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PubMed2005

The opportunistic pathogen Toxoplasma gondii deploys a diverse legion of invasion and survival proteins.

Host cell invasion is an essential step during infection by Toxoplasma gondii, an intracellular protozoan that causes the severe opportunistic disease toxoplasmosis in humans. Recent evidence strongly suggests that proteins discharged from Toxoplasma apical secretory organelles (micronemes, dense granules, and rhoptries) play key roles in host cell invasion and survival during infection. However, to date, only a limited number of secretory proteins have been discovered, and the full spectrum of effector molecules involved in parasite invasion and survival remains unknown. To address these issues, we analyzed a large cohort of freely released Toxoplasma secretory proteins by using two complementary methodologies, two-dimensional electrophoresis/mass spectrometry and liquid chromatography/electrospray ionization-tandem mass spectrometry (MudPIT, shotgun proteomics). Visualization of Toxoplasma secretory products by two-dimensional electrophoresis revealed approximately 100 spots, most of which were successfully identified by protein microsequencing or matrix-assisted laser desorption ionization-mass spectrometry analysis. Many proteins were present in multiple species suggesting they are subjected to substantial post-translational modification. Shotgun proteomic analysis of the secretory fraction revealed several additional products, including novel putative adhesive proteins, proteases, and hypothetical secretory proteins similar to products expressed by other related parasites including Plasmodium, the etiologic agent of malaria. A subset of novel proteins were re-expressed as fusions to yellow fluorescent protein, and this initial screen revealed shared and distinct localizations within secretory compartments of T. gondii tachyzoites. These findings provided a uniquely broad view of Toxoplasma secretory proteins that participate in parasite survival and pathogenesis during infection.

Animals↗
PubMed1999

Mycobacterium tuberculosis expresses a novel pH-dependent divalent cation transporter belonging to the Nramp family.

Mammalian natural resistance-associated macrophage protein (Nramp) homologues are important determinants of susceptibility to infection by diverse intracellular pathogens including mycobacteria. Eukaryotic Nramp homologues transport divalent cations such as Fe(2+), Mn(2+), Zn(2+), and Cu(2+). Mycobacterium tuberculosis and Mycobacterium bovis (bacillus Calmette-Guérin [BCG]) also encode an Nramp homologue (Mramp). RNA encoding Mramp induces approximately 20-fold increases in (65)Zn(2+) and (55)Fe(2+) uptake when injected into Xenopus laevis oocytes. Transport is dependent on acidic extracellular pH and is maximal between pH 5.5 and 6.5. Mramp-mediated (65)Zn(2+) and (55)Fe(2+) transport is abolished by an excess of Mn(2+) and Cu(2+), confirming that Mramp interacts with a broad range of divalent transition metal cations. Using semiquantitative reverse transcription PCR, we show that Mramp mRNA levels in M. tuberculosis are upregulated in response to increases in ambient Fe(2+) and Cu(2+) between <1 and 5 microM concentrations and that this upregulation occurs in parallel with mRNA for y39, a putative metal-transporting P-type ATPase. Using a quantitative ratiometric PCR technique, we demonstrate a fourfold decrease in Mramp/y39 mRNA ratios from organisms grown in 5-70 microM Cu(2+). M. bovis BCG cultured axenically and within THP-1 cells also expresses mRNA encoding Mramp. Mramp exemplifies a novel prokaryotic class of metal ion transporter. Within phagosomes, Mramp and Nramp1 may compete for the same divalent cations, with implications for intracellular survival of mycobacteria.

Amino Acid Sequence↗
PubMed2004

Evaluation of universal pre-enrichment broth for isolation of Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes from dairy farm environmental samples.

Use of universal pre-enrichment broth (UPB) as a primary enrichment medium for detection of Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes from dairy farm environmental samples was evaluated. There were no differences in bacterial growth between UPB and selective primary enrichment broths for each pathogen inoculated individually or in combination at 10(1) and 10(2) colony forming units/mL. In addition, no differences were observed when UPB and selective primary enrichment broths were compared for detection efficiency of pathogens in artificially contaminated raw milk and fecal samples. Listeria enrichment broth (LEB) was compared with UPB to support growth of L. monocytogenes from naturally contaminated environmental samples. Listeria monocytogenes was isolated from seven of 30 samples enriched in UPB and six of 30 samples enriched in LEB. Dairy farm environmental samples were examined for recovery of the three pathogens using UPB. Subsequent isolation was achieved using selective secondary enrichment of each pathogen. Listeria monocytogenes, Salmonella spp., and E. coli O157:H7 were isolated in 13.4% (30 of 224), 8.9% (20 of 224), and 2.2% (five of 224) of samples, respectively. Isolation rates of the three pathogens were somewhat higher than in previous reports. Overall, UPB supported growth of test pathogens to detectable levels within 24 h. Our results demonstrate that UPB has potential for routine use in isolation of foodborne pathogens from diverse environmental samples.

