bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin 18 Contributes to Host Resistance and Gamma Interferon Production in Mice Infected with VirulentSalmonella typhimurium
Authors: Mastroeni Pietro , Clare S. , Khan S. , Harrison J. A. , Hormaeche C. E. , Okamura H. , Kurimoto M. , Dougan G.
Published: December 2019

Spleen and peritoneal macrophages obtained from innately resistant A/J mice released low levels of interleukin 18 (IL-18) upon infection withSalmonella typhimuriumC5 RP4. Incubating the cells with recombinant gamma interferon (rIFN-γ) enhanced IL-18 production. A/J mice treated in vivo with anti-IL-18 antibodies showed impaired resistance to infection, with increased bacterial loads in the liver and spleen. Administration of rIL-18 could protect A/J mice from challenge with a lethal dose of virulent salmonellae, with a dramatic reduction in bacterial numbers in the tissues. rIL-18 administration did not ameliorate the disease in IFN-γ-R−/−mice. IL-18 proved to be required for IFN-γ production by mouse splenocytes from conventional,scid, andrag-1−/−mice; in vivo IL-18 neutralization caused a decrease in circulating IFN-γ levels. Thus, IL-18 is a key factor in early host resistance toSalmonellaand probably acts via IFN-γ.
Role of Adult Worm Antigen-Specific Immunoglobulin E in Acquired Immunity toSchistosoma mansoniInfection in Baboons
Authors: Nyindo Mramba , Kariuki Thomas M. , Mola Paul W. , Farah Idle O. , Elson Lynne , Blanton Ronald E. , King Christopher L.
Published: December 2019

Allergic-type immune responses, particularly immunoglobulin E (IgE), correlate with protective immunity in human schistosomiasis. To better understand the mechanisms of parasite elimination we examined the immune correlates of protection in baboons (Papio cynocephalus anubis), which are natural hosts forSchistosoma mansoniand also develop allergic-type immunity with infection. In one experiment, animals were exposed to a single infection (1,000 cercariae) or were exposed multiple times (100 cercariae per week for 10 weeks) and subsequently were cured with praziquantel prior to challenge with 1,000 cercariae. Singly and multiply infected animals mounted 59 and 80% reductions in worm burden, respectively (P< 0.01). In a second experiment, animals were inoculated withS. mansoniova and recombinant human interleukin 12 (IL-12). This produced a 37 to 39% reduction in adult worm burden after challenge (P< 0.05). Parasite-specific IgG, IgE, IgM, and peripheral blood cytokine production were evaluated. The only immune correlate of protection in both experiments was levels of soluble adult worm antigen (SWAP)-specific IgE in serum at the time of challenge infection and/or 6 weeks later. Baboons repeatedly infected with cercariae or immunized with ova and IL-12 developed two- to sixfold-greater levels of SWAP-specific IgE in serum than did controls, and this correlated with reductions in worm burden (r2, −0.40 to −0.64;P, <0.01). Thus, in baboons and unlike mice, adult worm-specific IgE is uniquely associated with acquired immunity toS. mansoniinfection. This similar association of parasite-specific IgE and protection among primates infected with schistosomiasis, along with similar pathology, anatomy, and genetic make-up, indicates that baboons provide an excellent permissive experimental model for better understanding the mechanisms of innate and acquired immunity to schistosomiasis in humans.
Increased Type 1 Fimbrial Expression among CommensalEscherichia coliIsolates in the Murine Cecum following Catabolic Stress
Authors: Hendrickson Barbara A. , Guo Jun , Laughlin Robert , Chen Yimei , Alverdy John C.
Published: December 2019

Although indigenous bacteria intimately colonize the intestinal mucosa, under normal conditions the intestinal epithelial cell is free of adherent bacteria. Nonetheless, commensal bacteria such asEscherichia coliadhere to and translocate across the intestinal epithelium in association with a number of pathologic states including hemorrhagic shock, immunosuppression, traumatic tissue injury, and lack of enteral feedings. The adhesins involved in the adherence of indigenousE. colito the intestinal epithelium in vivo following catabolic stress are unknown. We have developed a mouse model to study the bacterial adhesins which mediate the increased intestinal adherence ofE. coliafter partial hepatectomy and short-term starvation. Our studies demonstrated that hepatectomy and starvation in the mouse were associated with a 7,500-fold increase in the numbers ofE. colibacteria adhering to the cecum. In addition, erythrocyte agglutination studies, as well as immunostaining of fimbrial preparations and electron micrographs of the bacteria, revealed that surface type 1 fimbriae were more abundant in the commensalE. coliharvested from the ceca of the stressed mice. TheseE. coliisolates adhered to a mouse colon cell line and injected cecal loops in a mannose-inhibitable manner, which suggests a role for type 1 fimbriae in the adherence of theE. coliisolates to the cecum in vivo following host catabolic stress.
Characterization of the Avian PathogenicEscherichia coliHemagglutinin Tsh, a Member of the Immunoglobulin A Protease-Type Family of Autotransporters
Authors: Stathopoulos Christos , Provence David L. , Curtiss Roy
Published: December 2019

