Báo cáo khoa học: Comparison of human RNase 3 and RNase 7 bactericidal action at the Gram-negative and Gram-positive bacterial cell wall pot

Comparison of human RNase and RNase bactericidal action at the Gram-negative and Gram-positive bacterial cell wall ` ´ Marc Torrent, Marina Badia, Mohammed Moussaoui, Daniel Sanchez, M Victoria Nogues and Ester Boix ` ` ` ´ Departament de Bioquımica i Biologia Molecular, Facultat Biociencies, Universitat Autonoma de Barcelona, Cerdanyola del Valles, Spain Keywords antimicrobial proteins; cell wall; ECP; immunity; RNase Correspondence ´ E Boix, Departament de Bioquımica i ` Biologia Molecular, Facultat de Biociencies, ` Universitat Autonoma de Barcelona, 08193 ` Cerdanyola del Valles, Spain Fax: +34 93 5811264 Tel: +34 93 5814147 E-mail: ester.boix@uab.cat (Received 19 November 2009, revised 25 January 2010, accepted 27 January 2010) doi:10.1111/j.1742-4658.2010.07595.x The eosinophil cationic protein ⁄ RNase and the skin-derived RNase are two human antimicrobial RNases involved in host innate immunity Both belong to the RNase A superfamily and share a high cationicity and a common structural architecture However, they present significant divergence at their primary structures, displaying either a high number of Arg or Lys residues, respectively Previous comparative studies with a membrane model revealed two distinct mechanisms of action for lipid bilayer disruption We have now compared their bactericidal activity, identifying some features that confer specificity at the bacterial cell wall level RNase displays a specific Escherichia coli cell agglutination activity, which is not shared by RNase The RNase agglutination process precedes the bacterial death and lysis event In turn, RNase can trigger the release of bacterial cell content without inducing any cell aggregation process We hypothesize that the RNase agglutination activity may depend on its high affinity for lipopolysaccharides and the presence of an N-terminal hydrophobic patch, and thus could facilitate host clearance activity at the infection focus by phagocytic cells The present study suggests that the membrane disruption abilities not solely explain the protein bacterial target preferences and highlights the key role of antimicrobial action at the bacterial cell wall level An understanding of the interaction between antimicrobial proteins and their target at the bacterial envelope should aid in the design of alternative peptide-derived antibiotics Introduction Human antimicrobial RNase and RNase are members of the RNase A superfamily that participate in the host immune response against pathogen infection RNase was first identified as an eosinophil secretion product and named as eosinophil cationic protein (ECP) ECP is secreted by activated eosinophils during inflammation and its levels in biological fluids are considered to be a marker for the diagnosis and monitor- ing of allergy and eosinophilia disorders [1] Recently, it was reported that eosinophils can mediate their antibacterial effect through the release of cationic granule proteins [2] RNase was first reported as a skin antimicrobial protein [3] and is considered to be one of the main components of the innate immunity first-line protection against infections at the epithelial level [4,5] RNase is expressed in several epithelial tissues, Abbreviations CFU, colony-forming unit; ECP, eosinophil cationic protein; MAC, minimal agglutination concentration; PGN, peptidoglycan; SEM, scanning electron microscopy FEBS Journal 277 (2010) 1713–1725 ª 2010 The Authors Journal compilation ª 2010 FEBS 1713 RNase and RNase bactericidal activity M Torrent et al including skin, gut and the respiratory and genitourinary tracts, and its expression can be induced by inflammatory agents and bacterial infection [6] Both RNases display a wide range anti-pathogen activity, with toxicity being reported against viruses, bacteria, fungi, protozoans and, in the case of RNase 3, even helminthic parasites [7] Although both proteins belong to the RNase A superfamily and have conserved their catalytic RNase activity [3,8], studies indicate that their antimicrobial mechanism of action is strongly dependent on their membrane destabilizing mechanism of action [9,10] The RNase A superfamily includes other members with antimicrobial properties [7] and recent evolution studies suggest that the family may have started with an ancestral antipathogen physiological function [11,12] Previous experimental data on both RNases, using lipid vesicles as a membrane model, revealed that the lipid bilayer disruption event takes place with a distinct timing [10,13] However, the data obtained also indicate that mechanic action at the cytoplasmic membrane does not solely explain the protein bactericidal properties Therefore, we also characterized RNase activity at the surface of bacteria, identifying significant differences with respect to its action on both Gram-negative and Gram-positive strains A key distinctive feature of RNase is its high affinity for lipopolysaccharides (LPS) and Escherichia coli cell agglutination activity [14] Despite the fact RNase that both RNases show a high cationicity, they share approximately 40% amino acid identity; careful inspection reveals a distinct evolutionary pressure that leads to the accumulation of an unusual number of either Arg (18 Arg out of 133 amino acids) or Lys (18 Lys out of 128 amino acids) at the mature protein (Fig 1) Mutagenesis studies indicated the involvement of positive and aromatic surface-exposed residues for RNase [15] and some surface lysine clusters for RNase [9] On the other hand, a binding domain for heparin in RNase [16] may account for its high affinity for heterosaccharides at the bacterial cell wall Indeed, recent studies using RNase 3-derived peptides revealed a key domain at the protein N-terminus, which retained most of the protein bactericidal activity and a considerable LPS binding capacity [17] Moreover, screening of the RNase N-terminal sequence predicts a hydrophobic aggregation patch [9] and an antimicrobial prone sequence [18] We have now compared the activity of both RNases at the bacterial cell wall level Although RNase displays remarkable affinity for peptidoglycan (PGN) and LPS at the Gram-positive and Gram-negative outer surface, the very high LPS binding and cell agglutination activities represent a distinctive feature of RNase By contrast, RNase displays a high leakage activity and a high capacity for binding PGN The comparison of both antimicrobial RNases conducted RNase A B C 1714 Fig (A) Ribbon representation of the 3D structures of RNase (1DYT.pdb) [43] and RNase (2HKY.pdb) [9] Molecules are coloured from the N- to C-terminus The