Migration of Salmonella typhi through Intestinal

Microbiol. Immunol., 40(11), 799-811, 1996

Migration of Salmonella typhi through Intestinal Epithelial Monolayers: An In Vitro Study

Sandra Kraeuter Kops*, Daniel K. Lowe, William M. Bement, and A. Brian West

Section of Clinical Microbiology, Department of Laboratory Medicine, and Departments of Pathology, Surgery, and Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, U.S.A.

Received May 7, 1996; in revised form, July 22, 1996. Accepted July 24, 1996

Abstract: This study characterizes the transmigration of enteroinvasive Salmonella typhi in vitro, using a human intestinal epithelial cell line as a model of small intestinal epithelium. C2BBe cells, a subclone of CACO-2 with a highly differentiated enterocytic phenotype, were grown to maturity on Transwell filters. S. typhi Ty2 and the vaccine strain, Ty21a, the S. typhi mutant X7344 and parent strain SB130, and S.

typhimurium 5771 in logarithmic phase were introduced to the upper chamber of the filter units. Numbers of bacteria in the lower chamber, TER and permeability of the monolayer to mannitol were measured over time. Monolayers were examined by light, electron and confocal microscopy to determine the pathway of bacterial transmigration, and intracellular bacteria were estimated by gentamicin assay. Epithelial cell injury was quantified by light microscopy. S. typhi transmigrated earlier and in larger numbers than S. typhimuri-um, inducing marked changes in electrical resistance and permeability. Unlike S. typhimurium, S. typhi selected epithelial cells in small number and caused their death and extrusion from the monolayers leav-ing holes through which S. typhi transmigrated. Ty2 consistently transmigrated in larger numbers and with more injury to monolayers than Ty21a. S. typhi crosses the monolayers of C2BBe cells by a paracellular route in contrast to the transcellular pathway described for other Salmonellae. This may be related to the unique

pathophysiology of S. typhi infection and the restricted host specificity of this pathogen. In these assays the vaccine strain, Ty21a, is slightly less invasive than its parent, though more invasive than S. typhimurium.

Key words: Salmonella typhi, Intestinal cells, Transmigration

Salmonella typhi belongs to the group of enteroinva-sive pathogens that cause systemic disease (25). These organisms enter the mucosa of the host gastrointestinal tract from the lumen, and are subsequently phagocy-tosed by macrophages in which they survive and are distributed throughout the body in lymph and blood. A critical step in this process is penetration through the intestinal epithelium into the mucosal lamina propria, since the epithelial monolayer acts as a continuous mem-brane barrier separating the luminal flora of the intestinal tract from the internal milieu of the host.

Studies of the interactions of enteric pathogens with epithelial cells have been greatly facilitated by the use of in vitro cultures of intestinal cell lines and in particular CACO-2 cells (5, 8). These cells, though derived from a human colonic adenocarcinoma, are columnar and usually express some features of enterocytic differenti-ation, though they are typically of heterogeneous phe-notype. In the absence of non-neoplastic adult human

intestinal epithelial cell lines they are more appropriate models for investigating the interactions of enteric pathogens with host epithelial cells than are HEp-2, HeLa, and MDCK cells which are of human larynx, human cervix, and canine kidney origin respectively, and other non-intestinal cell lines that are sometimes used to model bacterial-host cell interactions (6, 13, 14, 18,23). In the present study we have used C2BBe cells, a

line subcloned from CACO-2 by selection for features of enterocytic differentiation (18). Cells of this line form uniform polarized monolayers on solid substrate, devel-op tight junctions and electrical resistance, and express a well developed microvillus brush border (18). Indeed, C2BBe is unique among human intestinal epithelial cell lines so far examined in expressing the late marker of enterocytic differentiation, brush border myosin I (18).

*Address correspondence to Dr. Sandra Kraeuter Kops, Section

of Clinir.' ",iicrobiology, CB650, Department of Laboratory Med 'ale University School of Medicine, 333 Cedar Street, Ne en, CT 06520, U.S.A.

Abbreviations: ANOVA, analysis of variance; CaCl,, calcium

chloride; CFU, colony forming unit; DMEM, Dulbecco's mini-

mum essential medium; EDTA, ethylenediaminetetraacetic acid;

FCS, fetal calf serum; PBS, phosphate buffered saline, SEM,

standard error of the mean; TER, transmonolayer electrical resis-

tance.

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800 S.K. KOPS ET AL

The mature enterocytic differentiation of this cell line make it especially suitable for investigating transmigra-tion of S. typhi. Use of a polarized monolayer of C2BBe cells grown on a microporous filter substrate enabled us to study this process in vitro, thereby modeling the first critical step in invasion of the small bowel.

While Salmonella typhimurium infection in mice has traditionally served as a model for typhoid fever because of its characteristic hepatosplenomagaly and fever, studies of S. typhi interactions with intestinal epithelium in vivo are few. Kohbata et al (15) did, however report the ultrastructural effects of S. typhi GIFU 10007 on epi-thelium overlying ileal Peyer's Patches in mice. S. typhi specifically affected M cells within the epithelium. The bacteria did not invade the M cells but the apical cyto-plasm of the M cells was pinched off into the luminal spaces, surrounded by bacteria.

