Factors affecting motility and morphology of Cryptosporidium sporozoites in vitro

In vitro motility and morphology of Cryptosporidium sporozoites were examined in the presence of various solutions. Crude preparations of the bile salt, taurocholic acid, maintained both motility and morphology in a dose-dependent manner. These effects appeared to be due to the taurocholic acid itself, and not simply due to pH variations, osmotic factors, or contaminants. Lysis of sporozoites was also observed and was found to be dependent on pH, with acidic conditions (pH less than 6.2) triggering the lysis.     

Studies on the Motility of Plasmodium Sporozoites*

Albumin was found to have a striking stimulatory effect on motility of Plasmodium sporozoites, while serum globulins had an inhibitory effect. Albumin also preserved viability of sporozoites in vitro at 4 C for several days. P. berghei, P. cynomolgi, and P. falciparum sporozoites each had a distinct and characteristic type of motility. P. berghei sporozoites from oocysts had a different type of motility from that of salivary gland sporozoites, each type presumably associated with different invasive capacities at different times during the life cycle of the parasite. This change in sporozoite motility during development was also associated with other physiologic developmental changes in the sporozoite. The degree of motility of a given pool of sporozoites was to some degree associated with other parameters of metabolic activity of these sporozoites, i.e. infectivity, immunogenicity, and secretory activity. Secretions of the rhoptry-microneme complex may play a role in sporozoite motility.     

The Effects of pH,Buffers, Bile and Bile Acids on Excystation of Sporozoites of Various Eimeria Species*

SYNOPSIS. Oocysts of Eimeria bovis were found to undergo excystation when subjected at 39 C to a pretreatment consisting of exposure for 24 hr to CO₂ and air (50–50), and a treatment for 7 hr with a mixture of bile and trypsin. At pH's of 6.0 thru 10.0 with tris-maleate buffer, excystation occurred over the entire range of pH tested, with the highest levels at pH 7.5-8.5. No adverse or inhibitive effect on excystation or the viability of the sporozoites was observed. Disintegration of sporozoites occurred within the sporocysts of intact oocysts at each of the pH levels studied when boric acid-borax, ammediol, and glycine-sodium hydroxide buffers were used in the treatment medium. Phosphate buffer inhibited excystation when used in the excysting medium. Excystation occurred at levels above 90% in all dilutions of taurocholic, glycocholic, glycotaurocholic, and cholic acids included in the study (0.5-10.0%) except for the 10% and 5% dilutions of cholic acid and the 10% dilution of glycotaurocholic acid. In the latter 3 dilutions, sporozoites within the sporocysts of intact oocysts disintegrated. Excystation levels above 90% were observed in the 50% and 10% dilutions of fresh bovine bile, and in the 5% dilution of lyophilized bovine bile. Lower levels of excystation occurred in greater dilutions of both kinds of bile. No excystation occurred when any of the bile acids, fresh bovine bile or lyophilized bile were used without trypsin, except for fresh bile that contained a heavy suspension of bacteria and fungi. In a medium containing trypsin and heat-treated bile, heat-treated bile acids, or no bile, 2.5–8% of the oocysts excysted. The findings indicate that satisfactory excystation can be obtained with a treatment medium containing tris-maleate at pH 7.5–8.5, 0.25% trypsin, and 1% of one of the bile acids.     

Palmitoyl transferases have critical roles in the development of mosquito and liver stages of Plasmodium

As the Plasmodium parasite transitions between mammalian and mosquito host, it has to adjust quickly to new environments. Palmitoylation, a reversible and dynamic lipid post‐translational modification, plays a central role in regulating this process and has been implicated with functions for parasite morphology, motility and host cell invasion. While proteins associated with the gliding motility machinery have been described to be palmitoylated, no palmitoyl transferase responsible for regulating gliding motility has previously been identified. Here, we characterize two palmityol transferases with gene tagging and gene deletion approaches. We identify DHHC3, a palmitoyl transferase, as a mediator of ookinete development, with a crucial role for gliding motility in ookinetes and sporozoites, and we co‐localize the protein with a marker for the inner membrane complex in the ookinete stage. Ookinetes and sporozoites lacking DHHC3 are impaired in gliding motility and exhibit a strong phenotype in vivo; with ookinetes being significantly less infectious to their mosquito host and sporozoites being non‐infectious to mice. Importantly, genetic complementation of the DHHC3‐ko parasite completely restored virulence. We generated parasites lacking both DHHC3, as well as the palmitoyl transferase DHHC9, and found an enhanced phenotype for these double knockout parasites, allowing insights into the functional overlap and compensational nature of the large family of PbDHHCs. These findings contribute to our understanding of the organization and mechanism of the gliding motility machinery, which as is becoming increasingly clear, is mediated by palmitoylation.     

