ATP-specificity of succinyl-CoA synthetase from Blastocystis hominis.

Succinyl-CoA synthetase (SCS) catalyzes the only step of the tricarboxylic acid cycle that leads to substrate-level phosphorylation. Some forms of SCS are specific for ADP/ATP or for GDP/GTP, while others can bind all of these nucleotides, generally with different affinities. The theory of `gatekeeper' residues has been proposed to explain the nucleotide-specificity. Gatekeeper residues lie outside the binding site and create specific electrostatic interactions with incoming nucleotides to determine whether the nucleotides can enter the binding site. To test this theory, the crystal structure of the nucleotide-binding domain in complex with Mg2+-ADP was determined, as well as the structures of four proteins with single mutations, K46ßE, K114ßD, V113ßL and L227ßF, and one with two mutations, K46ßE/K114ßD. The crystal structures show that the enzyme is specific for ADP/ATP because of interactions between the nucleotide and the binding site. Nucleotide-specificity is provided by hydrogen-bonding interactions between the adenine base and Gln20ß, Gly111ß and Val113ß. The O atom of the side chain of Gln20ß interacts with N6 of ADP, while the side-chain N atom interacts with the carbonyl O atom of Gly111ß. It is the different conformations of the backbone at Gln20ß, of the side chain of Gln20ß and of the linker that make the enzyme ATP-specific. This linker connects the two subdomains of the ATP-grasp fold and interacts differently with adenine and guanine bases. The mutant proteins have similar conformations, although the L227ßF mutant shows structural changes that disrupt the binding site for the magnesium ion. Although the K46ßE/K114ßD double mutant of Blastocystis hominis SCS binds GTP better than ATP according to kinetic assays, only the complex with Mg2+-ADP was obtained.

[Genotype analysis and isoenzyme patterns of ten isolates of Blastocystis hominis from Guangxi].

OBJECTIVE: To analyze genotypes and lactate dehydrogenase (LDH) and esterase (EST) patterns in 10 isolates of Blastocystis hominis collected from Guangxi. METHODS: Ten B. hominis isolates (BhGX1-BhGX10) were obtained from the fecal specimens of patients and cultivated in vitro, and then the genomic DNA was extracted. The isolates were genotyped by PCR using seven pairs of known sequenced-tagged site (STS) primers. Isoenzyme patterns of LDH and EST were investigated by SDS-PAGE. RESULTS: Out of the 10 isolates, 8 were identified as genotype I and the genotypes of the other two (BhGX4 and BhGX7) were unknown which were negative to all the STS primers. Among the ten isolates, 10 LDH bands were found, more with Rm37, Rm49, Rm57, Rm68 and Rm92. 12 bands showed in EST patterns: Rm14, Rm18, Rm23, Rm27, Rm35, Rm41, Rm45, Rm50, Rm55, Rm68, Rm77 and Rm82. Difference existed with the LDH and EST patterns among the isolates. CONCLUSION: Genotype I is the major one in the 10 B. hominis isolates from Guangxi, and the isolates show different isoenzyme patterns for LDH and EST.

Proteaese activity of Blastocystis hominis subtype3 in symptomatic and asymptomatic patients.

Despite accumulating evidence indicating that Blastocystis hominis is pathogenic and that cysteine proteases are involved in its pathogenesis, few researches discussed the protease activity of B. hominis genetic subtypes. Therefore, the present study aims to identify the underlying pathogenic role of the proteases of B. hominis subtype 3 at different molecular weights in correlation to gastrointestinal symptoms. Of 65 patients with various clinical presentations referred to our laboratory for stool examination, 26 (40%) were B. hominis positive by stool culture. Of 26 (group I) B. hominis patients, 18 (69.2%) were symptomatic (group IA) and 8(30.8%) were asymptomatic (group IB). Of 25 normal control group (group II), 5 (20%) were B. hominis positive. Subtype 3 was the only genotype recovered by polymerase chain reaction. Of 26 patients in group I, 19 (73.1%) were immunocompetent and 7 (26.9%) were immunocompromised. Protease activities of B. hominis subtype 3 were recognized at 32-kDa (46.2%), 39-kDa (7.7%), 120-kDa (38.5%), 140-kDa (11.5%), and 215-kDa (19.2%) bands in gelatin sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteases were recognized in 17 (94.4%) out of 18 symptomatic Blastocystis patients versus 2 (25.0%) out of 8 asymptomatic patients. Proteases at 32 kDa were reported in 61.1% of symptomatic versus 12.5% of asymptomatic patients. It was concluded that proteases of B. hominis genetic subtype 3, particularly those at 32 kDa, could be considered a virulence factor that is responsible for protein degradation and have a possible pathogenic role in host immune evasion.

[Isoenzyme patterns of the isolates of Blastocystis hominis from Guangxi].

OBJECTIVE: To observe malate dehydrogenase and alkaline phosphatase patterns of ten isolates of Blastocystis hominis (Bh) from Guangxi. METHODS: Blastocystis hominis agents were isolated from the fecal specimens of patients and cultivated in vitro. The samples were prepared for polyacrylamide gel slab electrophoresis (PAGSE). Sodium malate and 1-Naphthyl phosphoric acid sodium were used as substrates. NBT and fast blue RR salt were used to stain MDH and ALP respectively. The isoenzyme bands were recorded with relative mobility (Rm). RESULTS: Among the 10 isolates, 7 MDH bands were found, more with Rm34, Rm47, Rm51, Rm55, and Rm59. All the isolates showed Rm34 and Rm51 bands. 5 bands showed ALP patterns: Rm22, Rm25, Rm28, Rm35, and Rm38. Difference existed with the MDH and ALP patterns among the isolates. CONCLUSION: MDH and ALP patterns may indicate a genetic difference among the isolates, which might play a role in its classification.

