Daria Augustyniak

Metalloid tolerance based on phytochelatins is not functionally equivalent to the arsenite transporter Acr3p

Active transport of metalloids by Acr3p and Ycf1p in Saccharomyces cerevisiae and chelation by phytochelatins in Schizosaccharomyces pombe, nematodes, and plants represent distinct strategies of metalloid detoxification. In this report, we present results of functional comparison of both resistance mechanisms. The S. pombe and wheat phytochelatin synthase (PCS) genes, when expressed in S. cerevisiae, mediate only modest resistance to arsenite and thus cannot functionally compensate for Acr3p. On the other hand, we show for the first time that phytochelatins also contribute to antimony tolerance as PCS fully complement antimonite sensitivity of ycf1Delta mutant. Remarkably, heterologous expression of PCS sensitizes S. cerevisiae to arsenate, while ACR3 confers much higher arsenic resistance in pcsDelta than in wild-type S. pombe. The analysis of PCS and ACR3 homologues distribution in various organisms and our experimental data suggest that separation of ACR3 and PCS genes may lead to the optimal tolerance status of the cell.

Characterising the biology of novel lytic bacteriophages infecting multidrug resistant Klebsiella pneumoniae

BackgroundMembers of the genus Klebsiella are among the leading microbial pathogens associated with nosocomial infection. The increased incidence of antimicrobial resistance in these species has propelled the need for alternate/combination therapeutic regimens to aid clinical treatment. Bacteriophage therapy forms one of these alternate strategies.MethodsElectron microscopy, burst size, host range, sensitivity of phage particles to temperature, chloroform, pH, and restriction digestion of phage DNA were used to characterize Klebsiella phages.Results and conclusionsOf the 32 isolated phages eight belonged to the family Myoviridae, eight to the Siphoviridae whilst the remaining 16 belonged to the Podoviridae. The host range of these phages was characterised against 254 clinical Enterobacteriaceae strains including multidrug resistant Klebsiella isolates producing extended-spectrum beta-lactamases (ESBLs). Based on their lytic potential, six of the phages were further characterised for burst size, physicochemical properties and sensitivity to restriction endonuclease digestion. In addition, five were fully sequenced. Multiple phage-encoded host resistance mechanisms were identified. The Siphoviridae phage genomes (KP16 and KP36) contained low numbers of host restriction sites similar to the strategy found in T7-like phages (KP32). In addition, phage KP36 encoded its own DNA adenine methyltransferase. The φKMV-like KP34 phage was sensitive to all endonucleases used in this study. Dam methylation of KP34 DNA was detected although this was in the absence of an identifiable phage encoded methyltransferase. The Myoviridae phages KP15 and KP27 both carried Dam and Dcm methyltransferase genes and other anti-restriction mechanisms elucidated in previous studies. No other anti-restriction mechanisms were found, e.g. atypical nucleotides (hmC or glucosyl hmC), although Myoviridae phage KP27 encodes an unknown anti-restriction mechanism that needs further investigation.

Isolation and characterisation of KP34—a novel φKMV-like bacteriophage for Klebsiella pneumoniae

Bacteriophage KP34 is a novel virus belonging to the subfamily Autographivirinae lytic for extended-spectrum β-lactamase-producing Klebsiella pneumoniae strains. Its biological features, morphology, susceptibility to chemical and physical agents, burst size, host specificity and activity spectrum were determined. As a potential antibacterial agent used in therapy, KP34 molecular features including genome sequence and protein composition were examined. Phylogenetic analyses and clustering of KP34 phage genome sequences revealed its clear relationships with “phiKMV-like viruses”. Simultaneously, whole-genome analyses permitted clustering and classification of all phages, with completely sequenced genomes, belonging to the Podoviridae.

Phenotypic characterization of an international Pseudomonas aeruginosa reference panel: strains of cystic fibrosis (CF) origin show less in vivo virulence than non-CF strains

Pseudomonas aeruginosa causes chronic lung infections in people with cystic fibrosis (CF) and acute opportunistic infections in people without CF. Forty-two P. aeruginosa strains from a range of clinical and environmental sources were collated into a single reference strain panel to harmonise research on this diverse opportunistic pathogen. To facilitate further harmonized and comparable research on P. aeruginosa, we characterized the panel strains for growth rates, motility, virulence in the Galleria mellonella infection model, pyocyanin and alginate production, mucoid phenotype, LPS pattern, biofilm formation, urease activity, and antimicrobial and phage susceptibilities. Phenotypic diversity across the P. aeruginosa panel was apparent for all phenotypes examined, agreeing with the marked variability seen in this species. However, except for growth rate, the phenotypic diversity among strains from CF versus non-CF sources was comparable. CF strains were less virulent in the G. mellonella model than non-CF strains (P = 0.037). Transmissible CF strains generally lacked O-antigen, produced less pyocyanin and had low virulence in G. mellonella. Furthermore, in the three sets of sequential CF strains, virulence, O-antigen expression and pyocyanin production were higher in the earlier isolate compared to the isolate obtained later in infection. Overall, this full phenotypic characterization of the defined panel of P. aeruginosa strains increases our understanding of the virulence and pathogenesis of P. aeruginosa and may provide a valuable resource for the testing of novel therapies against this problematic pathogen.