S. typhimurium is the most widely studied species of Salmonella and serves as a model for the invasion of mammalian cells by other Salmonella species, including S. typhi. In essence, invasion of polarized epithelial cells by S. typhimurium involves the interaction of bacil-li with the apical membrane of the epithelial cell with ruf-fling of the brush border, and penetration of the cell by enclosure in phagosomes, in which intracellular repli-cation subsequently occurs. These bacilli-containing phagosomes move through the cytoplasm by actin micro-filaments (4, 5) and discharge their living pathogens through the basolateral surface. However, in human gastrointestinal infections S. typhimurium and S. typhi have different natural histories, the latter organism being more invasive and leading to more severe systemic infection. We, therefore, studied the transmigration of two strains of S. typhi across C2BBe epithelial cells in vitro: Ty2, the virulent strain which causes typhoid fever, and Ty2la, an avirulent strain used as an oral vaccine (7, 27). We briefly compare the transmigration activity of these strains with another strain SB130 and its mutant derivative which lacks genes for invasion, X7344 (9). In addition, comparisons are made with characteristics of S. typhimurium invasion.

Materials and Methods

Culture of C2BBe cell monolayers. C2BBe cells, a subclone of the human colonic adenocarcinoma cell line CACO-2 selected for its ability to form polarized mono-layers and a highly differentiated apical brush border (18), were obtained from Dr. Mark Mooseker, Yale Uni-versity. Cells of passage 21-40 were maintained in confluent layers in flasks in high-glucose (25 mm) DMEM (Sigma, St. Louis, Mo., U.S.A.) supplemented with 2 mm glutamine, 10% FCS (Sigma), and 10 µg/ml

human transferrin (Boehringer Mannheim, Indianapolis, Ind., U.S.A.). For routine culture, penicillin (100 U/ml), streptomycin (100 .g/ml) and amphotericin (1 µg/ml) were added. Flasks were treated with trypsin-EDTA (Sigma) to split the cells. Following two washes with complete medium the concentration of the cells was adjusted by counting in a hemocytometer and 2 X 105 cells were applied to collagen-coated 3 tm pore, 6.5 mm diameter Transwell filter units (Costar, Cambridge, Mass., U.S.A.). Collagen coating of the filters was accomplished by incubating in 100 ltl of Type I collagen solution (Sigma) (100 tg/ml collagen in 0.1 M acetic acid, stored over chloroform to maintain sterility) for 3 hr at 37 C, and irradiating with UV light overnight. Medi-um was added to both lower and upper chambers of the Transwell filter units, the volumes being kept constant at 1.0 ml and 200 ul respectively throughout experiments so that fluid levels were equal.

Cell cultures were maintained at 37 C in an atmosphere of 5% CO, Cells were fed with fresh medium every other day for three to four weeks. Confluent monolayers formed within one week of plating, but two to three additional weeks were necessary to allow full differen-tiation of the apical brush border and intercellular tight junctions in the C2BBe monolayers (18).

Culture of bacteria and their application to C2BBe cell monolayers. S. typhi Ty2 was obtained from Amer-ican Type Culture Collection (NCTC 8385). S. typhi Ty21 a was grown from the Vivotif Berne oral vaccine produced by the Swiss Serum and Vaccine Institute, Berne, Switzerland, and distributed by Berne Products Corp., Coral Gables, Fla., U.S.A. The invasion mutant strain X7344 and its parent strain SB130 was obtained from Dr. Jorges Gal'an, State Univerity of New York, Stonybrook, New York (9). S. typhimurium 5771 was obtained from Dr. Keith Joiner of the Section of Infec-tious Diseases at Yale University. E. coli DH5a was obtained from Dr. Stephen Fischer, Department of Lab-oratory Medicine, Section of Clinical Microbiology at Yale University. Bacteria were stored in LB broth (pre-pared according to Tartera and Metcalf (29) with bacto-tryptone, yeast extract, 0.17 M NaCl, and dextrose: DIFCO, MacAlaster-Bicknell, New Haven, Conn., U.S.A.) and 85% glycerol at -80 C. When grown in logarithmic phase for up to 6 hr, the growth curves of S. typhi Ty2 and Ty2la, and S. typhimurium were indistin-guishable. All monolayer-containing filters in each experiment contained C2BBe cells of the same passage number and stage of maturation. Four to twelve hours before the start, the medium in both lower and upper chambers of the filter units was replaced with antibiotic-free complete cell culture medium. S. typhi grown in overnight cul-

MIGRATION OF S. TYPHI THROUGH EPITHELIUM 801

tures, were diluted either antibiotic-free media or in fresh LB broth. Logarithmic growth phase was deter-mined spectrophotometrically at 595 nm. When cultures reached log phase and an optical density equivalent to between 10' and 10' organisms/ml, the medium in the upper chamber of the Transwell unit was withdrawn whereupon 10 µl of the bacterial suspension applied directly to the monolayer, and 190 µl fresh antibiotic-free medium were replaced over the infected C2BBe cells. Identical numbers of Salmonella strains were introduced at the start of the experiments. As a control for the effect of the monolayer on the transmigration of bacteria, equivalent numbers of Salmonellae were applied to monolayer-free collagen-coated filters. In cotransmi-gration experiments, equivalent numbers of E. coli DH5a and Salmonella were used to inoculate the mono-layers. In most experiments, three to six filters were assigned to each treatment group.

