No, Is the Subject Area "Tetracyclines" applicable to this article? Shorter duration (24-hour) thioglycolate elicitation yielding peritoneal neutrophils showed genotypically similar results to macrophages (Fig 5D), and heat inactivation of complement by heating at 56°C for 30 minutes completely abrogated uptake in primary peritoneal neutrophils and RAW 264.7 macrophages (Fig 5D and 5E). The upper aqueous phase was transferred to a new 1-mL tube and 200 μL sterile water was added. Writing – review & editing, Affiliation Furthermore, serially diluting the serum present in macrophage uptake assays decreased bacterial uptake only at ≲1% serum. RAW 264.7 cells were passaged in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Thermo Fisher Scientific, Waltham, MA USA #11875135) supplemented with 10% Fetal Bovine Serum (FBS) (Atlanta Biologicals Inc, Flowery Branch, GA USA #S11150) at 37°C with 5% CO2 to a minimum of three and no more than 15 passages. We found that capsule extracted from gtr6-intact strains inhibited uptake of ATCC 17978 whereas capsule from gtr6-disrupted strains did not alter uptake of bacteria (Fig 4B). Formal analysis, Next, the plasmid pSC1 (S2D Fig) was isolated from JM101/pSC1 to enable amplification of the chimeric gtr6-hygromycin-FRT cassette using the primers Gtr6-Hyg 5 and Gtr6-Hyg 3 (S3 Text) and Phusion HotStart II DNA Polymerase as described previously. No, Is the Subject Area "Complement system" applicable to this article? Wide bars denote median, error bars denote IQR. Division of Infectious Diseases, Department of Medicine, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Veterans Administration, Buffalo, New York, United States of America, Roles (3A) Bacterial burden in the blood at 1-hour post-infection with 1.0 ×108 CFUs of ATCC 17978 WT and Δgtr6 (left) and NIH1 WT and Δgtr6 (right). The electrocompetent cells were transformed with 500 ng linearized PCR product using a BioRad Pulse Controller at 2.5 kV, 25 μF, and 200 Ω. For example, genetic lesions in capsule assembly genes resulting in an acapsular phenotype typically result in absence of strain virulence in vivo [9,10]. Yes RNA sequencing analysis of wild-type HUMC1 and HUMC1::gtr6 revealed no differential gene expression outside of the capsule locus (S1A Fig). We have identified a single glycosyltransferase gene in the capsule locus that was capable of significantly modifying virulence in A. baumannii. The tetracycline resistance gene cassette was amplified from the plasmid pBS-Tetr (S1 Text) using Q5 High-Fidelity Master Mix (NEB) using primer sets TetF and TetR (S3 Text). The ATCC 17978 Δgtr6 mutant showed almost identical CFU levels in the bacteria-only and bacteria plus macrophage groups compared to a decrease in the bacteria plus macrophage group with wild type ATCC 17978, suggesting that the entire Δgtr6 bacterial inoculum was present in the assay supernatant upon plating with no bacteria adherent to or sequestrated in the RAW 264.7 cells. This led us to evaluate complement deposition on the bacterial surface. Writing – review & editing, Affiliation Since the gtr6 neighborhood of A. baumannii strain 15827 has the identical sequence as that in HUMC1 and possess wild-type gtr6, purified Ab15827 DNA was used as template for amplification of gtr6, starting from the 5’ end of the ORF up to 100 bases flanking the 3’ end. Formal analysis, We previously found that A. baumannii strain 15827 was nonlethal at an inoculum of 2×108 CFU whereas HUMC1—which has an identical KL22 capsule locus except for the gtr6 disruption—was 100% lethal at an inoculum 10-fold lower [12]. Replicates were pooled and statistical significance was established via log-rank (Mantel-Cox) survival tests. 1,704 Likes, 64 Comments - Mitch Herbert (@mitchmherbert) on Instagram: âExcited to start this journey! ATCC 17978, a lab-adapted avirulent reference strain originally isolated from cerebrospinal fluid more than 50 years ago, is a KL3-type strain. Underline–first/last 126bp of the gtr6 ORF at the 5’ end. Previous work has described surface charge as being important in both promoting and inhibiting complement deposition on bacterial and artificial surfaces [26,27], and the higher acetylation in pgt1+ strains may be minimally protective against complement deposition via this model by having an overall higher negative surface charge than pgt1- strains. The Jackson Laboratory for Genomic Medicine, Farmington, Connecticut, United States of America, Affiliation (2C) RAW 264.7 cells were co-incubated with ATCC 17978 wild type, Δgtr6, Δgtr6/pSC1a (the knockout mutant with a plasmid-borne functional copy) in the presence of complement-active serum, and Δgtr6/pSC1a in the presence of heat-inactivated serum. A high dose rate of this radioisotope can be used in brachytherapy to treat tumors by selectively delivering a cytotoxic dose of radiation to the tumor site. Thus, a small amount of complement deposition on the bacterial surface is sufficient to mediate phagocytic uptake in vitro. https://doi.org/10.1371/journal.ppat.1009291, Editor: David S. Weiss, Emory University School of Medicine, UNITED STATES, Received: May 27, 2020; Accepted: January 7, 2021; Published: February 2, 2021. Yes To quantify total capsule carbohydrate content, bacterial cells were prepared as above and diluted to OD600 0.5 and plated to count CFUs. In contrast, pgt1 was present in strains that had both low uptake (HUMC1) and high uptake (15827 and NIH1), and could therefore not be principally responsible for phagocytic phenotype. Validation, Writing – review & editing, Roles All strains used in this study are described according to Kenyon classification capsule assembly locus type, genotype by gtr6 and pgt1, and relative phagocytic potential. 