A multiomic approach to defining the essential genome of the globally important pathogen Corynebacterium diphtheriae.

Diphtheria is a respiratory disease caused by Corynebacterium diphtheriae. While the toxin-based vaccine has helped control outbreaks of the disease since the mid-20th century there has been an increase in cases in recent years, including systemic infections caused by non-toxigenic C. diphtheriae strains. Here we describe the first study of gene essentiality in C. diphtheriae, providing the most-dense Transposon Directed Insertion Sequencing (TraDIS) library in the phylum Actinobacteriota. This high-density library has allowed the identification of conserved genes across the genus and phylum with essential function and enabled the elucidation of essential domains within the resulting proteins including those involved in cell envelope biogenesis. Validation of these data through protein mass spectrometry identified hypothetical and uncharacterized proteins in the proteome which are also represented in the vaccine. These data are an important benchmark and useful resource for the Corynebacterium, Mycobacterium, Nocardia and Rhodococcus research community. It enables the identification of novel antimicrobial and vaccine targets and provides a basis for future studies of Actinobacterial biology.

Tuning the Polarity of Antibiotic-Cy5 Conjugates Enables Highly Selective Labeling of Binding Sites.

Multidrug-resistant bacteria pose a major threat to global health, even as newly introduced antibiotics continue to lose their therapeutic value. Against this background, deeper insights into bacterial interaction with antibiotic drugs are urgently required, whereas fluorescently labeled drug conjugates can serve as highly valuable tools. Herein, the preparation and biological evaluation of 13 new fluorescent antibiotic-Cy5 dye conjugates is described, in which the tuning of the polarity of the Cy5 dye proved to be a key element to achieve highly favorable properties for various fields of application.

Latest Review Papers in Molecular Microbiology.

This Special Issue-dedicated to high-quality review papers in molecular microbiology-is highlighting two important developments in the field: (i) the analysis of microbiome data in health and disease and (ii) the search for strategies against bacteria showing antimicrobial resistance [...].

Photodynamic Activity of Chlorophyllin and Polyethylenimine on Pseudomonas aeruginosa Planktonic, Biofilm and Persister Cells.

Antimicrobial photodynamic inactivation is considered a promising antimicrobial approach that may not develop resistance in the near future. Here, we investigate the influence of the photosensitizer chlorophyllin (CHL) and the cationic permeabilizer polyethylenimine (PEI), exposed to a red light-emitting diode, on the human pathogen Pseudomonas aeruginosa free-living planktonic cells, the sessile biofilm and persister cells. The broth microdilution checkerboard method was used to test antimicrobial susceptibility. As a substrate for biofilms, the Calgary biofilm device was used, and the quantification of the biofilm biomass was carried out using a crystal violet assay. Serine hydroxamate was used for the induction of persisters. Our findings reveal that PEI ameliorates the antimicrobial activity of CHL against P. aeruginosa planktonic and biofilm states, and the concentration required to eradicate the bacteria in the biofilm is more than fourfold that is required to eradicate planktonic cells. Interestingly, the persister cells are more susceptible to CHL/PEI (31.25/100 µg mL-1) than the growing cells by 1.7 ± 0.12 and 0.4 ± 0.1 log10 reduction, respectively, after 15 min of illumination. These data demonstrate that CHL excited with red light together with PEI is promising for the eradication of P. aeruginosa, and the susceptibility of P. aeruginosa to CHL/PEI is influenced by the concentrations and the exposure time.

Host-Pathogen Interaction 3.0.

Microorganisms can interact with plants, animals and humans in many different ways, e [...].

Interactions between the Re-Emerging Pathogen Corynebacterium diphtheriae and Host Cells.

Corynebacterium diphtheriae, the etiological agent of diphtheria, is a re-emerging pathogen, responsible for several thousand deaths per year. In addition to diphtheria, systemic infections, often by non-toxigenic strains, are increasingly observed. This indicates that besides the well-studied and highly potent diphtheria toxin, various other virulence factors may influence the progression of the infection. This review focuses on the known components of C. diphtheriae responsible for adhesion, invasion, inflammation, and cell death, as well as on the cellular signaling pathways activated upon infection.

Newly Isolated Animal Pathogen Corynebacterium silvaticum Is Cytotoxic to Human Epithelial Cells.

