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David A Haake

Publications and source records attributed to David A Haake.

At least 19 recordsLinked to original sources
PubMed2006

Lipoprotein computational prediction in spirochaetal genomes.

Lipoproteins are of great interest in understanding the molecular pathogenesis of spirochaetes. Because spirochaete lipobox sequences exhibit more plasticity than those of other bacteria, application of existing prediction algorithms to emerging sequence data has been problematic. In this paper a novel lipoprotein prediction algorithm is described, designated SpLip, constructed as a hybrid of a lipobox weight matrix approach supplemented by a set of lipoprotein signal peptide rules allowing for conservative amino acid substitutions. Both the weight matrix and the rules are based on a training set of 28 experimentally verified spirochaetal lipoproteins. The performance of the SpLip algorithm was compared to that of the hidden Markov model-based LipoP program and the rules-based algorithm Psort for all predicted protein-coding genes of Leptospira interrogans sv. Copenhageni, L. interrogans sv. Lai, Borrelia burgdorferi, Borrelia garinii, Treponema pallidum and Treponema denticola. Psort sensitivity (13-35 %) was considerably less than that of SpLip (93-100 %) or LipoP (50-84 %) due in part to the requirement of Psort for Ala or Gly at the -1 position, a rule based on E. coli lipoproteins. The percentage of false-positive lipoprotein predictions by the LipoP algorithm (8-30 %) was greater than that of SpLip (0-1 %) or Psort (4-27 %), due in part to the lack of rules in LipoP excluding unprecedented amino acids such as Lys and Arg in the -1 position. This analysis revealed a higher number of predicted spirochaetal lipoproteins than was previously known. The improved performance of the SpLip algorithm provides a more accurate prediction of the complete lipoprotein repertoire of spirochaetes. The hybrid approach of supplementing weight matrix scoring with rules based on knowledge of protein secretion biochemistry may be a general strategy for development of improved prediction algorithms.

Algorithms↗
PubMed2006

LipL46 is a novel surface-exposed lipoprotein expressed during leptospiral dissemination in the mammalian host.

Leptospirosis is a widespread zoonosis caused by invasive spirochaetes belonging to the genus Leptospira. Pathogenic leptospires disseminate via the bloodstream to colonize the renal tubules of reservoir hosts. Little is known about leptospiral outer-membrane proteins expressed during the dissemination stage of infection. In this study, a novel surface-exposed lipoprotein is described; it has been designated LipL46 to distinguish it from a previously described 31 kDa peripheral membrane protein, P31(LipL45), which is exported as a 45 kDa probable lipoprotein. The lipL46 gene encodes a 412 aa polypeptide with a 21 aa signal peptide. Lipid modification of cysteine at the lipoprotein signal peptidase cleavage site FSISC is supported by the finding that Leptospira interrogans intrinsically labels LipL46 during incubation in medium containing [(14)C]palmitate. LipL46 appears to be exported to the leptospiral outer membrane as a 46 kDa lipoprotein, based on Triton X-114 solubilization and phase partitioning studies, which included the outer and inner membrane controls LipL32 and LipL31, respectively. Surface immunoprecipitation and whole-cell ELISA experiments indicate that LipL46 is exposed on the leptospiral surface. Immunohistochemistry studies demonstrated expression of LipL46 by leptospires found in the bloodstream of acutely infected hamsters. Leptospires expressing LipL46 were also found in the intercellular spaces of the liver, within splenic phagocytes, and invading the glomerular hilum of the kidney. Infection-associated expression is supported by the finding that LipL46 is a major antigen recognized by sera from infected hamsters. These findings indicate that LipL46 may be important in leptospiral dissemination, and that it may serve as a useful serodiagnostic antigen.

Animals↗
PubMed2006

Effects of temperature on gene expression patterns in Leptospira interrogans serovar Lai as assessed by whole-genome microarrays.

