Ayub Darji

Oral Somatic Transgene Vaccination Using Attenuated S. typhimurium

An attenuated strain of S. typhimurium has been used as a vehicle for oral genetic immunization. Eukaryotic expression vectors containing truncated genes of ActA and listeriolysin--two virulence factors of Listeria monocytogenes--have been used to transform S. typhimurium aroA. Multiple or even single oral immunizations with such transformants induced excellent cellular and humoral responses. In addition, protective immunity was induced with listeriolysin transformants. The quality of the responses suggested a transfer of plasmid DNA from the bacterial carrier to the host. Such transfer was unequivocally shown in vitro with primary peritoneal macrophages. We describe a highly versatile system for antigen delivery, identification of protective antigens for vaccination, and efficient generation of antibodies against the product of open reading frames present on virtually any DNA segment.

Apoptosis of mouse dendritic cells is triggered by listeriolysin, the major virulence determinant of Listeria monocytogenes.

Infection of a murine‐spleen dendritic cell line by Listeria monocytogenes was found to induce cell death through apoptosis. To characterize the bacterial product(s) involved in induction of apoptosis, dendritic cells were infected with the L. monocytogenes EGD strain and several isogenic mutants deficient in the production of individual listerial virulence factors. The ability to induce cellular apoptosis was retained by all mutants tested, except the prfA and Δhly mutants, both of which are unable to produce listeriolysin. Apoptosis was also induced by purified listeriolysin suggesting that this protein directly induces apoptosis. Purified recombinant listeriolysins rendered either weakly haemolytic by a C‐484 to S mutation, or non‐haemolytic by a W‐491 to A mutation exhibited little or no capacity to induce apoptosis, indicating that both activities are associated within the same protein region. Treatment with purified listeriolysin or L. monocytogenes infection also triggers apoptosis in explanted bone‐marrow dendritic cells. Thus invasion of dendritic cells by L. monocytogenes, which results in cell death, may play an important role in the pathogenesis of listerial infections by impairing immune responses, hindering bacterial clearance and promoting spread of the infection.

INTERNALIN B IS ESSENTIAL FOR ADHESION AND MEDIATES THE INVASION OF LISTERIA MONOCYTOGENES INTO HUMAN ENDOTHELIAL CELLS

Listeria monocytogenes causes rhombencephalitis in humans and animals and also affects the fetus in utero, causing disseminated sepsis. In both instances, the infection occurs by the crossing of endothelial cells lining a physiological barrier, the blood–brain barrier or the transplacental barrier. In this study, the ability of L. monocytogenes wild‐type EGD to invade human umbilical vein endothelial cells (HUVECs) was evaluated using wild‐type bacteria and isogenic Listeria mutants. Here, we show that invasion of HUVECs by L. monocytogenes is dependent on the expression of the internalin B gene product. This was demonstrated in several ways. First, L. monocytogenes strains lacking the inlB gene did not invade HUVECs. Secondly, avid invasion was obtained when a strain deleted for inlAB was complemented with a plasmid harbouring inlB only, whereas strains expressing inlA did not enter HUVECs. Thirdly, entry of wild‐type EGD could be blocked effectively with antibodies to InlB. Fourthly, cell binding assays and flow cytometry with HUVECs showed binding of purified InlB, but not InlA, suggesting a tropism of InlB for this cell type. Finally, physical association of purified native InlB with the surface of non‐invasive mutants dramatically increased their ability to invade HUVECs. In laser‐scanning confocal microscopy, binding of InlB was observed as focal and localized patches on the cell surface of HUVECs. Qualitative examination of the entry process by scanning electron microscopy revealed that both wild‐type EGD and a recombinant strain overexpressing only InlB enter HUVECs in a similar fashion. The entry process was polarized, involved single bacteria and occurred over the entire surface of endothelial cells.

