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80800.9408Exposure of Legionella pneumophila to low-shear modeled microgravity: impact on stress response, membrane lipid composition, pathogenicity to macrophages and interrelated genes expression. Here, we studied the effect of low-shear modeled microgravity (LSMMG) on cross stress resistance (heat, acid, and oxidative), fatty acid content, and pathogenicity along with alteration in expression of stress-/virulence-associated genes in Legionella pneumophila. The stress resistance analysis result indicated that bacteria cultivated under LSMMG environments showed higher resistance with elevated D-values at 55 °C and in 1 mM of hydrogen peroxide (H(2)O(2)) conditions compared to normal gravity (NG)-grown bacteria. On the other hand, there was no significant difference in tolerance (p < 0.05) toward simulated gastric fluid (pH-2.5) acid conditions. In fatty acid analysis, our result showed that a total amount of saturated and cyclic fatty acids was increased in LSMMG-grown cells; as a consequence, they might possess low membrane fluidity. An upregulated expression level was noticed for stress-related genes (hslV, htrA, grpE, groL, htpG, clpB, clpX, dnaJ, dnaK, rpoH, rpoE, rpoS, kaiB, kaiC, lpp1114, ahpC1, ahpC2, ahpD, grlA, and gst) under LSMMG conditions. The reduced virulence (less intracellular bacteria and less % of induce apoptosis in RAW 264.7 macrophages) of L. pneumophila under LSMMG conditions may be because of downregulation related genes (dotA, dotB, dotC, dotD, dotG, dotH, dotL, dotM, dotN, icmK, icmB, icmS, icmT, icmW, ladC, rtxA, letA, rpoN, fleQ, fleR, and fliA). In the LSMMG group, the expression of inflammation-related factors, such as IL-1α, TNF-α, IL-6, and IL-8, was observed to be reduced in infected macrophages. Also, scanning electron microscopy (SEM) analysis showed less number of LSMMG-cultivated bacteria attached to the host macrophages compared to NG. Thus, our study provides understandings about the changes in lipid composition and different genes expression due to LSMMG conditions, which apparently influence the alterations of L. pneumophila' stress/virulence response.202438305908
604610.9404Safety Evaluations of Bifidobacterium bifidum BGN4 and Bifidobacterium longum BORI. Over the past decade, a variety of lactic acid bacteria have been commercially available to and steadily used by consumers. However, recent studies have shown that some lactic acid bacteria produce toxic substances and display properties of virulence. To establish safety guidelines for lactic acid bacteria, the Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO) has suggested that lactic acid bacteria be characterized and proven safe for consumers’ health via multiple experiments (e.g., antibiotic resistance, metabolic activity, toxin production, hemolytic activity, infectivity in immune-compromised animal species, human side effects, and adverse-outcome analyses). Among the lactic acid bacteria, Bifidobacterium and Lactobacillus species are probiotic strains that are most commonly commercially produced and actively studied. Bifidobacterium bifidum BGN4 and Bifidobacterium longum BORI have been used in global functional food markets (e.g., China, Germany, Jordan, Korea, Lithuania, New Zealand, Poland, Singapore, Thailand, Turkey, and Vietnam) as nutraceutical ingredients for decades, without any adverse events. However, given that the safety of some newly screened probiotic species has recently been debated, it is crucial that the consumer safety of each commercially utilized strain be confirmed. Accordingly, this paper details a safety assessment of B. bifidum BGN4 and B. longum BORI via the assessment of ammonia production, hemolysis of blood cells, biogenic amine production, antimicrobial susceptibility pattern, antibiotic resistance gene transferability, PCR data on antibiotic resistance genes, mucin degradation, genome stability, and possession of virulence factors. These probiotic strains showed neither hemolytic activity nor mucin degradation activity, and they did not produce ammonia or biogenic amines (i.e., cadaverine, histamine or tyramine). B. bifidum BGN4 and B. longum BORI produced a small amount of putrescine, commonly found in living cells, at levels similar to or lower than that found in other foods (e.g., spinach, ketchup, green pea, sauerkraut, and sausage). B. bifidum BGN4 showed higher resistance to gentamicin than the European Food Safety Authority (EFSA) cut-off. However, this paper shows the gentamicin resistance of B. bifidum