bluuebunny/crossref_metadata_2025 huggingface dataset:
Claude Bernard, the first systems biologist, and the future of physiology
Authors: Noble Denis
Published: October 2007

The first systems analysis of the functioning of an organism was Claude Bernard's concept of the constancy of the internal environment (le milieu intérieur), since it implied the existence of control processes to achieve this. He can be regarded, therefore, as the first systems biologist. The new vogue for systems biology today is an important development, since it is time to complement reductionist molecular biology by integrative approaches. Claude Bernard foresaw that this would require the application of mathematics to biology. This aspect of Claude Bernard's work has been neglected by physiologists, which is why we are not as ready to contribute to the development of systems biology as we should be. In this paper, I outline some general principles that could form the basis of systems biology as a truly multilevel approach from a physiologist's standpoint. We need the insights obtained from higher‐level analysis in order to succeed even at the lower levels. The reason is that higher levels in biological systems impose boundary conditions on the lower levels. Without understanding those conditions and their effects, we will be seriously restricted in understanding the logic of living systems. The principles outlined are illustrated with examples from various aspects of physiology and biochemistry. Applying and developing these principles should form a major part of the future of physiology.
Pathway‐specific use‐dependent dynamics of excitatory synaptic transmission in rat intracortical circuits
Authors: Williams Stephen R. , Atkinson Susan E.
Published: October 2007

Information processing in neuronal networks is determined by the use‐dependent dynamics of synaptic transmission. Here we characterize the dynamic properties of excitatory synaptic transmission in two major intracortical pathways that target the output neurons of the neocortex, by recording unitary EPSPs from layer 5 pyramidal neurons evoked in response to action potential trains of increasing complexity in presynaptic layer 2/3 or layer 5 pyramidal neurons. We find that layer 2/3 to layer 5 synaptic transmission is dominated by frequency‐dependent depression when generated at fixed frequencies of > 10 Hz. Synaptic depression evolved on a spike‐by‐spike basis in response to action potential trains that possessed a broad range of interspike intervals, but a low mean frequency (10 Hz). Layer 2/3 to layer 2/3 and layer 2/3 to layer 5 synapses were incapable of sustained release during prolonged, complex trains of presynaptic action potential firing (mean frequency, 48 Hz). By contrast, layer 5 to layer 5 synapses operated effectively across a wide range of frequencies, exhibiting increased efficacy at frequencies > 10 Hz. Furthermore, layer 5 to layer 5 synapses sustained release throughout the duration of prolonged, complex spike trains. The use‐dependent properties of synaptic transmission could be modulated by pharmacologically changing the probability of release and by induction of long‐term depression. The dynamic properties of intracortical excitatory synapses are therefore pathway‐specific. We suggest that the synaptic output of layer 5 pyramidal neurons is ideally suited to control the neocortical network across a wide range of frequencies and for sustained periods of time, a behaviour that helps to explain the pivotal role played by layer 5 neurons in the genesis of periods of network ‘up’ states and epileptiform activity in the neocortex.
Aerobic metabolism underlies complexity and capacity
Authors: Koch Lauren G. , Britton Steven L.
Published: October 2007

The evolution of biological complexity beyond single‐celled organisms was linked temporally with the development of an oxygen atmosphere. Functionally, this linkage can be attributed to oxygen ranking high in both abundance and electronegativity amongst the stable elements of the universe. That is, reduction of oxygen provides for close to the largest possible transfer of energy for each electron transfer reaction. This suggests the general hypothesis that the steep thermodynamic gradient of an oxygen environment was permissive for the development of multicellular complexity. A corollary of this hypothesis is that aerobic metabolism underwrites complex biological function mechanistically at all levels of organization. The strong contemporary functional association of aerobic metabolism with both physical capacity and health is presumably a product of the integral role of oxygen in our evolutionary history. Here we provide arguments from thermodynamics, evolution, metabolic network analysis, clinical observations and animal models that are in accord with the centrality of oxygen in biology.
A Bifunctional Bacterial Protein Links GDI Displacement to Rab1 Activation
Authors: Machner Matthias P. , Isberg Ralph R.
Published: October 2007

Rab guanosine triphosphatases (GTPases) regulate vesicle trafficking in eukaryotic cells by reversibly associating with lipid membranes. Inactive Rab GTPases are maintained in the cytosol by binding to GDP-dissociation inhibitor (GDI). It is believed that specialized proteins are required to displace GDI from Rab GTPases before Rab activation by guanosine diphosphate–guanosine 5′-triphosphate (GDP-GTP) exchange factors (GEFs). Here, we found that SidM from
Legionella pneumophila
could act as both GEF and GDI-displacement factor (GDF) for Rab1. Rab1 released from GDI was inserted into liposomal membranes and was used as a substrate for SidM-mediated nucleotide exchange. During host cell infection, recruitment of Rab1 to
Legionella
-containing vacuoles depended on the GDF activity of SidM. Thus, GDF and GEF activity can be promoted by a single protein, and GDF activity can coordinate Rab1 recruitment from the GDI-bound pool.
Resveratrol Modulates Phagocytosis of Bacteria through an NF-κB-Dependent Gene Program
Authors: Iyori Mitsuhiro , Kataoka Hideo , Shamsul Haque Mohammad , Kiura Kazuto , Yasuda Motoaki , Nakata Takashi , Hasebe Akira , Shibata Ken-ichiro
Published: October 2007

