bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and Temperature Regulation of
Legionella pneumophila
Genes Involved in Type IV Pilus Biogenesis and Type II Protein Secretion
Authors: Liles Mark R. , Viswanathan V. K. , Cianciotto Nicholas P.
Published: December 2019

Previously, we had isolated by transposon mutagenesis a
Legionella pneumophila
mutant that appeared defective for intracellular iron acquisition. While sequencing in the proximity of the mini-Tn
10
insertion, we found a locus that had a predicted protein product with strong similarity to PilB from
Pseudomonas aeruginosa
. PilB is a component of the type II secretory pathway, which is required for the assembly of type IV pili. Consequently, the locus was cloned and sequenced. Within this 4-kb region were three genes that appeared to be organized in an operon and encoded homologs of
P. aeruginosa
PilB, PilC, and PilD, proteins essential for pilus production and type II protein secretion. Northern blot analysis identified a transcript large enough to include all three genes and showed a substantial increase in expression of this operon when
L. pneumophila
was grown at 30°C as opposed to 37°C. The latter observation was then correlated with an increase in piliation when bacteria were grown at the lower temperature. Southern hybridization analysis indicated that the
pilB
locus was conserved within
L. pneumophila
serogroups and other
Legionella
species. These data represent the first isolation of type II secretory genes from an intracellular parasite and indicate that the legionellae express temperature-regulated type IV pili.
Proteasome-Independent Activation of Nuclear Factor κB in Cytoplasmic Extracts from Human Endothelial Cells by
Rickettsia rickettsii
Authors: Sahni Sanjeev K. , Van Antwerp Daniel J. , Eremeeva Marina E. , Silverman David J. , Marder Victor J. , Sporn Lee Ann
Published: December 2019

Interaction of many infectious agents with eukaryotic host cells is known to cause activation of the ubiquitous transcription factor nuclear factor κB (NF-κB) (U. Siebenlist, G. Franzoso, and K. Brown, Annu. Rev. Cell Biol. 10:405–455, 1994). Recently, we reported a biphasic pattern of NF-κB activation in cultured human umbilical vein endothelial cells consequent to infection with
Rickettsia rickettsii
, an obligate intracellular gram-negative bacterium and the etiologic agent of Rocky Mountain spotted fever (L. A. Sporn, S. K. Sahni, N. B. Lerner, V. J. Marder, D. J. Silverman, L. C. Turpin, and A. L. Schwab, Infect. Immun. 65:2786–2791, 1997). In the present study, we describe activation of NF-κB in a cell-free system, accomplished by addition of partially purified
R. rickettsii
to endothelial cell cytoplasmic extracts. This activation was rapid, reaching maximal levels at 60 min, and was dependent on the number of
R. rickettsii
organisms added. Antibody supershift assays using monospecific antisera against NF-κB subunits (p50 and p65) confirmed the authenticity of the gel-shifted complexes and identified both p50-p50 homodimers and p50-p65 heterodimers as constituents of the activated NF-κB pool. Activation occurred independently of the presence of endothelial cell membranes and was not inhibited by removal of the endothelial cell proteasome. Lack of involvement of the proteasome was further confirmed in assays using the peptide-aldehyde proteasome inhibitor MG 132. Activation was not ATP dependent since no change in activation resulted from addition of an excess of the unhydrolyzable ATP analog ATPγS, supplementation with exogenous ATP, or hydrolysis of endogenous ATP with ATPase. Furthermore, Western blot analysis before and after in vitro activation failed to demonstrate phosphorylation of serine 32 or degradation of the cytoplasmic pool of IκBα. This lack of IκBα involvement was supported by the finding that
R. rickettsii
can induce NF-κB activation in cytoplasmic extracts prepared from T24 bladder carcinoma cells and human embryo fibroblasts stably transfected with a superrepressor phosphorylation mutant of IκBα, rendering NF-κB inactivatable by many known signals. Thus, evidence is provided for a potentially novel NF-κB activation pathway wherein
R. rickettsii
may interact with and activate host cell transcriptional machinery independently of the involvement of the proteasome or known signal transduction pathways.
Evidence that Surface Proteins Sn14 and Sn16 ofSarcocystis neuronaMerozoites Are Involved in Infection and Immunity
Authors: Liang Fang Ting , Granstrom David E. , Zhao Xiao Min , Timoney John F.
Published: December 2019

Sarcocystis neuronais the etiologic agent of equine protozoal myeloencephalitis (EPM). Based on an analysis of 25,000 equine serum and cerebrospinal fluid (CSF) samples, including samples from horses with neurologic signs typical of EPM or with histologically or parasitologically confirmed EPM, four major immunoblot band patterns have been identified. Twenty-three serum and CSF samples representing each of the four immunoblot patterns were selected from 220 samples from horses with neurologic signs resembling EPM and examined for inhibitory effects on the infectivity ofS. neuronaby an in vitro neutralization assay. A high correlation between immunoblot band pattern and neutralizing activity was detected. Two proteins, Sn14 and Sn16 (14 and 16 kDa, respectively), appeared to be important for in vitro infection. A combination of the results of surface protein labeling, immunoprecipitation, Western blotting, and trypsin digestion suggests that these molecules are surface proteins and may be useful components of a vaccine againstS. neuronainfection. AlthoughS. neuronais an obligate intracellular parasite, it is potentially a target for specific antibodies which may lyse merozoites via complement or inhibit their attachment and penetration to host cells.
Identification of the Aspartate-β-Semialdehyde Dehydrogenase Gene of
Legionella pneumophila
and Characterization of a Null Mutant
Authors: Harb Omar S. , Kwaik Yousef Abu
Published: December 2019