We reported earlier that a single gene,tsh, isolated from a strain of avian pathogenicEscherichia coli(APEC) was sufficient to confer onE. coliK-12 a hemagglutinin-positive phenotype and that the deduced sequence of the Tsh protein shared homology to the serine-type immunoglobulin A (IgA) proteases ofNeisseria gonorrhoeaeandHaemophilus influenzae. In this report we show thatE. coliK-12 containing the recombinanttshgene produced two proteins, a 106-kDa extracellular protein and a 33-kDa outer membrane protein, and was also able to agglutinate chicken erythrocytes. N-terminal sequence data indicated that the 106-kDa protein, designated Tshs, was derived from the N-terminal end of Tsh after the removal of a 52-amino-acid N-terminal signal peptide, while the 33-kDa protein, designated Tshβ, was derived from the C-terminal end of Tsh starting at residue N1101. The Tshsdomain contains the 7-amino-acid serine protease motif that includes the active-site serine (S259), found also in the secreted domains of the IgA proteases. However, site-directed mutagenesis of S259did not abolish the hemagglutinin activity or the extracellular secretion of Tshsindicating that host-directed proteolysis was mediating the release of Tshs. Studies with anE. coliK-12ompTmutant strain showed that the surface protease OmpT was not needed for the secretion of Tshs. Tsh belongs to a subclass of the IgA protease family, which also includes EspC of enteropathogenicE. coli, EspP of enterohemorragicE. coli, and SepA and VirG ofShigella flexneri, which seem to involve a host endopeptidase to achieve extracellular release of their N-terminal domains. In proteolytic studies conducted in vitro, Tshsdid not cleave the substrate of the IgA proteases, human IgA1 or chicken IgA, and did not show proteolytic activity in a casein-based assay. Correlation of Tsh expression and hemagglutination activity appears to be a very complex phenomenon, influenced by strain and environmental conditions. Nevertheless, for both APEC and recombinantE. coliK-12 strains containing thetshgene, it was only the whole bacterial cells and not the cell-free supernatants that could confer hemagglutinin activity. Our results provide insights into the expression, secretion, and proteolytic features of the Tsh protein, which belongs to the growing family of gram-negative bacterial extracellular virulence factors, named autotransporters, which utilize a self-mediated mechanism to achieve export across the bacterial cell envelope.
Cytotoxic Action of
Serratia marcescens
Hemolysin on Human Epithelial Cells
Authors: Hertle Ralf , Hilger Martina , Weingardt-Kocher Sandra , Walev Iwan
Published: December 2019

Incubation of human epithelial cells with nanomolar concentrations of chromatographically purified
Serratia marcescens
hemolysin (ShlA) caused irreversible vacuolation and subsequent lysis of the cells. Vacuolation differed from vacuole formation by
Helicobacter pylori
VacA. Sublytic doses of ShlA led to a reversible depletion of intracellular ATP. Restoration to the initial ATP level was presumably due to the repair of the toxin damage and was inhibited by cycloheximide. Pores formed in epithelial cells and fibroblasts without disruption of the plasma membrane, and the pores appeared to be considerably smaller than those observed in artificial lipid membranes and in erythrocytes and did not allow the influx of propidium iodide or trypan blue. All cytotoxic effects induced by isolated recombinant ShlA were also obtained with exponentially growing
S. marcescens
cells. The previously suggested role of the hemolysin in the pathogenicity of
S. marcescens
is supported by these data.
Roles of PilC and PilE Proteins in Pilus-Mediated Adherence of
Neisseria gonorrhoeae
and
Neisseria meningitidis
to Human Erythrocytes and Endothelial and Epithelial Cells
Authors: Scheuerpflug Ina , Rudel Thomas , Ryll Roland , Pandit Jasmine , Meyer Thomas F.
Published: December 2019

Unlike other type 4 pili, the neisserial pili consist of at least two distinct proteins, the highly variable major subunit PilE forming the pilus fiber and the tip-associated adhesin PilC. PilC protein purified either from gonococci or from
Escherichia coli
interacted with different human epithelial cell lines, primary epithelial and endothelial cells. The binding of PilC protein efficiently prevented the attachment of piliated
Neisseria gonorrhoeae
and
Neisseria meningitidis
to these cell types. Fluorescent beads coated with pili prepared from piliated wild-type
N. gonorrhoeae
also adhered to these cells, in contrast to beads coated with pili prepared from a piliated PilC-deficient mutant. In the latter case, the binding of fluorescent beads was restored after pretreatment of the pilus-loaded beads with purified PilC. Piliated wild-type
N. gonorrhoeae
, the piliated PilC-deficient mutant, and
N. gonorrhoeae
pili assembled in
Pseudomonas aeruginosa
agglutinated human erythrocytes, while nonpiliated gonococci did not. Consistently, purified PilC did not agglutinate or bind to human erythrocytes, suggesting that
N. gonorrhoeae
PilE is responsible for pilus-mediated hemagglutination.
Apoptosis in Macrophages and Alveolar Epithelial Cells during Early Stages of Infection byLegionella pneumophilaand Its Role in Cytopathogenicity
Authors: Gao Lian-Yong , Abu Kwaik Yousef
Published: December 2019

The hallmark of Legionnaires’ disease is intracellular replication ofLegionella pneumophilawithin cells in the alveolar spaces. Cytopathogenicity of this bacterium to the host cell has been well demonstrated, but the mechanisms of host cell death due to infection byL. pneumophilaare not well understood. In this study, induction of apoptosis in macrophages and alveolar epithelial cells byL. pneumophiladuring early stages of infection was confirmed by using multiple criteria, including DNA fragmentation by agarose gel electrophoresis, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, surface exposure of phosphatidylserine, and cellular morphology by transmission electron microscopy. Induction of nuclear apoptosis inL. pneumophila-infected macrophages is mediated by activation of the caspase cascade death machinery. We provide genetic and biochemical evidence thatL. pneumophila-induced apoptosis in macrophages and alveolar epithelial cells does not require intracellular bacterial replication or new protein synthesis. In addition, extracellularL. pneumophilais capable of inducing apoptosis. Furthermore, induction of apoptosis byL. pneumophilacorrelates with cytopathogenicity. We conclude thatL. pneumophila-induced apoptosis in macrophages and alveolar epithelial cells plays an important role in cytopathogenicity to the host cell during early stages of infection.
Cryptococcus neoformansResides in an Acidic Phagolysosome of Human Macrophages
Authors: Levitz Stuart M. , Nong Shu-Hua , Seetoo Kurt F. , Harrison Thomas S. , Speizer Robert A. , Simons Elizabeth R.
Published: December 2019

