Novel capsular depolymerases-based strategy to kill multidrug-resistant pathogenic bacteria

Multidrug resistant pathogens represent one of the greatest threats to human health of the new millennium. ESKAPE bacterial pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and other Enterobacteriaceae species) are the leading group among these socalled superbugs, which rapidly acquire resistances to several (and sometimes all) available antibiotics and cause a variety of nosocomial infections (e.g. bacteraemia and wound infections). Our research has been leading an innovative approach based on bacteriophage-derived enzymes (called capsular depolymerases) against A. baumannii (see video at ref 1). Previously, we found that some bacteriophages (i.e. viruses that specifically infect bacteria) acquired the ability to infect different Acinetobacter hosts through acquisition of different capsular depolymerases (2). These enzymes located at the bacteriophage tails bind and degrade specific bacterial capsules types (2). Recently, recombinantly expressed capsular depolymerases showed to be active in several environment conditions, non-nontoxic to mammalian cells and able to make A. baumannii fully susceptible to host complement effect, namely in i) Galleria mellonella caterpillar, ii) murine and iii) human serum models (3, 4). A single intraperitoneal injection of depolymerase protect 60% of mice from dead, with significant reduction of proinflammatory cytokine profile (4). We show that capsular depolymerases fit the new trend of antimicrobials needed, as they are highly specific, stable and refractory to resistance as they do not kill bacteria per se, instead they remove bacterial surface polysaccharides, diminishing bacterial virulence and exposing them to the host immune system. This innovative antimicrobial approach can be applied to other pathogenic bacteria.info:eu-repo/semantics/publishedVersio

Erratum for Oliveira et al., "K2 Capsule Depolymerase Is Highly Stable, Is Refractory to Resistance, and Protects Larvae and Mice from Acinetobacter baumannii Sepsis"

Volume 85, no. 17, e00934-19, 2019, https://doi.org/10.1128/AEM.00934-19. Page 10, Acknowledgments, lines 4 and 5: POCI-01-0145-FEDER-016678 should read POCI-01-0145-FEDER-016643.info:eu-repo/semantics/publishedVersio

Insights into the antimicrobial activities and metabolomes of Aquimarina (Flavobacteriaceae, Bacteroidetes) species from the rare marine biosphere

Two novel natural products, the polyketide cuniculene and the peptide antibiotic aquimarin, were recently discovered from the marine bacterial genus Aquimarina. However, the diversity of the secondary metabolite biosynthetic gene clusters (SM-BGCs) in Aquimarina genomes indicates a far greater biosynthetic potential. In this study, nine representative Aquimarina strains were tested for antimicrobial activity against diverse human-pathogenic and marine microorganisms and subjected to metabolomic and genomic profiling. We found an inhibitory activity of most Aquimarina strains against Candida glabrata and marine Vibrio and Alphaproteobacteria species. Aquimarina sp. Aq135 and Aquimarina muelleri crude extracts showed particularly promising antimicrobial activities, amongst others against methicillin-resistant Staphylococcus aureus. The metabolomic and functional genomic profiles of Aquimarina spp. followed similar patterns and were shaped by phylogeny. SM-BGC and metabolomics networks suggest the presence of novel polyketides and peptides, including cyclic depsipeptide-related compounds. Moreover, exploration of the ‘Sponge Microbiome Project’ dataset revealed that Aquimarina spp. possess low-abundance distributions worldwide across multiple marine biotopes. Our study emphasizes the relevance of this member of the microbial rare biosphere as a promising source of novel natural products. We predict that future metabologenomics studies of Aquimarina species will expand the spectrum of known secondary metabolites and bioactivities from marine ecosystems.info:eu-repo/semantics/publishedVersio

Trimeric autotransporter adhesins in members of the Burkholderia cepacia complex: a multifunctional family of proteins implicated in virulence

Trimeric autotransporter adhesins (TAAs) are multimeric surface proteins, involved in various biological traits of pathogenic Gram-negative bacteria including adherence, biofilm formation, invasion, survival within eukaryotic cells, serum resistance and cytotoxicity. TAAs have a modular architecture composed by a conserved membrane-anchored C-terminal domain and a variable number of stalk and head domains. In this study, a bioinformatic approach has been used to analyze the distribution and architecture of TAAs among Burkholderia cepacia complex (Bcc) genomes. Fifteen genomes were probed revealing a total of 74 encoding sequences. Compared with other bacterial species, the Bcc genomes contain a disproportionately large number of TAAs (two genes to up to 8 genes, such as in B.cenocepacia). Phylogenetic analysis showed that the TAAs grouped into at least eight distinct clusters. TAAs with serine-rich repeats are clearly well separated from others, thereby representing a different evolutionary lineage. Comparative gene mapping across Bcc genomes reveals that TAA genes are inserted within conserved synteny blocks. We further focused our analysis on the epidemic strain B. cenocepacia J2315 in which 7 TAAs were annotated. Among these, 3 TAA-encoding genes (BCAM019, BCAM0223 and BCAM0224) are organized into a cluster and are candidates for multifunctional virulence factors. Here we review the current insights into the functional role of BCAM0224 as a model locus

