bluuebunny/crossref_metadata_2025 huggingface dataset:
Shiga Toxin Binds Human Platelets via Globotriaosylceramide (PkAntigen) and a Novel Platelet Glycosphingolipid
Authors: Cooling Laura L. W. , Walker Katherine E. , Gille Theresa , Koerner Theodore A. W.
Published: March 2020

Hemolytic-uremic syndrome is a clinical syndrome characterized by acute renal failure, microangiopathic hemolytic anemia, and thrombocytopenia that often follows infection by Shiga toxin- or verotoxin-producing strains ofEscherichia coli. Because thrombocytopenia and platelet activation are hallmark features of hemolytic-uremic syndrome, we examined the ability of Shiga toxin to bind platelets by flow cytometry and high-performance thin-layer chromatography (HPTLC) of isolated platelet glycosphingolipids. By HPTLC, Shiga toxin was shown to bind globotriaosylceramide (Gb3) and a minor platelet glycolipid with anRfof 0.03, band 0.03. In a survey of 20 human tissues, band 0.03 was identified only in platelets. In individuals, band 0.03 was expressed by 20% of donors and was specifically associated with increased platelet Gb3expression. Based on glycosidase digestion and epitope mapping, band 0.03 was hypothesized to represent a novel glycosphingolipid, IV3-β-Galα1-4galactosylglobotetraosylceramide. Based on incidence, structure, and association with increased Gb3expression, band 0.03 may represent the antithetical Luke blood group antigen. By flow cytometry, Shiga toxin bound human platelets, although the amount of Shiga toxin bound varied in donors. Differences in Shiga toxin binding to platelet membranes did not reflect differences in platelet Gb3expression. In contrast, there was a loose association between Shiga toxin binding and decreasing forward scatter, suggesting that Shiga toxin and verotoxins bind more efficiently to smaller, older platelets. In summary, Shiga and Shiga-like toxins may bind platelets via specific glycosphingolipid receptors. Such binding may contribute to the thrombocytopenia, platelet activation, and microthrombus formation observed in hemolytic-uremic syndrome.
Importance of Interleukin-10 in Genetic Susceptibility of Mice toCoccidioides immitis
Authors: Fierer Joshua , Walls Lorraine , Eckmann Lars , Yamamoto Tomoko , Kirkland Theo N.
Published: March 2020

Inbred strains of mice vary in their susceptibility toCoccidioides immitis. We infected resistant DBA/2 (D2) mice and three susceptible strains of mice (C57BL/6 [B6], BALB/c, and CAST/Ei) by intraperitoneal injection of arthroconidia and determined the severity of infection based on colony counts of fungus in the spleens and lungs 14 days after infection. We used quantitative reverse transcription-PCR to measure the amounts of cytokines made in the spleens and lungs of infected mice. Susceptible mice made 1,000-fold more interleukin-10 (IL-10) than resistant D2 mice and about 10-fold more IL-4. In contrast, D2 mice had more IL-12 p40 in their lungs than did B6 mice. Resistant and susceptible mice made equivalent amounts of gamma interferon, IL-6, and IL-2. In order to determine whether IL-10 adversely affected the response toC. immitis, we infected IL-10-deficient mice, and they were found to be as resistant as D2 mice. This result indicates that IL-10 plays a crucial role in determining susceptibility toC. immitisin inbred mice. Because IL-4 mRNA levels were higher in most strains of susceptible mice, we also infected IL-4-deficient B6 mice. They were more resistant than B6 controls but not as resistant as IL-10-deficient mice. Thus, both IL-10 and IL-4 adversely affect the ability of C57BL mice to resist infection withC. immitis, but IL-10 has a larger effect and is the cytokine that is consistently associated with susceptibility in all strains of inbred mice.
Immunological Relationship between the Class I Epitope of Streptococcal M Protein and Myosin
Authors: Quinn Anthony , Ward Kent , Fischetti Vincent A. , Hemric Mark , Cunningham Madeleine W.
Published: March 2020

The class I epitope of streptococcal M protein is an epidemiological marker for acute rheumatic fever (ARF)-associated serotypes of group A streptococci and is recognized by anti-M protein monoclonal antibody (MAb) 10B6. Using MAb 10B6, we determined the relationship between the class I epitope of M protein and the α-helical coiled-coil protein myosin. MAb 10B6 reacted by enzyme-linked immunosorbent assay and Western blotting with human cardiac myosin and rabbit skeletal myosin and its heavy meromyosin (HMM) subfragment. Overlapping synthetic peptides of M5 protein were used to identify the region of M5 protein recognized by MAb 10B6. Two C repeat peptides (C2A and C3) containing the amino acid sequence KGLRRDLDASREAK reacted with MAb 10B6. Partial sequence identity, RRDL, was found in the HMM fragment of myosin, which reacted with MAb 10B6. However, not all peptides of M5 protein and myosin containing the RRDL sequence reacted with MAb 10B6. ARF sera and sera from uncomplicated pharyngitis (UNC) reacted with C repeat region peptides of M protein, while acute glomerulonephritis sera were not as reactive. Affinity-purified human antibody to peptide C3 reacted with myosin. The data demonstrate that the class I epitope of M protein is immunologically cross-reactive with myosin and the HMM subfragment, and antibodies to peptide C3 and myosin were present in ARF and UNC sera.
Antibody Responses in Humans against Coli Surface Antigen 6 of Enterotoxigenic
Escherichia coli
Authors: Helander Anna , Wennerås Christine , Qadri Firdausi , Svennerholm Ann-Mari
Published: March 2020

