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Role of overexpressed CFA/I fimbriae in bacterial swimming - PubMed

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Role of overexpressed CFA/I fimbriae in bacterial swimming

Ling Cao et al. Phys Biol. 2012 Jun.

Abstract

Enterotoxigenic Escherichia coli CFA/I is a protective antigen and has been overexpressed in bacterial vectors, such as Salmonella Typhimurium H683, to generate vaccines. Effects that overexpressed CFA/I may engender on the bacterial host remain largely unexplored. To investigate, we constructed a high CFA/I expression strain, H683-pC2, and compared it to a low CFA/I expression strain, H683-pC, and to a non-CFA/I expression strain, H683-pY. The results showed that H683-pC2 was less able to migrate into semisolid agar (0.35%) than either H683-pC or H683-pY. Bacteria that migrated showed motility halo sizes of H683-pC2 < H683-pC < H683-pY. In the liquid culture media, H683-pC2 cells precipitated to the bottom of the tube, while those of H683-pY did not. In situ imaging revealed that H683-pC2 bacilli tended to auto-agglutinate within the semisolid agar, while H683-pY bacilli did not. When the cfaBE fimbrial fiber encoding genes were deleted from pC2, the new plasmid, pC2(-), significantly recovered bacterial swimming capability. Our study highlights the negative impact of overexpressed CFA/I fimbriae on bacterial swimming motility.

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Figures

Figure 1
Figure 1

Effect of overexpression of CFA/I on bacterial phenotypes. (a) Schematic plasmid maps. The cfaABCE is regulated by PtetA~PphoP in pC2 (i) and by PtetA in pC (ii), and control pY does not harbor cfaABCE (iii). (b) Overexpression of CFA/I resulting in cell auto-precipitation. The overnight cell culture of H683-pC2 precipitated to the bottom of the tube (i), while those of -pC (ii) and -pY (iii) did not. The arrow indicates the precipitated cell pellets. (c) Western blot of anti-flagella and -CFA/I fimbriae. The anti-FliC blots showed no differences for flagellum expression among H683-pC2 (i), -pC (ii) and -pY(iii), while anti-CFA/I blots showed that H683-pC2 (i) produced more CFA/I fimbriae than -pC (ii) but -pY (iii) did not synthesize CFA/I. (d) AFM imaging of bacterial flagella and CFA/I fimbriae. The H683-pC2 (i), -pC (ii) and -pY (iii) images all revealed flagella associated with the bacterial organisms, but CFA/I fimbriae were associated with H683-pC2 and -pC, but not with -pY. The wide and narrow arrows indicate flagella and CFA/I fimbriae, respectively. (e) Light microscope images of agglutinated bacteria. Imaging revealed that a majority of H683-pC2 cells (i) were auto-agglutinated within the liquid LB medium, while a great number of H683-pC (ii) and all -pY (iii) cells existed in the planktonic form.

Figure 2
Figure 2

Effects of overexpression of CFA/I fimbriae on bacterial swimming. (a) Not all inoculants were able to start swimming in 0.35% agar at 8 h post-inoculation. Images of H683-pC2 (i), -pC (iii) and -pY (v) inoculants which started swimming versus those unable to start swimming, (ii), (iv) and (vi), respectively. The scale bar, 10 mm, is applicable to all the images (i–vi). (b) CFA/I fimbriae counteract bacterial swimming capability. (i) The migrating inoculants rates were determined for H683-pC2, -pC and -pY, and the statistical differences were evaluated and are indicated as * P < 0.05 for -pC2 versus -pY and P < 0.05 for -pC2 versus -pC. Values are the mean ± SEM (n = 6). (ii) The motility halo diameters at 8 h post-inoculation were recorded, and the statistical differences were evaluated and are indicated as *** P < 0.001 for H683-pC2 or -pC versus -pY, and P < 0.05 for -pC2 versus -pC. Values are the mean ± SEM (n = 3).

