Lack of Clinical Manifestations in Asymptomatic Dengue Infection Is Attributed to Broad Down-Regulation and Selective Up-Regulation of Host Defence Response Genes

Objectives

Dengue represents one of the most serious life-threatening vector-borne infectious diseases that afflicts approximately 50 million people across the globe annually. Whilst symptomatic infections are frequently reported, asymptomatic dengue remains largely unnoticed. Therefore, we sought to investigate the immune correlates conferring protection to individuals that remain clinically asymptomatic.

Methods

We determined the levels of neutralizing antibodies (nAbs) and gene expression profiles of host immune factors in individuals with asymptomatic infections, and whose cognate household members showed symptoms consistent to clinical dengue infection.

Results

We observed broad down-regulation of host defense response (innate, adaptive and matrix metalloprotease) genes in asymptomatic individuals as against symptomatic patients, with selective up-regulation of distinct genes that have been associated with protection. Selected down-regulated genes include: TNF α (TNF), IL8, C1S, factor B (CFB), IL2, IL3, IL4, IL5, IL8, IL9, IL10 and IL13, CD80, CD28, and IL18, MMP8, MMP10, MMP12, MMP15, MMP16, and MMP24. Selected up-regulated genes include: RANTES (CCL5), MIP-1α (CCL3L1/CCL3L3), MIP-1β (CCL4L1), TGFβ (TGFB), and TIMP1.

Conclusion

Our findings highlight the potential association of certain host genes conferring protection against clinical dengue. These data are valuable to better explore the mysteries behind the hitherto poorly understood immunopathogenesis of subclinical dengue infection.     

Specific Role of Each Human Leukocyte Type in Viral Infections I. Monocyte as Host Cell for Vesicular Stomatitis Virus Replication In Vitro

Each major leukocyte type of the peripheral blood of healthy donors was studied in vitro for its ability to support vesicular stomatitis virus (VSV) replication. Purified cultures of each white blood cell type were prepared by the selective adsorption and elution of cells from silicone-treated glass beads. It was found that monocytes and macrophages (derived from the rapid transformation of monocytes in vitro) were the principal host cells for VSV replication. Interferon added to mixed leukocyte cultures, prior to virus inoculation, reduced virus yields and prevented destruction of macrophages. Cultures of small lymphocytes, containing no detectable monocytes or macrophages, produced amounts of virus equivalent to 1% of that produced in leukocyte cultures which contained 7% monocytes. Small lymphocytes did not undergo demonstrable cytopathic alterations in virus-infected cultures. VSV neither replicated nor produced cytopathic effects in polymorphonuclear leukocytes.     

Mutations in Complement Factor H Impair Alternative Pathway Regulation on Mouse Glomerular Endothelial Cells in Vitro

Complement factor H (FH) inhibits complement activation and interacts with glomerular endothelium via its complement control protein domains 19 and 20, which also recognize heparan sulfate (HS). Abnormalities in FH are associated with the renal diseases atypical hemolytic uremic syndrome and dense deposit disease and the ocular disease age-related macular degeneration. Although FH systemically controls complement activation, clinical phenotypes selectively manifest in kidneys and eyes, suggesting the presence of tissue-specific determinants of disease development. Recent results imply the importance of tissue-specifically expressed, sulfated glycosaminoglycans (GAGs), like HS, in determining FH binding to and activity on host tissues. Therefore, we investigated which GAGs mediate human FH and recombinant human FH complement control proteins domains 19 and 20 (FH19–20) binding to mouse glomerular endothelial cells (mGEnCs) in ELISA. Furthermore, we evaluated the functional defects of FH19–20 mutants during complement activation by measuring C3b deposition on mGEnCs using flow cytometry. FH and FH19–20 bound dose-dependently to mGEnCs and TNF-α treatment increased binding of both proteins, whereas heparinase digestion and competition with heparin/HS inhibited binding. Furthermore, 2-O-, and 6-O-, but not N-desulfation of heparin, significantly increased the inhibitory effect on FH19–20 binding to mGEnCs. Compared with wild type FH19–20, atypical hemolytic uremic syndrome-associated mutants were less able to compete with FH in normal human serum during complement activation on mGEnCs, confirming their potential glomerular pathogenicity. In conclusion, our study shows that FH and FH19–20 binding to glomerular endothelial cells is differentially mediated by HS but not other GAGs. Furthermore, we describe a novel, patient serum-independent competition assay for pathogenicity screening of FH19–20 mutants.     

