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Harry Smith

Publications and source records attributed to Harry Smith.

18 recordsLinked to original sources
PubMed2006

Lactate acquisition promotes successful colonization of the murine genital tract by Neisseria gonorrhoeae.

Previous studies on Neisseria gonorrhoeae have demonstrated that metabolism of lactate in the presence of glucose increases the growth rate of the bacterium and enhances its resistance to complement-mediated killing. Although these findings in vitro suggest that the acquisition of lactate promotes gonococcal colonization, the significance of this carbon source to the survival of the gonococcus in vivo remains unknown. To investigate the importance of lactate utilization during Neisseria gonorrhoeae genital tract infection, we identified the gene lctP, which encodes the gonococcal lactate permease. A mutant that lacks a functional copy of lctP was unable to take up exogenous lactate and did not grow in defined medium with lactate as the sole carbon source, in contrast to the wild-type and complemented strains; the mutant strain exhibited no growth defect in defined medium containing glucose. In defined medium containing physiological concentrations of lactate and glucose, the lctP mutant demonstrated reduced early growth and increased sensitivity to complement-mediated killing compared with the wild-type strain; the enhanced susceptibility to complement was associated with a reduction in lipopolysaccharide sialylation of the lctP mutant. The importance of lactate utilization during colonization was evaluated in the murine model of lower genital tract infection. The lctP mutant was significantly attenuated in its ability to colonize and survive in the genital tract, while the complemented mutant exhibited no defect for colonization. Lactate is a micronutrient in the genital tract that contributes to the survival of the gonococcus.

Animals↗
PubMed2006

Coordinated regulation of the Neisseria gonorrhoeae-truncated denitrification pathway by the nitric oxide-sensitive repressor, NsrR, and nitrite-insensitive NarQ-NarP.

Neisseria gonorrhoeae survives anaerobically by reducing nitrite to nitrous oxide catalyzed by the nitrite and nitric oxide reductases, AniA and NorB. P(aniA) is activated by FNR (regulator of fumarate and nitrate reduction), the two-component regulatory system NarQ-NarP, and induced by nitrite; P(norB) is induced by NO independently of FNR by an uncharacterized mechanism. We report the results of microarray analysis, bioinformatic analysis, and chromatin immunoprecipitation, which revealed that only five genes with readily identified NarP-binding sites are differentially expressed in narP(+) and narP strains. These include three genes implicated in the truncated gonococcal denitrification pathway: aniA, norB, and narQ. We also report that (i) nitrite induces aniA transcription in a narP mutant; (ii) nitrite induction involves indirect inactivation by nitric oxide of a gonococcal repressor, NsrR, identified from a multigenome bioinformatic study; (iii) in an nsrR mutant, aniA, norB, and dnrN (encoding a putative reactive nitrogen species response protein) were expressed constitutively in the absence of nitrite, suggesting that NsrR is the only NO-sensing transcription factor in N. gonorrhoeae; and (iv) NO rather than nitrite is the ligand to which NsrR responds. When expressed in Escherichia coli, gonococcal NarQ and chimaeras of E. coli and gonococcal NarQ are ligand-insensitive and constitutively active: a "locked-on" phenotype. We conclude that genes involved in the truncated denitrification pathway of N. gonorrhoeae are key components of the small NarQP regulon, that NarP indirectly regulates P(norB) by stimulating NO production by AniA, and that NsrR plays a critical role in enabling gonococci to evade NO generated as a host defense mechanism.

Antigens, Bacterial↗
PubMed2006

Phytochrome-mediated agravitropism in Arabidopsis hypocotyls requires GIL1 and confers a fitness advantage.

Plants use specialized photoreceptors to detect the amount, quality, periodicity and direction of light and to modulate their growth and development accordingly. These regulatory light signals often interact with other environmental cues. Exposure of etiolated Arabidopsis seedlings to red (R) or far-red (FR) light causes hypocotyls to grow in random orientations with respect to the gravitational vector, thus overcoming the signal from gravity to grow upwards. This light response, mediated by either phytochrome A or phytochrome B, represents a prime example of cross-talk between environmental signalling systems. Here, we report the isolation the mutant gil1 (for gravitropic in the light) in which hypocotyls continue to grow upwards after exposure of seedlings to R or FR light. The gil1 mutant displays no other phenotypic alterations in response to gravity or light. Cloning of GIL1 has identified a novel gene that is necessary for light-dependent randomization of hypocotyl growth orientation. Using gil1, we have demonstrated that phytochrome-mediated randomization of Arabidopsis hypocotyl orientation provides a fitness advantage to seedlings developing in patchy, low-light environments.

