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Anti-invasive activity of bovine lactoferrin towards group A streptococci Maria Ajello, Rita Greco, Francesco Giansanti, Maria Teresa Massucci, Giovanni Antonini, and Piera Valenti
Abstract: Group A streptococci (GAS) are able to invade cultured epithelial and endothelial cells without evidence of intracellular replication. GAS, like other facultative intracellular bacterial pathogens, evolved such ability to enter and to survive within host cells avoiding the host defences, and bacterial intracellular survival could explain the recurrence of infections. We report here that 1 mg bovine lactoferrin (bLf)/mL significantly hindered the in vitro invasion of cultured epithelial cells by GAS isolated from patients suffering from pharyngitis and completely inhibited the invasiveness of GAS pretreated with subinhibiting concentrations of erythromycin or ampicillin. One milligram of bLf per millilitre was also able to increase the number of epithelial cells undergoing apoptosis following GAS invasion, although the number of intracellular GAS in the presence of bLf decreased by about 10-fold. The ability of bLf to decrease GAS invasion was confirmed by an in vivo trial carried out on 12 children suffering from pharyngitis and already scheduled for tonsillectomy. In tonsil specimens from children treated for 15 days before tonsillectomy with both oral erythromycin (500 mg t.i.d. (three times daily)) and bLf gargles (100 mg t.i.d.), a lower number of intracellular GAS was found in comparison with that retrieved in tonsil specimens from children treated with erythromycin alone (500 mg t.i.d.). Key words: lactoferrin, group A streptococci, invasiveness, anti-invasive activity, apoptosis. Résumé : Les streptocoques de groupe A (SGA) peuvent envahir des cellules endothéliales etAjello épithéliales et al. en culture, sans évidence de réplication intracellulaire. Les SGA, comme d’autres bactéries pathogènes intracellulaires, ont acquis la capacité d’entrer et de survivre dans les cellules hôtes en évitant les défenses de l’hôte, et la survie de ces bactéries dans les cellules pourrait expliquer la récurrence des infections. Dans cet article, nous montrons que 1 mg/mL de lactoferrine bovine entrave significativement l’invasion de cellules épithéliales en culture par des SGA isolés de personnes ayant une pharyngite et inhibe complètement l’invasion des cellules par des SGA prétraités avec de l’érythromycine ou de l’ampicilline à des concentrations non inhibitrices. Un mg/mL de lactoferrine bovine augmente également le nombre de cellules épithéliales entrant en apoptose à la suite d’une invasion par des SGA, même si le nombre de SGA dans les cellules est diminué environ 10 fois en présence de lactoferrine bovine. La capacité de la lactoferrine bovine d’entraver l’invasion des cellules par les SGA a été confirmée par un test in vivo effectué sur 12 enfants ayant une pharyngite et en attente d’une amygdalectomie. Le nombre de SGA intracellulaires dans les échantillons d’amygdales d’enfants traités avec de l’érythromycine (500 mg t.i.d.) par voie orale et des gargarismes de lactoferrine bovine (100 mg t.i.d.) pendant 15 jours avant l’amygdalectomie est inférieur à celui mesuré dans les échantillons d’amygdales d’enfants traités seulement avec de l’érythromycine (500 mg t.i.d.). Mots clés : lactoferrine, streptocoques de groupe A, pouvoir envahissant, activité anti-invasive, apoptose. [Traduit par la Rédaction]
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Introduction Several mucosal pathogenic bacteria are capable of entering into nonprofessional phagocytes, such as epithelial cells. Inside the host cells, they are immediately localised within
the endosome where some bacteria are killed, others are capable of surviving, and some others escape from the vesicles and multiply in the cytoplasm. Generally, invasive bacteria adhere to the host cells by specific molecules called invasins that, in some cases, can also mediate the entry into host
Received 23 July 2001. Revised 26 October 2001. Accepted 31 October 2001. Published on the NRC Research Press Web site at http://bcb.nrc.ca on 23 January 2002. M. Ajello, R. Greco, and P. Valenti.1 Department of Experimental Medicine, II University of Naples, Napoli, I-80135, Italy. F. Giansanti and M.T. Massucci. Department of Pure and Applied Biology, University of L’Aquila, L’Aquila, I-67010, Italy. G. Antonini. Department of Pure and Applied Biology, University of L’Aquila, L’Aquila, I-67010, Italy, and Department of Biology, University of Rome TRE, Rome, I-00146, Italy. 1
Corresponding author (e-mail:
[email protected]).
