1 Centro Nacional de Microbiología, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid; 2 Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain
Received 16 July 2003; returned 15 September 2003; revised 18 September 2003; accepted 25 September 2003
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Abstract |
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Methods: Purified Pal amidase and/or Cpl-1 lysozyme were used alone or in combination. These enzymes were injected intraperitoneally at different times after challenge with 5 x 107 cfu of a type 6B, antibiotic-resistant S. pneumoniae clinical isolate.
Results: Animals challenged with 5 x 107 cfu of this strain alone died within 72 h, whereas a single intraperitoneal injection of Cpl-1 or Pal (200 µg; 1100 U) administered 1 h after the bacterial challenge was sufficient to effectively protect the mice, according to unpaired t-test (P < 0.0001). Bacteraemia in unprotected mice reached colony counts >107 cfu/mL, whereas the mean colony count in lysin-protected animals was <106 cfu/mL over time and ultimately became undetectable. Interestingly, a synergic effect in vivo was observed with the combined use of 2.5 µg each of Cpl-1 and Pal.
Conclusions: Our findings suggest strongly that phage lysins protect animals from bacteraemia and death. Moreover, the simultaneous attack of the pneumococcal peptidoglycan by a lysozyme and an amidase leads to a remarkable effect through enhanced destruction of the bacterial cell wall. The benefits of therapy with enzybiotics against pneumococcus reported here might warrant the examination of alternative strategies for the treatment of diseases caused by clinically relevant pathogens.
Keywords: enzybiotics, pneumococcus, phage, lytic enzymes
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Introduction |
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The last step of phage infection is the release of mature phage particles by hydrolysis of the bacterial cell wall. The biological properties of several lytic and temperate phages infecting S. pneumoniae have been reviewed.5 Most often, phages encode murein-degrading enzymes that hydrolyse either the glycosidic linkages between the amino sugars of the peptidoglycan (glucosaminidases, lysozymes), the N-acetylmuramoylL-alanine amide bond between the glycan strand and the cross-linking peptide (amidases), or the inter-peptide bridge linkages (endopeptidases).6 Peptidoglycan (murein) hydrolases represent one of the most widely distributed enzyme families in nature, and their participation in a series of important biological events (cell division, daughter cell separation, the irreversible effects caused by ß-lactam antibiotics, etc.) is well recognized.
In S. pneumoniae, we have analysed in detail the lytic enzymes encoded by four different bacteriophages: Cp-1,7 HB-3,8 EJ-19 and Dp-1.10 Genes encoding these lytic enzymes have been cloned by taking advantage of their homology with the lytA gene encoding the host LytA amidase. Sequence comparisons revealed that the host- and phage-coded enzymes have a highly similar C-terminal domain that is responsible for the binding to the choline residues present in the teichoic acids of the cell wall substrate.1113 We proposed that this aminoalcohol served as an element of selective pressure to preserve the substrate-recognition domain of most of the pneumococcal lytic enzymes characterized so far. The enzyme coded by Cp-1 has been identified as a lysozyme (Cpl-1), whereas those coded by Dp-1, HB-3 and EJ-1 (Pal, Hbl and Ejl, respectively) are amidases (Figure 1).
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Materials and methods |
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Bacteria, bacteriophages and growth conditions
The strain used in this study was S. pneumoniae 541, a serotype 6B clinical isolate resistant to ß-lactam antibiotics (penicillin MIC and MBC, 2 and 4 mg/L, respectively), was selected for this study on the basis of its resistance to ß-lactams and its virulence in mice.18 S. pneumoniae was grown in C medium19 supplemented with yeast extract (0.8 mg/mL; Difco Laboratories) (C+Y medium) at 37°C without shaking and the growth was monitored with a Hach 2100N nephelometer. Escherichia coli DH5 (pCIP100)20 and DH5
(pMSP11),10 grown in LuriaBertani (LB) medium at 37°C with shaking, were the sources for production of Cpl-1 and Pal, respectively.
