Influence of Certain Indigenous Gastrointestinal Microorganisms on Duodenal Alkaline Phosphatase in Mice

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1976, p Copyright 1976 American Society for Microbiology Vol. 31, No. 6 Printed in U.S.A. Influence of Certain Indigenous Gastrointestinal Microorganisms on Duodenal Alkaline Phosphatase in Mice DIANE P. YOLTON' AND DWAYNE C. SAVAGE* Department of Microbiology, and School of Basic Medical Sciences, Urbana, Illinois Received for publication 21 January 1976 Alkaline phosphatase activity was assayed in duodenal homogenates or extracts from adult specific pathogen-free (SPF) and germfree mice and gnotobiotic mice monoassociated with a Lactobacillus sp., a Bacteroides sp., or a coliform strain indigenous to SPF mice. Activity levels of the enzyme were much higher in the preparations from germfree mice than in those from the SPF controls. In the gnotobiotes monoassociated either with a freshly isolated Lactobacillus sp. or a Bacteroides sp., the levels of alkaline phosphatase activity were intermediate between the values for germfree and SPF mice. By contrast, in the gnotobiotes monoassociated with a coliform strain, alkaline phosphatase activity remained at high germfree levels. Butanol extracts of duodenal tissue from SPF mice, germfree mice, and exgermfree mice associated with an indigenous microflora from SPF mice (conventionalized) were subjected to acrylamide gel electrophoresis. A stain for alkaline phosphatase activity revealed three major bands in the gels prepared with extracts from SPF and conventionalized mice, but only two in the gels prepared with extracts from germfree mice. All three bands may have been present in the latter gels. One of the bands (the middle one) may have been obscured, however, by high activity in the slowest moving band. As determined by densitometric scanning, the slowest moving band had much higher activity in the preparations from germfree animals than in those from SPF or conventionalized mice. These findings suggest that the indigenous microbial flora affects not only quantitatively, but also qualitatively, the activity of alkaline phosphatases in the mouse intestinal mucosa. The indigenous microbiota is known to affect the development and maintenance of the normal morphology of the small intestine. The gastrointestinal tracts of germfree rodents appear to be anatomically anomalous when compared with those of conventional animals. For example, the tracts of germfree rats and mice consistently weigh less than those of conventional animals (11). The lamina propria of the intestines of the germfree animals is thinner and contains fewer plasma and lymphoid cells than that of the bowels of conventional rats and mice (1). The lamina propria provides important structural support for the intestinal epithelium. Thus, with little lamina propria, germfree mice have slender villi with shallow crypts of Lieberkuhn (1). Indigenous microorganisms also affect epithelial cell function in the intestinal canal. The activity of the alkaline phosphatases in duodenal epithelial cells is several-fold higher in germfree mice than in animals with a conventional indigenous microflora (25). By contrast, 'Present address: 2010 Laurel St., Forest Grove, Ore in exgermfree animals colonized by an entire indigenous microflora, the levels of alkaline phosphatase in the duodenum fall well within the range for adult specific pathogen-free (SPF) animals (25). Alkaline phosphatases may be involved in the absorption from the intestinal lumen of monosaccharides and amino acids (6), cholesterol and lipids (10), and vitamin D (5). Thus, the microbial flora may alter intestinal absorption by affecting the alkaline phosphatase activity found in these epithelial cells. We are interested, therefore, in the mechanisms by which the indigenous microbiota exerts its influence on alkaline phosphatase activity. In an effort to establish models to study these mechanisms, we monoassociated Ha/ICR germfree mice with an indigenous Lactobacillus sp. The lactobacilli depressed the high level of alkaline phosphatase activity found in the germfree animals to a level intermediate between the high values and the low values of SPF mice (25). In similar experiments, indigenous bacteroides in monoassociated exgermfree mice also depressed the alkaline phosphatase activity to an intermediate level (14). Thus, specific indi-

