Murine Ribonuclease 6 Limits Bacterial Dissemination During Experimental Urinary Tract Infection.

INTRODUCTION: The Ribonuclease (RNase) A superfamily encodes cationic antimicrobial proteins with potent microbicidal activity toward uropathogenic bacteria. Ribonuclease 6 (RNase6) is an evolutionarily conserved, leukocyte-derived antimicrobial peptide with potent microbicidal activity toward uropathogenic Escherichia coli (UPEC), the most common cause of bacterial urinary tract infections (UTI). In this study, we generated Rnase6 deficient mice to investigate the hypothesis that endogenous RNase 6 limits host susceptibility to UTI. METHODS: We generated a Rnase6EGFP knock-in allele to identify cellular sources of Rnase6 and determine the consequences of homozygous Rnase6 deletion on antimicrobial activity and UTI susceptibility. RESULTS: We identified monocytes and macrophages as the primary cellular sources of Rnase6 in bladders and kidneys of Rnase6EGFP/+ mice. Rnase6 deficiency (i.e., Rnase6EGFP/EGFP) resulted in increased upper urinary tract UPEC burden during experimental UTI, compared to Rnase6+/+ controls. UPEC displayed increased intracellular survival in Rnase6 deficient macrophages. CONCLUSION: Our findings establish that RNase6 prevents pyelonephritis by promoting intracellular UPEC killing in monocytes and macrophages and reinforce the overarching contributions of endogenous antimicrobial RNase A proteins to host UTI defense.

Association of symptom severity and cerebrospinal fluid alterations in recent onset psychosis in schizophrenia-spectrum disorders - An individual patient data meta-analysis.

Neuroinflammation and blood-cerebrospinal fluid barrier (BCB) disruption could be key elements in schizophrenia-spectrum disorders(SSDs) etiology and symptom modulation. We present the largest two-stage individual patient data (IPD) meta-analysis, investigating the association of BCB disruption and cerebrospinal fluid (CSF) alterations with symptom severity in first-episode psychosis (FEP) and recent onset psychotic disorder (ROP) individuals, with a focus on sex-related differences. Data was collected from PubMed and EMBASE databases. FEP, ROP and high-risk syndromes for psychosis IPD were included if routine basic CSF-diagnostics were reported. Risk of bias of the included studies was evaluated. Random-effects meta-analyses and mixed-effects linear regression models were employed to assess the impact of BCB alterations on symptom severity. Published (6 studies) and unpublished IPD from n = 531 individuals was included in the analyses. CSF was altered in 38.8 % of individuals. No significant differences in symptom severity were found between individuals with and without CSF alterations (SMD = -0.17, 95 %CI -0.55-0.22, p = 0.341). However, males with elevated CSF/serum albumin ratios or any CSF alteration had significantly higher positive symptom scores than those without alterations (SMD = 0.34, 95 %CI 0.05-0.64, p = 0.037 and SMD = 0.29, 95 %CI 0.17-0.41p = 0.005, respectively). Mixed-effects and simple regression models showed no association (p > 0.1) between CSF parameters and symptomatic outcomes. No interaction between sex and CSF parameters was found (p > 0.1). BCB disruption appears highly prevalent in early psychosis and could be involved in positive symptoms severity in males, indicating potential difficult-to-treat states. This work highlights the need for considering BCB breakdownand sex-related differences in SSDs clinical trials and treatment strategies.

Structural determinants for tRNA selective cleavage by RNase 2/EDN.

tRNA-derived fragments (tRFs) have emerged as key players of immunoregulation. Some RNase A superfamily members participate in the shaping of the tRFs population. By comparing wild-type and knockout macrophage cell lines, our previous work revealed that RNase 2 can selectively cleave tRNAs. Here, we confirm the in vitro protein cleavage pattern by screening of synthetic tRNAs, single-mutant variants, and anticodon-loop DNA/RNA hairpins. By sequencing of tRF products, we identified the cleavage selectivity of recombinant RNase 2 with base specificity at B1 (U/C) and B2 (A) sites, consistent with a previous cellular study. Lastly, protein-hairpin complexes were predicted by MD simulations. Results reveal the contribution of the α1, loop 3 and loop 4, and ß6 RNase 2 regions, where residues Arg36/Asn39/Gln40/Asn65/Arg68/Arg132 provide interactions, spanning from P-1 to P2 sites that are essential for anticodon loop recognition. Knowledge of RNase 2-specific tRFs generation might guide new therapeutic approaches for infectious and immune-related diseases.

