bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fibronectin Binding Protein and Host Cell Tyrosine Kinase Are Required for Internalization ofStaphylococcus aureusby Epithelial Cells
Authors: Dziewanowska Katarzyna , Patti Joseph M. , Deobald Claudia F. , Bayles Kenneth W. , Trumble William R. , Bohach Gregory A. 
Published: December 2019

ABSTRACTStaphylococcus aureusexpresses several surface proteins that promote adherence to host cell extracellular matrix proteins, including fibronectin (Fn). Since this organism has recently been shown to be internalized by nonprofessional phagocytes, a process that typically requires high-affinity binding to host cell receptors, we investigated the role of its Fn binding proteins (FnBPs) and other surface proteins in internalization by the bovine mammary gland epithelial cell line (MAC-T). Efficient internalization ofS. aureus8325-4 required expression of FnBPs; an isogenic mutant (DU5883), not expressing FnBPs, was reduced by more than 95% in its ability to invade MAC-T cells. Moreover, D3, a synthetic peptide derived from the ligand binding domain of FnBP, inhibited the internalization of the 8325-4 strain in a dose-dependent fashion and the efficiency of staphylococcal internalization was partially correlated with Fn binding ability. Interestingly, Fn also inhibited the internalization and adherence ofS. aureus8325-4 in a dose-dependent manner. In contrast to internalization, adherence of DU5883 to MAC-T was reduced by only approximately 40%, suggesting that surface binding proteins, other than FnBPs, can mediate bacterial adherence to cells. Adherence via these proteins, however, does not necessarily result in internalization of the staphylococci. An inhibitor of protein tyrosine kinase, genistein, reduced MAC-T internalization ofS. aureusby 95%, indicating a requirement for a host signal transduction system in this process. Taken together, these results indicate thatS. aureusinvades nonprofessional phagocytes by a mechanism requiring interaction between FnBP and the host cell, leading to signal transduction and subsequent rearrangement of the host cell cytoskeleton.
Pneumococcal Surface Protein A Inhibits Complement Activation byStreptococcus pneumoniae
Authors: Tu Anh-Hue T. , Fulgham Robert L. , McCrory Mark A. , Briles David E. , Szalai Alexander J. 
Published: December 2019

ABSTRACTPneumococcal surface protein A (PspA) is a surface-exposed protein virulence factor forStreptococcus pneumoniae. In this study, no significant depletion of serum complement was observed for the serum of mice infected with pneumococci that express PspA. In contrast, in mice infected with an isogenic strain of pneumococci lacking PspA, significant activation of serum complement was detected within 30 min after infection. Also, the PspA-deficient strain but not the PspA-expressing strain was cleared from the blood within 6 h. The contribution of PspA to pneumococcal virulence was further investigated by using mice deficient for C5, C3, or factor B. In mice deficient for C3 or factor B, PspA-negative pneumococci became fully virulent. In contrast, in C5-deficient mice as in wild-type mice, PspA-deficient pneumococci were avirulent. These in vivo data suggest that, in nonimmune mice infected with pneumococci, PspA interferes with complement-dependent host defense mechanisms mediated by factor B. Immunoblots of pneumococci opsonized in vitro suggested that more C3b was deposited on PspA-negative than on PspA-positive pneumococci. This was observed with and without anticapsular antibody. Furthermore, processing of the α chain of C3b was reduced in the presence of PspA. We propose that PspA exerts its virulence function by interfering with deposition of C3b onto pneumococci and/or by inhibiting formation of a fully functional alternative pathway C3 convertase. By blocking recruitment of the alternative pathway, PspA reduces the amount of C3b deposited onto pneumococci, thereby reducing the effectiveness of complement receptor-mediated pathways of clearance.
Interactions ofPenicillium marneffeiwith Human Leukocytes In Vitro
Authors: Rongrungruang Yong , Levitz Stuart M. 
Published: December 2019

