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PubMed2003

Novel approaches to the treatment of pneumonia.

As the prevalence of resistance to multiple antibiotics increases it is progressively more difficult to treat pneumonia in hospitalized patients. Therefore, anti-infectious agents that have new modes of action are needed urgently. Recent advances in DNA sequencing technology make it possible to elucidate the sequences of the entire genomes of pathogenic bacteria. This allows many novel, non-traditional targets for therapeutic intervention to be identified, such as those involved in disease pathogenesis, and in adaptation and growth at sites of infection. In the past few years, inhibitors of new bacterial targets have been developed, including inhibitors of genes that are required for either virulence or pathogenesis. The challenge is to optimize and develop these agents to provide novel approaches to the treatment of pneumonia in hospitalized patients.

Adjuvants, Immunologic↗
PubMed2005

Transcriptional response signature of human lymphoid cells to staphylococcal enterotoxin B.

Two shock-inducing toxins that result in similar eventual outcome of disease were studied to determine host gene expression responses, for correlation of both similar and unique gene patterns. We initially used differential display (DD)-PCR and identified 859 cDNA fragments that were differentially expressed after 16 h of in vitro exposure of human peripheral blood mononuclear cells (PBMC) to staphylococcal enterotoxin B (SEB). Upon further examination using custom cDNA microarrays and RT-PCR analysis, we found unique set of genes to each toxin (SEB or lipopolysaccharide (LPS)), especially at early time periods. By 16 h, there was a convergence of some gene expression responses and many of those genes code for proteins such as proteinases, transcription factors, vascular tone regulators, and respiratory distress. In an attempt to replicate the findings in vivo, monkeys were challenged with SEB and the resultant gene expression responses indicated a pattern typical of SEB exposure when compared to LPS, with a similar outcome. We provide evidence that vastly diverse global gene analysis techniques used in unison can not only effectively identify pathogen-specific genomic markers and provide a solid foundation to mechanistic insights but also explain some of the toxin-related symptoms through gene functions.

Animals↗
PubMed2005

Pathophysiology, pleiotrophy and paradigm shifts: genetic lessons from Parkinson's disease.

PD (Parkinson's disease) is an aetiologically heterogeneous disorder characterized by a clinical phenotype consisting of resting tremor, rigidity and bradykinesia. Motor symptoms are associated with a progressive loss of dopaminergic neurons, with Lewy body inclusions within surviving neurons. Although heritability studies have shown evidence of familial aggregation, twin studies have provided limited support for a genetic aetiology. Nevertheless, classical linkage methods have nominated 11 regions of the genome and pathogenic mutations have been identified in several genes, including alpha-synuclein, parkin, ubiquitin C-terminal hydrolase L1, oncogene DJ-1, PTEN-induced protein kinase 1 and microtubule-associated protein tau. Most recently, heterozygous mutations in LRRK2 (leucine-rich repeat kinase 2) were found to cause late-onset, autosomal-dominant PD. Despite their consistent clinical phenotype, family members with LRRK2 mutations can have variable alpha-synuclein and tau pathologies. Lrrk2 is a member of the Roc (Ras of complex proteins) family, with Ras GTPase and MAPKKK (mitogen-activated protein kinase kinase kinase) catalytic domains. Thus its discovery highlights vesicle dynamics and secondary-messenger signalling in disease pathophysiology. To diagnose a disease accurately and effectively treat it, requires an understanding of its molecular pathogenesis. Herein, we provide an overview of the genetics of PD, how these discoveries are revolutionizing long-held beliefs and more importantly how this knowledge may be translated into patient therapy.

Humans↗
PubMed1983

Loss of unstably amplified dihydrofolate reductase genes from mouse cells is greatly accelerated by hydroxyurea.

