26 p.-6 fig.-3 tab., Antibiotic resistance is a global current threat of increasing importance. Moreover, biofilms represent a medical challenge since the inherent antibiotic resistance of their producers demands the use of high doses of antibiotics over prolonged periods. Frequently, these therapeutic measures fail, contributing to bacterial persistence, therefore demanding the development of novel antimicrobials. Esters of bicyclic amines (EBAs), which are strong inhibitors of Streptococcus pneumoniae growth, were initially designed as inhibitors of pneumococcal choline-binding proteins on the basis of their structural analogy to the choline residues in the cell wall. However, instead of mimicking the characteristic cell chaining phenotype caused by exogenously added choline on planktonic cultures of pneumococcal cells, EBAs showed an unexpected lytic activity. In this work we demonstrate that EBAs display a second, and even more important, function as cell membrane destabilizers. We then assayed the inhibitory and disintegrating activity of these molecules on pneumococcal biofilms. The selected compound (EBA 31) produced the highest effect on S. pneumoniae (encapsulated and non-encapsulated) biofilms at very low concentrations. EBA 31 was also effective on mixed biofilms of non-encapsulated S. pneumoniae plus non-typeable Haemophilus influenzae, two pathogens frequently forming a self-produced biofilm in the human nasopharynx. These results support the role of EBAs as a promising alternative for the development of novel, broad-range antimicrobial drugs encompassing both Gram-positive and Gram-negative pathogens., This work was supported by grants from the Spanish Ministry of Economy, Industry and Competitiveness (BIO2013-47684-R, BIO2016-79323-R and SAF2017-88664-R), and the RETICS-FEDER RICET RD16/0027/0010., Peer reviewed

12 p.-2 tab., Lower respiratory tract infections and tuberculosis are responsible for the death of about 4.5 million people each year and are the main causes of mortality in children under 5 years of age. Streptococcus pneumoniae is the most common bacterial pathogen associated with severe pneumonia, although other Gram-positive and Gram-negative bacteria are involved in respiratory infections as well. The ability of these pathogens to persist and produce infection under the appropriate conditions is also associated with their capacity to form biofilms in the respiratory mucous membranes. Adding to the difficulty of treating biofilm-forming bacteria with antibiotics, many of these strains are becoming multidrug resistant, and thus the alternative therapeutics available for combating this kind of infections are rapidly depleting. Given these concerns, it is urgent to consider other unconventional strategies and, in this regard, phage lysins represent an attractive resource to circumvent some of the current issues in infection treatment. When added exogenously, lysins break specific bonds of the peptidoglycan and have potent bactericidal effects against susceptible bacteria. These enzymes possess interesting features, including that they do not trigger an adverse immune response and raise of resistance is very unlikely. Although Gram-negative bacteria had been considered refractory to these compounds, strategies to overcome this drawback have been developed recently. In this review we describe the most relevant in vitro and in vivo results obtained to date with lysins against bacterial respiratory pathogens., The authors are supported by a grant from the Ministerio de Economía, Industria y Competitividad (MEICOM) (SAF2017-88664-R). Additional funding was provided by CIBER de Enfermedades Respiratorias (CIBERES), an initiative of the Instituto de Salud Carlos III (ISCIII)., Peer reviewed

