Grants: KBN 6P207 033; "Listeria monocytogenes - universal biological vector induced intracellular responce"; KBN 4 P05A 067 11; "Bacillus subtilis as a biological vector  induced intracellular immunity"

Schoolarships: Germany , 1984, University of Tuebingen, 10 months; Germany, 1985, Max Planck Institute 8 months; USA, 1989, University of Pennsylvania post doc, 2,5 years; USA, 1993, University of Pennsylvania, visiting prof., 3 months;USA, 1999, University of California, Berkeley, visiting prof. 2 monthes

Awards: 2000, National Agency of Technique and Technology for universal vaccine 1995, award from Ministry of Education for research; 1987, 1988, 1989, 1990, 1992, 1997 – award from Rector of Warsaw University for research programs

Research intrests:
The facultative intracellular bacterium L. monocytogenes, a gram-positive food-borne human pathogen, successfully inhabits a variety of environments including contaminated foods, the gut and the intracellular environment of the mammalian cell. Once it has entered the cell, L. monocytogenes is found initially within the host vacuole and subsequently directly in the cytoplasm. Several bacterial gene products have been suggested to play a role in the pathogenicity of L. monocytogenes. The virulence of L. monocytogenes is determined by a cluster of five genes in the order plcA, hly, mpl, actA and plcB, which are coordinately regulated by a transcriptional activator, termed PrfA. Other determinants: catalase, superoxide dismutase, LmaA, proteases, siderophores, p60 protein and toxins, which are not under the control of PrfA and partially regulated internalins are necessary for full activity to cause disease by L. monocytogenes. The ability of L. monocytogenes to escape a phagocytic vesicle is due to the production of a hemolysin, listeriolysin O (LLO). LLO is a 58 kDa sulphydryl-activated, pore-forming cytotoxin, which has considerable amino acid sequence similarity to hemolysin-cytotoxins produced by two other gram-positive pathogens. LLO^- mutants have an LD₅₀ for mice that is five logs higher than that of the wild type. LLO^- mutants also do not survive in macrophages. This suggests the survival of L. monocytogenes in macrophages could be due to its ability to escape the phagosome before phagosomal fusion occurs. 

Bacillus subtilis hly entering Int407 cell
(J. Wiśniewski, J. Bielecki and M. Sobolewska SEM)

Listeria monocytogenes entering J774 cells
(J. Wiśniewski and J. Bielecki)

The importance of LLO for escape from the phagocytic vesicle was shown by an experiment in which hly was expressed in B. subtilis. Although wild-type B. subtilis remained inside the phagocytic vesicle, B. subtilis expressing LLO escaped it and entered the cytoplasm. The consumption of contaminated food is the most frequent mode of transmission of Listeria in humans, and bacteria must spread from the intestinal lumen to the gut. In previous work we showed that B. subtilis strain invades epithelial intestinal cells line Int407 and causes cytoskeletal reorganization. Our observations showed that LLO caused actin cytoskeleton reorganization in infected Int407 cells and for this process no contact of LLO expressing bacteria with eukaryotic cells is necessary. Accumulation of wide actin layer around the nucleus zone was observed. A comparison of our observations of live cells stained with acridine orange, and early changes in cytoskeleton rearrangements caused by LLO showed that induced by listeriolysin concentration of actin cytoskeleton was found in the region of the presence of acid organelles - Golgi apparatus and endoplasmic reticulum.

Publication:

1.Bielecki J., P. Youngman, P. Connelly, and D.A. Portnoy. (1990). Bacillus subtilis expressing a hemolysin gene from Listeria monocytogenes can grow in mammalian cells. Nature (London), 345 : 175 - 176.

2. Portnoy D.A., R.K. Tweten, M. Kechoe, J. Bielecki (1992). The capacity of listeriolysinO, streptolysinO and perfringolysinO to mediate growth of Bacillus subtilis within mammalian cells. Infect. Immun., 60, 2710-2717.

3. Wiśniewski J. M. i Bielecki J. (1999), Intracellular Growth of Listeria monocytogenes Insertional Mutants Deprived of Protein p60. Acta Microbiol. Pol. 48, 4, 317-329.

4. Wiśniewski J., Hrebenda J., Bielecki J. (2000) Insertional knock-out of the Protein Translocation System Common for Yersinia enterocolitica and Listeria monocytogenes. Acta Microbiol. Pol. 49, 1, 31-42.

5. Stachowiak R. and Bielecki J. (2001) Contribution of Hemolysin and Phosholipase Activity to Cytolytic Properties and Viability of Listeria monocytogenes. Acta Microbiol. Polon. 50, 3-4, 243-250.