Biochemical and cellular activity of chemically synthesized elastase inhibitor (S-AFUEI) from Aspergillus fumigatus.

PURPOSE: Elastase, produced by Aspergillus fumigatus and A. flavus, is an important pathogenic factor in pulmonary aspergillosis. We investigated the possibility of using A. fumigatus-derived A. fumigatus elastase inhibitor (AFUEI) as a therapeutic agent. As native-AFUEI (N-AFUEI) has an extremely low yield, we generated a synthetic-AFUEI (S-AFUEI) and investigated whether S-AFUEI has a biological activity against A. fumigatus elastase (AFUE) and inhibits cytotoxicity. METHODOLOGY: A. fumigatus was cultured in Yeast Carbon Base (YCB) -elastin culture medium for 3-7 days, and AFUE was purified by chromatography using DE52 cellulose and Sephadex G-75 column. Elastolytic activity was examined using Glt-Ala-Ala-Pro-Leu-pNA (GAAPLNA) as the substrate. The hydrolytic activity of AFUE was determined using the characteristic substrates, fibrinogen and collagen (Type IV), and human cell cytotoxicity was measured colorimetrically. Furthermore, the inhibitory effect of S-AFUEI on these activities was examined. RESULTS: We confirmed that S-AFUEI demonstrated elastase inhibitory activity and heat stability equivalent to that demonstrated by N-AFUEI, and inhibited human collagen hydrolytic activity and human fibrinogen hydrolytic activity. Further, S-AFUEI inhibited cytotoxicity in AFUE human pulmonary artery endothelial cells (HPAEC), human small airway epithelial cells (HSAEC), and human pulmonary alveolar epithelial cells (HPAEpiC). CONCLUSION: As S-AFUEI strongly inhibited cytotoxicity induced by elastase in human-derived cells, it could prove beneficial for the treatment of pulmonary aspergillosis.

A Salmonella virulence protein that inhibits cellular trafficking.

Salmonella enterica requires a type III secretion system, designated Spi/Ssa, to survive and proliferate within macrophages. The Spi/Ssa system is encoded within the SPI-2 pathogenicity island and appears to function intracellularly. Here, we establish that the SPI-2-encoded SpiC protein is exported by the Spi/Ssa type III secretion system into the host cell cytosol where it interferes with intracellular trafficking. In J774 macrophages, wild-type Salmonella inhibited fusion of Salmonella-containing phagosomes with lysosomes and endosomes, and interfered with trafficking of vesicles devoid of the microorganism. These inhibitory activities required living Salmonella and a functional spiC gene. Purified SpiC protein inhibited endosome-endosome fusion in vitro. A Sindbis virus expressing the SpiC protein interfered with normal trafficking of the transferrin receptor in vivo. A spiC mutant was attenuated for virulence, suggesting that the ability to interfere with intracellular trafficking is essential for Salmonella pathogenesis.

Identification and characterization of phoN-Sf, a gene on the large plasmid of Shigella flexneri 2a encoding a nonspecific phosphatase.

A gene encoding a nonspecific phosphatase, named PhoN-Sf, was identified on the large virulence plasmid (pMYSH6000) of Shigella flexneri 2a YSH6000. The phosphatase activity in YSH6000 was observed under high-phosphate conditions. However, it was found that low-phosphate conditions induced a slightly higher level of activity. The nucleotide sequence of the phoN-Sf region cloned from pMYSH6000 possessing the phoN-Sf gene encoded 249 amino acids with a typical signal sequence at the N terminus. The deduced amino acid sequence of the PhoN-Sf protein revealed significant homology to sequences of nonspecific acid phosphatases of other bacteria, such as Providencia stuartii (PhoN, 83.2%), Morganella morganii (PhoC, 80.6%), Salmonella typhimurium (PhoN, 47.8%), and Zymomonas mobilis (PhoC, 34.8%). The PhoN-Sf protein was purified, and its biochemical properties were characterized. The apparent molecular mass of the protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was calculated to be 27 kDa. The 20 amino acids at the N terminus corresponded to the 20 amino acid residues following the putative signal sequence of PhoN-Sf protein deduced from the nucleotide sequence. The PhoN-Sf activity had a pH optimum of 6.6, and the optimum temperature was 37 degrees C. The enzymatic activity was inhibited by diisopropyl fluorophosphate, N-bromosuccinimide, or dithiothreitol but not by EDTA. The subcellular localization of the PhoN-Sf protein in YSH6000 revealed that the protein was found predominantly in the periplasm. Examination of Shigella and enteroinvasive Escherichia coli strains for PhoN-Sf production by immunoblotting with the PhoN-specific antibody and for the presence of phoN-Sf DNA by using a phoN-Sf probe indicated that approximately one-half of the strains possessed the phoN-Sf gene on the large plasmid and expressed the PhoN-Sf protein. The Tn5 insertion mutants of YSH6000 possessing phoN-Sf::Tn5 still retained wild-type levels of invasiveness, as well as the subsequent spreading capacity in MK2 epithelial cell monolayers, thus suggesting that the PhoN-Sf activity is not involved in expression of the virulence phenotypes of Shigella strains under in vitro conditions.