7 pags., 1 fig., 2 tabs., Streptococcus pneumoniae is responsible for severe infections, causing millions of deaths yearly. Immunoglobulin G (IgG) antibodies against the capsular polysaccharide (CPS) offer S. pneumoniae serotype-specific protection. In this work, we examined the applicability of the microarray technology to detect CPS type-specific IgGs in serum, using a collection of 22 microarray-printed S. pneumoniae CPSs. First, printing of five CPSs onto nitrocellulose-coated glass slides was tested. Successful printing was only achieved for certain CPS types and concentrations. This behavior was tentatively related with diverse viscosities of the CPS solutions. Measurement of dynamic viscosities fully supported this assumption and helped to establish suitable CPS type-and concentration-dependent printing conditions. Next, the potential of CPS microarrays for detecting recognition by anti-CPS IgGs was examined using well-defined rabbit pneumococcal antisera. In all cases, the expected antiserum-CPS binding signals were detected, prompting a proof-of-concept analysis of human serum samples. Clearly distinct serum-and CPS-specific binding patterns and intensities were observed, evidencing selective detection of CPS type-specific IgGs. Compared to the ELISA assay commonly used to quantitate CPS type-specific IgGs in serum, the newly developed S. pneumoniae CPS microarrays offer the advantage of enabling the simultaneous analysis of multiple CPS-serum interactions using minute CPS amounts and significantly reduced serum volumes. Therefore, the approach could be particularly valuable for gauging the presence of CPS type-specific IgGs in human serum when sample volumes are limited and/or numerous serum samples are being examined., We gratefully acknowledge financial support from the Spanish
Ministry of Economy, Industry, and Competitiveness (Grants
BFU2015-70052-R and SAF2017-88664-R), the Spanish
Ministry of Science, Innovation, and Universities, the Spanish
State Research Agency, and the European Regional Development Fund (Grant RTI2018-099985-B-I00, MCIU/AEI/
FEDER, UE), and the CIBER of Respiratory Diseases
(CIBERES), an initiative from the Spanish Institute of Health
Carlos III (ISCIII).

16 p.-5 fig.-1 tab., The molecular basis of the pathogenesis of the opportunistic invasive infections caused by isolates of the Gemella genus remains largely unknown. Moreover, inconsistencies in the current species assignation were detected after genome-level comparison of 16 public Gemella isolates. A literature search detected that, between the two most pathogenic species, Gemella morbillorum causes about twice the number of cases compared to Gemella haemolysans. These two species shared their mean diseases - sepsis and endocarditis - but differed in causing other syndromes. A number of well-known virulence factors were harbored by all species, such as a manganese transport/adhesin sharing 83% identity from oral endocarditis-causing streptococci. Likewise, all Gemellae carried the genes required for incorporating phosphorylcholine into their cell walls and encoded some choline-binding proteins. In contrast, other proteins were species-specific, which may justify the known epidemiological differences. G. haemolysans, but not G. morbillorum, harbor a gene cluster potentially encoding a polysaccharidic capsule. Species-specific surface determinants also included Rib and MucBP repeats, hemoglobin-binding NEAT domains, peptidases of C5a complement factor and domains that recognize extracellular matrix molecules exposed in damaged heart valves, such as collagen and fibronectin. Surface virulence determinants were associated with several taxonomically dispersed opportunistic genera of the oral microbiota, such as Granulicatella, Parvimonas, and Streptococcus, suggesting the existence of a horizontally transferrable gene reservoir in the oral environment, likely facilitated by close proximity in biofilms and ultimately linked to endocarditis. The identification of the Gemella virulence pool should be implemented in whole genome-based protocols to rationally predict the pathogenic potential in ongoing clinical infections caused by these poorly known bacterial pathogens., This research was supported by grants MPY 509/19 from the Instituto de Salud Carlos III (ISCIII) and SAF2017-88664-R from the Spanish Ministerio de Economía, Industria y Competitividad (MEICOM)., Peer reviewed

23 p.-12 fig.-2 tab., Phage (endo)lysins are thought to be a viable alternative to usual antibiotic chemotherapy to fight resistant bacterial infections. However, a landscape view of lysins’ structure and properties regarding their function, with an applied focus, is somewhat lacking. Current literature suggests that specific features typical of lysins from phages infecting Gram-negative bacteria (G−) (higher net charge, amphipathic helices) are responsible for improved interaction with the G−envelope. Such antimicrobial peptide (AMP)-like elements are also of interest for antimicrobial molecules design. Thus, this study aims to provide an updated view on the primary structural landscape of phage lysins to clarify the evolutionary importance of several sequence-predicted properties, particularly for the interaction with the G−surface. A database of 2,182 lysin sequences was compiled, containing relevant information such as domain architectures, data on the phages’ host bacteria, and sequence
-predicted physicochemical properties. Based on such classifiers, an investigation of the differential appearance of certain features was conducted. Such analyses revealed different lysin architectural variants that are preferably found in phages infecting certain bacterial hosts. Particularly, some physicochemical properties (higher net charge,hydrophobicity, hydrophobic moment, and aliphatic index) were associated with G−phage lysins, appearing specifically at their C-terminal end. Evidence on the remarkable genetic specialization of lysins regarding the features of the bacterial hosts has been provided, specifically supporting the nowadays common hypothesis that lysins from G− usually contain AMP-like regions., This study was funded by a grant from the Ministerio de Economía y Competitividad (MINECO-FEDER, SAF2017-88664-R). Additional funding was provided by the Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), an initiative of the Instituto de Salud Carlos III., Peer reviewed

