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Identification of a CD4+ T cell-stimulating antigen of pathogenic bacteria by expression cloning.

Identifying the immunogenic proteins that elicit pathogen-specific T cell responses is key to rational vaccine design. While several approaches have succeeded in identifying major histocompatibility complex (MHC) class I bound peptides that stimulate CD8+ T cells, these approaches have been difficult to extend to peptides presented by MHC class II molecules that stimulate CD4+ T cells. We describe here a novel strategy for identifying CD4+ T cell-stimulating antigen genes. Using Listeria monocytogenes-specific, lacZ-inducible T cells as single-cell probes, we screened a Listeria monocytogenes genomic library as recombinant Escherichia coli that were fed to macrophages. The antigen gene was isolated from the E. coli clone that, when ingested by the macrophages, allowed generation of the appropriate peptide/MHC class II complex and T cell activation. We show that the antigenic peptide is derived from a previously unknown listeria gene product with characteristics of a membrane-bound protein.

Amino Acid Sequence↗

Complex transmission dynamics of clonally related virulent Mycobacterium tuberculosis associated with barhopping by predominantly human immunodeficiency virus-positive gay men.

Limited data suggest that measures to reduce tuberculosis transmission should be based on locations rather than on personal contacts. Molecular epidemiologic methods (analysis of IS6110 patterns, spoligotypes, variable numbers of tandem DNA repeats, and automated DNA sequence data) identified a cohort of 48 persons who were infected with progeny of the same Mycobacterium tuberculosis strain. Epidemiologic investigation documented that a large proportion of the patients were gay white human immunodeficiency virus-positive men. Most practiced barhopping, an activity that involved patronizing many bars in the same neighborhood each night. Few subjects were directly linked to more than 1 or 2 other persons by conventional investigation methods, which shows that the transmission dynamics were unusually complex compared with most previously described episodes of strain spread. The data support the concept that identification of locations where pathogen dissemination likely occurs may provide additional strategies for targeted tuberculosis control.

Alcohol Drinking↗

Detection of HIV-1 in injection paraphernalia: risk in an era of heightened awareness.

Links between AIDS and self-injection of drugs were first recognized more than 20 years ago, but identification of a specific pathogen and ways to neutralize it has not led to complete success in preventing transmission of HIV-1 infection among injecting drug users (IDUs). A street ethnographer identified active risk locales (places where IDUs go to inject drugs) and recruited their proprietors into a study of contaminated injection paraphernalia. Collected paraphernalia from locales were analyzed for contamination by HIV-1 using real-time polymerase chain reaction (PCR). Proprietors and clientele of 16 risk locales participated by contributing used paraphernalia and/or agreeing to a blood test (0.5-1 mL drawn by finger stick). Realtime PCR was the primary measure used to determine contamination of injection paraphernalia and blood samples with HIV-1. Of 130 samples collected at baseline, a total of 8 were found to have evidence of HIV-1 contamination by detection of either HIV-1 RNA or DNA. The most serious contamination (up to 600,000 copies per milliliter) was found in ancillary paraphernalia, rather than needle/syringe (N/S) specimens. Only 4 of 74 N/S specimens had any evidence of HIV-1 contamination at all (with very low viral loads), and none had both HIV-1 DNA and RNA. Although IDUs in risk locales in Miami/Dade appear to be taking care of their N/S with regard to contamination by HIV-1, important evidence of contamination in ancillary paraphernalia, especially cookers and cottons, indicates that IDUs may still incur serious risk regardless of how well they care for their N/S. Our observations indicated that IDUs rinsed their N/S before returning them to the proprietors, from whom we eventually collected them, and this rinsing would have masked the contamination to which they were exposed through use of unrinsed cookers and reused cottons.

Anthropology, Cultural↗

Seqwin: ultrafast identification of signature sequences in microbial genomes.

