bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the Role of the Divalent Metal Ion-Dependent Transcriptional Repressor MntR in the Virulence of
Staphylococcus aureus
Authors: Ando Masaru , Manabe Yukari C. , Converse Paul J. , Miyazaki Eishi , Harrison Robert , Murphy John R. , Bishai William R.
Published: April 2003

DtxR-type metal ion-dependent repressors, present in many bacterial pathogens, may regulate expression of virulence genes such as that encoding diphtheria toxin. SirR, a DtxR homologue initially identified in
Staphylococcus epidermidis
, governs the expression of the adjacent
sitABC
operon encoding a putative metal ion ABC transporter system. We identified a
sirR
homologue,
mntR
, in
Staphylococcus aureus
and demonstrated by gel shift assay that the corynebacterial repressor DtxR binds to the
S. aureus mntABC
operator in the presence of Fe
2+
or Mn
2+
. Since a mutant DtxR, DtxR(E175K), functions as an iron-independent hyperrepressor in certain settings, we constructed a heterodiploid
S. aureus
strain expressing
dtxR
(
E175K
) from the native
mntR
promoter. Transcription of the
S. aureus mntABC
operon was repressed in the presence of Fe
2+
or Mn
2+
in wild-type and heterodiploid
S. aureus
strains. Under metal ion-limiting conditions,
mntABC
transcription was reduced but not abolished in
S. aureus
isolates expressing
dtxR
(
E175K
) compared with an isogenic control, suggesting that DtxR(E175K) binds the
S. aureus
MntR box in vivo. Under all conditions tested,
mntABC
transcription in the
dtxR
(
E175K
)-expressing strain was reduced relative to the isogenic control, indicating that DtxR(E175K) function was constitutively active. In the mouse skin abscess model,
dtxR
(
E175K
)-expressing
S. aureus
recombinants showed significantly reduced CFU levels compared with the isogenic wild-type control. We conclude that the
S. aureus
MntR box is recognized by corynebacterial DtxR proteins and thus belongs to the DtxR family of metal-dependent operator sites. Moreover, constitutive repression by DtxR(E175K) reduces the virulence of
S. aureus
in the mouse skin abscess model.
Increased Adherence to Caco-2 Cells Caused by Disruption of the
yhiE
and
yhiF
Genes in Enterohemorrhagic
Escherichia coli
O157:H7
Authors: Tatsuno Ichiro , Nagano Keiji , Taguchi Kazuki , Rong Li , Mori Hiroshi , Sasakawa Chihiro
Published: April 2003

Adherence of enterohemorrhagic
Escherichia coli
(EHEC) to intestinal epithelium is essential for initiation of infections, including diarrhea, and expression of the genes of the locus of enterocyte effacement (LEE) is thought to be crucial for adherence. To identify genes involved in modulating the adherent capacity, bacteria collected from an EHEC O157:H7 strain (O157Sakai) mutagenized by mini-Tn
5
Km2 were screened for their ability to increase the number of microcolonies (MC) on Caco-2 cells and eight mutants with increased adherence were isolated. Analysis of the mini-Tn
5
Km2-flanked DNA sequences indicated that one possessed the insertion within an O157 antigen gene cluster, another possessed the insertion within the
yhiF
gene, and the remaining six mutants had their insertions in the
yhiE
gene.
yhiE
and
yhiF
products share amino acid homology (23% identity) to each other and with members of the LuxR family, which are known as transcriptional regulatory proteins. The mutant having the insertion within the O157 antigen gene cluster did not express the O157 side chain (as determined by agglutination test and immunoblotting with polyclonal O157-specific antiserum), unlike the other seven mutants. Importantly, the other mutants showed enhanced type III secretion. Levels of the related mRNAs of genes of the LEE, but not that of
ler
mRNA, were also increased compared with those in the wild type. Indeed, when we introduced an in-frame deletion into the
yhiE
or
yhiF
gene in O157Sakai, the capacity of the resultant mutants to adhere to Caco-2 cells was greatly increased. When one of the
yhiE
insertion mutants was orally inoculated into ICR mice, the number of bacteria shed into feces by day 14 was greater than that for the wild type. These results suggest that
yhiE
and
yhiF
are involved in the adherence of O157Sakai to epithelial cells as negative regulators for the expression of the genes required for the type III secretion system.
Induction of Proinflammatory Responses in the Human Monocytic Cell Line THP-1 byCampylobacter jejuni
Authors: Jones Michael A. , Tötemeyer Sabine , Maskell Duncan J. , Bryant Clare E. , Barrow Paul A.
Published: April 2003

Campylobacter jejunican cause an enteritis that is associated with an acute inflammatory response at the gut epithelial surface. The signals inducing inflammation are unknown.C. jejunican penetrate the intestinal epithelial barrier and may then interact with leucocytes, potentially inducing proinflammatory responses. To investigate this, we studied the interaction ofC. jejuniwith the human monocytic cell line THP-1 and show that a range of proinflammatory cytokines and chemokines is induced. These include interleukin-1α (IL-1α), IL-1β, IL-6, IL-8, and tumor necrosis factor alpha. Responses can be induced by killedCampylobacteras well as live bacteria and do not depend on the cytolethal distending toxin.C. jejuniinfection of THP-1 cells triggers both nuclear translocation of functional NF-κB and stimulation of IL-1α, indicating that NF-κB-dependent and -independent stimulation is occurring. The extent of proinflammatory cytokine stimulation suggests that monocytes might significantly contribute to intestinal inflammation and disease pathology.
CC Chemokines Mediate Leukocyte Trafficking into the Central Nervous System during Murine Neurocysticercosis: Role of γδ T Cells in Amplification of the Host Immune Response
Authors: Cardona Astrid E. , Gonzalez Paula A. , Teale Judy M.
Published: April 2003