A Common Polymorphism in RNASE6 Impacts Its Antimicrobial Activity toward Uropathogenic Escherichia coli.

Human Ribonuclease (RNase) 6 is a monocyte and macrophage-derived protein with potent antimicrobial activity toward uropathogenic bacteria. The RNASE6 gene is heterogeneous in humans due to the presence of single nucleotide polymorphisms (SNPs). RNASE6 rs1045922 is the most common non-synonymous SNP, resulting in a G to A substitution that determines an arginine (R) to glutamine (Q) transversion at position 66 in the protein sequence. By structural analysis we observed that R66Q substitution significantly reduces the positive electrostatic charge at the protein surface. Here, we generated both recombinant RNase 6-R66 and -Q66 protein variants and determined their antimicrobial activity toward uropathogenic Escherichia coli (UPEC), the most common cause of UTI. We found that the R66 variant, encoded by the major SNP rs1045922 allele, exhibited superior bactericidal activity in comparison to the Q66 variant. The higher bactericidal activity of R66 variant correlated with an increase in the protein lipopolysaccharide binding and bacterial agglutination abilities, while retaining the same enzymatic efficiency. These findings encourage further work to evaluate RNASE6 SNP distribution and its impact in UTI susceptibility.

Human Ribonuclease 6 Has a Protective Role during Experimental Urinary Tract Infection.

Mounting evidence suggests that antimicrobial peptides and proteins (AMPs) belonging to the RNase A superfamily have a critical role in defending the bladder and kidney from bacterial infection. RNase 6 has been identified as a potent, leukocyte-derived AMP, but its impact on urinary tract infection (UTI) in vivo has not been demonstrated. To test the functional role of human RNase 6, we generated RNASE6 transgenic mice and studied their susceptibility to experimental UTI. In addition, we generated bone marrow-derived macrophages to study the impact of RNase 6 on antimicrobial activity within a cellular context. When subjected to experimental UTI, RNASE6 transgenic mice developed reduced uropathogenic Escherichia coli (UPEC) burden, mucosal injury, and inflammation compared to non-transgenic controls. Monocytes and macrophages were the predominant cellular sources of RNase 6 during UTI, and RNASE6 transgenic macrophages were more proficient at intracellular UPEC killing than non-transgenic controls. Altogether, our findings indicate a protective role for human RNase 6 during experimental UTI.

Neural correlates of referential/persecutory delusions in schizophrenia: examination using fMRI and a virtual reality underground travel paradigm.

BACKGROUND: The brain functional correlates of delusions have been relatively little studied. However, a virtual reality paradigm simulating travel on the London Underground has been found to evoke referential ideation in both healthy subjects and patients with schizophrenia, making brain activations in response to such experiences potentially identifiable. METHOD: Ninety patients with schizophrenia/schizoaffective disorder and 28 healthy controls underwent functional magnetic resonance imaging while they viewed virtual reality versions of full and empty Barcelona Metro carriages. RESULTS: Compared to the empty condition, viewing the full carriage was associated with activations in the visual cortex, the cuneus and precuneus/posterior cingulate cortex, the inferior parietal cortex, the angular gyrus and parts of the middle and superior temporal cortex including the temporoparietal junction bilaterally. There were no significant differences in activation between groups. Nor were there activations associated with referentiality or presence of delusions generally in the patient group. However, patients with persecutory delusions showed a cluster of reduced activation compared to those without delusions in a region in the right temporal/occipital cortex. CONCLUSIONS: Performance of the metro task is associated with a widespread pattern of activations, which does not distinguish schizophrenic patients and controls, or show an association with referentiality or delusions in general. However, the finding of a cluster of reduced activation close to the right temporoparietal junction in patients with persecutory delusions specifically is of potential interest, as this region is believed to play a role in social cognition.

Exploring the RNase A scaffold to combine catalytic and antimicrobial activities. Structural characterization of RNase 3/1 chimeras.