2 VOL. 31, 1976 vidual indigenous microorganisms can influence duodenal alkaline phosphatase activity in monoassociated germfree mouse models. We have used these models to begin to explore the mechanisms by which the indigenous microbiota depresses intestinal alkaline phosphatase activity. In this report, we expand our information on the influences of lactobacilli and bacteroides on the activity and add evidence on the influence on the activity of a third microbial type, a coliform, also isolated from the gastrointestinal tract of an SPF mouse. In addition, we report on the influence of the indigenous microbiota on the heterogeneity of intestinal alkaline phosphatase in mice, as assessed by acrylamide gel electrophoresis of the "isozymes" of of duodenal alkaline phosphatase in germfree, conventionalized exgermfree, and SPF mice. MATERIALS AND METHODS Animals. Animals used were germfree ARS/Ha/ ICR mice and their conventional counterparts, Ha/ ICR SPF mice (A. R. Gibco, Madison, Wis.). Also used were germfree CD-1 mice and their conventional counterparts, CD-1 SPF mice (Charles River, Wilmington, Mass.). The SPF mice were housed in groups of eight adults in plastic boxes with paper covers (Isocage, Carworth, New City, N.Y.) with Ab-Sorb-Dri (Ab-Sorb-Dri, Inc., Garfield, N.J.) for bedding. They were maintained on Wayne Lab-Blox (Allied Mills, Inc., Chicago, Ill.). Their drinking water was acidified with M HCI (17). The germfree and monoassociated mice were maintained in inflatable vinyl isolators (23) with autoclaved-sterilized Ab-Sorb-Dri for bedding, and either autoclave-sterilized sterilizable Wayne Lab- Blox or sterile Charles River mouse food and sterile water. Sterility tests on the germfree mice were performed by established procedures (24). Monoassociation of germfree mice. The bacteria were introduced into the germfree isolator as pure broth cultures. The cultures were always introduced in the morning. Food was usually withheld from the mice from the evening before. The cultures were poured on a small amount of the food of the mice to be associated. The procedure used to insure that the mice were properly monoassociated has been described (25). The Lactobacillus strains used for monoassociation of germfree mice were isolated from SPF mice on a medium, 10A, developed for selection of lactobacilli (18) and were always propagated on this medium. Similarly, Bacteroides strains were isolated on a medium, -S, developed for the selection of bacteroides, and coliform strains were isolated on a medium, T7T, developed for the selection of coliforms (18). Both kinds of organisms were propagated on the medium used for their isolation. All strains were partially characterized (21) to insure their inclusion into the expected genus. Enumeration of bacteria. Segments of the gastrointestinal tract, weighing approximately 0.5 g INDIGENOUS GASTROINTESTINAL MICROORGANISMS 881 including contents, were homogenized either with Teflon grinders in 4.5 ml of sterile charcoal water (17) or by mixing with a Vortex for 30 s in 4.5 ml of sterile charcoal water to which glass beads had been added. The homogenates were diluted in charcoal water in 10-fold steps. Calibrated loopfuls of each dilution were then spread on the surface of agar media and incubated appropriately. Estimates of the bacterial population levels were made by reported procedures (18). Histology. Segments of the gastrointestinal tract with contents intact were frozen in a 2% solution of methyl cellulose (15) on the freezing shelf of a microtome-cryostat (International Equipment Co., Needham Heights, Mass.). Sections of frozen tissues cut at 4 zm were fixed for 60 s in absolute methyl alcohol and stained with a modified tissue Gram stain (3). Tissue preparation for enzymatic assay. The mice were killed at the same time each day by cervical separation. In some cases, the animals were lightly anesthetized with acetone-chloroform-ether (1:2:3) before they were killed. A 2-cm section of intestine just posterior to the pylorus was cut and freed of surrounding connective tissue and lumenal contents. Three procedures (methods one, two and three) were used to prepare the tissue for enzyme activity measurement. Method one involved only homogenization of the tissue, and yielded a tissue preparation unsuitable for electrophoresis (see below). Methods two and three involved butanol extraction as well as homogenization of the tissue. These two methods were used to prepare extracts used for electrophoresis. Extracts produced by method three were most effectively electrophoresed (see below). The extracts produced by methods two and three were also used in assays for enzymatic activity. In method one (25), each duodenum was homogenized in 1 ml of 0.05 M tris(hydroxymethyl)aminomethane (Tris), ph 7.0, in a tissue grinder. The homogenate was centrifuged at approximately 2,500 x g for 10 min, and the supernatant solution was taken for enzyme analysis. All procedures were carried out at 0 C. In method two, a modification of the method of Smith et al. (22), the