Malaria Sporozoites Leave Behind Trails of Circumsporozoite Protein During Gliding Motility1

As Plasmodium sporozoites undergo gliding motility in vitro, they leave behind trails of circumsporozoite (CS) protein that correspond to their patterns of movement. This light microscopic observation was made using Plasmodium berghei sporozoites, a monoclonal antibody (MAb H4) directed against the immunodominant repetitive epitope of the CS protein of P. berghei, and an immunogold-silver staining (IGSS) technique. Sporozoites pretreated with agents that inhibit sporozoite motility and invasiveaess did not produce trails. Sporozoites that glided on microscope slides coated with MAb H4 left behind considerably longer CS prolem trails than those on uncoated slides, and the staining of these trails was more intense. The fact that the CS protein is an exoantigen continuously released as trails by motile sporozoites, together with our previous finding that anti-CS protein antibodies inhibit sporozoite motility, strongly suggests that the CS protein plays a role in gliding motility. The sensitive IGSS technique used in this study may be a useful tool in the study of the translocation of surface proteins during gliding of other apicomplexans, other protists, and bacteria.     

Vital role for the Plasmodium actin capping protein (CP) beta‐subunit in motility of malaria sporozoites

Successful malaria transmission from the mosquito vector to the mammalian host depends crucially on active sporozoite motility. Sporozoite locomotion and host cell invasion are driven by the parasite's own actin/myosin motor. A unique feature of this motor machinery is the presence of very short subpellicular actin filaments. Therefore, F‐actin stabilizing proteins likely play a central role in parasite locomotion. Here, we investigated the role of the Plasmodium berghei actin capping protein (PbCP), an orthologue of the heterodimeric regulator of filament barbed end growth, by reverse genetics. Parasites containing a deletion of the CP beta‐subunit developed normally during the pathogenic erythrocytic cycle. However, due to reduced ookinete motility, mutant parasites form fewer oocysts and sporozoites in the Anopheles vector. These sporozoites display a vital deficiency in forward gliding motility and fail to colonize the mosquito salivary glands, resulting in complete attenuation of life cycle progression. Together, our results show that the CP beta‐subunit exerts an essential role in the insect vector before malaria transmission to the mammalian host. The vital role is restricted to fast locomotion, as displayed by Plasmodium sporozoites.     

Involvement of Trihydroxyconjugated Bile Salts in Cholesterol Assimilation by Bifidobacteria

To determine the conditions of cholesterol assimilation, various strains of Bifidobacterium species were cultured in the presence of cholesterol and bile salts. During culturing, Bifidobacterium breve ATCC 15700 assimilates cholesterol in the presence of oxgall at pH values lower than 6. This strain was selected to study the influence of conjugated (taurocholic acid) and deconjugated (cholic acid) bile salts on cholesterol assimilation. B. breve ATCC 15700 assimilated cholesterol (up to 51%) when cultures were undertaken in the presence of taurocholic acid, whereas less than 13% of the initial amount of cholesterol was measured in the cells in the presence of cholic acid. Cultured in the presence of six individual di- or trihydroxyconjugated bile salts, bifidobacteria strains assimilated cholesterol. This assimilation appeared to be more important in the presence of trihydroxyconjugated bile salts (tauro- and glycocholic acids). It is concluded that trihydroxyconjugated bile salts are involved in the assimilation of cholesterol by bifidobacteria. Received: 20 June 1996 / Accepted: 19 July 1996     

Rhoptry Secretion of Membranous Whorls by Plasmodium berghei Sporozoites1

Electron microscopy of sporozoites of the rodent malaria parasite, Plasmodium berghei, reveals electron-dense multi-laminate membranous whorls within components of the rhoptry-microneme complex after fixation with tannic acid in conjunction with glutaraldehyde. This multilaminate material, which has a dark line to dark line periodicity of approximately 5 nm, appears to be secreted from the sporozoite since it is also found adhering to the sporozoite's external surface. The material may function in sporozoite gliding motility and in invasion of host cells.     