active site is marked by a circle (B) Molecular surface representation of RNase and RNase Hydrophobic residues are labelled in grey, cationic residues in blue, anionic residues in red, cysteine residues in yellow, proline residues in orange and noncharged polar residues in cyan (C) Sequence alignment of RNase and RNase primary sequences Secondary structure elements of RNase are depicted at the top The sequence alignment was performed using ESPRIPT software (http://espript.ibcp.fr/ESPript/ESPript/) and molecular representations were drawn using PYMOL (DeLano Scientific, Palo Alto, CA, USA, http://www.pymol.org ⁄ ) FEBS Journal 277 (2010) 1713–1725 ª 2010 The Authors Journal compilation ª 2010 FEBS M Torrent et al RNase and RNase bactericidal activity in the present study therefore contributes towards elucidating the main determinants of their distinct potential in vivo anti-pathogen properties Results Studies on the bacterial cell viability We have compared the RNase and RNase antimicrobial activities with respect to E coli and Staphylococcus aureus cells, which are representative Gram-negative and Gram-positive strains Both proteins display comparable activity, as indicated by the reduction of colony-forming units (CFUs) as a function of protein concentration (Fig 2) On the other hand, kinetic profiles of bacterial viability show a similar overall pattern, although there were significant differences in the respective activities for the two tested strains The bactericidal activity profiles were monitored by staining of bacteria with a Live ⁄ Dead kit (BacLightÔ; Molecular Probes, Carlsbad, CA, USA), using syto and propidium iodide to determine bacterial viability Although syto dye can cross the cytoplasmic membrane and label all bacterial cells, propidium iodide can only access the content of membrane damaged cells, competing and displacing the bound syto Therefore, the integration of syto and propidium iodide fluorescence provides an estimate of the percentage viability for monitoring the kinetics of the bactericidal process (Fig 3) Although RNase shows a similar live ⁄ dead progression for both studied bacterial species, RNase is significantly more active on the E coli population, as reflected by the ED50 values (Fig and Table 1) The relative percentage survival, as evaluated by the viability assay, also correlated with the reduction in the percentage of remaining CFUs (Table 1) To determine the morphological changes in bacterial cell population upon incubation with both RNase and 7, the process was also visualized using confocal microscopy, where live ⁄ dead cells are also labelled with the syto and propidium iodide dyes, respectively A careful inspection on the culture population behaviour by confocal microscopy reveals how RNase aggregates E coli cells, and how bacterial cell death is a later event in relation to the aggregation process (Fig 4) By comparison, RNase 3-treated S aureus cells display a distinct behaviour, where bacterial death takes place at only a slightly lower rate but without a significant aggregation pattern (Fig S1) Therefore, we conclude that the results obtained for RNase indicate that the key bactericidal events take place at different times First, we observe an enlargement on the filaments formed by E coli cells The Fig Remaining CFUs after exposure of bacterial cultures to (A) E coli and (B) S aureus The response is registered as a function of the protein concentration RNase (triangles) and RNase (squares) were dissolved in 10 mM sodium phosphate (Na2HPO4 ⁄ NaH2PO4) buffer, pH 7.5, and serially diluted from 10 lM to 0.2 lM In each assay, protein solutions were added to each dilution of bacteria, incubated for h, plated in Petri dishes and the colonies counted after overnight incubation structures formed (after 10–20 of incubation) are only stained by syto 9, indicating that these filaments are formed by live bacteria From 30 onward, the bacterial population stained by propidium iodide is rapidly increased Subsequently, the aggregates begin to bind propidium iodide and recruit new dead clusters of bacteria (Video S1) For S aureus, this aggregation mechanism cannot be observed and only an increase in the propidium iodide-stained bacteria is detected Although some small clusters of bacteria can be observed, they are not comparable to the aggregates obtained in the case of E coli For RNase 7, aggregation is neither observed in E coli, nor in S aureus (Figs and S2) To quantify the bacterial aggregation ability, the minimal agglutination concentration (MAC) was calculated, with an estimated value of 1.5 lm for RNase activity with E coli cells, whereas no agglutination FEBS Journal 277 (2010) 1713–1725 ª 2010 The Authors Journal compilation ª 2010 FEBS 1715 RNase and RNase bactericidal activity M Torrent et al A B C RNase 10 60 D 120 E RNase F 120 Fig Study of bacterial viability kinetics for (A) RNase and (B) RNase Cell viability for Gram-positive S aureus (filled squares) and Gram-negative E coli (filled circles) was analysed using syto (for live bacteria) and propidium iodide (for dead bacteria) An aliquot of mL of exponential phase cells was incubated with lM of each protein Duplicates were performed for each condition activity was detected in the presence of S aureus cells, nor for RNase with the two tested strains, even with a 10 lm protein concentration The results obtained Fig Study of E coli viability and population morphology visualized by confocal microscopy E coli cells (A) before protein addition; (B–D) after lM of RNase at 10 (B), h (C) and h (D); and (E, F) after adding lM of RNase at and h, respectively Bacterial cells were stained using a : syto ⁄ propidium iodide mixture The left-hand panels correspond to the propidium iodidestained cells (dead cells), excited using an orange diode The central panels correspond to the syto 9-stained cells (live cells), excited using a 488 nm argon laser The right-hand panels correspond to the overlay of both signals Scale bar = 50 lm show that RNase lacks the ability to agglutinate bacteria but retains bactericidal activity To better understand the correlation between aggregation and bacterial leakage, the release of cell content was monitored using activity staining gels (Fig 5) With this technique, the endogenous bacterial Table Kinetic analysis on the antimicrobial activity of RNases and using the Live ⁄ Dead bacterial viability kit as described in the Materials and methods One millilitre of exponential phase cells was incubated with lM of protein during a total period of 150 ED50 (measured as the time needed to achieve a 50% decrease in live bacteria) and percentage survival were calculated by exponential fitting to the data presented in Fig The percentage