bluuebunny/crossref_metadata_2025_split huggingface dataset:
Reduced Susceptibility of
Haemophilus influenzae
to the Peptide Deformylase Inhibitor LBM415 Can Result from Target Protein Overexpression Due to Amplified Chromosomal
def
Gene Copy Number
Authors: Dean Charles R. , Narayan Shubha , Richards Joel , Daigle Denis M. , Esterow Stacy , Leeds Jennifer A. , Kamp Heather , Puyang Xiaoling , Wiedmann Brigitte , Mueller Dieter , Voshol Hans , van Oostrum Jan , Wall Daniel , Koehn James , Dzink-Fox JoAnn , Ryder Neil S. 
Published: January 2007

ABSTRACT

Previous genetic analysis of
Haemophilus influenzae
revealed two mechanisms associated with decreased susceptibility to the novel peptide deformylase inhibitor LBM415: AcrAB-TolC-mediated efflux and Fmt bypass, resulting from mutations in the pump repressor gene
acrR
and in the
fmt
gene, respectively. We have isolated an additional mutant, CDS23 (LBM415 MIC, 64 μg/ml versus 4 μg/ml against the parent strain NB65044) that lacks mutations in the
acrR
or
fmt
structural genes or in the gene encoding Def, the intracellular target of LBM415. Western immunoblot analysis, two-dimensional gel electrophoresis, and tryptic digestion combined with mass spectrometric identification showed that the Def protein was highly overexpressed in the mutant strain. Consistent with this, real-time reverse transcription-PCR revealed a significant increase in
def
transcript titer. No mutations were found in the region upstream of
def
that might account for altered expression; however, pulsed-field gel electrophoresis suggested that a genetic rearrangement of the region containing
def
had occurred. Using a combination of PCR, sequencing, and Southern blot analyses, it was determined that the
def
gene had undergone copy number amplification, explaining the high level of target protein expression. Inactivation of the AcrAB-TolC efflux pump in this mutant increased susceptibility 16-fold, highlighting the role of efflux in exacerbating the overall reduced susceptibility resulting from target overexpression.
In Planta Horizontal Transfer of a Major Pathogenicity Effector Gene
Authors: El Yacoubi B. , Brunings A. M. , Yuan Q. , Shankar S. , Gabriel D. W. 
Published: January 2007

ABSTRACTXanthomonas citripv. citri is a clonal group of strains that causes citrus canker disease and appears to have originated in Asia. A phylogenetically distinct clonal group that causes identical disease symptoms on susceptible citrus,X. citripv. aurantifolii, arose more recently in South America. Genomes ofX. citripv. aurantifolii strains carry two DNA fragments that hybridize topthA, anX. citripv. citri gene which encodes a major type III pathogenicity effector protein that is absolutely required to cause citrus canker. Marker interruption mutagenesis and complementation revealed thatX. citripv. aurantifolii strain B69 carried one functionalpthAhomolog, designatedpthB, that was required to cause cankers on citrus. GenepthBwas found among 38 open reading frames on a 37,106-bp plasmid, designated pXcB, which was sequenced and annotated. No additional pathogenicity effectors were found on pXcB, but 11 out of 38 open reading frames appeared to encode a type IV transfer system. pXcB transferred horizontally in planta, without added selection, from B69 to a nonpathogenicX. citripv. citri (pthA::Tn5) mutant strain, fully restoring canker. In planta transfer efficiencies were very high (>0.1%/recipient) and equivalent to those observed for agar medium with antibiotic selection, indicating thatpthBconferred a strong selective advantage to the recipient strain. A single pathogenicity effector that can confer a distinct selective advantage in planta may both facilitate plasmid survival following horizontal gene transfer and account for the origination of phylogenetically distinct groups of strains causing identical disease symptoms.
Effect of Superoxide Dismutase Gene Inactivation on Virulence of
Pseudomonas aeruginosa
PAO1 toward the Silkworm,
Bombyx mori
Authors: Iiyama Kazuhiro , Chieda Yuuka , Lee Jae Man , Kusakabe Takahiro , Yasunaga-Aoki Chisa , Shimizu Susumu 
Published: January 2007

