JAC Advance Access originally published online on March 31, 2004
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Journal of Antimicrobial Chemotherapy (2004) 53, 863-866
© 2004 The British Society for Antimicrobial Chemotherapy
Effect of listeriolysin O-loaded erythrocytes on Mycobacterium avium replication within macrophages
1 Institute of Biochemistry ‘G. Fornaini’, 2 Institute of Hygiene and 3 Institute of Histology and Laboratory Analyses, University of Urbino, Via Saffi, 2, 61029 Urbino (PU), Italy
Received 24 September 2003; returned 18 December 2003; revised 19 January 2004; accepted 4 February 2004
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Abstract |
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Objective: To evaluate the efficacy of erythrocytes loaded with the haemolytic toxin listeriolysin O against Mycobacterium avium replication within human macrophages.
Methods: Recombinant listeriolysin O was loaded in human erythrocytes by a procedure of hypotonic dialysis and isotonic resealing. Loaded erythrocytes were modified to allow them to be recognized and taken up by human macrophages infected with M. avium. The antimycobacterial activity of the erythrocytes loaded with listeriolysin O was evaluated by supernatant and intracellular cfu counts on days 4 and 7 post-erythrocyte administration.
Results: Recombinant listeriolysin O was encapsulated in human erythrocytes to reach final concentrations ranging from 1 to 4 ng/mL of erythrocytes. Erythrocytes loaded with increasing quantities of recombinant protein were able to reduce (at most by 50%) M. avium replication in a dose-dependent fashion when administered to infected macrophages.
Conclusions: Erythrocytes loaded with listeriolysin O are effective against M. avium replication within macrophages. We are confident that the strategy presented could be useful against mycobacteria other than M. avium (such as Mycobacterium tuberculosis and Mycobacterium leprae) by itself or as part of an antimycobacterial treatment.
Keywords: mycobacteria, mycobacterial phagosome, encapsulation, drug-targeting, phagocytosis
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Introduction |
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Like other pathogenic mycobacteria (e.g. Mycobacterium tuberculosis and Mycobacterium leprae), Mycobacterium avium is phagocytosed by macrophages where it survives and multiplies within a specialized vacuole, the mycobacterial phagosome, which does not fuse with lysosomes but remains in an immature form, thus evading the potent antimicrobial mechanisms of the host cells. One of the hallmark characteristics of M. avium is its resistance to most antituberculosis drugs, likely due to their limited access to the phagosomes containing Mycobacterium avium complex (MAC). Therefore new strategies to deliver drugs selectively to M. avium vacuoles within macrophages should be developed.
Since the mycobacterial vacuole retains its ability to fuse with endosomes1 and furthermore, the fusion of two vacuoles in macrophages co-infected with M. avium and Coxiella burnetti has been observed,2 we reasoned that administering drug in a way that it is phagocytosed by macrophages and retained inside a phagosome, we can reach the mycobacterial vacuole. Moreover, it is known that erythrocytes can be used as a drug-targeting system allowing the selective administration of substances to macrophages upon loaded erythrocyte phagocytosis.3 In this study, we show that erythrocytes loaded with the haemolytic toxin listeriolysin O and modified to increase their phagocytosis by M. avium-infected macrophages, are effective against M. avium replication. This strategy could be useful by itself or as part of an antimycobacterial regimen and prove to be a feasible approach in delivering new biologicals to the mycobacterial vacuole.
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Materials and methods |
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Recombinant listeriolysin O (rLLO) was obtained as described previously.4
rLLO was radio-iodinated by the chloramines-T procedure,5 using carrier-free Na125I obtained from Amersham Radiochemicals, to a specific activity of 1 192 351 cpm/µg.
The haemolytic activity of purified rLLO or 125I-rLLO was determined essentially as described previously.4
The M. avium strain was isolated and cultured as described previously.6
To evaluate the antimycobacterial activity of recombinant listeriolysin O, M. avium was cultured for 10–12 days in Middlebrook agar 7H10 and then a typical smooth transparent colony was diluted in 2 mL of supplemented Middlebrook 7H9 broth, incubated for 6 days at 35°C, diluted 1:10 in the same medium and then incubated again at 35°C overnight. Afterwards, bacteria were harvested and diluted in culture medium at two different pH values (5.5 and 7.4) and in the presence of two different rLLO quantities (10 or 30 µg/mL) at a final concentration of 500 000 cells/mL. After 18 and 42 h incubation at 35°C, aliquots (1 and 2 mL of suspension) were plated onto Middlebrook agar 7H10 to assess the cfu.