The ability of
Legionella pneumophila
to cause Legionnaires’ disease is dependent on its capacity to survive in the intracellular environment of its host cells. Furthermore, outbreaks of this disease have been associated with contaminated water sources where
L. pneumophila
survives as a parasite of protozoa. In this study, we determined the effect of nutritional auxotrophy on the ability of
L. pneumophila
to survive in the intracellular environment of its host cells. We generated a diaminopimelic acid (DAP) auxotroph (AA400) of
L. pneumophila
by disruption of the aspartate-β-semialdehyde (
asd
) gene. The ability of AA400 to survive within macrophages and protozoa was found to be defective. This defect was due solely to the
asd
disruption since complementation of the mutant with the wild-type
asd
gene restored its capacity for intracellular survival. Furthermore, the defect was not completely complemented by DAP supplementation to the culture media. Thus, our results suggest that disruption of the
asd
gene may prove to be useful in the design of attenuated vaccines against Legionnaires’ disease.
Acquired Resistance ofEscherichia colito Complement Lysis by Binding of Glycophosphoinositol-Anchored Protectin (CD59)
Authors: Rautemaa Riina , Jarvis Gary A. , Marnila Pertti , Meri Seppo
Published: December 2019

Protectin (CD59) is a glycophosphoinsitol (GPI)-anchored defender of human cells against lysis by the membrane attack complex of complement. In this study, we examined whether protectin released from human cell membranes can incorporate into the surface of gram-negative bacteria. Analysis by using radiolabeled protectin, immunofluorescence, flow cytometry, and whole-cell enzyme-linked immunosorbent assay demonstrated that protectin bound to nonencapsulatedEscherichia coliEH237 (Re) and EH234 (Ra) in a calcium-dependent manner. The incorporation required the GPI-phospholipid moiety since no binding of a phospholipid-free soluble form of protectin was observed. Mg2+did not enhance the binding, and a polysialic acid capsule prevented it (strain IH3080 [O18:K1:H8]). Bound protectin inhibited the C5b-9 neoantigen expression on complement-treated bacteria. Protection against complement lysis was observed in both a colony counting assay and a bioluminescence assay, where viable EH234 bacteria expressing the luciferase gene emitted green light in the presence of the luciferine substrate. In general, two- to four-times-higher serum concentrations were needed to obtain 50% lysis of protectin-coated versus noncoated bacteria. The results indicate that protectin can incorporate in a functionally active form into the cell membranes of the two nonencapsulated deep roughE. colistrains studied.
Integrins αvβ3and α5β1Mediate Attachment of Lyme Disease Spirochetes to Human Cells
Authors: Coburn Jenifer , Magoun Loranne , Bodary Sarah C. , Leong John M.
Published: December 2019

Borrelia burgdorferi(sensu lato), the agent of Lyme disease, is able to cause chronic, multisystemic infections in human and animal hosts. Attachment of the spirochete to host cells is likely to be important for the colonization of diverse tissues. The platelet-specific integrin αIIbβ3was previously identified as a receptor for all three species of Lyme disease spirochetes (B. burgdorferisensu stricto,B. garinii, andB. afzelii). Here we show thatB. burgdorferialso recognizes the widely expressed integrins αvβ3and α5β1, known as the vitronectin and fibronectin receptors, respectively. Three representatives of each species of Lyme disease spirochete were tested for the ability to bind to purified αvβ3and α5β1. All of the strains tested bound to at least one integrin. Binding to one integrin was not always predictive of binding to other integrins, and several different integrin preference profiles were identified. Attachment of the infectiousB. burgdorferistrain N40 to purified αvβ3and α5β1was inhibited by RGD peptides and the appropriate receptor-specific antibodies. Binding to αvβ3was also shown by using a transfected cell line that expresses this receptor but not αIIbβ3. Attachment ofB. burgdorferiN40 to human erythroleukemia cells and to human saphenous vein endothelial cells was mediated by both α5β1and αvβ3. Our results show that multiple integrins mediate attachment of Lyme disease spirochetes to host cells.
Cell-Contact-Stimulated Formation of Filamentous Appendages by
Salmonella typhimurium
Does Not Depend on the Type III Secretion System Encoded by
Salmonella
Pathogenicity Island 1
Authors: Reed Katharine A. , Clark M. Ann , Booth Trevor A. , Hueck Christoph J. , Miller Samuel I. , Hirst Barry H. , Jepson Mark A.
Published: December 2019

The formation of filamentous appendages on
Salmonella typhimurium
has been implicated in the triggering of bacterial entry into host cells (C. C. Ginocchio, S. B. Olmsted, C. L. Wells, and J. E. Galán, Cell 76:717–724, 1994). We have examined the roles of cell contact and
Salmonella
pathogenicity island 1 (SPI1) in appendage formation by comparing the surface morphologies of a panel of
S. typhimurium
strains adherent to tissue culture inserts, to cultured epithelial cell lines, and to murine intestine. Scanning electron microscopy revealed short filamentous appendages 30 to 50 nm in diameter and up to 300 nm in length on many wild-type
S. typhimurium
bacteria adhering to both cultured epithelial cells and to murine Peyer’s patch follicle-associated epithelia. Wild-type
S. typhimurium
adhering to cell-free culture inserts lacked these filamentous appendages but sometimes exhibited very short appendages which might represent a rudimentary form of the cell contact-stimulated filamentous appendages. Invasion-deficient
S. typhimurium
strains carrying mutations in components of SPI1 (
invA
,
invG
,
sspC
, and
prgH
) exhibited filamentous appendages similar to those on wild-type
S. typhimurium
when adhering to epithelial cells, demonstrating that formation of these appendages is not itself sufficient to trigger bacterial invasion. When adhering to cell-free culture inserts, an
S. typhimurium invG
mutant differed from its parent strain in that it lacked even the shorter surface appendages, suggesting that SPI1 may be involved in appendage formation in the absence of epithelia. Our data on
S. typhimurium
strains in the presence of cells provide compelling evidence that SPI1 is not an absolute requirement for the formation of the described filamentous appendages. However, appendage formation is controlled by PhoP/PhoQ since a PhoP-constitutive mutant very rarely possessed such appendages when adhering to any of the cell types examined.
Antibodies to RecombinantClostridium difficileToxins A and B Are an Effective Treatment and Prevent Relapse ofC. difficile-Associated Disease in a Hamster Model of Infection
Authors: Kink John A. , Williams Jim A.
Published: December 2019