Enterotoxigenic
Escherichia coli
(ETEC) strains expressing only coli surface antigen 6 (CS6) have previously been isolated from patients with diarrhea, but the immunogenicity of CS6 has not been established in humans. We have detected CS6-specific immunoglobulin A responses in the feces and blood of patients convalescing from natural ETEC disease and of volunteers given an oral ETEC vaccine.
Elevated Concentrations of Eotaxin and Interleukin-5 in Human Neurocysticercosis
Authors: Evans Carlton A. W. , Garcia Hector H. , Hartnell Adele , Gilman Robert H. , Jose Peter J. , Martinez Manuel , Remick Daniel G. , Williams Timothy J. , Friedland Jon S.
Published: March 2020

Symptomatic neurocysticercosis, a major cause of epilepsy worldwide, results from inflammation aroundTaenia soliumlarvae, but the mechanisms are unknown. Eotaxin, not previously reported in cases of human infection, and interleukin-5 (IL-5) but not IL-8 concentrations were elevated in patient serum, and IL-5 levels were also elevated in cerebrospinal fluid (CSF). Eosinophil-selective mediators may be involved in the pathogenesis of cysticercosis. IL-6 concentrations were also elevated in patient CSF, possibly indicative of an acute-phase response.
Clostridium perfringensType E Animal Enteritis Isolates with Highly Conserved, Silent Enterotoxin Gene Sequences
Authors: Billington Stephen J. , Wieckowski Eva U. , Sarker Mahfuzur R. , Bueschel Dawn , Songer J. Glenn , McClane Bruce A.
Published: March 2020

SeveralClostridium perfringensgenotype E isolates, all associated with hemorrhagic enteritis of neonatal calves, were identified by multiplex PCR. These genotype E isolates were demonstrated to express α and ι toxins, but, despite carrying sequences for the gene (cpe) encodingC. perfringensenterotoxin (CPE), were unable to express CPE. These silentcpesequences were shown to be highly conserved among type E isolates. However, relative to the functionalcpegene of type A isolates, these silent type Ecpesequences were found to contain nine nonsense and two frameshift mutations and to lack the initiation codon, promoters, and ribosome binding site. The type E animal enteritis isolates carrying these silentcpesequences do not appear to be clonally related, and their silent type Ecpesequences are always located, near the ι toxin genes, on episomal DNA. These findings suggest that the highly conserved, silentcpesequences present in most or all type E isolates may have resulted from the recent horizontal transfer of an episome, which also carries ι toxin genes, to several different type AC. perfringensisolates.
The Macrophage-Induced Gene
mig
as a Marker for Clinical Pathogenicity and In Vitro Virulence of
Mycobacterium avium
Complex Strains
Authors: Meyer Martin , von Grünberg Philipp W. R. , Knoop Tim , Hartmann Pia , Plum Georg
Published: March 2020

The capacity of 20
Mycobacterium avium
complex isolates to multiply intracellularly in human monocyte-derived macrophages was assessed and correlated to the clinical relevance of each isolate and its reactivity with several candidate genetic virulence markers. The strongest correlation with a virulence phenotype was found for a conserved coding sequence of the macrophage-induced gene
mig
identified by a specific
mig
restriction fragment length polymorphism type.
Chemokine Secretion of Human Cells in Response toToxoplasma gondiiInfection
Authors: Denney Carolyn F. , Eckmann Lars , Reed Sharon L.
Published: March 2020

The ubiquitous protozoan parasiteToxoplasma gondiiis a major cause of morbidity and mortality in neonates and immunocompromised hosts. Both acute invasion and reactivation of latent infection result in an inflammatory reaction with lymphocytes, macrophages, and neutrophils. The mechanisms responsible for triggering the local host response to toxoplasmosis are not fully understood. Infection of monolayers of human HeLa epithelial cells and fibroblasts withT. gondiiresulted in a marked increase in the expression of interleukin-8 (IL-8)-specific mRNA and secretion of the proinflammatory and chemoattractant cytokines interleukin-8 (IL-8), GROα, and MCP-1. Host cell invasion and lysis were required for this response, as tachyzoite lysates alone had no effect on IL-8 secretion. IL-8 release was dependent on the release of soluble host cell factors: IL-1α in HeLa cells and an additional mediator in fibroblasts. HT-29 epithelial cells, which lack IL-1α or another IL-8-inducing activity, did not release IL-8 after infection, although they were efficiently infected withT. gondiiand increased IL-8 secretion in response to added IL-1α. These data suggest that proinflammatory chemokine secretion is an important host cell response to toxoplasmosis and that the release of IL-1α and other mediators from lysed host cells is critical for this chemokine response.
Immunity toChlamydia trachomatisMouse Pneumonitis Induced by Vaccination with Live Organisms Correlates with Early Granulocyte-Macrophage Colony-Stimulating Factor and Interleukin-12 Production and with Dendritic Cell-Like Maturation
Authors: Zhang Dongji , Yang Xi , Lu Hang , Zhong Guangming , Brunham Robert C.
Published: March 2020

As is true for other intracellular pathogens, immunization with liveChlamydia trachomatisgenerally induces stronger protective immunity than does immunization with inactivated organism. To investigate the basis for such a difference, we studied immune responses in BALB/c mice immunized with viable or UV-killedC. trachomatismouse pneumonitis (MoPn). Strong, acquired resistance toC. trachomatisinfection was elicited by immunization with viable but not dead organisms. Immunization with viable organisms induced high levels of antigen-specific delayed-type hypersensitivity (DTH), gamma interferon production, and immunoglobulin A (IgA) responses. Immunization with inactivated MoPn mainly induced interleukin-10 (IL-10) production and IgG1 antibody without IgA or DTH responses. Analysis of local early cytokine and cellular events at days 3, 5, and 7 after peritoneal cavity immunization showed that high levels of granulocyte-macrophage colony-stimulating factor and IL-12 were detected with viable but not inactivated organisms. Furthermore, enrichment of a dendritic cell (DC)-like population was detected in the peritoneal cavity only among mice immunized with viable organisms. The results suggest that early differences in inducing proinflammatory cytokines and activation and differentiation of DCs may be the key mechanism underlying the difference between viable and inactivated organisms in inducing active immunity toC. trachomatisinfection.
TransformedToxoplasma gondiiTachyzoites Expressing the Circumsporozoite Protein ofPlasmodium knowlesiElicit a Specific Immune Response in Rhesus Monkeys
Authors: di Cristina Manlio , Ghouze Firman , Kocken Clemens H. M. , Naitza Silvia , Cellini Pamela , Soldati Dominique , Thomas Alan W. , Crisanti Andrea
Published: March 2020

