bluuebunny/crossref_metadata_2025_split huggingface dataset:
AgtA, the Dicarboxylic Amino Acid Transporter of
Aspergillus nidulans
, Is Concertedly Down-Regulated by Exquisite Sensitivity to Nitrogen Metabolite Repression and Ammonium-Elicited Endocytosis
Authors: Apostolaki Angeliki , Erpapazoglou Zoi , Harispe Laura , Billini Maria , Kafasla Panagiota , Kizis Dimosthenis , Peñalva Miguel Angel , Scazzocchio Claudio , Sophianopoulou Vicky 
Published: January 2009

ABSTRACT

We identified
agtA
, a gene that encodes the specific dicarboxylic amino acid transporter of
Aspergillus nidulans
. The deletion of the gene resulted in loss of utilization of aspartate as a nitrogen source and of aspartate uptake, while not completely abolishing glutamate utilization. Kinetic constants showed that AgtA is a high-affinity dicarboxylic amino acid transporter and are in agreement with those determined for a cognate transporter activity identified previously. The gene is extremely sensitive to nitrogen metabolite repression, depends on AreA for its expression, and is seemingly independent from specific induction. We showed that the localization of AgtA in the plasma membrane necessitates the ShrA protein and that an active process elicited by ammonium results in internalization and targeting of AgtA to the vacuole, followed by degradation. Thus, nitrogen metabolite repression and ammonium-promoted vacuolar degradation act in concert to downregulate dicarboxylic amino acid transport activity.
Towards New Antifolates Targeting Eukaryotic Opportunistic Infections
Authors: Liu Jieying , Bolstad David B. , Bolstad Erin S. D. , Wright Dennis L. , Anderson Amy C. 
Published: January 2009

ABSTRACT

Trimethoprim, an antifolate commonly prescribed in combination with sulfamethoxazole, potently inhibits several prokaryotic species of dihydrofolate reductase (DHFR). However, several eukaryotic pathogenic organisms are resistant to trimethoprim, preventing its effective use as a therapeutic for those infections. We have been building a program to reengineer trimethoprim to more potently and selectively inhibit eukaryotic species of DHFR as a viable strategy for new drug discovery targeting several opportunistic pathogens. We have developed a series of compounds that exhibit potent and selective inhibition of DHFR from the parasitic protozoa
Cryptosporidium
and
Toxoplasma
as well as the fungus
Candida glabrata
. A comparison of the structures of DHFR from the fungal species
Candida glabrata
and
Pneumocystis
suggests that the compounds may also potently inhibit
Pneumocystis
DHFR.
Role of CpSUB1, a Subtilisin-Like Protease, in
Cryptosporidium parvum
Infection In Vitro
Authors: Wanyiri Jane W. , Techasintana Patsharaporn , O'Connor Roberta M. , Blackman Michael J. , Kim Kami , Ward Honorine D. 
Published: January 2009

ABSTRACT

The apicomplexan parasite
Cryptosporidium
is a significant cause of diarrheal disease worldwide. Previously, we reported that a
Cryptosporidium parvum
subtilisin-like serine protease activity with furin-type specificity cleaves gp40/15, a glycoprotein that is proteolytically processed into gp40 and gp15, which are implicated in mediating infection of host cells. Neither the enzyme(s) responsible for the protease activity in
C. parvum
lysates nor those that process gp40/15 are known. There are no furin or other proprotein convertase genes in the
C. parvum
genome. However, a gene encoding CpSUB1, a subtilisin-like serine protease, is present. In this study, we cloned the CpSUB1 genomic sequence and expressed and purified the recombinant prodomain. Reverse transcriptase PCR analysis of RNA from
C. parvum
-infected HCT-8 cells revealed that CpSUB1 is expressed throughout infection in vitro. In immunoblots, antiserum to the recombinant CpSUB1 prodomain revealed two major bands, of ∼64 kDa and ∼48 kDa, for
C. parvum
lysates and proteins “shed” during excystation. In immunofluorescence assays, the antiserum reacted with the apical region of sporozoites and merozoites. The recombinant prodomain inhibited protease activity and processing of recombinant gp40/15 by
C. parvum
lysates but not by furin. Since prodomains are often selective inhibitors of their cognate enzymes, these results suggest that CpSUB1 may be a likely candidate for the protease activity in
C. parvum
and for processing of gp40/15. Importantly, the recombinant prodomain inhibited
C. parvum
infection of HCT-8 cells. These studies indicate that CpSUB1 plays a significant role in infection of host cells by the parasite and suggest that this enzyme may serve as a target for intervention.
Dual Sorting of the
Saccharomyces cerevisiae
Vacuolar Protein Sna4p
Authors: Pokrzywa W. , Guerriat B. , Dodzian J. , Morsomme P. 
Published: January 2009