Listeriolysin O: cholesterol inhibits cytolysis but not binding to cellular membranes

Listeriolysin O (LLO) binds to cholesterol‐containing membranes in which it oligomerizes to form pores. Preincubation of the toxin with cholesterol is known to inhibit haemolysis, whereas the oxidized form of cholesterol has no inhibitory effect. Using immunoblot analyses and flow cytometry we demonstrate that preincubation with cholesterol does not influence binding of the listeriolysin–cholesterol complex to red blood cells, eukaryotic cells or artificial membranes. Lytic activity of membrane‐bound LLO inactivated by cholesterol can be restored by enzymatic treatment with cholesterol oxidase. To determine the step at which cholesterol inhibits lytic activity, we looked for pore formation using electron microscopy. Pores formed by purified listeriolysin could be directly visualized using erythrocyte ghosts. This property was lost upon incubation of the toxin with cholesterol. Quantitative analysis strongly suggest that inhibition of lysis by cholesterol is not due to decreased binding of listeriolysin to target membranes, but rather to an interference with a subsequent step leading to polymerization of the toxin.

Listeriolysin of Listeria monocytogenes forms Ca2+-permeable pores leading to intracellular Ca2+ oscillations.

Listeriolysin (LLO) is a major virulence factor of Listeria monocytogenes, a Gram‐positive bacterium that can cause life‐threatening diseases. Various signalling events and cellular effects, including modulation of gene expression, are triggered by LLO through unknown mechanisms. Here, we demonstrate that LLO applied extracellularly at sublytic concentrations causes long‐lasting oscillations of the intracellular Ca2+ level of human embryonic kidney cells; resulting from a pulsed influx of extracellular Ca2+ through pores that are formed by LLO in the plasma membrane. Calcium influx does not require the activity of endogenous Ca2+ channels. LLO‐formed pores are transient and oscillate between open and closed states. Pore formation and Ca2+ oscillations were also observed after exposure of cells to native Listeria monocytogenes. Our data identify LLO as a tool used by Listeria monocytogenes to manipulate the intracellular Ca2+ level without direct contact of the bacterium with the target cell. As Ca2+ oscillations modulate cellular signalling and gene expression, our findings provide a potential molecular basis for the broad spectrum of Ca2+‐dependent cellular responses induced by LLO during Listeria infection.

Identification and Characterization of a Peptidoglycan Hydrolase, MurA, of Listeria monocytogenes, a Muramidase Needed for Cell Separation

A novel cell wall hydrolase encoded by the murA gene of Listeria monocytogenes is reported here. Mature MurA is a 66-kDa cell surface protein that is recognized by the well-characterized L. monocytogenes-specific monoclonal antibody EM-7G1. MurA displays two characteristic features: (i) an N-terminal domain with homology to muramidases from several gram-positive bacterial species and (ii) four copies of a cell wall-anchoring LysM repeat motif present within its C-terminal domain. Purified recombinant MurA produced in Escherichia coli was confirmed to be an authentic cell wall hydrolase with lytic properties toward cell wall preparations of Micrococcus lysodeikticus. An isogenic mutant with a deletion of murA that lacked the 66-kDa cell wall hydrolase grew as long chains during exponential growth. Complementation of the mutant strain by chromosomal reintegration of the wild-type gene restored expression of this murein hydrolase activity and cell separation levels to those of the wild-type strain. Studies reported herein suggest that the MurA protein is involved in generalized autolysis of L. monocytogenes.

Hyperexpression of listeriolysin in the nonpathogenic species Listeria innocua and high yield purification.

Listeriolysin, the hemolysin of the pathogenic species Listeria monocytogenes, was expressed in the non-pathogenic species Listeria innocua. Coexpression of the positive regulatory factor prfA in the plasmid vector in conjunction with the structural gene hly increased the expression over 500-fold. Purification from supernatant fluids was achieved by two steps of ion exchange chromatography. The procedure resulted in over 60% yield of a hemolytically active, homogeneous 58 kDa protein which was used to produce monospecific antibodies. As shown by immunoblot the purified listeriolysin was free of p60, a highly immunogenic protein of similar size also produced by Listeria spp., which otherwise would interfere with immunoassays. Listeriolysin retained full activity for more than 6 months at -70 degrees C.