Animals↗
PubMed2001

Evidence for recurrent paralogous gene conversion and exceptional allelic divergence in the Attacin genes of Drosophila melanogaster.

Insects produce a limited variety of antibacterial peptides to combat a wide diversity of pathogens. These peptides are often conserved across evolutionarily distant taxa, but little is known about the level and structure of polymorphism within species. We have surveyed naturally occurring genetic variation in the promoter and coding regions of three Attacin antibacterial peptide genes from 12 lines of Drosophila melanogaster. These genes exhibit high levels of silent nucleotide variations (1-3% per nucleotide heterozygosity), but are not excessively polymorphic at the amino acid level. There is extensive variation in the Attacin promoters, some of which may affect transcriptional efficiency, and one line carries a deletion in the Attacin A coding region that renders this gene nonfunctional. Two of the genes, Attacins A and B, are arranged in tandem and show evidence of repeated interlocus gene conversion. Attacin C, more divergent and located 1.3 Mbp upstream of Attacins A and B, does not appear to have been involved in such exchanges. All three genes are characterized by divergent haplotypes, and one Attacin AB allele appears to have recently increased rapidly in frequency in the population.

Alleles↗
PubMed2006

Nonhost resistance of barley is successfully manifested against Magnaporthe grisea and a closely related Pennisetum-infecting lineage but is overcome by Magnaporthe oryzae.

Magnaporthe oryzae is a major pathogen of rice (Oryza sativa L.) but is also able to infect other grasses, including barley (Hordeum vulgare L.). Here, we report a study using Magnaporthe isolates collected from other host plant species to evaluate their capacity to infect barley. A nonhost type of resistance was detected in barley against isolates derived from genera Pennisetum (fontaingrass) or Digitaria (crabgrass), but no resistance occurred in response to isolates from rice, genus Eleusine (goosegrass), wheat (Triticum aestivum L.), or maize (Zea mays L.), respectively. Restriction of pathogen growth in the nonhost interaction was investigated microscopically and compared with compatible interactions. Real-time polymerase chain reaction was used to quantify fungal biomass in both types of interaction. The phylogenetic relationship among the Magnaporthe isolates used in this study was investigated by inferring gene trees for fragments of three genes, actin, calmodulin, and beta-tubulin. Based on phylogenetic analysis, we could distinguish different species that were strictly correlated with the ability of the isolates to infect barley. We demonstrated that investigating specific host interaction phenotypes for a range of pathogen isolates can accurately highlight genetic diversity within a pathogen population.

Actins↗
PubMed2000

Plants secrete substances that mimic bacterial N-acyl homoserine lactone signal activities and affect population density-dependent behaviors in associated bacteria.

In gram-negative bacteria, many important changes in gene expression and behavior are regulated in a population density-dependent fashion by N-acyl homoserine lactone (AHL) signal molecules. Exudates from pea (Pisum sativum) seedlings were found to contain several separable activities that mimicked AHL signals in well-characterized bacterial reporter strains, stimulating AHL-regulated behaviors in some strains while inhibiting such behaviors in others. The chemical nature of the active mimic compounds is currently unknown, but all extracted differently into organic solvents than common bacterial AHLs. Various species of higher plants in addition to pea were found to secrete AHL mimic activities. The AHL signal-mimic compounds could prove to be important in determining the outcome of interactions between higher plants and a diversity of pathogenic, symbiotic, and saprophytic bacteria.

4-Butyrolactone↗
PubMed1997

Media and tests to simplify the recognition and identification of members of the Proteeae.

Several important and diverse human pathogens are found in the tribe Proteeae. By identifying and concentrating on key biochemical reactions, it has been possible to devise six simple media that permit the identification of all the important members of the tribe with ease, speed and accuracy. This was confirmed by optional additional confirmatory media and tests.

Amino Acids↗
PubMed1995

Identification, cloning, and characterization of PWL2, a gene for host species specificity in the rice blast fungus.

