Structural insights into CodY activation and DNA recognition.

Virulence factors enable pathogenic bacteria to infect host cells, establish infection, and contribute to disease progressions. In Gram-positive pathogens such as Staphylococcus aureus (Sa) and Enterococcus faecalis (Ef), the pleiotropic transcription factor CodY plays a key role in integrating metabolism and virulence factor expression. However, to date, the structural mechanisms of CodY activation and DNA recognition are not understood. Here, we report the crystal structures of CodY from Sa and Ef in their ligand-free form and their ligand-bound form complexed with DNA. Binding of the ligands-branched chain amino acids and GTP-induces conformational changes in the form of helical shifts that propagate to the homodimer interface and reorient the linker helices and DNA binding domains. DNA binding is mediated by a non-canonical recognition mechanism dictated by DNA shape readout. Furthermore, two CodY dimers bind to two overlapping binding sites in a highly cooperative manner facilitated by cross-dimer interactions and minor groove deformation. Our structural and biochemical data explain how CodY can bind a wide range of substrates, a hallmark of many pleiotropic transcription factors. These data contribute to a better understanding of the mechanisms underlying virulence activation in important human pathogens.

A Highly Substituted Ring-Fused 2-Pyridone Compound Targeting PrfA and the Efflux Regulator BrtA in Listeria monocytogenes.

Listeria monocytogenes is a facultative Gram-positive bacterium that causes listeriosis, a severe foodborne disease. We previously discovered that ring-fused 2-pyridone compounds can decrease virulence factor expression in Listeria by binding and inactivating the PrfA virulence activator. In this study, we tested PS900, a highly substituted 2-pyridone that was recently discovered to be bactericidal to other Gram-positive pathogenic bacteria, such as Staphylococcus aureus and Enterococcus faecalis. We show that PS900 can interact with PrfA and reduce the expression of virulence factors. Unlike previous ring-fused 2-pyridones shown to inactivate PrfA, PS900 had an additional antibacterial activity and was found to potentiate sensitivity toward cholic acid. Two PS900-tolerant mutants able to grow in the presence of PS900 carried mutations in the brtA gene, encoding the BrtA repressor. In wild-type (WT) bacteria, cholic acid binds and inactivates BrtA, thereby alleviating the expression of the multidrug transporter MdrT. Interestingly, we found that PS900 also binds to BrtA and that this interaction causes BrtA to dissociate from its binding site in front of the mdrT gene. In addition, we observed that PS900 potentiated the effect of different osmolytes. We suggest that the increased potency of cholic acid and osmolytes to kill bacteria in the presence of PS900 is due to the ability of the latter to inhibit general efflux, through a yet-unknown mechanism. Our data indicate that thiazolino 2-pyridones constitute an attractive scaffold when designing new types of antibacterial agents. IMPORTANCE Bacteria resistant to one or several antibiotics are a very large problem, threatening not only treatment of infections but also surgery and cancer treatments. Thus, new types of antibacterial drugs are desperately needed. In this work, we show that a new generation of substituted ring-fused 2-pyridones not only inhibit Listeria monocytogenes virulence gene expression, presumably by inactivating the PrfA virulence regulator, but also potentiate the bactericidal effects of cholic acid and different osmolytes. We identified a multidrug repressor as a second target of 2-pyridones. The repressor-2-pyridone interaction displaces the repressor from DNA, thus increasing the expression of a multidrug transporter. In addition, our data suggest that the new class of ring-fused 2-pyridones are efficient efflux inhibitors, possibly explaining why the simultaneous addition of 2-pyridones together with cholic acid or osmolytes is detrimental for the bacterium. This work proves conclusively that 2-pyridones constitute a promising scaffold to build on for future antibacterial drug design.

Identification of amino acid domains of Borrelia burgdorferi P66 that are surface exposed and important for localization, oligomerization, and porin function of the protein.

