Michael J. Moser

High-Throughput, Sensitive, and Accurate Multiplex PCR-Microsphere Flow Cytometry System for Large-Scale Comprehensive Detection of Respiratory Viruses

ABSTRACT Human respiratory viruses are a diverse group of pathogens composed of hundreds of virus strains, and this presents a major challenge for diagnostic laboratories. To efficiently detect numerous viruses in a large epidemiologic study, we developed a fast, multitarget, sensitive, and specific assay named the Respiratory MultiCode-PLx Assay (RMA). The RMA utilizes improved multiplex PCR chemistry (EraGen MultiCode-PLx technology) coupled with high-throughput microsphere flow cytometry (Luminex). Eighteen sets of virus-specific multiplex PCR primers were developed based on the conserved sequences of all available respiratory-virus sequences for eight distinct groups: human rhinovirus (HRV), respiratory syncytial virus (RSV), parainfluenza virus (PIV), influenza virus (InfV), metapneumovirus, adenovirus (Ad), coronavirus, and enterovirus. Each primer set detected 20 cDNA copies of the intended target per sample and had no reaction with 60,000 copies of human genomic DNA. The accuracy and sensitivity of the RMA for detecting respiratory viruses in human samples were tested with two sets of clinical specimens. First, 101 nasal-wash specimens that were positive for HRV, RSV, InfV, PIV, or Ad by traditional techniques were reanalyzed by RMA, and all target viruses were detected with an overall sensitivity of 94% and specificity of 99%. Second, 103 nasal-wash samples from 5-year-old children with asthma and respiratory symptoms were analyzed; RMA detected viruses in 74 specimens (71.8%) compared to only 24 (23.3%) by traditional culture and immunofluorescent-staining techniques. These results show that RMA is an accurate, sensitive, and practical test for respiratory-virus infections.

CA2+-CALMODULIN PROMOTES SURVIVAL OF PHEROMONE-INDUCED GROWTH ARREST BY ACTIVATION OF CALCINEURIN AND CA2+-CALMODULIN-DEPENDENT PROTEIN KINASE

The cmd1-6 allele contains three mutations that block Ca2+ binding to calmodulin from Saccharomyces cerevisiae. We find that strains containing cmd1-6 lose viability during cell cycle arrest induced by the mating pheromone alpha-factor. The 50% lethal dose (LD50) of alpha-factor for the calmodulin mutant is almost fivefold below the LD50 for a wild-type strain. The calmodulin mutants are not more sensitive to alpha-factor, as measured by activation of a pheromone-responsive reporter gene. Two observations indicate that activation of the Ca2+-calmodulin-dependent protein phosphatase calcineurin contributes to survival of pheromone-induced arrest. First, deletion of the gene encoding the calcineurin regulatory B subunit, CNB1, from a wild-type strain decreases the LD50 of alpha-factor but has no further effect on a cmd1-6 strain. Second, a dominant constitutive calcineurin mutant partially restores the ability of the cmd1-6 strain to survive exposure to alpha-factor. Activation of the Ca2+-calmodulin-dependent protein kinase (CaMK) also contributes to survival, thus revealing a new function for this enzyme. Deletion of the CMK1 and CMK2 genes, which encode CaMK, decreases the LD50 of pheromone compared with that for a wild-type strain but again has no effect in a cmd1-6 strain. Furthermore, the LD50 of alpha-factor for a mutant in which the calcineurin and CaMK genes have been deleted is the same as that for the calmodulin mutant. Finally, the CaMK and calcineurin pathways appear to be independent since the ability of constitutive calcineurin to rescue a cmd1-6 strain is not blocked by deletion of the CaMK genes.

WRN mutations in Werner syndrome

Werner syndrome (WS) is one of a group of human genetic diseases that have recently been linked to deficits in cellular helicase function. We review the spectrum of WS‐associated WRN mutations, the organization and potential functions of the WRN protein, and potential mechanistic links between the loss of WRN function and pathogenesis of the WS clinical and cellular phenotypes. Hum Mutat 13:271–279, 1999.

Following the Path of a Twin-arginine Precursor along the TatABC Translocase of Escherichia coli

The twin-arginine translocation (Tat) machinery present in bacterial and thylakoidal membranes is able to transport fully folded proteins. Consistent with previous in vivo data, we show that the model Tat substrate TorA-PhoA is translocated by the TatABC translocase of Escherichia coli inner membrane vesicles, only if the PhoA moiety was allowed to fold by disulfide bond formation. Although even unfolded TorA-PhoA was found to physically associate with the Tat translocase of the vesicles, site-specific cross-linking revealed a perturbed interaction of the signal sequence of unfolded TorA-PhoA with the TatBC receptor site. Some of the folded TorA-PhoA precursor accumulated in a partially protease-protected membrane environment, from where it could be translocated into the lumen of the vesicles upon re-installation of an H+-gradient. Translocation arrest occurred in immediate vicinity to TatA. Consistent with a neighborhood to TatA, TorA-PhoA remained protease-resistant in the presence of detergents that are known to preserve the oligomeric structures of TatA. Moreover, entry of TorA-PhoA to the protease-protected environment strictly required the presence of TatA. Collectively, our results are consistent with some degree of quality control by TatBC and a recruitment of TatA to a folded substrate that has functionally engaged the twin-arginine translocase.