Purification of Cpl-1 and Pal
Cpl-1 and Pal enzymes were purified by affinity chromatography in DEAE-cellulose as previously described for other choline-binding proteins.21 Typically, E. coli cultures harbouring either pCIP100 or pMSP11 were grown in LB medium supplemented with ampicillin (100 mg/L) and 0.4 mM isopropyl-ß-D-thiogalactopyranoside at 37°C for 24 h with shaking, harvested by centrifugation and broken in a French press cell. The crude extracts were ultracentrifuged (100 000g, for 1 h at 4°C) and the total soluble protein in the supernatant was loaded onto a DEAE-cellulose column (8 x 2.5 cm) and washed with three volumes of 20 mM sodium phosphate buffer pH 6.9 (or pH 6.0 for Cpl-1) containing 1.5 M NaCl. The same buffer containing 2% choline was then added and fractions were recovered. The purity of the lytic enzymes was checked by SDSPAGE (Figure 1d) and by western blot analysis (not shown), as already reported.10,20 The purified enzymes were dialysed against 20 mM sodium phosphate buffer pH 6.9 (or pH 6.0 for Cpl-1). Finally, the enzyme solutions were adjusted to 1 mg/mL, and filter-sterilized using 0.20 µm Iwaki membrane filters. Purified Cpl-1 and Pal enzymes had approximately the same specific activity (110 000 U/mg) (see below).
Preparation of radioactively labelled cell walls and assay for enzymatic activity
Pneumococcal cell walls labelled with [methyl-3H]choline were prepared by biosynthetic labelling of the bacteria. The standard assay conditions for the hydrolysis of the labelled pneumococcal cell walls, as well as the determination of the enzymatic activities, have been described elsewhere.22 Briefly, pneumococcal cell walls were incubated with enzymatic extracts or purified proteins at 37°C for 10 min and the reaction stopped upon the addition of formaldehyde and BSA. Non-digested murein was separated by centrifugation at 12 000g for 15 min, and the digested (solubilized) substrate was quantified by measuring the radioactivity in the supernatant. One unit of enzymatic activity is defined as the amount of enzyme that catalyses the hydrolysis of 1 µg (715 cpm) of pneumococcal cell walls in 10 min.
Animals
Eight- to 12-week-old female BALB/c mice weighing 19 to 22 g were used.
Determination of MLD and challenge dose
Groups of five mice per experiment were injected intraperitoneally with different inocula ranging from 105 to 108 cfu/mL to determine the minimal dose that produces 100% mortality rate over a 7 day follow-up period. Experimental details of this procedure have been documented recently.18
Immune response to bacteria and phage lytic enzymes
Immune response to specific 6B capsular polysaccharide was measured by a standardized ELISA protocol (Workshop at the CDC, 1996).18 Serum samples were obtained by cardiac puncture 10 days after bacterial challenge with S. pneumoniae 541 strain and enzybiotics. Specific IgG antibodies to capsular 6B polysaccharide were determined for preimmune and immune sera.
Statistical analysis
Unpaired t-test values (P) were calculated for survival of mice or not with the lytic enzymes using a program available from http://www.physics. csbsju.edu/stats/t-test.html. P values of <0.001 were considered highly significant.
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Results |
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The use of pneumococcal lysins to produce bacterial killing in in vitro assays, the so-called lysis from without, was previously shown to be successful in liberating the phage progeny from bacterial strains deficient in lytic enzymes.23 The effect of Cpl-1 lysozyme or Pal amidase on the pneumococcal strain 541 was tested here using the mouse sepsis model of infection. Cpl-1 (39.1 kDa) and Pal (34.6 kDa) enzymes have homologous C-terminal choline-binding domains, whereas their different N-terminal moieties determine the biochemical specificity (Figure 1). We first determined that inoculation intraperitoneally with 5 x 107 cfu (2x MLD) of the clinical isolate S. pneumoniae 541 was sufficient to produce a near 100% mortality rate over a 3 day follow-up period (Figure 2a) (mean survival rate per mouse and standard deviation of the mean were 7.14 x 102 and 0.26, respectively). Next, we performed a set of experiments with single doses of Cpl-1 or Pal administered intraperitoneally shortly (10 min) after the challenge with 2x MLD of bacteria. After 24 h a dose-dependent effect on the state of the animals was visible. When 40 µg of either Cpl-1 or Pal was used a pronounced therapeutic effect was observed: all mice were fully recovered and the establishment of bacteraemia was greatly reduced (unpaired t-test, P < 0.001) (unpublished data), indicating that both enzymes were capable of destroying the infecting bacteria in the peritoneal cavity. This test shows that, in the short-term, the intraperitoneal cavity mimics the intranasal model of infection previously developed for S. pneumoniae.15
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Rescue is associated with a significant decrease in bacterial titre
The mean bacterial titre in the blood of mice inoculated with 5 x 107 cfu S. pneumoniae 541 was 107108 cfu/mL until death (Figure 2b). Since the rise in the colony counts in blood is directly related to an increase in the probability of death,17 the ability of Pal or Cpl-1 to reduce colony counts in blood was investigated. When 200 µg of either lysin were injected intraperitoneally 1 h after the challenge, a drop of
4 log units was already observed 2 h later, followed by small fluctuations of bacterial titre, whereas after 45 days, bacterial clearance was complete, or nearly so (Figure 2c).