Toxoplasma gondiitachyzoites were transformed with the coding sequence of the circumsporozoite (CS) protein of the primate malaria parasitePlasmodium knowlesi. A single inoculation of live transformed tachyzoites elicited an antibody response directed against the immunodominant repeat epitope (EQPAAGAGG)2of theP. knowlesiCS protein in rhesus monkeys. Notably, these animals failed to show a positive serum conversion againstT. gondii. Antibodies againstToxoplasmaantigens were detected only after a second inoculation with a higher number of transformed tachyzoites. This boost induced an increased antibody response against theP. knowlesiCS protein associated with immunoglobulin class switching, thus demonstrating the establishment of immunological memory. These results indicate that theToxoplasma-derived CS protein is efficiently recognized by the monkey immune system and represents an immunodominant antigen in transformed parasites.
Identification of Pneumococcal Surface Protein A as a Lactoferrin-Binding Protein of
Streptococcus pneumoniae
Authors: Hammerschmidt Sven , Bethe Gesina , H. Remane Petra , Chhatwal Gursharan S.
Published: March 2020

Lactoferrin (Lf), an iron-sequestering glycoprotein, predominates in mucosal secretions, where the level of free extracellular iron (10
−18
M) is not sufficient for bacterial growth. This represents a mechanism of resistance to bacterial infections by prevention of colonization of the host by pathogens. In this study we were able to show that
Streptococcus pneumoniae
specifically recognizes and binds the iron carrier protein human Lf (hLf). Pretreatment of pneumococci with proteases reduced hLf binding significantly, indicating that the hLf receptor is proteinaceous. Binding assays performed with 63 clinical isolates belonging to different serotypes showed that 88% of the tested isolates interacted with hLf. Scatchard analysis showed the existence of two hLf-binding proteins with dissociation constants of 5.7 × 10
−8
and 2.74 × 10
−7
M. The receptors were purified by affinity chromatography, and internal sequence analysis revealed that one of the
S. pneumoniae
proteins was homologous to pneumococcal surface protein A (PspA). The function of PspA as an hLf-binding protein was confirmed by the ability of purified PspA to bind hLf and to competitively inhibit hLf binding to pneumococci.
S. pneumoniae
may use the hLf-PspA interaction to overcome the iron limitation at mucosal surfaces, and this might represent a potential virulence mechanism.
Interleukin-12 Production Is Required for Chlamydial Antigen-Pulsed Dendritic Cells To Induce Protection against LiveChlamydia trachomatisInfection
Authors: Lu Hang , Zhong Guangming
Published: March 2020

Immunization with dendritic cells pulsed ex vivo with antigens has been successfully used to elicit primary antigen-specific immune responses. We report that mouse bone marrow-derived dendritic cells pulsed with inactivated chlamydial organisms induced strong protection against live chlamydial infection in a mouse lung infection model. Either the dendritic cells or chlamydial organisms alone or macrophages similarly pulsed with chlamydial organisms failed to induce any significant protection. These observations suggest that dendritic cells can efficiently process and present chlamydial antigens to naive T cells in vivo. Mice immunized with the chlamydia-pulsed dendritic cells preferentially developed a Th1 cell-dominant response while mice immunized with the other immunogens did not, suggesting a correlation between a Th1 cell-dominant response and protection against chlamydial infection. We further found that dendritic cells produced a large amount of interleukin 12 (IL-12) upon ex vivo pulsing with inactivated chlamydial organisms, which may allow the dendritic cells to direct a Th1 cell-dominant response. Dendritic cells from mice deficient in the IL-12 p40 gene failed to produce IL-12 after a similar ex vivo pulse with chlamydial organisms, and more importantly, immunization with these dendritic cells failed to induce a Th1 cell-dominant response and did not induce strong protection against chlamydial infection. Thus, the ability of dendritic cells to efficiently process and present chlamydial antigens and to produce IL-12 upon chlamydial-organism stimulation are both required for the induction of protection against chlamydial infection. This information may be useful for the further design of effective chlamydial vaccines.
Rapid Local Expression of Interleukin-12, Tumor Necrosis Factor Alpha, and Gamma Interferon after CutaneousFrancisella tularensisInfection in Tularemia-Immune Mice
Authors: Stenmark Stephan , Sunnemark Dan , Bucht Anders , Sjöstedt Anders
Published: March 2020

Francisella tularensisLVS is an effective live vaccine strain used for cutaneous vaccination against tularemia in man. In mice, injection of LVS causes invasive disease and subsequent development of immunity that is characterized by effective control of otherwise lethal doses of the organism. In the present investigation, it is shown that LVS-immune mice controlled an intradermal infection much more effectively than did naive mice; bacterial counts in skin samples were 1.5 to 2.0 log10lower 24 h after injection and 6 log10lower 72 h after injection in immune mice. Moreover, in contrast to naive mice, no bacteria were demonstrated in samples from livers and spleens of immune mice. By immunohistochemistry, skin samples from immune mice showed an intense staining for interleukin-12 (IL-12) and a moderate staining for tumor necrosis factor alpha (TNF-α) at 24 h postinoculation, after which staining for both cytokines faded. In naive mice, the staining for IL-12 was weak at all time points and no staining for TNF-α was observed. No staining for gamma interferon (IFN-γ) was observed in any group before 72 h. At that time point, skin samples from immune mice showed moderate staining and skin samples from naive mice showed weak staining. Reverse transcriptase PCR showed an induction of mRNA of the three cytokines in the skin within the first day after injection. A quantitative analysis demonstrated higher IFN-γ and TNF-α mRNA levels in immune mice at 24 h postinoculation. In conclusion, immunization withF. tularensisLVS conferred a capability to respond to cutaneous reinfection, with rapid local expression of IL-12, TNF-α, and IFN-γ, and this expression was paralleled by containment and mitigation of the infection. The cytokine response may be part of a local barrier function of the skin, important to host protection against tularemia.
Identification of a Receptor-Binding Region within Domain 4 of the Protective Antigen Component of Anthrax Toxin
Authors: Varughese Mini , Teixeira Avelino V. , Liu Shihui , Leppla Stephen H.
Published: March 2020