Quantitation of bacteria. At selected time intervals aliquots of medium were withdrawn from the lower chambers of the Transwell units and plated onto Mac-Conkey agar either directly and after ten-fold serial dilu-tion in LB broth. Plates were incubated at 37 C for 24 hr at which time colony forming units (CFUs) were count-ed. For cotransmigration experiments, aliquots were plated onto XLD (xylose-lysine-deoxycholate) (Remel) medium enabling Salmonellae (black colonies) and E. coli (pink colonies) to be counted separately.

Gentamicin killing assay for intracellular bacteria. Monolayers were cultured to 21 day maturity in 6-well petri dishes, and pre-treated for 24 hr with antibiotic-free medium. Then 106 bacteria in logarithmic growth phase were placed on the monolayers for either 30 min or 2 hr, followed by extensive washing with PBS and treatment with 100 µg/ml gentamicin (Sigma) for 30 min. After the gentamicin was washed from the monolayers with PBS the cells were lysed with 0.5% Triton in PBS. Bacteria in the lysates were counted as described above. Experiments were run in triplicate on three separate days. Transmonolayer electrical resistance (TER). Electri-cal resistance across the monolayers was measured with an ohmvoltmeter (World Precision Instruments, New Haven, Conn., U.S.A.) by inserting electrodes into the medium in the upper and lower chambers of the Trans-well filter units (16). Baseline measurements were made on experimental monolayer-containing filters and control monolayer-free filters prior to the application of the bacteria. At specified time intervals the measurements were repeated. The readings were corrected for the sur-face area of the monolayer and the data expressed as S2.cm'. Background readings of the monolayer-free control filters were subtracted from the corrected readings

to determine monolayer TER. Between readings the monolayers were kept at 37 C in an incubator with 5% CO2. Great care was taken to avoid injuring the mono-layers with the probe during measurement of TER, and they were subsequently examined with an inverted microscope to check for possible mechanical disrup-tions from manipulations during measurements. Fil-ters with any suspicion of mechanical injury were dis-carded. Mannitol flux. Passage of mannitol from the apical to the basal side of monolayers was measured using a modification of the method of Rutten et al (24). Trans-well inserts with C2BBe monolayers were pre-incubat-ed in antibiotic-free medium and placed in fresh 24-well plates containing 1 ml medium with 150 mm man-nitol in the lower chamber. Baseline measurements were made over 1 hr after introducing 100 µl of similar medium, trace-labeled with 1 µCi ['H]-mannitol having a specific activity of 30 µCi/mM (Amersham, Arlington Heights, Ill., U.S.A.) to the upper chamber. Following the baseline period, 101-106S. typhi Ty2 and Ty2la were applied to the monolayers. As a maximum per-meability control, 25 mm EDTA was applied to addi-tional monolayers which were processed with bacterial samples. At 15, 30, 60, and 120 min intervals the inserts were removed to fresh wells containing non-radioac-tive mannitol-enriched incubation medium. At the end of the experiment, 0.5 ml aliquots were withdrawn from the lower chambers and added to 5 ml Optifluor scintillation medium. Radioactivity was determined by counting in a Beckman S1801 scintillation counter. All remaining fluid from the upper chambers was withdrawn for scin-tillation counting and calculation of net mannitol fluxes. Net flux was determined by the following equation:

cpm Flux= cpm* X Area X Cone;

where Flux is expressed as nmol/cm2, cpm is counts per minute sampled in the lower chamber, cpm* is counts per minute sampled in the upper chamber, Area is the sur-face area of the monolayer-covered filter (0.33 cm'), and Conc is the total mannitol concentration (150 mm) in the upper chamber.

Histology and electron microscopy. Following expo-sure to bacteria at various time intervals monolayers were fixed in formalin, embedded in paraffin and pre-pared for histological examination. For electron microscopy, C2BBe cell monolayers were fixed in situ on the filters in glutaraldehyde (2% glutaraldehyde in 0.1 m sodium phosphate buffer, pH 7.0, containing 0.2% tan-nic acid) for 1 hr at 4 C. Samples were processed at 60 and 120 min after the application of bacteria. For these studies, samples exposed to S. typhimurium and bacteria-

802 S.K. KOPS ET AL

free monolayers were fixed and processed as controls. Following four washes in cold 0.1 M sodium phosphate buffer, the samples were post-fixed in 1 % osmium tetroxide in 0.1 M sodium phosphate buffer (pH 6.0). Preparations were dehydrated by passage through a graded series of ethanol (25-100%), rinsed in propylene oxide, and embedded in Epon 812 (Electron Microscopy Sciences, Washington, Pa., U.S.A.). 300 A thin sections were cut on an LKB ultramicrotome with a diamond knife and mounted on copper grids. The grids were stained with 1% uranyl acetate-lead citrate (22) and examined with a Zeiss EM10B electron microscope.

