bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes in Cytokine Levels during Reactivation ofToxoplasma gondiiInfection in Lungs
Authors: Filice Gregory A. , Clabots Connie R. , Riciputi Paul E. , Goñi-Laguardia Oscar , Pomeroy Claire
Published: December 2019

We studied cytokine proteins and mRNAs in mice with two forms ofToxoplasma gondiipneumonia resulting from reactivation of infection. In the first form, mice were infected withT. gondii, developed and recovered from systemic disease, and then developed pneumonia 3 weeks later. As pulmonary inflammation developed, levels of cytokine mRNAs for gamma interferon (IFN-γ), interleukin-2 (IL-2), IL-4, and IL-10 increased in bronchoalveolar lavage (BAL) cells or lung tissue, and the level of IFN-γ protein increased in BAL fluid. The second form of pneumonia occurred as a complication of primary cytomegalovirus (CMV) disease in mice with dormantT. gondiiinfection. During CMV disease, IL-2 mRNA levels decreased in lung tissue, IL-10 protein levels increased in lung tissue, and IL-10 protein levels increased in BAL fluid. As the mice recovered from CMV disease,T. gondiiinfection was reactivated in the lungs and was manifested asT. gondiipneumonia. During CMV-inducedT. gondiipneumonia, IFN-γ, IL-2, IL-4, and IL-10 mRNA levels increased in BAL cells or lung tissue, and both IFN-γ and IL-2 protein levels increased in BAL fluid. We concluded that both forms ofT. gondiipneumonia are accompanied by increases in both type 1 T-helper and type 2 T-helper cytokine levels in lungs. The mechanism of CMV-induced reactivation ofT. gondiiinfection in lungs may involve local decreases in IL-2 levels and/or increases in IL-10 levels.
Down Regulation ofEntamoeba histolyticaVirulence by Monoxenic Cultivation withEscherichia coliO55 Is Related to a Decrease in Expression of the Light (35-Kilodalton) Subunit of the Gal/GalNAc Lectin
Authors: Padilla-Vaca Felipe , Ankri Serge , Bracha Rivka , Koole Lucy Anna , Mirelman David
Published: December 2019

Entamoeba histolyticavirulence is related to a number of amebic components (lectins, cysteine proteinases, and amebapore) and host factors, such as intestinal bacterial flora. Trophozoites are selective in their interactions with bacteria, and the parasite recognition of glycoconjugates plays an important role in amebic virulence. Long-term monoxenic cultivation of pathogenicE. histolyticatrophozoites, strains HK-9 or HM-1:IMSS, withEscherichia coliserotype O55, which binds strongly to the Gal/GalNAc amebic lectin, markedly reduced the trophozoites’ adherence and cytopathic activity on cell monolayers of baby hamster kidney (BHK) cells. Specific probes prepared fromE. histolyticalectin genes as well as antibodies directed against the light (35-kDa) and heavy (170-kDa) subunits of the Gal/GalNAc lectin revealed a decrease in the transcription and expression of the light subunit in trophozoites grown monoxenically withE. coliO55. This effect was not observed whenE. histolyticawas grown withE. coli346, a mannose-binding type I pilated bacteria. Our results suggest that the light subunit of the amebic lectin is involved in the modulation of parasite adherence and cytopathic activity.
Characterization of the
Escherichia coli
AF/R1 Pilus Operon: Novel Genes Necessary for Transcriptional Regulation and for Pilus-Mediated Adherence
Authors: Cantey J. Robert , Blake R. K. , Williford J. R. , Moseley Steve L.
Published: December 2019

We isolated the genetic determinant of AF/R1 pilus production in attaching/effacing
Escherichia coli
RDEC-1 and identified seven genes required for pilus expression and function. DNA sequence analysis of the structural subunit gene
afrA
corrected an error in the published sequence and extended homology with the F18 pilus subunit of pig edema
E. coli
strains. AfrB and AfrC, encoded downstream from AfrA, were required for pilus expression. AfrB was related to the usher protein PefC of
Salmonella typhimurium
plasmid-encoded fimbriae, and AfrC was related to PefD, a chaperone protein. AfrD and AfrE, encoded downstream from AfrC, were not necessary for the expression of AF/R1 pili but were required for ileal adherence as assayed by ileal brush border aggregation. Thus, the adhesive subunit of the AF/R1 pilus is distinct from the structural subunit, as is the case for Pap pili and type 1 pili. AfrD was related to FedE of the F18 fimbrial operon of the
E. coli
strain that causes edema disease in pigs. AfrE was a novel protein. AfrR and AfrS are encoded upstream from AfrA, in the opposite orientation. AfrR is related to the AraC family of transcriptional regulators, and AfrR and AfrS interact to function in a novel mode of transcriptional activation of
afrA
. AF/R1 pili mediate the adherence to Peyer’s patch M cells, ileal mucosa, and colonic mucosa in a rabbit model of diarrhea caused by enteropathogenic
E. coli
. Our observations will facilitate the further study of the phenomena of M-cell adherence.
Distinct Immunoglobulin Class and Immunoglobulin G Subclass Patterns against Ganglioside GQ1b in Miller Fisher Syndrome following Different Types of Infection
Authors: Schwerer Beatrix , Neisser Andrea , Bernheimer Hanno
Published: December 2019