Clostridium difficilecauses antibiotic-associated diarrhea and colitis in humans through the actions of toxin A and toxin B on the colonic mucosa. At present, broad-spectrum antibiotic drugs are used to treat this disease, and patients suffer from high relapse rates after termination of treatment. This study examined the role of both toxins in pathogenesis and the ability of orally administered avian antibodies against recombinant epitopes of toxin A and toxin B to treatC. difficile-associated disease (CDAD). DNA fragments representing the entire gene of each toxin were cloned, expressed, and affinity purified. Hens were immunized with these purified recombinant-protein fragments of toxin A and toxin B. Toxin-neutralizing antibodies fractionated from egg yolks were evaluated by a toxin neutralization assay in Syrian hamsters. The carboxy-terminal region of each toxin was most effective in generating toxin-neutralizing antibodies. With a hamster infection model, antibodies to both toxins A and B (CDAD antitoxin) were required to prevent morbidity and mortality from infection. In contrast to vancomycin, CDAD antitoxin prevented relapse and subsequentC. difficilereinfection in the hamsters. These results indicate that CDAD antitoxin may be effective in the treatment and management of CDAD in humans.
Delivery of the p67 Sporozoite Antigen of
Theileria parva
by Using Recombinant
Salmonella dublin
: Secretion of the Product Enhances Specific Antibody Responses in Cattle
Authors: Gentschev Ivo , Glaser Ines , Goebel Werner , McKeever Declan J. , Musoke Anthony , Heussler Volker T.
Published: December 2019

The p67 sporozoite antigen of
Theileria parva
has been fused to the C-terminal secretion signal of
Escherichia coli
hemolysin and expressed in secreted form by attenuated
Salmonella dublin aroA
strain SL5631. The recombinant p67 antigen was detected in the supernatant of transformed bacterial cultures. Immunization trials in cattle revealed that SL5631 secreting the antigen provoked a 10-fold-higher antibody response to p67 than recombinant SL5631 expressing but not secreting p67. Immunized calves were challenged with a 80% lethal dose of
T. parva
sporozoites and monitored for the development of infection. Two of three calves immunized intramuscularly with the p67-secreting SL5631 strain were found to be protected, whereas only one of three animals immunized with the nonsecreting p67-expressing SL5631 strain was protected. This is the first demonstration that complete eukaryotic antigens fused to the C-terminal portion of
E. coli
hemolysin can be exported from attenuated
Salmonella
strains and that such exported antigens can protect cattle against subsequent parasite challenge.
Clearance ofBorrelia burgdorferiMay Not Be Required for Resistance to Experimental Lyme Arthritis
Authors: Brown Charles R. , Reiner Steven L.
Published: December 2019

Infection of inbred mouse strains withBorrelia burgdorferiresults in the development of experimental Lyme arthritis. The degree of arthritic pathology has been suggested to correlate with the level of spirochete burden within tissues. To investigate this further, we infected resistant DBA/2 (DBA) and susceptible C3H/HeJ (C3H) mice in the hind footpads and monitored arthritis development for 21 days. To quantitate levels of spirochetes within tissues, we created a competitive PCR molecule containing modifiedospAandflagene segments. C3H mice developed severe arthritis of the tibiotarsal joints, while DBA mice developed only mild inflammation throughout the experimental period. At day 21, when the gross size and histologic composition of ankles revealed significant differences in arthritis between the strains, there was little difference in levels of spirochete DNA as determined by competitive PCR. Cultures of ankle tissue at day 21 were also uniformly positive in both C3H and DBA animals and contained relatively similar levels of spirochetes. These results indicate that the presence of spirochetes in the ankles of experimental animals is not sufficient for arthritis development. Since arthritic and nonarthritic animals can harbor relatively equal spirochete burdens yet retain their distinct phenotypic outcomes, an aberrant or overly exuberant immune response may be an additional requirement for pathology in arthritis-prone mice.
A Tumor Necrosis Factor Mimetic Peptide Activates a Murine Macrophage Cell Line To Inhibit Mycobacterial Growth in a Nitric Oxide-Dependent Fashion
Authors: Britton W. J. , Meadows N. , Rathjen D. A. , Roach D. R. , Briscoe H.
Published: December 2019