Many studies have shown that the pharmacological effects of resveratrol, a phytoalexin polyphenolic compound, include protective effects against cancer and inflammation as well as enhancement of stress resistance. In this study, we examined whether resveratrol affected the phagocytosis of bacteria by macrophages and the activation of the transcription factor NF-κB after stimulation with or without the ligand FSL-1 for Toll-like receptor 2 (TLR2). Phagocytosis of
Escherichia coli
and of
Staphylococcus aureus
by THP-1 cells and RAW264.7 cells was inhibited by resveratrol in a dose-dependent manner regardless of stimulation with FSL-1. The NF-κB activity in HEK293 cells stably expressing TLR2 was also inhibited by resveratrol after stimulation with FSL-1. Resveratrol also inhibited both the translocation of p65 of NF-κB into nuclei in the transfectant and tumor necrosis factor alpha production by THP-1 cells or RAW264.7 cells. It has recently been reported that TLR-mediated signaling pathways lead to the upregulation of mRNAs of phagocytic receptors, including scavenger receptors and C-type lectin receptors. This study also demonstrated that FSL-1 induced the upregulation of mRNAs of phagocytic receptors such as macrophage scavenger receptor-1, CD36, DC-SIGN, and Dectin-1 and that the FSL-1-induced upregulation of their mRNAs was inhibited by resveratrol. In addition, it was found that the expression of DC-SIGN in HEK293 cells stably expressing DC-SIGN was reduced by resveratrol and that the phagocytic activity was significantly inhibited by resveratrol. Thus, this study suggests that resveratrol inhibited bacterial phagocytosis by macrophages by downregulating the expression of phagocytic receptors and NF-κB activity.
Inhibition of Quorum Sensing-Controlled Virulence Factor Production in
Pseudomonas aeruginosa
by South Florida Plant Extracts
Authors: Adonizio Allison , Kong Kok-Fai , Mathee Kalai
Published: October 2007

Quorum sensing (QS) is a key regulator of virulence and biofilm formation in
Pseudomonas aeruginosa
and other medically relevant bacteria. Aqueous extracts of six plants,
Conocarpus erectus
,
Chamaesyce hypericifolia
,
Callistemon viminalis
,
Bucida buceras
,
Tetrazygia bicolor
, and
Quercus virginiana
, were examined in this study for their effects on
P. aeruginosa
virulence factors and the QS system.
C. erectus
,
B. buceras
, and
C. viminalis
caused a significant inhibition of LasA protease, LasB elastase, pyoverdin production, and biofilm formation. Additionally, each plant presented a distinct effect profile on the
las
and
rhl
QS genes and their respective signaling molecules, suggesting that different mechanisms are responsible for efficacy. Extracts of all plants caused the inhibition of QS genes and QS-controlled factors, with marginal effects on bacterial growth, suggesting that the quorum-quenching mechanisms are unrelated to static or cidal effects.
Complex Class 1 Integrons with Diverse Variable Regions, Including
aac
(
6
′)-
Ib
-
cr
, and a Novel Allele,
qnrB10
, Associated with IS
CR1
in Clinical Enterobacterial Isolates from Argentina
Authors: Quiroga María Paula , Andres Patricia , Petroni Alejandro , Soler Bistué Alfonso J. C. , Guerriero Leonor , Vargas Liliana Jordá , Zorreguieta Angeles , Tokumoto Marta , Quiroga Cecilia , Tolmasky Marcelo E. , Galas Marcelo , Centrón Daniela
Published: October 2007

Transferable quinolone resistance has not previously been reported in Argentina. Here we describe three complex class 1 integrons harboring the novel allele
qnrB10
in a unique region downstream of
orf513
, one of them also containing
aac
(
6
′)-
Ib
-
cr
within the variable region of integrons. The three arrays differed from
bla
CTX-M-2
-bearing integrons, which are broadly distributed in Argentina.
Molecular Epidemiology and Mechanisms of Carbapenem Resistance in
Pseudomonas aeruginosa
Isolates from Spanish Hospitals
Authors: Gutiérrez O. , Juan C. , Cercenado E. , Navarro F. , Bouza E. , Coll P. , Pérez J. L. , Oliver A.
Published: October 2007

All (236)
Pseudomonas aeruginosa
isolates resistant to imipenem and/or meropenem collected during a multicenter (127-hospital) study in Spain were analyzed. Carbapenem-resistant isolates were found to be more frequently resistant to all β-lactams and non-β-lactam antibiotics than carbapenem-susceptible isolates (
P
< 0.001), and up to 46% of the carbapenem-resistant isolates met the criteria used to define multidrug resistance (MDR). Pulsed-field gel electrophoresis revealed remarkable clonal diversity (165 different clones were identified), and with few exceptions, the levels of intra- and interhospital dissemination of clones were found to be low. Carbapenem resistance was driven mainly by the mutational inactivation of OprD, accompanied or not by the hyperexpression of AmpC or MexAB-OprM. Class B carbapenemases (metallo-β-lactamases [MBLs]) were detected in a single isolate, although interestingly, this isolate belonged to one of the few epidemic clones documented. The MBL-encoding gene (
bla
VIM-2
), along with the aminoglycoside resistance determinants, was transferred to strain PAO1 by electroporation, demonstrating its plasmid location. The class 1 integron harboring
bla
VIM-2
was characterized as well, and two interesting features were revealed:
intI1
was found to be disrupted by a 1.1-kb insertion sequence, and a previously undescribed aminoglycoside acetyltransferase-encoding gene [designated
aac(6′)-32
] preceded
bla
VIM-2
. AAC(6′)-32 showed 80% identity to AAC(6′)-Ib′ and the recently described AAC(6′)-31, and when
aac(6′)-32
was cloned into
Escherichia coli
, it conferred resistance to tobramycin and reduced susceptibility to gentamicin and amikacin. Despite the currently low prevalence of epidemic clones with MDR, active surveillance is needed to detect and prevent the dissemination of these clones, particularly those producing integron- and plasmid-encoded MBLs, given their additional capacity for the intra- and interspecies spread of MDR.
In Vitro and In Vivo Antibacterial Activities of DC-159a, a New Fluoroquinolone
Authors: Hoshino Kazuki , Inoue Kazue , Murakami Yoichi , Kurosaka Yuichi , Namba Kenji , Kashimoto Yoshinori , Uoyama Saori , Okumura Ryo , Higuchi Saito , Otani Tsuyoshi
Published: October 2007