Corynebacterium silvaticum is a newly identified animal pathogen of forest animals such as roe deer and wild boars. The species is closely related to the emerging human pathogen Corynebacterium ulcerans and the widely distributed animal pathogen Corynebacterium pseudotuberculosis. In this study, Corynebacterium silvaticum strain W25 was characterized with respect to its interaction with human cell lines. Microscopy, measurement of transepithelial electric resistance and cytotoxicity assays revealed detrimental effects of C. silvaticum to different human epithelial cell lines and to an invertebrate animal model, Galleria mellonella larvae, comparable to diphtheria toxin-secreting C. ulcerans. Furthermore, the results obtained may indicate a considerable zoonotic potential of this newly identified species.

Treat Me Well or Will Resist: Uptake of Mobile Genetic Elements Determine the Resistome of Corynebacterium striatum.

Corynebacterium striatum, a bacterium that is part of the normal skin microbiota, is also an opportunistic pathogen. In recent years, reports of infections and in-hospital and nosocomial outbreaks caused by antimicrobial multidrug-resistant C. striatum strains have been increasing worldwide. However, there are no studies about the genomic determinants related to antimicrobial resistance in C. striatum. This review updates global information related to antimicrobial resistance found in C. striatum and highlights the essential genomic aspects in its persistence and dissemination. The resistome of C. striatum comprises chromosomal and acquired elements. Resistance to fluoroquinolones and daptomycin are due to mutations in chromosomal genes. Conversely, resistance to macrolides, tetracyclines, phenicols, beta-lactams, and aminoglycosides are associated with mobile genomic elements such as plasmids and transposons. The presence and diversity of insertion sequences suggest an essential role in the expression of antimicrobial resistance genes (ARGs) in genomic rearrangements and their potential to transfer these elements to other pathogens. The present study underlines that the resistome of C. striatum is dynamic; it is in evident expansion and could be acting as a reservoir for ARGs.

Phylogenomic characterisation of a novel corynebacterial species pathogenic to animals.

The genus Corynebacterium includes species of biotechnological, medical and veterinary importance. An atypical C. ulcerans strain, W25, was recently isolated from a case of necrotizing lymphadenitis in a wild boar. In this study, we have analysed the genome sequence of this strain and compared the phenotypic and virulence properties with other corynebacterial pathogens. Phylogenomic analyses revealed that strain W25 belongs to a novel species along with PO100/5 and KL1196. The latter strains were isolated from a pig and a roe deer, respectively; hence, this species appears to be associated to animals. The isolate W25 is likely a non-toxigenic tox gene bearing strain and may have compromised abilities to adhere to pharyngeal and laryngeal epithelial cells due to potential loss of the gene functions in spaBC and spaDEF pilus gene clusters. A number of corynebacterial virulence genes are present including pld encoding phospholipase D. Therefore, this strain may be able to cause severe invasive infections in animals and zoonotic infections in humans.

Beyond diphtheria toxin: cytotoxic proteins of Corynebacterium ulcerans and Corynebacterium diphtheriae.

Diphtheria toxin is one of the best investigated bacterial toxins and the major virulence factor of toxigenic Corynebacterium diphtheriae and Corynebacterium ulcerans strains. However, also diphtheria toxin-free strains of these two species can cause severe infections in animals and humans, indicating the presence of additional virulence factors. In this study, we present a first characterization of two proteins with cytotoxic effect in corynebacteria. A putative ribosome-binding protein (AEG80717, CULC809_00177), first annotated in a genome sequencing project of C. ulcerans strain 809, was investigated in detail together with a homologous protein identified in C. diphtheriae strain HC04 (AEX80148, CDHC04_0155) in this study. The corresponding proteins show striking structural similarity to Shiga-like toxins. Interaction of wild-type, mutant and complementation as well as overexpression strains with invertebrate model systems and cell lines were investigated. Depending on the presence of the corresponding genes, detrimental effects were observed in vivo in two invertebrate model systems, Caenorhabditis elegans and Galleria mellonella, and on various animal and human epithelial and macrophage cell lines in vitro. Taken together, our results support the idea that pathogenicity of corynebacteria is a multifactorial process and that new virulence factors may influence the outcome of potentially fatal corynebacterial infections.

Detection and virulence potential of a phospholipase D-negative Corynebacterium ulcerans from a concurrent diphtheria and infectious mononucleosis case.

