The intracellular pathogen Listeria monocytogenes is distinguished by its ability to invade and replicate within mammalian cells. Remarkably, of the 15 serovars within the genus, strains belonging to serovar 4b cause the majority of listeriosis clinical cases and outbreaks. The Listeria O-antigens are defined by subtle structural differences amongst the peptidoglycan-associated wall-teichoic acids (WTAs), and their specific glycosylation patterns. Here, we outline the genetic determinants required for WTA decoration in serovar 4b L. monocytogenes, and demonstrate the exact nature of the 4b-specific antigen. We show that challenge by bacteriophages selects for surviving clones that feature mutations in genes involved in teichoic acid glycosylation, leading to a loss of galactose from both wall teichoic acid and lipoteichoic acid molecules, and a switch from serovar 4b to 4d. Surprisingly, loss of this galactose decoration not only prevents phage adsorption, but leads to a complete loss of surface-associated Internalin B (InlB),the inability to form actin tails, and a virulence attenuation in vivo. We show that InlB specifically recognizes and attaches to galactosylated teichoic acid polymers, and is secreted upon loss of this modification, leading to a drastically reduced cellular invasiveness. Consequently, these phage-insensitive bacteria are unable to interact with cMet and gC1q-R host cell receptors, which normally trigger cellular uptake upon interaction with InlB. Collectively, we provide detailed mechanistic insight into the dual role of a surface antigen crucial for both phage adsorption and cellular invasiveness, demonstrating a trade-off between phage resistance and virulence in this opportunistic pathogen.

Staphylococcus aureus is a major concern in human health care, mostly due to the increasing prevalence of antibiotic resistance. Intracellular localization of S. aureus plays a key role in recurrent infections by protecting the pathogens from antibiotics and immune responses. Peptidoglycan hydrolases (PGHs) are highly specific bactericidal enzymes active against both drug-sensitive and -resistant bacteria. However, PGHs able to effectively target intracellular S. aureus are not yet available. To overcome this limitation, we first screened 322 recombineered PGHs for staphylolytic activity under conditions found inside eukaryotic intracellular compartments. The most active constructs were modified by fusion to different cell-penetrating peptides (CPPs), resulting in increased uptake and enhanced intracellular killing (reduction by up to 4.5 log units) of various S. aureus strains (including methicillin-resistant S. aureus [MRSA]) in different tissue culture infection models. The combined application of synergistic PGH-CPP constructs further enhanced their intracellular efficacy. Finally, synergistically active PGH-CPP cocktails reduced the total S. aureus by more than 2.2 log units in a murine abscess model after peripheral injection. Significantly more intracellular bacteria were killed by the PGH-CPPs than by the PGHs alone. Collectively, our findings show that CPP-fused PGHs are effective novel protein therapeutics against both intracellular and drug-resistant S. aureus. IMPORTANCE The increasing prevalence of antibiotic-resistant bacteria is one of the most urgent problems of our time. Staphylococcus aureus is an important human pathogen that has acquired several mechanisms to evade antibiotic treatment. In addition, S. aureus is able to invade and persist within human cells, hiding from the immune response and antibiotic therapies. For these reasons, novel antibacterial strategies against these pathogens are needed. Here, we developed lytic enzymes which are able to effectively target drug-resistant and intracellular S. aureus. Fusion of these so-called enzybiotics to cell-penetrating peptides enhanced their uptake and intracellular bactericidal activity in cell culture and in an abscess mouse model. Our results suggest that cell-penetrating enzybiotics are a promising new class of therapeutics against staphylococcal infections.

