bluuebunny/crossref_metadata_2025 huggingface dataset:
Antibody Response to Fibronectin-Binding Adhesin FnbpA in Patients with
Staphylococcus aureus
Infections
Authors: Casolini Fabrizia , Visai Livia , Joh Danny , Conaldi Pier Giulio , Toniolo Antonio , Höök Magnus , Speziale Pietro
Published: December 2019

We have analyzed antibody reactivity to a fibronectin-binding microbial surface component that recognizes adhesive matrix molecules (MSCRAMM) in blood plasma collected from patients with staphylococcal infections. All patients had elevated levels of anti-MSCRAMM antibodies compared to those of young children who, presumably, had not been exposed to staphylococcal infections. The anti-MSCRAMM antibodies preferentially reacted with the ligand-binding repeat domain of the adhesin. However, these antibodies did not inhibit fibronectin binding. Essentially, all patients had antibodies which specifically recognized the fibronectin-MSCRAMM complex but not the isolated components. Epitopes recognized by these anti-ligand-induced binding sites antibodies were found in each repeat unit of the MSCRAMM. These results demonstrate that staphylococci have bound fibronectin some time during infection and that each repeat unit in the MSCRAMM can engage in ligand binding. Furthermore, our previously proposed model, suggesting that an unordered structure in the MSCRAMM undergoes a conformational change upon ligand binding (K. House-Pompeo, Y. Xu, D. Joh, P. Speziale, and M. Höök, J. Biol. Chem. 271:1379–1384, 1996), is presumably operational in patients during infections.
Transmission and Serial Propagation of
Enterocytozoon bieneusi
from Humans and Rhesus Macaques in Gnotobiotic Piglets
Authors: Kondova Ivanela , Mansfield Keith , Buckholt Michael A. , Stein Barry , Widmer Giovanni , Carville Angela , Lackner Andrew , Tzipori Saul
Published: December 2019

For over a decade
Enterocytozoon bieneusi
infections in people with AIDS have been linked with chronic diarrhea and wasting. The slow scientific progress in treating these infections is attributed to the inability of investigators to cultivate the parasite, which has also precluded evaluation of effective therapies. We report here successful serial transmissions of
E. bieneusi
from patients with AIDS and from macaques with AIDS to immunosuppressed gnotobiotic piglets. One infected piglet was still excreting spores at necropsy 50 days after an oral challenge. Spores in feces were detected microscopically by trichrome stain and by PCR and within enterocytes by in situ hybridization and immunohistochemistry.
E. bieneusi
infection induced no symptoms. The development of an animal model for
E. bieneusi
will open up new opportunities for investigating this parasite.
Protection against Exotoxin A (ETA) and
Pseudomonas aeruginosa
Infection in Mice with ETA-Specific Antipeptide Antibodies
Authors: El-Zaim Haissam S. , Chopra Ashok K. , Peterson Johnny W. , Vasil Michael L. , Heggers John P.
Published: December 2019

Pseudomonas aeruginosa
is an opportunistic pathogen that causes serious and sometimes fatal infections in the compromised host, especially in patients with major trauma or thermal injuries. Exotoxin A (ETA) is the major and most lethal virulence factor produced by this ubiquitous microorganism. In a recent study (H. S. Elzaim, A. K. Chopra, J. W. Peterson, R. Goodheart, and J. P. Heggers, Infect. Immun. 66:2170–2179, 1998), we identified two major epitopes, one within the translocation domain (amino acid [aa] residues 289 to 333) of ETA and another within the enzymatic domain (aa 610 to 638), by using a panel of antipeptide antibodies. Synthetic peptides representing these two epitopes induced ETA-specific antibodies which were able to abrogate the cytotoxic activity of ETA, as measured by incorporation of [
3
H]leucine into 3T3 fibroblasts. In the present study, these antibodies were tested for the ability to provide protection against ETA and infection with a toxin-producing strain of
P. aeruginosa
in a mouse model. Antibodies to either of the synthetic peptides conferred protection against ETA. Also, when used for immunization, both peptides induced active immunity to ETA in mice. Antibodies to the peptide representing a region within the enzymatic domain of ETA, in combination with the antibiotic amikacin, enhanced the survival of mice infected with a toxin-producing strain of
P. aeruginosa
. Thus, antipeptide antibodies specific for ETA might be paired with antibiotic treatment for passive immunization of patients suffering from
P. aeruginosa
infection.
Effect of P39 Gene Deletion in Live
Brucella
Vaccine Strains on Residual Virulence and Protective Activity in Mice
Authors: Tibor Anne , Jacques Isabelle , Guilloteau Laurence , Verger Jean-Michel , Grayon Maggy , Wansard Valerie , Letesson Jean-Jacques
Published: December 2019

The 39-kilodalton protein (P39) has previously been shown to be an immunodominant protein in
Brucella
infections. P39 gene deletion mutants of vaccine strains
Brucella abortus
S19 and
Brucella melitensis
Rev.1 were constructed by gene replacement. This deletion did not significantly modify the residual virulence of both vaccine strains in CD-1 mice. CD-1 mice vaccinated with the parent or mutant strains were protected against a virulent challenge. Mutant vaccine strains devoid of P39 could provide a means for differentiating vaccinated from infected animals.
A Globally Disseminated M1 Subclone of Group A Streptococci Differs from Other Subclones by 70 Kilobases of Prophage DNA and Capacity for High-Frequency Intracellular Invasion
Authors: Cleary P. P. , LaPenta D. , Vessela R. , Lam H. , Cue D.
Published: December 2019