P66, a bifunctional integral outer membrane protein, is necessary for Borrelia burgdorferi to establish initial infection and to disseminate in mice. The integrin binding function of P66 facilitates extravasation and dissemination, but the role of its porin function during murine infection has not been investigated. A limitation to studying P66 porin function during mammalian infection has been the lack of structural information for P66. In this study, we experimentally characterized specific domains of P66 with regard to structure and function. First, we aligned the amino acid sequences of P66 from Lyme disease-causing Borrelia and relapsing fever-causing Borrelia to identify conserved and unique domains between these disease-causing clades. Then, we examined whether specific domains of P66 are exposed on the surface of the bacteria by introducing c-Myc epitope tags into each domain of interest. The c-Myc epitope tag inserted C-terminally to E33 (highly conserved domain), to T187 (integrin binding region domain and a non-conserved domain), and to E334 (non-conserved domain) were all detected on the surface of Borrelia burgdorferi. The c-Myc epitope tag inserted C-terminally to E33 and D303 in conserved domains disrupted P66 oligomerization and porin function. In a murine model of infection, the E33 and D303 mutants exhibited decreased infectivity and dissemination. Taken together, these results suggest the importance of these conserved domains, and potentially P66 porin function, in vivo.

Ring-fused 2-pyridones effective against multidrug-resistant Gram-positive pathogens and synergistic with standard-of-care antibiotics.

The alarming rise of multidrug-resistant Gram-positive bacteria has precipitated a healthcare crisis, necessitating the development of new antimicrobial therapies. Here we describe a new class of antibiotics based on a ring-fused 2-pyridone backbone, which are active against vancomycin-resistant enterococci (VRE), a serious threat as classified by the Centers for Disease Control and Prevention, and other multidrug-resistant Gram-positive bacteria. Ring-fused 2-pyridone antibiotics have bacteriostatic activity against actively dividing exponential phase enterococcal cells and bactericidal activity against nondividing stationary phase enterococcal cells. The molecular mechanism of drug-induced killing of stationary phase cells mimics aspects of fratricide observed in enterococcal biofilms, where both are mediated by the Atn autolysin and the GelE protease. In addition, combinations of sublethal concentrations of ring-fused 2-pyridones and standard-of-care antibiotics, such as vancomycin, were found to synergize to kill clinical strains of VRE. Furthermore, a broad range of antibiotic resistant Gram-positive pathogens, including those responsible for the increasing incidence of antibiotic resistant healthcare-associated infections, are susceptible to this new class of 2-pyridone antibiotics. Given the broad antibacterial activities of ring-fused 2-pyridone compounds against Gram-positive (GmP) bacteria we term these compounds GmPcides, which hold promise in combating the rising tide of antibiotic resistant Gram-positive pathogens.

Complete Genome Sequence of the Uropathogenic Methicillin-Resistant Staphylococcus aureus Strain MRSA-1369.

MRSA-1369 is a uropathogenic methicillin-resistant Staphylococcus aureus (MRSA) strain. Here, we present the complete genome sequence of MRSA-1369, which consists of one chromosome (2.87 Mb) and two plasmids (16.68 kb and 3.13 kb). This will serve as a reference genome for future Staphylococcus aureus pathogenesis and multiomic studies.

K2S2O8-mediated coupling of 6-amino-7-aminomethyl-thiazolino-pyridones with aldehydes to construct amyloid affecting pyrimidine-fused thiazolino-2-pyridones.

We herein present the synthesis of diversely functionalized pyrimidine fused thiazolino-2-pyridones via K2S2O8-mediated oxidative coupling of 6-amino-7-(aminomethyl)-thiazolino-2-pyridones with aldehydes. The developed protocol is mild, has wide substrate scope, and does not require transition metal catalyst or base. Some of the synthesized compounds have an ability to inhibit the formation of Amyloid-ß fibrils associated with Alzheimer's disease, while others bind to mature amyloid-ß and α-synuclein fibrils.

The oxylipin and endocannabidome responses in acute phase Plasmodium falciparum malaria in children.

