Structure and function of Campylobacter jejuni polynucleotide phosphorylase (PNPase): Insights into the role of this RNase in pathogenicity.

Ribonucleases are in charge of the processing, degradation and quality control of all cellular transcripts, which makes them crucial factors in RNA regulation. This post-transcriptional regulation allows bacteria to promptly react to different stress conditions and growth phase transitions, and also to produce the required virulence factors in pathogenic bacteria. Campylobacter jejuni is the main responsible for human gastroenteritis in the world. In this foodborne pathogen, exoribonuclease PNPase (CjPNP) is essential for low-temperature cell survival, affects the synthesis of proteins involved in virulence and has an important role in swimming, cell adhesion/invasion ability, and chick colonization. Here we report the crystallographic structure of CjPNP, complemented with SAXS, which confirms the characteristic doughnut-shaped trimeric arrangement and evaluates domain arrangement and flexibility. Mutations in highly conserved residues were constructed to access their role in RNA degradation and polymerization. Surprisingly, we found two mutations that altered CjPNP into a protein that is only capable of degrading RNA even in conditions that favour polymerization. These findings will be important to develop new strategies to combat C. jejuni infections.

Increased Production of Pathogenic, Airborne Fungal Spores upon Exposure of a Soil Mycobiota to Chlorinated Aromatic Hydrocarbon Pollutants.

Organic pollutants are omnipresent and can penetrate all environmental niches. We evaluated the hypothesis that short-term (acute) exposure to aromatic hydrocarbon pollutants could increase the potential for fungal virulence. Specifically, we analyzed whether pentachlorophenol and triclosan pollution results in the production of airborne fungal spores with greater virulence than those derived from an unpolluted (Control) condition. Each pollutant altered the composition of the community of airborne spores compared to the control, favoring an increase in strains with in vivo infection capacity (the wax moth Galleria mellonella was used as an infection model). Fungi subsisting inside larvae at 72 h postinjection with airborne spore inocula collected in polluted and unpolluted conditions exhibited comparable diversity (mainly within Aspergillus fumigatus). Several virulent Aspergillus strains were isolated from larvae infected with the airborne spores produced in a polluted environment. Meanwhile, strains isolated from larvae injected with spores from the control, including one A. fumigatus strain, showed no virulence. Potential pathogenicity increased when two Aspergillus virulent strains were assembled, suggesting the existence of synergisms that impact pathogenicity. None of the observed taxonomic or functional traits could separate the virulent from the avirulent strains. Our study emphasizes pollution stress as a possible driver of phenotypic adaptations that increase Aspergillus pathogenicity, as well as the need to better understand the interplay between pollution and fungal virulence. IMPORTANCE Fungi colonizing soil and organic pollutants often meet. The consequences of this encounter constitute an outstanding question. We scrutinized the potential for virulence of airborne fungal spores produced under unpolluted and polluted scenarios. The airborne spores showed increased diversity of strains with higher infection capacity in Galleria mellonella whenever pollution is present. Inside the larvae injected with either airborne spore community, the surviving fungi demonstrated a similar diversity, mainly within Aspergillus fumigatus. However, the isolated Aspergillus strains greatly differ since virulence was only observed for those associated with a polluted environment. The interplay between pollution and fungal virulence still hides many unresolved questions, but the encounter is costly: pollution stress promotes phenotypic adaptations that may increase Aspergillus pathogenicity.

RNase R, a New Virulence Determinant of Streptococcus pneumoniae.

Pneumococcal infections have increasingly high mortality rates despite the availability of vaccines and antibiotics. Therefore, the identification of new virulence determinants and the understanding of the molecular mechanisms behind pathogenesis have become of paramount importance in the search of new targets for drug development. The exoribonuclease RNase R has been involved in virulence in a growing number of pathogens. In this work, we used Galleria mellonella as an infection model to demonstrate that the presence of RNase R increases the pneumococcus virulence. Larvae infected with the RNase R mutant show an increased expression level of antimicrobial peptides. Furthermore, they have a lower bacterial load in the hemolymph in the later stages of infection, leading to a higher survival rate of the larvae. Interestingly, pneumococci expressing RNase R show a sudden drop in bacterial numbers immediately after infection, resembling the eclipse phase observed after intravenous inoculation in mice. Concomitantly, we observed a lower number of mutant bacteria inside larval hemocytes and a higher susceptibility to oxidative stress when compared to the wild type. Together, our results indicate that RNase R is involved in the ability of pneumococci to evade the host immune response, probably by interfering with internalization and/or replication inside the larval hemocytes.

The nsp15 Nuclease as a Good Target to Combat SARS-CoV-2: Mechanism of Action and Its Inactivation with FDA-Approved Drugs.