We studied serum antibodies against gangliosides GQ1b and GM1 in 13 patients with Miller Fisher syndrome (MFS) and in 18 patients with Guillain-Barré syndrome (GBS) with cranial nerve involvement. Anti-GQ1b titers were elevated in all patients with MFS cases (immunoglobulin G [IgG] > IgA, IgM), and in 8 of the 18 with GBS. Lower frequencies of increased anti-GM1 titers were observed in MFS patients (3 of 13), as well as in GBS patients (5 of 18). During the course of MFS, anti-GQ1b titers of all Ig classes decreased within 3 weeks after onset. By contrast, anti-GM1 titers (mainly IgM) transiently increased during the course of MFS in five of six patients, suggesting a nonspecific secondary immune response. In patients with MFS following respiratory infections, IgG was the major anti-GQ1b Ig class (six of six patients) and IgG3 was the major subclass (five of six). In contrast, four of five patients with MFS following gastrointestinal infections showed predominance of anti-GQ1b IgA or IgM over IgG and predominance of the IgG2 subclass; anti-GQ1b IgG (IgG3) prevailed in one patient only. These distinct Ig patterns strongly suggest that different infections may trigger different mechanisms of anti-GQ1b production, such as via T-cell-dependent as opposed to T-cell-independent pathways. Thus, the origin of antibodies against GQ1b in MFS may be determined by the type of infectious agent that precipitates the disease.
Membrane Permeabilization by Thrombin-Induced Platelet Microbicidal Protein 1 Is Modulated by Transmembrane Voltage Polarity and Magnitude
Authors: Koo Su-Pin , Bayer Arnold S. , Kagan Bruce L. , Yeaman Michael R.
Published: December 2019

Thrombin-induced platelet microbicidal protein 1 (tPMP-1) is a small, cationic peptide generated from rabbit platelets when they are exposed to thrombin in vitro. It has potent microbicidal activity against a broad spectrum of bacterial and fungal pathogens, including
Staphylococcus aureus
. Previous in vitro studies involving whole staphylococcal cells and planar lipid bilayers (as artificial bacterial membrane models) suggested that membrane permeabilization by tPMP-1 is voltage dependent (S.-P. Koo, M. R. Yeaman, and A. S. Bayer, Infect. Immun. 64:3758–3764, 1996; M. R. Yeaman, A. S. Bayer, S. P. Koo, W. Foss, and P. M. Sullam, J. Clin. Investig. 101:178–187, 1998). Thus, the aims of the present study were to specifically characterize the electrophysiological events associated with membrane permeabilization by tPMP-1 by using artificial planar lipid bilayer membranes. We assessed the influence of transmembrane voltage polarity and magnitude on the initiation and modulation of tPMP-1 membrane permeabilization at various concentrations of tPMP-1 (range, 1 to 100 ng/ml) added to the
cis
side of the membranes. The incidence of membrane permeabilization induced by tPMP-1 at all of the concentrations tested was more frequent at −90 mV than at +90 mV. It is noteworthy that membrane permeabilization due to 1-ng/ml tPMP-1 was successfully initiated at −90 mV but not at +90 mV. Further, the mean onset times of induction of tPMP-1 activity were comparable under the various conditions. Modulation of ongoing membrane permeabilization was dependent on voltage and tPMP-1 concentration. Membrane permeabilization at a low tPMP-1 concentration (1 ng/ml) was directly correlated with
trans
-negative voltages, while a higher tPMP-1 concentration (100 ng/ml) induced conductance which was more dependent on
trans
-positive voltages. Collectively, these data indicate that the mechanism of tPMP-1 microbicidal activity at the bacterial cytoplasmic membrane may involve distinct induction and propagation stages of membrane permeabilization which, in turn, are modulated by transmembrane potential, as well as peptide concentration.
Alpha C Protein as a Carrier for Type III Capsular Polysaccharide and as a Protective Protein in Group B Streptococcal Vaccines
Authors: Gravekamp Claudia , Kasper Dennis L. , Paoletti Lawrence C. , Madoff Lawrence C.
Published: December 2019

The alpha C protein, a protective surface protein of group B streptococci (GBS), is present in most non-type III GBS strains. Conjugate vaccines composed of the alpha C protein and type III capsular polysaccharide (CPS) might be protective against most GBS infections. In this study, the type III CPS was covalently coupled to full-length, nine-repeat alpha C protein (resulting in III-α9r conjugate vaccine) or to two-repeat alpha C protein (resulting in III-α2r conjugate vaccine) by reductive amination. Initial experiments with the III-α9r vaccine showed that it was poorly immunogenic in mice with respect to both vaccine antigens and was suboptimally efficacious in providing protection in mice against challenge with GBS. Therefore, modified vaccination protocols were used with the III-α2r vaccine. Female mice were immunized three times with 0.5, 5, or 20 μg of the III-α2r vaccine with an aluminum hydroxide adjuvant and bred. Ninety-five percent of neonatal mice born to dams immunized with the III-α2r vaccine survived challenge with GBS expressing type III CPS, and 60% survived challenge with GBS expressing wild-type (nine-repeat) alpha C protein; 18 and 17%, respectively, of mice in the negative control groups survived (P, <0.0001). These protection levels did not differ significantly from those obtained with the type III CPS-tetanus toxoid conjugate vaccine and the unconjugated two-repeat alpha C protein, which protected 98 and 58% of neonates from infection with GBS expressing type III CPS or the alpha C protein, respectively. Thus, the two-repeat alpha C protein in the vaccine was immunogenic and simultaneously enhanced the immunogenicity of type III CPS. III-α vaccines may be alternatives to GBS polysaccharide-tetanus toxoid vaccines, eliciting additional antibodies protective against GBS infection.
A Murine Model for the Study of
Chlamydia trachomatis
Genital Infections during Pregnancy
Authors: Pal Sukumar , Peterson Ellena M. , de la Maza Luis M.
Published: December 2019

Pregnant BALB/c mice were inoculated intravaginally on day 5 of gestation with the
Chlamydia trachomatis
mouse pneumonitis biovar. Animals that received 10
5
, 10
6
, or 10
7
inclusion-forming units (IFU) of
C. trachomatis
delivered prematurely on days 15 to 16 of gestation. A focal inflammatory infiltrate was observed in the wall of the uterus on the day 14 of gestation in animals inoculated with 10
5
IFU. In this group of mice, immunohistochemical analysis showed chlamydial inclusions in the endometrium and fetal membranes.
The Siderophore 2,3-Dihydroxybenzoic Acid Is Not Required for Virulence of
Brucella abortus
in BALB/c Mice
Authors: Bellaire Bryan H. , Elzer Philip H. , Baldwin Cynthia L. , Roop R. Martin
Published: December 2019

