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Invasion of cultured human cells by Streptococcus pyogenes
Res. Microbiol.
~) INSTITUTPASTEUR/ELSEVIER Paris 1995
1995, 146. 551-560
Invasion of cultured human cells by Streptococcus pyogenes R. Greco (I), L. De Martino (i), G. Donnarumma (1) M.P. Conte (2), L. Seganti (2) and P. Valenti (I) (*)
to Institute of Microbiology, University of Naples, Naples (Italy), and ¢2~Institute of Microbiology, University of Rome "La Sapienza ", Roma
SUMMARY
The invasive capacity of streptococcal strains belonging to groups A and B was evaluated by infecting human epithelial and endothelialcells and monitoring the number of viable intracollular bacteria at diffomnt times postinf~tion. All strains tested entered eukaryotic cells (HeLa, HEp2 and HUVE), with Streptococcus pyogonoc exhibiting a higher invasion efficiency than group B streptococci (GBS). No il~racellular multiplicalion was observed, and GBS remained viable 24 h postinfection, where-.; S. pyogenes were gradually killed. We found that cytochalasin 0 almost completely inhibited internalization of all bacterial strains, whereas colchialne had no effect, indicating that host microfllaments play a major role in bacterial internalization. Moreover, the use of the lysosomotropic agent ammonium chloride enabled us to demonstrate that a pH increase in the intracellular vesicles did not affect streptococcal entry. These results were documented by electron microscopic observations which revealed the different steps in the invasion pathway, including a fos'mn event between phagosomes c,~ntaining S. pyogenes and lysesemes.
Key-words: Adhesion, Invasion, Streptococcus pyogenes, Phagosome, Lysosome; Streptococci, Endothelial cells, Epithelial cells, Endosomes, Virulence.
INTRODUCTION The first step in the infectious process is usually represented by adhesion of microorganisms to target cells, and some pathogens have evolved a specialized strategy to survive and avoid the immune system, consisting o f the ability to enter the cells (Wich et al., 1991 ; Falkow, 1991). Invasive bacteria are internal-
i z e d into m e m b r a n e - b o u n d p h a g o s o m e s . Within a few hours, some are able to escape into the cytoplasm by disrupting the phagosome membrane, whereas others remain entrapped in these compartments. In the first case, bacteria survive, multiply and eventually spre.d to adjacent cells; in the second case, the fusion of 9hagosomes with vacuoles creates a degradative environment in which bacteria are
Submitted January 24. 1995, accepted May 12, 1995. (*) Correspondingauthor: PieraValenti,Instituteof Microbiology,Facultyof Medicine,SecondUniversityof Naples,Larghetto S. Anielloa Caponapoli2. 80138 Naples.Italy.
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R. GRECO E T A L
digested. Occasionally, phagosome-lysosome fusion does not occur, and consequently bacteria can survive in intracellular vesicles. Among various pathogenic bacteria, streptococci, responsible for severe diseases, have stimulated the need to increase our knowledge of the bacterium/host interaction process. The virulence strategy of Streptococcus agalactiae (group B streptococci: GBS) has been studied in an in utero model of a n e w b o r n M a c a c a nemestrina (Rubens et aL, 1991). It has been demonstrated that these bacteria are able to i n v a d e a l v e o l a r e p i t h e l i a l cells, interstitial f i b r o b l a s t s and lung c a p i l l a r y e n d o t h e l i u m (Rubens et aL, 1992). Recently, the invasion of cultured human umbilical vein endothelial (HUVE) cells by GBS has been also described: bacteria penetrate and injure endothelial cells; microfilaments are necessary for uptake, but no evidence for intracellular replication was found (Gibson et ol., 1993). On the basis of these reports, it would be interesting to determine whether Streptococcus pyogenes may overcome the host barriers by passing by local defences. It is well k n o w n that S. pyogenes colonizes epithelial surfaces of the nasopharynx and the skin of humans, causing several diseases, such as pharyngitis, acute rheumatic fever, scarlet fever, poststreptococcal glomerulonephritis and toxic shocklike syndrome (Bartter et aL, 1988; Stevens, 1992; Spencer, 1995). This bacterium synthesizes a variety of virulence factors and possesses different surface-binding proteins which promote its a d h e r e n c e to epithelial tissues, including the mucosa of the upper respiratory tract (Hasty et al., 1992). Bacterial surface c o m p o n e n t s i n v o l v e d in the m e c h a n i s m o f adhesion to epithelial cells include M and F proteins (Tylewska et al., 1988; Hanski and C a p a r o n , 1992), c o m p l e x e s of l i p o t e i c h o i c acid and M protein (Courtney et al., 1992), the
FCS GBS HUVE MEM
= = = =
foetalcalf serum, groupB streptococci. humanumbilicalveinendothelial(ccIl). minimalessentialmedium.
fibronectin-binding protein and the polysaccharide (Botta, 1981). Thus, in an attempt to gain further i n s i g h t into the m e c h a n i s m o f p a t h o g e n e s i s of s t r e p t o c o c c a l diseases, we have hypothesized that, similarly to GBS, S. p y o g e n e s could e x h i b i t an i n v a s i v e ability. This report describes the invasion of cultured e p i t h e l i a l and e n d o t h e l i a l c e l l s ( H e L a $3, HEp2 and HUVE cells) by both S. pyogenes and GBS strains, without evidence of intracellular replication.
MATERIALS AND METHODS Organisms and media Clinical isolates of group B (Streptococcus agalactiae 103 and 123) and group A (S. pyogenes 63, type M5 and S. pyogenes 54, type M12) streptococci, obtained from patients with communityacquired cases of pharyngitis, were cultured in Todd-Hewitt medium (Difco Lab.). Bacto agar (Difco Lab.) was added at a final concentration of 1.5 % in solid media. All strains possessed betahaemolytic activity and were identified as belonging to group A or B using commercially available diagnostic test kits (Slidex Strepto kit, Biom~rieux). Cell cultures HeLa $3 and HEp2 cells were cultured in minimal essential medium (MEM; Seromed) supplemented with 1.2 g/! NaHCO. 2 mM glutamine 100 U/ml penicillin, 0.1 mg/ml streptomycin and 10% heat-inactivated foetal calf serum (FCS), in a 5 % CO 2 incubator. Human umbilical vein endothelial (HUVE) cells were isolated from fresh umbilical cords by using collagenase solution according to a modification of the method of Schwartz (1978). The HUVE cells (used at second passage in all the experiments) were cultured in medium 199 (Seromed) supplemented with 2.0 g/l NaHCO 3, 2 mM glutamine, 100 U/ml penicillin,
CFU PBS p.i.
= colony-formingunit, = phosphate-bufferedsaline. = postinfection.
