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Haemophilus influenzae is a human-adapted bacterial pathogen that causes airway infections. Bacterial and host elements associated with the fitness of H. influenzae within the host lung are not well understood. Here, we exploited the strength of in vivo -omic analyses to study host-microbe interactions during infection. We used in vivo transcriptome sequencing (RNA-seq) for genome-wide profiling of both host and bacterial gene expression during mouse lung infection. Profiling of murine lung gene expression upon infection showed upregulation of lung inflammatory response and ribosomal organization genes, and downregulation of cell adhesion and cytoskeleton genes. Transcriptomic analysis of bacteria recovered from bronchoalveolar lavage fluid samples from infected mice showed a significant metabolic rewiring during infection, which was highly different from that obtained upon bacterial in vitro growth in an artificial sputum medium suitable for H. influenzae. In vivo RNA-seq revealed upregulation of bacterial de novo purine biosynthesis, genes involved in non-aromatic amino acid biosynthesis, and part of the natural competence machinery. In contrast, the expression of genes involved in fatty acid and cell wall synthesis and lipooligosaccharide decoration was downregulated. Correlations between upregulated gene expression and mutant attenuation in vivo were established, as observed upon purH gene inactivation leading to purine auxotrophy. Likewise, the purine analogs 6-thioguanine and 6-mercaptopurine reduced H. influenzae viability in a dose-dependent manner. These data expand our understanding of H. influenzae requirements during infection. In particular, H. influenzae exploits purine nucleotide synthesis as a fitness determinant, raising the possibility of purine synthesis as an anti-H. influenzae target. IMPORTANCE In vivo -omic strategies offer great opportunities for increased understanding of host-pathogen interplay and for identification of therapeutic targets. Here, using transcriptome sequencing, we profiled host and pathogen gene expression during H. influenzae infection within the murine airways. Lung pro-inflammatory gene expression reprogramming was observed. Moreover, we uncovered bacterial metabolic requirements during infection. In particular, we identified purine synthesis as a key player, highlighting that H. influenzae may face restrictions in purine nucleotide availability within the host airways. Therefore, blocking this biosynthetic process may have therapeutic potential, as supported by the observed inhibitory effect of 6-thioguanine and 6-mercaptopurine on H. influenzae growth. Together, we present key outcomes and challenges for implementing in vivo -omics in bacterial airway pathogenesis. Our findings provide metabolic insights into H. influenzae infection biology, raising the possibility of purine synthesis as an anti-H. influenzae target and of purine analog repurposing as an antimicrobial strategy against this pathogen., Competing Interests: The authors declare no conflict of interest.

Expediting drug discovery to fight antibacterial resistance requires holistic approaches at system levels. In this study, we focused on the human-adapted pathogen Haemophilus influenzae, and by constructing a high-quality genome-scale metabolic model, we rationally identified new metabolic drug targets in this organism. Contextualization of available gene essentiality data within in silico predictions identified most genes involved in lipid metabolism as promising targets. We focused on the β-ketoacyl-acyl carrier protein synthase III FabH, responsible for catalyzing the first step in the FASII fatty acid synthesis pathway and feedback inhibition. Docking studies provided a plausible three-dimensional model of FabH in complex with the synthetic inhibitor 1-(5-(2-fluoro-5-(hydroxymethyl)phenyl)pyridin-2-yl)piperidine-4-acetic acid (FabHi). Validating our in silico predictions, FabHi reduced H. influenzae viability in a dose- and strain-dependent manner, and this inhibitory effect was independent of fabH gene expression levels. fabH allelic variation was observed among H. influenzae clinical isolates. Many of these polymorphisms, relevant for stabilization of the dimeric active form of FabH and/or activity, may modulate the inhibitory effect as part of a complex multifactorial process with the overall metabolic context emerging as a key factor tuning FabHi activity. Synergies with antibiotics were not observed and bacteria were not prone to develop resistance. Inhibitor administration during H. influenzae infection on a zebrafish septicemia infection model cleared bacteria without signs of host toxicity. Overall, we highlight the potential of H. influenzae metabolism as a source of drug targets, metabolic models as target-screening tools, and FASII targeting suitability to counteract this bacterial infection. IMPORTANCE Antimicrobial resistance drives the need of synergistically combined powerful computational tools and experimental work to accelerate target identification and drug development. Here, we present a high-quality metabolic model of H. influenzae and show its usefulness both as a computational framework for large experimental data set contextualization and as a tool to discover condition-independent drug targets. We focus on β-ketoacyl-acyl carrier protein synthase III FabH chemical inhibition by using a synthetic molecule with good synthetic and antimicrobial profiles that specifically binds to the active site. The mechanistic complexity of FabH inhibition may go beyond allelic variation, and the strain-dependent effect of the inhibitor tested supports the impact of metabolic context as a key factor driving bacterial cell behavior. Therefore, this study highlights the systematic metabolic evaluation of individual strains through computational frameworks to identify secondary metabolic hubs modulating drug response, which will facilitate establishing synergistic and/or more precise and robust antibacterial treatments.