Previous work has shown that mammalian cells that carry unstably amplified genes for dihydrofolate reductase (DHFR) gradually lose the amplified DHFR genes when grown in the absence of the DHFR inhibitor methotrexate (MTX). Unstably amplified genes occur on small acentric chromosomes called double minutes (DMs) or even smaller chromatin fragments, in contrast to stably amplified genes, which reside in centromere-containing chromosomes. We have found that the rate of loss of the unstably amplified DHFR genes can be greatly oncreased by growing the cells in the presence of a nonlethal concentration of hydroxyurea. For example, in one MTX-resistant subline studied, approximately equal to 90% of the original DHFR gene dosage is lost in 25-30 cell doublings in the absence of MTX. The same degree of loss is achieved, however, in less than 4 doublings if cells are grown in the presence of 50 microM hydroxyurea. This new effect of hydroxyurea does not appear to be due to changes in plating efficiency or selective cytotoxicity. In particular, no increase in cell death occurs at 50 microM hydroxyurea, and cells continue to multiply, albeit 1/2 to 2/3 as fast as in the absence of hydroxyurea. The ability to selectively accelerate the loss of amplified genes from mammalian cells as shown in the present work may have important implications both for the problem of drug resistance in cancer chemotherapy and for curing mammalian cells of extrachromosomally maintained DNA genomes of pathogenic viruses.

Animals↗
PubMed1996

Functional and molecular mitochondrial abnormalities associated with a C --> T transition at position 3256 of the human mitochondrial genome. The effects of a pathogenic mitochondrial tRNA point mutation in organelle translation and RNA processing.

We have previously identified a mitochondrial DNA polymorphism (a C --> T transition at position 3256, within the mitochondrial tRNALeu(UUR) gene in a patient with a multisystem disorder. Although there were several indicators suggesting a pathogenetic role for this mtDNA polymorphism, its heteroplasmic nature made functional and molecular studies difficult to interpret. We have now fused enucleated fibroblasts from the patient with a mtDNA-less cell line to generate transmitochondrial cybrids harboring different proportions of mutated and wild-type mtDNA. Individual clones harboring essentially 100% wild-type or > 99% mutated mtDNAs were characterized and studied for respiratory capacity, respiratory chain enzymes activity, mitochondrial protein synthesis, and RNA steady-state levels and processing. Our results showed that cell lines containing exclusively mutated mtDNAs respire poorly, overproduce lactic acid, and have significantly impaired activity of respiratory complexes I and IV. Molecular studies showed that mutant clones have a decrease in steady-state levels of mitochondrial tRNALeu(UUR), and a partial impairment of mitochondrial protein synthesis and steady-state levels, suggesting that these molecular abnormalities are involved in the pathogenetic mechanism of the mtDNA 3256 mutation.

Blotting, Northern↗
PubMed2003

Biochemical characterization of proline racemases from the human protozoan parasite Trypanosoma cruzi and definition of putative protein signatures.

Proline racemase catalyzes the interconversion of L- and D-proline enantiomers and has to date been described in only two species. Originally found in the bacterium Clostridium sticklandii, it contains cysteine residues in the active site and does not require co-factors or other known coenzymes. We recently described the first eukaryotic amino acid (proline) racemase, after isolation and cloning of a gene from the pathogenic human parasite Trypanosoma cruzi. Although this enzyme is intracellularly located in replicative non-infective forms of T. cruzi, membrane-bound and secreted forms of the enzyme are present upon differentiation of the parasite into non-dividing infective forms. The secreted form of proline racemase is a potent host B-cell mitogen supporting parasite evasion of specific immune responses. Here we describe that the TcPRAC genes in T. cruzi encode functional intracellular or secreted versions of the enzyme exhibiting distinct kinetic properties that may be relevant for their relative catalytic efficiency. Although the Km of the enzyme isoforms were of a similar order of magnitude (29-75 mM), Vmax varied between 2 x 10(-4 )and 5.3 x 10(-5) mol of L-proline/s/0.125 microM of homodimeric recombinant protein. Studies with the enzyme-specific inhibitor and abrogation of enzymatic activity by site-directed mutagenesis of the active site Cys330 residue reinforced the potential of proline racemase as a critical target for drug development against Chagas' disease. Finally, we propose a protein signature for proline racemases and suggest that the enzyme is present in several other pathogenic and non-pathogenic bacterial genomes of medical and agricultural interest, yet absent in mammalian host, suggesting that inhibition of proline racemases may have therapeutic potential.

Amino Acid Isomerases↗
PubMed2005

Structural and genetic analyses reveal a key role in prophage excision for the TorI response regulator inhibitor.

