Evolutionarily stable gene clusters shed light on the common grounds of pathogenicity in the Acinetobacter calcoaceticus-baumannii complex.

Nosocomial pathogens of the Acinetobacter calcoaceticus-baumannii (ACB) complex are a cautionary example for the world-wide spread of multi- and pan-drug resistant bacteria. Aiding the urgent demand for novel therapeutic targets, comparative genomics studies between pathogens and their apathogenic relatives shed light on the genetic basis of human-pathogen interaction. Yet, existing studies are limited in taxonomic scope, sensing of the phylogenetic signal, and resolution by largely analyzing genes independent of their organization in functional gene clusters. Here, we explored more than 3,000 Acinetobacter genomes in a phylogenomic framework integrating orthology-based phylogenetic profiling and microsynteny conservation analyses. We delineate gene clusters in the type strain A. baumannii ATCC 19606 whose evolutionary conservation indicates a functional integration of the subsumed genes. These evolutionarily stable gene clusters (ESGCs) reveal metabolic pathways, transcriptional regulators residing next to their targets but also tie together sub-clusters with distinct functions to form higher-order functional modules. We shortlisted 150 ESGCs that either co-emerged with the pathogenic ACB clade or are preferentially found therein. They provide a high-resolution picture of genetic and functional changes that coincide with the manifestation of the pathogenic phenotype in the ACB clade. Key innovations are the remodeling of the regulatory-effector cascade connecting LuxR/LuxI quorum sensing via an intermediate messenger to biofilm formation, the extension of micronutrient scavenging systems, and the increase of metabolic flexibility by exploiting carbon sources that are provided by the human host. We could show experimentally that only members of the ACB clade use kynurenine as a sole carbon and energy source, a substance produced by humans to fine-tune the antimicrobial innate immune response. In summary, this study provides a rich and unbiased set of novel testable hypotheses on how pathogenic Acinetobacter interact with and ultimately infect their human host. It is a comprehensive resource for future research into novel therapeutic strategies.

InvL, an Invasin-Like Adhesin, Is a Type II Secretion System Substrate Required for Acinetobacter baumannii Uropathogenesis.

Acinetobacter baumannii is an opportunistic pathogen of growing concern, as isolates are commonly multidrug resistant. While A. baumannii is most frequently associated with pulmonary infections, a significant proportion of clinical isolates come from urinary sources, highlighting its uropathogenic potential. The type II secretion system (T2SS) of commonly used model Acinetobacter strains is important for virulence in various animal models, but the potential role of the T2SS in urinary tract infection (UTI) remains unknown. Here, we used a catheter-associated UTI (CAUTI) model to demonstrate that a modern urinary isolate, UPAB1, requires the T2SS for full virulence. A proteomic screen to identify putative UPAB1 T2SS effectors revealed an uncharacterized lipoprotein with structural similarity to the intimin-invasin family, which serve as type V secretion system (T5SS) adhesins required for the pathogenesis of several bacteria. This protein, designated InvL, lacked the ß-barrel domain associated with T5SSs but was confirmed to require the T2SS for both surface localization and secretion. This makes InvL the first identified T2SS effector belonging to the intimin-invasin family. InvL was confirmed to be an adhesin, as the protein bound to extracellular matrix components and mediated adhesion to urinary tract cell lines in vitro. Additionally, the invL mutant was attenuated in the CAUTI model, indicating a role in Acinetobacter uropathogenesis. Finally, bioinformatic analyses revealed that InvL is present in nearly all clinical isolates belonging to international clone 2, a lineage of significant clinical importance. In all, we conclude that the T2SS substrate InvL is an adhesin required for A. baumannii uropathogenesis. IMPORTANCE While pathogenic Acinetobacter can cause various infections, we recently found that 20% of clinical isolates come from urinary sources. Despite the clinical relevance of Acinetobacter as a uropathogen, few virulence factors involved in urinary tract colonization have been defined. Here, we identify a novel type II secretion system effector, InvL, which is required for full uropathogenesis by a modern urinary isolate. Although InvL has predicted structural similarity to the intimin-invasin family of autotransporter adhesins, InvL is predicted to be anchored to the membrane as a lipoprotein. Similar to other invasin homologs, however, we demonstrate that InvL is a bona fide adhesin capable of binding extracellular matrix components and mediating adhesion to urinary tract cell lines. In all, this work establishes InvL as an adhesin important for Acinetobacter's urinary tract virulence and represents the first report of a type II secretion system effector belonging to the intimin-invasin family.