Leptospirosis is an important zoonosis of worldwide distribution. Humans become infected via exposure to pathogenic Leptospira spp. from infected animals or contaminated water or soil. The availability of genome sequences for Leptospira interrogans, serovars Lai and Copenhageni, has opened up opportunities to examine global transcription profiles using microarray technology. Temperature is a key environmental factor known to affect leptospiral protein expression. Leptospira spp. can grow in artificial media at a range of temperatures reflecting conditions found in the environment and the mammalian host. Therefore, transcriptional changes were compared between cultures grown at 20 degrees C, 30 degrees C, 37 degrees C, and 39 degrees C to represent ambient temperatures in the environment, growth under laboratory conditions, and temperatures in healthy and febrile hosts. Data from direct pairwise comparisons of the four temperatures were consolidated to examine transcriptional changes at two generalized biological conditions representing mammalian physiological temperatures (37 degrees C and 39 degrees C) versus environmental temperatures (20 degrees C and 30 degrees C). Additionally, cultures grown at 30 degrees C then shifted overnight to 37 degrees C were compared with those grown long-term at 30 degrees C and 37 degrees C to identify genes potentially expressed in the early stages of infection. Comparison of data sets from physiological versus environmental experiments with upshift experiments provided novel insights into possible transcriptional changes at different stages of infection. Changes included differential expression of chemotaxis and motility genes, signal transduction systems, and genes encoding proteins involved in alteration of the outer membrane. These findings indicate that temperature is an important factor regulating expression of proteins that facilitate invasion and establishment of disease.

Environment↗
PubMed2006

Use of electrochemical DNA biosensors for rapid molecular identification of uropathogens in clinical urine specimens.

We describe the first species-specific detection of bacterial pathogens in human clinical fluid samples using a microfabricated electrochemical sensor array. Each of the 16 sensors in the array consisted of three single-layer gold electrodes-working, reference, and auxiliary. Each of the working electrodes contained one representative from a library of capture probes, each specific for a clinically relevant bacterial urinary pathogen. The library included probes for Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Enterocococcus spp., and the Klebsiella-Enterobacter group. A bacterial 16S rRNA target derived from single-step bacterial lysis was hybridized both to the biotin-modified capture probe on the sensor surface and to a second, fluorescein-modified detector probe. Detection of the target-probe hybrids was achieved through binding of a horseradish peroxidase (HRP)-conjugated anti-fluorescein antibody to the detector probe. Amperometric measurement of the catalyzed HRP reaction was obtained at a fixed potential of -200 mV between the working and reference electrodes. Species-specific detection of as few as 2,600 uropathogenic bacteria in culture, inoculated urine, and clinical urine samples was achieved within 45 min from the beginning of sample processing. In a feasibility study of this amperometric detection system using blinded clinical urine specimens, the sensor array had 100% sensitivity for direct detection of gram-negative bacteria without nucleic acid purification or amplification. Identification was demonstrated for 98% of gram-negative bacteria for which species-specific probes were available. When combined with a microfluidics-based sample preparation module, the integrated system could serve as a point-of-care device for rapid diagnosis of urinary tract infections.

Biosensing Techniques↗
PubMed2005

Electrochemical molecular analysis without nucleic acid amplification.

Electrochemical biosensors have revolutionized glucose monitoring but have not yet fulfilled their promise of a low cost, direct detection replacement for genetic amplification tests such as PCR [K. Kerman, M. Kobayashi, E. Tamiya, Recent trends in electrochemical DNA biosensor technology, Meas. Sci. Technol. 15 (2004) R1-R11; A. Chaubey, B.D. Malhotra, Mediated biosensors. Biosens. Bioelectron. 17 (6-7) (2002) 441-456]. It has been anticipated that the integration of nanoscale chemical structures such as self-assembled monolayers with electrochemical biosensors would increase sensitivity by decreasing inherent system noise. We have designed a novel biosensing approach incorporating such integration and achieved rapid, ultra-low concentration sensitivities without target amplification. Raw samples are mixed with lysis buffer to allow hybridization of nucleic acid targets with anchor and signal probes before immobilizing a signaling enzyme proximate to the biosensor surface. A bias potential is subsequently applied and the secondary byproduct of a cyclic peroxidase reaction measured. Further studies have demonstrated the application of our approach in protein, clinical chemistry, and ionic assays.

Biosensing Techniques↗
PubMed2005

Osmolarity, a key environmental signal controlling expression of leptospiral proteins LigA and LigB and the extracellular release of LigA.

The high-molecular-weight leptospiral immunoglobulin-like repeat (Lig) proteins are expressed only by virulent low-passage forms of pathogenic Leptospira species. We examined the effects of growth phase and environmental signals on the expression, surface exposure, and extracellular release of LigA and LigB. LigA was lost from stationary-phase cells, while LigB expression was maintained. The loss of cell-associated LigA correlated with selective release of a lower-molecular-weight form of LigA into the culture supernatant, while LigB and the outer membrane lipoprotein LipL41 remained associated with cells. Addition of tissue culture medium to leptospiral culture medium induced LigA and LigB expression and caused a substantial increase in released LigA. The sodium chloride component of tissue culture medium was primarily responsible for the enhanced release of LigA. Addition of sodium chloride, potassium chloride, or sodium sulfate to leptospiral medium to physiological osmolarity caused the induction of both cell-associated LigA and LigB, indicating that osmolarity regulates the expression of Lig proteins. Osmotic induction of Lig expression also resulted in enhanced release of LigA and increased surface exposure of LigB, as determined by surface immunofluorescence. Osmolarity appears to be a key environmental signal that controls the expression of LigA and LigB.

