Hemolytic activity and biofilm-formation among clinical isolates of group B streptococcus causing acute urinary tract infection and asymptomatic bacteriuria.

Streptococcus agalactiae, also known as group B Streptococcus, is an aetiological agent of urinary tract infection (UTI) in adults, including cystitis, pyelonephritis and asymptomatic bacteriuria (ABU). Whereas ABU-causing S. agalactiae (ABSA) have been shown to grow and achieve higher culture denstity in human urine compared to uropathogenic S. agalactiae (UPSA) other phenotypic distinctions between S. agalactiae isolated from different forms of UTI are not known. Here, we define the hemolytic activities and biofilm-formation of a collection of clinical isolates of UPSA, ABSA and recurrent S. agalactiae bacteriuria (rSAB) strains to explore these phenotypes in the context of clinical history of isolates. A total of 61 UPSA, 184 ABSA, and 47 rSAB isolates were analyzed for relative hemolytic activity by spot assay on blood agar, which was validated using a erythrocyte lysis suspension assay. Biofilm formation was determined by microtiter plate assay with Lysogeny and Todd-Hewitt broths supplemented with 1% glucose to induce biofilm formation. We also used multiplex PCR to analyze isolates for the presence of genes encoding adhesive pili, which contribute to biofilm formation. Comparing the hemolytic activities of 292 isolates showed, surprisingly, that ABSA strains were significantly more likely to be highly hemolytic compared to other strains. In contrast, there were no differences between the relative abilities of strains from the different clinical history groups to form biofilms. Taken together, these findings demonstrate a propensity of S. agalactiae causing ABU to be highly hemolytic but no link between clinical history of UTI strains and ability to form biofilm.

An Updated Conceptual Model on the Pathogenesis of Bacterial Vaginosis.

Bacterial vaginosis (BV) is the most common cause of vaginal discharge. It is associated with an increased risk of preterm delivery, pelvic inflammatory disease, and an increased risk of acquisition of sexually transmitted infections including human immunodeficiency virus (HIV). The epidemiology of BV supports sexual transmission. However, its etiology remains unknown. At the center of the debate is whether BV is caused by a primary pathogen or a polymicrobial consortium of microorganisms that are sexually transmitted. We previously published a conceptual model hypothesizing that BV is initiated by sexual transmission of Gardnerella vaginalis. Critics of this model have iterated that G. vaginalis is found in virginal women and in sexually active women with a normal vaginal microbiota. In addition, colonization does not always lead to BV. However, recent advances in BV pathogenesis research have determined the existence of 13 different species within the genus Gardnerella. It may be that healthy women are colonized by nonpathogenic Gardnerella species, whereas virulent strains are involved in BV development. Based on our results from a recent prospective study, in addition to an extensive literature review, we present an updated conceptual model for the pathogenesis of BV that centers on the roles of virulent strains of G. vaginalis, as well as Prevotella bivia and Atopobium vaginae.

Mosquito Bite-Induced Controlled Human Malaria Infection with Plasmodium vivax or P. falciparum Generates Immune Responses to Homologous and Heterologous Preerythrocytic and Erythrocytic Antigens.

Seroepidemiological studies on the prevalence of antibodies to malaria antigens are primarily conducted on individuals from regions of endemicity. It is therefore difficult to accurately correlate the antibody responses to the timing and number of prior malaria infections. This study was undertaken to assess the evolution of antibodies to the dominant surface antigens of Plasmodium vivax and P. falciparum following controlled human malaria infection (CHMI) in malaria-naive individuals. Serum samples from malaria-naive adults, collected before and after CHMI with either P. vivax (n = 18) or P. falciparum (n = 18), were tested for the presence of antibodies to the circumsporozoite protein (CSP) and the 42-kDa fragment of merozoite surface protein 1 (MSP-142) of P. vivax and P. falciparum using an enzyme-linked immunosorbent assay (ELISA). Approximately 1 month following CHMI with either P. vivax or P. falciparum, >60% of subjects seroconverted to homologous CSP and MSP-1. More than 50% of the subjects demonstrated reactivity to heterologous CSP and MSP-142, and a similar proportion of subjects remained seropositive to homologous MSP-142 >5 months after CHMI. Computational analysis provides insight into the presence of cross-reactive responses. The presence of long-lived and heterologous reactivity and its functional significance, if any, need to be taken into account while evaluating malaria exposure in field settings.

