Dataset Information

Structural Organizations of Q? and MS2 Phages Affect Capsid Protein Modifications by Oxidants Hypochlorous Acid and Peroxynitrite.

ABSTRACT: Pathogenic enteric viruses and bacteriophages such as Q? and MS2 are transmitted through the fecal-oral route. However, oxidants such as peroxynitrite (ONOOH) and hypochlorous acid (HClO) can prevent new infection by inactivating infectious viruses. Their virucidal effect is well recognized, and yet predicting the effects of oxidants on viruses is currently impossible because the detailed mechanisms of viral inactivation remain unclear. Our data show that ONOOH and HClO cross-linked the capsid proteins and RNA genomes of Q? and MS2 phages. Consistently, the capsids appeared intact by transmission electron microscopy (TEM) even when 99% of the phages were inactivated by oxidation. Moreover, a precise molecular study of the capsid proteins shows that ONOOH and HClO preferentially targeted capsid protein regions containing the oxidant-sensitive amino acid C, Y, or W. Interestingly, the interaction of these amino acids was a crucial parameter defining whether they would be modified by the addition of O, Cl, or NO2 or whether it induced the loss of the protein region detected by mass spectrometry, together suggesting potential sites for cross-link formation. Together, these data show that HClO and ONOOH consistently target oxidant-sensitive amino acids regardless of the structural organization of Q? and MS2, even though the phenotypes change as a function of the interaction with adjacent proteins/RNA. These data also indicate a potential novel mechanism of viral inactivation in which cross-linking may impair infectivity.

SUBMITTER: Bastin G

PROVIDER: S-EPMC7283501 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Structural Organizations of Qβ and MS2 Phages Affect Capsid Protein Modifications by Oxidants Hypochlorous Acid and Peroxynitrite.

Bastin Guillaume G Loison Pauline P Vernex-Loset Lionel L Dupire François F Challant Julie J Majou Didier D Boudaud Nicolas N Krier Gabriel G Gantzer Christophe C

Frontiers in microbiology 20200603

Pathogenic enteric viruses and bacteriophages such as Qβ and MS2 are transmitted through the fecal-oral route. However, oxidants such as peroxynitrite (ONOOH) and hypochlorous acid (HClO) can prevent new infection by inactivating infectious viruses. Their virucidal effect is well recognized, and yet predicting the effects of oxidants on viruses is currently impossible because the detailed mechanisms of viral inactivation remain unclear. Our data show that ONOOH and HClO cross-linked the capsid p ...[more]

PMID: 32582098

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Project description:Pseudomonas aeruginosa is a significant nosocomial pathogen and is associated with lung infections in cystic fibrosis (CF). Once established, P. aeruginosa infections persist and are rarely eradicated despite host immune cells producing antimicrobial oxidants, including hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). There is limited knowledge as to how P. aeruginosa senses, responds to, and protects itself against HOCl and HOSCN and the contribution of such responses to its success as a CF pathogen. To investigate the P. aeruginosa response to these oxidants, we screened 707 transposon mutants, with mutations in regulatory genes, for altered growth following HOCl exposure. We identified regulators of antibiotic resistance, methionine biosynthesis, catabolite repression, and PA14_07340, the homologue of the Escherichia coli HOCl-sensor RclR (30% identical), which are required for protection against HOCl. We have shown that RclR (PA14_07340) protects specifically against HOCl and HOSCN stress and responds to both oxidants by upregulating the expression of a putative peroxiredoxin, rclX (PA14_07355). Transcriptional analysis revealed that while there was specificity in the response to HOCl (231 genes upregulated) and HOSCN (105 genes upregulated), there was considerable overlap, with 74 genes upregulated by both oxidants. These included genes encoding the type 3 secretion system, sulfur and taurine transport, and the MexEF-OprN efflux pump. RclR coordinates part of the response to both oxidants, including upregulation of pyocyanin biosynthesis genes, and, in the presence of HOSCN, downregulation of chaperone genes. These data indicate that the P. aeruginosa response to HOCl and HOSCN is multifaceted, with RclR playing an essential role.IMPORTANCE The bacterial pathogen Pseudomonas aeruginosa causes devastating infections in immunocompromised hosts, including chronic lung infections in cystic fibrosis patients. To combat infection, the host's immune system produces the antimicrobial oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). Little is known about how P. aeruginosa responds to and survives attack from these oxidants. To address this, we carried out two approaches: a mutant screen and transcriptional study. We identified the P. aeruginosa transcriptional regulator, RclR, which responds specifically to HOCl and HOSCN stress and is essential for protection against both oxidants. We uncovered a link between the P. aeruginosa transcriptional response to these oxidants and physiological processes associated with pathogenicity, including antibiotic resistance and the type 3 secretion system.

Project description:Purpose: The aim of this study was to determine the transcriptional response of P. aeruginosa PA14 to HOCl and HOSCN stress, and determine genes dependent on RclR (PA14_07340) regulation. Methods: PA14 WT and ∆rclR exponential phase cultures were exposed to sub-lethal amounts of HOCl or HOSCN (2.2 or 0.8 mM, respectively), or untreated, and after 20 minutes of exposure, RNA was extracted and gene expression analysed by RNA-seq. Reads Per Kilobase per Million mapped reads (RPKM) were determined and the log2 fold change of the RPKM values of treated WT compared to untreated WT, or treated WT compared to treated ∆rclR was calculated. Genes with a log2 fold change of >1.5 were considered upregulated and genes with a log2 fold change of <-1.5 were considered downregulated. Results: In the WT strain, HOCl exposure resulted in upregulation of 231 genes and downregulation of 20 genes, and HOSCN exposure caused upregulation of 105 genes and downregulation of 16 genes. There was considerable overlap in the transcriptional response to HOCl and HOSCN, with 74 genes being induced in response to both oxidants. Genes upregulated by HOCl and HOSCN included those involved in sulphur and taurine transport, those encoding the type 3 secretion system, the MexEF-OprN drug efflux pump, and the putative peroxiredoxin RclX (PA14_07355). Comparison of the treated WT and treated ∆rclR strains revealed that in the presence of HOCl RclR upregulated 42 genes and downregulated 7 genes, and in the presence of HOSCN upregulated 132 genes and downregulated 213 genes. In the presence of both HOCl and HOSCN, RclR was required for upregulation of rclX as well as pyocyanin biosynthesis genes. In the presence of HOSCN, RclR downregulated a large number of genes, including those encoding chaperones. Conclusions: Our data have identified that the transcriptional response of P. aeruginosa to HOCl and HOSCN is multifaceted and appears to be linked to antibiotic resistance and the virulence factors pyocyanin and type 3 secretion. Determination of the regulon of the transcriptional regulator RclR after HOCl and HOSCN exposure suggests RclR has a role in coordinating the P. aeruginosa response to these oxidants.

Dataset Information

Structural Organizations of Q? and MS2 Phages Affect Capsid Protein Modifications by Oxidants Hypochlorous Acid and Peroxynitrite.

Publications

Structural Organizations of Qβ and MS2 Phages Affect Capsid Protein Modifications by Oxidants Hypochlorous Acid and Peroxynitrite.

Similar Datasets

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