20 pags, 9 figs, 2 tabs. -- The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2021.740914/full#supplementary-material, We have structurally and functionally characterized Skl and Pal endolysins, the latter being the first endolysin shown to kill effectively Streptococcus pneumoniae, a leading cause of deathly diseases. We have proved that Skl and Pal are cysteine-amidases whose catalytic domains, from CHAP and Amidase_5 families, respectively, share an α3β6-fold with papain-like topology. Catalytic triads are identified (for the first time in Amidase_5 family), and residues relevant for substrate binding and catalysis inferred from in silico models, including a calcium-binding site accounting for Skl dependence on this cation for activity. Both endolysins contain a choline-binding domain (CBD) with a β-solenoid fold (homology modeled) and six conserved choline-binding loci whose saturation induced dimerization. Remarkably, Pal and Skl dimers display a common overall architecture, preserved in choline-bound dimers of pneumococcal lysins with other catalytic domains and bond specificities, as disclosed using small angle X-ray scattering (SAXS). Additionally, Skl is proved to be an efficient anti-pneumococcal agent that kills multi-resistant strains and clinical emergent-serotype isolates. Interestingly, Skl and Pal time-courses of pneumococcal lysis were sigmoidal, which might denote a limited access of both endolysins to target bonds at first stages of lysis. Furthermore, their DTT-mediated activation, of relevance for other cysteine-peptidases, cannot be solely ascribed to reversal of catalytic-cysteine oxidation., This work was supported by grants from the Ministry of Economy and Competitiveness (BFU2015-70072-R) and the Ministry of Science, Innovation and Universities (RTI2018-099985-B-I00/AEI/10.13039/501100011033) to MM, and by a grant from the Ministry of Economy and Competitiveness (MINECO-FEDER, SAF2017-88664-R) to PG. Additional funding was provided by the Centro de Investigacion Biomedica en Red de Enfermedades Respiratorias (CIBERES), an initiative of the Instituto de Salud Carlos III (ISCIII), to MM and PG., Peer reviewed

9 p.-3 fig., The recruitment and activation of polymorphonuclear neutrophils (PMNs) are of central importance for the elimination of pathogens in bacterial infections. We investigated the Streptococcus pneumoniae-dependent induction of the transcription factor Krüppel-like factor (KLF) 4 in PMNs as a potential regulator of PMN activation. We found that KLF4 expression is induced in human blood-derived PMNs in a time- and dose-dependent manner by wild-type S. pneumoniae and capsule knockout mutants. Unencapsulated knockout mutants induced stronger KLF4 expression than encapsulated wild types. The presence of autolysin LytA-competent (thus viable) pneumococci and LytA-mediated bacterial autolysis were required for KLF4 induction in human and murine PMNs. LyzMcre-mediated knockdown of KLF4 in murine blood-derived PMNs revealed that KLF4 influences pneumococci killing and increases the release of the proinflammatory cytokines tumor necrosis factor α and keratinocyte chemoattractant and decreases the release of the anti-inflammatory cytokine interleukin-10. Thus, S. pneumoniae induces KLF4 expression in PMNs, which contributes to PMN activation in S. pneumoniae infection., This work was supported by DFG SFB-TR84 grants to JZ, SH, and NS (projects C2, B1, and B6), Jürgen Manchot Stiftung to AB and TH and SAF2017-88664-R and CIBER de Enfermedades Respiratorias [an initiative of the Instituto de Salud Carlos III (ISCIII)] to PG. The publication was supported by the German Research Foundation (DFG) and the Open Access Publication Fund of Charité – Universitätsmedizin Berlin., Peer reviewed

