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Lmb is a 34 kDa laminin binding surface adhesin of Streptococcus agalactiae. The structure of Lmb reported by us recently has shown that it consists of a metal binding crevice, in which a zinc ion is coordinated to three highly conserved histidines. To elucidate the structural and functional significance of the metal ion in Lmb, these histidines have been mutated to alanine and single, double and triple mutants were generated. These mutations resulted in insolubility of the protein and revealed altered secondary and tertiary structures, as evidenced by circular dichroism and fluorescence spectroscopy studies. The mutations also significantly decreased the binding affinity of Lmb to laminin, implicating the role played by the metal binding residues in maintaining the correct conformation of the protein for its binding to laminin. A highly disordered loop, proposed to be crucial for metal acquisition in homologous structures, was deleted in Lmb by mutation (ΔLmb) and its crystal structure was solved at 2.6 Å. The ΔLmb structure was identical to the native Lmb structure with a bound zinc ion and exhibited laminin binding activity similar to wild type protein, suggesting that the loop might not have an important role in metal acquisition or adhesion in Lmb. Targeted mutations of histidine residues confirmed the importance of the zinc binding crevice for the structure and function of the Lmb adhesin. [ABSTRACT FROM AUTHOR]

S. agalactiae (group B streptococci, GBS) is a major microbial pathogen in human neonates and causes invasive infections in pregnant women and immunocompromised individuals. The S. agalactiae β-hemolysin is regarded as an important virulence factor for the development of invasive disease. To examine the role of β-hemolysin in the interaction with professional phagocytes, the THP-1 monocytic cell line and human granulocytes were infected with a serotype Ia S. agalactiae wild type strain and its isogenic nonhemolytic mutant. We could show that the nonhemolytic mutants were able to survive in significantly higher numbers than the hemolytic wild type strain, in THP-1 macrophage-like cells and in assays with human granulocytes. Intracellular bacterial multiplication, however, could not be observed. The hemolytic wild type strain stimulated a significantly higher release of Tumor Necrosis Factor-α than the nonhemolytic mutant in THP-1 cells, while similar levels of the chemokine Interleukin-8 were induced. In order to investigate bacterial mediators of IL-8 release in this setting, purified cell wall preparations from both strains were tested and found to exert a potent proinflammatory stimulus on THP-1 cells. In conclusion, our results indicate that the β-hemolysin has a strong influence on the intracellular survival of S. agalactiae and that a tightly controlled regulation of β-hemolysin expression is required for the successful establishment of S. agalactiae in different host niches. [ABSTRACT FROM AUTHOR]

Group B streptococcus (GBS) is the main cause of neonatal sepsis and meningitis. Bacterial surface proteins play a major role in GBS binding to and invasion of different host surfaces. The scpB and lmb genes, coding for fibronectin-binding and laminin-binding surface proteins, are present in almost all human GBS isolates. The scpB-lmb intergenic region is a hot spot for integration of two mobile genetic elements (MGEs): the insertion element IS1548 or the group II intron GBSi1. We studied the structure of scpB-lmb intergenic region in 111 GBS isolates belonging to the intraspecies major clonal complexes (CCs). IS1548 was mostly found (72.2%) in CC19 serotype III strains recovered more specifically (92.3%) from neonatal meningitis. GBSi1 was principally found (70.6%) in CC17 strains, mostly (94.4%) of serotype III, but also (15.7%) in CC19 strains, mostly (87.5%) of serotype II. No MGE was found in most strains of the other CCs (76.0%), notably CC23, CC10 and CC1. Twenty-six strains representing these three genetic configurations were selected to investigate the transcription and expression levels of scpB and lmb genes. Quantitative RT-PCR showed that lmb transcripts were 5.0- to 9.6-fold higher in the group of strains with IS1548 than in the other two groups of strains (P,0.001). Accordingly, the binding ability to laminin was 3.8- to 6.6-fold higher in these strains (P#0.001). Moreover, Lmb amount expressed on the cell surface was 2.4- to 2.7-fold greater in these strains (P,0.001). By contrast, scpB transcript levels and fibronectin binding ability were similar in the three groups of strains. Deletion of the IS1548 sequence between scpB and lmb genes in a CC19 serotype III GBS strain substantially reduced the transcription of lmb gene (13.5-fold), the binding ability to laminin (6.2-fold), and the expression of Lmb protein (5.0-fold). These data highlight the importance of MGEs in bacterial virulence and demonstrate the upregulation of lmb gene by IS1548; the increased lmb gene expression observed in CC19 serotype III strains with IS1548 may play a role in their ability to cause neonatal meningitis and endocarditis. [ABSTRACT FROM AUTHOR]

