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Infection and Immunity, February 2007, p. 932-940, Vol. 75, No. 2
0019-9567/07/$08.00+0     doi:10.1128/IAI.00736-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Reduced Phase Switch Capacity and Functional Adhesin Expression of Type 1-Fimbriated Escherichia coli from Immunoglobulin A-Deficient Individuals

Forough L. Nowrouzian,^1* Vanda Friman,² Ingegerd Adlerberth,¹ and Agnes E. Wold¹

Department of Clinical Bacteriology,¹ Department of Infectious Diseases, Göteborg University, Göteborg, Sweden²

Received 8 May 2006/ Returned for modification 12 September 2006/ Accepted 2 November 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
The mannose-specific adhesin of type 1 fimbriae is the most common adhesin in Escherichia coli. One receptor for this adhesin is the carbohydrate chains of secretory immunoglobulin A (S-IgA), and intestinal E. coli from IgA-deficient individuals has a reduced capacity to adhere to mannose-containing receptors. Here, we investigated the expression of the mannose-specific adhesin and its capacity to switch to the fimbriated phenotype in colonic resident and transient E. coli strains isolated from control (n = 16) and IgA-deficient (n = 17) persons. Resident E. coli strains from IgA-deficient individuals displayed weaker mannose-specific adherence to colonic cells than resident strains from control individuals (21 versus 44 bacteria/cell, P = 0.0009) due to three mechanisms: a lower carriage rate of the fimH gene (90% versus 97%, not significant), more frequent failure to switch on the fim genes (30% versus 6%, P = 0.02), and the reduced adhesive potential of fimH⁺ isolates capable of phase switch (26 versus 46 bacteria/cell, P = 0.02). On the other hand, resident strains from IgA-deficient individuals displayed stronger mannose-resistant adherence than resident strains from control individuals (P = 0.04) and transient strains from IgA-deficient individuals (P = 0.01). The presence of S-IgA appears to favor the establishment of E. coli clones which readily express mannose-specific adhesins in the bowel microbiota.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Type 1 fimbriae are the most common adherence structures in Escherichia coli and are also found in other enterobacterial species (9, 11). Type 1 fimbriae carry adhesins that recognize terminal mannose residues in the Man1-3(Man1-6)Manß conformation (10). This trisaccharide is exposed on many glycoproteins, and type 1 fimbriae mediate adherence to, e.g., human small and large intestinal (3) and urinary tract (30) epithelial cells. The adherence is abolished in the presence of mannose and hence is termed mannose sensitive (MS). The role of the E. coli MS adhesin in virulence has been debated, but it may play a role in urinary tract infection (7, 18, 28). Other E. coli adhesins, including those associated with P and S fimbriae, confer mannose-resistant (MR) adherence to uroepithelial and colonic epithelial cells (3, 30, 45). MR adhesins are well-known virulence factors in urinary tract infection, septicemia, and meningitis (23, 27, 37). Furthermore, P fimbriae seem to facilitate colonization of the human bowel. Thus, strains that persist in the human intestinal microbiota (so-called resident strains) are more often P fimbriated and display MR adherence to colonic epithelial cells than strains that appear only transiently in the microbiota (4, 32, 33, 35, 43).

Bacteria can switch between a fimbriated and a nonfimbriated state, a process termed phase variation (13). Phase variation of type 1 fimbriae is mediated by a 314-bp invertible DNA element (fim switch) which contains the promoter for fimA (2) and whose position is regulated by two site-specific recombinases, FimB and FimE (26). Several environmental factors influence phase switch of type 1 fimbriae, including temperature and osmolarity (16, 36, 39). To maximize type 1 fimbriation, E. coli strains are usually cultured in static broth (7), in which case the hydrophobic fimbriae allow the bacteria to form a pellicle on the liquid-air interface and get full access to atmospheric oxygen. With successive passages in static broth, the proportion of fimbriated bacteria therefore increases (9, 36).

