CELL SURFACE PROTEINS OF CRYPTOCOCCUS NEOFORMANS MEDIATE ADHERENCE AND INTERNALISATION INTO MAMMALIAN CELLS

@#Cryptococcus neoformans is an encapsulated fungal pathogen that causes severe disease primarily in immunocompromised patients. Adherence and internalisation of microbial pathogens into host cells often begin with engagement of microbes to the surface receptors of host. However, the mechanisms involved remain poorly understood. In this study, we investigated the association of cell surface determinants of C. neoformans with mammalian cells. Our results showed that treatment with trypsin, but not paraformaldehyde or heat killing, could reduce host-cryptococci interaction, suggesting the involvement of cell surface proteins (CSPs) of C. neoformans in the interaction. We extended our investigations to determine the roles of CSPs during cryptococci-host cells interaction by extracting and conjugating CSPs of C. neoformans to latex beads. Conjugation of CSPs with both encapsulated and acapsular C. neoformans increased the association of latex beads with mammalian alveolar epithelial cells, alveolar macrophages and monocyte-derived macrophages. Further examination on the actin organisation of the host cells implied the involvement of actin-dependent phagocytosis in the internalisation of C. neoformans in CSP-conjugated latex beads. We hypothesised that CSPs present on the cell wall of C. neoformans mediate the adherence and actin-dependent phagocytosis of cryptococci by mammalian cells. Our results warrant further studies on the exact role of CSPs in the pathogenesis of cryptococcosis

Cell surface proteins play an important role in probiotic activities of Lactobacillus reuteri

BACKGROUND: Eight Lactobacillus reuteri strains, previously isolated from breast-fed human infant feces, were selected to assess the potential contribution of their surface proteins in probiotic activity. These strains were treated with 5 M LiCl to remove their surface proteins, and their tolerance to simulated stomach-duodenum passage, cell surface characteristics, auto aggregation, adhesion, and inhibition of pathogen adhesion to Caco-2 cells were compared with untreated strains. RESULTS: The survival rates, auto aggregation, and adhesion abilities of the LiCl-treated L. Reuteri strains decreased significantly (p< 0.05) compared to that of the untreated cells. The inhibition ability of selected L. reuteri strains, untreated or LiCl treated, against adherence of Escherichia coli 25922 and Salmonella typh iNCDC113 to Caco-2 was evaluated in vitro with L. reuteri ATCC55730 strain as a positive control. Among the selected eight strains of L.reuteri, LR6 showed maximum inhibition against the E. Coli ATCC25922 and S. typhiNCDC113. After treatment with 5 M LiCl to remove surface protein, the inhibition activities of the lactobacilli against pathogens decreased significantly (p< 0.05). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated thatLR6 strains had several bands with molecular weight ranging from 10 to 100 KDa, and their characterization and functions need to be confirmed. CONCLUSIONS: The results revealed that the cell surface proteins of L. reuteri play an important role in their survivability, adhesion, and competitive exclusion of pathogen to epithelial cells.

Murine pro-tumor necrosis factor expressed in Saccharomyces cerevisiae HF7c localizes to membrane/particulate

Tumor necrosis factor (TNF) is a cytokine that is produced by immune cells in response to bacterial and viral stimuli and plays important roles in various inflammatory diseases. TNF is produced as a membrane-bound precursor, which is then cleaved to release soluble mature protein. We expressed murine pro-TNF in Saccharomyces cerevisiae and examined processing and cellular localization of the recombinant protein. Yeast cells were transformed with an expression construct carrying the pro-TNF gene under the control of alcohol dehydrogenase promoter. Immunoblotting analysis of cell homogenate revealed expression of 26 kD pro-TNF in transformed cells. Upon centrifugation, pro-TNF transformed cells fractionated into the membrane/particulate. In a clone that expresses a high level of pro-TNF, mature 17 kD TNF was detected in the culture medium, although the amount was far smaller than that of cell-associated pro-TNF. Flow cytometric analysis of yeast spheroplasts demonstrated the presence of TNF on the cell surface. Our results show that pro-TNF expressed in yeast mainly resides in the cellular membrane with an orientation similar to that of pro-TNF produced in mammalian cells. Our data suggest that the transformed yeast cells can be used for the genetic analysis of pro-TNF processing machinery in immune cells.