Deficient Strain Research Articles

The Detrimental Role of Regulatory T Cells in Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is induced by steatosis and metabolic inflammation. While involvement of the innate immune response has been shown, the role of the adaptive immune response in NASH remains controversial. Likewise, the role of regulatory T cells (Treg) in NASH remains unclear although initial clinical trials aim to target these regulatory responses. High‐fat high‐carbohydrate (HF‐HC) diet feeding of NASH‐resistant BALB/c mice as well as the corresponding recombination activating 1 (Rag)‐deficient strain was used to induce NASH and to study the role of the adaptive immune response. HF‐HC diet feeding induced strong activation of intrahepatic T cells in BALB/c mice, suggesting an antigen‐driven effect. In contrast, the effects of the absence of the adaptive immune response was notable. NASH in BALB/c Rag1−/− mice was substantially worsened and accompanied by a sharp increase of M1‐like macrophage numbers. Furthermore, we found an increase in intrahepatic Treg numbers in NASH, but either adoptive Treg transfer or anti‐cluster of differentiation (CD)3 therapy unexpectedly increased steatosis and the alanine aminotransferase level without otherwise affecting NASH. Conclusion: Although intrahepatic T cells were activated and marginally clonally expanded in NASH, these effects were counterbalanced by increased Treg numbers. The ablation of adaptive immunity in murine NASH led to marked aggravation of NASH, suggesting that Tregs are not regulators of metabolic inflammation but rather enhance it.

An Efficient Strategy for Obtaining Mutants by Targeted Gene Deletion in Ophiostoma novo-ulmi.

The dimorphic fungus Ophiostoma novo-ulmi is the highly aggressive pathogen responsible for the current, highly destructive, pandemic of Dutch elm disease (DED). Genome and transcriptome analyses of this pathogen previously revealed that a large set of genes expressed during dimorphic transition were also potentially related to plant infection processes, which seem to be regulated by molecular mechanisms different from those described in other dimorphic pathogens. Then, O. novo-ulmi can be used as a representative species to study the lifestyle of dimorphic pathogenic fungi that are not shared by the “model species” Candida albicans and Ustilago maydis. In order to gain better knowledge of molecular aspects underlying infection process and symptom induction by dimorphic fungi that cause vascular wilt disease, we developed a high-throughput gene deletion protocol for O. novo-ulmi. The protocol is based on transforming a Δmus52 O. novo-ulmi mutant impaired for non-homologous end joining (NHEJ) as the recipient strain, and transforming this strain with the latest version of OSCAR plasmids. The latter are used for generating deletion constructs containing the toxin-coding Herpes simplex virus thymidine kinase (HSVtk) gene which prevents ectopic integration of the T-DNA in Ophiostoma DNA. The frequency of gene deletion by homologous recombination (HR) at the ade1 locus associated with purine nucleotide biosynthesis was up to 77.8% in the Δmus52 mutant compared to 2% in the wild-type (WT). To validate the high efficiency of our deletion gene methodology we deleted ade7, which also belongs to the purine nucleotide pathway, as well as bct2, ogf1, and opf2 which encode fungal binuclear transcription factors (TFs). The frequency of gene replacement by HR for these genes reached up to 94%. We expect that our methodology combining the use of NHEJ deficient strains and OSCAR plasmids will function with similar high efficiencies for other O. novo-ulmi genes and other filamentous fungi.

Influence of sub-inhibitory concentrations of antimicrobials on micrococcal nuclease and biofilm formation in Staphylococcus aureus