of remaining CFUs is also indicated for each condition Values are the average of three replicates Protein E coli RNase RNase ED50 (min) 35 ± 56 ± S aureus Survival (%) 12.0 ± 0.8 17 ± Remaining CFUs (%) ± 4* 13 ± 3* ED50 (min) 60 ± 56 ± Survival (%) 20.1 ± 0.8 13 ± Remaining CFUs (%) 22 ± 3* 14 ± 2* *P < 0.05 (Student’s t-test) 1716 FEBS Journal 277 (2010) 1713–1725 ª 2010 The Authors Journal compilation ª 2010 FEBS M Torrent et al RNase and RNase bactericidal activity A B Fig Record of bacterial lysis process by the detection of the release of endogeneous bacterial RNase by activity staining gel (A) The clearance area corresponding to the bacterial RNase substrate degradation is indicated The intensity of the areas showing substrate degradation was analysed by densitometry as described in the Materials and methods The intensity values are referred to the h incubation density area The bacterial lysis activity of RNase (filled symbols) and RNase (empty symbols) on both E coli (triangles) and S aureus (squares) is shown (B) Polycytidylic acid SDS-PAGE (15%) activity staining gel from the time course of E coli cell incubation with RNase Left lanes: control cells; right lanes: cells incubated with lM of RNase at 0, 1, 2, and h ribonuclease released upon membrane leakage can be detected and the leakage kinetics can be monitored The bacterial cells were incubated with lm of each RNase and aliquots were taken at 1-h intervals For RNase 3, an important difference between E coli and S aureus is found Whereas leakage in E coli cells can be observed as soon as after h of incubation, no release is detected for S aureus, not even after h of incubation These results demonstrate that, even though RNase is able to kill 80% of S aureus cells after h of incubation, the damage at the membrane level is insufficient to allow the release of a detectable amount of endogenous ribonucleases In the case of RNase 7, both E coli and S aureus endogenous RNases are released (Fig 5) Nevertheless, RNase leakage in S aureus cells appears to be triggered later than in E coli cells The activity corresponding to the endogenous ribonucleases that are released by the bacteria is only registered after h of incubation Finally, membrane depolarizing activity was also studied using the DiSC3(5) marker (Table S1) The results obtained show that RNase is able to depolarize E coli cells more rapidly than S aureus cells When comparing membrane depolarization activities, we can observe that ECP easily accesses the Gramnegative cytoplasmic membrane, without any EDTA treatment being necessary to destabilize the cell outer membrane RNase activity on E coli cells is inde- pendent of EDTA chelation This is not applicable to RNase 7, which has a lower membrane depolarization activity without EDTA treatment On the other hand, RNase appears to alter more easily the S aureus cytoplasmic membrane than RNase The distinct abilities of both RNases to access and alter the cytoplasmic membrane may reflect their action at the outer envelope level Studies at the bacterial cell wall The bactericidal activity of both RNases is precluded by the protein binding to the cells Proteins incubated with both E coli and S aureus cultures are recovered in the cell pellet fraction (Fig S3) To gain insight on the bactericidal properties of both RNases, binding studies on different elements of the bacterial cell wall were carried out Binding to PGN and LPS has already been studied in detail for RNase [14] The results obtained are now compared with RNase binding affinities The new data (Figs and 7) indicate that RNase can also interact with both Gramnegative and Gram-positive heteropolysaccharides Affinity binding studies on LPS and PGN were complemented with scanning electron microscopy (SEM) microscopy to visualize the structural damage induced by the protein–cell wall interactions (Fig 8) Binding to LPS was assessed using the Bodipy TR cadaverine marker (Invitrogen, Carlsbad, CA, USA) FEBS Journal 277 (2010) 1713–1725 ª 2010 The Authors Journal compilation ª 2010 FEBS 1717 RNase and RNase bactericidal activity M Torrent et al Fig Displacement of LPS-bound Bodipy TR cadaverine by RNase (triangles), RNase (circles) and polymyxin B (squares); [LPS]: 10 lgỈmL)1; [BODIPY TR Cadaverine]: 10 lM in mM Hepes-KOH (pH 7.5) The results obtained show that RNase is able to bind with higher affinity to LPS compared to RNase In any case, RNase still retains a high LPS binding affinity because it displays an effective displacement activity similar to that for polymyxin B, a powerful LPS binder, which was selected as a positive control (Fig 6) We also assessed and compared RNase binding to PGN, the main component of Gram-positive bacteria, with our previous results obtained for RNase [14] Microfluidic gel electrophoresis showed that, after RNase incubation in the presence of S aureus PGN, most of the protein sample is recovered together with the insoluble PGN fraction, as also observed for lysozyme, the positive control, and previously for RNase [14] A slight anomalous displacement in the virtual gel is observed for RNase 7, with a higher apparent molecular weight, as a result of its cationic nature This behaviour is frequently observed for RNase A family members By contrast, BSA, the negative control, does not bind to the PGN fraction and is fully recovered in the supernatant fraction (Fig 7A) Moreover, a PGN binding assay using Alexa fluorophor-labelled RNase also indicates a high binding affinity A Kd value of · 10)8 m was determined using the Scatchard plot as shown in Fig 7B, which is a value considerably higher than that calculated for RNase (2 · 10)7 m) [14] SEM data were previously shown to be useful for assessing bacterial surface damage upon RNase A 100.0 75.0 50.0 37.0 25.0 20.0 B Fig (A) Analysis by a microfluidic electrophoresis system of the binding of RNase to PGN Lysozyme and BSA were taken as positive and negative controls, respectively, for PGN binding Molecular mass markers are indicated on the left For each protein, the first lane corresponds to pellet (P) and the second lane to the supernatant fractions (S) PGN were incubated with each protein and the soluble and insoluble fractions were collected as described in the Materials and methods Supernatant represents the soluble fraction, which contains the unbounded protein, whereas the pellet represents the insoluble fraction containing the PGN bound protein (B) Scatchard plot and the corresponding binding curve of RNase interaction with PGN RNase labelled with the fluorophor Alexa Fluor 488 at a concentration in the range 0.01–100 nM was incubated in the presence of 0.02 lg PGN in 200 lL of mM Hepes-KOH (pH 7.5) and the free and bound fractions were