Marcia B. Goldberg

Complete Genome Sequence and Comparative Genomics of Shigella flexneri Serotype 2a Strain 2457T

ABSTRACT We determined the complete genome sequence of Shigella flexneri serotype 2a strain 2457T (4,599,354 bp). Shigella species cause >1 million deaths per year from dysentery and diarrhea and have a lifestyle that is markedly different from those of closely related bacteria, including Escherichia coli. The genome exhibits the backbone and island mosaic structure of E. coli pathogens, albeit with much less horizontally transferred DNA and lacking 357 genes present in E. coli. The strain is distinctive in its large complement of insertion sequences, with several genomic rearrangements mediated by insertion sequences, 12 cryptic prophages, 372 pseudogenes, and 195 S. flexneri-specific genes. The 2457T genome was also compared with that of a recently sequenced S. flexneri 2a strain, 301. Our data are consistent with Shigella being phylogenetically indistinguishable from E. coli. The S. flexneri-specific regions contain many genes that could encode proteins with roles in virulence. Analysis of these will reveal the genetic basis for aspects of this pathogenic organisms distinctive lifestyle that have yet to be explained.

N-WASP deficiency reveals distinct pathways for cell surface projections and microbial actin-based motility.

The Wiskott–Aldrich syndrome protein (WASP) family of molecules integrates upstream signalling events with changes in the actin cytoskeleton. N-WASP has been implicated both in the formation of cell-surface projections (filopodia) required for cell movement and in the actin-based motility of intracellular pathogens. To examine N-WASP function we have used homologous recombination to inactivate the gene encoding murine N-WASP. Whereas N-WASP-deficient embryos survive beyond gastrulation and initiate organogenesis, they have marked developmental delay and die before embryonic day 12. N-WASP is not required for the actin-based movement of the intracellular pathogen Listeria but is absolutely required for the motility of Shigella and vaccinia virus. Despite these distinct defects in bacterial and viral motility, N-WASP-deficient fibroblasts spread by using lamellipodia and can protrude filopodia. These results imply a crucial and non-redundant role for N-WASP in murine embryogenesis and in the actin-based motility of certain pathogens but not in the general formation of actin-containing structures.

Actin-Based Motility of Intracellular Microbial Pathogens

SUMMARY A diverse group of intracellular microorganisms, including Listeria monocytogenes, Shigella spp., Rickettsia spp., and vaccinia virus, utilize actin-based motility to move within and spread between mammalian host cells. These organisms have in common a pathogenic life cycle that involves a stage within the cytoplasm of mammalian host cells. Within the cytoplasm of host cells, these organisms activate components of the cellular actin assembly machinery to induce the formation of actin tails on the microbial surface. The assembly of these actin tails provides force that propels the organisms through the cell cytoplasm to the cell periphery or into adjacent cells. Each of these organisms utilizes preexisting mammalian pathways of actin rearrangement to induce its own actin-based motility. Particularly remarkable is that while all of these microbes use the same or overlapping pathways, each intercepts the pathway at a different step. In addition, the microbial molecules involved are each distinctly different from the others. Taken together, these observations suggest that each of these microbes separately and convergently evolved a mechanism to utilize the cellular actin assembly machinery. The current understanding of the molecular mechanisms of microbial actin-based motility is the subject of this review.

Complete DNA Sequence and Analysis of the Large Virulence Plasmid of Shigella flexneri

ABSTRACT The complete sequence analysis of the 210-kb Shigella flexneri 5a virulence plasmid was determined.Shigella spp. cause dysentery and diarrhea by invasion and spread through the colonic mucosa. Most of the knownShigella virulence determinants are encoded on a large plasmid that is unique to virulent strains of Shigella and enteroinvasive Escherichia coli; these known genes account for approximately 30 to 35% of the virulence plasmid. In the complete sequence of the virulence plasmid, 286 open reading frames (ORFs) were identified. An astonishing 153 (53%) of these were related to known and putative insertion sequence (IS) elements; no known bacterial plasmid has previously been described with such a high proportion of IS elements. Four new IS elements were identified. Fifty putative proteins show no significant homology to proteins of known function; of these, 18 have a G+C content of less than 40%, typical of known virulence genes on the plasmid. These 18 constitute potentially unknown virulence genes. Two alleles of shet2 and five alleles ofipaH were also identified on the plasmid. Thus, the plasmid sequence suggests a remarkable history of IS-mediated acquisition of DNA across bacterial species. The complete sequence will permit targeted characterization of potential new Shigellavirulence determinants.

Disruption of IcsP, the major Shigella protease that cleaves IcsA, accelerates actin‐based motility

Shigella pathogenesis involves bacterial invasion of colonic epithelial cells and movement of bacteria through the cytoplasm and into adjacent cells by means of actin‐based motility. The Shigella protein IcsA (VirG) is unipolar on the bacterial surface and is both necessary and sufficient for actin‐based motility. IcsA is inserted into the outer membrane as a 120‐kDa polypeptide that is subsequently slowly cleaved, thereby releasing the 95‐kDa amino‐terminal portion into the culture supernatant. IcsP, the major Shigella protease that cleaves IcsA, was identified and cloned. It has significant sequence similarity to the E. coli serine proteases, OmpP and OmpT. Disruption of icsP in serotype 2a S. flexneri leads to a marked reduction in IcsA cleavage, increased amounts of IcsA associated with the bacterium and altered distribution of IcsA on the bacterial surface. The icsP mutant displays significantly increased rates of actin‐based motility, with a mean speed 27% faster than the wild‐type strain; moreover, a significantly greater percentage of the icsP mutant moves in the cytoplasm. Yet, plaque formation on epithelial monolayers by the mutant was not altered detectably. These data suggest that IcsA, and not a host protein, is limiting in the rate of actin‐based motility of wild‐type serotype 2a S. flexneri.