Correction: Characterization of the Newly Isolated Lytic Bacteriophages KTN6 and KT28 and Their Efficacy against Pseudomonas aeruginosa Biofilm

We here describe two novel lytic phages, KT28 and KTN6, infecting Pseudomonas aeruginosa, isolated from a sewage sample from an irrigated field near Wroclaw, in Poland. Both viruses show characteristic features of Pbunalikevirus genus within the Myoviridae family with respect to shape and size of head/tail, as well as LPS host receptor recognition. Genome analysis confirmed the similarity to other PB1-related phages, ranging between 48 and 96%. Pseudomonas phage KT28 has a genome size of 66,381 bp and KTN6 of 65,994 bp. The latent period, burst size, stability and host range was determined for both viruses under standard laboratory conditions. Biofilm eradication efficacy was tested on peg-lid plate assay and PET membrane surface. Significant reduction of colony forming units was observed (70-90%) in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages. Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms. Diffusion and goniometry experiments revealed the increase of diffusion rate through the biofilm matrix after phage application. These characteristics indicate these phages could be used to prevent Pseudomonas aeruginosa infections and biofilm formation. It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants.

The effect of Saccharomyces boulardii on Candida albicans-infected human intestinal cell lines Caco-2 and Intestin 407

Saccharomyces boulardii is a probiotic strain that confers many benefits to human enterocolopathies and is used against a number of enteric pathogens. Candida albicans is an opportunistic pathogen that causes intestinal infections in immunocompromised patients, and after translocation into the bloodstream, is responsible for serious systemic candidiasis. In this study, we investigated the influence of S. boulardii cells and its culture extract on C. albicans adhesion to Caco-2 and Intestin 407 cell lines. We also tested the proinflammatory IL-1beta, IL-6 and IL-8 cytokine expression by C. albicans-infected Caco-2 cells, using real-time RT-PCR. We found that both S. boulardii and its extract significantly inhibited C. albicans adhesion to epithelial cell lines. The IL-8 gene expression by C. albicans-infected Caco-2 cells was suppressed by the addition of S. boulardii extract. Our results indicate that S. boulardii affects C. albicans adhesion and reduces cytokine-mediated inflammatory host response.

Modern Therapeutic Approaches Against Pseudomonas aeruginosa Infections.

Despite the enormous progress that has been made in the last few decades in the field of drug design as well as virulence of pathogenic bacteria, the gradual spread of drug resistance can be observed. Only two new classes of antibiotics have been brought to medicine in the last 30 years. The need for novel antibacterial drugs is especially pressing when considering infections caused by multidrug-resistant (MDR) pathogens such as Pseudomonas aeruginosa. The discovery and development of new anti-pseudomonal therapies is one of the main challenges of modern pharmaceutical sciences. The great variety of innovative approaches presented in the current literature is astonishing. In this review, modern, promising strategies against P. aeruginosa infections are described. Antimicrobials, including new antibiotics, β-lactamase and efflux pump inhibitors, quorum quenching molecules and nanoparticles with antibacterial activity are currently being intensively studied. Methods of prevention of infection through vaccines, therapeutic antibodies and development of antimicrobial peptides are discussed as approaches that support the human immunological system. Finally, development of alternative/ supportive therapies such as phage therapy and photodynamic therapy, in which the mechanism of action is completely different from current antibiotic therapy, is of great importance.

Innate immune properties of selected human neuropeptides against Moraxella catarrhalis and nontypeable Haemophilus influenzae