ABSTRACT

To investigate the role of superoxide dismutase (SOD) in virulence against the silkworm,
Bombyx mori
, mutants of
Pseudomonas aeruginosa
PAO1 lacking manganese-SOD (PAO1
sodM
), iron-SOD (PAO1
sodB
), or both (PAO1
sodMB
) were generated. The mutants were injected into the hemocoel of
B. mori
. The virulence decreased in the order PAO1 = PAO1
sodM
> PAO1
sodB
> PAO1
sodMB
. In particular, PAO1
sodMB
was avirulent at a dose of 10
5
cells or less. The
sod
double mutant PAO1
sodMB
was then complemented with either pSodM or pSodB in
trans
. In both the complemented strains, the virulence was partially restored. Of the two plasmids, pSodB contributed more to the virulence of
P. aeruginosa
against
B. mori
. The results of growth in
B. mori
hemolymph broth and microscopic analysis suggested that a longer lag phase and superoxide sensitivity correlated with decreased virulence in
sod
mutants. In conclusion, the SODs are required for full virulence of
P. aeruginosa
against
B. mori
and Fe-SOD is more important than Mn-SOD in the infection process.
Autotransporter-Encoding Sequences Are Phylogenetically Distributed amongEscherichia coliClinical Isolates and Reference Strains
Authors: Restieri Concetta , Garriss Geneviève , Locas Marie-Claude , Dozois Charles M. 
Published: January 2007

ABSTRACTAutotransporters are secreted bacterial proteins exhibiting diverse virulence functions. Various autotransporters have been identified amongEscherichia coliassociated with intestinal or extraintestinal infections; however, the specific distribution of autotransporter sequences among a diversity ofE. colistrains has not been investigated. We have validated the use of a multiplex PCR assay to screen for the presence of autotransporter sequences. Herein, we determined the presence of 13 autotransporter sequences and five allelic variants of antigen 43 (Ag43) among 491E. coliisolates from human urinary tract infections, diarrheagenicE. coli, and avian pathogenicE. coli(APEC) andE. colireference strains belonging to the ECOR collection. Clinical isolates were also classified into established phylogenetic groups. The results indicated that Ag43 alleles were significantly associated with clinical isolates (93%) compared to commensal isolates (56%) and thatagn43K12was the most common and widely distributed allele.agn43allelic variants were also phylogenetically distributed. Sequences encodingespC,espP, andsepAandagn43allelesEDL933andRS218were significantly associated with diarrheagenicE. colistrains compared to other groups.tshwas highly associated with APEC strains, whereassatwas absent from APEC.vat,sat, andpicwere associated with urinary tract isolates and were identified predominantly in isolates belonging to either group B2 or D of the phylogenetic groups based on the ECOR strain collection. Overall, the results indicate that specific autotransporter sequences are associated with the source and/or phylogenetic background of strains and suggest that, in some cases, autotransporter gene profiles may be useful for comparative analysis ofE. colistrains from clinical, food, and environmental sources.
Evolutionary Relationships of “CandidatusRiesia spp.,” EndosymbioticEnterobacteriaceaeLiving within Hematophagous Primate Lice
Authors: Allen Julie M. , Reed David L. , Perotti M. Alejandra , Braig Henk R. 
Published: January 2007

