Functions of the Unique N-terminus of a GCN5 Histone Acetylase in Toxoplasma gondii

Indiana University-Purdue University Indianapolis (IUPUI)GCN5 is a histone acetyltransferase (HAT) that remodels chromatin by acetylating lysine residues of histones. The GCN5 HAT identified in Toxoplasma gondii (TgGCN5) contains a unique N-terminal “extension” that bears no similarity to known proteins and is devoid of known protein motifs. The hypothesis of this thesis is the N-terminal extension is critical to the function of TgGCN5. Three possible roles of the N-terminus were investigated: nuclear localization, protein-protein interactions, and substrate recognition. Subcellular localization was determined via immunocytochemistry using parasites expressing recombinant forms of TgGCN5 fused to a FLAG tag. Initial studies performed with parasites expressing full length FLAG-TgGCN5 were positive for nuclear localization. Without the N-terminal extension (FLAG-ΔNT-TgGCN5) the protein remains cytoplasmic. Additional studies mapped a six amino acid motif (RKRVKR) as the nuclear localization signal (NLS). When RKRVKR is fused to a cytoplasmic protein, it gains access to the nucleus. Furthermore, we have established the NLS interacts with Toxoplasma importin α, a protein involved in nuclear trafficking. Interaction with importin α provides evidence that the TgGCN5 N-terminal extension is involved in mediating protein-protein interactions. In order to identify additional interacting proteins, FLAG affinity purification was performed on parasites expressing full length FLAG-TgGCN5 and FLAG-ΔNT-TgGCN5. Upon comparing the results of the two purifications, proteins captured with only full length TgGCN5 may be interacting with the N-terminal extension. Full length TgGCN5 affinity purification indicates an interaction with histone proteins, two different homologues of Ada2 (adapter protein reported to interact with GCN5 homologues), and several heat shock proteins. With regard to substrate recognition, the N-terminal extension of TgGCN5 is dispensable for the acetylation of non-nucleosomal histones in vitro. However, the lysine acetylated by TgGCN5 is surprisingly unique. Other GCN5 homologues preferentially acetylate lysine 14 in histone H3, but TgGCN5 exclusively acetylates lysine 18 in histone H3 and has no activity on lysine 14. Taken together, these results argue that the N-terminal extension of TgGCN5 is critical for mediating protein-protein interactions, including those responsible for trafficking the HAT to the parasite nucleus but does not appear to be required for the acetylation of non-nucleosomal histones

Regions of Intrinsic Disorder Help Identify a Novel Nuclear Localization Signal in \u3cem\u3etoxoplasma Gondii\u3c/em\u3e Histone Acetyltransferase Tggcn5-b

We have previously shown that protozoan parasites, such as Toxoplasma gondii, contain a high prevalence of intrinsically disordered regions in their predicted proteins. Here, we determine that both TgGCN5-family histone acetyltransferases (HATs) contain unusually high levels of intrinsic disorder. A previously identified basic-rich nuclear localization signal (NLS) in the N-terminus of TgGCN5-A is located within such a region of predicted disorder, but this NLS is not conserved in TgGCN5-B. We therefore analyzed the intrinsically disordered regions of TgGCN5-B for basic-rich sequences that could be indicative of a functional NLS, and this led to the identification of a novel NLS for TgGCN5-B, RPAENKKRGR. The functionality of the GCN5-B NLS was validated experimentally and has predictive value. These studies demonstrate that basic-rich sequences within regions predicted to be intrinsically disordered constitute criteria for a candidate NLS

Erratum: evaluation of the performance of manual antimicrobial susceptibility testing methods and disk breakpoints for stenotrophomonas maltophilia