ABSTRACTPenicillium marneffei, a dimorphic fungus endemic in parts of Asia, causes disease in those with impaired cell-mediated immunity, especially persons with AIDS. The histopathology of penicilliosis marneffei features the intracellular infection of macrophages. We studied the interactions between human leukocytes and heat-killed yeast-phaseP. marneffei. Monocyte-derived macrophages bound and internalizedP. marneffeiin the presence of complement-sufficient pooled human serum (PHS). Binding and phagocytosis were still seen if PHS was heat inactivated or omitted altogether. The binding of unopsonizedP. marneffeito monocyte-derived macrophages occurred in the absence of divalent cations and was not affected by inhibitors of mannose and β-glucan receptors or monoclonal antibodies directed against CD14 and CD11/CD18. Binding was profoundly inhibited by wheat germ agglutinin. A vigorous respiratory burst was seen in peripheral blood mononuclear cells (PBMC) stimulated withP. marneffei, regardless of whether the fungi were opsonized. However, tumor necrosis factor alpha (TNF-α) release from PBMC stimulated withP. marneffeioccurred only if serum was present. These data demonstrate that (i) monocyte-derived macrophages bind and phagocytoseP. marneffeieven in the absence of opsonization, (ii) binding is divalent cation independent but is inhibited by wheat germ agglutinin, suggesting that the major receptor(s) recognizingP. marneffeiis a glycoprotein with exposedN-acetyl-β-d-glucosaminyl groups, (iii)P. marneffeistimulates the respiratory burst regardless of whether opsonins are present, and (iv) serum factors are required forP. marneffeito stimulate TNF-α release. The ability of unopsonizedP. marneffeito parasitize mononuclear phagocytes without stimulating the production of TNF-α may be critical for the virulence of this intracellular parasite.
The Gene Locus
yijP
Contributes to
Escherichia coli
K1 Invasion of Brain Microvascular Endothelial Cells
Authors: Wang Ying , Huang Sheng-He , Wass Carol A. , Stins Monique F. , Kim Kwang Sik 
Published: December 2019

ABSTRACT

Most cases of
Escherichia coli
meningitis develop as a result of hematogenous spread, but it is not clear how circulating
E. coli
crosses the blood-brain barrier. A Tn
phoA
mutant of
E. coli
K1 RS218 was shown to be significantly less invasive than its parent strain in bovine and human brain microvascular endothelial cells (BMEC), which constitute the blood-brain barrier. More importantly, traversal of the blood-brain barrier was significantly less with this mutant than with the parent strain in newborn rats with experimental hematogenous meningitis. A DNA segment containing the Tn
phoA
insertion site was cloned from RS218, and the cloned DNA complemented the Tn
phoA
mutant in invasion of BMEC. Nucleotide sequence revealed a near identity to that of a hypothetical
yijP
gene (also called
f577
) in the
E. coli
K-12 genome. Sequence analysis indicated that the
E. coli
K1
yijP
gene likely encodes a 66.6-kDa membrane protein. Deletion and complementation experiments indicated that the
yijP
gene was involved in
E. coli
K1 invasion of BMEC, i.e., the invasive ability of
E. coli
K1 was significantly reduced after
yijP
was deleted and was restored by complementation with a plasmid containing the
yijP
open reading frame. This is the first demonstration that the
yijP
gene locus plays a role in the pathogenesis of
E. coli
K1 meningitis.
Phage-Displayed T-Cell Epitope Grafted into Immunoglobulin Heavy-Chain Complementarity-Determining Regions: an Effective Vaccine Design Tested in Murine Cysticercosis
Authors: Manoutcharian Karen , Terrazas Luis Ignacio , Gevorkian Goar , Acero Gonzalo , Petrossian Pavel , Rodriguez Miriam , Govezensky Tzipe 
Published: December 2019

