The risk of seizures associated with tramadol

Several substances including proteases and restrictocin have been suggested as candidates for virulence determinants in invasive pulmonary aspergillosis. However, the roles of such substances are not well understood. This study compared the in vitro suppressive effects of Aspergillus fumigatus culture filtrates (ACFs), on the functions of human polymorphonuclear leukocytes (PMNLs), the principal cells in the host defence against aspergillus hyphae, from a clinically isolated wild-type and isogenic mutant strains which lack production of elastolytic alkaline protease (Alp) and/or restrictocin. ACFs were obtained by culturing conidia of each strain in Medium- 199 at 37 degrees C for 5 days. ACFs of the wild-type significantly (p<0.01) suppressed chemotaxis, superoxide anion (O2-) release and PMNL-mediated hyphal damage, compared with the control (Medium-199). ACFs of the mutant strains that lack Alp or restrictocin significantly (p<0.01) suppressed chemotaxis and O2(-)-release, but did not suppress hyphal damage, compared with the control. The wild-type significantly (p<0.01) suppressed chemotaxis of PMNLs compared with the mutant strains lacking Alp or restrictocin, whereas there were no significant differences in suppression of O2(-)-release and hyphal damage by PMNLs. ACF of a mutant strain that lacks both Alp and restrictocin had much less activity, but significantly (p<0.01) suppressed chemotaxis of PMNLs compared with the control. In conclusion, alkaline protease and restrictocin may play roles in the suppressive effect of Aspergillus fumigatus culture filtrates on the functions of human polymorphonuclear leukocytes. Other antiphagocytic substances produced by Aspergillus fumigatus remain to be identified.     

Pharmacokinetic changes of M1, M2, M3 and M4 after intravenous administration of a new anthracycline, DA-125, to rats pretreated with phenobarbital, 3-methylcholanthrene, chloramphenicol, or SKF-525A

Aspergillus fumigatus is an important pathogen of the immunocompromised host causing pneumonia and invasive disseminated disease with high mortality. Previously, we identified a mutant strain (white, W) lacking conidial pigmentation and, in addition, the conidia showed a smooth surface morphology, whereas wild-type (WT) conidia are grey-green and have a typical ornamentation. W conidia appeared to be less protected against killing by the host defence, e.g., were more susceptible to oxidants in vitro and more efficiently damaged by human monocytes in vitro than WT conidia. When compared to the WT, the W mutant strain showed reduced virulence in a murine animal model. Genetic analysis suggested that the W mutant carried a single mutation which caused all of the observed phenotypes. Here. we report the construction of a genomic cosmid library of A. fumigatus and its use for complementation of the W mutant. Transformation of the W mutant was facilitated by co-transformation with plasmid pHELP1 carrying the autonomously replicating ama1 sequence of A. nidulans which also increased the transformation efficiency of A. fumigatus by a factor of 10. Using this cosmid library a putative polyketide synthase gene, designated pksP (polyketide synthase involved in pigment biosynthesis) was isolated. The pksP gene has a size of 6660 bp. pksP consists of five exons separated by short (47-73 bp) introns. Its deduced open reading frame is composed of 2146 amino acids. The pksP gene complemented both the white phenotype and the surface morphology of the W mutant conidia to wild type. Whereas W mutant conidia caused a strong reactive oxygen species (ROS) release by polymorphonuclear leukocytes, the ability of pksP-complemented W mutant conidia to stimulate ROS release was significantly reduced and comparable to that of WT conidia. In addition, the complemented strains showed restored virulence in a mouse model.     

The alkaline protease of Aspergillus fumigatus is not a virulence determinant in two murine models of invasive pulmonary aspergillosis

Little is known of the pathophysiology of invasive pulmonary aspergillosis (IPA), an opportunistic fungal infection usually caused by Aspergillus fumigatus. It has been suggested that the ability of the fungus to degrade elastin may aid its invasion and growth in lung tissue. We have described previously the construction of a strain of A. fumigatus in which the gene encoding an alkaline protease, AFAlp, had been disrupted (C.M. Tang, J. Cohen, and D.W. Holden, Mol. Microbiol. 6:1663-1671, 1992); this mutant is deficient in extracellular proteolytic and elastinolytic activity over a broad pH range. In this study, we compared the pathogenicity of this and another AFAlp disruptant with their isogenic, elastase-producing parental strains in two murine models of IPA. In both models, animals were inoculated via the respiratory tract. In the first model, the inoculum was delivered as airborne conidia and animals developed signs of respiratory distress within 2 to 4 days. In the second model, conidia were administered intranasally as a suspension and the disease developed over a 2-week period. No difference was observed between the wild-type and AFAlp disruptants in terms of mortality, and elastin breakdown was detected in lung tissue from animals inoculated with all four strains. We conclude that AFAlp is not a virulence determinant in these models of IPA.     