Chromobacterium Csp_P Reduces Malaria and Dengue Infection in Vector Mosquitoes and Has Entomopathogenic and In Vitro Anti-pathogen Activities

Plasmodium and dengue virus, the causative agents of the two most devastating vector-borne diseases, malaria and dengue, are transmitted by the two most important mosquito vectors, Anopheles gambiae and Aedes aegypti, respectively. Insect-bacteria associations have been shown to influence vector competence for human pathogens through multi-faceted actions that include the elicitation of the insect immune system, pathogen sequestration by microbes, and bacteria-produced anti-pathogenic factors. These influences make the mosquito microbiota highly interesting from a disease control perspective. Here we present a bacterium of the genus Chromobacterium (Csp_P), which was isolated from the midgut of field-caught Aedes aegypti. Csp_P can effectively colonize the mosquito midgut when introduced through an artificial nectar meal, and it also inhibits the growth of other members of the midgut microbiota. Csp_P colonization of the midgut tissue activates mosquito immune responses, and Csp_P exposure dramatically reduces the survival of both the larval and adult stages. Ingestion of Csp_P by the mosquito significantly reduces its susceptibility to Plasmodium falciparum and dengue virus infection, thereby compromising the mosquito''s vector competence. This bacterium also exerts in vitro anti-Plasmodium and anti-dengue activities, which appear to be mediated through Csp_P -produced stable bioactive factors with transmission-blocking and therapeutic potential. The anti-pathogen and entomopathogenic properties of Csp_P render it a potential candidate for the development of malaria and dengue control strategies.     

In Vitro Studies on the Mechanism of Acquired Resistance to Tuberculous Infection

Immune lymph node cells were obtained from mice immunized with bovine gamma globulin (BGG) in complete Freund's adjuvant or allogeneic MH134 tumor cells. They showed the capacity of conferring bactericidal activity on macrophages infected with Mycobacterium tuberculosis, H37Rv, when they were incubated on macrophage monolayers together with the corresponding antigen, i.e., BGG or solubilized cellular antigen of the tumor cells. However, such capacity was lower than that of tubercle bacilli-immune lymph node cells. Culture supernatants were harvested after incubation of tubercle bacilli-immune, BGG-immune or allogeneic tumor-immune lymph node cells with the corresponding antigen for 24 hr. Macrophages were altered so as to suppress intracellular bacillary growth when macrophage monolayers were exposed to the supernatants for more than 2 days. When normal lymph node cells were incubated on normal macrophage monolayers together with a mitogen such as PHA or concanavalin A, growth of tubercle bacilli within the macrophages was slightly but difinitely suppressed. The mechanism of elicitation of cellular immunity to the infection with tubercle bacilli is discussed on the basis of results presented in this and the preceding paper.     

In Vitro Studies on the Mechanism of Acquired Resistance to Tuberculous Infection

The relationship between lymphocytes and macrophages in cellular immunity against tuberculous infection was studied by means of an in vitro cell culture system without addition of streptomycin. The peritoneal macrophages were obtained from normal mice or mice immunized with heat-killed tubercle bacilli in paraffin oil, boosted with live BCG and infected with H37Rv cells in vitro. The infected monolayers of macrophages were cultivated for 48 hr with immune lymphoid cells obtained from immunized mice. The intracellular growth of H37Rv cells 3,5 and 7 days after infection was examined by counting tubercle bacilli within infected macrophages under a microscope. 1) The increase of bacilli within macrophages derived from immunized mice was slightly smaller than that in normal macrophages. 2) The addition of immune lymph node cells to the macrophage monolayers resulted in a marked decrease in the number of bacilli within both normal and “immune” macrophages. Conversely, normal lymph node cells exhibited an enhancing effect on the intracellular bacillary growth. 3) Immune lymph node cells showed a higher capacity to cause macrophages to suppress intracellular growth of bacilli than that of splenic lymphoid cells or thymocytes after addition to macrophage monolayers. 4) The treatment of lymphoid cells with inhibitors of protein synthesis, cycloheximide or streptovitacin A, resulted in a remarkable reduction of the ability of sensitized lymphocytes to cause macrophages to suppress multiplication of intracellular bacilli.     