DC-159a is a new 8-methoxy fluoroquinolone that possesses a broad spectrum of antibacterial activity, with extended activity against gram-positive pathogens, especially streptococci and staphylococci from patients with community-acquired infections. DC-159a showed activity against
Streptococcus
spp. (MIC
90
, 0.12 μg/ml) and inhibited the growth of 90% of levofloxacin-intermediate and -resistant strains at 1 μg/ml. The MIC
90
s of DC-159a against
Staphylococcus
spp. were 0.5 μg/ml or less. Against quinolone- and methicillin-resistant
Staphylococcus aureus
strains, however, the MIC
90
of DC-159a was 8 μg/ml. DC-159a was the most active against
Enterococcus
spp. (MIC
90
, 4 to 8 μg/ml) and was more active than the marketed fluoroquinolones, such as levofloxacin, ciprofloxacin, and moxifloxacin. The MIC
90
s of DC-159a against
Haemophilus influenzae, Moraxella catarrhalis
, and
Klebsiella pneumoniae
were 0.015, 0.06, and 0.25 μg/ml, respectively. The activity of DC-159a against
Mycoplasma pneumoniae
was eightfold more potent than that of levofloxacin. The MICs of DC-159a against
Chlamydophila pneumoniae
were comparable to those of moxifloxacin, and DC-159a was more potent than levofloxacin. The MIC
90
s of DC-159a against
Peptostreptococcus
spp.,
Clostridium difficile
, and
Bacteroides fragilis
were 0.5, 4, and 2 μg/ml, respectively; and among the quinolones tested it showed the highest level of activity against anaerobic organisms. DC-159a demonstrated rapid bactericidal activity against quinolone-resistant
Streptococcus pneumoniae
strains both in vitro and in vivo. In vitro, DC-159a showed faster killing than moxifloxacin and garenoxacin. The bactericidal activity of DC-159a in a murine muscle infection model was revealed to be superior to that of moxifloxacin. These activities carried over to the in vivo efficacy in the murine pneumonia model, in which treatment with DC-159a led to bactericidal activity superior to those of the other agents tested.
The PhoQ-Activating Potential of Antimicrobial Peptides Contributes to Antimicrobial Efficacy and Is Predictive of the Induction of Bacterial Resistance
Authors: Kindrachuk Jason , Paur Nicole , Reiman Carla , Scruten Erin , Napper Scott
Published: October 2007

Antimicrobial peptides (AMPs) are among the leading candidates to replace antibiotics which have been rendered ineffective by the evolution of resistant bacterial strains. Concerns do exist, however, that the therapeutic administration of AMPs may also select for resistant strains but with much more dire consequences, as these peptides represent an endogenous and essential component of host immune defense. The recent demonstration that AMPs function as ligands for the bacterial sensory kinase PhoQ for the initiation of virulence and adaptive responses lends credence to these concerns. While the ability to serve as PhoQ ligands suggests that the therapeutic administration of AMPs could (i) exacerbate infections by promoting bacterial virulence and (ii) select resistant mutants by encouraging adaptive behaviors, it also provides a rational basis for AMP selection and optimization. Here, we demonstrate that derivatives of a representative AMP have differential abilities to serve as PhoQ ligands and that this correlates with the ability to induce bacterial adaptive responses. We propose that PhoQ-activating potential is a logical parameter for AMP optimization and introduce a novel strategy for the treatment of minimal bactericidal concentration data that permits the discrimination and quantification of the contributions of PhoQ-activating potential and direct antimicrobial activity to net antimicrobial efficiency.
Differential Activities of Newer Antifungal Agents against
Candida albicans
and
Candida parapsilosis
Biofilms
Authors: Katragkou Αspasia , Chatzimoschou Athanasios , Simitsopoulou Maria , Dalakiouridou Maria , Diza-Mataftsi Eudoxia , Tsantali Chaido , Roilides Emmanuel
Published: October 2007

The activities of voriconazole, posaconazole, caspofungin, and anidulafungin against
Candida albicans
and
Candida parapsilosis
biofilms were evaluated. In contrast to planktonic cells, the MICs for voriconazole and posaconazole for the biofilms of the two species were high (≥256 and >64 mg/liter, respectively) but relatively low for the echinocandins caspofungin and anidulafungin (≤1 and ≤2 mg/liter, respectively).
Comparative Antianaerobic Activities of Doripenem Determined by MIC and Time-Kill Analysis
Authors: Credito Kim L. , Ednie Lois M. , Appelbaum Peter C.
Published: October 2007

Against 447 anaerobe strains, the investigational carbapenem doripenem had an MIC50of 0.125 μg/ml and an MIC90of 1 μg/ml. Results were similar to those for imipenem, meropenem, and ertapenem. Time-kill studies showed that doripenem had very good bactericidal activity compared to other carbapenems, with 99.9% killing of 11 strains at 2× MIC after 48 h.
In Vitro Activity of DC-159a, a New Broad-Spectrum Fluoroquinolone, Compared with That of Other Agents against Drug-Susceptible and -Resistant Pneumococci
Authors: Clark Catherine , Smith Kathy , Ednie Lois , Bogdanovich Tatiana , Dewasse Bonifacio , McGhee Pamela , Appelbaum Peter C.
Published: October 2007