Immunoperoxidase localization of bacteria was per-formed by fixing monolayer-bearing filters in paraformaldehyde-lysine fixative. Prior to dehydration and embedding the monolayers were incubated with an anti-Salmonella rabbit IgG antibody to 0 or H antigen (Difco), diluted 1:200, followed by an incubation with a peroxidase conjugated goat anti-rabbit IgG (Molecular Probes, Inc.) secondary antibody. Following the perox-idase reaction the monolayers were post-fixed in reduced osmium tetroxide, dehydrated with ethanol, rinsed with propylene oxide, and embedded in Epon and sectioned for examination by electron microscopy.

For examination of C2BBe cell nuclei, filters bearing monolayers were fixed in 10% neutral buffered formal in after exposure to S. typhi Ty2 and Ty2la for 2 hr. Fol-lowing 24 hr fixation they were stained with Feulgen reagent (21), dehydrated, cleared in xylene and mounted on slides. The filter preparations were then examined en face by light microscopy at 400 X and the numbers of mitotic figures and necrotic cells (detected by the pres-ence of either pyknosis or karyorrhexis) counted in each of 50 fields per treatment (5 fields from each of ten fil-ters). The total area examined was 8 mm'.

Immunofluorescent localization and confocal microscopy. C2BBe monolayers were grown for three weeks on glass cover slips. Bacteria in logarithmic growth phase were applied to the monolayers as described for monolayers grown on filters. After 60 min, coverslips exposed to bacteria and controls were fixed in 4% paraformaldehyde for 20 min and perme-abilized with 0.5% Triton in PBS containing 0.1% CaCl2. After two washes with PBS/CaC12 the monolay-ers were incubated in a blocking solution containing PBS/CaC12, 0.1% bovine serum albumin, and normal goat serum (1:1,000) for 30 min. Salmonella were local-ized by incubating monolayers with rabbit IgG anti-body to Salmonellae H-antigen (Difco), diluted 1:200 in blocking solution, for 1 hr. Following three washes with PBS/CaC12, samples were labeled for 2 hr with 1:400 fluorescein conjugated goat anti-rabbit IgG (Mol-ecular Probes, Eugene, Ore., U.S.A.) and either 1:50

rhodamine conjugated phalloidin (Molecular Probes) for actin localization, or propidium iodide (1 pg/ml) for localization of degenerate nuclei. Monolayers were then washed three times and mounted on glass slides with DABCO mounting medium (Sigma). Slides were stored in UV protectant containers under refrigeration and examined with a Biorad dual channel laser scanning confocal microscope within 24 hr.

Statistical analysis. The means and standard error for each treatment group were calculated in all experi-ments. The significance of differences between the means was evaluated by using STATGRAPHICS (Gate-way 2000), for Student t-test. Analysis of variance (ANOVA) and repeated measures analysis (26, 30) were also used to evaluate data from some experiments.

Results

Passage of S. typhi through C2BBe Monolayers In order to determine how rapidly S. typhi cross

C2BBe cell monolayers, 105-106 bacteria of strains Sal-monella (Ty2 and Ty2la, and S. typhimurium 5771) in logarithmic phase of growth were applied to the upper chamber of Transwell filter units containing confluent monolayers of C2BBe cells, and the number of bacteria in the lower chamber were subsequently counted at intervals over 120 min (Fig. 1). In control Transwells

Fig. 1. Comparison of transmigration of Salmonella strains across C2BBe cell monolayers, measured as CFUs/ml in the lower chambers of Transwell filter units over 2 hr. Results are expressed as means ± SEM of several replicates . For No mono-layer+Ty2, n=6; No monolayer+Ty21a , n=6; C2BBe+Ty2, n=20; C2BBe+Ty2la, n=12; C2BBe+5771 , n=6.


which had not been seeded with C2BBe cells, bacteria of both strains crossed the filters into the lower chamber in large numbers within 15 min of their introduction into the upper chamber. At any time point the numbers of Ty2 and Ty2la present were not significantly different in these controls. In the presence of confluent C2BBe monolayers, bacteria of both strains also transmigrated in significant numbers even within 15 min, though fewer Ty2 and Ty2la were always present in the lower cham-bers than in the absence of the monolayers (P<0.05 and P<0.001, respectively). There was no transmi-gration of any of the strains at 5 and 10 min, but there was rapid passage of the S. ryphi strains (Ty2 and Ty2la) at 15 min. The numbers of the virulent strain Ty2 in the lower chamber were approximately 10 fold greater than the numbers of Ty2la (P<0.05 throughout the first hour), and the numbers of S. typhimurium were 1,000 fold lower than Ty2 bacteria up until 60 min (P<_0.01), although equal numbers of all strains were seeded in the upper chambers at the start of the experiment.

In order to differentiate between the mechanism of passage for S. typhi and S. typhimurium, cotransmigration of E. coli DH5a with selected Salmonellae strains was studi€ a 30 min. E. coli did not cross the C2BBe me ,rs when used alone as an inoculum (Fig. 2). In thr, , ;•ence of S. typhimurium, E. coli crossed the

Fig. 2. Cotransmigration studies of S. ryphi Ty2 or S. typhimuri-um 5771 with E. coli DH5a. E. coli alone did not invade the monolayer. Cotransmigration resulted with either Ty2 or 5771, although significantly less S. typhimurium 5771 transmigrated than Ty2 (P<0.03).