Design of novel antibiotics to fight antimicrobial resistance is one of the first global health priorities. Novel protein-based strategies come out as alternative therapies. Based on the structure-function knowledge of the RNase A superfamily we have engineered a chimera that combines RNase 1 highest catalytic activity with RNase 3 unique antipathogen properties. A first construct (RNase 3/1-v1) was successfully designed with a catalytic activity 40-fold higher than RNase 3, but alas in detriment of its anti-pathogenic activity. Next, two new versions of the original chimeric protein were created showing improvement in the antimicrobial activity. Both second generation versions (RNases 3/1-v2 and -v3) incorporated a loop characteristic of RNase 3 (L7), associated to antimicrobial activity. Last, removal of an RNase 1 flexible loop (L1) in the third version enhanced its antimicrobial properties and catalytic efficiency. Here we solved the 3D structures of the three chimeras at atomic resolution by X-ray crystallography. Structural analysis outlined the key functional regions. Prediction by molecular docking of the protein chimera in complex with dinucleotides highlighted the contribution of the C-terminal region to shape the substrate binding cavity and determine the base selectivity and catalytic efficiency. Nonetheless, the structures that incorporated the key features related to RNase 3 antimicrobial activity retained the overall RNase 1 active site conformation together with the essential structural elements for binding to the human ribonuclease inhibitor (RNHI), ensuring non-cytotoxicity. Results will guide us in the design of the best RNase pharmacophore for anti-infective therapies.

Negative schizophrenic symptoms as prefrontal cortex dysfunction: Examination using a task measuring goal neglect.

BACKGROUND: The negative symptoms of schizophrenia have been proposed to reflect prefrontal cortex dysfunction. However, this proposal has not been consistently supported in functional imaging studies, which have also used executive tasks that may not capture key aspects of negative symptoms such as lack of volition. METHOD: Twenty-four DSM-5 schizophrenic patients with high negative symptoms (HNS), 25 with absent negative symptoms (ANS) and 30 healthy controls underwent fMRI during performance of the Computerized Multiple Elements Test (CMET), a task designed to measure poor organization of goal directed behaviour or 'goal neglect'. Negative symptoms were rated using the PANSS and the Clinical Assessment Interview for Negative Symptoms (CAINS). RESULTS: On whole brain analysis, the ANS patients showed no significant clusters of reduced activation compared to the healthy controls. In contrast, the HNS patients showed hypoactivation compared to the healthy controls in the left anterior frontal cortex, the right dorsolateral prefrontal cortex (DLPFC), the anterior insula bilaterally and the bilateral inferior parietal cortex. When compared to the ANS patients, the HNS patients showed reduced activation in the left anterior frontal cortex, the left DLPFC and the left inferior parietal cortex. After controlling for disorganization scores, differences remained in clusters in the left anterior frontal cortex and the bilateral inferior parietal cortex. CONCLUSIONS: This study provides evidence that reduced prefrontal activation, perhaps especially in the left anterior frontal cortex, is a brain functional correlate of negative symptoms in schizophrenia. The simultaneous finding of reduced inferior parietal cortex activation was unexpected, but could reflect this region's involvement in cognitive control, particularly the 'regulative' component of this.

Essential Role of Enzymatic Activity in the Leishmanicidal Mechanism of the Eosinophil Cationic Protein (RNase 3).

The recruitment of eosinophils into Leishmania lesions is frequently associated with a favorable evolution. A feasible effector for this process is eosinophil cationic protein (ECP, RNase 3), one of the main human eosinophil granule proteins, endowed with a broad spectrum of antimicrobial activity, including parasites. ECP was active on Leishmania promastigotes and axenic amastigotes (LC50's = 3 and 16 µM, respectively) but, in contrast to the irreversible membrane damage caused on bacteria and reproduced by its N-terminal peptides, it only induced a mild and transient plasma membrane destabilization on Leishmania donovani promastigotes. To assess the contribution of RNase activity to the overall leishmanicidal activity of ECP, parasites were challenged in parallel with a single-mutant version, ECP-H15A, devoid of RNase activity, that fully preserves the conformation and liposome permeabilization ability. ECP-H15A showed a similar uptake to ECP on promastigotes, but with higher LC50's (>25 µM) for both parasite stages. ECP-treated promastigotes showed a degraded RNA pattern, absent in ECP-H15A-treated samples. Moreover ECP, but not ECP-H15A, reduced more than 2-fold the parasite burden of infected macrophages. Altogether, our results suggest that ECP enters the Leishmania cytoplasm by an endocytic pathway, ultimately leading to RNA degradation as a key contribution to the leishmanicidal mechanism. Thus, ECP combines both membrane destabilization and enzymatic activities to effect parasite killing. Taken together, our data highlight the microbicidal versatility of ECP as an innate immunity component and support the development of cell-penetrating RNases as putative leishmanicidal agents.

