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PubMed1997

Potato cysteine proteinase inhibitor gene family: molecular cloning, characterisation and immunocytochemical localisation studies.

Potato cysteine proteinase inhibitors (PCPIs) represent a distinct group of proteins as they show no homology to any other known cysteine proteinase inhibitor superfamilies, but they all belong to the Kunitz-type soybean trypsin inhibitor family. cDNA clones for five PCPIs have been isolated and sequenced. Amino acid substitutions occurring in the limited regions forming loops on the surface of these proteins suggest a further classification of PCPIs into three subgroups. Accumulation of PCPI was observed in vacuoles of stems after treatment with jasmonic acid (JA) using immunocytochemical localisation, implying that these inhibitors are part of a potato defence mechanism against insects and pathogens. Genomic DNA analysis show that PCPIs form a multigene family and suggest that their genes do not possess any introns.

Amino Acid Sequence↗
PubMed2026

Scalable near-real-time Bayesian phylogenetics for outbreaks with Delphy.

Pathogen genomic analysis is central to tracking, understanding and containing outbreaks1-13, but the complexity and cost of state-of-the-art phylogenetic tools limit global access and impact. Here we introduce Delphy, an exact reformulation of Bayesian phylogenetics14-17 designed to transform its speed, scalability and accessibility while retaining Bayesian state-of-the-art accuracy. Delphy's central data structure, an explicit mutation-annotated tree, takes advantage of the high sequence similarity of large-scale epidemic datasets18-20 for efficient tree exploration and convergence. By reproducing key analyses from recent major epidemics, including Ebola1,21, Zika2, SARS-CoV-2 (ref. 22), mpox3,4 and H5N1 (refs. 23,24), we demonstrate state-of-the-art accuracy with up to 2-3 orders of magnitude improvements in speed. Assessing Delphy's scalability, we show that a simulated dataset of 100,000 sequences can be analysed within a day. We distribute Delphy as a client-side web application that enables local, interactive analysis of raw data on the user's machine. Delphy automatically identifies key viral lineages and mutations, as well as their emergence and prevalence through time, with quantified uncertainties grounded in Bayesian theory. Delphy establishes Bayesian phylogenetics as a fast, accessible frontline tool for future outbreak response.

Journal Article↗
PubMed2005

Electron transport through nitrate and nitrite reductases in Campylobacter jejuni.

Campylobacter jejuni is a small genome pathogen that is incapable of growing strictly anaerobically due to its dependence on an oxygen-requiring ribonucleotide reductase for DNA synthesis. Nevertheless, it has a complex branched respiratory chain, which allows the use of several alternative electron acceptors for growth under oxygen-limited conditions. C. jejuni is equipped with both nitrate reductase (Nap) and nitrite reductase (Nrf) located in the periplasm, each predicted to receive electrons from menaquinol through distinct redox proteins. The pathways of electron transport to nitrate and nitrite are reviewed in this paper. Nitrate is considered as a potential in vivo electron acceptor and a role for nitrite reductase in NO detoxification is suggested.

Campylobacter jejuni↗
PubMed1996

Fluorescence correlation analysis of probe diffusion simplifies quantitative pathogen detection by PCR.

A sensitive, labor-saving, and easily automatable nonradioactive procedure named APEX-FCS (amplified probe extension detected by fluorescence correlation spectroscopy) has been established to detect specific in vitro amplification of pathogen genomic sequences. As an example, Mycobacterium tuberculosis genomic DNA was subjected to PCR amplification with the Stoffel fragment of Thermus aquaticus DNA polymerase in the presence of nanomolar concentrations of a rhodamine-labeled probe (third primer), binding to the target in between the micromolar amplification primers. The probe becomes extended only when specific amplification occurs. Its low concentration avoids false-positives due to unspecific hybridization under PCR conditions. With increasing portion of extended probe molecules, the probe's average translational diffusion properties gradually change over the course of the reaction, reflecting amplification kinetics. Following PCR, this change from a stage of high to a stage of low mobility can directly be monitored during a 30-s measurement using a fluorescence correlation spectroscopy device. Quantitation down to 10 target molecules in a background of 2.5 micrograms unspecific DNA without post-PCR probe manipulations could be achieved with different primer/ probe combinations. The assay holds the promise to concurrently perform amplification, probe hybridization, and specific detection without opening the reaction chamber, if sealable foils are used.

