bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic and Physiologic Characterization of Urease of
Actinomyces naeslundii
Authors: Morou-Bermudez Evangelia , Burne Robert A.
Published: December 2019

Ammonia production from urea by ureolytic oral bacteria is believed to have a significant impact on oral health and the ecological balance of oral microbial populations. In this study we cloned and characterized the urease gene cluster of
Actinomyces naeslundii
, which is one of the pioneer organisms in the oral cavity and a significant constituent of supragingival and subgingival dental plaque in children and adults. An internal fragment of the
ureC
gene of
A. naeslundii
WVU45 was initially amplified by PCR with degenerate primers derived from conserved amino acid sequences of the large catalytic subunit of urease in bacteria and plants. The PCR product was then used as a probe to identify recombinant bacteriophages carrying the
A. naeslundii
urease gene cluster and roughly 30 kbp of flanking DNA. Nucleotide sequence analysis demonstrated that the gene cluster was comprised of seven contiguously arranged open reading frames with significant homologies at the protein and nucleotide sequence levels to the
ureABCEFGD
genes from other organisms. By using primer extension, a putative transcription initiation site was mapped at 66 bases 5′ to the start codon of
ureA
. A urease-deficient strain was constructed by insertion of a kanamycin resistance determinant within the
ureC
gene via allelic replacement. In contrast to the wild-type organism, the isogenic mutant was unable to grow in a semidefined medium supplemented with urea as the nitrogen source and was not protected by the addition of urea against killing in moderately acidic environments. These data indicated that urea can be effectively utilized as a nitrogen source by
A. naeslundii
via a urease-dependent pathway and that ureolysis can protect
A. naeslundii
against environmental acidification at physiologically relevant pH values. Therefore, urease could confer to
A. naeslundii
critical selective advantages over nonureolytic organisms in dental plaque, constituting an important determinant of plaque ecology.
Consequences of Reduction of
Klebsiella pneumoniae
Capsule Expression on Interactions of This Bacterium with Epithelial Cells
Authors: Favre-Bonte Sabine , Joly Bernard , Forestier Christiane
Published: December 2019

Most
Klebsiella pneumoniae
clinical isolates are fully encapsulated and adhere in vitro to intestinal cell lines with an aggregative pattern. In this study, the influence of the capsule on interactions with epithelial cells was investigated by creating an isogenic mutant defective in the synthesis of the capsule. Determination of the uronic acid content of bacterial extracts confirmed that the mutant did not produce capsular polysaccharides whereas, with the wild-type strain, the level of encapsulation was growth phase dependent and reached a maximum during the lag and early log phases. Assays performed with different epithelial cell lines, Int-407, A-549, and HEp-2, showed that the capsule-defective mutant demonstrated greater adhesion than did the wild-type strain and that the aggregative pattern was maintained, indicating that the capsule was not related to the adhesion phenotype. In contrast, when the mucus-producing HT-29-MTX cells were used, the encapsulated wild-type strain adhered more strongly than did the capsule-defective mutant. No invasion properties were observed with any of the capsular phenotypes or cell lines used. The
K. pneumoniae
adhesin CF29K was detected by Western blot analysis and enzyme-linked immunosorbent assay on the surface of transconjugants obtained after transfer of a conjugative plasmid harboring the CF29K-encoding genes into both the wild-type and the capsule-defective strains. The amounts of adhesin detected were greater in the capsule-defective background strain than in the wild-type encapsulated strain and were associated with an increase in the level of adhesion to Caco-2 cells. Moreover, RNA slot blot experiments showed that transcription of the adhesin-encoding gene was markedly increased in the capsule-defective mutant compared to the wild-type encapsulated background. These results suggest (i) that the capsule plays an active role during the initial steps of the pathogenesis by interacting with mucus-producing cells but is subsequently not required for the adhesin-related interaction with the epithelial cell surface and (ii) that the expression of the adhesin is modulated by the presence of a capsule at a transcriptional level.
Binding and Utilization of Human Transferrin by
Prevotella nigrescens
Authors: Duchesne Pascale , Grenier Daniel , Mayrand Denis
Published: December 2019

