Bacteriomimetic invasin-functionalized nanocarriers for intracellular delivery.

Intracellular bacteria invade mammalian cells to establish an infectious niche. The current work models adhesion and subsequent internalization strategy of pathogenic bacteria into mammalian cells to design a bacteriomimetic bioinvasive delivery system. We report on the surface functionalization of liposomes with a C-terminal fragment of invasin (InvA497), an invasion factor in the outer membrane of Yersinia pseudotuberculosis. InvA497-functionalized liposomes adhere to mammalian epithelial HEp-2 cell line at different infection stages with a significantly higher efficiency than liposomes functionalized with bovine serum albumin. Covalent attachment of InvA497 results in higher cellular adhesion than liposomes with physically adsorbed InvA497 with non-specific surface protein alignment. Uptake studies in HEp-2 cells indicate active internalization of InvA497-functionalized liposomes via ß1-integrin receptor-mediated uptake mechanism mimicking the natural invasion strategy of Y. pseudotuberculosis. Uptake studies in Caco-2 cells at different polarization states demonstrate specific targeting of the InvA497-functionalized liposomes to less polarized cells reflecting the status of inflamed cells. Moreover, when loaded with the anti-infective agent gentamicin and applied to HEp-2 cells infected with Y. pseudotuberculosis, InvA497-functionalized liposomes are able to significantly reduce the infection load relative to non-functionalized drug-loaded liposomes. This indicates a promising application of such a bacteriomimetic system for drug delivery to intracellular compartments.

The cytotoxic necrotizing factor of Yersinia pseudotuberculosis (CNFY) enhances inflammation and Yop delivery during infection by activation of Rho GTPases.

Some isolates of Yersinia pseudotuberculosis produce the cytotoxic necrotizing factor (CNFY), but the functional consequences of this toxin for host-pathogen interactions during the infection are unknown. In the present study we show that CNFY has a strong influence on virulence. We demonstrate that the CNFY toxin is thermo-regulated and highly expressed in all colonized lymphatic tissues and organs of orally infected mice. Most strikingly, we found that a cnfY knock-out variant of a naturally toxin-expressing Y. pseudotuberculosis isolate is strongly impaired in its ability to disseminate into the mesenteric lymph nodes, liver and spleen, and has fully lost its lethality. The CNFY toxin contributes significantly to the induction of acute inflammatory responses and to the formation of necrotic areas in infected tissues. The analysis of the host immune response demonstrated that presence of CNFY leads to a strong reduction of professional phagocytes and natural killer cells in particular in the spleen, whereas loss of the toxin allows efficient tissue infiltration of these immune cells and rapid killing of the pathogen. Addition of purified CNFY triggers formation of actin-rich membrane ruffles and filopodia, which correlates with the activation of the Rho GTPases, RhoA, Rac1 and Cdc42. The analysis of type III effector delivery into epithelial and immune cells in vitro and during the course of the infection further demonstrated that CNFY enhances the Yop translocation process and supports a role for the toxin in the suppression of the antibacterial host response. In summary, we highlight the importance of CNFY for pathogenicity by showing that this toxin modulates inflammatory responses, protects the bacteria from attacks of innate immune effectors and enhances the severity of a Yersinia infection.

Bacterial invasion factors: tools for crossing biological barriers and drug delivery?

The oral route is the preferential route of drug delivery in humans. However, effective delivery through the gastrointestinal tract is often hampered by the low permeability of the intestinal epithelium. One possibility to overcome this problem is the encapsulation of drugs inside nanoparticulate systems, containing targeting moieties with cell invasive properties. The bioinvasive features of the delivery system could be provided by the attachment of bacterial invasion factors, which promote efficient uptake into host cells and mediate rapid transcytosis of the pathogen through the intestinal epithelium. This review gives an overview of bacterial invasion systems. The molecular structure and function of suitable bacterial invasins, their relative values as targeting agents and possible pitfalls of their use are described. The potential of bioinvasive drug delivery systems is mainly presented on the basis of the well-characterized Yersinia invasin protein, which enters M cells to gain access to subepithelial layers of the gastrointestinal tract, but alternative approaches and future prospects for oral drug delivery are also discussed.

ß7 integrin controls immunogenic and tolerogenic mucosal B cell responses.