quantified 1718 FEBS Journal 277 (2010) 1713–1725 ª 2010 The Authors Journal compilation ª 2010 FEBS M Torrent et al RNase and RNase bactericidal activity E coli S aureus Fig Scanning electron micrographs of E coli and S aureus incubated in the absence (top) and presence (bottom) of lM RNase for h The magnification scale is indicated at the bottom of each micrograph treatment [14], where severe damage on E coli cells and the ability of protein to trigger cell population agglutination was reported Accordingly, SEM was used to visualize changes in bacterial cell cultures upon incubation with RNase The addition of RNase at a final concentration of lm is unable to induce either E coli or S aureus cell culture aggregation and all cells retain their characteristic morphology Nevertheless, several blebs can be observed on the bacterial cell surface in both E coli and S aureus, suggesting that local cell surface disturbance is taking place (Fig 8) Discussion RNases and are the main representatives of the cytotoxic antimicrobial members of the RNase A superfamily Both are cationic proteins with a high pI, and display a broad antimicrobial action against Gram-positive and Gram-negative strains [6,19–21] The two RNases present, respectively, a high number of either Arg or Lys surface-exposed residues (Fig 1) that may contribute to their distinct bactericidal mechanisms of action Previous work revealed that the RNase bactericidal mechanism was not dependent on its RNase enzymatic activity but on direct membrane disruptive action [9,10,15,22] The contribution of bacterial wall determinants was also suggested [15] and recent studies on RNase indicated a high affinity for bacterial heterosaccharides [14] Indeed, the present comparative characterization of both the action of RNase and RNase at the bacterial wall level revealed some particular features that could explain their distinct abilities with respect to Gram-negative and Gram-positive strains We previously compared the mechanism of action of both RNases on model membranes [10,13] RNase has no significant membrane aggregation capacity compared to RNase 3, although it displays a much higher leakage capacity On the other hand, initial studies on RNase by sitedirected mutagenesis indicated that the membrane disruption ability could not solely explain the protein bactericidal properties [15] Indeed, strain selectivity was reported for RNase [3,9] We have now analysed the time course profile of bacterial cell viability for both RNases (Fig 3) The rapid decay during the first 30 may reflect a rapid direct lytic process We can differentiate between an initial active exponential growth phase, where the protein may have easy access to the cell membrane during duplication, and a later stage, where protein action at the wall envelope may acquire a critical role On the other hand, the viability assay, performed at a salt concentration close to physiological levels, rejects a mere unspecific electrostatic interaction and provides further corroboration for both proteins retaining their properties in vivo and being regarded as effective antimicrobial agents As noted by Hancock and Sahl [23], many cationic peptides with few hydrophobic residues at crucial positions are prone to having some antimicrobial activity at low ionic strength, although the term ‘antimicrobial’ should only be reserved for those that are able to kill microbes under physiological conditions The results obtained in the present study reveal distinct behaviours not only on lipid bilayers, but also at the bacterial cell wall In both strains, E coli and S aureus, RNase displays a restricted disturbance causing local blebs, whereas no agglutination is FEBS Journal 277 (2010) 1713–1725 ª 2010 The Authors Journal compilation ª 2010 FEBS 1719 RNase and RNase bactericidal activity M Torrent et al observed (Fig 8) These observations are much different from those observed in the case of RNase 3, where global cell damage has been observed in E coli cells after complete bacterial agglutination [14] Interestingly, the in vivo record of the RNase treated E coli culture assessed by confocal microscopy illustrates how the cells first aggregate but still retain an intact cytoplasmic membrane Cell death, as observed by the propidium iodide uptake, is then a later event (Fig and Video S1) We have further analysed RNase bacterial agglutination activity and estimated a MAC of 1.5 lm on E coli cell cultures Cell agglutination comprises a characteristic feature that also is reported for other antimicrobial peptides [24] and proteins, as lectin RNases, which are amphibian members of the RNase A superfamily with a particular ability for binding heterosaccharides [25] In turn, RNase could follow another bacterial process The ability to induce the bacterial cell content, as assayed by activity-staining gel analysis, has shown that, in S aureus, RNase presents an important leakage activity, whereas no significant activity is detected for RNase at the assayed conditions (Fig 5) This fact may be explained by the higher capacity of RNase to cause leakage of membranes at low concentrations These effects are in good agreement with the results observed in model membranes, where RNase is able to trigger leakage at a lower protein : lipid ratio before any aggregation event takes place, suggesting a local membrane disturbance process [10] Moreover, the higher binding affinity for PGN displayed by RNase may also partially account for the higher membrane depolarization activity observed against the S aureus strain (Table S1) RNase was previously reported to display a particularly high bactericidal activity for the Gram-positive Enterococcus faecium [3] Our membrane depolarizing assays confirm a distinct mechanism of action for both RNases on each of the two tested strains Mainly for Gram-negative cells, RNase does not require EDTA pretreatment EDTA pretreatment would sequester the divalent cations that hold LPS together and secure the outer membrane structure The higher affinity of RNase for LPS (Fig 6) could by itself facilitate outer membrane disturbance and access to the cytoplasmic membrane RNase displays a similar capacity for depolarizing cell membranes, as observed in RNase 3, when E coli cells are pretreated with EDTA, thus suggesting that the main differences may be restricted to the bacterial outer barrier These results confirm that the capacity to bind bacterial cell wall structures is of special importance for the antimicrobial properties of both RNases, as also 1720 reported for other antimicrobial proteins and peptides [26,27] If we compare the sequences and 3D structures available for both RNases (Fig 1), we can identify some of the features that may account