Occurrence of Prototheca Microalgae in Aquatic Ecosystems with a Description of Three New Species, Prototheca fontanea, Prototheca lentecrescens, and Prototheca vistulensis.

Prototheca species are unicellular, nonphotosynthetic, saprophytic, and occasionally pathogenic, microalgae, with an extensive environmental reservoir. This study explores, for the first time, the occurrence of Prototheca in aquatic ecosystems by using a molecular profiling approach. A total of 362 samples were collected from 80 natural and artificial waterbodies at 88 sampling sites in 26 localities across Poland during a 1.5-year period. The overall isolation rate of Prototheca from water environments was 14.1%. Prototheca were most prevalent in rivers of urbanized areas, indicating that the algae are primarily adapted to lotic ecosystems with a high input of organic matter. Interestingly, it is not the amount of organic matter per se but its quality that seems to shape the habitat potential of the protothecae. The two most frequently isolated species were P. wickerhamii and P. pringsheimii, representing a third and a fourth of the strains, respectively. Additionally, three novel species were described, namely, P. fontanea, P. lentecrescens, and P. vistulensis. The high species diversity of the genus Prototheca may reflect the complexity of water ecosystems along with ecological and functional adaptations of the algae to such environments. For further investigations, the study provides a revised scheme for identification of all 18 Prototheca species currently recognized. IMPORTANCE The study investigates the occurrence of very rare and poorly studied microalgae of the genus Prototheca, potentially pathogenic to humans and animals, in different water environments. Given the potential hazard to human and animal health from exposure to water-inhabiting protothecae, the prevalence of the algae in aquatic habitats deserves an insightful examination. The study is the first since the 1980s to explore the aquatic habitat of Prototheca spp. and the first ever performed to do this by molecular methods. Although the Prototheca isolation rate was low, a high species diversity was observed. The algae appear to represent allochthonous microflora, brought into waterbodies from various anthropogenic sources. Large rivers of urbanized areas were the most Prototheca-abundant. The study provides a description of three new Prototheca species, namely, P. fontanea, P. lentecrescens, and P. vistulensis. The study also delivers a new identification scheme for all Prototheca species currently recognized.

Drug Susceptibility Profiling and Genetic Determinants of Drug Resistance in Mycobacterium kansasii.

Very few studies have examined drug susceptibility of Mycobacterium kansasii, and they involve a limited number of strains. The purpose of this study was to determine drug susceptibility profiles of M. kansasii isolates representing a spectrum of species genotypes (subtypes) with two different methodologies, i.e., broth microdilution and Etest assays. To confirm drug resistance, drug target genes were sequenced. A collection of 85 M. kansasii isolates, including representatives of eight different subtypes (I to VI, I/II, and IIB) from eight countries, was used. Drug susceptibility against 13 and 8 antimycobacterial agents was tested by using broth microdilution and Etest, respectively. For drug-resistant or high-MIC isolates, eight structural genes (rrl, katG, inhA, embB, rrs, rpsL, gyrA, and gyrB) and one regulatory region (embCA) were PCR amplified and sequenced in the search for resistance-associated mutations. All isolates tested were susceptible to rifampin (RIF), amikacin (AMK), co-trimoxazole (SXT), rifabutin (RFB), moxifloxacin (MXF), and linezolid (LZD) according to the microdilution method. Resistance to ethambutol (EMB), ciprofloxacin (CIP), and clarithromycin (CLR) was found in 83 (97.7%), 17 (20%), and 1 (1.2%) isolate, respectively. The calculated concordance between the Etest and dilution method was 22.6% for AMK, 4.8% for streptomycin (STR), 3.2% for CLR, and 1.6% for RIF. For EMB, INH, and SXT, not even a single MIC value determined by one method equaled that by the second method. The only mutations disclosed were A2266C transversion at the rrl gene (CLR-resistant strain) and A128G transition at the rpsL gene (strain with STR MIC of >64 mg/liter). In conclusion, eight drugs, including RIF, CLR, AMK, SXT, RFB, MXF, LZD, and ethionamide (ETO), showed high in vitro activity against M. kansasii isolates. Discrepancies of the results between the reference microdilution method and Etest preclude the use of the latter for drug susceptibility determination in M. kansasii Drug resistance in M. kansasii may have different genetic determinants than resistance to the same drugs in M. tuberculosis.