BGN4 was not transferred via conjugation with L. acidophilus ATCC 4356, the latter of which is highly susceptible to gentamicin. The entire genomic sequence of B. bifidum BGN4 has been published in GenBank (accession no.: CP001361.1), documenting the lack of retention of plasmids capable of transferring an antibiotic-resistant gene. Moreover, there was little genetic mutation between the first and 25th generations of B. bifidum BGN4. Tetracycline-resistant genes are prevalent among B. longum strains; B. longum BORI has a tet(W) gene on its chromosome DNA and has also shown resistance to tetracycline. However, this research shows that its tetracycline resistance was not transferred via conjugation with L. fermentum AGBG1, the latter of which is highly sensitive to tetracycline. These findings support the continuous use of B. bifidum BGN4 and B. longum BORI as probiotics, both of which have been reported as safe by several clinical studies, and have been used in food supplements for many years.201829747442
80720.9390Transcriptomic analysis of Saccharomyces cerevisiae upon honokiol treatment. Honokiol (HNK), one of the main medicinal components in Magnolia officinalis, possesses antimicrobial activity against a variety of pathogenic bacteria and fungi. However, little is known of the molecular mechanisms underpinning the antimicrobial activity. To explore the molecular mechanism of its antifungal activity, we determined the effects of HNK on the mRNA expression profile of Saccharomyces cerevisiae using a DNA microarray approach. HNK markedly induced the expression of genes related to iron uptake and homeostasis. Conversely, genes associated with respiratory electron transport were downregulated, mirroring the effects of iron starvation. Meanwhile, HNK-induced growth deficiency was partly rescued by iron supplementation and HNK reacted with iron, producing iron complexes that depleted iron. These results suggest that HNK treatment induced iron starvation. Additionally, HNK treatment resulted in the upregulation of genes involved in protein synthesis and drug resistance networks. Furthermore, the deletion of PDR5, a gene encoding the plasma membrane ATP binding cassette (ABC) transporter, conferred sensitivity to HNK. Overexpression of PDR5 enhanced resistance of WT and pdr5Δ strains to HNK. Taken together, these findings suggest that HNK, which can be excluded by overexpression of Pdr5, functions in multiple cellular processes in S. cerevisiae, particularly in inducing iron starvation to inhibit cell growth.201728499955
19430.9380Possible Role of CHAD Proteins in Copper Resistance. Conserved Histidine Alpha-helical Domain (CHAD) proteins attached to the surface of polyphosphate (PolyP) have been studied in some bacteria and one archaeon. However, the activity of CHAD proteins is unknown beyond their interaction with PolyP granules. By using bioinformatic analysis, we report that several species of the biomining acidophilic bacteria contain orthologs of CHAD proteins with high sequence identity. Furthermore, the gene coding for the CHAD protein is in the same genetic context of the enzyme polyphosphate kinase (PPK), which is in charge of PolyP synthesis. Particularly, the group of ppk and CHAD genes is highly conserved. Metallosphaera sedula and other acidophilic archaea used in biomining also contain CHAD proteins. These archaea show high levels of identity in genes coding for a cluster having the same organization. Amongst these genes are chad and ppx. In general, both biomining bacteria and archaea contain high PolyP levels and are highly resistant to heavy metals. Therefore, the presence of this conserved genetic organization suggests a high relevance for their metabolism. It has been formerly reported that a crystallized CHAD protein contains a copper-binding site. Based on this previous knowledge, in the present report, it was determined that all analyzed CHAD proteins are very conserved at their structural level. In addition, it was found that the lack of YgiF, an Escherichia coli CHAD-containing protein, decreases copper resistance in this bacterium. This phenotype was not only complemented by transforming E. coli with YgiF but also by expressing CHAD from Acidithiobacillus ferrooxidans in it. Interestingly, the strains in which the possible copper-binding sites were mutated were also more metal sensitive. Based on these results, we propose that CHAD proteins are involved in copper resistance in microorganisms. These findings are very interesting and may eventually improve biomining operations in the future.202438399813
