Plasmid maintenance functions of the large virulence plasmid of Shigella flexneri.

The large virulence plasmid pMYSH6000 of Shigella flexneri contains a replicon and a plasmid maintenance stability determinant (Stb) on adjacent SalI fragments. The presence of a RepFIIA replicon on the SalI C fragment was confirmed, and the complete sequence of the adjacent SalI O fragment was determined. It shows homology to part of the transfer (tra) operon of the F plasmid. Stb stabilizes a partition-defective P1 miniplasmid in Escherichia coli. A 1.1-kb region containing a homolog of the F trbH gene was sufficient to confer stability. However, the trbH open reading frame could be interrupted without impairing stability. Deletion analysis implicated the involvement of two small open reading frames, STBORF1 and STBORF2, that fully overlap trbH in the opposite direction. These open reading frames are closely related to the vagC and vagD genes of the Salmonella dublin virulence plasmid and to open reading frame pairs in the F trbH region and in the chromosomes of Dichelobacter nodosus and Haemophilus influenzae. Stb appears to promote better-than-random distribution of plasmid copies and is a plasmid incompatibility determinant. The F homolog does not itself confer stability but exerts incompatibility against the activity of the Stb system. Stb is likely to encode either an active partition system or a postsegregational killing system. It shows little similarity to previously studied plasmid stability loci, but the genetic organization of STBORF1 and STBORF2 resembles that of postsegregational killing mechanisms.     

Regulation of O-antigen chain length is required for Shigella flexneri virulence

It is shown that Shigella flexneri maintains genetic control over the modal chain length of the O-antigen polysaccharide chains of its lipopolysaccharide (LPS) molecules because such a distribution is required for virulence. The effect of altering O-antigen chain length on S. flexneri virulence was investigated by inserting a kanamycin (Km)-resistance cassette into the rol gene (controlling the modal O-antigen chain length distribution), and into the rfbD gene, whose product is needed for synthesis of dTDP-rhamnose (the precursor of rhamnose in the O-antigen). The mutations had the expected effect on LPS structure. The rol ::Km mutation was impaired in the ability to elicit keratoconjunctivitis, as determined by the Serény test. The rol ::Km and rfbD ::Km mutations prevented plaque formation on HeLa cells, but neither mutation affected the ability of S. flexneri to invade and replicate in HeLa cells. Microscopy of bacteria-infected HeLa cells stained with fluorescein isothiocyanate (FITC)-phalloidin demonstrated that both the rol ::Km and rfbD ::Km mutants were defective in F-actin tail formation: the latter mutant showed distorted F-actin tails. Plasma-membrane protrusions were occasionally observed. Investigation of the location of IcsA (required for F-actin tail formation) on the cell surface by immunofluorescence and immunogold electron microscopy showed that while most rol mutant bacteria produced little or no cell-surface IcsA, 10% resembled the parental bacterial cell (which had IcsA at one cell pole; the rfbD mutant had IcsA located over its entire cell surface although it was more concentrated at one end of the cell). That the O-antigen chains of the rol ::Km mutant did not mask the IcsA protein was demonstrated by using the endorhamnosidase activity of Sf6c phage to digest the O-antigen chains, and comparing untreated and Sf6c-treated cells by immunofluorescence with anti-IcsA serum.     

The virulence plasmid pWR100 and the repertoire of proteins secreted by the type III secretion apparatus of Shigella flexneri

Bacteria of Shigella spp. are the causative agents of shigellosis. The virulence traits of these pathogens include their ability to enter into epithelial cells and induce apoptosis in macrophages. Expression of these functions requires the Mxi-Spa type III secretion apparatus and the secreted IpaA-D proteins, all of which are encoded by a virulence plasmid. In wild-type strains, the activity of the secretion apparatus is tightly regulated and induced upon contact of bacteria with epithelial cells. To investigate the repertoire of proteins secreted by Shigella flexneri in conditions of active secretion, we determined the N-terminal sequence of 14 proteins that are secreted by a mutant in which secretion was deregulated. Sequencing of the virulence plasmid pWR100 of the S. flexneri strain M90T (serotype 5) has allowed us to identify the genes encoding these secreted proteins and suggests that approximately 25 proteins are secreted by the type III secretion apparatus. Analysis of the G+C content and the relative positions of genes and open reading frames carried by the plasmid, together with information concerning the localization and function of encoded proteins, suggests that pWR100 contains blocks of genes of various origins, some of which were initially carried by four different plasmids.     