Characterization of Mutations Conferring Resistance to Rifampin in Mycobacterium tuberculosis Clinical Strains.

Resistance of Mycobacterium tuberculosis to rifampin (RMP), mediated by mutations in the rpoB gene coding for the beta-subunit of RNA polymerase, poses a serious threat to the efficacy of clinical management and, thus, control programs for tuberculosis (TB). The contribution of many individual rpoB mutations to the development and level of RMP resistance remains elusive. In this study, the incidence of mutations throughout the rpoB gene among 115 Mycobacterium tuberculosis clinical isolates, both resistant and susceptible to RMP, was determined. Of the newly discovered rpoB mutations, the role of three substitutions in the causation of RMP resistance was empirically tested. The results from in vitro mutagenesis experiments were combined with the assessment of the prevalence of rpoB mutations, and their reciprocal co-occurrences, across global M. tuberculosis populations. Twenty-two different types of mutations in the rpoB gene were identified and distributed among 58 (89.2%) RMP-resistant strains. The MICs of RMP were within the range of 40 to 800 mg/liter, with MIC50 and MIC90 values of 400 and 800 mg/liter, respectively. None of the mutations (Gln429His, Met434Ile, and Arg827Cys) inspected for their role in the development of RMP resistance produced an RMP-resistant phenotype in isogenic M. tuberculosis H37Rv strain-derived mutants. These mutations are supposed to compensate for fitness impairment incurred by other mutations directly associated with drug resistance.

Mutation profiling for detection of isoniazid resistance in Mycobacterium tuberculosis clinical isolates.

OBJECTIVES: Progress in the detection of drug-resistant TB has been underpinned by the development and implementation of new, reliable and rapid diagnostic tools. These rely mostly on the detection of specific mutations conferring resistance to anti-TB drugs. The aim of this study was to search for mutations associated with isoniazid resistance among Mycobacterium tuberculosis clinical isolates. METHODS: A collection of 150 M. tuberculosis strains, including 50 MDR, 50 isoniazid-monoresistant and 50 pan-susceptible strains, was used. For all the strains, seven structural genes (katG, inhA, ahpC, kasA, ndh, nat and mshA) and two regulatory regions (mabA-inhA promoter and oxyR-ahpC intergenic region) were PCR amplified and sequenced in their entirety. RESULTS: Sixty-six distinct mutations were detected at all nine loci investigated, accounting for 109 (72.7%) of the strains tested. The number of strains with any mutation among the MDR, isoniazid-monoresistant and pan-susceptible groups was 49 (98%), 37 (74%) and 23 (46%), respectively. Mutations in the katG gene predominated, with 29 different types distributed among 46 (92%) MDR, 31 (62%) isoniazid-monoresistant and 2 (4%) pan-susceptible strains. Twenty-nine and 19 mutations were found exclusively in MDR and isoniazid-monoresistant strains, respectively. CONCLUSIONS: This study revealed 17 mutations, previously unreported, that might be of potential use as new surrogate markers of isoniazid resistance. Their diagnostic accuracy needs to be confirmed on larger strain samples and from different geographical settings. For isoniazid resistance detection, molecular approaches should still be a complement to rather than a replacement for conventional drug susceptibility testing. This is supported by the lack of mutations in any of the nine genetic loci investigated in 18 isoniazid-resistant strains from this study.

Lmo0171, a novel internalin-like protein, determines cell morphology of Listeria monocytogenes and its ability to invade human cell lines.

Internalins comprise a class of Listeria monocytogenes proteins responsible for activation of signalling pathways leading to phagocytic uptake of the bacterium by the host cell. In this paper, a possible role of Lmo0171-a new member of the internalin family was investigated. Disruption of the lmo0171 gene resulted in important cell morphology alterations along with a decrease in the ability to invade three eukaryotic cell lines, that is Int407, Hep-2 and HeLa and diminished adhesion efficiency to int407, thereby suggesting bifunctionality of the newly characterised Lmo0171 internalin.