The M1inv+ subclone of M1 group A streptococci that spread globally in the late 1980s and early 1990s was previously identified by restriction fragment length polymorphism (RFLP), M protein, and SpeA exotoxin sequence analyses. Strains representing this subclone were characterized with regard to carriage of bacteriophage and capacity to invade cultured human epithelial cells. The M1inv+ subclone was found to harbor two entirely different prophages, phage T13 and phage T14, which together supplement its genome with nearly 70 kb of DNA. Phage T14 encodes the SpeA exotoxin and is closely related to the classic converting phage T12. Plaque-forming characteristics and RFLP analyses of phages T13 and T14 were compared to each other and to phage T12. Other subclones of M1, isolated in the 1970s to the early 1980s, lacked both prophages. The M1inv+ subclone was previously reported to be efficiently internalized by human epithelial cells. This potential was confirmed and expanded by comparing a variety of clinical isolates. The capacity for high-frequency invasion of epithelial cells was not transmitted to a laboratory strain of group A streptococci by the above-mentioned bacteriophages.
Identification of Immunodominant Regions within the C-Terminal Cell Binding Domain of Intimin α and Intimin β from Enteropathogenic
Escherichia coli
Authors: Adu-Bobie Jeannette , Trabulsi Luiz R. , Carneiro-Sampaio Magda M. S. , Dougan Gordon , Frankel Gad
Published: December 2019

Enteropathogenic
Escherichia coli
(EPEC) strains are a common cause of infantile diarrhea in developing countries. EPEC strains induce a characteristic attaching and effacing (A/E) lesion on epithelial cells. A/E lesion formation requires intimin, an outer membrane adhesin protein. The cell-binding activity of intimin is localized at the C-terminal 280 amino acids of the polypeptide (Int280). So far, four distinct Int280 types (α, β, γ, and δ) have been identified. The aim of this study was to identify immunodominant regions within the Int280α and Int280β domains. Recombinant DNA was used to construct and express overlapping polypeptides spanning these domains. Rabbit anti-Int280 antisera and human colostral immunoglobulin A were reacted with these polypeptides in Western blots and enzyme-linked immunosorbent assays. The results obtained with the rabbit antisera showed the presence of two separate immunodominant regions which are common to both Int280α and Int280β. The first localized within the N-terminal region of Int280, and the second localized between amino acids 80 and 130. The results with the human colostra revealed one reactivity pattern against the Int280α fragments but two different reactivity patterns against the Int280β domain.
Environmental Growth Conditions Influence the Ability of
Escherichia coli
K1 To Invade Brain Microvascular Endothelial Cells and Confer Serum Resistance
Authors: Badger Julie L. , Kim Kwang Sik
Published: December 2019

A major limitation to advances in prevention and therapy of neonatal meningitis is our incomplete understanding of the pathogenesis of this disease. In an effort to understand the pathogenesis of meningitis due to
Escherichia coli
K1, we examined whether environmental growth conditions similar to those that the bacteria might be exposed to in the blood could influence the ability of
E. coli
K1 to invade brain microvascular endothelial cells (BMEC) in vitro and to cross the blood-brain barrier in vivo. We found that the following bacterial growth conditions enhanced
E. coli
K1 invasion of BMEC 3- to 10-fold: microaerophilic growth, media buffered at pH 6.5, and media supplemented with 50% newborn bovine serum (NBS), magnesium, or iron. Growth conditions that significantly repressed invasion (i.e., 2- to 250-fold) included iron chelation, a pH of 8.5, and high osmolarity. More importantly,
E. coli
K1 traversal of the blood-brain barrier was significantly greater for the growth condition enhancing BMEC invasion (50% NBS) than for the condition repressing invasion (osmolarity) in newborn rats with experimental hematogenous meningitis. Of interest, bacterial growth conditions that enhanced or repressed invasion also elicited similar serum resistance phenotype patterns. This is the first demonstration that bacterial ability to enter the central nervous system can be affected by environmental growth conditions.
Helicobacter pylori
Alters Exogenous Antigen Absorption and Processing in a Digestive Tract Epithelial Cell Line Model
Authors: Matysiak-Budnik Tamara , Terpend Kathleen , Alain Sophie , Sanson le Pors Marie-José , Desjeux Jehan-Francois , Mégraud Francis , Heyman Martine
Published: December 2019

To study the influence of
Helicobacter pylori
on epithelial barrier function, bacteria, bacterial sonicates, or broth culture supernatants were incubated for 24 h with HT29-19A intestinal cells grown as monolayers. Subsequently, the monolayers were mounted in Ussing chambers, and electrical resistance (R), fluxes of
22
Na (JNa) and
14
C-mannitol (JMan) (markers of the paracellular pathway), and fluxes of horseradish peroxidase (HRP) in total (J
3
H-HRP), intact (JHRPi), and degraded forms were measured.
H. pylori
did not induce any modification of the paracellular pathway (R = 148 ± 10 versus 174 ± 16 Ω · cm
2
; JNa = 4.16 ± 0.44 versus 3.51 ± 0.41 μEq/h · cm
2
; JMan = 0.081 ± 0.01 versus 0.058 ± 0.009 μmol/h · cm
2
), nor did it modify J
3
H-HRP (2,201 ± 255 versus 2,110 ± 210 ng/h · cm
2
for
H. pylori
-infected and control cells, respectively). However, in the presence of
H. pylori
, we observed a significant increase in JHRPi (520 ± 146 versus 171 ± 88 ng/h · cm
2
). This effect was not dependent of the
cag
status of the strain and was not reproduced by the sonicates or the culture supernatants. It was related to the presence of urease, since a urease-negative mutant of
H. pylori
did not induce this effect. Ammonia and bafilomycin A
1
, two agents known to increase the endolysosomal pH, reproduced the increase in JHRPi. In conclusion,
H. pylori
does not affect directly the integrity of intercellular junctions of epithelial cells in vitro, but it increases the passage of intact HRP, probably by inhibition of the intralysosomal degradation due to the release of ammonia. The increased transport of intact macromolecules may contribute to the induction and maintenance of gastric inflammation by
H. pylori
.
Modulation of Lipopolysaccharide-Induced Monocyte Activation by Heparin-Binding Protein and Fucoidan
Authors: Heinzelmann Michael , Polk Hiram C. , Miller Frederick N.
Published: December 2019