Recently, we demonstrated that human monocyte-derived macrophages (MDM) treated with chloroquine or ammonium chloride had markedly increased antifungal activity against the AIDS-related pathogenCryptococcus neoformans. Both of these agents raise the lysosomal pH, which suggested that the increased antifungal activity was a function of alkalinizing the phagolysosome. Moreover, there was an inverse correlation between growth ofC. neoformansin cell-free media and pH. These data suggested thatC. neoformanswas well adapted to survive within acidic compartments. To test this hypothesis, we performed studies to determine the pH of human MDM and neutrophil phagosomes containingC. neoformans. Fungi were labeled with the isothiocyanate derivatives of two pH-sensitive probes: fluorescein and 2′,7′-difluorofluorescein (Oregon Green). These probes have pKas of 6.4 and 4.7, respectively, allowing sensitive pH detection over a broad range. The phagosomal pH averaged approximately 5 after ingestion of either live or heat-killed fungi and remained relatively constant over time, which suggested thatC. neoformansdoes not actively regulate the pH of its phagosome. The addition of 10 and 100 μM chloroquine resulted in increases in the phagosomal pH from a baseline of 5.1 up to 6.5 and 7.3, respectively. Finally, by immunofluorescence, colocalization ofC. neoformansand the MDM lysosomal membrane protein LAMP-1 was demonstrated, establishing that fusion ofC. neoformans-laden phagosomes with lysosomal compartments takes place. Thus, unlike many other intracellular pathogens,C. neoformansdoes not avoid fusion with macrophage lysosomal compartments but rather resides and survives in an acidic phagolysosome.
A Melanin Pigment Purified from an Epidemic Strain of
Burkholderia cepacia
Attenuates Monocyte Respiratory Burst Activity by Scavenging Superoxide Anion
Authors: Zughaier Susu M. , Ryley Henry C. , Jackson Simon K.
Published: December 2019

The acquisition of
Burkholderia cepacia
in some cystic fibrosis patients is associated with symptoms of acute pulmonary inflammation that may be life threatening. The ability of lipopolysaccharide (LPS) from
B. cepacia
to prime a monocyte cell line for enhanced superoxide anion generation was investigated and compared with the priming activities of LPSs from
Pseudomonas aeruginosa
,
Stenotrophomonas maltophilia
, and
Escherichia coli
. The human monocyte cell line MonoMac-6 (MM6) was primed overnight with different LPSs (100 ng/ml), and the respiratory burst was triggered by exposure to opsonized zymosan (125 μg/ml). Superoxide generation was detected by enhanced chemiluminescence with Lucigenin.
B. cepacia
LPS was found to prime MM6 cells to produce more superoxide anion than
P. aeruginosa
or
S. maltophilia
LPS, and this priming response was CD14 dependent. In addition, the inhibition of respiratory burst responses in monocytes by a bacterial melanin-like pigment purified from an epidemic
B. cepacia
strain was investigated. The melanin-like pigment was isolated from tyrosine-enriched media on which
B. cepacia
had been grown and was purified by gel filtration, anion ion-exchange chromatography, and ethanol precipitation. The scavenging potential of the melanin-like pigment for superoxide anion radical (
•
O
2
−
) generated during the respiratory burst was confirmed with superoxide produced from a cell-free system with xanthine-xanthine oxidase and detected by electron paramagnetic resonance spectroscopy with the spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline-
n
-oxide. The addition of melanin during the LPS priming stage had no effect on the subsequent triggering of the respiratory burst, but melanin inhibited
•
O
2
−
detection when added at the triggering stage of the respiratory burst. We conclude that melanin-producing
B. cepacia
may derive protection from the free-radical-scavenging properties of this pigment.
Role of Lipopolysaccharide Sialylation in Serum Resistance of Serogroup B and C Meningococcal Disease Isolates
Authors: Vogel Ulrich , Claus Heike , Heinze Gabriele , Frosch Matthias
Published: December 2019

α-2,3-Sialyltransferase mutants of three genetically and phenotypically diverse
Neisseria meningitidis
strains were compared with regard to resistance to human serum and systemic spread in the infant rat. Lipopolysaccharide sialylation was found to be of minor importance for the resistance of serogroup B and C meningococcal disease isolates to complement attack.
Production of Enterotoxin by
Yersinia bercovieri
, a Recently Identified
Yersinia enterocolitica
-Like Species
Authors: Sulakvelidze Alexander , Kreger Arnold , Joseph Aaron , Robins-Browne Roy M. , Fasano Alessio , Wauters Georges , Harnett Norma , DeTolla Louis , Morris J. Glenn
Published: December 2019

Yersinia bercovieri
, a recently identified
Y. enterocolitica
-like species, produces a heat-stable enterotoxin (designated YbST) which has biologic activity in infant mice and increases short circuit current in Ussing chambers. Although YbST has some properties in common with the heat-stable enterotoxins of
Y. enterocolitica
(YST I and YST II), it appears to be a novel toxin because (i) it was not neutralized by anti-YST I antiserum, (ii) YbST-neutralizing antiserum did not neutralize YST I, and (iii)
Y. bercovieri
strains did not hybridize with genetic probes for
yst
I,
yst
II, and other known enterotoxins.
Vaccination with LiveEscherichia coliExpressingBrucella abortusCu/Zn Superoxide Dismutase Protects Mice against VirulentB. abortus
Authors: Oñate Angel A. , Vemulapalli Ramesh , Andrews Edilia , Schurig Gerhardt G. , Boyle Stephen , Folch Hugo
Published: December 2019

