No abstract

We have previously shown that integration of the virulence plasmid pINV into the chromosome of enteroinvasive Escherichia coli and of Shigella flexneri makes these strains noninvasive (C. Zagaglia, M. Casalino, B. Colonna, C. Conti, A. Calconi, and M. Nicoletti, Infect. Immun. 59:792-799, 1991). In this work, we have studied the transcription of the virulence regulatory genes virB, virF, and hns (virR) in wild-type enteroinvasive E. coli HN280 and in its pINV-integrated derivative HN280/32. While transcription of virF and of hns is not affected by pINV integration, transcription of virB is severely reduced even if integration does not occur within the virB locus. This indicates that VirF cannot activate virB transcription when pINV is integrated, and this lack of expression accounts for the noninvasive phenotype of HN280/32. Virulence gene expression in strains HN280 and HN280/32, as well as in derivatives harboring a mxiC::lacZ operon fusion either on the autonomously replicating pINV or on the integrated pINV, was studied. The effect of the introduction of plasmids carrying virB (pBN1) or virF (pHW745 and pMYSH6504), and of a ⌬hns deletion, in the different strains was evaluated by measuring ␤-galactosidase activity, virB transcription, and virB-regulated virulence phenotypes like synthesis of Ipa proteins, contactmediated hemolysis, and capacity to invade HeLa cells. The introduction of pBN1 or of the ⌬hns deletion in pINV-integrated strains induces temperature-regulated expression or temperature-independent expression, respectively, of ␤-galactosidase activity and of all virulence phenotypes, while an increase in virF gene dosage does not, in spite of a high-level induction of virB transcription. Moreover, a wild-type hns gene placed in trans fully reversed the induction of ␤-galactosidase activity due to the ⌬hns deletion. These results indicate that virB transcription is negatively regulated by H-NS both at 30 and at 37؇C in pINV-integrated strains and that there is also a dosedependent effect of VirF on virB transcription. The negative effect of H-NS on virB transcription at the permissive temperature of 37؇C could be due to changes in the DNA topology occurring upon pINV integration that favor more stable binding of H-NS to the virB promoter DNA region. At 30؇C, the introduction of the high-copy-number plasmid pMYSH6504 (but not of the low-copy-number pHW745) or of the ⌬hns deletion induces, in strains harboring an autonomously replicating pINV, ␤-galactosidase activity, virB transcription, and expression of the virulence phenotypes, indicating that, as for HN280/32, the increase in virF gene dosage overcomes the negative regulatory effect of H-NS on virB transcription. Moreover, we have found that virF transcription is finely modulated by temperature and, with E. coli K-12 strains containing a virF-lacZ gene fusion, by H-NS. This leads us to speculate that, in enteroinvasive bacteria, the level of VirF inside the cell controls the temperature-regulated expression of invasion genes.Shigella flexneri and ...

The transcription of the virulence plasmid (pINV)-carried invasion genes of Shigella flexneri and enteroinvasiveEscherichia 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-Sfgene. 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 theapy 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 Δhns 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 virBand by the presence of the intact apy locus or intactapy 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 clonedvirF (pMYSH6504) or virB (pBN1) into Crb− mutants of HN280 and SFZM53 lacking virFor 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.

We have established a generic method for studying the effect of RNA interference in HBEC-ALI using standard lentiviral vectors. Down-regulation of ENaCα by lentiviral shRNA expression vectors as shown in the absence off-target effects has potential therapeutic value in the treatment of cystic fibrosis.

Lactobacilli are Gram-positive rod-shaped bacteria that inhabit the oral cavity, gastrointestinal tract, vagina and nasal cavity. In this report, a rare case of Lactobacillus jensenii endocarditis in a 47-year-old immunocompetent patient is described. Blood cultures and a replaced mitral valve were positive for L. jensenii as assessed by 16S rRNA gene sequencing. Based on susceptibility tests the patient was successfully treated with a mixture of teicoplanin and meropenem antimicrobial therapy.