Biochem. Cell Biol. 80: 119–124 (2002)
DOI: 10.1139/O01-211
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cells. Bacterial invasion of nonprofessional phagocytes is an active event requiring viable bacteria and cells, and this phenomenon ensures the bacteria a protective niche to replicate, persist, and avoid the host defences (Falkow 1991; Finlay and Cossart 1997; Finlay and Falkow 1997). Among facultative intracellular bacteria, group A streptococcus (GAS) is an important human pathogen that causes pharingitis, impetigo, cellulitis, and deeper infections such as rheumatic fever, scarlet fever, necrotizing fasciitis, and streptococcal toxic shock syndrome. GAS is capable of entering into endothelial cells (Greco et al. 1995), and the invasiveness could explain the recurrence of clinically observed GAS infections. Streptococcal structures involved in the adhesion to host cells are well known (Hasty et al. 1992), while surface molecules that mediate the invasion remain to be detected (Greco et al. 1998). In addition to the capability of GAS to invade host cells, it has been demonstrated that this pathogen causes apoptosis of infected human epithelial cells (Tsai et al. 1999). Lactoferrin, an iron-binding protein synthesised by neutrophils and glandular epithelial cells, exerts an important role in human defense against microbial infections. In addition to the well-known antibacterial activity (Vorland 1999), we have previously reported the protective effect of both bovine lactoferrin (bLf) (Longhi et al. 1993) and lactoferricin (Longhi et al. 1994) against the invasion of a recombinant enteroinvasive Escherichia coli strain. We have also reported the anti-invasive activity of bLf against a gram-positive intracellular facultative bacterium, Listeria monocytogenes (Antonini et al. 1997; Valenti et al. 1999). In this paper, in order to add further information on the protective effect of bLf towards invasive bacteria, we tested, in the presence and absence of bLf, the invasiveness of GAS isolated from pharingitis-suffering patients. The ability of bLf to decrease the in vitro invasiveness of GAS was confirmed by an in vivo trial carried out on 12 children undergoing tonsillectomy.
Materials and methods Bacterial strains, media, and growth conditions GAS were isolated from patients suffering from recurrent pharyngitis in Todd–Hewitt medium supplemented with 2% yeast extract (THY) (Difco, Detroit, Mich.) and Bacto agar (Difco) at 37°C and cultured in the same medium without agar in flasks with airtight sealed tops without shaking. Host cells HeLa S3 cells (from an epithelioid carcinoma of the human cervix) were grown as monolayers at 37°C in minimal essential medium (MEM) (Eurobio, Paris) supplemented with 1.2 g NaHCO3/L, 2 mM glutamine, 100 U penicillin/mL, 0.1 mg streptomycin/mL, and 10% heat-inactivated fetal calf serum (FCS) in a 5% CO2 incubator. During the invasive experiments, FCS was added at 2%. Lactoferrin The bLf was kindly supplied by Morinaga Milk Industry (Kanasawa, Japan). The purity of the bLf was checked by SDS-PAGE stained with silver nitrate. To obtain the apoform, bound iron was removed by extensive dialysis against
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50 mM pyrophosphate buffer, pH 4.0, followed by dialysis against phoshate buffer solution (PBS), pH 7.2. Iron-, manganese-, and zinc-saturated lactoferrins were obtained adding an adequate molar ratio of metal ions in form of sulphate or chloride salts in 0.1 M Tris-HCl, pH 8.0, containing 5 mM NaHCO3. Metal binding was performed overnight at 4°C under stirring in the same buffer; then, unbound ions were removed by dialysis against metal-free PBS, pH 7.2, for 72 h at 4°C. Hen’s ovotransferrin and human serotransferrin were purchased from the Sigma Chem. Co. (St. Louis, Mo.) and used without further purification. The iron saturation of ovotransferrin and serotransferrin was about 10–20% as determined