Antibiotic resistance is one of the major challenges that humankind shall face in the short term. (Bacterio)phage therapy is a valuable therapeutic alternative to antibiotics and, although the concept is almost as old as the discovery of phages, its wide application was hindered in the West by the discovery and development of antibiotics in the mid-twentieth century. However, research on phage therapy is currently experiencing a renaissance due to the antimicrobial resistance problem. Some countries are already adopting new ad hoc regulations to favor the short-term implantation of phage therapy in clinical practice. In this regard, the Phage Therapy Work Group from FAGOMA (Spanish Network of Bacteriophages and Transducing Elements) recently contacted the Spanish Drugs and Medical Devices Agency (AEMPS) to promote the regulation of phage therapy in Spain. As a result, FAGOMA was asked to provide a general view on key issues regarding phage therapy legislation. This review comes as the culmination of the FAGOMA initiative and aims at appropriately informing the regulatory debate on phage therapy.

Zebrafish embryo tumor transplant models are widely utilized in cancer research. Compared with traditional murine models, the small size and transparency of zebrafish embryos combined with large clutch sizes that increase statistical power and cheap husbandry make them a cost-effective and versatile tool for in vivo drug discovery. However, the lack of a comprehensive analysis of key factors impacting the successful use of these models impedes the establishment of basic guidelines for systematic screening campaigns. Thus, we explored the following crucial factors: (i) user-independent inclusion criteria, focusing on sample homogeneity; (ii) metric definition for data analysis; (iii) tumor engraftment criteria; (iv) image analysis versus quantification of human cancer cells using qPCR (RNA and gDNA); (v) tumor implantation sites; (vi) compound distribution (intratumoral administration versus alternative inoculation sites); and (vii) efficacy (intratumoral microinjection versus compound solution in media). Based on these analyses and corresponding assessments, we propose the first roadmap for systematic drug discovery screening in zebrafish xenograft cancer models using a melanoma cell line as a case study. This study aims to help the wider cancer research community to consider the adoption of this versatile model for cancer drug screening projects.

Haemophilus influenzae has contributed to key bacterial genome sequencing hallmarks, as being not only the first bacterium to be genome-sequenced, but also starring the first genome-wide analysis of chromosomes directly transformed with DNA from a divergent genotype, and pioneering Tn-seq methodologies. Over the years, the phenomenal and constantly evolving development of -omic technologies applied to a whole range of biological questions of clinical relevance in the H. influenzae -host interplay, has greatly moved forward our understanding of this human-adapted pathogen, responsible for multiple acute and chronic infections of the respiratory tract. In this way, essential genes, virulence factors, pathoadaptive traits, and multi-layer gene expression regulatory networks with both genomic and epigenomic complexity levels are being elucidated. Likewise, the unstoppable increasing whole genome sequencing information underpinning H. influenzae great genomic plasticity, mainly when referring to non-capsulated strains, poses major challenges to understand the genomic basis of clinically relevant phenotypes and even more, to clearly highlight potential targets of clinical interest for diagnostic, therapeutic or vaccine development. We review here how genomic, transcriptomic, proteomic and metabolomic-based approaches are great contributors to our current understanding of the interactions between H. influenzae and the human airways, and point possible strategies to maximize their usefulness in the context of biomedical research and clinical needs on this human-adapted bacterial pathogen., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)