Anthrax toxin from
Bacillus anthracis
is a three-component toxin consisting of lethal factor (LF), edema factor (EF), and protective antigen (PA). LF and EF are the catalytic components of the toxin, whereas PA is the receptor-binding component. To identify residues of PA that are involved in interaction with the cellular receptor, two solvent-exposed loops of domain 4 of PA (amino acids [aa] 679 to 693 and 704 to 723) were mutagenized, and the altered proteins purified and tested for toxicity in the presence of LF. In addition to the intended substitutions, novel mutations were introduced by errors that occurred during PCR. Substitutions within the large loop (aa 704 to 723) had no effect on PA activity. A mutated protein, LST-35, with three substitutions in the small loop (aa 679 to 693), bound weakly to the receptor and was nontoxic. A mutated protein, LST-8, with changes in three separate regions did not bind to receptor and was nontoxic. Toxicity was greatly decreased by truncation of the C-terminal 3 to 5 aa, but not by their substitution with nonnative residues or the extension of the terminus with nonnative sequences. Comparison of the 28 mutant proteins described here showed that the large loop (aa 704 to 722) is not involved in receptor binding, whereas residues in and near the small loop (aa 679 to 693) play an important role in receptor interaction. Other regions of domain 4, in particular residues at the extreme C terminus, appear to play a role in stabilizing a conformation needed for receptor-binding activity.
CD40 Ligation PreventsTrypanosoma cruziInfection through Interleukin-12 Upregulation
Authors: Chaussabel Damien , Jacobs Frédérique , de Jonge Jan , de Veerman Marijke , Carlier Yves , Thielemans Kris , Goldman Michel , Vray Bernard
Published: March 2020

Because of the critical role of the CD40-CD40 ligand (CD40L) pathway in the induction and effector phases of immune responses, we investigated the effects of CD40 ligation on the control ofTrypanosoma cruziinfection. First, we observed that supernatants of murine spleen cells stimulated by CD40L-transfected 3T3 fibroblasts (3T3-CD40L transfectants) prevent the infection of mouse peritoneal macrophages (MPM) byT. cruzi. This phenomenon depends on de novo production of nitric oxide (NO) as it is prevented by the addition ofN-nitro-l-arginine methyl ester, a NO synthase inhibitor. NO production requires interleukin (IL)-12-mediated gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) synthesis as demonstrated by inhibition experiments using neutralizing anti-IL-12, anti-IFN-γ, and anti-TNF-α monoclonal antibodies (MAb). We found that an activating anti-CD40 MAb also directly stimulates IFN-γ-activated MPM to produce NO and thereby to controlT. cruziinfection. To determine the in vivo relevance of these in vitro findings, mice were injected with 3T3-CD40L transfectants or 3T3 control fibroblasts at the time ofT. cruziinoculation. We observed that in vivo CD40 ligation dramatically reduced both parasitemia and the mortality rate ofT. cruzi-infected mice. A reduced parasitemia was still observed when the injection of 3T3-CD40L transfectants was delayed 8 days postinfection. It was abolished by injection of anti-IL-12 MAb. Taken together, these data establish that CD40 ligation facilitates the control ofT. cruziinfection through a cascade involving IL-12, IFN-γ, and NO.
Vaccinated Mice Remain More Susceptible to
Mycobacterium tuberculosis
Infection Initiated via the Respiratory Route than via the Intravenous Route
Authors: North Robert J. , LaCourse Ronald , Ryan Lynn
Published: March 2020

Mice given
Mycobacterium tuberculosis
bacilli via the respiratory route succumbed much sooner to infection than mice given 1,000 times more bacilli via the intravenous route. Vaccination provided increased protection to an
M. tuberculosis
challenge infection; however, mice infected via the respiratory route remained much more susceptible.
β
1
-Chain Integrins Are Not Essential for Intimin-Mediated Host Cell Attachment and Enteropathogenic
Escherichia coli
-Induced Actin Condensation
Authors: Liu Hui , Magoun Loranne , Leong John M.
Published: March 2020

Intimin is a bacterial outer membrane protein required for intimate attachment of enterohemorrhagic and enteropathogenic
Escherichia coli
(EHEC and EPEC) to mammalian cells. β
1
-chain integrins have been proposed as candidate receptors for intimin. We found that binding of mammalian cells to immobilized intimin was not detectable unless mammalian cells were preinfected with EPEC or EHEC. β
1
-chain integrin antagonists or inactivation of the gene encoding the β
1
-chain did not affect binding of preinfected mammalian cells to intimin or the actin condensation associated with the attachment of EPEC. The results indicate that β
1
-chain integrins are not essential for intimin-mediated cell attachment or EPEC-mediated actin polymerization.
Structural and Functional Characterization of IS
1358
from
Vibrio cholerae
Authors: Dumontier Sandrine , Trieu-Cuot Patrick , Berche Patrick
Published: March 2020