Diphtheria by Corynebacterium ulcerans is increasingly occurring in children, adolescents and adults. In addition to diphtheria toxin (DT), phospholipase D (PLD) is considered a virulence factor of C. ulcerans. In the present study, a first case of concurrent diphtheria by a PLD-negative C. ulcerans and infectious mononucleosis (IM) was verified. Clinical and microbiological profiles and binding properties to human Fibrinogen (Fbg), Fibronectin (Fn) and type I collagen (col I) biotinylated proteins and virulence to Caenorhabditis elegans were investigated for C. ulcerans strain 2590 (clinical isolate) and two control strains, including PLD-positive BR-AD22 wild type and PLD-negative ELHA-1 PLD mutant strains. MALDI-TOF assays and a multiplex PCR of genes coding for potentially toxigenic corynebacteria identified strain 2590 as non-DT producing. Interestingly, strain 2590 did not express PLD activity in the CAMP test although the presence of the pld gene was verified. PLD-negative 2590 and a PLD-positive 210932 strains showed similar affinity to Fbg, Fn and type I collagen. C. elegans were able to escape from C. ulcerans strains, independent of PLD and DT production. Higher mortality of nematodes was verified for PLD-negative strains. Additional studies concerning multifactorial virulence potential of C. ulcerans, including environmental conditions remain necessary.

Induction of Necrosis in Human Macrophage Cell Lines by Corynebacterium diphtheriae and Corynebacterium ulcerans Strains Isolated from Fatal Cases of Systemic Infections.

When infecting a human host, Corynebacterium diphtheriae and Corynebacterium ulcerans are able to impair macrophage maturation and induce cell death. However, the underlying molecular mechanisms are not well understood. As a framework for this project, a combination of fluorescence microscopy, cytotoxicity assays, live cell imaging, and fluorescence-activated cell sorting was applied to understand the pathogenicity of two Corynebacterium strains isolated from fatal cases of systemic infections. The results showed a clear cytotoxic effect of the bacteria. The observed survival of the pathogens in macrophages and, subsequent, necrotic lysis of cells may be mechanisms explaining dissemination of C. diphtheriae and C. ulcerans to distant organs in the body.

Elimination of bacterial contaminations by treatment of water with boron-doped diamond electrodes.

Boron-doped diamond electrodes can be used to generate reactive oxygen species directly at the electrode's surface. This property was used in this study for in-situ electrochemical oxidation to eliminate different bacteria, i.e. Escherichia coli, Pseudomonas fluorescens and Pseudomonas aeruginosa, as well as Bacillus subtilis spores from water samples. Application of low voltages in the rage from 4 to 10 V and short incubation times in the range of minutes allowed a complete disinfection of water contaminated with enterobacteria and freshwater microbes including nosocomial pathogens as well as a significant reduction of spores. A pilot reactor was constructed, which allowed to decrease microbial contamination of sewage plant effluent drastically. Boron-doped diamond electrodes allow efficient reduction of bacterial contaminations in water samples.

The C-terminal coiled-coil domain of Corynebacterium diphtheriae DIP0733 is crucial for interaction with epithelial cells and pathogenicity in invertebrate animal model systems.

BACKGROUND: Corynebacterium diphtheriae is the etiologic agent of diphtheria and different systemic infections. The bacterium has been classically described as an extracellular pathogen. However, a number of studies revealed its ability to invade epithelial cells, indicating a more complex pathogen-host interaction. The molecular mechanisms controlling and facilitating internalization of C. diphtheriae still remains unclear. Recently, the DIP0733 transmembrane protein was found to play an important role in the interaction with matrix proteins and cell surfaces, nematode colonization, cellular internalization and induction of cell death. RESULTS: In this study, we identified a number of short linear motifs and structural elements of DIP0733 with putative importance in virulence, using bioinformatic approaches. A C-terminal coiled-coil region of the protein was considered particularly important, since it was found only in DIP0733 homologs in pathogenic Corynebacterium species but not in non-pathogenic corynebacteria. Infections of epithelial cells and transepithelial resistance assays revealed that bacteria expressing the truncated form of C. diphtheriae DIP0733 and C. glutamicum DIP0733 homolog are less virulent, while the fusion of the coiled-coil sequence to the DIP0733 homolog from C. glutamicum resulted in increased pathogenicity. These results were supported by nematode killing assays and experiments using wax moth larvae as invertebrate model systems. CONCLUSIONS: Our data indicate that the coil-coiled domain of DIP0733 is crucial for interaction with epithelial cells and pathogenicity in invertebrate animal model systems.