INVASION OF CULTURED HUMAN CELLS BY STREPTOCOCCUS PYOGENES
0.1 m g / m l streptomycin, 200 [.tg/ml gentamicin, 2 0 % heat-inactivated FCS, in a 5% CO 2 incubator. Invasion assays Invasion of cultured cells was assayed by a modification o f the technique of lsberg and Falkow (1985). Briefly, semiconfluent monolayers of HeLa, HEp2 and HUVE cells grown without antibiotics in 24-well plates were infected with bacterial suspensions (100 bacteria per call) in early exponential phase, corresponding to a subculture of 120 min at 37°C. Infection was performed for 2 h at 37°C. Then, cells were w a s h e d extensively with PBS, and 1 ml of fresh medium containing 200 I.tg/ml g e n t a m i c i n w a s a d d e d to e a c h w e l l . W h e n 500 ~tg/ml gentamicin were used, no significant difference in the number of intracellular colonyforming units (CFU) was d ".ected. After a further 2-h incubation period at 37°C, infected cells were treated with t r y p s i n - E D T A (mixture o f 0.05 % trypsin (11250) and 0 . 0 2 % E V ' ~ ) for 5 min at 37°C and lysed by addition of ; ,, ml of cold 0.1% Triton-X100. Cell lysates were diluted in phosphate buffered saline (PBS) and plated on ToddHewitt agar to quantify the number of viable intracellular bacteria. In all experiments, the invasion was controlled by the microscopic observation of Giemsa-stained slides.
Assay for intracellular replication of GBS and $. pyogenes strains A f t e r b a c t e r i a l i n f e c t i o n , H e L a , HEp2 and H U V E cells were washed and fresh medium cont a i n i n g 200 I.tg/ml g e n t a m i c i n w a s added. The monolayers were then incubated for 4, 6, 8, 10, 12 and 24 h at 37°C in the presence of gentamicin. At these times, cells were lysed and the number of v i a b l e i n t r a c e l l u l a r b a c t e r i a w a s e v a l u a t e d as described above. Effect of a m m o n i u m chloride on bacterial invasion Prior to the invasion assays, HeLa, HEp2 and H U V E cell m o n o l a y e r s were p r e i n c u b a t e d for 30 m i n w i t h o r w i t h o u t 20 m M NH4Ci a n d infected with GBS or S. pyogenes strains ( 5 x 107 CFU/well). After removal of bacterial inoculum, gentamicin- and NH4Cl-containing m e d i u m was added to cell monolayers. At different times, cultured cells were lysed and viable intracellular bacteria were counted, as describea above.
553
Effect of c y t o s k e l e t o n i n h i b i t o r s on b a c t e r i a l invasion Stock solutions of cytochalasin D and colchicine (Sigma Chem.Co.) were dissolved in ethanol and PBS, respectively. HeLa and HUVE cell monolayers were preincubated for 45 min with MEM containing 0.5 I.tg/ml cytochalasin D or colchicine. Treated and untreated cells were then infected with streptococci (5× 107 CFU/well) and incubated for 2 h in the presence of hue cytoskeleton inhibitors. The n u m b e r of intracellular bacteria was determined. Electron microscopy Twenty-four-well tissue culture plates seeded with I x 106 epithelial or endothelial cells/well were infected with 1 x 10 s bacteria for 2 h at 37°C. Before infection, monolayers were pulsed with ferr i t i n for l a b e l l i n g s e c o n d a r y i y s o s o m e s as described by D'Arey et al. (1975), Ferritin was utilized as the lysosomal marker. After infection, monolayers were washed five times with M E M and once more with PBS without Ca z+ and Mg z+. T h e n , they were incubated with t r y p s i n - E D T A solution (0.05 and 0.02 %, respectively), washed a g a i n g e n t l y w i t h PBS, p e l l e t e d at 6 0 0 g for 10 rain and fixed with 2.5 m M glutaraldehyde in cacodylate buffer for 2 h at 4°C, post-fixed with unbuffered 1% OsO 4, dehydrated by using increasing concentrations of ethyl alcohol and embedded in Epon 812. The ultrathin sections, after staining with nranyl acetate and lead citrate, were examined with a "Zeiss EM109" transmission electron microscope. Statistics Data concerning invasion assays, derived from five independent experiments, were performed in triplicate. The mean value and the standard deviation were determined for each assay carded out.
RESULTS B a c t e r i a l invasion o f c u l t u r e d cells T h e capacity o f G B S 103, G B S 123 a n d S. pyogenes 54 and 63 to invade HeLa, HEp2 and H U V E cells, s h o w n in table I, was a s s a y e d following the procedure described above. The i n v a s i o n e f f i c i e n c y o f S. p y o g e n e s s t r a i n s
554
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Table I. Invasion efficiency of streptococcal strains.
HELA
Human cultured cells Bacterial strain GBS 103 GBS 123
HeLa
HEp2
HUVE
0.40±0.02 0.10±0.01 0.12±0.01 0.30 ± 0.02 0.10 ± 0.01 0.20 ± 0.01
1O00O
S. pyogenes 54 5.00 + 0.20 1.00 ± 0.02 1.00 ± 0.02 S. pyogenes 63 2.00 ± 0.20 1.00 ± 0.02 1.00 ± 0.02
Inoculated and internalized viable bacteria were evaluated by counting on agar plates. Invasion efficiency is the percent of the inoculatedCFU which were internalized. The data are presented as means +/- standard deviation of at least five experiments. o
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'rIME (houri}
appeared to be f r o m 5- to 10-fold-increased in comparison with that observed with GBS strains. The number of internalized bacteria b e l o n g i n g to both g r o u p s w a s a l w a y s h i g h e r in H e L a cells t h a n that d e t e c t a b l e in H E p 2 a n d H U V E c e l l s . To e n s u r e t h a t t h e n u m b e r o f intracellular bacteria w a s n o t affected by a d h e sive bacteria, e x p e r i m e n t s in t h e p r e s e n c e o f g e n t a m i c i n were also p e r f o r m e d at 4°C, a t e m perature permitting only the a d h e s i o n process. A f t e r cellular lysis, no C F U s were found, indicating that the e x p e r i m e n t a l m o d e l w a s correct.
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A s s a y for i n t r a c e l l u i a r replication o f b a c t e r i a
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S. p y o g e n e s 63 a n d G B S 103 w e r e t e s t e d f o r t h e i r ability to r e p l i c a t e w i t h i n c u l t u r e d cells (fig. 1). G B S 103 s h o w e d no s i g n i f i c a n t i n c r e a s e in i n t r a c e l l u l a r C F U b e t w e e n 2 a n d 24 h o f infection, indicating that this strain did n o t m u l t i p l y w i t h i n the h o s t cells. Indeed, for
HOVE
I
1o0oo
Fig. 1. Intracenular replication of streptococcal strains at different times of infection in HeLa, HEp2 and H U V E cells. Abscissa = number of viable intracellular bacteria/ml; ordinate = time p.i.; [] = GBS 103; 0 = S. p y o g e n e s 63. Data are presented as means + / - standard deviations.