Chronic obstructive pulmonary disease (COPD) is characterized by abnormal inflammatory responses and impaired airway immunity, which provides an opportunistic platform for nontypeable Haemophilus influenzae (NTHi) infection. Clinical evidence supports that the COPD airways present increased concentrations of glucose, which may facilitate proliferation of pathogenic bacteria able to use glucose as a carbon source. NTHi metabolizes glucose through respiration-assisted fermentation, leading to the excretion of acetate, formate, and succinate. We hypothesized that such specialized glucose catabolism may be a pathoadaptive trait playing a pivotal role in the NTHi airway infection. To find out whether this is true, we engineered and characterized bacterial mutant strains impaired to produce acetate, formate, or succinate by inactivating the ackA , pflA , and frdA genes, respectively. While the inactivation of the pflA and frdA genes only had minimal physiological effects, the inactivation of the ackA gene affected acetate production and led to reduced bacterial growth, production of lactate under low oxygen tension, and bacterial attenuation in vivo . Moreover, bacterially produced acetate was able to stimulate the expression of inflammatory genes by cultured airway epithelial cells. These results back the notion that the COPD lung supports NTHi growth on glucose, enabling production of fermentative end products acting as immunometabolites at the site of infection. Thus, glucose catabolism may contribute not only to NTHi growth but also to bacterially driven airway inflammation. This information has important implications for developing nonantibiotic antimicrobials, given that airway glucose homeostasis modifying drugs could help prevent microbial infections associated with chronic lung disease.

Chronic obstructive pulmonary disease (COPD) is characterized by abnormal inflammation and impaired airway immunity, providing an opportunistic platform for nontypeable Haemophilus influenzae (NTHi) infection. In this context, therapies targeting not only overactive inflammation without significant adverse effects, but also infection are of interest. Increasing evidence suggests that polyphenols, plant secondary metabolites with anti-inflammatory and antimicrobial properties, may be protective. Here, a Cistus salviifolius plant extract containing quercetin, myricetin, and punicalagin was shown to reduce NTHi viability. Analysis of these polyphenols revealed that quercetin has a bactericidal effect on NTHi, does not display synergies, and that bacteria do not seem to develop resistance. Moreover, quercetin lowered NTHi airway epithelial invasion through a mechanism likely involving inhibition of Akt phosphorylation, and reduced the expression of bacterially-induced proinflammatory markers il-8 , cxcl-1 , il-6 , pde4b , and tnfα . We further tested quercetin's effect on NTHi murine pulmonary infection, showing a moderate reduction in bacterial counts and significantly reduced expression of proinflammatory genes, compared to untreated mice. Quercetin administration during NTHi infection on a zebrafish septicemia infection model system showed a bacterial clearing effect without signs of host toxicity. In conclusion, this study highlights the therapeutic potential of the xenohormetic molecule quercetin against NTHi infection.

Zebrafish ( Danio rerio ) is an ideal in vivo model to study a wide variety of human cancer types. In this review, we provide a comprehensive overview of zebrafish in the cancer drug discovery process, from (i) approaches to induce malignant tumors, (ii) techniques to monitor cancer progression, and (iii) strategies for compound administration to (iv) a compilation of the 355 existing case studies showing the impact of zebrafish models on cancer drug discovery, which cover a broad scope of scenarios. Finally, based on the current state-of-the-art analysis, this review presents some highlights about future directions using zebrafish in cancer drug discovery and the potential of this model as a prognostic tool in prospective clinical studies. Cancer Res; 78(21); 6048-58. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Aim: To test the synergistic effect of Cpl-711 endolysin and antibiotics for antipneumococcal activity., Materials & Methods: A combination of Cpl-711 and different antibiotics (amoxicillin, cefotaxime, levofloxacin and vancomycin) was tested in a checkerboard assay against several multidrug-resistant Streptococcus pneumoniae strains. Mouse and zebrafish models of pneumococcal sepsis were used to confirm the in vitro data., Results: The activity of Cpl-711 combined with amoxicillin or cefotaxime was synergistic in the bactericidal effect against a serotype 23F multiresistant clinical isolate of S. pneumoniae. Synergy between Cpl-711 and cefotaxime was validated using both mouse and zebrafish models., Conclusion: Combination of Cpl-711 and cefotaxime may help in the treatment of diseases caused by multiresistant pneumococcal strains.