Identification of a novel virulence gene, virA, on the large plasmid of Shigella, involved in invasion and intercellular spreading.

A novel virulence gene (virA) was identified upstream of the virG gene on the large plasmid of Shigella flexneri 2a YSH6000. Characterization of virA mutants infecting MK2 epithelial cell monolayers revealed that their invasive capacity was decreased to less than one fifth of the wild-type level. Nevertheless, the bacteria were capable of expressing and secreting IpaB, IpaC and IpaD proteins. The virA mutants were also impaired in their ability to spread intercellularly, since the bacteria gave rise to a small number of foci in a focus-plaque-forming test with MK2 cells. Although virG expression was slightly decreased in the virA mutants, introduction of a cloned virG gene into a virA mutant, N1945, failed to restore spreading ability. Although, introduction of a cloned virA gene into N1945 restored invasiveness and spreading ability, the reduced virG transcription level was not affected, indicating that the reduced virG expression in virA mutants does not play a major role in defective intercellular spreading. The nucleotide sequence of the virA region revealed that the virA gene was located 528 bp upstream of the virG gene, in the opposite orientation. The deduced amino acid sequence of the VirA protein indicated a 44.7 kDa protein with no homology to known proteins. The VirA protein was secreted into the culture supernatant, a process that required the Mxi and Spa loci. The expression of virA was under the control of the virB gene, the positive regulator of the ipa, mxi and spa operons. These results indicate that virA is a new member of the invasion regulon directed by virB and that the VirA function is involved in invasion and intercellular spreading.

Expression of the O9 polysaccharide of Escherichia coli: sequencing of the E. coli O9 rfb gene cluster, characterization of mannosyl transferases, and evidence for an ATP-binding cassette transport system.

The rfb gene cluster of Escherichia coli O9 directs the synthesis of the O9-specific polysaccharide which has the structure -->2-alpha-Man-(1-->2)-alpha-Man-(1-->2)-alpha-Man-(1-->3)-alpha- Man-(1-->. The E. coli O9 rfb cluster has been sequenced, and six genes, in addition to the previously described rfbK and rfbM, were identified. They correspond to six open reading frames (ORFs) encoding polypeptides of 261, 431, 708, 815, 381, and 274 amino acids. They are all transcribed in the counter direction to those of the his operon. No gene was found between rfb and his. A higher G+C content indicated that E. coli O9 rfb evolved independently of the rfb clusters from other E. coli strains and from Shigella and Salmonella spp. Deletion mutagenesis, in combination with analysis of the in vitro synthesis of the O9 mannan in membranes isolated from the mutants, showed that three genes (termed mtfA, -B, and -C, encoding polypeptides of 815, 381, and 274 amino acids, respectively) directed alpha-mannosyl transferases. MtfC (from ORF274), the first mannosyl transferase, transfers a mannose to the endogenous acceptor. It critically depended on a functional rfe gene (which directs the synthesis of the endogenous acceptor) and initiates the growth of the polysaccharide chain. MtfB (from ORF381) then transfers two mannoses into the 3 position of the previous mannose, and MtfA (from ORF815) transfers three mannoses into the 2 position. Further chain growth needs only the two transferases MtfA and MtfB. Thus, there are fewer transferases needed than the number of sugars in the repeating unit. Analysis of the predicted amino acid sequence of the ORF261 and ORF431 proteins indicated that they function as components of an ATP-binding cassette transport system. A possible correlation between the mechanism of polymerization and mode of membrane translocation of the products is discussed.

Isolation and characterization of fibrinogenase from Candida albicans NH-1.