23340.9376Deletion of ArmPT, a LamB-like protein, increases cell membrane permeability and antibiotic sensitivity in Vibrio alginolyticus. Vibrio bacterial species are dominant pathogens in mariculture animals. However, the extensive use of antibiotics and other chemicals has increased drug resistance in Vibrio bacteria. Despite rigorous investigative studies, the mechanism of drug resistance in Vibrio remains a mystery. In this study, we found that a gene encoding LamB-like outer membrane protein, named ArmPT, was upregulated in Va under antibiotic stress by RT-qPCR. We speculated that ArmPT might play a role in Va's drug resistance. Subsequently, using ArmPT gene knockout and gene complementation experiments, we confirmed its role in resistance against a variety of antibiotics, particularly kanamycin (KA). Transcriptomic and proteomic analyses identified 188 and 83 differentially expressed genes in the mutant strain compared with the wild-type (WT) before and after KA stress, respectively. Bioinformatic analysis predicted that ArmPT might control cell membrane permeability by changing cadaverine biosynthesis, thereby influencing the cell entry of antibiotics in Va. The higher levels of intracellular reactive oxygen species and the infused content of KA showed that antibiotics are more likely to enter the Va mutant strain. These results uncover the drug resistance mechanism of Va that can also exist in other similar pathogenic bacteria.202438157797
819550.9373Comparative proteomics reveals essential mechanisms for osmotolerance in Gluconacetobacter diazotrophicus. Plant growth-promoting bacteria are a promising alternative to improve agricultural sustainability. Gluconacetobacter diazotrophicus is an osmotolerant bacterium able to colonize several plant species, including sugarcane, coffee, and rice. Despite its biotechnological potential, the mechanisms controlling such osmotolerance remain unclear. The present study investigated the key mechanisms of resistance to osmotic stress in G. diazotrophicus. The molecular pathways regulated by the stress were investigated by comparative proteomics, and proteins essential for resistance were identified by knock-out mutagenesis. Proteomics analysis led to identify regulatory pathways for osmotic adjustment, de novo saturated fatty acids biosynthesis, and uptake of nutrients. The mutagenesis analysis showed that the lack of AccC protein, an essential component of de novo fatty acid biosynthesis, severely affected G. diazotrophicus resistance to osmotic stress. Additionally, knock-out mutants for nutrients uptake (Δtbdr and ΔoprB) and compatible solutes synthesis (ΔmtlK and ΔotsA) became more sensitive to osmotic stress. Together, our results identified specific genes and mechanisms regulated by osmotic stress in an osmotolerant bacterium, shedding light on the essential role of cell envelope and extracytoplasmic proteins for osmotolerance.202133035671
10360.9372IL-1 receptor regulates S100A8/A9-dependent keratinocyte resistance to bacterial invasion. Previously, we reported that epithelial cells respond to exogenous interleukin (IL)-1α by increasing expression of several genes involved in the host response to microbes, including the antimicrobial protein complex calprotectin (S100A8/A9). Given that S100A8/A9 protects epithelial cells against invading bacteria, we studied whether IL-1α augments S100A8/A9-dependent resistance to bacterial invasion of oral keratinocytes. When inoculated with Listeria monocytogenes, human buccal epithelial (TR146) cells expressed and released IL-1α. Subsequently, IL-1α-containing media from Listeria-infected cells increased S100A8/A9 gene expression in naïve TR146 cells an IL-1 receptor (IL-1R)-dependent manner. Incubation with exogenous IL-1α decreased Listeria invasion into TR146 cells, whereas invasion increased with IL-1R antagonist. Conversely, when S100A8/A9 genes were knocked down using short hairpin RNA (shRNA), TR146 cells responded to exogenous IL-1α with increased intracellular bacteria. These data strongly suggest that infected epithelial cells release IL-1α to signal neighboring keratinocytes in a paracrine manner, promoting S100A8/A9-dependent resistance to invasive L. monocytogenes.201222031183