ABSTRACTThe primary endosymbiotic bacteria from three species of parasitic primate lice were characterized molecularly. We have confirmed the characterization of the primary endosymbiont (P-endosymbiont) of the human head/body lousePediculus humanusand provide new characterizations of the P-endosymbionts fromPediculus schaeffifrom chimpanzees andPthirus pubis, the pubic louse of humans. The endosymbionts show an average percent sequence divergence of 11 to 15% from the most closely related known bacterium “CandidatusArsenophonus insecticola.” We propose that two additional species be added to the genus “CandidatusRiesia.” The new species proposed within “CandidatusRiesia” have sequence divergences of 3.4% and 10 to 12% based on uncorrected pairwise differences. Our Bayesian analysis shows that the branching pattern for the primary endosymbionts was the same as that for their louse hosts, suggesting a long coevolutionary history between primate lice and their primary endosymbionts. We used a calibration of 5.6 million years to date the divergence between endosymbionts from human and chimpanzee lice and estimated an evolutionary rate of nucleotide substitution of 0.67% per million years, which is 15 to 30 times faster than previous estimates calculated forBuchnera, the primary endosymbiont in aphids. Given the evidence for cospeciation with primate lice and the evidence for fast evolutionary rates, this lineage of endosymbiotic bacteria can be evaluated as a fast-evolving marker of both louse and primate evolutionary histories.
Carbon Conversion Efficiency and Limits of Productive Bacterial Degradation of Methyl
tert
-Butyl Ether and Related Compounds
Authors: Müller Roland H. , Rohwerder Thore , Harms Hauke 
Published: January 2007

ABSTRACT

The utilization of the fuel oxygenate methyl
tert
-butyl ether (MTBE) and related compounds by microorganisms was investigated in a mainly theoretical study based on the Y
ATP
concept. Experiments were conducted to derive realistic maintenance coefficients and
K

s

values needed to calculate substrate fluxes available for biomass production. Aerobic substrate conversion and biomass synthesis were calculated for different putative pathways. The results suggest that MTBE is an effective heterotrophic substrate that can sustain growth yields of up to 0.87 g g
−1
, which contradicts previous calculation results (N. Fortin et al., Environ. Microbiol. 3:407-416, 2001). Sufficient energy equivalents were generated in several of the potential assimilatory routes to incorporate carbon into biomass without the necessity to dissimilate additional substrate, efficient energy transduction provided. However, when a growth-related kinetic model was included, the limits of productive degradation became obvious. Depending on the maintenance coefficient
m

s

and its associated biomass decay term
b
, growth-associated carbon conversion became strongly dependent on substrate fluxes. Due to slow degradation kinetics, the calculations predicted relatively high threshold concentrations,
S
min
, below which growth would not further be supported.
S
min
strongly depended on the maximum growth rate
μ
ma

x

, and
b
and was directly correlated with the half maximum rate-associated substrate concentration
K

s

, meaning that any effect impacting this parameter would also change
S
min
. The primary metabolic step, catalyzing the cleavage of the ether bond in MTBE, is likely to control the substrate flux in various strains. In addition, deficits in oxygen as an external factor and in reduction equivalents as a cellular variable in this reaction should further increase
K

s

and
S
min
for MTBE.
A 368-Base-Pair
cis
-Acting
HWP1
Promoter Region, HCR, of
Candida albicans
Confers Hypha-Specific Gene Regulation and Binds Architectural Transcription Factors Nhp6 and Gcf1p
Authors: Kim Samin , Wolyniak Michael J. , Staab Janet F. , Sundstrom Paula 
Published: January 2007

ABSTRACT

To elucidate the molecular mechanisms controlling the expression of the hypha-specific adhesin gene
HWP1
of
Candida albicans
, its promoter was dissected and analyzed using a green fluorescent protein reporter gene. A 368-bp region, the
HWP1
control region (HCR), was critical for activation under hypha-inducing conditions and conferred developmental regulation to a heterologous
ENO1
promoter. A more distal region of the promoter served to amplify the level of promoter activation. Using gel mobility shift assays, a 249-bp subregion of HCR, HCRa, was found to bind at least four proteins from crude extracts of yeasts and hyphae with differing binding patterns dependent on cell morphology. Four proteins with DNA binding activities were identified by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis after separation by anion-exchange and heparin-Sepharose chromatography. One protein with high similarity to Nhp6, an HMG1 family member in
Saccharomyces cerevisiae
, and another with weak similarity to an HMG-like condensation factor from
Physarum polycephalum
implicated changes in chromatin structure as a critical process in hypha-specific gene regulation. Proteins with strong homology to histones were also found. These studies are the first to identify proteins that bind to a DNA segment that confers developmental gene regulation in
C. albicans
and suggest a new model for hypha-specific gene regulation.
Genome-Wide Analysis of Cellular Response to Bacterial Genotoxin CdtB in Yeast
Authors: Kitagawa Takao , Hoshida Hisashi , Akada Rinji 
Published: January 2007

