Axel Schubert

A fibrinogen receptor from group B Streptococcus interacts with fibrinogen by repetitive units with novel ligand binding sites

Group B Streptococcus (GBS) is a frequent cause of bacterial sepsis and meningitis in neonates. During the course of infection, GBS colonizes and invades a number of host compartments, thereby interacting with different host proteins. In the present report, we describe the isolation of the fbsA gene, which encodes a fibrinogen receptor from GBS. The deduced FbsA protein is characterized by repetitive units, each 16 amino acids in length. Sequencing of the fbsA gene from five different GBS strains revealed significant variation in the number of repeat‐encoding units. The deletion of the fbsA gene in the genome of GBS 6313 completely abolished fibrinogen binding, suggesting that FbsA is the major fibrinogen receptor in this strain. Growth of the fbsA deletion mutant in human blood was significantly impaired, indicating that FbsA protects GBS from opsonophagocytosis. In Western blot experiments with truncated FbsA proteins, the repeat region of FbsA was identified as mediating fibrinogen binding. Using synthetic peptides, even a single repeat unit of FbsA was demonstrated to bind to fibrinogen. Spot membrane analysis and competitive binding experiments with peptides carrying single amino acid substitutions allowed the prediction of a fibrinogen‐binding motif with the consensus sequence G‐N/S/T‐V‐L‐A/E/M/Q‐R‐R‐X‐K/R/W‐A/D/E/N/Q‐A/F/I/L/V/Y‐X‐X‐K/R‐X‐X.

Identification and Molecular Analysis of PcsB, a Protein Required for Cell Wall Separation of Group B Streptococcus

Group B streptococcus (GBS) is the leading cause of bacterial sepsis and meningitis in neonates. N-terminal sequencing of major proteins in the culture supernatant of a clinical isolate of GBS identified a protein of about 50 kDa which could be detected in all of 27 clinical isolates tested. The corresponding gene, designated pcsB, was isolated from a GBS cosmid library and subsequently sequenced. The deduced PcsB polypeptide consists of 447 amino acid residues (M(r), 46,754), carries a potential N-terminal signal peptide sequence of 25 amino acids, and shows significant similarity to open reading frames of unknown function from different organisms and to the murein hydrolase P45 from Listeria monocytogenes. Northern blot analysis revealed a monocistronic transcriptional organization for pcsB in GBS. Insertional inactivation of pcsB in the genome of GBS resulted in mutant strain Sep1 exhibiting a drastically reduced growth rate compared to the parental GBS strain and showing an increased susceptibility to osmotic pressure and to various antibiotics. Electron microscopic analysis of GBS mutant Sep1 revealed growth in clumps, cell separation in several planes, and multiple division septa within single cells. These data suggest a pivotal role of PcsB for cell division and antibiotic tolerance of GBS.

The fibrinogen receptor FbsA promotes adherence of Streptococcus agalactiae to human epithelial cells

ABSTRACT Streptococcus agalactiae is a major cause of bacterial pneumonia, sepsis, and meningitis in human neonates. During the course of infection, S. agalactiae adheres to a variety of epithelial cells but the underlying mechanisms are only poorly understood. The present report demonstrates the importance of the fibrinogen receptor FbsA for the streptococcal adherence and invasion of epithelial cells. Deletion of the fbsA gene in various S. agalactiae strains substantially reduced their binding of soluble fibrinogen and their adherence to and invasion of epithelial cells, indicating a role of FbsA in these different processes. The adherence and invasiveness of an fbsA deletion mutant were partially restored by reintroducing the fbsA gene on an expression vector. Heterologous expression of fbsA in Lactococcus lactis enabled this bacterium to adhere to but not to invade epithelial cells, suggesting that FbsA is a streptococcal adhesin. Flow cytometry experiments revealed a dose-dependent binding of FbsA to the surface of epithelial cells. Furthermore, tissue culture experiments exhibited an intimate contact of FbsA-coated latex beads with the surfaces of human epithelial cells. Finally, host cell adherence and invasion were significantly blocked in competition experiments with either purified FbsA protein or a monoclonal antibody directed against the fibrinogen-binding epitope of FbsA. Taken together, our studies demonstrate that FbsA promotes the adherence of S. agalactiae to epithelial cells but that FbsA does not mediate the bacterial invasion into host cells. Our results also indicate that fibrinogen-binding epitopes within FbsA are involved in the adherence of S. agalactiae to epithelial cells.