Selective cleavage of ncRNA and antiviral activity by RNase2/EDN in THP1-induced macrophages.

RNase2 is the member of the RNaseA family most abundant in macrophages. Here, we knocked out RNase2 in THP-1 cells and analysed the response to Respiratory Syncytial Virus (RSV). RSV induced RNase2 expression, which significantly enhanced cell survival. Next, by cP-RNAseq sequencing, which amplifies the cyclic-phosphate endonuclease products, we analysed the ncRNA population. Among the ncRNAs accumulated in WT vs KO cells, we found mostly tRNA-derived fragments (tRFs) and second miRNAs. Differential sequence coverage identified tRFs from only few parental tRNAs, revealing a predominant cleavage at anticodon and D-loops at U/C (B1) and A (B2) sites. Selective tRNA cleavage was confirmed in vitro using the recombinant protein. Likewise, only few miRNAs were significantly more abundant in WT vs RNase2-KO cells. Complementarily, by screening of a tRF & tiRNA array, we identified an enriched population associated to RNase2 expression and RSV exposure. The results confirm the protein antiviral action and provide the first evidence of its cleavage selectivity on ncRNAs.

In Vivo Evaluation of ECP Peptide Analogues for the Treatment of Acinetobacter baumannii Infection.

Antimicrobial peptides (AMPs) are alternative therapeutics to traditional antibiotics against bacterial resistance. Our previous work identified an antimicrobial region at the N-terminus of the eosinophil cationic protein (ECP). Following structure-based analysis, a 30mer peptide (ECPep-L) was designed that combines antimicrobial action against Gram-negative species with lipopolysaccharides (LPS) binding and endotoxin-neutralization activities. Next, analogues that contain non-natural amino acids were designed to increase serum stability. Here, two analogues were selected for in vivo assays: the all-D version (ECPep-D) and the Arg to Orn version that incorporates a D-amino acid at position 2 (ECPep-2D-Orn). The peptide analogues retained high LPS-binding and anti-endotoxin activities. The peptides efficacy was tested in a murine acute infection model of Acinetobacter baumannii. Results highlighted a survival rate above 70% following a 3-day supervision with a single administration of ECPep-D. Moreover, in both ECPep-D and ECPep-2D-Orn peptide-treated groups, clinical symptoms improved significantly and the tissue infection was reduced to equivalent levels to mice treated with colistin, used as a last resort in the clinics. Moreover, treatment drastically reduced serum levels of TNF-α inflammation marker within the first 8 h. The present results support ECP-derived peptides as alternative candidates for the treatment of acute infections caused by Gram-negative bacteria.

Rationally Modified Antimicrobial Peptides from the N-Terminal Domain of Human RNase 3 Show Exceptional Serum Stability.

Multidrug resistance against conventional antibiotics poses an important threat to human health. In this context, antimicrobial peptides (AMPs) have been extensively studied for their antibacterial activity and promising results have been shown so far. However, AMPs tend to be rather vulnerable to protease degradation, which offsets their therapeutic appeal. Here, we demonstrate how replacing functional residues in the antimicrobial region of human RNase 3-also named eosinophil cationic protein-by non-natural amino acids increases stability in human serum. These changes were also shown to reduce the hemolytic effect of the peptides in general terms, whereas the antimicrobial activity was reasonably preserved. Digestion profiles enabled us to design new peptides with superior stability and lower toxicity that could become relevant candidates to reach clinical stages.

Correction: Lu et al. Weighted Gene Co-Expression Network Analysis Identifies Key Modules and Hub Genes Associated with Mycobacterial Infection of Human Macrophages. Antibiotics 2021, 10, 97.

The authors wish to make the following corrections to this paper [...].

Host Defence RNases as Antiviral Agents against Enveloped Single Stranded RNA Viruses.

