Residence-time dependent cell wall deformation of different Staphylococcus aureus strains on gold measured using surface-enhanced-fluorescence.

Bacterial adhesion to surfaces is accompanied by cell wall deformation that may extend to the lipid membrane with an impact on the antimicrobial susceptibility of the organisms. Nanoscale cell wall deformation upon adhesion is difficult to measure, except for Δpbp4 mutants, deficient in peptidoglycan cross-linking. This work explores surface enhanced fluorescence to measure the cell wall deformation of Staphylococci adhering on gold surfaces. Adhesion-related fluorescence enhancement depends on the distance of the bacteria from the surface and the residence-time of the adhering bacteria. A model is forwarded based on the adhesion-related fluorescence enhancement of green-fluorescent microspheres, through which the distance to the surface and cell wall deformation of adhering bacteria can be calculated from their residence-time dependent adhesion-related fluorescence enhancement. The distances between adhering bacteria and a surface, including compression of their extracellular polymeric substance (EPS)-layer, decrease up to 60 min after adhesion, followed by cell wall deformation. Cell wall deformation is independent of the integrity of the EPS-layer and proceeds fastest for a Δpbp4 strain.

Detachment and successive re-attachment of multiple, reversibly-binding tethers result in irreversible bacterial adhesion to surfaces.

Bacterial adhesion to surfaces occurs ubiquitously and is initially reversible, though becoming more irreversible within minutes after first contact with a surface. We here demonstrate for eight bacterial strains comprising four species, that bacteria adhere irreversibly to surfaces through multiple, reversibly-binding tethers that detach and successively re-attach, but not collectively detach to cause detachment of an entire bacterium. Arguments build on combining analyses of confined Brownian-motion of bacteria adhering to glass and their AFM force-distance curves and include the following observations: (1) force-distance curves showed detachment events indicative of multiple binding tethers, (2) vibration amplitudes of adhering bacteria parallel to a surface decreased with increasing adhesion-forces acting perpendicular to the surface, (3) nanoscopic displacements of bacteria with relatively long autocorrelation times up to several seconds, in absence of microscopic displacement, (4) increases in Mean-Squared-Displacement over prolonged time periods according to tα with 0 < α ≪ 1, indicative of confined displacement. Analysis of simulated position-maps of adhering particles using a new, in silico model confirmed that adhesion to surfaces is irreversible through detachment and successive re-attachment of reversibly-binding tethers. This makes bacterial adhesion mechanistically comparable with the irreversible adsorption of high-molecular-weight proteins to surfaces, mediated by multiple, reversibly-binding molecular segments.

Implications for directionality of nanoscale forces in bacterial attachment.

Adhesion and friction are closely related and play a predominant role in many natural processes. From the wall-clinging feet of the gecko to bacteria forming a biofilm, in many cases adhesion is a necessity to survive. The direction in which forces are applied has shown to influence the bond strength of certain systems tremendously and can mean the difference between adhesion and detachment. The spatula present on the extension of the feet of the gecko can either attach or detach, based on the angle at which they are loaded. Certain proteins are known to unfold at different loads, depending on the direction at which the load is applied and some bacteria have specific receptors which increase their bond strength in the presence of shear. Bacteria adhere to any man-made surface despite the presence of shear forces due to running fluids, air flow, and other causes. In bacterial adhesion research, however, adhesion forces are predominantly measured perpendicularly to surfaces, whereas other directions are often neglected. The angle of shear forces acting on bacteria or biofilms will not be at a 90° angle, as shear induced by flow is often along the surface. Measuring at different angles or even lateral to the surface will give a more complete overview of the adhesion forces and mechanism, perhaps even resulting in alternative means to discourage bacterial adhesion or promote removal.

A highly attenuated strain of Japanese encephalitis virus induces a protective immune response in mice.

A pair of virulent (RP-9) and attenuated (RP-2ms) mutants of Japanese encephalitis virus (JEV) were generated from a Taiwanese isolate NT109. The mutants differed in several aspects in vitro and in vivo. RP-2ms exhibited smaller plaque than RP-9 on BHK-21 cells, and when intracerebrally injected, RP-2ms was much less neurovirulent than RP-9. As peripherally inoculated, RP-2ms lost neuroinvasiveness while RP-9 penetrated blood-brain barrier, replicated in mouse brain, and killed all the mice. Single RP-2ms immunization completely protected C3H and ICR mice from a lethal challenge with RP-9; the sera from such mice contained antibodies against JEV envelope and nonstructural 1 proteins, indicating RP-2ms had replicated in the mice Neutralizing activity against NT109 in such sera was further demonstrated by plaque reduction neutralization test. In addition, significant lymphoproliferation was detected in spleen cells from the RP-2ms-immunized mice, and cytotoxic activity in these cells specific for the MHC-matched, JEV-infected cells, but not mock cells, was also observed. Altogether, these results demonstrate that RP-2ms, a highly attenuated JEV strain, can induce a protective immunity in mice.