BACKGROUND: Oxylipins and endocannabinoids are low molecular weight bioactive lipids that are crucial for initiation and resolution of inflammation during microbial infections. Metabolic complications in malaria are recognized contributors to severe and fatal malaria, but the impact of malaria infection on the production of small lipid derived signalling molecules is unknown. Knowledge of immunoregulatory patterns of these molecules in malaria is of great value for better understanding of the disease and improvement of treatment regimes, since the action of these classes of molecules is directly connected to the inflammatory response of the organism. METHODS: Detection of oxylipins and endocannabinoids from plasma samples from forty children with uncomplicated and severe malaria as well as twenty controls was done after solid phase extraction followed by chromatography mass spectrometry analysis. The stable isotope dilution method was used for compound quantification. Data analysis was done with multivariate (principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA®) and univariate approaches (receiver operating characteristic (ROC) curves, t tests, correlation analysis). RESULTS: Forty different oxylipin and thirteen endocannabinoid metabolites were detected in the studied samples, with one oxylipin (thromboxane B2, TXB2) in significantly lower levels and four endocannabinoids (OEA, PEA, DEA and EPEA) at significantly higher levels in infected individuals as compared to controls according to t test analysis with Bonferroni correction. Three oxylipins (13-HODE, 9-HODE and 13-oxo-ODE) were higher in severe compared to uncomplicated malaria cases according to the results from multivariate analysis. Observed changes in oxylipin levels can be connected to activation of cytochrome P450 (CYP) and 5-lipoxygenase (5-LOX) metabolic pathways in malaria infected individuals compared to controls, and related to increased levels of all linoleic acid oxylipins in severe patients compared to uncomplicated ones. The endocannabinoids were extremely responsive to malaria infection with majority of this class of molecules found at higher levels in infected individuals compared to controls. CONCLUSIONS: It was possible to detect oxylipin and endocannabinoid molecules that can be potential biomarkers for differentiation between malaria infected individuals and controls and between different classes of malaria. Metabolic pathways that could be targeted towards an adjunctive therapy in the treatment of malaria were also pinpointed.

Lipid response patterns in acute phase paediatric Plasmodium falciparum malaria.

INTRODUCTION: Several studies have observed serum lipid changes during malaria infection in humans. All of them were focused at analysis of lipoproteins, not specific lipid molecules. The aim of our study was to identify novel patterns of lipid species in malaria infected patients using lipidomics profiling, to enhance diagnosis of malaria and to evaluate biochemical pathways activated during parasite infection. METHODS: Using a multivariate characterization approach, 60 samples were representatively selected, 20 from each category (mild, severe and controls) of the 690 study participants between age of 0.5-6 years. Lipids from patient's plasma were extracted with chloroform/methanol mixture and subjected to lipid profiling with application of the LCMS-QTOF method. RESULTS: We observed a structured plasma lipid response among the malaria-infected patients as compared to healthy controls, demonstrated by higher levels of a majority of plasma lipids with the exception of even-chain length lysophosphatidylcholines and triglycerides with lower mass and higher saturation of the fatty acid chains. An inverse lipid profile relationship was observed when plasma lipids were correlated to parasitaemia. CONCLUSIONS: This study demonstrates how mapping the full physiological lipid response in plasma from malaria-infected individuals can be used to understand biochemical processes during infection. It also gives insights to how the levels of these molecules relate to acute immune responses.

Metabolic Signature Profiling as a Diagnostic and Prognostic Tool in Pediatric Plasmodium falciparum Malaria.

Background. Accuracy in malaria diagnosis and staging is vital to reduce mortality and post infectious sequelae. In this study, we present a metabolomics approach to diagnostic staging of malaria infection, specifically Plasmodium falciparum infection in children. Methods. A group of 421 patients between 6 months and 6 years of age with mild and severe states of malaria with age-matched controls were included in the study, 107, 192, and 122, individuals, respectively. A multivariate design was used as basis for representative selection of 20 patients in each category. Patient plasma was subjected to gas chromatography-mass spectrometry analysis, and a full metabolite profile was produced from each patient. In addition, a proof-of-concept model was tested in a Plasmodium berghei in vivo model where metabolic profiles were discernible over time of infection. Results. A 2-component principal component analysis revealed that the patients could be separated into disease categories according to metabolite profiles, independently of any clinical information. Furthermore, 2 subgroups could be identified in the mild malaria cohort who we believe represent patients with divergent prognoses. Conclusions. Metabolite signature profiling could be used both for decision support in disease staging and prognostication.