2,3-Dihydroxybenzoic acid (DHBA) is the only siderophore described for
Brucella
, and previous studies suggested that DHBA might contribute to the capacity of these organisms to persist in host macrophages. Employing an isogenic siderophore mutant (
ΔentC
) constructed from virulent
Brucella abortus
2308, however, we found that production of DHBA is not required for replication in cultured murine macrophages or for the establishment and maintenance of chronic infection in the BALB/c mouse model.
Characterization ofStaphylococcus epidermidisPolysaccharide Intercellular Adhesin/Hemagglutinin in the Pathogenesis of Intravascular Catheter-Associated Infection in a Rat Model
Authors: Rupp Mark E. , Ulphani Joseph S. , Fey Paul D. , Mack Dietrich
Published: December 2019

Biofilm production is thought to be a crucial factor in the ability ofStaphylococcus epidermidisto produce a biomaterial-based infection. A rat central venous catheter (CVC)-associated infection model was used to assess the importance of biofilm production, mediated by polysaccharide intercellular adhesin/hemagglutinin (PIA/HA), in the pathogenesis of intravascular catheter-associated infection. PIA/HA-positiveS. epidermidis1457 was significantly more likely to cause a CVC-associated infection (71 versus 14%,P< 0.03) resulting in bacteremia and metastatic disease than its isogenic PIA/HA-negative mutant. These results confirm the importance of biofilm production, mediated by PIA/HA, in the pathogenesis ofS. epidermidisexperimental CVC-associated infection.
Profiles of Th1 and Th2 Cytokines after Primary and Secondary Infection bySchistosoma mansoniin the Semipermissive Rat Host
Authors: Cêtre Catherine , Pierrot Christine , Cocude Cécile , Lafitte Sophia , Capron André , Capron Monique , Khalife Jamal
Published: December 2019

In contrast to most mouse strains, rats eliminate the primary schistosome burden around 4 weeks postinfection and subsequently develop protective immunity to reinfection. In rat schistosomiasis, we have shown predominant expression of a Th2-type cytokine response at the mRNA level after primary infection. In the present study, we showed a significant increase in interleukin-4 (IL-4) mRNA expression in inguinal lymph nodes early after a secondary infection. IL-5 mRNA expression showed a significant increase at days 2 and 4 postreinfection in the spleen and lymph nodes, respectively. We did not detect any gamma interferon (IFN-γ) mRNA after a challenge infection. Analysis of cytokine secretion by stimulated spleen cells after a primary infection showed predominant expression of IL-4 with maximum production on day 21, accompanied by production of IL-5 from day 11 to day 67. A significant increase in IFN-γ secretion was detected at day 21. Analysis of immunoglobulin G2b (IgG2b) and IgG2c (Th1-related isotypes) showed undetectable levels of IgG2b, but detectable levels of specific IgG2c antibodies were observed from day 42. The analysis of Th2-related isotypes showed high specific IgG1 and IgG2a antibody titers from day 29. After a secondary infection, only IL-4 and IL-5 secretion was sustained. This is supported by the increased production of Th2-related isotypes. These findings showed thatS. mansoniinfection can drive Th2 responses in rats in the absence of egg production which is required to induce a Th2 response in mice and are in favor of the role of Th2-type cytokines in protective immunity against reinfection.
Effect of Multiple Mutations in the Hemoglobin- and Hemoglobin-Haptoglobin-Binding Proteins, HgpA, HgpB, and HgpC, of
Haemophilus influenzae
Type b
Authors: Morton Daniel J. , Whitby Paul W. , Jin Hongfan , Ren Zhen , Stull Terrence L.
Published: December 2019

Haemophilus influenzae
requires heme for growth and can utilize hemoglobin and hemoglobin-haptoglobin as heme sources. We previously identified two hemoglobin- and hemoglobin-haptoglobin-binding proteins, HgpA and HgpB, in
H. influenzae
HI689. Insertional mutation of
hgpA
and
hgpB
, either singly or together, did not abrogate the ability to utilize or bind either hemoglobin or the hemoglobin-haptoglobin complex. A hemoglobin affinity purification method was used to isolate a protein of approximately 120 kDa from the
hgpA hgpB
double mutant. We have cloned and sequenced the gene encoding this third hemoglobin/hemoglobin-haptoglobin binding protein and designate it
hgpC
. Insertional mutation of
hgpC
did not affect the ability of the strain to utilize either hemoglobin or hemoglobin-haptoglobin. An
hgpA hgpB hgpC
triple mutant constructed by insertional mutagenesis showed a reduced ability to use the hemoglobin-haptoglobin complex but was unaltered in the ability to use hemoglobin. A second class of mutants was constructed in which the entire structural gene of each of the three proteins was deleted. The
hgpA hgpB hgpC
complete-deletion triple mutant was unable to utilize the hemoglobin-haptoglobin complex and showed a reduced ability to use hemoglobin. We have identified three hemoglobin/hemoglobin-haptoglobin-binding proteins in
Haemophilus influenzae
. Any one of the three proteins is sufficient to support growth with hemoglobin-haptoglobin as the heme source, and expression of at least one of the three is essential for hemoglobin-haptoglobin utilization. Although the three proteins play a role in hemoglobin utilization, an additional hemoglobin acquisition mechanism(s) exists.
Requirement of MrpH for Mannose-Resistant
Proteus
-Like Fimbria-Mediated Hemagglutination by
Proteus mirabilis
Authors: Li Xin , Johnson David E. , Mobley Harry L. T.
Published: December 2019