Animals↗
PubMed2005

LruA and LruB, novel lipoproteins of pathogenic Leptospira interrogans associated with equine recurrent uveitis.

Recurrent uveitis as a sequela to Leptospira infection is the most common infectious cause of blindness and impaired vision of horses worldwide. Leptospiral proteins expressed during prolonged survival in the eyes of horses with lesions of chronic uveitis were identified by screening a phage library of Leptospira interrogans DNA fragments with eye fluids from uveitic horses. Inserts of reactive phages encoded several known leptospiral proteins and two novel putative lipoproteins, LruA and LruB. LruA was intrinsically labeled during incubation of L. interrogans in medium containing [14C]palmitic acid, confirming that it is a lipoprotein. lruA and lruB were detected by Southern blotting in infectious Leptospira interrogans but not in nonpathogenic Leptospira biflexa. Fractionation data from cultured Leptospira indicate that LruA and LruB are localized in the inner membrane. Uveitic eye fluids contained significantly higher levels of immunoglobulin A (IgA) and IgG specific for each protein than did companion sera, indicating strong local antibody responses. Moreover, LruA- and LruB-specific antisera reacted with equine ocular components, suggesting an immunopathogenic role in leptospiral uveitis.

Acylation↗
PubMed2005

Surfaceome of Leptospira spp.

The identification of the subset of outer membrane proteins exposed on the surface of a bacterial cell (the surfaceome) is critical to understanding the interactions of bacteria with their environments and greatly narrows the search for protective antigens of extracellular pathogens. The surfaceome of Leptospira was investigated by biotin labeling of viable leptospires, affinity capture of the biotinylated proteins, two-dimensional gel electrophoresis, and mass spectrometry (MS). The leptospiral surfaceome was found to be predominantly made up of a small number of already characterized proteins, being in order of relative abundance on the cell surface: LipL32 > LipL21 > LipL41. Of these proteins, only LipL32 had not been previously identified as surface exposed. LipL32 surface exposure was subsequently verified by three independent approaches: surface immunofluorescence, whole-cell enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy. Three other proteins, Q8F8Q0 (a putative transmembrane outer membrane protein) and two proteins of 20 kDa and 55 kDa that could not be identified by MS, one of which demonstrated a high degree of labeling potentially representing an additional, as-yet-uncharacterized, surface-exposed protein. Minor labeling of p31(LipL45), GroEL, and FlaB1 was also observed. Expression of the surfaceome constituents remained unchanged under a range of conditions investigated, including temperature and the presence of serum or urine. Immunization of mice with affinity-captured surface components stimulated the production of antibodies that bound surface proteins from heterologous leptospiral strains. The surfaceomics approach is particularly amenable to protein expression profiling using small amounts of sample (<10(7) cells) offering the potential to analyze bacterial surface expression during infection.

Animals↗
PubMed2005

Diagnostic and therapeutic pitfalls associated with primaquine-tolerant Plasmodium vivax.

We describe a U.S. Army Ranger returning from duty in Afghanistan and Iraq with life-threatening infection due to Plasmodium vivax. Morphological variants were observed in blood films prepared using samples collected by venipuncture. The patient's multiple relapses indicate infection with primaquine-tolerant P. vivax. Strategies for relapse prevention using primaquine are reviewed.

Adult↗
PubMed2005

Leptospirosis after recreational exposure to water in the Yaeyama islands, Japan.

Leptospirosis is a global zoonotic disease with a variety of clinical manifestations. We report an outbreak of leptospirosis in the Yaeyama Islands, Japan, in the summer of 1999 associated with heavy rainfall. Fourteen people were diagnosed with leptospirosis and required hospitalization. All cases were found to have exposure to contaminated soil or water. A history of recreational activities involving water sports was more frequent (71%) than occupational risk factors related to agriculture or construction (29%). Fever was the primary symptom in all cases, followed by chills (93%), headache (86%), myalgias (57%) and conjunctival suffusion (57%). All cases were successfully treated with antimicrobial therapy except one patient who improved spontaneously. Jarisch-Herxheimer reactions were seen in six cases (43%). The increasing incidence of leptospirosis related to recreational sports is an important public health problem in resort areas. A high-index of suspicion, early treatment, and prevention are crucial in this latently endemic area.