Bacteriophage-borne lytic enzymes, also named lysins or enzybiotics, are efficient agents for the killing of bacterial pathogens. The colonization of the respiratory tract by Streptococcus pneumoniae is a prerequisite for the establishment of the infection process. Hence, we have evaluated the antibacterial activities of three different lysins against pneumococcal colonization using human nasopharyngeal and lung epithelial cells as well as a mouse model of nasopharyngeal colonization. The lysins tested were the wild-type Cpl-1, the engineered Cpl-7S, and the chimera Cpl-711. Moreover, we included amoxicillin as a comparator antibiotic. Human epithelial cells were infected with three different multidrug-resistant clinical isolates of S. pneumoniae followed by a single dose of the corresponding lysin. The antimicrobial activities of these lysins were also evaluated using a mouse nasopharyngeal carriage model. The exposure of the infected epithelial cells to Cpl-7S did not result in the killing of any of the pneumococcal strains investigated. However, the treatment with Cpl-1 or Cpl-711 increased the killing of S. pneumoniae organisms adhered to both types of human epithelial cells, with Cpl-711 being more effective than Cpl-1, at subinhibitory concentrations. In addition, a treatment with amoxicillin had no effect on reducing the carrier state, whereas mice treated by the intranasal route with Cpl-711 showed significantly reduced nasopharyngeal colonization, with no detection of bacterial load in 20 to 40% of the mice. This study indicates that Cpl-1 and Cpl-711 lysins might be promising antimicrobial candidates for therapy against pneumococcal colonization., (Copyright © 2018 American Society for Microbiology.)

The respiratory pathogen nontypeable Haemophilus influenzae (NTHi) is an important cause of acute exacerbation of chronic obstructive pulmonary disease (AECOPD) that requires efficient treatments. A previous screening for host genes differentially expressed upon NTHi infection identified sirtuin-1, which encodes a NAD-dependent deacetylase protective against emphysema and is activated by resveratrol. This polyphenol concomitantly reduces NTHi viability, therefore highlighting its therapeutic potential against NTHi infection at the COPD airway. In this study, resveratrol antimicrobial effect on NTHi was shown to be bacteriostatic and did not induce resistance development in vitro. Analysis of modulatory properties on the NTHi-host airway epithelial interplay showed that resveratrol modulates bacterial invasion but not subcellular location, reduces inflammation without targeting phosphodiesterase 4B gene expression, and dampens β defensin-2 gene expression in infected cells. Moreover, resveratrol therapeutics against NTHi was evaluated in vivo on mouse respiratory and zebrafish septicemia infection model systems, showing to decrease NTHi viability in a dose-dependent manner and reduce airway inflammation upon infection, and to have a significant bacterial clearing effect without signs of host toxicity, respectively. This study presents resveratrol as a therapeutic of particular translational significance due to the attractiveness of targeting both infection and overactive inflammation at the COPD airway.

Drug-loaded nanoparticles (NPs) can improve infection treatment by ensuring drug concentration at the right place within the therapeutic window. Poly(lactic-co-glycolic acid) (PLGA) NPs are able to enhance drug localization in target site and to sustainably release the entrapped molecule, reducing the secondary effects caused by systemic antibiotic administration. We have loaded auranofin, a gold compound traditionally used for treatment of rheumatoid arthritis, into PLGA NPs and their efficiency as antibacterial agent against two Gram-positive pathogens, Streptococcus pneumoniae and Streptococcus pyogenes was evaluated. Auranofin-PLGA NPs showed a strong bactericidal effect as cultures of multiresistant pneumococcal strains were practically sterilized after 6 h of treatment with such auranofin-NPs at 0.25 μM. Moreover, this potent bactericidal effect was also observed in S. pneumoniae and S. pyogenes biofilms, where the same concentration of auranofin-NPs was capable of decreasing the bacterial population about 4 logs more than free auranofin. These results were validated using a zebrafish embryo model demonstrating that treatment with auranofin loaded into NPs achieved a noticeable survival against pneumococcal infections. All these approaches displayed a clear superiority of loaded auranofin PLGA nanocarriers compared to free administration of the drug, which supports their potential application for the treatment of streptococcal infections.

