Biomedical subjects

Martin Wiedmann

Publications and source records attributed to Martin Wiedmann.

68 records · Page 4Linked to original sources

PubMed2003

Molecular subtyping to detect human listeriosis clusters.

We analyzed the diversity (Simpson's Index, D) and distribution of Listeria monocytogenes in human listeriosis cases in New York State (excluding New York City) from November 1996 to June 2000 by using automated ribotyping and pulsed-field gel electrophoresis (PFGE). We applied a scan statistic (p<or=0.05) to detect listeriosis clusters caused by a specific Listeria monocytogenes subtype. Among 131 human isolates, 34 (D=0.923) ribotypes and 74 (D=0.975) PFGE types were found. Nine (31% of cases) clusters were identified by ribotype or PFGE; five (18% of cases) clusters were identified by using both methods. Two of the nine clusters (13% of cases) corresponded with investigated multistate listeriosis outbreaks. While most human listeriosis cases are considered sporadic, highly discriminatory molecular subtyping approaches thus indicated that 13% to 31% of cases reported in New York State may represent single-source clusters. Listeriosis control and reduction efforts should include broad-based subtyping of human isolates and consider that a large number of cases may represent outbreaks.

Aged↗

PubMed2003

Listeria monocytogenes contamination patterns for the smoked fish processing environment and for raw fish.

Reliable data on the sources of Listeria monocytogenes contamination in cold-smoked fish processing are crucial in designing effective intervention strategies. Environmental samples (n = 512) and raw fish samples (n = 315) from two smoked fish processing facilities were screened for L. monocytogenes, and all isolates were subtyped by automated ribotyping to examine the relationship between L. monocytogenes contamination from raw materials and that from environmental sites. Samples were collected over two 8-week periods in early spring and summer. The five types of raw fish tested included lake whitefish, sablefish, farm-raised Norwegian salmon, farm-raised Chilean salmon, and feral (wild-caught) salmon from the U.S. West Coast. One hundred fifteen environmental samples and 46 raw fish samples tested positive for L. monocytogenes. Prevalence values for environmental samples varied significantly (P < 0.0001) between the two plants; plant A had a prevalence value of 43.8% (112 of 256 samples), and plant B had a value of 1.2% (3 of 256 samples). For plant A, 62.5% of drain samples tested positive for L. monocytogenes, compared with 32.3% of samples collected from other environmental sites and 3.1% of samples collected from food contact surfaces. Ribotyping identified 11 subtypes present in the plant environments. Multiple subtypes, including four subtypes not found on any raw fish, were found to persist in plant A throughout the study. Contamination prevalence values for raw fish varied from 3.6% (sablefish) to 29.5% (U.S. West Coast salmon), with an average overall prevalence of 14.6%. Sixteen separate L. monocytogenes subtypes were present on raw fish, including nine that were not found in the plant environment. Our results indicate a disparity between the subtypes found on raw fish and those found in the processing environment. We thus conclude that environmental contamination is largely separate from that of incoming raw materials and includes strains persisting, possibly for years, within the plant. Operational and sanitation procedures appear to have a significant impact on environmental contamination, with both plants having similar prevalence values for raw materials but disparate contamination prevalence values for the environmental sites. We also conclude that regular L. monocyrogenes testing of drains, combined with molecular subtyping of the isolates obtained, allows for efficient monitoring of persistent L. monocytogenes contamination in a processing plant.

Animals↗

PubMed2002

The organizing principle in the formation of the T cell receptor-CD3 complex.

The T cell receptor (TCR) serves a critical function in the immune system and represents one of the most complex receptor structures. A striking feature is the presence of nine highly conserved, potentially charged residues in the transmembrane helices. Previous models have attempted to explain assembly based on pairwise interactions of these residues. Using a novel method for the isolation of intact radiolabeled protein complexes, we demonstrate that one basic and two acidic transmembrane residues are required for the assembly of each of the three signaling dimers with the TCR. This remarkable three-helix arrangement applies to all three assembly steps and represents the organizing principle for the formation of this intricate receptor structure.

Amino Acids↗

PubMed2002

The N-terminal domain of the reticulocyte-type 15-lipoxygenase is not essential for enzymatic activity but contains determinants for membrane binding.