Adolescent↗
PubMed2004

Rapid, species-specific detection of uropathogen 16S rDNA and rRNA at ambient temperature by dot-blot hybridization and an electrochemical sensor array.

Development of rapid molecular approaches for pathogen detection is key to improving treatment of infectious diseases. For this study, the kinetics and temperature-dependence of DNA probe hybridization to uropathogen species-specific sequences were examined. A set of oligonucleotide probes were designed based on variable regions of the 16S gene of the Escherichia coli, Proteus mirabilis, Klebsiella oxytoca, and Pseudomonas aeruginosa. A universal bacterial probe and probes-specific for gram-positive and gram-negative organisms were also included. The oligonucleotide probes discriminated among 16S genes derived from 11 different species of uropathogenic bacteria applied to nylon membranes in a dot-blot format. Significant binding of oligonucleotide probes to target DNA and removal of nonspecific binding by membrane washing could both be achieved rapidly, requiring as little as 10 min. An oligonucleotide probe from the same species-specific region of the E. coli 16S gene was used as a capture probe in a novel electrochemical 16-sensor array based on microfabrication technology. Sequence-specific hybridization of target uropathogen 16S rDNA was detected through horseradish peroxidase acting as an electrochemical transducer via a second, detector probe. The sensor array demonstrated rapid, species-specific hybridization in a time course consistent with the rapid kinetics of the dot-blot hybridization studies. As in the dot-blot hybridization studies, species-specific detection of bacterial nucleic acids using the sensor array approach was demonstrated both at 65 degrees C and at room temperature. These results demonstrate that molecular hybridization approaches can be adapted to rapid, room temperature conditions ideal for an electrochemical sensor array platform.

Bacteria↗
PubMed2004

Outer membrane proteins of pathogenic spirochetes.

Pathogenic spirochetes are the causative agents of several important diseases including syphilis, Lyme disease, leptospirosis, swine dysentery, periodontal disease and some forms of relapsing fever. Spirochetal bacteria possess two membranes and the proteins present in the outer membrane are at the site of interaction with host tissue and the immune system. This review describes the current knowledge in the field of spirochetal outer membrane protein (OMP) biology. What is known concerning biogenesis and structure of OMPs, with particular regard to the atypical signal peptide cleavage sites observed amongst the spirochetes, is discussed. We examine the functions that have been determined for several spirochetal OMPs including those that have been demonstrated to function as adhesins, porins or to have roles in complement resistance. A detailed description of the role of spirochetal OMPs in immunity, including those that stimulate protective immunity or that are involved in antigenic variation, is given. A final section is included which covers experimental considerations in spirochetal outer membrane biology. This section covers contentious issues concerning cellular localization of putative OMPs, including determination of surface exposure. A more detailed knowledge of spirochetal OMP biology will hopefully lead to the design of new vaccines and a better understanding of spirochetal pathogenesis.

Amino Acid Sequence↗
PubMed2004

Molecular evolution and mosaicism of leptospiral outer membrane proteins involves horizontal DNA transfer.

Leptospires belong to a genus of parasitic bacterial spirochetes that have adapted to a broad range of mammalian hosts. Mechanisms of leptospiral molecular evolution were explored by sequence analysis of four genes shared by 38 strains belonging to the core group of pathogenic Leptospira species: L. interrogans, L. kirschneri, L. noguchii, L. borgpetersenii, L. santarosai, and L. weilii. The 16S rRNA and lipL32 genes were highly conserved, and the lipL41 and ompL1 genes were significantly more variable. Synonymous substitutions are distributed throughout the ompL1 gene, whereas nonsynonymous substitutions are clustered in four variable regions encoding surface loops. While phylogenetic trees for the 16S, lipL32, and lipL41 genes were relatively stable, 8 of 38 (20%) ompL1 sequences had mosaic compositions consistent with horizontal transfer of DNA between related bacterial species. A novel Bayesian multiple change point model was used to identify the most likely sites of recombination and to determine the phylogenetic relatedness of the segments of the mosaic ompL1 genes. Segments of the mosaic ompL1 genes encoding two of the surface-exposed loops were likely acquired by horizontal transfer from a peregrine allele of unknown ancestry. Identification of the most likely sites of recombination with the Bayesian multiple change point model, an approach which has not previously been applied to prokaryotic gene sequence analysis, serves as a model for future studies of recombination in molecular evolution of genes.

Amino Acid Sequence↗
PubMed2003

Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily.