Fig. 3. Cotransmigration studies of S. typhi Ty2, SB130, and the

invasion-deficient mutant X7344. E. coli alone did not invade the

monolayer. X7344 was totally deficient in its ability to cross

the monolayer. SB130 showed somewhat less transmigration

ability than Ty2 but this difference was not significant. SB130 did

give a healthy transmigration response, compared to S. typhimuri-

um in Fig. 2.

Fig. 4. Numbers of intracellular S. ryphi Ty2, S. typhi Ty2la, and S. typhimurium within C2BBe cell monolayers 30 and 120 min after application of 106 bacteria (gentamicin killing assay). Results are expressed as CFUs per ml cell lysate derived from the mono-layer in a single well. In comparison with S. typhimurium, sig-nificantly fewer intracellular S. typhi of either strain were found

(P<0.01), though the numbers for all three organisms increased between 30 and 120 min.

804 S.K. KOPS ET AL

epithelial barrier. Likewise, when co-inoculated with S. typhi Ty2, the normally noninvasive E. coli passed into the lower chambers of the Transwell units.

Cotransmigration comparing Ty2 with another strain of S. ryphi, SB130, and a mutant strain derived from it which lacks genetic loci important to the invasion process, designated as X7344, was also studied at 30 min, the time demonstrating the greatest difference between strains in Fig. 1. The invasion mutant failed to migrate across the C2BBe monolayers, and SB130 demonstrat-ed less, but significant transmigration compared to Ty2 at 30 min (Fig. 3).

A gentamicin assay was performed to quantify intra-cellular bacteria. S. ryphi was isolated from the mono-layers by this method, but in numbers that were about two orders of magnitude smaller than for S. typhimurium (P<0.01; Fig. 4). Moreover, only about 100 intracellular Ty2 were present per monolayer after 30 min, whereas by this time approximately 105 would be expected to be present in the lower chamber (Fig. 1).

Effects of S. typhi on Monolayer Permeability To determine the effects of the two strains of S. ryphi

on permeability of the C2BBe cell monolayer we mea-sured two parameters, TER and mannitol flux. The TER of control C2BBe cell monolayers was 256± 25 0-cm'. After introduction of Ty2 into the upper chamber the TER fell precipitously, reaching 24% of

baseline within 30 min after which it leveled off at 15% (Fig. 5). In contrast, Ty2la caused a more gradual decline in TER, the level not differing significantly from baseline values until 60 min when it had fallen to 63%. The differences between TERs of monolayers inocu-lated with Ty2 and Ty2la was highly significant at all time points (P<0.0005).

Marked increases in tnannitol flux were observed after inoculation with Ty2 (Fig. 6) (P<0.00001 vs. con-trol). A significant increase in flux was seen with Ty2la also (P<0.0004 vs. control). There was no significant difference in fluxes between Ty2 and Ty2la, reflecting the lower sensitivity of this technique than TER.

Morphological Changes in C2BBe Cell Monolayers Induced by S. typhi Ty2

Light microscopic examination of vertical sections of C2BBe cell monolayers exposed to Ty2 for 0-60 min revealed severe damage in the form of numerous discrete spaces, some of which contained fragments of cell debris, although the monolayers were not disrupted

(Fig. 7). Such spaces were not present in controls of similar age. Serial sections demonstrated that these spaces communicate between the apical and basal sides of the monolayers. Large spaces were rarely observed in sections of monolayers exposed to Ty2la for the same length of time, though smaller spaces were evident in both apical and basal cytoplasm of monolayers exposed to Ty21a for 2 hr. Monolayers exposed to S. typhimuri-

Fig. 5. Changes in TER across C2BBe cell monolayers with time after seeding with similar numbers of S. typhi Ty2 or Ty2la. Baseline for confluent monolayers derived from the pre-seeding measurements was 256 ± 25 Q.cm2. The results are shown as a % of the baseline (mean ± SEM). The differences between Ty2 and Ty2la were significant at P<0.0005 for all times from 30 to 120 min.

Fig. 6. Permeability of C2BBe monolayers to mannitol over 2 hr of exposure to S. ryphi Ty2 and Ty2la. Monolayers treated with 25 mm EDTA were included as indicators of maximum perme-ability. Ty2 induced a marked increase in permeability over the 2 hr period (P<0.00001). Ty2la induced a smaller but signifi-cant increase in permeability (P<0.0004). Permeability to man-nitol was not significantly different between Ty2 and Ty2la.


urn did not show formation of these spaces at any time. Electron microscopic examination confirmed that the large spaces observed by light microscopy in monolay-ers exposed to Ty2 were intercellular.

Histologic sections of monolayers fixed after exposure to S. typhi and S. typhimurium for 2 hr were immuno-stained with antibodies to H and 0 antigens of Salmo-nella revealed patchy distribution of bacteria along the apical surface, but only rare immunoreactive forms were detected within the monolayers, and it was unclear if they were within cells or in intercellular spaces.