Two new genes,
mrpH
and
mrpJ
, were identified downstream of
mrpG
in the
mrp
gene cluster encoding mannose-resistant
Proteus
-like (MR/P) fimbriae of uropathogenic
Proteus mirabilis
. Since the predicted MrpH has 30% amino acid sequence identity to PapG, the Galα(1-4)Gal-binding adhesin of
Escherichia coli
P fimbriae, we hypothesized that
mrpH
encodes the functional MR/P hemagglutinin. MR/P fimbriae, expressed in
E. coli
DH5α, conferred on bacteria both the ability to cause mannose-resistant hemagglutination and the ability to aggregate to form pellicles on the broth surface. Both a Δ
mrpH
mutant expressed in
E. coli
DH5α and an isogenic
mrpH
::
aphA
mutant of
P. mirabilis
were unable to produce normal MR/P fimbriae efficiently, suggesting that MrpH was involved in fimbrial assembly. Amino acid residue substitution of the N-terminal cysteine residues (C66S and C128S) of MrpH abolished the receptor-binding activity (hemagglutinating ability) of MrpH but allowed normal fimbrial assembly, supporting the notion that MrpH was the functional MR/P hemagglutinin. Immunogold electron microscopy of
P. mirabilis
HI4320 revealed that MrpH was located at the tip of MR/P fimbriae, also consistent with its role in receptor binding. The isogenic
mrpH
::
aphA
mutant of HI4320 was less able to colonize the urine, bladder, and kidneys in a mouse model of ascending urinary tract infection (
P
< 0.01), and therefore MR/P fimbriae contribute significantly to bacterial colonization in mice. While there are similarities between
P. mirabilis
MR/P and
E. coli
P fimbriae, there are more notable differences: (i) synthesis of the MrpH adhesin is required to initiate fimbrial assembly, (ii) MR/P fimbriae confer an aggregation phenotype, (iii) site-directed mutation of specific residues can abolish receptor binding but allows fimbrial assembly, and (iv) mutation of the adhesin gene abolishes virulence in a mouse model of ascending urinary tract infection.
Persistence and Protective Efficacy of a
Mycobacterium tuberculosis
Auxotroph Vaccine
Authors: Jackson Mary , Phalen Susan W. , Lagranderie Micheline , Ensergueix Danielle , Chavarot Pierre , Marchal Gilles , McMurray David N. , Gicquel Brigitte , Guilhot Christophe
Published: December 2019

New vaccines against tuberculosis are urgently required because of the impressive incidence of this disease worldwide and the highly variable protective efficacy of the current vaccine. The possibility of creating new live vaccines by the rational attenuation of strains from the
Mycobacterium tuberculosis
complex was investigated. Two auxotrophic mutants of
M. tuberculosis
and
M. bovis
BCG were constructed by disruption of one of their purine biosynthetic genes. These mutants appeared unable to multiply in vitro within mouse bone-marrow derived macrophages. They were also attenuated in vivo in the mouse and guinea pig animal models. In guinea pigs, the two mutants induced strong delayed-type hypersensitivity response to purified protein derivative. In a preliminary experiment, the two mutants were compared to the BCG vaccine for their protective efficacy in a challenge against aerosolized virulent
M. tuberculosis
in the guinea pig model. Both mutants conferred some level of protection. These experiments demonstrate that the rational attenuation of
M. tuberculosis
could lead to the design of new candidate live vaccines against tuberculosis.
Characterization of a Murine Model of Melioidosis: Comparison of Different Strains of Mice
Authors: Hoppe I. , Brenneke B. , Rohde M. , Kreft A. , Häußler S. , Reganzerowski A. , Steinmetz I.
Published: December 2019

Melioidosis is an infectious disease caused by the saprophytic gram-negative rod
Burkholderia pseudomallei
. The aim of this study was to establish and characterize a murine model of melioidosis to provide a basis for further investigations on the pathogenesis of the disease. After intravenous infection with
B. pseudomallei
, C57BL/6 mice were found to be significantly more resistant than BALB/c mice. There was a marked organotropism of
B. pseudomallei
for the spleen and liver in both strains of mice, with the highest bacterial load in the spleen. Electron microscopic investigations of the spleen clearly demonstrated intracellular replication within membrane-bound phagosomes. Electron micrographs of the liver provided evidence that
B. pseudomallei
-containing phagosomes in hepatocytes fuse with lysosomes, leading to degradation of bacteria. In both strains of mice, the course of infection was highly dependent on the infective dose and the bacterial strain used, ranging from death within a few days to death after several weeks. In comparison with BALB/c mice, the bacterial counts in C57BL/6 mice were decreased 12 h after infection, which is suggestive of an innate immune mechanism against
B. pseudomallei
in this early phase of infection contributing to the lower susceptibility of C57BL/6 mice. BALB/c mice developed a more pronounced lymphopenia, granulocytosis, and splenomegaly at a lower infective dose compared to C57BL/6 mice. Analysis of the antibody response against
B. pseudomallei
11 days after infection revealed a significantly higher immunoglobulin G2A (IgG2a)/IgG1 ratio in C57BL/6 mice than in BALB/c mice, indicating that a T helper type 1 immune response is associated with resistance to infection with
B. pseudomallei.
Identification and Molecular Analysis of Rough-Colony-Specific Outer Membrane Proteins of
Actinobacillus actinomycetemcomitans
Authors: Haase Elaine M. , Zmuda Joyce L. , Scannapieco Frank A.
Published: December 2019