Synergic effect of a combination of phage lytic enzymes
A recent study using S. pneumoniae cells revealed the existence of a synergic killing effect between Cpl-1 and Pal in vitro.24 We decided to analyse the combined effect of these enzymes in the pneumococcal sepsis model. The combined use of 2.5 µg of each Cpl-1 and Pal injected intraperitoneally 1 h after the challenge with the S. pneumoniae 541 strain completely rescued the mice from infection (Figure 3a), whereas a single injection of 5 µg of either Cpl-1 or Pal did not protect the animals, as shown above (Figure 2a). These findings were statistically significant according to an unpaired t-test (P < 0.0001). This synergic effect of the lysozyme and the amidase has been tested using various amounts and application times, with similar results. Again, mice rescue was associated with a drop in bacterial counts in blood a few hours after injection of both lysins (Figure 3b). When the mice were treated with 2.5 µg of both Cpl-1 and Pal, a near complete bacterial clearance was achieved by the sixth day, although a transitory increase in bacterial counts took place by day 23.
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We carried out additional experiments to determine whether Cpl-1 and/or Pal were capable of protecting mice when injected several hours after the bacterial lethal challenge. Thus, at various time points, ranging from 1 to 6 h, mice received a single intraperitoneal injection of 200 µg of Cpl-1 or Pal. The results of these treatments demonstrated that Cpl-1 and Pal cured mice even when inoculated up to 2 h after S. pneumoniae 541 injection. When the enzymes were injected with a more prolonged delay the protective effect of both enzybiotics was not statistically significant according to unpaired t-test (P > 0.05). When injection was performed 4 h after the challenge, the bacteraemia was already too high and the protective effect was limited to prolonging the life of the severely ill mice for several days, after which time all the animals died (not shown). When treated with 200 µg of either lysin 2 h after challenge, three out of four animals treated with Pal had no bacteraemia (or very low levels) by the sixth day (Figure 4b), corroborating the healthy state of these mice. In the case of Cpl-1, the protective effect extended to the four animals assayed although the bacteraemia level was maintained for a longer time (up to 8 days in some cases) (Figure 4a).
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To address the question of an eventual immune response of mice as a result of protein injection, we inoculated mice with a second dose of Cpl-1 or Pal at different times after the bacterial challenge. The immediate consequence of this second intraperitoneal injection was a decreasing bacteraemia profile of 23 log units, although complete recovery or death appears to depend on the clinical outcome of the animals at the time of second injection (data not shown). Furthermore, we checked the titre of antibodies 10 days after injection with the standard challenge dose of 541 strain and, immediately, 200 µg of Cpl-1 or Pal. Titres of specific IgG antibodies to serotype 6B polysaccharide were measured in parallel for the hyperimmune serum, raised against the 6B S. pneumoniae strain, and preimmune serum. ELISA titres were eight times higher in hyperimmune than preimmune serum (not shown). Immune serum was reactive to the corresponding lysin and several pneumococcal proteins as indicated by western blot analysis (Figure 5). Interestingly, preliminary experiments suggested that when these animals received a second dose of bacterial challenge and enzybiotics 10 days after the first injections, all mice recovered fully. Moreover, no signs of anaphylaxis or adverse side effects were observed (data not shown).
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Discussion |
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The ability of Cpl-1 lysozyme and Pal amidase to cure bacteraemic animals is a protein-specific function, as indicated by the observation that rescue did not occur with heat-inactivated enzymes. Irrespective of the enzymatic activity of the enzyme used, the experiments presented here revealed that a single intraperitoneal injection of the corresponding lysin rescued mice from death due to S. pneumoniae even when bacteraemia was already well established (Figure 2). Under the experimental conditions used, we found that effective protection takes place with both enzymes through a wide range of enzyme concentrations or time of intraperitoneal injection after challenge. We observed that effective protection was achieved in mice when a single dose of at least 10 µg of Cpl-1 or Pal was administered 1 h after the MLD of S. pneumoniae 541 strain was inoculated (Figure 2a). The protective effect of both enzybiotics was notable even when the enzymes were injected 2 h after pneumococcal challenge, although the extent of this effect was related to the pre-treatment outcome. Moreover, a complete pneumococcal clearance was observed in most surviving mice after a 68 day follow-up period (Figure 4). A single intraperitoneal injection of either lytic enzyme, delayed up to 4 h after challenge (Figure 4), has a partial protective effect by prolonging the life of the severely ill mice for several days. Provided that mice are most susceptible to pneumococcal infections,25 the results obtained in these experiments are encouraging, since our findings and those of others16,26 support the development in a near future of modified procedures to improve the use of lysins in a more efficient way. For example, intravenous administration of the enzymes may confer a superior protection to animals when used later after the challenge.
