Live cell imaging of macrophage/bacterium interaction demonstrates cell lysis induced by Corynebacterium diphtheriae and Corynebacterium ulcerans.

OBJECTIVES: In frame of a study to characterize the interaction of human macrophage-like cells with pathogenic corynebacteria, Corynebacterium diphtheriae and Corynebacterium ulcerans, live cell imaging experiments were carried out and time lapse fluorescence microscopy videos were generated, which are presented here. DATA DESCRIPTION: The time lapse fluorescence microscopy data revealed new insights in the interaction of corynebacteria with human macrophage-like THP-1 cells. In contrast to uninfected cells and infections with non-pathogenic C. glutamicum used as a control, pathogenic C. diphtheriae and C. ulcerans showed highly detrimental effects towards human cells and induction of cell death of macrophages.

Second-generation IL-2 receptor-targeted diphtheria fusion toxin exhibits antitumor activity and synergy with anti-PD-1 in melanoma.

Denileukin diftitox (DAB-IL-2, Ontak) is a diphtheria-toxin-based fusion protein that depletes CD25-positive cells including regulatory T cells and has been approved for the treatment of persistent or recurrent cutaneous T cell lymphoma. However, the clinical use of denileukin diftitox was limited by vascular leak toxicity and production issues related to drug aggregation and purity. We found that a single amino acid substitution (V6A) in a motif associated with vascular leak induction yields a fully active, second-generation biologic, s-DAB-IL-2(V6A), which elicits 50-fold less human umbilical vein endothelial cell monolayer permeation and is 3.7-fold less lethal to mice by LD50 analysis than s-DAB-IL-2. Additionally, to overcome aggregation problems, we developed a production method for the fusion toxin using Corynebacterium diphtheriae that secretes fully folded, biologically active, monomeric s-DAB-IL-2 into the culture medium. Using the poorly immunogenic mouse B16F10 melanoma model, we initiated treatment 7 days after tumor challenge and observed that, while both s-DAB-IL-2(V6A) and s-DAB-IL-2 are inhibitors of tumor growth, the capacity to treat with higher doses of s-DAB-IL-2(V6A) could provide a superior activity window. In a sequential dual-therapy study in tumors that have progressed for 10 days, both s-DAB-IL-2(V6A) and s-DAB-IL-2 given before checkpoint inhibition with anti-programmed cell death-1 (anti-PD-1) antibodies inhibited tumor growth, while either drug given as monotherapy had less effect. s-DAB-IL-2(V6A), a fully monomeric protein with reduced vascular leak, is a second-generation diphtheria-toxin-based fusion protein with promise as a cancer immunotherapeutic both alone and in conjunction with PD-1 blockade.

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.

Multilocus Sequencing of Corynebacterium pseudotuberculosis Biotype Ovis Strains.

Thirteen Corynebacterium pseudotuberculosis biotype ovis strains isolated from clinical cases of caseous lymphadenitis in Hungary were characterised using multilocus sequencing and their phylogenetic comparison was carried out on the basis of four housekeeping genes (groEL1, infB, dnaK, and leuA). The in silico analysis of the 16 frequently studied housekeeping genes showed that C. pseudotuberculosis strains could be readily distinguished from C. diphtheriae and C. ulcerans strains; however, sequences of the same genes in the two biotypes of the C. pseudotuberculosis were highly similar; the heterogeneity values were low. Genes dnaK, infB, groEL1, and leuA showed marked genetic variation within C. pseudotuberculosis, and strains of the two biotypes of C. pseudotuberculosis could be differentiated. Analysis of the individual genes showed a fairly conservative nature of C. pseudotuberculosis biotype ovis strains. The greatest genetic differentiation was seen in the dnaK and infB genes and concatenations of these two genes were very useful in the genetic separation of the studied strains.

Informe especial: Actualización epidemiológica de difteria / Special report: Epidemiological update of diphtheria

La difteria es una enfermedad infecciosa grave, con potencial epidémico, para la que existe una vacuna eficaz. Es una infección bacteriana aguda causada por la liberación de una exotoxina específica, producida por el bacilo gram positivo, Corynebacterium diphtheriae. Sólo las cepas toxigénicas causan enfermedad. La enfermedad se presenta principalmente en menores de 15 años de edad sin vacunación o con vacunación incompleta; no obstante puede presentarse en adultos en áreas con baja cobertura de vacunación. En este informe se presentan distintos aspectos de la enfermedad y la situación epidemiológica en las Américas y en Argentina: definiciones de caso, medidas de prevención y de control

Analysis of Corynebacterium diphtheriae macrophage interaction: Dispensability of corynomycolic acids for inhibition of phagolysosome maturation and identification of a new gene involved in synthesis of the corynomycolic acid layer.