60970.9372A metazoan ortholog of SpoT hydrolyzes ppGpp and functions in starvation responses. In nutrient-starved bacteria, RelA and SpoT proteins have key roles in reducing cell growth and overcoming stresses. Here we identify functional SpoT orthologs in metazoa (named Mesh1, encoded by HDDC3 in human and Q9VAM9 in Drosophila melanogaster) and reveal their structures and functions. Like the bacterial enzyme, Mesh1 proteins contain an active site for ppGpp hydrolysis and a conserved His-Asp-box motif for Mn(2+) binding. Consistent with these structural data, Mesh1 efficiently catalyzes hydrolysis of guanosine 3',5'-diphosphate (ppGpp) both in vitro and in vivo. Mesh1 also suppresses SpoT-deficient lethality and RelA-induced delayed cell growth in bacteria. Notably, deletion of Mesh1 (Q9VAM9) in Drosophila induces retarded body growth and impaired starvation resistance. Microarray analyses reveal that the amino acid-starved Mesh1 null mutant has highly downregulated DNA and protein synthesis-related genes and upregulated stress-responsible genes. These data suggest that metazoan SpoT orthologs have an evolutionarily conserved function in starvation responses.201020818390
880.9370The hawthorn CpLRR-RLK1 gene targeted by ACLSV-derived vsiRNA positively regulate resistance to bacteria disease. Virus-derived small interfering RNAs (vsiRNAs) can target not only viruses but also plant genes. Apple chlorotic leaf spot virus (ACLSV) is an RNA virus that infects Rosaceae plants extensively, including apple, pear and hawthorn. Here, we report an ACLSV-derived vsiRNA [vsiR1360(-)] that targets and down-regulates the leucine-rich repeat receptor-like kinase 1 (LRR-RLK1) gene of hawthorn (Crataegus pinnatifida). The targeting and cleavage of the CpLRR-RLK1 gene by vsiR1360(-) were validated by RNA ligase-mediated 5' rapid amplification of cDNA ends and tobacco transient transformation assays. And the CpLRR-RLK1 protein fused to green fluorescent protein localized to the cell membrane. Conserved domain and phylogenetic tree analyses showed that CpLRR-RLK1 is closely related to the proteins of the LRRII-RLK subfamily. The biological function of CpLRR-RLK1 was explored by heterologous overexpression of CpLRR-RLK1 gene in Arabidopsis. The results of inoculation of Pst DC3000 in Arabidopsis leaves showed that the symptoms of CpLRR-RLK1 overexpression plants infected with Pst DC3000 were significantly reduced compared with the wild type. In addition, the detection of reactive oxygen species and callose deposition and the expression analysis of defense-related genes showed that the CpLRR-RLK1 gene can indeed enhance the resistance of Arabidopsis to bacteria disease.202033180701
61090.9366Queuine Is a Nutritional Regulator of Entamoeba histolytica Response to Oxidative Stress and a Virulence Attenuator. Queuosine is a naturally occurring modified ribonucleoside found in the first position of the anticodon of the transfer RNAs for Asp, Asn, His, and Tyr. Eukaryotes lack pathways to synthesize queuine, the nucleobase precursor to queuosine, and must obtain it from diet or gut microbiota. Here, we describe the effects of queuine on the physiology of the eukaryotic parasite Entamoeba histolytica, the causative agent of amebic dysentery. Queuine is efficiently incorporated into E. histolytica tRNAs by a tRNA-guanine transglycosylase (EhTGT) and this incorporation stimulates the methylation of C38 in [Formula: see text] Queuine protects the parasite against oxidative stress (OS) and antagonizes the negative effect that oxidation has on translation by inducing the expression of genes involved in the OS response, such as heat shock protein 70 (Hsp70), antioxidant enzymes, and enzymes involved in DNA repair. On the other hand, queuine impairs E. histolytica virulence by downregulating the expression of genes previously associated with virulence, including cysteine proteases, cytoskeletal proteins, and small GTPases. Silencing of EhTGT prevents incorporation of queuine into tRNAs and strongly impairs methylation of C38 in [Formula: see text], parasite growth, resistance to OS, and cytopathic activity. Overall, our data reveal that queuine plays a dual role in promoting OS resistance and reducing parasite virulence.IMPORTANCEEntamoeba histolytica is a unicellular parasite that causes amebiasis. The parasite resides in the colon and feeds on the colonic microbiota. The gut flora is implicated in the onset of symptomatic amebiasis due to alterations in the composition of bacteria. These bacteria modulate the physiology of the parasite and affect the virulence of the parasite through unknown mechanisms. Queuine, a modified nucleobase of queuosine, is exclusively produced by the gut bacteria and leads to tRNA modification at the anticodon loops of specific tRNAs. We found that queuine induces mild oxidative stress resistance in the parasite and attenuates its virulence. Our study highlights the importance of bacterially derived products in shaping the physiology of the parasite. The fact that queuine inhibits the virulence of E. histolytica may lead to new strategies for preventing and/or treating amebiasis by providing to the host queuine directly or via probiotics.202133688012