A major contributor to biomaterial associated infection (BAI) is Staphylococcus aureus. This pathogen produces a protective biofilm, making eradication difficult. Biofilms are composed of bacteria encapsulated in a matrix of extracellular polymeric substances (EPS) comprising polysaccharides, proteins and extracellular DNA (eDNA). S. aureus also produces micrococcal nuclease (MN), an endonuclease which contributes to biofilm composition and dispersion, mainly expressed by nuc1. MN expression can be modulated by sub-minimum inhibitory concentrations of antimicrobials. We investigated the relation between the biofilm and MN expression and the impact of the application of antimicrobial pressure on this relation. Planktonic and biofilm cultures of three S. aureus strains, including a nuc1 deficient strain, were cultured under antimicrobial pressure. Results do not confirm earlier findings that MN directly influences total biomass of the biofilm but indicated that nuc1 deletion stimulates the polysaccharide production per CFU in the biofilm in in vitro biofilms. Though antimicrobial pressure of certain antibiotics resulted in significantly increased quantities of polysaccharides per CFU, this did not coincide with significantly reduced MN activity. Erythromycin and resveratrol significantly reduced MN production per CFU but did not affect total biomass or biomass/CFU. Reduction of MN production may assist in the eradication of biofilms by the host immune system in clinical situations.

Comparison of the fertility of tumor suppressor gene-deficient C57BL/6 mouse strains reveals stable reproductive aging and novel pleiotropic gene

Tumor suppressor genes are involved in maintaining genome integrity during reproduction (e.g., meiosis). Thus, deleterious alleles in tumor suppressor-deficient mice would exhibit higher mortality during the perinatal period. A recent aging model proposes that perinatal mortality and age-related deleterious changes might define lifespan. This study aimed to quantitatively understand the relationship between reproduction and lifespan using three established tumor suppressor gene (p53, APC, and RECQL4)-deficient mouse strains with the same C57BL/6 background. Transgenic mice delivered slightly reduced numbers of 1st pups than wild-type mice [ratio: 0.81–0.93 (p = 0.1–0.61)] during a similar delivery period, which suggest that the tumor suppressor gene-deficient mice undergo relatively stable reproduction. However, the transgenic 1st pups died within a few days after birth, resulting in a further reduction in litter size at 3 weeks after delivery compared with that of wild-type mice [ratio: 0.35–0.68 (p = 0.034–0.24)] without sex differences, although the lifespan was variable. Unexpectedly, the significance of reproductive reduction in transgenic mice was decreased at the 2nd or later delivery. Because mice are easily affected by environmental factors, our data underscore the importance of defining reproductive ability through experiments on aging-related reproduction that can reveal a trade-off between fecundity and aging and identify RECQL4 as a novel pleiotropic gene.

Prevalence and characterization of pertactin deficient Bordetella pertussis strains in Brazil, a whole-cell vaccine country

Many countries have reported antigenic divergence among circulating Bordetella pertussis strains, mainly in those countries which introduced the acellular pertussis (aP) vaccine. This phenomenon can be seen, for example, with the recent rise of pertactin (Prn)-deficient B. pertussis strains, one of the antigens included in aP vaccine formulas. The whole cell pertussis (wP) vaccine has been used in Brazil since 1977 for the primary pertussis, diphtheria and tetanus immunization series. In 2014, the aP vaccine was recommended for women during pregnancy to protect infants in the first months of life. Our objective was to determine the prevalence of Prn-deficiency in 511 isolates of B. pertussis collected in Brazil during 2010–2016. All isolates were characterized, through PFGE and serotyping, and screened for the loss of Prn by ELISA. Prn-deficiency was confirmed by immunoblotting, and identification of the possible genetic markers was performed with PCR and Sanger sequencing. Results indicate that 110 PFGE profiles are currently circulating, with five profiles representing the majority, and the predominant serotype 3, has been gradually replaced by serotype 2 and serotype 2,3. ELISA screening and immunoblotting identified three Prn-deficient isolates. Genotypic characterization by PCR and sequencing indicated that one isolate had a promoter mutation in prn, while the other two did not have an obvious genetic explanation for their deficiency. While the lack of Prn was identified in a few isolates, this study did not detect a relevant occurrence of Prn-deficiency, until 2016, confirming previous observations that Prn-deficiency is likely aP vaccine-driven.

Modified Monosaccharides Content of Xanthan Gum Impairs Citrus Canker Disease by Affecting the Epiphytic Lifestyle of Xanthomonas citri subsp. citri.