Activated polymorphonuclear leukocytes release heparin-binding protein (HBP; also known as CAP37 or azurocidin) from azurophilic granules. HBP is a strong chemoattractant for monocytes that also prolongs monocyte survival and potentiates endotoxin (lipopolysaccharide [LPS])-induced production of tumor necrosis factor alpha (TNF-α). We investigated the binding of fluorescein isothiocyanate-conjugated HBP to human monocytes in the presence of EDTA and the polysaccharide fucoidan. EDTA, which chelates divalent cations, has been widely used to study the role of divalent cations in receptor-ligand interactions or enzyme activity. Fucoidan has been used to inhibit the binding of various ligands to scavenger receptors or selectins. Scavenger receptors are multiligand receptors that mediate endocytosis of proteases, protease-inhibitor complexes, lipoproteins, and LPS-lipid A. Fucoidan also interferes with leukocyte rolling by binding to L-selectins (expressed on leukocytes) and P-selectins (expressed on platelets and endothelium). We demonstrate that the binding of the neutrophil-derived protein HBP to monocytes is inhibited in the presence of EDTA and fucoidan. In addition, fucoidan and EDTA abrogate the enhancing effect of HBP on LPS-induced TNF-α production. These data provide supporting evidence that HBP binds to a receptor expressed on monocytes. This receptor is dependent on divalent cations and is possibly related to the scavenger receptor. Furthermore, we demonstrate that fucoidan, by itself, stimulates TNF-α release from isolated monocytes in a CD14-independent fashion. This is an important finding for the interpretation of results from studies that use fucoidan to “block” either scavenger receptors or L- or P-selectins.
Phosphatidylcholine-Specific Phospholipase C fromListeria monocytogenesIs an Important Virulence Factor in Murine Cerebral Listeriosis
Authors: Schlüter Dirk , Domann Eugen , Buck Christine , Hain Torsten , Hof Herbert , Chakraborty Trinad , Deckert-Schlüter Martina
Published: December 2019

Meningoencephalitis is a serious and often fatal complication ofListeria monocytogenesinfection. The aim of the present study was to analyze the role of internalin A (InlA) and B, which are involved in the invasion ofL. monocytogenesinto cultivated host tissue cells, and that of phosphatidylcholine-specific phospholipase C (PlcB), which mainly promotes the direct cell-to-cell spread ofL. monocytogenes, in murine cerebral listeriosis by use of an InlA/B (ΔinlAB2)- and a PlcB (ΔplcB2)-deficient isogenic deletion mutant strain and the wild-type (WT)L. monocytogenesEGD.Listeriastrains were directly applied to the brain, a technique which has been employed previously to study the pathogenesis of cerebral listeriosis (D. Schlüter, S. B. Oprisiu, S. Chahoud, D. Weiner, O. D. Wiestler, H. Hof, and M. Deckert-Schlüter, Eur. J. Immunol. 25:2384–2391, 1995). We demonstrated that PlcB, but not InlA or InlB, is an important virulence factor in cerebral listeriosis. Nonimmunized mice infected intracerebrally with the ΔplcB2strain survived significantly longer and had a reduced intracerebral bacterial load compared to mice infected with the ΔinlAB2strain or WT bacteria. In addition, immunization with the WT prior to intracerebral infection significantly increased the survival rate of mice challenged intracerebrally with theΔplcB2strain compared to that of mice infected with the WT or ΔinlAB2strain. Histopathology revealed that the major difference between the various experimental groups was a significantly delayed intracerebral spread of the ΔplcB2mutant strain, indicating that cell-to-cell spread is an important pathogenic feature of cerebral listeriosis. Interestingly, irrespective of theListeriamutant used, the apoptosis of hippocampal and cerebellar neurons and an internal hydrocephalus developed in surviving mice, indicating that these complications are not dependent on the virulence factors InlA/B and PlcB. In conclusion, this study points to PlcB as a virulence factor important for the intracerebral pathogenesis of murineL. monocytogenesmeningoencephalitis.
Contrasting Roles of Mannan-Specific Monoclonal Immunoglobulin M Antibodies in the Activation of Classical and Alternative Pathways byCandida albicans
Authors: Zhang Mason X. , Cutler Jim E. , Han Yongmoon , Kozel Thomas R.
Published: December 2019

Polyclonal antimannan immunoglobulin G (IgG) activates the classical complement pathway and accelerates initiation of the alternative pathway byCanidida albicans. This dual role was assessed for two antimannan IgM monoclonal antibodies (MAbs). MAb B6.1 is specific for an epitope on the acid-labile portion ofC. albicansphosphomannan; MAb B6 is specific for an epitope on the acid-stable region. Both MAbs were potent activators of the classical pathway but poor facilitators of alternative pathway initiation.
Persistence of
Anaplasma ovis
Infection and Conservation of the
msp-2
and
msp-3
Multigene Families within the Genus
Anaplasma
Authors: Palmer Guy H. , Abbott Jeffrey R. , French Dorothy M. , McElwain Terry F.
Published: December 2019