To survive and multiply within their hosts, pathogens must possess efficient iron-scavenging mechanisms. In the present study, we investigate the capacity of
Prevotella nigrescens
and
Prevotella intermedia
to use various sources of iron for growth and characterize the transferrin-binding activity of
P. nigrescens
. Iron-saturated human transferrin and lactoferrin, but not ferric chloride and the iron-free form of transferrin, could be used as sources of iron by
P. nigrescens
and
P. intermedia
. Neither siderophore activity nor ferric reductase activity could be detected in
P. nigrescens
and
P. intermedia
. However, both species showed transferrin-binding activity as well as the capacity to proteolytically cleave transferrin. To various extents, all strains of
P. nigrescens
and
P. intermedia
tested demonstrated transferrin-binding activity. The activity was heat and protease sensitive. The capacity of
P. nigrescens
to bind transferrin was decreased when cells were grown in the presence of hemin. Preincubation of bacterial cells with hemin, hemoglobin, lactoferrin, fibrinogen, immunoglobulin G, or laminin did not affect transferrin-binding activity. The transferrin-binding protein could be extracted from the cell surface of
P. nigrescens
by treatment with a zwitterionic detergent. Subjecting the cell surface extract to affinity chromatography on an agarose-transferrin column revealed that it contained a protein having an estimated molecular mass of 37 kDa and possessing transferrin-binding activity. The transferrin-binding activity of
P. nigrescens
and
P. intermedia
may permit the bacteria to obtain iron for survival and growth in periodontal pockets.
Pathophysiology of Antigen 85 in Patients with Active Tuberculosis: Antigen 85 Circulates as Complexes with Fibronectin and Immunoglobulin G
Authors: Bentley-Hibbert Stuart I. , Quan Xin , Newman Thomas , Huygen Kris , Godfrey Henry P.
Published: December 2019

Antigen 85 (Ag85) complex proteins are major secretory products ofMycobacterium tuberculosisand induce strong cellular and humoral immune responses in infected experimental animals and human beings. We have previously shown that nanogram doses of these 30- to 32-kDa fibronectin-binding proteins inhibit local expression of delayed hypersensitivity by a T-cell fibronectin-dependent mechanism. Circulating levels of Ag85 might be expected to be elevated in patients with active tuberculosis and possibly to play a role in systemic anergy in these patients. To test this hypothesis, Ag85 was measured in serum and urine by a monoclonal antibody-based dot immunobinding assay in 56 patients and controls with known skin test reactivity. Median serum Ag85 levels were 50- to 150-fold higher in patients with active tuberculosis than in patients with activeM. avium-intracellularedisease or other nontuberculous pulmonary disease or in healthy controls (P< 0.001). The median and range of serum Ag85 in patients with active tuberculosis was not significantly different between skin test-positive and -negative subjects. Patients with activeM. aviumdisease could be distinguished from those with disease due toM. tuberculosisby monoclonal anti-Ag85 antibodies of appropriate specificities. No increases in urinary Ag85 were detected in any patient, regardless of the Ag85 level in serum. Chromatographic analysis and immunoprecipitation studies of serum revealed that Ag85 existed in the serum of these patients complexed to either fibronectin or immunoglobulin G (IgG). Uncomplexed circulating Ag85 was demonstrable in serum from fewer than 20% of patients with active tuberculosis. In patients with active tuberculosis, Ag85 is therefore likely to circulate primarily as complexes with plasma fibronectin and IgG rather than in unbound form. The existence of Ag85 complexes with plasma proteins would account for its lack of urinary clearance.
Tumor Necrosis Factor Alpha Receptor I Is Important for Survival fromStreptococcus pneumoniaeInfections
Authors: O’Brien David P. , Briles David E. , Szalai Alexander J. , Tu Anh-Hue , Sanz Inaki , Nahm Moon H.
Published: December 2019