z
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INVASION OF CULTURED HUMAN CELLS BY STREPTOCOCCUS PYOGENES S. pyogenes 63, there is evidence of g~'adual
ferent times, cells were lysed and the intracellular viable bacteria were counted. Results from a typical invasion experiment performed w i t h N H 4 C l - t r e a t e d c e l l s are r e p o r t e d in figure 2. The invasion efficiency of GBS 103 and S. pyogenes 63 in HeLa cells, exposed to NH4C1, was slightly lower than that observed in untreated cells. W h e n e x p e r i m e n t s with GBS 103 or S. pyogenes 63 strains were perf o r m e d in HEp2 and H U V E cells, s i m i l a r behaviour was observed.
intracellular killing, and no internalized viable
S. pyogenes cells were found 24 h p.i.
Effect of endosome acidification on bacterial invasion Acidification o f vesicles is an important event of several endoeytie pathways. Internali z a t i o n o f a m e m b r a n e - b o u n d v a c u o l e is i m m e d i a t e l y f o l l o w e d by p u m p i n g protons into the endosome by vacuolar ATPase, causing a decrease in vesicle pH (Schneider, 1987). NH4CI, a lipophilic amine, is a lysosomotropic agent which enters vesicles readily, preventing endosome acidification. The cell monolayers were treated with 20 mM NH4CI for 30 min prior to infection, for 2 h during the infection period and for 2, 4, 8, 12 and 24 h p.i. At dif-
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Effect of cytoskeleton inhibitors on bacterial invasion The effect of 0.5 I.tg/ml cytochalasin D and colchicine, which impair microfilament structures and microtubule organization, respectively (Axline and Raven, 1974; Schlima,
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(hours)
Fig. 2. Effect of 20 mM NH4CI on the invasion of streptococcal strains in HeLa cells. Abscissa = number o f viable intraccllular bacreri~ml; ordinate = time p.i. The inoculum
(CFUIml) of streptococciis reportedas time 0, while the values at the correspondingtimes refer to intraceilular bacteria. •--- • = GBS 103 (+NH4CI); A--- A = S. pyogenes 63 (+NH4CI); -- --- = GBS 103 (- NH4CI); A--,a = S. pyogenes 63 (- NH4CI). Data are presentedas means +/- standard deviations.
556
R. GRECO E T AL.
1986), upon the i n v a s i o n o f epithelial and endothelial cultured cells by GBS 103 and S. p y o g e n e s 63 strains was tested. The invasion efficiency o f infected cultured cells treated with cytoskeleton inhibitors (see "Materials and Methods") is shown in table II. Cytochalasin D significantly reduced the number o f bacteria recovered from different cell lysates, whereas colchicine was found to be ineffective.
Table II. Effect of eytochalasin D and colchicine (0.5 ug/ml) on the invasion efficiency of streptococcal strains. Bacterial strain
Host cells
GBS 103
HeLa
Drug
Invasion efficiency
none 0.35 +-0.02 Cytochalasin D 0.001 ± 0.001 Colchicine 0.40 ± 0.02 HUVE none 0.10 ± 0.01 Cytochalasin D 0.001 + 0.001 Colchicine 0.10 ± 0.01
$. pyogenes 63 HeLa
Electron microscopy study of GBS- and S. pyogenes.infected HeLa cells To verify that S. p y o g e n e s and G B S were internalized in HeLa cultured cells, transrmssion electron microscopy was performed from a 2-h invasion assay (fig. 3A and B). The bacterial attachment to the epithelial surface with early invagination and bacteria containing vacuoles are evident in both figures. H e L a cells c l e a r l y a p p e a r e d to be m o r e strongly invaded by S. p y o g e n e s (fig. 3A) than by GBS cells (fig. 3B). At 4 h, ultrathin sections of the infected cells showed the endocytic nature of bacteria containing vacuoles (fig. 3C and D). At that time of infection, no relevant damage to bacterial cells was detectable. After 8 h, cultured cells contained damaged or intact bacteria. Figure 4A shows an S. p y o g e n e s cell just starting to undergo degradation within the phagosome, according to the data of intracellular killing, reported in figure 1. Figure 4B represents a GBS intact cell inside the phagocytic vacuole whose surrounding membrane appears intact. This finding is well correlated with the capacity of survival in an intracellular environment of GBS cells (fig. 1).
none 4.50 ± 0.20 Cytochalasin D 0.002 ± 0.001 Colchicine 5.00 + 0.30 HUVE none 1.00 ± 0.04 Cytochalasin D 0.002 ± 0.001 Colchicine 1.20 ± 0.02
Inoculated and intemalizedviablebacteria were evaluated by countingCTU on agar plates.Invasionefficiencyis the percent of inoculatedCFU whichwereinternalized. The data are presentedas means+/- standarddeviationof at leastfiveexperiments.
In order to associate the damage of $. p y o . g e n e s c e l l s w i t h the p h a g o s o m e - l y s o s o m e fusion, ferritin was used as a lysosomal marker. Figure 4C illustrates the fusion o f lysosomes with a phagosome containing an S. p y o g e n e s cell. In figure 4D, a GBS-containing vesicle surrounded by lysosomes is depicted; the phagosome-lysosome fusion was not observed.
DISCUSSION Previous reports indicated that GBS penetrate and injure different cells such as alveolar epithelial and umbilical vein endothelial cells ( R u b e n s et al., 1992 ; G i b s o n , 1993). The
Fig. 3. Electron micrographs of HeLa cells infected with S. pyogenes 63 and with GBS 103 2 h (A and B, magnificationx g,800) and 4 h p.i. ((2 and D, magnification× 40,000). A) S. pyogenes 2 h p.i.; B) GBS 2 h p.i.; several bacteria were visualized within membrane bound vacuoles or in the process of ingestion.C) S. pyogenes 4 h p.i.; D) GBS 4 h p.i.; enlarged views of bacteria-containingvacuoles.
B~
558
R. GRECO ET AL.
results presented here confirm these data and demonstrate that S. pyogenes strains are also able to invade cultured human epithelial and endothelial cells. In fact, all four strains of Streptococcus spp. t e s t e d e n t e r e u k a r y o t i c cells, irrespective of the m o n o l a y e r type (HeLa, HEp2 and HUVE), with a higher invasion efficiency observed for S. pyogenes. After internalization, bacteria do not multiply within the infected cells: S. pyogenes is gradually damaged in the phagosomes, whereas GBS is still viable 24 h p.i. I n v a s i o n of H e L a cells by s t r e p t o c o c c a l strains was visualized by electron microscopy; micrographs of infected cells correlate well with results obtained in invasion assays. Internalized bacteria were seen within membranebound vacuoles, and enlarged views of thin sections indicated that damage of S. pyogenes cells was related to the phagosome-lysosome fusion. In an attempt to clarify the mechanism of this i n f e c t i o u s process, a m o n g c o m p o u n d s k n o w n to s u p p r e s s p a r t i c l e e n g u l f m e n t by phagocytic cells, cytochalasin D and colchicine, i n h i b i t o r s of c y t o s k e l e t o n s t r u c t u r e s , were tested for their potential effect on the infection of epithelial and endothelial-like cells by streptococcal strains. At concentrations which disrupt actin polymers of mierofilaments in both phagocytic and non-phagocytic cells and suppress cell motility and phagoeytosis (Axline and Raven, 1974), cytochalasin D inhibited the uptake of GBS and S. pyogenes by HeLa, HEp2 and H U V E cells. Conversely, the microtubule inhibitor, colchici'ae, had no effect on bacterial invasiveness. These results s u g g e s t that i n f e c t i o n o f n o n - p r o f e s s i o n a l phagocytic cells is dependent upon functional host cell microfilaments, whereas integrity of microtubules is not required.