Figure 3
Figure 3

CFA/I-inhibited bacterial swimming depends on agar concentration. (a) The migrating inoculant rates of the fimbriated bacteria were elevated with the decrease in agar concentration. The migrating inoculants rates of H683-pC2 and -pY were statistically compared for 0.45%, 0.35% and 0.25% agar, and the differences are indicated as *** P < 0.001 for H683-pC2 versus -pY, ¶¶¶ P < 0.001 for H683-pC2 at 0.35% versus 0.45% agar, and ††† P < 0.001 for H683-pY at 0.35% versus 0.45% agar. Values are the mean ± SEM (n = 6). (b) The motility halo sizes of the fimbriated bacteria increase with the decrease in agar concentration. H683-pC2 was compared with -pY for halo diameters at 8 h post-inoculation, and the statistical differences between H683-pC2 and -pY were evaluated and are indicated as *** P < 0.001 at each of the three agar concentrations; ¶¶¶ P < 0.001 for -pC2 at 0.35% versus 0.45% agar, and 0.25% versus 0.35% agar; and ††† P < 0.001 for -pY at 0.35% versus 0.45% agar, and 0.25% versus 0.35% agar. Values are the mean ± SEM (n = 3).

Figure 4
Figure 4

Evaluation of growth rate and observation of in situ auto-agglutination of H683-pC2. (a) Assay of the growth rates of H683-pC2 and -pY via OD600. The statistical differences in growth rate between H683-pC2(−) and -pY were evaluated and are indicated as * P < 0.05, ** P < 0.01 and *** P < 0.001 at each time point. Values are the mean ± SEM (n = 3). (b) Assay of the growth rates of H683-pC2 and -pY via CFU. The statistical differences in CFU between H683-pC2(−) and -pY were evaluated and are indicated as * P < 0.05, ** P < 0.01 and *** P < 0.001 at each time point. Values are the mean ± SEM (n = 3). (c) Observation of H683-pC2 auto-agglutination inside agar. The motility halos formed within 0.35% agar by strains H683-pC2 (i) and -pY (ii) were observed for individual cell behavior using an optical microscope in phase contrast mode. The arrows indicate cell auto-agglutination. No aggregates were detected for control H683-pY.

Figure 5
Figure 5

Impact of the CFA/I fimbrial fibers on bacterial swimming. (a) CFA/I fimbrial fibers hinder bacterial swimming. (i) The migration inoculants rates at 8 h post-inoculation were determined for H683-pC2(−), -pTP2cfaB(−), -pC2 and -pY, and the statistical differences were evaluated and are indicated as *** P < 0.001 for H683-pC2 versus -pY, ¶¶¶ P < 0.001 for -pC2 versus -pC2(−) and ††† P < 0.001 for H683 -pC2 versus -pTP2cfaB(−). Values are the mean ± SEM (n = 6). (ii) The motility halo sizes at 8 h post-inoculation were recorded, and the statistical differences were evaluated and are indicated as *** P < 0.001 for H683-pC2 versus -pY, ** P < 0.01 for H683-pC2(−) versus -pY, * P < 0.05 for H683-pTP2cfaB(−) versus -pY, ¶¶¶ P < 0.001 for H683-pC2 versus -pC2(−), ††† P < 0.001 for H683-pC2 versus -pTP2cfaB(−) and ‡ P < 0.05 for -pC2(−) versus -pTP2cfaB(−). Values are the mean ± SEM (n = 3). (b) Growth rates of the non-fimbriated strains versus control. The OD600 was recorded and the statistical differences were evaluated and are indicated as * P < 0.05, ** P < 0.01 and *** P < 0.001 for H683-pC2(−) versus -pY, P < 0.05, ¶¶ P < 0.01 and ¶¶¶ P < 0.001 for -pTP2cfaB(−) versus -pY. Values are the mean ± SEM (n = 3).

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