Human erythrocytes were loaded with rLLO by a procedure of hypotonic dialysis, isotonic resealing and reannealing as reported previously,3 except that the washing buffer, the hypotonic solution and the resealing solution were used at pH 8.4 instead of 7.4. After the hypotonic dialysis, increasing quantities of rLLO (range 0–50 ng) were added to 250 µL aliquots of dialysed erythrocytes. Once resealed and washed to remove the non-entrapped rLLO, cells were processed further to increase their recognition by macrophages as described previously.3 To encapsulate 125I-rLLO in erythrocytes, the same procedure was used except that, after the dialysis step, 125I-rLLO in the range 25–500 ng was added.
Peripheral blood mononuclear cells were obtained as described previously.7
Each human macrophage monolayer was infected with M. avium as reported6 except that 3 x 106 bacilli were used, in order to obtain an average of six bacteria per macrophage. Engineered red blood cells (RBC) were added overnight at a ratio of 100 RBC per macrophage in a medium without added antibiotics and, at the 4th and the 7th day, total viable bacilli were determined as described previously.6
Samples for electron microscopy (EM) analysis were prepared for conventional ultrastructural morphology as described previously.8
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Results |
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Haemolytic activity of rLLO
LLO shows optimum activity at pH 5.5 and is almost entirely inactivated at pH 7.0. To characterize rLLO, we verified the effect of pH on haemolytic activity by testing three different pH values: 5.5, 7.4 and 8.4. Different concentrations (range 0.5–10 ng) of rLLO were used in the presence of 50 x 106 erythrocytes. As expected, the haemolysin showed its maximum activity at the acidic pH whereas the haemolytic activity rapidly decreased when basic pH values were reached. The concentrations of rLLO at which 50% haemolysis was obtained (haemolytic units, HU) were approximately 1, 2 and 6 ng at pH 5.5, 7.4 and 8.4, respectively. In contrast, when 125I-rLLO was used, no haemolysis was observed, at least up to 10 ng of protein.
Antimycobacterial activity of rLLO
The antimycobacterial activity of rLLO was evaluated by incubating M. avium in enriched Middlebrook 7H9 broth in the presence of two different quantities of protein (10 and 30 µg/mL) and at two different pH values (5.5 and 7.4). Mycobacterium replication was assessed after 18 and 42 h of incubation at 35°C and compared to that of mycobacteria grown in the absence of the haemolytic toxin. The results obtained (Figure 1a) show that rLLO is able to interfere with M. avium replication in a concentration-, time- and pH-dependent way. In fact, the highest inhibition percentage (about 50%) was observed after 42 h in the presence of 30 µg of rLLO at pH 5.5.
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Loading of rLLO in human erythrocytes
The optimum conditions for loading a haemolytic toxin in erythrocytes, while preserving their entirety, were tested. For this purpose, 250 µL of dialysed erythrocytes were incubated in the presence of increasing quantities of rLLO (0–50 ng). At the end of incubation, erythrocytes were resealed and treated for macrophage recognition and successively the number of recovered erythrocytes was evaluated. The results obtained show that addition of increasing amounts of protein to a fixed aliquot of dialysed erythrocytes permits the recovery of a decreasing number of resealed erythrocytes. To calculate the percentage of encapsulated rLLO, 125I-rLLO (A. S. 1 192 351 c.p.m./µg) serving as a tracer was co-entrapped in RBC and radioactivity evaluated. On average, a 1.43 ± 0.13% entrapment (mean ± S.D. of five experiments) was observed.
