Silibinin inhibits hepatitis C virus entry into hepatocytes by hindering clathrin‐dependent trafficking

Hepatitis C virus (HCV) is a global health concern infecting 170 million people worldwide. Previous studies indicate that the extract from milk thistle known as silymarin and its main component silibinin inhibit HCV infection. Here we investigated the mechanism of anti‐HCV action ofsilymarin‐derived compounds at the molecular level. By using live‐cell confocal imaging, single particle tracking, transmission electron microscopy and biochemical approaches on HCV‐infected human hepatoma cells and primary hepatocytes, we show that silibinin potently inhibits HCV infection and hinders HCV entry by slowing down trafficking through clathrin‐coated pits and vesicles. Detailed analyses revealed that silibinin altered the formation of both clathrin‐coated pits and vesicles in cells and caused abnormal uptake and trafficking of transferrin, a well‐known cargo of the clathrin endocytic pathway. Silibinin also inhibited infection by other viruses that enter cells by clathrin‐mediated endocytosis including reovirus, vesicular stomatitis and influenza viruses. Our study demonstrates that silibinin inhibits HCV early steps of infection by affecting endosomal trafficking of virions. It provides new insights into the molecular mechanisms of action of silibinin against HCV entry and also suggests that silibinin is a potential broad‐spectrum antiviral therapy.     

A highly conserved mycobacterial cholesterol catabolic pathway

Degradation of the cholesterol side‐chain in Mycobacterium tuberculosis is initiated by two cytochromes P450, CYP125A1 and CYP142A1, that sequentially oxidize C26 to the alcohol, aldehyde and acid metabolites. Here we report characterization of the homologous enzymes CYP125A3 and CYP142A2 from Mycobacterium smegmatis mc² 155. Heterologously expressed, purified CYP125A3 and CYP142A2 bound cholesterol, 4‐cholesten‐3‐one, and antifungal azole drugs. CYP125A3 or CYP142A2 reconstituted with spinach ferredoxin and ferredoxin reductase efficiently hydroxylated 4‐cholesten‐3‐one to the C‐26 alcohol and subsequently to the acid. The X‐ray structures of both substrate‐free CYP125A3 and CYP142A2 and of cholest‐4‐en‐3‐one‐bound CYP142A2 reveal significant differences in the substrate binding sites compared with the homologous M. tuberculosis proteins. Deletion only of cyp125A3 causes a reduction of both the alcohol and acid metabolites and a strong induction of cyp142 at the mRNA and protein levels, indicating that CYP142A2 serves as a functionally redundant back up enzyme for CYP125A3. In contrast to M. tuberculosis, the M. smegmatis Δcyp125Δcyp142 double mutant retains its ability to grow on cholesterol albeit with a diminished capacity, indicating an additional level of redundancy within its genome.     

Characterization of water and wildlife strains as a subgroup of Campylobacter jejuni using DNA microarrays

Campylobacter jejuni is the leading cause of human bacterial gastroenteritis worldwide, but source attribution of the organism is difficult. Previously, DNA microarrays were used to investigate isolate source, which suggested a non‐livestock source of infection. In this study we analysed the genome content of 162 clinical, livestock and water and wildlife (WW) associated isolates combined with the previous study. Isolates were grouped by genotypes into nine clusters (C1 to C9). Multilocus sequence typing (MLST) data demonstrated that livestock associated clonal complexes dominated clusters C1–C6. The majority of WW isolates were present in the C9 cluster. Analysis of previously reported genomic variable regions demonstrated that these regions were linked to specific clusters. Two novel variable regions were identified. A six gene multiplex PCR (mPCR) assay, designed to effectively differentiated strains into clusters, was validated with 30 isolates. A further five WW isolates were tested by mPCR and were assigned to the C7‐C9 group of clusters. The predictive mPCR test could be used to indicate if a clinical case has come from domesticated or WW sources. Our findings provide further evidence that WW C. jejuni subtypes show niche adaptation and may be important in causing human infection.     

Unimodal size scaling of phytoplankton growth and the size dependence of nutrient uptake and use

Phytoplankton size structure is key for the ecology and biogeochemistry of pelagic ecosystems, but the relationship between cell size and maximum growth rate (μ_max) is not yet well understood. We used cultures of 22 species of marine phytoplankton from five phyla, ranging from 0.1 to 10⁶ μm³ in cell volume (V_cell), to determine experimentally the size dependence of growth, metabolic rate, elemental stoichiometry and nutrient uptake. We show that both μ_max and carbon‐specific photosynthesis peak at intermediate cell sizes. Maximum nitrogen uptake rate (V_maxN) scales isometrically with V_cell, whereas nitrogen minimum quota scales as V_cell^0.84. Large cells thus possess high ability to take up nitrogen, relative to their requirements, and large storage capacity, but their growth is limited by the conversion of nutrients into biomass. Small species show similar volume‐specific V_maxN compared to their larger counterparts, but have higher nitrogen requirements. We suggest that the unimodal size scaling of phytoplankton growth arises from taxon‐independent, size‐related constraints in nutrient uptake, requirement and assimilation.     