Adhesion/invasion of pathogenic bacteria is a critical step in infection and is mediated by surface-exposed proteins termed adhesins. The crystal structure of recombinant Lmb, a laminin-binding adhesin from Streptococcus agalactiae, has been determined at 2.5 A resolution. Based on sequence and structural homology, Lmb was placed into the cluster 9 family of the ABC (ATP-binding cassette) transport system. The structural organization of Lmb closely resembles that of ABC-type solute-binding proteins (SBPs), in which two structurally related globular domains interact with each other to form a metal-binding cavity at the interface. The bound zinc in Lmb is tetrahedrally coordinated by three histidines and a glutamate from both domains. A comparison of Lmb with other metal transporters revealed an interesting feature of the dimerization of molecules in the crystallographic asymmetric unit in all zinc-binding transporters. A closer comparison of Lmb with the zinc-binding ZnuA from Escherichia coli and Synechocystis 6803 suggested that Lmb might undergo a unique structural rearrangement upon metal binding and release. The crystal structure of Lmb provides an impetus for further investigations into the molecular basis of laminin binding by human pathogens. Being ubiquitous in all serotypes of group B streptococcus (GBS), the structure of Lmb may direct the development of an efficient vaccine. [ABSTRACT FROM AUTHOR]

Abstract: Streptococcus agalactiae (S. agalactiae) can cause severe pneumonia, sepsis and meningitis in neonates and remains one of the most prevalent causes of invasive neonatal infections. During the course of infection, S. agalactiae colonizes and invades a number of host compartments, thereby interacting with different host tissues. Deletion of the scpB-lmb region, coding for the C5a peptidase and the laminin-binding protein Lmb, respectively, resulted in a 64% decreased invasion of S. agalactiae into human brain microvascular endothelial cells (HBMEC). Decreased invasion was also seen in lmb mutant strains lmb-k1 and lmb-k2 (74% and 69% reduction, respectively). Finally, host cell invasion was significantly reduced in competition experiments with either purified recombinant laminin-binding protein by 46% or a polyclonal antibody directed against the laminin-binding protein of S. agalactiae by 45%. The S. agalactiae scpB-lmb mutant induced an equal amount of the neutrophil chemoattractant interleukin (IL)-8 release in comparison to the wild-type. Taken together, our studies support the conclusion that Lmb promotes invasion of S. agalactiae into HBMEC but does not play a role in IL-8 release from HBMEC. [Copyright &y& Elsevier]

Laminin-binding protein (Lmb), a surface-exposed lipoprotein from Streptococcus agalactiae (group B streptococcus), mediates attachment to human laminin and plays a crucial role in the adhesion/invasion of eukaryotic host cells. However, the structural basis of laminin binding still remains unclear. In the context of detailed structural analysis, the lmb gene has been cloned, expressed in Escherichia coli, purified and crystallized. The crystals diffracted to a resolution of 2.5 Å and belonged to the monoclinic space group P21, with unit-cell parameters a = 56.63, b = 70.60, c = 75.37 Å, β = 96.77°. [ABSTRACT FROM AUTHOR]

