Identification of a virulence locus encoding a second type III secretion system in Salmonella typhimurium.

Mapping the insertion points of 16 signature-tagged transposon mutants on the Salmonella typhimurium chromosome led to the identification of a 40-kb virulence gene cluster at minute 30.7. This locus is conserved among all other Salmonella species examined but is not present in a variety of other pathogenic bacteria or in Escherichia coli K-12. Nucleotide sequencing of a portion of this locus revealed 11 open reading frames whose predicted proteins encode components of a type III secretion system. To distinguish between this and the type III secretion system encoded by the inv/spa invasion locus known to reside on a pathogenicity island, we refer to the inv/spa locus as Salmonella pathogenicity island (SPI) 1 and the new locus as SPI2. SPI2 has a lower G+C content than that of the remainder of the Salmonella genome and is flanked by genes whose products share greater than 90% identity with those of the E. coli ydhE and pykF genes. Thus SPI2 was probably acquired horizontally by insertion into a region corresponding to that between the ydhE and pykF genes of E. coli. Virulence studies of SPI2 mutants have shown them to be attenuated by at least five orders of magnitude compared with the wild-type strain after oral or intraperitoneal inoculation of mice.

Enteropathogenic Escherichia coli contains a putative type III secretion system necessary for the export of proteins involved in attaching and effacing lesion formation.

Enteropathogenic Escherichia coli (EPEC) causes a characteristic histopathology in intestinal epithelial cells called the attaching and effacing lesion. Although the histopathological lesion is well described the bacterial factors responsible for it are poorly characterized. We have identified four EPEC chromosomal genes whose predicted protein sequences are similar to components of a recently described secretory pathway (type III) responsible for exporting proteins lacking a typical signal sequence. We have designated the genes sepA, sepB, sepC, and sepD (sep, for secretion of E. coli proteins). The predicted Sep polypeptides are similar to the Lcr (low calcium response) and Ysc (yersinia secretion) proteins of Yersinia species and the Mxi (membrane expression of invasion plasmid antigens) and Spa (surface presentation of antigens) regions of Shigella flexneri. Culture supernatants of EPEC strain E2348/69 contain several polypeptides ranging in size from 110 kDa to 19 kDa. Proteins of comparable size were recognized by human convalescent serum from a volunteer experimentally infected with strain E2348/69. A sepB mutant of EPEC secreted only the 110-kDa polypeptide and was defective in the formation of attaching and effacing lesions and protein-tyrosine phosphorylation in tissue culture cells. These phenotypes were restored upon complementation with a plasmid carrying an intact sepB gene. These data suggest that the EPEC Sep proteins are components of a type III secretory apparatus necessary for the export of virulence determinants.

Complete protection against P. berghei malaria upon heterologous prime/boost immunization against circumsporozoite protein employing Salmonella type III secretion system and Bordetella adenylate cyclase toxoid

Sterile immunity against malaria can be achieved by the induction of IFNgamma-producing CD8(+) T cells that target infected hepatocytes presenting epitopes of the circumsporozoite protein (CSP). In the present study we evaluate the protective efficacy of a heterologous prime/boost immunization protocol based on the delivery of the CD8(+) epitope of Plasmodium berghei CSP into the MHC class I presentation pathway, by either a type III secretion system of live recombinant Salmonella and/or by direct translocation of a recombinant Bordetella adenylate cyclase toxoid fusion (ACT-CSP) into the cytosol of professional antigen-presenting cells (APCs). A single intraperitoneal application of the recombinant ACT-CSP toxoid, as well as a single oral immunization with the Salmonella vaccine, induced a specific CD8(+) T cell response, which however conferred only a partial protection on mice against a subsequent sporozoite challenge. In contrast, a heterologous prime/boost vaccination with the live Salmonella followed by ACT-CSP led to a significant enhancement of the CSP-specific T cell response and induced complete protection in all vaccinated mice.

The finite range Wiener-Hopf integral equation and a boundary value problem in a waveguide /

"Contract No. AF33(616)-6079 Project No. 9-(13-6278) Task 40572. Sponsored by: Wright Air Development Center"

A priori prediction of roundoff error accumulation in the solution of a super-large geodetic normal equation system /

"June 1980."