The new epidemic serovar O139 of
Vibrio cholerae
has emerged from the pandemic serovar O1 biotype El Tor through the replacement of a 22-kbp DNA region by a 40-kbp O139-specific DNA fragment. This O139-specific DNA fragment contains an insertion sequence that was described previously (U. H. Stroeher, K. E. Jedani, B. K. Dredge, R. Morona, M. H. Brown, L. E. Karageorgos, J. M. Albert, and P. A. Manning, Proc. Natl. Acad. Sci. USA 92:10374–10378, 1995) and designated IS
1358
O139
. We studied the distribution of the IS
1358
element in strains from various serovars by Southern analysis. Its presence was detected in strains from serovars O1, O2, O22, O139, and O155 but not in strains from serovars O15, O39, and O141. Furthermore, IS
1358
was present in multiple copies in strains from serovars O2, O22, and O155. We cloned and sequenced four copies of IS
1358
from
V. cholerae
O22 and one copy from
V. cholerae
O155. A comparison of their nucleotide sequences with those of O1 and O139 showed that they were almost identical. We constructed a transposon consisting of a kanamycin resistance gene flanked by two directly oriented copies of IS
1358
to study the functionality of this element. Transposition of this element from a nonmobilizable plasmid onto the conjugative plasmid pOX38-Gen was detected in an
Escherichia coli recA
donor at a frequency of 1.2 × 10
−8
. Sequence analysis revealed that IS
1358
duplicates 10 bp at its insertion site.
H-NS and StpA Proteins Stimulate Expression of the Maltose Regulon in
Escherichia coli
Authors: Johansson Jörgen , Dagberg Björn , Richet Evelyne , Uhlin Bernt Eric
Published: March 2020

The nucleoid-associated protein H-NS is a major component of the chromosome-protein complex, and it is known to influence the regulation of many genes in
Escherichia coli
. Its role in gene regulation is manifested by the increased expression of several gene products in
hns
mutant strains. Here we report findings showing that H-NS and the largely homologous protein StpA play a positive role in the expression of genes in the maltose regulon. In studies with
hns
mutant strains and derivatives also deficient in the
stpA
gene, we found that expression of the LamB porin was decreased. Our results showed that the amounts of both LamB protein and
lamB
mRNA were greatly reduced in
hns
and
hns-stpA
mutant strains. The same results were obtained when we monitored the amount of transcription from the
malEFG
operon. The
lamB
gene is situated in the
malKlamBmalM
operon, which forms a divergent operon complex together with the
malEFG
operon. The activation of these genes depends on the action of the maltose regulon activator MalT and the global activator cyclic AMP receptor protein. Using a
malT-lacZ
translational fusion and antiserum raised against MalT to measure the expression of MalT, we detected reduced MalT expression in
hns
and
hns-stpA
mutant strains in comparison with the wild-type strain. Our results suggest that the H-NS and StpA proteins stimulate MalT translation and hence play a positive role in the control of the maltose regulon.
Molecular Characterization of a Stress-Inducible Gene from
Lactobacillus helveticus
Authors: Smeds Andrèas , Varmanen Pekka , Palva Airi
Published: March 2020

A gene (
htrA
) coding for a stress-inducible HtrA-like protein from
Lactobacillus helveticus
CNRZ32 was cloned, sequenced, and characterized. The deduced amino acid sequence of the gene exhibited 30% identity with the HtrA protein from
Escherichia coli
; the putative catalytic triad and a PDZ domain that characterize the HtrA family of known bacterial serine proteases were also found in the sequence. Expression of the
L. helveticus htrA
gene in a variety of stress conditions was analyzed at the transcriptional level. The strongest induction, resulting in over an eightfold increase in the
htrA
transcription level, was found in growing CNRZ32 cells exposed to 4% (wt/vol) NaCl. Enhanced
htrA
mRNA expression was also seen in CNRZ32 cells after exposure to puromycin, ethanol, or heat. The reporter gene
gusA
was integrated in the
Lactobacillus
chromosome downstream of the
htrA
promoter by a double-crossover event which also interrupted the wild-type gene. The expression of
gusA
in the stress conditions tested was similar to that of
htrA
itself. In addition, the presence of an intact
htrA
gene facilitated growth under heat stress but not under salt stress.
Catabolite Regulation of the
Bacillus subtilis ctaBCDEF
Gene Cluster
Authors: Liu Xuemin , Taber Harry W.
Published: March 2020

Bacillus subtilis
cytochrome
c
oxidase
caa
3
is encoded by the
ctaCDEF
genes at the
ctaABCDEF
locus, with the
ctaBCDEF
genes organized as an operon-like unit. A dyad symmetry sequence and a catabolite response element homolog can be recognized in the 240-bp intercistronic region between
ctaB
and
ctaC. ctaB′-lacZ
and
ctaBCD′-lacZ
transcriptional fusions integrated at the native locus were used to study catabolite effects on transcription of the
ctaB
and
ctaCDEF
genes. In Schaeffer’s medium lacking glucose,
ctaBCD′-lacZ
was expressed at a very low level during the exponential phase, and expression increased about 30-fold 2 h after entry into the stationary phase. In the presence of 0.5% glucose,
ctaBCD′-lacZ
expression was totally repressed. In contrast to
ctaBCD′-lacZ
,
ctaB′-lacZ
was constitutively expressed regardless of carbon source. The
ctaCDEF
genes were separated from
ctaB
by insertion of plasmids carrying selectable markers in such a way that the
ctaCDEF
and
ctaB
transcription units remained intact. Enzymatic assays of
caa
3
with these constructs, showed that
ctaCDEF
was not expressed independently of
ctaB
. Also, when a ′
ctaB-ctaC′-lacZ
fusion (containing the
ctaB-ctaC
intercistronic region) was placed at a remote nonessential locus, β-galactosidase activity could not be detected. The absence of a promoter in the
ctaB-ctaC
intercistronic space also was indicated by the inability to detect
ctaC
-specific transcripts with RNase protection assays, primer extension, and rapid amplification of 5′ cDNA ends. Direct mRNA measurements showed that, in the presence of 0.5% glucose,
ctaBCDEF
transcripts terminated at the 3′ end of the putative stem-loop structure and the distal portion was down-regulated. A possible mechanism for
ctaCDEF
gene regulation is suggested. Catabolite repression of
ctaBCD′-lacZ
was partly dependent on CcpA but was independent of HPr. The expression of
ctaBCDEF
also appears to require the
strC
,
ctaA
, and
resD-resE
gene products.
Gene Cloning and Characterization of Recombinant RNase HII from a Hyperthermophilic Archaeon
Authors: Haruki Mitsuru , Hayashi Keiko , Kochi Takayuki , Muroya Ayumu , Koga Yuichi , Morikawa Masaaki , Imanaka Tadayuki , Kanaya Shigenori
Published: March 2020