According to a previous report, the degree of the host immune response highly correlates with severity of the disease in the murine model for neurocysticercosis. In wild-type mice,Mesocestoides cortiinfection induced a rapid and extensive accumulation of γδ T cells and macrophages in the brain. NK cells, dendritic cells, αβ T cells, and B cells were also recruited to the brain but at lower levels. In contrast, γδ T-cell-deficient mice exhibited decreased cellular infiltration and reduced central nervous system (CNS) pathology. To understand the mechanisms of leukocyte recruitment into the CNS, chemokine expression was analyzed in infected brains in the present study. MCP-1 (CCL2), MIP-1α (CCL3), and MIP-1β (CCL4) were up-regulated within 2 days afterM. cortiinfection. Protein expression of RANTES (CCL5), eotaxin (CCL11), and MIP-2 was detected later, at 1 week postinfection. Correlating with the decreased cellular infiltration, delta chain T-cell receptor-deficient (TCRδ−/−) mice exhibited substantially reduced levels of most of the chemokines analyzed (with the exception of eotaxin). The results suggest that γδ T cells play an important role in the CNS immune response by producing chemokines such as MCP-1 and MIP-1α, enhancing leukocyte trafficking into the brain during murine neurocysticercosis.
Persistence of Mucosal Mast Cells and Eosinophils inShigella-Infected Children
Authors: Raqib Rubhana , Moly Pricila Khan , Sarker Protim , Qadri Firdausi , Alam Nurul Haque , Mathan Minnie , Andersson Jan
Published: April 2003

Cells of the innate immune system and their mediators were studied at the single-cell level in the rectums of pediatric and adult patients withShigellainfection to better understand why children are at higher risk for severe infection. Adult patients had increased infiltration of mucosal mast cells (MMC) at the acute stage (3 to 5 days after the onset of diarrhea) and eosinophils in early convalescence (14 to 16 days after onset). Increased expression of stem cell factor and prostaglandin H synthase-1 (PGHS-1) was associated with increased tryptase-Ki67-double-positive MMC in the acute stage and increased apoptosis of MMC, which led to a rapid decline in early convalescence. The eosinophils demonstrated increased expression of major basic protein (MBP), eotaxin, and CCR3, as well as increased necrotic death. The neutrophils showed enhanced α-defensin and lactoferrin expression in the acute phase. In contrast to adults, the pediatric patients demonstrated delayed accumulation of mast cells and eosinophils, while α-defensin expression persisted during convalescence. In contrast, neutrophil counts and lactoferrin expression were reduced in children compared to adults. The results suggest that children with shigellosis have a persistent activation of the innate immune response in the convalescent phase, indicating delayed elimination ofShigellaantigens compared to adults.
Rapid Apoptosis Induced by Shiga Toxin in HeLa Cells
Authors: Fujii Jun , Matsui Takashi , Heatherly Daniel P. , Schlegel Kailo H. , Lobo Peter I. , Yutsudo Takashi , Ciraolo Georgianne M. , Morris Randal E. , Obrig Tom
Published: April 2003

Apoptosis was induced rapidly in HeLa cells after exposure to bacterial Shiga toxin (Stx1 and Stx2; 10 ng/ml). Approximately 60% of HeLa cells became apoptotic within 4 h as detected by DNA fragmentation, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and electron microscopy. Stx1-induced apoptosis required enzymatic activity of the Stx1A subunit, and apoptosis was not induced by the Stx2B subunit alone or by the anti-globotriaosylceramide antibody. This activity was also inhibited by brefeldin A, indicating the need for toxin processing through the Golgi apparatus. The intracellular pathway leading to apoptosis was further defined. Exposure of HeLa cells to Stx1 activated caspases 3, 6, 8, and 9, as measured both by an enzymatic assay with synthetic substrates and by detection of proteolytically activated forms of these caspases by Western immunoblotting. Preincubation of HeLa cells with substrate inhibitors of caspases 3, 6, and 8 protected the cells against Stx1-dependent apoptosis. These results led to a more detailed examination of the mitochondrial pathway of apoptosis. Apoptosis induced by Stx1 was accompanied by damage to mitochondrial membranes, measured as a reduced mitochondrial membrane potential, and increased release of cytochromecfrom mitochondria at 3 to 4 h. Bid, an endogenous protein known to permeabilize mitochondrial membranes, was activated in a Stx1-dependent manner. Caspase-8 is known to activate Bid, and a specific inhibitor of caspase-8 prevented the mitochondrial damage. Although these data suggested that caspase-8-mediated cleavage of Bid with release of cytochromecfrom mitochondria and activation of caspase-9 were responsible for the apoptosis, preincubation of HeLa cells with a specific inhibitor of caspase-9 did not protect against apoptosis. These results were explained by the discovery of a simultaneous Stx1-dependent increase in endogenous XIAP, a direct inhibitor of caspase-9. We conclude that the primary pathway of Stx1-induced apoptosis and DNA fragmentation in HeLa cells is unique and includes caspases 8, 6, and 3 but is independent of events in the mitochondrial pathway.
Global Effects of Virulence Gene Regulators in aBacillus anthracisStrain with Both Virulence Plasmids
Authors: Bourgogne Agathe , Drysdale Melissa , Hilsenbeck Susan G. , Peterson Scott N. , Koehler Theresa M.
Published: April 2003