ABSTRACTThe cytolethal distending toxins (CDTs) are secreted virulence proteins produced by several bacterial pathogens, and the subunit CdtB has the ability to create DNA lesions, primarily DNA single-strand breaks (SSBs) in vitro, and cause cell cycle arrest, cellular distension, and cell death in both mammalian and yeast cells. To elucidate the components of the mechanisms underlying the response to CdtB-induced DNA lesions, a CdtB expression plasmid was transformed into a series of diploid yeast strains harboring deletions in 4,708 nonessential genes. A total of 4,706 of these clones were successfully transformed, which we have now designated as a systematic transformation array (STA), and were subsequently screened. We identified 61 sensitive strains from the STA whose deleted genes can be categorized into a number of groups, including DNA metabolism, chromosome segregation, vesicular traffic, RNA catabolism, protein translation, morphogenesis, and nuclear transport, as well as one unknown open reading frame. However, only 28 of these strains were found to be sensitive to HO endonuclease, which is known to create a DNA double-strand break (DSB), suggesting that CdtB-induced DNA lesion is not similar to the direct DSB. Amazingly, CdtB expression elicits severe growth defects in haploid yeast cells, but only marginal defects in diploid yeast cells. The presence and absence of genes known to be involved in DNA repair in these genome-wide data reveal that CdtB-induced DNA damage is specifically repaired well in the diploid by homologous recombination but not by other repair mechanisms. Our present results provide insights into how CdtB pathogenesis is linked to eukaryotic cellular functions.
In Vitro Induction of Immunoglobulin A (IgA)- and IgM-Secreting Plasma Blasts by Cholera Toxin Depends on T-Cell Help and Is Mediated by CD154 Up-Regulation and Inhibition of Gamma Interferon Synthesis
Authors: Arce Sergio , Nawar Hesham F. , Muehlinghaus Gwendolin , Russell Michael W. , Connell Terry D. 
Published: January 2007

ABSTRACTCholera toxin (CT) and the type II heat-labile enterotoxins (LT-IIa and LT-IIb) are potent immunological adjuvants which are hypothesized to enhance the production of antibody (Ab)-secreting cells, although their mechanisms of action are not fully understood. The treatment of splenic cells with concanavalin A (ConA) plus CT enhanced the production of immunoglobulin A (IgA) and IgM by dividing cells that expressed high levels of major histocompatibility complex class II (MHC-II), CD19, and CD138 and low levels of B220 a phenotype characteristic of plasma blasts. LT-IIa or LT-IIb moderately enhanced IgA and IgM production without enhancing plasma blast differentiation. CT up-regulated CD25, CD69, CD80, CD86, and MHC-II in isolated B cells but failed to induce proliferation or differentiation. The treatment of unfractionated splenic cells with ConA plus CT induced B-cell proliferation and differentiation, but the elimination of CD4+T cells inhibited this effect. CT treatment of ConA-activated CD4+T cells up-regulated CD134 and CD154, whereas the blockage of CD40-CD154 interactions inhibited the induction of plasma blasts and Ig synthesis. The treatment of unfractionated splenic cells with CT, LT-IIa, or LT-IIb enhanced the production of interleukin-6 (IL-6) and IL-10, whereas the production of gamma interferon was inhibited in both CD4+and CD8+T cells mostly by CT. Thus, major regulatory effects of CT on lymphocytes are likely exerted early during the induction of immune responses when B and T cells initially encounter antigen. Neither LT-IIa or LT-IIb had these effects, indicating that type II enterotoxins augment Ab responses by other mechanisms.
Pneumococcal Capsular Polysaccharide Vaccine-Mediated Protection against Serotype 3Streptococcus pneumoniaein Immunodeficient Mice
Authors: Tian Haijun , Groner Avi , Boes Marianne , Pirofski Liise-anne 
Published: January 2007