Automation↗
PubMed1998

H. pylori infection in non-ulcer patients--to treat or not to treat. The case against treatment.

Heretofore regarded as a strict pathogen, recent identification of multiple mutants of H. pylori, varying in pathogenicity, genomic composition, antigenic structure and other characteristics, has led to speculation that not all strains of the organism merit elimination. Affecting half the world's population, H. pylori appears to cause clinically significant disease in <20% of cases. The costs of eradicating harmless infection in over 2 billion people are prohibitive, particularly in countries lacking resources, and are questionable even in advanced countries where infection, gastritis and related diseases are declining as social conditions improve. Major controversies surround empiric eradication of helicobacter infection in patients with asymptomatic gastritis or non-specific dyspepsia. Apart from cost, and feasibility, there are concerns that widespread campaigns to eradicate H. pylori might cause major increases in esophageal reflux disease and esophageal adenocarcinoma, while causing some serious iatrogenic illness and increasing antibiotic resistance, with uncertain consequences to affected populations.

Dyspepsia↗
PubMed2006

Case studies in cost effectiveness of molecular diagnostics for infectious diseases: pulmonary tuberculosis, enteroviral meningitis, and BK virus nephropathy.

Pathogen genome amplification is used to detect and identify microorganisms, assess response to therapy, and detect mutations associated with drug resistance. Nucleic acid amplification tests have been shown to be superior to conventional culture-based testing methods in many circumstances. However, the enthusiasm for the technology in clinical laboratories may be decreased by the practical considerations of cost, complexity of the technology, and lack of US Food and Drug Administration-approved tests. The impact of nucleic acid amplification tests on the diagnosis and management of patients with tuberculosis, enteroviral meningitis, and BK virus transplant nephropathy will be examined, with an emphasis on the potential for health care cost savings.

BK Virus↗
PubMed2025

CREMSA: compressed indexing of (ultra) large multiple sequence alignments.

MOTIVATION: Recent viral outbreaks motivate the systematic collection of pathogenic genomes in order to accelerate their study and monitor the apparition/spread of variants. Due to their limited length and temporal proximity of their sequencing, viral genomes are usually organized, and analyzed as oversized Multiple Sequence Alignments (MSAs). Such MSAs are largely ungapped, and mostly homogeneous on a column-wise level but not at a sequential level due to local variations, hindering the performances of sequential compression algorithms. RESULTS: In order to enable an efficient handling of MSAs, including subsequent statistical analyses, we introduce CREMSA (Column-wise Run-length Encoding for MSAs), a new index that builds on sparse bitvector representations to compress an existing or streamed MSA, all the while allowing for an expressive set of accelerated requests to query the alignment without prior decompression. Using CREMSA, a 65 GB MSA consisting of 1.9M SARS-CoV 2 genomes could be compressed into 22 MB using less than half a gigabyte of main memory, while executing access requests in the order of 100&#x2009;ns. Such a speed up enables a comprehensive analysis of covariation over this very large MSA. We further assess the impact of the sequence ordering on the compressibility of MSAs and propose a resorting strategy that, despite the proven NP-hardness of an optimal sort, induces greatly increased compression ratios at a marginal computational cost. AVAILABILITY AND IMPLEMENTATION: CREMSA is freely accessible at https://gitlab.univ-lille.fr/cremsa/cremsa. The Snakemake workflow for the benchmarks is available at: https://gitlab.univ-lille.fr/cremsa/bench. The data used in the paper is on Zenodo at https://zenodo.org/records/14698859 and https://zenodo.org/records/15100011.

SARS-CoV-2↗
PubMed2005

Draft versus finished sequence data for DNA and protein diagnostic signature development.