Vaccination of mice withEscherichia coliexpressingBrucellaCu/Zn superoxide dismutase (SOD) [E. coli(pBSSOD)] induced a significant level of protection against virulentBrucella abortuschallenge, although this level was not as high as the one reached withB. abortusvaccine strain RB51. In addition, vaccination withE. coli(pBSSOD) induced antibodies to Cu/Zn SOD and a strong proliferative response of splenocytes when stimulated in vitro with a thioredoxin-Cu/Zn SOD fusion protein.
Polysaccharide Binding Potential of the Human A2 or A18 Kappa Light Chain Homologues
Authors: Reason Donald C. , Wagner T. Charis , Tang Vanessa R. , Moulton Karen D. , Lucas Alexander H.
Published: December 2019

Antibodies having light (L) chains encoded by the κII-A2 variable region gene segment predominate in the human response to theHaemophilus influenzaetype b polysaccharide (Hib PS). To determine whether the closely related homologue of the A2 gene, the κII-A18 gene, has the potential to contribute to the repertoire, we examined Hib PS binding to a series of recombinant Fab fragments having either A2 or A18 L chains isolated from a Hib PS-vaccinated adult. The ability to bind Hib PS resided exclusively with those Fab fragments having A2 and containing an insertional arginine at the variable-joining junction. Thus, despite the sequence similarity between A2 and A18, only A2 contributes to the canonical Hib PS paratope.
Role ofBordetella pertussisVirulence Factors in Adherence to Epithelial Cell Lines Derived from the Human Respiratory Tract
Authors: van den Berg Bernard M. , Beekhuizen Henry , Willems Rob J. L. , Mooi Frits R. , van Furth Ralph
Published: December 2019

During colonization of the respiratory tract byBordetella pertussis, virulence factors contribute to adherence of the bacterium to the respiratory tract epithelium. In the present study, we examined the roles of the virulence factors filamentous hemagglutinin (FHA), fimbriae, pertactin (Prn), and pertussis toxin (PT) in the adherence ofB. pertussisto cells of the human bronchial epithelial cell line NCI-H292and of the laryngeal epithelial cell line HEp-2. UsingB. pertussismutant strains and purified FHA, fimbriae, Prn, and PT, we demonstrated that both fimbriae and FHA are involved in the adhesion ofB. pertussisto laryngeal epithelial cells, whereas only FHA is involved in the adherence to bronchial epithelial cells. For PT and Prn, no role as adhesion factor was found. However, purified PT bound to both bronchial and laryngeal cells and as such reduced the adherence ofB. pertussisto these cells. These data may imply that fimbriae play a role in infection of only the laryngeal mucosa, while FHA is the major factor in colonization of the entire respiratory tract.
Non-Serum-Dependent Chemotactic Factors Produced byCandida albicansStimulate Chemotaxis by Binding to the Formyl Peptide Receptor on Neutrophils and to an Unknown Receptor on Macrophages
Authors: Edens Heather A. , Parkos Charles A. , Liang Tony W. , Jesaitis Algirdas J. , Cutler Jim E. , Miettinen Heini M.
Published: December 2019

Serum-free culture filtrates of sixCandidaspecies andSaccharomyces cerevisiaewere found to contain chemoattractants for human polymorphonuclear leukocytes (PMNs) and a mouse macrophage-like cell line, J774. The chemotactic factors differed for the PMN and J774 cells, however, in terms of heat stability, kinetics of liberation by the yeast cells, and divalent cation requirements for production. The chemoattractant inCandida albicansculture filtrates appeared to act through the formyl peptide receptor (FPR) of PMNs, since it was found to induce chemotaxis of Chinese hamster ovary (CHO) cells that were expressing the human FPR but did not induce chemotaxis of wild-type CHO cells. TheC. albicansculture filtrates also induced migration of PMNs across confluent monolayers of a human gastrointestinal epithelial cell line, T84; migration occurred in the basolateral-to-apical direction but not the reverse direction, unless the epithelial tight junctions were disrupted. J774 cells did not migrate toward the formylated peptide (fMet-Leu-Phe; fMLF), and chemotaxis toward theC. albicansculture filtrate was not inhibited by an FPR antagonist (t-butoxycarbonyl-Met-Leu-Phe), suggesting that a different receptor mediated J774 cell chemotaxis. In conclusion, we have identified a receptor by which a non-serum-dependent chemotactic factor (NSCF) produced byC. albicansinduced chemotaxis of PMNs. Additionally, we have shown that NSCF was active across epithelial monolayers. These findings suggest that NSCFs produced byC. albicansand other yeast species may influence host-pathogen interactions at the gastrointestinal tract mucosal surface by inducing phagocytic-cell infiltration.
Purification and Cloning of a Streptokinase from
Streptococcus uberis
Authors: Johnsen Laust B. , Poulsen Knud , Kilian Mogens , Petersen Torben E.
Published: December 2019

A bovine plasminogen activator was purified from the culture supernatant of the bovine pathogen
Streptococcus uberis
NCTC 3858. After the final reverse-phase high-performance liquid chromatography step a single protein with a molecular mass of 32 kDa was detected in the active fraction. A partial peptide map was established, and degenerate primers were designed and used for amplification of fragments of the gene encoding the activator. Inverse PCR was subsequently used for obtaining the full-length gene. The
S. uberis
plasminogen activator gene (
skc
) encodes a protein consisting of 286 amino acids including a signal peptide of 25 amino acids. In an amino acid sequence comparison the cloned activator showed an identity of approximately 26% to the streptokinases isolated from
Streptococcus equisimilis
and
Streptococcus pyogenes
. Interestingly, the activator from
S. uberis
was found to lack the C-terminal domain possessed by the streptokinase from
S. equisimilis
. This is apparently a general feature of the streptokinases of this species; biochemical and genetic analysis of 10 additional strains of
S. uberis
revealed that 9 of these were highly similar to strain NCTC 3858. Sequencing of the
skc
gene from three of these strains indicated that the amino acid sequence of the protein is highly conserved within the species.
A Sustained Rat Model for Studying the Long-Lasting Catabolic State of Sepsis
Authors: Breuille Denis , Voisin Laure , Contrepois Michel , Arnal Maurice , Rose Francis , Obled Christiane
Published: December 2019