14p.-7 fig.-2 tab., Phage (endo)lysins are nowadays one of the most promising ways out of the current antibiotic resistance crisis. Either as sole therapeutics or as a complement to common antibiotic chemotherapy, lysins are already entering late clinical phases to get regulatory agencies’ authorization. Even the old paradigm of the inability of lysins to attack Gram-negative bacteria from without has already been overcome in a variety of ways: either by engineering approaches or investigating the natural mechanisms by which some wild-type lysins are able to interact with the bacterial surface. Such inherent ability of some lysins has been linked to antimicrobial peptide (AMP)-like regions, which are, on their own, a significant source for novel antimicrobials. Currently, though, many of the efforts for searching novel lysin-based antimicrobial candidates rely on experimental screenings. In this work, we have bioinformatically analyzed the C-terminal end of a collection of lysins from phages infecting the Gram-negative genus Pseudomonas. Through the computation of physicochemical properties, the probability of such regions to be an AMP was estimated by means of a predictive k-nearest neighbors (kNN) model. This way, a subset of putatively membrane-interacting lysins was obtained from the original database. Two of such candidates (named Pae87 and Ppl65) were prospectively tested in terms of muralytic, bacteriolytic, and bactericidal activity. Both of them were found to possess an activity against Pseudomonas aeruginosa and other Gram-negative bacterial pathogens, implying that the prediction of AMP-like regions could be a useful approach toward the mining of phage lysins to design and develop antimicrobials or antimicrobial parts for further engineering., This work was funded by a grant from the Ministerio de Economía y Competitividad (MINECO-FEDER, SAF2017-88664-R). Additional funding was provided by the Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), an initiative of the Instituto de Salud Carlos III., Peer reviewed

21 p.-6 fig.-4 tab., Bacteriophages (phages) are viruses that infect bacteria. They are the most abundant biological entity on Earth (current estimates suggest there to be perhaps 1031 particles) and are found nearly everywhere. Temperate phages can integrate into the chromosome of their host, and prophages have been found in abundance in sequenced bacterial genomes. Prophages may modulate the virulence of their host in different ways, e.g., by the secretion of phage-encoded toxins or by mediating bacterial infectivity. Some 70% of Streptococcus pneumoniae (the pneumococcus)—a frequent cause of otitis media, pneumonia, bacteremia and meningitis—isolates harbor one or more prophages. In the present study, over 4000 S. pneumoniae genomes were examined for the presence of prophages, and nearly 90% were found to contain at least one prophage, either defective (47%) or present in full (43%). More than 7000 complete putative integrases, either of the tyrosine (6243) or serine (957) families, and 1210 full-sized endolysins (among them 1180 enzymes corresponding to 318 amino acid-long N-acetylmuramoyl-L-alanine amidases [LytAPPH]) were found. Based on their integration site, 26 different pneumococcal prophage groups were documented. Prophages coding for tRNAs, putative virulence factors and different methyltransferases were also detected. The members of one group of diverse prophages (PPH090) were found to integrate into the 3’ end of the host lytASpn gene encoding the major S. pneumoniae autolysin without disrupting it. The great similarity of the lytASpnand lytAPPH genes (85–92% identity) allowed them to recombine, via an apparent integrase-independent mechanism, to produce different DNA rearrangements within the pneumococcal chromosome. This study provides a complete dataset that can be used to further analyze pneumococcal prophages, their evolutionary relationships, and their role in the pathogenesis of pneumococcal disease., This research was supported by grants MPY 509/19 from the Instituto de Salud Carlos III (ISCIII) and SAF2017-88664-R from the Spanish Ministerio de Economía, Industria y Competitividad (MEICOM)., Peer reviewed