ABSTRACT
Sna4p, a vacuolar membrane protein, belongs to a small family of proteins conserved in plants and fungi. It is transported to the vacuolar membrane via the alkaline phosphatase (ALP) pathway, which bypasses the multivesicular bodies (MVBs). Here, we show that transfer of Sna4p by the ALP route involves the AP-3 adaptor protein complex, which binds to an acidic dileucine sorting signal in the cytoplasmic region of Sna4p. In addition, Sna4p can use the MVB pathway by using a PPPY motif, which is involved in the interaction with ubiquitin ligase Rsp5p. Deletion or mutation of the Sna4p PPPY motif or a low level of Rsp5p inhibits the entrance of Sna4p into MVBs. Sna4p is polyubiquitylated on its only lysine, and Sna4p lacking this lysine shows defective MVB sorting. These data indicate that Sna4p has two functional motifs, one for interaction with the AP-3 complex, followed by entry into the ALP pathway, and one for binding Rsp5p, which directs the protein to the MVB pathway. The presence of these two motifs allows Sna4p to localize to both the vacuolar membrane and the lumen.
Killed but Metabolically Active
Bacillus anthracis
Vaccines Induce Broad and Protective Immunity against Anthrax
Authors: Skoble Justin , Beaber John W. , Gao Yi , Lovchik Julie A. , Sower Laurie E. , Liu Weiqun , Luckett William , Peterson Johnny W. , Calendar Richard , Portnoy Daniel A. , Lyons C. Rick , Dubensky Thomas W. 
Published: January 2009

ABSTRACT

Bacillus anthracis
is the causative agent of anthrax. We have developed a novel whole-bacterial-cell anthrax vaccine utilizing
B. anthracis
that is killed but metabolically active (KBMA). Vaccine strains that are asporogenic and nucleotide excision repair deficient were engineered by deleting the
spoIIE
and
uvrAB
genes, rendering
B. anthracis
extremely sensitive to photochemical inactivation with S-59 psoralen and UV light. We also introduced point mutations into the
lef
and
cya
genes, which allowed inactive but immunogenic toxins to be produced. Photochemically inactivated vaccine strains maintained a high degree of metabolic activity and secreted protective antigen (PA), lethal factor, and edema factor. KBMA
B. anthracis
vaccines were avirulent in mice and induced less injection site inflammation than recombinant PA adsorbed to aluminum hydroxide gel. KBMA
B. anthracis
-vaccinated animals produced antibodies against numerous anthrax antigens, including high levels of anti-PA and toxin-neutralizing antibodies. Vaccination with KBMA
B. anthracis
fully protected mice against challenge with lethal doses of toxinogenic unencapsulated Sterne 7702 spores and rabbits against challenge with lethal pneumonic doses of fully virulent Ames strain spores. Guinea pigs vaccinated with KBMA
B. anthracis
were partially protected against lethal Ames spore challenge, which was comparable to vaccination with the licensed vaccine anthrax vaccine adsorbed. These data demonstrate that KBMA anthrax vaccines are well tolerated and elicit potent protective immune responses. The use of KBMA vaccines may be broadly applicable to bacterial pathogens, especially those for which the correlates of protective immunity are unknown.
Impaired Innate and Adaptive Immunity to
Streptococcus pneumoniae
and Its Effect on Colonization in an Infant Mouse Model
Authors: Bogaert Debby , Weinberger Daniel , Thompson Claudette , Lipsitch Marc , Malley Richard 
Published: January 2009

