Ctenocephalides orientis and Ctenocephalides felis in Thailand: Head geometry by species, sex and geography.

Fleas in the genus Ctenocephalides serve as biological vectors or intermediate hosts of microorganisms such as bacteria, rickettsia, protozoa and helminths. Ctenocephalides felis has a worldwide distribution, while C. orientis has long been considered as a subspecies of C. felis in Asia. To help the morphological recognition of these two species and further explore their differences, we used the geometric morphometric approach applied to the head. Both sexes were examined. Five anatomical landmarks of the head were used, and to capture the curvature of the front head, 10 semilandmarks were added. There was a consistent difference in species classification accuracy when considering landmarks only versus their combination with semilandmarks, suggesting the importance of the curve of the head as a taxonomic signal. Using or not the labels in the reclassification analyses, the head shape allowed by itself almost perfect recognition of the two species, in both sexes, even after adjustment for prior probabilities. The same approach disclosed a high level of sexual size and shape dimorphism in both species. The contribution of size variation to the discrimination by shape was much more important between sexes (from 27% to 45%) than between species (from 0.7% to 7.1%). Nevertheless, in our data, size never could represent a way to reliably recognise the sex of an individual, even less its species. Geographical variation in head shape could only be explored for the C. orientis sample. No significant correlation of morphometric variation with geography could be detected, which would be consistent with gene flow between Thai provinces. The geometric morphometric approach of the flea head, when it incorporates head curves, is a promising tool for rapid, economical, and accurate species and sex identification. It is, therefore, a useful tool for future epidemiological and demographic studies.

Central Role of the Trehalose Biosynthesis Pathway in the Pathogenesis of Human Fungal Infections: Opportunities and Challenges for Therapeutic Development.

Invasive fungal infections cause significant morbidity and mortality in part due to a limited antifungal drug arsenal. One therapeutic challenge faced by clinicians is the significant host toxicity associated with antifungal drugs. Another challenge is the fungistatic mechanism of action of some drugs. Consequently, the identification of fungus-specific drug targets essential for fitness in vivo remains a significant goal of medical mycology research. The trehalose biosynthetic pathway is found in a wide variety of organisms, including human-pathogenic fungi, but not in humans. Genes encoding proteins involved in trehalose biosynthesis are mechanistically linked to the metabolism, cell wall homeostasis, stress responses, and virulence of Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus. While there are a number of pathways for trehalose production across the tree of life, the TPS/TPP (trehalose-6-phosphate synthase/trehalose-6-phosphate phosphatase) pathway is the canonical pathway found in human-pathogenic fungi. Importantly, data suggest that proteins involved in trehalose biosynthesis play other critical roles in fungal metabolism and in vivo fitness that remain to be fully elucidated. By further defining the biology and functions of trehalose and its biosynthetic pathway components in pathogenic fungi, an opportunity exists to leverage this pathway as a potent antifungal drug target. The goal of this review is to cover the known roles of this important molecule and its associated biosynthesis-encoding genes in the human-pathogenic fungi studied to date and to employ these data to critically assess the opportunities and challenges facing development of this pathway as a therapeutic target.

Attenuation of UV-B exposure-induced inflammation by abalone hypobranchial gland and gill extracts.