We have cloned the gene encoding RNase HII (RNase HII
Pk
) from the hyperthermophilic archaeon
Pyrococcus kodakaraensis
KOD1 by screening of a library for clones that suppressed the temperature-sensitive growth phenotype of an
rnh
mutant strain of
Escherichia coli
. This gene was expressed in an
rnh
mutant strain of
E. coli
, the recombinant enzyme was purified, and its biochemical properties were compared with those of
E. coli
RNases HI and HII. RNase HII
Pk
is composed of 228 amino acid residues (molecular weight, 25,799) and acts as a monomer. Its amino acid sequence showed little similarity to those of enzymes that are members of the RNase HI family of proteins but showed 40, 31, and 25% identities to those of
Methanococcus jannaschii
,
Saccharomyces cerevisiae
, and
E. coli
RNase HII proteins, respectively. The enzymatic activity was determined at 30°C and pH 8.0 by use of an M13 DNA-RNA hybrid as a substrate. Under these conditions, the most preferred metal ions were Co
2+
for RNase HII
Pk
, Mn
2+
for
E. coli
RNase HII, and Mg
2+
for
E. coli
RNase HI. The specific activity of RNase HII
Pk
determined in the presence of the most preferred metal ion was 6.8-fold higher than that of
E. coli
RNase HII and 4.5-fold lower than that of
E. coli
RNase HI. Like
E. coli
RNase HI, RNase HII
Pk
and
E. coli
RNase HII cleave the RNA strand of an RNA-DNA hybrid endonucleolytically at the P-O3′ bond. In addition, these enzymes cleave oligomeric substrates in a similar manner. These results suggest that RNase HII
Pk
and
E. coli
RNases HI and HII are structurally and functionally related to one another.
Methionine Induces Sexual Development in the Fission Yeast
Schizosaccharomyces pombe
via an
ste11
-Dependent Signalling Pathway
Authors: Schweingruber Anne-Marie , Hilti Norma , Edenharter Eleonore , Schweingruber M. Ernst
Published: March 2020

Methionine added to minimal medium overcomes the repressing effects of ammonium and cyclic AMP (cAMP) on sexual development and efficiently induces mating and sporulation in homothallic strains of
Schizosaccharomyces pombe
. In heterothallic strains it induces G
1
arrest when cells enter stationary phase. We show that methionine reduces the intracellular cAMP pool and induces the expression of at least two cAMP-repressible genes, including
fbp1
and
ste11
. The easiest interpretation of the results is that methionine induces sexual development via a cAMP-dependent
ste11
signalling pathway.
A Family of Stability Determinants in Pathogenic Bacteria
Authors: Hayes Finbarr
Published: March 2020

A novel segregational stability system was identified on plasmid R485, which originates from
Morganella morganii
. The system is composed of two overlapping genes,
stbD
and
stbE
, which potentially encode proteins of 83 and 93 amino acids, respectively. Homologs of the
stbDE
genes were identified on the enterotoxigenic plasmid P307 from
Escherichia coli
and on the chromosomes of
Vibrio cholerae
and
Haemophilus influenzae
biogroup
aegyptius
. The former two homologs also promote plasmid stability in
E. coli
. Furthermore, the
stbDE
genes share homology with components of the
relBEF
operon and with the
dnaT
gene of
E. coli
. The organization of the
stbDE
cassette is reminiscent of toxin-antitoxin stability cassettes.
secG
and Temperature Modulate Expression of Azide-Resistant and Signal Sequence Suppressor Phenotypes of
Escherichia coli secA
Mutants
Authors: Ramamurthy Visvanathan , Dapíc Vesna , Oliver Donald
Published: March 2020

SecA is a dynamic protein that undergoes ATP-dependent membrane cycling to drive protein translocation across the
Escherichia coli
inner membrane. To understand more about this process, azide-resistant (
azi
) and signal sequence suppressor (
prlD
) alleles of
secA
were studied. We found that azide resistance is cold sensitive because of a direct effect on protein export, suggesting that SecA-membrane interaction is regulated by an endothermic step that is azide inhibitable.
secG
function is required for expression of azide-resistant and signal sequence suppressor activities of
azi
and
prlD
alleles, and in turn, these alleles suppress cold-sensitive and export-defective phenotypes of a
secG
null mutant. These remarkable genetic observations support biochemical data indicating that SecG promotes SecA membrane cycling and that this process is dependent on an endothermic change in SecA conformation.
Isolation in Endothelial Cell Cultures of
Chlamydia trachomatis
LGV (Serovar L2) from a Lymph Node of a Patient with Suspected Cat Scratch Disease
Authors: Maurin M. , Raoult D.
Published: March 2020