Control of anthrax toxin and capsule synthesis, the two major virulence factors ofBacillus anthracis, has been associated with two regulatory genes,atxAandacpA, located on virulence plasmids pXO1 and pXO2, respectively. We used transcriptional profiling to determine whetheratxAand/oracpAcontrol genes other than those already described and to investigate functional similarities of the regulators. Transcription was assessed in a pXO1+pXO2+parent strain and in isogenic mutants in which one or both regulatory genes were deleted. We determined that in addition to the toxin and capsule genes,atxAcontrols expression of numerous other genes on both plasmids and the chromosome. Generally, plasmid-encoded genes were more highly regulated than chromosomal genes, and both positive and negative effects were observed. CertainatxA-regulated genes were affected synergistically in anatxA acpAmutant. Yet overall,acpAappears to be a minor regulator with fewer targets thanatxA. In contrast to previous reports ofacpAfunction in attenuated strains,acpAhad a minimal influence on capsule gene transcription and capsule synthesis in a genetically complete strain. Surprisingly,acpAexpression was positively affected byatxA,althoughatxA-activated capsule gene transcription is notacpAdependent. The newly discoveredatxA-regulated targets include genes predicted to encode secreted proteins and proteins with roles in transcriptional regulation and signaling. Regulation of chromosomal genes byatxAis particularly intriguing, given that many of the target genes have homologues in otherBacillusspecies that lackatxAhomologues. Given the global effect ofatxAon gene expression inB. anthracis, previous assumptions regarding reduced virulence of strains harboring single plasmids must be reassessed and the potential roles of newly identifiedatxA-regulated genes should be investigated.
Inactivation of thesrtAGene Affects Localization of Surface Proteins and Decreases Adhesion ofStreptococcus pneumoniaeto Human Pharyngeal Cells In Vitro
Authors: Kharat Arun S. , Tomasz Alexander
Published: April 2003

Inactivation of sortase genesrtAinStreptococcus pneumoniaestrain R6 caused the release of β-galactosidase and neuraminidase A (NanA) from the cell wall into the surrounding medium. Both of these surface proteins contain the LPXTG motif in the C-terminal domain. Complementation with plasmid-bornesrtAreversed protein release. Deletion ofmurM, a gene involved in the branching of pneumococcal peptidoglycan, also caused partial release of β-galactosidase, suggesting preferential attachment of the protein to branched muropeptides in the cell wall. Inactivation ofsrtAcaused decreased adherence to human pharyngeal cells in vitro but had no effect on the virulence of a capsular type III strain ofS. pneumoniaein the mouse intraperitoneal model. The observations suggest that—as in other gram-positive bacteria—sortase-dependent display of proteins occurs inS. pneumoniaeand that some of these proteins may be involved in colonization of the human host.
Modulation of Myosin Light-Chain Phosphorylation by p21-Activated Kinase 1 in
Escherichia coli
Invasion of Human Brain Microvascular Endothelial Cells
Authors: Rudrabhatla Rajyalakshmi S. , Sukumaran Sunil K. , Bokoch Gary M. , Prasadarao Nemani V.
Published: April 2003

Cytoskeletal dynamics, modulated by actin-myosin interactions, play an important role in
Escherichia coli
K1 invasion of human brain microvascular endothelial cells (HBMEC). Herein, we show that inhibitors of myosin function, butanedione monoxide and ML-7, significantly blocked the
E. coli
invasion of HBMEC. The invasive
E. coli
induces myosin light-chain (MLC) phosphorylation during the invasion process, which gets recruited to the site of actin condensation beneath the bacteria. We also show that invading
E. coli
downregulates the activity of p21-activated kinase 1 (PAK1), which is an upstream regulator of MLC kinase (MLCK). Overexpression of wild-type PAK1 and constitutively active PAK1 in HBMEC inhibits
E. coli
invasion significantly with a concomitant decrease in MLC phosphorylation. The inhibition of
E. coli
invasion by these PAK1 mutants is due to the absence of phospho-MLC at the actin condensation points. In contrast, the dominant-negative PAK1 shows no effect either on the invasion or on MLC phosphorylation or phospho-MLC recruitment to the actin focal points, suggesting that activated PAK1 inactivates MLCK. Taken together, these results suggest that
E. coli
invasion of HBMEC induces MLC phosphorylation by inhibiting the activity of PAK1 and the recruitment of phosphorylated MLC to the site of actin condensation beneath the bacteria for efficient internalization of
E. coli
into HBMEC.
Deletion of the
Aspergillus fumigatus
Gene Encoding the Ras-Related Protein RhbA Reduces Virulence in a Model of Invasive Pulmonary Aspergillosis
Authors: Panepinto John C. , Oliver Brian G. , Fortwendel Jarrod R. , Smith Darcey L. H. , Askew David S. , Rhodes Judith C.
Published: April 2003

Aspergillus fumigatus
is the predominant mold pathogen in patients who lack functional innate immunity. The
A. fumigatus rhbA
gene was first identified as a transcript that was upregulated when the organism was grown in the presence of mammalian cells. To gain insight into the function of
rhbA
in the growth and pathogenesis of
A. fumigatus
, we constructed a strain that lacks a functional
rhbA
gene. The Δ
rhbA
mutant showed a significant reduction in virulence compared to the virulence of the wild type in a mouse model of invasive aspergillosis. Complementation of the deletion with the wild-type gene restored full virulence. Although the Δ
rhbA
mutant grew as well as the wild type on solid medium containing the rich nitrogen source ammonium, the growth of the mutant was impaired on medium containing poor nitrogen sources. Like the
Saccharomyces cerevisiae rhb1
mutant, the Δ
rhbA
mutant exhibited increased uptake of arginine. In addition, the Δ
rhbA
strain underwent asexual development in submerged cultures, even under ammonium-excess conditions. Growth of the mutant with poor nitrogen sources eliminated both the arginine uptake and submerged asexual development phenotypes. The mutant showed enhanced sensitivity to the TOR kinase inhibitor rapamycin. These findings establish the importance of
rhbA
for
A. fumigatus
virulence and suggest a role for
rhbA
in nutrient sensing.
Pretreatment of Mice with Streptomycin Provides aSalmonella entericaSerovar Typhimurium Colitis Model That Allows Analysis of Both Pathogen and Host
Authors: Barthel Manja , Hapfelmeier Siegfried , Quintanilla-Martínez Leticia , Kremer Marcus , Rohde Manfred , Hogardt Michael , Pfeffer Klaus , Rüssmann Holger , Hardt Wolf-Dietrich
Published: April 2003