Genetic analysis of host specificity in the rice blast fungus (Magnaporthe grisea) identified a single gene, PWL2 (for Pathogenicity toward Weeping Lovegrass), that exerts a major effect on the ability of this fungus to infect weeping lovegrass (Eragrostis curvula). The allele of the PWL2 gene conferring nonpathogenicity was genetically unstable, with the frequent appearance of spontaneous pathogenic mutants. PWL2 was cloned based on its map position. Large deletions detected in pathogenic mutants guided the gene cloning efforts. Transformants harboring the cloned PWL2 gene lost pathogenicity toward weeping lovegrass but remained fully pathogenic toward other host plants. Thus, the PWL2 host species specificity gene has properties analogous to classical avirulence genes, which function to prevent infection of certain cultivars of a particular host species. The PWL2 gene encodes a glycine-rich, hydrophilic protein (16 kD) with a putative secretion signal sequence. The pathogenic allele segregating in the mapping population, pwl2-2, differed from PWL2 by a single base pair substitution that resulted in a loss of function. The PWL2 locus is highly polymorphic among rice pathogens from diverse geographic locations.

Alleles↗
PubMed2005

Mammalian Toll-like receptors: to immunity and beyond.

Toll-like receptors (TLRs) constitute an archetypal pattern recognition system. Their sophisticated biology underpins the ability of innate immunity to discriminate between highly diverse microbial pathogens and self. However, the remarkable progress made in describing this biology has also revealed new immunological systems and processes previously hidden to investigators. In particular, TLRs appear to have a fundamental role in the generation of clonal adaptive immune responses, non-infectious disease pathogenesis and even in the maintenance of normal mammalian homeostasis. Although an understanding of TLRs has answered some fundamental questions at the host-pathogen interface, further issues, particularly regarding therapeutic modulation of these receptors, have yet to be resolved.

Animals↗
PubMed2004

Intraspecific diversity of the marine fish pathogen Tenacibaculum maritimum as determined by randomly amplified polymorphic DNA-PCR.

AIM: The aim of the present study was to evaluate the intraspecific genetic variability within Tenacibaculum maritimum strains isolated from different species of marine fish. METHODS AND RESULTS: Twenty-nine strains isolated from five different fish species and three reference strains were characterized by randomly amplified polymorphic DNA (RAPD) method. Cluster analysis of RAPD-PCR profiles showed that the strains, regardless of the oligonucleotide primer employed (P2 and P6), were separated into two main groups that strongly correlated with the host species and/or O-serotypes described for this pathogen. One group composed all strains isolated from sole (Solea senegalensis and S. solea) and gilthead seabream (Sparus aurata), and the other compiled the T. maritimum isolates from yellowtail (Seriola quinqueradiata), Atlantic salmon (Salmo salar) and turbot (Scophthalmus maximus). An important exception was observed in the RAPD patterns of the reference strains, which were included in different genetic groups depending on the primer employed. CONCLUSIONS: The results obtained demonstrated genetic variability within the T. maritimum isolated from different marine fish. Such genetic variability proved to be strongly associated with the host and/or serogroups described for this pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: The RAPD analysis constitutes a valuable molecular technique for epidemiological studies of T. maritimum. Interestingly, this is the first report of intraspecific differentiation and characterization of T. maritimum strains isolated from cultured fish.

Animals↗
PubMed2002

Distribution of human virus contamination in shellfish from different growing areas in Greece, Spain, Sweden, and the United Kingdom.

Viral pollution in shellfish has been analyzed simultaneously across a wide range of geographical regions, with emphasis on the concomitant variations in physicochemical characteristics and social features. The methods for sample treatment and for the detection of human enteric viruses were optimized by the participating laboratories. The second part of this study involves the selection of a protocol for virus detection, which was validated by analyzing the distribution and concentration of human viral pathogens under diverse conditions during an 18-month period in four European countries. Shellfish-growing areas from diverse countries in the north and south of Europe were defined and studied, and the microbiological quality of the shellfish was analyzed. Human adenovirus, Norwalk-like virus, and enterovirus were identified as contaminants of shellfish in all the participating countries. Hepatitis A virus was also isolated in all areas except Sweden. The seasonal distribution of viral contamination was also described. Norwalk-like virus appeared to be the only group of viruses that demonstrated seasonal variation, with lower concentrations occurring during warm months. The depuration treatments currently applied were shown to be adequate for reducing Escherichia coli levels but ineffective for the elimination of viral particles. The human adenoviruses detected by PCR correlate with the presence of other human viruses and could be useful as a molecular index of viral contamination in shellfish.

Adenoviruses, Human↗
PubMed2006

Effect of transport at ambient temperature on detection and isolation of Vibrio cholerae from environmental samples.