Amino Acid Sequence↗
PubMed2005

Mutational and biochemical analysis of cytochrome c', a nitric oxide-binding lipoprotein important for adaptation of Neisseria gonorrhoeae to oxygen-limited growth.

Neisseria gonorrhoeae is a prolific source of c-type cytochromes. Five of the constitutively expressed cytochromes are predicted, based on in silico analysis of the N. gonorrhoeae genome, to be components of the cytochrome bc1 complex, cytochrome c oxidase cbb3 or periplasmic cytochromes involved in electron transfer reactions typical of a bacterium with a microaerobic physiology. Cytochrome c peroxidase was previously shown to be a lipoprotein expressed only during oxygen-limited growth. The final c-type cytochrome, cytochrome c', similar to cytochrome c peroxidase, includes a lipobox required for targeting to the outer membrane. Maturation of cytochrome c' was partially inhibited by globomycin, an antibiotic that specifically inhibits signal peptidase II, resulting in the accumulation of the prolipoprotein in the cytoplasmic membrane. Disruption of the gonococcal cycP gene resulted in an extended lag phase during microaerobic growth in the presence but not in the absence of nitrite, suggesting that cytochrome c' protects the bacteria from NO generated by nitrite reduction during adaptation to oxygen-limited growth. The cytochrome c' gene was overexpressed in Escherichia coli and recombinant cytochrome c' was shown to be targeted to the outer membrane. Spectroscopic evidence is presented showing that gonococcal cytochrome c' is similar to previously characterized cytochrome c' proteins and that it binds NO in vitro. The demonstration that two of the seven gonococcal c-type cytochromes fulfil specialized functions and are outer membrane lipoproteins suggests that the localization of these lipoproteins close to the bacterial surface provides effective protection against external assaults from reactive oxygen and reactive nitrogen species.

Adaptation, Physiological↗
PubMed2005

Available carbon source influences the resistance of Neisseria meningitidis against complement.

Neisseria meningitidis is an important cause of septicaemia and meningitis. To cause disease, the bacterium must acquire essential nutrients for replication in the systemic circulation, while avoiding exclusion by host innate immunity. Here we show that the utilization of carbon sources by N. meningitidis determines its ability to withstand complement-mediated lysis, through the intimate relationship between metabolism and virulence in the bacterium. The gene encoding the lactate permease, lctP, was identified and disrupted. The lctP mutant had a reduced growth rate in cerebrospinal fluid compared with the wild type, and was attenuated during bloodstream infection through loss of resistance against complement-mediated killing. The link between lactate and complement was demonstrated by the restoration of virulence of the lctP mutant in complement (C3(-/-))-deficient animals. The underlying mechanism for attenuation is mediated through the sialic acid biosynthesis pathway, which is directly connected to central carbon metabolism. The findings highlight the intimate relationship between bacterial physiology and resistance to innate immune killing in the meningococcus.

Animals↗
PubMed2005

Influenza A virus-induced apoptosis is a multifactorial process: exploiting reverse genetics to elucidate the role of influenza A virus proteins in virus-induced apoptosis.