TorI (Tor inhibition protein) has been identified in Escherichia coli as a protein inhibitor acting through protein-protein interaction with the TorR response regulator. This interaction, which does not interfere with TorR DNA binding activity, probably prevents the recruitment of RNA polymerase to the torC promoter. In this study we have solved the solution structure of TorI, which adopts a prokaryotic winged-helix arrangement. Despite no primary sequence similarity, the three-dimensional structure of TorI is highly homologous to the (lambda)Xis, Mu bacteriophage repressor (MuR-DBD), and transposase (MuA-DBD) structures. We propose that the TorI protein is the structural missing link between the (lambda)Xis and MuR proteins. Moreover, in vivo assays demonstrated that TorI plays an essential role in prophage excision. Heteronuclear NMR experiments and site-directed mutagenesis studies have pinpointed out key residues involved in the DNA binding activity of TorI. Our findings suggest that TorI-related proteins identified in various pathogenic bacterial genomes define a new family of atypical excisionases.

Amino Acid Sequence↗
PubMed2004

YODA: selecting signature oligonucleotides.

MOTIVATION: Selecting oligonucleotide probes for use in microarray design, and other applications requiring signature sequences, involves identifying sequences which will bind strongly to their intended target, while binding only weakly (or preferably, not at all) to non-target sequences which may be present in the hybridization reaction. While many tools to assist in selection of such sequences exist, all the ones we examined lack important oligo design and software features. RESULTS: YODA is an application for assisting biological researchers in selecting signature sequences. It incorporates a custom sequence similarity search to find potential cross-hybridizing non-target sequences. For this task, most oligo design tools rely on BLAST, which is ill suited for it due to an unacceptable risk of false negatives. YODA supports multiple probe design goals including single-genome, multiple-genome, pathogen-host and species/strain-identification. A graphical interface is provided as well as a command-line interface, both of which support many user-controlled parameters. YODA is easy to install and use and runs on Windows, Mac OS X and Linux platforms. AVAILABILITY: Freely available (LGLP) along with source code and additional documentation at http://pathport.vbi.vt.edu/YODA CONTACT: enordber@vbi.vt.edu.

Algorithms↗
PubMed2006

In silico predicted robustness of viroids RNA secondary structures. I. The effect of single mutations.

Viroids are plant subviral pathogens whose genomes are constituted by a single-stranded and covalently closed small RNA molecule that does not encode for any protein. Despite this genomic simplicity, they are able of inducing devastating symptoms in susceptible plants. Most of the 29 described viroid species fold into a rodlike or quasi-rodlike structure, whereas a few of them fold as branched structures. The shape of these RNA structures is perhaps one of the most characteristic properties of viroids and sometimes is considered their only phenotype. Here we use RNA thermodynamic secondary structure prediction algorithms to compare the mutational robustness of all viroid species. After characterizing the statistical properties of the distribution of mutational effects on structure stability and the wideness of neutral neighborhood for each viroid species, we show an evolutionary trend toward increased structural robustness during viroid radiation, giving support to the adaptive value of robustness. Differences in robustness among the 2 viroid families can be explained by the larger fragility of branched structures compared with the rodlike ones. We also show that genomic redundancy can contribute to the robustness of these simple RNA genomes.

Algorithms↗
PubMed2006

In silico predicted robustness of viroid RNA secondary structures. II. Interaction between mutation pairs.

Viroids are plant subviral pathogens whose genomes are constituted by a single-stranded and covalently closed small RNA molecule that does not encode for any protein. Most of the 29 described viroid species fold into a rodlike or quasi-rodlike structure, whereas a few of them fold as highly branched structures. In a previous study, we used RNA thermodynamic secondary structure prediction algorithms to compare the mutational robustness of all viroid species. Here we used the same approach to explore the sign and strength of epistasis among pairs of random mutations. We found that antagonistic interactions were more abundant than synergistic ones. However, despite their lower frequency, synergistic interactions tended to be more intense. Mutational robustness and the intensity of epistasis were correlated such that viroid species with large average mutational effects showed stronger antagonistic epistasis, whereas viroids with mild average mutational effects showed weaker antagonistic interactions. The strength of antagonistic epistasis decreased with genome complexity as a consequence of the gained robustness of duplicated genomes. In good agreement with our previous finding of an evolutionary trend toward increased robustness, we now found a trend toward reduced antagonistic epistasis.