Small Molecule Inhibition of the Ubiquitin-specific Protease USP2 Accelerates cyclin D1 Degradation and Leads to Cell Cycle Arrest in Colorectal Cancer and Mantle Cell Lymphoma Models.

Deubiquitinases are important components of the protein degradation regulatory network. We report the discovery of ML364, a small molecule inhibitor of the deubiquitinase USP2 and its use to interrogate the biology of USP2 and its putative substrate cyclin D1. ML364 has an IC50 of 1.1 µm in a biochemical assay using an internally quenched fluorescent di-ubiquitin substrate. Direct binding of ML364 to USP2 was demonstrated using microscale thermophoresis. ML364 induced an increase in cellular cyclin D1 degradation and caused cell cycle arrest as shown in Western blottings and flow cytometry assays utilizing both Mino and HCT116 cancer cell lines. ML364, and not the inactive analog 2, was antiproliferative in cancer cell lines. Consistent with the role of cyclin D1 in DNA damage response, ML364 also caused a decrease in homologous recombination-mediated DNA repair. These effects by a small molecule inhibitor support a key role for USP2 as a regulator of cell cycle, DNA repair, and tumor cell growth.

Uropathogenic Escherichia coli Engages CD14-Dependent Signaling to Enable Bladder-Macrophage-Dependent Control of Acute Urinary Tract Infection.

BACKGROUND: CD14, a coreceptor for several pattern recognition receptors and a widely used monocyte/macrophage marker, plays a key role in host responses to gram-negative bacteria. Despite the central role of CD14 in the inflammatory response to lipopolysaccharide and other microbial products and in the dissemination of bacteria in some infections, the signaling networks controlled by CD14 during urinary tract infection (UTI) are unknown. METHODS: We used uropathogenic Escherichia coli (UPEC) infection of wild-type (WT) C57BL/6 and Cd14(-/-) mice and RNA sequencing to define the CD14-dependent transcriptional signature and the role of CD14 in host defense against UTI in the bladder. RESULTS: UPEC induced the upregulation of Cd14 and the monocyte/macrophage-related genes Emr1/F4/80 and Csf1r/c-fms, which was associated with lower UPEC burdens in WT mice, compared with Cd14(-/-) mice. Exacerbation of infection in Cd14(-/-) mice was associated with the absence of a 491-gene transcriptional signature in the bladder that encompassed multiple host networks not previously associated with this receptor. CD14-dependent pathways included immune cell trafficking, differential cytokine production in macrophages, and interleukin 17 signaling. Depletion of monocytes/macrophages in the bladder by administration of liposomal clodronate led to higher UPEC burdens. CONCLUSIONS: This study identifies new host protective and signaling roles for CD14 in the bladder during UPEC UTI.

Binding of undamaged double stranded DNA to vaccinia virus uracil-DNA Glycosylase.

BACKGROUND: Uracil-DNA glycosylases are evolutionarily conserved DNA repair enzymes. However, vaccinia virus uracil-DNA glycosylase (known as D4), also serves as an intrinsic and essential component of the processive DNA polymerase complex during DNA replication. In this complex D4 binds to a unique poxvirus specific protein A20 which tethers it to the DNA polymerase. At the replication fork the DNA scanning and repair function of D4 is coupled with DNA replication. So far, DNA-binding to D4 has not been structurally characterized. RESULTS: This manuscript describes the first structure of a DNA-complex of a uracil-DNA glycosylase from the poxvirus family. This also represents the first structure of a uracil DNA glycosylase in complex with an undamaged DNA. In the asymmetric unit two D4 subunits bind simultaneously to complementary strands of the DNA double helix. Each D4 subunit interacts mainly with the central region of one strand. DNA binds to the opposite side of the A20-binding surface on D4. Comparison of the present structure with the structure of uracil-containing DNA-bound human uracil-DNA glycosylase suggests that for DNA binding and uracil removal D4 employs a unique set of residues and motifs that are highly conserved within the poxvirus family but different in other organisms. CONCLUSION: The first structure of D4 bound to a truly non-specific undamaged double-stranded DNA suggests that initial binding of DNA may involve multiple non-specific interactions between the protein and the phosphate backbone.