MOTIVATION: Polymerase chain reaction (PCR) enables rapid, cost-effective diagnostics but requires prior identification of genomic regions that allow sensitive and specific detection of target microbial groups, herein referred to as microbial signature sequences. We introduce Seqwin, an open-source framework designed to automate microbial genome signature discovery. Tens of thousands of microbial genomes are now available for a single species, limiting the application of existing manual and automated approaches for identifying signatures. Modern approaches that are capable of leveraging all available microbial genomes will ensure sensitive and accurate DNA signature identification and enable robust pathogen detection for clinical, environmental, and public health applications. RESULTS: Seqwin builds weighted pan-genome minimizer graphs and uses a traversal algorithm to identify signature sequences that occur frequently in target genomes but remain rare in non-targets. Unlike earlier tools that depend on strict presence or absence of sequences, Seqwin accommodates natural sequence variation and scales to very large genome collections. When applied to genomes from C. difficile, M. tuberculosis, and S. enterica, Seqwin recovered more high-quality signatures than alternative methods with lower computational burden. Seqwin's analysis of nearly 15 000 S. enterica genomes yielded over 200 candidate signatures in three minutes. Seqwin provides an open-source solution for the long-standing need for scalable microbial signature discovery and diagnostic assay design. AVAILABILITY AND IMPLEMENTATION: Seqwin is available on GitHub (https://github.com/treangenlab/Seqwin) and can be installed via Bioconda (https://bioconda.github.io/recipes/seqwin/README.html). Benchmarking datasets, outputs, and scripts are available on Zenodo (https://doi.org/10.5281/zenodo.19874011).

Software↗

Clinical correlates of anaerobic bacteriology in peritonitis.

Recent surgical reports indicate that for patients with secondary bacterial peritonitis, surgeons do not routinely use the identification of the bacterial pathogens and determination of their antimicrobial susceptibilities in choosing antimicrobial therapy. Further, some surgeons advocate abandoning the routine practice of obtaining culture specimens from patients with complicated appendicitis, because the data from the clinical laboratory have not been found to have an impact on postoperative care. We review the rationale for continued surveillance of and implementation of bacteriological data in treatment of secondary peritonitis. We also describe in detail the anaerobic flora of secondary peritonitis, the unique susceptibility patterns of these organisms, and the specific virulence factors of anaerobes, particularly Bacteroides fragilis. The fact that clinical investigations sometimes result in treatment failure when gram-negative anaerobes are resistant to the antimicrobials used or when complete antimicrobial susceptibility data are not available emphasizes the need for accurate and early knowledge of the bacteriologic characteristic of the flora of the operative site. We emphasize the relationship of in vitro susceptibility of intraoperative isolates with clinical outcome. We propose a cooperative trial that would demonstrate that successful antimicrobial therapy should be based on the susceptibility of the flora of the operative site, which correlates with clinical outcome.

Animals↗

Encephalitogenic activity of truncated myelin oligodendrocyte glycoprotein (MOG) peptides and their recognition by CD8+ MOG-specific T cells on oligomeric MHC class I molecules.

We have previously demonstrated that the 21-residue peptide pMOG(35-55) from myelin oligodendrocyte glycoprotein (MOG) contains an antigenic epitope that activates CD8(+) encephalitogenic T cells in C57BL/6 (B6) mice. To identify the core encephalitogenic epitope of CD8(+) MOG-specific T cells, we have prepared a panel of highly purified peptides of varying lengths, which span the entire length of pMOG(35-55), and tested their binding to recombinant H-2D(b) dimers and their ability to induce EAE. Two of the truncated peptides, pMOG(40-54) and pMOG(44-54), strongly bound recombinant H-2D(b) protein and this complex bound MOG-specific CD8(+) T cells. Interestingly, pMOG(40-54) retained the full capability of inducing paralytic disease, whereas only a part of the B6 mice immunized with pMOG(44-54) developed clinical paralysis and central nervous system (CNS) inflammation. Further deletion of 1 amino acid from either the N- or C-terminus of the peptide pMOG(44-54) dramatically reduced binding to recombinant H-2D(b), and abolished the induction of paralysis and CNS inflammation. Our results demonstrate that the ability of truncated pMOG(35-55) peptides to bind recombinant H-2D(b) protein does not always correlate with their ability of inducing encephalomyelitis. This approach enables the further identification of the core pathogenic epitope within the pMOG(35-55) that activates MOG-specific encephalitogenic CD8(+) T cells.