Three influenza viruses, A/Puerto Rico/8/34-A/England/939/69 clone 7a (H3N2), A/Fiji/15899/83 (H1N1), and A/Victoria/3/75 (H3N2), induce different levels of apoptosis in vitro at equal moi; Clone 7a > A/Victoria > A/Fiji. Previous studies have shown that several viral proteins from clone 7a and A/Fiji, including PB2, NA, NS1, M1, and M2, induce apoptosis when expressed individually fused to the herpes simplex virus tegument protein, VP22. However, this did not reflect viral protein-protein-RNA interactions known to occur within infected cells. To explore the role of viral proteins in apoptosis under infection conditions, recombinant viruses with single or triple gene exchanges were generated using A/Victoria or clone 7a as the background virus. Inserting the A/Fiji NS or PB2 gene into A/Victoria or clone 7a significantly reduced the level of apoptosis compared to the parent virus while clone 7a PA or NP genes increased apoptosis. Inserting A/Fiji NA or HA or clone 7a NS, M, NA, or HA genes individually into A/Victoria had no significant effect on apoptosis. Surprisingly, inserting the M, NA, and HA genes of A/Fiji together into clone 7a reduced apoptosis, whereas inserting clone 7a M, NA, and HA together into A/Fiji increased apoptosis. These results suggest that no single virus protein induces apoptosis and that the combination of genes required may be strain specific, highlighting the difficulty of predicting the virulence of new strains that arise in nature. No support for the view that apoptosis is essential for high virus yields was obtained as high virus yields were obtained with viruses that induced both high and low levels of apoptosis.

Amino Acid Sequence↗
PubMed2005

Identification of novel antigens that protect against systemic meningococcal infection.

Systemic infection with Neisseria meningitidis leads to a devastating, septicaemic illness with a high mortality, particularly in children. Currently, there are no vaccines to prevent disease caused by strains of N. meningitidis serogroup B, the commonest isolate in developed countries. Here, we describe the identification of vaccine candidates that protect mice against lethal challenge with this bacterium. A total 11 meningococcal proteins that are necessary for establishing systemic infection were expressed as recombinant antigens and assessed for their ability to protect animals against live bacterial challenge; the lactate permease (LctP) and ExbB, which is required for iron acquisition, elicited protective immunity. Both LctP and ExbB are expressed by a wide range of pathogenic isolates of N. meningitidis. Targeting bacterial factors required for pathogenesis may lead to new vaccines to protect individuals from this important human pathogen.

Bacterial Proteins↗
PubMed2005

Neisseria meningitidis lactate permease is required for nasopharyngeal colonization.

Neisseria meningitidis is a human specific pathogen that is part of the normal nasopharyngeal flora. Little is known about the metabolic constraints on survival of the meningococcus during colonization of the upper airways. Here we show that glucose and lactate, both carbon energy sources for meningococcal growth, are present in millimolar concentrations within nasopharyngeal tissue. We used a mutant defective for the uptake of lactate (C311DeltalctP) to investigate the contribution of this energy source during colonization. Explants of nasopharyngeal tissue were inoculated with the wild-type strain (C311) and C311DeltalctP; the mutant was recovered at significantly lower levels (P = 0.01) than C311 18 h later. This defect was not due to changes in the expression of adhesins or initial adhesion in C311DeltalctP to epithelial cells. Instead, lactate appears to be important energy source for the bacterium during colonization and is necessary for growth of the bacterium in nasopharyngeal tissue. Studies with other strains defective for the uptake of specific nutrients should provide valuable information about the environment in which N. meningitidis persists during carriage.

Adhesins, Bacterial↗
PubMed2003

A novel cytochrome c peroxidase from Neisseria gonorrhoeae: a lipoprotein from a Gram-negative bacterium.

A cytochrome c peroxidase (CCP) produced by Neisseria gonorrhoeae has been shown to have novel characteristics by investigating its location, expression and role in Neisseria gonorrhoeae and by expression in Escherichia coli. Analysis of the N-terminus of CCP indicated that it is a lipoprotein with a signal peptide for cleavage by signal peptidase II. Expression of the gonococcal CCP in E. coli revealed that it is first synthesized as a pro-apo-cytochrome that is translocated across the cytoplasmic membrane. The signal peptide is cleaved and haem is attached in the periplasm. The gonococcal CCP was associated with the membrane of both E. coli and N. gonorrhoeae. The expression of a MalE-CCP fusion protein has allowed characterization of CCP in vitro. Evidence is presented that CCP protects gonococci from hydrogen peroxide, presumably in the periplasmic compartment of the cell. The expression of CCP is dependent on the transcription factor FNR, but is repressed by nitrite, indicating that it could be most important in the stationary-phase response. These data support the hypothesis that the gonococcal lipoprotein CCP is anchored to the membrane in the periplasm, where it might be responsible for the reduction of hydrogen peroxide. Other putative CCP lipoproteins have been identified, representing a new subclass of bacterial CCP proteins.