The normal niche for E. coli is the bowel microbiota of humans and animals (8). The gut contents are a rich source of secretory immunoglobulin A (S-IgA), which is produced at a rate of 2 to 5 g per day in an adult human being (1). S-IgA is heavily glycosylated, and many of its carbohydrate chains terminate with mannose and act as receptors for the MS adhesin of type 1-fimbriated E. coli (44). Thus, independent of the specificity of the S-IgA, type 1-fimbriated E. coli will interact with S-IgA antibodies through a lectin-carbohydrate interaction (44). Our previous findings indicate that the lectin-carbohydrate interaction is the main mechanism for the agglutinating activity of S-IgA against type 1-fimbriated E. coli in vitro (44).

About 1 individual in 600 lacks IgA in both serum and secretions but has normal levels of the other immunoglobulin isotypes (19). Approximately one-third of IgA-deficient individuals suffer from recurrent respiratory tract infections (5), but most are healthy and their IgA deficiencies are discovered accidentally, e.g., at blood donor screening. We have previously shown that E. coli isolated from IgA-deficient individuals displays reduced mannose-specific adherence to colonic epithelial cells in comparison with E. coli from age-matched controls (14). Two factors contributed to this effect. First, E. coli from IgA-deficient individuals carried the fim operon less often than did E. coli from control individuals. Second, fimH⁺ E. coli from IgA-deficient individuals displayed reduced mannose-specific adherence in comparison with fimH⁺ E. coli from control individuals (14).

In the present study we decided to further explore the differences in MS adhesin expression between IgA-deficient and control individuals. One aim was to investigate whether differences between E. coli strains obtained from IgA-deficient and control individuals were foremost evident among resident or transient strains. The second aim was to explore whether differences in capacity to switch to a fimbriated phenotype could underlie the difference in MS adherence capacity between E. coli strains from IgA-deficient and control individuals. For this purpose, the rectal microbiota of IgA-deficient and control individuals was sampled monthly over a period of 6 months. E. coli strains were isolated and characterized as resident (i.e., present in consecutive samples) or transient (i.e., present only on a single sampling occasion) in the microbiota. Resident and transient strains from IgA-deficient and control individuals were then compared with respect to possession of fimH and other adhesin genes, capacity to adhere via MS or MR mechanisms to the colonic cell line HT-29, and capacity to switch the type 1-fimbrial gene promoter to the "on" position.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Subjects. Seventeen individuals (nine males, eight females) with selective IgA deficiency were included in the study (median age, 43 years; range, 18 to 68 years). IgA deficiency was defined as a serum IgA level of <0.05 g/liter in the presence of IgM at a level higher than 0.5 g/liter and IgG at a level higher than 7 g/liter, the lower limits of the normal ranges. They also had the following normal levels of the IgG subclasses (in grams/liter): IgG1, >4.22; IgG2, >1.17; IgG3, >0.41; and IgG4, >0.01 (19, 38).

Sixteen individuals (seven males, nine females) with normal levels of serum immunoglobulins served as a control group (median age, 46 years; range, 28 to 73 years). None of the individuals included had consumed antibiotics during the 3 months preceding the study. The study was approved by the Medical Ethics Committee of Göteborg University, Göteborg, Sweden.

Sampling of rectal microbiota and species identification. Rectal swabs were obtained monthly over a period of 6 months. The swabs were transported in Stuart's transportation medium to the laboratory, where they were streaked in a three-step manner on Drigalski agar, a medium selective for Enterobacteriaceae (41). After aerobic culture overnight at 37°C, the last three free-lying colonies were picked, which gives a 97% probability of including the dominant E. coli strain (31). After subculture on Drigalski agar for purity, the isolates were identified to the species level using API 20E (API Systems SA, La Balme Les Grottes, Montalieu-Vercieu, France), and those identified as E. coli were selected for study.