Complete DNA sequence and gene analysis of the virulence plasmid pCP301 of Shigella flexneri 2a

Bacteria Shigella spp. are highly contagious, severely harmful and gram-negative facultative intracellular pathogens. They may cause shigellosis characterized by fever, dehydration and hematochezia in clinic, and shigellosis has been remaining a leading cause of infant mortality in the world. Shigella belongs to the family Enterobacteriaceae and the group Escherichiaeae, which are divided into four species and at least 47 serotypes: Shigella dysenteriae (13 serotypes), Shigella flexneri (15 …     

The stability region of the large virulence plasmid of Shigella flexneri encodes an efficient postsegregational killing system

The large virulence plasmid pMYSH6000 of Shigella flexneri contains a determinant that is highly effective in stabilizing otherwise unstable plasmids in Escherichia coli. Expression of two small contiguous genes, mvpA and mvpT (formerly termed STBORF1 and STBORF2), was shown to be sufficient for stability. Mutations in mvpT abolished plasmid stability, and plasmids expressing only mvpT killed the cells unless mvpA was supplied from a separate plasmid or from the host chromosome. When replication of a plasmid carrying the minimal mvp region was blocked, growth of the culture stopped after a short lag and virtually all of the surviving cells retained the plasmid. Thus, the mvp system stabilizes by a highly efficient postsegregational killing (PSK) mechanism, with mvpT encoding a cell toxin and mvpA encoding an antidote. The regions that surround the mvp genes in their original context have an inhibitory effect that attenuates plasmid stabilization and PSK. The region encompassing the mvp genes also appears to contain an additional element that can aid propagation of a pSC101-based plasmid under conditions where replication initiation is marginal. However, this appears to be a relatively nonspecific effect of DNA insertion into the plasmid vector.     

Plasmid partition system of the P1par family from the pWR100 virulence plasmid of Shigella flexneri

P1par family members promote the active segregation of a variety of plasmids and plasmid prophages in gram-negative bacteria. Each has genes for ParA and ParB proteins, followed by a parS partition site. The large virulence plasmid pWR100 of Shigella flexneri contains a new P1par family member: pWR100par. Although typical parA and parB genes are present, the putative pWR100parS site is atypical in sequence and organization. However, pWR100parS promoted accurate plasmid partition in Escherichia coli when the pWR100 Par proteins were supplied. Unique BoxB hexamer motifs within parS define species specificities among previously described family members. Although substantially different from P1parS from the P1 plasmid prophage of E. coli, pWR100parS has the same BoxB sequence. As predicted, the species specificity of the two types proved identical. They also shared partition-mediated incompatibility, consistent with the proposed mechanistic link between incompatibility and species specificity. Among several informative sequence differences between pWR100parS and P1parS is the presence of a 21-bp insert at the center of the pWR100parS site. Deletion of this insert left much of the parS activity intact. Tolerance of central inserts with integral numbers of helical DNA turns reflects the critical topology of these sites, which are bent by binding the host IHF protein.     

Surprising dependence on postsegregational killing of host cells for maintenance of the large virulence plasmid of Shigella flexneri

Low-copy-number plasmids all encode multiple systems to ensure their propagation, including replication, partition (active segregation), and postsegregational killing (PSK) systems. PSK systems kill those rare cells that lose the plasmid due to replication or segregation errors. PSK systems should not be used as the principle means of maintaining the plasmid. The metabolic cost of killing the many cured cells that would arise from random plasmid segregation is far too high. Here we describe an interesting exception to this rule. Maintenance of the large virulence plasmid of Shigella flexneri is highly dependent on one of its PSK systems, mvp, at 37 degrees C, the temperature experienced during pathogenesis. At 37 degrees C, the plasmid is very unstable and mvp efficiently kills the resulting cured bacterial cells. This imposes a major growth disadvantage on the virulent bacterial population. The systems that normally ensure accurate plasmid replication and segregation are attenuated or overridden at 37 degrees C. At 30 degrees C, a temperature encountered by Shigella in the outside environment, the maintenance systems function normally and the plasmid is no longer dependent on mvp. We discuss why the virulent pathogen tolerates this self-destructive method of propagation at the temperature of infection.     

Identification of functional regions within invasion plasmid antigen C (IpaC) of Shigella flexneri

Shigella flexneri causes bacillary dysentery with symptoms resulting from the inflammation that accompanies bacterial entry into the cells of the colonic epithelium. The effectors of S. flexneri invasion are the Ipa proteins, particularly IpaB and IpaC, which are secreted at the host-pathogen interface following bacterial contact with a host cell. Of the purified Ipa proteins, only IpaC has been shown to possess quantifiable in vitro activities that are related to cellular invasion. In this study, ipaC deletion mutants were generated to identify functional regions within the IpaC protein. From these data, we now know that the N-terminus and an immunogenic central region are not required for IpaC-dependent enhancement of cellular invasion by S. flexneri. However, to restore invasiveness to an ipaC null mutant of S. flexneri, the N-terminus is essential, because IpaC mutants lacking the N-terminus are not secreted by the bacterium. Deletion of the central hydrophobic region eliminates IpaC's ability to interact with phospholipid membranes, and fusion of this region to a modified form of green fluorescent protein converts it into an efficient membrane-associating protein. Meanwhile, deletion of the C-terminus eliminates the mutant protein's ability to establish protein-protein contacts with full-length IpaC. Interestingly, the mutant form of ipaC that restores partial invasiveness to the S. flexneri ipaC null mutant also restores full contact-mediated haemolysis activity to this bacterium. These data support a model in which IpaC possesses a distinct functional organization that is important for bacterial invasion. This information will be important in defining the precise role of IpaC in S. flexneri pathogenesis and in exploring the potential effects of purified IpaC at mucosal surfaces.     