by optical spectroscopy. Before biological assays, all proteins were sterilised by filtration on 0.45-µm Millex HV at low protein retention (Millipore Corp., Bedford, Mass). Cytotoxicity of lactoferrin Cells propagated in tissue culture were incubated with different concentrations (from 10 µg/mL to 10 mg/mL) of the different forms of bLf or with different concentrations of ovotransferrin and serotransferrin (from 10 µg/mL to 10 mg/mL) at 37°C for 120 min in MEM. Cell monolayers were washed in Earle’s balanced salt solution, with fresh medium, and examined after a 24-h incubation period at 37°C. Cell morphology, viability (as determined by neutral red staining), and yield were evaluated. In addition, a 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide based assay (Cell Proliferation Kit I) (Boering Mannheim, GmbH, Germany) was employed for the nonradioactive measurements of cell proliferation, viability, and cytoxicity. Antibacterial activity of lactoferrin and antibiotics Bacterial cells, cultured overnight at 37°C on THY, were suspended (about 1 × 107 cells/mL) in MEM in the presence of apo-, iron-, manganese-, or zinc-lactoferrin or in the presence of ovotransferrin and serotransferrin. After 2 h of contact with different concentrations of the proteins (from 10 µg/mL to 10 mg/mL), the samples were centrifuged and the pellets obtained were washed three times in PBS. The number of viable bacteria was detected by counting colonyforming units (CFU) on the selective medium. The same protocol was applied for evaluating the maximal concentration of erythromycin and ampicillin that did not inhibit the viability of the bacteria. The antibiotics were tested at concentrations from 10 ng/mL to 10 µg/mL. Invasion and intracellular survival assay Semiconfluent monolayers of HeLa S3 cells (5 × 105 cells/mL) grown without antibiotics in 12-well plates (Costar) were infected with GAS in the logarithmic phase of growth at a multiplicity of infection of 100 bacteria/cell. The infection was performed for 2 h at 37°C in the presence and absence of apo-, iron-, zinc-, or manganese-lactoferrin at 2 or 1 mg/mL. Cells were then washed extensively with PBS without Ca2+ and Mg2+, and 1 mL of fresh medium containing 200 µg gentamicin/mL was added to each well in order to kill extracellular bacteria. After a further 2-h incubation period at 37°C, infected cells were washed extensively with PBS without Ca2+ and Mg2+ and subsequently treated with trypsin–EDTA (a mixture of 0.05% trypsin (1/250) and 0.02% EDTA) for 5 min at 37°C and lysed © 2002 NRC Canada
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121 Table 1. Effect of bLf on the HeLa cells invasion ability of GAS untreated or pretreated with subinhibiting concentrations of antibiotics. Lactoferrin
Pretreatment of inoculum before monolayer infection
Intracellular bacteria (CFU)
None Apo-bLf, 1 mg/mL Apo-bLf, 2 mg/mL Native bLf, 1 mg/mL Fe-bLf, 1 mg/mL Zn-bLf, 1 mg/mL Mn-bLf, 1 mg/mL Ovotransferrin, 1 mg/mL Serum transferrin, 1 mg/mL None Apo-bLf, 1 mg/mL Apo-bLf, 2 mg/mL None Apo-bLf, 1 mg/mL Apo-bLf, 2 mg/mL
None None None None None None None None None Erythromycin, 200 ng/mL Erythromycin, 200 ng/mL Erythromycin, 200 ng/mL Ampicillin, 100 ng/mL Ampicillin, 100 ng/mL Ampicillin, 100 ng/mL
2.5 6.0 1.0 7.2 6.4 8.8 7.6 2.2 3.4 1.0 3.0 0 6.0 0 0
± ± ± ± ± ± ± ± ± ± ±
0.5 0.8 0.5 0.2 0.3 0.2 0.4 0.6 0.5 0.4 0.6
× × × × × × × × × × ×
105 103 103 103 103 103 103 105 105 105 10
± 0.5 × 104
Note: The number of intracellular bacteria was evaluated by counting CFU on selective media after cell lysis. When indicated, bacterial inoculum (5 × 107/mL) was pretreated with subinhibiting concentrations of the antibiotics before the monolayer infection (5 × 105 cell/mL). Lactoferrins, ovotransferrin, and serotransferrin were present in the media during the infection (see text for details). The data are presented as mean ± SD of at least five experiments.
