Salmonella Enteritidis RfbD enhances bacterial colonization and virulence through inhibiting autophagy.

Autophagy is a crucial innate immune response that clears pathogens intracellularly. Salmonella enterica serovar Enteritidis (S.E) has emerged as one of the most important food-borne pathogens. Here, we reported that dTDP-4-dehydro-ß-ւ-rhamnose reductase (RfbD) was able to enhance bacterial colonization in vivo and in vitro by regulating autophagy. We screened the transposon mutant library of Salmonella Enteritidis strain Z11 by High-Content Analysis System, found that rfbD gene has an effect on autophagy. The Z11ΔrfbD-infected group showed greater expression of LC3-II than the Z11-infected group in HeLa, RAW264.7, and J774A.1 cells. Overall, the survival of Z11ΔrfbD in RAW264.7 cells was reduced after 8 h of infection compared to that of the Z11 wild-type strain. In addition, we observed that inhibition of autophagic flux significantly increased the survival of Z11ΔrfbD in RAW264.7 cells. Mice infection experiments revealed that Z11ΔrfbD virulence was significantly reduced, and bacterial load was reduced in the liver and cecum in mice model, and LC3-II expression was significantly increased. These findings indicate an important role of Salmonella Enteritidis protein as a strategy to suppress autophagy and provides new ideas for manipulating autophagy as a novel strategy to treat infectious diseases.

A near-infrared fluorescent probe based on photostable Si-rhodamine for imaging hypochlorous acid during lysosome-involved inflammatory response.

Hypochloric acid (HClO) is mainly distributed in acidic lysosomes of phagocytes and closely associated with numerous physiological and pathological processes, especially inflammatory response. Fluorescent probe has become an important tool for imaging HClO in lysosomes, but suffered from interference from autofluorescence in vivo, phototoxicity to biosamples and photobleaching phenomenon due to their short-wavelength excitation and emission. Unfortunately, up to now, no near-infrared (NIR) lysosome-targetable fluorescent probe has been reported for imaging HClO. In this paper, a near-infrared fluorescent probe Lyso-NIR-HClO for imaging lysosomal HClO was reported for the first time. Lyso-NIR-HClO based on Si-rhodamine is consisted of a morpholine unit as a lysosome-targetable group and a HClO-mediated cyclization reaction site as a response group, which was applied for highly selective and sensitive detection and imaging for endogenous and exogenous HClO in lysosomes, with a linear range from 5.0 × 10-8 to 1.0 × 10-5 M and a detection limit of 2.0 × 10-8 M in vitro. Attributed to NIR emission and excellent photostability of Si-rhodamine, Lyso-NIR-HClO exhibits excellent performances in vivo, such as low interference from intracellular autofluorescence, stable and persistent fluorescence signal and good tissue penetration, which are in favor of accurate, time-lapse and long-term imaging for HClO. Finally, we applied the probe Lyso-NIR-HClO to visualize endogenous HClO during lysosome-involved inflammatory response including bacteria-infected cells and inflamed mouse model with satisfactory results. The above results proved that Lyso-NIR-HClO would be a potentially useful tool for the study of biological functions and pathological roles of HClO in lysosomes, especially role of lysosome in the inflammatory response.

Secondary metabolites from the fermented rice of the fungus Monascus purpureus and their bioactivities.

Phytochemical investigation of the EtOAc-soluble fraction of the ethanolic extract of a yellow mutant of the fungus Monascus purpureus BCRC 38110 (Eurotiaceae) grown on rice resulted in the isolation of one new azaphilone derivative, monapurpureusone (1), one acetophenone metabolite isolated for the first time from natural source, monapurpureusin (2), along with four known compounds, TW94a (3), ergosterol (4), monascin (5), and ankaflavin (6). The structures and relative configurations of these compounds were elucidated by spectroscopic analyses, including 1D- and 2D-NMR spectroscopy and mass spectrometry, and by the comparison of their NMR data with those of related compounds. Some phytochemicals were evaluated for both anti-inflammatory activity through the measurement of nitric oxide (NO) production levels in lipopolysaccharide (LPS)-stimulated murine-derived macrophages RAW264.7 cell lines and antioxidant activities.

Global gene-expression profiles of intracellular survival of the BruAb2_1031 gene mutated Brucella abortus in professional phagocytes, RAW 264.7 cells.