A double approach was followed in the search of novel inhibitors of the surface choline-binding proteins (CBPs) of Streptococcus pneumoniae (pneumococcus) with antimicrobial properties. First, a library of 49 rationally-designed esters of alkyl amines was screened for their specific binding to CBPs. The best binders, being esters of bicyclic amines (EBAs), were then tested for their in vitro effect on pneumococcal growth and morphology. Second, the efficiency of EBA-induced CBP inhibition was enhanced about 45,000-fold by multivalency effects upon synthesizing a poly(propylene imine) dendrimer containing eight copies of an atropine derivative. Both approaches led to compounds that arrest bacterial growth, dramatically decrease cell viability, and exhibit a protection effect in animal disease models, demonstrating that the pneumococcal CBPs are adequate targets for the discovery of novel antimicrobials that overcome the currently increasing antimicrobial resistance issues., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Streptococcus pneumoniae is a major cause of life-threatening diseases worldwide. Here we provide an in-depth functional characterization of LytB, the peptidoglycan hydrolase responsible for physical separation of daughter cells. Identified herein as an N-acetylglucosaminidase, LytB is involved also in colonization and invasion of the nasopharynx, biofilm formation and evasion of host immunity as previously demonstrated. We have shown that LytB cleaves the GlcNAc-β-(1,4)-MurNAc glycosidic bond of peptidoglycan building units. The hydrolysis occurs at sites with fully acetylated GlcNAc moieties, with preference for uncross-linked muropeptides. The necessity of GlcN acetylation and the presence of a single acidic moiety (Glu585) essential for catalysis strongly suggest a substrate-assisted mechanism with anchimeric assistance of the acetamido group of GlcNAc moieties. Additionally, modelling of the catalytic region bound to a hexasaccharide tripentapeptide provided insights into substrate-binding subsites and peptidoglycan recognition. Besides, cell-wall digestion products and solubilisation rates might indicate a tight control of LytB activity to prevent unrestrained breakdown of the cell wall. Choline-independent localization at the poles of the cell, mediated by the choline-binding domain, peptidoglycan modification, and choline-mediated (lipo)teichoic-acid attachment contribute to the high selectivity of LytB. Moreover, so far unknown chitin hydrolase and glycosyltransferase activities were detected using GlcNAc oligomers as substrate.