duodenal sample was stored in 1 ml of 0.25 M sucrose or 1 ml of 0.05 M Tris, ph 7.0, for at least 24 h at -20 C after removal from the animal. After thawing, the tissue was minced with scissors and homogenized in 2 ml of 0.25 M sucrose in a tissue grinder.'the homogenate was centrifuged for 10 min at 15,000 x g. A 1.9-ml amount of the supernatant was stirred while 0.38 ml of n-butanol was slowly added over a 15-min period. The stirring was continued for a further 30 min. The mixture was centrifuged for 30 min at 38,000 x g, and the aqueous layer was taken for enzyme analysis. All procedures were carried out at 0 C. In method three, a modification of the method of Morton (9), the tissue was also stored for at least 24 h at -20 C in 0.05 M Tris, ph 7.0. After thawing, the tissues was minced with scissors and homogenized in 2 ml of 0.05 M Tris, ph 7.0, in a tissue grinder. The homogepate was stirred and 1 ml of n-butanol

3 882 YOLTON AND SAVAGE was slowly added over a 10-min period. The stirring was continued for a further 20 min. The mixture was centrifuged at 18,000 x g for 10 min, and the aqueous layer was taken for analysis. Here all procedures were done at room temperature except for the centrifugation which was done at 0 C. In all three procedures, the protein concentration was determined by the method of Lowry et al. (7) with bovine serum albumin as the standard. Alkaline phosphatase assay. Alkaline phosphatase activity was assayed with p-nitrophenol phosphate as substrate (2). The reaction mixture contained 54 mm MgCl2, 2.4 mm p-nitrophenol phosphate, and 85 mm Tris, ph 9.0. The reaction was started with the addition of 0.1 ml of duodenal homogenate or butanol extract, depending upon which procedure was used to prepare the tissue for enzymatic assay. (See the footnotes to the tables for specific information). The mixture was incubated at 37 C. The results were read at 415 nm on a Beckman DU spectrophotometer with or without a Gilford multiple sample absorbance recorder. Acrylamide gel disc electrophoresis. Electrophoresis was carried out in a Canalco disc electrophoresis apparatus using the formulations and procedures for making and running a 7% standard running gel at ph 9.5 with a stacking gel as outlined in their instruction manual (4). The running gel was polymerized first, then the stacking gel, and finally the sample gel. Extracts of duodenal tissue from CD-1 germfree, conventionalized, and SPF mice, prepared using method three, were used for electrophoresis. With extracts from germfree mice, 0.5 ml of the extract was mixed with 0.35 ml of the sample gel used for dilute protein solutions (4); two-tenths milliliter of the mixture was polymerized on the stacking gel. With extracts from conventionalized and SPF mice, 0.12 ml of the extract was mixed with 0.20 ml of the sample gel used for very dilute protein solutions (4); two-tenths milliliter of this mixture was polymerized on the stacking gel. The samples were electrophoresed for 70 min at 3 ma per tube. The location of alkaline phosphatase activity was determined using the method of Smith et al. (22), but with a-naphthyl phosphate as the substrate. The gels were incubated with the staining solution for 30 min. After staining, the gels were stored in 7% acetic acid. Densitometric scans of the gels were made using a Gilford linear transport attachment on a Beckman DU spectrophotometer with a Gilford recorder. The scanner speed was 2.0 cm/min and the chart speed was 1.25 mm/s. The wavelength was 600 nm. Statistics. The means are arithmetic. Significance of any difference between mean values was determined using the Mann-Whitney U test (20). RESULTS Monoassociation of germfree mice with lactobacilli. Germfree mice were monoassociated with lactobacilli in several different experiments. In previously described experiments (25), Ha/ICR germfree mice were associated with a Lactobacillus strain freshly isolated APPL. ENVIRON. MICROBIOL. from the stomach of an SPF Ha/ICR mouse. The lactobacilli colonized the stomachs and intestines of the monoassociated mice in the same pattern as they colonized those areas in the SPF mice. Further, a layer of lactobacilli was found associated with the nonsecreting epithelium of the stomachs of the monoassociated mice (25) similar to the layer found in the stomachs of SPF mice (15). Three years after these first experiments, Ha/ICR germfree mice were again monoassociated with this strain oflactobacillus. This time, the microbe had been revived from a lyophilized culture. Again, it colonized the animals as indicated by the high population levels estimated to be present in the stomachs (Table 1). CD-1 germfree mice also were associated with the Lactobacillus sp. from the