Cytotoxicity of purified listeriolysin O on mouse and human leukocytes and leukaemia cells.

BACKGROUND: Listeriolysin O (LLO) is the main virulence factor of Listeria monocytogenes and facilitates the intracellular survival of the pathogen. Some of its characteristics endorse the growing popularity of LLO for use in biotechnology, particularly in the development of novel vaccines. Here, we evaluate the use of LLO to eradicate leukaemia cells. RESULTS: A purified LLO preparation was obtained by affinity chromatography. The LLO preparation procedure was optimized and purified LLO was tested for optimal conditions of storage including temperature, application of proteinase inhibitors and serum components. We demonstrated the possibility of regulating LLO activity by adjusting cell membrane cholesterol content. The LLO preparation had haemolytic activity and had a cytotoxic effect on the human T-leukaemia Jurkat cell line as well as mouse and human peripheral blood mononuclear cells. CONCLUSIONS: LLO has a very potent cytotoxic activity towards human leukocytes. Importantly, the cytotoxic activity was easily regulated in vitro and could be restricted to areas containing malignant cells, raising the possibility of future clinical application of LLO for leukaemia treatment.

Detection of mutations associated with isoniazid resistance in multidrug-resistant Mycobacterium tuberculosis clinical isolates.

OBJECTIVES: To determine the prevalence of isoniazid resistance-conferring mutations among multidrug-resistant (MDR) isolates of Mycobacterium tuberculosis from Poland. METHODS: Nine genetic loci, including structural genes (katG, inhA, ahpC, kasA, ndh, nat and mshA) and regulatory regions (i.e. the mabA-inhA promoter and oxyR-ahpC intergenic region) of 50 MDR M. tuberculosis isolates collected throughout Poland were PCR-amplified in their entirety and screened for mutations by direct sequencing methodology. RESULTS: Forty-six (92%) MDR M. tuberculosis isolates had mutations in the katG gene, and the katG Ser315Thr substitution predominated (72%). Eight (16%) isolates (six with a mutated katG allele) had mutations in the inhA promoter region and two such isolates also had single inhA structural gene mutations. Mutations in the oxyR-ahpC locus were found in five (10%) isolates, of which all but one had at least one additional mutation in katG. Mutations in the remaining genetic loci (kasA, ndh, nat and mshA) were detected in 12 (24%), 4 (8%), 5 (10%) and 17 (34%) MDR isolates, respectively. All non-synonymous mutants for these genes harboured mutations in katG. One isolate had no mutations in any of the analysed loci. CONCLUSIONS: This study accentuates the usefulness of katG and inhA promoter mutations as predictive markers of isoniazid resistance. Testing only for katG 315 and inhA -15 mutations would detect isoniazid resistance in 84% of the MDR M. tuberculosis sample. This percentage would increase to 96% if the sequence analysis was extended to the entire katG gene. Analysis of the remaining genetic loci did not contribute greatly to the identification of isoniazid resistance.

Distribution of Malassezia species on the skin of patients with atopic dermatitis, psoriasis, and healthy volunteers assessed by conventional and molecular identification methods.