Corynebacterium diphtheriae is the causative agent of diphtheria, a toxin mediated disease of upper respiratory tract, which can be fatal. As a member of the CMNR group, C. diphtheriae is closely related to members of the genera Mycobacterium, Nocardia and Rhodococcus. Almost all members of these genera comprise an outer membrane layer of mycolic acids, which is assumed to influence host-pathogen interactions. In this study, three different C. diphtheriae strains were investigated in respect to their interaction with phagocytic murine and human cells and the invertebrate infection model Caenorhabditis elegans. Our results indicate that C. diphtheriae is able to delay phagolysosome maturation after internalization in murine and human cell lines. This effect is independent of the presence of mycolic acids, as one of the strains lacked corynomycolates. In addition, analyses of NF-κB induction revealed a mycolate-independent mechanism and hint to detrimental effects of the different strains tested on the phagocytic cells. Bioinformatics analyses carried out to elucidate the reason for the lack of mycolates in one of the strains led to the identification of a new gene involved in mycomembrane formation in C. diphtheriae.

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.

Caso inusual de difteria cutánea con compromiso mucoso y síndrome del vértex orbitario / Unusual clinical presentation of cutaneous diphteria with mucosal involvement and orbital Vertex syndrome

La difteria es una enfermedad prevenible por vacunas. En la actualidad existe un brote en nuestro país que se inició en abril de 2016. Desde entonces se han descrito casos en diferentes sitios del país predominantemente de la forma faríngea. La difteria cutánea no ha sido reportada en este brote. A nivel mundial el compromiso simultáneo en piel y mucosas, no ha sido descrito. La paciente cuya enfermedad describimos aquí es un caso de difteria de presentación atípica que se inicia con expresión cutánea y evoluciona a compromiso nasal, ocular y faríngeo con complicaciones severas en la órbita ocular expresadas como un síndrome del vértex orbitario, en una paciente con diabetes mellitus tipo 2 no controlada, que tuvo una evolución tórpida que la llevó a la muerte. Se revisa la literatura(AU)

Diphtheria is a vaccine-preventable disease. Actually, there is an outbreak in Venezuela since April 2016. Cases in different parts of the country have been described, predominantly pharyngeal. Cutaneous diphtheria has not been reported in this outbreak. The simultaneous presentation of the cutaneous form with mucosal involvement has not been described in the revised literature. We present a case of diphtheria with atypical presentation that begins with cutaneous expression and evolves to nasal, ocular and pharyngeal compromise with severe complications of the orbit, expressed in an orbital vertex syndrome in a patient with type 2 diabetes mellitus, who had a torpid evolution that led her to death. The literature is reviewed(AU)

Pathogenic properties of a Corynebacterium diphtheriae strain isolated from a case of osteomyelitis.

Corynebacterium diphtheriae is typically recognized as a colonizer of the upper respiratory tract (respiratory diphtheria) and the skin (cutaneous diphtheria). However, different strains of Corynebacteriumdiphtheriae can also cause invasive infections. In this study, the characterization of a non-toxigenic Corynebacteriumdiphtheriae strain (designated BR-INCA5015) isolated from osteomyelitis in the frontal bone of a patient with adenoid cystic carcinoma was performed. Pathogenic properties of the strain BR-INCA5015 were tested in a Caenorhabditis elegans survival assay showing strong colonization and killing by this strain. Survival rates of 3.8±2.7 %, 33.6±7.3 % and 0 % were observed for strains ATCC 27010T, ATCC 27012 and BR-INCA5015, respectively, at day 7. BR-INCA5015 was able to colonize epithelial cells, showing elevated capacity to adhere to and survive within HeLa cells compared to other Corynebacteriumdiphtheriae isolates. Intracellular survival in macrophages (THP-1 and RAW 264.7) was significantly higher compared to control strains ATCC 27010T (non-toxigenic) and ATCC 27012 (toxigenic). Furthermore, the ability of BR-INCA5015 to induce osteomyelitis was confirmed by in vivo assay using Swiss Webster mice.