Animals↗

Mitochondria and the heart.

Since the identification of the first pathogenic mutations of mitochondrial DNA in 1988, a plethora of information about human mitochondrial diseases has been brought to light. Not surprisingly, many of these disorders affect the myocardium, because this tissue relies heavily upon oxidative metabolism. This review focuses on disorders of the respiratory chain, the only area of mammalian cellular metabolism under the control of two genomes, nuclear and mitochondrial. Consequently, defects of aerobic synthesis of adenosine triphosphate (ATP) can be due to mutations of either genome. We describe genetic mitochondrial cardiomyopathies and briefly review mouse models and the mitochondrial theory of presbycardia.

Animals↗

Lyme disease.

The number of reported cases of Lyme disease in the United States has increased steadily since 1982, and the increase coincides with a growth in deer populations in endemic regions. Application of careful epidemiologic principles has led to the identification of two new pathogens that produce syndromes closely related to Lyme disease. Coinfection with other pathogens such as Babesia has been shown to alter the clinical course of Lyme disease. Better understanding of the pathogenesis of Lyme disease has provided clues into the mechanisms responsible for persistent symptoms and will serve as a foundation for the design and implementation of appropriate therapic recommendations. A safe vaccine for the prevention of Lyme disease in humans has been developed, and clinical vaccine efficacy trials are close to completion.

Humans↗

Lyme disease.

A record number of cases of Lyme disease was reported in the United States in 1996. The steady increase in Lyme disease cases coincides with a growth in deer populations in endemic regions. Application of careful epidemiologic principles have led to the identification of two new pathogens that produce syndromes closely related to Lyme disease. Coinfection with Borrelia burgdorferi and the agent of human granulocytic ehrlichiosis have been documented. Coinfection with multiple organisms may alter the clinical course of Lyme disease. Better understanding of the pathogenesis of neuroborreliosis has provided clues into mechanisms responsible for persistent symptoms and will serve as a foundation for the design and implementation of appropriate therapeutic recommendations. A safe vaccine for the prevention of Lyme disease in humans has been developed, and preliminary reports from large clinical vaccine efficacy trials look promising.

Animals↗

Phenotype of Pseudomonas aeruginosa isolates causing corneal infection between 1997 and 2000.

PURPOSE: To investigate the relationship between functional phenotype of and the associated human corneal infection. METHODS: This was an experimental pilot study of patients presenting with corneal infections at the Jules Stein Eye Institute with presumed infection during the period from 12/30/97 to 9/1/00. Thirteen patients were admitted to the study based on positive identification of the causative pathogen as and patient consent. Data were collected (including bacterial cultures, lens wear schedule and care, gender and age, completed history questionnaire, clinical photographs). Statistical analysis of possible correlations was performed. Phenotypes of were determined, and clinical factors associated with infection were explored. RESULTS: Both invasive and cytotoxic phenotypes of were isolated in equal proportion. Cytotoxic strains and invasive strains were found to be associated with patients younger than 50 years of age and older than 50 years of age, respectively. CONCLUSIONS: remains a significant pathogen in corneal infection, especially during contact lens wear. The age of the patient may influence the phenotype of causing infection. Since invasive and cytotoxic strains have different effects on corneal cells, treatment of the infection might require different approaches depending on this phenotype of the causative bacteria.

Adolescent↗

Sternoclavicular septic arthritis due to haemophilus influenzae.

We have reported a case of septic sternoclavicular arthritis illustrating a rare complication of infection with a relatively common pathogen, Haemophilus influenze. Early identification of the infecting organism and appropriate antibiotic therapy are essential to prevent further morbidity from the complications of this unusual pyoarthritis.

Arthritis, Infectious↗

Genomic and phenotypic characterization of Xanthomonas cynarae sp. nov., a new species that causes bacterial bract spot of artichoke (Cynara scolymus L.).