Actinobacillus actinomycetemcomitans
, a gram-negative bacterium isolated from the human mouth, has been implicated in the pathogenesis of early-onset periodontitis. Primary isolates cultured from subgingival plaque exhibit an adherent, rough colony phenotype which spontaneously converts to a nonadherent, smooth phenotype upon in vitro subculture. The rough colony variant produces abundant fimbriae and autoaggregates, while the smooth colony variant is planktonic and produces scant fimbriae. To begin to understand the significance of colony variation in biofilm formation by
A. actinomycetemcomitans
, outer membrane protein profiles of four isogenic rough and smooth colony variants were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two proteins with relative molecular masses of 43 and 20 kDa were expressed by the rough colony variants exclusively. Expression of these proteins was not found to be dependent on growth phase, oxygen tension, or type of complex medium. N-terminal amino acid sequences of these proteins obtained by Edman degradation were compared with sequences from the University of Oklahoma
A. actinomycetemcomitans
genome database. Two contiguous open reading frames (ORFs) encoding proteins having sequence homology with these proteins were identified. The 43-kDa protein (RcpA [rough colony protein A]) was similar to precursor protein D of the general secretion pathway of gram-negative bacilli, while the 20-kDa protein (RcpB [rough colony protein B]) appeared to be unique. The genes encoding these proteins have been cloned from
A. actinomycetemcomitans
283 and sequenced. A BLASTX (gapped BLAST) search of the surrounding ORFs revealed homology with other fimbria-related proteins. These data suggest that the genes encoding the 43-kDa (
rcpA
) and 20-kDa (
rcpB
) proteins may be functionally related to each other and to genes that may encode fimbria-associated proteins.
Helicobacter pylori
-Associated Gastritis in Mice is Host and Strain Specific
Authors: van Doorn Nathalie E. M. , Namavar Ferry , Sparrius Marion , Stoof Jeroen , van Rees Emmelien P. , van Doorn Leen-Jan , Vandenbroucke-Grauls Christina M. J. E.
Published: December 2019

The
vacA
and
cagA
geno- and phenotypes of two mouse-adapted strains of
Helicobacter pylori
, SS1 and SPM326, were determined. The SS1 strain, which had the
cagA
+
and
vacA
s2-m2 genotype, induced neither vacuole formation in HeLa cells nor interleukin-8 (IL-8) production in KATO III cells. In contrast,
H. pylori
SPM326, with the
cagA
+
and
vacA
s1b-m1 genotype, induced vacuoles as well as IL-8 production in vitro. Furthermore, a spontaneous mutant of SPM326, which produced a vacuolating cytotoxin but was not able to induce IL-8 production (SPM326/IL-8
−
), was detected. C57Bl/6 and BALB/c mice were infected with these three strains to investigate the colonization pattern and the effect on the immune response in vivo. The SS1 strain colonized the stomachs of all mice in large numbers which remained constant over time. Colonization with the SPM326/IL-8
+
and SPM326/IL-8
−
strains was lesser, or even absent, and decreased over time. At 5 weeks postinoculation all three
H. pylori
strains induced a mild increase of neutrophil count in the gastric corpus of C57Bl/6 mice, which disappeared by 12 weeks. At both 5 and 12 weeks postinoculation C57Bl/6 mice colonized with SPM326/IL-8
+
showed an increased expression of major histocompatibility complex (MHC) class II antigen in the cardia which was accompanied by an increased number of T cells. C57Bl/6 mice that were infected with SS1 and SPM326/IL-8
−
did not show chronic inflammation. BALB/c mice colonized with SS1 and SPM326/IL-8
−
also showed an increase in neutrophil count at 5 weeks, which normalized again by 12 weeks postinoculation. At this time point SS1-infected mice showed inflammation in the corpus and antrum. At these sites an increased expression of MHC class II antigens and an increased number of T cells were observed. Although small lymphoid follicles were already observed 5 weeks after inoculation with SS1, their incidence as well as their number was increased at 12 weeks. These results show that inflammation induced by
H. pylori
depends both on the bacterial strain and the host.
Proteasome Activity Is Required for Anthrax Lethal Toxin To Kill Macrophages
Authors: Tang Guangqing , Leppla Stephen H.
Published: December 2019

Anthrax lethal toxin (LeTx), consisting of protective antigen (PA) and lethal factor (LF), rapidly kills primary mouse macrophages and macrophage-like cell lines such as RAW 264.7. LF is translocated by PA into the cytosol of target cells, where it acts as a metalloprotease to cleave mitogen-activated protein kinase kinase 1 (MEK1) and possibly other proteins. In this study, we show that proteasome inhibitors such as acetyl-Leu-Leu-norleucinal, MG132, and lactacystin efficiently block LeTx cytotoxicity, whereas other protease inhibitors do not. The inhibitor concentrations that block LF cytotoxicity are similar to those that inhibit the proteasome-dependent IκB-α degradation induced by lipopolysaccharide. The inhibitors did not interfere with the proteolytic cleavage of MEK1 in LeTx-treated cells, indicating that they do not directly block the proteolytic activity of LF. However, the proteasome inhibitors did prevent ATP depletion, an early effect of LeTx. No overall activation of the proteasome by LeTx was detected, as shown by the cleavage of fluorogenic substrates of the proteasome. All of these results suggest that the proteasome mediates a toxic process initiated by LF in the cell cytosol. This process probably involves degradation of unidentified molecules that are essential for macrophage homeostasis. Moreover, this proteasome-dependent process is an early step in LeTx intoxication, but it is downstream of the cleavage by LF of MEK1 or other putative substrates.
Transcriptional Activation of mRNA of Intercellular Adhesion Molecule 1 and Induction of Its Cell Surface Expression in Normal Human Gingival Fibroblasts byMycoplasma salivariumandMycoplasma fermentans
Authors: Dong Li , Shibata Ken-Ichiro , Sawa Yoshihiko , Hasebe Akira , Yamaoka Yuji , Yoshida Shigemitsu , Watanabe Tsuguo
Published: December 2019