Most animal models of sepsis induced high mortality or early recovery and do not mimic the long-lasting catabolic state observed in patients. The purpose of this study is to develop a model of sepsis which reproduces these disorders, especially the long-lasting muscle wasting. This report summarizes our observations in a series of seven experiments using this model with rats to study the route of live
Escherichia coli
administration, dose of bacteria, reproducibility of the model, bacterial count in tissues, comparison of injection of live or dead bacteria, metabolic perturbations linked to infection, and potential role of tumor necrosis factor alpha (TNF-α) in muscle wasting. After intravenous infection, animals were anorexic and the catabolic state was long-lasting: body weight loss for 2 to 3 days followed by a chronic wasting state for several days. Liver, spleen, lung protein content, and plasma concentration of α
2
-macroglobulin were increased 2 and 6 days after infection. At 6 days, muscle protein content was substantially (−40%) reduced. The plasma TNF-α level measured 1.5 h after infection correlated with body weight loss observed 9 days later. The inhibition of TNF-α secretion by administration of pentoxifylline 1 h before infection reduced muscle wasting and activation of proteolysis at day 2 and abolished them at day 6. This septic model mimics in rats the prolonged protein metabolism alterations and muscle atrophy characteristics of infected patients and thus is useful for studying the impact of nutritional support on outcome.
Pathogenicity Island 2 Mutants ofSalmonella typhimuriumAre Efficient Carriers for Heterologous Antigens and Enable Modulation of Immune Responses
Authors: Medina Eva , Paglia Paola , Nikolaus Thomas , Müller Astrid , Hensel Michael , Guzmán Carlos A.
Published: December 2019

The potential use as vaccine delivery system ofSalmonella typhimuriumstrains harboring defined mutations in thesseC(HH104) andsseD(MvP101) genes, which encode putative effector proteins of the type III secretion system ofSalmonellapathogenicity island 2, was evaluated and compared with that of the well-characterizedaroAmutant strain SL7207 by using β-galactosidase (β-Gal) as a model antigen. When orally administered to immune-competent or gamma interferon-deficient (IFN-γ−/−) BALB/c mice, both mutants were found to be highly attenuated (50% lethal dose, >109bacteria). Both strains were also able to efficiently colonize and persist in Peyer’s patches. Immunization with HH104 and MvP101 triggered β-Gal-specific serum and mucosal antibody responses equivalent to or stronger than those observed in SL7207-immunized mice. Although immunoglobulin G2 (IgG2) serum antibodies were dominant in all groups, IgG1 was also significantly increased in mice vaccinated with MvP101 and SL7207. Comparable β-Gal-specific IgA and IgG antibodies were detected in intestinal lavages from mice immunized with the different strains. Antigen-specific CD4+T-helper cells were generated after vaccination with all vaccine prototypes; however, responses were significantly more efficient when HH104 and MvP101 were used (P< 0.05). Significantly higher levels of IFN-γ were produced by restimulated spleen cells from mice immunized with HH104 than from those vaccinated with the MvP101 or SL7207 derivatives (P≤ 0.05). Interestingly, the three strains induced major histocompatibility complex class I-restricted CD8+cytotoxic T cells against β-Gal; however, cytotoxic T-lymphocyte responses were significantly stronger after immunization with HH104 (P< 0.05). These novelS. typhimuriumattenuated strains constitute promising delivery systems for vaccine antigens. The qualitative differences observed in the obtained responses with different carriers may be useful for those applications in which a targeted immunomodulation is required.
Vibrio parahaemolyticus
Thermostable Direct Hemolysin Modulates Cytoskeletal Organization and Calcium Homeostasis in Intestinal Cultured Cells
Authors: Fabbri Alessia , Falzano Loredana , Frank Claudio , Donelli Gianfranco , Matarrese Paola , Raimondi Francesco , Fasano Alessio , Fiorentini Carla
Published: December 2019

Vibrio parahaemolyticus
is a marine bacterium known to be the leading cause of seafood gastroenteritis worldwide. A 46-kDa homodimer protein secreted by this microorganism, the thermostable direct hemolysin (TDH), is considered a major virulence factor involved in bacterial pathogenesis since a high percentage of strains of clinical origin are positive for TDH production. TDH is a pore-forming toxin, and its most extensively studied effect is the ability to cause hemolysis of erythrocytes from different mammalian species. Moreover, TDH induces in a variety of cells cytotoxic effects consisting mainly of cell degeneration which often leads to loss of viability. In this work, we examined the cellular changes induced by TDH in monolayers of IEC-6 cells (derived from the rat crypt small intestine), which represent a useful cell model for studying toxins from enteric bacteria. In experimental conditions allowing cell survival, TDH induces a rapid transient increase in intracellular calcium as well as a significant though reversible decreased rate of progression through the cell cycle. The morphological changes seem to be dependent on the organization of the microtubular network, which appears to be the preferential cytoskeletal element involved in the cellular response to the toxin.
Virulence of a
spaP
Mutant of
Streptococcus mutans
in a Gnotobiotic Rat Model
Authors: Crowley Paula J. , Brady L. Jeannine , Michalek Suzanne M. , Bleiweis Arnold S.
Published: December 2019