A c i d i f i c a t i o n o f e n d o s o m e s and vesicles occurs soon after internalization in processes such as receptor-mediated endocytosis (Schneider, 1987). The weak base, a m m o n i u m chloride, which readily crosses the cell membranes and raises the low pH of intracellular compartments, did not modify the bacterial invasive capability, d e m o n s t r a t i n g that e n d o s o m e pH does not i n f l u e n c e the i n v a s i o n p r o c e s s o f streptococci. Taken together, the data presented here provide further information on the pathogenesis m e e h a - . ' s m of S. pyogenes infections. T h i s research strongly supports the hypothesis that S. pyogenes may have a strategy for gaining entry into the host tissues which involves internalization into susceptible cells, thus evading the innate host defences. Bacterial products involved in the entry step are not yet known, and further investigations are in progress to correlate this invasion property with external structures of S. pyogenes cells.
Acknowledgments This work was supported by MURST and CNR grants.
Invasion de cellules humaines en culture par Streptococcus pyogenes
Nous avons ~tudi~ l ' i n v a s i o n de cellules humaines ~pith~liales et endothfliales en culture (HeLa, HEp2 et HUVE) par des souches de stteptotoques des groupes A et B, en ~valuant ~ difffrents intervalles de temps apr~s l'infection le hombre des baetfries intracellulaires vivants. Toutes les souehes test~s entrent darts les cellules eukaryotes bien qne les souches de Streptococcus pyogenes montrent une
Fig. 4. Enlarged views of HeLa cells: ultrathin sections infected with S. pyogenes 63 and with GBS 103, 8 h p.i. A and B, magnification x 60,000; C and D, magnification x 40,000). A) S. pyogenes 8 h p.i., damaged bacterium within a vacuole; B) GBS 8 h p.i., intact bacterium within a vacuole; C) S. pyogenes 24 h p.i., phagoaome-lysosomefusion; D) GBS 24 h p.i., lysosomes surrounding a phagosome.
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plus grande capacit6 d'invasion par rapport/t celle qu'on observe pour les souches des streptocoques de groupe B (GBS). Les bact6ries, une fois entr6es, ne se multiplient pas et les cellules GBS sont encore vivantes apr~s 48 h tandis que ceiles de S. pyogenes sont lentement alt6r6es. Nous avons aussi d6montr6 que la cytochalasine D inhibe presque compl~tement l'internalisation des souches test6es, tandis que la colchicine n'a aucun effet, d6montrant ainsi que les microf'daments jouent un r61e essentiel darts le processus d'invasion des cellules par les streptocoques. En utilisant le chlorure d'ammonium, un agent lysosomotropique, nous avons montr6 qu'avee une 616ration du pH des v6sicules intracellulaires l'internalisation des streptocoques n'est pas influenc6e. Les r6sultats obtenus sont en accord avee les observations au microscope 61eetronique montrant les 6tapes de l'invasion des cellules HeLa par les streptotoques, y compris la fusion des lysosomes et des phagosomes contenant les eellules de S. pyogenes. Mots-clds : Adh6rence, Invasion, Streptococcus pyogenes, Phagosome, Lysosome; Streptocoques, Cellules endoth61iales, Cellules 6pith61iales, Endosomes, Virulence.
References Axline, S.G. & Raven, E.P. (1974), Inhibition of phagocytosis and plasma membrane mobility of cultivated macrophages by cytochalasin B. J. Cell Biol., 62, 647-653. Battler, T., Dascal, A., Carroll, K. & Curley, F.J. (1988), "Toxic strep syndrome": a manifestation of group A streptococcal infection. Arch. Intern. Med., 148, 1421-1424. Botta, G. (1981), Surface components in adhesion of group A streptococci to pharyngeal epithelial cells. Curr. Microbiol., 6, 101-104. Courtney, H.S., yon Hunolstein, C., Dale, J.B., Bronze, M.S., Beachey, E.H. & Hasty, D.L. (1992), Lipoteichoic acid and M protein: dual adhesins of group A streptococci. Microb. Pathog., 12, 199-208.
D'Arcy, P., Hart, J. & Young, M.R. (1975), Interference with normal phagosome-lysosome fusion in macrophages, using ingested yeast cells arid suramin. Nature (Lond.), 256, 47-49. Falkow, S. (1991), Bacterial entry into eukaryotic cells. Cell 65, 1099-1102. Gibson, R.L., Lee, M.K., Soderland, C., Chi, E.Y. & Rubens, G.E. (1993), Group B streptococci invade endothelial cells : type III capsular polysaceharide attenuates invasion. Infect. lmman., 61, 478-485. Hanski, E. & Caparon, M. (1992), Protein F, a fibronectlnbinding protein, is an adhesin of the group A streptococcus Streptococcus pyogenes. PICAS, 89, 61726176. Hasty, D.L., Ofek, I., Courtney, H.S. & Doyle, R.J. (1992), Multiple adhesins of streptococci. Infect. lmmur~, 601, 2147-2152. Isberg, R.R. & Falcow, S.A. (1985), A single genetic locus encoded by Yersinia pseudotuberculosis permits invasion of culr~tred animal cells by Escherichia coil K12. Nature (Lond.), 317, 262-264. Rubens, C.E., Raft, H.V., Jackson, LC., Chi, E.Y., Bielltaki, J.T. & Hillier, S.L. (1991), Pathophysiology and histopathology of group B streptococcal sepsis in Macaca nemestrina primates induced after intraamniotic inoculation: evidence for bacterial cellular invasion. Infect. lmmun., 164, 320-330. Rubens, C.E., Smith, S., Hulse, M., Chi, E.Y. & van Belle, G. (1992), Respiratory epithefial cell invasion by group B streptococci. Infect. Immun., 60, 51575163. Schlima, M. (1986), The cytoskeleton: an introductory survey. Spnnger ~,erlag, Wian, New York, p. 326. Schneider, D.L. (1987), The proton pump ATPase of lysosomes and related m~anelles of vacuolar apparatus. Biochim. Biophys. Acta, 895,1-10. Schwartz, S.M. (1978), Selection and characterization of bovine aortic endothefial cells. In vitro, 14, 966-980. Spencer, R.C. (1995), Invasive streptococci. Fur. J. Clin. Microbiol. Infect. Dis., 14, suppl. 1, 26-32. Stevens, D.L. (1992), Invasive group A streptococcus infections. Clin. Infect. Dis., 14, 2-13. Tylewska, S.K., Fischetri, V.A. & Gibbons, R.J. (1988), Binding selectivity of Streptococcus pyogenes and M protein to epithefial cells differs from that of fipoteichnic acid. Curr. Microbiol., 16, 209-216. Wich, M.J., Madara, J.L., Fields, B.N. & Normak, SJ. (1991), Molecular cross talk between epithelial cells and pathogenic micreorganlsms. Ceil, 67, 651-659.