BACKGROUND: The Malassezia yeasts which belong to the physiological microflora of human skin have also been implicated in several dermatological disorders, including pityriasis versicolor (PV), atopic dermatitis (AD), and psoriasis (PS). The Malassezia genus has repeatedly been revised and it now accommodates 14 species, all but one being lipid-dependent species. The traditional, phenotype-based identification schemes of Malassezia species are fraught with interpretative ambiguities and inconsistencies, and are thus increasingly being supplemented or replaced by DNA typing methods. The aim of this study was to explore the species composition of Malassezia microflora on the skin of healthy volunteers and patients with AD and PS. METHODS: Species characterization was performed by conventional, culture-based methods and subsequently molecular techniques: PCR-RFLP and sequencing of the internal transcribed spacer (ITS) 1/2 regions and the D1/D2 domains of the 26S rRNA gene. The Chi-square test and Fisher's exact test were used for statistical analysis. RESULTS: Malassezia sympodialis was the predominant species, having been cultured from 29 (82.9%) skin samples collected from 17 out of 18 subjects under the study. Whereas AD patients yielded exclusively M. sympodialis isolates, M. furfur isolates were observed only in PS patients. The isolation of M. sympodialis was statistically more frequent among AD patients and healthy volunteers than among PS patients (P < 0.03). Whether this mirrors any predilection of particular Malassezia species for certain clinical conditions needs to be further evaluated. The overall concordance between phenotypic and molecular methods was quite high (65%), with the discordant results being rather due to the presence of multiple species in a single culture (co-colonization) than true misidentification. All Malassezia isolates were susceptible to cyclopiroxolamine and azole drugs, with M. furfur isolates being somewhat more drug tolerant than other Malassezia species. CONCLUSIONS: This study provides an important insight into the species composition of Malassezia microbiota in human skin. The predominance of M. sympodialis in both normal and pathologic skin, contrasts with other European countries, reporting M. globosa and M. restricta as the most frequently isolated Malassezia species.

Increase in resistance to fluconazole and itraconazole in Trichophyton rubrum clinical isolates by sequential passages in vitro under drug pressure.

Trichophyton rubrum, an anthropophilic dermatophyte fungus, is the predominant causative agent of superficial skin infections in human population. There are only scanty reports on drug susceptibility profiling of T. rubrum. Neither mechanisms for drug resistance development nor correlation between in vitro drug susceptibility and in vivo response to treatment is known for that species. In this study, changes in the in vitro susceptibilities to fluconazole (FLZ) and itraconazole (ITZ) among thirty T. rubrum clinical strains subjected to sequential passages in the presence or absence of the azoles were investigated. Each strain was passaged 12 times at 4-week intervals as three parallel cultures, maintained on a drug-free medium (1), and a medium containing FLZ (2) or ITZ (3) at subinhibitory concentrations. Susceptibility to FLZ and ITZ of the original strain and its 3 subcultures was determined by microdilution method. The MIC values of the two azoles remained unaltered for all T. rubrum strains tested, after 12 passages on a drug-free medium. Among the strains grown with FLZ, an increase in the MICs of FLZ and ITZ was noted in 17 (56.7 %) and 19 (63.3 %) strains, respectively. Increased MICs of ITZ and FLZ were demonstrated for 24 (80 %) and 20 (66.7 %) strains that were propagated with ITZ. The results indicate the capacity of T. rubrum to develop resistance toward the azoles after prolonged exposure to these drugs. Resistance of T. rubrum to azoles plays an important role in therapy failures and consequently contributes to persistence and chronicity of the infections.

Identification of Scopulariopsis species by partial 28S rRNA gene sequence analysis.

The genus Scopulariopsis contains over 30 species of mitosporic moulds, which although usually saprophytic may also act as opportunistic pathogens in humans. They have mainly been associated with onychomycosis, and only sporadically reported as a cause of deep tissue infections or systemic disease. Identification of Scopulariopsis species still largely relies on phenotype-based methods. There is a need for a molecular diagnostic approach, that would allow to reliably discriminate between different Scopulariopsis species. The aim of this study was to apply sequence analysis of partial 28S rRNA gene for species identification of Scopulariopsis clinical isolates. Although the method employed did reveal some genetic polymorphism among Scopulariopsis isolates tested, it was not enough for species delineation. For this to be achieved, other genetic loci, within and beyond the rDNA operon, need to be investigated.

Identification and analysis of mutations in the katG gene in multidrug-resistant Mycobacterium tuberculosis clinical isolates.