Timing and intensity of bush cricket predation on egg batches of pine processionary moth: no evidence of population control

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Regulation of ICAM‐1 expression in gingival fibroblasts infected with high‐glucose‐treated P. gingivalis

Porphyromonas gingivalis is a major pathogen in the initiation and progression of periodontal disease, which is recognized as a common complication of diabetes. ICAM‐1 expression by human gingival fibroblasts (HGFs) is crucial for regulating local inflammatory responses in inflamed periodontal tissues. However, the effect of P. gingivalis in a high‐glucose situation in regulating HGF function is not understood. The P. gingivalis strain CCUG25226 was used to study the mechanisms underlying the modulation of HGF ICAM‐1 expression by invasion of high‐glucose‐treated P. gingivalis (HGPg). A high‐glucose condition upregulated fimA mRNA expression in P. gingivalis and increased its invasion ability in HGFs. HGF invasion with HGPg induced increases in the expression of ICAM‐1. By using specific inhibitors and short hairpin RNA (shRNA), we have demonstrated that the activation of p38 MAPK and Akt pathways is critical for HGPg‐induced ICAM‐1 expression. Luciferase reporters and chromatin immunoprecipitation assays suggest that HGPg invasion increases NF‐κB‐ and Sp1‐DNA‐binding activities in HGFs. Inhibition of NF‐κB and Sp1 activations blocked the HGPg‐induced ICAM‐1 promoter activity and expression. The effect of HGPg on HGF signalling and ICAM‐1 expression is mediated by CXC chemokine receptor 4 (CXCR4). Our findings identify the molecular pathways underlying HGPg‐dependent ICAM‐1 expression in HGFs, providing insight into the effect of P. gingivalis invasion in HGFs.     

Lipoarabinomannan mannose caps do not affect mycobacterial virulence or the induction of protective immunity in experimental animal models of infection and have minimal impact on in vitro inflammatory responses

Mannose‐capped lipoarabinomannan (ManLAM) is considered an important virulence factor of Mycobacterium tuberculosis. However, while mannose caps have been reported to be responsible for various immunosuppressive activities of ManLAMobserved in vitro, there is conflicting evidence about their contribution to mycobacterial virulence in vivo. Therefore, we used Mycobacterium bovis BCG and M. tuberculosis mutants that lack the mannose cap of LAM to assess the role of ManLAM in the interaction of mycobacteria with the host cells, to evaluate vaccine‐induced protection and to determine its importance in M. tuberculosis virulence. Deletion of the mannose cap did not affect BCG survival and replication in macrophages, although the capless mutant induced a somewhat higher production of TNF. In dendritic cells, the capless mutant was able to induce the upregulation of co‐stimulatory molecules and the only difference we detected was the secretion of slightly higher amounts of IL‐10 as compared to the wild type strain. In mice, capless BCG survived equally well and induced an immune response similar to the parental strain. Furthermore, the efficacy of vaccination against a M. tuberculosis challenge in low‐dose aerosol infection models in mice and guinea pigs was not affected by the absence of the mannose caps in the BCG. Finally, the lack of the mannose cap in M. tuberculosis did not affect its virulence in mice nor its interaction with macrophages in vitro. Thus, these results do not support a major role for the mannose caps of LAM in determining mycobacterial virulence and immunogenicity in vivo in experimental animal models of infection, possibly because of redundancy of function.     

A perforin‐like protein mediates disruption of the erythrocyte membrane during egress of Plasmodium berghei male gametocytes

Successful gametogenesis of the malaria parasite depends on egress of the gametocytes from the erythrocytes within which they developed. Egress entails rupture of both the parasitophorous vacuole membrane and the erythrocyte plasma membrane, and precedes the formation of the motile flagellated male gametes in a process called exflagellation. We show here that egress of the male gametocyte depends on the function of a perforin‐like protein, PPLP2. A mutant of Plasmodium berghei lacking PPLP2 displayed abnormal exflagellation; instead of each male gametocyte forming eight flagellated gametes, it produced gametocytes with only one, shared thicker flagellum. Using immunofluorescence and transmission electron microscopy analysis, and phenotype rescue with saponin or a pore‐forming toxin, we conclude that rupture of the erythrocyte membraneis blocked in the mutant. The parasitophorous vacuole membrane, on the other hand, is ruptured normally. Some mutant parasites are still able to develop in the mosquito, possibly because the vigorous motility of the flagellated gametes eventually leads to escape from the persisting erythrocyte membrane. This is the first example of a perforin‐like protein in Plasmodium parasites having a role in egress from the host cell and the first parasite protein shown to be specifically required for erythrocyte membrane disruption during egress.