Borrelia burgdorferi BBK32 Inhibits the Classical Pathway by Blocking Activation of the C1 Complement Complex

Pathogens that traffic in blood, lymphatics, or interstitial fluids must adopt strategies to evade innate immune defenses, notably the complement system. Through recruitment of host regulators of complement to their surface, many pathogens are able to escape complement-mediated attack. The Lyme disease spirochete, Borrelia burgdorferi, produces a number of surface proteins that bind to factor H related molecules, which function as the dominant negative regulator of the alternative pathway of complement. Relatively less is known about how B. burgdorferi evades the classical pathway of complement despite the observation that some sensu lato strains are sensitive to classical pathway activation. Here we report that the borrelial lipoprotein BBK32 potently and specifically inhibits the classical pathway by binding with high affinity to the initiating C1 complex of complement. In addition, B. burgdorferi cells that produce BBK32 on their surface bind to both C1 and C1r and a serum sensitive derivative of B. burgdorferi is protected from killing via the classical pathway in a BBK32-dependent manner. Subsequent biochemical and biophysical approaches localized the anti-complement activity of BBK32 to its globular C-terminal domain. Mechanistic studies reveal that BBK32 acts by entrapping C1 in its zymogen form by binding and inhibiting the C1 subcomponent, C1r, which serves as the initiating serine protease of the classical pathway. To our knowledge this is the first report of a spirochetal protein acting as a direct inhibitor of the classical pathway and is the only example of a biomolecule capable of specifically and noncovalently inhibiting C1/C1r. By identifying a unique mode of complement evasion this study greatly enhances our understanding of how pathogens subvert and potentially manipulate host innate immune systems.     

Characterization of Wolbachia Host Cell Range via the In Vitro Establishment of Infections

Maternally transmitted bacteria of the genus Wolbachia are obligate, intracellular symbionts that are frequently found in insects and cause a diverse array of reproductive manipulations, including cytoplasmic incompatibility, male killing, parthenogenesis, and feminization. Despite the existence of a broad range of scientific interest, many aspects of Wolbachia research have been limited to laboratories with insect-rearing facilities. The inability to culture these bacteria outside of the invertebrate host has also led to the existing bias of Wolbachia research toward infections that occur in host insects that are easily reared. Here, we demonstrate that Wolbachia infections can be simply established, stably maintained, and cryogenically stored in vitro using standard tissue culture techniques. We have examined Wolbachia host range by introducing different Wolbachia types into a single tissue culture. The results show that an Aedes albopictus (Diptera: Culicidae) cell line can support five different Wolbachia infection types derived from Drosophila simulans (Diptera: Drosophilidae), Culex pipiens (Culicidae), and Cadra cautella (Lepidoptera: Phycitidae). These bacterial types include infection types that have been assigned to two of the major Wolbachia clades. As an additional examination of Wolbachia host cell range, we demonstrated that a Wolbachia strain from D. simulans could be established in host insect cell lines derived from A. albopictus, Spodoptera frugiperda (Lepidoptera: Noctuidae), and Drosophila melanogaster. These results will facilitate the development of a Wolbachia stock center, permitting novel approaches for the study of Wolbachia infections and encouraging Wolbachia research in additional laboratories.     

A Non Mouse-Adapted Dengue Virus Strain as a New Model of Severe Dengue Infection in AG129 Mice

The spread of dengue (DEN) worldwide combined with an increased severity of the DEN-associated clinical outcomes have made this mosquito-borne virus of great global public health importance. Progress in understanding DEN pathogenesis and in developing effective treatments has been hampered by the lack of a suitable small animal model. Most of the DEN clinical isolates and cell culture-passaged DEN virus strains reported so far require either host adaptation, inoculation with a high dose and/or intravenous administration to elicit a virulent phenotype in mice which results, at best, in a productive infection with no, few, or irrelevant disease manifestations, and with mice dying within few days at the peak of viremia. Here we describe a non-mouse-adapted DEN2 virus strain (D2Y98P) that is highly infectious in AG129 mice (lacking interferon-α/β and -γ receptors) upon intraperitoneal administration. Infection with a high dose of D2Y98P induced cytokine storm, massive organ damage, and severe vascular leakage, leading to haemorrhage and rapid death of the animals at the peak of viremia. In contrast, very interestingly and uniquely, infection with a low dose of D2Y98P led to asymptomatic viral dissemination and replication in relevant organs, followed by non-paralytic death of the animals few days after virus clearance, similar to the disease kinetic in humans. Spleen damage, liver dysfunction and increased vascular permeability, but no haemorrhage, were observed in moribund animals, suggesting intact vascular integrity, a cardinal feature in DEN shock syndrome. Infection with D2Y98P thus offers the opportunity to further decipher some of the aspects of dengue pathogenesis and provides a new platform for drug and vaccine testing.     