Virulence, immunogenicity and reactivation of bovine herpesvirus 1 mutants with a deletion in the gC, gG, gI, gE, or in both the gI and gE gene

Within the framework of developing a marker vaccine against bovine herpesvirus 1 (BHV1), several mutants with deletions in non-essential glycoprotein genes were constructed. Glycoprotein gC, gG, gI and gE single deletion mutants, a gI/gE double deletion mutant and a gE frame-shift mutant were made. The virulence and immunogenicity of these mutants were evaluated in specific-pathogen-free calves. Except for the gC deletion mutant, all mutants were significantly less virulent than the parental wild-type (wt) BHV1 strain Lam. The virulence of the gI and the gI-/gE- mutants was almost completely reduced. Upon challenge infection, the calves of the control group became severely ill, whereas all other calves remained healthy. The reduction of the virus shedding after challenge infection was related to the virulence of the strain of primary inoculation. Virus shedding was almost completely reduced in calves first inoculated with Lam-wt or with gC- and the least reduced in calves inoculated with gI- or gI-/gE-. Six weeks after challenge, all calves were treated with dexamethasone to study whether mutant or challenge virus or both could be reactivated. The gC- and the gG- mutants were reactivated, whereas none of the other mutants were reisolated. Reactivation of challenge virus was reduced in all calves inoculated with mutant viruses. The gC deletion mutant was too virulent and the gI and the gI/gE deletion mutants were the least immunogenic, but based on residual virulence and immunogenicity, both the gG and the gE deletion mutants are candidates for incorporation in live BHV1 vaccines. However, it also depends on the kinetics of the anti-gG and anti-gE antibody response after wild-type virus infection, whether these deletion mutants are really suitable to be incorporated in a marker vaccine.     

Identification of tandem genes involved in lipooligosaccharide expression by Haemophilus ducreyi

A transposon insertion mutant of Haemophilus ducreyi 35000 possessing a truncated lipooligosaccharide (LOS) failed to bind the LOS-specific monoclonal antibody 3E6 (M. K. Stevens, L. D. Cope, J. D. Radolf, and E. J. Hansen, Infect. Immun. 63:2976-2982, 1995). This transposon was found to have inserted into the first of two tandem genes and also caused a deletion of chromosomal DNA upstream of this gene. These two genes, designated lbgA and lbgB, encoded predicted proteins with molecular masses of 25,788 and 40,236 Da which showed homology with proteins which function in lipopolysaccharide biosynthetic in other gram-negative bacteria. The tandem arrangement of the lbgA and lbgB genes was found to be conserved among H. ducreyi strains. Isogenic LOS mutants, constructed by the insertion of a cat cartridge into either the lbgA or the lbgB gene, expressed an LOS phenotype indistinguishable from that of the original transposon-derived LOS mutant. The wild-type LOS phenotype could be restored by complementation with the appropriate wild-type allele. These two LOS mutants proved to be as virulent as the wild-type parent strain in an animal model. A double mutant with a deletion of the lbgA and lbgB genes yielded equivocal results when its virulence was tested in an animal model.     

Yersinia-induced apoptosis in vivo aids in the establishment of a systemic infection of mice

