Deep Intraclonal Analysis for the Development of Vaccines against Drug-Resistant Klebsiella pneumoniae Lineages.

Despite its medical relevance, there is no commercial vaccine that protects the population at risk from multidrug-resistant (MDR) Klebsiella pneumoniae infections. The availability of massive omic data and novel algorithms may improve antigen selection to develop effective prophylactic strategies. Up to 133 exposed proteins in the core proteomes, between 516 and 8666 genome samples, of the six most relevant MDR clonal groups (CGs) carried conserved B-cell epitopes, suggesting minimized future evasion if utilized for vaccination. Antigens showed a range of epitopicity, functional constraints, and potential side effects. Eleven antigens, including three sugar porins, were represented in all MDR-CGs, constitutively expressed, and showed limited reactivity with gut microbiota. Some of these antigens had important interactomic interactions and may elicit adhesion-neutralizing antibodies. Synergistic bivalent to pentavalent combinations that address expression conditions, interactome location, virulence activities, and clone-specific proteins may overcome the limiting protection of univalent vaccines. The combination of five central antigens accounted for 41% of all non-redundant interacting partners of the antigen dataset. Specific antigen mixtures represented in a few or just one MDR-CG further reduced the chance of microbiota interference. Rational antigen selection schemes facilitate the design of high-coverage and "magic bullet" multivalent vaccines against recalcitrant K. pneumoniae lineages.

Cross-Recognition of SARS-CoV-2 B-Cell Epitopes with Other Betacoronavirus Nucleoproteins.

The B and T lymphocytes of the adaptive immune system are important for the control of most viral infections, including COVID-19. Identification of epitopes recognized by these cells is fundamental for understanding how the immune system detects and removes pathogens, and for antiviral vaccine design. Intriguingly, several cross-reactive T lymphocyte epitopes from SARS-CoV-2 with other betacoronaviruses responsible for the common cold have been identified. In addition, antibodies that cross-recognize the spike protein, but not the nucleoprotein (N protein), from different betacoronavirus have also been reported. Using a consensus of eight bioinformatic methods for predicting B-cell epitopes and the collection of experimentally detected epitopes for SARS-CoV and SARS-CoV-2, we identified four surface-exposed, conserved, and hypothetical antigenic regions that are exclusive of the N protein. These regions were analyzed using ELISA assays with two cohorts: SARS-CoV-2 infected patients and pre-COVID-19 samples. Here we describe four epitopes from SARS-CoV-2 N protein that are recognized by the humoral response from multiple individuals infected with COVID-19, and are conserved in other human coronaviruses. Three of these linear surface-exposed sequences and their peptide homologs in SARS-CoV-2 and HCoV-OC43 were also recognized by antibodies from pre-COVID-19 serum samples, indicating cross-reactivity of antibodies against coronavirus N proteins. Different conserved human coronaviruses (HCoVs) cross-reactive B epitopes against SARS-CoV-2 N protein are detected in a significant fraction of individuals not exposed to this pandemic virus. These results have potential clinical implications.

Determination of Ligand Profiles for Pseudomonas aeruginosa Solute Binding Proteins.

Solute binding proteins (SBPs) form a heterogeneous protein family that is found in all kingdoms of life. In bacteria, the ligand-loaded forms bind to transmembrane transporters providing the substrate. We present here the SBP repertoire of Pseudomonas aeruginosa PAO1 that is composed of 98 proteins. Bioinformatic predictions indicate that many of these proteins have a redundant ligand profile such as 27 SBPs for proteinogenic amino acids, 13 proteins for spermidine/putrescine, or 9 proteins for quaternary amines. To assess the precision of these bioinformatic predictions, we have purified 17 SBPs that were subsequently submitted to high-throughput ligand screening approaches followed by isothermal titration calorimetry studies, resulting in the identification of ligands for 15 of them. Experimentation revealed that PA0222 was specific for γ-aminobutyrate (GABA), DppA2 for tripeptides, DppA3 for dipeptides, CysP for thiosulphate, OpuCC for betaine, and AotJ for arginine. Furthermore, RbsB bound D-ribose and D-allose, ModA bound molybdate, tungstate, and chromate, whereas AatJ recognized aspartate and glutamate. The majority of experimentally identified ligands were found to be chemoattractants. Data show that the ligand class recognized by SPBs can be predicted with confidence using bioinformatic methods, but experimental work is necessary to identify the precise ligand profile.

Systematic mapping of chemoreceptor specificities for Pseudomonas aeruginosa.