Strain typing by RAPD. E. coli isolates were typed to the strain level by random amplified polymorphic DNA (RAPD) (33, 34). In brief, a small amount of bacteria from an overnight culture was mixed with 6.0 µM of the primer GTGATCGCAG and 25 µl HotStarTaq master mix (QIAGEN, Spånga, Sweden). The PCR started with a 15-min incubation step at 95°C to activate the polymerase and continued with the following temperature profile: 94°C for 45 s, 30°C for 120 s, and 72°C for 60 s for four cycles; followed by 94°C for 5 s, 36°C for 30 s, and 72°C for 30 s for 26 cycles; with the extension step being increased by 1 s for every new cycle. The reaction was terminated at 72°C for 10 min and cooled to 4°C. The PCR products were separated on 8% ready-made Tris-glycine gels and visualized by silver staining (34).

All E. coli isolates from one individual were assayed together, and their PCR products were, when possible, separated on the same gel. Two isolates with identical profiles from the same individual were considered to belong to the same strain. Isolates were not compared between individuals.

Multiplex PCR for identification of adhesin genes in E. coli strains. The carriage of the fimH gene (the MS adhesin of type 1 fimbriae) was analyzed by PCR using previously published primers (25) (Table 1). In addition, each strain was characterized by multiplex PCR with respect to carriage of the following virulence genes: papC (P fimbriae); the class I, II, and III varieties of the P-fimbrial adhesin gene papG (recognizing subtle differences in receptor conformation); sfaD and sfaE (S fimbriae, F1C fimbriae); and draA (Dr hemagglutinin) (32). The primers used are listed in Table 1. Bacteria from colonies grown on tryptic soy agar (TSA) were added to a mixture containing HotStarTaq master mix (QIAGEN) and 0.45 µM of each primer pair in a final volume of 50 µl. The PCR program was started with an initial heat activation step for the Taq polymerase (95°C for 15 min). Thereafter, the PCR was run as described previously (32, 33). PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide.

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TABLE 1. Primers used for detection of E. coli virulence factor genes and the phase switch position

Determination of the E. coli fim switch position (on/off). The orientation of the invertible fim switch element was determined by PCR amplification followed by restriction cleavage. When the element is in the "on" position, the promoter is active and the fimbrial genes are transcribed, leading to formation of functional type 1 fimbriae with mannose-specific adhesins. When the element is in the "off" position, the fimbrial genes are not transcribed and the bacteria are nonfimbriated. A culture of a bacterial strain generally contains a mixture of fimbriated and nonfimbriated cells.

To determine the position of the promoter in fimH⁺ strains, a 559-bp fragment containing the fimA promoter was generated by PCR and cleaved by endonuclease enzyme HinfI (40). Since the position of the cutting site depends on the position of the invertible fim switch element, bacteria whose fim promoter is in the "on" position give rise to different cleavage fragments than bacteria whose promoter is in the "off" position.

E. coli strains were grown aerobically on TSA at 37°C overnight and thereafter passaged three times in Luria broth. Bacteria were harvested by centrifugation at 1,500 rpm for 20 min at 4°C, washed in phosphate-buffered saline (PBS) at room temperature, and thereafter centrifuged at 1,500 rpm for 4 min at 4°C. The bacterial pellet was suspended in 500 µl PBS and incubated for 12 min at 95°C to release bacterial DNA. The suspension was centrifuged at 13,400 rpm for 5 min, and the supernatant was frozen at –20°C until used as a template for PCR.

A previously described PCR assay (40) was used with some modifications. The primers used are shown in Table 1. The PCR mixture was prepared in a total volume of 50 µl consisting of 25 µl HotStarTaq master mix (QIAGEN), a 0.2 µM concentration of each primer, and distilled water. The PCR program started with 15 min at 95°C for activation of Taq polymerase, followed by 30 cycles of 94°C for 1 min (denaturation), 61°C for 70 s (annealing), and 72°C for 70 s (extension). The program terminated with a 3-min final extension at 72°C and cooling to 4°C.