Urinary hyaluronidase activity in rats infected with Blastocystis hominis--evidence for invasion?

The fact whether Blastocystis hominis can invade has always been in question. Apart from a few sporadic studies such as that done on gnotobiotic guinea pigs which showed surface invasion and mucosal inflammation of the host's intestine caused by B. hominis infection, no real documentation of invasion has been proven. Studies have shown that hyaluronidase is secreted during the penetration into the host's skin and gut by nematode parasites. Hyaluronidase activity in protozoa namely Entamoeba histolytica has also been described previously. This study attempts to determine hyaluronidase in urine samples of B. hominis-infected rats. The presence of hyaluronidase in urine provides an indirect evidence of invasion by B. hominis into colonic epithelium causing the degradation of extracellular matrix proteins namely hyaluronic acid (HA). HA is depolymerized by hyaluronidase which may be used by organisms to invade one another. In this study, the levels of urinary hyaluronidase of Sprague-Dawley rats infected with B. hominis were monitored for 30 days. Hyaluronidase levels in the infected rats were significantly higher on days 28 and 30 compared to the day before inoculation (P < 0.01 and P < 0.05, respectively). During this stage, parasitic burden in infected stools was also at a high level. Proinflammatory cytokines, interleukin-6 and interleukin-8, were also significantly higher (P < 0.05) in the serum of infected rats. The study demonstrates that since no other pathogen was present and that amoeboid forms of the parasites have been shown to exist previously, the elevated levels of hyaluronidase in this preliminary finding suggests that the organism is capable of having invasion or penetration activity in the hosts' intestine.

Protease activity of Blastocystis hominis.

Parasite-derived proteases are important for the parasite life cycle and the pathogenesis of the disease they produce. Proteases of intestinal protozoan parasite Blastocystis hominis were studied for the first time with azocasein assays and gelatin SDS-PAGE analysis. Parasitic lysates were found to have high protease activity and nine protease bands of low (20-33 kDa) and high (44-75 kDa) molecular weights were reported. Proteases were found to be pH-dependent and highest proteolytic activity was observed at neutral pH. Inhibition studies showed that B. hominis isolate B, like many other protozoan parasites, contains mainly cysteine proteases.

Degradation of human secretory immunoglobulin A by Blastocystis.

Microbial immunoglobulin A (IgA) proteases cleave human secretory IgA, promoting the mucosal adhesion of pathogens. To investigate if the enteric protozoan Blastocystis degrades human secretory IgA, cell lysate and conditioned medium from two species were exposed to immunoglobulin A. Secretory IgA was cleaved by both cell lysate and conditioned medium with mainly cysteine proteinase activity in B. hominis B isolate and aspartic proteinase activity in B. ratii WR1 isolate. These findings suggest that Blastocystis proteases may play a role in parasite survival in vivo.

Blastocystis hominis: evidence for caspase-3-like activity in cells undergoing programmed cell death.

We have shown previously that the human intestinal protozoan, Blastocystis hominis, undergoes apoptosis-like programmed cell death (PCD) when exposed to a cytotoxic monoclonal antibody (mAb), 1D5. In the present study, ELISA and immunoblot assays employing chicken anti-CPP32 antibody suggest that caspase-3-like antigens exist in B. hominis. Using colorimetric and flow cytometric assays for caspase-3 activity, we also observed an increase in activity between 1 h and 6 h after exposure to mAb 1D5, with greatest activity at 6 h. These findings suggest that caspase-3-like proteases play an important role in B. hominis undergoing PCD, similar to the phenomenon in higher eukaryotic organisms.

Isoenzyme patterns of Blastocystis hominis patient isolates derived from symptomatic and healthy carriers.

Isolates of Blastocystis hominis derived from patients with intestinal symptoms and from healthy carriers were cultured in vitro and isoenzyme patterns of hexokinase (E.C. 2.7.1.1), phosphoglucomutase (E.C. 2.7.5.1) and glucose phosphate isomerase (E.C. 5.3.1.9) were investigated to find evidence for pathogenic and non-pathogenic subspecies of B. hominis. For this purpose we examined 2000 patients of the out-patient department of the Institute for Tropical Medicine in Tübingen. From these, we obtained 232 B. hominis patient isolates; 119 isolates could be tested for the three isoenzymes. Blastocystis hominis possesses 5 patterns for hexokinase, 11 for phosphoglucomutase and 35 for glucose phosphate isomerase, showing that B. hominis is highly polymorphic. However, there was no correlation between isoenzyme patterns and disease of patients.

Biochemical characterisation of human isolates of Blastocystis hominis.

SDS-PAGE and iso-enzyme analysis of 11 human isolates of Blastocystis hominis revealed at least two variants with different polypeptide patterns and two zymodemes, respectively. This is the first iso-enzyme and the second protein analysis to indicate strain differences in B. hominis.