for the specific ability of RNase to aggregate both lipid vesicles and bacterial cells Scanning of both RNases with aggregscan software [28] reveals a distinct aggregation profile, in particular at the N-terminal zone In the case of RNase 3, we can observe a hydrophobic patch in one side of the molecule, surrounded by polar residues Indeed, a hydrophobic patch at the RNase N-terminus that retains most of the protein antimicrobial activity, and may be responsible for the protein vesicle aggregation ability, was recently characterized by synthetic-derived peptides in our laboratory [17] Bacteria agglutinating efficiency was also correlated with the presence of hydrophobic patches for de novo designed antimicrobial peptides In the case of RNase 7, no hydrophobic patches on the protein surface can be observed The protein cationicity, as a result of the high number of lysines present in the structure, is distributed uniformly on the protein surface The absence of hydrophobic patches may be responsible for the lack of agglutinating capacity of RNase Although both RNases contain a high number of cationic residues, the bias on either Arg or Lys content (18 Arg for RNase and 18 Lys for RNase 7) suggests that the cationicity of both proteins has been acquired independently during their evolution A comparison with other RNase A family members indicates that most Lys residues are retained in the RNase A lineage group that includes RNases 6, and [29] Phylogenetic studies suggest the recent divergence of RNase and RNase as a result of a duplication event [29] However, no homologues were identified in rodents [12] as described for the RNase2 ⁄ RNase group, where members with antimicrobial activity were reported in both rat and mouse In turn, RNase acquired many Arg residues during its divergence from RNase [12,29] However, a comparison of antimicrobial RNases suggests that local positive clusters, rather than their overall pI, are key for protein bactericidal activities [30,31] For example, a comparison of the primary sequences for fish, chicken and human antimicrobial RNases revealed a distinct Lys ⁄ Arg ratio but a similar total number of positive residues [30] On the other hand, arginine residues are implied in carbohydrate binding proteins because they display hydrogen bonding between the guanidinium group and sulphates or phosphates [32,33] This fact may explain the higher binding affinity of RNase for LPS (Fig 6) FEBS Journal 277 (2010) 1713–1725 ª 2010 The Authors Journal compilation ª 2010 FEBS M Torrent et al The tissue distribution of both RNases also suggests some functional differences Whereas RNase is mostly present in eosinophils and, to a less extent, in other cells of the immune system (e.g neutrophils and basophils) [34,35], RNase is expressed in multiple somatic tissues, especially the skin, where it is described as a major antimicrobial agent [3,6] Although both RNases are secreted, they may respond to distinct challenges RNase is stored in secretion granules and is depleted at the site of inflammation where these cells are recruited [36] RNase represents one of the major contributors to the antimicrobial activity involved in first-line host defence at the human skin barrier [37] In the skin, basal RNase secretion is detected but mRNA overexpression is observed as a result of bacterial challenge [37] A correlation between a dysfunction in antimicrobial protein expression at the skin level during dermatitis and a predisposition to skin infections also highlights their contribution to a host defence role [38–40] In conclusion, in the present study, we have shown that RNase and RNase have particular antimicrobial activities that are modulated by their action at the bacterial cell wall We observed that RNase displays a mechanism based on local membrane disturbance, in contrast to RNase that demonstrated global action Accordingly, we have shown that RNase displays an E coli agglutinating activity (not shared by RNase 7), which would probably be dependent on both the presence of a hydrophobic patch and the capacity of the protein to bind LPS An understanding of the molecular mechanism that is responsible for the high binding affinity of antimicrobial protein for unique heterosaccharide structures at the bacterial envelope would also contribute to the development of new peptide-derived antibiotics, which would overcome the increasing emergence of antibiotic resistant strains Materials and methods RNase and RNase bactericidal activity WI, USA) and S aureus 502 A (ATCC, Rockville, MD, USA) strains were used PD-10 columns were purchased from GE Healthcare (Milwaukee, WI, USA) Expression and purification of recombinant RNase and RNase Wild-type RNase was expressed using a synthetic gene for human coding sequence RNase was expressed starting from a cDNA subcloned in the pET11c plasmid vector Protein expression in E coli BL21(DE3) strain, folding of the protein from inclusion bodies, and the purification steps, were carried out as described previously [8,10] Fluorescent labelling of proteins RNases were labelled with the Alexa Fluor 488 fluorophor, in accordance with the manufacturer’s instructions To 0.5 mL of a mgỈmL)1 protein solution in NaCl ⁄ Pi, 50 lL of m sodium bicarbonate (pH 8.3) was added The protein was incubated for h at room temperature with the reactive dye, with stirring, in accordance with the manufacturer’s instructions The labelled protein was separated from the free dye by a PD-10 desalting column Antibacterial activity Antimicrobial activity was calculated by assessing the number of CFUs as a function of protein concentration Values were averaged from two independent experiments performed in triplicate for each protein concentration Proteins were dissolved in 10 mm sodium phosphate (Na2HPO4 ⁄ NaH2PO4) buffer (pH 7.5) and serially diluted from 10 lm to 0.2 lm Bacteria were incubated at 37 °C overnight in LB broth and diluted to give approximately · 105 CFmL)1 In each assay, protein solutions were added to each dilution of bacteria, incubated for h, and samples were plated on Petri dishes and incubated at 37 °C overnight The number of CFUs in each Petri dish was counted and the average values were represented in a semi-logarithmic plot Materials Bacterial viability Bodipy TR cadaverine, BC [5-(((4-(4,4-difluoro-5-(2-thienyl)4-bora-3a,4a-diaza-s-indacene-3-yl)phe-noxy)acetyl)amino) pentylamine, hydrochloride], 3,3-dipropylthiacarbocyanine [DiSC3(5)], Gramicidin D, Alexa Fluor 488 protein labelling kit and the Live ⁄ Dead bacterial viability kit were all purchased from Molecular Probes (Eugene, OR, USA) LPSs from E coli serotype 0111:B4, Polymyxin B sulfate, PGN from S aureus, polycytidylic acid and lysozyme from chicken