Goats which have recovered from acute
Anaplasma ovis
infection remain seropositive, although infected erythrocytes cannot be detected by microscopic examination. Persistence of
A. ovis
17 to 21 months following experimental infection was demonstrated by PCR detection of the
msp-5
gene. Quantitative analysis of persistent rickettsemia over time showed that all levels were below the limit of microscopic detection and ranged from a low of 10
2
organisms/ml to peaks of 10
6
organisms/ml. Two patterns of persistent rickettsemia were observed: the first was characterized by cyclic fluctuations at 6- to 9-week intervals, similar to the pattern described for
A. marginale
-infected cattle, while in the second pattern, repetitive cycles did not occur and the rickettsemia levels were relatively constant. The
msp-2
and
msp-3
multigene families, which provide the genetic capacity for outer membrane protein antigenic variation during persistent
A. marginale
rickettsemia, were identified in the
A. ovis
genome by Southern blot analysis, and expression of an MSP-2 homologue was confirmed by using immunoblots.
The Fimbria Gene Cluster of Nonencapsulated
Haemophilus influenzae
Authors: Geluk Forien , Eijk Paul P. , van Ham S. Marieke , Jansen Henk M. , van Alphen Loek
Published: December 2019

The occurrence of fimbria gene clusters in nonencapsulated
Haemophilus influenzae
strains from chronic bronchitis patients (
n
= 58), patients with acute otitis media (
n
= 13), and healthy carriers (
n
= 12) was determined by DNA hybridization and PCR, based on sequences of fimbriate
H. influenzae
type b. Although an average of 18% of all nonencapsulated strains had a fimbria gene cluster consisting of
hifA
to
hifE
inserted in the chromosome between
purE
and
pepN
, differences in the frequency of fimbria cluster-positive strains were observed, depending on the source of isolates. The compositions of the fimbria gene clusters of seven strains from chronic bronchitis patients and one strain from an otitis media patient were analyzed in more detail. After enrichment for fimbria expression, the promoter of the gene cluster contained 10 TA repeats (
n
= 2), leading to optimal positioning between the −10 and −35 promoter regions. The promoter regions of five fimbria-negative strains were sequenced; four were found to have nine TA repeats, and one had only four TA repeats. The protein sequence of three ganglioside GM1-specific HifA adhesins consisted of conserved regions intermingled with regions of sequence diversity.
hifA
appeared to be flanked by intergenic regions that varied between strains and contained both direct and inverted DNA repeats. Since noncoding DNA between
hifA
and
purE
has not been found in
H. influenzae
type b, these DNA sequences are probably not essential for fimbria expression. An analysis of strains lacking the gene cluster revealed the presence of similar sequences in 13 of 15 strains from chronic bronchitis patients, 5 of 5 strains from otitis media patients, and 3 of 5 strains from healthy carriers. The lengths of these intergenic regions were the same for multiple isolates of strains obtained during persistent infections. The presence or absence and the composition of the fimbria gene cluster and other sequences between the flanking genes
purE
and
pepN
suggest that the fimbria gene cluster was originally contained on a mobile element.
Prevalence of the “High-Pathogenicity Island” of
Yersinia
Species among
Escherichia coli
Strains That Are Pathogenic to Humans
Authors: Schubert S. , Rakin A. , Karch H. , Carniel E. , Heesemann J.
Published: December 2019

The
fyuA-irp
gene cluster contributes to the virulence of highly pathogenic
Yersinia
(
Yersinia pestis
,
Yersinia pseudotuberculosis
, and
Yersinia enterocolitica
1B). The cluster encodes an iron uptake system mediated by the siderophore yersiniabactin and reveals features of a pathogenicity island. Two evolutionary lineages of this “high pathogenicity island” (HPI) can be distinguished on the basis of DNA sequence comparison: a
Y. pestis
group and a
Y. enterocolitica
group. In this study we demonstrate that the HPI of the
Y. pestis
evolutionary group is disseminated among species of the family
Enterobacteriaceae
which are pathogenic to humans. It prevails in enteroaggregative
Escherichia coli
and in
E. coli
blood culture isolates (93 and 80%, respectively), but is rarely found in enteropathogenic
E. coli
, enteroinvasive
E. coli
, and enterotoxigenic
E. coli
isolates. In contrast, the HPI was absent from enterohemorrhagic
E. coli
,
Shigella
, and
Salmonella enterica
strains investigated. Polypeptides encoded by the
fyuA
,
irp1
, and
irp2
genes located on the HPI could be detected in
E. coli
strains pathogenic to humans. However, these
E. coli
strains showed a reduced sensitivity to the bacteriocin pesticin, whose uptake is mediated by the FyuA receptor.
Escherichia
strains do not possess the
hms
gene locus thought to be a part of the HPI of
Y. pestis
. Deletions of the
fyuA-irp
gene cluster affecting solely the
fyuA
part of the HPI were identified in 3% of the
E. coli
strains tested. These results suggest horizontal transfer of the HPI between
Y. pestis
and some pathogenic
E. coli
strains.
Identification and Characterization of a Novel Extracellular Ferric Reductase from
Mycobacterium paratuberculosis
Authors: Homuth Matthias , Valentin-Weigand Peter , Rohde M. , Gerlach Gerald-F.
Published: December 2019

A novel extracellular mycobacterial enzyme was identified in the ruminant pathogen
Mycobacterium paratuberculosis
. The enzyme was capable of mobilizing iron from different sources such as ferric ammonium citrate, ferritin, and transferrin by reduction of the metal. The purified reductase had a calculated
M
r
of 17,000, was sensitive to proteinase K treatment, and had an isoelectric point of pH 9. Analysis of the amino acid composition revealed glycine, serine, asparagine (or aspartic acid), and glutamine (or glutamic acid) as the most frequently occurring residues. Enzymatic activity was highest at 37°C and between pH 5 and 10. The calculated