The PCR products were digested by HinfI (New England Biolabs, Hitchin, Hertfordshire, United Kingdom) according to the manufacturer's instructions, and the digested PCR products were separated electrophoretically on an agarose gel (32).

Adherence to the colonic cell line HT-29. Adherence to the colonic cell line HT-29 was tested after culture of bacteria in static Luria broth to promote type 1-fimbrial expression (9, 36). Preliminary experiments were performed to determine the number of passages in static Luria broth required for expression of type 1-fimbrial MS adhesins in fimH⁺ isolates. Six different fimH⁺ strains were cultured in duplicate on TSA plates overnight and thereafter passaged up to 10 times in static Luria broth. Adherence was assessed after growth on TSA and after 1, 3, 5, and 10 passages in static Luria broth. Three passages were selected as the standard procedure. Thus, all isolates of E. coli were passaged three times in static Luria broth to select for bacteria expressing the MS adhesin and thereafter assessed for adherence to the human colonic cell line HT-29 in the absence and presence of mannose (14, 45). Briefly, a mixture of 5 x 10⁸ bacteria, 5 x 10⁵ HT-29 cells, and Hanks' balanced salt solution with or without 1% (final concentration) of methyl--D-mannoside was incubated for 30 min at 4°C with end-over-end rotation. The cells were washed and fixed with formalin, and at least 40 epithelial cells were examined by interference contrast microscopy (x500 magnification) (Nikon Optiphot; Bergström Instruments AB, Göteborg, Sweden). The number of bacteria adhering to each cell was counted, and the average number of adherent bacteria per cell was calculated. In each experiment, all isolates from one IgA-deficient and one control individual were assayed, and the person examining adherence was blinded as to their identity. The MS adherence was calculated by subtracting the mean number of bacteria per cell adhering in the presence of methyl--D-mannoside (MR adherence) from the mean number of bacteria per cell adhering in the absence of methyl--D-mannoside (total adherence). The transformant E. coli strains 506 MS (type 1 fimbriated) and 506 P (adhesin negative) (17, 22) were included as controls in each adhesion experiment. The control strains were cultivated on TSA plates supplemented with 20 µg/ml chloramphenicol.

Statistical methods. Proportions were compared using Fisher's exact test. Adherence data were compared using the Mann-Whitney U test.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Identification of resident and transient E. coli strains. Figure 1 shows the RAPD profiles of the E. coli isolates obtained from an individual who carried four different E. coli strains over the 6-month study period. Strain A appeared in the first sample only, whereas strains B, C, and D were each present on two consecutive sampling occasions. Strains found in an individual on at least two sampling occasions were defined as resident. Strains isolated on a single sampling occasion were defined as transient, provided that this was not the first or last sampling occasion. Thus, strains B, C, and D were all defined as resident, while strain A could not be classified as either resident or transient. RAPD patterns were not compared between strains from different individuals.

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FIG. 1. RAPD patterns of 10 intestinal E. coli isolates obtained from a single individual over a period of 6 months. The subject carried four different strains during this period. Strain A appeared in the first sample only and was classified as neither resident nor transient, while strains B, C, and D were present on two sampling occasions and thus defined as resident strains. No E. coli was found in the sample obtained at 4 months.

Persons with normal IgA levels carried an average of 4.3 different E. coli strains during the 6-month period, while the corresponding figure for IgA-deficient individuals was 3.8 strains (P = 0.27). Of the 68 strains isolated from the 16 control individuals, 36 were defined as resident and 18 as transient, while 14 could not be classified. Of the 64 strains isolated from the 17 IgA-deficient individuals, 31 were resident and 17 were transient, while 16 were unclassified.

Adhesin gene carriage. The frequencies of adhesin genes were compared between resident and transient E. coli strains from control and IgA-deficient individuals (Table 2). The MS adhesin gene was somewhat more common among resident than transient strains in both controls and IgA-deficient individuals, but the differences were not significant. In IgA-deficient individuals, genes encoding P and S fimbriae tended to be more common in resident than in transient strains (Table 2).