ABSTRACT

Streptococcus pneumoniae
colonization and invasive disease peak around the third and first birthdays, respectively, and decline thereafter. While these declines are attributable in part to immunity acquired via natural exposure, maturation of innate immune responses may also be involved. A mucosally administered candidate whole-cell pneumococcal vaccine (WCV) containing killed pneumococcal antigen (WCA) plus a cholera toxin adjuvant protects against intranasal carriage of pneumococci by a mechanism that is antibody independent and CD4
+
TH17 cell dependent. Because infants and children are a key target population for this vaccine, we sought to evaluate the immune responses of neonatal and infant mice to
S. pneumoniae
and to assess whether the WCV would be effective in these mice. Like human infants, infant mice showed impaired clearance of nasopharyngeal colonization with
S. pneumoniae
. Macrophages from neonatal and infant mice stimulated with killed pneumococci in vitro showed significantly reduced cytokine production, including that of KC, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, macrophage chemoattractant protein 1, interleukin-6 (IL-6), IL-1α, tumor necrosis factor alpha, and gamma interferon, whereas IL-10 expression was significantly increased compared to that in macrophages from adult mice. IL-17A production from adult immune CD4
+
T cells was significantly delayed when neonatal macrophages instead of adult macrophages were used as antigen-presenting cells. Moreover, whole blood from mice immunized as neonates with WCV produced significantly less IL-17A after stimulation with WCA than did blood from mice immunized as adults. Nonetheless, a single immunization of neonatal mice with WCV significantly reduced colonization density. Overall, our data suggest an impairment of both innate and acquired cellular immune responses in neonatal and infant mice. However, WCV confers a significant reduction in colonization following pneumococcal challenge, suggesting that it may still be effective in the setting of immature immune responses.
Role of Deoxyribose Catabolism in Colonization of the Murine Intestine by PathogenicEscherichia coliStrains
Authors: Martinez-Jéhanne Vanessa , du Merle Laurence , Bernier-Fébreau Christine , Usein Codruta , Gassama-Sow Amy , Wane Abdul-Aziz , Gouali Malika , Damian Maria , Aïdara-Kane Awa , Germani Yves , Fontanet Arnaud , Coddeville Bernadette , Guérardel Yann , Le Bouguénec Chantal 
Published: January 2009

ABSTRACTWe previously suggested that the ability to metabolize deoxyribose, a phenotype encoded by thedeoKoperon, is associated with the pathogenic potential ofEscherichia colistrains. Carbohydrate metabolism is thought to provide the nutritional support required forE. colito colonize the intestine. We therefore investigated the role of deoxyribose catabolism in the colonization of the gut, which acts as a reservoir, by pathogenicE. colistrains. Molecular and biochemical characterization of 1,221E. coliclones from various collections showed this biochemical trait to be common in theE. colispecies (33.6%). However, multivariate analysis evidenced a higher prevalence of sugar-metabolizingE. coliclones in the stools of patients from countries in which intestinal diseases are endemic. Diarrhea processes frequently involve the destruction of intestinal epithelia, so it is plausible that such clones may be positively selected for in intestines containing abundant DNA, and consequently deoxyribose. Statistical analysis also indicated that symptomatic clinical disorders and the presence of virulence factors specific to extraintestinal pathogenicE. coliwere significantly associated with an increased risk of biological samples and clones testing positive for deoxyribose. Using the streptomycin-treated-mouse model of intestinal colonization, we demonstrated the involvement of thedeoKoperon in gut colonization by two pathogenic isolates (one enteroaggregative and one uropathogenic strain). These results, indicating that deoxyribose availability promotes pathogenicE. coligrowth during host colonization, suggest that the acquisition of this trait may be an evolutionary step enabling these pathogens to colonize and persist in the mammalian intestine.
Host Complement Regulatory Protein CD59 Is Transported to the Chlamydial Inclusion by a Golgi Apparatus-Independent Pathway
Authors: Hasegawa Ayako , Sogo L. Farah , Tan Ming , Sütterlin Christine 
Published: January 2009