Volume 65, no. 5, e02631-20, 2021, https://doi-org.ezproxy.unbosque.edu.co/10.1128/AAC.02631-20. We found several typographical errors in our recently published article. The overarching conclusions for the paper remain the same, but some of the data should be changed numerically, as described herein. Table 3 should appear as shown below. The “Gradient strip performance” section in Results should read as follows: The performances of two brands of gradient strips were evaluated against BMD (Table 3). CLSI document M100 breakpoints were used for SXT, MIN, LEV, and CAZ, while EUCAST PK/PD breakpoints were used for CIP and TGC (Table 1). Etest performance met overall acceptance criteria for SXT, MIN, and LEV (Table 3). Overall values for CA with Etest for SXT, MIN, LEV, and CAZ were 99%, 93%, 81%, and 71%, respectively (Table 3). Etest for SXT yielded 1 VME within the acceptable error range for an isolate with an MIC at the breakpoint (4 mg/ml) by BMD. All SXT MEs were resolved with repeat testing. Etest for LEV yielded 1 VME, 18 MIs, and 5 MEs, 3 of which were resolved upon repeat testing. One ME was within 1 doubling dilution of the intermediate MIC breakpoint (Table 3). The majority of MIs (17/18) were within 1 doubling dilution of the intermediate breakpoint, while 1 had an MIC lower than 2 doubling dilutions of the intermediate breakpoint, yielding results that were in the acceptance range (1.7%) (Table 3). The LEV Etest yielded a more resistant result for 17 of the 18 MIs, calling 9 isolates as intermediate when they had a BMD at the susceptible breakpoint (2 mg/ml) (Table S2). Eight of the MIs were called resistant by the LEV Etest when they had a BMD MIC at the intermediate breakpoint (4mg/ml) (Table 3). Initial testing for MIN yielded 1 ME, which was resolved with repeat testing, 0 VME, and 8 MIs. All MI were intermediate by Etest but susceptible by BMD (Table S2). The CAZ Etest strip yielded 9 VMEs, 7 MEs, and 16 MIs, none of which resolved on repeat testing. Of these, 6 VMEs (17%), 1 ME (3%), and 13 MIs (37%) were within 1 doubling dilution of the intermediate breakpoint (Table 3). Six MEs were isolates with an MIC lower than 1 doubling dilution of the intermediate breakpoint (Table 3). The MIC test strip (MTS; Liofilchem, Roseto degli Abruzzi, Italy) performance met the acceptance criteria for SXT, LEV, and MIN (Table 3). Values for CA with MTS for SXT, MIN, LEV, and CAZ were 97%, 99%, 83%, and 72%, respectively. Initial testing with SXT yielded 3 ME, one which resolved with repeat testing, and 1 VME, which was within 1 doubling dilution of the susceptible breakpoint MIC (error rates of 4%) (Table 3). The (Table Presented) MIN MTS yielded 1 MI and no VMEs or MEs. The LEV MTS yielded 0 VME, 18 MIs, and 0 MEs (Table 3). Sixteen of the LEV MIs were within 1 doubling dilution of the intermediate breakpoint MIC. Eight MIs were susceptible by BMD and intermediate by MTS, 2 were resistant by BMD and intermediate by MTS, 5 were intermediate by BMD and resistant by MTS, and 3 were intermediate by BMD and susceptible by MTS (Table S2). Eleven out of 18 MIs had MICs within essential agreement between BMD and MTS. CAZ MTS did not have an acceptable performance (72% CA) and yielded 4 VMEs, 8 MEs, and 19 MIs (Table 3). Six of the MEs were MICs lower than 1 doubling dilution from the intermediate breakpoint (15% error rate), which fell outside the acceptable performance criteria (Table 3). Copyright © 2021 American Society for Microbiology. All Rights Reserved

Evaluation of the vitek 2, phoenix, and microscan for antimicrobial susceptibility testing of stenotrophomonas maltophilia