An inguinal lymph node, removed from a 21-year-old Romanian man suspected of having cat scratch disease, was sent to our laboratory for
Bartonella
culture. Lymph node specimens were inoculated on blood-enriched agar and in an endothelial cell culture system using the centrifugation shell vial technique. Bacteria were grown in cell monolayers and detected as positive with an anti-
Bartonella henselae
rabbit serum. However, such bacteria were identified as
Chlamydia trachomatis
biovar LGV serovar L2 by PCR sequencing techniques. Pathological examination of tissue biopsies was compatible with either lymphogranuloma venereum or cat scratch disease. The shell vial system is suitable for isolation of intracellular pathogens responsible for chronic lymphadenopathies, including
C. trachomatis
,
Bartonella
species,
Francisella tularensis
, and mycobacteria. However, care should be taken when identifying
Chlamydia
spp. and
Bartonella
spp. using polyclonal antibodies, since species of both genera have common antigens which are responsible for cross-reactions.
Persistence of Human Papillomavirus DNA in Benign and (Pre)malignant Skin Lesions from Renal Transplant Recipients
Authors: Berkhout Ron J. M. , Bouwes Bavinck Jan N. , ter Schegget Jan
Published: March 2020

An extremely diverse group of human papillomavirus (HPV) types consisting of epidermodysplasia verruciformis (EV)-associated HPV types and other cutaneous HPV types (e.g., HPV types 2 and 3) is associated with nonmelanoma cancers and benign lesions of the skin. The frequent presence of multiple HPV types in single skin biopsy specimens of renal transplant recipients prompted us to develop PCR techniques for the detection of distinct (sub)groups of genotypically related cutaneous HPV types, i.e., three subgroups of EV-associated HPV types and two groups (A2 and A4) of other cutaneous HPV types. This approach generally allowed a reliable identification of HPV genotypes by direct sequencing of the PCR products, despite the frequent occurrence of multiple infections. The targeted spectrum of HPV types comprises 66 cutaneous HPV types including 21 putative novel HPV types. We also detected 17 putative novel HPV subtypes. We demonstrated that the skin of nearly all renal transplant recipients who developed various benign and (pre)malignant skin lesions was persistently infected with one or more EV-associated HPV types and/or HPV types belonging to groups A2 and A4. The frequency and distribution of EV-associated HPV and HPV types belonging to groups A2 and A4 were similar in biopsy specimens from hyperkeratotic papillomas (77.5%), squamous cell carcinomas (77.8%), and actinic keratoses (67.9%) but appeared to be lower in specimens of basal cell carcinomas (35.7%), benign lesions (38.5%), and clinically normal skin (32.3%). These findings suggest that renal transplant recipients are prone to persistent cutaneous HPV infection. Our data do not support the existence of high-risk cutaneous HPV types.
A European Multicenter Study of Immunoblotting in Serodiagnosis of Lyme Borreliosis
Authors: Robertson J. , Guy E. , Andrews N. , Wilske B. , Anda P. , Granström M. , Hauser U. , Moosmann Y. , Sambri V. , Schellekens J. , Stanek G. , Gray J.
Published: March 2020

A European multicenter study of immunoblotting for the serodiagnosis of Lyme borreliosis showed considerable variation in results obtained from tests with a panel of 227 serum samples. Six laboratories used different immunoblot methods, and a wide range of bands was detected in all the assays. Multivariable logistic regression analysis of data from individual laboratories was used to determine the most discriminatory bands for reliable detection of antibodies to
Borrelia burgdorferi
sensu lato. These bands were used to construct individual interpretation rules for the immunoblots used in the six laboratories. Further analysis identified a subset of eight bands, which were important in all the laboratories, although with variations in significance. Possible European rules, all closely related, were formulated from these bands, although there was no single rule that gave high levels of sensitivity and specificity for all the laboratories. This is a reflection of the wide range of methodologies used, especially the use of different species and strains of
B. burgdorferi
sensu lato. The panel of European rules provides a framework for immunoblot interpretation which may be adapted in relation to the characteristics of Lyme borreliosis in local areas.
Accuracy and Precision of Quantitative Calibrated Loops in Transfer of Bronchoalveolar Lavage Fluid
Authors: Jacobs J. A. , De Brauwer E. I. G. B. , Cornelissen E. I. M. , Drent M.
Published: March 2020

Quantitative cultures of bronchoalveolar lavage (BAL) fluid are important in the diagnosis of ventilator-associated pneumonia, and calibrated loops are commonly used to set up these cultures. In this study, the performances of calibrated 0.010- and 0.001-ml loops in the transfer of BAL fluid were determined. Five loops of one lot from seven manufacturers were tested. Calibrations were performed by the gravimetric method (0.010-ml loops) and the colorimetric method (0.001-ml loops). Most of the 0.010-ml loops displayed a precision that was less than 10%, but six of them showed very poor accuracies as they transferred a deficiency (nichrome loops) or an excess (disposable loops) of BAL fluid that exceeded ±10%. The mean maximum and minimum BAL fluid volumes delivered by the 0.010-ml loops differed by a factor 3. The 0.001-ml loops displayed acceptable precision. Five of them showed inaccuracies of ≤±10%, and mean maximum and minimum BAL fluid volumes had a range of a factor of 2. For all loops, the volumes of BAL fluid sampled were larger than the volumes of reagent-grade water sampled. Results of the colony counting experiments confirmed these findings and revealed a high intra-assay variability for the 0.001-ml loops. We conclude that, when BAL fluid samples are cultured with calibrated loops, (i) proper verification of the calibration of these loops is mandatory, (ii) calibrations should be performed with BAL fluid as the test solution, and (iii) borderline quantitative culture results should be interpreted with knowledge of the inaccuracy values of these loops.
Comparison of Quantitative Cytomegalovirus (CMV) PCR in Plasma and CMV Antigenemia Assay: Clinical Utility of the Prototype AMPLICOR CMV MONITOR Test in Transplant Recipients
Authors: Caliendo Angela M. , St. George Kirsten , Kao Shaw-Yi , Allega Jessica , Tan Ban-Hock , LaFontaine Robert , Bui Larry , Rinaldo Charles R.
Published: March 2020