587100.9365The Nramp (Slc11) proteins regulate development, resistance to pathogenic bacteria and iron homeostasis in Dictyostelium discoideum. The Dictyostelium discoideum genome harbors two genes encoding members of the Nramp superfamily, which is conserved from bacteria (MntH proteins) to humans (Slc11 proteins). Nramps are proton-driven metal ion transporters with a preference for iron and manganese. Acquisition of these metal cations is vital for all cells, as they act as redox cofactors and regulate key cellular processes, such as DNA synthesis, electron transport, energy metabolism and oxidative stress. Dictyostelium Nramp1 (Slc11a1), like its mammalian ortholog, mediates resistance to infection by invasive bacteria. We have extended the analysis to the nramp2 gene, by generating single and double nramp1/nramp2 knockout mutants and cells expressing GFP fusion proteins. In contrast to Nramp1, which is recruited to phagosomes and macropinosomes, the Nramp2 protein is localized exclusively in the membrane of the contractile vacuole, a vesicular tubular network regulating cellular osmolarity. Both proteins colocalize with the V-H(+)-ATPase, which can provide the electrogenic force for vectorial transport. Like nramp1, nramp2 gene disruption affects resistance to Legionella pneumophila. Disrupting both genes additionally leads to defects in development, with strong delay in cell aggregation, formation of large streams and multi-tipped aggregates. Single and double mutants display differential sensitivity to cell growth under conditions of iron overload or depletion. The data favor the hypothesis that Nramp1 and Nramp2, under control of the V-H(+)-ATPase, synergistically regulate iron homeostasis, with the contractile vacuole possibly acting as a store for metal cations.201322992462
542110.9365Role of Yops and adhesins in resistance of Yersinia enterocolitica to phagocytosis. Yersinia enterocolitica is a pathogen endowed with two adhesins, Inv and YadA, and with the Ysc type III secretion system, which allows extracellular adherent bacteria to inject Yop effectors into the cytosol of animal target cells. We tested the influence of all of these virulence determinants on opsonic and nonopsonic phagocytosis by PU5-1.8 and J774 mouse macrophages, as well as by human polymorphonuclear leukocytes (PMNs). The adhesins contributed to phagocytosis in the absence of opsonins but not in the presence of opsonins. In agreement with previous results, YadA counteracted opsonization. In every instance, the Ysc-Yop system conferred a significant level of resistance to phagocytosis. Nonopsonized single-mutant bacteria lacking either YopE, -H, -T, or -O were phagocytosed significantly more by J774 cells and by PMNs. Opsonized bacteria were phagocytosed more than nonopsonized bacteria, and mutant bacteria lacking either YopH, -T, or -O were phagocytosed significantly more by J774 cells and by PMNs than were wild-type (WT) bacteria. Opsonized mutants lacking only YopE were phagocytosed significantly more than were WT bacteria by PMNs but not by J774 cells. Thus, YopH, -T, and -O were involved in all of the phagocytic processes studied here but YopE did not play a clear role in guarding against opsonic phagocytosis by J774. Mutants lacking YopP and YopM were, in every instance, as resistant as WT bacteria. Overexpression of YopE, -H, -T, or -O alone did not confer resistance to phagocytosis, although it affected the cytoskeleton. These results show that YopH, YopT, YopO, and, in some instances, YopE act synergistically to increase the resistance of Y. enterocolitica to phagocytosis by macrophages and PMNs.200212117925