Pathogenic Yersinia cause a systemic infection in mice that is dependent on the presence of a large plasmid encoding a number of secreted virulence proteins called Yops. We previously demonstrated that a plasmid-encoded Yop, YopJ, was essential for inducing apoptosis in cultured macrophages. Here we report that YopJ is a virulence factor in mice and is important for the establishment of a systemic infection. The oral LD50 for a yopJ mutant Yersinia pseudotuberculosis increases 64-fold compared with wild-type. Although the yopJ mutant strain is able to reach the spleen of infected mice, the mutant strain seldom reaches the same high bacterial load that is seen with wild-type Yersinia strain and begins to be cleared from infected spleens on day 4 after infection. Furthermore, when in competition with wild-type Yersinia in a mixed infection, the yopJ mutant strain is deficient for spread from the Peyer's patches to other lymphoid tissue. We also show that wild-type Yersinia induces apoptosis in vivo of Mac-1(+) cells from infected mesenteric lymph nodes or spleens, as measured by quantitative flow cytometry of TUNEL (Tdt-mediated dUTP-biotin nick-end labeling)-positive cells. The levels of Mac-1(+), TUNEL+ cells from tissue infected with the yopJ mutant strain were equivalent to the levels detected in cells from uninfected tissue. YopJ is necessary for the suppression of TNF-alpha production seen in macrophages infected with wild-type Yersinia, based on previous in vitro studies (Palmer, L.E., S. Hobbie, J.E. Galan, and J.B. Bliska. 1998. Mol. Microbiol. 27:953-965). We conclude here that YopJ plays a role in the establishment of a systemic infection by inducing apoptosis and that this is consistent with the ability to suppress the production of the proinflammatory cytokine tumor necrosis factor alpha.     

Role of the negative charges in the cytosolic domain of TOM22 in the import of precursor proteins into mitochondria

TOM22 is an essential mitochondrial outer membrane protein required for the import of precursor proteins into the organelles. The amino-terminal 84 amino acids of TOM22 extend into the cytosol and include 19 negatively and 6 positively charged residues. This region of the protein is thought to interact with positively charged presequences on mitochondrial preproteins, presumably via electrostatic interactions. We constructed a series of mutant derivatives of TOM22 in which 2 to 15 of the negatively charged residues in the cytosolic domain were changed to their corresponding amido forms. The mutant constructs were transformed into a sheltered Neurospora crassa heterokaryon bearing a tom22::hygromycin R disruption in one nucleus. All constructs restored viability to the disruption-carrying nucleus and gave rise to homokaryotic strains containing mutant tom22 alleles. Isolated mitochondria from three representative mutant strains, including the mutant carrying 15 neutralized residues (strain 861), imported precursor proteins at efficiencies comparable to those for wild-type organelles. Precursor binding studies with mitochondrial outer membrane vesicles from several of the mutant strains, including strain 861, revealed only slight differences from binding to wild-type vesicles. Deletion mutants lacking portions of the negatively charged region of TOM22 can also restore viability to the disruption-containing nucleus, but mutants lacking the entire region cannot. Taken together, these data suggest that an abundance of negative charges in the cytosolic domain of TOM22 is not essential for the binding or import of mitochondrial precursor proteins; however, other features in the domain are required.     

Salmonella typhimurium cob mutants are not hyper-virulent

It was previously reported that Salmonella typhimurium LT2 cob mutants defective in the biosynthesis of vitamin B12 (cobalamin) are more virulent than the wild type in mice. Here we show that the strains used previously are non-isogenic and that the proposed increase in virulence of the cob mutant strain results from an uncharacterized mutation in the "wild type" which attenuates virulence, most likely by decreasing expression of the spv genes on the virulence plasmid. As a result the cob mutant will appear as hyper-virulent. Examination of the virulence of reconstructed wild-type and cob mutant strains showed that their growth rates were similar in mice, and we conclude that vitamin B12 does not affect the virulence of S. typhimurium LT2.     

Reevaluation of the virulence phenotype of the inv yadA double mutants of Yersinia pseudotuberculosis

Yersinia pseudotuberculosis and Yersinia enterocolitica are closely related human pathogens causing gastroenteritis. Invasin and YadA are two of the most extensively studied virulence factors of the Yersinia genus. Invasin is the primary invasion factor encoded by the inv gene on the chromosome and is required for the penetration of the epithelial cells. YadA is encoded by the yadA gene on the 70-kb virulence plasmid and has multiple functions. Previous studies indicate that an inv yadA double mutant of Y. enterocolitica is avirulent while an inv yadA mutant of Y. pseudotuberculosis is hypervirulent. In this study, we investigated this unexpected difference. New constructs of the inv yadA mutants of Y. pseudotuberculosis were made and tested in mice. These new constructs were not hypervirulent; rather, they maintained the same virulence as the wild-type strain. Further examination of the inv mutant used for the previous study revealed that it carries an aberrant inv phenotype and has an altered outer membrane profile and an altered colony morphology. Therefore, the mutants used previously were not isogenic to the parental wild-type strain, which may in part account for the difference in the results obtained.     