Salmonella entericasubspecies 1 serovar Typhimurium is a principal cause of human enterocolitis. For unknown reasons, in mice serovar Typhimurium does not provoke intestinal inflammation but rather targets the gut-associated lymphatic tissues and causes a systemic typhoid-like infection. The lack of a suitable murine model has limited the analysis of the pathogenetic mechanisms of intestinal salmonellosis. We describe here how streptomycin-pretreated mice provide a mouse model for serovar Typhimurium colitis. Serovar Typhimurium colitis in streptomycin-pretreated mice resembles many aspects of the human infection, including epithelial ulceration, edema, induction of intercellular adhesion molecule 1, and massive infiltration of PMN/CD18+cells. This pathology is strongly dependent on protein translocation via the serovar Typhimurium SPI1 type III secretion system. Using a lymphotoxin β-receptor knockout mouse strain that lacks all lymph nodes and organized gut-associated lymphatic tissues, we demonstrate that Peyer's patches and mesenteric lymph nodes are dispensable for the initiation of murine serovar Typhimurium colitis. Our results demonstrate that streptomycin-pretreated mice offer a unique infection model that allows for the first time to use mutants of both the pathogen and the host to study the molecular mechanisms of enteric salmonellosis.
Activation of Transcription Factors AP-1 and NF-κB in Murine Chagasic Myocarditis
Authors: Huang Huan , Petkova Stefka B. , Cohen Alex W. , Bouzahzah Boumediene , Chan John , Zhou Jian-nian , Factor Stephen M. , Weiss Louis M. , Krishnamachary Mohan , Mukherjee Shankar , Wittner Murray , Kitsis Richard N. , Pestell Richard G. , Lisanti Michael P. , Albanese Chris , Tanowitz Herbert B.
Published: April 2003

The myocardium of CD1 mice was examined for the activation of signal transduction pathways leading to cardiac inflammation and subsequent remodeling duringTrypanosoma cruziinfection (Brazil strain). The activity of three pathways of the mitogen-activated protein kinases (MAPKs) was determined. Immunoblotting revealed a persistent elevation of phosphorylated (activated) extracellular-signal-regulated kinase (ERK), which regulates cell proliferation. During infection there was a transient activation of p38 MAPK but no activation of Jun N-terminal kinase. Early targets of activated ERK, c-Jun and c-Fos, were elevated during infection, as demonstrated by semiquantitative reverse transcription-PCR. Immunostaining revealed that the endothelium and the interstitial cells were most intensely stained with antibodies to c-Jun and c-Fos. Soon after infection, AP-1 and NF-κB DNA binding activity was increased. Protein levels of cyclin D1, the downstream target of ERK and NF-κB, were induced during acute infection. Immunostaining demonstrated increased expression of cyclin D1 in the vascular and endocardial endothelium, inflammatory cells, and the interstitial areas. Increased expression of the cyclin D1-specific phosphorylated retinoblastoma protein (Ser780) was also evident. Immunoblotting and immunostaining also demonstrated increased expression of proliferating cellular nuclear antigen that was predominantly present in the inflammatory cells, interstitial areas (i.e., fibroblasts), and endothelium. These data demonstrate thatT. cruziinfection results in activation of the ERK-AP-1 pathway and NF-κB. Cyclin D1 expression was also increased. These observations provide a molecular basis for the activation of pathways involved in cardiac remodeling in chagasic cardiomyopathy.
Limited Role of Lipopolysaccharide Lewis Antigens in Adherence ofHelicobacter pylorito the Human Gastric Epithelium
Authors: Mahdavi Jafar , Borén Thomas , Vandenbroucke-Grauls Christina , Appelmelk Ben J.
Published: April 2003

In vitro and in vivo studies from various groups have suggested thatHelicobacter pylorilipopolysaccharide (LPS) Lewis x (Lex) antigens mediate bacterial adhesion. We have now reevaluated this hypothesis by studying the adherence in situ ofH. pyloristrain 11637 and its corresponding Lex-negativerfbMmutant to human gastric mucosa from patients (n= 22) with various gastric pathologies. Significant binding of the parent strain was observed in only 8 out of 22 sections; in four out of eight patients, the Lex-negative mutant bound less well. One of these four patients displayed no gastric abnormalities, and the other three showed dysplasia, metaplasia, and adenocarcinoma, respectively; hence, we are unable to define the circumstances under which LPS-mediated adhesion takes place. We conclude thatH. pyloriLPS plays a distinct but minor role in adhesion.
Differential Expression of Genes That Harbor a Common Regulatory Element in
Neisseria meningitidis
upon Contact with Target Cells
Authors: Deghmane Ala-Eddine , Larribe Mireille , Giorgini Dario , Sabino Dora , Taha Muhamed-Kheir
Published: April 2003

The expression of several genes in
Neisseria meningitidis
upon contact with epithelial cells was associated with the presence of the contact regulatory elements of
Neisseria
. These genes are involved in various aspects of meningococcal biology and could be coordinately regulated upon contact with target cells.
Influence of Lewis Antigen Expression byHelicobacter pylorion Bacterial Internalization by Gastric Epithelial Cells
Authors: Lozniewski Alain , Haristoy Xavier , Rasko David A. , Hatier Renée , Plénat François , Taylor Diane E. , Angioi-Duprez Karine
Published: April 2003

The role ofHelicobacter pylorilipopolysaccharide (LPS) Lewis antigens in infection is still not well known. We investigated the influence of Lewis antigen expression byH. pylorion its internalization by AGS cells and the epithelium of human gastric xenografts in nude mice using isogenic mutants in LPS biosynthetic genes. In vivo, colonization rates were unaffected by the change inH. pyloriLewis antigen expression, whereas the number of viable intracellular bacteria was significantly higher with wild-typeH. pyloristrains expressing Lewis antigens when compared to the isogenic mutants in both models.H. pyloristrains expressing more Lewis X antigens (Lex) were internalized at a higher rate than those expressing less Lex, type II Lewis antigens (Leaor Leb) alone, or no Lewis antigens. Thus, Lewis antigens appear to be involved in the internalization ofH. pyloriby the gastric epithelium.
Characterization of
Staphylococcus aureus
Enterotoxin L
Authors: Orwin Paul M. , Fitzgerald J. Ross , Leung Donald Y. M. , Gutierrez Juan A. , Bohach Gregory A. , Schlievert Patrick M.
Published: April 2003