608120.9363Entamoeba histolytica Adaption to Auranofin: A Phenotypic and Multi-Omics Characterization. Auranofin (AF), an antirheumatic agent, targets mammalian thioredoxin reductase (TrxR), an important enzyme controlling redox homeostasis. AF is also highly effective against a diversity of pathogenic bacteria and protozoan parasites. Here, we report on the resistance of the parasite Entamoeba histolytica to 2 µM of AF that was acquired by gradual exposure of the parasite to an increasing amount of the drug. AF-adapted E. histolytica trophozoites (AFAT) have impaired growth and cytopathic activity, and are more sensitive to oxidative stress (OS), nitrosative stress (NS), and metronidazole (MNZ) than wild type (WT) trophozoites. Integrated transcriptomics and redoxomics analyses showed that many upregulated genes in AFAT, including genes encoding for dehydrogenase and cytoskeletal proteins, have their product oxidized in wild type trophozoites exposed to AF (acute AF trophozoites) but not in AFAT. We also showed that the level of reactive oxygen species (ROS) and oxidized proteins (OXs) in AFAT is lower than that in acute AF trophozoites. Overexpression of E. histolytica TrxR (EhTrxR) did not protect the parasite against AF, which suggests that EhTrxR is not central to the mechanism of adaptation to AF.202134439488
106130.9362Genomic evidence of the illumination response mechanism and evolutionary history of magnetotactic bacteria within the Rhodospirillaceae family. BACKGROUND: Magnetotactic bacteria (MTB) are ubiquitous in natural aquatic environments. MTB can produce intracellular magnetic particles, navigate along geomagnetic field, and respond to light. However, the potential mechanism by which MTB respond to illumination and their evolutionary relationship with photosynthetic bacteria remain elusive. RESULTS: We utilized genomes of the well-sequenced genus Magnetospirillum, including the newly sequenced MTB strain Magnetospirillum sp. XM-1 to perform a comprehensive genomic comparison with phototrophic bacteria within the family Rhodospirillaceae regarding the illumination response mechanism. First, photoreceptor genes were identified in the genomes of both MTB and phototrophic bacteria in the Rhodospirillaceae family, but no photosynthesis genes were found in the MTB genomes. Most of the photoreceptor genes in the MTB genomes from this family encode phytochrome-domain photoreceptors that likely induce red/far-red light phototaxis. Second, illumination also causes damage within the cell, and in Rhodospirillaceae, both MTB and phototrophic bacteria possess complex but similar sets of response and repair genes, such as oxidative stress response, iron homeostasis and DNA repair system genes. Lastly, phylogenomic analysis showed that MTB cluster closely with phototrophic bacteria in this family. One photoheterotrophic genus, Phaeospirillum, clustered within and displays high genomic similarity with Magnetospirillum. Moreover, the phylogenetic tree topologies of magnetosome synthesis genes in MTB and photosynthesis genes in phototrophic bacteria from the Rhodospirillaceae family were reasonably congruent with the phylogenomic tree, suggesting that these two traits were most likely vertically transferred during the evolution of their lineages. CONCLUSION: Our new genomic data indicate that MTB and phototrophic bacteria within the family Rhodospirillaceae possess diversified photoreceptors that may be responsible for phototaxis. Their genomes also contain comprehensive stress response genes to mediate the negative effects caused by illumination. Based on phylogenetic studies, most of MTB and phototrophic bacteria in the Rhodospirillaceae family evolved vertically with magnetosome synthesis and photosynthesis genes. The ancestor of Rhodospirillaceae was likely a magnetotactic phototrophic bacteria, however, gain or loss of magnetotaxis and phototrophic abilities might have occurred during the evolution of ancestral Rhodospirillaceae lineages.201931117953
33140.9361Transgenic Silkworms Overexpressing Relish and Expressing Drosomycin Confer Enhanced Immunity to Multiple Pathogens. The sericulture industry faces substantial economic losses due to severe pathogenic infections caused by fungi, viruses, and bacteria. The development of transgenic silkworms against specific pathogens has been shown to enhance disease resistance against a particular infection. A single gene or its products that can confer protection against multiple pathogens is required. In an attempt to develop silkworms with enhanced immunity against multiple pathogens, we generated transgenic silkworm lines with an overexpressed NF-kB transcription factor, Relish 1, under two different promoters. Separately, a potent anti-fungal gene, Drosomycin, was also expressed in transgenic silkworms. Both Relish 1 and Drosomycin transgenic silkworms had single copy genomic integration, and their mRNA expression levels were highly increased after infection with silkworm pathogens. The overexpression of the Relish 1 in transgenic silkworms resulted in the upregulation of several defense-related genes, Cecropin B, Attacin, and Lebocin, and showed enhanced resistance to Nosema bombycis (microsporidian fungus), Nucleopolyhedrovirus (BmNPV), and bacteria. The Drosomycin expressing transgenic silkworms showed elevated resistance to N. bombycis and bacteria. These findings demonstrate the role of Relish 1 in long-lasting protection against multiple pathogens in silkworms. Further, the successful introduction of a foreign gene, Drosomycin, also led to improved disease resistance in silkworms.202235098482