Xanthomonas citri subsp. citri (X. citri) is a plant pathogenic bacterium causing citrus canker disease. The xanA gene encodes a phosphoglucomutase/phosphomannomutase protein that is a key enzyme required for the synthesis of lipopolysaccharides and exopolysaccharides in Xanthomonads. In this work, firstly we isolated a xanA transposon mutant (xanA::Tn5) and analyzed its phenotypes as biofilm formation, xanthan gum production, and pathogenesis on the sweet orange host. Moreover, to confirm the xanA role in the impaired phenotypes we further produced a non-polar deletion mutant (ΔxanA) and performed the complementation of both xanA mutants. In addition, we analyzed the percentages of the xanthan gum monosaccharides produced by X. citri wild-type and xanA mutant. The mutant strain had higher ratios of mannose, galactose, and xylose and lower ratios of rhamnose, glucuronic acid, and glucose than the wild-type strain. Such changes in the saccharide composition led to the reduction of xanthan yield in the xanA deficient strain, affecting also other important features in X. citri, such as biofilm formation and sliding motility. Moreover, we showed that xanA::Tn5 caused no symptoms on host leaves after spraying, a method that mimetics the natural infection condition. These results suggest that xanA plays an important role in the epiphytical stage on the leaves that is essential for the successful interaction with the host, including adaptive advantage for bacterial X. citri survival and host invasion, which culminates in pathogenicity.

Identification of the Genes Related to the Glycogen Metabolism in Hyperthermophilic Archaeon, Sulfolobus acidocaldarius.

Glycogen is a polysaccharide that comprises α-1,4-linked glucose backbone and α-1,6-linked glucose polymers at the branching points. It is widely found in organisms ranging from bacteria to eukaryotes. The physiological role of glycogen is not confined to being an energy reservoir and carbon source but varies depending on organisms. Sulfolobus acidocaldarius, a thermoacidophilic archaeon, was observed to accumulate granular glycogen in the cell. However, the role of glycogen and genes that are responsible for glycogen metabolism in S. acidocaldarius has not been identified clearly. The objective of this study is to identify the gene cluster, which is composed of enzymes that are predicted to be involved in the glycogen metabolism, and confirm the role of each of these genes by constructing deletion mutants. This study also compares the glycogen content of mutant and wild type and elucidates the role of glycogen in this archaeon. The glycogen content of S. acidocaldarius MR31, which is used as a parent strain for constructing the deletion mutant in this study, was increased in the early and middle exponential growth phases and decreased during the late exponential and stationary growth phases. The pattern of the accumulated glycogen was independent to the type of supplemented sugar. In the comparison of the glycogen content between the gene deletion mutant and MR31, glycogen synthase (GlgA) and α-amylase (AmyA) were shown to be responsible for the synthesis of glycogen, whereas glycogen debranching enzyme (GlgX) and glucoamylase (Gaa) appeared to affect the degradation of glycogen. The expressions of glgC–gaa–glgX and amyA–glgA were detected by the promoter assay. This result suggests that the gradual decrease of glycogen content in the late exponential and stationary phases occurs due to the increase in the gene expression of glgC–gaa–glgX. When the death rate in nutrient limited condition was compared among the wild type strain, the glycogen deficient strain and the strain with increased glycogen content, the death rate of the glycogen deficient strain was found to be higher than any other strain, thereby suggesting that the glycogen in S. acidocaldarius supports cell maintenance in harsh conditions.

Pathogenesis and chemotherapy of a thymidine kinase deficient mutant strain of HSV‐1 in a murine zosteriform infection model.