Lipoproteins in the cell membranes of bothMycoplasma salivariumandMycoplasma fermentanswere demonstrated to trigger the transcription of intercellular adhesion molecule-1 mRNA in normal fibroblasts isolated from human gingival tissue and to induce its cell surface expression by a mechanism distinct from that ofEscherichia colilipopolysaccharide. The lipid moiety of the lipoproteins was suggested to play a key role in the expression of the activity.
Macrophages and Epithelial Cells Respond Differently to the
Pseudomonas aeruginosa
Type III Secretion System
Authors: Coburn Jenifer , Frank Dara W.
Published: December 2019

The multiple effects of
Pseudomonas aeruginosa
type III secretion have largely been attributed to variations in cytotoxin expression between strains. Here we show that the target cell type is also important. While lung epithelial cells showed significant changes in morphology but not viability when infected with
P. aeruginosa
, macrophages were efficiently killed by
P. aeruginosa
. Both responses were dependent on the type III secretion system.
Genetic Characterization of Wild-Type and Mutant
fur
Genes of
Bordetella avium
Authors: Murphy Erin R. , Dickenson Amy , Militello Kevin T. , Connell Terry D.
Published: December 2019

For most, if not all, organisms, iron (Fe) is an essential element. In response to the nutritional requirement for Fe, bacteria evolved complex systems to acquire the element from the environment. The genes encoding these systems are often coordinately regulated in response to the Fe concentration. Recent investigations revealed that
Bordetella avium
, a respiratory pathogen of birds, expressed a number of Fe-regulated genes (T. D. Connell, A. Dickenson, A. J. Martone, K. T. Militello, M. J. Filiatraut, M. L. Hayman, and J. Pitula, Infect. Immun. 66:3597–3605, 1998). By using manganese selection on an engineered strain of
B. avium
that carried an Fe-regulated alkaline phosphatase reporter gene, a mutant was obtained that was affected in expression of Fe-regulated genes. To determine if Fe-dependent regulation in
B. avium
was mediated by a
fur
-like gene, a fragment of the
B. avium
chromosome, corresponding to the
fur
locus of
B. pertussis
, was cloned by PCR. Sequencing revealed that the fragment from
B. avium
encoded a polypeptide with 92% identity to the Fur protein of
B. pertussis
. In vivo experiments showed that the cloned gene complemented H1780, a
fur
mutant of
Escherichia coli
. Southern hybridizations and PCRs demonstrated that the manganese mutant had a deletion of 2 to 3 kbp of nucleotide sequence in the region located immediately 5′ of the
fur
open reading frame. A spontaneous PCR-derived mutant of the
B. avium fur
gene was isolated that encoded a Fur protein in which a histidine was substituted for an arginine at amino acid position 18 (R18H). Genetic analysis showed that the R18H mutant gene when cloned into a low-copy-number vector did not complement the
fur
mutation in H1780. However, the R18H mutant gene was able to complement the
fur
mutation when cloned into a high-copy-number vector. The cloned wild-type
fur
gene will be useful as a genetic tool to identify Fur-regulated genes in the
B. avium
chromosome.
Experimental Lyme Arthritis in the Absence of Interleukin-4 or Gamma Interferon
Authors: Brown Charles R. , Reiner Steven L.
Published: December 2019

Genetic resistance and susceptibility to experimental Lyme arthritis have been linked with the production of interleukin-4 (IL-4) or gamma interferon (IFN-γ), respectively. To determine the absolute requirement for these cytokines in disease outcome, we compared arthritis development in wild-type, IL-4-deficient (IL-4°), and IFN-γ-deficient (IFN-γ°) mice. While susceptible C3H mice developed swelling of ankle joints during the second week of infection, this swelling was exacerbated in C3H IFN-γ° mice. Their arthritis severity scores at day 21, however, were similar. Resolution of arthritis was also similar between C3H and C3H IFN-γ° mice. Arthritis-resistant DBA mice did not develop ankle swelling during the experimental period. There were no differences in ankle swelling or arthritis severity scores between control DBA mice and DBA IL-4° mice at any of the time points tested. While the presence of spirochetes in various tissues was similar among all strains at day 21, DBA IL-4° mice had a higher presence of spirochetes in blood, heart, and spleen than the DBA, C3H, and C3H IFN-γ° mice did at day 60. DBA IL-4° mice also had impaired ability to produceBorrelia-specific antibody responses, especially immunoglobulin G1. Thus, while IFN-γ and IL-4 are not absolutely required for arthritis susceptibility or resistance, the production of IL-4 does appear to play an important role inBorrelia-specific antibody production and spirochete clearance.
Interleukin-10 Responses to Liver-Stage Antigen 1 Predict Human Resistance toPlasmodium falciparum
Authors: Kurtis Jonathan D. , Lanar David E. , Opollo Malachi , Duffy Patrick E.
Published: December 2019

The design of an effective vaccine againstPlasmodium falciparum, the most deadly malaria parasite of humans, requires a careful definition of the epitopes and the immune responses involved in protection. Liver-stage antigen 1 (LSA-1) is specifically expressed during the hepatic stage ofP. falciparumand elicits cellular and humoral immune responses in naturally exposed individuals. We report here that interleukin-10 (IL-10) production in response to LSA-1 predicts resistance toP. falciparumafter eradication therapy. Resistance was not related to gamma interferon or tumor necrosis factor alpha production. This is the first report that human IL-10 responses are associated with resistance after eradication therapy, and our findings support the inclusion of LSA-1 in a vaccine against malaria.
Pretreatment with Granulocyte Colony-Stimulating Factor Attenuates the Inflammatory Response but Not the Bacterial Load in Cerebrospinal Fluid during Experimental Pneumococcal Meningitis in Rabbits
Authors: Østergaard Christian , Benfield Thomas , Gesser Borbola , Kharazmi Arsalan , Frimodt-Møller Niels , Espersen Frank , Lundgren Jens D.
Published: December 2019