Induction of apoptosis by Sindbis virus occurs at cell entry and does not require virus replication.

Sindbis virus (SV) is an alphavirus that causes encephalitis in mice and can lead to the apoptotic death of infected cells. To determine the step in virus replication during which apoptosis is triggered, we used UV-inactivated SV, chemicals that block virus fusion or protein synthesis, and cells that do and do not express heparan sulfate, the initial binding molecule for SV infection of many cells. In initial experiments, UV-inactivated neuroadapted SV (NSV) induced apoptosis in Chinese hamster ovary (CHO) cells lacking heparan sulfate in the presence of cycloheximide. When fusion of prebound UV-inactivated NSV was rapidly induced at the plasma membrane by exposure to acidic pH, apoptosis was induced in CHO cells with or without heparan sulfate in the presence or absence of cycloheximide in a virus dose-dependent manner. In N18 neuroblastoma cells, the relative virulence of the virus strain was an important determinant of apoptosis induced by UV-inactivated SV. Treatment of N18 cells with monensin to prevent endosomal acidification an hour before, but not 2 h after, exposure to live NSV blocked the induction of cell death, as did treatment with NH(4)Cl or bafilomycin A1. These studies indicate that SV can induce apoptosis at the time of fusion with the cell membrane and that virus replication is not required.

Sindbis virus entry into cells triggers apoptosis by activating sphingomyelinase, leading to the release of ceramide.

Sindbis virus (SV) causes acute encephalomyelitis by infecting and inducing the death of neurons. Induction of apoptosis occurs during virus entry and involves acid-induced conformational changes in the viral surface glycoproteins and sphingomyelin (SM)-dependent fusion of the virus envelope with the endosomal membrane. We have studied neuroblastoma cells to determine how this entry process triggers cell death. Acidic sphingomyelinase was activated during entry followed by activation of neutral sphingomyelinase, SM degradation, and a sustained increase in ceramide. Ceramide-induced apoptosis and SV-induced apoptosis could be inhibited by treatment with Z-VAD-fmk, a caspase inhibitor, and by overexpression of Bcl-2, an antiapoptotic cellular protein. Acid ceramidase, expressed in a recombinant SV, decreased intracellular ceramide and protected cells from apoptosis. The data suggest that acid-induced SM-dependent virus fusion initiates the apoptotic cascade by inducing SM degradation and ceramide release.

Characterization of a Chinese hamster ovary cell line developed by retroviral insertional mutagenesis that is resistant to Sindbis virus infection.

The alphavirus Sindbis virus (SV) has a wide host range and infects many types of cultured cells in vitro. The outcome of infection is dependent on the strain of virus used for infection and the properties of the cells infected. To identify cellular determinants of susceptibility to SV infection we mutagenized Chinese hamster ovary (CHO) cells by retroviral insertion with a vector containing the neomycin resistance gene that allowed selection for integration into transcriptionally active genes. Cells were then selected for survival after infection with SV. The most resistant cell line (CHO-18.4m) exhibited delayed virus replication and virus-induced cell death, had a single retroviral insertion, and was defective in SV binding to the cell surface. Further analysis revealed that CHO-18.4m cells were deficient in the expression of the sulfated glycosaminoglycans heparan sulfate and chondroitin sulfate. This further confirms the importance of heparan sulfate as an attachment molecule for SV in vitro and demonstrates the usefulness of this technique for identifying cellular genes that are important for virus replication.

Identification of B-cell epitope of dengue virus type 1 and its application in diagnosis of patients.

