Biomedical subjects

Michael Hensel

Publications and source records attributed to Michael Hensel.

At least 19 recordsLinked to original sources

PubMed2006

Regulation of Salmonella pathogenicity island 2 genes by independent environmental signals.

The function of a type III secretion system (T3SS) encoded by Salmonella pathogenicity island 2 (SPI2) is essential for the intracellular lifestyle of Salmonella enterica serovar Typhimurium. Expression of SPI2 genes is induced within the Salmonella-containing vacuole (SCV) inside host cells and is controlled by the SsrAB two-component system. However, the nature of the signals leading to expression of SPI2 genes is controversial. Here we report that expression of SPI2 genes can be induced independently by two different environmental stimuli. Exposure of bacteria to slightly acidic pH was sufficient to induce a rapid up-regulation of SPI2 genes. In contrast, limitation of inorganic phosphate (P(i)) in the growth media led to activation of SPI2 genes in the late exponential growth phase and was independent of the media pH. Limitation of P(i) induced an over-expression of sensor protein SsrB. Response to both environmental stimuli required a functional SsrAB system. In vivo analyses indicated that limitation of P(i) is also encountered by intracellular Salmonella. Our observations allow the integration of previous disparate reports on the induction of SPI2 genes.

Adaptation, Physiological↗

PubMed2006

Functional dissection of SseF, a type III effector protein involved in positioning the salmonella-containing vacuole.

Intracellular replication of Salmonella enterica requires the formation of a unique organelle termed Salmonella-containing vacuole (SCV). The type III secretion system (T3SS) encoded by Salmonella Pathogenicity Island 2 (SPI2-T3SS) has a crucial role in the formation and maintenance of the SCV. The SPI2-T3SS translocates a large number of effector proteins that interfere with host cell functions such as microtubule-dependent transport. We investigated the function of the effector SseF and observed that this protein is required to maintain the SCV in a juxtanuclear position in infected epithelial cells. The formation of juxtanuclear clusters of replicating Salmonella required the recruitment of dynein to the SCV but SseF-deficient strains were highly reduced in dynein recruitment to the SCV. We performed a functional dissection of SseF and defined domains that were important for translocation and the specific effector functions of this protein. Of particular importance was a hydrophobic domain in the C-terminal half that contains three putative transmembrane (TM) helices. Deletion of one of these TM helices ablated the effector functions of SseF. We observed that this domain was essential for the proper intracellular positioning of the SCV to a juxtanuclear, Golgi-associated localization. These data show that SseF, in concert with the effector proteins SifA and SseG mediate the precise positioning of the SCV by differentially modulating the recruitment of microtubule motor proteins to the SCV.

Bacterial Proteins↗

PubMed2006

Intracellular Salmonella enterica redirect exocytic transport processes in a Salmonella pathogenicity island 2-dependent manner.

During intracellular life, Salmonella enterica proliferate within a specialized membrane compartment, the Salmonella-containing vacuole (SCV), and interfere with the microtubule cytoskeleton and cellular transport. To characterize the interaction of intracellular Salmonella with host cell transport processes, we utilized various model systems to follow microtubule-dependent transport. The vesicular stomatitis virus glycoprotein (VSVG) is a commonly used marker to follow protein transport from the Golgi to the plasma membrane. Using a VSVG-GFP fusion protein, we observed that virulent intracellular Salmonella alter exocytotic transport and recruit exocytotic transport vesicles to the SCV. This virulence function was dependent on the function of the type III secretion system encoded by Salmonella Pathogenicity Island 2 (SPI2) and more specifically on a subset of SPI2 effector proteins. Furthermore, the Golgi to plasma membrane traffic of the shingolipid C(5)-ceramide was redirected to the SCV by virulent Salmonella. We propose that Salmonella modulates the biogenesis of the SCV by deviating this compartment from the default endocytic pathway to an organelle that interacts with the exocytic pathway. This observation might reveal a novel element of the intracellular survival and replication strategy of Salmonella.

Animals↗

PubMed2006

Manipulating cellular transport and immune responses: dynamic interactions between intracellular Salmonella enterica and its host cells.

Intracellular survival and replication within eukaryotic host cells is of central importance for the pathogenesis of infections caused by Salmonella enterica. Intracellular Salmonella translocates a set of effector proteins by means of a type III secretion system (T3SS) encoded by Salmonella pathogenicity island 2 (SPI2) that manipulates normal host-cell functions. Intracellular survival and replication is linked to the function of the SPI2-T3SS, but recent observations show that many additional cellular functions are targeted by this virulence system. In this review, we focus on the recent observations on the interference of intracellular Salmonella with functions of the innate and adaptive immune system and the modification of endocytic and exocytic cellular transport. The common molecular basis of the different SPI2-dependent phenotypes could be the interference with cellular transport along microtubules.