A Theileria annulata parasite with a single mutation, methionine 128 to isoleucine (M128I), in cytochrome B is resistant to buparvaquone.

Tropical theileriosis is a fatal leukemic-like disease of cattle caused by the tick-transmitted protozoan parasite Theileria annulata. The economics of cattle meat and milk production is severely affected by theileriosis in endemic areas. The hydroxynaphtoquinone buparvaquone (BPQ) is the only available drug currently used to treat clinical theileriosis, whilst BPQ resistance is emerging and spreading in endemic areas. Here, we chronically exposed T. annulata-transformed macrophages in vitro to BPQ and monitored the emergence of drug-resistant parasites. Surviving parasites revealed a significant increase in BPQ IC50 compared to the wild type parasites. Drug resistant parasites from two independent cloned lines had an identical single mutation, M128I, in the gene coding for T. annulata cytochrome B (Tacytb). This in vitro generated mutation has not been reported in resistant field isolates previously, but is reminiscent of the methionine to isoleucine mutation in atovaquone-resistant Plasmodium and Babesia. The M128I mutation did not appear to exert any deleterious effect on parasite fitness (proliferation and differentiation to merozoites). To gain insight into whether drug-resistance could have resulted from altered drug binding to TaCytB we generated in silico a 3D-model of wild type TaCytB and docked BPQ to the predicted 3D-structure. Potential binding sites cluster in four areas of the protein structure including the Q01 site. The bound drug in the Q01 site is expected to pack against an alpha helix, which included M128, suggesting that the change in amino acid in this position may alter drug-binding. The in vitro generated BPQ resistant T. annulata is a useful tool to determine the contribution of the various predicted docking sites to BPQ resistance and will also allow testing novel drugs against theileriosis for their potential to overcome BPQ resistance.

Crystallization and preliminary X-ray diffraction analysis of three recombinant mutants of Vaccinia virus uracil DNA glycosylase.

Amino-acid residues located at a highly flexible area in the uracil DNA glycosylase of Vaccinia virus were mutated. In the crystal structure of wild-type D4 these residues lie at the dimer interface. Specifically, three mutants were generated: (i) residue Arg167 was replaced with an alanine (R167AD4), (ii) residues Glu171, Ser172 and Pro173 were substituted with three glycine residues (3GD4) and (iii) residues Glu171 and Ser172 were deleted (Δ171-172D4). Mutant proteins were expressed, purified and crystallized in order to investigate the effects of these mutations on the structure of the protein.

Crystal structure and induced stability of trimeric BxpB: implications for the assembly of BxpB-BclA complexes in the exosporium of Bacillus anthracis.

The outermost exosporium layer of Bacillus anthracis spores, the causative agents of anthrax, is comprised of a basal layer and an external hair-like nap. The nap includes filaments composed of trimers of the collagen-like glycoprotein BclA. Essentially all BclA trimers are attached to the spore in a process in which part of the 38-residue amino-terminal domain (NTD) of BclA forms an extremely stable interaction with the basal layer protein BxpB. Evidence indicates that the BclA-BxpB interaction is direct and requires trimeric BxpB. To further investigate the nature of the BclA-BxpB interaction, we determined the crystal structure of BxpB. The structure was trimeric with each monomer consisting of 11 ß strands with connecting loops. The structure did not include apparently disordered amino acids 1-19, which contain the only two cysteine residues of the 167-residue BxpB. The orientation of the structure reveals regions of BxpB that could be involved in interacting with the BclA NTD and with adjacent cysteine-rich proteins in the basal layer. Furthermore, the BxpB structure closely resembles that of the 134-residue carboxyl-terminal domain of BclA, which forms trimers that are highly resistant to heat and detergent. We demonstrated that BxpB trimers do not share this resistance. However, when BxpB trimers are mixed with a peptide containing residues 20-38 of BclA, they form a complex that is as stable as BclA-BxpB complexes extracted from spores. Together, our results provide new insights into the mechanism of BclA-BxpB attachment and incorporation into the exosporium. IMPORTANCE The B. anthracis exosporium plays major roles in spore survival and infectivity, but the complex mechanism of its assembly is poorly understood. Key steps in this process are the stable attachment of collagen-like BclA filaments to the major basal layer structural protein BxpB and the insertion of BxpB into an underlying basal layer scaffold. The goal of this study is to further elucidate these interactions thereby advancing our understanding of exosporium assembly, a process shared by many spore-forming bacteria including important human pathogens.