ABSTRACTChlamydiais an obligate intracellular bacterium that grows and replicates inside a cytoplasmic inclusion. We report that a host protein, CD59, which regulates complement function at the surfaces of uninfected cells, can be detected at the membrane of the chlamydial inclusion. This localization to the inclusion membrane was specific for CD59 and not a general feature of other glycosylphosphatidylinositol (GPI)-anchored proteins or representative cell surface proteins. Using differential permeabilization studies, we showed that CD59 is localized to the luminal but not the cytoplasmic face of the inclusion membrane, consistent with membrane association via its GPI anchor. Furthermore, CD59 was present at the inclusion even when we prevented it from associating with membrane microdomains via the GPI anchor or when we inhibited general protein transport to the cell surface, indicating that a conventional Golgi apparatus-dependent trafficking mechanism was not involved. Based on these findings, we propose that selected host proteins are trafficked to the inclusion by a Golgi apparatus-independent pathway during aChlamydiainfection.
Efficacy of Opsonic and Nonopsonic Serotype 3 Pneumococcal Capsular Polysaccharide-Specific Monoclonal Antibodies against Intranasal Challenge withStreptococcus pneumoniaein Mice
Authors: Tian Haijun , Weber Sarah , Thorkildson Peter , Kozel Thomas R. , Pirofski Liise-anne 
Published: January 2009

ABSTRACTSerotype-specific antibodies to pneumococcal capsular polysaccharide (PPS) are a critical component of vaccine-mediated immunity toStreptococcus pneumoniae. In this study, we investigated the in vitro opsonophagocytic activities of three PPS-specific mouse immunoglobulin G1 monoclonal antibodies (MAbs), 1E2, 5F6, and 7A9, and determined their in vivo efficacies against intranasal challenge with WU2, a serotype 3 pneumococcal strain, in normal and immunodeficient mice. The MAbs had different in vitro activities in a pneumococcal killing assay: 7A9 enhanced killing by mouse neutrophils and J774 cells in the presence of a complement source, whereas 5F6 promoted killing in the absence, but not the presence, of complement, and 1E2 did not promote killing under any conditions. Nonetheless, all three MAbs protected normal and complement component 3-deficient mice from a lethal intranasal challenge with WU2 in passive-immunization experiments in which 10 μg of the MAbs were administered intraperitoneally before intranasal challenge. In contrast, only 1E2 protected Fcγ receptor IIB knockout (FcγRIIB KO) mice and mice that were depleted of neutrophils with the MAb RB6, whereas 7A9 and 5F6 required neutrophils and FcγRIIB to mediate protection. Conversely, 7A9 and 5F6 protected FcγR KO mice, but 1E2 did not. Hence, the efficacy of 1E2 required an activating FcγR(s), whereas 5F6 and 7A9 required the inhibitory FcγR (FcγRIIB). Taken together, our data demonstrate that both MAbs that do and do not promote pneumococcal killing in vitro can mediate protection in vivo, although their efficacies depend on different host receptors and/or components.
Direct Regulation by theVibrio choleraeRegulator ToxT To Modulate Colonization and Anticolonization Pilus Expression
Authors: Hsiao Ansel , Xu Xiao , Kan Biao , Kulkarni Rahul V. , Zhu Jun 
Published: January 2009