Automated classification of Plasmodium sporozoite movement patterns reveals a shift towards productive motility during salivary gland infection

The invasive stages of malaria and other apicomplexan parasites use a unique motility machinery based on actin, myosin and a number of parasite-specific proteins to invade host cells and tissues. The crucial importance of this motility machinery at several stages of the life cycle of these parasites makes the individual components potential drug targets. The different stages of the malaria parasite exhibit strikingly diverse movement patterns, likely reflecting the varied needs to achieve successful invasion. Here, we describe a Tool for Automated Sporozoite Tracking (ToAST) that allows the rapid simultaneous analysis of several hundred motile Plasmodium sporozoites, the stage of the malaria parasite transmitted by the mosquito. ToAST reliably categorizes different modes of sporozoite movement and can be used for both tracking changes in movement patterns and comparing overall movement parameters, such as average speed or the persistence of sporozoites undergoing a certain type of movement. This allows the comparison of potentially small differences between distinct parasite populations and will enable screening of drug libraries to find inhibitors of sporozoite motility. Using ToAST, we find that isolated sporozoites change their movement patterns towards productive motility during the first week after infection of mosquito salivary glands.     

Bile salt alteration of ion transport across jejunal mucosa

The effect of conjugated dihydroxy and trihydroxy bile salts on electrolyte transport across isolated rabbit jejunal mucosa was studied. Both taurochenodeoxycholic acid and taurocholic acid increased the short-circuit current (I_sc) in bicarbonate-Ringer solution but not in a bicarbonate-free, chloride-free solution. Taurochenodeoxycholic acid was significantly more effective than taurocholic acid in increasing I_sc. The presence of theophylline prevented the taurochenodeoxycholic acid-and taurocholic acid-induced increase in I_sc. Transmural ion fluxes across jejunal mucosa demonstrated that 2 mM taurochenodeoxycholic acid decreased net Na⁺ absorption, increased net Cl⁻ secretion and increased the residual flux (which probably represents HCO₃⁻ secretion). These studies support the hypothesis that cyclic AMP may be a mediator of intestinal electrolyte secretion.     

Effects of oleic acid and bile salts on canine villous motility

The effects on canine villous motility of mucosal Tyrodes solution containing oleic acid (10 mM) and/or either taurocholic or cholic acid (15 mM) in the presence or absence of IV atropine (1 mg/kg) was used to assess the neural mediation of the effects of luminal nutrients. Villous motility was measured over 12 min periods by in vivo videomicroscopy of segments of jejunum. Neither bile salt had effects alone but villous motility increased after oleic acid was added to taurocholate and decreased after oleic acid was added to cholate. Villous motility increased when taurocholate and oleic acid were present initially and returned to control levels when removed. Villous motility was not affected by cholate and oleic acid but villous motility decreased when they were removed from the Tyrodes solution. Atropine blocked the increase in villous motility caused by taurocholate and oleic acid. Bile salts can modify the effect of oleic acid on villous motility and a cholinergic step is involved in the stimulation of motility.     