Genomic analysis of a nontoxigenic, invasive Corynebacterium diphtheriae strain from Brazil

We report the complete genome sequence and analysis of an invasive Corynebacterium diphtheriae strain that caused endocarditis in Rio de Janeiro, Brazil. It was selected for sequencing on the basis of the current relevance of nontoxigenic strains for public health. The genomic information was explored in the context of diversity, plasticity and genetic relatedness with other contemporary strains.

A thiol-disulfide oxidoreductase of the Gram-positive pathogen Corynebacterium diphtheriae is essential for viability, pilus assembly, toxin production and virulence.

The Gram-positive pathogen Corynebacterium diphtheriae exports through the Sec apparatus many extracellular proteins that include the key virulence factors diphtheria toxin and the adhesive pili. How these proteins attain their native conformations after translocation as unfolded precursors remains elusive. The fact that the majority of these exported proteins contain multiple cysteine residues and that several membrane-bound oxidoreductases are encoded in the corynebacterial genome suggests the existence of an oxidative protein-folding pathway in this organism. Here we show that the shaft pilin SpaA harbors a disulfide bond in vivo and alanine substitution of these cysteines abrogates SpaA polymerization and leads to the secretion of degraded SpaA peptides. We then identified a thiol-disulfide oxidoreductase (MdbA), whose structure exhibits a conserved thioredoxin-like domain with a CPHC active site. Remarkably, deletion of mdbA results in a severe temperature-sensitive cell division phenotype. This mutant also fails to assemble pilus structures and is greatly defective in toxin production. Consistent with these defects, the ΔmdbA mutant is attenuated in a guinea pig model of diphtheritic toxemia. Given its diverse cellular functions in cell division, pilus assembly and toxin production, we propose that MdbA is a component of the general oxidative folding machine in C. diphtheriae.

Corynebacterium diphtheriae putative tellurite-resistance protein (CDCE8392_0813) contributes to the intracellular survival in human epithelial cells and lethality of Caenorhabditis elegans

Corynebacterium diphtheriae, the aetiologic agent of diphtheria, also represents a global medical challenge because of the existence of invasive strains as causative agents of systemic infections. Although tellurite (TeO3^2-) is toxic to most microorganisms, TeO3^2--resistant bacteria, including C. diphtheriae, exist in nature. The presence of TeO3^2--resistance (Te^R) determinants in pathogenic bacteria might provide selective advantages in the natural environment. In the present study, we investigated the role of the putative Te^R determinant (CDCE8392_813gene) in the virulence attributes of diphtheria bacilli. The disruption of CDCE8392_0813 gene expression in the LDCIC-L1 mutant increased susceptibility to TeO3^2- and reactive oxygen species (hydrogen peroxide), but not to other antimicrobial agents. The LDCIC-L1 mutant also showed a decrease in both the lethality of Caenorhabditis elegansand the survival inside of human epithelial cells compared to wild-type strain. Conversely, the haemagglutinating activity and adherence to and formation of biofilms on different abiotic surfaces were not regulated through the CDCE8392_0813 gene. In conclusion, the CDCE8392_813 gene contributes to the Te^R and pathogenic potential of C. diphtheriae.

Corynebacterium diphtheriae putative tellurite-resistance protein (CDCE8392_0813) contributes to the intracellular survival in human epithelial cells and lethality of Caenorhabditis elegans.

Corynebacterium diphtheriae, the aetiologic agent of diphtheria, also represents a global medical challenge because of the existence of invasive strains as causative agents of systemic infections. Although tellurite (TeO32-) is toxic to most microorganisms, TeO32--resistant bacteria, including C. diphtheriae, exist in nature. The presence of TeO32--resistance (TeR) determinants in pathogenic bacteria might provide selective advantages in the natural environment. In the present study, we investigated the role of the putative TeR determinant (CDCE8392_813gene) in the virulence attributes of diphtheria bacilli. The disruption of CDCE8392_0813 gene expression in the LDCIC-L1 mutant increased susceptibility to TeO32- and reactive oxygen species (hydrogen peroxide), but not to other antimicrobial agents. The LDCIC-L1 mutant also showed a decrease in both the lethality of Caenorhabditis elegans and the survival inside of human epithelial cells compared to wild-type strain. Conversely, the haemagglutinating activity and adherence to and formation of biofilms on different abiotic surfaces were not regulated through the CDCE8392_0813 gene. In conclusion, the CDCE8392_813 gene contributes to the TeR and pathogenic potential of C. diphtheriae.

Characterization of DIP0733, a multi-functional virulence factor of Corynebacterium diphtheriae.