INTRODUCTION: A major role in the development of resistance of Mycobacterium tuberculosis to isoniazid (INH) is attributed to mutations in the katG gene coding for the catalase/peroxidase, an enzyme required for obtaining a pharmacologically active form of the drug. Analysis of mutations in the katG gene in M. tuberculosis strains may contribute to the development of reliable and rapid tests for detection of INH resistance. The aim of the study was to identify and characterize mutations in the katG gene in multidrug-resistant M. tuberculosis clinical isolates. MATERIAL AND METHODS: The study included 46 strains of M. tuberculosis, recovered from MDR-TB patients in Poland in 2004. Mutations in the katG gene were detected by comparing DNA sequences with the corresponding sequence of a wild-type reference laboratory strain (M. tuberculosis H37Rv). The obtained results were interpreted in the context of MIC values of INH and catalase activity of the strains tested. RESULTS: A total of 43 (93%) strains contained mutations in the katG gene. The most frequently observed were mutations at codon 315, found in 34 (74%) strains. Mutations at other codons were rare: 4 strains contained mutations at codon 463, 2 at codon 131 and another 2 at codon 234. Mutations at codons 68, 91, 101, 126, 128 and 194 were found in single strains only. Two strains, for which no mutations at codon 315 of the katG gene were identified, had a unique translation termination mutation, which would invariably result in polypeptide truncation leading to the generation of dysfunctional catalase polypeptides. Both these strains presented the highest MIC values for INH (80 and 100 µg/mL) and showed a complete loss of catalase activity. For the remaining 41 strains with katG mutations, the MICs of INH were within the range 0.2-10 µg/mL. Thirty-six (88%) of those strains retained their catalase activity. CONCLUSIONS: Mutations at codon 315 within the katG gene, depending on their type might be useful for the prediction of INH resistance. Whereas the missense mutations do not affect the catalase activity or the level of INH resistance, the nonsense mutations result in high-level resistance to INH and a total loss of catalase activity.

Multicentre Etest evaluation of in vitro activity of conventional antifungal drugs against European bovine mastitis Prototheca spp. isolates.

OBJECTIVES: Bovine mammary protothecosis is a serious pathology that entails high economic losses in the dairy industry. The disease, the frequency of which has recently been increasing worldwide, is caused by unicellular, achlorophyllous, yeast-like algae of two species: Prototheca zopfii and Prototheca blaschkeae. The objective of this study was to investigate the in vitro activity of a panel of conventional antifungal drugs against Prototheca spp. isolates. METHODS: A total of 144 P. zopfii genotype 2 and P. blaschkeae strains isolated from milk of mastitic cows were subjected to drug susceptibility testing by Etest methodology. RESULTS: Five out of ten antifungal drugs tested exhibited no activity against Prototheca spp. isolates. The best activity against Prototheca spp. was demonstrated by amphotericin B (MIC90 of 1.5 mg/L). The MICs differed significantly (P < 0.01) between P. zopfii genotype 2 and P. blaschkeae, with the latter species being more susceptible to amphotericin B and azoles. Marked differences (P < 0.05) in azole and amphotericin B activities were noted among Prototheca spp. isolates originating from different European countries. Based on the correlation coefficients, a considerable cross-interaction was found among MICs of azoles and between MICs of azoles and amphotericin B for Prototheca spp. (P < 0.03). CONCLUSIONS: This study represents the largest, cross-European evaluation of antifungal activity against Prototheca spp. to date. The activity of amphotericin B against Prototheca spp. validates its potential use as a therapeutic agent against bovine protothecosis. For laboratory testing of drug activity against Prototheca spp., the Etest method is encouraged, due to its technical simplicity, rapidity and high intra- and inter-laboratory reproducibility.

Stability of tandemly repetitive subelement PCR patterns in Trichophyton rubrum over serial passaging and with respect to drug pressure.

Trichophyton rubrum is the most significant agent of dermatomycoses worldwide, primarily causing tinea pedis and tinea unguium. PCR analysis of tandemly repetitive subelements (TRS) within the rDNA nontranscribed spacer region is a major tool for molecular typing of T. rubrum. The aim of this study was to investigate the stability of TRS PCR patterns by analyzing isogenic strains of T. rubrum. Twenty-seven groups of isogenic T. rubrum strains were examined, each composed of an original clinical isolate and its 3 subcultures, maintained on a drug-free medium, a medium containing fluconazole and itraconazole. TRS typing was performed for the original strains and their subcultures grown after 12 passages, at 4-week intervals, on respective media. To add more objectivity to the results, TRS typing for each of the isogenic strain was performed three times, using DNA isolated from three different colonies. Among 27 groups of isogenic strains, all but one were exclusively composed of strains with identical TRS-1 and TRS-2 PCR patterns. In one group, 3 isolates from the last, twelfth passage had identical TRS-1 PCR profiles (type 1), yet different TRS-2 PCR profiles, as compared with the original strain (type I vs. type II). The mechanism underlying the genotype switch was a deletion of a single repeat unit in the TRS-2 locus, as evidenced by sequence analysis. In the interpretation of TRS typing results, microevolutionary events need to be taken into account, urging drawing epidemiological conclusions with caution and in conjunction with other genotyping data and traditional contact tracing information.

