Possible mechanisms underlying the slow lactose fermentation phenotype in Shigella spp.

A Southern hybridization analysis revealed that the region homologous to Escherichia coli lacZ was present on the chromosomal DNAs of beta-galactosidase-positive Shigella strains, such as Shigella dysenteriae serovar 1 and Shigella sonnei strains, whereas this region was absent from chromosomal DNAs of beta-galactosidase-negative strains of Shigella flexneri and Shigella boydii. We found that the lacY-A region was deficient in S. dysenteriae serovar 1 and believe that this is the reason for the slow fermentation of lactose by this strain. S. sonnei strains possessed the region which hybridized with E. coli lacY-A despite their slow hydrolysis of lactose. The whole lactose-fermenting region was cloned from S. sonnei and compared with the cloned lac operon of E. coli K-12. Both clones directed the synthesis of beta-galactosidase in an E. coli K-12 strain lacking indigenous beta-galactosidase activity (strain JM109-1), and we observed no difference in the expression of beta-galactosidase activity in S. sonnei and E. coli. However, E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei exhibited the slow lactose fermentation phenotype like the parental strain. S. sonnei strains had no detectable lactose permease activities. E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei had a detectable permease activity, possibly because of the multicopy nature of the cloned genes, but this permease activity was much lower than that of strain JM109-1 harboring the lac operon of E. coli K-12. From these results we concluded that slow lactose fermentation by S. sonnei is due to weak lactose permease activity.     

Possible mechanisms underlying the slow lactose fermentation phenotype in Shigella spp.

A Southern hybridization analysis revealed that the region homologous to Escherichia coli lacZ was present on the chromosomal DNAs of beta-galactosidase-positive Shigella strains, such as Shigella dysenteriae serovar 1 and Shigella sonnei strains, whereas this region was absent from chromosomal DNAs of beta-galactosidase-negative strains of Shigella flexneri and Shigella boydii. We found that the lacY-A region was deficient in S. dysenteriae serovar 1 and believe that this is the reason for the slow fermentation of lactose by this strain. S. sonnei strains possessed the region which hybridized with E. coli lacY-A despite their slow hydrolysis of lactose. The whole lactose-fermenting region was cloned from S. sonnei and compared with the cloned lac operon of E. coli K-12. Both clones directed the synthesis of beta-galactosidase in an E. coli K-12 strain lacking indigenous beta-galactosidase activity (strain JM109-1), and we observed no difference in the expression of beta-galactosidase activity in S. sonnei and E. coli. However, E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei exhibited the slow lactose fermentation phenotype like the parental strain. S. sonnei strains had no detectable lactose permease activities. E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei had a detectable permease activity, possibly because of the multicopy nature of the cloned genes, but this permease activity was much lower than that of strain JM109-1 harboring the lac operon of E. coli K-12. From these results we concluded that slow lactose fermentation by S. sonnei is due to weak lactose permease activity.     

The two-component regulatory system ompR-envZ controls the virulence of Shigella flexneri.

In Shigella flexneri, the ompB locus (containing the ompR and envZ genes) was found to modulate expression of the vir genes, which are responsible for invasion of epithelial cells. vir gene expression was markedly enhanced under conditions of high osmolarity (300 mosM), similar to that encountered in tissues both extra- and intracellularly. Two ompB mutants were constructed and tested for virulence and for osmotic regulation of vir genes. An envZ::Tn10 mutant remained invasive, although its virulence was significantly decreased as a result of its inability to survive intracellularly. By using a vir::lac operon fusion, this mutation was shown to decrease beta-galactosidase expression both in low- and high-osmolarity conditions but did not affect vir expression in response to changes in osmolarity. A delta ompB deletion mutant was also constructed via allelic exchange with an in vitro-mutagenized ompB locus of Escherichia coli. This mutation severely impaired virulence and abolished expression of the vir::lac fusion in both low- and high-osmolarity conditions. Therefore, a two-component regulatory system modulates virulence according to environmental conditions. In addition, the mutation affecting a spontaneous avirulent variant of S. flexneri serotype 5, M90T, has been mapped at the ompB locus and was complemented by the cloned E. coli ompB locus. Introduction of the vir::lac fusion into this mutant did not result in the expression of beta-galactosidase (Lac-).     

Attenuation of the sensing capabilities of PhoQ in transition to obligate insect-bacterial association

Sodalis glossinidius, a maternally inherited endosymbiont of the tsetse fly, maintains genes encoding homologues of the PhoP-PhoQ two-component regulatory system. This two-component system has been extensively studied in facultative bacterial pathogens and is known to serve as an environmental magnesium sensor and a regulator of key virulence determinants. In the current study, we show that the inactivation of the response regulator, phoP, renders S. glossinidius sensitive to insect derived cationic antimicrobial peptides (AMPs). The resulting mutant strain displays reduced expression of genes involved in the structural modification of lipid A that facilitates resistance to AMPs. In addition, the inactivation of phoP alters the expression of type-III secretion system (TTSS) genes encoded within three distinct chromosomal regions, indicating that PhoP-PhoQ also serves as a master regulator of TTSS gene expression. In the absence of phoP, S. glossinidius is unable to superinfect either its natural tsetse fly host or a closely related hippoboscid louse fly. Furthermore, we show that the S. glossinidius PhoQ sensor kinase has undergone functional adaptations that result in a substantially diminished ability to sense ancestral signals. The loss of PhoQ's sensory capability is predicted to represent a novel adaptation to the static symbiotic lifestyle, allowing S. glossinidius to constitutively express genes that facilitate resistance to host derived AMPs.     