lyophilized culture. The organisms established in high numbers throughout the gastrointestinal tract of these animals (Table 1). After 7 days of exposure of the mice to the bacteria, the number of lactobacilli per gram of tissue ranged from 10" to 108. In frozen sections, lactobacilli also could be seen layered on the nonsecreting epithelium in the stomachs of these CD-1 mice. CD-1 germfree mice were monoassociated as well with a Lactobacillus sp. isolated from the gastrointestinal tract of an SPF CD-1 mouse. Population estimates were made at 1 and at 5 weeks after the animals were exposed to the bacteria. At 1 week, the population levels of lactobacilli estimated to be present in the gastrointestinal tracts of the monoassociated animals were 1 to 2 logs lower than the levels in corresponding areas of the tracts of SPF mice (Table 1) (25). After 5 weeks of exposure to the bacteria, however, the levels in the tracts of the monoassociated mice corresponded to the levels of lactobacilli found in SPF CD-1 mice. Analysis of frozen sections of the stomachs of the monoassociated animals substantiated the culture results. At 1 week the lactobacillus layer on the nonsecreting epithelium was sparse and and irregular, whereas at 5 weeks the layer was thick and continuous. Monoassociation of germfree mice with bacteroides. CD-1 germfree mice were monoassociated with a Bacteroides sp. isolated from a CD-1 SPF mouse. Population estimates were made 7 days after the animals were exposed to the bacteria. This Bacteroides sp. established at high levels throughout the gastrointestinal tract of the gnotobiotic mice (Table 2). In SPF mice, the bacteroides localize in the large bowel and are found only in low numbers in the stomach and duodenum (18, 19). The estimated high levels of 10" to 108 bacteroides per gram of stomach or duodenal tissue found in the monoasso-

4 VOL. 31, 1976 INDIGENOUS GASTROINTESTINAL MICROORGANISMS 883 TABLE 1. Population estimates of lactobacilli in the digestive tract of monoassociated exgermfree mice Micea Gastrointestinal segment culturedb Kind No. Stomach Duodenum Jejunum Ileum Cecum Colon Ha/ICRc 6 8d NDe ND ND ND ND CD ND 7 (6-8) ND 8 (8-9) ND CD (3-7) 6 (<3-6) ND 6 (5-7) 7 6 (6-7) CD-lh 5 8 (6-8) 6 (6-7) ND 8 (6-8) 8 8 (7-8) a All males, 5 to 12 weeks old. b Cultured anaerobically (18) at 37 C on 1OA medium (18). c Cultured 1 week after the germfree mice were exposed to a Lactobacillus sp. indigenous to Ha/ICR specific pathogen-free mice; tissue contents gently removed before homogenization. d Median and range (in parentheses) of log10 of the numbers of bacteria per gram of fresh tissue. e ND, Not done. f Cultured 1 week after the germfree mice were exposed to a Lactobacillus sp. indigenous to Ha/ICR specific pathogen-free mice. " Cultured 1 week after the germfree mice were exposed to a Lactobacillus sp. indigenous to CD-1 specific pathogen-free mice. h Cultured 5 weeks after the germfree mice were exposed to alactobacillus sp. indigenous to CD-1 specific pathogen-free mice. TABLE 2. Population estimates ofbacteroides and coliforms in the digestive tract of monoassociated CD-1 exgermfree mice Micea Gastrointestinal segment culturedb Type No. Stomach Duodenum Ileum Cecum Colon Monoassociated bacteroidesc Monoassociated coliforms, (3-8)d 6 (6-7) 6 (3-7) 6 (4-6) 7 (7-8) (8-9) 8 (7-8) a All CD-1 males; 5 to 8 weeks old. b Bacteroides cultured anaerobically (18) at 37 C on -S medium (19); coliforms cultured aerobically at 37 C on T7T medium (18). c Cultured 7 days after the germfree mice were exposed to a Bacteroides sp. indigenous to CD-1 specific pathogen-free mice. d Median and range (in parentheses) of log,,, of the numbers of bacteria per gram of fresh tissue. e Cultured 7 days after the germfree mice were exposed to a coliform strain indigenous to CD-1 specific pathogen-free mice. TABLE 3. Differences in levels of alkaline phosphatase activity in the duodenums of CD-1 specific pathogen-free (SPF) mice killed in the morning or at night Micea Sp act0 SPFc SPFd a Individual mice assayed; all males, 5 to 8 weeks old ḃ Homogenate prepared using method one; micromoles of protein at 37 C. e Mice killed at 11 a.m. d Mice killed at 9 p.m. ciated mice contrast sharply with this normal localization pattern. Bacteroides, even though they are found in large numbers in the lower gastrointestinal tract, do not form layers on any epithelium (15, 16). When colons of the monoassociated mice were examined by using the frozen section technique, bacteria were seen only in the lumen; no layer on the epithelium was evident. Monoassociation of germfree mice with coliforms. CD-1 germfree mice were monoassociated with a coliform strain indigenous to CD-1 SPF mice. The coliforms