The control of mycobacterial infections depends on the cytokine-mediated activation of mononuclear phagocytes to inhibit the growth of intracellular mycobacteria. Optimal activation requires the presence of T-cell-derived gamma interferon (IFN-γ) and other signals, including tumor necrosis factor (TNF). Recently, an 11-mer peptide based on amino acids 70 to 80 of the human TNF sequence, TNF(70-80), was found to have TNF mimetic properties, which include the activation of human and mouse neutrophils to killPlasmodiaspp. Therefore, we investigated the capacity of TNF(70-80)to activate the murine macrophage cell line RAW264.7 infected with the vaccine strainMycobacterium bovisbacillus Calmette-Guérin (BCG). When RAW264.7 cells were pretreated with human TNF or TNF(70-80)in the presence of IFN-γ, there was a dose-dependent reduction in the replication of BCG as measured by the uptake of3H-labeled uracil and a concomitant release of nitric oxide as measured by the nitrite in the culture supernatants. TNF- or TNF(70-80)-induced macrophage activation was dependent on IFN-γ and was inhibited by neutralizing monoclonal antibody to human TNF and by anti-IFN-γ antisera. Both nitrite release and BCG growth inhibition were abrogated by competitive inhibitors ofl-arginine, which blocked the activation of inducible nitric oxide synthase. A soluble form of the Type 1 TNF receptor blocked the activation of BCG-infected macrophages by human TNF and TNF(70-80), demonstrating that the effect of TNF(70-80)is dependent on signaling through TNF receptor I. The mimetic effects of TNF(70-80)on macrophage activation in vitro suggest that treatment with TNF(70-80)may modulate mycobacterial infections in vivo.
Kinetics of Infection and Effects on Placental Cell Populations in a Murine Model ofChlamydia psittaci-Induced Abortion
Authors: Buendía Antonio J. , Sánchez Joaquín , Martínez María C. , Cámara Paulina , Navarro Jose A. , Rodolakis Annie , Salinas Jesus
Published: December 2019

The anatomical progression of chlamydial infection was studied in different areas of the placenta, using a mouse model and two inoculation times: early pregnancy (day 7, group A) and midpregnancy (day 11, group B). The first population cells affected were decidual cells and neutrophils located just at the limits of the maternal and fetal placenta. The following invaded area was the layer of giant cells. Complete colonization of the maternal placenta occurred after day 15 of pregnancy independently of the inoculation time, the metrial gland being the last area to be invaded; numerous granulated metrial gland (GMG) cells were infected. Finally, chlamydial inclusions were observed in labyrinthine trophoblastic cells from day 18 of pregnancy onward. Since no fetal damage was observed, it seems that an indirect mechanism involving the lysis of GMG cells and neutrophil infiltration of the decidua and metrial gland may be the pathogenic mechanism that leads to abortion.
Proteins Encoded by thecagPathogenicity Island ofHelicobacter pyloriAre Required for NF-κB Activation
Authors: Glocker Erik , Lange Christina , Covacci Antonello , Bereswill Stefan , Kist Manfred , Pahl Heike L.
Published: December 2019

Helicobacter pyloriis the etiological agent in the development of chronic gastritis, duodenal ulceration, and gastric adenocarcinoma. The difference in virulence between individual strains is reflected in their ability to induce interleukin-8 (IL-8) secretion from gastric epithelial cells. It has been shown that virulence is associated with the presence of a bacterial gene cluster (a pathogenicity island). We have recently demonstrated thatH. pylori-mediated IL-8 secretion requires activation of the transcription factor NF-κB. Here, we show that NF-κB induction requires six membrane proteins encoded within the pathogenicity island.
Lethal Factor Active-Site Mutations Affect Catalytic Activity In Vitro
Authors: Hammond S. E. , Hanna P. C.
Published: December 2019

The lethal factor (LF) protein ofBacillus anthracislethal toxin contains the thermolysin-like active-site and zinc-binding consensus motif HEXXH (K. R. Klimpel, N. Arora, and S. H. Leppla, Mol. Microbiol. 13:1093–1100, 1994). LF is hypothesized to act as a Zn2+metalloprotease in the cytoplasm of macrophages, but no proteolytic activities have been previously shown on any target substrate. Here, synthetic peptides are hydrolyzed by LF in vitro. Mass spectroscopy and peptide sequencing of isolated cleavage products separated by reverse-phase high-pressure liquid chromatography indicate that LF seems to prefer proline-containing substrates. Substitution mutations within the consensus active-site residues completely abolish all in vitro catalytic functions, as does addition of 1,10-phenanthroline, EDTA, and certain amino acid hydroxamates, including the novel zinc metalloprotease inhibitor ZINCOV. In contrast, the protease inhibitors bestatin and lysine CMK, previously shown to block LF activity on macrophages, did not block LF activity in vitro. These data provide the first direct evidence that LF may act as an endopeptidase.
Cryptosporidium parvum
Infection of Human Intestinal Xenografts in SCID Mice Induces Production of Human Tumor Necrosis Factor Alpha and Interleukin-8
Authors: Seydel Karl B. , Zhang Tonghai , Champion Gretchen A. , Fichtenbaum Carl , Swanson Paul E. , Tzipori Saul , Griffiths Jeffrey K. , Stanley Samuel L.
Published: December 2019

The protozoan parasite
Cryptosporidium parvum
invades intestinal epithelial cells and can cause life-threatening diarrhea in immunocompromised individuals. Despite the clinical importance of this organism, much remains to be learned about the pathogenesis of
C. parvum
-induced diarrhea. To explore the role of the intestinal inflammatory response in
C. parvum
disease, using
C. parvum
oocysts we infected human intestinal xenografts in severe combined immunodeficient (SCID) mice. Seven days after infection, we found levels of human tumor necrosis factor alpha and interleukin-8 in
C. parvum
-infected human intestinal xenografts that were significantly higher than those seen in uninfected control xenografts. These results demonstrate that human intestinal cells produce proinflammatory cytokines in response to
C. parvum
infection and establish SCID-HU-INT mice as a model system to study the interactions of
C. parvum
with the human intestine.
Cytochalasin-Induced Actin Disruption of Polarized Enterocytes Can Augment Internalization of Bacteria
Authors: Wells Carol L. , van de Westerlo Elisabeth M. A. , Jechorek Robert P. , Haines Holly M. , Erlandsen Stanley L.
Published: December 2019