Immunostained monolayers processed for electron microscopy, showed that while S. typhimurium were readily seen in membrane-bound vacuoles within C2BBe cell cytoplasm (Fig. 8), including forms undergoing division, S. typhi were concentrated on the luminal sur-face of cells. In several hundred micrographs reviewed only rare intracellular forms were found, invariably in vacuoles in the superficial cytoplasm near the apical membrane (Fig. 9B) and after exposure to the bacilli for 2 hr. Replicating S. typhi were never seen within or bet'- C2BBe cells.

injury (pyknosis and karyorrhexis) and mitosis in ;onolayers, evaluated by examination of en face

Fig. 7. Photomicrographs of paraffin-embedded monolayers, sectioned vertically and stained with hematoxylin-eosin. A: C2BBe mono-layer exposed to S. typhi Ty2 for 10 min, showing no change in the ordering of monolayer cells. This section looked similar to mono-layers not exposed to bacteria (control) and monolayers exposed to Ty21 a and S. typhimurium 5771 for periods up to 2 hr. Bar= 30 µm. B: C2BBe monolayer exposed to S. typhi Ty2 for 30 min. Large spaces have formed in the monolayer, some containing cells. Bar= 30 µm.

Fig. 8. Electron micrograph of C2BBe cells exposed to Salmo-

nella for 2 hr and immunostained with antibody raised against the

Salmonella H antigen. These cells have been inoculated with S.

typhimurium. Numerous bacilli are present within endocytic vacuoles. Bar= 1 µm.

806 S.K. KOPS ET AL

preparations of filters stained with Feulgen's reagent, demonstrated that Ty2 and Ty2la induced highly sig-nificant increases in the number of cells in the mono-layers showing degenerative nuclear changes of this type (Table 1 and Fig. 10), features that are typically indicative of cell injury and death. As expected, the

effect of Ty2 was more marked than that of Ty2la. The number of cells in mitosis was significantly increased also in monolayers exposed to both strains of S. typhi.

To elucidate the relationship between transmigration of S. typhi and injury of C2BBe cells we examined en face preparations of monolayers double stained for bac-teria and either filamentous actin or DNA, by confocal microscopy (Fig. 11). After 60 min exposure Ty2 was present in scattered distribution on the apical surface of the monolayers, but with focal localization of the bacilli around extruded cells that were above the normal plane of the monolayer (Fig. 11d). Extruded cells were and parts of cells were seen along the surface of mono-layers examined by electron microscopy as well (Fig. 12, A and B). Bacilli were present within the monolayer beneath some of these partially extruded cells (Fig. 10C). This pattern was never seen in monolayers exposed to Ty2la (not shown) or S. typhimurium (Fig. 11, e and f). The association of Ty2la with the mono-layers was similar to that observed for S. typhimurium, which is at the apical surface of the C2BBe cells. Repli-cating forms were observed for monolayers seeded with

Fig. 9. Electron micrographs of C2BBe cells immunostained for Salmonella H antigen. A: Control monolayer cell with no bacterial inocu-lum or background staining, showing an intact brush border. Bar= 2 µm. B: A monolayer inoculated with S. typhi Ty2. Bacteria are local-ized on the apical surface with rare forms in the apical cytoplasm (arrow). The brush border is lost and the brush border membrane appears to be ruffling at the point of bacterial contact. It also appears as if some of the apical cytoplasm is lost or contracted. Bar=2 µm.

Table 1. The effects of S. typhi Ty2 and Ty2la on nuclear degen-

eration and mitotic activity in C2BBe monolayers

Results are the average of counts in five fields of standard area in each of 8 or 10 filters of confluent cells, means±SEM. Cells exhibiting pyknosis or karyorrhexis were identified by intense staining of shrunken nuclei and fragmentation of nuclear material. Filters were fixed in formalin after 2 hr exposure to bac-teria, stained by Feulgen reaction, cleared and examined en face.

°' P< 0.0005 by Student t-test for monolayers exposed to bac-teria vs. controls.

°' P<0 .03 for S. typhi Ty2 vs. Ty2la (Student t-test).


S. typhimurium but were never seen in sections of mono-layers seeded with S. typhi Ty2 or Ty2la. It was difficult to distinguish whether the apically localized bacteria are within the monolayer cells or between the cells.

Discussion

Using this in vitro system, we have characterized several features of transmigration through the intestinal epithelial cell monolayers by S. typhi Ty2 (NCTC 8385). Specifically, we have shown that this virulent strain of S. typhi transmigrates with great rapidity, substantial pas-sage through the polarized monolayer having occurred within 15 min of application to its apical surface (Fig. 1). As might be expected, this process is associated with a marked increase in permeability of the monolayer as demonstrated by decrease in TER (Fig. 5) and increase in mannitol flux (Fig. 6). Further evidence of increased per-meability of the monolayer after exposure to S. typhi is the significant transepithelial passage of non-invasive E. coli in the presence of Ty2, but not in its absence (Figs. 2 and 3). Interestingly, when we sought evidence of transcellular passage of S. typhi Ty2 by light and electron microscopy we were disappointed, although intracellular organisms were easily demonstrated in large numbers in control monolayers exposed to S. typhimurium under

the same experimental conditions. Rare S. typhi Ty2 were seen in phagosomes in the apical cytoplasm after 2 hr exposure to the bacteria, but none were seen at earli-er times, although transmigration and increased manni-tol flux were evident at 15 min, and TER had decreased to 25% of its baseline value at 30 min (the earliest time point measured). Moreover, no S. typhi Ty2 were seen deep in the cytoplasm or in division within the C2BBe cells, though this was commonly seen in S. typhimurium.