Molecular snapshot of drug-resistant Mycobacterium tuberculosis strains from the Plateau State, Nigeria.

Nigeria ranks 1st in Africa and 6th globally with the highest burden of tuberculosis (TB). However, only a relatively few studies have addressed the molecular epidemiology of Mycobacterium tuberculosis in this country. The aim of this work was to analyze the genetic structure of drug-resistant (DR) M. tuberculosis population in the Plateau State (central Nigeria), with the results placed in the broader context of West Africa. The study sample included 67 DR M. tuberculosis isolates, recovered from as many TB patients between November 2015 and January 2016, in the Plateau State. The isolates were subjected to spoligotyping and MIRU-VNTR typing. A total of 20 distinct spoligotypes were obtained, split into 3 clusters (n = 50, 74.6%, 2-33 isolates per cluster) and 17 (25.4%) unique patterns. The Cameroon clade was the largest lineage (62.7%) followed by T (28.3%), LAM (3%), and Haarlem (3%) clades. Upon MIRU-VNTR typing, the isolates produced 31 profiles, i.e. 7 clusters (n = 43, 64.2%, 2-17 isolates per cluster) and 24 singletons. A combined spoligotyping and MIRU-VNTR typing analysis showed 20.9% of the cases clustered and estimated the recent transmission rate at 11.9%. In conclusion, two lineages, namely Cameroon, and T accounted for the majority (91%) of cases. No association was observed between the most prevalent Cameroon lineage and drug resistance, including multidrug resistant (MDR) phenotype, or any of the patient demographic characteristics.

OmpR controls Yersinia enterocolitica motility by positive regulation of flhDC expression.

Flagella and invasin play important roles during the early stages of infection by the enteric pathogen Yersinia enterocolitica. Our previous study demonstrated that OmpR negatively regulates invasin gene expression at the transcriptional level. The present study focused on the role of OmpR in the regulation of flagella expression. Motility assays and microscopic observations revealed that an ompR mutant strain exhibits a non-motile phenotype due to the lack of flagella. An analysis of flhDC::lacZYA chromosomal fusions demonstrated a decrease in flhDC expression in ompR mutant cells, suggesting a role for OmpR in the positive control of flagellar master operon flhDC, which is in contrast to the negative role it plays in Escherichia coli. Moreover, high temperature or osmolarity and low pH decreased flhDC expression and OmpR was not required for the response to these factors. Evidence from an examination of the DNA binding properties of OmpR in vitro indicated that the mechanism by which OmpR regulates flhDC is direct. Electrophoretic mobility shift assays confirmed that OmpR binds specifically to the flhDC promoter region and suggested the presence of more than one OmpR-binding site. In addition, phosphorylation of OmpR by acetyl-P appeared to stimulate the binding abilities of OmpR. Together with the results of our previous studies revealing the negative role of OmpR in the regulation of invasin expression, these findings support a model in which invasion and motility might be reciprocally regulated by OmpR.

A two-step strategy for molecular typing of multidrug-resistant Mycobacterium tuberculosis clinical isolates from Poland.