Base Pairing↗
PubMed2025

Candida glabrata replicating within macrophages experiences amino acid deprivation, DNA damage, and chromosome instability.

Macrophages, the central players of innate immunity, control invading microbes by encapsulating them inside the phagosome, a nutrient-poor, reactive oxidant species-rich organelle. Nevertheless, some microbes, including the opportunistic yeast pathogen Candida glabrata, noted for its karyotype diversity, rapid evolution of antifungal drug resistance, and lack of meiosis, can survive and even replicate inside macrophages. However, it is not fully understood how C. glabrata responds to macrophage engulfment, and it is unknown how this presumably DNA-damaging environment influences the pathogen's genome stability. In this study, we used comparative transcriptomics to identify amino acid starvation and DNA damage as conditions eliciting C. glabrata responses most similar to macrophage engulfment. Consistent with this, we found that C. glabrata intra-macrophage survival and replication require master regulator of amino acid biosynthesis GCN4 and functional DNA double-strand break repair. Furthermore, comet assays provided the first direct evidence for increased DNA breaks in intra-macrophage yeast, and pulse-field gel electrophoresis showed that chromosomal alterations occur frequently in macrophage-passaged C. glabrata. Interestingly, these alterations could not be resolved by long read DNA sequencing, suggesting that they involved highly complex repetitive regions. Finally, we identified several point mutations emerging during macrophage passaging and showed that among them, a frameshift in RME1 (repressor of meiosis in Saccharomyces cerevisiae), increased C. glabrata intra-macrophage fitness. Together, these analyses point to amino acid deprivation, reveal elevated DNA breakage and chromosome instability, and raise intriguing questions about the role of meiotic gene orthologs in C. glabrata persisting and replicating within macrophages.

Journal Article↗
PubMed2006

Structure of the conserved hypothetical protein MAL13P1.257 from Plasmodium falciparum.

The structure of a conserved hypothetical protein, PlasmoDB sequence MAL13P1.257 from Plasmodium falciparum, Pfam sequence family PF05907, has been determined as part of the structural genomics effort of the Structural Genomics of Pathogenic Protozoa consortium. The structure was determined by multiple-wavelength anomalous dispersion at 2.17 A resolution. The structure is almost entirely beta-sheet; it consists of 15 beta-strands and one short 3(10)-helix and represents a new protein fold. The packing of the two monomers in the asymmetric unit indicates that the biological unit may be a dimer.

Amino Acid Sequence↗
PubMed2006

Sensitive and rapid detection of Flavobacterium columnare in channel catfish Ictalurus punctatus by a loop-mediated isothermal amplification method.

AIMS: To evaluate the loop-mediated isothermal amplification method (LAMP) for rapid detection of Flavobacterium columnare and determine the suitability of LAMP for rapid diagnosis of columnaris infection in channel catfish, Ictalurus punctatus. METHODS AND RESULTS: A set of four primers, two outer and two inner, were designed specifically to recognize 16S ribosomal RNA gene of this pathogen. Bacterial genomic DNA templates were prepared by hot lysis in a lysis buffer. Amplification of the specific gene segments was carried out at 65 degrees C for 1 h. The amplified gene products were analysed by agarose gel electrophoresis and detected by staining gels with ethidium bromide. A PCR assay was also included in this study. Our results demonstrate that the ladder-like pattern of bands from 204 bp specific to the Fl. columnare 16S ribosomal RNA gene was amplified. The detection limit of the LAMP assay was comparable to that of PCR in prepared genomic DNA reactions. In addition, this optimized LAMP assay was able to detect the Fl. columnare 16S ribosomal RNA gene in experimentally infected channel catfish. CONCLUSIONS: The LAMP assay for Fl. columnare detection in channel catfish was established. SIGNIFICANCE AND IMPACT OF THE STUDY: Because LAMP assay is a rapid, sensitive, specific, simple and cost-effective assay for Fl. columnare detection in channel catfish, it is useful for rapid diagnosis of Fl. columnare in fish hatcheries and the field.