The Lectin Pathway of Complement Activation Is a Critical Component of the Innate Immune Response to Pneumococcal Infection

The complement system plays a key role in host defense against pneumococcal infection. Three different pathways, the classical, alternative and lectin pathways, mediate complement activation. While there is limited information available on the roles of the classical and the alternative activation pathways of complement in fighting streptococcal infection, little is known about the role of the lectin pathway, mainly due to the lack of appropriate experimental models of lectin pathway deficiency. We have recently established a mouse strain deficient of the lectin pathway effector enzyme mannan-binding lectin associated serine protease-2 (MASP-2) and shown that this mouse strain is unable to form the lectin pathway specific C3 and C5 convertases. Here we report that MASP-2 deficient mice (which can still activate complement via the classical pathway and the alternative pathway) are highly susceptible to pneumococcal infection and fail to opsonize Streptococcus pneumoniae in the none-immune host. This defect in complement opsonisation severely compromises pathogen clearance in the lectin pathway deficient host. Using sera from mice and humans with defined complement deficiencies, we demonstrate that mouse ficolin A, human L-ficolin, and collectin 11 in both species, but not mannan-binding lectin (MBL), are the pattern recognition molecules that drive lectin pathway activation on the surface of S. pneumoniae. We further show that pneumococcal opsonisation via the lectin pathway can proceed in the absence of C4. This study corroborates the essential function of MASP-2 in the lectin pathway and highlights the importance of MBL-independent lectin pathway activation in the host defense against pneumococci.     

In Vitro Studies on the Biosynthesis of the Surface Glycoprotein of Trypanosoma congolense1,2

Tritiated leucine, glucosamine, mannose, and galactose were incorporated into the variant specific surface glycoprotein (VSG) of Trypanosoma congolense in vitro. The uptake of the precursors is shown by SDS-polyacrylamide electrophoresis and fluorography, by assay of the radioactivity in immunoprecipitates obtained with specific antisera, and by the isolation of the labeled antigens by affinity chromatography on concanavalin A-sepharose and isoelectric focusing. The in vitro labeled VSG exhibits the same degree of microheterogeneity as that observed in the VSG isolated from trypanosomes grown in animals. Analysis of the incorporated sugars after hydrolysis of the glycoprotein showed that glucosamine and mannose were utilized in biosynthesis of the carbohydrate moiety directly whereas galactose was converted possibly to other intermediates before being incorporated into the antigen. Tunicamycin completely prevented the incorporation of the radiolabeled sugars into the surface glycoprotein. The unglycosylated VSG with a molecular weight of 47 kDa had completely lost its size heterogeneity.     

In Vitro Reconstitution Reveals Key Intermediate States of Trimer Formation by the Dengue Virus Membrane Fusion Protein