Staphylococcus aureus
causes a wide variety of diseases. Major virulence factors of this organism include enterotoxins (SEs) that cause both food poisoning and toxic shock syndrome. Recently, a novel SE, tentatively designated SEL, was identified in a pathogenicity island from a bovine mastitis isolate. The toxin had a molecular weight of 26,000 and an isoelectric point of 8.5. Recombinant SEL shared many biological activities with SEs, including superantigenicity, pyrogenicity, enhancement of endotoxin shock, and lethality in rabbits when administered in subcutaneous miniosmotic pumps, but the protein lacked emetic activity. T cells bearing the T-cell receptor β chain variable regions 5.1, 5.2, 6.7, 16, and 22 were significantly stimulated by recombinant SEL.
The Entner-Doudoroff Pathway Has Little Effect on
Helicobacter pylori
Colonization of Mice
Authors: Wanken Amy E. , Conway Tyrrell , Eaton Kathryn A.
Published: April 2003

Helicobacter pylori
mutants deficient in 6-phosphogluconate dehydratase (6PGD) were constructed. Colonization densities were lower and minimum infectious doses were higher for mutant strains than for wild-type strains. In spite of better colonization, however, wild-type strains did not displace the mutant in cocolonization experiments. Loss of 6PGD diminishes the fitness of
H. pylori
in vivo, but the pathway is nonessential for colonization.
Production of the Siderophore 2,3-Dihydroxybenzoic Acid Is Required for Wild-Type Growth of
Brucella abortus
in the Presence of Erythritol under Low-Iron Conditions In Vitro
Authors: Bellaire Bryan H. , Elzer Philip H. , Baldwin Cynthia L. , Roop R. Martin
Published: April 2003

Production of the siderophore 2,3-dihyroxybenzoic acid (2,3-DHBA) is required for the wild-type virulence of
Brucella abortus
in cattle. A possible explanation for this requirement was uncovered when it was determined that a
B. abortus dhbC
mutant (BHB1) defective in 2,3-DHBA production displays marked growth restriction in comparison to its parent strain,
B. abortus
2308, when cultured in the presence of erythritol under low-iron conditions. This phenotype is not displayed when these strains are cultured under low-iron conditions in the presence of other readily utilizable carbon and energy sources. The addition of either exogenous 2,3-DHBA or FeCl
3
relieves this growth defect, suggesting that the inability of the
B. abortus dhbC
mutant to display wild-type growth in the presence of erythritol under iron-limiting conditions is due to a defect in iron acquisition. Restoring 2,3-DHBA production to the
B. abortus dhbC
mutant by genetic complementation abolished the erythritol-specific growth defect exhibited by this strain in low-iron medium, verifying the relationship between 2,3-DHBA production and efficient growth in the presence of erythritol under low-iron conditions. The positive correlation between 2,3-DHBA production and growth in the presence of erythritol was further substantiated by the observation that the addition of erythritol to low-iron cultures of
B. abortus
2308 stimulated the production of 2,3-DHBA by increasing the transcription of the
dhbCEBA
operon. Correspondingly, the level of exogenous iron needed to repress
dhbCEBA
expression in
B. abortus
2308 was also greater when this strain was cultured in the presence of erythritol than that required when it was cultured in the presence of any of the other readily utilizable carbon and energy sources tested. The tissues of the bovine reproductive tract are rich in erythritol during the latter stages of pregnancy, and the ability to metabolize erythritol is thought to be important to the virulence of
B. abortus
in pregnant ruminants. Consequently, the experimental findings presented here offer a plausible explanation for the attenuation of the
B. abortus
2,3-DHBA-deficient mutant BHB1 in pregnant ruminants.
Immune Response Induced by RecombinantMycobacterium bovisBCG Producing the Cholera Toxin B Subunit
Authors: Biet Franck , Kremer Laurent , Wolowczuk Isabelle , Delacre Myriam , Locht Camille
Published: April 2003

The pentameric form of the cholera toxin B subunit (CTB) is known to be a strong mucosal adjuvant and stimulates antigen-specific secretory immunoglobulin A (IgA) and systemic antibody responses to antigens when given by mucosal routes. To deliver CTB for prolonged periods of time to the respiratory mucosa, we constructed aMycobacterium bovisbacillus Calmette-Guérin (BCG) strain that produces and secretes assembled pentameric CTB. Mice immunized intranasally (i.n.) with recombinant BCG (rBCG) developed a stronger anti-BCG IgA response in bronchoalveolar lavage fluids (BALF) than mice immunized with nonrecombinant BCG. The total IgA response in the BALF of mice immunized with rBCG was also stronger than that in BALF of mice immunized with the nonrecombinant strain. The induction of IgA was well correlated with an increased production of transforming growth factor β1. Simultaneous administration of intraperitoneally delivered ovalbumin and of i.n. delivered CTB-producing BCG induced a long-lasting ovalbumin-specific mucosal IgA response as well as a systemic IgG response, both of which were significantly higher than those in mice immunized with nonrecombinant BCG together with ovalbumin. These results suggest that the CTB-producing BCG may be a powerful adjuvant to be considered for future mucosal vaccine development.
Humoral Immunity Reflects Altered T Helper Cell Bias inBorrelia burgdorferi-Infected γδ T-Cell-Deficient Mice
Authors: Bockenstedt Linda K. , Shanafelt Marie-Claude , Belperron Alexia , Mao Jialing , Barthold Stephen W.
Published: April 2003