Tumor necrosis factor alpha (TNF-α) is important in resistance to various microorganisms and provides signals to the target cells through two different receptors, TNF-α receptor I (TNFRI) (p55 receptor) and TNFRII (p75 receptor). To delineate the significance of the two different signaling pathways in resisting infections with extracellular bacteria, we examined the resistance of mice toStreptococcus pneumoniae(serotype 6B). TNF-α needs to be present early in infections, since one injection of wild-type mice with anti-TNF-α leads to an increased susceptibility of these mice toS. pneumoniae. TNF-α signaling through the p55 receptor (but not the p75 receptor) is crucial in resistingS. pneumoniaeinfections, because intraperitoneal injection of 100 CFU/mouse killed p55-deficient mice by day 2 of infection, whereas 1,000,000 CFU/mouse was needed to kill half of the control mice. p55-deficient mice do not show evidence of a deficient acute-phase response. All three types of mice (p55 deficient, p75 deficient, and normal) showed comparable rises in the levels of two acute-phase proteins (serum amyloid P and C3) at 24, 48, and 72 h after the experimental infections, and all of the mice showed comparable influxes of neutrophils to the site of infection. Finally, it was demonstrated that p55-deficient mice can be protected from the lethal effects ofS. pneumoniaeinfection by injection of antibodies specific forS. pneumoniaepolysaccharide capsule.
Orchestration of Neutrophil Movement by Intestinal Epithelial Cells in Response toSalmonella typhimuriumCan Be Uncoupled from Bacterial Internalization
Authors: Gewirtz Andrew T. , Siber Andrew M. , Madara James L. , McCormick Beth A.
Published: December 2019

Intestinal epithelial cells respond toSalmonella typhimuriumby internalizing this pathogen and secreting, in a polarized manner, an array of chemokines which direct polymorphonuclear leukocyte (PMN) movement. Notably, interleukin-8 (IL-8) is secreted basolaterally and directs PMN through the lamina propria, whereas pathogen-elicited epithelial chemoattractant (PEEC) is secreted apically and directs PMN migration across the epithelial monolayer to the intestinal lumen. While most studies ofS. typhimuriumpathogenicity have focused on the mechanism by which this bacterium invades its host, the enteritis characteristically associated with salmonellosis appears to be more directly attributable to the PMN movement that occurs in response to this pathogen. Therefore, we sought to better understand the relationship betweenS. typhimuriuminvasion and epithelial promotion of PMN movement. First, we investigated whetherS. typhimuriumbecoming intracellular was necessary or sufficient to induce epithelial promotion of PMN movement. BlockingS. typhimuriuminvasion by preventing, with cytochalasin D, the epithelial cytoskeletal rearrangements which mediate internalization did not reduce the epithelial promotion of PMN movement. Conversely, bacterial attainment of an intracellular position was not sufficient to induce model epithelia to direct PMN transmigration, since neither basolateral invasion byS. typhimuriumnor apical internalization of an invasion-deficient mutant (achieved by inducing membrane ruffling with epidermal growth factor) induced this epithelial cell response. These results indicate that specific interactions between the apical surface of epithelial cells andS. typhimurium, rather than simply bacterial invasion, mediate the epithelial direction of PMN transmigration. To further investigate the means by whichS. typhimuriuminduces epithelia to direct PMN movement, we investigated whether the same signaling pathways regulate secretion of IL-8 and PEEC. IL-8 secretion, but not PEEC secretion, was activated by phorbol myristate acetate and blocked by an inhibitor (mg-132) of the proteosome which mediates NF-κβ activation. Further, secretion of IL-8, but not PEEC, was activated by an entry-deficient (HilΔ)S. typhimuriummutant or by basolateral invasion of a wild-type strain. Together, these results indicate that distinct signaling pathways mediateS. typhimuriuminvasion, induction of IL-8 secretion, and induction of PEEC secretion in model intestinal epithelia.
Genetic Vaccination againstCoccidioides immitis: Comparison of Vaccine Efficacy of Recombinant Antigen 2 and Antigen 2 cDNA
Authors: Jiang Chengyong , Magee D. Mitchell , Quitugua Teresa N. , Cox Rebecca A.
Published: December 2019