ABSTRACTA new type of immunogenic molecule was engineered by replacing all three complementarity-determining-region (CDR) loops of the human immunoglobulin (Ig) heavy-chain variable (VH) domain with theTaenia crassicepsepitope PT1 (PPPVDYLYQT) and by displaying this construct on the surfaces of M13 bacteriophage. When BALB/c mice were immunized with such phage particles (PIgphage), a strong protection against challenge infection in very susceptible female hosts was obtained. When specifically stimulated, the in vivo-primed CD4+and CD8+T cells isolated from mice immunized with PT1, both as a free peptide and as the PIgphage construct, proliferated in vitro, indicating efficient epitope presentation by both major histocompatibility complex class II and class I molecules in the specifically antigen-pulsed macrophages used as antigen-presenting cells. These data demonstrate the immunogenic potential of recombinant phage particles displaying CDR epitope-grafted Ig VHdomains and establish an alternative approach to the design of an effective subunit vaccine for prevention of cysticercosis. The key advantage of this type of immunogen is that no adjuvant is required for its application. The proposed strategy for immunogen construction is potentially suitable for use in any host-pathogen interaction.
The EspD Protein of Enterohemorrhagic
Escherichia coli
Is Required for the Formation of Bacterial Surface Appendages and Is Incorporated in the Cytoplasmic Membranes of Target Cells
Authors: Kresse Andreas U. , Rohde Manfred , Guzmán Carlos A. 
Published: December 2019

ABSTRACT

The formation of EspA-containing surface appendages in pathogenic
Escherichia coli
strains, both enteropathogenic
E. coli
(EPEC) and Shiga toxin-producing
E. coli
strains, is essential for critical events in the infective process, e.g., localized bacterial adherence to host cells with formation of microcolonies and induction of attaching and effacing lesions. It has been reported that EPEC mutants deficient in the production of EspD, which is encoded by the
esp
operon, are unable to accumulate actin underneath adherent bacteria but exhibit an attachment similar to that of the wild type. Here, we report the construction and characterization of an in-frame
espD
deletion mutant of the enterohemorrhagic
E. coli
(EHEC) strain EDL933. In contrast to what was observed in EPEC mutants, the EDL933
espD
mutant not only lacked the capacity to accumulate actin but also exhibited an impaired attachment to HeLa cells. The synthesis of the EspD protein was also essential for the formation of EspA-containing filaments. Finally, localization studies demonstrated that the EspD protein is transferred to the cytoplasm and integrated into the cytoplasmic membranes of infected cells. These results help to elucidate the underlying molecular events in infections caused by EHEC.
Molecular Cloning and Physical Mapping of the Daptomycin Gene Cluster from
Streptomyces roseosporus
Authors: Mchenney Margaret A. , Hosted Thomas J. , Dehoff Bradley S. , Rosteck Paul R. , Baltz Richard H. 
Published: December 2019

ABSTRACT

The daptomycin biosynthetic gene cluster of
Streptomyces roseosporus
was analyzed by Tn
5099
mutagenesis, molecular cloning, partial DNA sequencing, and insertional mutagenesis with cloned segments of DNA. The daptomycin biosynthetic gene cluster spans at least 50 kb and is located about 400 to 500 kb from one end of the ∼7,100-kb linear chromosome. We identified two peptide synthetase coding regions interrupted by a 10- to 20-kb region that may encode other functions in lipopeptide biosynthesis.
Differential Expression of the OmpF and OmpC Porin Proteins in
Escherichia coli
K-12 Depends upon the Level of Active OmpR
Authors: Lan Chung-Yu , Igo Michele M. 
Published: December 2019

ABSTRACT
We have generated a mutant form of the OmpR regulatory protein, OmpRD55E, that is active independent of the EnvZ kinase. Notably, the pattern of OmpF and OmpC expression can be altered simply by changing the level of this mutant protein in the cell. This result supports a key prediction of the current model of porin regulation, which states that the differential regulation of OmpF and OmpC is a direct consequence of the cellular level of the active form of OmpR.
Identification and Cloning of
waaF
(
rfaF
) from
Bordetella pertussis
and Use To Generate Mutants of
Bordetella
spp. with Deep Rough Lipopolysaccharide
Authors: Allen Andrew G. , Isobe Tomoko , Maskell Duncan J. 
Published: December 2019