by addition of 1.0 mL of cold 0.1% Triton-X100. Cell lysates were diluted in PBS and plated on THY agar medium to quantify the number of viable intracellular bacteria by CFU counts. In another set of experiments, to detect intracellular survival, cell monolayers were incubated for 24 h at 37°C in fresh medium containing 50 µg gentamicin/mL, 2% FCS, 1.2 g NaHCO3/L, and 2 mM glutamine. After this period, cells were washed and lysed and the number of viable intracellular bacteria was evaluated by CFU counts. In the invasion assays, carried out with GAS pretreated with subinhibiting concentrations of antibiotic, bacteria were precultured overnight in the presence of either 200 ng erythromycin/mL or 100 ng ampicillin/mL. Then, bacteria were extensively washed before infection of monolayers. Evaluation of cell death After incubation for 2 or 24 h in culture medium, in the presence and absence of GAS and 1 mg bLf/mL, floating and enzymatically detached cells were collected by centrifugation at 800 g for 5 min. Apoptosis was estimated in all experiments using cytofluorimetric analysis (Melino et al. 1994); a DNA ladder (Martin et al. 1995) was also used to assess DNA fragmentation. Briefly, cells were fixed with an equal volume of 1:4 methanol–acetone at –20°C, stored at 4°C, incubated with 50 mM propidium iodide, and 10 U Rnase/mL, and analysed on a FACScan cytofluorometer (Becton-Dickinson, Palo Alto, Calif.) using an electronic gate on the forward scatter (FSCh/FSCa) to avoid doublets. Ten thousand events were collected for each point and analysed with the Lysia II program (Martin et al. 1995). Representative experiments were also confirmed by standard Hoechst staining (Hoechst 33258, 0.1 µg PBS/mL, at 37°C for 1 h, washed, air-dried, and mounted) or Tunel (TdTmediated dUTP-biotin nick end labeling) (Melino et al. 1994, 1997). Lactate dehydrogenase (LDH) release was also
simultaneoulsy measured on the supernatant of infected cell monolayers to estimate cell lysis (Valenti et al. 1999). Evaluation of the number of GAS within tonsil cells In order to count the number of GAS inside the tonsil cells of children who underwent tonsillectomy, five specimens of tonsils (100 mg) for each patient were incubated with 1 mg collagenase/mL under agitation for 2 h. Cell suspensions were then collected by centrifugation at 1100 rpm for 10 min and the pellets were incubated in MEM plus 200 µg gentamicin/mL and 20 µg penicillin/mL for 2 h to kill extracellular bacteria. After incubation and extensive washings, the cells were lysed by addition of 1.0 mL of cold 0.1% Triton-X100 and the number of intracellular GAS was evaluated by means of CFU counts. Statistics Data derived from at least three to five independent experiments were gathered from triplicate assays. The mean value and standard deviation were determined for each assay carried out.