Sequencing pathogen genomes is costly, demanding careful allocation of limited sequencing resources. We built a computational Sequencing Analysis Pipeline (SAP) to guide decisions regarding the amount of genomic sequencing necessary to develop high-quality diagnostic DNA and protein signatures. SAP uses simulations to estimate the number of target genomes and close phylogenetic relatives (near neighbors or NNs) to sequence. We use SAP to assess whether draft data are sufficient or finished sequencing is required using Marburg and variola virus sequences. Simulations indicate that intermediate to high-quality draft with error rates of 10(-3)-10(-5) (approximately 8x coverage) of target organisms is suitable for DNA signature prediction. Low-quality draft with error rates of approximately 1% (3x to 6x coverage) of target isolates is inadequate for DNA signature prediction, although low-quality draft of NNs is sufficient, as long as the target genomes are of high quality. For protein signature prediction, sequencing errors in target genomes substantially reduce the detection of amino acid sequence conservation, even if the draft is of high quality. In summary, high-quality draft of target and low-quality draft of NNs appears to be a cost-effective investment for DNA signature prediction, but may lead to underestimation of predicted protein signatures.

Computational Biology↗
PubMed2000

Molecular analysis of thermoregulation of phaseolotoxin-resistant ornithine carbamoyltransferase (argK) from Pseudomonas syringae pv. phaseolicola.

The phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) and phaseolotoxin are produced by Pseudomonas syringae pv. phaseolicola at 18 degrees C but not at 28 degrees C. At 28 degrees C, the pathogen produces a protein(s) that binds (in vitro) to a 485-bp fragment (thermoregulatory region, TRR) from a heterologous clone from the pathogen genomic library, which in multiple copies overrides thermoregulation of phaseolotoxin production in wild-type cells (K. B. Rowley, D. E. Clements, M. Mandel, T. Humphreys, and S. S. Patil, Mol. Microbiol. 8:625-635, 1993). We report here that DNase I protection analysis of the 485-bp fragment shows that a single site is protected from cleavage by the protein in the 28 degrees C extract and that this site contains two repeats of a core motif G/C AAAG separated by a 5-bp spacer. Partially purified binding protein forms specific complexes with a synthetic oligonucleotide containing four tandem repeats of this motif. A 492-bp upstream fragment from argK encoding ROCT also forms specific complexes with the protein in the 28 degrees C crude extract, and a 260-bp subfragment from the TRR containing the binding site cross competes with the argk fragment, indicating that the same protein binds to nucleotides in both fragments. DNase I protection analysis of the fragment from argK revealed four separate protected sequence elements, with element III containing half of the core motif sequence (CTTTG), and the other elements containing similar sequences. Gel shift assays were done with DNA fragments from which one or all of the sites were removed as competitor DNAs against the argK probe. The results of these experiments confirmed that the binding sites (in argK) are necessary for the protein to bind to the argK fragment in a specific manner. Taken together, the results of studies presented here suggest that in cells of P. syringae pv. phaseolicola grown at high temperature argK may be negatively regulated by the protein produced at this temperature.

Bacterial Proteins↗
PubMed1999

Ribotyping of Vibrio parahaemolyticus isolates obtained from food poisoning outbreaks in Taiwan.

Vibrio parahaemolyticus is a prevalent food-borne pathogen in Taiwan, Japan and other Asian countries. This work presents a novel ribotyping method for the molecular epidemiological examination of this pathogen. Genomic DNA was fragmented by HindIII digestion and hybridized with cDNA probe for Escherichia coli 16S and 23S RNA genes. A total of 121 isolates obtained from outbreaks during 1992 and 1994 in Taiwan were characterized by this ribotyping method. Four to seventeen restricted fragments were visualized in these isolates. After hierarchical cluster analysis, these isolates were grouped into thirty different ribotypes. In addition, A3, A7, E3 and F1 were the major ribotypes, consisting of 22.3, 13.2, 9.1, and 8.3% of the isolates, respectively. A, E, F, G and B were the major groups, consisting of 46.2, 14.0, 9.1, 6.7, and 6.7% of the isolates, respectively. The discriminatory ability of this ribotyping method, as determined by Simpson's index of diversity, was 0.93, which closely resembled that of a previously reported pulsed-field gel electrophoresis method.