Our results demonstrate that the simultaneous use of both enzymes, rather than being competitive, enhanced the destruction of the cell wall, and hence shows a synergic lytic action on S. pneumoniae in the murine sepsis model used here. The positive interaction of the two muralytic enzymes could be due to the increased access of these enzymes, with different catalytic domains, to their cleavage sites (Figure 1), as recently suggested.24 Survival rates in the animals that received both enzymes were significantly higher than those of animals receiving only Cpl-1 or Pal (Figure 3). Both enzymes, as most of the rest of cell wall lytic enzymes of the pneumococcal system, specifically recognize the choline component of the envelope of this bacterium through their C-terminal domain.5 The experiments reported here extend these observations to the sepsis model, and also reveal the lack of toxicity of the pneumococcal enzybiotics in mice. Furthermore, the combined use of two lytic enzymes would make more unlikely the intrinsic resistance of the bacteria to these proteins that target essential cell wall molecules, as previously suggested.16 These findings, together with the observations12 that these enzymes exhibit a modular design that facilitates the construction of chimeric enzymes in which bacterial specificities and activities can be combined by enzymatic swapping,27 suggest that these lysins are adaptable for use with different pathogens.
Antibiotic therapeutic effect in infected mice requires an appropriate dosing interval of administration to maintain pharmacological concentrations in serum. Most interestingly, a single intraperitoneal injection of a lytic enzyme(s) was sufficient for a complete clearance of invading pneumococci from surviving mice (Figures 24). This property, referred to as therapeutic efficacy, provides a rapid and specific lytic activity, making these proteins very promising candidates in current antimicrobial therapies.
It has recently been emphasized that during the long history of using phages administered by different routes in several countries there have been virtually no reports of serious complications related to their use.28 As phages are common entities in the environment and regularly consumed in foods, in theory the development of neutralizing antibodies should not be a significant obstacle during the initial treatment of acute infections because the kinetics of phage action or lytic enzymes, as illustrated in this work (Figure 5), are much faster than is the hosts production of neutralizing antibodies.28 In addition, as reported here, improved survival upon lysin injection may enable the host to synthesize neutralizing anti-capsular antibodies that should be protective against a new challenge with a pneumococcal strain of the same or of a cross-reactive serotype. However, it would be prudent to further ensure the safety of lytic enzymes as therapeutic tools before using them as therapeutic agents.
The emergence of antibiotic-resistant bacteria, such as many pneumococci that have spread over several continents, has highlighted the need to explore the potential therapeutic applications of alternative therapies, such as the utilization of bacteriophages,26 or the use of phage-coded lytic enzymes.14 A better understanding of the biology of phages, combined with the availability of highly purified phage lytic enzymes and the experience attained in several Eastern European countries where rigorously controlled trials are currently being carried out,4,28 should facilitate the development of these new therapies.
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Acknowledgements |
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Spanish Pneumococcal Infection Study Network (G03/103). General coordination: Román Pallarés (rpallares{at}bell.ub.es). Participants and centres: Ernesto García (Centro de Investigaciones Biológicas, Madrid); Julio Casal, Asunción Fenoll, Adela G. de la Campa (Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid); Emilio Bouza (Hospital Gregorio Marañón, Madrid); Fernando Baquero (Hospital Ramón y Cajal, Madrid); Francisco Soriano, José Prieto (Fundación Jiménez Díaz y Facultad de Medicina de la Universidad Complutense, Madrid); Román Pallarés, Josefina Liñares (Hospital Universitari de Bellvitge, Barcelona); Javier Garau, Javier Martínez Lacasa (Hospital Mutua de Terrassa, Barcelona); Cristina Latorre (Hospital Sant Joan de Deu, Barcelona); Emilio Pérez-Trallero (Hospital Donostia, San Sebastián); Juan García de Lomas (Hospital Clínico, Valencia); and Ana Fleites (Hospital Central de Asturias).
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Footnotes |
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Members of the Spanish Pneumococcal Infection Study Network are listed in the Acknowledgements.
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