Previous studies from our laboratory established that C-ASWS, an alkali-soluble, water-soluble extract from cell walls ofCoccidioides immitis, protects mice against lethal challenge with this fungus. The C-ASWS extract contains a glycosylated protein, designated antigen 2 (Ag2), and a polysaccharide antigen. We recently cloned Ag2 cDNA and showed that the recombinant fusion protein elicited strong delayed-type hypersensitivity responses in immunized mice. This investigation was undertaken to determine if the recombinant Ag2 protein, expressed as an Ag2-glutathioneS-transferase (GST) fusion protein, or Ag2 cDNA would protect mice against lethal challenge withC. immitis. The recombinant Ag2-GST protein protected BALB/c mice against intraperitoneal challenge with 250 arthroconidia, as assessed by a decrease in fungal CFU in tissues. The Ag2-GST-immunized mice did not show, however, an increased survival during a 30-day period postinfection. By contrast, immunization of mice with Ag2 cDNA ligated into the pVR1012 plasmid engendered protection against intraperitoneal challenge with 2,500 arthroconidia and against pulmonary challenge with 50 arthroconidia. Vaccine efficacy paralleled the development of delayed-type hypersensitivity responses toC. immitisantigen. Whereas mice vaccinated with the recombinant Ag2-GST protein did not mount footpad hypersensitivity to C-ASWS or the recombinant Ag2-GST protein, mice vaccinated with the pVR1012-Ag2 construct mounted a strong footpad hypersensitivity and their spleen cells secreted gamma interferon upon in vitro stimulation with the Ag2-containing C-ASWS extract. This is the first investigation to show that genetic immunization can protect against lethal challenge withC. immitis.
An In Vitro Tissue Culture Bilayer Model To Examine Early Events inMycobacterium tuberculosisInfection
Authors: Birkness K. A. , Deslauriers M. , Bartlett J. H. , White E. H. , King C. H. , Quinn F. D.
Published: December 2019

A tissue culture bilayer system that mimics some aspects of early alveolar infection byMycobacterium tuberculosiswas developed. This model incorporates human lung epithelial type II pneumocyte (A549) (upper chamber) and endothelial cell (lower chamber) layers separated by a microporous membrane. This construction makes it possible to observe and quantify the passage of bacteria through the two layers, to observe the interaction of the bacteria with the various cell types, and to examine the basic mechanisms of immune cell recruitment to the site of infection. After 107organisms were added to the upper chamber we microscopically observed large numbers of bacteria attached to and within the pneumocytes and we determined by viable-cell counting that a small percentage of the inoculum (0.02 to 0.43%) passed through the bilayer into the lower chamber. When peripheral blood mononuclear cells were added to the lower chamber, microscopic examination indicated a migration of the mononuclear cells through the bilayer to the apical surface, where they were seen associated with the mycobacteria on the pneumocytes. The added complexity of the bilayer system offers an opportunity to define more precisely the roles of the various lung cell types in the pathogenesis of early tuberculosis.
Induction of CD18-Mediated Passage of Neutrophils byPasteurella haemolyticain Pulmonary Bronchi and Bronchioles
Authors: Ackermann Mark R. , Brogden Kim A. , Florance Allison F. , Kehrli Marcus E.
Published: December 2019

Pasteurella haemolyticais an important respiratory pathogen of cattle that incites extensive infiltrates of neutrophils into the lung. In addition to the parenchymal damage caused by factors released byP. haemolytica, neutrophils contribute to the pathologic changes in the lungs. Molecules which mediate neutrophil infiltration into the lungs duringP. haemolyticapneumonia are poorly characterized. To determine whether the CD18 family (β2-integrin) of leukocyte adhesion molecules mediates initial passage of neutrophils into the pulmonary bronchi and bronchioles of lungs infected withP. haemolytica, three Holstein calves homozygous for bovine leukocyte adhesion deficiency (BLAD) (CD18-deficient neutrophils), and three age- and breed-matched control calves (normal CD18 expression) were inoculated withP. haemolyticaA1 via a fiberoptic bronchoscope and euthanized at 2 h postinoculation. Sections of lung were stained for neutrophils, and the intensity of neutrophilic infiltration was determined by computerized image analysis. Significantly fewer (P< 0.05) neutrophils infiltrated the lumen, epithelium, and adventitia of bronchioles and bronchi in lungs of calves with BLAD compared to normal calves, which had dense infiltrates within these sites at 2 h postinoculation. The reduced infiltration in calves with BLAD occurred despite the presence of an extremely large number of neutrophils in peripheral blood that is typical for these calves. The large number of neutrophils in the blood of calves with BLAD is probably a physiologic response that can occur without microbial colonization, since one calf with BLAD that was raised under germ-free conditions had large numbers of neutrophils in the blood that were similar to those in a calf with BLAD that was raised conventionally. Neutrophil counts in the germ-free and conventionally reared calves with BLAD were much higher than those in the three normal calves raised under germ-free conditions. The work in this study demonstrates that during the initial inflammatory response, neutrophils with normal CD18 expression pass more readily than CD18-deficient neutrophils into the walls and lumen of bronchi and bronchioles. It suggests that CD18 is needed for initial passage through the extensive extracellular matrix of the bronchi and bronchioles. This has potential importance for the development of therapies to direct or inhibit neutrophil infiltration into conducting airways rather than alveolar spaces.
Bacterial Invasion Is Not Required for Activation of NF-κB in Enterocytes
Authors: Eaves-Pyles Tonyia , Szabó Csaba , Salzman Andrew L.
Published: December 2019