Gentamicin assays also failed to demonstrate sub-stantial numbers of S. typhi Ty2 within the monolayers after 30 min and even after 2 hr (Fig. 4) and the number at both time points was an order of magnitude less than for S. typhimurium, although consistently at these time

points in transmigration experiments S. typhi Ty2 had crossed the monolayer in significantly greater numbers than S. typhimurium. These data also suggest that a transcellular pathway may be less important for Ty2 than for S. typhimurium. Detailed microscopic exami-nation of the monolayers following exposure to S. typhi Ty2 by a variety of morphological techniques demon-strated the presence of intracellular spaces, increased numbers of degenerate nuclei and of cells in mitosis (Table 1 and Fig. 10), and extrusion of degenerate cells from the luminal surface of the monolayer (Fig. 12). These findings are indicative of cytotoxic injury to

Fig. 10. En face preparations of C2BBe monolayers fixed on Transwell microporous filters (A, B, C) and an electron micrograph of a Ty2 inoculated monolayer (D). A: Control monolayer showing normal cell nuclei. Bar=50 µm. B: Ty2 inoculated monolayer showing numerous pyknotic and karyorrhexic nuclei (arrow). Bar= 50 tum. C: Mitotic figure (arrow) within a Ty2 inoculated C2BBe monolayer. Bar= 50 µm. D: Degenerative nucleus (arrow) within a Ty2 inoculated epithelium. In this preparation S. typhi, which do not adhere and

penetrate the monolayer cells, were washed away in the fixation process. Bar=5 µm.

808 S.K. KOPS ET AL

C2BBe cells by S. typhi Ty2 with compensatory increase in proliferation (mitosis). They were not seen in mono-layers exposed to S. typhimurium.

The simplest explanation of these findings is that S. typhi Ty2 causes rapid direct injury to certain of the C2BBe cells in the monolayer some of which die and are extruded, and that bacteria pass through the spaces in the monolayer caused by injury to or extrusion of cells, before such spaces have time to reseal, a process which occurs rapidly in vitro (1). We are not aware of any

precedent for this model, and data from S. typhimurium (5, 17, 29 and present study), S. choleraesuis (5) and from a few recent studies of S. typhi (3, 17) have been interpreted differently. However, in natural infections of the human intestinal tract, which our system models more closely, S, typhi behaves differently from S. typhimurium and S. choleraesuis. While S. typhi is an

extremely effective enteroinvasive organism that causes systemic infection (25), S. typhimurium is a localized superficial invader that causes an acute and usually self-limiting mucosal infection with or without transient bac-teremia (in contrast to its pattern of systemic infection in mice). S. choleraesuis is an uncommon cause of mucosa-limiting infection (though it is common in pigs, causing a cholera-like diarrhea). The typically different behavior of S. typhi may be attributed in part to its exceptional ability to survive and proliferate within human macrophages. However, unlike the enteritidis group (to which S. typhimurium belongs) and S. choler-aesuis, S. typhi exhibits an extremely restricted host specificity, infecting humans almost exclusively. It is possible that this restricted specificity results from a mechanism of interaction with enterocytes that is dif-ferent from that of the other Salmonellae, and that while

Fig. 11. Confocal micrographs of C2BBe monolayers, transformed from en face views to vertical section. All photomicrographs are 120 x . a: Control C2BBe monolayer without bacteria, showing staining of actin at the upper and lower surfaces of the monolayer. b: Con-trol monolayer without bacteria. The signal from the C2BBe cell nuclei appears brighter than in the other micrographs as it has been enhanced to demonstrate the faint staining of normal nuclei. c: Monolayer exposed to Ty2 for 2 hr. Bacteria are present at the apical sur-face of the monolayer and are associated with one extruded cell and one cell within the monolayer that appears injured. d: Monolayer exposed to Ty2 for 2 hr showing congregation of bacteria around abnormal cells that are extruded above the surface of the monolayer. e: Monolayer exposured to S. typhimurium for 2 hr. Note the ruffling of the apical surface. S. ryphimurium were localized within the mono-layer. f: Monolayer exposed to S. ryphimurium for 2 hr. Note the absence of abnormal nuclei within the monolayer. Bacteria were local-ized to the apical cytoplasm.


S. typhi is highly adapted for penetrating human intesti-nal epithelium it is effective in other hosts. Our data sup-

port the concept that S. typhi and S. typhimurium have different mechanisms of transmigration through entero-cytic monolayers.

Comparison of these results with those reported by

others is made difficult by the use of different cell lines.

The apical membrane of enterocytes are exquisitely spe-

cialized and highly differentiated structures, and while use of squamous lines as HeLa and HEp-2 cells which

bear little resemblance to enterocytes may provide inter-

esting data on potential mechanisms of molecular inter-

action between enteropathogens and mammalian cells, the pathophysiologic relevance of such interactions

needs to be demonstrated in cells with enterocytic dif-

ferentiation. We chose to use the most highly differen-

tiated human enterocytic cell line available to us for our

studies: C2BBe cells are more differentiated than the parent CACO-2 cell line and than embryonic Henle 407 cells, and we attribute some of the differences between our results and those of others (3, 17) to the use of these different cell lines. There is no doubt that certain strains of S. typhi can invade epithelial cells in vitro- indeed Elsinghorst et al have cloned a gene from S. typhi that determines invasiveness in Henle 407 cells and have expressed it in E. coli rendering them invasive also (3)-but we question whether this mechanism contributes to epithelial transmigration in vivo.