~) INSTITUTPASTEUR/ELSEVIER Paris 1995
1995, 146. 551-560
Invasion of cultured human cells by Streptococcus pyogenes R. Greco (I), L. De Martino (i), G. Donnarumma (1) M.P. Conte (2), L. Seganti (2) and P. Valenti (I) (*)
to Institute of Microbiology, University of Naples, Naples (Italy), and ¢2~Institute of Microbiology, University of Rome "La Sapienza ", Roma
SUMMARY
The invasive capacity of streptococcal strains belonging to groups A and B was evaluated by infecting human epithelial and endothelialcells and monitoring the number of viable intracollular bacteria at diffomnt times postinf~tion. All strains tested entered eukaryotic cells (HeLa, HEp2 and HUVE), with Streptococcus pyogonoc exhibiting a higher invasion efficiency than group B streptococci (GBS). No il~racellular multiplicalion was observed, and GBS remained viable 24 h postinfection, where-.; S. pyogenes were gradually killed. We found that cytochalasin 0 almost completely inhibited internalization of all bacterial strains, whereas colchialne had no effect, indicating that host microfllaments play a major role in bacterial internalization. Moreover, the use of the lysosomotropic agent ammonium chloride enabled us to demonstrate that a pH increase in the intracellular vesicles did not affect streptococcal entry. These results were documented by electron microscopic observations which revealed the different steps in the invasion pathway, including a fos'mn event between phagosomes c,~ntaining S. pyogenes and lysesemes.
Key-words: Adhesion, Invasion, Streptococcus pyogenes, Phagosome, Lysosome; Streptococci, Endothelial cells, Epithelial cells, Endosomes, Virulence.
INTRODUCTION The first step in the infectious process is usually represented by adhesion of microorganisms to target cells, and some pathogens have evolved a specialized strategy to survive and avoid the immune system, consisting o f the ability to enter the cells (Wich et al., 1991 ; Falkow, 1991). Invasive bacteria are internal-
i z e d into m e m b r a n e - b o u n d p h a g o s o m e s . Within a few hours, some are able to escape into the cytoplasm by disrupting the phagosome membrane, whereas others remain entrapped in these compartments. In the first case, bacteria survive, multiply and eventually spre.d to adjacent cells; in the second case, the fusion of 9hagosomes with vacuoles creates a degradative environment in which bacteria are
Submitted January 24. 1995, accepted May 12, 1995. (*) Correspondingauthor: PieraValenti,Instituteof Microbiology,Facultyof Medicine,SecondUniversityof Naples,Larghetto S. Anielloa Caponapoli2. 80138 Naples.Italy.
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digested. Occasionally, phagosome-lysosome fusion does not occur, and consequently bacteria can survive in intracellular vesicles. Among various pathogenic bacteria, streptococci, responsible for severe diseases, have stimulated the need to increase our knowledge of the bacterium/host interaction process. The virulence strategy of Streptococcus agalactiae (group B streptococci: GBS) has been studied in an in utero model of a n e w b o r n M a c a c a nemestrina (Rubens et aL, 1991). It has been demonstrated that these bacteria are able to i n v a d e a l v e o l a r e p i t h e l i a l cells, interstitial f i b r o b l a s t s and lung c a p i l l a r y e n d o t h e l i u m (Rubens et aL, 1992). Recently, the invasion of cultured human umbilical vein endothelial (HUVE) cells by GBS has been also described: bacteria penetrate and injure endothelial cells; microfilaments are necessary for uptake, but no evidence for intracellular replication was found (Gibson et ol., 1993). On the basis of these reports, it would be interesting to determine whether Streptococcus pyogenes may overcome the host barriers by passing by local defences. It is well k n o w n that S. pyogenes colonizes epithelial surfaces of the nasopharynx and the skin of humans, causing several diseases, such as pharyngitis, acute rheumatic fever, scarlet fever, poststreptococcal glomerulonephritis and toxic shocklike syndrome (Bartter et aL, 1988; Stevens, 1992; Spencer, 1995). This bacterium synthesizes a variety of virulence factors and possesses different surface-binding proteins which promote its a d h e r e n c e to epithelial tissues, including the mucosa of the upper respiratory tract (Hasty et al., 1992). Bacterial surface c o m p o n e n t s i n v o l v e d in the m e c h a n i s m o f adhesion to epithelial cells include M and F proteins (Tylewska et al., 1988; Hanski and C a p a r o n , 1992), c o m p l e x e s of l i p o t e i c h o i c acid and M protein (Courtney et al., 1992), the
FCS GBS HUVE MEM
= = = =
foetalcalf serum, groupB streptococci. humanumbilicalveinendothelial(ccIl). minimalessentialmedium.
fibronectin-binding protein and the polysaccharide (Botta, 1981). Thus, in an attempt to gain further i n s i g h t into the m e c h a n i s m o f p a t h o g e n e s i s of s t r e p t o c o c c a l diseases, we have hypothesized that, similarly to GBS, S. p y o g e n e s could e x h i b i t an i n v a s i v e ability. This report describes the invasion of cultured e p i t h e l i a l and e n d o t h e l i a l c e l l s ( H e L a $3, HEp2 and HUVE cells) by both S. pyogenes and GBS strains, without evidence of intracellular replication.
MATERIALS AND METHODS Organisms and media Clinical isolates of group B (Streptococcus agalactiae 103 and 123) and group A (S. pyogenes 63, type M5 and S. pyogenes 54, type M12) streptococci, obtained from patients with communityacquired cases of pharyngitis, were cultured in Todd-Hewitt medium (Difco Lab.). Bacto agar (Difco Lab.) was added at a final concentration of 1.5 % in solid media. All strains possessed betahaemolytic activity and were identified as belonging to group A or B using commercially available diagnostic test kits (Slidex Strepto kit, Biom~rieux). Cell cultures HeLa $3 and HEp2 cells were cultured in minimal essential medium (MEM; Seromed) supplemented with 1.2 g/! NaHCO. 2 mM glutamine 100 U/ml penicillin, 0.1 mg/ml streptomycin and 10% heat-inactivated foetal calf serum (FCS), in a 5 % CO 2 incubator. Human umbilical vein endothelial (HUVE) cells were isolated from fresh umbilical cords by using collagenase solution according to a modification of the method of Schwartz (1978). The HUVE cells (used at second passage in all the experiments) were cultured in medium 199 (Seromed) supplemented with 2.0 g/l NaHCO 3, 2 mM glutamine, 100 U/ml penicillin,
CFU PBS p.i.
= colony-formingunit, = phosphate-bufferedsaline. = postinfection.