Pathogenic enteric microorganisms induce the NF-κB-dependent expression of proinflammatory genes in intestinal epithelial cells. The purpose of the present study was to clarify the contribution of microbial invasion to the degradation of the regulatory protein IκBα and the subsequent activation of NF-κB in cultured intestinal epithelial cells. Caco-2BBe cells were incubated withSalmonella dublin,Salmonella typhimurium, or a weakly invasive strain ofE. coli.S. dublinandS. typhimurium(107organisms/ml) induced equivalent concentration-dependent gel mobility shifts of an NF-κB consensus sequence that was preceded by IκBα degradation.E. coli(107organisms/ml) did not induce IκBα degradation or NF-κB translocation. Pretreatment with cytochalasin D blocked invasion of all three strains but had no effect on IκBα degradation or NF-κB activation.S. dublinandS. typhimuriumadhered to Caco-2BBe cells 3- to 10-fold more thanE. coli. NF-κB activation was prevented by physical separation ofS. dublinfrom Caco-2BBe cells by a 0.4-μm-pore-size filter. Our results imply that bacterial adhesion, rather than invasion or release of a secreted factor, is sufficient to induce IκBα degradation and NF-κB activation in intestinal epithelial cells. Our data suggest that strategies to reduce enteric inflammation should be directed to the reduction of bacterial enterocyte adhesion.
A Role for Host Phosphoinositide 3-Kinase and Cytoskeletal Remodeling during
Cryptosporidium parvum
Infection
Authors: Forney John R. , DeWald Daryll B. , Yang Shiguang , Speer Clarence A. , Healey Mark C.
Published: December 2019

Cryptosporidium parvum
preferentially infects epithelial cells lining the intestinal mucosa of mammalian hosts. Parasite development and propagation occurs within a unique intracellular but extracytoplasmic parasitophorous vacuole at the apical surface of infected cells. Parasite-induced host cell signaling events and subsequent cytoskeletal remodeling were investigated by using cultured bovine fallopian tube epithelial (BFTE) cells inoculated with
C. parvum
sporozoites. Indirect-immunofluorescence microscopy detected host tyrosine phosphorylation within 30 s of inoculation. At >30 min postinoculation, actin aggregates were detected at the site of parasite attachment by fluorescein isothiocyanate-conjugated phalloidin staining as well as by indirect immunolabeling with monoclonal anti-actin. The actin-binding protein villin was also detected in focal aggregates at the site of attachment. Host cytoskeletal rearrangement persisted for the duration of the parasitophorous vacuole and contributed to the formation of long, branched microvilli clustered around the cryptosporidial vacuole. The phosphoinositide 3-kinase inhibitor wortmannin significantly inhibited (
P
< 0.05)
C. parvum
infection when BFTE cells were pretreated for 60 min at 37°C prior to inoculation. Similarly, treatment of BFTE cells with the protein kinase inhibitors genistein and staurosporine and the cytoskeletally acting compounds 1-(5-iodonaphthalene-1-sulfonyl)-1
H
-hexahydro-1,4-diazapine, cytochalasin D, and 2,3-butanedione monoxime significantly inhibited (
P
< 0.05) in vitro infection at 24 h postinoculation. These findings demonstrate a prominent role for phosphoinositide 3-kinase activity during the early
C. parvum
infection process and suggest that manipulation of host signaling pathways results in actin rearrangement at the site of sporozoite attachment.
Lmb, a Protein with Similarities to the LraI Adhesin Family, Mediates Attachment of
Streptococcus agalactiae
to Human Laminin
Authors: Spellerberg Barbara , Rozdzinski Eva , Martin Simone , Weber-Heynemann Josephine , Schnitzler Norbert , Lütticken Rudolf , Podbielski Andreas
Published: December 2019

