bluuebunny/crossref_metadata_2025_split huggingface dataset:
Novel Quantitative Biosystem for Modeling Physiological Fluid Shear Stress on Cells
Authors: Nauman Eric A. , Ott C. Mark , Sander Ed , Tucker Don L. , Pierson Duane , Wilson James W. , Nickerson Cheryl A. 
Published: December 2006

ABSTRACT

The response of microbes to changes in the mechanical force of fluid shear has important implications for pathogens, which experience wide fluctuations in fluid shear in vivo during infection. However, the majority of studies have not cultured microbes under physiological fluid shear conditions within a range commonly encountered by microbes during host-pathogen interactions. Here we describe a convenient batch culture biosystem in which (i) the levels of fluid shear force can be varied within physiologically relevant ranges and quantified via mathematical models and (ii) large numbers of cells can be planktonically grown and harvested to examine the effect of fluid shear levels on microbial genomic and phenotypic responses. A quantitative model based on numerical simulations and in situ imaging analysis was developed to calculate the fluid shear imparted by spherical beads of different sizes on bacterial cell cultures grown in a rotating wall vessel (RWV) bioreactor. To demonstrate the application of this model, we subjected cultures of the bacterial pathogen
Salmonella enterica
serovar Typhimurium to three physiologically-relevant fluid shear ranges during growth in the RVW and demonstrated a progressive relationship between the applied fluid shear and the bacterial genetic and phenotypic responses. By applying this model to different cell types, including other bacterial pathogens, entire classes of genes and proteins involved in cellular interactions may be discovered that have not previously been identified during growth under conventional culture conditions, leading to new targets for vaccine and therapeutic development.
Role of Protein
O
-Mannosyltransferase Pmt4 in the Morphogenesis and Virulence of
Cryptococcus neoformans
Authors: Olson Gillian M. , Fox Deborah S. , Wang Ping , Alspaugh J. Andrew , Buchanan Kent L. 
Published: December 2006

ABSTRACT

Protein O mannosylation is initiated in the endoplasmic reticulum by protein
O
-mannosyltransferases (Pmt proteins) and plays an important role in the secretion, localization, and function of many proteins, as well as in cell wall integrity and morphogenesis in fungi. Three Pmt proteins, each belonging to one of the three respective Pmt subfamilies, are encoded in the genome of the human fungal pathogen
Cryptococcus neoformans
. Disruption of the
C. neoformans PMT4
gene resulted in abnormal growth morphology and defective cell separation. Transmission electron microscopy revealed defective cell wall septum degradation during mother-daughter cell separation in the
pmt4
mutant compared to wild-type cells. The
pmt4
mutant also demonstrated sensitivity to elevated temperature, sodium dodecyl sulfate, and amphotericin B, suggesting cell wall defects. Further analysis of cell wall protein composition revealed a cell wall proteome defect in the
pmt4
mutant, as well as a global decrease in protein mannosylation. Heterologous expression of
C. neoformans PMT4
in a
Saccharomyces cerevisiae pmt1pmt4
mutant strain functionally complemented the deficient Pmt activity. Furthermore, Pmt4 activity in
C. neoformans
was required for full virulence in two murine models of disseminated cryptococcal infection. Taken together, these results indicate a central role for Pmt4-mediated protein O mannosylation in growth, cell wall integrity, and virulence of
C. neoformans
.
Transmembrane Molecules for Phylogenetic Analyses of Pathogenic Protists:
Leishmania
-Specific Informative Sites in Hydrophilic Loops of Trans- Endoplasmic Reticulum
N
-Acetylglucosamine-1-Phosphate Transferase
Authors: Waki Kayoko , Dutta Sujoy , Ray Debalina , Kolli Bala Krishna , Akman Leyla , Kawazu Shin-Ichiro , Lin Chung-Ping , Chang Kwang-Poo 
Published: December 2006