ABSTRACT

A DNA locus from
Bordetella pertussis
capable of reconstituting lipopolysaccharide (LPS) O-antigen biosynthesis in
Salmonella typhimurium
SL3789 (
rfaF511
) has been isolated, by using selection with the antibiotic novobiocin. DNA within the locus encodes a protein with amino acid sequence similarity to heptosyltransferase II, encoded by
waaF
(previously
rfaF
) in other gram-negative bacteria. Mutation of this gene in
B. pertussis
,
Bordetella parapertussis
, and
Bordetella bronchiseptica
by allelic exchange generated bacteria with deep rough LPS phenotypes consistent with the proposed function of the gene as an inner core heptosyltransferase. These are the first LPS mutants generated in
B. parapertussis
and
B. bronchiseptica
and the first deep rough mutants of any of the bordetellae.
Stimulation of Transcription by Mutations Affecting Conserved Regions of RNA Polymerase II
Authors: Archambault Jacques , Jansma David B. , Kawasoe Jean H. , Arndt Kim T. , Greenblatt Jack , Friesen James D. 
Published: December 2019

ABSTRACT

Mutations that increase the low-level transcription of the
Saccharomyces cerevisiae HIS4
gene, which results from deletion of the genes encoding transcription factors BAS1, BAS2, and GCN4, were isolated previously in
SIT1
(also known as
RPO21
,
RPB1
, and
SUA8
), the gene encoding the largest subunit of RNA polymerase II (RNAPII). Here we show that
sit1
substitutions cluster in two conserved regions of the enzyme which form part of the active site. Six
sit1
mutations, affect region F, a region that is involved in transcriptional elongation and in resistance to α-aminatin. Four
sit1
substitutions lie in another region involved in transcriptional elongation, region D, which binds Mg
2+
ions essential for RNA catalysis. One region D substitution is lethal unless suppressed by a substitution in region G and interacts genetically with
PPR2
, the gene encoding transcription elongation factor IIS. Some
sit1
substitutions affect the selection of transcriptional start sites at the
CYC1
promoter in a manner reminiscent of that of
sua8
(
sua
stands for suppression of upstream ATG) mutations. Together with previous findings which indicate that regions D and G are in close proximity to the 3′ end of the nascent transcript and that region F is involved in the translocation process, our results suggest that transcriptional activation by the
sit1
mutations results from alteration of the RNAPII active center.
(Methyl)ammonium Transport in the Nitrogen-Fixing Bacterium
Azospirillum brasilense
Authors: Van Dommelen Anne , Keijers Veerle , Vanderleyden Jos , de Zamaroczy Miklos 
Published: December 2019

ABSTRACT

An ammonium transporter of
Azospirillum brasilense
was characterized. In contrast to most previously reported putative prokaryotic NH
4
+
transporter genes,
A. brasilense amtB
is not part of an operon with
glnB
or
glnZ
which, in
A. brasilense
, encode nitrogen regulatory proteins P
II
and P
Z
, respectively. Sequence analysis predicts the presence of 12 transmembrane domains in the deduced AmtB protein and classifies AmtB as an integral membrane protein. Nitrogen regulates the transcription of the
amtB
gene in
A. brasilense
by the Ntr system.
amtB
is the first gene identified in
A. brasilense
whose expression is regulated by NtrC. The observation that ammonium uptake is still possible in mutants lacking the AmtB protein suggests the presence of a second NH
4
+
transport mechanism. Growth of
amtB
mutants at low ammonium concentrations is reduced compared to that of the wild type. This suggests that AmtB has a role in scavenging ammonium at low concentrations.
The Wzz (Cld) Protein in
Escherichia coli
: Amino Acid Sequence Variation Determines O-Antigen Chain Length Specificity
Authors: Franco Agustin V. , Liu Dan , Reeves Peter R. 
Published: December 2019

