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In the current scenario of high antibiotic resistance, the search for therapeutic options against Pseudomonas aeruginosa must be approached from different perspectives: cell-wall biology as source of bacterial weak points and our immune system as source of weapons. Our recent study suggests that once the permeability barrier has been overcome, the activity of our cell-wall-targeting immune proteins is notably enhanced, more in mutants with impaired peptidoglycan recycling. The present work aims at analyzing the activity of these proteins [lysozyme and Peptidoglycan-Recognition-Proteins (PGLYRPs)], alone or with a permeabilizer (subinhibitory colistin) in clinical strains, along with other features related to the cell-wall. We compared the most relevant and complementary scenarios: acute (bacteremia) and chronic infections [early/late isolates from lungs of cystic fibrosis (CF) patients]. Although a low activity of lysozyme/PGLYRPs per se (except punctual highly susceptible strains) was found, the colistin addition significantly increased their activity regardless of the strains' colistin resistance levels. Our results show increased susceptibility in late CF isolates, suggesting that CF adaptation renders P. aeruginosa more vulnerable to proteins targeting the cell-wall. Thus, our work suggests that attacking some P. aeruginosa cell-wall biology-related elements to increase the activity of our innate weapons could be a promising therapeutic strategy.

In the current scenario of high antibiotic resistance, the search for therapeutic options against Pseudomonas aeruginosa must be approached from different perspectives: cell-wall biology as source of bacterial weak points and our immune system as source of weapons. Our recent study suggests that once the permeability barrier has been overcome, the activity of our cell-wall-targeting immune proteins is notably enhanced, more in mutants with impaired peptidoglycan recycling. The present work aims at analyzing the activity of these proteins [lysozyme and Peptidoglycan-Recognition-Proteins (PGLYRPs)], alone or with a permeabilizer (subinhibitory colistin) in clinical strains, along with other features related to the cell-wall. We compared the most relevant and complementary scenarios: acute (bacteremia) and chronic infections [early/late isolates from lungs of cystic fibrosis (CF) patients]. Although a low activity of lysozyme/PGLYRPs per se (except punctual highly susceptible strains) was found, the colistin addition significantly increased their activity regardless of the strains' colistin resistance levels. Our results show increased susceptibility in late CF isolates, suggesting that CF adaptation renders P. aeruginosa more vulnerable to proteins targeting the cell-wall. Thus, our work suggests that attacking some P. aeruginosa cell-wall biology-related elements to increase the activity of our innate weapons could be a promising therapeutic strategy., This work was supported by the Ministerio de Economia y Competitividad of Spain and the Instituto de Salud Carlos III, cofinanced by the European Regional Development Fund (ERDF; A way to achieve Europe) through the Spanish Network for the Research in Infectious Diseases (RD12/0015 and RD16/0016), and grants CP12/03324, PI15/00088, and PI15/02212. I.M.B. is funded by the project SOIB Jove: Qualificats Sector Public (JQ-SP 18/17), cofinanced by SOIB, Garantia Juvenil, and the European Social Fund. Authors thank Jonathan McFarland for his assistance in English corrections.

This review deeply addresses for the first time the diversity, regulation and mechanisms leading to mutational overexpression of intrinsic β-lactamases from non-fermenting and other non-Enterobacteriaceae Gram-negative opportunistic pathogens. After a general overview of the intrinsic β-lactamases described so far in these microorganisms, including circa. 60 species and 100 different enzymes, we review the wide array of regulatory pathways of these β-lactamases. They include diverse LysR-type regulators, which control the expression of β-lactamases from relevant nosocomial pathogens such as Pseudomonas aeruginosa or Stenothrophomonas maltophilia or two-component regulators, with special relevance in Aeromonas spp., along with other pathways. Likewise, the multiple mutational mechanisms leading to β-lactamase overexpression and β-lactam resistance development, including AmpD (N-acetyl-muramyl-L-alanine amidase), DacB (PBP4), MrcA (PPBP1A) and other PBPs, BlrAB (two-component regulator) or several lytic transglycosylases among others, are also described. Moreover, we address the growing evidence of a major interplay between β-lactamase regulation, peptidoglycan metabolism and virulence. Finally, we analyse recent works showing that blocking of peptidoglycan recycling (such as inhibition of NagZ or AmpG) might be useful to prevent and revert β-lactam resistance. Altogether, the provided information and the identified gaps should be valuable for guiding future strategies for combating multidrug-resistant Gram-negative pathogens.