Streptococcus agalactiae
is a leading cause of neonatal sepsis and meningitis. Adherence to extracellular matrix proteins is considered an important factor in the pathogenesis of infection, but the genetic determinants of this process remain largely unknown. We identified and sequenced a gene which codes for a putative lipoprotein that exhibits significant homology to the streptococcal LraI protein family. Mutants of this locus were demonstrated to have substantially reduced adherence to immobilized human laminin. The nucleotide sequence of the gene was subsequently designated
lmb
(laminin binding) and shown to be present in all of the common serotypes of
S. agalactiae
. To determine the role of Lmb in the adhesion of
S. agalactiae
wild-type strains to laminin, a recombinant Lmb protein harboring six consecutive histidine residues at the C terminus was cloned, expressed, and purified from
Escherichia coli
. Preincubation of immobilized laminin with recombinant Lmb significantly reduced adherence of the wild-type strain O90R to laminin. These results indicate that Lmb mediates the attachment of
S. agalactiae
to human laminin, which may be essential for the bacterial colonization of damaged epithelium and translocation of bacteria into the bloodstream.
rfb
Mutations in
Vibrio cholerae
Do Not Affect Surface Production of Toxin-Coregulated Pili but Still Inhibit Intestinal Colonization
Authors: Chiang Su L. , Mekalanos John J.
Published: December 2019

The toxin-coregulated pilus (TCP) of
Vibrio cholerae
is essential for colonization. It was recently reported that
rfb
mutations in
V. cholerae
569B cause the translocation arrest of the structural subunit of TCP, raising the possibility that the colonization defects of lipopolysaccharide mutants are due to effects on TCP biogenesis. However, an
rfbB
gene disruption in either
V. cholerae
O395 or 569B has no apparent effect on surface TCP production as assessed by immunoelectron microscopy and CTX phage transduction, and an
rfbD
::Tn
5lac
mutant of O395 also shows no defect in TCP expression. We conclude that the colonization defect associated with
rfb
mutations is unrelated to defects in TCP assembly.
All Accessible Epitopes in theSalmonellaLipopolysaccharide Core Are Associated with Branch Residues
Authors: Nnalue Ndubisi Anthony
Published: December 2019

Antisera generated against each of the nine known chemotypes ofSalmonellalipopolysaccharide (LPS) core were characterized in order to delineate cross-reactive epitopes and define the bases for their accessibility. Strongly cross-reactive epitopes were associated with three chemotypes: Ra and Rb4, which recognized α-GlcNAc-1→2-α-Glc, and Rd1, which recognizedl-α-d-heptose-1→7-l-α-d-heptose. Both these disaccharides and the more weakly cross-reactive α-Gal-1→6-α-Glc terminal in Rb3LPS represent branch points along the core oligosaccharide. Therefore, branch points in endotoxin core oligosaccharides may generally be cross-reactive.
Transcutaneous Immunization with Bacterial ADP-Ribosylating Exotoxins as Antigens and Adjuvants
Authors: Glenn Gregory M. , Scharton-Kersten Tanya , Vassell Russell , Matyas Gary R. , Alving Carl R.
Published: December 2019