The flavivirus dengue virus (DV) infects cells through a low-pH-triggered membrane fusion reaction mediated by the viral envelope protein E. E is an elongated transmembrane protein with three domains and is organized as a homodimer on the mature virus particle. During fusion, the E protein homodimer dissociates, inserts the hydrophobic fusion loop into target membranes, and refolds into a trimeric hairpin in which domain III (DIII) packs against the central trimer. It is clear that E refolding drives membrane fusion, but the steps in hairpin formation and their pH requirements are unclear. Here, we have used truncated forms of the DV E protein to reconstitute trimerization in vitro. Protein constructs containing domains I and II (DI/II) were monomeric and interacted with membranes to form core trimers. DI/II-membrane interaction and trimerization occurred efficiently at both neutral and low pH. The DI/II core trimer was relatively unstable and could be stabilized by binding exogenous DIII or by the formation of mixed trimers containing DI/II plus E protein with all three domains. The mixed trimer had unoccupied DIII interaction sites that could specifically bind exogenous DIII at either low or neutral pH. Truncated DV E proteins thus reconstitute hairpin formation and define properties of key domain interactions during DV fusion.Dengue virus (DV) is a flavivirus that is spread by mosquitoes and causes millions of cases of disease each year worldwide (2, 9, 17). DV infection can result in dengue hemorrhagic fever, a more lethal disease that leads to ∼500,000 hospitalizations and ∼12,500 deaths per year (10, 39). DV is currently endemic in more than 100 countries, including the United States (17), and the World Health Organization estimates that about 40% of the world''s population lives in areas where dengue fever is endemic (39). As yet, there is no licensed DV vaccine or antiviral therapy. Studies of the molecular mechanisms of the virus life cycle are important to the development of new antiviral strategies.Flaviviruses such as DV are small, highly organized enveloped viruses with plus-sense single-stranded RNA genomes (reviewed in references 21 and 25). The flavivirus particle contains 3 structural proteins: a capsid protein, which associates with the genomic RNA to form the viral core, and two membrane proteins, the M protein and the membrane fusion protein E. Like many enveloped viruses, flaviviruses infect cells via endocytic uptake and a membrane fusion reaction triggered by the low pH within endosomes (38). Low-pH-triggered membrane fusion is mediated by conformational changes in the viral E protein, which converts from a prefusion E homodimer to a target membrane-inserted homotrimer. The structure of the DV E ectodomain in the prefusion form shows an elongated finger-like molecule with three domains (DI, DII, and DIII) composed primarily of β-sheets (22, 24, 42) (Fig. ​(Fig.1A;1A; see also Fig. ​Fig.7).7). The central DI is connected to DII. The distal tip of DII contains the hydrophobic fusion loop, the region of E that inserts into the target membrane during fusion. On the other side, DI connects via a short linker to DIII, an immunoglobulin-like domain. In the full-length viral E protein, DIII is followed by the stem, which contains 2 helical regions (H1 and H2) connected by a conserved sequence (CS). The stem connects to the C-terminal transmembrane (TM) anchor. The E-protein homodimer is arranged in a head-to-tail fashion, with the fusion loop on DII of each E protein hidden in a pocket formed by DI and DIII of its dimeric E partner.Open in a separate windowFIG. 1.Production and characterization of truncated DV2 E proteins. (A) Constructs used to express truncated forms of the DV2 E protein. At the top is a linear diagram of the full-length DV2 E protein, with DI indicated in red, DII in yellow, the fusion loop in green, the DI-DIII linker in cyan, DIII in dark blue, and the stem and TM regions in gray. L indicates the linker, and H1, CS, and H2 indicate the stem regions helix1, conserved sequence, and helix2, respectively. The residue numbers of the domain boundaries are listed below the diagram. The four S2 expression constructs primarily used in this work are shown in the middle rows. The E′-ST protein is truncated at residue 395 (DV2-NGC E-protein numbering), DI/II is truncated at residue 291, DI/II-L is truncated at residue 301, and the sequences are joined to the Strep or His tag (underlined) used for protein purification. The four DIII constructs are shown in the bottom rows, where LDIII comprises E residues 289 to 395, DIIIH1 residues 296 to 415, LDIIIH1 residues 289 to 415, and LDIIIH1CS residues 289 to 430. (B) Purified truncated E proteins were electrophoresed on SDS gels (left, 4 to 20% acrylamide; right, 10% acrylamide) under nonreducing conditions unless indicated and stained with Coomassie blue. The calculated mass of each protein (without modifications) is shown in kDa below each lane. DTT, dithiothreitol. (C) Sedimentation analysis of E proteins. Samples of purified E proteins were separated on sucrose sedimentation gradients in TAN buffer, pH 8.0, without detergent. Fractions were analyzed by SDS-PAGE, Western blotting, and Licor quantitation, all as described in Materials and Methods. Fraction 1 is the top of the gradient. (D) Inhibition of DV2 fusion by DIII proteins. Serial dilutions of DV2 were bound to BHK cells on ice and treated at pH 5.7 in the presence of the indicated DIII proteins at a final concentration of 50 μM or in buffer alone (control). Cells infected by virus fusion with the plasma membrane were quantitated by immunofluorescence. The data shown are the averages and standard deviations of three independent experiments.Open in a separate windowFIG. 