Owing to the recent outbreak of Coronavirus Disease of 2019 (COVID-19), it is urgent to develop effective and safe drugs to treat the present pandemic and prevent other viral infections that might come in the future. Proteins from our own innate immune system can serve as ideal sources of novel drug candidates thanks to their safety and immune regulation versatility. Some host defense RNases equipped with antiviral activity have been reported over time. Here, we try to summarize the currently available information on human RNases that can target viral pathogens, with special focus on enveloped single-stranded RNA (ssRNA) viruses. Overall, host RNases can fight viruses by a combined multifaceted strategy, including the enzymatic target of the viral genome, recognition of virus unique patterns, immune modulation, control of stress granule formation, and induction of autophagy/apoptosis pathways. The review also includes a detailed description of representative enveloped ssRNA viruses and their strategies to interact with the host and evade immune recognition. For comparative purposes, we also provide an exhaustive revision of the currently approved or experimental antiviral drugs. Finally, we sum up the current perspectives of drug development to achieve successful eradication of viral infections.

Weighted Gene Co-Expression Network Analysis Identifies Key Modules and Hub Genes Associated with Mycobacterial Infection of Human Macrophages.

Tuberculosis (TB) is still a leading cause of death worldwide. Treatments remain unsatisfactory due to an incomplete understanding of the underlying host-pathogen interactions during infection. In the present study, weighted gene co-expression network analysis (WGCNA) was conducted to identify key macrophage modules and hub genes associated with mycobacterial infection. WGCNA was performed combining our own transcriptomic results using Mycobacterium aurum-infected human monocytic macrophages (THP1) with publicly accessible datasets obtained from three types of macrophages infected with seven different mycobacterial strains in various one-to-one combinations. A hierarchical clustering tree of 11,533 genes was built from 198 samples, and 47 distinct modules were revealed. We identified a module, consisting of 226 genes, which represented the common response of host macrophages to different mycobacterial infections that showed significant enrichment in innate immune stimulation, bacterial pattern recognition, and leukocyte chemotaxis. Moreover, by network analysis applied to the 74 genes with the best correlation with mycobacteria infection, we identified the top 10 hub-connecting genes: NAMPT, IRAK2, SOCS3, PTGS2, CCL20, IL1B, ZC3H12A, ABTB2, GFPT2, and ELOVL7. Interestingly, apart from the well-known Toll-like receptor and inflammation-associated genes, other genes may serve as novel TB diagnosis markers and potential therapeutic targets.

Structure-Based Design of an RNase Chimera for Antimicrobial Therapy.

Bacterial resistance to antibiotics urges the development of alternative therapies. Based on the structure-function of antimicrobial members of the RNase A superfamily, we have developed a hybrid enzyme. Within this family, RNase 1 exhibits the highest catalytic activity and the lowest cytotoxicity; in contrast, RNase 3 shows the highest bactericidal action, alas with a reduced catalytic activity. Starting from both parental proteins, we designed a first RNase 3/1-v1 chimera. The construct had a catalytic activity much higher than RNase 3, unfortunately without reaching an equivalent antimicrobial activity. Thus, two new versions were created with improved antimicrobial properties. Both of these versions (RNase 3/1-v2 and -v3) incorporated an antimicrobial loop characteristic of RNase 3, while a flexible RNase 1-specific loop was removed in the latest construct. RNase 3/1-v3 acquired both higher antimicrobial and catalytic activities than previous versions, while retaining the structural determinants for interaction with the RNase inhibitor and displaying non-significant cytotoxicity. Following, we tested the constructs' ability to eradicate macrophage intracellular infection and observed an enhanced ability in both RNase 3/1-v2 and v3. Interestingly, the inhibition of intracellular infection correlates with the variants' capacity to induce autophagy. We propose RNase 3/1-v3 chimera as a promising lead for applied therapeutics.

Human RNase3 immune modulation by catalytic-dependent and independent modes in a macrophage-cell line infection model.