established in high numbers throughout the gastrointestinal tracts of the monoassociated mice (Table 2). In all areas of the tract, the coliforms were found in much higher numbers than in the corresponding portions of the tracts of SPF mice (18). Influence of time of sampling on duodenal alkaline phosphatase activity. Alkaline phosphatase activity was assayed in duodenal homogenates (method one) from CD-1 SPF mice killed at 11 a.m. and 9 p.m. (Table 3). The mean activity level in the mice killed at 9 p.m. was more than twofold higher than the activity found in the mice at 11 a.m. Therefore, the levels differed substantially, depending upon the time of day the animals were sacrificed. As a consequence, in all our experiments, the mice

5 884 YOLTON AND SAVAGE were always killed during the same period between 10 a.m. and 2 p.m. The levels of alkaline phosphatase activity and some other enzymatic activities rise and fall in circadian rhythms in the mucosa of the small bowels of rats (13). In those animals the levels of alkaline phosphatase activity are highest at midnight and lowest at noon (13). Our findings suggest a similar circadian fluctuation in the activity of the enzyme in the duodenums of mice. Effect of a germfree environment on the alkaline phosphatase activity of SPF mice. The effect of the germfree environment on SPF alkaline phosphatase activity was determined in Ha/ICR SPF mice housed under SPF conditions from birth to 3 weeks of age and then housed under conditions usually used for germfree animals for 3 weeks. The mean level of alkaline phosphatase activity in duodenal homogenates (method one) from 11 such animals was 2.0 units. This compares well with the mean level of activity of 2.8 units (Table 3) found in SPF mice living in the open animal colony (25). Thus, under the conditions of these experiments, the environment in an isolator and sterilized food do not influence duodenal alkaline phosphatase activity in mice. Alkaline phosphatase activity in CD-1 SPF and germfree mice. Alkaline phosphatase activity was measured in butanol extracts of duodenal tissue (method two) from CD-1 SPF and germfree mice (Table 4). Similar to results with TABLE 4. Alkaline phosphatase activity in the duodenums of CD-I specific pathogen-free mice, germfree mice, and ex-germfree mice monoassociated with a Lactobacillus sp. isolated from CD-I SPF mice Mice' Sp actb Germfree Monoassociated lactoba " cillus' Monoassociated lactoba f cillus' Specific pathogen-free " Individual mice assayed; all males, 5 to 12 weeks old. b Extract prepared using method two; micromoles of protein at 37 C. ' Associated for 1 week with a Lactobacillus sp. indigenous to CD-1 SPF mice. " Not significantly different from value for germfree (P > 0.05,f. ' Associated for 5 weeks with the Lactobacillus sp. indigenous to CD-1 SPF mice. f Significantly different from value for germfree (P < 0.05). APPL. ENVIRON. MICROBIOL. Ha/ICR mice (14, 25), the level of alkaline phosphatase activity in germfree CD-1 mice was many-fold higher than the level in SPF animals. Alkaline phosphatase activity in germfree mice monoassociated with lactobacilli. CD-1 germfree mice were monoassicated with a freshly isolated Lactobacillus sp. indigenous to CD-1 SPF mice. One or 5 weeks after the initial exposure to the bacteria, duodenal tissues from the monoassociated mice were assayed for alkaline phosphatase activity after butanol extraction (niethod two) (Table 4). Although not significantly different from values in the germfree controls at 1 week, the activity levels had declined significantly by 5 weeks after the animals had been monoassociated. As with Ha/ ICR mice monoassociated with a Lactobacillus sp. isolated from Ha/ICR SPF mice (25), the level of activity in these CD-1 gnotobiotes was intermediate between the germfree and SPF levels. CD-1 germfree mice also were associated with the Lactobacillus sp. indigenous to Ha/ ICR mice. As noted above (Table 1), a lyophilized culture served as the source of microorganisms. Method two was used in extracting the duodenal tissues of these gnotobiotes when they had been associated with the lactobacilli for 7 days. Such extracts were made also of the duodena of germfree and SPF controls. The level of alkaline phosphatase activity in the extracts from the gnotobiotes was significantly higher than the levels detected in extracts from the germfree controls (Table 5). This is the only case in which duodenal alkaline phosphatase activity was stimulated in gnotobiotic mice monoassociated with any microorganism. This surprising result led us to reexamine the effect of this Lactobacillus sp. isolated from Ha/ICR SPF mice on alkaline phosphatase activity in germfree Ha/ICR mice. In earlier