7.Model for the steps in rearrangement of the dengue virus E protein during membrane fusion. DI, DII, and DIII are colored red, yellow, and blue, respectively. The hydrophobic fusion loop at the tip of DII is shown as a green star. The stem region is shown in gray and the TM domains in black. The virus membrane is shown in pink and the target membrane in blue. (I) At the top is shown the prefusion E-protein dimer, with the orientation looking down on the virus membrane. During the initial step of the fusion protein conformational change, the dimer dissociates upon exposure to low pH (bottom). (II to V) Side views of the trimerization reaction with the target membrane at the top. (II) The E fusion loops insert into the target membrane, and initial trimerization occurs between the DII tips. (III) Trimerization continues with contacts between DI and the β-strand exchange reaction. (IV) The DI-DIII linker inserts into the groove formed by strand exchange. DIII folds back against the core trimer, locking the linker into place. The trimer is now irreversible and stable in detergent. (V) In the final postfusion trimer, the stem has packed against the core trimer. The exact disposition of the fusion loops versus the stem and TM domains is not known, except that they are at the same end of the trimer, as shown in the model.Upon exposure to low pH, the homodimer dissociates and the E proteins insert their fusion loops into the target membrane and form very stable homotrimers (reviewed in reference 12). The structure of the DV E ectodomain trimer reveals that trimerization is mediated by dramatic domain movements (23, 26). The central region of the trimer is composed of DI and DII. DIII rotates by about 70°, folds back toward the target membrane, and packs against the grooves formed by DI and DII in the central trimer. During this refolding, part of the DI-DIII linker region inserts into a β-sheet of DI. These linker-DI rearrangements produce significant intersubunit contacts at the membrane-distal region of the trimer. The DV E protein stem region is not present in the trimer structure, but its length is sufficient to extend along the central trimer and connect with the TM domain. The final postfusion trimer thus has a hairpin-like conformation with the fusion loops and TM domains at the same end of the molecule. The pre- and postfusion structures of the alphavirus E1 protein (8, 18, 29) are very similar to those of the flavivirus E proteins, suggesting common features of membrane fusion between the two virus groups.Biogenesis of flavivirus particles occurs by budding into the endoplasmic reticulum (ER) and transit through the secretory pathway. The M protein is synthesized in the ER as a precursor protein termed prM, which forms a heterodimer with the E protein in the ER and on the nascent immature virus particle (19, 40, 41). Exposure to low pH in the trans-Golgi network mediates rearrangement of the viral envelope proteins and allows furin processing of prM to produce pr peptide and the mature M protein (32). The pr peptide remains associated with E throughout the low-pH environment of the secretory pathway, thereby protecting the virus from premature fusion until it is released from the cell (19, 40, 41). In the mature virus particle, the prefusion E homodimers are oriented tangentially to the virus membrane and form a herringbone-like pattern on the virus surface, essentially covering the virus membrane (16, 25).Thus, extensive structural information is available for both the DV E protein homodimer and the low-pH-induced E homotrimer. In contrast, the intermediates and mechanisms involved in the dramatic conformational transition from prefusion to postfusion E are relatively undefined. Recent studies of the flavivirus West Nile virus (WNV) suggest that an early fusion intermediate involves an extension of the stem region prior to dimer dissociation (15). Studies of the flavivirus tick-borne encephalitis (TBE) virus at pH 10 suggest that initial membrane insertion occurs via an E monomer (36). DV fusion and infection are inhibited by the addition of exogenous DIII during the E conformational change (20), implying that the central trimer region is formed before complete foldback of DIII. The presence of stem peptides can inhibit infection by DV and WNV, indicating the importance of stem interactions during hairpin formation (13). The dissociation of the TBE virus E dimer at low pH is dependent on a key histidine residue on DIII (H323; TBE virus numbering), which also promotes formation of the stable E trimer (5). However, studies of WNV indicate that viral E triggering is not controlled by protonation of a critical histidine residue (27). A better understanding of E-protein conformational changes during trimerization is important to define such intermediate steps and to evaluate their usefulness as targets for fusion inhibitors.Toward this end, in this study we expressed truncated forms of the DV E protein and used them to reconstitute steps in the trimerization reaction. This in vitro system allowed us to characterize the features of E protein involved in the formation of a stable central trimer and in DIII foldback. Our results suggest that monomeric DI/II proteins insert their fusion loops into target membranes and form a core trimer at either neutral or low pH. This core trimer is relatively unstable and can be stabilized by the binding of DIII, thus reconstituting hairpin formation.     