Antiviral treatment of cytomegalovirus infection and resistant strains

This review discusses the management of resistant cytomegalovirus and prevention strategies for fatal therapy failures. Five drugs, ganciclovir/valganciclovir, cidofovir, foscarnet and fomivirsen, have been approved so far for the treatment of human cytomegalovirus (HCMV) diseases. Except for fomivirsen, all of the approved drugs share the same target molecule, the viral DNA polymerase. The emergence of drug-resistant HCMV has also been reported for all of them. For optimal care of patients, the clinical virologist has to provide the most meaningful assays for monitoring of therapy and early detection of emerging drug-resistant HCMV. Additionally, a quantitative drug monitoring would be helpful. New antiviral agents are urgently needed with less adverse effects, good oral bioavailability and possibly novel targets or mechanisms of action to avoid cross-resistance and to improve the ability to suppress the selection of resistant virus strains by combination therapy. Compounds like maribavir, leflunomide and artesunate, which exhibit anti-HCMV activity in vitro and in patients need to be evaluated in clinical studies. Besides these, new therapy approaches like immunotherapy or new diagnostic techniques like pyrosequencing have to be considered in the future.

Intravenous Zanamivir for Patients with Pneumonitis due to Pandemic (H1N1) 2009 Influenza Virus

We report on 2 critically ill patients with pneumonitis and acute respiratory distress syndrome due to pandemic (H1N1) 2009 influenza A virus who were treated with intravenous zanamivir and had favorable clinical outcomes. Zanamivir given intravenously may be a therapeutic option in patients with critical illness and mechanical ventilation.

Single nucleotide polymorphisms of thymidine kinase and DNA polymerase genes in clinical herpes simplex virus type 1 isolates associated with different resistance phenotypes.

The role of mutations in the thymidine kinase (TK, UL23) and DNA polymerase (pol, UL30) genes of herpes simplex virus (HSV) for development of different resistance phenotypes has to be exactly determined before genotypic resistance testing can be implemented in patients care. Furthermore, the occurrence of cross-resistance is of utmost clinical importance. In this study, clinical HSV-1 isolates obtained between 2004 and 2011 from 26 patients after stem cell transplantation were examined in parallel by phenotypic and genotypic resistance testing. Thirteen isolates, which were phenotypically cross-resistant to acyclovir (ACV), penciclovir (PCV) and brivudin (BVDU), exhibited consistently frameshift or non-synonymous mutations in the TK gene known to confer resistance. One of these mutations (insertion of C at the nucleotide positions 1061-1065) has not been described before. Seven strains, phenotypically resistant to ACV and PCV and, except one each, sensitive to BVDU and resistant to foscarnet (FOS), carried uniformly resistance-related substitutions in the DNA pol gene. Finally, 3 isolates, resistant to ACV, PCV and 2 out of these also resistant to BVDU, had known but also unclear substitutions in the TK and DNA pol genes, and 3 isolates were completely sensitive. In conclusion, clinical ACV-resistant HSV-1 isolates, carrying resistance-associated mutations in the TK gene, can be regarded as cross-resistant to other nucleoside analogs such as BVDU. In contrast, clinical FOS-resistant HSV-1 strains which are cross-resistant to ACV may be sensitive to BVDU. This has to be considered for drug changes in antiviral treatment in case of ACV resistance.

Fast selection of maribavir resistant cytomegalovirus in a bone marrow transplant recipient