ABSTRACT

A sequence database was created for the
Leishmania N
-acetylglucosamine-1-phosphate transferase (
nagt
) gene from 193 independent isolates. PCR products of this single-copy gene were analyzed for restriction fragment length polymorphism based on seven
nagt
sequences initially available. We subsequently sequenced 77 samples and found 19 new variants (genotypes). Alignment of all 26
nagt
sequences is gap free, except for a single codon addition or deletion. Phylogenetic analyses of the sequences allow grouping the isolates into three subgenera, each consisting of recognized species complexes, i.e., subgenus
Leishmania
(
L. amazonensis-L. mexicana
,
L. donovani-L. infantum
,
L. tropica
,
L. major
, and
L. turanica-L. gerbilli
), subgenus
Viannia
(
L. braziliensis
,
L. panamensis
), and one unclassified (
L. enriettii
) species. This hierarchy of grouping is also supported by sequence analyses of selected samples for additional single-copy genes present on different chromosomes. Intraspecies divergence of
nagt
varies considerably with different species complexes. Interestingly, species complexes with less subspecies divergence are more widely distributed than those that are more divergent. The relevance of this to
Leishmania
evolutionary adaptation is discussed. Heterozygosity of subspecies variants contributes to intraspecies diversity, which is prominent in
L. tropica
but not in
L. donovani-L. infantum.
This disparity is thought to result from the genetic recombination of the respective species at different times as a rare event during their predominantly clonal evolution. Phylogenetically useful sites of
nagt
are restricted largely to several extended hydrophilic loops predicted from hypothetical models of
Leishmania
NAGT as an endoplasmic reticulum transmembrane protein. In silico analyses of
nagt
from fungi and other protozoa further illustrate the potential value of this and, perhaps, other similar transmembrane molecules for phylogenetic analyses of single-cell eukaryotes.
Expression of
Magnaporthe grisea
Avirulence Gene
ACE1
Is Connected to the Initiation of Appressorium-Mediated Penetration
Authors: Fudal Isabelle , Collemare Jérôme , Böhnert Heidi U. , Melayah Delphine , Lebrun Marc-Henri 
Published: December 2006

ABSTRACT

Magnaporthe grisea
is responsible for a devastating fungal disease of rice called blast. Current control of this disease relies on resistant rice cultivars that recognize
M. grisea
signals corresponding to specific secreted proteins encoded by avirulence genes. The
M. grisea ACE1
avirulence gene differs from others, since it controls the biosynthesis of a secondary metabolite likely recognized by rice cultivars carrying the
Pi33
resistance gene. Using a transcriptional fusion between
ACE1
promoter and
eGFP
, we showed that
ACE1
is only expressed in appressoria during fungal penetration into rice and barley leaves, onion skin, and cellophane membranes.
ACE1
is almost not expressed in appressoria differentiated on Teflon and Mylar artificial membranes.
ACE1
expression is not induced by cellophane and plant cell wall components, demonstrating that it does not require typical host plant compounds. Cyclic AMP (cAMP) signaling mutants Δ
cpkA
and Δ
mac1 sum1
-
99
and tetraspanin mutant Δ
pls1
::
hph
differentiate melanized appressoria with normal turgor but are unable to penetrate host plant leaves.
ACE1
is normally expressed in these mutants, suggesting that it does not require cAMP signaling or a successful penetration event.
ACE1
is not expressed in appressoria of the
buf1
::
hph
mutant defective for melanin biosynthesis and appressorial turgor. The addition of hyperosmotic solutes to
buf1
::
hph
appressoria restores appressorial development and
ACE1
expression. Treatments of young wild-type appressoria with actin and tubulin inhibitors reduce both fungal penetration and
ACE1
expression. These experiments suggest that
ACE1
appressorium-specific expression does not depend on host plant signals but is connected to the onset of appressorium-mediated penetration.
Rapid Evolution of Diminished Transformability in
Acinetobacter baylyi
Authors: Bacher Jamie M. , Metzgar David , de Crécy-Lagard Valérie 
Published: December 2006

ABSTRACT

The reason for genetic exchange remains a crucial question in evolutionary biology.
Acinetobacter baylyi
strain ADP1 is a highly competent and recombinogenic bacterium. We compared the parallel evolution of wild-type and engineered noncompetent lineages of
A. baylyi
in the laboratory. If transformability were to result in an evolutionary benefit, it was expected that competent lineages would adapt more rapidly than noncompetent lineages. Instead, regardless of competency, lineages adapted to the same extent under several laboratory conditions. Furthermore, competent lineages repeatedly evolved a much lower level of transformability. The loss of competency may be due to a selective advantage or the irreversible transfer of loss-of-function alleles of genes required for transformation within the competent population.
Proteolytic Processing Is Not Essential for Multiple Functions of the
Escherichia coli
Autotransporter Adhesin Involved in Diffuse Adherence (AIDA-I)
Authors: Charbonneau Marie-Ève , Berthiaume Frédéric , Mourez Michael 
Published: December 2006