Tuberculosis (TB) continues to be one of the most challenging public health problems in the world. An important contributor to the global burden of the disease is the emergence and spread of drug-resistant and particularly multidrug-resistant Mycobacterium tuberculosis strains (MDR), defined as being resistant to at least isoniazid and rifampicin. In recent years, the introduction of different DNA-based molecular typing methods has substantially improved the knowledge of the epidemiology of TB. The purpose of this study was to employ a combination of two PCR-based genotyping methods, namely spoligotyping and IS6110-Mtb1/Mtb2 PCR to investigate the clonal relatedness of MDR M. tuberculosis clinical isolates recovered from pulmonary TB patients from Poland. Among the 50 isolates examined, 28 (56%) were clustered by spoligotyping, whereas IS6110-Mtb1/Mtb2 PCR resulted in 16 (32%) clustered isolates. The isolates that clustered in both typing methods were assumed to be clonally related. A two-step strategy consisting of spoligotyping as a first-line test, performed on the entire pool of isolates, and IS6110-Mtb1/Mtb2 PCR typing as a confirmatory subtyping method, performed only within spoligotype-defined clusters, is an efficient approach for determining clonal relatedness among M. tuberculosis clinical isolates.

Polyelectrolyte Membrane with Hydroxyapatite and Silver Nanoparticles as a Material for Modern Wound Dressings.

Modern wound dressings not only play a covering role but also facilitate the function of the wound, contributing to a faster healing process. In this paper, we present a polyelectrolyte system with nanosized elements that could stimulate the growth of eukaryotic cells while providing antimicrobial properties, which may be recommended as a potential dressing material. The proposed platform consisted of polyethyleneimine, hydroxyapatite, and silver nanoparticles and was characterized using various macroscopic techniques. The constructed membrane scaffold was evaluated with immobilized WEHI 164 cells as a model system for cells sustained at the interface of bone and skin. Moreover, the bacteriostatic function of the designed membrane material was evaluated using different bacterial strains.

Contribution of cysteine residue to the properties of Listeria monocytogenes listeriolysin O.

Listeriolysin (LLO) is the key virulence factor critical for Listeria monocytogenes pathogenesis. Listerial cytolysin belongs to the family of cholesterol-dependent cytolysins (CDCs), a group of pore-forming toxins produced by related gram-positive bacteria. Most CDCs contain a cysteine residue in the conserved undecapeptide - a sequence that is highly preserved among this group of proteins. Substitutions of cysteine do not always lead to loss of hemolytic activity, questioning the purpose of such strong conservation of this amino acid in the sequence of CDC. The properties of 3 L. monocytogenes strains, a wild type and 2 mutants expressing modified LLO within the cysteine residue, were analyzed in this work. The first of these mutants producing a toxin with cysteine to alanine substitution showed similar features to the wild type except that a thiol-reducing agent was not necessary for hemolytic activity. Another strain secreting LLO containing serine instead of cysteine exhibited strikingly different properties than the wild type. Modified toxin is independent of the reducing reagents, less stable, and shows accelerated kinetics of cytolysis in comparison with the unchanged protein. However, both mutant strains are less invasive in the cell culture model showing the important role of cysteine in L. monocytogenes virulence.

Delivery of Chicken Egg Ovalbumin to Dendritic Cells by Listeriolysin O-Secreting Vegetative Bacillus subtilis.

Listeriolysin O (LLO), one of the most immunogenic proteins of Listeria monocytogenes and its main virulence factor, mediates bacterial escape from the phagosome of the infected cell. Thus, its expression in a nonpathogenic bacterial host may enable effective delivery of heterologous antigens to the host cell cytosol and lead to their processing predominantly through the cytosolic MHC class I presentation pathway. The aim of this project was to characterize the delivery of a model antigen, chicken egg ovalbumin (OVA), to the cytosol of dendritic cells by recombinant Bacillus subtilis vegetative cells expressing LLO. Our work indicated that LLO produced by non-sporulating vegetative bacteria was able to support OVA epitope presentation by MHC I molecules on the surface of antigen presenting cells and consequently influence OVA-specific cytotoxic T cell activation. Additionally, it was proven that the genetic context of the epitope sequence is of great importance, as only the native full-sequence OVA fused to the N-terminal fragment of LLO was sufficient for effective epitope delivery and activation of CD8⁺ lymphocytes. These results demonstrate the necessity for further verification of the fusion antigen potency of enhancing the MHC I presentation, and they prove that LLO-producing B. subtilis may represent a novel and attractive candidate for a vaccine vector.