Mutual Exclusivity of Hyaluronan and Hyaluronidase in Invasive Group A Streptococcus

A recent analysis of group A Streptococcus (GAS) invasive infections in Australia has shown a predominance of M4 GAS, a serotype recently reported to lack the antiphagocytic hyaluronic acid (HA) capsule. Here, we use molecular genetics and bioinformatics techniques to characterize 17 clinical M4 isolates associated with invasive disease in children during this recent epidemiology. All M4 isolates lacked HA capsule, and whole genome sequence analysis of two isolates revealed the complete absence of the hasABC capsule biosynthesis operon. Conversely, M4 isolates possess a functional HA-degrading hyaluronate lyase (HylA) enzyme that is rendered nonfunctional in other GAS through a point mutation. Transformation with a plasmid expressing hasABC restored partial encapsulation in wild-type (WT) M4 GAS, and full encapsulation in an isogenic M4 mutant lacking HylA. However, partial encapsulation reduced binding to human complement regulatory protein C4BP, did not enhance survival in whole human blood, and did not increase virulence of WT M4 GAS in a mouse model of systemic infection. Bioinformatics analysis found no hasABC homologs in closely related species, suggesting that this operon was a recent acquisition. These data showcase a mutually exclusive interaction of HA capsule and active HylA among strains of this leading human pathogen.     

Circadian rhythm does not affect responses to chemical cues in the red swamp crayfish,Procambarus clarkii

Studies of crayfish chemical ecology have been conducted in both day and night conditions. This variation may hinder the comparison of data among studies, if the responses by crayfish to chemical cues are dependent upon the time at which the cues are encountered. We tested the hypothesis that responses to chemical cues are dependent on observation time using the red swamp crayfish, Procambarus clarkii. Procambarus clarkii is known to exhibit a light-regulated circadian rhythm, with nocturnal activity peaks. Habitat use differed significantly between non-stimulated periods and periods of exposure to a food stimulus, but no effects of photoperiod (normal vs. reversed) or laboratory conditions (dark vs. light) were observed. The results suggest that, all else being equal, (1) studies of crayfish chemical ecology can be successfully conducted in a variety of experimental conditions, and (2) previous studies conducted at various times of the day should have comparable results.     

Toxoplasma gondii Rab6 mediates a retrograde pathway for sorting of constitutively secreted proteins to the Golgi complex

Toxoplasma gondii relies on protein secretion from specialized organelles for invasion of host cells and establishment of a parasitophorous vacuole. We identify T. gondii Rab6 as a regulator of protein transport between post-Golgi dense granule organelles and the Golgi. Toxoplasma Rab6 was localized to cisternal rims of the late Golgi and trans-Golgi network, associated transport vesicles, and microdomains of dense granule and endosomal membranes. Overexpression of wild-type Rab6 or GTP-activated Rab6(Q70L) rerouted soluble dense granule secretory proteins to the Golgi and endoplasmic reticulum and augmented the effect of brefeldin A on Golgi resorption to the endoplasmic reticulum. Parasites expressing a nucleotide-free (Rab6(N124I)) or a GDP-bound (Rab6(T25N)) mutant accumulated dense granule proteins in the Golgi and associated transport vesicles and displayed reduced secretion of GRA4 and a delay in glycosylation of GRA2. Activated Rab6 on Golgi membranes colocalized with centrin during mitosis, and parasite clones expressing Rab6 mutants displayed a partial shift in cytokinesis from endodyogeny (formation of two daughter cells) to endopolygeny (multiple daughter cells). We propose that Toxoplasma Rab6 regulates retrograde transport from post-Golgi secretory granules to the parasite Golgi.     

Do Healthy Monarchs Migrate Farther? Tracking Natal Origins of Parasitized vs. Uninfected Monarch Butterflies Overwintering in Mexico

Long-distance migration can lower parasite prevalence if strenuous journeys remove infected animals from wild populations. We examined wild monarch butterflies (Danaus plexippus) to investigate the potential costs of the protozoan Ophryocystis elektroscirrha on migratory success. We collected monarchs from two wintering sites in central Mexico to compare infection status with hydrogen isotope (δ ²H) measurements as an indicator of latitude of origin at the start of fall migration. On average, uninfected monarchs had lower δ ²H values than parasitized butterflies, indicating that uninfected butterflies originated from more northerly latitudes and travelled farther distances to reach Mexico. Within the infected class, monarchs with higher quantitative spore loads originated from more southerly latitudes, indicating that heavily infected monarchs originating from farther north are less likely to reach Mexico. We ruled out the alternative explanation that lower latitudes give rise to more infected monarchs prior to the onset of migration using citizen science data to examine regional differences in parasite prevalence during the summer breeding season. We also found a positive association between monarch wing area and estimated distance flown. Collectively, these results emphasize that seasonal migrations can help lower infection levels in wild animal populations. Our findings, combined with recent declines in the numbers of migratory monarchs wintering in Mexico and observations of sedentary (winter breeding) monarch populations in the southern U.S., suggest that shifts from migratory to sedentary behavior will likely lead to greater infection prevalence for North American monarchs.     