ABSTRACT

The O antigen is a polymer with a repeated unit. The chain length in most
Escherichia coli
strains has a modal value of 10 to 18 O units, but other strains have higher or lower modal values.
wzz
(
cld/rol
) mutants have a random chain length distribution, showing that the modal distribution is determined by the Wzz protein. Cloned
wzz
genes from
E. coli
strains with short (7 to 16), intermediate (10 to 18), and long (16 to 25) modal chain lengths were transferred to a model system, and their effects on O111 antigen were studied. The O111 chain length closely resembled that of the parent strains. We present data based on the construction of chimeric
wzz
genes and site-directed mutagenesis of the
wzz
gene to show that the modal value of O-antigen chain length of
E. coli
O1, O2, O7, and O157 strains can be changed by specific amino acid substitutions in
wzz
. It is concluded that the O-antigen chain length heterogeneity in
E. coli
strains is the result of amino acid sequence variation of the Wzz protein.
In Vitro and In Vivo Oxidation of Methionine Residues in Small, Acid-Soluble Spore Proteins from
Bacillus
Species
Authors: Hayes Christopher S. , Illades-Aguiar Berenice , Casillas-Martinez Lilliam , Setlow Peter 
Published: December 2019

ABSTRACT

Methionine residues in α/β-type small, acid-soluble spore proteins (SASP) of
Bacillus
species were readily oxidized to methionine sulfoxide in vitro by
t
-butyl hydroperoxide (tBHP) or hydrogen peroxide (H
2
O
2
). These oxidized α/β-type SASP no longer bound to DNA effectively, but DNA binding protected α/β-type SASP against methionine oxidation by peroxides in vitro. Incubation of an oxidized α/β-type SASP with peptidyl methionine sulfoxide reductase (MsrA), which can reduce methionine sulfoxide residues back to methionine, restored the α/β-type SASP’s ability to bind to DNA. Both tBHP and H
2
O
2
caused some oxidation of the two methionine residues of an α/β-type SASP (SspC) in spores of
Bacillus subtilis
, although one methionine which is highly conserved in α/β-type SASP was only oxidized to a small degree. However, much more methionine sulfoxide was generated by peroxide treatment of spores carrying a mutant form of SspC which has a lower affinity for DNA. MsrA activity was present in wild-type
B. subtilis
spores. However,
msrA
mutant spores were no more sensitive to H
2
O
2
than were wild-type spores. The major mechanism operating for dealing with oxidative damage to α/β-type SASP in spores is DNA binding, which protects the protein’s methionine residues from oxidation both in vitro and in vivo. This may be important in vivo since α/β-type SASP containing oxidized methionine residues no longer bind DNA well and α/β-type SASP-DNA binding is essential for long-term spore survival.
Generation ofEscherichia coliO9a Serotype, a Subtype ofE. coliO9, by Transfer of thewb* Gene Cluster ofKlebsiellaO3 intoE. colivia Recombination
Authors: Sugiyama Tsuyoshi , Kido Nobuo , Kato Yutaka , Koide Naoki , Yoshida Tomoaki , Yokochi Takashi 
Published: December 2019

ABSTRACTGenetic characterization of thewb* gene in a series ofEscherichia coliandKlebsiellastrains possessing the mannose homopolymer as the O-specific polysaccharide was carried out. The partial nucleotide sequences and PCR-restriction fragment length polymorphism analysis suggested thatE. coliserotype O9a, a subtype ofE. coliO9, might have been generated by the insertion of theKlebsiellaO3wb* gene into a certainE. colistrain.
Menaquinone (Vitamin K
2
) Biosynthesis: Localization and Characterization of the
menA
Gene from
Escherichia coli
Authors: Suvarna K. , Stevenson D. , Meganathan R. , Hudspeth M. E. S. 
Published: December 2019