The rabbit reticulocyte-type 15-lipoxygenase is capable of oxygenating biomembranes and lipoproteins without the preceding action of ester lipid cleaving enzymes. This reaction requires an efficient membrane binding, and the N-terminal beta-barrel domain of the enzyme has been implicated in this process. To obtain detailed information on the structural requirements for membrane oxygenation, we expressed the rabbit wild-type 15-lipoxygenase, its beta-barrel deletion mutant (catalytic domain), and several lipoxygenase point mutations as His-tagged fusion proteins in Escherichia coli and tested their membrane binding characteristics. We found that: (i) the beta-barrel deletion mutant was catalytically active and its enzymatic properties (K(M), V(max), pH optimum, substrate specificity) were similar to those of the wild-type enzyme; (ii) when compared with the wild-type lipoxygenase, the membrane binding properties of the N-terminal truncation mutant were impaired but not abolished, suggesting a role of the catalytic domain in membrane binding; and (iii) Phe-70 and Leu-71 (constituents of the beta-barrel domain) but also Trp-181, which is located in the catalytic domain, were identified as sequence determinants for membrane binding. Mutation of these amino acids to more polar residues (F70H, L71K, W181E) impaired the membrane binding capacity of the recombinant enzyme. These data indicate that the C-terminal catalytic domain of the rabbit 15-lipoxygenase is enzymatically active and that the membrane binding properties of the enzyme are determined by a concerted action of the N-terminal beta-barrel and the C-terminal catalytic domain.

Animals↗

PubMed2002

The in vivo function of the ribosome-associated Hsp70, Ssz1, does not require its putative peptide-binding domain.

Two proteins of the Hsp70 class (Ssb and Ssz1) and one of the J-type class (Zuo1) of molecular chaperones reside on the yeast ribosome, with Ssz1 forming a stable heterodimer with Zuo1. We designed experiments to address the roles of these two distantly related ribosome-associated Hsp70s and their functional relationship to Zuo1. Strains lacking all three proteins have the same phenotype as those lacking only one, suggesting that these chaperones all function in the same pathway. The Hsp70 Ssb, whose peptide-binding domain is essential for its in vivo function, can be crosslinked to nascent chains on ribosomes that are as short as 54 amino acids, suggesting that Ssb interacts with nascent chains that extend only a short distance beyond the tunnel of the ribosome. A ssz1 mutant protein lacking its putative peptide-binding domain allows normal growth. Thus, binding of unfolded protein substrates in a manner similar to that of typical Hsp70s is not critical for Ssz1's in vivo function. The three chaperones are present in cells in approximately equimolar amounts compared with ribosomes. The level of Ssb can be reduced only a few-fold before growth is affected. However, a 50- to 100-fold reduction of Ssz1 and Zuo1 levels does not have a substantial effect on cell growth. On the basis of these results, we propose that Ssbs function as the major Hsp70 chaperone for nascent chains on the ribosome, and that Ssz1 has evolved to perform a nonclassical function, perhaps modulating Zuo1's ability to function as a J-type chaperone partner of Ssb.

Animals↗

PubMed2002

Interaction of the eukaryotic elongation factor 1A with newly synthesized polypeptides.

eEF1A, the eukaryotic homologue of bacterial elongation factor Tu, is a well characterized translation elongation factor responsible for delivering aminoacyl-tRNAs to the A-site at the ribosome. Here we show for the first time that eEF1A also associates with the nascent chain distal to the peptidyltransferase center. This is demonstrated for a variety of nascent chains of different lengths and sequences. Interestingly, unlike other ribosome-associated factors, eEF1A also interacts with polypeptides after their release from the ribosome. We demonstrate that eEF1A does not bind to correctly folded full-length proteins but interacts specifically with proteins that are unable to fold correctly in a cytosolic environment. This association was demonstrated both by photo-cross-linking and by a functional refolding assay.

Animals↗

PubMed2002

Hormonally regulated alpha(4)beta(2)delta GABA(A) receptors are a target for alcohol.

Here we report that low concentrations of alcohol (1-3 mM) increased Cl(-) currents gated by a recombinant GABA(A) receptor, alpha(4)beta(2)delta, by 40-50% in Xenopus laevis oocytes. We also found greater hippocampal expression of receptors containing alpha(4) and delta subunits, using a rat model of premenstrual syndrome (PMS) in which 1-3 mM alcohol preferentially enhanced GABA-gated currents, and low doses of alcohol attenuated anxiety and behavioral reactivity. The alcohol sensitivity of delta-containing receptors may underlie the reinforcing effects of alcohol during PMS, when eye saccade responses to low doses of alcohol are increased.

Animals↗

PubMed2002

Sigma B contributes to PrfA-mediated virulence in Listeria monocytogenes.

Transcription of the Listeria monocytogenes positive regulatory factor A protein (PrfA) is initiated from either of two promoters immediately upstream of prfA (prfAp(1) and prfAp(2)) or from the upstream plcA promoter. We demonstrate that prfAp(2) is a functional sigma(B)-dependent promoter and that a sigB deletion mutation affects the virulence phenotype of L. monocytogenes. Thus, the alternative sigma factor sigma(B) contributes to virulence in L. monocytogenes.

Animals↗

PubMed2002

Rational design of DNA sequence-based strategies for subtyping Listeria monocytogenes.