Stenotrophomonas maltophilia causa infecciones de alta mortalidad en huéspedes inmunocomprometidos con opciones terapéuticas limitadas. Muchos laboratorios de EE. UU. confían en las pruebas comerciales automatizadas de susceptibilidad a los antimicrobianos (cAST) y utilizan los puntos de corte CLSI (BP) para S. maltophilia. Sin embargo, faltan datos contemporáneos sobre estos sistemas. Evaluamos el rendimiento de Vitek 2, MicroScan WalkAway y Phoenix en relación con la microdilución en caldo de referencia para trimetoprima-sulfametoxazol (SXT), levofloxacina (LEV), minociclina (MIN) y ceftazidima (CAZ) con 109 aislados del torrente sanguíneo de S. maltophilia. Usando los puntos de corte CLSI, el acuerdo categórico (CA) estuvo por debajo del 90 % en todos los sistemas y medicamentos, con la excepción de SXT de MicroScan (98,1 %) y Phoenix (98,1 %) y MIN de MicroScan (100 %) y Phoenix (99,1 %). . Para SXT, Vitek 2 produjo un 77,1 % de CA. LEV y CAZ CA oscilaron entre 67% y 85%. Los errores muy importantes (VME) fueron del 3 % para SXT (MicroScan, Phoenix), LEV (MicroScan) y CAZ (todos los sistemas). Los errores principales (ME) fueron del 3 % para SXT (Vitek 2), LEV (Phoenix) y CAZ (MicroScan, Phoenix). Los errores menores fueron .10% para CAZ y LEV en todos los sistemas. Los datos se analizaron con los puntos de corte farmacocinéticos/farmacodinámicos CAZ, LEV, ciprofloxacina (CIP) y tigeciclina (TGC) de EUCAST cuando fue posible. CA fue, 90% para todos. VME fue del 3 % para CAZ (todos los sistemas), LEV (MicroScan) y TGC (Vitek 2), y ME fue del 3 % para LEV (MicroScan), CAZ (todos los sistemas), ciprofloxacina (Vitek 2 y MicroScan), y TGC (Vitek 2, Phoenix). Los errores menores (MI) fueron .10% para todos los agentes y sistemas, por puntos de corte de EUCAST con una categoría intermedia (LEV, CAZ, CIP). Los laboratorios deben tener cuidado con los cAST para S. maltophilia, ya que se puede observar una alta tasa de errores.Stenotrophomonas maltophilia causes high-mortality infections in immunocompromised hosts with limited therapeutic options. Many U.S. laboratories rely on commercial automated antimicrobial susceptibility tests (cASTs) and use CLSI breakpoints (BPs) for S. maltophilia. However, contemporary data on these systems are lacking. We assessed performance of Vitek 2, MicroScan WalkAway, and Phoenix relative to that of reference broth microdilution for trimethoprim-sulfamethoxazole (SXT), levofloxacin (LEV), minocycline (MIN), and ceftazidime (CAZ) with 109 S. maltophilia bloodstream isolates. Using CLSI breakpoints, categorical agreement (CA) was below 90% on all systems and drugs, with the exception of SXT by MicroScan (98.1%) and Phoenix (98.1%) and MIN by MicroScan (100%) and Phoenix (99.1%). For SXT, Vitek 2 yielded a 77.1% CA. LEV and CAZ CA ranged from 67% to 85%. Very major errors (VME) were.3% for SXT (MicroScan, Phoenix), LEV (MicroScan), and CAZ (all systems). Major errors (ME) were.3% for SXT (Vitek 2), LEV (Phoenix), and CAZ (MicroScan, Phoenix). Minor errors were.10% for CAZ and LEV on all systems. Data were analyzed with EUCAST pharmacokinetic/ pharmacodynamic CAZ, LEV, ciprofloxacin (CIP), and tigecycline (TGC) breakpoints when possible. CA was,90% for all. VME were.3% for CAZ (all systems), LEV (MicroScan), and TGC (Vitek 2), and ME were.3% for LEV (MicroScan), CAZ (all systems), ciprofloxacin (Vitek 2 and MicroScan), and TGC (Vitek 2, Phoenix). Minor errors (MI) were.10% for all agents and systems, by EUCAST breakpoints with an intermediate category (LEV, CAZ, CIP). Laboratories should use caution with cASTs for S. maltophilia, as a high rate of errors may be observed