Assembly and Functional Role of PACE Transporter PA2880 from Pseudomonas aeruginosa.

The recently identified proteobacterial antimicrobial compound efflux (PACE) transporters are multidrug transporters energized by the electrochemical gradient of protons. Here, we present the results of phylogenetic and functional studies on the PACE family transporter PA2880 from Pseudomonas aeruginosa. A phylogenetic analysis of the PACE family revealed that PA2880 and AceI from Acinetobacter baumannii are classified into evolutionarily distinct clades, although they both transport chlorhexidine. We demonstrate that PA2880 mainly exists as a dimer in solution, which is independent of pH, and its dimeric state is essential for its proper function. Electrogenicity studies revealed that the chlorhexidine/H+ antiport process is electrogenic. The function of several highly conserved residues was investigated. These findings provide further insights into the functional features of PACE family transporters, facilitating studies on their transport mechanisms. IMPORTANCE Pseudomonas aeruginosa is a pathogen that causes hospital-acquired (nosocomial) infections, such as ventilator-associated pneumonia and sepsis syndromes. Chlorhexidine diacetate is a disinfectant used for bacterial control in various environments potentially harboring P. aeruginosa. Therefore, investigation of the mechanism of the efflux of chlorhexidine mediated by PA2880, a PACE family transporter from P. aeruginosa, is of significance to combat bacterial infections. This study improves our understanding of the transport mechanism of PACE family transporters and will facilitate the effective utilization of chlorhexidine for P. aeruginosa control.

An Update on "Reverse Vaccinology": The Pathway from Genomes and Epitope Predictions to Tailored, Recombinant Vaccines.

In this chapter, we review the computational approaches that have led to a new generation of vaccines in recent years. There are many alternative routes to develop vaccines based on the concept of reverse vaccinology. They all follow the same basic principles-mining available genome and proteome information for antigen candidates, and recombinantly expressing them for vaccine production. Some of the same principles have been used successfully for cancer therapy approaches. In this review, we focus on infectious diseases, describing the general workflow from bioinformatic predictions of antigens and epitopes down to examples where such predictions have been used successfully for vaccine development.

Peptidoglycan editing provides immunity to Acinetobacter baumannii during bacterial warfare.

Peptidoglycan (PG) is essential in most bacteria. Thus, it is often targeted by various assaults, including interbacterial attacks via the type VI secretion system (T6SS). Here, we report that the Gram-negative bacterium Acinetobacter baumannii strain ATCC 17978 produces, secretes, and incorporates the noncanonical d-amino acid d-lysine into its PG during stationary phase. We show that PG editing increases the competitiveness of A. baumannii during bacterial warfare by providing immunity against peptidoglycan-targeting T6SS effectors from various bacterial competitors. In contrast, we found that d-Lys production is detrimental to pathogenesis due, at least in part, to the activity of the human enzyme d-amino acid oxidase (DAO), which degrades d-Lys producing H2O2 toxic to bacteria. Phylogenetic analyses indicate that the last common ancestor of A. baumannii had the ability to produce d-Lys. However, this trait was independently lost multiple times, likely reflecting the evolution of A. baumannii as a human pathogen.