egg white were purchased from Sigma-Aldrich (St Louis, MO, USA) E coli BL21DE3 (Novagen, Madison, Kinetics of bacterial survival were carried out using the Live ⁄ Dead bacterial viability kit in accordance with the manufacturer’s instructions Bacteria were stained using a syto ⁄ propidium iodide : mix as provided with the kit E coli and S aureus cells were grown at 37 °C to the midexponential phase (D600 = 0.4), centrifuged at 5000 g for and resuspended in a 0.75% NaCl solution in accordance with the manufacturer’s instructions One millilitre of stained E coli or S aureus bacteria (D600 = 0.2) was mixed with lm of RNase or and the fluorescence intensity FEBS Journal 277 (2010) 1713–1725 ª 2010 The Authors Journal compilation ª 2010 FEBS 1721 RNase and RNase bactericidal activity M Torrent et al was continuously measured using a Cary Eclipse Spectrofluorimeter (Varian Inc., Palo Alto, CA, USA) RNase A was used in all cases as a negative control The excitation wavelength was 470 nm and the emission was recorded in the range 490–700 nm To calculate bacterial viability, the signal in the range 510–540 nm was integrated to obtain the syto signal (live bacteria) and from 620–650 nm to obtain the propidium iodide signal (dead bacteria) Then, the percentage of live bacteria was represented as a function of time ED50 was calculated by fitting the data to a simple exponential decay function Protein affinity to PGN was calculated using a fluorescence-based method, employing a microtitre plate as described previously [14] Protein labelled with the fluorophor Alexa Fluor 488 was incubated with insoluble PGN Proteins at different concentrations, in the range 1–100 nm, were incubated in the presence of 0.02 lg of peptidoglycans in a mm Hepes buffer at pH 7.5 in a final volume of 200 lL The reaction mixture was kept at °C for h with gentle shaking Next, the remaining soluble protein was removed from the insoluble PGN fraction by a centrifugation step at 13 000 g for 30 and quantified with Victor (Perkin-Elmer, Boston, MA, USA) Agglutination activity Agglutination activity was evaluated by calculating the MAC An aliquot of mL of E coli cells was grown at 37 °C to the mid-exponential phase (D600 = 0.6), centrifuged at 5000 g for and resuspended in Tris-HCl buffer, 0.15 m NaCl (pH 7.5) until D600 of 10 was reached An aliquot of 200 lL of the bacterial suspension was incubated in microtitre plates with an increasing protein concentration at 0.1 and 0.5 lm intervals up to 10 lm and left overnight at room temperature The aggregation behaviour was observed by visual inspection and checked with a binocular microscope at ·50 magnification The agglutinating activity is expressed as the minimum agglutinating concentration of the sample tested, corresponding to the first condition where bacterial aggregates are visible by the naked eye, as described previously [41] Affinity binding assay for LPS LPS binding was assessed using the fluorescent probe Bodipy TR cadaverine as described previously [14] Briefly, the displacement assay was performed by the addition of 1–2 lL aliquots of a solution of Polymyxin B, RNase 3, RNase or RNase A to mL of a continuously stirred mixture of LPS (10 lgỈmL)1) and Bodipy TR cadaverine (10 lm) in mm Hepes buffer at pH 7.5 Fluorescence measurements were performed on a Cary Eclipse spectrofluorimeter The BC excitation wavelength was 580 nm and the emission wavelength was 620 nm The excitation slit was set at 2.5 nm and the emission slit was set at 20 nm Final values correspond to an average of four replicates and were the mean of a 0.3 s continuous measurement Quantitative effective displacement values (ED50) were calculated Protein binding to bacterial cells RNase was incubated at lm with E coli bacterial cells grown to the exponential phase (D600 = 0.6) in mL of NaCl ⁄ Pi buffer at 37 °C for h After centrifugation at 13 000 g, proteins from the pellet were extracted with electrophoresis loading buffer Supernatant fractions were lyophilized and dissolved in loading buffer Samples were analysed by SDS-PAGE (15%) and Coomassie blue staining Affinity binding assay for PGN Protein binding to PGN was first analysed by electrophoresis as described previously [14] PGN at 0.4 mgỈmL)1 in 10 mm Tris-HCl (pH 7.5) was incubated with the protein at a protein ⁄ PGN ratio of : 20 (w ⁄ w) Samples were kept at °C for h with gentle mixing and centrifuged at 13 000 g for 15 to separate the soluble and insoluble fractions Lysozyme and BSA were chosen as positive and negative controls, respectively Samples were resuspended directly in the electrophoresis loading buffer and evaluated using an Experion automated microfluidic electrophoresis system (Bio-Rad, Hercules, CA, USA) 1722 SEM E coli and S aureus cell cultures of mL were grown at 37 °C to the mid-exponential phase (D600 = 0.4) and incubated with lm RNase or RNase in NaCl ⁄ Pi at room temperature Aliquots were taken up to h of incubation and were prepared for analysis by SEM, as described previously [14] The cell suspensions were fixed with 2.5% gluteraldehyde in 100 mm Na-cacodylate buffer (pH 7.4) for h at room temperature Next, the cells were pelleted, a drop of each suspension was transferred to a nucleopore filter, which was kept in a hydrated chamber for 30 allowing the cells to adhere, and then washed to remove the gluteraldehyde, and resuspended in the same 100 mm Na-cacodylate buffer at pH 7.4 Attached cells were post-fixed by immersing the filters in 1% osmium tetroxide in cacodylate buffer for 30 min, rinsed in the same buffer, and dehydrated in ethanol in ascending percentage concentrations [31, 70, 90 (·2) and 100 (·2)] for 15 each The filters were mounted on aluminum stubs and coated with goldpalladium in a sputter coater (K550; Emitech, East Grinsted, UK) The filters were viewed at 15 kV accelerating voltage in a Hitachi S-570 scanning electron microscope FEBS Journal 277 (2010) 1713–1725 ª 2010 The Authors Journal compilation ª 2010 FEBS M Torrent et al (Hitachi, Tokyo, Japan) and a secondary electron image of cells for topography contrast was collected at several magnifications A total of ten micrographs were collected at random for each condition, and the number of isolated cells and aggregates was registered RNase and RNase bactericidal activity recorded for each condition, and the time required to achieve half of total membrane depolarization was estimated from the nonlinear regression curve E coli cells were also incubated in the presence of EDTA, allowing loss of the LPS outer membrane surface layer, as previously described [14] Confocal microscopy Experiments were carried out in a plate-coverslide system Five hundred microlitres of E coli or S aureus bacteria (D600 = 