Effects of mannan oligosaccharide dietary supplementation on performances of the tropical spiny lobsters juvenile (Panulirus ornatus,Fabricius 1798)

The effects of dietary mannan oligosaccharide (MOS) (Bio-Mos^®, Alltech, USA) on the growth, survival, physiology, bacteria and morphology of the gut and immune response to bacterial infection of tropical rock lobsters (Panulirus ornatus) juvenile were investigated. Dietary inclusion level of MOS at 0.4% was tested against the control diet (trash fish) without MOS inclusion. At the end of 56 days of rearing period, a challenged test was also conducted to evaluate the bacterial infection resistant ability of the lobsters fed the two diets. Lobster juvenile fed MOS diet attained 2.86 ± 0.07 g of total weigh and 66.67 ± 4.76% survival rate which were higher (P < 0.05) than the lobsters fed control diet (2.35 ± 0.14 g total weight and 54.76 ± 2.38% survival rate, respectively) thus providing the higher (P < 0.05) specific growth rate (SGR) and average weekly gain (AWG) of lobsters fed MOS diet. Physiological condition indicators such as wet tail muscle index (Tw/B), wet hepatosomatic index (Hiw) and dry tail muscle index (Td/B) of the lobsters fed MOS supplemented diet were higher (P < 0.05) than that of the lobsters fed the control diet. Bacteria in the gut (both total aerobic and Vibrio spp.) and gut's absorption surface indicated by the internal perimeter/external perimeter ratio were also higher (P < 0.05) when the lobsters were fed MOS diet. Lobsters fed MOS diet were in better immune condition showed by higher THC and GC, and lower bacteraemia. Survival, THC, GC were not different among the lobsters fed either MOS or control diet after 3 days of bacterial infection while bacteraemia was lower in the lobsters fed MOS diet. After 7 days of bacterial infection the lobsters fed MOS diet showed higher survival, THC, GC and lower bacteraemia than the lobsters fed the control diet. The experimental trial demonstrated the ability of MOS to improve the growth performance, survival, physiological condition, gut health and immune responses of tropical spiny lobsters juveniles.     

Comparative determination of the rates of Kluyveromyces bulgaricus protoplast formation by the coulter counter technique and spectrophotometry

Kluyveromyces bulgaricus protoplast formation was studied by the determination of the remaining intact cells with the Coulter counter, the decrease of the absorbance of the cell suspension upon dilution into an hypoosmotic medium and the liberation of intracellular proteins after protoplast lysis. Due to the scattering of light by cells and ghosts, spectrophotometric readings were not enough specific and led to the underestimation of the reaction rates (about 40% less). The Coulter counter technique and the determination of the liberation of intracellular proteins gave the same rates over a large range of cell concentrations and in the presence of various cell lytic enzymes. The importance of corrected etermination of the time course of the reaction was illustrated by the good correlation obtained between protoplast formation and ATP turnover.     

Transport of phage P22 DNA across the cytoplasmic membrane

Although a great deal is known about the life cycle of bacteriophage P22, the mechanism of phage DNA transport into Salmonella is poorly understood. P22 DNA is initially ejected into the periplasmic space and subsequently transported into the host cytoplasm. Three phage-encoded proteins (gp16, gp20, and gp7) are coejected with the DNA. To test the hypothesis that one or more of these proteins mediate transport of the DNA across the cytoplasmic membrane, we purified gp16, gp20, and gp7 and analyzed their ability to associate with membranes and to facilitate DNA uptake into membrane vesicles in vitro. Membrane association experiments revealed that gp16 partitioned into the membrane fraction, while gp20 and gp7 remained in the soluble fraction. Moreover, the addition of gp16, but not gp7 or gp20, to liposomes preloaded with a fluorescent dye promoted release of the dye. Transport of ³²P-labeled DNA into liposomes occurred only in the presence of gp16 and an artificially created membrane potential. Taken together, these results suggest that gp16 partitions into the cytoplasmic membrane and mediates the active transport of P22 DNA across the cytoplasmic membrane of Salmonella.     