13 p.-5 fig.3 tab. Martínez, Isidoro et al., Lower respiratory tract infections are among the top five leading causes of human death. Fighting these infections is therefore a world health priority. Searching for induced alterations in host gene expression shared by several relevant respiratory pathogens represents an alternative to identify new targets for wide-range host-oriented therapeutics. With this aim, alveolar macrophages were independently infected with three unrelated bacterial (Streptococcus pneumoniae, Klebsiella pneumoniae, and Staphylococcus aureus) and two dissimilar viral (respiratory syncytial virus and influenza A virus) respiratory pathogens, all of them highly relevant for human health. Cells were also activated with bacterial lipopolysaccharide (LPS) as a prototypical pathogen-associated molecular pattern. Patterns of differentially expressed cellular genes shared by the indicated pathogens were searched by microarray analysis. Most of the commonly up-regulated host genes were related to the innate immune response and/or apoptosis, with Toll-like, RIG-I-like and NOD-like receptors among the top 10 signaling pathways with over-expressed genes. These results identify new potential broad-spectrum targets to fight the important human infections caused by the bacteria and viruses studied here., Research activities in the participating laboratories received further funding from the following sources: Centro Nacional de Microbiología, ISCIII, PI15CIII/00024 and MINECO (SAF2015- 67033-R); Centro Nacional de Biotecnología, MINECO (BFU2014-57797-R); Hospital Universitari Germans Trias I Pujol, Spanish Society of Pneumology and Thoracic Surgery (SEPAR 054/2011); Departamento de Bioquímica y Biología Molecular I, MINECO (SAF2015-65307-R); Centro de Investigaciones Biológicas, MINECO (SAF2012-39444-C01/02); Fundación de Investigación Sanitaria de las Islas Baleares, MINECO (SAF2012-39841); Instituto de Agrobiotecnología, MINECO (SAF2015-66520-R); Instituto de Química Física Rocasolano, MINECO (BFU2015-70052-R) and the Marie Curie Initial Training Network GLYCOPHARM (PITN-GA- 2012-317297). Subprograma Estatal de Formación (BES-2013- 065355).

Klebsiella pneumoniae is an etiologic agent of community-acquired and nosocomial pneumonia. It has been shown that K. pneumoniae infections are characterized by reduced early inflammatory response. Recently our group has shown that K. pneumoniae dampens the activation of inflammatory responses by antagonizing the activation of the NF-kappa B canonical pathway. Our results revealed that K. pneumoniae capsule polysaccharide (CPS) was necessary but not sufficient to attenuate inflammation. To identify additional Klebsiella factors required to dampen inflammation, we standardized and applied a high-throughput gain-of-function screen to examine a Klebsiella transposon mutant library. We identified 114 mutants that triggered the activation of NF-kappa B. Two gene ontology categories accounted for half of the loci identified in the screening: metabolism and transport genes (32% of the mutants) and envelope-related genes (17%). Characterization of the mutants revealed that the lack of the enterobactin siderophore was linked to a reduced CPS expression, which in turn underlined the NF-kappa B activation induced by the mutant. The lipopolysaccharide (LPS) O-polysaccharide and the pullulanase (PulA) type 2 secretion system (T2SS) are required for full effectiveness of the immune evasion. Importantly, these factors do not play a redundant role. The fact that LPS O-polysaccharide and T2SS mutant-induced responses were dependent on TLR2-TLR4-MyD88 activation suggested that LPS O-polysaccharide and PulA perturbed Toll-like receptor (TLR)-dependent recognition of K. pneumoniae. Finally, we demonstrate that LPS O-polysaccharide and pulA mutants are attenuated in the pneumonia mouse model. We propose that LPS O-polysaccharide and PulA T2SS could be new targets for the design of new antimicrobials. Increasing TLR-governed defense responses might provide also selective alternatives for the management of K. pneumoniae pneumonia., This work was supported by a grant from ERA-Net Pathogenomic 2008 (to P. J. S. and J. A. B.), Marie Curie Career Integration Grant (U-KARE) PCIG13-GA-2013-618162, Biotechnology and Biological Sciences Research Council (BBSRC) Grant BB/L007223/1, and Queen's University Belfast start-up funds (to J. A. B.). The authors declare that they have no conflicts of interest with the contents of this article.; Recipient of a predoctoral fellowship from the Programa Nacional de Proyectos de Investigacion Fundamental (SAF2012-39841) del Ministerio de Ciencia e Innovacion.