experiments (25), the microbe had been freshly isolated from the stomachs of the SPF animals at the time it was associated with the germfree animals. In the experiment just described with CD-1 mice, the culture had been revived from a 3-year-old lyophilized specimen. Therefore, we decided to check the effect of this revived culture on alkaline phosphatase activity in mice of the same strain from which it was isolated. Accordingly, germfree Ha/ICR mice were monoassociated with it (Table 1). This time, however, alkaline phosphatase activity in the duodenal tissue was assayed in butanol extracts prepared by method three. Such extracts were prepared from SPF and germfree Ha/ICR mice, as well as from gnotobiotes monoassociated for 7 days with the lactobacilli (Table 6). Even

6 VOL. 31, 1976 TABLE 5. Alkaline phosphatase activity in the duodenums ofcd-i specific pathogen-free, germfree, and exgermfree mice monoassociated with a Lactobacillus sp. isolated from specific pathogen-free HalICR mice Mice Sp act Germfree Monoassociated lactoba " cillus' Specific pathogen-free " Individual mice assayed; all males, 5 to 8 weeks old ḃ Extract prepared using method two; micromoles protein. ' Associated for 1 week with a Lactobacillus sp. indigenous to specific pathogen-free Ha/ICR mice. Microbe revived from a 3-year-old lyophilized culture. d Significantly different from germfree (P < 0.05). TABLE 6. Alkaline phosphatase activity in the duodenums of HalICR specific pathogen-free mice, germfree mice, and exgermfree mice monoassociated with a Lactobacillus sp. Mice' Sp act' Germfree Monoassociated lac tobacillus' Specific pathogen free "1 Individual mice were assayed; all males, 5 to 8 weeks old. b Extract prepared using method three; micromoles of protein. ' Associated for 1 week with a Lactobacillus sp. indigenous to Ha/ICR mice. Microbe revived from a 3-year-old lyophilized culture. INDIGENOUS GASTROINTESTINAL MICROORGANISMS 885 though the method of preparing the extracts differed from procedures used earlier, the levels of alkaline phosphatase activity in the germfree mice were again significantly higher than the levels found in the SPF mice. In this case, however, the lactobacilli neither stimulated nor reduced the high level of alkaline phosphatase activity found in the germfree animals. Alkaline phosphatase activity in germfree CD-1 mice monoassociated with bacteroides. CD-1 germfree mice were monoassociated with a Bacteroides sp. indigenous to CD-1 SPF mice. Alkaline phosphatase activity in duodenal homogenates (method one) was assayed 7 days after the germifree mice had been exposed to the bacteria. Similarly, to freshly isolated lactobacilli (Table 4) (25), the bacteroides reduced the germfree level of alkaline phosphatase activity but not completely to the SPF level (Table 7). A similar intermediate value of alkaline phosphatase activity was found when Ha/ICR germfree mice were monoassociated with a Bacteroides sp. indigenous to Ha/ICR SPF mice (14). Alkaline phosphatase activity in germfree mice monoassociated with coliforms. Alkaline phosphatase activity (method one) was assayed in CD-1 germfree mice monoassociated for 7 days with a coliform indigenous to CD-1 SPF mice (Table 7). The level of alkaline phosphatase activity in these gnotobiotes was not significantly different from the germfree level. Acrylamide gel electrophoresis of duodenal extracts from germfree, conventionalized, and SPF mice. Butanol extracts of homogenized duodenal mucosa from germfree, convenalized, and SPF CD-1 mice were electrophoresed in acrylamide gel. All material in the extracts did not uniformly enter the gels. As a consequence, in most gels, some activity remained at or near the top as is evidenced by activity peaks detected by densitometer scanning at the cathodal end. (See non-numbered peaks to the extreme right in Fig. 1A, B, C.) Extracts produced by method three were more effective in entering the gels than those produced by method two. In such extracts derived from SPF and conventionalized mice three bands (labeled I, II TABLE 7. Alkaline phosphatase activity in the duodenums of CD-I specific pathogen-free (SPF), germfree, and exgermfree mice monoassociated with a coliform orbacteroides sp. isolated from CD-I SPF mice Mice Sp act' Germfree Monoassociated bac " teroides' Monoassociated coli ' form' Specific pathogen free " Individual mice assayed; all males, 5 to 8 weeks old ḃhomogenate prepared using method one; micromoles protein. ' Associated for 7 days with a Bacteroides sp. indigenous to the SPF mice. d Significantly different from germfree (P < 0.05). Associated for 7 days with a coliform strain indigenous to the SPF mice. f Not significantly different from germfree (P > 0.05).