Protective or Deleterious Role of Scavenger Receptors SR-A and CD36 on Host Resistance to Staphylococcus aureus Depends on the Site of Infection

Staphylococcus aureus is a major human opportunistic pathogen responsible for a broad spectrum of infections ranging from benign skin infection to more severe life threatening disorders (e.g. pneumonia, sepsis), particularly in intensive care patients. Scavenger receptors (SR-A and CD36) are known to be involved in S. aureus recognition by immune cells in addition to MARCO, TLR2, NOD2 and α5β1 integrin. In the present study, we further deciphered the contribution of SR-A and CD36 scavenger receptors in the control of infection of mice by S. aureus. Using double SR-A/CD36 knockout mice (S/C-KO) and S. aureus strain HG001, a clinically relevant non-mutagenized strain, we showed that the absence of these two scavenger receptors was protective in peritoneal infection. In contrast, the deletion of these two receptors was detrimental in pulmonary infection following intranasal instillation. For pulmonary infection, susceptible mice (S/C-KO) had more colony-forming units (CFU) in their broncho-alveolar lavages fluids, associated with increased recruitment of macrophages and neutrophils. For peritoneal infection, susceptible mice (wild-type) had more CFU in their blood, but recruited less macrophages and neutrophils in the peritoneal cavity than resistant mice. Exacerbated cytokine levels were often observed in the susceptible mice in the infected compartment as well as in the plasma. The exception was the enhanced compartmentalized expression of IL-1β for the resistant mice (S/C-KO) after peritoneal infection. A similar mirrored susceptibility to S. aureus infection was also observed for MARCO and TLR2. Marco and tlr2 -/- mice were more resistant to peritoneal infection but more susceptible to pulmonary infection than wild type mice. In conclusion, our results show that innate immune receptors can play distinct and opposite roles depending on the site of infection. Their presence is protective for local pulmonary infection, whereas it becomes detrimental in the peritoneal infection.     

Stability of C3 Convertase in the Rat Classical Complement Pathway

It has been reported that rat serum complement causes efficient hemolysis of antibody-sensitized sheep erythrocytes (EA) at 20 C but not at 37 C. In connection with this, we demonstrated that C3 convertase of rat complement was significantly unstable at 37 C using purified components of rat complement.     

Mucociliary Clearance Defects in a Murine In Vitro Model of Pneumococcal Airway Infection

Mucociliary airway clearance is an innate defense mechanism that protects the lung from harmful effects of inhaled pathogens. In order to escape mechanical clearance, airway pathogens including Streptococcus pneumoniae (pneumococcus) are thought to inactivate mucociliary clearance by mechanisms such as slowing of ciliary beating and lytic damage of epithelial cells. Pore-forming toxins like pneumolysin, may be instrumental in these processes. In a murine in vitro airway infection model using tracheal epithelial cells grown in air-liquid interface cultures, we investigated the functional consequences on the ciliated respiratory epithelium when the first contact with pneumococci is established. High-speed video microscopy and live-cell imaging showed that the apical infection with both wildtype and pneumolysin-deficient pneumococci caused insufficient fluid flow along the epithelial surface and loss of efficient clearance, whereas ciliary beat frequency remained within the normal range. Three-dimensional confocal microscopy demonstrated that pneumococci caused specific morphologic aberrations of two key elements in the F-actin cytoskeleton: the junctional F-actin at the apical cortex of the lateral cell borders and the apical F-actin, localized within the planes of the apical cell sides at the ciliary bases. The lesions affected the columnar shape of the polarized respiratory epithelial cells. In addition, the planar architecture of the entire ciliated respiratory epithelium was irregularly distorted. Our observations indicate that the mechanical supports essential for both effective cilia strokes and stability of the epithelial barrier were weakened. We provide a new model, where - in pneumococcal infection - persistent ciliary beating generates turbulent fluid flow at non-planar distorted epithelial surface areas, which enables pneumococci to resist mechanical cilia-mediated clearance.     

Effect of Puromycin and Actinomycin D on a Persistent Mumps Virus Infection In Vitro

Puromycin and actinomycin D were used to treat a line of human conjunctiva cells persistently infected with mumps virus (C-M cells) in order to determine where virus synthesis is inhibited. Although 90% of the cells in C-M cultures are infected, little or no infectious virus is produced by most cells in a growing culture. Adding puromycin to inhibit protein synthesis resulted in the production of infectious virus. Thus, all the viral proteins needed for virus completion were made in the growing cells. When actinomycin D was added to growing cells, infectious virus was again produced. Since mumps virus synthesis is actinomycin D-insensitive, this suggested a host control of the virus. Interferon was not detected. The possible mechanisms of host control are discussed.