Effector ExoU from the type III secretion system is an important modulator of gene expression in lung epithelial cells in response to Pseudomonas aeruginosa infection

Pseudomonas aeruginosa is an important pathogen in immunocompromised patients and secretes a diverse set of virulence factors that aid colonization and influence host cell defenses. An important early step in the establishment of infection is the production of type III-secreted effectors translocated into host cells by the bacteria. We used cDNA microarrays to compare the transcriptomic response of lung epithelial cells to P. aeruginosa mutants defective in type IV pili, the type III secretion apparatus, or in the production of specific type III-secreted effectors. Of the 18,000 cDNA clones analyzed, 55 were induced or repressed after 4 It of infection and could be classified into four different expression patterns. These include (i) host genes that are induced or repressed in a type III secretion-independent manner (32 clones), (ii) host genes induced specifically by ExoU (20 clones), and (iii) host genes induced in an ExoU-independent but type III secretion dependent manner (3 clones). In particular, ExoU was essential for the expression of immediate-early response genes, including the transcription factor c-Fos. ExoU-dependent gene expression was mediated in part by early and transient activation of the AN transcription factor complex. In conclusion, the present study provides a detailed insight into the response of epithelial cells to infection and indicates the significant role played by the type III virulence mechanism in the initial host response.

On the numerical solution of a Cauchy problem for the Laplace equation via a direct integral equation approach

We investigate the problem of determining the stationary temperature field on an inclusion from given Cauchy data on an accessible exterior boundary. On this accessible part the temperature (or the heat flux) is known, and, additionally, on a portion of this exterior boundary the heat flux (or temperature) is also given. We propose a direct boundary integral approach in combination with Tikhonov regularization for the stable determination of the temperature and flux on the inclusion. To determine these quantities on the inclusion, boundary integral equations are derived using Green’s functions, and properties of these equations are shown in an L2-setting. An effective way of discretizing these boundary integral equations based on the Nystr¨om method and trigonometric approximations, is outlined. Numerical examples are included, both with exact and noisy data, showing that accurate approximations can be obtained with small computational effort, and the accuracy is increasing with the length of the portion of the boundary where the additionally data is given.

Fast reconstruction of harmonic functions from Cauchy data using integral equation techniques

We consider the problem of stable determination of a harmonic function from knowledge of the solution and its normal derivative on a part of the boundary of the (bounded) solution domain. The alternating method is a procedure to generate an approximation to the harmonic function from such Cauchy data and we investigate a numerical implementation of this procedure based on Fredholm integral equations and Nyström discretization schemes, which makes it possible to perform a large number of iterations (millions) with minor computational cost (seconds) and high accuracy. Moreover, the original problem is rewritten as a fixed point equation on the boundary, and various other direct regularization techniques are discussed to solve that equation. We also discuss how knowledge of the smoothness of the data can be used to further improve the accuracy. Numerical examples are presented showing that accurate approximations of both the solution and its normal derivative can be obtained with much less computational time than in previous works.

On an indirect integral equation approach for stationary heat transfer in semi-infinite layered domains in R3 with cavities

Regions containing internal boundaries such as composite materials arise in many applications.We consider a situation of a layered domain in IR3 containing a nite number of bounded cavities. The model is stationary heat transfer given by the Laplace equation with piecewise constant conductivity. The heat ux (a Neumann condition) is imposed on the bottom of the layered region and various boundary conditions are imposed on the cavities. The usual transmission (interface) conditions are satised at the interface layer, that is continuity of the solution and its normal derivative. To eciently calculate the stationary temperature eld in the semi-innite region, we employ a Green's matrix technique and reduce the problem to boundary integral equations (weakly singular) over the bounded surfaces of the cavities. For the numerical solution of these integral equations, we use Wienert's approach [20]. Assuming that each cavity is homeomorphic with the unit sphere, a fully discrete projection method with super-algebraic convergence order is proposed. A proof of an error estimate for the approximation is given as well. Numerical examples are presented that further highlights the eciency and accuracy of the proposed method.

Nonlinear integral equation methods for the reconstruction of an acoustically sound-soft obstacle

The problem considered is that of determining the shape of a planar acoustically sound-soft obstacle from knowledge of the far-field pattern for one time-harmonic incident field. Two methods, which are based on the solution of a pair of integral equations representing the incoming wave and the far-field pattern, respectively, are proposed and investigated for finding the unknown boundary. Numerical resultsare included which show that the methods give accurate numerical approximations in relatively few iterations.

On a Conditional Cauchy-type Functional Equation Involving Powers

We solve the functional equation f(x^m + y) = f(x)^m + f(y) in the realm of polynomials with integer coefficients.

An Abstract Second Kind Fredholm Integral Equation with Degenerated Kernel

Mathematics Subject Classification: 44A40, 45B05