BACKGROUND: Since recognizing the interaction between Brucella and host cells is crucial to the elucidation of the infectious process, Brucella researches have prioritized the investigation of genes related to pathogenicity. To demonstrate the roles of Brucella genes, RAW 264.7 cells were infected with the Brucella abortus wild-type and mutant strains (generated using transposon mutagenesis), after which the different transcriptional responses of the infected cells were determined using microarray. RESULTS: Following infection, enhanced strategies for intracellular survival, such as down-regulation of genes associated with cytokine responses and apoptosis, were observed in RAW 264.7 cells infected with C3 mutant strain when compared to the transcriptional responses of wild-type infected cells. Using sequence analysis, we determined the mutation site of a C3 mutant strain as the ATP-binding cassette transporter permease (BruAb2_1031). These results were evidenced by an increased level of intracellular survival of the C3 mutant strain. CONCLUSIONS: Characteristics of each mutant strain including bacterial growth rate, abilities to induce cytokine production in macrophages after infection, internalization, and levels of intracellular survival and replication, were investigated by performing RAW 264.7 cell infection experiments. Our results indicate that the BruAb2_1031 gene might be closely related with intracellular survival of B. abortus in RAW 264.7 cells.

Evaluation of a real-time impedance analysis platform on fungal infection.

End-point assays of in vitro cell proliferation and death have been employed to study the mechanisms of fungal pathogenesis and have shown the responses of host cells at individual time points. A new cell analysis technology has been developed that allows for the continuous measurement and quantification of cell activities, thus enabling the dynamic assessment of electrical impedance when various pathogens are cultured in vitro. In this study, this system was evaluated to determine the response of the cell line RAW264.7 to infection by several clinically relevant fungi in vitro, including Aspergillus fumigatus, Candida albicans, and melanized and albino mutant strains of Fonsecaea monophora. The results showed that infection resulted in rounding of the host cells with a loss of contact between individual cells and a decline in the electrical impedance of all test groups. However, changes in the electrical impedance were variable. Aspergillus fumigatus caused initial increases and later significant decreases in the electrical impedance, while for C. albicans and F. monophora, the effect was reduced. The melanized strain of F. monophora caused a faster change in the electrical impedance than the albino strain. Our data proved that this system can be used as an efficient tool for monitoring cellular responses to fungal infection.

Noncanonical Fungal Autophagy Inhibits Inflammation in Response to IFN-γ via DAPK1.

Defects in a form of noncanonical autophagy, known as LC3-associated phagocytosis (LAP), lead to increased inflammatory pathology during fungal infection. Although LAP contributes to fungal degradation, the molecular mechanisms underlying LAP-mediated modulation of inflammation are unknown. We describe a mechanism by which inflammation is regulated during LAP through the death-associated protein kinase 1 (DAPK1). The ATF6/C/EBP-ß/DAPK1 axis activated by IFN-γ not only mediates LAP to Aspergillus fumigatus but also concomitantly inhibits Nod-like receptor protein 3 (NLRP3) activation and restrains pathogenic inflammation. In mouse models and patient samples of chronic granulomatous disease, which exhibit defective autophagy and increased inflammasome activity, IFN-γ restores reduced DAPK1 activity and dampens fungal growth. Additionally, in a cohort of hematopoietic stem cell-transplanted patients, a genetic DAPK1 deficiency is associated with increased inflammation and heightened aspergillosis susceptibility. Thus, DAPK1 is a potential drugable player in regulating the inflammatory response during fungal clearance initiated by IFN-γ.

MicroRNA-125b-5p suppresses Brucella abortus intracellular survival via control of A20 expression.

BACKGROUND: Brucella may establish chronic infection by regulating the expression of miRNAs. However, the role of miRNAs in modulating the intracellular growth of Brucella remains unclear. RESULTS: In this study, we show that Brucella. abortus infection leads to downregulation of miR-125b-5p in macrophages. We establish that miR-125b-5p targets A20, an inhibitor of the NF-kB activation. Additionally, expression of miR-125b-5p decreases A20 expression in B. abortus-infected macrophages and leads to NF-kB activation and increased production of TNFα. Furthermore, B. abortus survival is attenuated in the presence of miR-125b-5p. CONCLUSIONS: These results uncover a role for miR-125b-5p in the regulation of B. abortus intracellular survival via the control of A20 expression.

Metabolism of myo-Inositol by Legionella pneumophila Promotes Infection of Amoebae and Macrophages.