Cytochalasin-induced actin disruption has often been associated with decreased bacterial internalization by cultured epithelial cells, although polarized enterocytes have not been systematically studied. In assays using confluent polarized HT-29 enterocytes, cytochalasin D appeared to increase internalization of wild-type
Salmonella typhimurium
,
Proteus mirabilis
, and
Escherichia coli
. HeLa and HEp-2 epithelial cells, as well as HT-29 and Caco-2 enterocytes, were used to clarify this unexpected observation. Resulting data showed that cytochalasin D was associated with increased internalization of
S. typhimurium
and
P. mirabilis
by both HT-29 and Caco-2 enterocytes and with increased internalization of
E. coli
by HT-29 enterocytes; with either HeLa or HEp-2 cells, cytochalasin was associated with no change or a decrease in internalization of these same bacterial strains. Cytochalasin caused decreased internalization of
Listeria monocytogenes
by HT-29, Caco-2, HeLa, and HEp-2 cells, indicating that cytochalasin did not consistently augment bacterial internalization by polarized enterocytes. Fluorescein-labeled phalloidin confirmed marked disruption of filamentous actin in cytochalasin-treated HT-29, Caco-2, HeLa, and HEp-2 cells. Cytochalasin had no noticeable effect on epithelial viability but caused distorted apical microvilli, cell rounding, and separation of adjacent enterocytes in confluent cultures (with a corresponding decrease in transepithelial electrical resistance). Scanning electron microscopy showed that cytochalasin-induced enhanced bacterial internalization was associated with preferential bacterial adherence on the exposed enterocyte lateral surface. Colchicine, used to disrupt microtubules, had no noticeable effect on bacterial internalization by HT-29 or Caco-2 enterocytes. These data indicated that for HT-29 and Caco-2 enterocytes, cytochalasin-induced disruption of filamentous actin might augment internalization of some bacterial species by a mechanism that appeared to involve exposure of the enterocyte lateral surface.
Differential Gene Expression in Response to Adjunctive Recombinant Human Interleukin-2 Immunotherapy in Multidrug-Resistant Tuberculosis Patients
Authors: Johnson Barbara J. , Estrada Iris , Shen Zhu , Ress Stan , Willcox Paul , Colston M. Joseph , Kaplan Gilla
Published: December 2019

Administration of low-dose recombinant human interleukin 2 (rhuIL-2) in combination with multidrug chemotherapy to patients with multidrug-resistant tuberculosis (MDR TB) induces measurable changes in in vitro immune response parameters which are associated with changes in the clinical and bacteriologic status of the patients. To determine the molecular basis of these changes, we have used semiquantitative reverse transcriptase-initiated PCR (RT-PCR) and differential display technology. During rhuIL-2 treatment of MDR TB patients, decreased levels of gamma interferon (IFN-γ) mRNA in peripheral blood mononuclear cells (PBMC) relative to baseline levels were observed. However, at the site of a delayed-type hypersensitivity (DTH) response to purified protein derivative of tuberculin (PPD), the expression of cellular IFN-γ and IL-2 mRNAs was increased during rhuIL-2 therapy. Levels of other cytokine mRNAs were not significantly affected by rhuIL-2 administration. Using differential-display RT-PCR, we identified several genes expressed at the DTH skin test site which were up- or down-regulated during rhuIL-2 treatment. Cytochrome oxidase type I mRNA was increased in response to rhuIL-2 therapy relative to baseline levels, as was heterogeneous nuclear ribonuclear protein G mRNA. CD63, clathrin heavy chain, and β-adaptin mRNAs, all of which encode proteins associated with the endocytic vacuolar pathway of cells, were also differentially regulated by rhuIL-2 administration. The differential effects of IL-2 were confirmed in vitro by using PBMC obtained from PPD-positive individuals stimulated withMycobacterium tuberculosisand IL-2. The differential expression of genes may provide a surrogate marker for leukocyte activation at a mycobacterial antigen-specific response site and for the development of an enhanced antimicrobial response which may result in improved outcomes in MDR TB patients.
Streptococcus pneumoniaeType 14 Polysaccharide-Conjugate Vaccines: Length Stabilization of Opsonophagocytic Conformational Polysaccharide Epitopes
Authors: Laferriere Craig A. , Sood Ramesh K. , de Muys Jean-Marc , Michon Francis , Jennings Harold J.
Published: December 2019