IMPORTANCE: Chemotaxis of motile bacteria has multiple physiological functions. It enables bacteria to locate optimal ecological niches, mediates collective behaviors, and can play an important role in infection. These multiple functions largely depend on ligand specificities of chemoreceptors, and the number and identities of chemoreceptors show high diversity between organisms. Similar diversity is observed for the spectra of chemoeffectors, which include not only chemicals of high metabolic value but also bacterial, plant, and animal signaling molecules. However, the systematic identification of chemoeffectors and their mapping to specific chemoreceptors remains a challenge. Here, we combined several in vivo and in vitro approaches to establish a systematic screening strategy for the identification of receptor ligands and we applied it to identify a number of new physiologically relevant chemoeffectors for the important opportunistic human pathogen P. aeruginosa. This strategy can be equally applicable to map specificities of sensory domains from a wide variety of receptor types and bacteria.

Role of peptidoglycan recycling enzymes AmpD and AnmK in Acinetobacter baumannii virulence features.

Acinetobacter baumannii is an important causative agent of hospital acquired infections. In addition to acquired resistance to many currently-available antibiotics, it is intrinsically resistant to fosfomycin. It has previously been shown that AmpD and AnmK contribute to intrinsic fosfomycin resistance in A. baumannii due to their involvement in the peptidoglycan recycling pathway. However, the role that these two enzymes play in the fitness and virulence of A. baumannii has not been studied. The aim of this study was to characterize several virulence-related phenotypic traits in A. baumannii mutants lacking AmpD and AnmK. Specifically, cell morphology, peptidoglycan thickness, membrane permeability, growth under iron-limiting conditions, fitness, resistance to disinfectants and antimicrobial agents, twitching motility and biofilm formation of the mutant strains A. baumannii ATCC 17978 ΔampD::Kan and ΔanmK::Kan were compared to the wild type strain. Our results demonstrate that bacterial growth and fitness of both mutants were compromised, especially in the ΔampD::Kan mutant. In addition, biofilm formation was decreased by up to 69%, whereas twitching movement was reduced by about 80% in both mutants. These results demonstrate that, in addition to increased susceptibility to fosfomycin, alteration of the peptidoglycan recycling pathway affects multiple aspects related to virulence. Inhibition of these enzymes could be explored as a strategy to develop novel treatments for A. baumannii in the future. Furthermore, this study establishes a link between intrinsic fosfomycin resistance mechanisms and bacterial fitness and virulence traits.

Chemotaxis of the Human Pathogen Pseudomonas aeruginosa to the Neurotransmitter Acetylcholine.

Acetylcholine is a central biological signal molecule present in all kingdoms of life. In humans, acetylcholine is the primary neurotransmitter of the peripheral nervous system; it mediates signal transmission at neuromuscular junctions. Here, we show that the opportunistic human pathogen Pseudomonas aeruginosa exhibits chemoattraction toward acetylcholine over a concentration range of 1 µM to 100 mM. The maximal magnitude of the response was superior to that of many other P. aeruginosa chemoeffectors. We demonstrate that this chemoattraction is mediated by the PctD (PA4633) chemoreceptor. Using microcalorimetry, we show that the PctD ligand-binding domain (LBD) binds acetylcholine with a equilibrium dissociation constant (KD) of 23 µM. It also binds choline and with lower affinity betaine. Highly sensitive responses to acetylcholine and choline, and less sensitive responses to betaine and l-carnitine, were observed in Escherichia coli expressing a chimeric receptor comprising the PctD-LBD fused to the Tar chemoreceptor signaling domain. We also identified the PacA (ECA_RS10935) chemoreceptor of the phytopathogen Pectobacterium atrosepticum, which binds choline and betaine but fails to recognize acetylcholine. To identify the molecular determinants for acetylcholine recognition, we report high-resolution structures of PctD-LBD (with bound acetylcholine and choline) and PacA-LBD (with bound betaine). We identified an amino acid motif in PctD-LBD that interacts with the acetylcholine tail. This motif is absent in PacA-LBD. Significant acetylcholine chemotaxis was also detected in the plant pathogens Agrobacterium tumefaciens and Dickeya solani. To the best of our knowledge, this is the first report of acetylcholine chemotaxis and extends the range of host signals perceived by bacterial chemoreceptors. IMPORTANCE P. aeruginosa causes a significant number of deaths annually worldwide. For many pathogens, chemotaxis plays an import role in the initial stages of infection, and deciphering the key chomoeffectors and their cognate chemoreceptors may permit the development of strategies to inhibit this process. Genome analyses have shown that many bacteria possess a large number of chemoreceptors. The chemoeffectors recognized by the large majority of chemoreceptors are unknown. However, identifying these chemoeffectors is crucial for deciphering the evolutionary forces that have shaped chemosensory signaling mechanisms in bacteria with different lifestyles. Our current understanding of the relationship between bacterial lifestyle and chemoreceptor repertoire is limited, and this work contributes to closing this gap in our knowledge. By expanding the list of known chemoeffectors and chemoreceptors, progress is made toward identifying functional receptor homologs in other bacteria.