A bacterial disease of artichoke (Cynara scolymus L.) was first observed in 1954 in Brittany and the Loire Valley, France. This disease causes water-soaked spots on bracts and depreciates marketability of the harvest. Ten strains of the pathogen causing bacterial spot of artichoke, previously identified as a member of the genus Xanthomonas, were characterized and compared with type and pathotype strains of the 20 Xanthomonas species using a polyphasic study including both phenotypic and genomic methods. The ten strains presented general morphological, biochemical and physiological traits and G+C content characteristic of the genus Xanthomonas. Sequencing of the 165 rRNA gene confirmed that this bacterium belongs to the genus Xanthomonas, and more precisely to the Xanthomonas campestris core. DNA-DNA hybridization results showed that the strains that cause bacterial spot of artichoke were 92-100% related to the proposed type strain CFBP 4188T and constituted a discrete DNA homology group that was distinct from the 20 previously described Xanthomonas species. The results of numerical analysis were in accordance with DNA-DNA hybridization data. Strains causing the bacterial bract spot of artichoke exhibited consistent determinative biochemical characteristics, which distinguished them from the 20 other Xanthomonas species previously described. Furthermore, pathogenicity tests allowed specific identification of this new phytopathogenic bacterium. Thus, it is concluded that this bacterium is a new species belonging to the genus Xanthomonas, for which the name Xanthomonas cynarae is proposed. The type strain, CFBP 4188T, has been deposited in the Collection Française des Bactéries Phytopathogènes (CFBP).

Base Composition↗

A new regulatory DNA motif of the gamma subclass Proteobacteria: identification of the LexA protein binding site of the plant pathogen Xylella fastidiosa.

Escherichia coli LexA protein is the repressor of a gene network whose members are directly involved in the repair of damaged DNA and in the survival of bacterial cells until DNA lesions have been eliminated. The lexA gene is widely present in bacteria, although the sequences of only three LexA-binding sites are known: Gram-positive, alpha Proteobacteria and some members of gamma Proteobacteria represented by E. coli. Taking advantage of the fact that the genome sequence of the plant-pathogenic bacterium Xylella fastidiosa has been determined, its lexA gene has been cloned and overexpressed in E. coli to purify its product. After demonstration that X. fastidiosa lexA and recA genes are co-transcribed, gel mobility shift assays and directed mutagenesis experiments using the promoter of the lexA-recA transcriptional unit demonstrated that the X. fastidiosa LexA protein specifically binds the imperfect palindrome TTAGN(6)TACTA. This is the first LexA binding sequence identified in the gamma Proteobacteria differing from the E. coli-like LexA box. Although a computational search has revealed the presence of TTAGN(6)TACTA-like motifs upstream of X. fastidiosa genes other than lexA, X. fastidiosa LexA only binds the promoter of one of them, XF2313, encoding a putative DNA-modification methylase. Moreover, X. fastidiosa LexA protein does not bind any of the other genes whose homologues are regulated by the LexA repressor in E. coli (uvrA, uvrB, ssb, ruvAB, ftsK, dinG, recN and ybfE). RT-PCR quantitative analysis has also demonstrated that lexA-recA and XF2313 genes, as well as the X. fastidiosa genes which are homologues to those of E. coli belonging to the LexA regulon, with the exception of ssb, are DNA damage-inducible in X. fastidiosa.

Bacterial Proteins↗

Molecular characterization of nocardioform actinomycetes in activated sludge by 16S rRNA analysis.