Evaluation of a Multiplexed PCR Assay for Detection of Respiratory Viral Pathogens in a Public Health Laboratory Setting

ABSTRACT There are numerous viral and bacterial causes of respiratory disease. To enable rapid and sensitive detection of even the most prevalent causes, there is a need for more-simplified testing systems that enable researchers and clinicians to perform multiplexed molecular diagnostics quickly and easily. To this end, a new multiplexed molecular test called the MultiCode-PLx respiratory virus panel (PLx-RVP) was developed and then implemented in a public health laboratory setting. A total of 687 respiratory samples were analyzed for the presence of 17 viruses that commonly cause respiratory disease. As a comparator, the samples were also tested using a standard testing algorithm that included the use of a real-time influenza virus A and B reverse transcription-PCR test and routine viral culture identification. The standard testing algorithm identified 503 (73%) samples as positive and 184 as negative. Analyzing the same 687 samples, the PLx-RVP assay detected one or more targets in 528 (77%) samples and found 159 samples negative for all targets. There were 25 discordant results between the two systems; 14 samples were positive for viruses not routinely tested for by the Wisconsin State Laboratory of Hygiene, and 13 of these were confirmed by real-time PCR. When the results of the standard testing algorithm were considered “true positives,” the PLx-RVP assay showed an overall sensitivity of 99% and an overall specificity of 87%. In total, the PLx-RVP assay detected an additional 40 viral infections, of which 11 were mixed infections.

Mapping Precursor-binding Site on TatC Subunit of Twin Arginine-specific Protein Translocase by Site-specific Photo Cross-linking

Background: TatA, TatB, and TatC are subunits of the Tat translocase allowing transport of folded pre-proteins across cellular membranes Results: We identified TatC sites that interact with pre-proteins, TatA, TatB, and TatC Conclusion: The cytosolic N terminus and first cytosolic TatC loop constitute part of a twin arginine recognition site Significance: We developed a working model of how twin arginine pre-protein inserts into Tat translocase. A number of secreted precursor proteins of bacteria, archaea, and plant chloroplasts stand out by a conserved twin arginine-containing sequence motif in their signal peptides. Many of these precursor proteins are secreted in a completely folded conformation by specific twin arginine translocation (Tat) machineries. Tat machineries are high molecular mass complexes consisting of two types of membrane proteins, a hexahelical TatC protein, and usually one or two single-spanning membrane proteins, called TatA and TatB. TatC has previously been shown to be involved in the recognition of twin arginine signal peptides. We have performed an extensive site-specific cross-linking analysis of the Escherichia coli TatC protein under resting and translocating conditions. This strategy allowed us to map the recognition site for twin arginine signal peptides to the cytosolic N-terminal region and first cytosolic loop of TatC. In addition, discrete contact sites between TatC, TatB, and TatA were revealed. We discuss a tentative model of how a twin arginine signal sequence might be accommodated in the Tat translocase.

Membrane Targeting of a Bacterial Virulence Factor Harbouring an Extended Signal Peptide

Filamentous haemagglutinin (FHA) is the major adhesin of Bordetella pertussis, the whooping cough agent. FHA is synthesized as a 367-kDa precursor harbouring a remarkably long signal peptide with an N-terminal extension that is conserved among related virulence proteins. FHA is secreted via the two-partner secretion pathway that involves transport across the outer membrane by a cognate transporter protein. Here we have analyzed the mechanism by which FHA is targeted to, and translocated across, the inner membrane. Studies were performed both in vitro using Escherichia coli inside-out inner membrane vesicles and in vivo by pulse-chase labelling of Bordetella pertussis cells. The data collectively indicate that like classical periplasmic and outer membrane proteins, FHA requires SecA and SecB for its export through the SecYEG translocon in the inner membrane. Although short nascent chains of FHA were found to cross-link to signal recognition particle (SRP), we did not obtain indication for an SRP-dependent, co-translational membrane targeting provoked by the FHA signal sequence. Our results rule out that the extended signal peptide of FHA determines a specific mode of membrane targeting but rather suggest that it might influence the export rate at the inner membrane.

TatB Functions as an Oligomeric Binding Site for Folded Tat Precursor Proteins

The TatABC subunits of the twin-arginine translocation machinery allow transport of folded proteins by an unknown mechanism. Here we show that the entire surfaces of folded Tat substrates contact TatB via both of its predicted helices. Our data suggest that TatB forms an oligomeric binding site that transiently accommodates folded Tat precursors.

Cell fusion corrects the 4-nitroquinoline 1-oxide sensitivity of Werner syndrome fibroblast cell lines

We have shown that Werner syndrome (WRN) fibroblast cell lines are unusually sensitive to the DNA-damaging agent 4-nitroquinoline 1-oxide (4NQO), though not to gamma radiation or to hydrogen peroxide. The fusion of 4NQO-sensitive WRN and 4NQO-resistant control fibroblast cell lines generated proliferating WRN × control cell hybrids that expressed WRN protein and were 4NQO-resistant. These results establish the recessive nature of 4NQO sensitivity in WRN cell lines and provide a cellular assay for WRN protein function.

A Derivative of Lipid A Is Involved in Signal Recognition Particle/SecYEG-dependent and -independent Membrane Integrations

A cell-free system was developed that allows the correct integration of single and multispanning membrane proteins of Escherichia coli into proteoliposomes. We found that physiological levels of diacylglycerol were required to prevent spontaneous integration into liposomes even of the polytopic mannitol permease. Using diacylglycerol-containing proteoliposomes, we identified a novel integration-stimulating factor. Integration of mannitol permease was dependent on both the SecYEG translocon and this factor and was mediated by signal recognition particle and signal recognition particle receptor. Integration of M13 procoat, which is independent of both signal recognition particle/signal recognition particle receptor and SecYEG, was also promoted by this factor. Furthermore, the factor stimulated the post-translational translocation of presecretory proteins, suggesting that it also mediates integration of a signal sequence. This factor was found to be a lipid A-derived membrane component possessing a peptide moiety.