1. Fibrinogenase was isolated from Candida albicans NH-1 by DEAE-Cellulose, Sephadex G-75 and Sephadex G-100 column chromatographies. 2. The purified fibrinogenase gave a single band on disc polyacrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis. 3. The enzyme preparation had a molecular weight of 13,000, isoelectric point of pH 4.2 and possessed 117 amino acid residues. 4. The purified fibrinogenase possessed capillary permeability-increasing activity. 5. The enzyme hydrolyzed fibrinogen, casein, hide powder azure, azocoll hydrolytic activities and also hydrolyzed the oxidized B chain of insulin. The cleavage sites in the oxidized B chain of insulin were identified as Asp(3)-Glu(4), Glu(13)-Ala(14), Ala(14)-Leu(15), Tyr(16)-Leu(17), Arg(22)-Gly(23), Phe(25)-Tyr(26) and Tyr(26)-Thr(27). 6. Fibrinogenase activity of this preparation was inhibited by alpha 2-macroglobulin antithrombin-III, o-phenanthroline, disodium ethylenediaminetetra acetic acid and dithiothreitol.

Isolation of rfb gene clusters directing the synthesis of O polysaccharides consisting of mannose homopolymers and serological analysis of lipopolysaccharides.

Four serotypes of two genera, Escherichia coli O8 and O9 and Klebsiella O3 and O5, produce the O polysaccharides consisting of mannose homopolymers. Previously, we reported the isolation and expression of E. coli O9 rfb in E. coli K-12 strains (Kido et al, J. Bacteriol., 171: 3629-3633, 1989). In this study, R' plasmids carrying his-rfb region of the other three strains were isolated and expressed in E. coli K-12 strain. Serological study of lipopolysaccharides (LPS) synthesized in E. coli K-12 strain was carried out. His-linked rfb genes from E. coli O9 and Klebsiella O3 directed the synthesis of O polysaccharides with the same antigenicity as those of the parental strains in E. coli K-12 strain. On the other hand, rfb genes from E. coli O8 and Klebsiella O5 directed the synthesis of O polysaccharides which were antigenically not identical but partially common to those of the parental strains. A rough strain derived from E. coli O8 synthesized LPS which showed the identical antigenicity as the wild strain when the his-rfb region of E. coli O8 was introduced. The results suggest that some genes located distantly from his are additionally required to complete the synthesis of O polysaccharides of E. coli O8 and Klebsiella O5.

Susceptibility to active oxygen species of protective and nonprotective strains on the challenge of Salmonella enteritidis by immunization with culture filtrate.

Protective ability against the challenge of different strains by immunization with culture filtrate (CF) obtained from Salmonella enteritidis was investigated. It was shown that the different strains of S. enteritidis can be separated into two distinct groups of protective (2547, 116M, 116-54, SR-98G, and 3775) and nonprotective strains (2822, 3975, and IID-604). Using a cell-free microbicidal system, the susceptibilities of these strains to active oxygen species was evaluated. S. enteritidis was found to be susceptible to these active oxygen species, however no differences between the protective and nonprotective strains were observed. Both catalase (H2O2 scavenger) and histidine (1O2 scavenger) inhibited the bactericidal activity of the xanthine-xanthine oxidase system. Therefore, among the various oxygen intermediates, H2O2 and 1O2 appears to be necessary for killing of S. enteritidis. In tests for the ability to trigger an oxidative burst in murine peritoneal macrophages, strain 2547 triggered O2 generation at levels as high as those observed with strain 2822. These studies indicate that the difference between the protective and nonprotective strains is not attributed to susceptibility against active oxygen species nor to the ability to trigger an oxidative burst. From these observations, it is suggested that the difference is not due to differences in resistance to the killing of different strains within macrophages.

Difference in the protection against infection with different challenge strains of Salmonella enteritidis by killed vaccine.

Differences in protection against the challenge of different strains of formalin-killed cells obtained from Salmonella enteritidis were investigated. When strains 2547, 116M, 116-54, SR-98G, and 3775 were used as the challenge strain, protective effects were apparent in groups of mice immunized with formalin-killed cells from S. enteritidis strains (protective strains). Conversely, no protective effects were observed with the challenge of strains 2822, 3975, and IID-604 (nonprotective strains). Electrophoretic banding patterns of lipopolysaccharides in SDS-PAGE were similar between the LPS obtained from the various strains used in this study. Additionally, no differences in sensitivity to macrophage intracellular killing were observed between the protective and nonprotective strains. Phagocytic experiments by macrophages in vitro indicated that immune serum used as the opsonin promotes phagocytosis of various strains by macrophages as compared to using normal serum as the opsonin, but the rate of phagocytosis enhanced by immune serum is higher in protective strains than nonprotective strains. In studies of passive transfer of immune serum, it was found that mice passively immunized with immune serum could only protect against infection by challenging with protective strains with the exception of strain SR-98G. These results suggest that the protective effect observed with the challenge of protective strains may be due to macrophage phagocytosis enhanced by opsonization with immune serum.

