mortality. Reductions in bioﬁlm formation capacity by

staphylo-cocci represent decreased virulence of these bacteria, imparting greater susceptibility to body defenses and antimicrobials drugs

[25,26]. Thus, theseﬁndings reinforce the potential application of

McLTP1in clinical settings, since it can act against both planktonic

cells and bioﬁlm formation.

Drug interaction strategies are alternatives being tested to overcome increases in microbial resistance. Particularly, a

prom-ising synergistic effect was found between McLTP1 and oxacillin

against S. aureusand S. epidermidis. Apart from re-establishing

microorganism sensitivity to antimicrobial drugs in clinical prac-tice, combination therapies can also lead to a reduction in thera-peutic doses and treatment durations, which are important to diminish adverse effects and minimize the recurrence of microbial resistance [8,27].

Concerning the mechanism of interaction between antibacterial drugs, several studies have reported that those with closely related mechanisms exhibit synergistic effects more readily than drug

combinations with an entirely different mode of action [28].

Moreover, it has been proposed that the pore-forming capacity of AMPs can also enhance the activity of bacterial peptidoglycan hy-drolases. This event compromises the integrity of the peptido-glycan layer, increasing the access of antibiotics that affect cell wall

synthesis (e.g.

b

-lactams), and resulting in a more pronounced

antimicrobial effect [28,29]. A mechanism such as this might

explain the positive interaction observed between McLTP1 and

oxacillin.

In order to study the protective effect ofMcLTP1in severe sepsis,

we used the CLP experimental model. This model has been widely used to study the complexity of sepsis pathophysiology and to search for new therapeutic agents. Over the past 45 years, since its

establishment in 1970 by Chadry et al. [20], it has been considered

the‘gold standard’in sepsis studies, because it most closely mimics

the progress of the syndrome in humans [30,31].

In an attempt to observe the prophylactic effect ofMcLTP1, mice

were pretreated with the peptide by intraperitoneal injection and by oral delivery. However, this pretreatment is not conducive to clinical settings. Therefore, a therapeutic group involving

contin-uous oral administration ofMcLTP1to the mice after sepsis

induc-tion was also included.

In the present study,McLTP1(8 mg/kg) displayed a potent

pro-tective effect against sepsis induced by CLP in mice. Treatment with the peptide completely prevented the lethality frequently observed in severe sepsis that is caused by systemic and pathological changes, such as alterations in water and food consumption, loss of body weight, hyperpyrexia, leukocytosis, and organ damage

[32e35]. Due to an imbalance in the release of proinﬂammatory

and anti-inﬂammatory mediators, sepsis is commonly described as

a“cytokine storm.”Among the proinﬂammatory mediators,

upre-gulation of TNF-

a

, IL-1

b

, IL-6, and IL-8 has been strongly associated

with the harmful effects of sepsis. In general, the CLP model pro-vides an exacerbated release of these mediators, leading to the

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McLTP1 administration was observed with pretreatment (intra-peritoneal and oral) and especially post-treatment (oral), which

supports the potential prophylactic and therapeutic beneﬁts of

McLTP1in the management of sepsis in the clinical setting.

Sepsis induced by CLP consists of an endogenous polymicrobial infection, promoted through perforation of the cecum, which al-lows leakage of gut bacteria into the intraperitoneal cavity, gener-ating peritonitis and leading to sepsis. Thus, high blood bacterial counts are observed ~20 h after CLP, consisting mainly of

Gram-negative bacteria, such asEnterococcus cloacae,E. coli,Proteus

mir-abilis, andAlcaligenes faecalis[36]. BecauseMcLTP1did not display

efﬁcacy against Gram-negative bacteriain vitro, includingE. coli, we

propose that its protective effect is related to the anti-inﬂammatory

properties of this peptide rather than its direct effect on

microor-ganisms. Using classical models of inﬂammation, Campos et al. [37]

have shown that McLTP1 is able to reduce the levels of

pro-inﬂammatory cytokines (i.e., TNF-

a

, IL-1, and IL-6) signiﬁcantly

and increase the levels of IL-10, an anti-inﬂammatory cytokine, by

both intraperitoneal and oral routes (8 mg/kg).

