Expression of the virulence plasmid-carried apyrase gene (apy) of enteroinvasive Escherichia coli and Shigella flexneri is under the control of H-NS and the VirF and VirB regulatory cascade

Abstract

The transcription of the virulence plasmid (pINV)-carried invasion genes of Shigella flexneri and enteroinvasive Escherichia coli (EIEC) is induced at 37 degreesC and repressed at 30 degreesC. In this work, we report that the O135: K-:H- EIEC strain HN280 and S. flexneri SFZM53, M90T, and 454, of serotypes 4, 5, and 2a, respectively, produce apyrase (ATP-diphosphohydrolase), the product of the apy gene. In addition, the S. flexneri strains, but not the EIEC strain, produce a nonspecific phosphatase encoded by the phoN-Sf gene. Both apy and phoN-Sf are pINV-carried loci whose contribution to the pathogenicity of enteroinvasive microorganisms has been hypothesized but not yet established. We found that, like that of virulence genes, the expression of both the apy and the phoN-Sf genes was temperature regulated. Strain HN280/32 (a pINV-integrated avirulent derivative of HN280 which has a severe reduction of virB transcription) expressed the apy gene in a temperature-regulated fashion but to a much lower extent than wild-type HN280, while the introduction of the Deltahns deletion in HN280 and in HN280/32 induced the wild-type temperature-independent expression of apyrase. These results indicated that a reduction of virB transcription, which is known to occur in the pINV-integrated strain HN280/32, accounts for reduced apyrase expression and that the histone-like protein H-NS is involved in this regulatory network. Independent spontaneously generated mutants of HN280 and of SFZM53 which had lost the capacity to bind Congo red dye (Crb-) were isolated, and the molecular alterations of pINV were evaluated by PCR analysis. Alterations of pINV characterized by the absence of virF or virB and by the presence of the intact apy locus or intact apy and phoN-Sf loci were detected among Crb- mutants of HN280 and SFZM53, respectively. While all Crb- apy+ mutants of HN280 failed to produce apyrase, Crb- apy+ phoN-Sf+ mutants of SFZM53 lacked apyrase activity but produced a nonspecific phosphatase, like parental SFZM53. Moreover, the introduction of recombinant plasmids carrying cloned virF (pMYSH6504) or virB (pBN1) into Crb- mutants of HN280 and SFZM53 lacking virF or virB, respectively, fully restored temperature-dependent apyrase expression to levels resembling those of the parental strains. Taken together, our results demonstrate that, as has already been shown for invasion genes, apy is another locus whose expression is controlled by temperature, H-NS, and the VirF and VirB regulatory cascade. In contrast, the temperature-regulated expression of the nonspecific phosphatase does not appear to be under the control of the same regulatory network. These findings led us to speculate that apyrase may play a role in the pathogenicity of enteroinvasive bacteria.

FIG. 1

Zymogram analysis of apyrase activity (A) and RNA hybridization analysis (B) of O135:K−:H− EIEC strain HN280 and S. flexneri SFZM53 (serotype 4), M90T (serotype 5), and 454 (serotype 2a) grown at 30 and 37°C. The conditions used to renature and to visualize apyrase activity are described in Materials and Methods. MW, prestained molecular weight markers (Bio-Rad, Hercules, Calif.); sizes (in thousands) are shown to the right of panel A. Northern blots were probed with the apy probe, a ³²P-labelled 672-bp PCR-generated DNA fragment (internal to the apy coding region) described in Materials and Methods. Each lane was loaded with 20 μg of total RNA. The specific β-emission value of each RNA sample (counts per minute per microgram of RNA), determined by electronic analysis of RNA dot blots, is given within a circle below each lane. The electrophoretic mobilities of the 23S, 16S, and 5S rRNA fractions are indicated to the right of panel B.

FIG. 2

Zymogram analysis of apyrase activity (A) and RNA hybridization analysis (B) of O135:K−:H− EIEC strain HN280 derivatives HN680 (Δhns), HN280/32 (pINV integrated), and HN680/32 (Δhns; pINV integrated) grown at 30 and 37°C. The conditions used to renature and to visualize apyrase activity are described in Materials and Methods. MW, prestained molecular weight markers (Bio-Rad); sizes (in thousands) are shown to the right of panel A. Northern blots were probed with the apy probe, a ³²P-labelled 672-bp PCR-generated DNA fragment (internal to the apy coding region) described in Materials and Methods. Each lane was loaded with 20 μg of total RNA. The specific β-emission value of each RNA sample (counts per minute per microgram of RNA), determined by electronic analysis of RNA dot blots, is given within a circle below each lane. The electrophoretic mobilities of the 23S, 16S, and 5S rRNA fractions are indicated to the right of panel B.

FIG. 3

Zymogram analysis of apyrase activity (A) and RNA hybridization analysis (B) of HN280-16 and SFZM53-15 (Crb⁻ mutants of O135:K−:H− EIEC strain HN280 and of S. flexneri SFZM53 [serotype 4], respectively) and of pBN1 (virB) transformants grown at 30 and 37°C. The conditions used to renature and to visualize apyrase activity are described in Materials and Methods. MW, prestained molecular weight markers (Bio-Rad); sizes (in thousands) are shown to the right of panel A. Northern blots were probed with the apy probe, a ³²P-labelled 672-bp PCR-generated DNA fragment (internal to the apy coding region) described in Materials and Methods. Each lane was loaded with 20 μg of total RNA. The specific β-emission value of each RNA sample (counts per minute per microgram of RNA), determined by electronic analysis of RNA dot blots, is given within a circle below each lane. The electrophoretic mobilities of the 23S, 16S, and 5S rRNA fractions are indicated to the right of panel B.