Proteins Encoded by Sphingomonas elodea ATCC 31461 rmlA and ugpG Genes, Involved in Gellan Gum Biosynthesis, Exhibit both dTDP- and UDP-Glucose Pyrophosphorylase Activities

The commercial gelling agent gellan is a heteropolysaccharide produced by Sphingomonas elodea ATCC 31461. In this work, we carried out the biochemical characterization of the enzyme encoded by the first gene (rmlA) of the rml 4-gene cluster present in the 18-gene cluster required for gellan biosynthesis (gel cluster). Based on sequence homology, the putative rml operon is presumably involved in the biosynthesis of dTDP-rhamnose, the sugar necessary for the incorporation of rhamnose in the gellan repeating unit. Heterologous RmlA was purified as a fused His(6)-RmlA protein from extracts prepared from Escherichia coli IPTG (isopropyl-β-d-thiogalactopyranoside)-induced cells, and the protein was proven to exhibit dTDP-glucose pyrophosphorylase (K(m) of 12.0 μM for dTDP-glucose) and UDP-glucose pyrophosphorylase (K(m) of 229.0 μM for UDP-glucose) activities in vitro. The N-terminal region of RmlA exhibits the motif G-X-G-T-R-X(2)-P-X-T, which is highly conserved among bacterial XDP-sugar pyrophosphorylases. The motif E-E-K-P, with the conserved lysine residue (K(163)) predicted to be essential for glucose-1-phosphate binding, was observed. The S. elodea ATCC 31461 UgpG protein, encoded by the ugpG gene which maps outside the gel cluster, was previously identified as the UDP-glucose pyrophosphorylase involved in the formation of UDP-glucose, also required for gellan synthesis. In this study, we demonstrate that UgpG also exhibits dTDP-glucose pyrophosphorylase activity in vitro and compare the kinetic parameters of the two proteins for both substrates. DNA sequencing of ugpG gene-adjacent regions and sequence similarity studies suggest that this gene maps with others involved in the formation of sugar nucleotides presumably required for the biosynthesis of another cell polysaccharide(s)

K2 capsule depolymerase is highly stable, is refractory to resistance, and protects larvae and mice from Acinetobacter baumannii Sepsis

"AEM Accepted Manuscript Posted Online 21 June 2019"Acinetobacter baumannii is emerging as a major nosocomial pathogen in intensive care units. The bacterial capsules are considered major virulence factors, and the particular A. baumannii capsular type K2 has been associated with high antibiotic resistance. In this study, we identified a K2 capsule-specific depolymerase in a bacteriophage tail spike C terminus, a fragment that was heterologously expressed, and its antivirulence properties were assessed by in vivo experiments. The K2 depolymerase is active under a broad range of environmental conditions and is highly thermostable, with a melting point (Tm ) at 67°C. In the caterpillar larva model, the K2 depolymerase protects larvae from bacterial infections, using either pretreatments or with single-enzyme injection after bacterial challenge, in a dose-dependent manner. In a mouse sepsis model, a single K2 depolymerase intraperitoneal injection of 50 μg is able to protect 60% of mice from an otherwise deadly infection, with a significant reduction in the proinflammatory cytokine profile. We showed that the enzyme makes bacterial cells fully susceptible to the host complement system killing effect. Moreover, the K2 depolymerase is highly refractory to resistance development, which makes these bacteriophage-derived capsular depolymerases useful antivirulence agents against multidrug-resistant A. baumannii infections.IMPORTANCEAcinetobacter baumannii is an important nosocomial pathogen resistant to many, and sometimes all, antibiotics. The A. baumannii K2 capsular type has been associated with elevated antibiotic resistance. The capsular depolymerase characterized here fits the new trend of alternative antibacterial agents needed against multidrug-resistant pathogens. They are highly specific, stable, and refractory to resistance, as they do not kill bacteria per se; instead, they remove bacterial surface polysaccharides, which diminish the bacterial virulence and expose them to the host immune system.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and the Project PTDC/BBB - BSS/6471/2014 and COMPETE 20202 (POCI -01 -0145 -FEDER -016678, POCI -01 -0145 -FEDER -006684). This work was also supported by BioTecNorte operation (NORTE -01 -0145 -FEDER -000004, NORTE -01 - 0145 -FEDER -000013 and NORTE -01 -0145 -FEDER -000023) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. This study was additionally supported by Infect -ERA grant Infect -ERA/0002/2015: BU_SPONT_HEAL. HO, AGF and DMH acknowledge the FCT grants SFRH/BPD/111653/2015, FRH/BPD/112903/2015, and SFRH/BDP/91831/2012. AM acknowledges the NORTE -08 -5369 -FSE -000041 project. Funding received by iBB -Institute for Bioengineering and Biosciences from FCT (UID/BIO/04565/2013) and from Programa Operacional Regional de Lisboa 2020 (Project N. 007317) is acknowledged