The role of corynomycolic acids in Corynebacterium-host interaction.

Within the Actinobacteria, the genera Corynebacterium, Mycobacterium, Nocardia and Rhodococcus form the so-called CMNR group, also designated as mycolic acid-containing actinomycetes. Almost all members of this group are characterized by a mycolic acid layer, the mycomembrane, which covers the cell wall and is responsible for a high resistance of these bacteria against chemical and antibiotic stress. Furthermore, components of the mycomembrane are crucial for the interaction of bacteria with host cells. This review summarizes the current knowledge of mycolic acid synthesis and interaction with components of the immune system for the genus Corynebacterium with an emphasis on the pathogenic species Corynebacterium diphtheriae, Corynebacterium pseudotuberculosis and Corynebacterium ulcerans as well as the biotechnology workhorse Corynebacterium glutamicum.

Synthesis and characterization of manganese containing mesoporous bioactive glass nanoparticles for biomedical applications.

Mesoporous bioactive glass (BG) nanoparticles based in the system: SiO2-P2O5-CaO-MnO were synthesized via a modified Stöber process at various concentrations of Mn (0-7 mol %). The synthesized manganese-doped BG nanoparticles were characterized in terms of morphology, composition, in vitro bioactivity and antibacterial activity. Scanning electron microscopy (SEM), transmission electron microscopy (TEM) and Brunauer-Emmett-Teller (BET) analysis confirmed that the particles had spherical morphology (mean particle size: 110 nm) with disordered mesoporous structure. Energy dispersive X-ray spectroscopy (EDX) confirmed the presence of Mn, Ca, Si and P in the synthesized Mn-doped BG particles. Moreover, X-ray diffraction (XRD) analysis showed that Mn has been incorporated in the amorphous silica network (bioactive glass). Moreover, it was found that manganese-doped BG particles form apatite crystals upon immersion in simulated body fluid (SBF). Inductively coupled plasma atomic emission spectroscopy (ICP-OES) measurements confirmed that Mn is released in a sustained manner, which provided antibacterial effect against Bacillus subtilis, Pseudomonas aeruginosa and Staphylococcus aureus. The results indicate that the incorporation of Mn in the bioactive glass network is an effective strategy to develop novel multifunctional BG nanoparticles for bone tissue engineering.

Toll-Like Receptor 2 and Mincle Cooperatively Sense Corynebacterial Cell Wall Glycolipids.

Nontoxigenic Corynebacterium diphtheriae and Corynebacterium ulcerans cause invasive disease in humans and animals. Host sensing of corynebacteria is largely uncharacterized, albeit the recognition of lipoglycans by Toll-like receptor 2 (TLR2) appears to be important for macrophage activation by corynebacteria. The members of the order Corynebacterineae (e.g., mycobacteria, nocardia, and rhodococci) share a glycolipid-rich cell wall dominated by mycolic acids (termed corynomycolic acids in corynebacteria). The mycolic acid-containing cord factor of mycobacteria, trehalose dimycolate, activates the C-type lectin receptor (CLR) Mincle. Here, we show that glycolipid extracts from the cell walls of several pathogenic and nonpathogenic Corynebacterium strains directly bound to recombinant Mincle in vitro Macrophages deficient in Mincle or its adapter protein Fc receptor gamma chain (FcRγ) produced severely reduced amounts of granulocyte colony-stimulating factor (G-CSF) and of nitric oxide (NO) upon challenge with corynebacterial glycolipids. Consistently, cell wall extracts of a particular C. diphtheriae strain (DSM43989) lacking mycolic acid esters neither bound Mincle nor activated macrophages. Furthermore, TLR2 but not TLR4 was critical for sensing of cell wall extracts and whole corynebacteria. The upregulation of Mincle expression upon encountering corynebacteria required TLR2. Thus, macrophage activation by the corynebacterial cell wall relies on TLR2-driven robust Mincle expression and the cooperative action of both receptors.

Genomic analysis of endemic clones of toxigenic and non-toxigenic Corynebacterium diphtheriae in Belarus during and after the major epidemic in 1990s.