In Vitro Excystation of Caryospora simplex (Apicomplexa: Eimeriorina)1

In vitro excystation of sporozoites of the heteroxenous coccidian Caryospora simplex Léger, 1904 (Apicomplexa: Eimeriorina) is described. Sporocysts freed mechanically from oocysts released a maximum of 51% of their sporozoites within 45 min at 25°C and a maximum of 74% within 20 min at 37°C when incubated in a 0.25% (w/v) trypsin–0.75% (w/v) sodium taurocholate (bile salt) excystation solution. At emergence from sporocysts, sporozoites were weakly motile then became highly active after about 2 min in excystation solution. Sporozoites within sporocysts exposed to bile salt only became highly motile within 25 min at 25°C and within 15 min at 37°C but did not excyst. When exposed only to trypsin at the above temperatures, the Stieda body dissolved; the substieda body remained intact, and the sporozoites exhibited only limited motility within sporocysts; only a few excysted. Intact, sporulated oocysts incubated at 25° or 37°C in 0.02 M cysteine-HC1 and a 50% CO₂ atmosphere for 18 h had no morphologic changes in the oocyst wall. Further incubation of these intact oocysts in excystation solution for 30 min at 37°C caused neither motility of sporozoites within sporocysts nor excystation. Grinding oocysts for 30 sec in a motor-driven, teflon-coated tissue grinder caused motility of some sporozoites within sporocysts but did not result in excystation.     

Identification,release and olfactory detection of bile salts in the intestinal fluid of the Senegalese sole (<Emphasis Type="Italic">Solea senegalensis</Emphasis>)

Olfactory sensitivity to bile salts is wide-spread in teleosts; however, which bile salts are released in sufficient quantities to be detected is unclear. The current study identified bile salts in the intestinal and bile fluids of Solea senegalensis by mass spectrometry–liquid chromatography and assessed their olfactory potency by the electro-olfactogram. The main bile salts identified in the bile were taurocholic acid (342 mM) and taurolithocholic acid (271 mM) plus a third, unidentified, bile salt of 532.3 Da. These three were also present in the intestinal fluid (taurocholic acid, 4.13 mM; taurolithocholic acid, 0.4 mM). In sole-conditioned water, only taurocholic acid (0.31 μM) was released in sufficient quantities to be measured (release rate: 24 nmol kg⁻¹ min⁻¹). Sole had high olfactory sensitivity to taurocholic acid but not to taurolithocholic acid. Furthermore, olfactory sensitivity was higher in the upper (right) olfactory epithelium than the lower (left). These two bile acids contribute about 40% of the olfactory potency of intestinal fluid and account for the difference in potency at the two epithelia. Taurocholic acid (but not taurolithocholic acid), and possibly other types of bile acid not tested, could be used as chemical signals and the upper olfactory epithelium is specialised for their detection.     

Human beta-glucuronidase. Studies on the effects of pH and bile acids in regard to its role in the pathogenesis of cholelithiasis

Human bile contains a considerable amount of endogenous beta-glucuronidase. The effects of pH and bile acids on its activity have been studied in regard to its role in the pathogenesis of cholelithiasis. beta-Glucuronidase, purified from human liver to homogeneity, was structurally stable between pH 4 and 10, but was active only over a much narrower range of pH, with a pH optimum of 5.2. The inactivation below pH 4 was due to its irreversible denaturation, whereas the inactivation at higher pH was due to a true reversible pH effect on the enzyme velocity. Kinetic studies revealed that hydrogen ion acted as a substrate-directed activator of the free enzyme, but not the enzyme-substrate complex, with a molecular dissociation constant of 4 X 10(-6). The enzyme activity was not affected by unconjugated bile acids, primarily due to their extremely low water solubility. Conjugated bile acids, on the other hand, exerted heterogeneous and pH-dependent effects on the enzyme. At pH 5.2, taurocholic acid and glycocholic acid were substrate-directed activators of the enzyme; taurochenodeoxycholic acid and taurodeoxycholic acid, competitive inhibitors; and glycochenodeoxycholic acid and glycodeoxycholic acid, mixed inhibitors. At pH 7.0 all taurine and glycine conjugates behaved as substrate-directed activators. Though beta-glucuronidase activity at pH 7 was only 23% of its maximal activity at pH 5.2, conjugated bile acids tended to restore its activity to a certain extent at pH 7. Thus, endogenous beta-glucuronidase could play a significant role in pigment cholelithiasis.     