Integrin binding by Borrelia burgdorferi†P66 facilitates dissemination but is not required for infectivity.

P66, a Borrelia burgdorferi surface protein with porin and integrin-binding activities, is essential for murine infection. The role of P66 integrin-binding activity in B. burgdorferi infection was investigated and found to affect transendothelial migration. The role of integrin binding, specifically, was tested by mutation of two amino acids (D205A,D207A) or deletion of seven amino acids (Del202-208). Neither change affected surface localization or channel-forming activity of P66, but both significantly reduced binding to αv ß3 . Integrin-binding deficient B. burgdorferi strains caused disseminated infection in mice at 4 weeks post-subcutaneous inoculation, but bacterial burdens were significantly reduced in some tissues. Following intravenous inoculation, the Del202-208 bacteria were below the limit of detection in all tissues assessed at 2 weeks post-inoculation, but bacterial burdens recovered to wild-type levels at 4 weeks post-inoculation. The delay in tissue colonization correlated with reduced migration of the Del202-208 strains across microvascular endothelial cells, similar to Δp66 bacteria. These results indicate that integrin binding by P66 is important to efficient dissemination of B. burgdorferi, which is critical to its ability to cause disease manifestations in incidental hosts and to its maintenance in the enzootic cycle.

Study of the protein complex, pore diameter, and pore-forming activity of the Borrelia burgdorferi P13 porin.

P13 is one of the major outer membrane proteins of Borrelia burgdorferi. Previous studies described P13 as a porin. In the present study some structure and function aspects of P13 were studied. P13 showed according to lipid bilayer studies a channel-forming activity of 0.6 nanosiemens in 1 m KCl. Single channel and selectivity measurements demonstrated that P13 had no preference for either cations or anions and showed no voltage-gating up to ±100 mV. Blue native polyacrylamide gel electrophoresis was used to isolate and characterize the P13 protein complex in its native state. The complex had a high molecular mass of about 300 kDa and was only composed of P13 monomers. The channel size was investigated using non-electrolytes revealing an apparent diameter of about 1.4 nm with a 400-Da molecular mass cut-off. Multichannel titrations with different substrates reinforced the idea that P13 forms a general diffusion channel. The identity of P13 within the complex was confirmed by second dimension SDS-PAGE, Western blotting, mass spectrometry, and the use of a p13 deletion mutant strain. The results suggested that P13 is the protein responsible for the 0.6-nanosiemens pore-forming activity in the outer membrane of B. burgdorferi.

Use of nonelectrolytes reveals the channel size and oligomeric constitution of the Borrelia burgdorferi P66 porin.

In the Lyme disease spirochete Borrelia burgdorferi, the outer membrane protein P66 is capable of pore formation with an atypical high single-channel conductance of 11 nS in 1 M KCl, which suggested that it could have a larger diameter than 'normal' Gram-negative bacterial porins. We studied the diameter of the P66 channel by analyzing its single-channel conductance in black lipid bilayers in the presence of different nonelectrolytes with known hydrodynamic radii. We calculated the filling of the channel with these nonelectrolytes and the results suggested that nonelectrolytes (NEs) with hydrodynamic radii of 0.34 nm or smaller pass through the pore, whereas neutral molecules with greater radii only partially filled the channel or were not able to enter it at all. The diameter of the entrance of the P66 channel was determined to be ≤1.9 nm and the channel has a central constriction of about 0.8 nm. The size of the channel appeared to be symmetrical as judged from one-sidedness of addition of NEs. Furthermore, the P66-induced membrane conductance could be blocked by 80-90% by the addition of the nonelectrolytes PEG 400, PEG 600 and maltohexaose to the aqueous phase in the low millimolar range. The analysis of the power density spectra of ion current through P66 after blockage with these NEs revealed no chemical reaction responsible for channel block. Interestingly, the blockage of the single-channel conductance of P66 by these NEs occurred in about eight subconductance states, indicating that the P66 channel could be an oligomer of about eight individual channels. The organization of P66 as a possible octamer was confirmed by Blue Native PAGE and immunoblot analysis, which both demonstrated that P66 forms a complex with a mass of approximately 460 kDa. Two dimension SDS PAGE revealed that P66 is the only polypeptide in the complex.