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TABLE 2. Prevalence of adhesin genes in resident and transient intestinal E. coli strains from control and IgA-deficient individualsa

Mannose-sensitive adherence to HT-29 cells. Type 1-fimbrial expression is regulated by phase variation, and culture in static Luria broth enhances expression of MS adhesins (9). To determine the number of passages that were needed to drive fimH⁺ strains into expression of MS adhesins, six fimH⁺ strains were cultured on TSA, passaged 1, 3, 5, and 10 times in static Luria broth, and thereafter tested for adherence to HT-29 cells. As seen in Fig. 2, only one out of six strains showed any MS adherence after culture on TSA. MS adherence increased during the first three passages in four of the strains. Three of these strains continued to increase their expression of MS adhesins for up to 5 passages, and one strain for even up to 10 passages. Two strains failed to express any MS adhesins, even after 10 passages. Three passages were selected as a reasonable compromise between promoting adhesin expression and being practically feasible. Patterns of attachment of E. coli strains to the HT-29 cells are shown in Fig. 3.

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FIG. 2. Mannose-sensitive adherence to HT-29 cells of six fimH-positive E. coli strains after 0 (agar-grown bacteria), 1, 3, 5, and 10 serial passages in static Luria broth. For each strain, the mean value and the standard deviation (shown by I bars) for two parallel experiments are presented. Each symbol represents a certain E. coli strain.

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FIG. 3. Attachment of E. coli strains to cells of the HT-29 cell line. (A) A type 1-fimbriated E. coli strain. (B) An adhesin-negative control strain.

All E. coli isolates from IgA-deficient and control individuals were passaged three times in static Luria broth and thereafter tested for adherence to HT-29 colonic epithelial cells in the presence and absence of methyl--D-mannoside, which permitted subdivision of the isolates into those with MS or MR adherence. For each resident strain, the average MS and MR adherence of all isolates of that strain was calculated. Transient strains contributed a single isolate, or in some cases more than one isolate from a single sampling occasion. In the latter case, their MS and MR adherence was averaged. The average MS adherence levels of resident and transient strains from control and IgA-deficient individuals are shown in Fig. 4.

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FIG. 4. Mannose-sensitive adherence to HT-29 cells of 50 E. coli strains (35 resident and 15 transient) obtained from 16 healthy controls and 47 strains (31 resident and 16 transient) obtained from 17 IgA-deficient (IgA-d) individuals. Each circle represents the mean adherence value for all isolates belonging to one strain. The median values are represented by horizontal bars, and the groups were compared using the Mann-Whitney U test. Data are missing for one resident and three transient strains from control individuals and for one transient strain from an IgA-deficient individual.

As shown in the figure, the average MS adherence of resident strains from control individuals was twice as high as the MS adherence of resident strains from IgA-deficient individuals (P = 0.0009). The MS adherence of transient strains was also higher in strains from controls than in those from IgA-deficient individuals, but the difference was not significant (P = 0.24). In both control and IgA-deficient individuals, resident strains displayed higher MS adherence than transient strains, but the differences were not significant (P = 0.19 and P = 0.10, respectively). When strains from IgA-deficient and control individuals were analyzed together, resident strains displayed significantly higher MS adherence than transient strains (P = 0.03).