An exo-poly-alpha-D-galacturonosidase, PehB, is required for wild-type virulence of Ralstonia solanacearum

Ralstonia solanacearum, which causes bacterial wilt disease of many plant species, produces several extracellular plant cell wall-degrading enzymes that are suspected virulence factors. These include a previously described endopolygalacturonase (PG), PehA, and two exo-PGs. A gene encoding one of the exo-PGs, pehB, was cloned from R. solanacearum K60. The DNA fragment specifying PehB contained a 2,103-bp open reading frame that encodes a protein of 74.2 kDa with a typical N-terminal signal sequence. The cloned pehB gene product cleaves polygalacturonic acid into digalacturonic acid units. The amino acid sequence of pehB resembles that of pehX, an exo-PG gene from Erwinia chrysanthemi, with 47.2% identity at the amino acid level. PehB also has limited similarity to plant exo-PGs from Zea mays and Arabidopsis thaliana. The chromosomal pehB genes in R. solanacearum wild-type strain K60 and in an endo-PG PehA- strain were replaced with an insertionally inactivated copy of pehB. The resulting mutants were deficient in the production of PehB and of both PehA and PehB, respectively. The pehB mutant was significantly less virulent than the wild-type strain in eggplant virulence assays using a soil inoculation method. However, the pehA mutant was even less virulent, and the pehA pehB double mutant was the least virulent of all. These results suggest that PehB is required for a wild-type level of virulence in R. solanacearum although its individual role in wilt disease development may be minor. Together with endo-PG PehA, however, PehB contributes substantially to the virulence of R. solanacearum.     

Inactivation of DsbA, but not DsbC and DsbD, affects the intracellular survival and virulence of Shigella flexneri

In this study, three mutants, dsbA::kan, dsbC-kan, and dsbD-kan, of Shigella flexneri serotype 5 were constructed and characterized to investigate the role of the periplasmic thiol:disulfide oxidoreductases in pathogenicity. In gentamicin protection assays and the Serény test, the dsbA mutant showed reduced virulence while the dsbC and dsbD mutants were similar to the wild type. That inactivation of dsbA was responsible for the reduced virulence was verified by complementation with the cloned wild-type gene in in vitro and in vivo assays. Despite the changed virulence behavior, the dsbA mutant could penetrate HeLa cells 15 min postinfection, consistent with the fact that it actively secretes Ipa proteins upon Congo red induction. Furthermore, the dsbA mutant was able to produce actin comets and protrusions, indicating its capacity for intra- and intercellular spread. However, a kinetic analysis of intracellular growth showed that the dsbA mutant barely grew in HeLa cells during a 4-h infection whereas the wild type had a doubling time of 41 min. Electron microscopy analysis revealed that dsbA mutant bacteria were trapped in protrusion-derived vacuoles surrounded by double membranes, resembling an icsB mutant reported previously. Moreover, the trapped bacteria appeared to be lysed simultaneously with the double membranes, resulting in characteristic empty vacuoles in the host cell cytosol. Thus, the attenuation mechanism for dsbA mutant appears to be more complicated than was previously suggested.     

Inactivation of glycoprotein gE and thymidine kinase or the US3-encoded protein kinase synergistically decreases in vivo replication of pseudorabies virus and the induction of protective immunity