The Acinetobacter trimeric autotransporter adhesin Ata controls key virulence traits of Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative pathogen that causes a multitude of nosocomial infections. The Acinetobacter trimeric autotransporter adhesin (Ata) belongs to the superfamily of trimeric autotransporter adhesins which are important virulence factors in many Gram-negative species. Phylogenetic profiling revealed that ata is present in 78% of all sequenced A. baumannii isolates but only in 2% of the closely related species A. calcoaceticus and A. pittii. Employing a markerless ata deletion mutant of A. baumannii ATCC 19606 we show that adhesion to and invasion into human endothelial and epithelial cells depend on Ata. Infection of primary human umbilical cord vein endothelial cells (HUVECs) with A. baumannii led to the secretion of interleukin (IL)-6 and IL-8 in a time- and Ata-dependent manner. Furthermore, infection of HUVECs by WT A. baumannii was associated with higher rates of apoptosis via activation of caspases-3 and caspase-7, but not necrosis, in comparison to ∆ata. Ata deletion mutants were furthermore attenuated in their ability to kill larvae of Galleria mellonella and to survive in larvae when injected at sublethal doses. This indicates that Ata is an important multifunctional virulence factor in A. baumannii that mediates adhesion and invasion, induces apoptosis and contributes to pathogenicity in vivo.

Identification of the novel class D ß-lactamase OXA-679 involved in carbapenem resistance in Acinetobacter calcoaceticus.

OBJECTIVES: The aim of this study was to characterize the Acinetobacter calcoaceticus clinical isolate AC_2117 with the novel carbapenem-hydrolysing class D ß-lactamase (CHDL) OXA-679. METHODS: Identification of the species and ß-lactamases was verified by genome sequencing (PacBio) and phylogenetic analyses. Antibiotic susceptibility of AC_2117 and transformants harbouring cloned blaOXA-679 was evaluated using antibiotic gradient strips and microbroth dilution. OXA-679 was purified heterologously and kinetic parameters were determined using spectrometry or isothermal titration calorimetry. The impact of OXA-679 production during imipenem therapy was evaluated in the Galleria mellonella infection model. RESULTS: Sequencing of the complete genome of the clinical A. calcoaceticus isolate AC_2117 identified a novel CHDL, termed OXA-679. This enzyme shared sequence similarity of 71% to each of the families OXA-143 and OXA-24/40. Phylogenetic analyses revealed that OXA-679 represents a member of a new OXA family. Cloning and expression of blaOXA-679 as well as measurement of kinetic parameters revealed the effective hydrolysis of carbapenems which resulted in reduced susceptibility to carbapenems in Escherichia coli and A. calcoaceticus, and high-level carbapenem resistance in Acinetobacter baumannii. Infection of larvae of G. mellonella with a sublethal dose of blaOXA-679-expressing A. baumannii could not be cured by high-dose imipenem therapy, indicating carbapenem resistance in vivo. CONCLUSIONS: We identified blaOXA-679 in a clinical A. calcoaceticus isolate that represents a member of the new OXA-679 family and that conferred high-level carbapenem resistance in vitro and in vivo.

Natural product diversity associated with the nematode symbionts Photorhabdus and Xenorhabdus.

Xenorhabdus and Photorhabdus species dedicate a large amount of resources to the production of specialized metabolites derived from non-ribosomal peptide synthetase (NRPS) or polyketide synthase (PKS). Both bacteria undergo symbiosis with nematodes, which is followed by an insect pathogenic phase. So far, the molecular basis of this tripartite relationship and the exact roles that individual metabolites and metabolic pathways play have not been well understood. To close this gap, we have significantly expanded the database for comparative genomics studies in these bacteria. Clustering the genes encoded in the individual genomes into hierarchical orthologous groups reveals a high-resolution picture of functional evolution in this clade. It identifies groups of genes-many of which are involved in secondary metabolite production-that may account for the niche specificity of these bacteria. Photorhabdus and Xenorhabdus appear very similar at the DNA sequence level, which indicates their close evolutionary relationship. Yet, high-resolution mass spectrometry analyses reveal a huge chemical diversity in the two taxa. Molecular network reconstruction identified a large number of previously unidentified metabolite classes, including the xefoampeptides and tilivalline. Here, we apply genomic and metabolomic methods in a complementary manner to identify and elucidate additional classes of natural products. We also highlight the ability to rapidly and simultaneously identify potentially interesting bioactive products from NRPSs and PKSs, thereby augmenting the contribution of molecular biology techniques to the acceleration of natural product discovery.