Adhesion and invasion of a mutant Shigella flexneri to an eukaryotic cell line in absence of the 220-kb virulence plasmid

A Shigella flexneri strain, cured of the large 220-kb virulence plasmid, expresses adhering and invading ability in confluent monolayers of HeLa cells similar to its parent strain. Invasion by both the parent and the cured strains resulted in alteration of the monomeric actin (G) in the total actin pool of HeLa cells. Other indicators of invasive characteristics of virulent Shigella strains such as production of keratoconjunctivitis in guinea pig eye in vivo, Congo red binding and expression of contact hemolysin however, indicated loss of invasive properties in the plasmid cured strain. Further, pretreatment of bacterial cells with para-bromophenacyl bromide (p-BPB), a specific chemical inhibitor of phospholipase A, adversely affected adhesion to and invasion of HeLa cells in vitro, irrespective of the presence of the 220-kb plasmid indicating the possible involvement of the enzyme phospholipase A in the invasion process. Adherence of both the strains to guinea pig colonic epithelial cells (CECs) in vitro was reduced significantly on pretreatment of bacteria or CECs with p-BPB. Expression of exocellular enzymes viz. protease, elastase, phospholipase A and phospholipase C were not related to the large plasmid.     

Multicopy icsA is able to suppress the virulence defect caused by the wzz(SF) mutation in Shigella flexneri

The lipopolysaccharides (LPS) of Shigella flexneri are important for virulence and their O antigen (Oag) polysaccharide chains affect IcsA (VirG)-mediated actin-based motility (ABM) within mammalian cells. S. flexneri 2a 2457T has smooth LPS whose Oag chains have two modal lengths (short (S)-type and very long (VL)-type), and has IcsA predominantly located at one pole on its cell surface. A S. flexneri 2457T wzz(SF) mutant (RMA696) has VL-type Oag but not S-type Oag chains, less IcsA detectable by immunofluorescence on its cell surface, reduced virulence and defective ABM. Introduction of a plasmid encoding IcsA into S. flexneri wzz(SF) showed that multicopy icsA could suppress the virulence defects (Sereny reaction, HeLa cell monolayer plaquing, and F-actin comet tail formation) caused by the wzz(SF) mutation suggesting that the VL-type Oag chains were masking IcsA and limiting the amount available to initiate ABM.     

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-).     

Intracellular mobility of plasmid DNA is limited by the ParA family of partitioning systems

The highly conserved ParA family of partitioning systems is responsible for positioning DNA and protein complexes in bacteria. In Escherichia coli , plasmids that rely upon these systems are positioned at mid-cell and are repositioned at the quarter-cell positions after replication. How they remain fixed at these positions throughout the cell cycle is unknown. We use fluorescence recovery after photobleaching and time-lapse microscopy to measure plasmid mobility in living E. coli cells. We find that a minimalized version of plasmid RK2 that lacks its Par system is highly mobile, that the intact RK2 plasmid is relatively immobile, and that the addition of a Par system to the minimalized RK2 plasmid limits its mobility to that of the intact RK2. Mobility is thus the default state, and Par systems are required not only to position plasmids, but also to hold them at these positions. The intervention of Par systems is required continuously throughout the cell cycle to restrict plasmid movement that would, if unrestricted, subvert the segregation process. Our results reveal an important function for Par systems in plasmid DNA segregation that is likely to be conserved in bacteria.     

Molecular cloning and characterization of chromosomal virulence region kcpA of Shigella flexneri

In Shigella flexneri, in addition to several well-recognized plasmid-borne virulence loci, at least three genetic loci implicated in pathogenesis have been recognized on the chromosome. To understand more about the pathogenesis of bacillary dysentery at a molecular level, the genetically recognized but previously unidentified KcpA region (one of the chromosomal regions near purE) was cloned and sequenced. A single translatable open reading frame encoding a 12310 Dalton protein corresponding to the minicell product was found. Immunofluorescence microscopy, as well as optical and electron microscopic comparison of tissue-cultured cells and guinea-pigs' eyes infected with wild-type or kcpA mutant bacteria, revealed that the kcpA product is required by invading bacteria for spread into adjacent cells.