Conformationally Defined Rexinoids for the Prevention of Inflammation and Nonmelanoma Skin Cancers.

Compound 1 is a potent rexinoid that is highly effective in cancer chemoprevention but elevates serum triglycerides. In an effort to separate the lipid toxicity from the anticancer activity of 1, we synthesized four new analogs of rexinoid 1, of which three rexinoids did not elevate serum triglycerides. Rexinoids 3 and 4 are twice as potent as rexinoid 1 in binding to Retinoid X receptor (RXR). All-trans retinoic acid (ATRA) plays a key role in maintaining skin homeostasis, and rexinoids 3-6 are highly effective in upregulating the genes responsible for the biosynthesis of ATRA. Inflammation plays a key role in skin cancer, and rexinoids 3 and 4 are highly effective in diminishing LPS-induced inflammation. Rexinoids 3 and 4 are highly effective in preventing UVB-induced nonmelanoma skin cancer (NMSC) without displaying any overt toxicities. Biophysical studies of rexinoids 3 and 5 bound to hRXRα-ligand binding domain (LBD) reveal important conformational and dynamical differences in the ligand binding domain.

Malaria immunoepidemiology in low transmission: correlation of infecting genotype and immune response to domains of Plasmodium falciparum merozoite surface protein 3.

Malaria caused by Plasmodium falciparum is a major cause of global infant mortality, and no effective vaccine currently exists. Multiple potential vaccine targets have been identified, and immunoepidemiology studies have played a major part in assessing those candidates. When such studies are carried out in high-transmission settings, individuals are often superinfected with complex mixtures of genetically distinct P. falciparum types, making it impossible to directly correlate the genotype of the infecting antigen with the antibody response. In contrast, in regions of low transmission P. falciparum infections are often genetically simple, and direct comparison of infecting genotype and antigen-specific immune responses is possible. As a test of the utility of this approach, responses against several domains and allelic variants of the vaccine candidate P. falciparum merozoite surface protein 3 (PfMSP3) were tested in serum samples collected near Iquitos, Peru. Antibodies recognizing both the conserved C-terminal and the more variable N-terminal domain were identified, but anti-N-terminal responses were more prevalent, of higher titers, and primarily of cytophilic subclasses. Comparing antibody responses to different PfMSP3 variants with the PfMSP3 genotype present at the time of infection showed that anti-N-terminal responses were largely allele class specific, but there was some evidence for responses that cross-reacted across allele classes. Evidence for cross-reactive responses was much stronger when variants within one allele class were tested, which has implications for the rational development of genotype-transcending PfMSP3-based vaccines.

Identification of protein-protein interaction inhibitors targeting vaccinia virus processivity factor for development of antiviral agents.

Poxvirus uracil DNA glycosylase D4 in association with A20 and the catalytic subunit of DNA polymerase forms the processive polymerase complex. The binding of D4 and A20 is essential for processive polymerase activity. Using an AlphaScreen assay, we identified compounds that inhibit protein-protein interactions between D4 and A20. Effective interaction inhibitors exhibited both antiviral activity and binding to D4. These results suggest that novel antiviral agents that target the protein-protein interactions between D4 and A20 can be developed for the treatment of infections with poxviruses, including smallpox.

Crystal structure of Plasmodium falciparum phosphoglycerate kinase: evidence for anion binding in the basic patch.