Resident strains may display increased MS adherence for either of two reasons. First, strains with an inherent tendency to express MS adhesins may be superior colonizers. Second, the capacity to adhere might increase progressively during persistence in the microbiota due to upregulation of fimbrial expression. In the latter case, resident strains would express more MS adhesins because they have, on average, spent longer time in the microbiota than transient strains. To examine these two possibilities, we calculated the MS adherence of the first and last isolates of resident strains carrying the fimH gene. fimH⁺ strains which were already present on the first sampling occasion were excluded, as their time of persistence in the microbiota was unknown. The result of this analysis is shown in Fig. 5. As is evident from the figure, fim⁺ strains resident in the microbiota of control individuals already had high MS adherence when they were first isolated, and their MS adherence increased only marginally over time (P = 0.30). Conversely, fimH⁺ strains resident in IgA-deficient individuals expressed lower MS adherence than the corresponding strains from control individuals at the outset (P = 0.02), and the difference remained (P = 0.04 for the last isolate) (Fig. 5). For transient fimH⁺ strains, the difference in MS adherence between those isolated from IgA-deficient and those isolated from control individuals was small (Fig. 5) and not significant (P = 0.60). We concluded that strains capable of long-term persistence in individuals with S-IgA in their secretions had an inherently strong capacity to express MS adhesins.

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FIG. 5. Mannose-sensitive adherence to HT-29 cells of fimH-positive E. coli strains (28 resident, 13 transient) isolated from 16 healthy controls and 35 strains (22 resident, 13 transient) from 22 IgA-deficient (IgA-d) individuals. The adherence of the first and last isolate of resident strains and of each transient strain is represented by a circle. The median adherence for each group of strains is indicated by a horizontal bar, and the groups were compared using the Mann-Whitney U test.

Orientation of the fim switch element in E. coli strains from IgA-deficient and control individuals. The weak capacity for MS adherence of fimH⁺ strains from IgA-deficient individuals could be due to a decreased capacity of the strains to switch to the fimbriated phase, a reduced binding capacity of the adhesins, or both. To test this, we examined the capacity of strains from IgA-deficient and control individuals to switch to the fimbriated phase after three passages in static broth. Bacterial DNA was obtained from the culture, and the position of the invertible element containing the fim promoter was examined by PCR followed by restriction cleavage. Figure 6 shows an example of four strains analyzed by this method. Different restriction fragments are obtained from the fimH promoter region depending on the position of the fim switch element. Strains A and B show the presence of the phase switch element in the "off" position as evidenced by the 200- and 359-bp fragments. In contrast, bacterial cultures from strains C and D display in both the "on" and "off" positions.

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FIG. 6. Detection of the phase switch orientation of fimH+ E. coli strains using PCR followed by restriction enzyme cleavage. The left lane contains DNA molecular size markers. The cleaved PCR product of strains A and B reveal fragments of 359 and 200 bp, generated from bacterial cultures only in the phase switch "off" position. Strains C and D show cleavage products representing bacterial cultures in both the "on" and "off" switch positions. The PCR products were digested by restriction enzyme HinfI, separated by agarose gel electrophoresis, and stained with ethidium bromide.

After three passages in static broth, bacterial cultures of fimH⁺ strains contained either cells that all had the promoter in the "off" position or a mixture of cells with the promoter in the "on" and "off" positions. If all cells were in the "off" position, we considered that the strain had a block in the capacity to switch to the fimbriated phase. Nine percent of the 53 fimH⁺ strains from control individuals and 27% of the 49 fimH⁺ strains from IgA-deficient individuals examined appeared to be locked in the "off" position (P = 0.04). Table 3 shows the proportions of resident and transient fimH⁺ strains from control and IgA-deficient individuals that revealed a phase switch block. A fairly large proportion of both resident and transient strains from IgA-deficient strains appeared incapable of switching to the fimbriated phase. When resident strains from control and IgA-deficient individuals were compared, the strains from the IgA-deficient individuals had a phase switch block significantly more often (Table 3).