The pandemic caused by SARS-CoV-2 is not over yet, despite all the efforts from the scientific community. Vaccination is a crucial weapon to fight this virus; however, we still urge the development of antivirals to reduce the severity and progression of the COVID-19 disease. For that, a deep understanding of the mechanisms involved in viral replication is necessary. nsp15 is an endoribonuclease critical for the degradation of viral polyuridine sequences that activate host immune sensors. This enzyme is known as one of the major interferon antagonists from SARS-CoV-2. In this work, a biochemical characterization of SARS-CoV-2 nsp15 was performed. We saw that nsp15 is active as a hexamer, and zinc can block its activity. The role of conserved residues from SARS-CoV-2 nsp15 was investigated, and N164 was found to be important for protein hexamerization and to contribute to the specificity to degrade uridines. Several chemical groups that impact the activity of this ribonuclease were also identified. Additionally, FDA-approved drugs with the capacity to inhibit the in vitro activity of nsp15 are reported in this work. This study is of utmost importance by adding highly valuable information that can be used for the development and rational design of therapeutic strategies.

New targets for drug design: importance of nsp14/nsp10 complex formation for the 3'-5' exoribonucleolytic activity on SARS-CoV-2.

SARS-CoV-2 virus has triggered a global pandemic with devastating consequences. The understanding of fundamental aspects of this virus is of extreme importance. In this work, we studied the viral ribonuclease nsp14, one of the most interferon antagonists from SARS-CoV-2. Nsp14 is a multifunctional protein with two distinct activities, an N-terminal 3'-to-5' exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), both critical for coronaviruses life cycle, indicating nsp14 as a prominent target for the development of antiviral drugs. In coronaviruses, nsp14 ExoN activity is stimulated through the interaction with the nsp10 protein. We have performed a biochemical characterization of nsp14-nsp10 complex from SARS-CoV-2. We confirm the 3'-5' exoribonuclease and MTase activities of nsp14 and the critical role of nsp10 in upregulating the nsp14 ExoN activity. Furthermore, we demonstrate that SARS-CoV-2 nsp14 N7-MTase activity is functionally independent of the ExoN activity and nsp10. A model from SARS-CoV-2 nsp14-nsp10 complex allowed mapping key nsp10 residues involved in this interaction. Our results show that a stable interaction between nsp10 and nsp14 is required for the nsp14-mediated ExoN activity of SARS-CoV-2. We studied the role of conserved DEDD catalytic residues of SARS-CoV-2 nsp14 ExoN. Our results show that motif I of ExoN domain is essential for the nsp14 function, contrasting to the functionality of these residues in other coronaviruses, which can have important implications regarding the specific pathogenesis of SARS-CoV-2. This work unraveled a basis for discovering inhibitors targeting specific amino acids in order to disrupt the assembly of this complex and interfere with coronaviruses replication.

Correction to: Isolation and Analysis of Bacterial Ribosomes Through Sucrose Gradient Ultracentrifugation.

This chapter was inadvertently published without including the author Cátia Bárria. The correct authorship for this chapter should have been Ricardo F. dos Santos, Cátia Bárria, Cecília M. Arraiano, and José M. Andrade. And the sentence before the final sentence in the acknowledgement section should have been printed as "R.F.dS. is recipient of an FCT Doctoral fellowship (PD/BD/105733/2014) and Cátia Bárria is recipient of a FCT Post-doctoral grant PTDC/BIA-BQM/28479/2017)". These corrections have been updated in the chapter.

Isolation and Analysis of Bacterial Ribosomes Through Sucrose Gradient Ultracentrifugation.

Ribosomes are large macromolecular complexes responsible for the translation process. During the course of ribosome biogenesis and protein synthesis, extra-ribosomal factors interact with the ribosome or its subunits to assist in these vital processes. Here we describe a method to isolate and analyze not only bacterial ribosomes but also their associated factors, providing insights into translation regulation. This detailed protocol allows the separation and monitoring of the ribosomal species and their interacting partners along a sucrose density gradient. Simultaneously, fractionation of the gradient allows for the recovery of 70S ribosomes and its subunits enabling a wide range of downstream applications. This protocol can be easily adapted to ribosome-related studies in other species or for separating other macromolecular complexes.

Pneumococcal RNase R globally impacts protein synthesis by regulating the amount of actively translating ribosomes.

Ribosomes are macromolecular machines that carry out protein synthesis. After each round of translation, ribosome recycling is essential for reinitiating protein synthesis. Ribosome recycling factor (RRF), together with elongation factor G (EF-G), catalyse the transient split of the 70S ribosome into subunits. This splitting is then stabilized by initiation factor 3 (IF3), which functions as an anti-association factor. The correct amount of these factors ensures the precise level of 70S ribosomes in the cell. RNase R is a highly conserved exoribonuclease involved in the 3' to 5' degradation of RNAs. In this work we show that pneumococcal RNase R directly controls the expression levels of frr, fusA and infC mRNAs, the corresponding transcripts of RRF, EF-G and IF3, respectively. We present evidences showing that accumulation of these factors leads to a decreased amount of 70S active particles, as demonstrated by the altered sucrose gradient ribosomal pattern in the RNase R mutant strain. Furthermore, the single deletion of RNase R is shown to have a global impact on protein synthesis and cell viability, leading to a ~50% reduction in bacterial CFU/ml. We believe that the fine-tuned regulation of these transcripts by RNase R is essential for maintaining the precise amount of active ribosomal complexes required for proper mRNA translation and thus we propose RNase R as a new auxiliary factor in ribosome reassociation. Considering the overall impact of RNase R on protein synthesis, one of the main targets of antibiotics, this enzyme may be a promising target for antimicrobial treatment.