Potent inhibitors of malarial P. Falciparum protein kinase G: Improving the cell activity of a series of imidazopyridines

Development of a class of bicyclic inhibitors of the Plasmodium falciparum cyclic GMP-dependent protein kinase (PfPKG), starting from known compounds with activity against a related parasite PKG orthologue, is reported. Examination of key sub-structural elements led to new compounds with good levels of inhibitory activity against the recombinant kinase and in vitro activity against the parasite. Key examples were shown to possess encouraging in vitro ADME properties, and computational analysis provided valuable insight into the origins of the observed activity profiles.     

Differential effects of corticotropin-releasing hormone on central dopaminergic and noradrenergic neurons

Corticotropin-releasing hormone (CRH) has been shown to be a central mediator for most, if not all, stress-induced responses. Since stressful stimuli may decrease hypothalamic tuberoinfundibular and tuberohypophysial dopaminergic neuronal activities, we aimed to determine whether CRH is involved. Using central administration of various doses of ovine CRH (oCRH; 1, 3 and 10 µg/rat) into the lateral cerebroventricle of either male or female rats, the neurochemical changes in various parts of the central nervous system, including the hypothalamus, were determined by high-performance liquid chromatography at various times after the injection (30, 60, 120 and 240 min). The concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) and 3-methoxy-4-hydroxy-phenylethyleneglycol (MHPG), two major metabolites of dopamine and norepinephrine, respectively, in discrete brain regions were used as indices for catecholaminergic neuron activity. Plasma corticosterone levels increased significantly after all doses of oCRH and at all time points studied. oCRH also exerted significant stimulatory effects on noradrenergic neuron terminals in the frontal cortex, and on dopaminergic neuron terminals in the nucleus accumbens, hypothalamic paraventricular and periventricular nuclei, and intermediate pituitary lobe. Dopaminergic neuron terminals in the median eminence and the neural lobe of the pituitary, however, were not affected. There was no major difference in the responses between male and female rats. We conclude that CRH has a differential effect on central catecholaminergic neurons.     

Assimilation of 13NH 4 + by Anthoceros grown with and without symbiotic Nostoc

The pathways of assimilation of ammonium by pure cultures of symbiont-free Anthoceros punctatus L. and the reconstituted Anthoceros-Nostoc symbiotic association were determined from time-course (5–300 s) and inhibitor experiments using ¹³NH ₄ ⁺ . The major product of assimilation after all incubation times was glutamine, whether the tissues were cultured with excess ammonium or no combined nitrogen. The ¹³N in glutamine was predominantly in the amide-nitrogen position. Formation of glutamine and glutamate by Anthoceros-Nostoc was strongly inhibited by either 1mM methionine sulfoximine (MSX) or 1 mM exogenous ammonium. These data are consistent with the assimilation of ¹³NH ₄ ⁺ and formation of glutamate by the glutamine synthetase (EC 6.3.1.2)-glutamate synthase (EC 1.4.7.1) pathway in dinitrogen-grown Anthoceros-Nostoc. However, in symbiont-free Anthoceros, grown with 2.5 mM ammonium, formation of glutamine, but not glutamate, was decreased by either MSX or exogenous ammonium. These results indicate that during short incubation times ammonium is assimilated in nitrogenreplete Anthoceros by the activities of both glutamine synthetase and glutamate dehydrogenase (EC 1.4.1.2). In-vitro activities of glutamine synthetase were similar in nitrogen-replete Anthoceros and Anthoceros-Nostoc, indicating that the differences in the routes of glutamate formation were not based upon regulation of synthesis of the initial enzyme of the glutamine synthetase-glutamate synthase pathway. When symbiont-free Anthoceros was cultured for 2 d in the absence of combined nitrogen, total ¹³NH ₄ ⁺ assimilation, and glutamine and glutamate formation in the presence of inhibitors, were similar to dinitrogen-grown Anthoceros-Nostoc. The routes of immediate (within 2 min) glutamate formation and ammonium assimilation in Anthoceros were apparently determined by the intracellular levels of ammonium; at low levels the glutamine synthetase-glutamate synthase pathway was predominant, while at high levels independent activities of both glutamine synthetase and glutamate dehydrogenase were expressed.     

X-ray absorption spectroscopic investigation of the resting ferrous and cosubstrate-bound active sites of phenylalanine hydroxylase

Previous studies of ferrous wild-type phenylalanine hydroxylase, [Fe(2+)]PAH(T)[], have shown the active site to be a six-coordinate distorted octahedral site. After the substrate and cofactor bind to the enzyme ([Fe(2+)]PAH(R)[L-Phe,5-deaza-6-MPH(4)]), the active site converts to a five-coordinate square pyramidal structure in which the identity of the missing ligand had not been previously determined. X-ray absorption spectroscopy (XAS) at the Fe K-edge further supports this coordination number change with the binding of both cosubstrates to the enzyme, and determines this to be due to the loss of a water ligand.