Animals↗
PubMed2006

Insights into the evolutionary history of tubercle bacilli as disclosed by genetic rearrangements within a PE_PGRS duplicated gene pair.

BACKGROUND: The highly homologous PE_PGRS (Proline-glutamic acid_polymorphic GC-rich repetitive sequence) genes are members of the PE multigene family which is found only in mycobacteria. PE genes are particularly abundant within the genomes of pathogenic mycobacteria where they seem to have expanded as a result of gene duplication events. PE_PGRS genes are characterized by their high GC content and extensive repetitive sequences, making them prone to recombination events and genetic variability. RESULTS: Comparative sequence analysis of Mycobacterium tuberculosis genes PE_PGRS17 (Rv0978c) and PE_PGRS18 (Rv0980c) revealed a striking genetic variation associated with this typical tandem duplicate. In comparison to the M. tuberculosis reference strain H37Rv, the variation (named the 12/40 polymorphism) consists of an in-frame 12-bp insertion invariably accompanied by a set of 40 single nucleotide polymorphisms (SNPs) that occurs either in PE_PGRS17 or in both genes. Sequence analysis of the paralogous genes in a representative set of worldwide distributed tubercle bacilli isolates revealed data which supported previously proposed evolutionary scenarios for the M. tuberculosis complex (MTBC) and confirmed the very ancient origin of "M. canettii" and other smooth tubercle bacilli. Strikingly, the identified polymorphism appears to be coincident with the emergence of the post-bottleneck successful clone from which the MTBC expanded. Furthermore, the findings provide direct and clear evidence for the natural occurrence of gene conversion in mycobacteria, which appears to be restricted to modern M. tuberculosis strains. CONCLUSION: This study provides a new perspective to explore the molecular events that accompanied the evolution, clonal expansion, and recent diversification of tubercle bacilli.

Evolution, Molecular↗
PubMed2004

Immunome-derived vaccines.

Immune response to a subset of antigens and epitopes derived from an infectious pathogen may be sufficient for competent protection; immune recognition of every potential epitope derived from the pathogen's genome does not appear to be required. The pneumococcal and hepatitis vaccines, both of which are subunit vaccines, illustrate this premise. Similarly, 'immunome-derived vaccines' are based on the concept that response to the subset of antigens and epitopes that interface with the host immune system (the immunome) and not the whole organism (represented by the proteome or genome) can be sufficient for protection. Competent immune responses to cancer are also probably restricted to the neoplasm's 'immunome', although the set of antigens that drive successful immune response to cancer cells has proven more difficult to uncover. Researchers are now using bioinformatics sequence analysis tools, epitope mapping tools, microarrays and high-throughput immunology assays to discover the components of the immunome, which are then used to compose these new vaccines. At least one immunome-derived vaccine is in clinical trials and many others are in the vaccine pipeline. Due to the rapid improvement of immunoinformatics tools and immunological assays, the era of immunome-derived vaccines has begun.

Animals↗
PubMed2000

Clonotypes of Staphylococcus aureus isolated from continuous ambulatory peritoneal dialysis patients: what is the vector between nares and infection site?

Staphylococcus aureus is frequently isolated from patients with infections related to continuous ambulatory peritoneal dialysis (CAPD). In many cases, the organism is also isolated simultaneously from the anterior nares. To clarify the transmission trail of S. aureus, we used DNA analysis to identify clonotypes of clinical strains. The nares and exit sites of 32 CAPD patients were swabbed, and PD fluid samples were taken for pathogen culture. Genome DNA of S. aureus was digested with restriction enzyme Sma I for pulsed-field gel electrophoresis. We also asked the patients how they usually performed the PD procedure. S. aureus was isolated from 4 patients, including 3 who hosted two strains isolated separately from different sites. The DNA patterns of the strains isolated from these latter 3 patients were identical. However, the clonotypes from all 4 patients were different. Most of the patients did not wash their hands and wear masks while exchanging PD bags and caring for their exit sites. After the patients were disinfected and re-educated in proper procedures, S. aureus was not detected in any of them. These data suggest that no outbreak occurred in our hospital and that the vectors of endogenous infection were the patients themselves, probably their hands. A bacteriological study presents an efficient opportunity to re-educate patients in PD procedure.

Adult↗