单增李斯特菌二硫键形成蛋白DsbG介导酸耐受及鞭毛运动性调控北大核心

【目的】本研究旨在通过构建单增李斯特菌(Listeria monocytogenes)二硫键形成蛋白编码基因dsbG缺失株和回补株,探究该蛋白在酸耐受和鞭毛介导的运动性中发挥的生物学功能。【方法】利用同源重组方法构建单增李斯特菌dsbG缺失株和回补株,比较野生株和突变株在不同pH梯度培养基条件下的生长速率和存活率;通过实时荧光定量PCR方法,比较致死酸应激条件下野生株和缺失株酸耐受基因转录水平。通过半固体培养基、荧光定量PCR方法和鞭毛负染色透射电镜观察,比较野生株和突变株的运动能力、鞭毛相关基因转录水平变化和鞭毛形态差异。【结果】与野生株相比,dsbG缺失株的生长能力和速率差异不显著;在pH 3.5(盐酸和柠檬酸)培养条件下,存活率显著降低;精氨酸合成途径基因argD和argF转录水平分别下调2.4和3.7倍。同时,dsbG缺失株的运动能力减弱,且鞭毛形成相关的flaA、flgB和flgD等基因转录水平显著下调(分别为29.7、6.7和6.9倍),鞭毛形成能力减弱。【结论】本研究首次证实了单增李斯特菌二硫键形成蛋白DsbG能感应低pH应激,并形成耐受;证实了DsbG通过调控鞭毛相关基因的转录进而影响细菌的鞭毛形成和运动性。本研究有助于深入了解二硫键形成蛋白家族介导单增李斯特菌环境适应的分子机制,为食源性致病菌的防控提供理论基础。     

Properties of Crystalline 3β-Hydroxysteroid Dehydrogenase from Pseudomonas putida

A crystalline 3α-hydroxysteroid: NAD⁺-oxidoreductase (EC 1 1.1.50) which had been obtained from the cell-free extracts of Pseudomonas putida NRRL B-11064 in the presence of added polyethylene glycol, was found to be a native monomer form with a specific activity of 63.0 and a molecular weight of 45,000. Isoelectric focusing exhibited the enzyme to be composed of two isoenzymes: one major part focusing at pH 4.75 and a minor part focusing at pH 5.10. Whereas the enzyme was changed from the monomeric form to a dimeric one with a considerable decrease in the specific activity during the course of crystallization in the absence of the added polyethylene glycol.

The enzyme showed an absolute specificity with regard to 3α-hydroxyl group besides a high requirement for cis A: B fusion of steroids. Typical substrates are cholic acid (Km = 1.33 × 10^?5 m), deoxycholic acid, chenodeoxycholic acid, 3α-hydroxy-12-keto-9,11-cholanoic acid, and etiocholan-3α-ol-17-one. Conjugated bile acids such as taurocholic acid and glycocholic acid are also rapidly oxidized. The pH optima for oxidation of cholic acid and reduction of etiocholan-3,17-dione were 11.5 and 7.0, respectively. The enzyme could be employed for the sensitive and specific assay of bile acids.     

Effects of Cultivation Parameters on the Morphology of Rhizopus arrhizus and the Lactic Acid Production in a Bubble Column Reactor

The use of filamentous Rhizopus for lactic acid production is facing a challenge due to its low yield mainly caused by the difficulty to control its morphology in submerged fermentation processes. This study was aimed at investigating the impacts of cultivation parameters on the morphology of Rhizopus arrhizus DAR 36017 and lactic acid production using waste potato starch in a laboratory scale bubble column reactor (BCR). The fungal morphology was significantly influenced by carbon sources, process pH, starch concentrations, sparger designs and aeration rates. The favorable morphology for lactic acid production was a freely dispersed small pellet, which was achieved under operation conditions at pH 5.0–6.0, starch concentrations of 60–120 g/L and aeration rates of 0.2–0.8 vvm using a sintered stainless steel disc sparger. Optimal cultivation conditions at pH 6.0 and an aeration rate of 0.4 vvm resulted in the formation of freely dispersed small pellets and 103.8 g/L lactic acid with a yield of 87 % from 120 g/L liquefied potato starch in 48 h. The overall results in terms of lactic acid yield and productivity are comparable to those reported in previous studies using immobilized Rhizopus cells in batch fermentations.     