INVASION OF CULTURED HUMAN CELLS BY STREPTOCOCCUS PYOGENES
0.1 m g / m l streptomycin, 200 [.tg/ml gentamicin, 2 0 % heat-inactivated FCS, in a 5% CO 2 incubator. Invasion assays Invasion of cultured cells was assayed by a modification o f the technique of lsberg and Falkow (1985). Briefly, semiconfluent monolayers of HeLa, HEp2 and HUVE cells grown without antibiotics in 24-well plates were infected with bacterial suspensions (100 bacteria per call) in early exponential phase, corresponding to a subculture of 120 min at 37°C. Infection was performed for 2 h at 37°C. Then, cells were w a s h e d extensively with PBS, and 1 ml of fresh medium containing 200 I.tg/ml g e n t a m i c i n w a s a d d e d to e a c h w e l l . W h e n 500 ~tg/ml gentamicin were used, no significant difference in the number of intracellular colonyforming units (CFU) was d ".ected. After a further 2-h incubation period at 37°C, infected cells were treated with t r y p s i n - E D T A (mixture o f 0.05 % trypsin (11250) and 0 . 0 2 % E V ' ~ ) for 5 min at 37°C and lysed by addition of ; ,, ml of cold 0.1% Triton-X100. Cell lysates were diluted in phosphate buffered saline (PBS) and plated on ToddHewitt agar to quantify the number of viable intracellular bacteria. In all experiments, the invasion was controlled by the microscopic observation of Giemsa-stained slides.
Assay for intracellular replication of GBS and $. pyogenes strains A f t e r b a c t e r i a l i n f e c t i o n , H e L a , HEp2 and H U V E cells were washed and fresh medium cont a i n i n g 200 I.tg/ml g e n t a m i c i n w a s added. The monolayers were then incubated for 4, 6, 8, 10, 12 and 24 h at 37°C in the presence of gentamicin. At these times, cells were lysed and the number of v i a b l e i n t r a c e l l u l a r b a c t e r i a w a s e v a l u a t e d as described above. Effect of a m m o n i u m chloride on bacterial invasion Prior to the invasion assays, HeLa, HEp2 and H U V E cell m o n o l a y e r s were p r e i n c u b a t e d for 30 m i n w i t h o r w i t h o u t 20 m M NH4Ci a n d infected with GBS or S. pyogenes strains ( 5 x 107 CFU/well). After removal of bacterial inoculum, gentamicin- and NH4Cl-containing m e d i u m was added to cell monolayers. At different times, cultured cells were lysed and viable intracellular bacteria were counted, as describea above.
553
Effect of c y t o s k e l e t o n i n h i b i t o r s on b a c t e r i a l invasion Stock solutions of cytochalasin D and colchicine (Sigma Chem.Co.) were dissolved in ethanol and PBS, respectively. HeLa and HUVE cell monolayers were preincubated for 45 min with MEM containing 0.5 I.tg/ml cytochalasin D or colchicine. Treated and untreated cells were then infected with streptococci (5× 107 CFU/well) and incubated for 2 h in the presence of hue cytoskeleton inhibitors. The n u m b e r of intracellular bacteria was determined. Electron microscopy Twenty-four-well tissue culture plates seeded with I x 106 epithelial or endothelial cells/well were infected with 1 x 10 s bacteria for 2 h at 37°C. Before infection, monolayers were pulsed with ferr i t i n for l a b e l l i n g s e c o n d a r y i y s o s o m e s as described by D'Arey et al. (1975), Ferritin was utilized as the lysosomal marker. After infection, monolayers were washed five times with M E M and once more with PBS without Ca z+ and Mg z+. T h e n , they were incubated with t r y p s i n - E D T A solution (0.05 and 0.02 %, respectively), washed a g a i n g e n t l y w i t h PBS, p e l l e t e d at 6 0 0 g for 10 rain and fixed with 2.5 m M glutaraldehyde in cacodylate buffer for 2 h at 4°C, post-fixed with unbuffered 1% OsO 4, dehydrated by using increasing concentrations of ethyl alcohol and embedded in Epon 812. The ultrathin sections, after staining with nranyl acetate and lead citrate, were examined with a "Zeiss EM109" transmission electron microscope. Statistics Data concerning invasion assays, derived from five independent experiments, were performed in triplicate. The mean value and the standard deviation were determined for each assay carded out.
RESULTS B a c t e r i a l invasion o f c u l t u r e d cells T h e capacity o f G B S 103, G B S 123 a n d S. pyogenes 54 and 63 to invade HeLa, HEp2 and H U V E cells, s h o w n in table I, was a s s a y e d following the procedure described above. The i n v a s i o n e f f i c i e n c y o f S. p y o g e n e s s t r a i n s
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Table I. Invasion efficiency of streptococcal strains.
HELA
Human cultured cells Bacterial strain GBS 103 GBS 123
HeLa
HEp2
HUVE
0.40±0.02 0.10±0.01 0.12±0.01 0.30 ± 0.02 0.10 ± 0.01 0.20 ± 0.01
1O00O
S. pyogenes 54 5.00 + 0.20 1.00 ± 0.02 1.00 ± 0.02 S. pyogenes 63 2.00 ± 0.20 1.00 ± 0.02 1.00 ± 0.02
Inoculated and internalized viable bacteria were evaluated by counting on agar plates. Invasion efficiency is the percent of the inoculatedCFU which were internalized. The data are presented as means +/- standard deviation of at least five experiments. o
2
4
|
10
l
12 "
'rIME (houri}
appeared to be f r o m 5- to 10-fold-increased in comparison with that observed with GBS strains. The number of internalized bacteria b e l o n g i n g to both g r o u p s w a s a l w a y s h i g h e r in H e L a cells t h a n that d e t e c t a b l e in H E p 2 a n d H U V E c e l l s . To e n s u r e t h a t t h e n u m b e r o f intracellular bacteria w a s n o t affected by a d h e sive bacteria, e x p e r i m e n t s in t h e p r e s e n c e o f g e n t a m i c i n were also p e r f o r m e d at 4°C, a t e m perature permitting only the a d h e s i o n process. A f t e r cellular lysis, no C F U s were found, indicating that the e x p e r i m e n t a l m o d e l w a s correct.
HSp2 1OOOO0
!
I
!
