Gelatinase is important for translocation of Enterococcus faecalis across polarized human enterocyte-like T84 cells - PubMed (original) (raw)

Gelatinase is important for translocation of Enterococcus faecalis across polarized human enterocyte-like T84 cells

Jing Zeng et al. Infect Immun. 2005 Mar.

Erratum in

Abstract

Previously, in our laboratory, we established a two-chamber system to study translocation of Enterococcus faecalis across monolayers of polarized human colon carcinoma-derived T84 cells. By using the same system in the present study, we now show that disruption of gelE of strain OG1RF, which also has a polar effect on the cotranscribed sprE, as well as disruption of its regulatory system (fsrA, fsrB, and fsrC) resulted in a loss of detectable translocation by E. faecalis OG1RF; these mutants lost gelatinase (GelE) and serine protease (SprE) production by standard assay. A gelE deletion mutant of OG1RF (GelE- SprE+) also showed that significantly reduced translocation and complementation with the gelE gene (pTEX5438) in trans restored gelatinase and translocation, demonstrating that gelatinase is important for E. faecalis translocation. Complementation of fsrA, fsrB, and fsrC mutants with all three fsr genes also resulted in production of gelatinase and translocation. Furthermore, introduction of fsr genes into two non-gelatinase-producing E. faecalis isolates, the well-characterized laboratory strain JH2-2 and a human-derived fecal isolate, TX1322 (both of which have gelE but not fsrA or fsrB, are gelatinase negative, and do not translocate), resulted in gelatinase production by these strains and restored translocation across T84 monolayers, while transformation with pTEX5438 (gelE) showed little or no translocation and no detectable gelatinase, confirming the importance of both fsr and gelatinase for E. faecalis translocation. The importance of gelatinase production was also corroborated among 20 E. faecalis human isolates (7 fecal, 7 endocarditis, and 6 urine isolates), which showed translocation by all gelatinase-positive isolates but little to no translocation for gelatinase nonproducers. These results indicate that gelatinase is important for the successful in vitro translocation of E. faecalis across human enterocyte-like T84 cells.

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Figures

FIG. 1.

FIG. 1.

A. Composition of fsr-gelE/sprE locus (15). Line, chromosome; boxes, genes and open reading frames; arrows, promoters indicating direction of transcription; Pa, fsrA promoter; Pb, fsrB promoter; Pc, gelE promoter. The fragments cloned into pAT18 for complementation are also shown. B. Translocation of OG1RF, gelE/fsr mutants, and their complementation derivatives across a T84 monolayer. Two to three combined experiments are shown, and 4 transwells were used for each strain in each experiment. The geometric mean bacterial cell counts (CFU) in the lower chamber at 6 and 8 h are shown. For mutants which had shown no translocation in pilot studies, we plated the whole 1-ml volume of the lower chamber, and for others, 100 μl out of 1 ml was plated; thus, for the latter experiments, the lower limit of detection is 10 CFU, which was used in our calculations for geometric mean. Error bars show the standard deviations. TX5128, TX5240, TX5241, TX5242, TX5264, and TX5266 are gelE, fsrA, fsrB, and fsrC disruption and gelE and fsrB deletion mutants, respectively, while TX5244, TX5245, TX5246, and TX5439 are _fsr_-complemented fsrA, fsrB, and fsrC disruption and _gelE_-complemented gelE deletion mutants. A significant difference in translocation was detected for OG1RF compared to that of TX5264 and for OG1RF compared to that of TX5244 (P < 0.001 by ANOVA with Bonferroni's posttest). Gelatinase activity (shown as absorption at 540 nm [A540]) at the bottom of the figure was measured with Azocoll and supernatant from bacteria grown under translocation conditions according to a method described previously by Nakayama et al. (12).

FIG. 2.

FIG. 2.

Correlation between Azocoll proteolytic activities and translocation capabilities among OG1RF derivatives. The points shown are from TX5128 (which, like TX5240, TX5241, TX5242, and TX5266, showed no detectable gelatinase activity or translocation), TX5264, TX5244, TX5245, TX5246, and TX5439; translocation at 8 h is shown. Correlation coefficients are 0.98 and 0.92 for Spearman's r and Pearson's r, respectively, and a significantly positive correlation was detected (P = 0.003 and P = 0.010, respectively).

FIG. 3.

FIG. 3.

Translocation of gelatinase nonproducers and their derivatives across a T84 monolayer compared to that of OG1RF. Two to three combined experiments are shown, and 4 transwells were used for each strain in each experiment. The geometric mean bacterial cell counts (CFU) and standard deviations in the lower chamber at 6 and 8 h are shown. Gelatinase activity is as described in the legend of Fig. 1B. JH2-2 and TX1322 were complemented with gelE (TX5440 and TX5442) and fsr (TX5441 and TX5443) genes, respectively. Translocation of TX5440, TX5441, or TX5443 was significantly lower than that of OG1RF (P < 0.001 by ANOVA with Bonferroni's posttest).

FIG. 4.

FIG. 4.

Translocation of human-derived E. faecalis isolates across T84 monolayers. Results represent two combined experiments with 4 transwells used for each strain in each experiment, and the percentages of CFU showing translocation relative to the CFU of OG1RF are shown. Gelatinase activity is as described in the legend of Fig. 1B. TX1317 and TX0231 (marked with *) showed 17 and 22 and 20 and 7 times more CFU in the lower chamber than did OG1RF at 6 and 8 h, respectively. †The translocation of TX0040 cannot be seen in the figure due to the low percentage compared to OG1RF (0.2% at 6 h and 0.8% at 8 h). The capability of translocation of the two groups (GelE+ and GelE− strains) is significantly different, with translocation occurring in a total of 76 out of 95 and 92 out of 95 transwells of GelE+ strains versus a total of 1 out of 63 and 1 out of 63 transwells of GelE− strains at 6 and 8 h (P < 0.001 by Fisher's exact test).

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