ABSTRACT

The
Escherichia coli
adhesin involved in diffuse adherence (AIDA-I), like many other autotransporter proteins, is released in the periplasm as a proprotein undergoing proteolytic processing after its translocation across the outer membrane. The proprotein is cleaved into a membrane-embedded fragment, AIDAc, and an extracellular fragment, the mature AIDA-I adhesin. The latter remains noncovalently associated with the outer membrane and can be released by heat treatment. The mechanism of cleavage of the proprotein and its role in the functionality of AIDA-I are not understood. Here, we show that cleavage is independent of the amount of AIDA-I in the outer membrane, suggesting an intramolecular autoproteolytic mechanism or a cleavage mediated by an unknown protease. We show that the two fragments, mature AIDA-I and AIDAc, can be cosolubilized and copurified in a folded and active conformation. We observed that the release by heat treatment results from the unfolding of AIDA-I and that the interaction of AIDA-I with AIDAc seems to be disturbed only by denaturation. We constructed an uncleavable point mutant of AIDA-I, where a serine of the cleavage site was changed into a leucine, and showed that adhesion, autoaggregation, and biofilm formation mediated by the mutant are indistinguishable from the wild-type levels. Lastly, we show that both proteins can mediate the invasion of cultured epithelial cells. Taken together, our experiments suggest that the proteolytic processing of AIDA-I plays a minor role in the functionality of this protein.
SarA Is a Repressor of
hla
(α-Hemolysin) Transcription in
Staphylococcus aureus
: Its Apparent Role as an Activator of
hla
in the Prototype Strain NCTC 8325 Depends on Reduced Expression of
sarS
Authors: Oscarsson Jan , Kanth Anna , Tegmark-Wisell Karin , Arvidson Staffan 
Published: December 2006

ABSTRACT

In most
Staphylococcus aureus
strains, inactivation of
sarA
increases
hla
transcription, indicating that
sarA
is a repressor. However, in
S. aureus
NCTC 8325 and its derivatives, used for most studies of
hla
regulation, inactivation of
sarA
resulted in decreased
hla
transcription. The disparate phenotype of strain NCTC 8325 seems to be associated with its
rsbU
mutation, which leads to σ
B
deficiency. This has now been verified by the demonstration that
sarA
repressed
hla
transcription in an
rsbU
+
derivative of strain 8325-4 (SH1000). That
sarA
could act as a repressor of
hla
in an 8325-4 background was confirmed by the observation that inactivation of
sarA
in an
agr sarS rot
triple mutant dramatically increased
hla
transcription to wild-type levels. However, the apparent role of
sarA
as an activator of
hla
in 8325-4 was not a result of the
rsbU
mutation alone, as inactivation of
sarA
in another
rsbU
mutant, strain V8, led to increased
hla
transcription. Northern blot analysis revealed much higher levels of
sarS
mRNA in strain V8 than in 8325-4, which was likely due to the mutation in the
sarS
activator,
tcaR
, in 8325-4, which was not found in strain V8. On the other hand, the relative increase in
sarS
transcription upon the inactivation of
sarA
was 15-fold higher in 8325-4 than in strain V8. Because of this, inactivation of
sarA
in 8325-4 means a net increase in repressor activity, whereas in strain V8, inactivation of
sarA
means a net decrease in repressor activity and, therefore, enhanced
hla
transcription.
Release of Immunity Protein Requires Functional Endonuclease Colicin Import Machinery
Authors: Duché Denis , Frenkian Aurélie , Prima Valérie , Lloubès Roland 
Published: December 2006