Streptococcus mutans
, the principal etiologic agent of dental caries in humans, possesses a variety of virulence traits that enable it to establish itself in the oral cavity and initiate disease. A 185-kDa cell surface-localized protein known variously as antigen I/II, antigen B, PAc, and P1 has been postulated to be a virulence factor in
S. mutans
. We showed previously that P1 expression is necessary for in vitro adherence of
S. mutans
to salivary agglutinin-coated hydroxyapatite as well as for fluid-phase aggregation. Since adherence of the organism is a necessary first step toward colonization of the tooth surface, we sought to determine what effect deletion of the gene for P1,
spaP
, has on the colonization and subsequent cariogenicity of this organism in vivo. Germ-free Fischer rats fed a diet containing 5% sucrose were infected with either
S. mutans
NG8 or an NG8-derived
spaP
mutant strain, PC3370, which had been constructed by allelic exchange mutagenesis. At 1-week intervals for 6 weeks after infection, total organisms recovered from mandibles were enumerated. At week 6, caries lesions also were scored. A significantly lower number of enamel and dentinal carious lesions was observed for the mutant-infected rats, although there was no difference between parent and mutant in the number of organisms recovered from teeth through 6 weeks postinfection. Coinfection of animals with both parent and mutant strains resulted in an increasing predominance of the mutant strain being recovered over time, suggesting that P1 is not a necessary prerequisite for colonization. These data do, however, suggest a role for P1 in the virulence of
S. mutans
, as reflected by a decrease in the cariogenicity of bacteria lacking this surface protein.
Linear Peptide Specificity of Bovine Antibody Responses to p67 of
Theileria parva
and Sequence Diversity of Sporozoite-Neutralizing Epitopes: Implications for a Vaccine
Authors: Nene Vishvanath , Gobright Elke , Bishop Richard , Morzaria Subhash , Musoke Antony
Published: December 2019

A stage-specific surface antigen of
Theileria parva
, p67, is the basis for the development of an anti-sporozoite vaccine for the control of East Coast fever (ECF) in cattle. By Pepscan analysis with a series of overlapping synthetic p67 peptides, the antigen was shown to contain five distinct linear peptide sequences recognized by sporozoite-neutralizing murine monoclonal antibodies. Three epitopes were located between amino acid positions 105 to 229 and two were located between positions 617 to 639 on p67. Bovine antibodies to a synthetic peptide containing one of these epitopes neutralized sporozoites, validating this approach for defining immune responses that are likely to contribute to immunity. Comparison of the peptide specificity of antibodies from cattle inoculated with recombinant p67 that were immune or susceptible to ECF did not reveal statistically significant differences between the two groups. In general, antipeptide antibody levels in the susceptible animals were lower than in the immune group and neither group developed high responses to all sporozoite-neutralizing epitopes. The bovine antibody response to recombinant p67 was restricted to the N- and C-terminal regions of p67, and there was no activity against the central portion between positions 313 and 583. So far, p67 sequence polymorphisms have been identified only in buffalo-derived
T. parva
parasites, but the consequence of these for vaccine development remains to be defined. The data indicate that optimizations of the current vaccination protocol against ECF should include boosting of relevant antibody responses to neutralizing epitopes on p67.
Complete Nucleotide Sequence of the 27-Kilobase Virulence Related Locus (
vrl
) of
Dichelobacter nodosus
: Evidence for Extrachromosomal Origin
Authors: Billington Stephen J. , Huggins Andrea S. , Johanesen Priscilla A. , Crellin Paul K. , Cheung Jackie K. , Katz Margaret E. , Wright Catherine L. , Haring Volker , Rood Julian I.
Published: December 2019

The
vrl
locus is preferentially associated with virulent isolates of the ovine footrot pathogen,
Dichelobacter nodosus
. The complete nucleotide sequence of this 27.1-kb region has now been determined. The data reveal that the locus has a G+C content much higher than the rest of the
D. nodosus
chromosome and contains 22 open reading frames (ORFs) encoding products including a putative adenine-specific methylase, two potential DEAH ATP-dependent helicases, and two products with sequence similarity to a bacteriophage resistance system. These ORFs are all in the same orientation, and most are either overlapping or separated by only a few nucleotides, suggesting that they comprise an operon and are translationally coupled. Expression vector studies have led to the identification of proteins that correspond to many of these ORFs. These data, in combination with evidence of insertion of
vrl
into the 3′ end of an
ssrA
gene, are consistent with the hypothesis that the
vrl
locus was derived from the insertion of a bacteriophage or plasmid into the
D. nodosus
genome.
Potent Immunoregulatory Effects ofSalmonella typhiFlagella on Antigenic Stimulation of Human Peripheral Blood Mononuclear Cells
Authors: Wyant Timothy L. , Tanner Michael K. , Sztein Marcelo B.
Published: December 2019

A key function of monocytes/macrophages (Mφ) is to present antigens to T cells. However, upon interaction with bacteria, Mφ lose their ability to effectively present soluble antigens. This functional loss was associated with alterations in the expression of adhesion molecules and CD14 and a reduction in the uptake of soluble antigen. Recently, we have demonstrated thatSalmonella typhiflagella (STF) markedly decrease CD14 expression and are potent inducers of proinflammatory cytokine production by human peripheral blood mononuclear cells (hPBMC). In order to determine whetherS. typhiand soluble STF also alter the ability of Mφ to activate T cells to proliferate to antigens and mitogens, hPBMC were cultured in the presence of tetanus toxoid (TT) or phytohemagglutinin (PHA) and either killed whole-cellS. typhior purified STF protein. Both whole-cellS. typhiand STF suppressed proliferation to PHA and TT. This decreased proliferation was not a result of increased Mφ production of nitric oxide, prostaglandin E2, or oxygen radicals or the release of interleukin-1β, tumor necrosis factor alpha, interleukin-6, or interleukin-10 following exposure to STF. However, the ability to take up soluble antigen, as determined by fluorescein isothiocyanate-labeled dextran uptake, was reduced in cells cultured with STF. Moreover, there was a dramatic reduction in the expression of CD54 on Mφ after exposure to STF. These results indicate that whole-cellS. typhiand STF have the ability to alter in vitro proliferation to soluble antigens and mitogens by affecting Mφ function.
Gamma Interferon Stimulates Rat Alveolar Macrophages To KillPneumocystis cariniibyl-Arginine- and Tumor Necrosis Factor-Dependent Mechanisms
Authors: Downing James F. , Kachel Diane L. , Pasula Rajamouli , Martin William J.
Published: December 2019