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TABLE 3. Proportion of fimH+ E. coli strains displaying the switch element only in the OFF position after three passages in static brothc

We next examined whether fimH⁺ strains that were able to switch on their fimbrial production differed in MS adherence depending on whether they were isolated from IgA-deficient or control individuals. Figure 6 shows the MS adherence of strains that displayed the fim promoter in both the "on" and "off" positions after three passages in static broth, indicating that they could produce fimbriae with MS adhesins. As shown in the figure, the difference in MS adherence between resident and transient strains from control individuals was now gone. Thus, strains that were able to switch their fimH promoter to the "on" position displayed equally strong adherence levels whether they had been resident or transient in the microbiota. However, the difference between strains from IgA-deficient and control individuals remained (Fig. 7). Thus, fimH⁺ resident strains from IgA-deficient individuals that could switch on their fimbrial expression displayed reduced MS adherence to HT-29 cells in comparison with corresponding strains from healthy controls (P = 0.02) (Fig. 7).

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FIG. 7. Mannose-sensitive adherence to colonic HT-29 cells of fimH+ strains capable of switching their promoter to the "on" phase switch position. Thirty-eight E. coli strains (28 resident, 10 transient) obtained from 13 control individuals and 25 strains (16 resident, 9 transient) from 14 IgA-deficient (IgA-d) individuals were included in the analysis. Each circle represents the mean value for all E. coli isolates belonging to one strain, and the horizontal bar represents the median. Adherence levels were compared between groups using the Mann-Whitney U test.

MR adherence to HT-29 cells. The MR adherence of resident and transient E. coli strains from control and IgA-deficient individuals is shown in Fig. 8. Resident strains from IgA-deficient individuals on average displayed higher MR adherence than resident strains from control individuals (P = 0.04). In IgA-deficient individuals, resident strains displayed higher MR adherence than did transient strains (P = 0.01). This was not seen in strains from control individuals. The MR adherence levels did not differ between the first and last isolates of resident strains retrieved from either control or IgA-deficient individuals (data not shown).

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FIG. 8. Mannose-resistant adherence to HT-29 cells of 50 strains (35 resident, 15 transient) obtained from 16 healthy controls and 47 strains (31 resident, 16 transient) obtained from 17 IgA-deficient (IgA-d) individuals. Adherence was assessed after three passages in static broth culture. Each circle represents the mean value for all E. coli isolates belonging to one strain. The median values are given in parentheses for each group of strains. The mannose-resistant adherence levels were compared using the Mann-Whitney U test.

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have earlier shown that E. coli isolated from IgA-deficient individuals has a reduced capacity to adhere via mannose-specific mechanisms to colonic epithelial cells in comparison with E. coli from individuals with normal IgA levels in serum and secretions (14). Two factors in IgA-deficient individuals contributed to this finding: a lower prevalence of E. coli carrying the genes for type 1 fimbriae and the MS adhesin and a reduced MS adherence to colonic cells by strains that had the genes for this adhesin (14).

Here, we confirm and extend these findings in a longitudinal study. We isolated E. coli from rectal swabs obtained monthly from IgA-deficient and control individuals. Strains found on more than one occasion in an individual were defined as resident, while strains found only in a single sample were defined as transient. We found that the differences in MS adherence between E. coli strains from IgA-deficient and control individuals were most marked and highly significant when resident strains were examined. Such strains are likely to be best adapted to the human colonic milieu. This reinforces the findings of our previous cross-sectional study (14) and suggests that a superior capacity to adhere to mannose-containing receptors is beneficial for long-term persistence in the colon, especially when S-IgA is present in the secretions. Since the capacity for strong adherence was evident in the first isolate of resident strains, strains with a superior capacity to produce mannose-binding adhesins may be positively selected for persistence. Strains that have a less prominent adherence capacity may not be able to establish residence in the microbiota and may disappear in a short time.