ABSTRACTThe pathogenVibrio choleraeuses a large number of coordinated transcriptional regulatory events to transition from its environmental reservoir to the host and establish itself at its preferred colonization site at the host intestinal mucosa. The key regulator in this process is the AraC/XylS family transcription factor, ToxT, which plays critical roles in pathogenesis, including the regulation of two type IV pili, the anticolonization factor mannose-sensitive hemagglutinin and the toxin-coregulated pilus. Previously, it was thought ToxT required dimerization in order to effect transcriptional regulation at its cognate promoters. Here, we present evidence that ToxT directly represses transcription of themshoperon by binding to three promoters within this operon and that dimerization may not be required for transcriptional repression of target promoters by ToxT, suggesting that this regulator uses different mechanisms to modulate the transcriptional repertoire ofV. cholerae.
Pseudomonas aeruginosa
Infection of Zebrafish Involves both Host and Pathogen Determinants
Authors: Clatworthy Anne E. , Lee Jenny See-Wai , Leibman Mark , Kostun Zachary , Davidson Alan J. , Hung Deborah T. 
Published: January 2009

ABSTRACT

Zebrafish (
Danio rerio
) have a number of strengths as a host model for infection, including genetic tractability, a vertebrate immune system similar to that of mammals, ease and scale of laboratory handling, which allows analysis with reasonable throughput, and transparency, which facilitates visualization of the infection. With these advantages in mind, we examined whether zebrafish could be used to study
Pseudomonas aeruginosa
pathogenesis and found that infection of zebrafish embryos with live
P. aeruginosa
(PA14 or PAO1) by microinjection results in embryonic death, unlike infection with
Escherichia coli
or heat-killed
P. aeruginosa
, which has no effect. Similar to studies with mice,
P. aeruginosa
mutants deficient in type three secretion (
pscD
) or quorum sensing (
lasR
and
mvfR
) are attenuated in zebrafish embryos infected at 50 h postfertilization (hpf), a developmental stage when both macrophages and neutrophils are present. In contrast, embryos infected at 28 hpf, when only macrophages are initially present, succumb to lethal challenge with far fewer
P. aeruginosa
cells than those required for embryos infected at 50 hpf, are susceptible to infection with
lasR
and
pscD
deletion mutants, and are moderately resistant to infection with an
mvfR
mutant. Finally, we show that we can control the outcome of infection through the use of morpholinos, which allow us to shift immune cell numbers, or small molecules (antibiotics), which rescue embryos from lethal challenge. Thus, zebrafish are a novel host model that is well suited for studying the interactions among individual pathogenic functions of
P. aeruginosa
, the role of individual components of host immune defense, and small-molecule modulators of infection.
Salmonella enterica
Serovar Typhimurium Mutants Unable To Convert Malate to Pyruvate and Oxaloacetate Are Avirulent and Immunogenic in BALB/c Mice
Authors: Mercado-Lubo Regino , Leatham Mary P. , Conway Tyrrell , Cohen Paul S. 
Published: January 2009

ABSTRACT

Previously, we showed that the
Salmonella enterica
serovar Typhimurium SR-11 tricarboxylic acid (TCA) cycle must operate as a complete cycle for full virulence after oral infection of BALB/c mice (M. Tchawa Yimga, M. P. Leatham, J. H. Allen, D. C. Laux, T. Conway, and P. S. Cohen, Infect. Immun.
74:
1130-1140, 2006). In the same study, we showed that for full virulence, malate must be converted to both oxaloacetate and pyruvate. Moreover, it was recently demonstrated that blocking conversion of succinyl-coenzyme A to succinate attenuates serovar Typhimurium SR-11 but does not make it avirulent; however, blocking conversion of succinate to fumarate renders it completely avirulent and protective against subsequent oral infection with the virulent serovar Typhimurium SR-11 wild-type strain (R. Mercado-Lubo, E. J. Gauger, M. P. Leatham, T. Conway, and P. S. Cohen, Infect. Immun.
76:
1128-1134, 2008). Furthermore, the ability to convert succinate to fumarate appeared to be required only after serovar Typhimurium SR-11 became systemic. In the present study, evidence is presented that serovar Typhimurium SR-11 mutants that cannot convert fumarate to malate or that cannot convert malate to both oxaloacetate and pyruvate are also avirulent and protective in BALB/c mice. These results suggest that in BALB/c mice, the malate that is removed from the TCA cycle in serovar Typhimurium SR-11 for conversion to pyruvate must be replenished by succinate or one of its precursors, e.g., arginine or ornithine, which might be available in mouse phagocytes.
Burkholderia mallei tssMEncodes a Putative Deubiquitinase That Is Secreted and Expressed inside Infected RAW 264.7 Murine Macrophages
Authors: Shanks John , Burtnick Mary N. , Brett Paul J. , Waag David M. , Spurgers Kevin B. , Ribot Wilson J. , Schell Mark A. , Panchal Rekha G. , Gherardini Frank C. , Wilkinson Keith D. , DeShazer David 
Published: January 2009