ABSTRACTBacteria producing endonuclease colicins are protected against the cytotoxic activity by a small immunity protein that binds with high affinity and specificity to inactivate the endonuclease. This complex is released into the extracellular medium, and the immunity protein is jettisoned upon binding of the complex to susceptible cells. However, it is not known how and at what stage during infection the immunity protein release occurs. Here, we constructed a hybrid immunity protein composed of the enhanced green fluorescent protein (EGFP) fused to the colicin E2 immunity protein (Im2) to enhance its detection. The EGFP-Im2 protein binds the free colicin E2 with a 1:1 stoichiometry and specifically inhibits its DNase activity. The addition of this hybrid complex to susceptible cells reveals that the release of the hybrid immunity protein is a time-dependent process. This process is achieved 20 min after the addition of the complex to the cells. We showed that complex dissociation requires a functional translocon formed by the BtuB protein and one porin (either OmpF or OmpC) and a functional import machinery formed by the Tol proteins. Cell fractionation and protease susceptibility experiments indicate that the immunity protein does not cross the cell envelope during colicin import. These observations suggest that dissociation of the immunity protein occurs at the outer membrane surface and requires full translocation of the colicin E2 N-terminal domain.
Mycoplasma genitalium
P140 and P110 Cytadhesins Are Reciprocally Stabilized and Required for Cell Adhesion and Terminal-Organelle Development
Authors: Burgos Raul , Pich Oscar Q. , Ferrer-Navarro Mario , Baseman Joel B. , Querol Enrique , Piñol Jaume 
Published: December 2006

ABSTRACT

Mycoplasma genitalium
is a human pathogen that mediates cell adhesion by a complex structure known as the attachment organelle. This structure is composed of cytadhesins and cytadherence-associated proteins, but few data are available about the specific role of these proteins in
M. genitalium
cytadherence. We have deleted by homologous recombination the mg191 and mg192 genes from the MgPa operon encoding the P140 and P110 cytadhesins. Molecular characterization of these mutants has revealed a reciprocal posttranslational stabilization between the two proteins. Loss of either P140 or P110 yields a hemadsorption-negative phenotype and correlates with decreased or increased levels of cytoskeleton-related proteins MG386 and DnaK, respectively. Scanning electron microscopy analysis reveals the absolute requirement of P140 and P110 for the proper development of the attachment organelle. The phenotype described for these mutants resembles that of the spontaneous class I and class II cytadherence-negative mutants [G. R. Mernaugh, S. F. Dallo, S. C. Holt, and J. B. Baseman, Clin. Infect. Dis.
17
(Suppl. 1)
:
S69-S78, 1993], whose genetic basis remained undetermined until now. Complementation assays and sequencing analysis demonstrate that class I and class II mutants are the consequence of large deletions affecting the mg192 and mg191-mg192 genes, respectively. These deletions originated from single-recombination events involving sequences of the MgPa operon and the MgPa island located immediately downstream. We also demonstrate the translocation of MgPa sequences to a particular MgPa island by double-crossover events. Based on these observations, we propose that in addition to being a source of antigenic variation, MgPa islands could be also involved in a general phase variation mechanism switching on and off, in a reversible or irreversible way, the adhesion properties of
M. genitalium
.
The OxyR Regulon in Nontypeable
Haemophilus influenzae
Authors: Harrison Alistair , Ray William C. , Baker Beth D. , Armbruster David W. , Bakaletz Lauren O. , Munson Robert S. 
Published: December 2006

ABSTRACT

Nontypeable
Haemophilus influenzae
(NTHi) is a gram-negative bacterium and a common commensal organism of the upper respiratory tract in humans. NTHi causes a number of diseases, including otitis media, sinusitis, conjunctivitis, exacerbations of chronic obstructive pulmonary disease, and bronchitis. During the course of colonization and infection, NTHi must withstand oxidative stress generated by insult due to multiple reactive oxygen species produced endogenously by other copathogens and by host cells. Using an NTHi-specific microarray containing oligonucleotides representing the 1821 open reading frames of the recently sequenced NTHi isolate 86-028NP, we have identified 40 genes in strain 86-028NP that are upregulated after induction of oxidative stress due to hydrogen peroxide. Further comparisons between the parent and an isogenic
oxyR
mutant identified a subset of 11 genes that were transcriptionally regulated by OxyR, a global regulator of oxidative stress. Interestingly, hydrogen peroxide induced the OxyR-independent upregulation of expression of the genes encoding components of multiple iron utilization systems. This finding suggested that careful balancing of levels of intracellular iron was important for minimizing the effects of oxidative stress during NTHi colonization and infection and that there are additional regulatory pathways involved in iron utilization.
Lysine Decarboxylase Expression by
Vibrio vulnificus
Is Induced by SoxR in Response to Superoxide Stress
Authors: Kim Ju-Sim , Choi Sang Ho , Lee Jeong K. 
Published: December 2006