Background: Auranofin is an FDA-approved, gold-containing compound in clinical use for the oral treatment of rheumatoid arthritis and has been recently granted by the regulatory authorities due to its antiprotozoal properties., Methods: A reprofiling strategy was performed with a Streptococcus pneumoniae phenotypic screen and a proprietary library of compounds, consisting of both FDA-approved and unapproved bioactive compounds. Two different multiresistant S. pneumoniae strains were employed in a sepsis mouse model of infection. In addition, an MRSA strain was tested using both the thigh model and a mesh-associated biofilm infection in mice., Results: The repurposing approach showed the high potency of auranofin against multiresistant clinical isolates of S. pneumoniae and Staphylococcus aureus in vitro and in vivo. Efficacy in the S. pneumoniae sepsis model was obtained using auranofin by the oral route in the dose ranges used for the treatment of rheumatoid arthritis. Thioglucose replacement by alkyl chains showed that this moiety was not essential for the antibacterial activity and led to the discovery of a new gold derivative (MH05) with remarkable activity in vitro and in vivo., Conclusions: Auranofin and the new gold derivative MH05 showed encouraging in vivo activity against multiresistant clinical isolates of S. pneumoniae and S. aureus. The clinical management of auranofin, alone or in combination with other antibiotics, deserves further exploration before use in patients presenting therapeutic failure caused by infections with multiresistant Gram-positive pathogens. Decades of clinical use mean that this compound is safe to use and may accelerate its evaluation in humans., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objectives: Streptococcus pneumoniae is becoming increasingly antibiotic resistant worldwide and new antimicrobials are urgently needed. Our aim was new chimeric phage endolysins, or lysins, with improved bactericidal activity by swapping the structural components of two pneumococcal phage lysozymes: Cpl-1 (the best lysin tested to date) and Cpl-7S., Methods: The bactericidal effects of four new chimeric lysins were checked against several bacteria. The purified enzymes were added at different concentrations to resuspended bacteria and viable cells were measured after 1 h. Killing capacity of the most active lysin, Cpl-711, was tested in a mouse bacteraemia model, following mouse survival after injecting different amounts (25-500 μg) of enzyme. The capacity of Cpl-711 to reduce pneumococcal biofilm formation was also studied., Results: The chimera Cpl-711 substantially improved the killing activity of the parental phage lysozymes, Cpl-1 and Cpl-7S, against pneumococcal bacteria, including multiresistant strains. Specifically, 5 μg/mL Cpl-711 killed ≥7.5 log of pneumococcal R6 strain. Cpl-711 also reduced pneumococcal biofilm formation and killed 4 log of the bacterial population at 1 μg/mL. Mice challenged intraperitoneally with D39_IU pneumococcal strain were protected by treatment with a single intraperitoneal injection of Cpl-711 1 h later, resulting in about 50% greater protection than with Cpl-1., Conclusions: Domain swapping among phage lysins allows the construction of new chimeric enzymes with high bactericidal activity and a different substrate range. Cpl-711, the most powerful endolysin against pneumococci, offers a promising therapeutic perspective for the treatment of multiresistant pneumococcal infections., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Phage endolysins are murein hydrolases that break the bacterial cell wall to provoke lysis and release of phage progeny. Recently, these enzymes have also been recognized as powerful and specific antibacterial agents when added exogenously. In the pneumococcal system, most cell wall associated murein hydrolases reported so far depend on choline for activity, and Cpl-7 lysozyme constitutes a remarkable exception. Here, we report the improvement of the killing activity of the Cpl-7 endolysin by inversion of the sign of the charge of the cell wall-binding module (from -14.93 to +3.0 at neutral pH). The engineered variant, Cpl-7S, has 15 amino acid substitutions and an improved lytic activity against Streptococcus pneumoniae (including multiresistant strains), Streptococcus pyogenes, and other pathogens. Moreover, we have demonstrated that a single 25-μg dose of Cpl-7S significantly increased the survival rate of zebrafish embryos infected with S. pneumoniae or S. pyogenes, confirming the killing effect of Cpl-7S in vivo. Interestingly, Cpl-7S, in combination with 0.01% carvacrol (an essential oil), was also found to efficiently kill Gram-negative bacteria such as Escherichia coli and Pseudomonas putida, an effect not described previously. Our findings provide a strategy to improve the lytic activity of phage endolysins based on facilitating their pass through the negatively charged bacterial envelope, and thereby their interaction with the cell wall target, by modulating the net charge of the cell wall-binding modules.

The emergence of Streptococcus pneumoniae strains displaying high levels of multidrug resistance is of great concern worldwide and a serious threat for the outcome of the infection. Modifications of the bacterial envelope by antibiotics may assist the recognition and clearance of the pathogen by the host immune system. Recognition of S. pneumoniae resistant strains by the complement component C3b was increased in the presence of specific anti-pneumococcal antibodies and subinhibitory concentrations of different macrolides and β-lactam antibiotics for all the strains investigated. However, C3b levels were unchanged in the presence of serum containing specific antibodies and sub-MICs of levofloxacin. To investigate whether LytA, the main cell wall hydrolase of S. pneumoniae, might be involved in this process, lytA-deficient mutants were constructed. In the presence of antibiotics, loss of LytA was not associated with enhanced C3b deposition on the pneumococcal surface, which confirms the importance of LytA in this interaction. The results of this study offer new insights into the development of novel therapeutic strategies using certain antibiotics by increasing the efficacy of the host immune response to efficiently recognize pneumococcal resistant strains.