To evaluate the contribution of glycoprotein E (gE), thymidine kinase (TK), and the US3-encoded protein kinase (PK) in the induction of protective immunity to pseudorabies virus (PRV), we intranasally inoculated pigs, the natural host of this virus, with mutant PRV strains in which the genes encoding these proteins were inactivated. Both single and double mutants were constructed. Of these proteins, gE has previously been demonstrated to induce antibodies (in mice and pigs), which require complement to neutralize the virus, and helper T cell responses (in mice). PK and TK have thus far not been reported to induce B or T cell responses. All mutants had a strongly reduced virulence for pigs in comparison with wild-type (wt) PRV. After primary infection, most virus was excreted by wt PRV-inoculated animals. Animals inoculated with gE-PK- and gE-TK- double mutants excreted less virus than animals inoculated with gE-, PK-, and TK- single mutants. After challenge infection with the virulent PRV strain NIA-3, no virus was excreted by wt PRV- and PK- mutant-immunized animals, indicating complete protective immunity. Only one of seven gE- and two of seven TK- mutant-immunized animals excreted virus after the challenge inoculation. In contrast, most animals immunized with the gE-PK- or gE-TK- double mutants excreted virus after the challenge inoculation. Daily mean virus excretion after challenge infection was inversely correlated with daily mean virus excretion after primary infection. In most animals, lack of virus excretion was associated with lack of secondary antibody responses, probably attributable to inadequate stimulation of memory B cells as a consequence of early elimination of viral antigen. Thus, inactivation of gE, TK, and PK all affected the immunogenicity of PRV and the effect of gE and TK and gE and PK inactivation appeared synergistic. We found no simple correlation between in vitro growth properties of the mutants and their immunogenic capacity. Strains lacking PK reached lower end titers in vitro than the other mutants. The most likely explanation for the lower protective capacity of some of the mutants appears their reduced replicative capacity in some cells or tissues in vivo, rather than a loss of particular epitopes.     

Structural roles of the spore coat proteins in Dictyostelium discoideum

The integrity of spores formed by mutant strains of Dictyostelium discoideum lacking the major spore coat proteins, SP96, SP70, or SP60, was compared to that of wild-type strains. Single, double, and triple knock-out strains developed normally and produced spores which were indistinguishable from wild-type spores by light or electron microscopy. However, the mutant strains were susceptable to staining with the lectin, ricin A, which recognizes a galactose-rich polysaccharide that is normally hidden by overlying spore coat proteins. The intensity of staining with fluorescently labeled ricinA increased as the spore coat proteins were incrementally lost. While these results indicate that the major outer spore coat proteins are not essential for the construction of a multi-layered spore coat in Dictyostelium, they show that the spores are more porous which might make them at risk to predators before germination.     

A K319N/E325Q double mutant of the lactose permease cotransports H+ with lactose. Implications for a proposed mechanism of H+/lactose symport

In this study, we have examined the transport characteristics of the wild-type lactose permease, single mutants in which Lys-319 was changed to asparagine or alanine or Glu-325 was changed to glutamine or alanine, and the corresponding double mutant strains. The wild-type and Asn-319 mutant showed high levels of lactose uptake, with Km values of 0.42 and 1.30 mM and Vmax values of 102.6 and 48.3 nmol of lactose/min/mg of protein, respectively. The Asn-319/Gln-325 strain had a normal Km of 0.36 mM and a moderate Vmax of 18.5 nmol of lactose/min/mg of protein. By comparison, the single E325Q strain had a normal Km of 0.27 mM but a very defective Vmax of 1.3 nmol of lactose/min/mg of protein. A similar trend was observed among the alanine substitutions at these positions, although the Vmax values were lower for the Ala-319 mutations. When comparing the Vmax values between the single position 325 mutants with those of the double mutants, these results indicate that neutral 319 mutations substantially alleviate a defect in Vmax caused by neutral 325 mutations. With regard to H+/lactose coupling, the wild-type permease is normally coupled and can transport lactose against a gradient. The position 325 single mutants showed no evidence of H+ transport with lactose or thiodigalactoside (TDG) and were unable to facilitate uphill lactose transport. The single Asn-319 mutant and double Asn-319/Gln-325 mutant were able to transport H+ upon the addition of lactose or TDG. In addition, both of these strains catalyzed a sugar-dependent H+ leak that inhibited cell growth in the presence of TDG. These two strains were also defective in uphill transport, which may be related to their sugar-dependent leak pathway. Based on these and other results in the literature, a model is presented that describes how the interactions among several ionizable residues within the lactose permease act in a concerted manner to control H+/lactose coupling. In this model, Lys-319 and Glu-325 play a central role in governing the ability of the lactose permease to couple the transport of H+ and lactose.     