Type I and II Herpesvirus simplex virus [HSV] are contagious and cause oral (cold sores) and genital ulcers (genital HSV) before becoming latent in trigeminal ganglia (oral HSV) or sacral ganglia (genital HSV), and can reactivate and cause disease almost at the same spot of the initial virus exposure. Acyclovir is one of the gold standard treatments for labial and genital HSV for decades. Topical Acyclovir and penciclovir and their prodrugs act on thymidine kinase (TK) and are viral DNA chain inhibitor agents and hence inhibit virus replication. In TK deletion mutants, drugs which interact with TK enzyme and inhibit virus replication do not have any inhibitory effect. TK deletion mutants also replicate at slower rates and therefore take longer to make plaques in tissue culture compared to wild type strains. Resistant virus could also arise due to suboptimal treatment regimens although it is not a great problem currently in immunocompetent patients. However resistant HSV could be a major challenge to treat in immunocompromised and immunosuppressed patients as not many options are available to use antiviral drugs whose mode of action is to work on TK inhibition. In this study pathogenesis of TK deletion mutant resistant strains of HSV were used in a immunocompetent neck-ear zosteriform infection mouse model and efficacy of prodrugs on zoster spread was studied. Resistant strains of HSV mutants received were originally raised in vitro and plaques purified. These strains were then grown in tissue culture and working stock was produced. We observed that rate of replication of these mutant viruses was slower as they took longer to make plaque in tissue culture compared to wild type strains. Resistant strain of HSV were used in the immunocompetent murine zosteriform infection model along with the wild strain and efficacy of prodrugs was studied to see if murine immunocompetent natural defense will have any effect on the pathogenicity and if antiviral agents have any effect in vivo. A murine neck ear zosteriform infection model was used and development of zosteriform lesion were noted on the ear pinna along with ear thickness and body weight. Mutant viruses show delayed zosteriform spread. It was noticed that though lesions were not completely inhibited by prodrugs, zosteriform lesion and intensity was lower after antiviral treatment. This was not expected so we tend to believe that either immunocompetent immune defense system of mice were providing extra help to reduce the virus lesion or a possibility of helper virus in the murine model. In order to see the effect of TK activity in resistant mutants original stock was further analyzed by radiolabeled C-14 TK assay to confirm the deletion mutants status. In this communication In vivo results of zoster spread, radiolabeled C-14 TK assays to identify TK deletion mutant, purified cloning, other antiviral which can have an effect on TK deletion resistant HSV in immunocompromised and immunosuppressed patients will be discussed.

Bacillus subtilis High Cell Density Fermentation Using a Sporulation-Deficient Strain for the Production of Surfactin

Bacillus subtilis 3NA is a strain capable of reaching high cell densities. A surfactin producing sfp+ variant of this strain, named JABs32, was utilized in fed-batch cultivation processes. Both a glucose and an ammonia solution were fed to set a steady growth rate μ of 0.1 h-1. In this process, a cell dry weight of up to 88 g L-1 was reached after 38 h of cultivation, and surfactin titers of up to 26.5 g L-1 were detected in this high cell density fermentation process, achieving a YP/X value of 0.23 g g-1 as well as a qP/X of 0.007 g g-1 h-1. In sum, a 21-fold increase in surfactin titer was obtained compared with cultivations in shake flasks. In contrast to fed-batch operations using Bacillus subtilis JABs24, an sfp+ variant derived from B. subtilis 168, JABs32, reached an up to fourfold increase in surfactin titers using the same fed-batch protocol. Additionally, a two-stage feed process was established utilizing strain JABs32. Using an optimized mineral salt medium in this high cell density fermentation approach, after 31 h of cultivation, surfactin titers of 23.7 g L-1 were reached with a biomass concentration of 41.3 g L-1, thus achieving an enhanced YP/X value of 0.57 g g-1 as well as a qP/X of 0.018 g g-1 h-1. The mutation of spo0A locus and an elongation of AbrB in the strain utilized in combination with a high cell density fed-batch process represents a promising new route for future enhancements on surfactin production.Key points• Utilization of a sporulation deficient strain for fed-batch operations• High cell density process with Bacillus subtilis for lipopeptide production was established• High titer surfactin production capabilities confirm highly promising future platform strain

