H-NS family proteins, bacterial xenogeneic silencers, play central roles in genome organization and in the regulation of foreign genes. It is thought that gene repression is directly dependent on the DNA binding modes of H-NS family proteins. These proteins form lateral protofilaments along DNA. Under specific environmental conditions they switch to bridging two DNA duplexes. This switching is a direct effect of environmental conditions on electrostatic interactions between the oppositely charged DNA binding and N-terminal domains of H-NS proteins. The Pseudomonas lytic phage LUZ24 encodes the protein gp4, which modulates the DNA binding and function of the H-NS family protein MvaT of Pseudomonas aeruginosa. However, the mechanism by which gp4 affects MvaT activity remains elusive. In this study, we show that gp4 specifically interferes with the formation and stability of the bridged MvaT-DNA complex. Structural investigations suggest that gp4 acts as an 'electrostatic zipper' between the oppositely charged domains of MvaT protomers, and stabilizes a structure resembling their 'half-open' conformation, resulting in relief of gene silencing and adverse effects on P. aeruginosa growth. The ability to control H-NS conformation and thereby its impact on global gene regulation and growth might open new avenues to fight Pseudomonas multidrug resistance.
The immune-escape strategy employed by human oncogenic adenovirus type 12 (Ad12) involves downregulation of major histocompatibility complex class I (MHC-I) transcription by disabling the transactivator NF-kappaB (p50/p65). This is accomplished by the Ad12 E1A protein (E1A-12), which prevents NF-kappaB from becoming phosphorylated by the protein kinase A catalytic subunit (PKAc). In this study, we examined the interactions between E1A-12 and NF-kappaB. Our data show that an E1A-12 mutant retaining the N-terminal 66 amino acids was as effective as the wild-type E1A-12 protein (266 amino acids) in binding p65, preventing phosphorylation of p65-Ser(276), and inhibiting transactivation. In contrast, the nontumorigenic adenovirus type 5 E1A protein (E1A-5) and other E1A-12 mutants lacking the N-terminal regions were severely defective in these activities. Further studies revealed that an N-terminal peptide consisting of residues 1 to 40 of E1A-12 was able to associate directly with p65 in vitro and prevent PKAc from phosphorylating p65-Ser(276). In the absence of the N terminus, there is an almost complete loss of E1A-12 binding to p65. These findings provide solid evidence for the role of the E1A-12 N terminus as an NF-kappaB binding domain. Significantly, this study indicates that the E1A-12 N terminus prevents PKAc from gaining access to p65 to account for Ser(276) hypophosphorylation. The E1A-12 N terminus interaction with p65 serves as a key explanation of how Ad12 downregulates MHC-I transcription and contributes to oncogenesis by escaping cytotoxic T lymphocytes.
To determine whether the herpes simplex virus type 1 (HSV-1) viral glycoprotein C (gC) plays a role in induction of keratitis in unscarified and scarified rabbit eyes.A gC deletion mutant (DeltagC) was constructed and then rescued back to wild type (wt) for use as a control. Following ocular infection with each virus in rabbit eyes, with or without prior corneal scarification, keratitis was compared.At low infection doses of 2 x 10(3) and 2 x 10(4) plaque-forming units (PFU)/eye, in unscarified cornea, DeltagC produced significantly less keratitis than did wt virus (p = 0.007 and 0.03, respectively). In contrast, the keratitis induced by DeltagC was similar to that induced by the wt virus (p > 0.60) in scarified cornea. At high infection dose (2 x 10(5) PFU/eye), keratitis induced by DeltagC was similar in scarified and unscarified cornea, and the severity of disease was similar to that seen in scarified eyes at the low-dose DeltagC infections. Interestingly, although DeltagC induced keratitis with or without corneal scarification at high infection doses, the severity of disease was significantly less than that induced by wt infection. At all infection doses, keratitis induced by wt infection was similar in scarified and unscarified eyes.These results suggest that (1) at low infection doses, in unscarified corneas, gC is required for HSV-1 induced keratitis; (2) corneal scarification prior to infection can circumvent the need for gC at low doses, but (3) at higher doses, gC is required for wild-type levels of keratitis even in scarified cornea.
ABSTRACT The PhoQ/PhoP two-component system is repressed by divalent cations, such as Mg 2+ and Ca 2+ , in the growth medium and stimulated by low pH and certain cationic antimicrobial peptides. In Escherichia coli , it was recently shown that the histidine kinase PhoQ is also modulated by at least two additional factors, the small membrane proteins SafA and MgrB. This raises the possibility that the PhoQ/PhoP circuit has additional regulatory components and integrates additional input signals. We screened E. coli transposon insertion mutants to look for proteins that modulate the activity of the PhoQ/PhoP system, and we uncovered a role for DsbA, a periplasmic oxidant that facilitates the formation of disulfide bonds. Deletion of dsbA or dsbB , which maintains a pool of oxidized DsbA, leads to increased transcription of at least two PhoP-regulated genes. Addition of the reducing agent dithiothreitol to wild-type cells had a similar effect, and treatment of a dsbA null strain with the oxidant Cu 2+ rescued the reporter gene expression phenotype. We also demonstrated that expression of an MgrB mutant that lacked cysteines blocked the effect of a dsbA null mutation on PhoQ/PhoP activity, suggesting that MgrB acts downstream of DsbA in this pathway. Taken together, these results demonstrate that a decrease in the oxidizing activity of the periplasm stimulates PhoQ/PhoP and may reveal a new input stimulus for this important two-component system.