Actin Cytoskeleton↗

PubMed2005

Recombinant vaccines based on translocated effector proteins of Salmonella Pathogenicity Island 2.

Attenuated live Salmonella enterica are useful carriers for the delivery of heterologous antigens for vaccination. Effector proteins translocated by type III secretion systems (T3SS) of Salmonella have been successfully utilized for antigen delivery. Here we investigated the use of effector proteins of the T3SS encoded by Salmonella Pathogenicity Island 2 (SPI2). We observed that the effector protein SseF is suitable for delivery of various fusion proteins with heterologous antigens. The efficiency of this carrier protein was demonstrated in vaccination experiments with fusion proteins with Listeria monocytogenes protective antigens. SseF can thus be used as a versatile vehicle for translocation of heterologous proteins for vaccination.

Animals↗

PubMed2005

Formation of a novel surface structure encoded by Salmonella Pathogenicity Island 2.

The type III secretion system (T3SS) encoded by Salmonella Pathogenicity Island 2 (SPI2) is essential for virulence and intracellular proliferation of Salmonella enterica. We have previously identified SPI2-encoded proteins that are secreted and function as a translocon for the injection of effector proteins. Here, we describe the formation of a novel SPI2-dependent appendage structure in vitro as well as on the surface of bacteria that reside inside a vacuole of infected host cells. In contrast to the T3SS of other pathogens, the translocon encoded by SPI2 is only present singly or in few copies at one pole of the bacterial cell. Under in vitro conditions, appendages are composed of a filamentous needle-like structure with a diameter of 10 nm that was sheathed with secreted protein. The formation of the appendage in vitro is dependent on acidic media conditions. We analyzed SPI2-encoded appendages in infected cells and observed that acidic vacuolar pH was not required for induction of SPI2 gene expression, but was essential for the assembly of these structures and their function as translocon for delivery of effector proteins.

Animals↗

PubMed2005

Evaluation of an intracellular-activated promoter for the generation of live Salmonella recombinant vaccines.

Salmonella enterica serovar Typhimurium is a versatile host organism for the generation of recombinant live vaccines for mucosal immunization. We investigated the performance of an intracellular-activated promoter for expression of heterologous antigens by Salmonella carrier strains. We observed that intracellular expressed antigens were more efficient in elicitation of humoral and cellular immune responses than constitutively expressed antigens. Furthermore, the intracellular-activated promoter was more efficient in combination with a carrier strain deficient in SPI2 gene sseC than with the conventional aroA strain. We propose that intracellular-activated promoters will be useful tools for construction of efficient recombinant live vaccines.

Animals↗

PubMed2005

Intracellular Salmonella inhibit antigen presentation by dendritic cells.

Dendritic cells (DC) are important APCs linking innate and adaptive immunity. During analysis of the intracellular activities of Salmonella enterica in DC, we observed that viable bacteria suppress Ag-dependent T cell proliferation. This effect was dependent on the induction of inducible NO synthase by DC and on the function of virulence genes in Salmonella pathogenicity island 2 (SPI2). Intracellular activities of Salmonella did not affect the viability, Ag uptake, or maturation of DC, but resulted in reduced presentation of antigenic peptides by MHC class II molecules. Increased resistance to reinfection was observed after vaccination of mice with SPI2-deficient Salmonella compared with mice vaccinated with SPI2-proficient Salmonella, and this correlated with an increased amount of CD4(+) as well as CD8(+) T cells. Our study is the first example of interference of an intracellular bacterial pathogen with Ag presentation by DC. The subversion of DC functions is a novel strategy deployed by this pathogen to escape immune defense, colonize host organs, and persist in the infected host.

Animals↗

PubMed2005

The Salmonella pathogenicity island (SPI)-2 and SPI-1 type III secretion systems allow Salmonella serovar typhimurium to trigger colitis via MyD88-dependent and MyD88-independent mechanisms.

Salmonella typhimurium can colonize the gut, invade intestinal tissues, and cause enterocolitis. In vitro studies suggest different mechanisms leading to mucosal inflammation, including 1) direct modulation of proinflammatory signaling by bacterial type III effector proteins and 2) disruption or penetration of the intestinal epithelium so that penetrating bacteria or bacterial products can trigger innate immunity (i.e., TLR signaling). We studied these mechanisms in vivo using streptomycin-pretreated wild-type and knockout mice including MyD88(-/-) animals lacking an adaptor molecule required for signaling via most TLRs. The Salmonella SPI-1 and the SPI-2 type III secretion systems (TTSS) contributed to inflammation. Mutants that retain only a functional SPI-1 (M556; sseD::aphT) or a SPI-2 TTSS (SB161; DeltainvG) caused attenuated colitis, which reflected distinct aspects of the colitis caused by wild-type S. typhimurium: M556 caused diffuse cecal inflammation that did not require MyD88 signaling. In contrast, SB161 induced focal mucosal inflammation requiring MyD88. M556 but not SB161 was found in intestinal epithelial cells. In the lamina propria, M556 and SB161 appeared to reside in different leukocyte cell populations as indicated by differential CD11c staining. Only the SPI-2-dependent inflammatory pathway required aroA-dependent intracellular growth. Thus, S. typhimurium can use two independent mechanisms to elicit colitis in vivo: SPI-1-dependent and MyD88-independent signaling to epithelial cells and SPI-2-dependent intracellular proliferation in the lamina propria triggering MyD88-dependent innate immune responses.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