In murine Lyme borreliosis, the absence of γδ T lymphocytes augments the T helper cell 2 type humoral response but does not alter disease susceptibility. Arthropod transmission ofBorrelia burgdorferispirochetes results in similar antibody isotypes when γδ T cells are present, suggesting that vector effects can negate γδ T-cell functions in vivo.
Expression of H
C
Subunits from
Clostridium botulinum
Types C and D and Their Evaluation as Candidate Vaccine Antigens in Mice
Authors: Woodward L. A. , Arimitsu H. , Hirst R. , Oguma K.
Published: April 2003

Two proteins representing the heavy-chain subunits of botulinum neurotoxin types C and D were expressed in
Escherichia coli
, and their vaccine potential was evaluated. Mice were vaccinated with doses ranging from 0.5 to 10 μg and were challenged with 10 to 10
5
50% lethal doses of toxin. For the type C subunit protein, C50, two doses of 2 μg were required for full protection, while, for type D subunit protein, D50, two 1-μg doses were required. A bivalent vaccine consisting of a mixture of these two proteins also provided protection against both botulinum neurotoxin type C and type D challenge. Antibody levels in serum were determined by both enzyme-linked immunosorbent assays and serum neutralization assays
Coordinated Expression of Tracheal Antimicrobial Peptide and Inflammatory-Response Elements in the Lungs of Neonatal Calves with Acute Bacterial Pneumonia
Authors: Caverly Jessica M. , Diamond Gill , Gallup Jack M. , Brogden Kim A. , Dixon Richard A. , Ackermann Mark R.
Published: April 2003

Lung tissue removed from neonatal calves with acuteMannheimia haemolyticapneumonia showed that rapid up-regulation of the basal mRNA expression of tracheal antimicrobial peptide (TAP), NF-κB, and intercellular adhesion molecule 1 occurred after infection; TAP and interleukin 8 expression were highly correlated. This work suggests that the coordinated expression of β-defensin and inflammatory elements occurs during bacterial pneumonia.
Pneumococcal Serotype 19F Conjugate Vaccine Induces Cross-Protective Immunity to Serotype 19A in a Murine Pneumococcal Pneumonia Model
Authors: Jakobsen Håvard , Sigurdsson Viktor D. , Sigurdardottir Sigurveig , Schulz Dominique , Jonsdottir Ingileif
Published: April 2003

Immunization with a pneumococcal conjugate vaccine (PNC) containing serotype 19F induces cross-reactive antibodies to 19A in mice and human infants. Active immunization with PNC and passive immunization with serum samples from infants vaccinated with PNC containing serotype 19F, but not serotype 19A, protected against lung infection caused by both serotypes in a murine model.
Immunization with Genetic Toxoids of theArcanobacterium pyogenesCholesterol-Dependent Cytolysin, Pyolysin, Protects Mice against Infection
Authors: Jost B. Helen , Trinh Hien T. , Songer J. Glenn , Billington Stephen J.
Published: April 2003

Pyolysin (PLO), a cholesterol-dependent cytolysin expressed byArcanobacterium pyogenes, is an important host-protective antigen. However, this molecule is toxic and requires inactivation prior to its use as a vaccine. Three genetically toxoided, nonhemolytic PLO molecules, HIS-PLO.F497, HIS-PLO.ΔP499, and HIS-PLO.A522, were found to be nontoxic, and vaccinated mice were protected from infection, indicating the potential of these toxoids as vaccines. Furthermore, in a mouse model of infection,A. pyogenescarrying the F497mutation was as attenuated as a PLO-deficient strain, indicating that the cytolytic activity of PLO is important in virulence.
Structural and Functional Cellular Changes Induced by
Burkholderia pseudomallei
Rhamnolipid
Authors: Häuβler S. , Rohde M. , von Neuhoff N. , Nimtz M. , Steinmetz I.
Published: April 2003

In this study we report that extracellular
Burkholderia pseudomallei
rhamnolipid induced cytopathic changes characterized by retraction, rounding up, and, finally, detachment in phagocytic and nonphagocytic cell lines. These changes were due to a progressive reorganization of the F-actin network resulting in impaired cell cycle progression and a reduced phagocytic function of macrophages.
Glycoconjugate Binding of Gastric and Enterohepatic
Helicobacter
spp
Authors: Hynes Sean O. , Teneberg Susann , Roche Niamh , Wadström Torkel
Published: April 2003

Helicobacter pylori
is able to utilize several lectin-like, protein-carbohydrate interactions for binding to mucins, cell surfaces, and extracellular matrix proteins. As determined by hemagglutination assays and binding of radiolabeled bacteria to glycosphingolipids on thin-layer chromatograms, strains of gastric helicobacters and enterohepatic helicobacters, including
Helicobacter canis, Helicobacter hepaticus
, and
Helicobacter bilis
, also demonstrated evidence for the presence of lectin-hemagglutinin adhesins. In addition, in
H. hepaticus
and
H. bilis
, binding may be sialic acid dependent. The presence or absence and differences in the levels of activity of lectin adhesins may reflect the species' ecological niche.
Advances in multimodal mass spectrometry for single‐cell analysis and imaging enhancement
Authors: Croslow Seth W. , Trinklein Timothy J. , Sweedler Jonathan V.
Published: January 2024

Multimodal mass spectrometry (MMS) incorporates an imaging modality with probe‐based mass spectrometry (MS) to enable precise, targeted data acquisition and provide additional biological and chemical data not available by MS alone. Two categories of MMS are covered; in the first, an imaging modality guides the MS probe to target individual cells and to reduce acquisition time by automatically defining regions of interest. In the second category, imaging and MS data are coupled in the data analysis pipeline to increase the effective spatial resolution using a higher resolution imaging method, correct for tissue deformation, and incorporate fine morphological features in an MS imaging dataset. Recent methodological and computational developments are covered along with their application to single‐cell and imaging analyses.
Insights into the pathogenesis of primary hyperoxaluria type I from the structural dynamics of alanine:glyoxylate aminotransferase variants
Authors: Vankova Pavla , Pacheco‐Garcia Juan Luis , Loginov Dmitry S. , Gómez‐Mulas Atanasio , Kádek Alan , Martín‐Garcia José Manuel , Salido Eduardo , Man Petr , Pey Angel L.
Published: January 2024