Transcutaneous immunization (TCI) is a new technique that uses the application of vaccine antigens in a solution on the skin to induce potent antibody responses without systemic or local toxicity. We have previously shown that cholera toxin (CT), a potent adjuvant for oral and nasal immunization, can induce both serum and mucosal immunoglobulin G (IgG) and IgA and protect against toxin-mediated mucosal disease when administered by the transcutaneous route. Additionally, CT acts as an adjuvant for coadministered antigens such as tetanus and diphtheria toxoids when applied to the skin. CT, a member of the bacterial ADP-ribosylating exotoxin (bARE) family, is most potent as an adjuvant when the A-B subunits are present and functional. We now show that TCI induces secondary antibody responses to coadministered antigens as well as to CT in response to boosting immunizations. IgG antibodies to coadministered antigens were also found in the stools and lung washes of immunized mice, suggesting that TCI may target mucosal pathogens. Mice immunized by the transcutaneous route with tetanus fragment C and CT developed anti-tetanus toxoid antibodies and were protected against systemic tetanus toxin challenge. We also show that bAREs, similarly organized as A-B subunits, as well as the B subunit of CT alone, induced antibody responses to themselves when given via TCI. Thus, TCI appears to induce potent, protective immune responses to both systemic and mucosal challenge and offers significant potential practical advantages for vaccine delivery.
Evolutionary Relationships of Pathogenic Clones of
Vibrio cholerae
by Sequence Analysis of Four Housekeeping Genes
Authors: Byun Roy , Elbourne Liam D. H. , Lan Ruiting , Reeves Peter R.
Published: December 2019

Studies of the
Vibrio cholerae
population, using molecular typing techniques, have shown the existence of several pathogenic clones, mainly sixth-pandemic, seventh-pandemic, and U.S. Gulf Coast clones. However, the relationship of the pathogenic clones to environmental
V. cholerae
isolates remains unclear. A previous study to determine the phylogeny of
V. cholerae
by sequencing the
asd
(aspartate semialdehyde dehydrogenase) gene of
V. cholerae
showed that the sixth-pandemic, seventh-pandemic, and U.S. Gulf Coast clones had very different
asd
sequences which fell into separate lineages in the
V. cholerae
population. As gene trees drawn from a single gene may not reflect the true topology of the population, we sequenced the
mdh
(malate dehydrogenase) and
hlyA
(hemolysin A) genes from representatives of environmental and clinical isolates of
V. cholerae
and found that the
mdh
and
hlyA
sequences from the three pathogenic clones were identical, except for the previously reported 11-bp deletion in
hlyA
in the sixth-pandemic clone. Identical sequences were obtained, despite average nucleotide differences in the
mdh
and
hlyA
genes of 1.52 and 3.25%, respectively, among all the isolates, suggesting that the three pathogenic clones are closely related. To extend these observations, segments of the
recA
and
dnaE
genes were sequenced from a selection of the pathogenic isolates, where the sequences were either identical or substantially different between the clones. The results show that the three pathogenic clones are very closely related and that there has been a high level of recombination in their evolution.
Activation of Murine Macrophages by Lipoprotein and Lipooligosaccharide ofTreponema denticola
Authors: Rosen Graciela , Sela Michael N. , Naor Ronit , Halabi Amal , Barak Vivian , Shapira Lior
Published: December 2019

We have recently demonstrated that the periodontopathogenic oral spirocheteTreponema denticolapossesses membrane-associated lipoproteins in addition to lipooligosaccharide (LOS). The aim of the present study was to test the potential of these oral spirochetal components to induce the production of inflammatory mediators by human macrophages, which in turn may stimulate tissue breakdown as observed in periodontal diseases. An enriched lipoprotein fraction (dLPP) fromT. denticolaATCC 35404 obtained upon extraction of the treponemes with Triton X-114 was found to stimulate the production of nitric oxide (NO), tumor necrosis factor alpha (TNF-α), and interleukin-1 (IL-1) by mouse macrophages in a dose-dependent manner. Induction of NO by dLPP was at 25% of the levels obtained bySalmonella typhosalipopolysaccharide (LPS) at similar concentrations, while IL-1 was produced at similar levels by both inducers. dLPP-mediated macrophage activation was unaffected by amounts of polymyxin B that neutralized the induction produced byS. typhosaLPS. dLPP also induced NO and TNF-α secretion from macrophages isolated from endotoxin-unresponsive C3H/HeJ mice to an extent similar to the stimulation produced in endotoxin-responsive mice. PurifiedT. denticolaLOS also produced a concentration-dependent activation of NO and TNF-α in LPS-responsive and -nonresponsive mouse macrophages. However, macrophage activation by LOS was inhibited by polymyxin B. These results suggest thatT. denticolalipoproteins and LOS may play a role in the inflammatory processes that characterize periodontal diseases.
Isolation and Chemical Characterization of a Capsular Polysaccharide Antigen Shared by Clinical Isolates of
Enterococcus faecalis
and Vancomycin-Resistant
Enterococcus faecium
Authors: Huebner Johannes , Wang Ying , Krueger Wolfgang A. , Madoff Lawrence C. , Martirosian Gayane , Boisot Saskia , Goldmann Donald A. , Kasper Dennis L. , Tzianabos Arthur O. , Pier Gerald B.
Published: December 2019