Distribution patterns of influenza virus receptors and viral attachment patterns in the respiratory and intestinal tracts of seven avian species

This study assessed the presence of sialic acid α-2,3 and α-2,6 linked glycan receptors in seven avian species. The respiratory and intestinal tracts of the chicken, common quail, red-legged partridge, turkey, golden pheasant, ostrich, and mallard were tested by means of lectin histochemistry, using the lectins Maackia amurensis agglutinin II and Sambucus nigra agglutinin, which show affinity for α-2,3 and α-2,6 receptors, respectively. Additionally, the pattern of virus attachment (PVA) was evaluated with virus histochemistry, using an avian-origin H4N5 virus and a human-origin seasonal H1N1 virus. There was a great variation of receptor distribution among the tissues and avian species studied. Both α-2,3 and α-2,6 receptors were present in the respiratory and intestinal tracts of the chicken, common quail, red-legged partridge, turkey, and golden pheasant. In ostriches, the expression of the receptor was basically restricted to α-2,3 in both the respiratory and intestinal tracts and in mallards the α-2,6 receptors were absent from the intestinal tract. The results obtained with the lectin histochemistry were, in general, in agreement with the PVA. The differential expression and distribution of α-2,3 and α-2,6 receptors among various avian species might reflect a potentially decisive factor in the emergence of new viral strains.

Pathogenesis and transmissibility of highly (H7N1) and low (H7N9) pathogenic avian influenza virus infection in red-legged partridge ( Alectoris rufa )

An experimental infection with highly pathogenic avian influenza virus (HPAIV) and low pathogenic avian influenza virus (LPAIV) was carried out in red-legged partridges (Alectoris rufa) in order to study clinical signs, gross and microscopic lesions, and viral distribution in tissues and viral shedding. Birds were infected with a HPAIV subtype H7N1 (A/Chicken/Italy/5093/1999) and a LPAIV subtype H7N9 (A/Anas crecca/Spain/1460/2008). Uninoculated birds were included as contacts in both groups. In HPAIV infected birds, the first clinical signs were observed at 3 dpi, and mortality started at 4 dpi, reaching 100% at 8 dpi. The presence of viral antigen in tissues and viral shedding were confirmed by immunohistochemistry and quantitative real time RT-PCR (qRRT-PCR), respectively, in all birds infected with HPAIV. However, neither clinical signs nor histopathological findings were observed in LPAIV infected partridges. In addition, only short-term viral shedding together with seroconversion was detected in some LPAIV inoculated animals. The present study demonstrates that the red-legged partridge is highly susceptible to the H7N1 HPAIV strain, causing severe disease, mortality and abundant viral shedding and thus contributing to the spread of a potential local outbreak of this virus. In contrast, our results concerning H7N9 LPAIV suggest that the red-legged partridge is not a reservoir species for this virus.

Induction of immune responses by attenuated isogenic mutant strains of Listeria monocytogenes

We have generated isogenic Listeria monocytogenes mutant strains to study the induction of protective immunity in mice. These strains harbored either a specific deletion within the actin nucleator (actA) and/or have multiple deletions within the actA and phospholipase B (plcB) genes. In comparison to the wild type parental L. monocytogenes EGDe strains, the mutant strains were extremely low in virulence and were rapidly eliminated by the host during the first days of infection. Nevertheless, a single immunization with both mutant strains (EGDe DeltaactA2 and DeltaactADeltaplcB) efficiently induced and maintained effector memory (CD8(+)) T cells and has provided animals with a state of long-lasting protective immunity against wild type L. monocytogenes. These mutant strains can be used as live vaccines against the corresponding virulent pathogen and as carriers for introducing heterologous protective antigens into animals and humans.