K
m

and
V
max
for ferric ammonium citrate were 0.213 mM and 0.345 mM min
−1
mg
−1
, respectively. Using a specific antireductase antibody in immunoelectron microscopy, we were able to detect the enzyme associated with intracellular mycobacteria in naturally
M. paratuberculosis
-infected bovine tissue. We propose that the reductase of
M. paratuberculosis
represents an alternative strategy of mycobacteria to mobilize ferric iron and discuss its potential role in bacterial evasion of intracellular defense mechanisms.
Identification of a Putative Precursor to the Major Surface Glycoprotein ofPneumocystis carinii
Authors: Sunkin Susan M. , Linke Michael J. , McCormack Francis X. , Walzer Peter D. , Stringer James R.
Published: December 2019

The major surface glycoprotein (MSG) ofPneumocystis cariniif. sp.cariniiis a family of proteins encoded by a family of heterogeneous genes. Messenger RNAs encoding different MSGs each begin with the same 365-bp sequence, called the Upstream Conserved Sequence (UCS), which is in frame with the contiguous MSG sequence. The UCS contains several potential start sites for translation. To determine if translation of MSG mRNAs begins in the UCS, polyclonal antiserum was raised against the 123-amino-acid peptide encoded by the UCS. The anti-UCS serum reacted with aP. cariniiprotein that migrated at 170 kDa; however, it did not react with the mature MSG protein, which migrates at 116 kDa. A 170-kDa protein was immunoprecipitated with anti-UCS serum and shown to react with a monoclonal antibody against a conserved MSG epitope. To explore the functional role of the UCS in the trafficking of MSG, the nucleotide sequence encoding the UCS peptide was ligated to the 5′ end of an MSG gene and incorporated into a recombinant baculovirus. Insect cells infected with the UCS-MSG hybrid gene expressed a 160-kDa protein which was N-glycosylated. By contrast, insect cells infected with a baculovirus carrying an MSG gene lacking the UCS expressed a nonglycosylated 130-kDa protein. These data suggest that inP. carinii, translation begins in the UCS to produce a pre-MSG protein, which is subsequently directed to the endoplasmic reticulum and processed to the mature form by proteolytic cleavage.
Involvement of Bradykinin Generation in Intravascular Dissemination ofVibrio vulnificusand Prevention of Invasion by a Bradykinin Antagonist
Authors: Maruo Keishi , Akaike Takaaki , Ono Tomomichi , Maeda Hiroshi
Published: December 2019

Involvement of bradykinin generation in bacterial invasion was examined by using a gram-negative bacillus,Vibrio vulnificus, which is known to invade the blood circulatory system and cause septicemia.V. vulnificuswas injected intraperitoneally (i.p.) into mice with or without bradykinin or a bradykinin (B2 receptor) antagonist. Dissemination ofV. vulnificusfrom peritoneal septic foci to the circulating blood was assessed by counting of viable bacteria in venous blood by use of the colony-forming assay. Intravascular dissemination ofV. vulnificusin mice was significantly potentiated by simultaneous injection with bradykinin but was markedly reduced by coadministration with the B2 antagonistd-Arg,[Hyp3, Thi5,8,d-Phe7]-bradykinin. Furthermore,V. vulnificuslethality was significantly increased when bradykinin was administered simultaneously with the bacillus, whereas it was definitely suppressed by treatment withd-Arg,[Hyp3, Thi5,8,d-Phe7]-bradykinin. Similarly, ovomacroglobulin, a potent inhibitor of theV. vulnificusprotease, showed a strong suppressive effect on theV. vulnificussepticemia. We also confirmed appreciable bradykinin production in the primary septic foci in the mouse peritoneal cavity after i.p. inoculation withV. vulnificus. It is thus concluded that bradykinin generation in infectious foci is critically involved in facilitation of intravascular dissemination ofV. vulnificus.
Protective Role of Nitric Oxide inStaphylococcus aureusInfection in Mice
Authors: Sasaki Sanae , Miura Tomisato , Nishikawa Shinsuke , Yamada Kyogo , Hirasue Mayuko , Nakane Akio
Published: December 2019

This study was carried out to determine the role of nitric oxide (NO) inStaphylococcus aureusinfection in mice. NO production in spleen cell cultures was induced by heat-killedS. aureus. Expression of mRNA of the inducible isoform of NO synthase (iNOS) was induced in the spleens and kidneys ofS. aureus-infected mice. When mice were treated with monoclonal antibodies (MAbs) against tumor necrosis factor alpha (TNF-α) or gamma interferon (IFN-γ) beforeS. aureusinfection, the induction of iNOS mRNA expression in the kidneys was inhibited. These MAbs also inhibited NO production in spleen cell cultures stimulated with heat-killedS. aureus. NO production in the spleen cell cultures and levels of urinary nitrate plus nitrite were suppressed by treatment with aminoguanidine (AG), a selective inhibitor of iNOS. The survival rates of AG-treated mice were significantly decreased by either lethal or sublethalS. aureusinfections. However, an effect of AG administration on bacterial growth was not observed in the spleens and kidneys of mice during either type of infection. Production of TNF-α and IFN-γ was not affected by AG treatment in vitro and in vivo. These results suggest that NO plays an important role in protection from lethality by the infection, but the protective role of NO in host resistance againstS. aureusinfection was not proved. Moreover, our results show that TNF-α and IFN-γ regulate NO production while NO may not be involved in the regulation of the production of these cytokines duringS. aureusinfection.
Antigenicity of Recombinant Proteins Derived from Rhoptry-Associated Protein 1 of
Plasmodium falciparum
Authors: Fonjungo Peter N. , Stüber Dietrich , McBride Jana S.
Published: December 2019