PubMed2005

Cloning vectors and fluorescent proteins can significantly inhibit Salmonella enterica virulence in both epithelial cells and macrophages: implications for bacterial pathogenesis studies.

Plasmid vectors and fluorescent protein reporter systems are commonly used in the study of bacterial pathogenesis. Here we show that they can impair the ability of Salmonella enterica serovar Typhimurium to productively infect either cultured mammalian cells or mice. This has significant implications for studies that rely on these systems.

Animals↗

PubMed2005

Rapid method for the construction of Salmonella enterica Serovar Typhimurium vaccine carrier strains.

Salmonella enterica serovar Typhimurium is a versatile organism for the generation of live recombinant vaccines for mucosal immunization. Various strategies have been devised for the stable and efficient expression of heterologous antigens by attenuated S. enterica strains, but these methods often require complex manipulations. Use of phage lambda Red recombinase has recently been devised for gene replacements in Escherichia coli and S. enterica after introduction of PCR products. Based on this method, we have developed an approach that allows the integration of recombinant expression cassettes for heterologous antigens in a single step. The recombinant construct is integrated into the chromosome and is devoid of any selective marker such as antibiotic resistance. We observed the stable expression of model antigens without selective pressure. In addition, the method allows the simultaneous generation of attenuating mutations by gene deletions. The novel "knock-in" approach allows the rapid and efficient construction of recombinant Salmonella strains as vaccine carriers.

Animals↗

PubMed2005

The Shiga toxin 1-converting bacteriophage BP-4795 encodes an NleA-like type III effector protein.

In this study, the complete DNA sequence of Shiga toxin 1-converting bacteriophage BP-4795 was determined. The genome of BP-4795 consists of 85 open reading frames, including two complete IS629 elements and three morons at the end of its late regulatory region. One of these morons encodes a type III effector that is translocated by the locus of enterocyte effacement-encoded type III secretion system into HeLa cells, where it localizes with the Golgi apparatus.

Base Sequence↗

PubMed2004

Evolution of pathogenicity islands of Salmonella enterica.

Virulence genes located on pathogenicity islands play a crucial role in the pathogenesis of Salmonella enterica infections. Salmonella pathogenicity islands (SPI) contribute to host cell invasion and intracellular pathogenesis. At present, 12 SPI have been described. Although size, structure and function of these SPI, as well as the distribution in Salmonella subspecies and serovars can be markedly different, several common motifs are present among SPI. In this review, the characteristics of SPI are described with focus on the evolution of these genetic elements.

Animals↗

PubMed2004

Effector proteins encoded by Salmonella pathogenicity island 2 interfere with the microtubule cytoskeleton after translocation into host cells.

The facultative intracellular pathogen Salmonella enterica has evolved strategies to modify its fate inside host cells. One key virulence factor for the intracellular pathogenesis is the type III secretion system encoded by Salmonella Pathogenicity Island 2 (SPI2). We have previously described SPI2-encoded SseF and SseG as effector proteins that are translocated by intracellular Salmonella. Detailed analysis of the subcellular localization of SseF and SseG within the host cell indicated that these effector proteins are associated with endosomal membranes as well as with microtubules. Specific association with microtubules was observed after translocation by intracellular Salmonella as well as after expression by transfection vectors. In epithelial cells infected with Salmonella, both SseF and SseG are required for the aggregation of endosomal compartments along microtubules and to induce the formation of massive bundles of microtubules. These observations demonstrate that SPI2 effectors interfere with the microtubule cytoskeleton and suggest that microtubule-dependent host cell functions such as vesicle transport or organelle positioning are altered by intracellular Salmonella.

Bacterial Proteins↗

PubMed2004

Pathogenicity islands in bacterial pathogenesis.