201150.9360Hyaluronic Acid--an "Old" Molecule with "New" Functions: Biosynthesis and Depolymerization of Hyaluronic Acid in Bacteria and Vertebrate Tissues Including during Carcinogenesis. Hyaluronic acid is an evolutionarily ancient molecule commonly found in vertebrate tissues and capsules of some bacteria. Here we review modern data regarding structure, properties, and biological functions of hyaluronic acid in mammals and Streptococcus spp. bacteria. Various aspects of biogenesis and degradation of hyaluronic acid are discussed, biosynthesis and degradation metabolic pathways for glycosaminoglycan together with involved enzymes are described, and vertebrate and bacterial hyaluronan synthase genes are characterized. Special attention is given to the mechanisms underlying the biological action of hyaluronic acid as well as the interaction between polysaccharide and various proteins. In addition, all known signaling pathways involving hyaluronic acid are outlined. Impaired hyaluronic acid metabolism, changes in biopolymer molecular weight, hyaluronidase activity, and enzyme isoforms often accompany carcinogenesis. The interaction between cells and hyaluronic acid from extracellular matrix that may be important during malignant change is discussed. An expected role for high molecular weight hyaluronic acid in resistance of naked mole rat to oncologic diseases and the protective role of hyaluronic acid in bacteria are discussed.201526555463
723160.9359Ail and PagC-related proteins in the entomopathogenic bacteria of Photorhabdus genus. Among pathogenic Enterobacteriaceae, the proteins of the Ail/OmpX/PagC family form a steadily growing family of outer membrane proteins with diverse biological properties, potentially involved in virulence such as human serum resistance, adhesion and entry into eukaryotic culture cells. We studied the proteins Ail/OmpX/PagC in the bacterial Photorhabdus genus. The Photorhabdus bacteria form symbiotic complexes with nematodes of Heterorhabditis species, associations which are pathogenic to insect larvae. Our phylogenetic analysis indicated that in Photorhabdus asymbiotica and Photorhabdus luminescens only Ail and PagC proteins are encoded. The genomic analysis revealed that the Photorhabdus ail and pagC genes were present in a unique copy, except two ail paralogs from P. luminescens. These genes, referred to as ail1Pl and ail2Pl, probably resulted from a recent tandem duplication. Surprisingly, only ail1Pl expression was directly controlled by PhoPQ and low external Mg2+ conditions. In P. luminescens, the magnesium-sensing two-component regulatory system PhoPQ regulates the outer membrane barrier and is required for pathogenicity against insects. In order to characterize Ail functions in Photorhabdus, we showed that only ail2Pl and pagCPl had the ability, when expressed into Escherichia coli, to confer resistance to complement in human serum. However no effect in resistance to antimicrobial peptides was found. Thus, the role of Ail and PagC proteins in Photorhabdus life cycle is discussed.201425333642
8192170.9358Resisting the Heat: Bacterial Disaggregases Rescue Cells From Devastating Protein Aggregation. Bacteria as unicellular organisms are most directly exposed to changes in environmental growth conditions like temperature increase. Severe heat stress causes massive protein misfolding and aggregation resulting in loss of essential proteins. To ensure survival and rapid growth resume during recovery periods bacteria are equipped with cellular disaggregases, which solubilize and reactivate aggregated proteins. These disaggregases are members of the Hsp100/AAA+ protein family, utilizing the energy derived from ATP hydrolysis to extract misfolded proteins from aggregates via a threading activity. Here, we describe the two best characterized bacterial Hsp100/AAA+ disaggregases, ClpB and ClpG, and compare their mechanisms and regulatory modes. The widespread ClpB disaggregase requires cooperation with an Hsp70 partner chaperone, which targets ClpB to protein