DC-159a yielded MICs of ≤1 μg/ml against 316 strains of both quinolone-susceptible and -resistant pneumococci (resistance was defined as a levofloxacin MIC ≥4 μg/ml). Although the MICs for DC-159a against quinolone-susceptible pneumococci were a few dilutions higher than those of gemifloxacin, the MICs of these two compounds against 28 quinolone-resistant pneumococci were identical. The DC-159a MICs against quinolone-resistant strains did not appear to depend on the number or the type of mutations in the quinolone resistance-determining region. DC-159a, as well as the other quinolones tested, was bactericidal after 24 h at 2× MIC against 11 of 12 strains tested. Two of the strains were additionally tested at 1 and 2 h, and DC-159a at 4× MIC showed significant killing as early as 2 h. Multistep resistance selection studies showed that even after 50 consecutive subcultures of 10 strains in the presence of sub-MICs, DC-159a produced only two mutants with maximum MICs of 1 μg/ml.
Failure of Current Cefepime Breakpoints To Predict Clinical Outcomes of Bacteremia Caused by Gram-Negative Organisms
Authors: Bhat Sunil V. , Peleg Anton Y. , Lodise Thomas P. , Shutt Kathleen A. , Capitano Blair , Potoski Brian A. , Paterson David L.
Published: October 2007

For commonly encountered gram-negative bacilli, a MIC of cefepime of 8 μg/ml or less was defined by the Clinical and Laboratory Standards Institute as “susceptible” prior to the commercial release of the antibiotic. We assessed 204 episodes of bacteremia caused by gram-negative organisms for which patients received cefepime (typically 1 to 2 g every 12 h) as the primary mode of therapy. The cefepime MIC breakpoint derived by classification and regression tree (CART) software analysis to delineate the risk of 28-day mortality was 8 μg/ml. Patients infected with gram-negative organisms treated with cefepime at a MIC of ≥8 μg/ml had a mortality rate of 54.8% (17/31 died), compared to 24.1% (35/145 died) for those treated with a cefepime MIC of <8 μg/ml. The rate of mortality for those treated with a cefepime MIC of 8 μg/ml was 56.3% (9/16 died), compared to 53.3% (8/15 died) for those treated with cefepime at a MIC of >8 μg/ml. A multivariable analysis including severity of illness indices showed that treating patients with bacteremia due to gram-negative organisms with a cefepime MIC of ≥8 μg/ml was an independent predictor of mortality (
P
≤ 0.001). There was no significant difference in outcome according to the dosage regimen utilized. Pharmacodynamic assessments that were presented previously would suggest that cefepime treatment (particularly a dosage of 1 g every 12 h) has a low probability of target attainment associated with successful in vivo outcome when the cefepime MIC is ≥8 μg/ml. It would appear reasonable, based on pharmacodynamic and clinical grounds, to lower the breakpoints for cefepime in countries where the cefepime dosage of 1 to 2 g every 12 h is the licensed therapy for serious infections, so that organisms with a cefepime MIC of 8 μg/ml are no longer regarded as susceptible to the antibiotic.
Nickel Resistance Determinants in
Bradyrhizobium
Strains from Nodules of the Endemic New Caledonia Legume
Serianthes calycina
Authors: Chaintreuil Clémence , Rigault Frédéric , Moulin Lionel , Jaffré Tanguy , Fardoux Joël , Giraud Eric , Dreyfus Bernard , Bailly Xavier
Published: October 2007

Bradyrhizobium
strains, isolated in New Caledonia from nodules of the endemic legume
Serianthes calycina
growing in nickel-rich soils, were able to grow in the presence of 15 mM NiCl
2
. The genomes of these strains harbored two Ni resistance determinants, the
cnr
and
nre
operons. By constructing a
cnrA
mutant, we demonstrated that the
cnr
operon determines the high nickel resistance in
Bradyrhizobium
strains.
Mat Formation in
Saccharomyces cerevisiae
Requires Nutrient and pH Gradients
Authors: Reynolds Todd B. , Jansen An , Peng Xin , Fink Gerald R.
Published: October 2007

The ability of
Saccharomyces cerevisiae
to form morphologically complex colony-like structures called mats requires expression of the cell surface glycoprotein Flo11p and growth on a semisolid surface. As the mat grows, it forms two visually distinct populations called the rim (edge of the mat) and the hub (interior of the mat), which can be physically separated from one another based on their agar adherence properties. Here, we show that growth of the mat on a semisolid agar surface creates concentric glucose and pH gradients in the medium that are required for the differentiation of the hub and rim. Disruption of the pathways that respond to changing levels of glucose block mat formation by decreasing
FLO11
expression. However, in wild-type cells, Flo11p is expressed in both portions of the structure. The difference in adherence between the rim and hub appears to be a consequence of the reduced adherence of Flo11p at the elevated pH of the rim.
Genome-Wide Transcriptional Profiling of the Cyclic AMP-Dependent Signaling Pathway during Morphogenic Transitions of
Candida albicans
Authors: Bahn Yong-Sun , Molenda Matthew , Staab Janet F. , Lyman Courtney A. , Gordon Laura J. , Sundstrom Paula
Published: October 2007

Candida albicans
is an opportunistic human fungal pathogen that causes systemic candidiasis as well as superficial mucosal candidiasis. In response to the host environment,
C. albicans
transitions between yeast and hyphal forms. In particular, hyphal growth is important in facilitating adhesion and invasion of host tissues, concomitant with the expression of various hypha-specific virulence factors. In previous work, we showed that the cyclic AMP (cAMP) signaling pathway plays a crucial role in morphogenic transitions and virulence of
C. albicans
by studying genes encoding adenylate cyclase-associated protein (
CAP1
) and high-affinity phosphodiesterase (
PDE2
) (Y. S. Bahn, J. Staab, and P. Sundstrom, Mol. Microbiol.
50:
391-409, 2003; and Y. S. Bahn and P. Sundstrom, J. Bacteriol.
183:
3211-3223, 2001). However, little is known about the downstream targets of the cAMP signaling pathway that are responsible for morphological transitions and the expression of virulence factors. Here, microarrays were probed with RNA from strains with hypoactive (
cap1/cap1
null mutant), hyperactive (
pde2/pde2
null mutant), and wild-type cAMP signaling pathways to provide insight into the molecular mechanisms of virulence that are regulated by cAMP and that are related to the morphogenesis of
C. albicans
. Genes controlling metabolic specialization, cell wall structure, ergosterol/lipid biosynthesis, and stress responses were modulated by cAMP during hypha formation. Phenotypic traits predicted to be regulated by cAMP from the profiling results correlated with the relative strengths of the mutants when tested for resistance to azoles and subjected to heat shock stress and oxidative/nitrosative stress. The results from this study provide important insights into the role of the cAMP signaling pathway not only in morphogenic transitions of
C. albicans
but also for adaptation to stress and for survival during host infections.
cis
-Acting Elements within the
Candida albicans ERG11
Promoter Mediate the Azole Response through Transcription Factor Upc2p
Authors: Oliver Brian G. , Song Jia L. , Choiniere Jake H. , White Theodore C.
Published: October 2007