ABSTRACTPneumococcal capsular polysaccharide (PPS) vaccines are less immunogenic in immunocompromised than immunocompetent individuals. However, neither the efficacy of PPS vaccines in immunocompromised individuals nor the host cellular subsets required for vaccine efficacy against pneumococcal disease have been directly investigated. In this study, we vaccinated CD4-deficient (CD4−/−), CD8-deficient (CD8−/−), and secretory immunoglobulin M-deficient (sIgM−/−) mice and wild-type C57BL/6 (Wt) mice with a conjugate of PPS of serotype 3 and tetanus toxoid (PPS3-TT) and determined the antibody response and efficacy of vaccination against systemic and pulmonary challenge with serotype 3 pneumococcus in immunized and control mice. Our results showed that the isotype and predominant IgG subclass of the PPS3 response differed between immunodeficient mouse strains and between immunodeficient and Wt mice, with CD8−/−mice having the most robust response. Vaccination protected Wt, CD4−/−, and sIgM−/−mice from death resulting from both systemic and pulmonary challenge, whereas CD8−/−mice were protected only from systemic and not from pulmonary challenge. Passive vaccination with PPS3-TT-induced sera from Wt, CD4−/−, CD8−/−, and sIgM−/−mice protected naïve Wt mice from death due to pulmonary challenge; however, CD8−/−mice were not protected by sera from Wt or CD8−/−mice. Our findings suggest that PPS-based vaccines can be effective in the setting of CD4 T-cell deficiency but that CD8 T cells could be required for vaccine-mediated protection against pulmonary challenge with serotype 3 pneumococcus.
Kgp and RgpB, but Not RgpA, Are Important for
Porphyromonas gingivalis
Virulence in the Murine Periodontitis Model
Authors: Pathirana Rishi D. , O'Brien-Simpson Neil M. , Brammar Gail C. , Slakeski Nada , Reynolds Eric C. 
Published: January 2007

ABSTRACT

The contributions of three proteinase genes (
rgpA
,
rgpB
, and
kgp
) to the virulence of
Porphyromonas gingivalis
W50 were investigated in the murine periodontitis model. Mice were orally inoculated with eight doses (1 × 10
10
cells per dose) of
rgpA
,
rgpB
,
kgp
,
rgpA rgpB
, or
rgpA rgpB kgp
isogenic mutants, and the level of alveolar bone loss, immune response induced, and number of bacterial cells per half maxilla were compared with those of animals inoculated with wild-type
P. gingivalis
. The
kgp
,
rgpB
,
rgpA rgpB
, and
rgpA rgpB kgp
isogenic mutants induced significantly (
P
< 0.05) less bone loss than the
rgpA
isogenic mutant and the wild type did, and the virulence of the
rgpA
isogenic mutant and the wild type were not significantly different. Mice inoculated with the wild type or the
rgpA
isogenic mutant exhibited significantly (
P
< 0.01) more
P. gingivalis
cells per half maxilla than mice inoculated with
rgpB
,
kgp
,
rgpA rgpB
, and
rgpA rgpB kgp
isogenic mutants or nonchallenged mice did, as determined using real-time PCR. A significant positive correlation was found between the number of
P. gingivalis
cells detected per half maxilla and the amount of alveolar bone loss induced. Enzyme-linked immunosorbent assay results showed that each isogenic mutant and the wild type induced a predominant
P. gingivalis
antigen-specific immunoglobulin G3 (IgG3) response. Furthermore, the
kgp
and
rgpA rgpB kgp
isogenic mutants induced significantly (
P
< 0.05) lower IgG3 antibody responses than the responses induced by the wild type or the
rgpA
,
rgpB
, and
rgpA rgpB
isogenic mutants. The results suggest that the order in which the proteinases contribute to the virulence of
P. gingivalis
in the murine periodontitis model is Kgp ≥ RgpB
≫
RgpA.
Evidence for Distinctive Mechanisms of
S
-Nitrosoglutathione Metabolism by AdhC in Two Closely Related Species,
Neisseria gonorrhoeae
and
Neisseria meningitidis
Authors: Potter Adam J. , Kidd Stephen P. , Jennings Michael P. , McEwan Alastair G. 
Published: January 2007