Exposure to solar ultraviolet B (UV-B) is a known causative factor for many skin complications such as wrinkles, black spots, shedding and inflammation. Within the wavelengths 280­320 nm, UV-B can penetrate to the epidermal level. This investigation aimed to test whether extracts from the tropical abalone [Haliotis asinina (H. asinina)] mucus-secreting tissues, the hypobranchial gland (HBG) and gills, were able to attenuate the inflammatory process, using the human keratinocyte HaCaT cell line. Cytotoxicity of abalone tissue extracts was determined using an AlamarBlue viability assay. Results showed that HaCaT cells could survive when incubated in crude HBG and gill extracts at concentrations between <11.8 and <16.9 µg/ml, respectively. Subsequently, cell viability was compared between cultured HaCaT cells exposed to serial doses of UV-B from 1 to 11 (x10) mJ/cm2 and containing 4 different concentrations of abalone extract from both the HBG and gill (0, 0.1, 2.5, 5 µg/ml). A significant increase in cell viability was observed (P<0.001) following treatment with 2.5 and 5 µg/ml extract. Without extract, cell viability was significantly reduced upon exposure to UV-B at 4 mJ/cm2. Three morphological changes were observed in HaCaT cells following UV-B exposure, including i) condensation of cytoplasm; ii) shrunken cells and plasma membrane bubbling; and iii) condensation of chromatin material. A calcein AM­propidium iodide live­dead assay showed that cells could survive cytoplasmic condensation, yet cell death occurred when damage also included membrane bubbling and chromatin changes. Western blot analysis of HaCaT cell COX­2, p38, phospho­p38, SPK/JNK and phospho­SPK/JNK following exposure to >2.5 µg/ml extract showed a significant decrease in intensity for COX­2, phospho­p38 and phospho­SPK/JNK. The present study demonstrated that abalone extracts from the HGB and gill can attenuate inflammatory proteins triggered by UV-B. Hence, the contents of abalone extract, including cellmetabolites and peptides, may provide new agents for skin anti­inflammation, preventing damage due to UV-B.

Preliminary Studies of Lactic Acid Bacteria Isolated from Feces of Thai Newborns.

Background: The microbiota of human plays an important role in the health improvement, and found abundant in the gastrointestinal tract. In recent years, probiotics have been increasingly used in prevention of certain intestinal diseases. The most important population to study the microbiome is probably in the healthy newborns. Objective: The preliminary study aimed to isolate and identify the gut microbiota of newborns for the assessment of prevalent Lactic Acid Bacteria (LAB) distribution. Material and Method: Thirty eight Thai newborns, 0-5 days old of both sexes were subjected for fecal samples collection. Isolated bacteria were cultivated on the MRS selective media and further phenotypically characterized by conventional methods including Gram stain, catalase, and lactic acid production. Genotypic identification was completed by 16S rRNA gene sequencing and phylogenetic analysis. Results: Forty five isolates of LAB and non-LAB bacteria were obtained from feces of newborns. The most prevalence LAB found in this population were 45% Enterococcus faecalis, 14% E. faecium, 11% E. hirae, 11% Lactobacillus paracasei, and 2% L. gasseri. Unusually, Gram-negative bacteria including Klebsiella pneumoniae, Enterobacter hormaechei, Escherichia fergusonii, Leclercia adecarboxylata, and Shigella flexneri were isolated among LAB strains on the selective MRS media. Conclusion: The gut microbiota was a great resource of beneficial LAB which was remarkably distributed among this population of Thai newborns. Further study on individual LAB isolates for the effective probiotics development would be essentially investigated for future alternative treatment of gastrointestinal diseases.

Dsc orthologs are required for hypoxia adaptation, triazole drug responses, and fungal virulence in Aspergillus fumigatus.

Hypoxia is an environmental stress encountered by Aspergillus fumigatus during invasive pulmonary aspergillosis (IPA). The ability of this mold to adapt to hypoxia is important for fungal virulence and genetically regulated in part by the sterol regulatory element binding protein (SREBP) SrbA. SrbA is required for fungal growth in the murine lung and to ultimately cause lethal disease in murine models of IPA. Here we identified and partially characterized four genes (dscA, dscB, dscC, and dscD, here referred to as dscA-D) with previously unknown functions in A. fumigatus that are orthologs of the Schizosaccharomyces pombe genes dsc1, dsc2, dsc3, and dsc4 (dsc1-4), which encode a Golgi E3 ligase complex critical for SREBP activation by proteolytic cleavage. A. fumigatus null dscA-D mutants displayed remarkable defects in hypoxic growth and increased susceptibility to triazole antifungal drugs. Consistent with the confirmed role of these genes in S. pombe, both ΔdscA and ΔdscC resulted in reduced cleavage of the SrbA precursor protein in A. fumigatus. Inoculation of corticosteroid immunosuppressed mice with ΔdscA and ΔdscC strains revealed that these genes are critical for A. fumigatus virulence. Reintroduction of SrbA amino acids 1 to 425, encompassing the N terminus DNA binding domain, into the ΔdscA strain was able to partially restore virulence, further supporting a mechanistic link between DscA and SrbA function. Thus, we have shown for the first time the importance of a previously uncharacterized group of genes in A. fumigatus that mediate hypoxia adaptation, fungal virulence, and triazole drug susceptibility and that are likely linked to regulation of SrbA function.