The azole antifungal drugs are used to treat infections caused by
Candida albicans
and other fungi. These drugs interfere with the biosynthesis of ergosterol, the major sterol in fungal cells, by inhibiting an ergosterol biosynthetic enzyme, lanosterol 14 α-demethylase, encoded by the
ERG11
gene. In vitro, these drugs as well as other ergosterol biosynthesis inhibitors increase
ERG11
mRNA expression by activation of the
ERG11
promoter. The signal for this activation most likely is the depletion of ergosterol, the end product of the pathway. To identify
cis
-acting regulatory elements that mediate this activation,
ERG11
promoter fragments have been fused to the luciferase reporter gene from
Renilla reniformis
. Promoter deletions and linker scan mutations localized the region important for azole induction to a segment from bp −224 to −251 upstream of the start codon, specifically two 7-bp sequences separated by 13 bp. These sequences form an imperfect inverted repeat. The region is recognized by the transcription factor Upc2p and functions as an enhancer of transcription, as it can be placed upstream of a heterologous promoter in either direction, resulting in the azole induction of that promoter. The promoter constructs are not azole inducible in the
upc2/upc2
homozygous deletion, demonstrating that Upc2p controls the azole induction of
ERG11
. These results identify an azole-responsive enhancer element (ARE) in the
ERG11
promoter that is controlled by the Upc2p transcription factor. No other ARE is present in the promoter. Thus, this ARE and Upc2p are necessary and sufficient for azole induction of
ERG11
.
Ssk2 Mitogen-Activated Protein Kinase Kinase Kinase Governs Divergent Patterns of the Stress-Activated Hog1 Signaling Pathway in
Cryptococcus neoformans
Authors: Bahn Yong-Sun , Geunes-Boyer Scarlett , Heitman Joseph
Published: October 2007

The stress-activated p38/Hog1 mitogen-activated protein kinase (MAPK) pathway is structurally conserved in many diverse organisms, including fungi and mammals, and modulates myriad cellular functions. The Hog1 pathway is uniquely specialized to control differentiation and virulence factors in a majority of clinical
Cryptococcus neoformans
serotype A and D strains. Here, we identified and characterized the Ssk2 MAPKKK that functions upstream of the MAPKK Pbs2 and the MAPK Hog1 in
C. neoformans
. The
SSK2
gene was identified as a potential component responsible for the difference in Hog1 phosphorylation between the serotype D f1 sibling strains B-3501 and B-3502 through comparative analysis of meiotic maps showing their meiotic segregation patterns of Hog1-dependent sensitivity to the antifungal drug fludioxonil. Ssk2 is the only component of the Hog1 MAPK cascade that is polymorphic between the two strains, and the B-3501 and B-3502
SSK2
alleles were distinguished by two coding sequence changes. Supporting this finding,
SSK2
allele exchange completely interchanged the Hog1-controlled signaling patterns, related phenotypes, and virulence levels of strains B-3501 and JEC21. In the serotype A strain H99, disruption of the
SSK2
gene enhanced capsule and melanin biosynthesis and mating efficiency, similar to
pbs2
and
hog1
mutations. Furthermore,
ssk2
Δ,
pbs2
Δ, and
hog1
Δ mutants were hypersensitive to a variety of stresses and resistant to fludioxonil. In agreement with these results, Hog1 phosphorylation was abolished in the
ssk2
Δ mutant, similar to what occurred in the
pbs2
Δ mutant. Taken together, these findings indicate that Ssk2 is a critical interface connecting the two-component system and the Pbs2-Hog1 MAPK pathway in
C. neoformans
.
Fibrotic Response as a Distinguishing Feature of Resistance and Susceptibility to Pulmonary Infection with
Mycobacterium tuberculosis
in Mice
Authors: Marquis Jean-François , Nantel André , LaCourse Ronald , Ryan Lynn , North Robert J. , Gros Philippe
Published: October 2007

The differential susceptibility of inbred mouse strains DBA/2J (susceptible) and C57BL/6J (resistant) to pulmonary tuberculosis following aerosol infection is under complex genetic control. In this report, transcriptional profiling with RNAs from
Mycobacterium tuberculosis
-infected lungs was used to investigate the physiological response, cell type, and biochemical pathways underlying differential susceptibility to infection. Statistical analysis of cDNA-based microarrays revealed that 1,097 transcripts showed statistically significant changes in abundance (changes of ≥1.5-fold) in at least one of four experimental group comparisons (C57BL/6J [day 0] versus DBA/2J [day 0] mice, C57BL/6J [day 90] versus DBA/2J [day 90] mice, C57BL/6J [day 90] versus C57BL/6J [day 0] mice, or DBA/2J [day 90] versus DBA/2J [day 0] mice). A group of genes showing very high degrees of significance (changes of ≥2.0-fold) displayed enrichment for transcripts associated with tissue remodeling and the fibrotic response. The differential expression of fibrotic response genes (
Sparc
,
Col1a1
,
Col1a2
,
Col4a1
, and
Col4a2
) in the infected lungs of the two mouse strains was validated by another microarray platform (Affymetrix oligonucleotide chips) and by reverse transcription-PCR. Furthermore, the differential expression of additional genes known to be associated with fibrosis (
Mmp2
,
Timp1
, and
Arg1
) was also validated by these approaches. Overall, these results identify the differential fibrotic response as a pathological basis for the high susceptibility of DBA/2J mice to pulmonary tuberculosis.
Impairment of Infectivity and Immunoprotective Effect of a
LYT1
Null Mutant of
Trypanosoma cruzi
Authors: Zago M. Paola , Barrio Alejandra B. , Cardozo Rubén M. , Duffy Tomás , Schijman Alejandro G. , Basombrío Miguel A.
Published: October 2007