ABSTRACT

The
adhC
gene from 11 strains of
Neisseria gonorrhoeae
was distinguished from its homologue in
Neisseria meningitidis
by the presence of a premature stop codon caused by a single base insertion. Mutational analysis showed that NADH
S
-nitrosoglutathione oxidoreductase activity was associated with
adhC
in
Neisseria meningitidis
but not in
Neisseria gonorrhoeae
.
Pneumolysin, PspA, and PspC Contribute to Pneumococcal Evasion of Early Innate Immune Responses during Bacteremia in Mice
Authors: Quin Lisa R. , Moore Quincy C. , McDaniel Larry S. 
Published: January 2007

ABSTRACTThe pneumococcal virulence factors include capsule, PspA, PspC, and Ply. Cytometric analysis demonstrated that the greatest levels of C3 deposition were on a ΔplyPspA−PspC−mutant. Also, Ply, PspA, and PspC expression resulted in C3 degradation in vitro and in vivo. Finally, blood clearance assays demonstrated that there was enhanced clearance of ΔplyPspA−PspC−pneumococci compared to the clearance of nonencapsulated pneumococci.
The HP0165-HP0166 Two-Component System (ArsRS) Regulates Acid-Induced Expression of HP1186 α-Carbonic Anhydrase in
Helicobacter pylori
by Activating the pH-Dependent Promoter
Authors: Wen Yi , Feng Jing , Scott David R. , Marcus Elizabeth A. , Sachs George 
Published: January 2007

ABSTRACT

The periplasmic α-carbonic anhydrase of
Helicobacter pylori
is essential for buffering the periplasm at acidic pH. This enzyme is an integral component of the acid acclimation response that allows this neutralophile to colonize the stomach. Transcription of the HP1186 α-carbonic anhydrase gene is upregulated in response to low environmental pH. A binding site for the HP0166 response regulator (ArsR) has been identified in the promoter region of the HP1186 gene. To investigate the mechanism that regulates the expression of HP1186 in response to low pH and the role of the HP0165-HP0166 two-component system (ArsRS) in this acid-inducible regulation, Northern blot analysis was performed with RNAs isolated from two different wild-type
H. pylori
strains (26695 and 43504) and mutants with HP0165 histidine kinase (
ArsS
) deletions, after exposure to either neutral pH or low pH (pH 4.5). ArsS-dependent upregulation of HP1186 α-carbonic anhydrase in response to low pH was found in both strains. Western blot analysis of
H. pylori
membrane proteins confirmed the regulatory role of ArsS in HP1186 expression in response to low pH. Analysis of the HP1186 promoter region revealed two possible transcription start points (TSP
1
and TSP
2
) located 43 and 11 bp 5′ of the ATG start codon, respectively, suggesting that there are two promoters transcribing the HP1186 gene. Quantitative primer extension analysis showed that the promoter from TSP
1
(43 bp 5′ of the ATG start codon) is a pH-dependent promoter and is regulated by ArsRS in combating environmental acidity, whereas the promoter from TSP
2
may be responsible for control of the basal transcription of HP1186 α-carbonic anhydrase.