3-Phosphoglycerate kinase (EC 2.7.2.3) is a key enzyme in the glycolytic pathway and catalyzes an important phosphorylation step leading to the production of ATP. The crystal structure of Plasmodium falciparum phosphoglycerate kinase (PfPGK) in the open conformation is presented in two different groups, namely I222 and P6(1)22. The structure in I222 space group is solved using MAD and refined at 3Å whereas that in P6(1)22A is solved using MR and refined at 2.7Å. I222 form has three monomers in asymmetric unit whereas P6(1)22 form has two monomers in the asymmetric unit. In both crystal forms a sulphate ion is located at the active site where ATP binds, but no Mg(2+) ion is observed. For the first time another sulphate ion is found at the basic patch where the 3-phosphate of 1,3-biphosphoglycerate normally binds. This was found in both chains of P6(1)22 form but only in chain A of I222 form.

Vaccinia virus D4 mutants defective in processive DNA synthesis retain binding to A20 and DNA.

Genome replication is inefficient without processivity factors, which tether DNA polymerases to their templates. The vaccinia virus DNA polymerase E9 requires two viral proteins, A20 and D4, for processive DNA synthesis, yet the mechanism of how this tricomplex functions is unknown. This study confirms that these three proteins are necessary and sufficient for processivity, and it focuses on the role of D4, which also functions as a uracil DNA glycosylase (UDG) repair enzyme. A series of D4 mutants was generated to discover which sites are important for processivity. Three point mutants (K126V, K160V, and R187V) which did not function in processive DNA synthesis, though they retained UDG catalytic activity, were identified. The mutants were able to compete with wild-type D4 in processivity assays and retained binding to both A20 and DNA. The crystal structure of R187V was resolved and revealed that the local charge distribution around the substituted residue is altered. However, the mutant protein was shown to have no major structural distortions. This suggests that the positive charges of residues 126, 160, and 187 are required for D4 to function in processive DNA synthesis. Consistent with this is the ability of the conserved mutant K126R to function in processivity. These mutants may help unlock the mechanism by which D4 contributes to processive DNA synthesis.

Structure of the catalytic domain of Plasmodium falciparum ARF GTPase-activating protein (ARFGAP).

The crystal structure of the catalytic domain of the ADP ribosylation factor GTPase-activating protein (ARFGAP) from Plasmodium falciparum has been determined and refined to 2.4 Å resolution. Multiwavength anomalous diffraction (MAD) data were collected utilizing the Zn(2+) ion bound at the zinc-finger domain and were used to solve the structure. The overall structure of the domain is similar to those of mammalian ARFGAPs. However, several amino-acid residues in the area where GAP interacts with ARF1 differ in P. falciparum ARFGAP. Moreover, a number of residues that form the dimer interface in the crystal structure are unique in P. falciparum ARFGAP.

Streptococcus agalactiae glyceraldehyde-3-phosphate dehydrogenase (GAPDH) elicits multiple cytokines from human cells and has a minor effect on bacterial persistence in the murine female reproductive tract.

Streptococcus agalactiae glyceraldehyde 3-phosphate dehydrogenase (GAPDH), encoded by gapC, is a glycolytic enzyme that is associated with virulence and immune-mediated protection. However, the role of GAPDH in cellular cytokine responses to S. agalactiae, bacterial phagocytosis and colonization of the female reproductive tract, a central host niche, is unknown. We expressed and studied purified recombinant GAPDH (rGAPDH) of S. agalactiae in cytokine elicitation assays with human monocyte-derived macrophage, epithelial cell, and polymorphonuclear leukocyte (PMN) co-culture infection models. We also generated a S. agalactiae mutant that over-expresses GAPDH (oeGAPDH) from gapC using a constitutively active promoter, and analyzed the mutant in murine macrophage antibiotic protection assays and in virulence assays in vivo, using a colonization model that is based on experimental infection of the reproductive tract in female mice. Human cell co-cultures produced interleukin (IL)-1ß, IL-6, macrophage inflammatory protein (MIP)-1, tumor necrosis factor (TNF)-α and IL-10 within 24 h of exposure to rGAPDH. PMNs were required for several of these cytokine responses. However, over-expression of GAPDH in S. agalactiae did not significantly affect measures of phagocytic uptake compared to an empty vector control. In contrast, oeGAPDH-S. agalactiae showed a small but statistically significant attenuation for persistence in the reproductive tract of female mice during the chronic phase of infection (10-28 days post-inoculation), relative to the vector control. We conclude that S. agalactiae GAPDH elicits production of multiple cytokines from human cells, and over-expression of GAPDH renders the bacterium more susceptible to host clearance in the female reproductive tract.One-sentence summary: This study shows Streptococcus agalactiae glyceraldehyde 3-phosphate dehydrogenase, an enzyme that functions in glycolysis, gluconeogenesis and virulence, modifies phagocytosis outcomes, including cytokine synthesis, and affects bacterial persistence in the female reproductive tract.