Amino Acid Sequence↗
PubMed2003

Lactate carbon does not enter the sugars of lipopolysaccharide when gonococci are grown in a medium containing glucose and lactate: implications in vivo.

In media containing glucose, lactate stimulates the metabolism of gonococci at concentrations that simulate conditions in vivo. Nuclear magnetic resonance (NMR) spectroscopy of (13)C-labelled lipids obtained from gonococci grown in a synthetic medium with (13)C-labelled lactate and unlabelled glucose (culture A), (13)C-labelled glucose alone (culture B) or (13)C-labelled glucose and unlabelled lactate (culture C) showed lactate carbon was not present in glycerol/ethanolamine residues of lipids from culture A. This indicated that, in the presence of glucose, lactate gluconeogenesis is shut down. Hence, the stimulation of metabolism could result from the production of extra energy because lactate is used solely for conversion to acetyl-CoA, the precursor of fatty acid synthesis and the components of the tricarboxylic acid cycle. In this paper, additional evidence for lack of gluconeogenesis has been sought using a different approach. The carbohydrate moieties of lipopolysaccharide (LPS) have been examined for lactate carbon after gonococci were grown with lactate and glucose. Two methods were used: NMR spectroscopy of (13)C-labelled lipopolysaccharide purified from the three cultures described above showed that, in the presence of glucose, lactate carbon, in contrast to glucose carbon, was not in the carbohydrate moiety. Also, (14)C-labelled lactate was added to a culture containing unlabelled glucose and lactate (culture A) and [(14)C]glucose to cultures containing unlabelled glucose without unlabelled lactate (culture B) and with unlabelled lactate (culture C). When LPS samples purified from these cultures were subjected to hydrazinolysis, the ratio of the radioactivity of water-soluble products (carbohydrate moieties) to those of chloroform-soluble products (fatty acids) was much lower when [(14)C]lactate was used in culture A, than when [(14)C]glucose was used in cultures B and C. Thus, in the presence of glucose, lactate carbon, unlike glucose carbon, is incorporated predominantly into fatty acids of LPS, not into its carbohydrate moieties. There is no doubt, therefore, that gluconeogenesis is shut off when lactate is present with glucose and there is a consequent stimulation of metabolism. This probably occurs in vivo on mucous surfaces, where gonococci are surrounded by a mixture of glucose and lactate in the secretions.

Carbohydrates↗
PubMed2003

Improved classification of recent HIV-1 infection by employing a two-stage sensitive/less-sensitive test strategy.

Current serologic techniques for the classification of recent HIV-1 infection produce some misclassifications, and, together with the loss to follow-up of individuals, results in decreased enrollment of HIV-infected persons into appropriate intervention programs. We report on the development of a sensitive/less sensitive (S/LS) test strategy that includes a rapid assay to quickly identify persons most likely to have recent infection, followed by an enzyme immunoassay (EIA) with exquisite specificity. The Uni-Gold Recombigen HIV rapid assay (UG; Trinity Biotech, Dublin, Ireland) was procedurally-modified and calibrated as an LS test to differentiate recent (<133 days) from established HIV infections using 178 samples from persons with known dates of infection. This method correctly classified 83.0% of recent infections, but with a high misclassification rate of persons with established infection. By performing the rapid test followed by a modified S/LS EIA, the positive predictive value of the combined results for recent infections was increased to 100%. This two-stage testing algorithm can result in an increased efficiency for the enrollment of recent infection cases over a standard EIA S/LS method alone due to provisional enrollment during an initial testing visit, and because of an increased accuracy for identifying truly recent infections. We conclude that the rapid S/LS assay provides a tool for capturing recent infection cases quickly and is particularly valuable in resource-limited settings, and that the two-stage strategy provides a more accurate identification of persons with recent HIV infection.

Algorithms↗
PubMed2003

Influenza A virus-induced apoptosis in bronchiolar epithelial (NCI-H292) cells limits pro-inflammatory cytokine release.