The H-NS-like proteins MvaT and MvaU act coordinately as global repressors in Pseudomonas aeruginosa by binding to AT-rich regions of the chromosome. Although cells can tolerate loss of either protein, identifying their combined regulatory effects has been challenging because the loss of both proteins is lethal due to induction of prophage Pf4 and subsequent superinfection of the cell. In other bacteria, H-NS promotes the cellular fitness by inhibiting intragenic transcription from AT-rich target regions, preventing them from sequestering RNA polymerase; however, it is not known whether MvaT and MvaU function similarly. Here, we utilize a parental strain that cannot be infected by Pf4 phage to define the collective MvaT and MvaU regulon and demonstrate that the combined loss of both MvaT and MvaU leads to increased intragenic transcription from loci directly controlled by these proteins. We further show that the loss of MvaT and MvaU leads to a striking redistribution of RNA polymerase containing σ70 to genomic regions vacated by these proteins. Our findings suggest that the ability of H-NS-like proteins to repress intragenic transcription is a universal function of these proteins and point to a second mechanism by which MvaT and MvaU may contribute to the growth of P. aeruginosa.
Abstract Background The H-NS-like proteins MvaT and MvaU act coordinately as global repressors in Pseudomonas aeruginosa by binding to AT-rich regions of the chromosome, which include horizontally acquired genes and numerous virulence factors. Although cells can tolerate the loss of either protein, identifying their combined regulatory effects has been challenging because the loss of both proteins is lethal due to induction of the prophage Pf4 and subsequent superinfection of the cell. Although in other bacteria, H-NS promotes cellular fitness by inhibiting intragenic transcription from AT-rich target regions, preventing them from sequestering RNA polymerase, a role for MvaT and MvaU in repressing transcription from intragenic promoters has not been demonstrated. Methods Here we utilize a parental strain that cannot be infected by Pf4 phage to identify the combined MvaT and MvaU regulon. RNA-seq was utilized to identify genes differentially expressed in cells lacking MvaU or both MvaU and MvaT. ChIP-seq was utilized to identify genes directly regulated by MvaT and MvaU in wild-type cells. Further, ChIP-seq was performed in cells of the parental strain and cells lacking both MvaT and MvaU to map genome-wide σ 70-dependent promoters that were active in the presence or absence of both H-NS-like proteins. Results We demonstrate that the loss of both MvaT and MvaU, but not MvaU alone, leads to increased sense, antisense, and intragenic transcription from loci directly controlled by these proteins. We further show that the loss of MvaT and MvaU leads to a striking redistribution of RNA polymerase containing σ 70 to those genomic regions vacated by these proteins. Loss of MvaT and MvaU causes global changes in gene expression Loss of MvaT and MvaU results in increased sense and antisense transcription Loss of both MvaT and MvaU results in genome-wide redistribution of RNA polymerase Conclusion Our findings suggest that the ability of H-NS-like proteins to repress intragenic transcription is a universal function of these proteins and describe a second mechanism by which MvaT and MvaU may contribute to the growth of P. aeruginosa. Disclosures All Authors: No reported disclosures
Sotrovimab 500 mg administered by a single intravenous (IV) infusion has been granted special approval for emergency use in Japan for treatment of SARS-CoV-2 infection in adults and children aged ≥ 12 years weighing ≥ 40 kg. This Phase 1, single-dose study investigated the pharmacokinetics, safety, and tolerability of IV or intramuscular (IM) sotrovimab 500 mg doses versus placebo in healthy Japanese and Caucasian volunteers. This was a two-part, Phase 1, randomized, placebo-controlled, single-blind study. In Part 1, participants received a single sotrovimab 500 mg IV infusion or matching placebo on Day 1. In Part 2, participants received a single sotrovimab 500 mg IM dose or matching placebo on Day 1, administered as two 4 mL injections. There was no effect of ethnicity on the peak or total serum exposure of IV sotrovimab through Week 18; after adjusting for body weight, the point estimate and 90 % confidence interval for the ratio of total exposure between Japanese and Caucasian participants fell within conventional bioavailability bounds (80–125%). Geometric mean Cmax and AUClast following a single IM administration of sotrovimab were higher in Japanese participants compared with Caucasian participants, even after adjustment for body weight. Overall, a single IV or IM dose of sotrovimab was well tolerated by both Japanese and Caucasian participants. After adjusting for body weight, exposures following a single IV dose of sotrovimab 500 mg were similar between Japanese and Caucasian participants, and higher in Japanese participants following IM administration. Higher exposures were not associated with any safety signals. ClinicalTrials.Gov: NCT04988152.
Abstract A 20‐year‐old male presented 3.5 years after intestinal transplantation with rapidly progressive sensorineural hearing loss. Initial brain imaging was consistent with inflammation and/or demyelination. Lumbar puncture was initially non‐diagnostic and a broad infectious workup was unrevealing. Three months after presentation, a repeat LP detected JC virus for which tests had not earlier been conducted. He continued to deteriorate despite withdrawal of prior immunosuppression and addition of mirtazapine, maraviroc, and steroids. He died of progressive neurologic decompensation 5 months after his initial presentation. This case highlights progressive multifocal leukoencephalopathy (PML) as a rare complication after solid organ transplantation and acute sensorineural hearing loss as an unusual first presenting symptom of PML. JC virus should be considered in the differential diagnosis of acute sensorineural hearing loss in any immunocompromised patient.
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