I
I
o
A s s a y for i n t r a c e l l u i a r replication o f b a c t e r i a
'rIME ~ m )
S. p y o g e n e s 63 a n d G B S 103 w e r e t e s t e d f o r t h e i r ability to r e p l i c a t e w i t h i n c u l t u r e d cells (fig. 1). G B S 103 s h o w e d no s i g n i f i c a n t i n c r e a s e in i n t r a c e l l u l a r C F U b e t w e e n 2 a n d 24 h o f infection, indicating that this strain did n o t m u l t i p l y w i t h i n the h o s t cells. Indeed, for
HOVE
I
1o0oo
Fig. 1. Intracenular replication of streptococcal strains at different times of infection in HeLa, HEp2 and H U V E cells. Abscissa = number of viable intracellular bacteria/ml; ordinate = time p.i.; [] = GBS 103; 0 = S. p y o g e n e s 63. Data are presented as means + / - standard deviations.
z
4
s
e lO TIME ( ~ w l )
1= "
INVASION OF CULTURED HUMAN CELLS BY STREPTOCOCCUS PYOGENES S. pyogenes 63, there is evidence of g~'adual
ferent times, cells were lysed and the intracellular viable bacteria were counted. Results from a typical invasion experiment performed w i t h N H 4 C l - t r e a t e d c e l l s are r e p o r t e d in figure 2. The invasion efficiency of GBS 103 and S. pyogenes 63 in HeLa cells, exposed to NH4C1, was slightly lower than that observed in untreated cells. W h e n e x p e r i m e n t s with GBS 103 or S. pyogenes 63 strains were perf o r m e d in HEp2 and H U V E cells, s i m i l a r behaviour was observed.
intracellular killing, and no internalized viable
S. pyogenes cells were found 24 h p.i.
Effect of endosome acidification on bacterial invasion Acidification o f vesicles is an important event of several endoeytie pathways. Internali z a t i o n o f a m e m b r a n e - b o u n d v a c u o l e is i m m e d i a t e l y f o l l o w e d by p u m p i n g protons into the endosome by vacuolar ATPase, causing a decrease in vesicle pH (Schneider, 1987). NH4CI, a lipophilic amine, is a lysosomotropic agent which enters vesicles readily, preventing endosome acidification. The cell monolayers were treated with 20 mM NH4CI for 30 min prior to infection, for 2 h during the infection period and for 2, 4, 8, 12 and 24 h p.i. At dif-
000000
i
" "~" ~
Effect of cytoskeleton inhibitors on bacterial invasion The effect of 0.5 I.tg/ml cytochalasin D and colchicine, which impair microfilament structures and microtubule organization, respectively (Axline and Raven, 1974; Schlima,
. .T
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- - ' ~ X X ,T
100
~l
lo
0
555
t
t
t
t
t
2
4
6
8
10
TIME
f II~ 12
24
(hours)
Fig. 2. Effect of 20 mM NH4CI on the invasion of streptococcal strains in HeLa cells. Abscissa = number o f viable intraccllular bacreri~ml; ordinate = time p.i. The inoculum
(CFUIml) of streptococciis reportedas time 0, while the values at the correspondingtimes refer to intraceilular bacteria. •--- • = GBS 103 (+NH4CI); A--- A = S. pyogenes 63 (+NH4CI); -- --- = GBS 103 (- NH4CI); A--,a = S. pyogenes 63 (- NH4CI). Data are presentedas means +/- standard deviations.
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R. GRECO E T AL.
1986), upon the i n v a s i o n o f epithelial and endothelial cultured cells by GBS 103 and S. p y o g e n e s 63 strains was tested. The invasion efficiency o f infected cultured cells treated with cytoskeleton inhibitors (see "Materials and Methods") is shown in table II. Cytochalasin D significantly reduced the number o f bacteria recovered from different cell lysates, whereas colchicine was found to be ineffective.
Table II. Effect of eytochalasin D and colchicine (0.5 ug/ml) on the invasion efficiency of streptococcal strains. Bacterial strain
Host cells
GBS 103
HeLa
Drug
Invasion efficiency
none 0.35 +-0.02 Cytochalasin D 0.001 ± 0.001 Colchicine 0.40 ± 0.02 HUVE none 0.10 ± 0.01 Cytochalasin D 0.001 + 0.001 Colchicine 0.10 ± 0.01
$. pyogenes 63 HeLa
Electron microscopy study of GBS- and S. pyogenes.infected HeLa cells To verify that S. p y o g e n e s and G B S were internalized in HeLa cultured cells, transrmssion electron microscopy was performed from a 2-h invasion assay (fig. 3A and B). The bacterial attachment to the epithelial surface with early invagination and bacteria containing vacuoles are evident in both figures. H e L a cells c l e a r l y a p p e a r e d to be m o r e strongly invaded by S. p y o g e n e s (fig. 3A) than by GBS cells (fig. 3B). At 4 h, ultrathin sections of the infected cells showed the endocytic nature of bacteria containing vacuoles (fig. 3C and D). At that time of infection, no relevant damage to bacterial cells was detectable. After 8 h, cultured cells contained damaged or intact bacteria. Figure 4A shows an S. p y o g e n e s cell just starting to undergo degradation within the phagosome, according to the data of intracellular killing, reported in figure 1. Figure 4B represents a GBS intact cell inside the phagocytic vacuole whose surrounding membrane appears intact. This finding is well correlated with the capacity of survival in an intracellular environment of GBS cells (fig. 1).
none 4.50 ± 0.20 Cytochalasin D 0.002 ± 0.001 Colchicine 5.00 + 0.30 HUVE none 1.00 ± 0.04 Cytochalasin D 0.002 ± 0.001 Colchicine 1.20 ± 0.02
Inoculated and intemalizedviablebacteria were evaluated by countingCTU on agar plates.Invasionefficiencyis the percent of inoculatedCFU whichwereinternalized. The data are presentedas means+/- standarddeviationof at leastfiveexperiments.
In order to associate the damage of $. p y o . g e n e s c e l l s w i t h the p h a g o s o m e - l y s o s o m e fusion, ferritin was used as a lysosomal marker. Figure 4C illustrates the fusion o f lysosomes with a phagosome containing an S. p y o g e n e s cell. In figure 4D, a GBS-containing vesicle surrounded by lysosomes is depicted; the phagosome-lysosome fusion was not observed.
DISCUSSION Previous reports indicated that GBS penetrate and injure different cells such as alveolar epithelial and umbilical vein endothelial cells ( R u b e n s et al., 1992 ; G i b s o n , 1993). The
Fig. 3. Electron micrographs of HeLa cells infected with S. pyogenes 63 and with GBS 103 2 h (A and B, magnificationx g,800) and 4 h p.i. ((2 and D, magnification× 40,000). A) S. pyogenes 2 h p.i.; B) GBS 2 h p.i.; several bacteria were visualized within membrane bound vacuoles or in the process of ingestion.C) S. pyogenes 4 h p.i.; D) GBS 4 h p.i.; enlarged views of bacteria-containingvacuoles.