Corynebacterium diphtheriae is typically recognized 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 Cor. diphtheriae are poorly understood. In this study, we investigated the role of DIP0733 as virulence factor to elucidate how it contributes to the process of pathogen-host cell interaction. Based on in vitro experiments, it was suggested recently that the DIP0733 protein might be involved in adhesion, invasion of epithelial cells and induction of apoptosis. A corresponding Cor. diphtheriae mutant strain generated in this study was attenuated in its ability to colonize and kill the host in a Caenorhabditis elegans infection model system. Furthermore, the mutant showed an altered adhesion pattern and a drastically reduced ability to adhere and invade epithelial cells. Subsequent experiments showed an influence of DIP0733 on binding of Cor. diphtheriae to extracellular matrix proteins such as collagen and fibronectin. Furthermore, based on its fibrinogen-binding activity, DIP0733 may play a role in avoiding recognition of Cor. diphtheriae by the immune system. In summary, our findings support the idea that DIP0733 is a multi-functional virulence factor of Cor. diphtheriae.

[Adhesion of corynebacterium diphtheriae: the role of surface structures and formation mechanism].

The paper is devoted to the study of surface structures including pili (fimbriae) 67-72p surface protein, DIP 1281 surface protein, lipoarabinomannan CdiLAM and their role in the adhesion and colonization of the mucous membrane of the throat by Corynebacterium diphtheriae. A description is offered for the main stages in the adhesion process of diphtheria causative agent and the ability of its adhesins to stimulate the effect of innate and acquired immunity factors. The paper stresses prospectiveness of the development of vaccines forming immunoprotection of the organism against adhesive activity of C. diphtheriae and also preventing their colonization and reproduction. That would facilitate a solution for the problem of diphtheria carrier state, which cannot be solved using the existing means of preventive vaccination.

Pilus gene pool variation and the virulence of Corynebacterium diphtheriae clinical isolates during infection of a nematode.

Toxigenic Corynebacterium diphtheriae strains cause diphtheria in humans. The toxigenic C. diphtheriae isolate NCTC13129 produces three distinct heterotrimeric pili that contain SpaA, SpaD, and SpaH, making up the shaft structure. The SpaA pili are known to mediate bacterial adherence to pharyngeal epithelial cells. However, to date little is known about the expression of different pili in various clinical isolates and their importance in bacterial pathogenesis. Here, we characterized a large collection of C. diphtheriae clinical isolates for their pilin gene pool by PCR and for the expression of the respective pilins by immunoblotting with antibodies against Spa pilins. Consistent with the role of a virulence factor, the SpaA-type pili were found to be prevalent among the isolates, and most significantly, corynebacterial adherence to pharyngeal epithelial cells was strictly correlated with isolates that were positive for the SpaA pili. By comparison, the isolates were heterogeneous for the presence of SpaD- and SpaH-type pili. Importantly, using Caenorhabditis elegans as a model host for infection, we show here that strain NCTC13129 rapidly killed the nematodes, the phenotype similar to isolates that were positive for toxin and all pilus types. In contrast, isogenic mutants of NCTC13129 lacking SpaA-type pili or devoid of toxin and SpaA pili exhibited delayed killing of nematodes with similar kinetics. Consistently, nontoxigenic or toxigenic isolates that lack one, two, or all three pilus types were also attenuated in virulence. This work signifies the important role of pili in corynebacterial pathogenesis and provides a simple host model to identify additional virulence factors.

Induction of the NFκ-B signal transduction pathway in response to Corynebacterium diphtheriae infection.

Corynebacterium diphtheriae, the causative agent of diphtheria, has been thoroughly studied with respect to toxin production and pili formation, while knowledge on host responses to C. diphtheriae infection is limited. In this study, we studied adhesion to and invasion of epithelial cells by different C. diphtheriae isolates. When NFκ-B reporter cell lines were used to monitor the effect of C. diphtheriae infection on human cells, strain-specific differences were observed. While adhesion to host cells had no effect, a correlation of invasion rate with NFκ-B induction was found, which indicates that internalization of bacteria is crucial for NFκ-B induction. Immunofluorescence microscopy experiments used to support the reporter assays showed that translocation of p65, as a hallmark of NFκ-B induction, was only observed in association with cell invasion by C. diphtheriae. Our data indicate that the response of epithelial cells to C. diphtheriae infection is determined by internalization of bacteria and that invasion of these cells is an active process; tetracycline-treated C. diphtheriae was still able to attach to host cells, but lost its ability to invade the cytoplasm. Recognition of pathogen-associated molecular patterns such as pili subunits by membrane-bound receptors facing the outside of the cell is not sufficient for NFκ-B induction.