Results Cytotoxicity and antibacterial activity of bLf and antibiotics An initial test, to evaluate the cytotoxicity of antibiotics and bLf in apo- or saturated forms, was carried out on cell morphology and viability of cultured cells. bLf in both apoand metal-saturated forms and human serotransferrin at concentrations of up to 4 mg/mL did not affect HeLa cells, while the maximal nontoxic concentration of ovotransferrin was 1 mg/mL. Both erythromycin and ampicillin did not show any cytotoxicity at concentrations 10-fold higher than those employed in the anti-invasive experiments, i.e., 2000 © 2002 NRC Canada
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Table 2. GAS-induced apoptosis in the presence and absence of bLf. Apoptotic cells (%) Time of infection (h) 2 24
Noninfected cells 3±1 3±1
Infected cells without bLf 5±2 15±5
Infected cells with bLf 3±1 25±5
Note: bLf was added at a concentration of 1 mg/mL. Apoptosis of cells was evaluated using cytofluorimetric analysis (see text for details). The data are presented as mean ± SD of at least three experiments.
and 1000 ng/mL for erythromycin and ampicillin, respectively. When lactoferrin in either apo- or metal-saturated forms at 2 or 1 mg/mL was tested together with erythromycin or ampicillin at 200 or 100 ng/mL, respectively, no cytotoxicity was observed. In order to assay the viability of bacterial cells in the invasion experiments, which require viable bacteria, GAS suspended in MEM were incubated for 2 h at 37°C with the different proteins and antibiotics. After centrifugation for collecting and washing bacteria with PBS, viable bacteria were quantified with CFU counts on selective media. Up to concentrations of 2 and 1 mg/mL for apo- and metalsaturated forms of the proteins, respectively, no alteration of streptococcal viability was observed. The maximal concentration of erythromycin and ampicillin not affecting bacterial viability was 200 and 100 ng/mL, respectively. Thus, concentrations of 200 and 100 ng/mL for erythromycin and ampicillin, respectively, were considered the subinhibiting concentrations. No synergistic effect on bacterial viability was observed when lactoferrin in either apo- or metalsaturated forms at 1 or 2 mg/mL was tested together with erythromycin or ampicillin at 200 or 100 ng/mL, respectively. Effect of lactoferrin against GAS invasion of HeLa cells HeLa cell monolayers were infected with GAS in the logarithmic phase of growth in the presence and absence of apo-, iron-, zinc-, or manganese-lactoferrin, ovotransferrin, and serotransferrin at 2 or 1 mg/mL for 120 min at 37°C. Extracellular bacteria were killed by the addition of gentamicin. The number of intracellular bacteria was then evaluated by CFU counts on selective media after the lysis of infected monolayers. The data obtained are reported in Table 1. The invasion efficiency of GAS was significantly inhibited by apo-lactoferrin at 2 or 1 mg/mL and by the metal-saturated forms; in contrast, ovotransferrin and serotransferrin were ineffective in inhibiting the invasion of bacteria. In agreement with previous observations, no differences were observed in the numbers of internalised bacteria in the experiments carried out with 2 or 24 h of infection, indicating that no intracellular replication occurred (Greco et al. 1995). Control experiments were performed by preincubating each of the forms of lactoferrin with bacteria or cells separately before the invasion assays. Under these experimental conditions, lactoferrin was ineffective in protecting against GAS invasion (data not shown).