DipA, a pore-forming protein in the outer membrane of Lyme disease spirochetes exhibits specificity for the permeation of dicarboxylates.

Lyme disease Borreliae are highly dependent on the uptake of nutrients provided by their hosts. Our study describes the identification of a 36 kDa protein that functions as putative dicarboxylate-specific porin in the outer membrane of Lyme disease Borrelia. The protein was purified by hydroxyapatite chromatography from Borrelia burgdorferi B31 and designated as DipA, for dicarboxylate-specific porin A. DipA was partially sequenced, and corresponding genes were identified in the genomes of B. burgdorferi B31, Borrelia garinii PBi and Borrelia afzelii PKo. DipA exhibits high homology to the Oms38 porins of relapsing fever Borreliae. B. burgdorferi DipA was characterized using the black lipid bilayer assay. The protein has a single-channel conductance of 50 pS in 1 M KCl, is slightly selective for anions with a permeability ratio for cations over anions of 0.57 in KCl and is not voltage-dependent. The channel could be partly blocked by different di- and tricarboxylic anions. Particular high stability constants up to about 28,000 l/mol (in 0.1 M KCl) were obtained among the 11 tested anions for oxaloacetate, 2-oxoglutarate and citrate. The results imply that DipA forms a porin specific for dicarboxylates which may play an important role for the uptake of specific nutrients in different Borrelia species.

Multiplex PCR as a tool for validating plasmid content of Borrelia burgdorferi.

Borrelia burgdorferi has an unusual genomic structure containing 21 plasmids. These plasmids carry genes that are essential for infectivity and survival of the spirochetes in vivo. Several plasmids are lost during cultivation in vitro, which might lead to a heterogeneous population after multiple passages and loss of infectivity in laboratory animals. Herein, we present a simple and inexpensive multiplex PCR method that detects the complete plasmid profile of B. burgdorferi B31 in just two PCR tubes.

P66 porins are present in both Lyme disease and relapsing fever spirochetes: a comparison of the biophysical properties of P66 porins from six Borrelia species.

The genus Borrelia is the cause of the two human diseases: Lyme disease (LD) and relapsing fever (RF). Both LD and RF Borrelia species are obligate parasites and are dependent on nutrients provided by their hosts. The first step of nutrient uptake across the outer membrane of these Gram-negative bacteria is accomplished by water-filled channels, so-called porins. The knowledge of the porin composition in the outer membranes of the different pathogenic Borrelia species is limited. Only one porin has been described in relapsing fever spirochetes to date, whereas four porins are known to be present in Lyme disease agents. From these, the Borrelia burgdorferi outer membrane channel P66 is known to act as an adhesin and was well studied as a porin. To investigate if P66 porins are expressed and similarly capable of pore formation in other Borrelia causing Lyme disease or relapsing fever three LD species (B. burgdorferi, B. afzelii, B. garinii) and three RF species (B. duttonii, B. recurrentis and B. hermsii) were investigated for outer membrane proteins homologous to P66. A search in current published RF genomes, comprising the ones of B. duttonii, B. recurrentis and B. hermsii, indicated that they all contained P66 homologues. The P66 homologues of the six Borrelia species were purified to homogeneity and their pore-forming abilities as well as the biophysical properties of the pores were analyzed using the black lipid bilayer assay.