Administration of rLLO-loaded erythrocytes to uninfected macrophages
In Figure 2(a), one opsonized erythrocyte phagocytosed by a macrophage is shown. Since our goal was to fight M. avium inside macrophages without cellular damage, the morphology of macrophages receiving erythrocytes loaded with two different amounts of rLLO (0.81 and 4.08 ng/mL RBC) was evaluated by electron microscopy. The results obtained reveal no appreciable differences between untreated macrophages (not shown), macrophages receiving unloaded (UL) erythrocytes (i.e. erythrocytes subjected to the procedure of loading without rLLO addition) (not shown) and macrophages receiving erythrocytes loaded with the lowest rLLO concentration (Figure 2b). In contrast, when erythrocytes loaded with the highest rLLO concentration were administered to macrophages, the appearance of numerous vesicles in the cytoplasm was observed (Figure 2c). For this reason, experiments to evaluate the antimycobacterial activity of loaded erythrocytes were continued with rLLO concentrations lower than 4 ng/mL RBC.
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Antimycobacterial efficacy of rLLO-loaded erythrocytes
To evaluate the antimycobacterial activity of rLLO-loaded erythrocytes, erythrocytes loaded with different quantities of rLLO (1.22, 1.63, 2.03 and 2.45 ng) were administered overnight to M. avium-infected macrophages. The total number of viable bacilli in the 4th and 7th day supernatants and in the adherent macrophages on day 7 was evaluated. The results obtained (Figure 1b) show an antimycobacterial efficacy of rLLO-loaded RBC protein which is concentration-dependent. The efficacy of rLLO-loaded erythrocytes was also confirmed by electron microscopy (Figure 2d). When administering erythrocytes loaded with rLLO, a degradation of many M. avium inside the phagosome was observed, whereas the cellular components appeared unaffected by the toxin.
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Discussion |
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In this study, it is demonstrated that through erythrocyte phagocytosis, it is possible to reach the phagosome containing pathogenic mycobacteria. Although M. avium inhibits phagosome–lysosome fusion in macrophages, it is known that the mycobacterial vacuole still retains its ability to fuse with endosomes.1 Furthermore, fusion between the M. avium vacuole and a phagosome containing Mycobacterium smegmatis was recently used as a strategy to target the lytic phage TH4 directly against M. avium.9 We exploited this fusion capability to deliver the recombinant toxin listeriolysin O (rLLO) inside the phagosome containing M. avium by means of rLLO-loaded erythrocyte phagocytosis. Furthermore, since rLLO has optimum activity at pH 5.5, we also exploited the toxin activation occurring after phagosomal acidification, that is, once rLLO-loaded erythrocytes are phagocytosed by macrophages. Until now, LLO has only been used as a strategy for the cytosolic delivery of macromolecules.10 Here, it is demonstrated that LLO can also be used as an antimicrobial agent when properly administered. The results obtained show the concentration-dependent antimycobacterial effect of rLLO delivered by RBC and confirm the antimycobacterial effect of rLLO observed during incubation of the free protein with M. avium suspensions. Furthermore, rLLO probably acts not only directly against the pathogen but also damages the membrane of vacuole in which M. avium is able to survive, this being particularly rich in cholesterol. Indeed, the rLLO concentration able to inhibit M. avium replication within macrophages is much lower compared with that necessary to obtain the same inhibition when M. avium was incubated with the free protein. This could suggest that rLLO is capable of favouring the antimicrobial activity of macrophages by exposing the pathogens directly to the components which constitute the macrophage’s defence. However, a complete inhibition of M. avium replication was never obtained (at most, approximately 50% inhibition was reached). Very likely this is because single erythrocyte administrations were tested and that, on average, each macrophage is able to phagocytose only 1.2 erythrocytes.3 Probably, repeated administrations of rLLO-loaded erythrocytes at intervals would improve results.
The strategy described here could also be useful against other pathogens, such as M. tuberculosis and M. leprae. Moreover, our results can constitute a basis for future studies on the use of rLLO-loaded erythrocytes in combination with traditional antimycobacterial drugs or alternatively, erythrocytes loaded with non-diffusible antimycobacterial drugs, other than listeriolysin O, could be synthesized as prodrugs and encapsulated into erythrocytes exerting a potent bactericidal action when selectively delivered to the mycobacterial phagosome.
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Acknowledgements |
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We thank Dr Camilla Giammarini from Diatheva (Italy) for kindly providing recombinant listeriolysin O. This work is partially supported by C.N.R. Target Project on Biotechnology and FIRB funds (PNR 2001–2003, Red blood cells as drug carriers, RBNE01TBTR).
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Footnotes |
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* Corresponding author. Tel: +39-722-305211; Fax: +39-722-320188; E-mail: magnani{at}uniurb.it
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References |
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