ABSTRACTBurkholderia mallei, a category B biothreat agent, is a facultative intracellular pathogen that causes the zoonotic disease glanders. TheB. malleiVirAG two-component regulatory system activates the transcription of ∼60 genes, including a large virulence gene cluster encoding a type VI secretion system (T6SS). TheB. mallei tssMgene encodes a putative ubiquitin-specific protease that is physically linked to, and transcriptionally coregulated with, the T6SS gene cluster. Mass spectrometry and immunoblot analysis demonstrated that TssM was secreted in avirAG-dependent manner in vitro. Surprisingly, the T6SS was found to be dispensable for the secretion of TssM. The C-terminal half of TssM, which contains Cys and His box motifs conserved in eukaryotic deubiquitinases, was purified and biochemically characterized. Recombinant TssM hydrolyzed multiple ubiquitinated substrates and the cysteine at position 102 was critical for enzymatic activity. ThetssMgene was expressed within 1 h after uptake ofB. malleiinto RAW 264.7 murine macrophages, suggesting that the TssM deubiquitinase is produced in this intracellular niche. Although the physiological substrate(s) is currently unknown, the TssM deubiquitinase may provideB. malleia selective advantage in the intracellular environment during infection.
The RgpA-Kgp Proteinase-Adhesin Complexes ofPorphyromonas gingivalisInactivate the Th2 Cytokines Interleukin-4 and Interleukin-5
Authors: Tam Vivian , O'Brien-Simpson Neil M. , Chen Yu-Yen , Sanderson Colin J. , Kinnear Beverley , Reynolds Eric C. 
Published: January 2009

ABSTRACTThe RgpA-Kgp proteinase-adhesin complexes are a primary virulence factor ofPorphyromonas gingivalis, a major pathogen in the development of chronic periodontitis. The RgpA-Kgp complexes have been suggested to bias the immune response to a Th2 phenotype in disease by hydrolysis of Th1 cytokines. Here, we show that the RgpA-Kgp complexes hydrolyze and inactivate interleukin-4 (IL-4) and IL-5 under physiologically relevant conditions. Using the IL-4/IL-5-dependent TF1.8 T-cell line, it was found that at equimolar ratios of cytokine to RgpA-Kgp complexes, IL-4 and IL-5 were inactivated in the culture medium. The inactivation of IL-4 and IL-5 was RgpA-Kgp concentration dependent, as at an enzyme-to-cytokine molar ratio of 1:8, the bioactivity of the cytokines was greater than at the higher concentration of RgpA-Kgp of 1:1. Furthermore, inactivation of the cytokines by the RgpA-Kgp complexes was time dependent, as longer preincubation times resulted in lower cytokine activity. IL-5 was found to be slightly more resistant to inactivation than IL-4. Mass spectrometric analyses of IL-4 and IL-5 showed that hydrolysis by RgpA-Kgp complexes was C terminal to Arg and Lys residues of the cytokines. The peptides released indicated that the regions of IL-4 and IL-5 important for bioactivity were being hydrolyzed in the first 15 min of incubation. The ability of the RgpA-Kgp complexes to degrade Th2 cytokines may contribute to immune dysregulation and may play a role in the pathology of chronic periodontitis.