Signal integration by the Cpx-envelope stress system

The Cpx-envelope stress system coordinates the expression and assembly of surface structures important for the virulence of Gram-negative pathogenic bacteria. It is comprised of the membrane-anchored sensor kinase CpxA, the cytosolic response regulator CpxR and the accessory protein CpxP. Characteristic of the group of two-component systems, the Cpx system responds to a broad range of stimuli including pH, salt, metals, lipids and misfolded proteins that cause perturbation in the envelope. Moreover, the Cpx system has been linked to inter-kingdom signalling and bacterial cell death. However, although signal specificity has been assumed, for most signals the mechanism of signal integration is not understood. Recent structural and functional studies provide the first insights into how CpxP inhibits CpxA and serves as sensor for misfolded pilus subunits, pH and salt. Here, we summarize and reflect on the current knowledge on signal integration by the Cpx-envelope stress system.     

Shigella chromosomal IpaH proteins are secreted via the type III secretion system and act as effectors

Shigella possess 220 kb plasmid, and the major virulence determinants, called effectors, and the type III secretion system (TTSS) are exclusively encoded by the plasmid. The genome sequences of S. flexneri strains indicate that several ipaH family genes are located on both the plasmid and the chromosome, but whether their chromosomal IpaH cognates can be secreted from Shigella remains unknown. Here we report that S. flexneri strain, YSH6000 encodes seven ipaH cognate genes on the chromosome and that the IpaH proteins are secreted via the TTSS. The secretion kinetics of IpaH proteins by bacteria, however, showed delay compared with those of IpaB, IpaC and IpaD. Expression of the each mRNA of ipaH in Shigella was increased after bacterial entry into epithelial cells, and the IpaH proteins were secreted by intracellular bacteria. Although individual chromosomal ipaH deletion mutants showed no appreciable changes in the pathogenesis in a mouse pulmonary infection model, the DeltaipaH-null mutant, whose chromosome lacks all ipaH genes, was attenuated to mice lethality. Indeed, the histological examination for mouse lungs infected with the DeltaipaH-null showed a greater inflammatory response than induced by wild-type Shigella, suggesting that the chromosomal IpaH proteins act synergistically as effectors to modulate the host inflammatory responses.     

IcsB,secreted via the type III secretion system,is chaperoned by IpgA and required at the post-invasion stage of Shigella pathogenicity

Shigella deliver a subset of effector proteins such as IpaA, IpaB and IpaC via the type III secretion system (TTSS) into host cells during the infection of colonic epithelial cells. Many bacterial effectors including some from Shigella require specific chaperones for protection from degradation and targeting to the TTSS. In this study, we have investigated the role of the icsB gene located upstream of the ipaBCDA operon in Shigella infection because the role of IcsB as a virulence factor remains unknown. Here, we found that the IcsB protein is secreted via the TTSS of Shigella in vitro and in vivo. We show that IpgA protein encoded by ipgA, the gene immediately downstream of icsB, serves as the chaperone required for the stabilization and secretion of IcsB. We have shown that IcsB binds to IpgA in bacterial cytosol and the binding site is in the middle of the IcsB protein. Intriguingly, although its significance in Shigella pathogenicity is as yet unclear, the icsB gene can be read-through into the ipgA gene to create a translational fusion protein. Furthermore, the contribution of IcsB to the pathogenicity of Shigella was demonstrated by plaque-forming assay and the Sereny test. The ability of the icsB mutant to form plaques was greatly reduced compared with that of the wild type in MDCK cell monolayers. Furthermore, when guinea pig eyes were infected with a non-polar icsB mutant, the bacteria failed to provoke keratoconjunctivitis. These results suggest that IcsB is secreted via the TTSS, chaperoned by IpgA, and required at the post-invasion stage of Shigella pathogenicity     

Accumulation of the F plasmid TraJ protein in cpx mutants of Escherichia coli.

We report here studies of the cellular control of F plasmid TraJ protein levels, focusing on the effects of chromosomal cpx mutations. The principal conclusion from our results is that the cpx mutations impair accumulation of the TraJ protein, thereby reducing tra gene expression. We measured TraJ activity in vivo by expression of a traY'-'lacZ fusion gene and TraJ protein by immuno-overlay blot. In strains with normal TraJ levels, traY expression and donor-related functions were reduced in cells carrying any of four cpxA mutations. In the strain background used to isolate cpx mutants, these reductions were especially evident in cells grown to high density, when traY expression and donor activity both increased in cpx+ cells. In each of the four cpxA mutants tested, TraJ levels were lower than in the otherwise isogenic cpxA+ strain. In cells grown to high density, the differences ranged from 4-fold in the cpxA6 strain to > 10-fold in the cpxA2, cpxA5, and cpxA9 strains. The cpxA2 mutation had little or no effect on traY expression or on donor-related functions when TraJ was present in excess of its limiting level in F' or Hfr cells or on a mutant traY promoter whose expression in vivo was independent of TraJ.