Rhoptry-associated protein 1 (RAP1) of
Plasmodium falciparum
is a potential component of a malaria vaccine. We have expressed in
Escherichia coli
eight recombinant RAP1 proteins representing almost the entire sequence of the mature protein and assessed the antigenicity of the proteins by immunization of mice. Antisera to six of the recombinant proteins reacted specifically with parasite-derived RAP1 (
Pf
RAP1), as determined by indirect immunofluorescence and by immunoblotting. These proteins were then used in enzyme-linked immunosorbent assays to evaluate human antibody responses to RAP1 during naturally transmitted infections in The Gambia. Immunoglobulin G (IgG) antibodies specifically reactive with the recombinant RAP1 proteins are directed mostly towards fragments containing the N-terminal sequences of mature
Pf
RAP1. The most N-terminal segment (residues 23 to 175) contains only minor epitopes, while major epitopes are outside this region. Antibodies from malaria patients do not compete for a linear epitope recognized by an inhibitory anti-RAP1 monoclonal antibody. Analysis of IgG subclass distribution shows that human anti-RAP1 antibodies are predominantly IgG1.
Analysis of the Organization of Multicopy Linear- and Circular-Plasmid-Carried Open Reading Frames in
Borrelia burgdorferi
Sensu Lato Isolates
Authors: Carlyon Jason A. , LaVoie Crystal , Sung Shian-Ying , Marconi Richard T.
Published: December 2019

Plasmid cp8.3 of
Borrelia afzelii
IP21 carries several open reading frames (ORFs) and a 184-bp inverted repeat (IR) element. It has been speculated that this plasmid may encode factors involved in virulence or infectivity. In this report, we have characterized the distribution, molecular variability, and organization of ORFs 1, 2, and 4 and the IR elements among isolates of the
Borrelia burgdorferi
sensu lato complex. ORFs 1 and 2 are contained within a segment of cp8.3 that is bordered by the IR elements, while ORF 4 resides just outside of the IR-bordered region. By PCR, ORF 4 was amplified from most isolates while ORFs 1 and 2 were amplified from only some
B. afzelii
isolates. However, Southern hybridization analyses with ORF 1, 2, and 4 probes detected related sequences even in some isolates that were PCR negative. The ORF restriction fragment length polymorphism patterns varied widely even among isolates of the same species. Two-dimensional contour-clamped homogeneous electric field–pulsed-field gel electrophoresis and Southern hybridization detected ORF 1-, 2-, and 4-related sequences on linear and circular plasmids. In addition, an ORF 4-related sequence was detected on a previously uncharacterized, circular plasmid that is greater than 70 kb in size. The IR elements originally identified on plasmid cp8.3 of
B. afzelii
IP21 were also analyzed by Southern hybridization. Related sequences were detected in some but not all
B. burgdorferi
sensu lato isolates. These sequences are carried on plasmids in addition to cp8.3 in some isolates. Single-primer PCR analyses demonstrated that in some isolates these sequences exist with IR orientation. The data presented here demonstrate that the IR elements and the ORF 1-, 2-, and 4-related sequences are multicopy and are variable in organization and in genomic location among isolates of the
B. burgdorferi
sensu lato complex. These analyses provide additional evidence for the highly variable organization of the plasmid component of the
B. burgdorferi
sensu lato genome.
Cell Activation Mediated by Glycosylphosphatidylinositol-Anchored or Transmembrane Forms of CD14
Authors: Pugin J. , Kravchenko V. V. , Lee J.-D. , Kline L. , Ulevitch R. J. , Tobias P. S.
Published: December 2019

CD14 is a glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein which functions as a receptor on myeloid cells for ligands derived from microbial pathogens such as lipopolysaccharide (LPS). We have studied the importance of the GPI tail of CD14 in signalling with the promonocytic cell line THP-1 expressing recombinant CD14 in a GPI-anchored form (THP1-wtCD14 cells) or in a transmembrane form (THP1-tmCD14). We found that, like other GPI-anchored molecules, GPI-anchored CD14 was recovered mainly from a Triton X-100-insoluble fraction, whereas transmembrane CD14 was fully soluble in Triton X-100. LPS induced cell activation of THP1-wtCD14 and of THP1-tmCD14 (protein tyrosine kinase phosphorylation, NF-κB activation, and cytokine production) in a very similar manner. However, anti-CD14 antibody-induced cross-linking caused a rapid calcium mobilization signal only in GPI-anchored CD14 cells. Studies with pharmacologic inhibitors of intracellular signalling events implicate phospholipase C and protein tyrosine kinases in the genesis of this antibody-induced calcium signal. Our results suggest that GPI anchoring and CD14 targeting to glycolipid-rich membrane microdomains are not required for LPS-mediated myeloid cell activation. GPI anchoring may however be important for other signalling functions, such as those events reflected by antibody cross-linking.
Specific Antibody toCryptococcus neoformansAlters Human Leukocyte Cytokine Synthesis and Promotes T-Cell Proliferation
Authors: Vecchiarelli Anna , Retini Cinzia , Monari Claudia , Casadevall Arturo
Published: December 2019