In vivo hypoxia and a fungal alcohol dehydrogenase influence the pathogenesis of invasive pulmonary aspergillosis.

Currently, our knowledge of how pathogenic fungi grow in mammalian host environments is limited. Using a chemotherapeutic murine model of invasive pulmonary aspergillosis (IPA) and (1)H-NMR metabolomics, we detected ethanol in the lungs of mice infected with Aspergillus fumigatus. This result suggests that A. fumigatus is exposed to oxygen depleted microenvironments during infection. To test this hypothesis, we utilized a chemical hypoxia detection agent, pimonidazole hydrochloride, in three immunologically distinct murine models of IPA (chemotherapeutic, X-CGD, and corticosteroid). In all three IPA murine models, hypoxia was observed during the course of infection. We next tested the hypothesis that production of ethanol in vivo by the fungus is involved in hypoxia adaptation and fungal pathogenesis. Ethanol deficient A. fumigatus strains showed no growth defects in hypoxia and were able to cause wild type levels of mortality in all 3 murine models. However, lung immunohistopathology and flow cytometry analyses revealed an increase in the inflammatory response in mice infected with an alcohol dehydrogenase null mutant strain that corresponded with a reduction in fungal burden. Consequently, in this study we present the first in vivo observations that hypoxic microenvironments occur during a pulmonary invasive fungal infection and observe that a fungal alcohol dehydrogenase influences fungal pathogenesis in the lung. Thus, environmental conditions encountered by invading pathogenic fungi may result in substantial fungal metabolism changes that influence subsequent host immune responses.

The small GTPase RacA mediates intracellular reactive oxygen species production, polarized growth, and virulence in the human fungal pathogen Aspergillus fumigatus.

Aspergillus fumigatus is the predominant mold pathogen in immunocompromised patients. In this study, we present the first characterization of the small GTPase RacA in A. fumigatus. To gain insight into the function of racA in the growth and pathogenesis of A. fumigatus, we constructed a strain that lacks a functional racA gene. The ΔracA strain showed significant morphological defects, including a reduced growth rate and abnormal conidiogenesis on glucose minimal medium. In the ΔracA strain, apical dominance in the leading hyphae is lost and, instead, multiple axes of polarity emerge. Intriguingly, superoxide production at the hyphal tips was reduced by 25% in the ΔracA strain. Treatment of wild-type hyphae with diphenylene iodonium, an inhibitor of NADPH oxidase, resulted in phenotypes similar to that of the ΔracA strain. These data suggest that ΔracA strain phenotypes may be due to a reduction or alteration in the production of reactive oxygen species. Most surprisingly, despite these developmental and growth abnormalities, the ΔracA strain retained at least wild-type virulence in both an insect model and two immunologically distinct murine models of invasive pulmonary aspergillosis. These results demonstrate that in vitro growth phenotypes do not always correlate with in vivo virulence and raise intriguing questions about the role of RacA in Aspergillus virulence.

Trehalose 6-phosphate phosphatase is required for cell wall integrity and fungal virulence but not trehalose biosynthesis in the human fungal pathogen Aspergillus fumigatus.

The trehalose biosynthesis pathway is critical for virulence in human and plant fungal pathogens. In this study, we tested the hypothesis that trehalose 6-phosphate phosphatase (T6PP) is required for Aspergillus fumigatus virulence. A mutant of the A. fumigatus T6PP, OrlA, displayed severe morphological defects related to asexual reproduction when grown on glucose (1%) minimal media. These defects could be rescued by addition of osmotic stabilizers, reduction in incubation temperature or increase in glucose levels (> 4%). Subsequent examination of the mutant with cell wall perturbing agents revealed a link between cell wall biosynthesis and trehalose 6-phosphate (T6P) levels. As expected, high levels of T6P accumulated in the absence of OrlA resulting in depletion of free inorganic phosphate and inhibition of hexokinase activity. Surprisingly, trehalose production persisted in the absence of OrlA. Further analyses revealed that A. fumigatus contains two trehalose phosphorylases that may be responsible for trehalose production in the absence of OrlA. Despite a normal growth rate under in vitro growth conditions, the orlA mutant was virtually avirulent in two distinct murine models of invasive pulmonary aspergillosis. Our results suggest that further study of this pathway will lead to new insights into regulation of fungal cell wall biosynthesis and virulence.