10 μL of the Gibson mix was transformed into electrocompetent E. coli JM101as described previously. Is the Subject Area "Phagocytosis" applicable to this article? Resources, Software, Cells were scraped from the wells using a pipette tip and 100uL of supernatant were plated for CFU measurement of internalized bacteria. Investigation, For all knockout mouse experiments, male mice aged 8–12 weeks on a C57BL/6 background (strain # 003991 for CR3 KO and # 012337 for Dectin-1) along with wild-type controls were purchased from The Jackson Laboratory. Thus, escape from innate immune effectors is a key driver of A. baumannii virulence. Investigation, For example, genetic lesions in capsule assembly genes resulting in an acapsular phenotype typically result in absence of strain virulence in vivo [9,10]. Furthermore, sub-inhibitory concentrations of chloramphenicol increase capsule thickness in A. baumannii, and increase both virulence and resistance to innate immune killing [11]. Flow cytometry revealed that C3b bound >40% of the gtr6+ ATCC 17978, >95% of an acapsular mutant (ATCC 17978 ΔitrA), and was almost undetectable on the gtr6− strain (ATCC 17978 Δgtr6). Methodology, Genomic DNA was extracted and 62 ng was used as template in a 25μL PCR reaction with outside primers 1128/1129 (S3 Text) (0.5nM each), dNTPs (0.2nM), 5% DMSO, GC buffer and Phusion Hotstart II DNA Polymerase (Thermo Scientific). In untreated mice, mortality primarily occurs via TLR-4 mediated toxicity and sepsis through the release of endogenous lipopolysaccharide (LPS), directly dependent upon bacterial density in the blood or lung [8]. Formal analysis, Formal analysis, 5 μL 20 μg/mL Proteinase K was then added and the solution was incubated at 56°C for 1 h. An equal volume of phenol-chloroform-isoamyl alcohol mix was added and the solution was vigorously vortexed for 30 s. The samples were centrifuged at 10,000 rpm for 15 min at 4°C and the aqueous phase was transferred to a new 1.7-mL tube. (A) Whole-genome sequencing of HUMC1 (a hypervirulent strain), 15827 (a hypovirulent strain) and ATCC 17978 (an avirulent strain) revealed distinct capsule loci organized into KL22 (HUMC1 and 15827) and KL3 (ATCC 17978) groups. 1×107 CFU were incubated with 10% complement-active mouse serum for 1 h at 37°C, washed three times with PBS, incubated with antibodies against mouse complement factor C3b (Thermo Fisher, clone 6C9) or an isotype control for 30 min, washed three times with PBS, and incubated with a secondary fluorescent antibody followed by three washes. The samples were spun at 10,000 rpm in a benchtop centrifuge at 4°C for 30 min, and resuspended in 50 μL sterile water. Electrocompetent E.coli JM101/pSIM5/pAT03 were prepared by growing the strain at 30°C to OD600 0.6–0.8 in 10 mL LB (chloramphenicol, carbenicillin). The plasmid pSC1 (S2E Fig) and the gtr6 indigenous promoter sequence (192 bp) were PCR amplified (S3 Text). Cell suspensions were plated on LB plates containing tetracycline at 20 μg/mL. The flippase encoding plasmid pAT03a (560ng) was transformed into HUMC1::gtr6-hygromycin via electroporation (100μL competent cells in a 0.2cm cuvette at 1.8 kV, 25 μF and 200 Ω). Laminarin, a branched 1,3- and 1,6-linked β-glucan fungal sugar, is known to bind a number of mammalian C-type lectins including Dectin-1 and Complement Receptor 3 (CR3) [15]. In summary, these results indicate that anti-virulence strategies specifically targeting the A. baumannii capsule or promoting complement deposition on bacteria (for example by antibody-based therapy) are promising means to prevent or treat serious infections caused by this deadly pathogen. Specifically, CR3 null macrophages mediated drastically less uptake than macrophages from wild type or Dectin-1 KO animals (Fig 5C). For phagocytosis assays, five images were taken per coverslip and all cells within each image were counted. The PCR conditions for hygromycin-FRT gene cassette were: initial denaturation at 98°C for 3 mins followed by 35 cycles of 96°C for 10 secs, 70°C for 30 secs, 72°C for 2 mins and the final extension was done at 72°C for 5 mins. e1009291. The gna gene, present in multiple strains including KL3 and KL22, is most likely responsible for the synthesis of GlcpNAcA from GlcpNAc while dgaA, dgaB, and dgaC (annotated as mnnA-C elsewhere) are responsible for the synthesis of GlcpNAc3NAcA [28], a modified side chain of which is present in both KL3 and KL22 but does not seem play a principal role in innate immune recognition given that both ATCC 17978 and HUMC1 contain this residue in their capsule structures (although its differential acetylation may play a minimal role as discussed above).
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