Bacterial Typing Techniques↗
PubMed2006

Relapsing fever spirochaetes produce a serine protease that provides resistance to oxidative stress and killing by neutrophils.

The spirochaetes that cause tick-borne relapsing fever and Lyme disease are closely related human pathogens, yet they differ significantly in their ecology and pathogenicity. Genome sequencing of two species of relapsing fever spirochaetes, Borrelia hermsii and Borrelia turicatae, identified a chromosomal open reading frame, designated bhpA, not present in the Lyme disease spirochaete Borrelia burgdorferi. The predicted amino acid sequence of bhpA was homologous with the HtrA serine proteases, which are involved with stress responses and virulence in other bacteria. B. hermsii produced an active serine protease that was recognized by BhpA antibodies and the recombinant BhpA protein-degraded beta-casein. bhpA was transcribed in vitro at all growth temperatures and transcription levels were slightly elevated at higher temperatures. These results correlated with the synthesis of BhpA during B. hermsii infection in mice. With the exception of Borrelia recurrentis, the bhpA gene, protein and enzymatic activity were found in all relapsing fever spirochaetes, but not in Lyme disease or related spirochaetes. Heterologous expression of bhpA in B. burgdorferi increased the spirochaete's resistance to both oxidative stress and killing by human neutrophils. Therefore, we propose that bhpA encodes a unique and functional serine protease in relapsing fever spirochaetes. This periplasmic enzyme may prevent the accumulation of proteins damaged by the innate immune response and contribute to the ability of the relapsing fever spirochaetes to achieve high cell densities in blood.

Animals↗
PubMed2000

Mitigation of bioterrorist threats in the 21st century.

There is a raising level of awareness and concern that agriculture and food supplies might serve as targets for bioterrorists. To minimize such threats a number of new initiates are urgently needed: 1. Greater levels of financial commitment from federal, state, and international organizations for research on highly infectious diseases and for emergency response infrastructure; 2. Wellorchestrated emergency response plans based on inter-agency, inter-departmental and stakeholder working groups; 3. Teams prepared for risk assessment and risk communication; 4. Modern systems of animal identification and accurate trace-back for animal movement; 5. Increased biosecurity in intensive production operations; 6. Accurate intelligence as to what pathogens pose the highest risks for economic and social impact; 7. Establishment of new international animal and plant disease research networks and partnerships; 8. Strengthen international disease surveillance networks for early detection and intervention; 9. New generations of rapid diagnostic tests for pathogen detection that are practical for field and diagnostic laboratories; 10. Increased level of commitment to pathogen genomics research for molecular epidemiology and vaccine development; 11. New chemical and immunological intervention strategies to prevent or control disease outbreaks; 12. Increased level of training on exotic and emerging animal diseases in basic veterinary education and through continuing education for veterinarians, state and federal field personnel and laboratory diagnosticians.

Agriculture↗
PubMed2000

Listeriolysin O as a reporter to identify constitutive and in vivo-inducible promoters in the pathogen Listeria monocytogenes.

Listeria monocytogenes is a facultative intracellular gram-positive bacterium capable of growing in the cytoplasm of infected host cells. Bacterial escape from the phagosomal vacuole of infected cells is mainly mediated by the pore-forming hemolysin listeriolysin O (LLO) encoded by hly. LLO-negative mutants of L. monocytogenes are avirulent in the mouse model. We have developed a genetic system with hly as a reporter gene allowing the identification of both constitutive and in vivo-inducible promoters of this pathogen. Genomic libraries were created by randomly inserting L. monocytogenes chromosomal fragments upstream of the promoterless hly gene cloned into gram-positive and gram-negative shuttle vectors and expressed in an LLO-negative mutant strain. With this hly-based promoter trap system, combined with access to the L. monocytogenes genome database, we identified 20 in vitro-transcribed genes, including genes encoding (i) p60, a previously known virulence gene, (ii) a putative new hemolysin, and (iii) two proteins of the general protein secretion pathway. By using the hly-based system as an in vivo expression technology tool, nine in vivo-induced loci of L. monocytogenes were identified, including genes encoding (i) the previously known in vivo-inducible phosphatidylinositol phospholipase C and (ii) a putative N-acetylglucosamine epimerase, possibly involved in teichoic acid biosynthesis. The use of hly as a reporter is a simple and powerful alternative to classical methods for transcriptional analysis to monitor promoter activity in L. monocytogenes.