A possible immunomodulatory role of granulocyte colony-stimulating factor (G-CSF) was investigated in an experimental pneumococcal meningitis model in rabbits. Animals were pretreated with G-CSF (10 μg/kg subcutaneously twice a day) starting 48 h before in vivo and ex vivo experiments, causing a five- to six-fold increase in the peripheral leukocyte level. Meningitis was induced by intracisternal inoculation of ∼4 × 105CFU ofStreptococcus pneumoniaetype 3. Neutrophil pleocytosis and interleukin-8 (IL-8) levels were significantly attenuated in G-CSF-pretreated animals compared to untreated animals (P< 0.05). Furthermore, G-CSF pretreatment significantly delayed alterations in cerebrospinal fluid (CSF) tumor necrosis factor alpha and IL-1β levels, as well as protein and glucose levels (P< 0.05). No difference in CSF bacterial concentrations was found, whereas the blood bacterial concentration was significantly decreased in G-CSF-pretreated animals (P< 0.05). Ex vivo chemotaxis of neutrophils isolated from G-CSF-pretreated animals was significantly decreased compared to that of neutrophils from untreated animals (P< 0.05). In conclusion, G-CSF pretreatment attenuates meningeal inflammation and enhances systemic bacterial killing. Further preclinical studies are required to investigate whether this may affect the clinical course of meningitis and thus whether G-CSF treatment may have a beneficial role in pneumococcal meningitis.
Vaccination against Shigellosis with Attenuated
Shigella flexneri
2a Strain SC602
Authors: Coster Trinka S. , Hoge Charles W. , VanDeVerg Lillian L. , Hartman Antoinette B. , Oaks Edwin V. , Venkatesan Malabi M. , Cohen Dani , Robin Guy , Fontaine-Thompson Annick , Sansonetti Philippe J. , Hale Thomas L.
Published: December 2019

The
Shigella flexneri
2a SC602 vaccine candidate carries deletions of the plasmid-borne virulence gene
icsA
(mediating intra- and intercellular spread) and the chromosomal locus
iuc
(encoding aerobactin) (S. Barzu, A. Fontaine, P. J. Sansonetti, and A. Phalipon, Infect. Immun. 64:1190–1196, 1996). Dose selection studies showed that SC602 causes shigellosis in a majority of volunteers when 3 × 10
8
or 2 × 10
6
CFU are ingested. In contrast, a dose of 10
4
CFU was associated with transient fever or mild diarrhea in 2 of 15 volunteers. All volunteers receiving single doses of ≥10
4
CFU excreted
S. flexneri
2a, and this colonization induced significant antibody-secreting cell and enzyme-linked immunosorbent assay responses against
S. flexneri
2a lipopolysaccharide in two-thirds of the vaccinees. Seven volunteers who had been vaccinated 8 weeks earlier with a single dose of 10
4
CFU and 7 control subjects were challenged with 2 × 10
3
CFU of virulent
S. flexneri
2a organisms. Six of the control volunteers developed shigellosis with fever and severe diarrhea or dysentery, while none of the vaccinees had fever, dysentery, or severe symptoms
(P
= 0.005). Three vaccinees experienced mild diarrhea, and these subjects had lower antibody titers than did the fully protected volunteers. Although the apparent window of safety is narrow, SC602 is the first example of an attenuated
S. flexneri
2a candidate vaccine that provides protection against shigellosis in a stringent, human challenge model.
Differential Sensitivity of Human Epithelial Cells to
Pseudomonas aeruginosa
Exoenzyme S
Authors: McGuffie Eileen M. , Fraylick Jennifer E. , Hazen-Martin Debra J. , Vincent Timothy S. , Olson Joan C.
Published: December 2019

Exoenzyme S (ExoS) is an ADP-ribosyltransferase produced and directly translocated into eukaryotic cells by the opportunistic pathogen
Pseudomonas aeruginosa
. Model systems that allow bacterial translocation of ExoS have found ExoS to have multiple effects on eukaryotic cell function, affecting DNA synthesis, actin cytoskeletal structure, and cell matrix adherence. To understand mechanisms underlying differences observed in cell sensitivities to ExoS, we examined the effects of bacterially translocated ExoS on multiple human epithelial cell lines. Of the cell lines examined, confluent normal kidney (NK) epithelial cells were most resistant to ExoS, while tumor-derived cell lines were highly sensitive to ExoS. Analysis of the mechanisms of resistance indicated that cell association as well as an intrinsic resistance to morphological alterations were associated with increased resistance to ExoS. Conversely, increased sensitivity to ExoS appeared to be linked to epithelial cell growth, with tumor cells capable of undergoing non-contact-inhibited, anchorage-independent growth all being sensitive to ExoS, and NK cells becoming sensitive to ExoS when subconfluent and growing. Consistent with the possibility that growth-related, actin-based structures are involved in sensitivity to ExoS, scanning electron microscopy revealed cellular extensions from sensitive, growing cells to bacteria, which were not readily evident in resistant cells. In all studies, the severity of effects of ExoS on cell function directly correlated with the degree of Ras modification, indicating that sensitivity to ExoS in some manner related to the efficiency of ExoS translocation and its ADP-ribosylation of Ras. Our results suggest that factors expressed by growing epithelial cells are required for the bacterial contact-dependent translocation of ExoS; as normal epithelial cells differentiate into polarized confluent monolayers, expression of these factors is altered, and cells in turn become more resistant to the effects of ExoS.
Protective Immunity of Microsphere-Based Mucosal Vaccines against Lethal Intranasal Challenge withStreptococcus pneumoniae
Authors: Seong Seung-Yong , Cho Nam-Hyuk , Kwon Ick Chan , Jeong Seo Young
Published: December 2019