7 886 YOLTON AND SAVAGE A Specif ic Pathogen- Free I fm B Conventionalized I Ium c Germfree FIG. 1. Densitometric scans of polyacrylamide gels stained after disc electrophoresis for alkaline phosphatase activity. All samples were taken from butanol extracts of duodenal tissue. The anodal end of the gels is to the left ofeach panel. The direction of run is from right to left. (A) specific pathogen-free mouse; (B) conventionalized exgermfree mouse; (C) germfree mouse. and III, starting from the anodal end of the gel) could be detected anodally from the activity at the top of the gels (Fig. 1A, B). Only one major and one minor band (labeled I and III, starting at the anodal end of the gel) could be detected anodally from the activity at the top of the gels in those prepared with extracts from germfree animals (Fig. 1C). Careful analysis of the scans revealed, however, that the gels prepared with the extracts from germfree mice probably gave three bands anodally from the activity at the top. These bands undoubtedly coincide with the bands in the gels prepared with extracts from SPF and conventionalized mice. The problem in differentiating the bands arises from the apparent amount of activity in each band. In the gels showing the activity from SPF mice, the most anodal band (I) had the least activity. Bands II and III did not distinctly separate; band II appeared as an irregular shoulder to band III (Fig. 1A). In the gels prepared with extracts from conventionalized mice, all three bands usually had strong activity (Fig. 1B). However, the proportion of activity in each of the three bands varied from mouse to mouse. In some, the activity was almost the same in all three bands. In others, band II appeared as a shoulder on band III or band III appeared as a shoulder on band II. By contrast, in gels prepared with extracts from germfree mice, band I, although always present, had only minor activity, whereas band III was distinct and always densely stained indicating strong activity (Fig. 1C). Although band II was not distinct in any I Im APPL. ENVIRON. MICROBIOL. preparation from germfree animals, we surmise that band II activity is largely obscured by band III which was always asymmetrically arranged with more activity on the leading than the trailing edge. The influence on these results of the material in the extracts that failed to enter the gels is unknown at this time. The results reported were consistently obtained, and compared well with similar findings of others (8, 12). Therefore, we believe they are valuable in revealing that germfree mice have in their duodenums a large amount of a particular molecular species of alkaline phosphatase that is found only at much lower levels in the duodenums of mice with indigenous microbiotas. DISCUSSION The effect of the indigenous gastrointestinal microbial flora on alkaline phosphatase activity has been studied in models in which germfree animals were monoassociated with individual microbial types. Three different types of microorganisms were selected to be tested for their ability to reduce alkaline phosphatase activity in monoassociated gnotobiotic mice. One of the microbes, a Lactobacillus sp., is known to colonize to high population levels the keratinized epithelium of the stomach and to be present in significant numbers throughout the gastrointestinal canal in conventionally colonized mice (15, 18). Another, a Bacteroides sp., although known to colonize to significant levels the large bowels of conventionally colonized mice (18, 19), is not known to associate with any epithelium (15, 16). The third, a coliform, colonizes in low numbers predominantly in the large bowel of conventional animals (18). These three bacterial types, then, constitute a representative sample of the various indigenous microbes inhabiting the alimentary canal of conventionally colonized mice. Both the freshly isolated Lactobacillus sp. and the Bacteroides sp. depressed alkaline phosphatase activity, but only to levels intermediate between the germfree and SPF values. A freshly isolated coliform species did not affect alkaline phosphatase activity. All three types of microorganisms were present, however, at about the same population levels throughout the gastrointestinal tracts of the monoassociated animals. Importantly, in conventionally colonized mice, of the three microbial types tested only lactobacilli are found normally at large population levels in the duodenum. In contrast to colonizing to high population levels throughout the tracts of monoassociated ani-