Platelet MHC class I mediates CD8+ T-cell suppression during sepsis

AbstractCirculating platelets interact with leukocytes to modulate host immune and thrombotic responses. In sepsis, platelet-leukocyte interactions are increased and have been associated with adverse clinical events, including increased platelet–T-cell interactions. Sepsis is associated with reduced CD8+ T-cell numbers and functional responses, but whether platelets regulate CD8+ T-cell responses during sepsis remains unknown. In our current study, we systemically evaluated platelet antigen internalization and presentation through major histocompatibility complex class I (MHC-I) and their effects on antigen-specific CD8+ T cells in sepsis in vivo and ex vivo. We discovered that both human and murine platelets internalize and proteolyze exogenous antigens, generating peptides that are loaded onto MHC-I. The expression of platelet MHC-I, but not platelet MHC-II, is significantly increased in human and murine platelets during sepsis and in human megakaryocytes stimulated with agonists generated systemically during sepsis (eg, interferon-γ and lipopolysaccharide). Upregulation of platelet MHC-I during sepsis increases antigen cross-presentation and interactions with CD8+ T cells in an antigen-specific manner. Using a platelet lineage–specific MHC-I–deficient mouse strain (B2Mf/f-Pf4Cre), we demonstrate that platelet MHC-I regulates antigen-specific CD8+ T-cell proliferation in vitro, as well as the number and functional responses of CD8+ T cells in vivo, during sepsis. Loss of platelet MHC-I reduces sepsis-associated mortality in mice in an antigen-specific setting. These data identify a new mechanism by which platelets, through MHC-I, process and cross-present antigens, engage antigen-specific CD8+ T cells, and regulate CD8+ T-cell numbers, functional responses, and outcomes during sepsis.

Comparative transcriptomics analysis of Streptococcus mutans with disruption of LuxS/AI-2 quorum sensing and recovery of methyl cycle

ObjectiveThe LuxS/AI-2 quorum sensing (QS) system has critical roles in Streptococcus mutans cariogenicity. Whereas the molecular and cellular mechanisms of the LuxS/AI-2 QS system are not thoroughly understood. Given that LuxS has roles in QS and methyl cycle, its mutation can cause QS deficiency and methyl cycle disruption. The aim of this study was to investigate effects of the LuxS/AI-2 QS system on gene expression in Streptococcus mutans when methyl cycle was recovered with exogenous sahH gene. MethodsOur previous study introduced the exogenous sahH gene from Pseudomonas aeruginosa into an S. mutans luxS-null strain to restore the disrupted methyl cycle, and this produced the solely the LuxS/AI-2 QS system deficient strain. Here, we analyzed the transcriptomics of this strain to get insights into the molecular mechanisms of the LuxS/AI-2 QS system applying RNA-seq. ResultsWith recovery of methyl cycle, 84 genes didn’t change in expression trends in S. mutans luxS-null strain. These genes mainly encode the ABC transporters, sugar transporter EII and enzymes of carbohydrate metabolism, and are rich in the Phosphotransferase system, Fructose and mannose metabolism, Amino sugar and nucleotide sugar metabolism, Galactose metabolism, Glycolysis/Gluconeogenesis, RNA degradation, Lysine biosynthesis, and Glycine, serine and threonine metabolism. ConclusionsThe LuxS/AI-2 QS system may mainly affect ABC transporters and carbohydrate transport, transformation and metabolism via EII subunits and enzymes to influence virulence-associated traits without effects of methyl cycle inStreptococcus mutans.

Preservation stress resistance of melanin deficient conidia from Paecilomyces variotii and Penicillium roqueforti mutants generated via CRISPR/Cas9 genome editing