A simple and convenient method was developed for the preparation ofStreptococcus pneumoniaetype 14 polysaccharide (Pn14PS)-tetanus toxoid (TT) conjugate vaccines, using terminally linked Pn14PS fragments of different lengths. Native Pn14PS was simultaneously depolymerized and activated for conjugation by partial N-deacetylation followed by nitrous acid deamination which yielded fragments (1.4 to 150.0 kDa) having a free aldehyde at the reducing end. These were then conjugated to TT through their terminal aldehydic groups, using the reductive amination procedure. All of the above conjugates, when injected in rabbits, induced anti-Pn14PS antibodies, whereas the native Pn14PS did not. The amounts of anti-Pn14PS antibodies elicited by these conjugates, as determined by enzyme-linked immunosorbent assay, followed a trend with conjugates containing the highest-molecular-weight Pn14PS eliciting the highest titers. The same trend was also observed in the ability of the antibodies to opsonize and kill live type 14 pneumococci, although the increase in opsonophagocytic activity was more pronounced and did not correlate linearly with increases in antibody titer. Competitive inhibition of the binding of different conjugate antisera to the native Pn14PS, using Pn14PS fragments as inhibitors, established that the conjugates induced antibodies with specificities for different lengths of Pn14PS beginning at 2 repeating units (RU). It was also established, both immunologically and antigenically, that at least 4 RU of Pn14PS were required to form an extended conformational epitope and that approximately 22 RU of Pn14PS were required to duplicate the same epitope on the same saccharide chain. The conformational epitope was found to be essential for the induction of antibodies with high opsonophagocytic activity and that augmentation of opsonophagocytic activity was also dependent on further chain extension.
Age-DependentNeisseria meningitidisSerogroup C Class-Specific Antibody Concentrations and Bactericidal Titers in Sera from Young Children from Montana Immunized with a Licensed Polysaccharide Vaccine
Authors: Maslanka Susan E. , Tappero Jordan W. , Plikaytis Brian D. , Brumberg Robert S. , Dykes Janet K. , Gheesling Linda L. , Donaldson Kimberley B. J. , Schuchat Anne , Pullman John , Jones MaryAnn , Bushmaker Julie , Carlone George M.
Published: December 2019

Neisseria meningitidisserogroup C bactericidal titers and class-specific enzyme-linked immunosorbent assay (ELISA) antibody concentrations were measured in sera from 173 children (1 to 5 years old) before and 6 weeks and 7 months following vaccination with a quadrivalent (A/C/Y/W-135) polysaccharide vaccine. The immune responses of the children were compared with those of 40 adults 6 weeks postvaccination. Both bactericidal titers and ELISA antibody concentrations were significantly higher in the adults than in the children (P< 0.05). In addition, the ratio of immunoglobulin G (IgG) to IgM was higher in the children than in the adults. With an ELISA total antibody concentration of ≥2 μg/ml used as a measure of seroconversion, ≥84% of the individuals from each age group responded to the serogroup C polysaccharide. However, with a ≥4-fold-increase in bactericidal titer used, only 18% of 1-year-olds, 32% of 2-year-olds, and 50 to 60% of 3-, 4-, and 5-year-olds seroconverted. The ELISA results suggest that >50% of all children retained ≥2 μg of total antibody per ml at 7 months postimmunization. However, the bactericidal titers suggest that <10% of children <4 years old retained a ≥4-fold increase at 7 months following vaccination. Of particular note, 59 of 79 sera (75%) from the 1- and 2-year-olds had high ELISA antibody concentrations (2 to 20 μg/ml) with no associated bactericidal titer (<1:8). Discordant results between bactericidal titers and ELISA antibody concentrations were not explained by the presence of IgA blocking antibody or relative levels of IgG and IgM. The bactericidal results show age-dependent differences in the production and retention of antibody in young children immunized with serogroup C polysaccharide; these differences are not evident with the ELISA data.
Analysis of Host Cells Associated with the Spv-Mediated Increased Intracellular Growth Rate ofSalmonella typhimuriumin Mice
Authors: Gulig Paul A. , Doyle Thomas J. , Hughes Jeffrey A. , Matsui Hidenori
Published: December 2019

The 90-kb virulence plasmid ofSalmonella typhimuriumencodes fivespvgenes which increase the growth rate of the bacteria within host cells within the first week of systemic infection of mice (P. A. Gulig and T. J. Doyle, Infect. Immun. 61:504–511, 1993). The presently described study was aimed at identifying the host cells associated with Spv-mediated virulence by manipulating the mouse host and the salmonellae. To test the effects of T cells and B cells on the Spv phenotype, salmonellae were orally inoculated into nude and SCID BALB/c mice. Relative to normal BALB/c mice, nude and SCID BALB/c mice were unaffected for splenic infection with either the Spv+or Spv−S. typhimuriumstrains at 5 days postinoculation. When mice were pretreated with cyclophosphamide to induce granulocytopenia, there was a variable increase in total salmonella infection, but the relative splenic CFU of Spv+versus Spv−S. typhimuriumwas not changed after oral inoculation. In contrast, depletion of macrophages from mice by treatment with cyclophosphamide plus liposomes containing dichloromethylene diphosphate resulted in equivalent virulence of Spv+and Spv−salmonellae. To examine if thespvgenes affected the growth of salmonellae in nonphagocytic cells, aninvA::aphTmutation was transduced into Spv+and Spv−S. typhimuriumstrains. InvA−Spv+salmonellae were not significantly affected for splenic infection after subcutaneous inoculation compared with the wild-type strain, and InvA−Spv−salmonellae were only slightly attenuated relative to InvA+Spv−salmonellae. Invasion-defective salmonellae still exhibited the Spv phenotype. Therefore, infection of nonphagocytes is not involved with the Spv virulence function. Taken together, these data demonstrate that macrophages are essential for suppressing the infection by Spv−S. typhimurium, by serving as the primary host cell for Spv-mediated intracellular replication and possibly by inhibiting the replication of salmonellae within other macrophages.
Identification of Persistent Infection in Experimental Syphilis by PCR
Authors: Wicher Konrad , Abbruscato Frank , Wicher Victoria , Collins Doris N. , Auger Ivan , Horowitz Harold W.
Published: December 2019