BackgroundHuman cytomegalovirus infections are still significant causes of morbidity and mortality in transplant recipients. The use of antiviral agents is limited by toxicity and evolving resistance in immunocompromised patients with ongoing viral replication during therapy. Here, we present the first documented case of genotypic resistance against maribavir in a bone marrow transplant (BMT) recipient.Case presentationThe female 13-year-old patient was suffering from a refractory cytopenia. Ganciclovir, foscarnet, cidofovir, leflunomide and maribavir, an inhibitor of the cytomegalovirus UL97 protein, were administered to treat a therapy-resistant cytomegalovirus infection. Viral mutations conferring resistance against nucleotide and pyrophosphate analogs as well as maribavir (MBV) have evolved sequentially. Particularly, impressive was the fast emergence of multiple mutations T409M, H411Y and H411N conferring maribavir resistance after less than 6 weeks.ConclusionWe describe the fast emergence of cytomegalovirus variants with different maribavir resistance associated mutations in a bone marrow transplant recipient treated with MBV 400 mg p.o. twice per day. The results suggest that a high virus load permitted a selection of several but distinct therapy-resistant HCMV mutants. Since a phase II study with MBV is intended for the treatment of resistant or refractory HCMV infections in transplant recipients this has to be kept in mind in patients with high viral loads during therapy (NCT01611974).

Fluorescence-Based Assay for Phenotypic Characterization of Human Cytomegalovirus Polymerase Mutations Regarding Drug Susceptibility and Viral Replicative Fitness

ABSTRACT One essential prerequisite for genotypic drug susceptibility testing of human cytomegalovirus (HCMV) is the phenotypic characterization of mutations identified in the viral protein kinase gene UL97 and the viral DNA polymerase gene UL54 regarding their quantitative impact on drug susceptibility. We developed a new method for phenotypic characterization of UL54 mutations with regard to polymerase activity, viral replication, and drug susceptibility. To determine the most suitable viral indicator gene, enhanced green fluorescence protein was C-terminally fused to the HCMV early-late protein UL83 (pp65) or the late proteins UL32 (pp150) and UL99 (pp28), resulting in reporter viruses vTB65g, vTB150g, and vTB28g. vTB65g proved to be superior to the other constructs due to its favorable signal-to-noise ratio and was therefore used to establish the optimum conditions for our assay. The UL54 E756K and D413E mutations were introduced into vTB65g by markerless bacterial artificial chromosome mutagenesis, resulting in virus strains vE756Kg and vD413Eg. The drug susceptibility phenotypes of vE756Kg and vD413Eg were comparable to those previously reported. Furthermore, we found a reduced replicative fitness of vE756Kg by measuring fluorescence intensity as well as by conventional virus growth kinetics. Decreased fluorescence signals of vE756Kg- and vD413Eg-infected cells at late times of infection suggested a reduced polymerase activity, which was confirmed by real-time PCR quantification of the newly synthesized viral DNAs. This new fluorescence-based assay is a highly reproducible method for the phenotypic characterization of mutations potentially influencing drug susceptibility, viral replicative fitness, and polymerase activity of HCMV after marker transfer.

Inactivation and Environmental Stability of Zika Virus.

To the Editor: Zika virus is an emerging virus that has spread to most countries in Latin America and the Caribbean (1,2). It is transmitted by mosquitoes and through sexual intercourse (3). Most persons infected with Zika virus are asymptomatic or experience mild symptoms (4). However, in a pregnant woman, infection may cause severe pregnancy and birth complications, most notably microcephaly in children infected in utero (5–7). Zika virus infection might also be associated with an increased incidence of Guillain-Barre syndrome (8). Thus, the virus represents a threat to healthcare workers who manage infected patients or diagnostic samples and researchers who work with infectious virus in laboratories.

Development of a high-throughput colorimetric Zika virus infection assay

Zika virus (ZIKV) is an emerging pathogen that causes congenital infections which may result in birth defects, such as microcephaly. Currently, no approved treatment or vaccination is available. ZIKV can be readily detected in cell culture where virally infected cells are normally stained by specific antibodies. As ZIKV regularly causes a cytopathic effect, we were wondering whether this viral property can be used to quantitatively determine viral infectivity. We here describe the use of an 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide-(MTT)-based cell viability assay that allows to determine ZIKV-induced cell death. We show that this colorimetric assay quantifies ZIKV infection over a broad range of viral dilutions in both monkey and human cells. It allows to determine inhibitory activities of antivirals that block ZIKV or to define the neutralizing antibody titers of ZIKV antisera. This MTT-based ZIKV detection assay can be evaluated by naked eye or computational tools, has a broad linear range, does not require large equipment or costly reagents, and thus represents a promising alternative to antibody-based assays, in particular in resource-poor settings. We propose to use this simple, fast, and cheap method for quantification of ZIKV neutralizing antibodies and testing of antiviral compounds.