In this review, we focus on a group of mobile genetic elements designated pathogenicity islands (PAI). These elements play a pivotal role in the virulence of bacterial pathogens of humans and are also essential for virulence in pathogens of animals and plants. Characteristic molecular features of PAI of important human pathogens and their role in pathogenesis are described. The availability of a large number of genome sequences of pathogenic bacteria and their benign relatives currently offers a unique opportunity for the identification of novel pathogen-specific genomic islands. However, this knowledge has to be complemented by improved model systems for the analysis of virulence functions of bacterial pathogens. PAI apparently have been acquired during the speciation of pathogens from their nonpathogenic or environmental ancestors. The acquisition of PAI not only is an ancient evolutionary event that led to the appearance of bacterial pathogens on a timescale of millions of years but also may represent a mechanism that contributes to the appearance of new pathogens within a human life span. The acquisition of knowledge about PAI, their structure, their mobility, and the pathogenicity factors they encode not only is helpful in gaining a better understanding of bacterial evolution and interactions of pathogens with eukaryotic host cells but also may have important practical implications such as providing delivery systems for vaccination, tools for cell biology, and tools for the development of new strategies for therapy of bacterial infections.

Bacteria↗

PubMed2004

Role of neutrophils in murine salmonellosis.

Gastrointestinal infections with Salmonella enterica serovars have different clinical outcomes that range from localized inflammation to a life-threatening systemic disease in the case of typhoid fever. Using a mouse model of systemic salmonellosis, we investigated the contribution of neutrophils to the innate immune defense against Salmonella after oral infection. Neutrophil infiltration was dependent on the bacterial burden in various infected organs (Peyer's patches, mesenteric lymph nodes, spleen, and liver). However, the massive infiltration of neutrophils did not allow clearance of an infection with wild-type Salmonella, presumably due to protection of intracellular Salmonella against neutrophil activities. A Salmonella mutant strain deficient in Salmonella pathogenicity island 2 (SPI2) was able to infect systemic sites, but its replication was highly restricted and it did not cause detectable attraction of neutrophils. Neutrophil depletion by antibody treatment of mice did not restore the virulence of SPI2 or auxotrophic mutant strains, supporting the hypothesis that attenuation of the strains is not due to greater susceptibility to neutrophil killing. Our observations reveal that neutrophils have completely different roles during systemic salmonellosis and localized gastrointestinal infections. In the latter conditions, rapid neutrophil attraction efficiently prevents the spread of the pathogen, whereas the neutrophil influx is delayed during systemic infections and cannot protect against lethal bacteremia.

Animals↗

PubMed2004

Functional transfer of Salmonella pathogenicity island 2 to Salmonella bongori and Escherichia coli.

The type III secretion system (T3SS) encoded by the Salmonella pathogenicity island 2 (SPI2) has a central role in systemic infections by Salmonella enterica and for the intracellular phenotype. Intracellular S. enterica uses the SPI2-encoded T3SS to translocate a set of effector proteins into the host cell, which modify host cell functions, enabling intracellular survival and replication of the bacteria. We sought to determine whether specific functions of the SPI2-encoded T3SS can be transferred to heterologous hosts Salmonella bongori and Escherichia coli Mutaflor, species that lack the SPI2 locus and loci encoding effector proteins. The SPI2 virulence locus was cloned and functionally expressed in S. bongori and E. coli. Here, we demonstrate that S. bongori harboring the SPI2 locus is capable of secretion of SPI2 substrate proteins under culture conditions, as well as of translocation of effector proteins under intracellular conditions. An SPI2-mediated cellular phenotype was induced by S. bongori harboring the SPI2 if the sifA locus was cotransferred. An interference with the host cell microtubule cytoskeleton, a novel SPI2-dependent phenotype, was observed in epithelial cells infected with S. bongori harboring SPI2 without additional effector genes. S. bongori harboring SPI2 showed increased intracellular persistence in a cell culture model, but SPI2 transfer was not sufficient to confer to S. bongori systemic pathogenicity in a murine model. Transfer of SPI2 to heterologous hosts offers a new tool for the study of SPI2 functions and the phenotypes of individual effectors.

Animals↗

PubMed2003

Acidic pH is required for the functional assembly of the type III secretion system encoded by Salmonella pathogenicity island 2.

Salmonella enterica employs two type III secretion systems (T3SS) for interactions with host cells during pathogenesis. The T3SS encoded by Salmonella pathogenicity island 2 (SPI2) is required for the intracellular replication of Salmonella and the survival inside phagocytes. During growth in vitro, acidic pH is a signal that promotes secretion of proteins by this T3SS. We analyzed protein levels and subcellular localization of various T3SS subunits under in vitro conditions at acidic or neutral pH, inducing or ablating secretion, respectively. Growth at acidic pH resulted in higher levels of SsaC, a protein forming the outer membrane secretin, without increasing expression of the operon containing ssaC. Acidic pH also induced oligomerization of SsaC subunits, a prerequisite for a functional secretin pore. It has previously been described that environmental stimuli resembling the intraphagosomal habitat of Salmonella control the expression of SPI2 genes. Here we propose that such stimuli also modulate the assembly of a functional T3SS that is capable of translocation of effector proteins into the host cell.

Bacterial Outer Membrane Proteins↗