Addition of a monoclonal antibody which binds theCryptococcus neoformanscapsule to suspensions of human monocytes, T lymphocytes, and cryptococcal cells (i) enhances interleukin-1β (IL-1β), tumor necrosis factor alpha, and IL-2 production; (ii) reduces IL-10 secretion; and (iii) promotes T-cell proliferation. The ability of specific antibody to influence cytokine production and lymphoproliferation suggests a mechanism by which humoral immunity can influence cell-mediated immunity.
Identification of Epitopes of Fibronectin Attachment Protein (FAP-A) ofMycobacterium aviumWhich Stimulate Strong T-Cell Responses in Mice
Authors: Holsti Matthew A. , Schorey Jeffrey S. , Brown Eric J. , Allen Paul M.
Published: December 2019

The T-cell response to fibronectin attachment protein (FAP-A) in BALB/c and B10.BR mice was examined. Both strains developed strong T-cell responses to FAP-A, directed to single, unique epitopes. T cells from mice infected withMycobacterium aviumresponded to FAP-A, suggesting a possible role in a protective immune response.
Acute Neutropenia Decreases Inflammation Associated with Murine Vaginal Candidiasis but Has No Effect on the Course of Infection
Authors: Black C. Allen , Eyers Fiona M. , Russell Adrian , Dunkley Margaret L. , Clancy Robert L. , Beagley Kenneth W.
Published: December 2019

We have used a mouse model of vaginal candidiasis to determine the effect of neutrophil depletion on (a) the clearance of
Candida albicans
and (b) the degree of inflammation associated with infection. No differences in recoverable yeast number or rate of clearance were observed between normal and neutrophil-depleted mice; however, vaginal inflammation was significantly decreased in neutrophil-depleted animals.
Chlamydia trachomatis
Infection in the Female Reproductive Tract of the Rat: Influence of Progesterone on Infectivity and Immune Response
Authors: Kaushic Charu , Murdin Andrew D. , Underdown Brian J. , Wira Charles R.
Published: December 2019

As the most common cause of sexually transmitted disease in women, chlamydial infections can lead to pelvic inflammatory disease, infertility, and ectopic pregnancy. To better understand the role played by sex hormones in modulating the immune response of the genital tract to microbial infections, we have developed a rat model to study
Chlamydia trachomatis
infection. Inbred female Lewis rats were primed with progesterone and inoculated by intrauterine instillation of
C. trachomatis
(mouse pneumonitis strain MoPn) into each uterine horn. When infected animals were examined for the presence of chlamydial antigens 14 days postinfection, both the uterus and vagina were found to be positive compared to those of saline-treated animals, which did not show specific staining. The involvement of local and systemic immune systems following chlamydial infection was determined by analyzing major histocompatibility complex (MHC) class II expression in the reproductive tract and lymphocyte proliferation in response to mitogenic and chlamydia-specific stimulation of cells from the spleen and lymph nodes (LN) draining the reproductive tract. Enhanced proliferation was observed in LN following mitogenic but not antigenic (MOMP [major outer membrane protein]) stimulation. In contrast, spleen cell proliferation was lower in chlamydia-infected rats than in saline-treated controls. MHC class II expression, an indicator of inflammatory responses, was upregulated in the uterus, on glandular epithelial cells, and adjacent to glands in response to chlamydial infection. In other experiments, when rats were infected at estrus and diestrus without prior progesterone priming, chlamydial inclusions were not detected in either the uterus or vagina. However, enhanced lymphocyte proliferation was observed in response to mitogenic and MOMP stimulation in the reproductive tract-draining LN from estrous and diestrous animals. These findings indicate that under appropriate endocrine conditions, the rat uterus is susceptible to
C. trachomatis
infection and that immune responses to this pathogen can be detected locally and systemically. Further, they suggest that clearance of the infection from the reproductive tract involves immune cells from the LN draining the reproductive tract.
Differences in the Frequency of Cytokine-Producing Cells in Antigenemic and Nonantigenemic Individuals with Bancroftian Filariasis
Authors: de Almeida Adriana B. , e Silva Maria Carmelita Maia , Braga Cynthia , Freedman David O.
Published: December 2019

Individuals with clinical manifestations of lymphatic filariasis may be currently infected or not. Twenty-five individuals from aWuchereria bancrofti-endemic area of Brazil were classified as being asymptomatic microfilaremic individuals, antigenemic individuals with clinical filariasis, or nonantigenemic individuals with clinical filariasis. Intracellular cytokine staining of mitogen-stimulated peripheral blood mononuclear cells (PBMC) showed that the frequency of either gamma interferon (IFN-γ)- or interleukin-4 (IL-4)-producing cells was higher in the nonantigenemic individuals with clinical filariasis than in the asymptomatic microfilaremic individuals (geometric means, 22.1 versus 10.7% [P= 0.02] and 2.9 versus 1.4% [P= 0.01], respectively). When the asymptomatic microfilaremic individuals and antigenemic individuals with clinical filariasis were grouped together to constitute all actively infected individuals, the frequency of IFN-γ-producing cells was also lower than in the nonantigenemic individuals with clinical filariasis (P= 0.04). Likewise, the frequency of IL-4-producing cells in the actively infected individuals was also lower than in the nonantigenemic individuals with clinical filariasis (P= 0.02). No differences in the frequency of IFN-γ-, IL-4-, or IL-5-producing cells in purified CD4 T lymphocytes were found among the groups. These findings suggest that the presence of antigenemia, which is an indicator of current active infection, is closely associated with the frequency of IFN-γ- and IL-4-producing cells in lymphatic filariasis. The differences found in the frequency of cytokine-producing cells among the three groups appear to be due to a subset of cells other than CD4 T cells.
Immunochemical Characterization of the MPB70/80 and MPB83 Proteins of
Mycobacterium bovis
Authors: Wiker Harald G. , Lyashchenko Konstantin P. , Aksoy A. Murat , Lightbody Kenneth A. , Pollock John M. , Komissarenko Sergiy V. , Bobrovnik Sergiy O. , Kolesnikova Irina N. , Mykhalsky Leonid O. , Gennaro Maria L. , Harboe Morten
Published: December 2019