IgA production in the gut-associated lymphoid tissue represents a pivotal defense mechanism against luminal pathogens. The other important challenge for the GALT is the induction of local and systemic hyporesponsiveness (tolerance) to dietary antigens and luminal bacterial flora to prevent allergies or deleterious immunologic reactions to food or environmental antigens. In this study we analyzed the impact of ß7 integrin on immunogenic and tolerogenic B cell responses in the gastrointestinal tract. ß7 integrin deficient mice failed to mount a normal intestinal IgA response to ovalbumin and cholera toxin, whereas the IgG response was unchanged in comparison to control mice. Oral B cell tolerance to ovalbumin, measured as the suppression of specific serum IgG responses, did not develop in the absence of ß7 integrin. After adoptive transfer of spleen cells from ß7 integrin +/+ mice into RAG-2 deficient or RAG-2/ß7 integrin double deficient mice, only RAG-2 deficient mice were able to develop oral B cell tolerance. These observations suggest that ß7 integrin expression on cells of the innate immune system contributes to the critical role of ß7 integrin in the process of B cell tolerance.

In vivo-induced InvA-like autotransporters Ifp and InvC of Yersinia pseudotuberculosis promote interactions with intestinal epithelial cells and contribute to virulence.

The Yersinia pseudotuberculosis Ifp and InvC molecules are putative autotransporter proteins with a high homology to the invasin (InvA) protein. To characterize the function of these surface proteins, we expressed both factors in Escherichia coli K-12 and demonstrated the attachment of Ifp- and InvC-expressing bacteria to human-, mouse-, and pig-derived intestinal epithelial cells. Ifp also was found to mediate microcolony formation and internalization into polarized human enterocytes. The ifp and invC genes were not expressed under in vitro conditions but were found to be induced in the Peyer's patches of the mouse intestinal tract. In a murine coinfection model, the colonization of the Peyer's patches and the mesenteric lymph nodes of mice by the ifp-deficient strain was significantly reduced, and considerably fewer bacteria reached liver and spleen. The absence of InvC did not have a severe influence on bacterial colonization in the murine infection model, and it resulted in only a slightly reduced number of invC mutants in the Peyer's patches. The analysis of the host immune response demonstrated that the presence of Ifp and InvC reduced the recruitment of professional phagocytes, especially neutrophils, in the Peyer's patches. These findings support a role for the adhesins in modulating host-pathogen interactions that are important for immune defense.

Monitoring of gene expression in bacteria during infections using an adaptable set of bioluminescent, fluorescent and colorigenic fusion vectors.

A family of versatile promoter-probe plasmids for gene expression analysis was developed based on a modular expression plasmid system (pZ). The vectors contain different replicons with exchangeable antibiotic cassettes to allow compatibility and expression analysis on a low-, midi- and high-copy number basis. Suicide vector variants also permit chromosomal integration of the reporter fusion and stable vector derivatives can be used for in vivo or in situ expression studies under non-selective conditions. Transcriptional and translational fusions to the reporter genes gfp(mut3.1), amCyan, dsRed2, luxCDABE, phoA or lacZ can be constructed, and presence of identical multiple cloning sites in the vector system facilitates the interchange of promoters or reporter genes between the plasmids of the series. The promoter of the constitutively expressed gapA gene of Escherichia coli was included to obtain fluorescent and bioluminescent expression constructs. A combination of the plasmids allows simultaneous detection and gene expression analysis in individual bacteria, e.g. in bacterial communities or during mouse infections. To test our vector system, we analyzed and quantified expression of Yersinia pseudotuberculosis virulence genes under laboratory conditions, in association with cells and during the infection process.

Mucosal addressin cell-adhesion molecule-1 controls plasma-cell migration and function in the small intestine of mice.

BACKGROUND & AIMS: Immunoglobulin (Ig) A secretion into the intestinal lumen is an important immune mechanism of the gastrointestinal (GI) tract. B cells proliferate and differentiate into IgA-secreting plasma cells (PC) within lymphoid organs then migrate directly into the intestinal lamina propria. We aimed to elucidate the in vivo role of the mucosal addressin cell-adhesion molecule-1 (MAdCAM-1), which is constitutively expressed in the GI-associated lymphoid tissue, in B-cell migration. METHODS: We generated MAdCAM-1-deficient mice (MAdCAM(Delta)) and evaluated the B-cell compartment of the GI-associated lymphoid tissue. We also assessed PC migration to the small intestine and the intestinal immune response after oral immunization. RESULTS: In MAdCAM(Delta) mice, the size of Peyer's patches was drastically reduced, compared with that of wild-type mice; this difference was detectable by 3 days after birth, indicating that MAdCAM-1 is dispensable for embryonic Peyer's patch development but mediates recruitment of lymphocytes into this lymphoid organ at later stages. Moreover, antigen-specific, IgA-positive PC were severely compromised in their migration to the small intestine; accordingly, there was a reduced number of IgA-secreting PC in the lamina propria of the small intestine. The MAdCAM(Delta) mice were unable to mount a normal intestinal IgA response after oral immunization with cholera toxin. CONCLUSION: These data provide in vivo evidence that MAdCAM-1 is required for the localization and function of IgA-secreting PC in the intestine.