Identification of temperature-sensitive mutations and characterization of thermolabile RNase II variants.

The RNase II family of ribonucleases is ubiquitous and critical for RNA metabolism. The rnb500 allele has been widely used for over 30 years; however, the underlying genetic changes which result in RNase II thermolabile activity remain unknown. Here, we combine molecular and biophysical studies to carry out an in vivo and in vitro investigation of RNase II mutation(s) that confer the rnb500 phenotype. Our findings indicate that RNase II thermolability is due to the Cys284Tyr mutation within the RNB domain, which abolishes activity by increasing protein kinetic instability at the nonpermissive temperature. These findings have important implications for the design of temperature-sensitive variants of other RNase II enzymes, namely those with yet unknown functions.

The Role of Ribonucleases and sRNAs in the Virulence of Foodborne Pathogens.

Contaminated food is the source of many severe infections in humans. Recent advances in food science have discovered new foodborne pathogens and progressed in characterizing their biology, life cycle, and infection processes. All this knowledge has been contributing to prevent food contamination, and to develop new therapeutics to treat the infections caused by these pathogens. RNA metabolism is a crucial biological process and has an enormous potential to offer new strategies to fight foodborne pathogens. In this review, we will summarize what is known about the role of bacterial ribonucleases and sRNAs in the virulence of several foodborne pathogens and how can we use that knowledge to prevent infection.

Ribonucleases, antisense RNAs and the control of bacterial plasmids.

In the last decade regulatory RNAs have emerged as powerful tools to regulate the expression of genes both in prokaryotes and in eukaryotes. RNases, by degrading these RNA molecules, control the right amount of regulatory RNAs, which is fundamental for an accurate regulation of gene expression in the cell. Remarkably the first antisense RNAs identified were plasmid-encoded and their detailed study was crucial for the understanding of prokaryotic antisense RNAs. In this review we highlight the role of RNases in the precise modulation of antisense RNAs that control plasmid replication, maintenance and transfer.

The role of RNase R in trans-translation and ribosomal quality control.

Gene expression not only depends on the rate of transcription but is also largely controlled at the post-transcriptional level. Translation rate and mRNA decay greatly influence the final protein levels. Surveillance mechanisms are essential to ensure the quality of the RNA and proteins produced. Trans-translation is one of the most important systems in the quality control of bacterial translation. This process guarantees the destruction of abnormal proteins and also leads to degradation of the respective defective RNAs through the action of Ribonuclease R (RNase R). This exoribonuclease hydrolyzes RNAs starting from their 3' end. Besides its involvement in trans-translation, RNase R also participates in the quality control of rRNA molecules involved in ribosomal biogenesis. RNase R is thus emerging as a key factor in ensuring translation accuracy. This review focuses on issues related to the quality control of translation, with special emphasis on the role of RNase R.

The role of RNases in the regulation of small RNAs.

Ribonucleases (RNases) are key factors in the control of biological processes, since they modulate the processing, degradation and quality control of RNAs. This review gives many illustrative examples of the role of RNases in the regulation of small RNAs (sRNAs). RNase E and PNPase have been shown to degrade the free pool of sRNAs. RNase E can also be recruited to cleave mRNAs when they are interacting with sRNAs. RNase III cleaves double-stranded structures, and can cut both the sRNA and its RNA target when they are hybridized. Overall, ribonucleases act as conductors in the control of sRNAs. Therefore, it is very important to further understand their role in the post-transcriptional control of gene expression.

Characterization of the RNase R association with ribosomes.

BACKGROUND: In this study we employed the TAP tag purification method coupled with mass spectrometry analysis to identify proteins that co-purify with Escherichia coli RNase R during exponential growth and after temperature downshift. RESULTS: Our initial results suggested that RNase R can interact with bacterial ribosomes. We subsequently confirmed this result using sucrose gradient ribosome profiling joined with western blot analysis. We found that RNase R co-migrates with the single 30S ribosomal subunits. Independent data involving RNase R in the rRNA quality control process allowed us to hypothesize that the RNase R connection with ribosomes has an important physiological role. CONCLUSIONS: This study leads us to conclude that RNase R can interact with ribosomal proteins and that this interaction may be a result of this enzyme involvement in the ribosome quality control.