Science against microbial pathogens: communicating current research and technological advances ______________________________________________________________________________ A. Mndez-Vilas (Ed.

Isolation of a New Antimicrobial/Antitumor Plant Peptide: Biotechnology Prospects for its Use in Cancer and Infectious Diseases Therapies
Mara G. Guevara*, Fernando F. Muoz, Mara B. Fernndez, Julieta R. Mendieta and Gustavo R. Daleo
Plant Biochemistry Laboratory, Biological Research Institute, National Scientific and Technical Research Council, University of Mar del Plata. Argentina. gguevara@mdp.edu.ar The immune system of multi-cellular organisms comprises a vast arsenal of mechanisms to protect the host from the continuous interactions with infectious microorganisms. Antimicrobial peptides (AMPs) are peptides which protect their hosts against a vast array of microorganisms. These peptides are produced by several species including bacteria, insects, plants, vertebrates and they have been recognized as ancient evolved molecules that have been effectively preserved in mammals. AMPs are expressed on the primary barriers of the organism such as skin and mucosal epithelia, preventing the colonization of host tissues by pathogens. We have previously reported the induction after infection and the cytotoxic activity of potato aspartic proteases (StAPs) towards plant pathogens. Here we show results on the antimicrobial/antitumor activities of these enzymes and of a domain of these enzymes named as StAsp-PSI. StAsp-PSI has structural homology with a family of proteins with antimicrobial/antitumor activity named as SAPLIPs family. Ours results show that StAspPSI is able to kill spores of two potato pathogens but not plant cells, in a dose dependent manner. As reported for StAPs (Solanum tuberosum aspartic proteases), StAsp-PSI ability to kill microbial pathogens is dependent on the direct interaction of the protein with the microbial cell wall/or membrane, leading to increased permeability and lysis. Additionally, we demonstrated that, like proteins of SAPLIP family, StAsp-PSI and StAPs are cytotoxic for Gram negative and Gram positive bacteria in a dose dependent manner. The amino acid residues conserved in SP_B (pulmonary surfactant protein B) and StAsp-PSI could explain the cytotoxic activity exerted by StAsp-PSI and StAPs against Gram positive bacteria. On the other hand, results obtained show that StAPs induce apoptosis on Jurkat T cells at short time of incubation in a dose dependent manner. However, not significative effect on the T lymphocytes viability was observed at any time and StAPs concentration assay. StAsp-PSI was able to induce DNA fragmentation, ROS induction and cell cytotoxicity on human breast cancer cells in a dose dependent manner. These results open a new perspective to test these proteins as possible candidates to develop new drugs that would be active against microbes but not against mammalian cells and considerer these proteins as conceptually promising agent in cancer therapy.

1. Introduction
Antimicrobial proteins and peptides (AMPPs) are important components of the natural defences against invading pathogens and are found in a wide range of eukaryotic organisms, from humans to plants [1-6]. The discovery of new groups of AMPPs as potential natural antibiotics represents a hit toward the discovery of a novel generation of drugs for the treatment of bacterial and fungal infections [7]. Moreover, the broad spectrum of antimicrobial activities reported for these molecules suggests their potential benefit in the treatment of viral or parasitic infections [8, 9] and cancer [10, 11]. In contrast to conventional antibiotics, they act by physical disturbance or destruction of the barrier function of the plasma membrane cell without involvement of a specific receptor [12, 13]. Some of them have been successfully used to eliminate, e.g., prostate tumour cells in vitro [14], and other were also very effective in vivo in the elimination of leukaemia, ascite and ovarian tumours [15, 16]. Plants, unlike mammals, lack mobile defensive cells and a somatic adaptive immune system. Instead, they rely on the innate immunity of each cell and on systemic signals emanating from infection sites [17-19]. In plants, in vitro antimicrobial activity has been demonstrated for the following peptides and proteins: (i) some of the so-called pathogenesis-related proteins, which were originally identified as pathogen-elicited proteins [20, 21]; (ii) a number of plant antimicrobial protein and peptide families [22]. Furthermore, plant proteins and peptides with cytotoxic activity and anticancer properties in vitro on human cancer cell lines have been reported [23-28]. During the last years we have reported the first evidence of the bifunctional activity - proteolytic and antimicrobialof monomeric aspartic proteases from Solanum tuberosum (StAPs) [29, 30]. Plant genome encodes hundreds of proteases, but little is known about the roles they play in the life of a plant [31, 32]. Plant aspartic proteases (APs) have common characteristics with aspartic protease A1 family, are active at acidic pH, are specifically inhibited by pepstatin A and have two aspartic acid residues responsible for the catalytic activity [33, 34].The typical plant AP sequences predict preproproteins similar to the animal and fungal aspartic proteases, with a signal peptide and a proregion at the amino-terminus of the mature protein. In contrast, plant APs genes, with the exception of nucellin, barley AP [35] and AP encoded by the cdr-1 gene from Arabidopsis [36], have an extra region of approximately 100 amino acids known as plant specific insert (PSI). This sequence has homology with the precursor of mammalian saposins, lysosomal sphingolipid-activating proteins (SAPLIPs) [37, 38].