Trypanosoma cruzi
infection of host cells is a complex process in which many proteins participate but only a few of these proteins have been identified experimentally. One parasite factor likely to be involved is the protein product of
LYT1
, a single-copy gene cloned, sequenced, and characterized by Manning-Cela et al. (Infect. Immun. 69:3916-3923, 2001). This gene was potentially associated with infectivity, since the deletion of both
LYT1
alleles in the CL Brenner strain (the wild type [WT]) resulted in a null mutant
T. cruzi
clone (L16) that shows an attenuated phenotype in cell culture models. The aim of this work was to characterize the infective behavior of L16 in the insect vector and murine models. The infection of adult Swiss mice with 10
3
trypomastigotes of both clones revealed a significant reduction in infective behavior of L16, as shown by direct parasitemia, spleen index, and quantitation of tissue parasite burden, suggesting the loss of virulence in the null mutant clone. Although L16 blood counts were almost undetectable, blood-based PCRs indicated the presence of latent and persistent infection during all of the study period and epimastigotes were reisolated from hemocultures until 12 months postinfection. Nevertheless, virulence was not restored in L16 by serial passages in mice, and reisolated parasites lacking the
LYT1
gene and bearing the antibiotic resistance genes revealed the stability of the genetic manipulation. Histopathological studies showed a strong diminution in the muscle inflammatory response triggered by L16 compared to that triggered by the WT group, consistent with a lower tissue parasite load. A strong protection against a virulent challenge in both L16- and WT-infected mice was observed; however, the immunizing infection by the genetically modified parasite was highly attenuated.
Essential Role of Chitinase in the Development of the Filarial Nematode
Acanthocheilonema viteae
Authors: Tachu Babila , Pillai Smitha , Lucius Richard , Pogonka Thomas
Published: October 2007

Chitinases of pathogens have been proposed as potential targets of vaccines or specific inhibitors. We studied the genomic organization, transcript levels, developmental expression, and biological function of chitinases in the rodent filarial nematode
Acanthocheilonema viteae
, a model organism for human-pathogenic filarial worms. Characterization of nine genomic clones from an
A. viteae
phage library and Southern blot experiments revealed the existence of three different chitinase genes, two of which could theoretically yield functional transcripts. The deduced proteins of these genes had the common modular organization of family 18 chitinases. Northern blot experiments and rapid amplification of cDNA ends-PCR with adult worms and larval stages showed that only one gene is expressed, with high variation in transcript levels, as determined by real-time PCR. Chitinase transcript levels were lowest in the late male stage 4 larva (L4) and peaked in the stage 3 larva (L3), which was corroborated by Western blotting. RNA interference (RNAi) experiments showed that treatment of L3 and adult female worms with double-stranded RNA of chitinase inhibited molting of L3 worms and hatching of microfilariae. RNAi also led to the death of 50% of female worms, revealing the essential role of chitinase in the life cycle of filarial nematodes.
Mechanisms of Dexamethasone-Mediated Inhibition of Toll-Like Receptor Signaling Induced byNeisseria meningitidisandStreptococcus pneumoniae
Authors: Mogensen Trine H. , Berg Randi S. , Paludan Søren R. , Østergaard Lars
Published: October 2007

Excessive inflammation contributes to the pathogenesis of bacterial meningitis, which remains a serious disease despite treatment with antibiotics. Therefore, anti-inflammatory drugs have important therapeutic potential, and clinical trials have revealed that early treatment with dexamethasone significantly reduces mortality and morbidity from bacterial meningitis. Here we investigate the molecular mechanisms behind the inhibitory effect of dexamethasone upon the inflammatory responses evoked byNeisseria meningitidisandStreptococcus pneumoniae, two of the major causes of bacterial meningitis. The inflammatory cytokine response was dependent on Toll-like receptor signaling and was strongly inhibited by dexamethasone. Activation of the NF-κB pathway was targeted at several levels, including inhibition of IκB phosphorylation and NF-κB DNA-binding activity as well as upregulation of IκBα synthesis. Our data also revealed that the timing of steroid treatment relative to infection was important for achieving strong inhibition, particularly in response toS. pneumoniae. Altogether, we describe important targets of dexamethasone in the inflammatory responses evoked byN. meningitidisandS. pneumoniae, which may contribute to our understanding of the clinical effect and the importance of timing with respect to corticosteroid treatment during bacterial meningitis.
Interaction ofBrucella suisandBrucella abortusRough Strains with Human Dendritic Cells
Authors: Billard Elisabeth , Dornand Jacques , Gross Antoine
Published: October 2007