Infections caused by yeasts of the genus Candida are likely to occur not only in immunocompromised patients but also in healthy individuals, leading to infections of the gastrointestinal tract, urinary tract, and respiratory tract. Due to the rapid increase in the frequency of reported Candidiasis cases in recent years, diagnostic research has become the subject of many studies, and therefore, we developed a polyclonal aptamer library-based fluorometric assay with high specificity and affinity towards Candida spec. to quantify the pathogens in clinical samples with high sensitivity. We recently obtained the specific aptamer library R10, which explicitly recognized Candida and evolved it by mimicking an early skin infection model caused by Candida using the FluCell-SELEX system. In the follow-up study presented here, we demonstrate that the aptamer library R10-based bioassay specifically recognizes invasive clinical Candida isolates, including not only C. albicans but also strains like C. tropcialis, C. krusei, or C. glabrata. The next-generation fluorometric bioassay presented here can reliably and easily detect an early Candida infection and could be used for further clinical research or could even be developed into a full in vitro diagnostic tool. [ABSTRACT FROM AUTHOR]

The diminishing portfolio of mankind's available antibiotics urges science to develop novel potent drugs. Here, we present a peptide fitting the typical blueprint of amphipathic and membrane-active antimicrobial peptides, denominated C14R. This 2 kDa peptide consists of 16 amino acid residues, with seven being either hydrophobic, aromatic, or non-polar, and nine being polar or positively charged, strictly separated on opposite sides of the predicted α-helix. The affinity of the peptide C14R to P. aeruginosa membranes and its intrinsic tendency to productively insert into membranes of such composition were analyzed by dynamic simulations. Its biological impact on the viability of two different P. aeruginosa reference strains was demonstrated by determining the minimal inhibitory concentrations (MICs), which were found to be in the range of 10–15 µg/mL. C14R's pore-forming capability was verified in a permeabilization assay based on the peptide-triggered uptake of fluorescent dyes into the bacterial cells. Finally, the peptide was used in radial diffusion assays, which are commonly used for susceptibility testing of antimicrobial peptides in clinical microbiology. In comparison to reference strains, six clinical P. aeruginosa isolates were clearly affected, thereby paving the way for further in-depth analyses of C14R as a promising new AMP drug in the future. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs) represent a promising class of therapeutic biomolecules that show antimicrobial activity against a broad range of microorganisms, including life-threatening pathogens. In contrast to classic AMPs with membrane-disrupting activities, new peptides with a specific anti-biofilm effect are gaining in importance since biofilms could be the most important way of life, especially for pathogens, as the interaction with host tissues is crucial for the full development of their virulence in the event of infection. Therefore, in a previous study, two synthetic dimeric derivatives (parallel Dimer 1 and antiparallel Dimer 2) of the AMP Cm-p5 showed specific inhibition of the formation of Candida auris biofilms. Here we show that these derivatives are also dose-dependently effective against de novo biofilms that are formed by the widespread pathogenic yeasts C. albicans and C. parapsilosis. Moreover, the activity of the peptides was demonstrated even against two fluconazole-resistant strains of C. auris. [ABSTRACT FROM AUTHOR]

Here we present for the first time a potential wound dressing material implementing aptamers as binding entities to remove pathogenic cells from newly contaminated surfaces of wound matrix-mimicking collagen gels. The model pathogen in this study was the Gram-negative opportunistic bacterium Pseudomonas aeruginosa, which represents a considerable health threat in hospital environments as a cause of severe infections of burn or post-surgery wounds. A two-layered hydrogel composite material was constructed based on an established eight-membered focused anti-P. aeruginosa polyclonal aptamer library, which was chemically crosslinked to the material surface to form a trapping zone for efficient binding of the pathogen. A drug-loaded zone of the composite released the C14R antimicrobial peptide to deliver it directly to the bound pathogenic cells. We demonstrate that this material combining aptamer-mediated affinity and peptide-dependent pathogen eradication can quantitatively remove bacterial cells from the "wound" surface, and we show that the surface-trapped bacteria are completely killed. The drug delivery function of the composite thus represents an extra safeguarding property and thus probably one of the most important additional advances of a next-generation or smart wound dressing ensuring the complete removal and/or eradication of the pathogen of a freshly infected wound. [ABSTRACT FROM AUTHOR]