Primary hyperoxaluria type I (PH1) is caused by deficient alanine:glyoxylate aminotransferase (AGT) activity. PH1‐causing mutations in AGT lead to protein mistargeting and aggregation. Here, we use hydrogen‐deuterium exchange (HDX) to characterize the wild‐type (WT), the LM (a polymorphism frequent in PH1 patients) and the LM G170R (the most common mutation in PH1) variants of AGT. We provide the first experimental analysis of AGT structural dynamics, showing that stability is heterogeneous in the native state and providing a blueprint for frustrated regions with potentially functional relevance. The LM and LM G170R variants only show local destabilization. Enzymatic transamination of the pyridoxal 5‐phosphate cofactor bound to AGT hardly affects stability. Our study, thus, supports that AGT misfolding is not caused by dramatic effects on structural dynamics.
Microenvironment‐induced restoration of cohesive growth associated with focal activation of P‐cadherin expression in lobular breast carcinoma metastatic to the colon
Authors: Gronewold Malte , Grote Isabel , Bartels Stephan , Christgen Henriette , Kandt Leonie D , Brito Maria Jose , Cserni Gàbor , Daemmrich Maximilian E , Fogt Franz , Helmke Burkhard M , ter Hoeve Natalie , Lang‐Schwarz Corinna , Vieth Michael , Wellmann Axel , Kuehnle Elna , Kulik Ulf , Riedel Gesa , Reineke‐Plaass Tanja , Lehmann Ulrich , Koorman Thijs , Derksen Patrick WB , Kreipe Hans , Christgen Matthias
Published: January 2024

Invasive lobular carcinoma (ILC) is a special breast cancer type characterized by noncohesive growth and E‐cadherin loss. Focal activation of P‐cadherin expression in tumor cells that are deficient for E‐cadherin occurs in a subset of ILCs. Switching from an E‐cadherin deficient to P‐cadherin proficient status (EPS) partially restores cell–cell adhesion leading to the formation of cohesive tubular elements. It is unknown what conditions control EPS. Here, we report on EPS in ILC metastases in the large bowel. We reviewed endoscopic colon biopsies and colectomy specimens from a 52‐year‐old female (index patient) and of 18 additional patients (reference series) diagnosed with metastatic ILC in the colon. EPS was assessed by immunohistochemistry for E‐cadherin and P‐cadherin. CDH1/E‐cadherin mutations were determined by next‐generation sequencing. The index patient's colectomy showed transmural metastatic ILC harboring a CDH1/E‐cadherin p.Q610* mutation. ILC cells displayed different growth patterns in different anatomic layers of the colon wall. In the tunica muscularis propria and the tela submucosa, ILC cells featured noncohesive growth and were E‐cadherin‐negative and P‐cadherin‐negative. However, ILC cells invading the mucosa formed cohesive tubular elements in the intercryptal stroma of the lamina propria mucosae. Inter‐cryptal ILC cells switched to a P‐cadherin‐positive phenotype in this microenvironmental niche. In the reference series, colon mucosa infiltration was evident in 13 of 18 patients, one of which showed intercryptal EPS and conversion to cohesive growth as described in the index patient. The large bowel is a common metastatic site in ILC. In endoscopic colon biopsies, the typical noncohesive growth of ILC may be concealed by microenvironment‐induced EPS and conversion to cohesive growth.
Total scalp irradiation: A study comparing multiple types of bolus and VMAT optimization techniques
Authors: Davis Tanisha M. , Luca Kirk , Sudmeier Lisa J. , Buchwald Zachary S. , Khan Mohammad K. , Yang Xiaofeng , Schreibmann Eduard , Zhang Jiahan , Roper Justin
Published: January 2024

Purpose

To investigate bolus design and VMAT optimization settings for total scalp irradiation.

Methods

Three silicone bolus designs (flat, hat, and custom) from .decimal were evaluated for adherence to five anthropomorphic head phantoms. Flat bolus was cut from a silicone sheet. Generic hat bolus resembles an elongated swim cap while custom bolus is manufactured by injecting silicone into a 3D printed mold. Bolus placement time was recorded. Air gaps between bolus and scalp were quantified on CT images. The dosimetric effect of air gaps on target coverage was evaluated in a treatment planning study where the scalp was planned to 60 Gy in 30 fractions. A noncoplanar VMAT technique based on gEUD penalties was investigated that explored the full range of gEUD alpha values to determine which settings achieve sufficient target coverage while minimizing brain dose. ANOVA and the t‐test were used to evaluate statistically significant differences (threshold = 0.05).

Results

The flat bolus took 32 ± 5.9 min to construct and place, which was significantly longer (p < 0.001) compared with 0.67 ± 0.2 min for the generic hat bolus or 0.53 ± 0.10 min for the custom bolus. The air gap volumes were 38 ± 9.3 cc, 32 ± 14 cc, and 17 ± 7.0 cc for the flat, hat, and custom boluses, respectively. While the air gap differences between the flat and custom boluses were significant (p = 0.011), there were no significant dosimetric differences in PTV coverage at V57Gy or V60Gy. In the VMAT optimization study, a gEUD alpha of 2 was found to minimize the mean brain dose.