Bacterial subversion of host innate immune pathways.

Defense and Counter-Defense Provided a pathogen can enter the body and survive coughing and spluttering, peristalsis, and mucus, the first active responses the host evokes to an invading organism will be at the level of the first cell encountered, well before classical cellular immunity and antibody responses are initiated. Randow et al. (p. 701) review the range of intracellular defenses against incoming pathogens and describe how compartmental boundaries within the cell provide multiple levels at which pathogens can be thwarted in their attempts to subjugate the cell to do their bidding. Baxt et al. (p. 697) review the range of evasion tactics that bacterial pathogens can summon to counter host repulsion and establish a niche in which to replicate and ensure onward transmission. The pathogenesis of infection is a continuously evolving battle between the human host and the infecting microbe. The past decade has brought a burst of insights into the molecular mechanisms of innate immune responses to bacterial pathogens. In parallel, multiple specific mechanisms by which microorganisms subvert these host responses have been uncovered. This Review highlights recently characterized mechanisms by which bacterial pathogens avoid killing by innate host responses, including autophagy pathways and a proinflammatory cytokine transcriptional response, and by the manipulation of vesicular trafficking to avoid the toxicity of lysosomal enzymes.

The unipolar Shigella surface protein IcsA is targeted directly to the bacterial old pole: IcsP cleavage of IcsA occurs over the entire bacterial surface

Shigella flexneri is an intracellular pathogen that is able to move within the cytoplasm of infected cells by the continual assembly of actin onto one pole of the bacterium. IcsA, an outer membrane protein, is localized to the old pole of the bacterium and is both necessary and sufficient for actin assembly. IcsA is slowly cleaved from the bacterial surface by the protease IcsP (SopA). Absence of IcsP leads to an alteration in the distribution of surface IcsA, such that the polar cap is maintained and some IcsA is distributed along the lateral walls of the bacillus. The mechanism of unipolar localization of IcsA and the role of IcsP in its unipolar localization are incompletely understood. Here, we demonstrate that cleavage of IcsA occurs exclusively in the outer membrane and that IcsP is localized to the outer membrane. In addition, we show that IcsA at the old pole is susceptible to cleavage by IcsP and that native IcsP is active at the pole. Taken together, these data indicate that IcsP cleaves IcsA over the entire bacterial surface. Finally, we show that, immediately after induction from a tightly regulated promoter, IcsA is expressed exclusively at the old pole in both the icsP−icsA− and the icsA− background. These data demonstrate that unipolar localization of IcsA results from its direct targeting to the pole, followed by its diffusion laterally in the outer membrane.

Requirement for YaeT in the Outer Membrane Assembly of Autotransporter Proteins

Autotransporters constitute the largest group of secreted proteins in gram-negative bacteria. Autotransporter secretion involves the insertion of a carboxy-terminal beta barrel into and the translocation of an amino-terminal domain across the outer membrane. Here, we demonstrate that secretion of autotransporters from several organisms requires the outer membrane assembly factor YaeT.

IcsA, a polarly localized autotransporter with an atypical signal peptide, uses the Sec apparatus for secretion, although the Sec apparatus is circumferentially distributed

Asymmetric localization of proteins is essential to many biological functions of bacteria. Shigella IcsA, an outer membrane protein, is localized to the old pole of the bacillus, where it mediates assembly of a polarized actin tail during infection of mammalian cells. Actin tail assembly provides the propulsive force for intracellular movement and intercellular dissemination. Localization of IcsA to the pole is independent of the amino‐terminal signal peptide (Charles, M., Perez, M., Kobil, J.H., and Goldberg, M.B., 2001, Proc Natl Acad Sci USA 98: 9871–9876) suggesting that IcsA targeting occurs in the bacterial cytoplasm and that its secretion across the cytoplasmic membrane occurs only at the pole. Here, we characterize the mechanism by which IcsA is secreted across the cytoplasmic membrane. We present evidence that IcsA requires the SecA ATPase and the SecYEG membrane channel (translocon) for secretion. Our data suggest that YidC is not required for IcsA secretion. Furthermore, we show that polar localization of IcsA is independent of SecA. Finally, we demonstrate that while IcsA requires the SecYEG translocon for secretion, components of this apparatus are uniformly distributed within the membrane. Based on these data, we propose a model for coordinate polar targeting and secretion of IcsA at the bacterial pole.

Periplasmic Transit and Disulfide Bond Formation of the Autotransported Shigella Protein IcsA

The Shigella outer membrane protein IcsA belongs to the family of type V secreted (autotransported) virulence factors. Members of this family mediate their own translocation across the bacterial outer membrane: the carboxy-terminal beta domain forms a beta barrel channel in the outer membrane through which the amino-terminal alpha domain passes. IcsA, which is localized at one pole of the bacterium, mediates actin assembly by Shigella, which is essential for bacterial intracellular movement and intercellular dissemination. Here, we characterize the transit of IcsA across the periplasm during its secretion. We show that an insertion in the dsbB gene, whose gene product mediates disulfide bond formation of many periplasmic intermediates, does not affect the surface expression or unipolar targeting of IcsA. However, IcsA forms one disulfide bond in the periplasm in a DsbA/DsbB-dependent fashion. Furthermore, cellular fractionation studies reveal that IcsA has a transient soluble periplasmic intermediate. Our data also suggest that IcsA is folded in a proteinase K-resistant state in the periplasm. From these data, we propose a novel model for the secretion of IcsA that may be applicable to other autotransported proteins.