Mollusks have been widely investigated for antimicrobial peptides because their humoral defense against pathogens is mainly based on these small biomolecules. In this report, we describe the identification of three novel antimicrobial peptides from the marine mollusk Nerita versicolor. A pool of N. versicolor peptides was analyzed with nanoLC-ESI-MS-MS technology, and three potential antimicrobial peptides (Nv-p1, Nv-p2 and Nv-p3) were identified with bioinformatical predictions and selected for chemical synthesis and evaluation of their biological activity. Database searches showed that two of them show partial identity to histone H4 peptide fragments from other invertebrate species. Structural predictions revealed that they all adopt a random coil structure even when placed near a lipid bilayer patch. Nv-p1, Nv-p2 and Nv-p3 exhibited activity against Pseudomonas aeruginosa. The most active peptide was Nv-p3 with an inhibitory activity starting at 1.5 µg/mL in the radial diffusion assays. The peptides were ineffective against Klebsiella pneumoniae, Listeria monocytogenes and Mycobacterium tuberculosis. On the other hand, these peptides demonstrated effective antibiofilm action against Candida albicans, Candida parapsilosis and Candida auris but not against the planktonic cells. None of the peptides had significant toxicity on primary human macrophages and fetal lung fibroblasts at effective antimicrobial concentrations. Our results indicate that N. versicolor-derived peptides represent new AMP sequences and have the potential to be optimized and developed into antibiotic alternatives against bacterial and fungal infections. [ABSTRACT FROM AUTHOR]

Streptococcus anginosus is a commensal of mucous membranes and an emerging human pathogen. Some strains, including the type strain, display a prominent β-hemolytic phenotype. A gene cluster ( sag), encoding a variant of streptolysin S (SLS) has recently been identified as the genetic background for β-hemolysin production in S. anginosus. In this study, we further characterized the hemolytic and cytolytic activity of the S. anginosus hemolysin in comparison with other streptococcal hemolysins. The results indicate that SLS of S. anginosus is a broad-range hemolysin able to lyse erythrocytes of different species, including horse, bovine, rabbit and even chicken. The hemolytic activity is temperature dependent, and a down-regulation of the hemolysin expression is induced in the presence of high glucose levels. Survival assays indicate that in contrast to other streptococcal species, S. anginosus does not require SLS for survival in the presence of human granulocytes. Cross-complementation studies using the sagB and sagD genes of Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis demonstrated functional similarities to the S. anginosus SLS. Nevertheless, distinct differences to other streptolysin S variants were noted and provide further insights into the molecular mechanisms of SLS pathogen host interactions. [ABSTRACT FROM AUTHOR]

Easy and reliable identification of pathogenic species such as yeasts, emerging as problematic microbes originating from the genus Candida, is a task in the management and treatment of infections, especially in hospitals and other healthcare environments. Aptamers are seizing an already indispensable role in different sensing applications as binding entities with almost arbitrarily tunable specificities and optimizable affinities. Here, we describe a polyclonal SELEX library that not only can specifically recognize and fluorescently label Candida cells, but is also capable to differentiate C. albicans, C. auris and C. parapsilosis cells in flow-cytometry, fluorometric microtiter plate assays and fluorescence microscopy from human cells, exemplified here by human dermal fibroblasts. This offers the opportunity to develop diagnostic tools based on this library. Moreover, these specific and robust affinity molecules could also serve in the future as potent binding entities on biomaterials and as constituents of technical devices and will thus open avenues for the development of cost-effective and easily accessible next generations of electronic biosensors in clinical diagnostics and novel materials for the specific removal of pathogenic cells from human bio-samples. [ABSTRACT FROM AUTHOR]