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Plant APs are found in either heterodimeric or monomeric forms [39]. Heterodimer formation is a result of posttranslational proteolytic modifications resulting in the removal of PSI prior to activation [40, 41]. In contrast, monomeric aspartic proteases retain their PSI domain [42]. Several possible functions have been associated with the presence of PSI domain into the mature plant APs. Trmkangas et al. [43] have related the presence of PSI domain with the protein sorting of heterodimeric APs precursors, during Golgi-mediated intracellular transport to vacuoles. On the other hand, the presence of PSI in the mature monomeric plant APs has been associated with the in vitro bifunctional activity, membrane-destabilizing/proteolytic, exerted by these enzymes [44, 45]. However, the real function/s of PSI in plant APs is/are still unclear. Previously we have reported the overexpression in E. coli of the PSI domain from AP potato cDNA clone, named StAsp (GeneBank accession no. AY672651). Here, we have summarized the results obtained in our laboratory about the analysis of StAsp-PSI amino acid and structure. Additionally, we demonstrate how it can determine its selective cytotoxic activity and mechanism of action to produce leakage of plasma cell membranes. StAsp-PSI amino acid sequence; structure and antimicrobial activity. StAsp-PSI contains 148 amino acids inserted into the C-terminal domain (271 to 419 positions) [46]. Analysis of StAspPSI by NCBI Conserved domain search [47] (Figure 1) showed a high homology with three domains present in saposinlike proteins (SAPLIPs) type B: smart00741 (93.4 and 67.1%); Pfam 03489 (87.14%) and Pfam 05184 (54,4%) [37]. The StAps-PSI amino-terminal portion aligned to Pfam05184 domain corresponding to SAPLIP type B, region 2 and StAps-PSI carboxy-terminal portion aligned to Pfam05184 domain, corresponding to SAPLIP type B, region 1 as aspartic protease insert do [48].

Fig. 1 StAsp- plant specific insert domain organization. Alignment of saposins domains: pfam 05184, Saposin-like type B, region 1 (gnlCDD16479) and pfam 03489, a Saposin-like type B, region 2 (gnlCDD26096) with the swaposin domain from StAsp. The alignment of saposin domains was automatically aligned using CLUSTAL W algorithm. Conserved cysteines are indicated by narrows, similar residues are highlighted in black and gaps are indicated by dashes.