ABSTRACT

A key reaction in the biosynthesis of menaquinone involves the conversion of the soluble bicyclic naphthalenoid compound 1,4-dihydroxy-2-naphthoic acid (DHNA) to the membrane-bound demethylmenaquinone. The enzyme catalyzing this reaction, DHNA-octaprenyltransferase, attaches a 40-carbon side chain to DHNA. The
menA
gene encoding this enzyme has been cloned and localized to a 2.0-kb region of the
Escherichia coli
genome between
cytR
and
glpK
. DNA sequence analysis of the cloned insert revealed a 308-codon open reading frame (ORF), which by deletion analyses was shown to restore anaerobic growth of a
menA
mutant. Reverse-phase high-performance liquid chromatography analysis of quinones extracted from the
orf
-complemented cells independently confirmed the restoration of menaquinone biosynthesis, and similarly, analyses of isolated cell membranes for DHNA octaprenyltransferase activity confirmed the introduction of the
menA
product into the
orf
-complemented
menA
mutant. The validity of an ORF-associated putative promoter sequence was confirmed by primer extension analyses.
Mutational Analysis of the Phosphate-Binding Loop of
Rhizobium meliloti
DctD, a ς
54
-Dependent Activator
Authors: Gao Yan , Wang Ying-Kai , Hoover Timothy R. 
Published: December 2019

ABSTRACT

The phosphate-binding loop of ς
54
-dependent activators is thought to participate in ATP binding and/or hydrolysis. Alanine substitutions at positions 3, 4, 6, 7, and 8 of this motif in
Rhizobium meliloti
DctD disrupted transcriptional activation and ATP hydrolysis. Interestingly, substitution of alanine at position 7 also affected DNA binding.
Activation and Repression of Transcription at the Double Tandem Divergent Promoters for the
xylR
and
xylS
Genes of the TOL Plasmid of
Pseudomonas putida
Authors: Marqués Silvia , Gallegos María-Trinidad , Manzanera Maximino , Holtel Andreas , Timmis Kenneth N. , Ramos Juan L. 
Published: December 2019

ABSTRACT

The
xylR
and
xylS
genes are divergent and control transcription of the TOL plasmid catabolic pathways for toluene metabolism. Four promoters are found in the 300-bp intergenic region: Pr1 and Pr2 are constitutive ς
70
-dependent tandem promoters that drive expression of
xylR
, while expression of the
xylS
gene is driven from Ps2, a constitutive ς
70
-dependent promoter, and by the regulatable ς
54
class Ps1 promoter. In Ps1 the XylR targets (upstream activator sequences [UASs]) overlap the Pr promoters, and two sites for integration host factor (IHF) binding are located at the region from positions −2 to −30 (−2/−30 region) and the −137/−156 region, the latter overlapping the Pr promoters. When the XylR protein binds to the UASs in the absence of effector, it represses expression from Pr promoters. In the XylR-plus background and in the absence of an effector, the level of expression from Ps1 is low, although detectable, whereas Ps2 is active. In this background and in the presence of an effector, XylR increases autorepression. In a ς
54
-deficient
Pseudomonas putida
background, no expression occurred from Ps1 regardless of the presence of an effector. However, in the presence of an effector, the amount of RNA produced from Pr promoters was almost undetectable. This finding suggests that when no transcription occurred at the Ps1 promoter, clearance of XylR from the UASs was almost negligible. In this background, expression from Ps2 was very high regardless of the presence of an effector; this finding suggests that RNA polymerase containing ς
54
modulates expression from the downstream Ps2 ς
70
-dependent promoter. In a
P. putida
IHF-minus background and in the presence of effector, Ps1 expression was the highest found; in contrast, the basal levels of this promoter were the lowest observed. This finding suggests that IHF acts in vivo as a repressor of the ς
54
-dependent Ps1 promoter. In an IHF-deficient host background, expression from Ps2 in the presence of effector was negligible. Thus, binding of RNA polymerase containing ς
54
at the upstream promoter may modulate expression from the Ps2 promoter.