BackgroundThe filamentous fungi Paecilomyces variotii and Penicillium roqueforti are prevalent food spoilers and are of interest as potential future cell factories. A functional CRISPR/Cas9 genome editing system would be beneficial for biotechnological advances as well as future (genetic) research in P. variotii and P. roqueforti.ResultsHere we describe the successful implementation of an efficient AMA1-based CRISPR/Cas9 genome editing system developed for Aspergillus niger in P. variotii and P. roqueforti in order to create melanin deficient strains. Additionally, kusA− mutant strains with a disrupted non-homologous end-joining repair mechanism were created to further optimize and facilitate efficient genome editing in these species. The effect of melanin on the resistance of conidia against the food preservation stressors heat and UV-C radiation was assessed by comparing wild-type and melanin deficient mutant conidia.ConclusionsOur findings show the successful use of CRISPR/Cas9 genome editing and its high efficiency in P. variotii and P. roqueforti in both wild-type strains as well as kusA− mutant background strains. Additionally, we observed that melanin deficient conidia of three food spoiling fungi were not altered in their heat resistance. However, melanin deficient conidia had increased sensitivity towards UV-C radiation.

Lyophilized yeast powder for adjuvant free thermostable vaccine delivery.

Thermolabile nature of commercially available vaccines necessitates their storage, transportation, and dissemination under refrigerated condition. Maintenance of continuous cold chain at every step increases the final cost of vaccines. Any breach in the cold chain even for a short duration results in the need to discard the vaccines. As a result, there is a pressing need for the development of thermostable vaccines. In this proof-of-concept study, we showed that E. coli curli-green fluorescent fusion protein remains stable in freeze-dried yeast powder for more than 18 and 12 months when stored at 30 °C and 37 °C respectively. Stability of the heterologous protein remains unaffected during the process of heat-inactivation and lyophilization. The mass of lyophilized yeast powder remains almost unchanged during the entire period of storage and expressed protein remains intact even after two cycles of freeze and thaws. The protease-deficient strain appears ideal for the development of whole recombinant yeast-based vaccines. The cellular abundance of expressed antigen in dry powder after a year was comparable to freshly lyophilized cells. Scanning electron microscopy showed the intact nature of cells in powdered form even after a year of storage at 30 °C. Observation made in this study showed that freeze-dry yeast powder can play a vital role in the development of thermostable vaccines.Key Points• Yeast-based vaccines can overcome problem of cold chain associated with conventional vaccines• Lyophilized yeast powder can be a simple way for long-term storage of immunogen(s)• Protease deficient strain is important for whole recombinant yeast-based vaccines

Biochemical characterization of a unique DNA polymerase A from the extreme radioresistant organism Deinococcus radiodurans

Deinococcus radiodurans survives extraordinary doses of ionizing radiation and desiccation that cause numerous DNA strand breaks. D. radiodurans DNA polymerase A (DrPolA) is essential for reassembling the shattered genome, while its biochemical property has not been fully demonstrated. In this study, we systematically examined the enzymatic activities of DrPolA and characterized its unique features. DrPolA contains an N-terminal nuclease domain (DrPolA-NTD) and a C-terminal Klenow fragment (KlenDr). Compared with the Klenow fragment of E. coli Pol I, KlenDr shows higher fidelity despite the lacking of 3′–5′ exonuclease proofreading activity and prefers double-strand DNA rather than Primer-Template substrates. Apart from the well-annotated 5′–3′ exonuclease and flap endonuclease activities, DrPolA-NTD displays approximately 140-fold higher gap endonuclease activity than its homolog in E. coli and Human FEN1. Its 5′–3′ exonuclease activity on ssDNA, gap endonuclease, and Holliday junction cleavage activities are greatly enhanced by Mn2+. The DrPolA-NTD deficient strain shows increased sensitivity to UV and gamma-ray radiation. Collectively, our results reveal distinct biochemical characteristics of DrPolA during DNA degradation and re-synthesis, which provide new insight into the outstanding DNA repair capacity of D. radiodurans.

A novel Huntington's disease mouse model to assess the role of neuroinflammation on disease progression and to develop human cell therapies.