Corrigendum: Brief Research Report: Virus-Specific Humoral Immunity at Admission Predicts the Development of Respiratory Failure in Unvaccinated SARS-CoV-2 Patients.

[This corrects the article DOI: 10.3389/fimmu.2022.878812.].

Brief Research Report: Virus-Specific Humoral Immunity at Admission Predicts the Development of Respiratory Failure in Unvaccinated SARS-CoV-2 Patients.

Introduction: There is robust evidence indicating that the SARS-CoV-2-specific humoral response is associated with protection against severe disease. However, relatively little data exist regarding how the humoral immune response at the time of hospital admission correlates with disease severity in unimmunized patients. Our goal was toidentify variables of the humoral response that could potentially serve as prognostic markers for COVID-19 progressionin unvaccinated SARS-CoV-2 patients. Methods: A prospective cross-sectional study was carried out in a cohort of 160 unimmunized, adult COVID-19 patients from the Hospital Universitario 12Octubre. Participants were classified into four clinical groups based on disease severity: non-survivors with respiratory failure (RF), RF survivors, patients requiring oxygen therapy and those not receiving oxygen therapy. Serum samples were taken on admission and IgM, IgG, IgG subclass antibody titers were determined by ELISA, and neutralizing antibody titersusing a surrogate neutralization assay. The differences in the antibody titers between groups and the association between the clinical and analytical characteristics of the patients and the antibody titers were analyzed. Results: Patients that developed RF and survived had IgM titers that were 2-fold higher than non-survivors (p = 0.001), higher levels of total IgG than those who developed RF and succumbed to infection (p< 0.001), and than patients who required oxygen therapy (p< 0.05), and had 5-fold higher IgG1 titers than RF non-survivors (p< 0.001) and those who needed oxygen therapy (p< 0.001), and 2-fold higher than patients that did not require oxygen therapy during admission (p< 0.05). In contrast, RF non-survivorshad the lowest neutralizing antibodylevels, which were significantly lower compared those with RF that survived (p = 0.03). A positive correlation was found between IgM, total IgG, IgG1 and IgG3 titers and neutralizing antibody titers in the total cohort (p ≤ 0.0036). Conclusions: We demonstrate that patients with RF that survived infection had significantly higher IgM, IgG, IgG1 and neutralizing titers compared to patients with RF that succumb to infection, suggesting that using humoral response variables could be used as a prognostic marker for guiding the clinical management of unimmunized patients admitted to the hospital for SARS-CoV-2 infection.

Subinhibitory Concentrations of Clinically-Relevant Antimicrobials Affect Resistance-Nodulation-Division Family Promoter Activity in Acinetobacter baumannii.

Efflux pumps contribute to multidrug resistance in Acinetobacter baumannii due to their ability to expel a wide variety of structurally unrelated compounds. This study aimed to characterize the effect of subinhibitory concentrations of clinically-relevant antibiotics and disinfectants on the promoter activity of members of the Resistance-Nodulation-Division (RND) family in A. baumannii. The promoter regions from three RND efflux pumps (AdeABC, AdeFGH and AdeIJK) and the AdeRS regulatory system from three different A. baumannii strains (ATCC 17961, ATCC 17978, and ATCC 19606) were cloned into a luciferase reporter system (pLPV1Z). Promoter activity was quantitatively assessed in both exponential and stationary phase cultures after exposure to subinhibitory concentrations of four antibiotics from different classes (rifampicin, meropenem, tigecycline and colistin) and two disinfectants (ethanol and chlorhexidine). Subinhibitory concentrations of the compounds tested had variable effects on promoter activity that were highly dependent on the A. baumannii strain, the compound tested and the growth phase. Fold changes in AdeABC promoter activity ranged from 1.97 to 113.7, in AdeFGH from -5.6 to 1.13, in AdeIJK from -2.5 to 2, and in AdeRS from -36.2 to -1.32. Taken together, these results indicate that subinhibitory concentrations of clinically-relevant antibiotics and disinfectants affect the promoter activity of RND family members in A. baumannii in a strain and growth phase dependent manner. These results may have important implications for the treatment of infections caused by A. baumannii.