To better mimic the in vivo streptococcal infections, we carried out a series of experiments in which GAS were pretreated, before the infection of cell monolayers, with erythromycin and ampicillin at subinhibiting concentrations. The data obtained (Table 1) indicate that the pretreatment of bacteria with subinhibiting concentrations of antibiotics partially influenced the GAS invasion, decreasing the number of intracellular bacteria by about fivefold, while greatly enhanced the efficiency of bLf, resulting in a complete inhibition towards GAS invasivity. Effect of bLf on GAS-induced apoptosis According to Tsai et al. (1999), we observed that GAS invasion partially brings infected cells to apoptosis. In our experiments, apoptotic cells in GAS-infected cell monolayers were determined by propidium iodide staining and quantified by flow cytometry. The data are reported in Table 2. It was observed that GAS, after 24 h of infection, induces apoptosis in 15% of the cell monolayers. To determine whether bLf may influence the GAS-induced apoptosis in cell monolayers infected by GAS, the percentage of apoptotic cells was quantified in the presence of bLf. Although the number of intracellular GAS was about 10-fold lower than in the absence of the protein, the percentage of the cells undergoing apoptosis increased from 15 to 25% (Table 2). In agreement with the data obtained with the cytofluorimeter, we observed that LDH release in the culture medium of infected cells slightly decreased from 15.0 to 9.7 mU/mL in the absence and presence of bLf, respectively. LDH release was about 10 mU/mL in uninfected cells, indicating that, even in the absence of bLf, intracellular GAS were not able to induce lysis of a relevant number of cells. Clinical trial From the data obtained in the in vitro models, we tried to confirm the protective activity of bLf towards GAS invasion in a small in vivo trial. GAS were isolated from 30 children suffering from recurrent pharyngitis and already scheduled for tonsillectomy, and these clinically isolated GAS strains were tested for their invasiveness using the protocols described above. Among the examined children, 12 were found to be colonised by invasive strains of GAS and those children were enrolled in the study. The children were randomly divided into two groups. Group A was treated with 500 mg of oral erythromycin three times daily for the 15 days preceding tonsillectomy, and Group B was treated with gargles of 100 mg of bLf three times daily for 10 days followed by gargles of 100 mg of bLf plus 500 mg of oral erythromycin three times daily for the 15 days preceding tonsillectomy. Treatments with bLf alone were excluded for ethical reasons. After tonsillectomy, five 100-mg tonsil samples for each child were treated as described under Materials and methods. The number of extracellular bacteria, putatively adherent to tonsil cells, was not assayed, since the antibiotic treatment did not allow a correct estimate of extracellular bacteria. Conversely, after the cell lysis, the number of intracellular GAS was counted on selective media (Table 3). It should be noted that in tonsil specimens from children treated with erythromycin plus bLf gargles, a lower number of GAS within host cells was found in comparison with the © 2002 NRC Canada
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123 Table 3. Number of intracellular GAS in tonsil specimens. Erythromycin alone
Erythromycin plus blf
Child
Intracellular GAS in 100 mg of tonsil specimen
Child
Intracellular GAS in 100 mg of tonsil specimen
4 5 6 10 11 12
7490±2015 9600±354 6730±727 8560±942 3850±936 2440±927
1 2 3 7 8 9
45±40 750±156 150±122 1420±429 265±145 355±63
Note: Children suffering from recurrent pharyngitis by GAS facultative intracellular strains and already scheduled for tonsillectomy were treated (Group A) with 500 mg of oral erythromycin three times daily for the 15 days preceding tonsillectomy or (Group B) with gargles of 100 mg of bLf three times daily for 10 days followed by gargles of 100 mg of bLf plus 500 mg of oral erythromycin three times daily for the 15 days preceding tonsillectomy. Cells were isolated from tonsil specimens and the number of intracellular bacteria was evaluated by counting CFU on selective media after cell lysis (see text for details). The number of intracellular GAS is the mean ± SD of five 100-mg tonsil samples for each child.
number of intracellular GAS retrieved in specimens from children treated with erythromycin alone.