The human RNase3 is a member of the RNaseA superfamily involved in host immunity. RNase3 is expressed by leukocytes and shows broad-spectrum antimicrobial activity. Together with a direct antimicrobial action, RNase3 exhibits immunomodulatory properties. Here, we have analysed the transcriptome of macrophages exposed to the wild-type protein and a catalytic-defective mutant (RNase3-H15A). The analysis of differently expressed genes (DEGs) in treated THP1-derived macrophages highlighted a common pro-inflammatory "core-response" independent of the protein ribonucleolytic activity. Network analysis identified the epidermal growth factor receptor (EGFR) as the main central regulatory protein. Expression of selected DEGs and MAPK phosphorylation were inhibited by an anti-EGFR antibody. Structural analysis suggested that RNase3 activates the EGFR pathway by direct interaction with the receptor. Besides, we identified a subset of DEGs related to the protein ribonucleolytic activity, characteristic of virus infection response. Transcriptome analysis revealed an early pro-inflammatory response, not associated to the protein catalytic activity, followed by a late activation in a ribonucleolytic-dependent manner. Next, we demonstrated that overexpression of macrophage endogenous RNase3 protects the cells against infection by Mycobacterium aurum and the human respiratory syncytial virus. Comparison of cell infection profiles in the presence of Erlotinib, an EGFR inhibitor, revealed that the receptor activation is required for the antibacterial but not for the antiviral protein action. Moreover, the DEGs related and unrelated to the protein catalytic activity are associated to the immune response to bacterial and viral infection, respectively. We conclude that RNase3 modulates the macrophage defence against infection in both catalytic-dependent and independent manners.

Magnetite Nanoparticles Functionalized with RNases against Intracellular Infection of Pseudomonas aeruginosa.

Current treatments against bacterial infections have severe limitations, mainly due to the emergence of resistance to conventional antibiotics. In the specific case of Pseudomonas aeruginosa strains, they have shown a number of resistance mechanisms to counter most antibiotics. Human secretory RNases from the RNase A superfamily are proteins involved in a wide variety of biological functions, including antimicrobial activity. The objective of this work was to explore the intracellular antimicrobial action of an RNase 3/1 hybrid protein that combines RNase 1 high catalytic and RNase 3 bactericidal activities. To achieve this, we immobilized the RNase 3/1 hybrid on Polyetheramine (PEA)-modified magnetite nanoparticles (MNPs). The obtained nanobioconjugates were tested in macrophage-derived THP-1 cells infected with Pseudomonas aeruginosa PAO1. The obtained results show high antimicrobial activity of the functionalized hybrid protein (MNP-RNase 3/1) against the intracellular growth of P. aeruginosa of the functionalized hybrid protein. Moreover, the immobilization of RNase 3/1 enhances its antimicrobial and cell-penetrating activities without generating any significant cell damage. Considering the observed antibacterial activity, the immobilization of the RNase A superfamily and derived proteins represents an innovative approach for the development of new strategies using nanoparticles to deliver antimicrobials that counteract P. aeruginosa intracellular infection.

Synergism between Host Defence Peptides and Antibiotics Against Bacterial Infections.

BACKGROUND: Antimicrobial resistance (AMR) to conventional antibiotics is becoming one of the main global health threats and novel alternative strategies are urging. Antimicrobial peptides (AMPs), once forgotten, are coming back into the scene as promising tools to overcome bacterial resistance. Recent findings have attracted attention to the potentiality of AMPs to work as antibiotic adjuvants. METHODS: In this review, we have tried to collect the currently available information on the mechanism of action of AMPs in synergy with other antimicrobial agents. In particular, we have focused on the mechanisms of action that mediate the inhibition of the emergence of bacterial resistance by AMPs. RESULTS AND CONCLUSION: We find in the literature many examples where AMPs can significantly reduce the antibiotic effective concentration. Mainly, the peptides work at the bacterial cell wall and thereby facilitate the drug access to its intracellular target. Complementarily, AMPs can also contribute to permeate the exopolysaccharide layer of biofilm communities, or even prevent bacterial adhesion and biofilm growth. Secondly, we find other peptides that can directly block the emergence of bacterial resistance mechanisms or interfere with the community quorum-sensing systems. Interestingly, the effective peptide concentrations for adjuvant activity and inhibition of bacterial resistance are much lower than the required for direct antimicrobial action. Finally, many AMPs expressed by innate immune cells are endowed with immunomodulatory properties and can participate in the host response against infection. Recent studies in animal models confirm that AMPs work as adjuvants at non-toxic concentrations and can be safely administrated for novel combined chemotherapies.