In order to determine whether cytokine modulation could be

involved in the beneﬁcial effects ofMcLTP1against sepsis, cytokine

(TNF-

a

, IFN-

g

, IL-6, IL-10, IL-12 p70, and MCP-1) levels were

quantiﬁed from serum samples of CLP-induced septic mice by the

CBA technique.McLTP1administration was able to downregulate

the proinﬂammatory cytokines, TNF-

a

, IL-6, and MCP-1, which was

consistent with the results found earlier with the paw edema

in-ﬂammatory model [37]. According to other investigators [5,33],

during the early phase of sepsis, TNF-

a

and IL-1 are the main

ini-tiators of septic responses, released predominantly from activated macrophages within 30 min after the infectious stimulus. Once in the systemic circulation, these cytokines stimulate different target cells, such as endothelial cells, which upregulate expression of adhesion molecules and enhance integrin adhesiveness to

neu-trophils, leading to excessive extravasation into tissues. TNF-

a

and

IL-1 are also responsible for amplifying later proinﬂammatory

cytokine production (e.g., IL-6 and IL-8). Elevated IL-6 blood levels are associated with mediation of the acute phase response in sepsis that involves fever, leukocytosis, and multiple organ failure. Moreover, IL-6 is an indicator of sepsis severity because it can be found at high levels for an extended period.

Several studies have also shown elevation of chemokines

(macrophage migration inhibitory factoreMIF; MCP-1) in the host

response to pathogens in sepsis [38,39]. MCP-1 also acts as a

proinﬂammatory mediator in sepsis, involved in the recruitment of

mononuclear cells and neutrophils. Therapies targeting MCP-1 synthesis have been shown to reduce lung and liver neutrophil

inﬁltration and to improve mouse survival during CLP-induced

sepsis [39]. Therefore, reﬁned regulation in the cytokine and

che-mokine proﬁles plays a pivotal role in eliminating invading

path-ogens and attenuating excessive tissue-damaging inﬂammation to

improve sepsis outcomes [5].

Regarding IL-10 production,McLTP1administration

(intraperi-toneal and oral) signiﬁcantly reduced the level of this

anti-inﬂammatory mediator compared to that in the control mice,

which differed from previous results where an increase in IL-10

production was found [37]. Despite the involvement of IL-10 in

the suppression of proinﬂammatory cytokines (e.g., TNF-

a

, 1,

IL-6, and IFN-

g

), its role in sepsis is controversial. Although IL-10

protected mice from lethal endotoxemia in another experimental sepsis model induced by lipopolysaccharide administration, its

inhibition 12 h after CLP markedly improved survival [5].

Although Gram-negative bacteria were originally described as the major causative organisms for sepsis, recent epidemiological data have revealed that septicemia due to Gram-positive bacteria

has become very common in the last 25 years, withStaphylococcus

spp. frequently involved in such cases [3,32]. SinceMcLTP1displays

antimicrobial activity against Gram-positive bacteria (S.aureusand

S. epidermidis), we hypothesized that, in systemic infections

involving these organisms,McLTP1could exert anti-inﬂammatory

and antimicrobial activity, making it even more effective in the management of sepsis. However, further investigations are needed to support this hypothesis.

In conclusion, the data presented herein extend the antibacterial

properties displayed by the LTP group of AMPs.McLTP1was active

not only against the planktonic growth of Gram-positive bacteria,

but also against bioﬁlm formation. To the best of our knowledge,

this is theﬁrst report that describes an LTP with protective effects

against polymicrobial sepsis by alleviating organ damage. The current investigation provides a novel candidate for the potential prevention and treatment of sepsis.

Conﬂicts of interest

The authors have declared that there is no conﬂict of interest,

includingﬁnancial, personal or any other relationships with other

people or organisations.

Acknowledgements

The authors thank the Brazilian institutions CNPq (National

Council for Scientiﬁc and Technological Development; grant

num-ber 482507/2013-6), FUNCAP (Ceara Research Foundation) and UFC

(Federal University of Ceara), for physical installation andﬁnancial

support of this research.

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