Infection of cells with influenza A virus results in cell death with apoptotic characteristics. Apoptosis is regarded as a non-inflammatory process. However, during influenza an inflammatory response occurs in the airway epithelium. An examination of this apparent paradox was made using influenza A virus infection of human nasal and bronchiolar epithelial cells. Some cytokine genes (IL-18, CCL2 and CCL5) were expressed constitutively in nasal cells but no cytokine was released. In bronchiolar cells, IL-1 beta, IL-6 and CXCL8 expression was constitutive, whilst CCL2 and CCL5 expression was upregulated following influenza virus infection. IL-6, CXCL8 and CCL5 were released but IL-1 beta and CCL2 were not. In bronchiolar cells, cell death was inhibited by the caspase-8 (Z-IETD-fmk) and pan-caspase (Z-VAD-fmk) inhibitors and these inhibitors enhanced expression of CCL5 and increased the levels of the three secreted cytokines significantly. Thus, the amount of each cytokine released from bronchiolar cells is reduced during cell death, implying that the observed inflammatory response in influenza would be greater if cell death did not occur. Reduced cytokine release is also associated with fragmentation of the Golgi body, as the caspase inhibitors also rescued influenza A virus-induced fragmentation of the Golgi ribbon.

Amino Acid Chloromethyl Ketones↗
PubMed2003

Transcription activation at Escherichia coli FNR-dependent promoters by the gonococcal FNR protein: effects of a novel S18F substitution and comparisons with the corresponding substitution in E. coli FNR.

The Neisseria gonorrhoeae genome encodes a homologue of the Escherichia coli FNR protein (the fumarate and nitrate reductase regulator). Despite its similarity to E. coli FNR, the gonococcal FNR only partially complemented an E. coli fnr mutation. After error-prone PCR mutagenesis of the gonococcal fnr gene, we identified four mutant fnr derivatives carrying the same S18F substitution, and we showed that the mutant FNR could activate transcription from a range of class I and class II FNR-dependent promoters in E. coli. Prompted by the similarities between gonococcal and E. coli FNR, we made changes in gonococcal fnr that created substitutions that are equivalent to previously characterized substitutions in E. coli FNR. First, our experiments showed that cysteine, C116, in the gonococcal FNR, equivalent to C122 in E. coli FNR, is essential, presumably because, as in E. coli FNR, it binds to an iron-sulfur center. Second, the L22H and D148A substitutions in gonococcal FNR were made. These changes are equivalent to the L28H and D154A changes in E. coli FNR, which had been shown to increase FNR activity in the presence of oxygen. We show that the effects of these substitutions in gonococcal FNR are distinct from those of the S18F substitution. Similarly, substitutions in the putative activating regions of gonococcal FNR were made. We show that the activity of gonococcal FNR in E. coli can be increased by transplanting certain activating regions from E. coli FNR. The effects of these substitutions are additive to those due to S18F. From these data, we conclude that the effects of the S18F substitution in gonococcal FNR are distinct from the effects of the other substitutions. S18 is immediately adjacent to one of three N-terminal cysteine residues that coordinate the iron-sulfur center, and thus the S18F substitution is most likely to stabilize this center. Support for this came from complementary experiments in which we created the S24F substitution in E. coli FNR, which is equivalent to the S18F substitution in gonococcal FNR. Our results show that the S24F substitution changes the activity of E. coli FNR and that the changes are distinct from those due to previously characterized substitutions.

Amino Acid Sequence↗
PubMed2002

Discovery of the anthrax toxin: the beginning of studies of virulence determinants regulated in vivo.

Anthrax kills many animal species. It was used to prove Koch's Postulates in 1876. Soon after that the classical bacterial toxins were produced in vitro, but up to 1950, a lethal toxin had not been demonstrated in either anthrax bacilli or filtrates from laboratory cultures. The cause of death had been an enigma for seventy years. During the 1950's, a toxin was recognized by examining bacteria and their products obtained from guinea pigs dying of anthrax. The toxin was found in their plasma and shown to contain two components. It was then produced in vitro and a third component recognized. The work reawakened interest in bacterial toxins after a period of dormancy and showed that toxins could be multicomponent. It demonstrated that previously unknown determinants of bacterial pathogenicity could be revealed by examining organisms grown in vivo. It was the beginning of such studies, which took a long time to evolve, but have now expanded greatly with the development of many new methods for examining bacterial activities in vivo. This paper is a personal account of the early work.