MPB70 and MPB80 (MPB70/80) and MPB83 are closely related antigens which are highly expressed in
Mycobacterium bovis
. MPB70/80 are soluble secreted antigens, while MPB83 is an exported lipoprotein associated with the bacterial surface. In the present study, these antigens had different mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. These differences may be explained by the fact that MPB70 and MPB83 both have two internal cysteine residues which would create ring structures by disulfide bonding. We analyzed the structures of MPB70/80 and MPB83 by using monoclonal antibodies (MAbs) raised against bovine purified protein derivative or whole
M. bovis
cells. MAb 1-5C reacted specifically with MPB70 and MPB80, and MAb MBS43 reacted specifically with MPB83, while the other antibodies, including several previously described MAbs, bound all three antigens. MAbs and polyclonal antibodies reacted strongly with reduced protein and less well with nonreduced protein, indicating involvement of linear epitopes. Epitopes of MAbs Bov-1, 2-6B, 1-5C, and 1-1D were mapped by using synthetic peptides of MPB70. Sequence comparison showed the peptide with the 1-5C-reactive epitope to have three residues different from those in the homologous region of MPB83. Exchanges of A for S in position 112 or Q for E in position 116 abolished the reactivity of MAb 1-5C. Polyclonal rabbit antibodies to native purified MPB70 reacted strongly with peptides 6, 7, and 8 of the N-terminal half of mature MPB70. Cattle sera of experimentally
M. bovis
-infected animals recognized a broader spectrum of peptides. These findings indicate that there is diagnostic potential for these proteins and that there is also a possible role for antibodies in elucidation of the host-mycobacterium relationship involving a surface-bound and exposed lipoprotein, MPB83, and its highly homologous soluble secreted MPB70/80 counterparts.
Identification of a Gene Involved in Assembly ofActinomyces naeslundiiT14V Type 2 Fimbriae
Authors: Yeung Maria K. , Donkersloot Jacob A. , Cisar John O. , Ragsdale Pamela A.
Published: December 2019

The nucleotide sequence of theActinomyces naeslundiiT14V type 2 fimbrial structural subunit gene,fimA, and the 3′ flanking DNA region was determined. ThefimAgene encoded a 535-amino-acid precursor subunit protein (FimA) which included both N-terminal leader and C-terminal cell wall sorting sequences. A second gene, designatedorf365, that encoded a 365-amino-acid protein which contained a putative transmembrane segment was identified immediately 3′ tofimA. Mutants in which eitherfimAororf365was replaced with a kanamycin resistance gene did not participate in type 2 fimbriae-mediated coaggregation withStreptococcus oralis34. Type 2 fimbrial antigen was not detected in cell extracts of thefimAmutant by Western blotting with anti-A. naeslundiitype 2 fimbrial antibody, but the subunit protein was detected in extracts of theorf365mutant. The subunit protein detected in this mutant also was immunostained by an antibody raised against a synthetic peptide representing the C-terminal 20 amino acid residues of the predicted FimA. The antipeptide antibody reacted with FimA isolated from the recombinantEscherichia coliclone containingfimAbut did not react with purified type 2 fimbriae in extracts of the wild-type strain. These results indicate that synthesis of type 2 fimbriae inA. naeslundiiT14V may involve posttranslational cleavage of both the N-terminal and C-terminal peptides of the precursor subunit and also the expression oforf365.
Maturation of the Arginine-Specific Proteases of
Porphyromonas gingivalis
W50 Is Dependent on a Functional
prR2
Protease Gene
Authors: Aduse-Opoku Joseph , Rangarajan Minnie , Young Katherine A. , Curtis Michael A.
Published: December 2019

The
prpR1
of
Porphyromonas gingivalis
codes for three distinct enzymes with specificity for arginyl peptide bonds termed RI, RIA, and RIB. These three isoforms comprise the majority of the extracellular, arginine-specific protease activity in
P. gingivalis
W50. RI is a heterodimer in which the catalytic α chain is noncovalently associated with a second chain involved in adherence phenomena. RIA and RIB are both monomeric species. RIA represents the free α chain, and RIB is a highly posttranslationally modified form of the α chain which is exclusively vesicle or membrane associated and migrates as a diffuse band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In previous studies, insertional inactivation of the
prpR1
demonstrated that arginine-specific protease activity can also arise from a closely related second gene,
prR2
. In the present work, the
prR2
was insertionally inactivated in
P. gingivalis
W50 in order to establish the contribution of this locus to the arginine-specific protease activity of this periodontal bacterium. Loss of
prR2
function had several effects on
prpR1
-derived enzymes. First, the total Arg-X activity was reduced by approximately 50% relative to that of the parent strain. The reduction in total activity was a consequence of decreased concentrations of the monomeric enzymes derived from the
prpR1
, while the heterodimeric enzyme, RI, was unaffected by this mutation. Second, the chromatographic behavior of both the soluble and vesicle- or membrane-associated monomeric enzymes was radically different from the behavior of RIA and RIB from the parent strain. Finally, the vesicle- or membrane-associated enzyme in the
prR2
mutant strain lacked the extensive posttranslational additions which are found on RIB in
P. gingivalis
W50. These data suggest that the product(s) of the
prR2
plays a significant role in the maturation pathway of
prpR1
-derived enzymes, and this may contribute to the coconservation of these two genes in
P. gingivalis.