It has long been assumed that prolonged holding of environmental samples at the ambient air temperature prior to bacteriological analysis is detrimental to isolation and detection of Vibrio cholerae, the causative agent of pandemic cholera. The present study was aimed at understanding the effect of transporting environmental samples at the ambient air temperature on isolation and enumeration of V. cholerae. For water and plankton samples held at ambient temperatures ranging from 31 degrees C to 35 degrees C for 20 h, the total counts did not increase significantly but the number of culturable V. cholerae increased significantly compared to samples processed within 1 h of collection, as measured by culture, acridine orange direct count, direct fluorescent-antibody-direct viable count (DFA-DVC), and multiplex PCR analyses. For total coliform counts, total bacterial counts, and DFA-DVC counts, the numbers did not increase significantly, but the culturable plate counts for V. cholerae increased significantly after samples were held at the ambient temperature during transport to the laboratory for analysis. An increase in the recovery of V. cholerae O1 and improved detection of V. cholerae O1 rfb and ctxA also occurred when samples were enriched after they were kept for 20 h at the ambient temperature during transport. Improved detection and isolation of toxigenic V. cholerae from freshwater ecosystems can be achieved by holding samples at the ambient temperature, an observation that has significant implications for tracking this pathogen in diverse aquatic environments.

Bacterial Proteins↗
PubMed1996

Nonopsonic binding of Mycobacterium tuberculosis to human complement receptor type 3 expressed in Chinese hamster ovary cells.

Nonopsonic invasion of mononuclear phagocytes by Mycobacterium tuberculosis is likely important in the establishment of a primary infection in the lung. M. tuberculosis binds to a variety of phagocyte receptors, of which the mannose receptor and complement receptor type 3 (CR3) may support nonopsonic binding. CR3, a beta2 integrin, is a target for diverse intracellular pathogens, but its role in nonopsonic binding remains uncertain. We have examined the binding of M. tuberculosis H37Rv to human CR3 heterologously expressed in Chinese hamster ovary (CHO) cells, thereby circumventing the problems of competing receptors and endogenously synthesized complement, which are inherent in studies with mononuclear phagocytes. The surface expression of CD11b and CD18 was assessed by immunofluorescence, immunobead binding, flow cytometry, and immunoprecipitation with anti-CD11b and anti-CD18 monoclonal antibodies (MAbs). The functional activity of the surface-expressed CD11b/CD18 (CR3) heterodimer was confirmed by rosetting with C3bi-coated microspheres. We found that M. tuberculosis bound four- to fivefold more avidly to CR3-expressing CHO cells than to wild-type cells and, importantly, that this binding was at similar levels in the presence of fresh or heat-inactivated human or bovine serum or no serum. In contrast, Mycobacterium smegmatis bound poorly to CR3-expressing CHO cells in the absence of serum, but after opsonization in serum, binding was comparable to that of M. tuberculosis. The binding of M. tuberculosis to the transfected CHO cells was CR3 specific, as it was inhibited by anti-CR3 MAbs, particularly the anti-CD11b MAbs LM2/1 (I domain epitope) and OKM1 (C-terminal epitope), neither of which inhibit C3bi binding. MAb 2LPM19c, which recognizes the C3bi-binding site on CD11b, had little or no effect on M. tuberculosis binding. The converse was found for the binding of opsonized M. smegmatis, which was strongly inhibited by 2LPM19c but unaffected by LM2/1 or OKM1. CR3-specific binding was also evidenced by the failure of M. tuberculosis to bind to CHO cells transfected with an irrelevant surface protein (angiotensin-converting enzyme) in the presence or absence of serum. We conclude that the binding of M. tuberculosis H37Rv to CR3 expressed in CHO cells is predominantly nonopsonic and that the organism likely expresses a ligand that binds directly to CR3.

Animals↗
PubMed1997

Characterization of a class II pilin expression locus from Neisseria meningitidis: evidence for increased diversity among pilin genes in pathogenic Neisseria species.