Alignment of saposins with StAsp-PSI shows a conserved position of all cysteine residues that bound in known disulphide linkages [37]. These results show that StAsp-PSI, as well as the most of PSI of plant aspartic proteases [49], does not consist of a single saposin domain. The insert sequence is thus equivalent to a circular permutation within the saposin gene, called swaposin domain [48]. As expected, comparison of the StAsp-PSI amino acid deduced sequence reveals a high degree of conservation with other PSI domains from plant aspartic proteases (Figure 2). StAsp-PSI shows the highest sequence identity with SlAspPSI from tomato [50] (94%), IbAsp-PSI from sweet potato (73%), and NaAsp1-PSI [51] from Nepenthes alata (65%). SlAP and StAPs have been considered as being a part of the defensive machinery against pathogens and/or an effector on cell death [34, 45]. On the other hand, NaAP1 has been associated with a contribution to prey digestion by destroying prey cell membranes, in carnivorous plants [51]. Lower identity percentages were found between StAsp-PSI and PSI domains from precursors of vacuolar aspartic proteases isolated from several species: NaAsp2-PSI (54%), NaAsp3-PSI (55%) and NaAsp4-PSI (57%) from N. alata; GmAsp1-PSI (57%) and GmAsp2-PSI (54%) both from Glycine max; ZmAsp-PSI (56%) from Zea mays; CaAsp-PSI (57%) from Cicer arietinum; OsAsp1-PSI (56%), OsAsp2-PSI (52%) and OsAsp3-PSI (51%) from Oryza sativa; TaAsp1-PSI (57%) and TaAsp2-PSI (53%) both from Triticum aestivum; HvAsp-PSI (56%) from Hordeum vulgare; FeAsp1-PSI (56%) and FeAsp2-PSI (51%) both from Fagopyrum esculentum; TcAsp1-PSI (54%) and TcAsp2-PSI (52%) both from Theobroma cacao; VvAsp-PSI (56%) from Vitis vinifera; AtAsp1-PSI (54%), AtAsp2-PSI (50%) and AtAsp3-PSI (43%) from Arabidopsis thaliana; CcAsp-PSI (52%) from Centaurea calcitrapa; BoAsp-PSI (53%) from Brassica oleracea; BnAsp-PSI (53%) from B. napus; HaAsp-PSI (48%) from Helianthus annuus; NtAsp-PSI (50%) from Nicotiana tabacum; CpAsp-PSI (50%) from Curcubita pepo; VuAsp-PSI (49%) from Vigna unguiculata; CysAsp1-PSI (52%), CysAsp2-PSI (47%), CysAsp3-PSI (47%), CysAsp4-PSI (44%) and CysAsp5-PSI (42%) from Cynara cardunculus; and ScAsp-PSI (49%) from Sonneratia caseolaris. In these APs, PSI domains have been associated with the vacuolar sorting of plant AP precursors [43, 52]. PSI functions in plant APs are still unclear; however, several roles have been suggested. These results could indicate that the amino acid composition would be related with the APs apoplastic function.

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Fig. 2 Unrooted neighbor-joining tree of the PSI domains from plant aspartic proteases. Sequences retrieved from databases were aligned using the CLUSTAL W algorithm [53] and protein sequence similarity searches were performed by using GeneDoc alignment editor version 2.6.002 [54]. Phylogenetic analysis were conducted using MEGA version 4.0 [55]. The phylogenetic tree was inferred using the Neighbor-Joining method [56] and the robustness of each node was assessed by bootstrap resampling (10000 replicates) [57]. The PSI amino acid sequences were either obtained from previously reported sequences or translated from the reported cDNA sequences at the NCBI. StAsp-PSI (S. toberosum, AY672651); SlAsp-PSI (S. lycopersicum, AAB18280); IbAsp-PSI (I. batatas, DQ903691); NaAsp1-PSI to NaAsp4-PSI (N. alata, AB045894, AB045891, AB045892, AB045893); GmAsp1-PSI and GmAsp2-PSI (G. max, AB070857, AB069959); ZmAsp-PSI (Z. mays, EU960771); CaAsp-PSI (C. arietinum, AB024999); OsAsp1PSI to OsAsp3-PSI (O. sativa, D32144, NP_001042785, AAS98423); TaAsp1-PSI and TaAsp2-PSI (T. aestivum, AB219968, AB219969); HvAsp-PSI (H. vulgare, X56136); FeAsp1-PSI and FeAsp2-PSI (F. esculentum, AY826351, AAV84086); TcAsp1-PSI and TcAsp2-PSI (T. cacao, AJ313384, AJ313385); VvAsp-PSI (V. vinifera, EF123256); AtAsp1-PSI to AtAsp3-PSI (A. thaliana, U51036, AAL49856, NP_192355); CcAsp-PSI (C. calcitrapa, Y09123); BoAsp-PSI (B. oleracea, X77260); BnAsp-PSI (B. napus, AAB03108); HaAsp-PSI (H. annuus, AB025359); NtAsp-PSI (N. tabacum, DQ648018); CpAsp-PSI (C. pepo, AB002695); VuAspPSI (V. unguiculata, U61396); CysAsp1-PSI to CysAsp5-PSI (C. cardunculus, CAA57510, CAL07969, CAA48939, AJ132884, AJ237674); and ScAsp-PSI (S. caseolaris, ABQ41931).