The ability to differentiate bacteria beyond the species level is essential for identifying and tracking infectious disease outbreaks and to improve our knowledge of the population genetics, epidemiology, and ecology of bacterial pathogens. Commonly used subtyping methods, such as serotyping, phage typing, ribotyping, and pulsed-field gel electrophoresis, can yield ambiguous results that are difficult to standardize and share among laboratories. DNA sequence-based subtyping strategies can reduce interpretation ambiguity. We report the development of a rational approach for designing sequence-based subtyping methods. Listeria monocytogenes was selected as the model organism for testing the efficacy of this approach. Two housekeeping genes (recA and prs), one stress response gene (sigB), two virulence genes (actA and inlA), and two intergenic regions (hly-mpl and plcA-hly) were sequenced for 15 L. monocytogenes isolates. Isolates were chosen from a representative collection of more than 1,000 L. monocytogenes isolates to reflect the genetic diversity of this species. DNA sequences were aligned, and sliding window analyses were performed for each gene to define 600-bp-long regions that were (i) most polymorphic (using ProSeq) or (ii) most discriminatory (using a new algorithm implemented in WINDOWMIN). Complete gene sequences for actA (1,929 bp) and inlA (2,235 bp) provided the highest discrimination (identifying 15 and 14 allelic types, respectively). WINDOWMIN allowed identification of 600-bp regions within these genes that provided similar discriminatory power (yielding 15 and 13 allelic types, respectively). The most discriminatory 600-bp fragments identified in the housekeeping and stress response genes differentiated the isolates into 8 to 10 subtypes; intergenic region sequences yielded 8 and 12 allelic types based on 335- and 242-bp sequences for hly-mpl and plcA-hly, respectively. Regions identified as most polymorphic were not necessarily most discriminatory; therefore, application of the WINDOWMIN algorithm provided a powerful tool for determining the best target regions for DNA sequence-based subtyping. Our specific results also show that inclusion of virulence gene target sequences in a DNA sequence-based subtyping scheme for L. monocytogenes is necessary to achieve maximum subtype differentiation.

Algorithms↗

PubMed2002

Subtyping of bacterial foodborne pathogens.

Phenotype-based and DNA-based subtyping methods allow for differentiation of bacterial isolates beyond the species and subspecies level. Bacterial subtyping methods not only have improved our ability to detect and track foodbome disease outbreaks, but also represent tools to track sources of bacterial contamination throughout the food system. The use of subtyping methods furthermore provides an opportunity to better understand the population genetics, epidemiology, and ecology of different foodbome pathogens. The last 5 years have seen tremendous advancement in the development of sensitive, rapid, automated, and increasingly easy-to-use molecular subtyping methods for a variety of different bacterial foodborne pathogens. This review will highlight key aspects of different subtyping methods for bacterial foodborne pathogens and provide examples of their applications in public health, food safety, epidemiology, and population genetics. Molecular subtyping and characterization methods may also facilitate the development of a novel framework for tracking, preventing, and regulating foodborne bacterial diseases, which is based on evolutionary relationships and genetic characteristics rather than traditional species definitions.

Bacterial Typing Techniques↗

PubMed2002

Detection of Listeria in crawfish processing plants and in raw, whole crawfish and processed crawfish (Procambarus spp.).

The foodborne pathogen Listeria monocytogenes represents a major concern to the food industry and particularly to producers of ready-to-eat (RTE) foods because of the severity of human listeriosis infections and because of the ubiquitous nature of this organism. Although several studies on the prevalence and sources of L monocytogenes in various RTE seafoods have been conducted, limited information is available on the presence and potential sources of this organism in RTE crawfish products. We thus monitored the presence of L monocytogenes and other Listeria spp. in the processing environment, in raw, whole crawfish, and in cooked crawfish meat from two processing plants. Samples were collected from the two plants throughout one crawfish season (April to June 2001) at 5 and 8 separate visits, respectively. At each visit, 6 raw, whole crawfish, 6 finished product samples (crawfish meat), and 14 mid- or end-of-processing environmental sponge samples were collected and tested for L. monocytogenes and Listeria spp. Of the 337 samples tested, 31 contained Listeria spp. Although Listeria innocua was the predominant Listeria spp. found (20 samples), four samples were positive for L monocytogenes. L. monocytogenes was detected in three raw material samples and in one environmental sample. Listeria spp. were found in 29.5% of raw, whole crawfish (n = 78) and in 4.4% of environmental samples (n = 181) but in none of the finished product samples. Among the environmental samples, Listeria spp. were found in 15.4% of the drains (n = 39) and in 5.1% of the employee contact surfaces (gloves and aprons) (n = 39) but in none of the samples from food contact surfaces. Even though a high prevalence of Listeria spp. was detected on raw materials, it appears that the heat treatment during the processing of crawfish and the practices preventing postprocessing recontamination can significantly reduce Listeria contamination of RTE crawfish meat.