Mucosal vaccination of capsular polysaccharide (PS) ofStreptococcus pneumoniaeand subsequent creation of the first line of immunological defense in mucosa were examined. Mucosal as well as systemic antibody responses to PS were evoked by peroral or intranasal immunization of BALB/c mice with PS-cholera toxin B subunit (CTB) conjugates entrapped in the alginate microspheres (AM). The bacterial colonization at the lung mucosa was most profoundly inhibited (<95%) by intranasal immunization with the naked conjugate (PS-CTB). The mice vaccinated orally with encapsulated conjugate [AM(PS-CTB)] showed significant reduction on the level of pneumococcal bacteremia (<99%). Eighty percent of the mice perorally immunized with AM (PS-CTB) were protected from lethal intranasal challenge withS. pneumoniae, whereas more than 60% of the mice in the other control groups died of infection. Our novel approach may prove to be important in the development of a mucosal vaccine that will provide protection of mucosal surfaces of host.
CD4+T Cells Which React to theLeishmania majorLACK Antigen Rapidly Secrete Interleukin-4 and Are Detrimental to the Host in Resistant B10.D2 Mice
Authors: Julia Valérie , Glaichenhaus Nicolas
Published: December 2019

Leishmania majorinduces the rapid production of interleukin-4 (IL-4) in both susceptible BALB/c and resistant B10.D2 mice. In both strains, IL-4 is produced by T cells which react to the parasite LACK (forLeishmaniahomolog of the receptor for activated C kinase) antigen. The rapid production of IL-4 in B10.D2 mice does not confer susceptibility but results in increased parasite burdens.
Antibody-Based Protection of Gnotobiotic Piglets Infected with
Escherichia coli
O157:H7 against Systemic Complications Associated with Shiga Toxin 2
Authors: Donohue-Rolfe Art , Kondova Ivanela , Mukherjee Jean , Chios Kerry , Hutto David , Tzipori Saul
Published: December 2019

Hemolytic-uremic syndrome (HUS) is a serious disease in children, attributable in the majority of cases to infection with Shiga toxin (Stx)-producing
Escherichia coli
. Using gnotobiotic piglets orally infected with
E. coli
O157:H7, which develop Stx-related cerebellar lesions and fatal neurological symptoms, we show that administration of Stx2-specific antiserum well after challenge protected, in a dose-response fashion, against these symptoms for at least 24 h after bacterial challenge. Twenty-six of 30 piglets given Stx2 antiserum survived the challenge, compared to only 4 of 16 animals given control serum or saline. Given our observations in piglets, Stx antibody of human origin may likewise prevent HUS in children.
Chlamydial Colonization of Multiple Mucosae following Infection by Any Mucosal Route
Authors: Perry Linda L. , Hughes Scott
Published: December 2019

Chlamydia trachomatisinoculated by any mucosal route colonized multiple murine mucosae and, in most cases, the spleen, liver, and kidneys. Cell-to-cell transmission, systemic dissemination, and autoinoculation of infectious fluids may have contributed to chlamydial spread. Intermucosal trafficking of protective T cells cannot be accurately evaluated by using live chlamydial challenges.
Immunostimulatory DNA as an Adjuvant in Vaccination againstLeishmania major
Authors: Stacey Katryn J. , Blackwell Jenefer M.
Published: December 2019

Oligodeoxynucleotides (ODN) which contain immunostimulatory CG motifs (CpG ODN) can promote T helper 1 (Th1) responses, an adjuvant activity that is desirable for vaccination against leishmaniasis. To test this, susceptible BALB/c mice were vaccinated with soluble leishmanial antigen (SLA) with or without CpG ODN as adjuvant and then challenged withLeishmania majormetacyclic promastigotes. CpG ODN alone gave partial protection when injected up to 5 weeks prior to infection, and longer if the ODN was bound to alum. To demonstrate an antigen-specific adjuvant effect, a minimum of 6 weeks between vaccination and infection was required. Subcutaneous administration of SLA alone, SLA plus alum, or SLA plus non-CpG ODN resulted in exacerbated disease compared to unvaccinated mice. Mice receiving SLA plus CpG ODN showed a highly significant (P< 5 × 10−5) reduction in swelling compared to SLA-vaccinated mice and enhanced survival compared to unvaccinated mice. The modulation of the response to SLA by CpG ODN was maintained even when mice were infected 6 months after vaccination. CpG ODN was not an effective adjuvant for antibody production in response to SLA unless given together with alum, when it promoted production of immunoglobulin G2a, a Th1-associated isotype. Our results suggest that with an appropriate antigen, CpG ODN would provide a stable, cost-effective adjuvant for use in vaccination against leishmaniasis.
Loss of Resistance to Ingestion and Phagocytic Killing by O−and K−Mutants of a UropathogenicEscherichia coliO75:K5 Strain
Authors: Burns Stacy M. , Hull Sheila I.
Published: December 2019

To determine the importance of the O75 O antigen and the K5 capsular antigen in resistance to phagocytosis and phagocytic killing, we used previously described O75−and K5−mutants from an O75+K5+wild-type uropathogenicEscherichia colistrain in phagocytosis assays with polymorphonuclear leukocytes (PMNs) and monocytes. At a 10-to-1 ratio of bacteria to phagocytes and in the presence of 10% serum, the parental strain GR-12 was resistant to both PMNs and monocytes over a 2-h incubation period. The O75−and K5−mutants were similar in sensitivity to killing by both PMNs and monocytes, decreasing in viability by 80% in the first hour. Yet, a significant difference in killing between the O75−and K5−mutants was observed in the first 15 min of incubation. The K5−mutant decreased in numbers by almost 60%, while the O75−mutant increased in numbers similarly to GR-12 in the first 15 min. The difference in killing was found not to be due to the rate of opsonization. To further determine the mechanism of resistance, a fluorescence assay was used to differentiate attached and internalized bacteria. The K5 capsule hindered the association of both the wild-type strain and the O75−mutant in the initial incubation time with PMNs. In conclusion, both the K5 capsule and O75 O antigen play crucial roles in resistance to phagocytosis over time.