8 VOL. 31, 1976 mals, the bacteroides and coliform species tested normally colonize only the large bowels of conventionally reared mice. A question remains, therefore, whether or not bacteroides have any influence on levels of alkaline phosphatase activity under normal circumstances. By contrast, it is apparent from these findings that indigenous coliforms, even at abnormally high population levels, do not influence the enzymatic activity. These findings argue that certain species of indigenous bacteria are intrinscially more important for "normal" functioning of the gastrointestinal tract than are others. Lactobacilli and bacteroides are both strict anaerobes in the metabolic sense of the term, whereas coliforms are facultative bacteria. Thus, these findings are consistent with our view (14) that strict anaerobes are most important in influencing physiological properties in the bowel. This ability to influence the enzymatic activity is dependent upon special properties of these microbes. One strain of Lactobacillus revived from lyophilization did not change the levels of alkaline phosphatase in monoassociated animals and under one set of conditions apparently even stimulated the levels. Nevertheless, these bacteria did form a layer on the nonsecreting gastric mucosa of the stomachs of the monoassociated animals. Thus, they had retained the elements necessary for attachment to the epithelium (D. C. Savage, R. W. Schaedler, and R. Dubos, Bacteriol. Proc., p. 67, 1967) and to colonize the tract to high population levels, but could no longer interact with the mouse to reduce alkaline phosphatase activity. Freshly isolated strains of lactobacilli can alter alkaline phosphatase activity as well as attach to the gastric epithelium. Therefore, the ability to alter the enzymatic level is due to a property that was lost by the lactobacilli revived from the lyophilized culture. Furthermore, as noted with the animals monoassociated with coliforms, high bacterial populations, even of lactobacilli, are not sufficient to depress the enzymatic activity if the microbes do not have special properties as well as the ability to colonize the tract. The mechanism by which certain kinds of bacteria reduce the levels of alkaline phosphatase activity in associated exgermfree mice involves either a change in the enzyme molecules themselves or a change in the numbers of enzyme molecules. The small intestine of the mouse is said to contain at least two alkaline phosphatases distinguishable by their catalytic properties (8). Purified material from rat intestine contains three isozymes, separable by electrophoresis in polyacrylamide gels (12). We were able to distinguish at least three bands INDIGENOUS GASTROINTESTINAL MICROORGANISMS 887 with alkaline phosphatase activity in the gels after electrophoresing duodenal extracts from SPF and conventionalized exgermfree mice. Two of the bands may be analogous to the known alkaline phosphatases in mice (8). The relationship of the third band of activity to the two known isozymes is obscure. Our interest at this time is focused on the shift of enzymatic activity from the dominant slow-moving band seen in gels prepared from extracts from germfree mice to faster-moving bands seen in gels prepared from extracts from conventionalized mice. This phenomenon indicates that the bacteria may be inducing changes in the enzyme molecules. Heterogeneity of alkaline phosphatase is thought to be associated with a glycoprotein, the carbohydrate moiety of which may be a sialic acid (22). Human intestinal tissue contains alkaline phosphatase with an attached carbohydrate moiety which can be removed with neuraminidase. When this putative sialic acid residue is removed, some of the activity in the treated sample is then found in electrophoresis in a slower-moving band not seen in the untreated sample (22). Thus, one possible explanation of the differences in alkaline phosphatase in germfree and conventional mice as detected by electrophoresis is that most of the alkaline phosphatase in the germfree mice does not contain a sialic acid and, therefore, runs as a slowmoving band. In this hypothesis, then, the flora would stimulate attachment of sialic acid to the protein enzyme, thereby causing it to run faster during gel electrophoresis. Thus, the intestinal microbial flora may affect not only quantitatively, but also qualitatively the alkaline phosphatase in the mouse intestinal mucosa. It is possible that the levels of alkaline phosphatase activity are the same in the duodenal mucosa of germfree and SPF mice and gnotobiotic animals associated with lactobacilli and bacteroides, and simply assay differently because inhibitors of the activity appear in the extracts from the latter two types of mice. Presumably, such inhibitors would have to derive in the lumen of the duodenum in vivo from microbes or their action on the animal's food. For such conditions to exist and be consonant with our findings, the inhibitors would have to be made by some microbes, but not all (e.g., some Lactobacillus and Escherichia coli strains); remain in the extracts even though the lumenal contents were washed from the duodenums before they were homogenized; and pass into the assay via all three extraction procedures used. We believe that these circumstances are possible, but taken together, not too likely. Therefore, our general working hypoth-

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