In Table 1 and Table 2 results obtained show that, StAP1, StAP3 and StAsp-PSI are able to kill human pathogenic bacteria in a dose dependent manner, but are not toxic to human red blood cells (hRBC) at the concentrations assayed [58, 59]. The amounts necessary to kill 50% of the microorganisms are in the same order of magnitude that those previously reported for NK-lysin and granulysin (Table 1).
Table 1 StAsp-PSI/StAPs antimicrobial activity.

Pathogen Plant pathogen Phytophthora infestans Fusarium solani

StAP1 0.005 0.80

LD50 (M) StAP3 StAsp-PSI 0.37 2.95 0.20 2.50

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Streptomyces scabies Erwinia carotovora Human pathogen Bacillus cereus Escherichia coli Staphylococcus aureus

1.50 3.70 3.20 4.25 1.01

1.20 3.75 1.73 2.87 3.85

0.24 0.30 2.67

LD50: concentration causing 50% of pathogen cell death -: not determined Figure 3 shows that this selective toxicity could be associated with the structural homology found between StAsp-PSI and NK-lysin and granulysin, since, in the correct folding; both SAPLIPs are unable to produce hRBC lysis [60]. Structural analysis revealed a high structural homology between StAsp-PSI, NK-lysin and granulysin. The location of the cysteine residues bonds in StAsp-PSI was a common feature with granulysin. 3-D predicted StAsp-PSI structure has a motif containing helix 1 - helix 5 similar to the motif composed by helix 2 - loop - helix 3 described for NK-lysin and granulysin. Like granulysin [61, 62], StAsp-PSI structure comprises five amphipathic -helices folded into globular domain and linked with each other by two disulfide bridges.

Fig. 3 Sequence and structural homology between StAsp-PSI, CardoA-PSI and SAPLIPs. (A) Amino acid sequence alignment of the PSI domain of StAsp-PSI (GeneBank acc. No. AY672651) and CardoA-PSI (GeneBank accession no. AJ132884) and permuted sequences of NK-lysin (GeneBank accession no. Q29075) and Granulysin (GeneBank acc. No. EAW99485). Sequences retrieved from databases were automatically aligned using CLUSTAL W algorithm. Conserved cysteines are highlighted in black, identical residues are highlighted in gray and gaps are indicated by dashes. (B) Ribbon representation of the model structure of the PSI domain of StAsp-PSI and CardoA-PSI based on the crystal structure of prophytepsin-PSI (PDB acc. No. 1QDM) and crystal structures of NK-lysin and Granulysin. Similar helixes are displayed in same color. The disordered part of the PSIs is marked by dotted line and disulfide bridges by dashed lines. Similarities between StAsp-PSI, CardoA-PSI, NK-lysin (PDB: 1NKL) and Granulysin (PDB: 1L9L) were determined using DeepView/Swiss-PdbViewer 3.7 program.

Unlike NK-lysin and granulysin and similar to SP_B [63], another SAPLIP, StAP and StAsp-PSI are cytotoxic in a dose dependent manner for Gram + bacteria. This cytotoxic effect could be explained by the presence in StAsp-PSI of three basic residues (Lys83, Lys88, and Lys90) in the domain constitute by helix 4 and helix 5. These residues are exposed in the StAsp-PSI structure in the same feature than the residues required for SP_B antibacterial activity (Arg12 and Lys16 form in helix 1) [63]. The StAPs/StAsp-PSI selective cytotoxic activity suggest that these proteins could be used to develop alternative drugs, that would be active against microbes but not against mammalian cells.

3. StAsp-PSI antitumor activity

Several studies on AMPPs have focused in their putative antitumoral activities on human and mammalian cancer cell lines in vitro [4, 7, 8, 14-16, 64-66]. In consequence, they have been characterized as alternative compounds to be employed in cancer treatment and/or prevention [7, 8]. Likewise, StAPs and StAsp-PSI have antitumor activity in vitro [67]. StAP1 and StAP3 exert cytotoxic effect on several human cell lines: Jurkat T cell, a leukaemia cell line [67]; an epithelial carcinoma cell line (A431); a colon adenocarcinoma cell line (CaCo-2); a neuroblastoma cell line (SH-SY5Y)