ABSTRACT

Lysine decarboxylase expression by
Vibrio vulnificus
, which is up-regulated by CadC in response to acid stress, is also induced by SoxR in response to superoxide stress. SoxR binds to the promoter region of the
cadBA
operon, coding for a lysine-cadaverine antiporter (CadB) and a lysine decarboxylase (CadA). The induction of
cadBA
transcription by SoxR is independent of CadC. Cadaverine, which neutralizes the external medium, also appears to scavenge superoxide radicals, since increasing cellular cadaverine by elevating the gene dosage of
cadBA
significantly diminished the induction of Mn-containing superoxide dismutase under methyl viologen-induced oxidative stress. Consistently, a lack of cadaverine caused by mutation in
cadA
resulted in low tolerance to oxidative stress compared with that of the wild type.
Proteomic Analysis and Identification of
Streptococcus pyogenes
Surface-Associated Proteins
Authors: Severin Anatoly , Nickbarg Elliott , Wooters Joseph , Quazi Shakey A. , Matsuka Yury V. , Murphy Ellen , Moutsatsos Ioannis K. , Zagursky Robert J. , Olmsted Stephen B. 
Published: December 2006

ABSTRACT

Streptococcus pyogenes
is a gram-positive human pathogen that causes a wide spectrum of disease, placing a significant burden on public health. Bacterial surface-associated proteins play crucial roles in host-pathogen interactions and pathogenesis and are important targets for the immune system. The identification of these proteins for vaccine development is an important goal of bacterial proteomics. Here we describe a method of proteolytic digestion of surface-exposed proteins to identify surface antigens of
S. pyogenes
. Peptides generated by trypsin digestion were analyzed by multidimensional tandem mass spectrometry. This approach allowed the identification of 79 proteins on the bacterial surface, including 14 proteins containing cell wall-anchoring motifs, 12 lipoproteins, 9 secreted proteins, 22 membrane-associated proteins, 1 bacteriophage-associated protein, and 21 proteins commonly identified as cytoplasmic. Thirty-three of these proteins have not been previously identified as cell surface associated in
S. pyogenes
. Several proteins were expressed in
Escherichia coli
, and the purified proteins were used to generate specific mouse antisera for use in a whole-cell enzyme-linked immunosorbent assay. The immunoreactivity of specific antisera to some of these antigens confirmed their surface localization. The data reported here will provide guidance in the development of a novel vaccine to prevent infections caused by
S. pyogenes
.
Overlapping and Specialized Functions of the Molybdenum-Dependent Regulators MopA and MopB in
Rhodobacter capsulatus
Authors: Wiethaus Jessica , Wirsing Andrea , Narberhaus Franz , Masepohl Bernd 
Published: December 2006

ABSTRACT

The phototrophic purple bacterium
Rhodobacter capsulatus
encodes two similar but functionally not identical molybdenum-dependent regulator proteins (MopA and MopB), which are known to replace each other in repression of the
modABC
genes (coding for an ABC-type high-affinity Mo transport system) and
anfA
(coding for the transcriptional activator of Fe-nitrogenase genes). We identified further Mo-regulated (
mor
) genes coding for a putative ABC-type transport system of unknown function (MorABC) and a putative Mo-binding protein (Mop). The genes coding for MopA and the ModABC transporter form part of a single transcriptional unit,
mopA
-
modABCD
, as shown by reverse transcriptase PCR. Immediately upstream of
mopA
and transcribed in the opposite direction is
mopB
. The genes coding for the putative MorABC transporter belong to two divergently transcribed operons,
morAB
and
morC
. Expression studies based on
lacZ
reporter gene fusions in mutant strains defective for either MopA, MopB, or both revealed that the regulators substitute for each other in Mo-dependent repression of
morAB
and
morC
. Specific Mo-dependent activation of the
mop
gene by MopA, but not MopB, was found to control the putative Mo-binding protein. Both MopA and MopB are thought to bind to conserved DNA sequences with dyad symmetry in the promoter regions of all target genes. The positions of these so-called Mo boxes relative to the transcription start sites (as determined by primer extension analyses) differed between Mo-repressed genes and the Mo-activated
mop
gene. DNA mobility shift assays showed that MopA and MopB require molybdenum to bind to their target sites with high affinity.