0.4) were mixed with 40 lL of 60 lm to achieve a final concentration of lm of RNase or 7, and images were immediately recorded RNase A was used in all cases as a negative control Bacteria were pre-stained using the syto ⁄ propidium iodide : mix provided in the Live ⁄ Dead staining kit Syto is a DNA green fluorescent dye that diffuses thorough intact cell membranes and propidium iodide comprises a DNA red fluorescent dye that can only access the nucleic acids of membrane damaged cells, displacing the DNA bound syto The method allows the labelling of intact viable cells and membrane compromised cells, which are labelled in green and red respectively, referred to as live and dead cells [42] Confocal images of the bacteria were captured using a laser scanning confocal microscope (Leica TCS SP2 AOBS equipped with a HCX PL APO 63, ·1.4 oil immersion objective; Leica Microsystems, Wetzlar, Germany) Syto was excited using a 488 nm argon laser (515–540 nm emission collected) and propidium iodide was excited using an orange diode (588– 715 nm emission collected) To record the time-lapse experiment, Life Data Mode software (Leica) was used, obtaining an image every in a experiment lasting 180 Bacteria cytoplasmic membrane depolarization assay Membrane depolarization was assayed by monitoring the DiSC3(5) fluorescence intensity change in response to changes in transmembrane potential as described previously [14] E coli and S aureus cells were grown at 37 °C to the mid-exponential phase and resuspended in mm HepesKOH, 20 mm glucose and 100 mm KCl at pH 7.2 until D600 of 0.05 was reached DiSC3(5) was added to a final concentration of 0.4 lm Changes in the fluorescence because of the alteration of the cytoplasmic membrane potential were continuously monitored at 20 °C at an excitation wavelength of 620 nm and an emission wavelength of 670 nm When the dye uptake was maximal, as indicated by a stable reduction in the fluorescence as a result of quenching of the accumulated dye in the membrane interior, protein in mm Hepes-KOH buffer at pH 7.2 was added at a final tested protein concentration of lm Gramicidin D was used as control reference protein All conditions were assayed in duplicate The time required to reach a stabilized maximum fluorescence reading was Bacteria leakage analysis by activity-staining gels Activity-staining gels (zymograms) were selected to analyse bacterial leakage upon incubation with ribonucleases E coli and S aureus cells were grown at 37 °C to the midexponential phase (D600 = 0.4) in LB medium, centrifuged at 5000 g for min, and resuspended in a 10 mm Na2HPO3 buffer at pH 7.2 Cells were incubated with lm of RNase or and 10 lL aliquots were collected at 1, 2, and h The aliquots collected were mixed with nonreducing loading buffer (60 mm Tris-HCl, 10% glycerol, 0.015% bromophenol blue, 3% SDS, pH 6.8) and analysed for RNase activity by zymogram on SDS-PAGE (15%) containing 0.6 mgỈmL)1 of polycytidylic acid as substrate After elimination of SDS by incubation with a pH 7.5 solution consisting of 10 mm Tris-HCl and 20% isopropanol, the gels were incubated at 25 °C for 90 in 100 mm Tris-HCl (pH 7.5) The relative intensity of the areas showing substrate degradation was analysed by densitometry Bacterial cell leakage was assessed by monitoring, as a function of time, the increase of the clearance area corresponding to polynucleotide cleavage by the released bacterial RNase Acknowledgements Confocal microscopy and scanning electron micros` copy were performed at the Servei de Microscopia of ` the Universitat Autonoma de Barcelona (UAB) We ` ´ ´ thank Monica Roldan and Helena Monton for their technical support with confocal microscopy, and Fran´ cisca Cardoso, Francesc Bohils and Alejandro Sanchez for their assistance with the electron microscopy samples Spectrofluorescence and densitometry assays ` were performed at the Laboratori d’Analisi i Fotodoc´ umentacio, UAB The work was supported by the ´ Ministerio de Educacion y Cultura (grant numbers BFU2006-15543-C02-01 and BFU2009-09371) and by ´ ´ the Fundacio La Marato de TV3 (grant number TV3031110) M.T was the recipient of a predoctoral fellowship from the Generalitat de Catalunya References Ahlstedt S (1995) Clinical application of eosinophilic cationic protein in asthma Allergy Proc 16, 59–62 FEBS Journal 277 (2010) 1713–1725 ª 2010 The Authors Journal compilation ª 2010 FEBS 1723 RNase and RNase bactericidal activity M Torrent et al Linch SN, Kelly AM, Danielson ET, Pero R, Lee JJ & Gold JA (2009) Mouse eosinophils possess potent antibacterial properties in vivo Infect Immun 77, 4976–4982 Harder J & Schroder JM (2002) RNase 7, a novel innate immune defense antimicrobial protein of healthy human skin J Biol Chem 277, 46779–46784 Schroder JM & Harder J (2006) Antimicrobial skin peptides and proteins Cell Mol Life Sci 63, 469–486 Koten B, Simanski M, Glaser R, Podschun R, Schroder JM & Harder J (2009) RNase contributes to the cutaneous defense against Enterococcus faecium PLoS ONE 4, e6424 Zhang J, Dyer KD & Rosenberg HF (2003) Human RNase 7: a new cationic ribonuclease of the RNase A superfamily Nucleic Acids Res 31, 602–607 Boix E & Nogues MV (2007) Mammalian antimicrobial proteins and peptides: overview on the RNase A superfamily members involved in innate host defence Mol Biosyst 3, 317–335 Boix E, Nikolovski Z, Moiseyev GP, Rosenberg HF, Cuchillo CM & Nogues MV (1999) Kinetic and product distribution analysis of human eosinophil cationic protein indicates a subsite arrangement that favors exonuclease-type activity J Biol Chem 274, 15605–15614 Huang YC, Lin YM, Chang TW, Wu SJ, Lee YS, Chang MD, Chen C, Wu SH & Liao YD (2007) The flexible and clustered lysine residues of human ribonuclease are critical for membrane permeability and antimicrobial activity J Biol Chem 282, 4626–4633 10 Torrent M, Sanchez D, Buzon V, Nogues MV, Cladera J & Boix E (2009) Comparison of the membrane interaction mechanism of two antimicrobial RNases: RNase ⁄ ECP and RNase Biochim Biophys Acta 1788, 1116–1125 11 Pizzo E & D’Alessio G (2007) The success of the RNase scaffold in the advance of biosciences and in evolution Gene 406, 8–12 12 Dyer KD & Rosenberg HF (2006) The RNase a superfamily: generation of diversity and innate host defense Mol Divers 10, 585–597 13 Torrent M, Cuyas E, Carreras E, Navarro S, Lopez O, de la Maza A, Nogues MV, Reshetnyak YK & Boix E (2007) Topography studies on the membrane interaction mechanism of the eosinophil cationic protein Biochemistry 46, 720–733 14 Torrent M, Navarro S, Moussaoui M, Nogues MV & Boix E (2008) Eosinophil cationic protein high affinity binding to bacteria wall lipopolysaccharides and peptidoglycans Biochemistry 47, 3544–3555 15 Carreras E, Boix