Amino Acid Sequence↗
PubMed1996

Subspecies typing of Vibrio parahaemolyticus by pulsed-field gel electrophoresis.

Vibrio parahaemolyticus is one of the most important food-borne pathogens in Taiwan, Japan, and other costal regions. We report on the development of a pulsed-field gel electrophoresis (PFGE) method for the molecular typing of this pathogen. Genomic DNA was digested with SfiI, and the fragments were resolved on 1% agarose with a contour-clamped homogeneous electric field apparatus set at 190 V and a pulse time of 3 to 80 s. A total of 130 selected isolates obtained from outbreaks during 1993 and 1994 on Taiwan were also characterized by this PFGE method. These isolates were grouped into 14 PFGE types which consisted on one to six patterns, and a total of 39 patterns were identified. Most of these domestic clinical isolates could be clustered into several major types (types A, B, C, and G). These major types showed relatively low degrees of similarity to several foreign strains and other domestic but environmental strains. Strain CCRC12863, which originated from Japan, was close to the group consisting of F, G, and H PFGE types, suggesting a clonal relationship between this Japanese strain and other domestic isolates.

Bacterial Typing Techniques↗
PubMed2002

The expression, processing and localization of polymorphic membrane proteins in Chlamydia pneumoniae strain CWL029.

BACKGROUND: Chlamydiae are obligate intracellular bacteria, which are important human pathogens. Genome sequences of C. trachomatis and C. pneumoniae have revealed the presence of a Chlamydia specific gene family encoding polymorphic outer membrane proteins, Pmps. In C. pneumoniae the family comprises twenty-one members, which are all transcribed. In the present study, the expression, processing and localisation of the sixteen full-length Pmps in C. pneumoniae strain CWL029 have been further investigated by two-dimensional gel electrophoresis and immunofluorescence microscopy. RESULTS: Ten Pmps were identified in elementary bodies (EBs). Eight of these were investigated with respect to time dependent expression and all were found to be up-regulated between 36 and 48 hours post infection. Antibodies against Pmp6, 8, 10, 11 and 21 reacted with chlamydiae when infected cells were formalin fixed. Pmp6, Pmp20 and Pmp21 were found in cleaved forms, and the cleavage sites of Pmp6 and Pmp21 were identified. CONCLUSIONS: The Pmps are heavily up-regulated at the time of conversion of RB to EB, and at least ten Pmps are present in EBs. Due to their reaction in formalin fixation it is likely that Pmp6, 8, 10, 11 and 21 are surface exposed. The identified cleavage sites of Pmp6 and Pmp21 are in agreement with the theory that the Pmps are autotransporters.

Bacterial Outer Membrane Proteins↗
PubMed2022

A deep learning approach to real-time HIV outbreak detection using genetic data.

Pathogen genomic sequence data are increasingly made available for epidemiological monitoring. A main interest is to identify and assess the potential of infectious disease outbreaks. While popular methods to analyze sequence data often involve phylogenetic tree inference, they are vulnerable to errors from recombination and impose a high computational cost, making it difficult to obtain real-time results when the number of sequences is in or above the thousands. Here, we propose an alternative strategy to outbreak detection using genomic data based on deep learning methods developed for image classification. The key idea is to use a pairwise genetic distance matrix calculated from viral sequences as an image, and develop convolutional neutral network (CNN) models to classify areas of the images that show signatures of active outbreak, leading to identification of subsets of sequences taken from an active outbreak. We showed that our method is efficient in finding HIV-1 outbreaks with R0 &#x2265; 2.5, and overall a specificity exceeding 98% and sensitivity better than 92%. We validated our approach using data from HIV-1 CRF01 in Europe, containing both endemic sequences and a well-known dual outbreak in intravenous drug users. Our model accurately identified known outbreak sequences in the background of slower spreading HIV. Importantly, we detected both outbreaks early on, before they were over, implying that had this method been applied in real-time as data became available, one would have been able to intervene and possibly prevent the extent of these outbreaks. This approach is scalable to processing hundreds of thousands of sequences, making it useful for current and future real-time epidemiological investigations, including public health monitoring using large databases and especially for rapid outbreak identification.