TmpL, a transmembrane protein required for intracellular redox homeostasis and virulence in a plant and an animal fungal pathogen.

The regulation of intracellular levels of reactive oxygen species (ROS) is critical for developmental differentiation and virulence of many pathogenic fungi. In this report we demonstrate that a novel transmembrane protein, TmpL, is necessary for regulation of intracellular ROS levels and tolerance to external ROS, and is required for infection of plants by the necrotroph Alternaria brassicicola and for infection of mammals by the human pathogen Aspergillus fumigatus. In both fungi, tmpL encodes a predicted hybrid membrane protein containing an AMP-binding domain, six putative transmembrane domains, and an experimentally-validated FAD/NAD(P)-binding domain. Localization and gene expression analyses in A. brassicicola indicated that TmpL is associated with the Woronin body, a specialized peroxisome, and strongly expressed during conidiation and initial invasive growth in planta. A. brassicicola and A. fumigatus DeltatmpL strains exhibited abnormal conidiogenesis, accelerated aging, enhanced oxidative burst during conidiation, and hypersensitivity to oxidative stress when compared to wild-type or reconstituted strains. Moreover, A. brassicicola DeltatmpL strains, although capable of initial penetration, exhibited dramatically reduced invasive growth on Brassicas and Arabidopsis. Similarly, an A. fumigatus DeltatmpL mutant was dramatically less virulent than the wild-type and reconstituted strains in a murine model of invasive aspergillosis. Constitutive expression of the A. brassicicola yap1 ortholog in an A. brassicicola DeltatmpL strain resulted in high expression levels of genes associated with oxidative stress tolerance. Overexpression of yap1 in the DeltatmpL background complemented the majority of observed developmental phenotypic changes and partially restored virulence on plants. Yap1-GFP fusion strains utilizing the native yap1 promoter exhibited constitutive nuclear localization in the A. brassicicola DeltatmpL background. Collectively, we have discovered a novel protein involved in the virulence of both plant and animal fungal pathogens. Our results strongly suggest that dysregulation of oxidative stress homeostasis in the absence of TmpL is the underpinning cause of the developmental and virulence defects observed in these studies.

A sterol-regulatory element binding protein is required for cell polarity, hypoxia adaptation, azole drug resistance, and virulence in Aspergillus fumigatus.

At the site of microbial infections, the significant influx of immune effector cells and the necrosis of tissue by the invading pathogen generate hypoxic microenvironments in which both the pathogen and host cells must survive. Currently, whether hypoxia adaptation is an important virulence attribute of opportunistic pathogenic molds is unknown. Here we report the characterization of a sterol-regulatory element binding protein, SrbA, in the opportunistic pathogenic mold, Aspergillus fumigatus. Loss of SrbA results in a mutant strain of the fungus that is incapable of growth in a hypoxic environment and consequently incapable of causing disease in two distinct murine models of invasive pulmonary aspergillosis (IPA). Transcriptional profiling revealed 87 genes that are affected by loss of SrbA function. Annotation of these genes implicated SrbA in maintaining sterol biosynthesis and hyphal morphology. Further examination of the SrbA null mutant consequently revealed that SrbA plays a critical role in ergosterol biosynthesis, resistance to the azole class of antifungal drugs, and in maintenance of cell polarity in A. fumigatus. Significantly, the SrbA null mutant was highly susceptible to fluconazole and voriconazole. Thus, these findings present a new function of SREBP proteins in filamentous fungi, and demonstrate for the first time that hypoxia adaptation is likely an important virulence attribute of pathogenic molds.