The correlation between the prototype AMPLICOR CMV MONITOR test (Roche Molecular Systems), a quantitative PCR assay, and the cytomegalovirus (CMV) pp65 antigenemia assay was evaluated in transplant recipients. Sequential blood specimens were collected on 29 patients (491 specimens), the leukocyte fraction was tested by CMV antigenemia, and quantitative PCR was performed on plasma specimens. None of the 15 patients (242 specimens) who were antigenemia negative were positive for CMV DNA by PCR, and none of these patients developed active CMV disease. There were 14 antigenemia-positive patients, 8 of whom developed active CMV disease. In all patients, there was a good association between the antigenemia and PCR assays. Ganciclovir-resistant virus was isolated from three patients with active CMV disease. These three patients had persistently elevated levels of antigenemia and CMV DNA by PCR when resistance to ganciclovir developed. This standardized, quantitative CMV PCR assay on plasma has clinical utility for the diagnosis of active disease and in monitoring the response to antiviral therapy in transplant recipients.
Macrorestriction Fingerprinting of “
Streptococcus milleri
” Group Bacteria by Pulsed-Field Gel Electrophoresis
Authors: Bartie K. L. , Wilson M. J. , Williams D. W. , Lewis M. A. O.
Published: March 2020

Although isolates of the “
Streptococcus milleri
” group (SMG) of bacteria are regarded as members of the commensal microflora of the body, they are frequently encountered in purulent infections from a range of body sites. The genetic diversity of 91 epidemiologically unrelated SMG isolates (including 37 commensal strains and 49 disease-associated strains) was analyzed by macrorestriction fingerprinting (MF). The genomes were digested with
Sma
I and
Apa
I independently, and fragments were resolved by pulsed-field gel electrophoresis. Similarities between banding profiles were determined, and strains were clustered on this basis into dendrograms. In common with other commensal species that have been examined by MF, considerable genetic diversity was revealed. In addition, the clustering of strains tended to support the current taxonomic position of this heterogeneous group. The present study has shown that MF is a powerful tool for characterization of SMG strains and that its use is likely to be of great value in epidemiological and population genetic studies of this group of bacteria.
Comparison of Pulsed-Field Gel Electrophoresis and Coagulase Gene Restriction Profile Analysis Techniques in the Molecular Typing of
Staphylococcus aureus
Authors: Chiou Chien-Shun , Wei Hsiao-Lun , Yang Li-Chu
Published: March 2020

Pulsed-field gel electrophoresis (PFGE) and coagulase gene restriction profile (CRP) analysis techniques were used to analyze 71
Staphylococcus aureus
isolates recovered from nine food-borne disease outbreaks. Twenty-two PFGE profiles and 11 CRPs were identified, with discrimination indices of 0.86 and 0.72, respectively. In addition, the variable regions of the coagulase genes of 39 isolates were sequenced and showed extensive identity, indicating that this is not an efficient alternative for the molecular typing of
S. aureus
.
Oral Colonization, Phenotypic, and Genotypic Profiles of
Candida
Species in Irradiated, Dentate, Xerostomic Nasopharyngeal Carcinoma Survivors
Authors: Leung W. Keung , Dassanayake Ranil S. , Yau Joyce Y. Y. , Jin Li Jian , Yam Wing Cheong , Samaranayake Lakshman P.
Published: March 2020

The aim of this study was to investigate oral yeast colonization and oral yeast strain diversity in irradiated (head and neck), dentate, xerostomic individuals. Subjects were recruited from a nasopharyngeal carcinoma clinic and were segregated into group A (age, <60 years [
n
= 25; average age ± standard deviation {SD}, 48 ± 6 years; average postirradiation time ± SD, 5 ± 5 years]) and group B (age, ≥60 years [
n
= 8; average age ± SD, 67 ± 4 years; average postirradiation time ± SD, 2 ± 2 years]) and were compared with age- and sex-matched healthy individuals in group C (age, <60 years [
n
= 20; average age ± SD, 44 ± 12 years] and group D (age, ≥60 years [
n
= 10; average age, 70 ± 3 years]). Selective culture of oral rinse samples was carried out to isolate, quantify, and speciate yeast recovery. All test subjects underwent a 3-month comprehensive oral and preventive care regimen plus topical antifungal therapy, if indicated. A total of 12 subjects from group A and 5 subjects from group B were recalled for reassessment of yeast colonization. Sequential (pre- and posttherapy)
Candida
isolate pairs from patients were phenotypically (all isolate pairs; biotyping and resistotyping profiles) and genotypically (
Candida albicans
isolate pairs only; electrophoretic karyotyping by pulsed-field gel electrophoresis, restriction fragment length polymorphism [RFLP], and randomly amplified polymorphic DNA [RAPD] assays) evaluated. All isolates were
Candida
species. Irradiated individuals were found to have a significantly increased yeast carriage compared with the controls. The isolation rate of
Candida
posttherapy remained unchanged. A total of 9 of the 12 subjects in group A and 3 of the 5 subjects in group B harbored the same
C. albicans
or
Candida tropicalis
phenotype at recall. Varying degrees of congruence in the molecular profiles were observed when these sequential isolate pairs of
C. albicans
were analyzed by RFLP and RAPD assays. Variations in the genotype were complementary to those in the phenotypic characteristics for some isolates. In conclusion, irradiation-induced xerostomia seems to favor intraoral colonization of
Candida
species, particularly
C. albicans
, which appeared to undergo temporal modifications in clonal profiles both phenotypically and genotypically following hygienic and preventive oral care which included topical antifungal therapy, if indicated. We postulate that the observed ability of
Candida
species to undergo genetic and phenotypic adaptation could strategically enhance its survival in the human oral cavity, particularly when salivary defenses are impaired.