Synthesis and characterization of potent inhibitors of Trypanosoma cruzi dihydrofolate reductase.

Dihydrofolate reductase (DHFR) of the parasite Trypanosoma cruzi (T. cruzi) is a potential target for developing drugs to treat Chagas' disease. We have undertaken a detailed structure-activity study of this enzyme. We report here synthesis and characterization of six potent inhibitors of the parasitic enzyme. Inhibitory activity of each compound was determined against T. cruzi and human DHFR. One of these compounds, ethyl 4-(5-[(2,4-diamino-6-quinazolinyl)methyl]amino-2-methoxyphenoxy)butanoate (6b) was co-crystallized with the bifunctional dihydrofolate reductase-thymidylate synthase enzyme of T. cruzi and the crystal structure of the ternary enzyme:cofactor:inhibitor complex was determined. Molecular docking was used to analyze the potential interactions of all inhibitors with T. cruzi DHFR and human DHFR. Inhibitory activities of these compounds are discussed in the light of enzyme-ligand interactions. Binding affinities of each inhibitor for the respective enzymes were calculated based on the experimental or docked binding mode. An estimated 60-70% of the total binding energy is contributed by the 2,4-diaminoquinazoline scaffold.

Structure of Plasmodium falciparum ADP-ribosylation factor 1.

Vesicular trafficking may play a crucial role in the pathogenesis and survival of the malaria parasite. ADP-ribosylation factors (ARFs) are among the major components of vesicular trafficking pathways in eukaryotes. The crystal structure of ARF1 GTPase from Plasmodium falciparum has been determined in the GDP-bound conformation at 2.5 Šresolution and is compared with the structures of mammalian ARF1s.

A simple and reliable method for determination of optimum pH in coupled enzyme assays.

Determination of the optimum pH in a coupled enzyme assay poses significant challenges because altering the pH of the reaction mixture can affect the performance of both enzymes. Here, we demonstrate a simple and reliable method to determine the pH optimum for pyruvate kinase using the pyruvate kinase/lactate dehydrogenase coupled enzyme assay. This simple and reliable method can be broadly adapted to determine the pH optimum for various enzymes that are assayed using a coupled enzyme assay.

Novel structure of the N-terminal helical domain of BibA, a group B streptococcus immunogenic bacterial adhesin.

BibA, a group B streptococcus (GBS) surface protein, has been shown to protect the pathogen from phagocytic killing by sequestering a complement inhibitor: C4b-binding protein (C4BP). Here, the X-ray crystallographic structure of a GBS BibA fragment (BibA126-398) and a low-resolution small-angle X-ray scattering (SAXS) structure of the full-length N-terminal domain (BibA34-400) are described. The BibA126-398 fragment crystal structure displayed a novel and predominantly helical structure. The tertiary arrangement of helices forms four antiparallel three-helix-bundle-motif repeats, with one long helix from a bundle extending into the next. Multiple mutations on recombinant BibA34-400 delayed the degradation of the protein, and circular dichroism spectroscopy of BibA34-400 suggested a similar secondary-structure composition to that observed in the crystallized BibA126-398 fragment. A model was generated for the 92 N-terminal residues (BibA34-125) using structural similarity prediction programs, and a BibA34-400 model was generated by combining the coordinates of BibA34-126 and BibA126-398. The X-ray structure of BibA126-398 and the model of BibA34-400 fitted well into the calculated SAXS envelope. One possible binding site for the BibA N-terminal domain was localized to the N-terminal CCP (complement-control protein) domains of the C4BP α-chain, as indicated by the decreased binding of BibA to a ΔCCP1 C4BP α-chain mutant. In summary, it is suggested that the GBS surface protein BibA, which consists of three antiparallel α-helical-bundle motifs, is unique and belongs to a new class of Gram-positive surface adhesins.