Hydrogen and formate are important electron carriers in methanogenic degradation in anoxic environments such as sediments, sewage sludge digestors and biogas reactors. Especially in the terminal steps of methanogenesis, they determine the energy budgets of secondary (syntrophically) fermenting bacteria and their methanogenic partners. The literature provides considerable data on hydrogen pool sizes in such habitats, but little data exist for formate concentrations due to technical difficulties in formate determination at low concentration. Recent evidence from biochemical and molecular biological studies indicates that several secondary fermenters can use both hydrogen and formate for electron release, and may do so even simultaneously. Numerous strictly anaerobic bacteria contain enzymes which equilibrate hydrogen and formate pools to energetically equal values, and recent measurements in sewage digestors and biogas reactors indicate that - beyond occasional fluctuations - the pool sizes of hydrogen and formate are indeed energetically nearly equivalent. Nonetheless, a thermophilic archaeon from a submarine hydrothermal vent, Thermococcus onnurineus, can obtain ATP from the conversion of formate to hydrogen plus bicarbonate at 80°C, indicating that at least in this extreme environment the pools of formate and hydrogen are likely to be sufficiently different to support such an unusual type of energy conservation.

Reports have been published on blood coagulation disturbances by valproate therapy. In the present prospective trial, blood samples were drawn before valproate therapy, after 6 weeks of therapy, after more than 6 weeks and after longer than 6 months of valproate therapy from 23 children newly treated with valproate. Two children developed thrombocytopenia, and six children with initial normal von Willebrand factor showed acquired von Willebrand's disease. Fibrinogen levels dropped below the lower limit in 12 patients and subnormal factor XIII plasma levels were observed in 17% of patients. No patient developed signs of hemorrhage. Eight percent of patients developed valproate-induced thrombocytopenia. Reduction in platelets did not reach statistic significance. Thrombelastography showed a 47% incidence of altered platelet function. We found a statistically significant, positive correlation between clotting time of collagen extrinsic pathway inhibitor and, accordingly, adenosindiphosphate and valproate level. Plasmatic coagulation investigations showed a significant decrease of prothrombin time. Activated partial thromboplastin time measurements also showed significant prolongation with valproate. Activity of von Willebrand factor antigen and von Willebrand factor ristocetin cofactor significantly decreased. Factor XIII activity significantly decreased after valproate therapy for longer than 6 months (17% of children). Fibrinogen was significantly reduced. In the coagulatory system a decrease in the main antiprotease antithrombin III activity was observed. Blood coagulation disturbances are common in patients with valproate, but rarely become clinically symptomatic. Acquired von Willebrand's disease and hypofibrinogenemia may become relevant in patients with surgery or trauma. Particular attention should be paid to factor-XIII deficiency, which is especially seen with valproate therapy.

Growth of the anaerobic thermophile Thermacetogenium phaeum with methanol, ethanol, ethanolamine, and acetate was investigated in axenic cultures and in syntrophic cultures with Methanothermobacter thermautotrophicus. Microcompartment genes were identified in the T. phaeum genome, and presence of microcompartments was confirmed by transmission electron microscopy and proteome analysis. These genes were expressed only during growth with ethanolamine. Proteome data were compared after growth with all four substrates, and activities of key enzymes of the Wood–Ljungdahl pathway and of enzyme systems leading to production or degradation of acetaldehyde such as alcohol dehydrogenase, aldehyde:ferredoxin oxidoreductase, acetate kinase, and phosphate acetyltransferase were measured in cytoplasmic fractions. Accounting of fermentation stoichiometries and growth yields with all four substrates showed that ethanol and methanol oxidation follow the same stoichiometries as in Acetobacterium woodii. On the other hand, the pathways of ethanol and methanol degradations vary between both organisms. Growth yields of T. phaeum were substantially lower than reported for A. woodii. Since T. phaeum has no Rnf complex encoded in its genome, the mechanisms of ATP synthesis have to be different from those of A. woodii. In addition to the central degradation pathways also found in A. woodii, T. phaeum maintains enzyme systems that compensate for the absence of an Rnf-complex but which on the other hand cause a loss of energy. On the basis of our data, pathways of methanol and ethanol degradation in T. phaeum are discussed.