Conclusions

Two challenging aspects of total scalp irradiation were investigated: bolus design and plan optimization. Results from this study show opportunities to shorten bolus fabrication time during simulation and create high quality treatment plans using a straightforward VMAT template with simple optimization settings.
Hydrogel Alginate Considerations for Improved 3D Matrix Stability and Cell Graft Viability and Function in Studying Type 1 Diabetes In Vitro
Authors: Quiroz Victor M. , Wang Yuanjia , Rakoski Amanda I. , Kasinathan Devi , Neshat Sarah Y. , Hollister‐Lock Jennifer , Doloff Joshua C.
Published: January 2024

Biomedical devices such as islet‐encapsulating systems are used for treatment of type 1 diabetes (T1D). Despite recent strides in preventing biomaterial fibrosis, challenges remain for biomaterial scaffolds due to limitations on cells contained within. The study demonstrates that proliferation and function of insulinoma (INS‐1) cells as well as pancreatic rat islets may be improved in alginate hydrogels with optimized gel%, crosslinking, and stiffness. Quantitative polymerase chain reaction (qPCR)‐based graft phenotyping of encapsulated INS‐1 cells and pancreatic islets identified a hydrogel stiffness range between 600 and 1000 Pa that improved insulin Ins and Pdx1 gene expression as well as glucose‐sensitive insulin‐secretion. Barium chloride (BaCl2) crosslinking time is also optimized due to toxicity of extended exposure. Despite possible benefits to cell viability, calcium chloride (CaCl2)‐crosslinked hydrogels exhibited a sharp storage modulus loss in vitro. Despite improved stability, BaCl2‐crosslinked hydrogels also exhibited stiffness losses over the same timeframe. It is believed that this is due to ion exchange with other species in culture media, as hydrogels incubated in dIH2O exhibited significantly improved stability. To maintain cell viability and function while increasing 3D matrix stability, a range of useful media:dIH2O dilution ratios for use are identified. Such findings have importance to carry out characterization and optimization of cell microphysiological systems with high fidelity in vitro.
Effect of the Glass Transition Temperature of Organic Materials on Exciton Recombination Region of Deep Blue OLED under Thermal Stress
Authors: Shi Wei , Yue Yuqing , Zhao Bingjia , Xu Hanfei , Cao Chenhui , Lan Weixia , Lin Yang , Wei Bin
Published: January 2024

The carrier transport and the exciton recombination region, which are strongly affected by temperature, are essential to organic light‐emitting diodes (OLEDs) under thermal stress. A different charge carrier transport materials with different glass transition temperature (Tg) is selected to investigate their effect on the thermal stability of OLEDs. By calculating the carrier mobility at different annealing temperatures and comparing the simulated values by the Arrhenius equation, the effect of the thermal stress on the performance of OLEDs is analyzed. Then, using the luminescent dye as a probe adjacent to the emitting layer and charge transporting layer to identify the exciton recombination region at different temperatures and driving voltages, it is revealed that replacing the functional material with low Tg with a high Tg one could effectively increase the thermal stability of the device to 100 °C. The optimized devices have a reduced turn‐on voltage, and the electron mobility is increased by more than one order of magnitude, which was conducive to exciton recombination. The work promotes the application of OLEDs in extreme environments by improving their thermal stability.
Trifluoromethyl‐Substituted Perylene Diimides for Rapid and Sensitive Film‐Based Fluorescence Detection of Methamphetamine and Cocaine Hydrochloride
Authors: Chen Ming , Chu Ronan , Babazadeh Mohammad , Kistemaker Jos C. M. , Burn Paul L. , Gentle Ian R. , Shaw Paul E.
Published: January 2024

Film‐based fluorescence sensors are attractive for rapid and on‐site detection of illicit drugs, due to their potential for portability, high sensitivity and reusability. Three new bay‐substituted perylene diimide derivatives are synthesized , P1‐CH3 (1‐methyl), P1‐CF3 (1‐trifluoromethyl), and P1‐(CF3)2 (1,7‐di‐trifluoromethyl) that can be solution processed into thin films capable of detecting methamphetamine hydrochloride (MA•HCl) and cocaine•HCl via a luminescence quenching mechanism. The ionization potentials of the materials are sufficiently high to enable photoinduced hole transfer to occur, which led to the luminescence quenching. The trifluoromethyl‐substituted PDIs exhibit superior sensitivity and reversibility of the luminescence quenching process. In particular, P1‐(CF3)2 shows a limit of detection to MA•HCl of ≈100 ng after a 15‐second exposure, and its luminescence can be fully recovered either through mild heating or being placed in an air flow. Furthermore, by using the luminescence quenching and recovery responses of four PDI sensing materials it is possible to differentiate MA•HCl, cocaine•HCl, from aspirin, caffeine, sugar, and (sebum) skin oil in around a minute.
High Energy Density Solid‐State Lithium Metal Batteries Enabled by In Situ Polymerized Integrated Ultrathin Solid Electrolyte/Cathode
Authors: Hu Jiang‐Kui , Gao Yu‐Chen , Yang Shi‐Jie , Wang Xi‐Long , Chen Xiang , Liao Yu‐Long , Li Shuai , Liu Jia , Yuan Hong , Huang Jia‐Qi
Published: January 2024

Solid‐state batteries (SSBs) are regarded as the most promising next‐generation energy storage devices due to their potential to achieve higher safety performance and energy density. However, the troubles in the preparation of ultrathin solid‐state electrolytes (SEs) as well as the resultant compromise in mechanical strength greatly limit the safety application of SSBs. Herein, a novel in situ polymerized integrated ultrathin SE/cathode design is developed. The ultrathin ceramic layer supported on the cathode serves not only as a rigid scaffold to prevent direct contact between the cathode and anode but also as active inorganic fillers to enhance the mechanical properties of in situ polymerized SE film. The unique Li‐ion coordination environments as well as the Li hopping mechanism profoundly promote fast ion transport in composite SEs. The in situ polymerized SEs simultaneously achieve the balance in ultrathin thickness (10 µm), fast ion transport (0.65 mS cm−1), superior Young's modulus (66.8 GPa), and excellent interface contact. The pouch cells with practical Li||LiNi0.8Co0.1Mn0.1O2 configuration achieve an ultrahigh volumetric energy density of 1018 Wh L−1 and safety performance. The in situ polymerized integrated ultrathin SE/cathode design exhibits great promise for the practical application of SSBs with high energy density and safety performance.