Huntington's disease (HD) is a fatal autosomal‐dominant neurodegenerative disease caused by a trinucleotide CAG repeat expansion of the huntingtin gene (HTT) that affects 1 in every 10 000 individuals in the United States. Our lab developed a novel immune deficient HD mouse strain, the YACNSG, from a commonly used line, the YAC128 mouse, to enable transplantation studies using engineered human cells in addition to studying the impact of the immune system on disease progression. The primary goal of this project was to characterize this novel immune deQficient HD mouse model, using behavioral assays and histology to compare this new model to the immune competent YAC128 and immune deficient mice that had engraftment of a human immune system. Flow cytometry was used to confirm that the YACNSG strain lacked immune cells, and in vivo imaging was used to assess human mesenchymal stem/stromal cell (MSC) retention compared with a commonly used immune deficient line, the NSG mouse. We found that YACNSG were able to retain human MSCs longer than the immune competent YAC128 mice. We performed behavioral assessments starting at 4 months of age and continued testing monthly until 12 months on the accelerod and in the open field. At 12 months, brains were isolated and evaluated using immunohistochemistry for striatal volume. Results from these studies suggest that the novel immune deficient YACNSG strain of mice could provide a good model for human stem‐cell based therapies and that the immune system appears to play an important role in the pathology of HD.

Enhancement of antibiotic production by co-cultivation of two antibiotic producing marine Vibrionaceae strains.

Deciphering the cues that stimulate microorganisms to produce their full secondary metabolic potential promises to speed up the discovery of novel drugs. Ecology-relevant conditions, including carbon-source(s) and microbial interactions, are important effectors of secondary metabolite production. Vice versa secondary metabolites are important mediators in microbial interactions, although their exact natural functions are not always completely understood. In this study, we investigated the effects of microbial interactions and in-culture produced antibiotics on the production of secondary metabolites by Vibrio coralliilyticus and Photobacterium galatheae, two co-occurring marine Vibrionaceae. In co-culture, production of andrimid by V. coralliilyticus and holomycin by P. galatheae, were, compared to monocultures, increased 4.3 and 2.7 fold, respectively. Co-cultures with the antibiotic deficient mutant strains (andrimid- and holomycin-) did not reveal a significant role for the competitor's antibiotic as stimulator of own secondary metabolite production. Furthermore, we observed that V. coralliilyticus detoxifies holomycin by sulphur-methylation. Results presented here indicate that ecological competition in Vibrionaceae is mediated by, and a cue for, antibiotic secondary metabolite production.

A red-letter day for RBC research

Hematology The study of primary human red blood cell (huRBC) disorders such as sickle cell disease (SCD) and infectious diseases such as malaria has been hampered by a lack of in vivo models of human erythropoiesis. Song et al. transferred human fetal liver cells into MISTRG mice, which are immunodeficient and are genetically engineered with several human genes involved in hematopoiesis. This approach was unsuccessful because mature huRBCs are rapidly destroyed in the mouse liver. They then used CRISPR-Cas9 to mutate these mice into a fumarylacetoacetate hydrolase–deficient strain, allowing them to replace the mouse liver with engrafted human hepatocytes. These mice exhibited enhanced human erythropoiesis and circulating huRBC survival and could recapitulate SCD pathology when reconstituted with SCD-derived HSCs. Science , this issue p. [1019][1] [1]: /lookup/doi/10.1126/science.abe2485

Study of quorum-sensing LasR and RhlR genes and their dependent virulence factors in Pseudomonas aeruginosa isolates from infected burn wounds

BackgroundPseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen responsible for burn-wound infection. High incidence, infection severity and increasing resistance characterize P. aeruginosa -induced burn infection.PurposeTo estimate quorum-sensing (QS)-dependent virulence factors of P. aeruginosa isolates from burn wounds and correlate it to the presence of QS genes.MethodsA cross-sectional descriptive study included 50 P . aeruginosa isolates from burn patients in Mansoura University Plastic and Burn Hospital, Egypt. Antibiotic sensitivity tests were done. All isolates were tested for their ability to produce biofilm using a micro-titration assay method. Protease, pyocyanin and rhamnolipid virulence factors were determined using skimmed milk agar, King’s A medium and CTAB agar test, respectively. The identity of QS lasR and rhlR genes was confirmed using PCR.Results In total, 86 % of isolates had proteolytic activity. Production of pyocyanin pigment was manifested in 66 % of isolates. Altogether, 76 % of isolates were rhamnolipid producers. Biofilm formation was detected in 96 % of isolates. QS lasR and rhlR genes were harboured by nearly all isolates except three isolates were negative for both lasR and rhlR genes and two isolates were positive for lasR gene and negative for rhlR gene. Forty-nine isolates were considered as extremely QS-proficient strains as they produced QS-dependent virulence factors. In contrast, one isolate was a QS deficient strain.ConclusionsQS affects P. aeruginosa virulence-factor production and biofilm in burn wounds. Isolates containing lasR and rhlR seem to be a crucial regulator of virulence factors and biofilm formation in P. aeruginosa whereas the lasR gene positively regulates biofilm formation, proteolytic activity, pyocyanin production and rhamnolipid biosurfactant synthesis. The QS regulatory RhlR gene affects protease and rhamnolipid production positively.