Ocean plankton. Structure and function of the global ocean microbiome.

Microbes are dominant drivers of biogeochemical processes, yet drawing a global picture of functional diversity, microbial community structure, and their ecological determinants remains a grand challenge. We analyzed 7.2 terabases of metagenomic data from 243 Tara Oceans samples from 68 locations in epipelagic and mesopelagic waters across the globe to generate an ocean microbial reference gene catalog with >40 million nonredundant, mostly novel sequences from viruses, prokaryotes, and picoeukaryotes. Using 139 prokaryote-enriched samples, containing >35,000 species, we show vertical stratification with epipelagic community composition mostly driven by temperature rather than other environmental factors or geography. We identify ocean microbial core functionality and reveal that >73% of its abundance is shared with the human gut microbiome despite the physicochemical differences between these two ecosystems.

Argyreia nervosa (Burm. f.): receptor profiling of lysergic acid amide and other potential psychedelic LSD-like compounds by computational and binding assay approaches.

ETHNOPHARMACOLOGICAL RELEVANCE: The convolvulacea Argyreia nervosa (Burm. f.) is well known as an important medical plant in the traditional Ayurvedic system of medicine and it is used in numerous diseases (e.g. nervousness, bronchitis, tuberculosis, arthritis, and diabetes). Additionally, in the Indian state of Assam and in other regions Argyreia nervosa is part of the traditional tribal medicine (e.g. the Santali people, the Lodhas, and others). In the western hemisphere, Argyreia nervosa has been brought in attention as so called "legal high". In this context, the seeds are used as source of the psychoactive ergotalkaloid lysergic acid amide (LSA), which is considered as the main active ingredient. AIM OF THE STUDY: As the chemical structure of LSA is very similar to that of lysergic acid diethylamide (LSD), the seeds of Argyreia nervosa (Burm. f.) are often considered as natural substitute of LSD. In the present study, LSA and LSD have been compared concerning their potential pharmacological profiles based on the receptor binding affinities since our recent human study with four volunteers on p.o. application of Argyreia nervosa seeds has led to some ambiguous effects. MATERIAL AND METHODS: In an initial step computer-aided in silico prediction models on receptor binding were employed to screen for serotonin, norepinephrine, dopamine, muscarine, and histamine receptor subtypes as potential targets for LSA. In addition, this screening was extended to accompany ergotalkaloids of Argyreia nervosa (Burm. f.). In a verification step, selected LSA screening results were confirmed by in vitro binding assays with some extensions to LSD. RESULTS: In the in silico model LSA exhibited the highest affinity with a pKi of about 8.0 at α1A, and α1B. Clear affinity with pKi>7 was predicted for 5-HT1A, 5-HT1B, 5-HT1D, 5-HT6, 5-HT7, and D2. From these receptors the 5-HT1D subtype exhibited the highest pKi with 7.98 in the prediction model. From the other ergotalkaloids, agroclavine and festuclavine also seemed to be highly affine to the 5-HT1D-receptor with pKi>8. In general, the ergotalkaloids of Argyreia nervosa seem to prefer serotonin and dopamine receptors (pKi>7). However, with exception of ergometrine/ergometrinine only for 5-HT3A, and histamine H2 and H4 no affinities were predicted. Compared to LSD, LSA exhibited lower binding affinities in the in vitro binding assays for all tested receptor subtypes. However, with a pKi of 7.99, 7.56, and 7.21 a clear affinity for 5-HT1A, 5-HT2, and α2 could be demonstrated. For DA receptor subtypes and the α1-receptor the pKi ranged from 6.05 to 6.85. CONCLUSION: Since the psychedelic activity of LSA in the recent human study was weak and although LSA from Argyreia nervosa is often considered as natural exchange for LSD, LSA should not be regarded as LSD-like psychedelic drug. However, vegetative side effects and psychotropic effects may be triggered by serotonin or dopamine receptor subtypes.