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(unpublished results). Additionally, StAsp-PSI has cytotoxic activity on a human breast adenocarcinoma (MCF7) and a murine melanoma (B16) cell lines (unpublished results). StAPs and StAsp-PSI were able to produce significant dosedependent decreases in the percentage of viable tumour cells, whereas only a slight decrease was detected in the viability of non-transformed T cells, red blood cells and a human epidermal keratinocytes (HaCaT) cell line (Table 2). The StAPs and StAsp-PSI concentrations needed to reduce tumour cell lines viability by 50% were in the same order of magnitude than those previously reported for plant peptides and proteins active against human tumour cells [23, 6870]. Results obtained show that StAPs induce apoptosis only on Jurkat T cells after short times of incubation at the highest concentration assayed; however, no significative apoptosis induction was observed on T lymphocytes at any time and StAPs concentrations assayed [67] (Table 2). These results are in accordance with previous reports showing that the capacity of many antitumoral compounds to cause necrosis or apoptosis in vitro could depend on the doses and/or the time of treatment. That is, some drugs could induce necrosis at high doses and prolonged times of treatment or, alternatively, could induce apoptosis after a short time of treatment or at subnecrotic doses [71, 72] . The capacity of StAPs to induce apoptosis on human leukemia Jurkat T cells provides an interesting starting point for further investigations on the molecular mechanisms underlying that effect, as well as on the suitable conditions to induce programmed cell death without necrosis, which consequently produces an inflammatory process. Because selective targeting for cancer cells is a fundamental requisite for potential chemotherapy agents, the lack of haemolytic activity of StAPs/StAsp-PSI in vitro [59] and its non significative cell toxicity on non-transformed T lymphocytes, could be important features. However, more assays involving other cell lines should be performed.

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Table 1 Apoptosis inducing activity of StAP1, StAP3 and StAsp-PSI in human and murine cancer and blood cells.
Ref.
[Unpublished] [Unpublished] [Unpublished] [Unpublished] [67] [Unpublished] [Unpublished] [67] [58]

Cell lines and types St Asp-PSI

St AP1 1 for 6h 9 for 72h 7 for 6h 0.18 for 6h 2 for 6h 85.0 2.3 for 1 M (24h) 91.2 0.9 for 1 M (24h)

IC50 (M ) St AP3 0.75 for 6h 8.9 for 72h 5 for 6h 1.9 for 6h 2.5 for 6h St AP1 68.10 5.30 for 10 M (6h) 19.00 3.80 for 10 M (6h) 98.20 0.09 for 10 M (24h) 99.00 0.80 for 10 M (6h) 60.10 2.30 for 1.5 M (24h)

St Asp-PSI

Cell viability (%) St AP3 75.30 2.70 for 10 M (6h) 21.00 3.80 for 10 M (6h) 97.10 2.80 for 10 M (24h) 97.00 0.70 for 10 M (6h) 90.00 0.70 for 3.75 M (24h)

Apoptotic cell (%) St AP1 StA P3 63.77 0.30 for 10 M (6h) 68.06 0.10 for 10 M (6h) 42.86 0.08 for 10 M (6h) 38.65 0.13 for 10 M (6h) 57.45 0.16 for 10 M (24h) 63.37 0.43 for 10 M (24h) 71.43 0.30 for 10 M (6h) 79.30 0.10 for 10 M (6h) 26.60 7.40 for 3.75 M (6h) 34.11 0.10 for 3.75 M (6h)

St Asp-PSI

0,38 for 24h 0.60 for 24h 7.10 2.30 for 3.75 M (48h) 1.30 0.87 for 10 M (6h) 1.30 0.87 for 10 M (6h)

Human epithelial carcinoma (A431) Human epidermal keratinocytes (HaCaT) Human colon adenocarcinoma (CaCo-2) Human neuroblastoma (SH-SY5Y) Leukemia cells (Jurkat T) Human breast adenocarcinoma (M CF7) M urine melanoma (B16) Human T lymphocyte Human erythrocyte 6.50 0.70 for 3.75 M (48h) 2.00 0.53 for 10 M (6h) 4.20 0.40 for 3.75 M (24h) 6.30 1.50 for 3.75 M (24h)

Not detected Not detected

Not detected Not detected

IC50:concentra ti on i nhi bi ti ng 50% of cel l growth.

: notdetermi ned

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Acknowledgements This research was supported by grants from National Scientific and Technical Research Council (CONICET), Scientific Research Commission of the Province of Buenos Aires (CIC), and University of Mar del Plata. Muoz, F.and .and Fernandez, MB are fellows of CONICET; Daleo, G. and Mendieta, J are established researchers of CIC and Guevara, G. is established researcher of CONICET.

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