20 p.-11 fig.-1 tab., Phage lysins are a source of novel antimicrobials to tackle the bacterial antibiotic-resistance crisis. The engineering of phage lysins is being explored as a game-changing technological strategy to introduce a more precise approach in the way in which antimicrobial therapy is applied. Such engineering efforts will benefit from a better understanding of lysin structure and function. In this work, the antimicrobial activity of the endolysin from Pseudomonas aeruginosa phage JG004, termed Pae87, has been characterized. This lysin had previously been identified as an antimicrobial agent candidate that is able to interact with the Gram-negative surface and disrupt it. Further evidence is provided here based on a structural and biochemical study. A high-resolution crystal structure of Pae87 complexed with a peptidoglycan fragment showed a separate substrate-binding region within the catalytic domain, 18 Å away from the catalytic site and located on the opposite side of the lysin molecule. This substrate-binding region was conserved among phylogenetically related lysins lacking an additional cell-wall-binding domain, but not among those containing such a module. Two glutamic acids were identified to be relevant for the peptidoglycan-degradation activity, although the antimicrobial activity of Pae87 was seemingly unrelated. In contrast, an antimicrobial peptide-like region within the Pae87 C-terminus, named P87, was found to be able to actively disturb the outer membrane and display antibacterial activity by itself. Therefore, an antimicrobial mechanism for Pae87 is proposed in which the P87 peptide plays the role of binding to the outer membrane and disrupting the cell-wall function, either with or without the participation of the catalytic activity of Pae87., The authors acknowledge the Spanish Ministry of Science and Innovation for grant Nos. SAF2017-88664-R (to PG) and BFU2017-87022-P (to MJvR) funded by MCIN/AEI/10.13039/501100011033 and by ERDF A Way of Making Europe, and for the Severo Ochoa program to the CNB-CSIC (SEV 2017–0712). Additional funding was provided by the Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES) to PG, an initiative of the Instituto de Salud Carlos III., Peer reviewed

This article belongs to the Special Issue Bacteriophages-Based Technologies for a One Health Society: Applications in Clinical, Veterinary, and Industrial Settings., 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., This work was supported by Grant RED2018-102589-T funded by MCIN/AEI/10.13039/501100011033. R.V. and D.G. were supported by the Research Foundation–Flanders (FWO) under grant [G066919N]. Pe. G. and R.V. were supported by the Spanish Ministry of Science and Innovation (Grant number SAF2017-88664-R), with additional funding provided by the Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), an initiative of the Instituto de Salud Carlos III. P.D.-C. was financially supported by a Ramón y Cajal contract RYC2019-028015-I and project PID2020-112835RA-I00 funded by MCIN/AEI/10.13039/501100011033, ESF Invest in your future, project SEJIGENT/2021/014, funded by Conselleria d’Innovació, Universitats, Ciència i Societat Digital (Generalitat Valenciana), and project AP2020-27 funded by VLC-Biomed (UV-La Fe 2020 Programa Acciones Preparatorias). M.M. was supported by project PID2020-113355GB-I00 of Spanish Ministry of Science and Innovation /Agencia Estatal de Investigación (AEI)/European regional fund (ERF). M.Á.T.-M. was supported by Spanish Ministry of Science and Innovation (Grant number SAF2017- 82251-R)., Peer reviewed

12 pags., 6 figs., Bacteriophage-derived endolysins and bacterial autolysins (hereinafter lysins) represent a completely new class of efficient antibacterials. They prevent the development of bacterial resistance and help protect commensal microbiota, producing cell wall lysis. Here we have investigated whether the acquisition of enzymatic active domains (EADs) and cell wall binding domains (CWBDs) of balancing efficiencies could be a way of tuning natural lysin activity. The concept was applied to produce a chimeric lysin of superior antibacterial capacity using the endolysin Skl and the major pneumococcal autolysin LytA. Combination of the Skl EAD and the cell wall choline-binding domain (CBD) of LytA in the chimera QSLA increased the bacterial killing by 2 logs or more compared to parental enzymes at an equal concentration and extended the substrate range to resistant and emergent pneumococci and other pathogens of the mitis group. Contrarily, QLAS, containing LytA EAD and Skl CBD, was inactive against all tested strains, although domain structures were preserved and hydrolysis of purified cell walls maintained in both chimeras. As a whole, our study provides a novel clue to design superior lysins to fight multidrug-resistant pathogens based on domain selection, and a powerful in-vivo active lysin (QSLA) with promising therapeutic perspectives., This work was supported by grants from the Ministry of Economy and Competitiveness (BFU2015-70072-R) and the Ministry of Science, Innovation and Universities (RTI2018-099985-B-I00/AEI/10.13039/501100011033) to M. Menéndez, and by a grant from the Ministry of Economy and Competitiveness (MINECO-FEDER, SAF2017-88664-R) to P. García. Additional funding was provided by the “CIBER de Enfermedades Respiratorias” (CIBERES), an initiative of the Instituto de Salud Carlos III (ISCIII), to M. Menéndez and P. García., Peer reviewed