Humans↗
PubMed2026

Beyond antibiotics: artificial intelligence-enabled anti-infective ecosystems for next-generation precision therapeutics against antimicrobial resistance.

The rapid global expansion of antimicrobial resistance (AMR) threatens to undermine decades of progress in infectious disease management and highlights the limitations of conventional antibiotic-centered therapeutic strategies. Although emerging technologies-including antimicrobial peptides, bacteriophage therapy, CRISPR-based antimicrobials, microbiome therapeutics, anti-virulence approaches, nanotechnology-enabled drug delivery, and artificial intelligence (AI)-have individually demonstrated considerable promise, they are predominantly being developed as independent interventions rather than as coordinated components of an integrated therapeutic strategy. This Perspective proposes the Intelligent Anti-Infective Ecosystem (IAIE) as a conceptual systems-level framework that computationally integrates multimodal diagnostics, pathogen genomics, microbiome profiling, AI-assisted decision support, programmable precision therapeutics, ecological monitoring, and longitudinal clinical feedback within a continuously learning dynamically optimized workflow. Unlike existing paradigms that primarily optimize individual technologies or therapeutic decisions, IAIE emphasizes closed-loop coordination among complementary antimicrobial approaches to support precision-guided infection management while preserving microbiome integrity and mitigating resistance selection pressure. We further outline the core components, operational principles, translational challenges, and technology readiness of the major therapeutic platforms that could contribute to such an ecosystem, while distinguishing clinically established interventions from emerging experimental strategies. Importantly, IAIE should be interpreted as a prospective conceptual architecture rather than an existing clinical platform. Its proposed clinical value remains to be established through sequential computational, preclinical, and prospective clinical investigations using standardized microbiological, ecological, and patient-centered outcome measures. By framing antimicrobial innovation within an responsive systems perspective, IAIE provides a roadmap for future multidisciplinary research aimed at integrating artificial intelligence and systems microbiology to enable sustainable management of antimicrobial resistance.

Humans↗
PubMed2006

Fine mapping of the keratoconus with cataract locus on chromosome 15q and candidate gene analysis.

PURPOSE: To report the fine mapping of the keratoconus with cataract locus on chromosome 15q and the mutational analysis of positional candidate genes. METHODS: Genotyping of two novel microsatellite markers and a single nucleotide polymorphism (SNP) in the critical region of linkage for keratoconus with cataract on 15q was performed. Positional candidate genes (MORF4L1, KIAA1055, ETFA, AWP1, REC14, KIAA1199, RCN2, FAH, IDH3A, MTHFS, ADAMTS7, MAN2C1, PTPN9, KIAA1024, ARNT2, BCL2A1, ISL2, C15ORF22 (P24B), DNAJA4, FLJ14594, CIB2 (KIP2), C15ORF5, and PSMA4) prioritized on the basis of ocular expression and probable function were screened by PCR-based DNA sequencing methods. RESULTS: We report the refinement of the linkage region for keratoconus with cataract to an interval of approximately 5.5 Mb flanked by the MAN2C1 gene and the D15S211 marker on chromosome 15q. Mutational analysis of positional candidate genes detected many sequence variations and single nucleotide polymorphisms. None of the sequence variants were considered pathogenic as they were also found in unaffected family members and normal control DNA samples. CONCLUSIONS: Fine mapping of the keratoconus with cataract locus on 15q has reduced the linked region to 5.5 Mb, thereby excluding 28 candidate genes. A further 23 candidate genes were excluded by direct sequencing methods, although a pathogenic genomic rearrangement or exonic deletion would not have been detected.

Case-Control Studies↗