The role of the proton-pumping and alternative respiratory chain NADH:ubiquinone oxidoreductases in overflow catabolism of Aspergillus niger

Mitochondria of fungi contain two respiratory chain enzymes concerned with the oxidation of matrix NADH. These are the proton-pumping NADH:ubiquinone oxidoreductase, also called complex I, which has a high affinity for NADH, and a non-proton-pumping NADH:ubiquinone oxidoreductase, called alternative NADH dehydrogenase, which has a low affinity for NADH. The role of these two enzymes in normal and overflow catabolism has been studied in Aspergillus niger. Three strains were investigated, the wild-type 732, the mutant nuo51 that was generated from the wild-type by disrupting the gene of the (51-kDa) NADH-binding subunit of complex I and the citric acid over-producing strain B60 that looses complex I concomitantly with the onset of the over-production. Under standard growth conditions, respiratory energy transduction in the mutant nuo51 was decreased by 40% compared to the parental wild-type and the strain B60. Respiratory electron transfer in the mutant nuo51, however, meets standard catabolic requirements. The intracellular levels of citric acid cycle intermediates in the mutant nuo51 were the same as in the other two strains. Under growth conditions which lead to uncontrolled catabolic flux through glycolysis, a dramatic catabolic overflow occurred in the mutant nuo51. Intracellular levels of citric acid cycle intermediates increased to 20-fold normal levels. The strain B60, likewise lacking complex I under these conditions, excretes large amounts of citrate to moderate the intracellular catabolic overflow.     

Construction and characterization of avian Escherichia coli cya crp mutants

We constructed delta cya delta crp mutants of two avian septicemic Escherichia coli strains and evaluated their attenuation in virulence. The P1 phage was used to transfer cya::Tn10 from an E. coli K-12 strain into virulent avian O78 and O2 E. coli isolates. Tetracycline-resistant transductants were plated on Bochner-Maloy Medium, and tetracycline-sensitive colonies were selected, then tested by polymerase chain reaction to confirm that they had deletions of the cya gene. Deletions of crp were created by the same technique in isolates with deletions in cya. The delta cya and delta cya delta crp derivatives had slower growth rates, smaller colonies, and impaired fermentation of carbohydrates compared with their wild parents, and they did not revert. Attenuation of the mutant strains was evaluated by subcutaneous (s.c.) inoculation of day-old chicks and by intratracheal (i.t.) inoculation of 9-day-old chicks previously inoculated intranasally with infectious bronchitis virus. For the wild O78 strain and its delta cya and delta cya delta crp derivatives, the percentages of chicks that died within 6 days of s.c. injection of approximately 5 x 10(7) organisms were 100, 60, and 0, respectively. The corresponding percentages for wild-type O2 and its delta cya and delta cya delta crp mutants were 100, 70, and 20 at a dose of approximately 2 x 10(5) organisms. Following i.t. inoculation, group scores based on pathologic and bacteriologic findings were 51%, 15%, and 9% for wild, delta cya, and delta crp O78 strains (inoculum approximately 2 x 10(7) organisms) and 98%, 31%, and 11%, respectively, for the corresponding O2 strains (inoculum approximately 4 x 10(6) organisms). This study demonstrated reduced virulence and stability of the double mutant, which may useful as a live attenuated vaccine against poultry colibacillosis.     

Leishmania mexicana cysteine proteinase-deficient mutants have attenuated virulence for mice and potentiate a Th1 response

Leishmania mexicana mutants lacking cysteine proteinase genes cpa (delta cpa), cpb (delta cpb), or both cpa and cpb (delta cpa/cpb) have been generated by targeted gene disruption. Delta cpa mutants produce a disease phenotype in BALB/c mice close to that of wild-type L. mexicana, but delta cpb mutants are much less infective, producing very slowly growing small lesions, and delta cpa/cpb double mutants do not induce lesion growth. Immunologic analysis of Ab isotype during infection and splenocyte IFN-gamma, IL-2, and IL-4 production following stimulation with Leishmania Ag or Con A indicates that there was a significant shift from a predominantly Th2-associated immune response in mice infected with wild-type L. mexicana to a Th1-associated response in mice inoculated with delta cpb or delta cpa/cpb. Significantly, delta cpa altered the balance of the immunologic response to a lesser extent than did the other mutants. Similar disease outcomes and switches in the Th1/Th2 balance were also observed when other L. mexicana-susceptible mouse strains were infected with the mutants. BALB/c and C57BL/6 mice vaccinated with delta cpa/cpb and CBA/Ca mice vaccinated with delta cpb or delta cpa/cpb were subsequently more resistant, to varying degrees, than were untreated mice to infection with wild-type parasites, as measured by development of lesions and parasite burden. These data implicate leishmanial cysteine proteinases not only as parasite virulence factors but also in modulation of the immune response and provide strong encouragement that cysteine proteinase-deficient L. mexicana mutants are candidate attenuated live vaccines.     