Propriedades adesivas a substratos abióticos e bióticos, invasão e indução de apoptose celular de Corynebacterium pseudodiphtheriticum / Adhesive properties to abiotic and biotic substrates, invasion and induction of apoptosis of Corynebacterium pseudodiphtheriticum

A ocorrência de fenótipos multirresistentes de Corynebacterium pseudodiphtheriticum e sua associação a infecções graves, com elevada mortalidade em pacientes imunocomprometidos, aliados ao escasso conhecimento da virulência e patogenia destas infecções, motivou esta pesquisa, que teve como objetivo investigar mecanismos de virulência e resistência microbiana deste agente entre pacientes de um hospital universitário brasileiro. Um total de 113 amostras de C. pseudodiphtheriticum identificadas por métodos bioquímicos convencionais e sistema API-Coryne isoladas de pacientes de diferentes grupos etários. Os micro-organismos eram, em sua maioria, relacionados a infecções no trato respiratório (27,45%), urinário (29,20%) e sitios intravenosos (18,60%) e cerca de 32,70% das amostras foram provenientes de pacientes com pelo menos uma das condições predisponentes: insuficiência renal; transplante renal, tuberculose em paciente HIV+, câncer, cirrose hepática, hemodiálise e uso de cateter. As amostras testadas revelaram-se multirresistentes sendo a maioria resistente à oxacilina, eritromicina e clindamicina. A adesão das cepas ao poliestireno e ao poliuretano indicou o envolvimento de hidrofobicidade da superfície celular na fase inicial da formação de biofilmes. O crescimento subsequente conduziu à formação de microcolônias, agregados bacterianos densos incorporados na matriz exopolimérica rodeada por espaços vazios, típica de biofilmes maduros...

The occurrence of multiresistant phenotypes and associated with severe infections, with high mortality in immunocompromised hosts due to Corynebacterium pseudodiphtheriticum, allied to little known about virulence and pathogenesis these infections, led to present investigation. The investigation aims to examine the virulence mechanisms and resistance to antimicrobial agents of C. pseudodiphtheriticum among patients with bacterial infections at a Brazilian teaching hospital. A total of 113 C. pseudodiphtheriticum strains identified by conventional biochemical methods and API-Coryne System were recovered from patients from different age groups. Micro-organisms were mostly related to infections in the respiratory tracts (27.45%), urinary (29.20%) and intravenous sites (18.60%) and approximately 32.70% samples were obtained of patients presenting at least one of the pre-disposing conditions: end-stage renal disease; renal transplant; AIDS and Mycobacterium tuberculosis infection; cancer, hepatic cirrhosis; haemodialysis and catheter use. Antimicrobial susceptibility tests identified multiresistant phenotypes. Most strains were resistant to oxacillin, erythromycin and clindamycin. Adherence to polystyrene and polyurethane indicated the involvement of cell surface hydrophobicity in the initial stage of biofilm formation. Further growth led to the formation of dense bacterial aggregates embedded in the exopolymeric matrix surrounded by voids, typical of mature biofilms...

Evaluation of invertebrate infection models for pathogenic corynebacteria.

For several pathogenic bacteria, model systems for host-pathogen interactions were developed, which provide the possibility of quick and cost-effective high throughput screening of mutant bacteria for genes involved in pathogenesis. A number of different model systems, including amoeba, nematodes, insects, and fish, have been introduced, and it was observed that different bacteria respond in different ways to putative surrogate hosts, and distinct model systems might be more or less suitable for a certain pathogen. The aim of this study was to develop a suitable invertebrate model for the human and animal pathogens Corynebacterium diphtheriae, Corynebacterium pseudotuberculosis, and Corynebacterium ulcerans. The results obtained in this study indicate that Acanthamoeba polyphaga is not optimal as surrogate host, while both Caenorhabtitis elegans and Galleria larvae seem to offer tractable models for rapid assessment of virulence between strains. Caenorhabtitis elegans gives more differentiated results and might be the best model system for pathogenic corynebacteria, given the tractability of bacteria and the range of mutant nematodes available to investigate the host response in combination with bacterial virulence. Nevertheless, Galleria will also be useful in respect to innate immune responses to pathogens because insects offer a more complex cell-based innate immune system compared with the simple innate immune system of C. elegans.