E, Rosenberg HF, Cuchillo CM & Nogues MV (2003) Both aromatic and cationic residues contribute to the membrane-lytic and bactericidal activity of eosinophil cationic protein Biochemistry 42, 6636–6644 1724 16 Fan TC, Fang SL, Hwang CS, Hsu CY, Lu XA, Hung SC, Lin SC & Chang MD (2008) Characterization of molecular interactions between eosinophil cationic protein and heparin J Biol Chem 283, 25468–25474 17 Torrent M, de la Torre BG, Nogues VM, Andreu D & Boix E (2009) Bactericidal and membrane disruption activities of the eosinophil cationic protein are largely retained in an N-terminal fragment Biochem J 421, 425–434 18 Torrent M, Nogues MV & Boix E (2009) A theoretical approach to spot active regions in antimicrobial proteins BMC Bioinformatics 10, 373–382 19 Boix E, Torrent M, Sanchez D & Nogues MV (2008) The antipathogen activities of eosinophil cationic protein Curr Pharm Biotechnol 9, 141–152 20 Domachowske JB, Dyer KD, Adams AG, Leto TL & Rosenberg HF (1998) Eosinophil cationic protein ⁄ RNase is another RNase A-family ribonuclease with direct antiviral activity Nucleic Acids Res 26, 3358–3363 21 Rosenberg HF (2008) RNase A ribonucleases and host defense: an evolving story J Leukoc Biol 83, 1079–1087 22 Rosenberg HF (1995) Recombinant human eosinophil cationic protein Ribonuclease activity is not essential for cytotoxicity J Biol Chem 270, 7876–7881 23 Hancock RE & Sahl HG (2006) Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies Nat Biotechnol 24, 1551–1557 24 Gorr SU, Sotsky JB, Shelar AP & Demuth DR (2008) Design of bacteria-agglutinating peptides derived from parotid secretory protein, a member of the bactericidal ⁄ permeability increasing-like protein family Peptides 29, 2118–2127 25 Irie M, Nitta K & Nonaka T (1998) Biochemistry of frog ribonucleases Cell Mol Life Sci 54, 775–784 26 Rosenfeld Y & Shai Y (2006) Lipopolysaccharide (endotoxin)-host defense antibacterial peptides interactions: role in bacterial resistance and prevention of sepsis Biochim Biophys Acta 1758, 1513–1522 27 Weidenmaier C, Kristian SA & Peschel A (2003) Bacterial resistance to antimicrobial host defenses – an emerging target for novel antiinfective strategies? Curr Drug Targets 4, 643–649 28 Conchillo-Sole O, de Groot NS, Aviles FX, Vendrell J, Daura X & Ventura S (2007) AGGRESCAN: a server for the prediction and evaluation of ‘hot spots’ of aggregation in polypeptides BMC Bioinformatics 8, 65 29 Cho S, Beintema JJ & Zhang J (2005) The ribonuclease A superfamily of mammals and birds: identifying new members and tracing evolutionary histories Genomics 85, 208–220 30 Cho S & Zhang J (2007) Zebrafish ribonucleases are bactericidal: implications for the origin of the FEBS Journal 277 (2010) 1713–1725 ª 2010 The Authors Journal compilation ª 2010 FEBS M Torrent et al 31 32 33 34 35 36 37 38 39 40 vertebrate RNase A superfamily Mol Biol Evol 24, 1259–1268 Nitto T, Dyer KD, Czapiga M & Rosenberg HF (2006) Evolution and function of leukocyte RNase A ribonucleases of the avian species, Gallus gallus J Biol Chem 281, 25622–25634 Hileman RE, Fromm JR, Weiler JM & Linhardt RJ (1998) Glycosaminoglycan–protein interactions: definition of consensus sites in glycosaminoglycan binding proteins Bioessays 20, 156–167 Gandhi NS & Mancera RL (2008) The structure of glycosaminoglycans and their interactions with proteins Chem Biol Drug Des 72, 455–482 Sur S, Glitz DG, Kita H, Kujawa SM, Peterson EA, Weiler DA, Kephart GM, Wagner JM, George TJ, Gleich GJ et al (1998) Localization of eosinophilderived neurotoxin and eosinophil cationic protein in neutrophilic leukocytes J Leukoc Biol 63, 715–722 Bystrom J, Garcia RC, Hakansson L, Karawajczyk M, Moberg L, Soukka J & Venge P (2002) Eosinophil cationic protein is stored in, but not produced by, peripheral blood neutrophils Clin Exp Allergy 32, 1082–1091 Venge P, Bystrom J, Carlson M, Hakansson L, Karawacjzyk M, Peterson C, Seveus L & Trulson A (1999) Eosinophil cationic protein (ECP): molecular and biological properties and the use of ECP as a marker of eosinophil activation in disease Clin Exp Allergy 29, 1172–1186 Schroder JM & Harder J (2006) Innate antimicrobial peptides in the skin Med Sci (Paris) 22, 153–157 Nomura I, Goleva E, Howell MD, Hamid QA, Ong PY, Hall CF, Darst MA, Gao B, Boguniewicz M, Travers JB et al (2003) Cytokine milieu of atopic dermatitis, as compared to psoriasis, skin prevents induction of innate immune response genes J Immunol 171, 3262–3269 Ong PY, Ohtake T, Brandt C, Strickland I, Boguniewicz M, Ganz T, Gallo RL & Leung DY (2002) Endogenous antimicrobial peptides and skin infections in atopic dermatitis N Engl J Med 347, 1151–1160 Ong PY & Leung DY (2002) Atopic dermatitis Clin Allergy Immunol 16, 355–379 RNase and RNase bactericidal activity 41 Kawabata SI & Iwanaga S (1997) Big defensin and tachylectins -1 and -2 In Methods in Molecular Biology: Antibacterial Peptide Protocols (Shafer WM ed) pp 51–61 Humana Press Inc, Totowa, NJ 42 Roth BL, Poot M, Yue ST & Millard PJ (1997) Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain Appl Environ Microbiol 63, 2421–2431 ´ 43 Mallorquı´ -Fernandez G, Pous J, Peracaula R, Aymamı´ J, Maeda T, Tada H, Yamada H, Seno M, de Llorens R, Gomis-Ruth FX et al (2000) Three-dimensional ă crystal structure of human eosinophil cationic protein (RNase 3) at 1.75 A resolution J Mol Biol 300, 1297–1307 Supporting information The following supplementary material is available: Fig S1 Study of S aureus viability and population morphology upon incubation with RNase visualized by confocal microscopy Fig S2 Study of S aureus viability and population morphology upon incubation with RNase visualized by confocal microscopy Fig S3 Analysis by SDS-PAGE of RNase cell binding to E coli and S aureus cells Table S1 RNase and RNase membrane depolarization activities Video S1 Register of E coli viability and population morphology upon incubation with RNase 3, stained with the Live ⁄ Dead kit and visualized by confocal microscopy This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 277 (2010) 1713–1725 ª 2010 The Authors Journal compilation ª 2010 FEBS 1725 ... characterization of both the action of RNase and RNase at the bacterial wall level revealed some particular features that could explain their distinct abilities with respect to Gram-negative and Gram-positive. .. reflect their action at the outer envelope level Studies at the bacterial cell wall The bactericidal activity of both RNases is precluded by the protein binding to the cells Proteins incubated with... Journal 277 (2010) 17 13? ?? 172 5 ª 2010 The Authors Journal compilation ª 2010 FEBS 171 5 RNase and RNase bactericidal activity M Torrent et al A B C RNase 10 60 D 120 E RNase F 120 Fig Study of bacterial