B~
558
R. GRECO ET AL.
results presented here confirm these data and demonstrate that S. pyogenes strains are also able to invade cultured human epithelial and endothelial cells. In fact, all four strains of Streptococcus spp. t e s t e d e n t e r e u k a r y o t i c cells, irrespective of the m o n o l a y e r type (HeLa, HEp2 and HUVE), with a higher invasion efficiency observed for S. pyogenes. After internalization, bacteria do not multiply within the infected cells: S. pyogenes is gradually damaged in the phagosomes, whereas GBS is still viable 24 h p.i. I n v a s i o n of H e L a cells by s t r e p t o c o c c a l strains was visualized by electron microscopy; micrographs of infected cells correlate well with results obtained in invasion assays. Internalized bacteria were seen within membranebound vacuoles, and enlarged views of thin sections indicated that damage of S. pyogenes cells was related to the phagosome-lysosome fusion. In an attempt to clarify the mechanism of this i n f e c t i o u s process, a m o n g c o m p o u n d s k n o w n to s u p p r e s s p a r t i c l e e n g u l f m e n t by phagocytic cells, cytochalasin D and colchicine, i n h i b i t o r s of c y t o s k e l e t o n s t r u c t u r e s , were tested for their potential effect on the infection of epithelial and endothelial-like cells by streptococcal strains. At concentrations which disrupt actin polymers of mierofilaments in both phagocytic and non-phagocytic cells and suppress cell motility and phagoeytosis (Axline and Raven, 1974), cytochalasin D inhibited the uptake of GBS and S. pyogenes by HeLa, HEp2 and H U V E cells. Conversely, the microtubule inhibitor, colchici'ae, had no effect on bacterial invasiveness. These results s u g g e s t that i n f e c t i o n o f n o n - p r o f e s s i o n a l phagocytic cells is dependent upon functional host cell microfilaments, whereas integrity of microtubules is not required.
A c i d i f i c a t i o n o f e n d o s o m e s and vesicles occurs soon after internalization in processes such as receptor-mediated endocytosis (Schneider, 1987). The weak base, a m m o n i u m chloride, which readily crosses the cell membranes and raises the low pH of intracellular compartments, did not modify the bacterial invasive capability, d e m o n s t r a t i n g that e n d o s o m e pH does not i n f l u e n c e the i n v a s i o n p r o c e s s o f streptococci. Taken together, the data presented here provide further information on the pathogenesis m e e h a - . ' s m of S. pyogenes infections. T h i s research strongly supports the hypothesis that S. pyogenes may have a strategy for gaining entry into the host tissues which involves internalization into susceptible cells, thus evading the innate host defences. Bacterial products involved in the entry step are not yet known, and further investigations are in progress to correlate this invasion property with external structures of S. pyogenes cells.
Acknowledgments This work was supported by MURST and CNR grants.
Invasion de cellules humaines en culture par Streptococcus pyogenes
Nous avons ~tudi~ l ' i n v a s i o n de cellules humaines ~pith~liales et endothfliales en culture (HeLa, HEp2 et HUVE) par des souches de stteptotoques des groupes A et B, en ~valuant ~ difffrents intervalles de temps apr~s l'infection le hombre des baetfries intracellulaires vivants. Toutes les souehes test~s entrent darts les cellules eukaryotes bien qne les souches de Streptococcus pyogenes montrent une
Fig. 4. Enlarged views of HeLa cells: ultrathin sections infected with S. pyogenes 63 and with GBS 103, 8 h p.i. A and B, magnification x 60,000; C and D, magnification x 40,000). A) S. pyogenes 8 h p.i., damaged bacterium within a vacuole; B) GBS 8 h p.i., intact bacterium within a vacuole; C) S. pyogenes 24 h p.i., phagoaome-lysosomefusion; D) GBS 24 h p.i., lysosomes surrounding a phagosome.
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plus grande capacit6 d'invasion par rapport/t celle qu'on observe pour les souches des streptocoques de groupe B (GBS). Les bact6ries, une fois entr6es, ne se multiplient pas et les cellules GBS sont encore vivantes apr~s 48 h tandis que ceiles de S. pyogenes sont lentement alt6r6es. Nous avons aussi d6montr6 que la cytochalasine D inhibe presque compl~tement l'internalisation des souches test6es, tandis que la colchicine n'a aucun effet, d6montrant ainsi que les microf'daments jouent un r61e essentiel darts le processus d'invasion des cellules par les streptocoques. En utilisant le chlorure d'ammonium, un agent lysosomotropique, nous avons montr6 qu'avee une 616ration du pH des v6sicules intracellulaires l'internalisation des streptocoques n'est pas influenc6e. Les r6sultats obtenus sont en accord avee les observations au microscope 61eetronique montrant les 6tapes de l'invasion des cellules HeLa par les streptotoques, y compris la fusion des lysosomes et des phagosomes contenant les eellules de S. pyogenes. Mots-clds : Adh6rence, Invasion, Streptococcus pyogenes, Phagosome, Lysosome; Streptocoques, Cellules endoth61iales, Cellules 6pith61iales, Endosomes, Virulence.
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D'Arcy, P., Hart, J. & Young, M.R. (1975), Interference with normal phagosome-lysosome fusion in macrophages, using ingested yeast cells arid suramin. Nature (Lond.), 256, 47-49. Falkow, S. (1991), Bacterial entry into eukaryotic cells. Cell 65, 1099-1102. Gibson, R.L., Lee, M.K., Soderland, C., Chi, E.Y. & Rubens, G.E. (1993), Group B streptococci invade endothelial cells : type III capsular polysaceharide attenuates invasion. Infect. lmman., 61, 478-485. Hanski, E. & Caparon, M. (1992), Protein F, a fibronectlnbinding protein, is an adhesin of the group A streptococcus Streptococcus pyogenes. PICAS, 89, 61726176. Hasty, D.L., Ofek, I., Courtney, H.S. & Doyle, R.J. (1992), Multiple adhesins of streptococci. Infect. lmmur~, 601, 2147-2152. Isberg, R.R. & Falcow, S.A. (1985), A single genetic locus encoded by Yersinia pseudotuberculosis permits invasion of culr~tred animal cells by Escherichia coil K12. Nature (Lond.), 317, 262-264. Rubens, C.E., Raft, H.V., Jackson, LC., Chi, E.Y., Bielltaki, J.T. & Hillier, S.L. (1991), Pathophysiology and histopathology of group B streptococcal sepsis in Macaca nemestrina primates induced after intraamniotic inoculation: evidence for bacterial cellular invasion. Infect. lmmun., 164, 320-330. Rubens, C.E., Smith, S., Hulse, M., Chi, E.Y. & van Belle, G. (1992), Respiratory epithefial cell invasion by group B streptococci. Infect. Immun., 60, 51575163. Schlima, M. (1986), The cytoskeleton: an introductory survey. Spnnger ~,erlag, Wian, New York, p. 326. Schneider, D.L. (1987), The proton pump ATPase of lysosomes and related m~anelles of vacuolar apparatus. Biochim. Biophys. Acta, 895,1-10. Schwartz, S.M. (1978), Selection and characterization of bovine aortic endothefial cells. In vitro, 14, 966-980. Spencer, R.C. (1995), Invasive streptococci. Fur. J. Clin. Microbiol. Infect. Dis., 14, suppl. 1, 26-32. Stevens, D.L. (1992), Invasive group A streptococcus infections. Clin. Infect. Dis., 14, 2-13. Tylewska, S.K., Fischetri, V.A. & Gibbons, R.J. (1988), Binding selectivity of Streptococcus pyogenes and M protein to epithefial cells differs from that of fipoteichnic acid. Curr. Microbiol., 16, 209-216. Wich, M.J., Madara, J.L., Fields, B.N. & Normak, SJ. (1991), Molecular cross talk between epithelial cells and pathogenic micreorganlsms. Ceil, 67, 651-659.