Brucellais a facultative intracellular pathogen of various mammals and the etiological agent of brucellosis. We recently demonstrated that dendritic cells (DCs), which are critical components of adaptive immunity, are highly susceptible toBrucellainfection. Furthermore,Brucellaprevented the infected DCs from engaging in maturation processes and impaired their capacity to present antigen to naive T cells and to secrete interleukin-12 (IL-12). The lipopolysaccharide (LPS) phenotype is largely associated with the virulence ofBrucella. Depending on whether they express the O-side chain of LPS or not, the bacteria display a smooth or rough phenotype. RoughBrucellamutants are attenuated and induce a potent protective T-cell-dependent immune response. Due to the essential role of DCs in the initiation of T-cell-dependent adaptive immune responses, it seemed pertinent to study the interaction between roughBrucellastrains and human DCs. In the present paper, we report that, in contrast to smooth bacteria, infection of DCs with rough mutants ofBrucella suisorBrucella abortusleads to both phenotypic and functional maturation of infected cells. Rough mutant-infected DCs then acquire the capacity to produce IL-12 and to stimulate naive CD4+T lymphocytes. Experiments with rough and smooth purified LPS ofBrucellasupported the hypothesis of an indirect involvement of the O-side chain. These results provide new data concerning the role of LPS inBrucellavirulence strategy and illuminate phenomena contributing to immune protection conferred by rough vaccine strains.
Phagocytosis ofBorrelia burgdorferi, the Lyme Disease Spirochete, Potentiates Innate Immune Activation and Induces Apoptosis in Human Monocytes
Authors: Cruz Adriana R. , Moore Meagan W. , La Vake Carson J. , Eggers Christian H. , Salazar Juan C. , Radolf Justin D.
Published: October 2007

We have previously demonstrated that phagocytosedBorrelia burgdorferiinduces activation programs in human peripheral blood mononuclear cells that differ qualitatively and quantitatively from those evoked by equivalent lipoprotein-rich lysates. Here we report that ingestedB. burgdorferiinduces significantly greater transcription of proinflammatory cytokine genes than do lysates and that liveB. burgdorferi, but notB. burgdorferilysate, is avidly internalized by monocytes, where the bacteria are completely degraded within phagolysosomes. In the course of these experiments, we discovered that liveB. burgdorferialso induced a dose-dependent decrease in monocytes but not a decrease in dendritic cells or T cells and that the monocyte population displayed morphological and biochemical hallmarks of apoptosis. Particularly noteworthy was the finding that apoptotic changes occurred predominantly in monocytes that had internalized spirochetes. Abrogation of phagocytosis with cytochalasin D prevented the death response. Heat-killedB. burgdorferi, which was internalized as well as live organisms, induced a similar degree of apoptosis of monocytes but markedly less cytokine production. Surprisingly, opsonophagocytosis ofTreponema pallidumdid not elicit a discernible cell death response. Our combined results demonstrate thatB. burgdorfericonfined to phagolysosomes is a potent inducer of cytosolic signals that result in (i) production of NF-κB-dependent cytokines, (ii) assembly of the inflammasome and activation of caspase-1, and (iii) induction of programmed cell death. We propose that inflammation and apoptosis represent mutually reinforcing components of the immunologic arsenal that the host mobilizes to defend itself against infection with Lyme disease spirochetes.
α-Enolase Resides on the Cell Surface of
Mycoplasma fermentans
and Binds Plasminogen
Authors: Yavlovich Amichai , Rechnitzer Hagai , Rottem Shlomo
Published: October 2007

Plasminogen (Plg) binding to the cell surface of
Mycoplasma fermentans
results in a marked increase in the maximal adherence of the organism to HeLa cells, enhanced Plg activation by the urokinase-type Plg activator, and the induction of the internalization of
M. fermentans
by eukaryotic host cells (A. Yavlovich, A. Katzenell, M. Tarshis, A. A. Higazi, and S. Rottem, Infect. Immun. 72:5004-5011, 2004). In this study, the
M. fermentans
Plg binding protein was isolated by affinity chromatography of Triton X-100-solubilized
M. fermentans
membranes by utilizing a column of a Plg-biotin complex attached to avidin that was eluted with ε-aminocaproic acid. The eluted ∼50-kDa protein was identified by mass spectrometric techniques as α-enolase. The possibility that α-enolase, a key cytoplasmatic glycolytic enzyme, resides also on the cell surface of
M. fermentans
was supported by an immunoblot analysis using polyclonal anti-α-enolase antiserum, which showed that α-enolase was present in a purified
M. fermentans
membrane preparation, as well as by immunochemical criteria and by immunoelectron microscopy analysis. Our observation that Plg blocked the binding of anti-α-enolase antibodies to a 50-kDa polypeptide band resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of
M. fermentans
membrane or soluble preparations further supports our notion that mycoplasmal surface α-enolase is a major Plg binding protein of
M. fermentans
.
Flagellin Suppresses the Inflammatory Response and Enhances Bacterial Clearance in a Murine Model ofPseudomonas aeruginosaKeratitis
Authors: Kumar Ashok , Hazlett Linda D. , Yu Fu-Shin X.
Published: October 2007

Pseudomonas aeruginosais a common organism associated with bacterial keratitis, especially in extended-wear contact lens users. In the present study, we determined that pretreatment of cultured human corneal epithelial cells with flagellin isolated from theP. aeruginosaPAO1 strain attenuated cytokine production when the cells were challenged with a cytotoxic strain (ATCC 19660), suggesting a potential use of bacterial flagellin to downregulate infection-associated inflammation in vivo. Administration of flagellin via the subconjunctival and intraperitoneal routes 24 h prior toPseudomonasinoculation significantly improved the disease outcome, preserved structural integrity and transparency, and thus maintained vision in otherwise perforated corneas of C57BL/6 (B6) mice. The flagellin pretreatment resulted in suppression of polymorphonuclear leukocyte infiltration at a late stage of infection but not at an early stage of infection, decreased the expression of proinflammatory cytokine genes (genes encoding interleukin-1β [IL-1β], macrophage inflammatory protein 2, IL-12, and gamma interferon), and greatly enhanced bacterial clearance in the corneas of B6 mice probably through induced expression of the cathelicidin-related antimicrobial peptide and inducible nitric oxide synthase. This is the first report that describes the protective mechanisms induced by a Toll-like receptor agonist that not only curbs the host inflammatory response but also eliminates invading bacteria in the B6 mouse cornea.
TheMycobacterium aviumsubsp.paratuberculosisMAP3464 Gene Encodes an Oxidoreductase Involved in Invasion of Bovine Epithelial Cells through the Activation of Host Cell Cdc42
Authors: Alonso-Hearn Marta , Patel Dilip , Danelishvili Lia , Meunier-Goddik Lisbeth , Bermudez Luiz E.
Published: October 2007

