The genome of Shigella, a gram negative bacterium which is the causative agent of bacillary dysentery, shares strong homologies with that of its commensal ancestor, Escherichia coli. The acquisition, by lateral gene transfer, of a large plasmid carrying virulence determinants has been a crucial event in the evolution towards the pathogenic lifestyle and has been paralleled by the occurrence of mutations affecting genes, which negatively interfere with the expression of virulence factors. In this context, we have analysed to what extent the presence of the plasmid-encoded virF gene, the major activator of the Shigella regulon for invasive phenotype, has modified the transcriptional profile of E. coli. Combining results from transcriptome assays and comparative genome analyses we show that in E. coli VirF, besides being able to up-regulate several chromosomal genes, which potentially influence bacterial fitness within the host, also activates genes which have been lost by Shigella. We have focused our attention on the speG gene, which encodes spermidine acetyltransferase, an enzyme catalysing the conversion of spermidine into the physiologically inert acetylspermidine, since recent evidence stresses the involvement of polyamines in microbial pathogenesis. Through identification of diverse mutations, which prevent expression of a functional SpeG protein, we show that the speG gene has been silenced by convergent evolution and that its inactivation causes the marked increase of intracellular spermidine in all Shigella spp. This enhances the survival of Shigella under oxidative stress and allows it to better face the adverse conditions it encounters inside macrophage. This is supported by the outcome of infection assays performed in mouse peritoneal macrophages and of a competitive-infection assay on J774 macrophage cell culture. Our observations fully support the pathoadaptive nature of speG inactivation in Shigella and reveal that the accumulation of spermidine is a key determinant in the pathogenicity strategy adopted by this microrganism.

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 role in virulence of the Shigella flexneri ospB-phoN2 operon has been evaluated. Here we confirm that OspB is an effector and show that apyrase, the product of phoN2, may be a virulence factor, since it is required for efficient intercellular spreading. Apyrase may be important in a deoxynucleoside triphosphate-hydrolyzing activity-independent manner, suggesting that it may act as an interaction partner in the process of IcsA localization.ospB and phoN2 (apy) are genes located on the virulence plasmid (pINV) of Shigella species and of related enteroinvasive Escherichia coli (EIEC) strains (3,6,35). Indirect evidence indicates that ospB and phoN2 may contribute to pathogenicity (1,3,5,8,17,23,25,35). ospB encodes a protein of unknown function (OspB) surmised to be an effector secreted by the type III secretion (TTS) apparatus of Shigella flexneri (6,20). The two genes are organized in a single highly conserved bicistronic operon. Although phoN2 encodes a periplasmic protein (5), their expression is regulated by the VirF-VirB cascade (35) and by MxiE in concert with IpgC so that they are expressed in a VirB-dependent, MxiE-independent manner under conditions of nonactivated secretion and are up-regulated, in a MxiEdependent manner, under conditions of activated secretion (8,17,20,26,30), indicating that these genes may be important in postinvasion events related to virulence.phoN2 encodes apyrase, a periplasmic enzyme which belongs to the family of ATP-hydrolyzing enzymes (3, 5, 18) able to sequentially hydrolyze nucleoside triphosphates to diphosphates and then to monophosphates. However, it is not active against monophosphates, a distinguishing feature from acid phosphatases (1,3,41). Due to its catalytic activity and primary structure (apyrase presents an exposed N-terminal poly-proline sequence), it has been implicated in the decrease of host cell intracellular ATP, a characteristic metabolic event following S. flexneri infection, as well as in the S. flexneri-induced actin-polymerization process (1,3,5,25,35). Thus, these findings strongly suggest that OspB and apyrase may be virulence factors. The purpose of this study was to investigate the role of both genes in the mechanism of pathogenicity of S. flexneri. OspB is an effector secreted by the TTS apparatus of S. flexneri. To confirm and extend previous reports indicatingOspB as an effector secreted by the TTS apparatus (6, 20), we transduced a nonpolar deletion encompassing mxiA into the S. flexneri M90T derivative strain HND549 (ospB::3ϫFLAG), thus generating HND5311 (mxiA ospB::3ϫFLAG) ( Table 1). C-terminal 3ϫ FLAG tagging was achieved essentially as described by Uzzau et al. (42) using the specific primer pair shown in Table S1 in the supplemental material. Whole-cell extracts and supernatants of exponentially growing bacteria were analyzed by immunoblotting using monoclonal antibodies against the 3ϫ FLAG epitope (Sigma) and mouse polyclonal antibodies preparation directed against apyrase (Fig. 1). Apyrase antiserum was obtained by immunizing BALB/...

Enteroinvasive E. coli (EIEC), like Shigella, is the etiological agent of bacillary dysentery, a particularly severe syndrome in children in developing countries. All EIEC strains share with Shigella the inability to synthesize lysine decarboxylase (the LDC phenotype). The lack of this function is considered a pathoadaptive mutation whose emergence was necessary to obtain the full expression of invasiveness. Cadaverine, the product of lysine decarboxylation, is a small polyamine which interferes mainly with the inflammatory process induced by dysenteric bacteria. Genes coding for lysine decarboxylase and its transporter constitute a single operon (cadBA) and are expressed at low pH under the positive control of CadC. This regulator is an inner membrane protein that is able to sense pH variation and to respond by transcriptionally activating the cadBA genes. In this study we show that, unlike in Shigella, mutations affecting the cad locus in the EIEC strains we have analyzed are not followed by a novel gene arrangement and that the LCD ؊ phenotype is dependent mainly on inactivation of the cadC gene. Introduction of a functional CadC restores cadaverine expression in all EIEC strains harboring either an IS2 element or a defective cadC promoter. Comparative analysis between the cad regions of S. flexneri and EIEC suggests that the LDC ؊ phenotype has been attained by different strategies within the E. coli species.

Stenotrophomonas maltophilia is an emerging nosocomial bacterial pathogen which is currently isolated with increasing frequency from the airways of cystic fibrosis (CF) patients. In this study 13 S. maltophilia strains (11 isolated from the airways of independent CF patients, and two non-CF respiratory reference strains) have been characterized for the expression of several virulence-associated factors. In particular, the ability to form biofilm on abiotic surfaces has been determined and correlated with different features, such as motility, adherence and the ability to invade A549 respiratory epithelial cells. Moreover, the presence of a flagellum-associated gene as well as that of the StmPr1 gene, which encodes an extra-cellular protease, have been determined by Southern blot hybridization. Our data indicate that the different degree of biofilm formation exhibited by the 11 CF isolates does not correlate with motility, ability to adhere to and invade A549 cells, or with the presence of flagella. On the other hand, among the CF isolates the StmPr1 gene was found only in two strains, both able to establish chronic lung infections in CF patients. Moreover, only four of the strains analyzed show a temperature-independent antibiotic-resistance profile, suggesting either a different origin of these strains or an intervening adaptation to host tissues.