Using a serotype-specific monoclonal antibody (MAb) of dengue virus type 1 (DEN-1), 15F3-1, we identified the B-cell epitope of DEN-1 from a random peptide library displayed on phage. Fourteen immunopositive phage clones that bound specifically to MAb 15F3-1 were selected. These phage-borne peptides had a consensus motif of HxYaWb (a = S/T, b = K/H/R) that mimicked the sequence HKYSWK, which corresponded to amino acid residues 111 to 116 of the nonstructural protein 1 (NS1) of DEN-1. Among the four synthetic peptides corresponding to amino acid residues 110 to 117 of the NS1 of DEN-1, -2, -3, and -4, only one peptide, EHKYSWKS (P14M) of DEN-1, was found to bind to 15F3-1 specifically. Furthermore, P14M was shown to inhibit the binding of phage particles to 15F3-1 in a competitive inhibition assay. Histidine(111) (His(111)) was crucial to the binding of P14M to 15F3-1, since its binding activity dramatically reduced when it changed to leucine(111) (Leu(111)). This epitope-based peptide demonstrated its clinical diagnostic potential when it reacted with a high degree of specificity with serum samples obtained from both DEN-1-infected rabbits and patients. Based on these observations, our DEN-1 epitope-based serologic test could be useful in laboratory viral diagnosis and in understanding the pathogenesis of DEN-1.

Generation and characterization of organ-tropism mutants of Japanese encephalitis virus in vivo and in vitro.

Using gamma-ray irradiation, a pair of virulent (RP-9) and attenuated (RP-2ms) variants of Japanese encephalitis virus (JEV) were generated from a Taiwanese isolate, NT109. The two variants differed in plaque morphology, virus adsorption, and growth properties in BHK-21 cells: (i) RP-2ms produced smaller plaques than RP-9; (ii) RP-2ms adsorbed less efficiently to host cells but yielded a higher virus titer (burst size); and (iii) RP-2ms virions were mostly accumulated intracellularly, whereas RP-9 was released extracellularly. In addition, in an in vitro binding assay, the envelope (E) protein of RP-9, but not that of RP-2ms, bound specifically to a cellular protein of 57-kDa derived from BHK-21 cells. When injected into mice intracerebrally, RP-2ms was much less virulent than RP-9, with 50% lethal doses of > 10(7) and 0.4 plaque forming units, respectively. Moreover, when inoculated intraperitoneally, their organ tropism differed in that the main target organ for RP-2ms was liver, whereas that for RP-9 was brain. These results suggest that RP-2ms was less neurovirulent and less neuroinvasive from peripheral routes. Molecular analysis of the virus structural proteins detected only two differences between RP-9 and RP-2ms: one in E protein, Glu-138 in RP-9 and Lys-138 in RP-2ms, and the other in prM, Tyr-43 in RP-9 and His-43 in RP-2ms. Since the N-terminal 92 amino acids of prM are cleaved and not present in mature JEV virions, the single-amino-acid change of the E protein at position 138 may account for the difference between the mutants in the in vitro binding assay. Such mutation in E protein, or perhaps in conjunction with the prM mutation, may be responsible, in part, for the phenotypic differences observed in vitro and in vivo between the two mutants.

Potential dengue virus-triggered apoptotic pathway in human neuroblastoma cells: arachidonic acid, superoxide anion, and NF-kappaB are sequentially involved.

Direct in vivo evidence for the susceptibility of human neuronal cells to dengue virus has not been reported. In this study, we demonstrated that type 2 dengue (DEN-2) virus infection induced extensive apoptosis in the human neuroblastoma cell line SK-N-SH. Phospholipase A(2) (PLA(2)) was activated by DEN-2 infection, which led to the generation of arachidonic acid (AA). Inhibition of PLA(2) activity by the PLA(2) inhibitors, AACOCF(3) and ONO-RS-082, diminished DEN-2 virus-induced apoptosis. In contrast, the cyclooxygenase inhibitors aspirin and indomethacin, thought to increase AA accumulation by blocking AA catabolism, enhanced apoptosis. Exogenous AA induced apoptosis in a dose-dependent manner. Superoxide anion, which is thought to be generated through the AA-activated NADPH oxidase, was increased after infection. Pretreatment with superoxide dismutase (SOD) protected cells against DEN-2 virus-induced apoptosis. Furthermore, generation of superoxide anion was blocked by AACOCF(3). In addition, the transcription factors, NF-kappaB and c-Jun, were found to be activated after DEN-2 virus infection. However, pretreatment of cells with oligodeoxynucleotides containing NF-kappaB, but not c-Jun, binding sites (transcription factor decoy) strongly prevented dengue virus-induced apoptosis. The finding that AACOCF(3) and SOD significantly block activation of NF-kappaB suggests that this activation is derived from the AA-superoxide anion pathway. Our results indicate that DEN-2 virus infection of human neuroblastoma cells triggers an apoptotic pathway through PLA(2) activation to superoxide anion generation and subsequently to NF-kappaB activation. This apoptotic effect can be either directly derived from the action of AA and superoxide anion on mitochondria or indirectly derived from the products of apoptosis-related genes activated by NF-kappaB.