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Infection and Immunity, October 1998, p. 4957-4964, Vol. 66, No. 10
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

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

Francesca Berlutti,1 Mariassunta Casalino,2 Carlo Zagaglia,3 Piera Assunta Fradiani,4 Paolo Visca,5 and Mauro Nicoletti6,*

Istituto di Microbiologia,1 Dipartimento di Medicina Sperimentale e Patologia,3 and Dipartimento di Biologia Cellulare e dello Sviluppo,4 Sezione di Scienze Microbiologiche, Università di Roma La Sapienza, 00185 Rome, Dipartimento di Biologia, Università Roma Tre, 00146 Rome,2 Istituto Superiore di Sanità, 00161 Rome,5 and Dipartimento di Scienze Biomediche, Sezione di Microbiologia, Università G. D'Annunzio, 66100 Chieti,6 Italy

Received 11 May 1998/Returned for modification 26 June 1998/Accepted 21 July 1998

The transcription of the virulence plasmid (pINV)-carried invasion genes of Shigella flexneri and enteroinvasive Escherichia coli (EIEC) is induced at 37°C and repressed at 30°C. 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 Delta hns 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.

* Corresponding author. Mailing address: Dipartimento di Scienze Biomediche, Sezione di Microbiologia, Università G. D'Annunzio, Via dei Vestini, 31, 66100 Chieti, Italy. Phone: 39-871-3555279. Fax: 39-871-3555282. E-mail: nicoletti{at}axrma.uniroma1.it.

Infection and Immunity, October 1998, p. 4957-4964, Vol. 66, No. 10
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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