A striking feature of transmigration by S. typhi in our system was the rapidity of the process. Approxi-mately 2 X 105 Ty2 and 2 X 10' Ty21a were detected in the lower chamber after exposure to the pathogen for only 15 min. In contrast, S. choleraesuis and S. typh-imurium did not transmigrate through a CACO-2 cell

Fig. 12. Electron photomicrographs of monolayers of C2BBe cells immunostained for Salmonella H antigen after inoculation with S. typhi Ty2 for 2 hr. They show pieces of extruded cells (arrows) on the monolayer surface. A: A fragment of nuclear material on the sur-face of a C2BBe monolayer, surrounded by Ty2 bacteria. Note the disruption of the tight junctions between cells and the absence of intra-cellular bacteria. Bar= 1 µm. B: C2BBe monolayer with a surface associated cell fragment. The fragment and the monolayer cell show immunoreactive matter which do not resemble whole bacteria (near nucleus). These fragments were not seen in control monolayers which had not been inoculated with bacteria, or in sections from monolayers inoculated with Ty2la or S. typhimurium 5771. Note the disruption in the brush border in the lower right of the micrograph. Perhaps these immunoreactive clusters of matter represent dead bacterial frag-ments that enter the cell when it is disrupted. Bar= 1.5 µm.

810 S.K. KOPS ET AL

monolayer in detectable numbers until after 2 hr o exposure (5). S. typhimurium required. about 2 hr t cause a fall in TER of the CACO-2 cell monolayer t 50% of the baseline (5). In our system S. typhi Ty reduced the TER to 25% of baseline in 15 min. It is o interest that both S. typhimurium and S. choleraesuis multiply within endosomes in the epithelial cell cyto-plasm (5, 6), whereas we never observed this with S. typhi, perhaps as a result of the rapidity of transcytosis. Intracellular multiplication was not noted by Takeuchi in his detailed ultrastructural study of S. typhimurium in guinea pigs (28).

If S. typhi does not become sequestered in the cyto-plasm of the enterocytes on its way into the lamina pro-pria, as our results suggest, the ability to pass extremely quickly through the epithelial monolayer might be of paramount importance in establishing infection. Gastric contents are usually propelled through the 10 meters of small intestine into the colon in 2 to 3 hr, the process being accompanied by vigorous mixing activity. Thus, organisms cannot colonize the lumen or enter the pro-tected environment of an epithelial cell cytoplasmic

phagosome may have to breach the epithelial barrier very rapidly to avoid being eliminated from the host. Circumstantial evidence in support of this concept comes from experimental testing of the vaccine strain S. typhi Ty2la in which the organisms were no longer detectable in the stools 24 hr after oral administration to volunteers (10-12). Our morphologic data indicate that S. typhi Ty2 and Ty2la have a selective toxic effect on a certain minority of cells in the C2BBe monolayer. It is possible that the affected cells all belong to a variant phenotype or to a particular stage of maturation, or are merely the cells with which the bacteria have come in close contact. How such injury is mediated is not evident, but it is more severe after exposure to Ty2 than with Ty2la , and the same pattern was not observed in studies of S. typhimuri-um by confocal and electron microscopy. Structural differences in the enterotoxin genes of S, typhi and S. typhimurium (2, 19, 20) could theoretically account for these different effects.

In so far as comparisons can be made, both the viru-lent Ty2 and avirulent Ty2la strains of S. typhi appear to traverse human enterocyte monolayers from the luminal surface more effectively than other Salmonellae and in particular S. typhimurium. In vivo, effective transep-ithelial migration and delivery of antigens to the small intestinal lamina propria, with its rich supply of lym-phatics and abundant lymphoid tissue, may account in part for the striking immunogenicity of these strains. However, although Ty2la is an outstanding oral vaccine and an important vector for delivery of engineered anti-

gens gens for immunization against other pathogens, our o results show that it is less effective at crossing the epithelial monolayer in vitro than Ty2: about one order of 2 magnitude fewer Ty2la transmigrated after the same f challenge as Ty2, and the effect on TER and mannitol

flux was less. There was less cell injury as determined by

nuclear changes, and cell extrusion was not as prominent in the confocal studies with Ty2. If this is confirmed in

vivo, it may have important implications for the devel- opment of optimal typhoid and polyvalent enteric vac-

cines. It will especially provide impetus for the con- struction of avirulent strains that still retain full potential

for mucosal invasion and immunogenicity by genetic manipulation, in contrast to avirulent strain such as

Ty2la that are derived by chemical mutagenesis.

The authors thank Drs. Michael Kashgarian, Mark Mooseker, Keith Joiner, William Marks, Stephen Fischer, and Jay Gates

for their advice on this project. The authors would also like to thank Corinne Simoes for facilitating communications during

these studies.

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Migration of Salmonella typhi through Intestinal