Factors associated with PTSD symptoms following treatment for breast cancer: test of the Andersen model

Glomerella graminicola transformants were generated by insertional plasmid mutagenesis. Five transformants with developmental mutant phenotypes that segregated in crosses as single-gene mutations were selected. In four transformants, the mutant phenotype cosegregated with the inserted plasmid DNA. At least three of the mutants result from gene disruption, as demonstrated by recovery of the mutant phenotypes after transformation of wild type with "rescued" plasmid DNA. Whereas the wild type produces uninucleate, salmon-colored conidia, the tagged mutant M26 has white conidia. After exposure to either UV light or singlet oxygen, the percentage germination of M26 conidia is reduced compared to that of the wild-type conidia, indicating that the spore pigment confers protection from UV light and singlet oxygen. The tagged mutant T30 has weakened walls; falcate conidia rupture and hyphae have swollen regions unless the medium is amended with an osmoticum. The tagged mutant T29 has falcate conidia with one to four nuclei; wild-type falcate conidia are uninucleate. Two other mutants, one which grows slowly and one having conidia with increased curvature, are also described.     

Evolution of virulence: a unified framework for coinfection and superinfection

Models of the evolution of parasite virulence have focused on computing the evolutionarily stable level of virulence favored by tradeoffs within a host and by competition for hosts, and deriving conditions under which strains with different virulence levels can coexist. The results depend on the type of interaction between disease strains, such as single infection (immunity of infected individuals to other strains), coinfection (simultaneous infection by two strains), and superinfection (instantaneous takeover of host by the more virulent strain). We present a coinfection model with two strains and derive the superinfection model as the limit where individuals are rapidly removed from the doubly-infectious class. When derived in this way, the superinfection model includes not only the takeover of hosts infected by the less virulent strain, but new terms which take into account the possibility of increased mortality of doubly-infected individuals. Coinfection tends to favor higher virulence and support more coexistence than the single infection model, but the detailed results depend sensitively on two factors: (1) whether and how the model is near the superinfection limit, and (2) the shape of the coinfection function (the function describing the rate at which a more virulent strain can infect a host). If the superinfection limit arises due to rapid mortality of doubly-infected hosts, there is a region of uninvadable virulence levels rather than coexistence. When the coinfection function is discontinuous, as in many previous models, neither the coinfection model nor the superinfection limit can support an evolutionarily stable virulence level. Piecewise differentiable and differentiable coinfection functions produce qualitatively different results, and we propose that these more general cases should be used to study evolution of virulence when other mechanisms like space, population dynamics, and stochasticity interact.     

Establishing equivalence of two treatments and sample size requirements in matched-pairs design

To investigate the role of putative virulence factors of Streptococcus pyogenes (group A streptococcus; GAS) in causing disease, we introduced specific mutations in GAS strain B514, a natural mouse pathogen, and tested the mutant strains in two models of infection. To study late stages of disease, we used our previously described mouse model (C3HeB/FeJ mice) in which pneumonia and systemic spread of the streptococcus follow intratracheal inoculation. To study the early stages of disease, we report here a model of long-term (at least 21 days) throat colonization following intranasal inoculation of C57BL/10SnJ mice. When the three emm family genes of GAS strain B514-Sm were deleted, the mutant showed no significant difference from the wild type in induction of long-term throat colonization or pneumonia. We inactivated the scpA gene, which encodes a complement C5a peptidase, by insertion of a nonreplicative plasmid and found no significant difference from the wild type in the incidence of throat colonization. However, there was a small but statistically significant decrease in the incidence of pneumonia caused by the scpA mutant. Finally, we demonstrated a very important effect of the hyaluronic acid capsule in both models. Following intranasal inoculation of mice with a mutant in which a nonreplicative plasmid was inserted into the hasA gene, which encodes hyaluronate synthase, we found that all bacteria recovered from the throats of the mice were encapsulated revertants. Following intratracheal inoculation with the hasA mutant, the incidence of pneumonia within 72 h was significantly reduced from that of the control strain (P = 0.006). These results indicate that the hyaluronic acid capsule of S. pyogenes B514 confers an important selective advantage for survival of the bacteria in the upper respiratory tract and is also an important determinant in induction of pneumonia in our model system.