UNLABELLED: Legionella pneumophila is a natural parasite of environmental amoebae and the causative agent of a severe pneumonia termed Legionnaires' disease. The facultative intracellular pathogen employs a bipartite metabolism, where the amino acid serine serves as the major energy supply, while glycerol and glucose are mainly utilized for anabolic processes. The L. pneumophila genome harbors the cluster lpg1653 to lpg1649 putatively involved in the metabolism of the abundant carbohydrate myo-inositol (here termed inositol). To assess inositol metabolism by L. pneumophila, we constructed defined mutant strains lacking lpg1653 or lpg1652, which are predicted to encode the inositol transporter IolT or the inositol-2-dehydrogenase IolG, respectively. The mutant strains were not impaired for growth in complex or defined minimal media, and inositol did not promote extracellular growth. However, upon coinfection of Acanthamoeba castellanii, the mutants were outcompeted by the parental strain, indicating that the intracellular inositol metabolism confers a fitness advantage to the pathogen. Indeed, inositol added to L. pneumophila-infected amoebae or macrophages promoted intracellular growth of the parental strain, but not of the ΔiolT or ΔiolG mutant, and growth stimulation by inositol was restored by complementation of the mutant strains. The expression of the Piol promoter and bacterial uptake of inositol required the alternative sigma factor RpoS, a key virulence regulator of L. pneumophila Finally, the parental strain and ΔiolG mutant bacteria but not the ΔiolT mutant strain accumulated [U-(14)C6]inositol, indicating that IolT indeed functions as an inositol transporter. Taken together, intracellular L. pneumophila metabolizes inositol through the iol gene products, thus promoting the growth and virulence of the pathogen. IMPORTANCE: The environmental bacterium Legionella pneumophila is the causative agent of a severe pneumonia termed Legionnaires' disease. The opportunistic pathogen replicates in protozoan and mammalian phagocytes in a unique vacuole. Amino acids are thought to represent the prime source of carbon and energy for L. pneumophila However, genome, transcriptome, and proteome studies indicate that the pathogen not only utilizes amino acids as carbon sources but possesses broader metabolic capacities. In this study, we analyzed the metabolism of inositol by extra- and intracellularly growing L. pneumophila By using genetic, biochemical, and cell biological approaches, we found that L. pneumophila accumulates and metabolizes inositol through the iol gene products, thus promoting the intracellular growth, virulence, and fitness of the pathogen. Our study significantly contributes to an understanding of the intracellular niche of a human pathogen.

Interactions of Streptococcus iniae with phagocytic cell line.

Streptococcus iniae has become one of the most serious aquatic pathogens in the last decade, causing large losses in wild and farmed fish worldwide. There is clear evidence that this pathogen is capable not only of causing serious disease in fish but also of being transferred to and infecting humans. In this study, we investigate the interaction of S. iniae with two murine macrophage cell lines, J774-A1 and RAW 264.7. Cytotoxicity assay demonstrated significant differences between live and UV-light killed IUSA-1 strains. The burst respiratory activity decreased to baseline after 1 and 4 h of exposure for J774-A1 and RAW 264.7, respectively. Immunofluorescent and ultrastructural study of infected cells confirmed the intracellular localization of bacteria at 1 h and 24 h post-infection. Using qRT-PCR arrays, we investigated the changes in the gene expression of immune relevant genes associated with macrophage activation. In this screening, we identified 11 of 84 genes up-regulated, we observed over-expression of pro-inflammatory response as IL-1α, IL-1ß, and TNF-α, without a good anti-inflammatory response. Present findings suggest a capacity of S. iniae to modulate a mammalian macrophages cell lines to their survival and replication intracellular, which makes this cell type as a reservoir for continued infection.

Functional high-throughput screening identifies the miR-15 microRNA family as cellular restriction factors for Salmonella infection.

Increasing evidence suggests an important role for miRNAs in the molecular interplay between bacterial pathogens and host cells. Here we perform a fluorescence microscopy-based screen using a library of miRNA mimics and demonstrate that miRNAs modulate Salmonella infection. Several members of the miR-15 miRNA family were among the 17 miRNAs that more efficiently inhibit Salmonella infection. We discovered that these miRNAs are downregulated during Salmonella infection, through the inhibition of the transcription factor E2F1. Analysis of miR-15 family targets revealed that derepression of cyclin D1 and the consequent promotion of G1/S transition are crucial for Salmonella intracellular proliferation. In addition, Salmonella induces G2/M cell cycle arrest in infected cells, further promoting its replication. Overall, these findings uncover a mechanism whereby Salmonella renders host cells more susceptible to infection by controlling cell cycle progression through the active modulation of host cell miRNAs.