Proteins with bacterial immunoglobulin-like (Big) domains, such as the Yersinia pseudotuberculosis invasin and Escherichia coli intimin, are surface-expressed proteins that mediate host mammalian cell invasion or attachment. Here, we report the identification and characterization of a new family of Big domain proteins, referred to as Lig (leptospiral Ig-like) proteins, in pathogenic Leptospira. Screening of L. interrogans and L. kirschneri expression libraries with sera from leptospirosis patients identified 13 lambda phage clones that encode tandem repeats of the 90 amino acid Big domain. Two lig genes, designated ligA and ligB, and one pseudogene, ligC, were identified. The ligA and ligB genes encode amino-terminal lipoprotein signal peptides followed by 10 or 11 Big domain repeats and, in the case of ligB, a unique carboxy-terminal non-repeat domain. The organization of ligC is similar to that of ligB but contains mutations that disrupt the reading frame. The lig sequences are present in pathogenic but not saprophytic Leptospira species. LigA and LigB are expressed by a variety of virulent leptospiral strains. Loss of Lig protein and RNA transcript expression is correlated with the observed loss of virulence during culture attenuation of pathogenic strains. High-pressure freeze substitution followed by immunocytochemical electron microscopy confirmed that the Lig proteins were localized to the bacterial surface. Immunoblot studies with patient sera found that the Lig proteins are a major antigen recognized during the acute host infection. These observations demonstrate that the Lig proteins are a newly identified surface protein of pathogenic Leptospira, which by analogy to other bacterial immunoglobulin superfamily virulence factors, may play a role in host cell attachment and invasion during leptospiral pathogenesis.

Amino Acid Sequence↗
PubMed2003

LipL21 is a novel surface-exposed lipoprotein of pathogenic Leptospira species.

Leptospira is the etiologic agent of leptospirosis, a bacterial zoonosis distributed worldwide. Leptospiral lipopolysaccharide is a protective immunogen, but the extensive serological diversity of leptospires has inspired a search for conserved outer membrane proteins (OMPs) that may stimulate heterologous immunity. Previously, a global analysis of leptospiral OMPs (P. A. Cullen, S. J. Cordwell, D. M. Bulach, D. A. Haake, and B. Adler, Infect. Immun. 70:2311-2318, 2002) identified pL21, a novel 21-kDa protein that is the second most abundant constituent of the Leptospira interrogans serovar Lai outer membrane proteome. In this study, we identified the gene encoding pL21 and found it to encode a putative lipoprotein; accordingly, the protein was renamed LipL21. Southern hybridization analysis revealed the presence of lipL21 in all of the pathogenic species but in none of the saprophytic species examined. Alignment of the LipL21 sequence from six strains of Leptospira revealed 96 to 100% identity. When specific polyclonal antisera to recombinant LipL21 were used, LipL21 was isolated together with other known leptospiral OMPs by both Triton X-114 extraction and sucrose density gradient membrane fractionation. All nine strains of pathogenic leptospires investigated by Western blotting, whether culture attenuated or virulent, were found to express LipL21. In contrast, the expression of LipL21 or an antigenically related protein could not be detected in nonpathogenic L. biflexa. Infected hamster sera and two of eight human leptospirosis sera tested were found to react with recombinant LipL21. Native LipL21 was found to incorporate tritiated palmitic acid, consistent with the prediction of a lipoprotein signal peptidase cleavage site. Biotinylation of the leptospiral surface resulted in selective labeling of LipL21 and the previously known OMPs LipL32 and LipL41. These findings show that LipL21 is a surface-exposed, abundant outer membrane lipoprotein that is expressed during infection and conserved among pathogenic Leptospira species.

Animals↗
PubMed2002

Gastrointestinal microflora studies in late-onset autism.

Some cases of late-onset (regressive) autism may involve abnormal flora because oral vancomycin, which is poorly absorbed, may lead to significant improvement in these children. Fecal flora of children with regressive autism was compared with that of control children, and clostridial counts were higher. The number of clostridial species found in the stools of children with autism was greater than in the stools of control children. Children with autism had 9 species of Clostridium not found in controls, whereas controls yielded only 3 species not found in children with autism. In all, there were 25 different clostridial species found. In gastric and duodenal specimens, the most striking finding was total absence of non-spore-forming anaerobes and microaerophilic bacteria from control children and significant numbers of such bacteria from children with autism. These studies demonstrate significant alterations in the upper and lower intestinal flora of children with late-onset autism and may provide insights into the nature of this disorder.

Age of Onset↗