The analysis of complex microbiota present in activated sludge is important for the understanding and possible control of severe separation problems in sewage treatment such as sludge bulking or sludge foaming. Previous studies have shown that nocardioform actinomycetes are responsible for these conditions, which not only affect the efficiency of sewage treatment but also represent a threat to public health due to spread of pathogens. However, isolation and identification of these filamentous, nocardioform actinomycetes is hampered by their fastidious nature. Most species are still uncultivable and their taxonomy is unresolved. To study the ecology of these micro-organisms at the molecular level, we have established a clone library of 16S rRNA gene fragments amplified from bulk sludge DNA. A rough indication of the predominant flora in the sludge was given by sequencing randomly chosen clones, which revealed a great diversity of bacteria from different taxa. Colony hybridization with oligonucleotide probe MNP1 detected 27 clones with 16S rDNA inserts from nocardioform actinomycetes and mycobacteria. The sequence data from these clones together with those from randomly chosen clones were used for comparative 16S rRNA analysis and construction of dendrograms. All sequences differed from those of previously sequenced species in the databases. Phenotypic characterization of isolates of nocardioform actinomycetes and mycobacteria cultivated in parallel from the same activated-sludge sample revealed a large discrepancy between the two approaches. Only one 16S rDNA sequence of a cultured isolate was represented in the clone library, indicating that culture conditions could select species which represent only a small fraction of the organisms in the activated sludge.

Actinomycetales↗

Magnaporthe grisea pth11p is a novel plasma membrane protein that mediates appressorium differentiation in response to inductive substrate cues.

Mutagenesis of Magnaporthe grisea strain 4091-5-8 led to the identification of PTH11, a pathogenicity gene predicted to encode a novel transmembrane protein. We localized a Pth11-green fluorescent protein fusion to the cell membrane and vacuoles. pth11 mutants of strain 4091-5-8 are nonpathogenic due to a defect in appressorium differentiation. This defect is reminiscent of wild-type strains on poorly inductive surfaces; conidia germinate and undergo early differentiation events, but appressorium maturation is impaired. Functional appressoria are formed by pth11 mutants at 10 to 15% of wild-type frequencies, suggesting that the protein encoded by PTH11 (Pth11p) is not required for appressorium morphogenesis but is involved in host surface recognition. We assayed Pth11p function in multiple M. grisea strains. These experiments indicated that Pth11p can activate appressorium differentiation in response to inductive surface cues and repress differentiation on poorly inductive surfaces and that multiple signaling pathways mediate differentiation. PTH11 genes from diverged M. grisea strains complemented the 4091-5-8 pth11 mutant, indicating functional conservation. Exogenous activation of cellular signaling suppressed pth11 defects. These findings suggest that Pth11p functions at the cell cortex as an upstream effector of appressorium differentiation in response to surface cues.

Amino Acid Sequence↗

A single gene encodes a selective toxin causal to the development of tan spot of wheat.

The identification and characterization of pathogenicity factors are essential to an understanding of the molecular events that regulate the interaction of plant-pathogenic microbes with their hosts. We have isolated the gene that encodes a host-selective toxic protein produced by the fungus Pyrenophora tritici-repentis and confirmed that this gene functions in the plant as the primary determinant of pathogenicity in the Pyrenophora-wheat interaction. These results demonstrate that a single gene encodes the production of a host-selective toxin and that transformation of this gene into a non-toxin-producing isolate of P. tritici-repentis leads to both toxin production and pathogenicity.

Amino Acid Sequence↗

Deoxyribonucleic acid hybridization studies of Exophiala dermatitidis and Exophiala jeanselmei.

Exophiala dermatitidis and Exophiala jeanselmei share similar morphological features and have been confused with each other. To clarify the relationship between the two fungi, we conducted a deoxyribonucleic acid (DNA)-DNA hybridization study using a dot blot method. Between E. dermatitidis and E. jeanselmei, only a very low level of DNA relatedness was seen and it was confirmed that these two fungi are distinct species based on DNA similarity. Close correspondence of DNA from the isolates of E. dermatitidis was obtained, whereas the isolates of E. jeanselmei were divided into 6 groups according to their DNA similarity and a possibility was shown that E. jeanselmei is composed of genetically heterogeneous groups. The subdivision of the species E. jeanselmei by the DNA-DNA hybridization method was in agreement with serotyping exoantigens. This result suggests that DNA-DNA hybridization studies provide an excellent tool for the identification and grouping of pathogenic dematiaceous fungi.

DNA, Fungal↗