The studies described herein were designed to evaluate the usefulness of the PCR in detecting persistent syphilitic infection. Three groups of animals were used: a nonimmune group infected withTreponema pallidum(NI/TP), a nonimmune group injected with heat-killed treponemes (NI/HKTP), and an immune and reinfected group (I/TP). All animals were inoculated with similar numbers of organisms distributed at 10 sites on the clipped back and in both testes. The persistence of the treponemes was examined by PCR and the rabbit infectivity test (RIT). The kinetic studies and statistical analysis of their results demonstrated that the rate of bacterial clearance from the NI/TP group was very low and incomplete at 4 months after infection. It was significantly different from those of both the NI/HKTP (P< 0.001) and I/TP (P< 0.05) groups. No statistically significant differences in treponemal elimination were found between the NI/HKTP and I/TP groups. PCR can detect the DNA of dead organisms, but the latter are eliminated by the host relatively quickly (15 to 30 days) as compared to elimination of live treponemes (>120 days). PCR results correlated well with RIT results. These data suggest that PCR-positive specimens obtained from an untreated patient(s) or collected weeks after treatment indicate persistent infection. They also show that the process of elimination ofT. pallidumfrom primary sites of infection is prolonged and incomplete.
In Vitro Expression of Adhesion Receptors and Diapedesis by Polymorphonuclear Neutrophils during Experimentally InducedStreptococcus uberisMastitis
Authors: Smits Elke , Burvenich Christian , Guidry Albert J. , Roets Eddy
Published: December 2019

The expression of adhesion receptors and diapedesis by polymorphonuclear neutrophils (PMN) were studied before and during experimentally inducedStreptococcus uberismastitis. Both quarters of the left half of the udders of five midlactation cows were inoculated with a suspension containing approximately 500 CFU ofS. uberis0140J. Clinical signs of an inflammatory reaction and leukocyte influx were observed 24 h after challenge. The expression of CD11b/CD18 adhesion receptors, determined by flow cytometry, was upregulated 24 h after challenge. A confluent monolayer of bovine secretory mammary epithelial cells on collagen-coated inserts was used to study PMN diapedesis. Bovine C5a was used as the chemoattractant. An 80% decrease in PMN diapedesis was observed 24 h after challenge. The decrease in diapedesis continued for 3 weeks after challenge.
Disparate Findings on the Role of Virulence Factors of
Enterococcus faecalis
in Mouse and Rat Models of Peritonitis
Authors: Dupont Herve , Montravers Philippe , Mohler Jacqueline , Carbon Claude
Published: December 2019

The role of
Enterococcus faecalis
in polymicrobial peritonitis is still debated. Virulence factors expressed in some enterococcal strains might be involved in the pathogenicity of these organisms. To clarify their role, three of these virulence factors (cytolysin, gelatinase, and aggregation substance) were studied in six isogenic strains of
E. faecalis
expressing various combinations of these factors. Since the pathogenic effects of enterococci are only moderate, the expression of their virulence might vary from one animal species to another and from one type of infection to another. Therefore, we evaluated these effects in two animal models, i.e., a systemic infection in mice in which we assessed the virulence of the strains in 50% lethal dose studies and a model of compartmentalized infection in rats in which the microbiologic and inflammatory effects of the strains were evaluated in monomicrobial or polymicrobial infection. In mice, significant differences were observed in the cumulative survival curves depending on the virulence factors (
P
< 0.0001 [log rank test]). In rats, monomicrobial infection induced only mild changes. In polymicrobial peritonitis, the virulence factors mainly increased the inflammatory response while the changes observed in the microbiologic response were minimal. The combination of two virulence factors did not significantly increase the severity of infection either in the mice model or the polymicrobial rat model. These data argue for species and model dependence of the role of the virulence factors studied here and suggest that other important factors may be involved in the pathogenicity of enterococci.
Volatile Fatty Acid, Metabolic By-Product of Periodontopathic Bacteria, Induces Apoptosis in WEHI 231 and RAJI B Lymphoma Cells and Splenic B Cells
Authors: Kurita-Ochiai Tomoko , Ochiai Kuniyasu , Fukushima Kazuo
Published: December 2019

The ability of butyric acid, an extracellular metabolite from periodontopathic bacteria, to induce apoptosis in murine WEHI 231 cells, splenic B cells, and human RAJI cells was examined. The culture filtrate ofPorphyromonas gingivalis,Prevotella loescheii, andFusobacterium nucleatum, which contains high a percentage of butyric acid, induced DNA fragmentation in WEHI 231 cells. Volatile fatty acid, especially butyric acid, significantly suppressed B-cell viability in a concentration-dependent fashion. The DNA fragmentation assay indicated that butyric acid rapidly induced apoptosis in WEHI 231 cells (with 1.25 mM butyric acid and 6 h after treatment), splenic B cells (with 1.25 mM butyric acid), and RAJI cells (with 2.5 mM butyric acid). Incubation of WEHI 231 cells with butyric acid for 16 h resulted in the typical ladder pattern of DNA fragmentation and the apoptoic change such as chromatin condensation and hypodiploid nuclei. Cell cycle analysis implied that butyric acid arrested the cells at the G1phase. The inhibitory assay suggested that butyric acid-induced apoptosis of WEHI 231 and splenic B cells was inhibited by W-7, a calmodulin inhibitor. These results suggest that calmodulin-dependent regulation is involved in the signal transduction pathway of butyric acid.
Mutacin Production by
Streptococcus mutans
May Promote Transmission of Bacteria from Mother to Child
Authors: Grönroos L. , Saarela M. , Mättö J. , Tanner-Salo U. , Vuorela A. , Alaluusua S.
Published: December 2019