The substantially reduced adherence to mannose-containing receptors on human colonic cells of the HT-29 cell line of E. coli from IgA-deficient versus age-matched control individuals derived from a combination of three factors. First, there was a slight and nonsignificant reduction in the proportion of strains in IgA-deficient individuals that carried the fimH⁺ gene cluster, which was also observed in our previous study, where this difference was significant (14). Second, in IgA-deficient individuals, the proportion of strains carrying the fimH gene cluster in their genome that appeared to be incapable of switching to the fimbriated phase was larger than that of fimH⁺ strains from control individuals. The fim switch experiments were performed after three passages of the bacteria in Luria broth which, according to our adherence experiments, was sufficient to induce expression of type 1 fimbriae in strains capable of expressing these adhesins. The third factor was the reduced MS adherence of fimH⁺ switch-capable strains from IgA-deficient individuals in comparison with corresponding strains from control individuals.

A limitation of this study was that we could not quantify the proportion of the bacteria that had switched to the fimbriated phase. Thus, we cannot exclude the possibility that there was a quantitative difference in the proportions of bacterial cells that had their promoter switched to the "on" position between switch-capable strains from IgA-deficient and control individuals. Using RT-PCR, it should be possible to quantify whether the proportion of bacterial cells that are in the "on" and "off" position differs between isolates retrieved from IgA-deficient and control individuals. Another possibility would be that the fimH adhesins of E. coli colonizing IgA-deficient individuals have reduced binding capacity to colonic receptors. Slight changes in adhesin conformation, conferring a broader receptor specificity for the MS adhesin, have been demonstrated among E. coli strains isolated from urinary tract infections, suggested to be due to "pathoadaptive mutations" (21). In a future study, we will attempt to examine whether fimH adhesin genes in E. coli from IgA-deficient and control individuals differ in sequence.

The reason E. coli with poor MS adherence can persist better in IgA-deficient than in control individuals can only be speculated upon. The MS adhesin allows the bacteria to adhere to colonic epithelial cells. S-IgA acts as a competing receptor blocking this adherence. Competition from S-IgA may force bacteria to produce more MS adhesins in order to adhere to mucosal receptors, while a moderate expression of the MS adhesin might be sufficient to attach to colonic cells in IgA-deficient individuals. Another possibility is that the interaction between MS adhesins and mannose residues on the carbohydrate chains of S-IgA is actually beneficial for the bacteria and that the bacteria which obtain a coat of S-IgA have an advantage over other strains. Coating of bacteria by IgA reduces their surface hydrophobicity (12), and other factors rendering the bacteria more hydrophilic, such as capsule and smooth O antigen, enhance the colonizing capacity of E. coli in rodent models (20, 42). It is also possible that the weak interaction between mucin molecules and S-IgA covering the bacteria would position the bacterium in a favorable niche in the mucus layer (6).

We noted that resident strains from IgA-deficient individuals displayed significantly higher mannose-resistant adherence than both resident strains from control individuals and transient strains from IgA-deficient individuals. We have previously reported that E. coli from IgA-deficient individuals more often carries genes for both S fimbriae and hemolysin than E. coli from control individuals (15).

In summary, our results indicate that the presence of S-IgA in colonic secretions modulates the colonic microbiota and determines which subgroups of strains may establish residence in the colon. Indeed, preliminary evidence indicates that the microbiota of IgA-deficient individuals is phylogenetically different from E. coli colonizing control individuals (F. Nowrouzian et al., unpublished observation). Taken together, our findings indicate that S-IgA plays a significant role in regulating large intestinal microbial ecology.

 
The skillful technical assistance of Jolanta Bonislawska is gratefully appreciated.

This study was supported by grant K2001-06X-14072-01 from the Swedish Medical Research Council. This study was not financially supported by any commercial or other association.

There are no conflicts of interest.

 
* Corresponding author. Mailing address: Department of Clinical Bacteriology, Guldhedsgatan 10, S-413 46 Göteborg, Sweden. Phone: 46-31-3424887. Fax: 46-31-3424975. E-mail: forough.nowrouzian{at}microbio.gu.se.

Published ahead of print on 13 November 2006.

Editor: V. J. DiRita

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Infection and Immunity, February 2007, p. 932-940, Vol. 75, No. 2
0019-9567/07/$08.00+0     doi:10.1128/IAI.00736-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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