Pneumocystis cariniipneumonia remains a serious complication for immunocompromised patients. In the present study,P. cariniiorganisms interacted with gamma interferon (IFN-γ)-stimulated alveolar macrophages (AMs) to activate thel-arginine-dependent cytocidal pathway involving reactive nitrogen intermediates (RNI) that were assayed as nitrite (NO2−). Unstimulated cultures of AMs produced negligible quantities of RNI. Addition ofP. cariniiorganisms to IFN-γ-primed AMs resulted in greatly enhanced production of RNI. NO2−levels increased from 0.8 ± 0.4 to 11.1 ± 3.8 μM as early as 6 h afterP. cariniiorganisms were incubated with IFN-γ-stimulated AMs and to 35.1 ± 8.9 μM after a 24-h incubation, a near-maximum level. High levels of NO2−were produced by AMs primed with as little as 10 U of IFN-γ per ml in the presence ofP. carinii, and a 20-fold increase in IFN-γ concentration resulted in only a further 65% increase in NO2−production. RNI-dependent killing ofP. cariniiwas demonstrated by both a51Cr release assay and a [35S]methionine pulse immunoprecipitation assay. Addition of either monoclonal tumor necrosis factor alpha (TNF-α) neutralizing antibody or 200 μMNG-monomethyl-l-arginine (l-NGMMA), a competitive inhibitor of thel-arginine-dependent pathway, significantly decreased NO2−production and reducedP. cariniikilling. TNF-α alone had no effect onP. cariniiviability. These results suggest that (i) the specific interaction ofP. cariniiorganisms with IFN-γ-primed AMs triggers the production of RNI, (ii) RNI are toxic toP. carinii, and (iii) TNF-α likely plays a central role in mediatingP. cariniikilling by IFN-γ-stimulated AMs.
Neutralization of Endotoxin In Vitro and In Vivo by a Human Lactoferrin-Derived Peptide
Authors: Zhang Gui-Hang , Mann David M. , Tsai Chao-Ming
Published: December 2019

Endotoxin (lipopolysaccharide [LPS]) is the major pathogenic factor of gram-negative septic shock, and endotoxin-induced death is associated with the host overproduction of tumor necrosis factor alpha (TNF-α). In the search for new antiendotoxin molecules, we studied the endotoxin-neutralizing capacity of a human lactoferrin-derived 33-mer synthetic peptide (GRRRRSVQWCAVSQPEATKCFQWQRNMRKVRGP; designated LF-33) representing the minimal sequence for lactoferrin binding to glycosaminoglycans. LF-33 inhibited the coagulation of theLimulusamebocyte lysate and the secretion of TNF-α by RAW 264.7 cells induced by lipid A and four different endotoxins with a potency comparable to that of polymyxin B. The first six residues at the N terminus of LF-33 were critical for its antiendotoxin activity. The endotoxin-neutralizing capacity of LF-33 and polymyxin B was attenuated by human serum. Coinjection ofEscherichia coliLPS (125 ng) with LF-33 (2.5 μg) dramatically reduced the lethality of LPS in the galactosamine-sensitized mouse model. Significant protection of the mice against the lethal LPS challenge was also observed when LF-33 (100 μg) was given intravenously after intraperitoneal injection of LPS. Protection was correlated with a reduction in TNF-α levels in the mouse serum. These results demonstrate the endotoxin-neutralizing capability of LF-33 in vitro and in vivo and its potential use for the treatment of endotoxin-induced septic shock.
Clearance ofChlamydia trachomatisfrom the Murine Genital Mucosa Does Not Require Perforin-Mediated Cytolysis or Fas-Mediated Apoptosis
Authors: Perry Linda L. , Feilzer Karen , Hughes Scott , Caldwell Harlan D.
Published: December 2019

The molecular mechanisms of resistance to genital infection with the mouse pneumonitis (MoPn) strain ofChlamydia trachomatisare unknown. A role for major histocompatibility complex class II-restricted, interleukin-12-dependent CD4+T cells has been established, but the functional activity of these cells does not depend on secretion of gamma interferon. Here we examined the potential contribution of T-cell-mediated cytotoxicity and apoptosis to mucosal clearance of MoPn by using mice deficient in the molecular mediators of target cell lysis. Animals lacking perforin, Fas, Fas ligand, or both perforin and Fas ligand were infected genitally withC. trachomatisMoPn and monitored for expression of immunity to chlamydial antigens and clearance of MoPn from the genital mucosa. In each case, the profile of spleen cytokine production, the magnitude of the host antibody response, and the kinetics of chlamydial clearance were similar to those of genetically intact controls. Compensatory overproduction of tumor necrosis factor alpha, an alternate mediator of apoptosis in certain cell types, did not appear to account for the ability of mutant mice to resolveChlamydiainfections. These results fail to support CD4+T-cell-mediated apoptosis or CD8+T-cell-mediated cytotoxicity as being critical to the clearance ofC. trachomatisMoPn urogenital infections.
Relationship between UDP-Glucose 4-Epimerase Activity and Oligoglucose Glycoforms in Two Strains of
Neisseria meningitidis
Authors: Lee Frank K. N. , Gibson Bradford W. , Melaugh William , Zaleski Anthony , Apicella Michael A.
Published: December 2019