BACKGROUND: Diphtheria remains a major public health concern with multiple recent outbreaks around the world. Moreover, invasive non-toxigenic strains have emerged globally causing severe infections. A diphtheria epidemic in the former Soviet Union in the 1990s resulted in ~5000 deaths. In this study, we analysed the genome sequences of a collection of 93 C. diphtheriae strains collected during and after this outbreak (1996 - 2014) in a former Soviet State, Belarus to understand the evolutionary dynamics and virulence capacities of these strains. RESULTS: C. diphtheriae strains from Belarus belong to ten sequence types (STs). Two major clones, non-toxigenic ST5 and toxigenic ST8, encompassed 76% of the isolates that are associated with sore throat and diphtheria in patients, respectively. Core genomic diversity is limited within outbreak-associated ST8 with relatively higher mutation rates (8.9 × 10-7 substitutions per strain per year) than ST5 (5.6 × 10-7 substitutions per strain per year) where most of the diversity was introduced by recombination. A variation in the virulence gene repertoire including the presence of tox gene is likely responsible for pathogenic differences between different strains. However, strains with similar virulence potential can cause disease in some individuals and remain asymptomatic in others. Eight synonymous single nucleotide polymorphisms were observed between the tox genes of the vaccine strain PW8 and other toxigenic strains of ST8, ST25, ST28, ST41 and non-toxigenic tox gene-bearing (NTTB) ST40 strains. A single nucleotide deletion at position 52 in the tox gene resulted in the frameshift in ST40 isolates, converting them into NTTB strains. CONCLUSIONS: Non-toxigenic C. diphtheriae ST5 and toxigenic ST8 strains have been endemic in Belarus both during and after the epidemic in 1990s. A high vaccine coverage has effectively controlled diphtheria in Belarus; however, non-toxigenic strains continue to circulate in the population. Recombination is an important evolutionary force in shaping the genomic diversity in C. diphtheriae. However, the relative role of recombination and mutations in diversification varies between different clones.

Functional characterization of the collagen-binding protein DIP2093 and its influence on host-pathogen interaction and arthritogenic potential of Corynebacterium diphtheriae.

Corynebacterium diphtheriae is typically recognized as the a etiological agent of diphtheria, a toxaemic infection of the respiratory tract; however, both non-toxigenic and toxigenic strains are increasingly isolated from cases of invasive infections. The molecular mechanisms responsible for bacterial colonization and dissemination to host tissues remain only partially understood. In this report, we investigated the role of DIP2093, described as a putative adhesin of the serine-aspartate repeat (Sdr) protein family in host-pathogen interactions of C. diphtheriae wild-type strain NCTC13129. Compared to the parental strain, a DIP2093 mutant RN generated in this study was attenuated in its ability to bind to type I collagen, to adhere to and invade epithelial cells, as well as to survive within macrophages. Furthermore, DIP2093 mutant strain RN had a less detrimental impact on the viability of Caenorhabditis elegans as well as in the clinical severity of arthritis in mice. In conclusion, DIP2093 functions as a microbial surface component recognizing adhesive matrix molecules, and may be included among the factors that contribute to the pathogenicity of C. diphtheriae strains, independently of toxin production.

Caenorhabditis elegans star formation and negative chemotaxis induced by infection with corynebacteria.

Caenorhabditis elegans is one of the major model systems in biology based on advantageous properties such as short life span, transparency, genetic tractability and ease of culture using an Escherichia coli diet. In its natural habitat, compost and rotting plant material, this nematode lives on bacteria. However, C. elegans is a predator of bacteria, but can also be infected by nematopathogenic coryneform bacteria such Microbacterium and Leucobacter species, which display intriguing and diverse modes of pathogenicity. Depending on the nematode pathogen, aggregates of worms, termed worm-stars, can be formed, or severe rectal swelling, so-called Dar formation, can be induced. Using the human and animal pathogens Corynebacterium diphtheriae and Corynebacterium ulcerans as well as the non-pathogenic species Corynebacterium glutamicum, we show that these coryneform bacteria can also induce star formation slowly in worms, as well as a severe tail-swelling phenotype. While C. glutamicum had a significant, but minor influence on survival of C. elegans, nematodes were killed after infection with C. diphtheriae and C. ulcerans. The two pathogenic species were avoided by the nematodes and induced aversive learning in C. elegans.