Animals↗
PubMed2002

N-acylhomoserine lactones undergo lactonolysis in a pH-, temperature-, and acyl chain length-dependent manner during growth of Yersinia pseudotuberculosis and Pseudomonas aeruginosa.

In gram-negative bacterial pathogens, such as Pseudomonas aeruginosa and Yersinia pseudotuberculosis, cell-to-cell communication via the N-acylhomoserine lactone (AHL) signal molecules is involved in the cell population density-dependent control of genes associated with virulence. This phenomenon, termed quorum sensing, relies upon the accumulation of AHLs to a threshold concentration at which target structural genes are activated. By using biosensors capable of detecting a range of AHLs we observed that, in cultures of Y. pseudotuberculosis and P. aeruginosa, AHLs accumulate during the exponential phase but largely disappear during the stationary phase. When added to late-stationary-phase, cell-free culture supernatants of the respective pathogen, the major P. aeruginosa [N-butanoylhomoserine lactone (C4-HSL) and N-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL)] and Y. pseudotuberculosis [N-(3-oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) and N-hexanoylhomoserine lactone (C6-HSL)] AHLs were inactivated. Short-acyl-chain compounds (e.g., C4-HSL) were turned over more extensively than long-chain molecules (e.g., 3-oxo-C12-HSL). Little AHL inactivation occurred with cell extracts, and no evidence for inactivation by specific enzymes was apparent. This AHL turnover was discovered to be due to pH-dependent lactonolysis. By acidifying the growth media to pH 2.0, lactonolysis could be reversed. By using carbon-13 nuclear magnetic resonance spectroscopy, we found that the ring opening of homoserine lactone (HSL), N-propionyl HSL (C3-HSL), and C4-HSL increased as pH increased but diminished as the N-acyl chain was lengthened. At low pH levels, the lactone rings closed but not via a simple reversal of the ring opening reaction mechanism. Ring opening of C4-HSL, C6-HSL, 3-oxo-C6-HSL, and N-octanoylhomoserine lactone (C8-HSL), as determined by the reduction of pH in aqueous solutions with time, was also less rapid for AHLs with more electron-donating longer side chains. Raising the temperature from 22 to 37 degrees C increased the rate of ring opening. Taken together, these data show that (i) to be functional under physiological conditions in mammalian tissue fluids, AHLs require an N-acyl side chain of at least four carbons in length and (ii) that the longer the acyl side chain the more stable the AHL signal molecule.

Body Fluids↗
PubMed2002

Diagnosis of epizootic bovine abortion in Nevada and identification of the vector.

In the 43 years since the first description in California, epizootic bovine abortion (EBA) has been considered but not definitively diagnosed as a cause of late-term abortions on Nevada ranches. Examination of aborted full-term bovine fetuses obtained from Nevada ranches revealed gross abnormalities consistent with EBA (enlarged lymph nodes, petechial hemorrhages of the oral mucosa and conjunctiva, ascites, and splenohepatomegaly), and EBA was confirmed by histologic examination of fetal tissues. The histologic thymic changes were characteristic of EBA and included severe histocytic thymusitis with depletion of thymocytes, interlobular hemorrhage, and fibrinocellular exudation. The gross enlargement of lymph nodes was the result of cortical follicular hyperplasia and histiocytic lymphadenitis. In addition, widespread, predominately nonsuppurative histologic lesions typical of EBA were observed in most organs, including the brain, lung, heart, liver, and spleen. Furthermore, the presence of Ornithodorus coriaceus, the argasid tick vector of EBA, was established by tick collection using CO2 traps. The tick was identified on ranches and in geographic areas (northern and northwestern counties of Nevada) coincident with diagnosis of multiple cases of EBA. This study establishes the presence of EBA as a cause of late-term abortion in Nevada. Additionally, identification of the EBA tick vector, O. coriaceus, in the same areas as the abortions provides strong evidence that the disease is endemic.

Abortion, Veterinary↗