Sodium dodecyl sulfate-polyacrylamide gel analysis of lipooligosaccharide (LOS) from
Neisseria meningitidis
has demonstrated considerable microheterogeneity in the variable region of LOS due to the presence of novel glycoforms. As a step toward understanding the basis for the expression of these novel glycoforms, we have examined the LOS structures and UDP-glucose 4-epimerase (epimerase) activity levels in two strains (NMB and MA-1) and their respective
galE
mutants. Strain NMB was found to have low epimerase activity and to contain multiple glycoforms, some of which appear to contain only glucose sugars. The
galE
mutant had only the oligoglucose glycoforms. Strain MA-1 had higher epimerase activity at both log and stationary phases (2- and 12.5-fold, respectively) and one glycoform with a putative lactosyl structure. Strain MA-1
galE
had two glycoforms that contained one or two glucose residues. To understand the molecular basis for the different epimerase activities, we examined the predicted amino acid sequences of the respective
galE
open reading frames and determined the relative amounts of GalE protein. We found no significant differences between the predicted amino acid sequence of the GalE protein in NMB and that in MA-1. We observed no significant differences in the level of GalE protein between MA-1 and NMB at exponential or stationary phase. We also observed an 8.2-fold drop in epimerase activity in NMB between the log and stationary phases that was not due to the GalE protein level or low glucose levels.
Effect of Transforming Growth Factor β on ExperimentalSalmonella typhimuriumInfection in Mice
Authors: Galdiero Massimiliano , Marcatili Antonella , Cipollaro de l’ero Gabriella , Nuzzo Immacolata , Bentivoglio Concetta , Galdiero Marilena , Carratelli Caterina Romano
Published: December 2019

We have investigated the effect of the in vivo administration of recombinant transforming growth factor β (rTGF-β) on the pathogenic mechanisms involved inSalmonella typhimuriumexperimental infection in mice. The protective response elicited by macrophages was induced by rTGF-β1by 2 days after experimental infection, as demonstrated by an increased NO production, while the humoral protective effect began with cytokine mRNA expression 2 days after the challenge and continued after 5 days with cytokine release and lymphocyte activation. We demonstrated that all mice who received rTGF-β1survived 7 days after infection. The number of bacteria recovered in the spleens and in the livers of rTGF-β1-treated mice 2 and 5 days after infection was significantly smaller than that found in the same organs after phosphate-buffered saline (PBS) inoculation. Furthermore, 2 and 5 days after infection, splenic macrophages from rTGF-β1-treated mice showed a greater NO production than did those from PBS-treated mice. The effect of rTGF-β1onS. typhimuriuminfection in mice was correlated with the expression of cell costimulatory CD28 molecules. Five days afterS. typhimuriuminfection, the percentage of CD28+-expressing T cells in splenic lymphocytes from rTGF-β1-treated mice increased with respect to that from control mice. Gamma interferon (IFN-γ) mRNA was present in a greater amount in spleen cells from rTGF-β1-treated mice after 2 days, although the intensity of the band decreased 5 days after the challenge. A similar pattern was obtained with the mRNAs for interleukin-1α (IL-1α), IL-6, TGF-β, and inducible nitric oxide synthase, which showed greater expression in cells obtained from rTGF-β1-treated andS. typhimurium-infected mice 2 days after challenge. The treatment with rTGF-β1induced an increase in IL-1α and IFN-γ release in the supernatant of splenocyte cultures 5 days after the experimental infection withS. typhimurium. Moreover, we demonstrated that 5 days after infection, the IFN-γ titer was significantly greater in the sera of rTGF-β-treated mice than in those of PBS-treated mice. Also, hsp60 showed greater expression 2 days after the challenge in splenocytes from rTGF-β1-treated mice. The role played by proinflammatory and immunoregulatory cytokines and by CD28 is discussed.
Safety and Immunogenicity of a
Pseudomonas aeruginosa
Hybrid Outer Membrane Protein F-I Vaccine in Human Volunteers
Authors: Mansouri Erfan , Gabelsberger Josef , Knapp Bernhard , Hundt Erika , Lenz Uwe , Hungerer Klaus-Dieter , Gilleland Harry E. , Staczek John , Domdey Horst , von Specht Bernd-Ulrich
Published: December 2019

A hybrid protein [Met-Ala-(His)
6
OprF
190–342
-OprI
21–83
] consisting of the mature outer membrane protein I (OprI) and amino acids 190 to 342 of OprF of
Pseudomonas aeruginosa
was expressed in
Escherichia coli
and purified by Ni
2+
chelate-affinity chromatography. After safety and pyrogenicity evaluations in animals, four groups of eight adult human volunteers were vaccinated intramuscularly three times at 4-week intervals and revaccinated 6 months later with either 500, 100, 50, or 20 μg of OprF-OprI adsorbed onto A1(OH)
3
. All vaccinations were well tolerated. After the first vaccination, a significant rise of antibody titers against
P. aeruginosa
OprF and OprI was measured in volunteers receiving the 100- or the 500-μg dose. After the second vaccination, significant antibody titers were measured for all groups. Elevated antibody titers against OprF and OprI could still be measured 6 months after the third vaccination. The capacity of the elicited antibodies to promote complement binding and opsonization could be demonstrated by a C1q-binding assay and by the in vitro opsonophagocytic uptake of
P. aeruginosa
bacteria. These data support the continued development of an OprF-OprI vaccine for use in humans.