Malaria IMC1 membrane skeleton proteins operate autonomously and participate in motility independently of cell shape

Plasmodium IMC1 (inner membrane complex 1) proteins comprise components of the subpellicular network, a lattice of intermediate filaments that form a structural part of the pellicle in the zoite stages of malaria parasites. Family members IMC1a and IMC1b are differentially expressed in sporozoites and ookinetes, respectively, but have functionally equivalent roles affecting cell morphology, strength, motility, and infectivity. Because of the coincident effects of previous imc1 gene disruptions on both zoite shape and locomotion, it has been impossible to ascribe a direct involvement in motility to these proteins. We show here that a third family member, IMC1h, has a distinct differential expression pattern and localizes to the pellicle of both ookinetes and sporozoites. Knock-out of IMC1h mimics the loss-of-function phenotypes of IMC1a and IMC1b in their respective life stages, indicating that IMC1 proteins could be operating co-dependently. By generating double null mutant parasites for IMC1h and IMC1b, we tested this hypothesis: double knock-out exacerbated the phenotypes of the single knock-outs in terms of ookinete strength, motility, and infectivity but did not further affect ookinete morphology. These findings provide the first genetic evidence that IMC1 proteins can function independently of each other and contribute to gliding motility independently of cell shape.     

Rhoptry neck protein 11 has crucial roles during malaria parasite sporozoite invasion of salivary glands and hepatocytes

The malaria parasite sporozoite sequentially invades mosquito salivary glands and mammalian hepatocytes; and is the Plasmodium lifecycle infective form mediating parasite transmission by the mosquito vector. The identification of several sporozoite-specific secretory proteins involved in invasion has revealed that sporozoite motility and specific recognition of target cells are crucial for transmission. It has also been demonstrated that some components of the invasion machinery are conserved between erythrocytic asexual and transmission stage parasites. The application of a sporozoite stage-specific gene knockdown system in the rodent malaria parasite, Plasmodium berghei, enables us to investigate the roles of such proteins previously intractable to study due to their essentiality for asexual intraerythrocytic stage development, the stage at which transgenic parasites are derived. Here, we focused on the rhoptry neck protein 11 (RON11) that contains multiple transmembrane domains and putative calcium-binding EF-hand domains. PbRON11 is localised to rhoptry organelles in both merozoites and sporozoites. To repress PbRON11 expression exclusively in sporozoites, we produced transgenic parasites using a promoter-swapping strategy. PbRON11-repressed sporozoites showed significant reduction in attachment and motility in vitro, and consequently failed to efficiently invade salivary glands. PbRON11 was also determined to be essential for sporozoite infection of the liver, the first step during transmission to the vertebrate host. RON11 is demonstrated to be crucial for sporozoite invasion of both target host cells – mosquito salivary glands and mammalian hepatocytes – via involvement in sporozoite motility.     

Plasmodium yoelii S4/CelTOS is important for sporozoite gliding motility and cell traversal

Gliding motility and cell traversal by the Plasmodium ookinete and sporozoite invasive stages allow penetration of cellular barriers to establish infection of the mosquito vector and mammalian host, respectively. Motility and traversal are not observed in red cell infectious merozoites, and we have previously classified genes that are expressed in sporozoites but not merozoites (S genes) in order to identify proteins involved in these processes. The S4 gene has been described as criticaly involved in Cell Traversal for Ookinetes and Sporozoites (CelTOS), yet knockout parasites (s4/celtos¯) do not generate robust salivary gland sporozoite numbers, precluding a thorough analysis of S4/CelTOS function during host infection. We show here that a failure of oocysts to develop or survive in the midgut contributes to the poor mosquito infection by Plasmodium yoelii (Py) s4/celtos¯ rodent malaria parasites. We rescued this phenotype by expressing S4/CelTOS under the ookinete‐specific circumsporozoite protein and thrombospondin‐related anonymous protein‐related protein (CTRP) promoter (S4/CelTOS^CTRP), generating robust numbers of salivary gland sporozoites lacking S4/CelTOS that were suitable for phenotypic analysis. Py S4/CelTOS^CTRP sporozoites showed reduced infectivity in BALB/c mice when compared to wild‐type sporozoites, although they appeared more infectious than sporozoites deficient in the related traversal protein PLP1/SPECT2 (Py plp1/spect2¯). Using in vitro assays, we substantiate the role of S4/CelTOS in sporozoite cell traversal, but also uncover a previously unappreciated role for this protein for sporozoite gliding motility.