Strains of Neisseria meningitidis elaborate one of two classes of pili. Meningococcal class I pili have many features in common with pili produced by N. gonorrhoeae, including the ability to bind monoclonal antibody SM1 and a common gene and protein structure consisting of conserved, semivariable, and hypervariable regions. Class II pili are SM1 nonreactive and display smaller subunit molecular weights than do gonococcal or meningococcal class I pili. In this study, we have determined the N-terminal amino acid sequence for class II pilin and isolated the expression locus encoding class II pilin from N. meningitidis FAM18. Meningococcal class II pilin displays features typical of type IV pili and shares extensive amino acid identity with the N-terminal conserved regions of other neisserial pilin proteins. However, the deduced class II pilin sequence displays several unique features compared with previously reported meningococcal class I and gonococcal pilin sequences. Class II pilin lacks several conserved peptide regions found within the semivariable and hypervariable regions of other neisserial pilins and displays a large deletion in a hypervariable region of the protein believed to be exposed on the pilus face in gonococcal pili. DNA sequence comparisons within all three regions of the coding sequence also suggest that the meningococcal class II pilin gene is the most dissimilar of the three types of neisserial pilE loci. Additionally, the class II locus fails to display flanking-sequence homology to class I and gonococcal genes and lacks a downstream Sma/Cla repeat sequence, a feature present in all other neisserial pilin genes examined to date. These data indicate meningococcal class II pili represent a structurally distinct class of pili and suggest that relationships among pilin genes in pathogenic Neisseria do not necessarily follow species boundaries.

Amino Acid Sequence↗
PubMed2004

The Salmonella enterica serovar typhimurium divalent cation transport systems MntH and SitABCD are essential for virulence in an Nramp1G169 murine typhoid model.

Nramp1 is a transporter that pumps divalent cations from the vacuoles of phagocytic cells and is associated with the innate resistance of mice to diverse intracellular pathogens. We demonstrate that sitA and mntH, genes encoding high-affinity metal ion uptake systems in Salmonella enterica serovar Typhimurium, are upregulated when Salmonella is internalized by Nramp1-expressing macrophages and play an essential role in systemic infection of congenic Nramp1-expressing mice.

Animals↗
PubMed2001

Differential roles of the Pseudomonas aeruginosa PA14 rpoN gene in pathogenicity in plants, nematodes, insects, and mice.

We cloned the rpoN (ntrA, glnF) gene encoding the alternate sigma factor sigma(54) from the opportunistic multihost pathogen Pseudomonas aeruginosa strain PA14. A marker exchange protocol was used to construct the PA14 rpoN insertional mutation rpoN::Gen(r). PA14 rpoN::Gen(r) synthesized reduced levels of pyocyanin and displayed a variety of phenotypes typical of rpoN mutants, including a lack of motility and the failure to grow on nitrate, glutamate, or histidine as the sole nitrogen source. Compared to wild-type PA14, rpoN::Gen(r) was ca. 100-fold less virulent in a mouse thermal injury model and was significantly impaired in its ability to kill the nematode Caenorhabditis elegans. In an Arabidopsis thaliana leaf infectivity assay, although rpoN::Gen(r) exhibited significantly reduced attachment to trichomes, stomata, and the epidermal cell surface, did not attach perpendicularly to or perforate mesophyll cell walls, and proliferated less rapidly in Arabidopsis leaves, it nevertheless elicited similar disease symptoms to wild-type P. aeruginosa PA14 at later stages of infection. rpoN::Gen(r) was not impaired in virulence in a Galleria mellonella (greater wax moth) pathogenicity model. These data indicate that rpoN does not regulate the expression of any genes that encode virulence factors universally required for P. aeruginosa pathogenicity in diverse hosts.

Amino Acids↗
PubMed2003

Cluster of type IV secretion genes in Helicobacter pylori's plasticity zone.

Some genes present in only certain strains of the genetically diverse gastric pathogen Helicobacter pylori may affect its phenotype and/or evolutionary potential. Here we describe a new 16.3-kb segment, 7 of whose 16 open reading frames are homologs of type IV secretion genes (virB4, virB7 to virB11, and virD4), the third such putative secretion gene cluster found in H. pylori. This segment, to be called tfs3, was discovered by subtractive hybridization and chromosome walking. Full-length and truncated tfs3 elements were found in 20 and 19%, respectively, of 94 strains tested, which were from Spain, Peru, India, and Japan. A tfs3 remnant (6 kb) was found in an archived stock of reference strain J99, although it was not included in this strain's published genome sequence. PCR and DNA sequence analyses indicated the following. (i) tfs3's ends are conserved. (ii) Right-end insertion occurred at one specific site in a chromosomal region that is varied in gene content and arrangement, the "plasticity zone." (iii) Left-end insertion occurred at different sites in each of nine strains studied. (iv) Sequences next to the right-end target in tfs3-free strains were absent from most strains carrying full-length tfs3 elements. These patterns suggested insertion by a transposition-like event, but one in which targets are chosen with little or no specificity at the left end and high specificity at the right end, thereby deleting the intervening DNA.

Bacterial Proteins↗