Discussion GAS, an important human pathogen that causes pharyngitis, impetigo, cellulitis, and deeper infections such as rheumatic fever, scarlet fever, necrotizing fasciitis, and streptococcal toxic shock syndrome, is capable of entering into epithelial and endothelial cells avoiding host defences (Greco et al. 1995). Since lactoferrin is present in most secretions and the activity of lactoferrin towards cell invasion by some facultative intracellular pathogens has been previously demonstrated (Longhi et al. 1993, 1994; Antonini et al. 1997; Valenti et al. 1999), we tested the anti-invasive activity of bLf towards GAS invasion of epithelial cells. We report here that bLf hinders GAS invasion of nonprofessional phagocytes. This effect was demonstrated at noncytotoxic and nonbactericidal concentrations. Under the conditions employed in vitro, bLf at 1 or 2 mg/mL decreased the number of internalised GAS by about 10- or 100-fold, respectively; however, a complete inhibition of GAS invasiveness was observed when GAS were pretreated with subinhibiting concentrations of erythromycin or ampicillin. Interestingly, the anti-invasive activity of bLf can be observed only when the protein is present during the infection assay, while it cannot be observed by pretreating bacteria or cells with bLf; in addition, there are no differences between apo- and differently metal-saturated bLf in the inhibition of GAS invasivity. It is also important to note that both ovotransferrin and human serotransferrin did not display any significant anti-invasive activity, despite their sequence and structural homologies. All together, these observations suggest that the anti-invasive activity of bLf towards GAS is specific in inhibiting the entry of bacteria into host cells. The facultative intracellular gram-positive GAS can induce apoptosis within infected epithelial cells (Tsai et al. 1999). We observed that, similar to that previously reported by Tsai et al. (1999), at 24 h postinfection, about 15% of GASinfected cells underwent apoptosis. Moreover, we report data demonstrating that the addition of bLf to cell monolayers,
while decreasing the number of internalised bacteria, enhanced the percentage of apoptotic cells from 15 to 25% and decreased the LDH content of the medium. This leads to the conclusion that, in the employed in vitro model, in the presence of bLf, the predominant mechanism of cell death is apoptosis. This effect of bLf was observed at concentrations that are unable to induce apoptosis in uninfected cells (Valenti et al. 1999), suggesting that bLf is able to amplify the apoptotic signals in infected cells, shifting the equilibrium between apoptosis and necrosis towards the programmed cell death. However, the meaning of the apoptosis in GASinfected cells remains to be elucidated, and it is not clear whether the ability of bacteria to induce apoptosis in nonprofessional phagocytes might be beneficial for the pathogen or is a defensive mechanism of the host. It is generally believed that the activation of the apoptotic cell death program with cell condensation, fragmentation, and neutrophil chemoattractant release is a critical step in nonspecific defence against systemic dissemination of L. monocytogenes, which is able to replicate intracellularly (Rogers et al. 1996; Valenti et al. 1999). On the contrary, in the case of microorganisms such as GAS that are not able to replicate intracellularly, apoptosis could be a strategy developed by GAS for survival and spreading of infection (Tsai et al. 1999). In this case, however, the addition of bLf, increasing the number of apoptotic cells, could allow a greater number of microorganisms to be exposed to the host defensive mechanism or to antibacterial drugs, contributing to eradication of the infection. This interpretation of the phenomenon appears to be sustained by the small clinical trial that we carried out on 12 children suffering from recurrent pharyngitis and already scheduled for tonsillectomy. In the pharynx of these enrolled children, we isolated GAS strains able to invade epithelial cells. After tonsillectomy, we found a lower number of intracellular GAS in the tonsil specimens from children treated with bLf plus antibiotics in comparison with that from children treated with antibiotics alone. This result could be due to both bLf hindering of GAS internalisation into host cells and increasing the number of apoptotic cells. As already discussed, such an increase in apoptotic events may allow a greater number of microorganisms to be released from infected cells and exposed to the administered © 2002 NRC Canada
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antibiotic, even though it has yet to be determined whether apoptosis induced by GAS also occurs in vivo. In conclusion, our data indicate that the long list of host defensive functions of lactoferrin towards bacterial infections could be increased by a further biological role consisting of the inhibitory effect towards invasion of epithelial cells by the important human pathogen GAS.
Acknowledgements This work was supported by grant CNR project on “Biotechnology” and by MURST PRINs “Role of metal ions in intracellular infections”.
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