BackgroundConsiderable evidence supports the concept of active communication between the nervous and immune systems. One class of such communicators are the neuropeptides (NPs). Recent reports have highlighted the antimicrobial activity of neuropeptides, placing them among the integral components of innate immune defense. This study examined the action of four human neuropeptides: calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), substance P (SP) and somatostatin (SOM), which are accessible in the upper respiratory tract, against two human-specific respiratory pathogens. We studied: (i) neuropeptide-mediated direct antibacterial activity exerted against Moraxella catarrhalis and nontypeable Haemophilus influenzae, and (ii) indirect immunomodulatory role of these neuropeptides in the neutrophil-mediated phagocytosis of indicated pathogens.ResultsWe found that 100 micromolar concentrations of CGRP, NPY, SP, and SOM effectively permeabilized bacterial membranes and showed (except SOM) bactericidal activity against both pathogens. SOM acted only bacteriostatically. However the killing efficacy was dependent on the bactericidal assay used. The rank order of killing NP effect was: NPY ≥ CGRP > SP >> SOM and correlated with their potency to permeabilize bacterial membranes. The killing and permeabilization activity of the analyzed NPs showed significant correlation with several physicochemical properties and amino acid composition of the neuropeptides. M. catarrhalis was more sensitive to neuropeptides than nontypeable H. influenzae.The immunomodulatory bimodal effect of physiological concentrations of CGRP, NPY, and SP on the phagocytic function of human neutrophils against M. catarrhalis and H. influenzae was observed both in the ingestion (pathogen uptake) and reactive oxygen species generation stages. This effect was also dependent on the distinct type of pathogen recognition (opsonic versus nonopsonic).ConclusionsThe present results indicate that neuropeptides such as CGRP, NPY, and SP can effectively participate in the direct and indirect elimination of human-specific respiratory pathogens. Because the studied NPs show both direct and indirect modulating antimicrobial potency, they seem to be important molecules involved in the innate host defense against M. catarrhalis and nontypeable H. influenzae.

Killing multiple myeloma cells with the small molecule 3-bromopyruvate: Implications for therapy

The small molecule 3-bromopyruvate (3-BP), which has emerged recently as the first member of a new class of potent anticancer agents, was tested for its capacity to kill multiple myeloma (MM) cancer cells. Human MM cells (RPMI 8226) begin to lose viability significantly within 8 h of incubation in the presence of 3-BP. The Km (0.3 mmol/l) for intracellular accumulation of 3-BP in MM cells is 24 times lower than that in control cells (7.2 mmol/l). Therefore, the uptake of 3-BP by MM cells is significantly higher than that by peripheral blood mononuclear cells. Further, the IC50 values for human MM cells and control peripheral blood mononuclear cells are 24 and 58 µmol/l, respectively. Therefore, specificity and selectivity of 3-BP toward MM cancer cells are evident on the basis of the above. In MM cells the transcription levels of the gene encoding the monocarboxylate transporter MCT1 is significantly amplified compared with control cells. The level of intracellular ATP in MM cells decreases by over 90% within 1 h after addition of 100 µmol/l 3-BP. The cytotoxicity of 3-BP, exemplified by a marked decrease in viability of MM cells, is potentiated by the inhibitor of glutathione synthesis buthionine sulfoximine. In addition, the lack of mutagenicity and its superior capacity relative to Glivec to kill MM cancer cells are presented in this study.

The immunogenicity of the liposome-associated outer membrane proteins (OMPs) of Moraxella catarrhalis

The outer membrane proteins (OMPs) are the most immunogenic and attractive of the Moraxella catarrhalis vaccine antigens that may induce the protective immune response. The aim of this study was to determine the effectiveness of two types of OMP-associated phosphatidylcholine (PC) liposomal formulations (OMPs-PC, PC-OMPs) and of Zwittergent-based proteomicelles (OMPs-Z) in potentiating an anti-OMP systemic immune response in mice. The immunogenicities of the above preparations were evaluated by assessing serum anti-OMP IgG and IgA reactivity in the post-immunized mouse antisera using ELISA and Western blotting. Additionally, the cross-reactivity of the most effective anti-OMP response was determined using heterologous sera from both humans and mice. Both the proteoliposomes and the proteomicelles showed high immunogenic properties and did not elicit any distinct quantitative differences in the antibody titer or qualitative differences in the pattern of the mouse antisera. The post-immunized mouse antisera predominantly recognized a ∼60-kDa OMP of M. catarrhalis. That protein was also found to be a highly cross-reactive antigen interacting with a panel of pooled mouse antisera produced by immunization either with whole cells or the purified OMPs of heterologous M. catarrhalis strains. Furthermore, normal sera collected from healthy children were found to be preferentially reactive with the 60-kDa OMP. The serum-specific IgG, IgA and IgM were respectively detected via immunoblotting in 90%, 85% and 30% of heterologous human sera. This similar immunogenic effectiveness of both OMP-associated liposomal formulations could contribute to the practical use of such formulations in the future in human vaccination. Moreover, the highly cross-reactive 60-kDa OMP seems to be an important antigenic marker of M. catarrhalis, and, as it is responsible for the induction of an antibody-mediated and long-lasting immune response, studying it may partially aid us in understanding the relatively low degree of pathogenicity of the bacterium in immunocompetent individuals.