Variability of protection against different strains induced by a culture filtrate (CF) obtained from Salmonella enteritidis.

Cross-protective ability against the challenge of different strains of culture filtrate (CF) obtained from Salmonella enteritidis was investigated. Immunization with CF from various smooth strains with the exception of strain 3551 which is a rough strain of S. enteritidis induced protective immunity against challenge with strains 2547 and 116M. However, when strains 2822, 3551 and 3975 were used as the challenge strain, no protective effect was observed. The agglutination titers against whole cells of strain 2547 or 2822 of sera taken from the 2547 CF-immunized mice were almost the same as those of sera from the 2822 CF-immunized mice. Furthermore, delayed footpad reactivity was observed when heat-killed cells of strain 2547 or 2822 were transferred into the footpad of 2547 CF- or 2822 CF-immunized mice. These results show that the capability of inducing protective immunity is similar between the 2547 CF and 2822 CF, and protection against infection with S. enteritidis differs in accordance with different challenge strains.

Immunological studies on soluble protective antigen (SPA) separated from culture supernatant fluids of Salmonella enteritidis.

Studies were carried out to analyze the antigenicity of Soluble Protective Antigen (SPA) separated from culture supernatant fluids of Salmonella enteritidis strain 2547. Mice injected with anti-SPA mouse serum were capable of tolerating a challenge dose of 100 LD50 S. enteritidis. After absorption of the anti-SPA mouse serum with lipopolysaccharide (LPS) prepared from strain 2547, no protective effect was observed. Ouchterlony immunodiffusion analysis showed that the P1 fraction obtained from Sephadex G-50 gel filtration of strain 2547 or 2822 LPS reacted with antiserum to SPA, but no reaction was observed with the P2 or P3 fraction. The LPS from strain 2547 gave 80% mouse protection against challenge with 100 LD50 of the homologous bacteria, while the P1 from strain 2547 LPS afforded 40% protective immunity. When P1, LPS and SPA were transferred into the footpad of SPA-immunized mice, a positive delayed footpad reaction was elicited. Similarly, macrophage migration inhibitory activity was observed when SPA-induced peritoneal exudate cells were cultured in medium containing P1, LPS and SPA. These results suggest that the antigenic determinant of SPA exists in the O-antigenic components of LPS.

Biological and chemical studies on soluble protective antigen (SPA) from culture supernatant fluids of Salmonella enteritidis.

The chemical and biological characteristics of Soluble Protective Antigen (SPA) separated from culture fluids of Salmonella enteritidis strain 2547 were analysed. It was shown that SPA has 3-hydroxy, nonpolar fatty acids by thin-layer chromatography. The fatty acids were identified as lauric, myristic, palmitic and 3-hydroxymyristic acid using gas chromatography-mass spectrometry and mass chromatography. These fatty acids are common constituents of the lipid A obtained from S. enteritidis. SPA was found to enhance the plaque-forming cell (PFC)-response to sheep erythrocytes in the recipients' spleen. In addition, SPA enhanced the clotting activity of Limulus amebocyte lysate. These results show that SPA possesses the properties of lipopolysaccharides isolated from strain 2547 by chemical procedures.

Enhancing effect of Salmonella enteritidis SPA on nonspecific resistance.

The peritoneal macrophages from mice treated with soluble protective antigen (SPA) or lipopolysaccharide (LPS) showed an increase in chemotactic activity, but muramyl dipeptide (MDP)-induced macrophages did not show any enhancing effect. Conversely, the chemotactic activity of mice peritoneal neutrophils was enhanced by SPA, LPS or MDP treatment. The superoxide anion (O2-)-generating activity of SPA-induced macrophages was higher than LPS or MDP. And even 30 days after SPA treatment, a significant increase of O2(-)-generating activity was evident as compared to the control. SPA-induced macrophages showed a higher degree of intracellular killing of Listeria monocytogenes, in vitro, as compared to macrophages obtained from normal mice. In the studies of in vivo protection, the number of bacteria in the liver after challenging with L. monocytogenes was smaller in SPA-treated mice than in untreated mice. Also, SPA-treated mice showed an increased resistance to L. monocytogenes infection.