The production of bacteriocin-like inhibitory substances, mutacins, by mutans streptococci varies among isolates. To find if the degree of mutacin activity of an isolate was related to its transmission between mother and her child, 19 mothers and their 18-month- to 3-year-old children were sampled for their oral mutans streptococci. In addition, the stability of mutacin activity was studied with isolates from the mothers and with isolates from five unrelated 5-year-old children in 5- to 7-year follow-up studies. A total of 145 oral mutans streptococcal isolates were serotyped by immunodiffusion, ribotyped, and mutacin typed by the stab culture technique. Mutacin was produced by 88% of the strains against more than 1 of the 14 indicator strains, representing mutans streptococci,
Streptococcus sanguis
,
Streptococcus salivarius
,
Streptococcus oralis
,
Streptococcus gordonii
, and
Streptococcus pyogenes. Streptococcus mutans
isolates showed more inhibitory activity than did
Streptococcus sobrinus
isolates. Identical ribotypes had similar mutacin activity profiles within a subject, initially and in the follow-up studies, in all but two cases. The mothers harbored a total of 37 different mutans streptococcal ribotypes. Six children were negative for mutans streptococci. Transmission was probable in 9 of 20 mother-child pairs on the basis of the presence of identical strains, as determined by ribotyping and bacteriocin (mutacin) typing.
S. mutans
strains shared between a mother and her child showed a broader spectrum of inhibitory activity than did nontransmitted strains. In conclusion, the mutacin activity of clinical isolates is reasonably stable, and this virulence factor seems to be of clinical importance in early colonization by
S. mutans.
Experimental
Helicobacter pylori
Infection Induces Antral Gastritis and Gastric Mucosa-Associated Lymphoid Tissue in Guinea Pigs
Authors: Shomer Nirah H. , Dangler Charles A. , Whary Mark T. , Fox James G.
Published: December 2019

Humans infected with
Helicobacter pylori
have abnormally low levels of the antioxidant vitamin C, which protects against the formation of carcinogenic nitrosamines, in gastric juice. Guinea pigs, like humans and nonhuman primates, have a dietary requirement for vitamin C. As such, these species have gastrointestinal vitamin C transport systems not found in other animals. We have developed and characterized a guinea pig model of chronic gastric
H. pylori
infection with the rodent-adapted Sydney strain of
H. pylori
. At 4 weeks postinfection, five of six animals of the infected group and zero of two animals of the control group were positive for
H. pylori
as determined by culture or PCR. At 15 weeks, six of six animals of the infected group and zero of two animals of the control group were positive.
H. pylori
-specific seroconversion was observed among infected animals. There were no histologic abnormalities in the gastric antra or fundi of control guinea pigs. In contrast, there was multifocal, mild to moderate lymphohistiocytic antral gastritis and formation of antral lymphoid follicles in
H. pylori
-infected animals. The lesion distribution in the gastric antra paralleled that observed in
H. pylori
-infected humans. The
H. pylori
-infected guinea pig should prove useful in modeling the interaction of helicobacter and vitamin C in gastric carcinogenesis.
Decorin-Binding Protein ofBorrelia burgdorferiIs Encoded within a Two-Gene Operon and Is Protective in the Murine Model of Lyme Borreliosis
Authors: Hagman Kayla E. , Lahdenne Pekka , Popova Taissia G. , Porcella Stephen F. , Akins Darrin R. , Radolf Justin D. , Norgard Michael V.
Published: December 2019

Isolated outer membranes ofBorrelia burgdorferiwere used in immunoblotting experiments with sera from immune mice to identify new putative Lyme disease vaccine candidates. One immunoreactive polypeptide migrated on polyacrylamide gels just proximal to outer surface protein C and comigrated with [3H]palmitate-labeled polypeptides. A degenerate oligonucleotide primer based upon internal amino acid sequence information was used to detect the corresponding gene within aB. burgdorferitotal genomic library. The relevant open reading frame (ORF) encoded a polypeptide comprised of a 24-amino-acid putative signal peptide terminated by LLISC, a probable consensus sequence for lipoprotein modification, and a mature protein of 163 amino acids. Immunoblots of a recombinant fusion protein corresponding to this ORF supported the idea that the encoded protein was a previously reported decorin-binding protein (DBP) ofB. burgdorferiN40 (B. P. Guo, S. J. Norris, L. C. Rosenberg, and M. Höök, Infect. Immun. 63:3467–3472, 1995). However, further DNA sequencing revealed the presence of a second ORF, designated ORF-1, whose termination codon was 119 bp upstream of thedbpgene. ORF-1 also encoded a putative lipoprotein with a mature length of 167 amino acids. Northern blots, Southern blots, and primer extension analyses indicated that ORF-1 anddbpcomprised a two-gene operon located on the 49-kb linear plasmid. Both proteins, which were 40% identical and 56% similar, partitioned into Triton X-114 detergent extracts ofB. burgdorferiisolated outer membranes. Mice infected withB. burgdorferiproduced high titers of antibodies against the ORF-1-encoded protein and DBP during both early and later stages of chronic infection. Both DBP and the ORF-1-encoded protein were sensitive to proteinase K treatment of intact borreliae, suggesting that they were surface exposed. In active immunization experiments, 78% of mice immunized with recombinant DBP were immune to challenge. While it is not clear whether the two lipoproteins encoded by the ORF-1-dbpoperon have analogous decorin-binding functions in vivo, the combined studies implicate DBP as a new candidate for a human Lyme disease vaccine.