Mycobacterium aviumsubsp.paratuberculosisinfection of cattle takes place through the intestinal mucosa. To identifyM.aviumsubsp.paratuberculosisgenes associated with the invasion of bovine epithelial cells in vitro, we screened a library of transposon mutants. Several mutants ofM.aviumsubsp.paratuberculosiswere identified which invaded Madin-Darby bovine kidney (MDBK) epithelial cells less efficiently than wild-type (wt)M.aviumsubsp.paratuberculosis. The ΔOx mutant had the transposon located in the MAP3464 gene, a putative oxidoreductase gene whose expression is upregulated upon bacterial contact with MDBK cells. Complete restoration of invasion comparable to that for the wt bacterium was achieved by introducing a copy of the complete oxidoreductase operon into the ΔOx mutant. Immunoprecipitation and Western blot analysis indicated that wtM.aviumsubsp.paratuberculosisactivates Cdc42 and RhoA pathways of internalization 15 and 60 min after infection of the host cell, respectively. The ΔOx mutant, however, failed to activate the Cdc42 pathway. To determine whether anM.aviumsubsp.paratuberculosisprotein delivered to the host cell mediates the entry of the wt bacterium by activation of the Cdc42 pathway, affinity precipitation of active Cdc42 from MDBK-infected cells followed by mass spectrometry was carried out. We identified a 17-amino-acid bacterial peptide associated with the Cdc42 of cells infected with wtM.aviumsubsp.paratuberculosisbut not with the ΔOx mutant. The sequence of the peptide matches MAP3985c, a hypothetical protein, possibly functioning as a putative Cdc42 effector. These findings reveal a novel signaling pathway activated duringM.aviumsubsp.paratuberculosisentry that links the product of MAP3464 gene to activation of Cdc42 in the host cell.
Genetic and Functional Analyses of PptA, a Phospho-Form Transferase Targeting Type IV Pili inNeisseria gonorrhoeae
Authors: Næssan Cecilia L. , Egge-Jacobsen Wolfgang , Heiniger Ryan W. , Wolfgang Matthew C. , Aas Finn Erik , Røhr Åsmund , Winther-Larsen Hanne C. , Koomey Michael
Published: October 2007

The PilE pilin subunit protein ofNeisseria gonorrhoeaeundergoes unique covalent modifications with phosphoethanolamine (PE) and phosphocholine (PC). Thepilinphospho-formtransferase A (PptA) protein, required for these modifications, shows sequence relatedness with and architectural similarities to lipopolysaccharide PE transferases. Here, we used regulated expression and mutagenesis as means to better define the relationships between PptA structure and function, as well as to probe the mechanisms by which other factors impact the system. We show here thatpptAexpression is coupled at the level of transcription to its distal gene,murF, in a division/cell wall gene operon and that PptA can act in a dose-dependent fashion in PilE phospho-form modification. Molecular modeling and site-directed mutagenesis provided the first direct evidence that PptA is a member of the alkaline phosphatase superfamily of metalloenzymes with similar metal-binding sites and conserved structural folds. Through phylogenetic analyses and sequence alignments, these conclusions were extended to include the lipopolysaccharide PE transferases, including members of the disparate Lpt6 subfamily, and the MdoB family of phosphoglycerol transferases. Each of these enzymes thus likely acts as a phospholipid head group transferase whose catalytic mechanism involves atrans-esterification step generating a protein-phospho-form ester intermediate. Coexpression of PptA with PilE inPseudomonas aeruginosaresulted in high levels of PE modification but was not sufficient for PC modification. This and other findings show that PptA-associated PC modification is governed by as-yet-undefined ancillary factors unique toN. gonorrhoeae.
O-Linked Glycosylation Ensures the Normal Conformation of the Autotransporter Adhesin Involved in Diffuse Adherence
Authors: Charbonneau Marie-Ève , Girard Victoria , Nikolakakis Anastasia , Campos Manuel , Berthiaume Frédéric , Dumas France , Lépine François , Mourez Michael
Published: October 2007

The
Escherichia coli
adhesin involved in diffuse adherence (AIDA-I) is one of the few glycosylated proteins found in
Escherichia coli
. Glycosylation is mediated by a specific heptosyltransferase encoded by the
aah
gene, but little is known about the role of this modification and the mechanism involved. In this study, we identified several peptides of AIDA-I modified by the addition of heptoses by use of mass spectrometry and N-terminal sequencing of proteolytic fragments of AIDA-I. One threonine and 15 serine residues were identified as bearing heptoses, thus demonstrating for the first time that AIDA-I is O-glycosylated. We observed that unglycosylated AIDA-I is expressed in smaller amounts than its glycosylated counterpart and shows extensive signs of degradation upon heat extraction. We also observed that unglycosylated AIDA-I is more sensitive to proteases and induces important extracytoplasmic stress. Lastly, as was previously shown, we noted that glycosylation is required for AIDA-I to mediate adhesion to cultured epithelial cells, but purified mature AIDA-I fused to GST was found to bind in vitro to cells whether or not it was glycosylated. Taken together, our results suggest that glycosylation is required to ensure a normal conformation of AIDA-I and may be only indirectly necessary for its cell-binding function.