Bacteria belonging to the group of ESKAPE pathogens are responsible for the majority of nosocomial infections. Due to the increase of antibiotic resistance, alternative treatment strategies are of high clinical relevance. In this context visible light as disinfection technique represents an interesting option as microbial pathogens can be inactivated without adjuvants. However cytotoxic effects of visible light on host cells have also been reported. We compared the cytotoxicity of violet and blue light irradiation on monocytic THP-1 and alveolar epithelium A549 cells with the inactivation effect on ESKAPE pathogens. THP-1 cells displayed a higher susceptibility to irradiation than A549 cells with first cytotoxic effects occurring at 300 J cm−2 (405 nm) and 400 J cm−2 (450 nm) in comparison to 300 J cm−2 and 1000 J cm−2, respectively. We could define conditions in which a significant reduction of colony forming units for all ESKAPE pathogens, except Enterococcus faecium, was achieved at 405 nm while avoiding cytotoxicity. Irradiation at 450 nm demonstrated a more variable effect which was species and medium dependent. In summary a significant reduction of viable bacteria could be achieved at subtoxic irradiation doses, supporting a potential use of visible light as an antimicrobial agent in clinical settings. [ABSTRACT FROM AUTHOR]

As a conserved defense mechanism, many bacteria produce antimicrobial peptides, called bacteriocins, which provide a colonization advantage in a multispecies environment. Here the first bacteriocin of Streptococcus anginosus, designated Angicin, is described. S. anginosus is commonly described as a commensal, however it also possesses a high pathogenic potential. Therefore, understanding factors contributing to its host colonization and persistence are important. A radial diffusion assay was used to identify S. anginosus BSU 1211 as a potent bacteriocin producer. By genetic mutagenesis the background of bacteriocin production and the bacteriocin gene itself were identified. Synthetic Angicin shows high activity against closely related streptococci, listeria and vancomycin resistant enterococci. It has a fast mechanism of action and causes a membrane disruption in target cells. Angicin, present in cell free supernatant, is insensitive to changes in temperature from − 70 to 90 °C and pH values from 2 to 10, suggesting that it represents an interesting compound for potential applications in food preservation or clinical settings. [ABSTRACT FROM AUTHOR]

Background: In non-pregnant adults, the incidence of invasive Group B Streptococcus (GBS) disease is continuously increasing. Elderly and immunocompromised persons are at increased risk of infection. GBS commonly colonizes the vaginal tract, though data on colonization in the elderly are scarce. It is unknown whether the prevalence of GBS colonization is increasing in parallel to the observed rise of invasive infection. We conducted a three-year (2017-2019) prospective observational cross-sectional study in two teaching hospitals in Switzerland to determine the rate of GBS vaginal colonization in women over 60 years and i) to compare the proportions of known risk factors associated with invasive GBS diseases in colonized versus non-colonized women and ii) to evaluate the presence of GBS clusters with specific phenotypic and genotypic patterns in this population.Methods: GBS screening was performed by using vaginal swabs collected during routine examination from women willing to participate in the study and to complete a questionnaire for risk factors. Isolates were characterized for antibiotic resistance profile, serotype and sequence type (ST).Results: The GBS positivity rate in the elderly was 17% (44/255 positive samples), and similar to the one previously reported in pregnant women (around 20%). We could not find any association between participants' characteristics, previously published risk factors and GBS colonization. All strains were susceptible to penicillin, 22% (8/36) were not susceptible to erythromycin, 14% (5/36) were not susceptible to clindamycin and 8% (3/36) showed inducible clindamycin resistance. Both M and L phenotypes were each detected in one isolate. The most prevalent serotypes were III (33%, 12/36) and V (31%, 11/36). ST1 and ST19 accounted for 11% of isolates each (4/36); ST175 for 8% (3/36); and ST23, ST249 and ST297 for 6% each (2/36). Significantly higher rates of resistance to macrolides and clindamycin were associated with the ST1 genetic background of ST1.Conclusions: Our findings indicate a similar colonization rate for pregnant and elderly women.Trial Registration: Current Controlled Trial ISRCTN15468519 ; 06/01/2017. [ABSTRACT FROM AUTHOR]