Toxoplasma gondii Cathepsin L Is the Primary Target of the Invasion-inhibitory Compound Morpholinurea-leucyl-homophenyl-vinyl Sulfone Phenyl

The protozoan parasite Toxoplasma gondii relies on post-translational modification, including proteolysis, of proteins required for recognition and invasion of host cells. We have characterized the T. gondii cysteine protease cathepsin L (TgCPL), one of five cathepsins found in the T. gondii genome. We show that TgCPL is the primary target of the compound morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS), which was previously shown to inhibit parasite invasion by blocking the release of invasion proteins from microneme secretory organelles. As shown by fluorescently labeled LHVS and TgCPL-specific antibodies, TgCPL is associated with a discrete vesicular structure in the apical region of extracellular parasites but is found in multiple puncta throughout the cytoplasm of intracellular replicating parasites. LHVS fails to label cells lacking TgCPL due to targeted disruption of the TgCPL gene in two different parasite strains. We present a structural model for the inhibition of TgCPL by LHVS based on a 2.0 Å resolution crystal structure of TgCPL in complex with its propeptide. We discuss possible roles for TgCPL as a protease involved in the degradation or limited proteolysis of parasite proteins involved in invasion.The recent completion of many genome-sequencing projects has allowed an unprecedented view of the complete set of proteases in biologically or medically important organisms (1). Of the five mechanistically distinct catalytic types (serine, cysteine, aspartyl, metallo, and threonine), cysteine proteases are the second largest group. In particular, cysteine proteases of the C1 papain family of “lysosomal” cathepsins have garnered intense scrutiny because of their key roles in cancer, embryogenesis, heart disease, osteoporosis, immunity, and infectious diseases. Microbial cathepsins, particularly those expressed by parasites, have also attracted attention recently because of their potential as targets for treatment of helminthic and protozoal infections (2, 3).The protozoan parasite Toxoplasma gondii infects virtually all warm-blooded animals and approximately one-third of the human population worldwide. Although most Toxoplasma infections are benign, severe opportunistic disease is seen in immunodeficient or immunosuppressed individuals or congenitally infected babies. T. gondii is an obligate intracellular organism that uses an actin-myosin-based motility system to actively invade nucleated host cells (4, 5). The parasite secretes a variety of proteins during and after cell invasion that contribute to recognition of the host cell, formation of an adhesive “moving” junction, modulation of host signaling pathways and gene expression, and remodeling of the parasitophorous vacuole in preparation for parasite growth (6, 7). Although it has been known for some time that many Toxoplasma secretory proteins are post-translationally modified by proteolysis before and/or after secretion, in most cases, the consequences of proteolysis or the specific protease involved are unclear.Analysis of the T. gondii genome indicates the existence of five genes encoding cathepsin proteases of the papain family, including three cathepsin C proteases (TgCPC1, TgCPC2, and TgCPC3), one cathepsin B (Toxopain-1 or TgCPB), and one cathepsin L (TgCPL). TgCPC1 and TgCPC2 are secreted into the parasitophorous vacuole after parasite invasion and are proposed to function in nutrient acquisition (8). TgCPC3 is not expressed in tachyzoites, a rapidly dividing form of the parasite that is most commonly studied in the laboratory. TgCPB is localized in club-shaped invasion organelles called rhoptries, where it may act as a maturase for rhoptry proteins involved in modulation of the host cell (9). TgCPL is predicted to be a type II membrane protein, and a recent report by Reed and co-workers (10) showed that it has enzymatic activity with a low pH optimum and that it occupies a membrane-bound structure in the apical region of extracellular parasites. This same study revealed that T. gondii expresses two endogenous inhibitors of cysteine proteases (TgICP1 and TgICP2), but their role in regulating parasite or host cysteine proteases remains to be determined. Similar inhibitors are expressed by other parasites, including Trypanosoma cruzi, that act on host proteases, and the crystal structure of an inhibitor (chagasin)-enzyme (human cathepsin L) complex was recently reported (11).In a recent study, we screened a small library of cathepsin and proteasome inhibitors and identified two compounds that substantially impair Toxoplasma cell invasion (12). The most effective of these compounds, morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS),² inhibited invasion with a 50% inhibitory concentration (IC₅₀) of ∼10 μm. Further analysis revealed that LHVS blocks parasite attachment and gliding motility by impairing the release of proteins from a distinct set of apical secretory organelles called micronemes. Here we definitively show, using a variety of biochemical, genetic, and structural approaches, that TgCPL is the primary target of LHVS in the parasite.