aggregates. Furthermore, Hsp70 activates ClpB by shifting positions of regulatory ClpB M-domains from a repressed to a derepressed state. ClpB activity remains tightly controlled during the disaggregation process and high ClpB activity states are likely restricted to initial substrate engagement. The recently identified ClpG (ClpK) disaggregase functions autonomously and its activity is primarily controlled by substrate interaction. ClpG provides enhanced heat resistance to selected bacteria including pathogens by acting as a more powerful disaggregase. This disaggregase expansion reflects an adaption of bacteria to extreme temperatures experienced during thermal based sterilization procedures applied in food industry and medicine. Genes encoding for ClpG are transmissible by horizontal transfer, allowing for rapid spreading of extreme bacterial heat resistance and posing a threat to modern food production.202134017857
711180.9358Non-specific, general and multiple stress resistance of growth-restricted Bacillus subtilis cells by the expression of the sigmaB regulon. Bacillus subtilis cells respond almost immediately to different stress conditions by increasing the production of general stress proteins (GSPs). The genes encoding the majority of the GSPs that are induced by heat, ethanol, salt stress or by starvation for glucose, oxygen or phosphate belong to the sigmaB-dependent general stress regulon. Despite a good understanding of the complex regulation of the activity of sigmaB and knowledge of a very large number of general stress genes controlled by sigmaB, first insights into the physiological role of this nonspecific stress response have been obtained only very recently. To explore the physiological role of this reguIon, we and others identified sigmaB-dependent general stress genes and compared the stress tolerance of wild-type cells with mutants lacking sigmaB or general stress proteins. The proteins encoded by sigmaB-dependent general stress genes can be divided into at least five functional groups that most probably provide growth-restricted B. subtilis cells with a multiple stress resistance in anticipation of future stress. In particular, sigB mutants are impaired in non-specific resistance to oxidative stress, which requires the sigmaB-dependent dps gene encoding a DNA-protecting protein. Protection against oxidative damage of membranes, proteins or DNA could be the most essential component of sigmaB mediated general stress resistance in growth-arrested aerobic gram-positive bacteria. Other general stress genes have both a sigmaB-dependent induction pathway and a second sigmaB-independent mechanism of stress induction, thereby partially compensating for a sigmaB deficiency in a sigB mutant. In contrast to sigB mutants, null mutations in genes encoding those proteins, such as cIpP or cIpC, cause extreme sensitivity to salt or heat.19989767581
8726190.9356CRISPR-dCpf1 mediated whole genome crRNA inhibition library for high-throughput screening of growth characteristic genes in Bacillus amyloliquefaciens LB1ba02. Bacillus amyloliquefaciens LB1ba02 is generally recognized as food safe (GRAS) microbial host and important enzyme-producing strain in the industry. However, autolysis affects the growth of bacteria, further affecting the yield of target products. Besides, the restriction-modification system, existed in B. amyloliquefaciens LB1ba02, results in a low transformation efficiency, which further leads to a lack of high-throughput screening tools. Here, we constructed a genome-wide crRNA inhibition library based on the CRISPR/dCpf1 system and high-throughput screening of related genes affecting the cell growth and autolysis using flow cytometry in B. amyloliquefaciens LB1ba02. The whole genome crRNA library was first validated for resistance to the toxic chemical 5-fluorouracil, and then used for validation of essential genes. In addition, seven gene loci (oppD, flil, tuaA, prmA, sigO, hslU, and GE03231) that affect the growth characteristics of LB1ba02 were screened. Among them, the Opp system had the greatest impact on growth. When the expression of operon oppA-oppB-oppC-oppD-oppF was inhibited, the cell growth difference was most significant. Inhibition of other sites could also promote rapid growth of bacteria to varying degrees; however, inhibition of GE03231 site accelerated cell autolysis. Therefore, the whole genome crRNA inhibition library is well suited for B. amyloliquefaciens LB1ba02 and can be further applied to high-throughput mining of other functional genes.202337802457