Animals↗

PubMed2001

Comparative genetic characterization of Listeria monocytogenes isolates from human and animal listeriosis cases.

Listeria monocytogenes isolates from human sporadic and epidemic cases (n=119) and from animal cases (n=76) were characterized by automated ribotyping and PCR-restriction fragment length polymorphism (PCR-RFLP) typing of the virulence genes actA and hly. This combination of typing methods differentiated 39 distinctive strains, each reflecting a unique combination of ribotypes, hly and actA alleles. Simpson's index of discrimination indicated a high discriminatory ability of ribotyping for both animal (0.867) and human isolates (0.857), which was further increased by the addition of hly and actA typing (0.916 and 0.904, respectively). Ribotype and hly allele data were further used to group isolates into three genetically distinct lineages. Each lineage is composed of several ribotype fragment subsets, each of which contains multiple ribotypes characterized by common ribotype fragments. To determine whether certain clones of L. monocytogenes show indications for unique pathogenic potential or host specificity, frequency distributions for five genetic characteristics (i.e. lineage, ribotype, ribotype fragment subset and hly and actA allele) were calculated for isolates from animal cases, human epidemic cases and human sporadic cases. Lineage III isolates were found less frequently in human cases (1 of 119 isolates) than in animal cases (8 of 76 isolates; P=0.003). These results suggest the possibility of host specificity for non-primate mammals among lineage III strains. In addition, lineage I strains were found more frequently among human cases than among animal cases (P<0.001). Among the eight hly alleles observed, hly allele 1 was more common among human isolates as compared to animal isolates (P=0.002). We also identified one ribotype (DUP-1030) which was significantly more common among animal isolates (P=0.005) and one ribotype (DUP-1038; lineage I) which was significantly more common among human epidemic isolates as compared to human sporadic isolates (P<0.001). These findings confirm the presence of clonal groups of L. monocytogenes, which appear to be characterized by unique virulence or host specificity patterns. This study also establishes baseline data describing the genetic diversity of human and animal L. monocytogenes isolates which can be utilized in future surveillance programmes to track the emergence of new strains.

Alleles↗

PubMed2000

Phylogeny and functional conservation of sigma(E) in endospore-forming bacteria.

Conservation of the sporulation processes between Bacillus spp. and Clostridium spp. was investigated through evolutionary and complementation analyses of sigma(E). Alignment of partial predicted sigma(E) amino acid sequences from three Bacillus spp., Paenibacillus polymyxa and five Clostridium spp. revealed that amino acid residues previously reported to be involved in promoter utilization (M124, E119 and N120) and strand opening (C117) are conserved among all these species. Phylogenetic analyses of various sigma factor sequences from endospore-forming bacteria revealed that homologues of sigma(E), sigma(K) and sigma(G) clustered together regardless of genus, suggesting a common origin of sporulation sigma factors. The functional equivalence between Clostridium acetobutylicum sigma(E) and Bacillus subtilis sigma(E) was investigated by complementing a non-polar B. subtilis sigma(E) null mutant with the spoIIG operon from either B. subtilis (spoIIG(Bs)) or C. acetobutylicum (spoIIG(Ca)). Single-copy integration of spoIIG(Bs) into the amyE locus of the sigma(E) null mutant completely restored the wild-type sporulation phenotype, while spoIIG(Ca) only partially restored sporulation. Maximal expression of spoIIG(Ca)-lacZ occurred approximately 12 h later than maximal expression of spoIIG(Bs)-lacZ. Differences in temporal expression patterns for spoIIG(Ca) and spoIIG(Bs) in the B. subtilis background may at least partially explain the observed sporulation complementation phenotypes. This study suggests a common phylogenetic ancestor for sigma(E) in Bacillus spp. and Clostridium spp., although regulation of sigma(E) expression may differ in these two genera.

Amino Acid Sequence↗

PubMedDate not supplied

Molecular subtyping methods for Listeria monocytogenes.

Conventional, phenotypic, and DNA-based subtyping methods allow differentiation of Listeria monocytogenes beyond the species and subspecies level. Bacterial subtyping methods not only improve our ability to detect and track human listeriosis outbreaks, but also provide tools to track sources of L. monocytogenes contamination throughout the food system. The use of subtyping methods also provides an opportunity to better understand the population genetics, epidemiology, and ecology of L. monocytogenes. The last 5 years have seen tremendous advancements in the development of sensitive, rapid, automated, and increasingly easy-to-use molecular subtyping methods for L. monocytogenes. This review highlights key aspects of different L. monocytogenes subtyping methods and provides examples of their application in public health, food safety, population genetics, and epidemiology. A significant focus is on the application of subtyping methods to define L. monocytogenes subtypes and clonal groups, which may differ in phenotypic characteristics and pathogenic potential.

Bacterial Typing Techniques↗