Development of epithelial cholinergic chemosensory cells of the urethra and trachea of mice

Cholinergic chemosensory cells (CCC) are infrequent epithelial cells with immunosensor function, positioned in mucosal epithelia preferentially near body entry sites in mammals including man. Given their adaptive capacity in response to infection and their role in combatting pathogens, we here addressed the time points of their initial emergence as well as their postnatal development from first exposure to environmental microbiota (i.e., birth) to adulthood in urethra and trachea, utilizing choline acetyltransferase (ChAT)-eGFP reporter mice, mice with genetic deletion of MyD88, toll-like receptor-2 (TLR2), TLR4, TLR2/TLR4, and germ-free mice. Appearance of CCC differs between the investigated organs. CCC of the trachea emerge during embryonic development at E18 and expand further after birth. Urethral CCC show gender diversity and appear first at P6-P10 in male and at P11-P20 in female mice. Urethrae and tracheae of MyD88- and TLR-deficient mice showed significantly fewer CCC in all four investigated deficient strains, with the effect being most prominent in the urethra. In germ-free mice, however, CCC numbers were not reduced, indicating that TLR2/4-MyD88 signaling, but not vita-PAMPs, governs CCC development. Collectively, our data show a marked postnatal expansion of CCC populations with distinct organ-specific features, including the relative impact of TLR2/4-MyD88 signaling. Strong dependency on this pathway (urethra) correlates with absence of CCC at birth and gender-specific initial development and expansion dynamics, whereas moderate dependency (trachea) coincides with presence of first CCC at E18 and sex-independent further development.

Cleaning the Cellular Factory-Deletion of McrA in Aspergillus oryzae NSAR1 and the Generation of a Novel Kojic Acid Deficient Strain for Cleaner Heterologous Production of Secondary Metabolites.

The use of filamentous fungi as cellular factories, where natural product pathways can be refactored and expressed in a host strain, continues to aid the field of natural product discovery. Much work has been done to develop host strains which are genetically tractable, and for which there are multiple selectable markers and controllable expression systems. To fully exploit these strains, it is beneficial to understand their natural metabolic capabilities, as such knowledge can rule out host metabolites from analysis of transgenic lines and highlight any potential interplay between endogenous and exogenous pathways. Additionally, once identified, the deletion of secondary metabolite pathways from host strains can simplify the detection and purification of heterologous compounds. To this end, secondary metabolite production in Aspergillus oryzae strain NSAR1 has been investigated via the deletion of the newly discovered negative regulator of secondary metabolism, mcrA (multicluster regulator A). In all ascomycetes previously studied mcrA deletion led to an increase in secondary metabolite production. Surprisingly, the only detectable phenotypic change in NSAR1 was a doubling in the yields of kojic acid, with no novel secondary metabolites produced. This supports the previous claim that secondary metabolite production has been repressed in A. oryzae and demonstrates that such repression is not McrA-mediated. Strain NSAR1 was then modified by employing CRISPR-Cas9 technology to disrupt the production of kojic acid, generating the novel strain NSARΔK, which combines the various beneficial traits of NSAR1 with a uniquely clean secondary metabolite background.