Enterococci are a common cause of serious infections, especially in newborns, severely immunocompromised patients, and patients requiring intensive care. To characterize enterococcal surface antigens that are targets of opsonic antibodies, rabbits were immunized with various gentamicin-killed
Enterococcus faecalis
strains, and immune sera were tested in an opsonophagocytic assay against a selection of clinical isolates. Serum raised against one strain killed the homologous strain (12030) at a dilution of 1:5,120 and mediated opsonic killing of 33% of all strains tested. In addition, this serum killed two (28%) of seven vancomycin-resistant
Enterococcus faecium
strains. Adsorption of sera with the homologous strain eliminated killing activity. The adsorbing antigens were resistant to treatment with proteinase K and to boiling for 1 h, but were susceptible to treatment with sodium periodate, indicating that the antigen inducing opsonic activity is a polysaccharide. Antibodies in immune rabbit sera reacted with a capsule-like structure visualized by electron microscopy both on the homologous
E. faecalis
strain and on a vancomycin-resistant
E. faecium
strain. The capsular polysaccharides from
E. faecalis
12030 and
E. faecium
838970 were purified, and chemical and structural analyses indicated they were identical glycerol teichoic acid-like molecules with a carbohydrate backbone structure of 6-α-
d
-glucose-1-2 glycerol-3-PO
4
with substitution on carbon 2 of the glucose with an α-2-1-
d
-glucose residue. The purified antigen adsorbed opsonic killing activity from immune rabbit sera and elicited high titers of antibodies (when used to immunize rabbits) that both mediated opsonic killing of bacteria and bound to a capsule-like structure visualized by electron microscopy. These results indicate that approximately one-third of a sample of 15
E. faecalis
strains and 7 vancomycin-resistant
E. faecium
strains possess shared capsular polysaccharides that are targets of opsonophagocytic antibodies and therefore are potential vaccine candidates.
Enhanced Adhesion ofPasteurella multocidato Cultured Turkey Peripheral Blood Monocytes
Authors: Pruimboom Ingrid M. , Rimler Richard B. , Ackermann Mark R.
Published: December 2019

Capsular hyaluronic acid (HA) mediates adhesion of serogroup A strains ofPasteurella multocidato elicited turkey air sac macrophages (TASM). In contrast, freshly isolated turkey peripheral blood monocytes (TPBM) do not bind serogroup A strains. Following culture of TPBM for 6 days in chamber slides, adhesion of the bacteria to TPBM increased gradually. Incubation in chamber slides coated with entactin-collagen IV-laminin (ECL) attachment matrix or exposure to phorbol myristate acetate (PMA) further enhanced the adhesion ofP. multocidato TPBM. Addition of HA, but not Arg-Gly-Asp peptide, to TPBM culture inhibited bacterial adherence similarly to the inhibition previously reported for TASM. Exposure of TPBM to monoclonal antibody directed against HA-binding cell surface proteoglycan (CD44) decreased binding ofP. multocida. Collectively, these findings indicate thatP. multocidaadhesion to TPBM is mediated by capsular HA and can be increased by culture on ECL attachment matrix or PMA exposure. Additionally, the findings suggest that the capsular mucopolysaccharide of serogroup A strains ofP. multocidarecognizes an isoform of CD44 expressed on cultured TPBM.
Genetic Characterization of a New Type IV-A Pilus Gene Cluster Found in Both Classical and El Tor Biotypes of
Vibrio cholerae
Authors: Fullner Karla Jean , Mekalanos John J.
Published: December 2019

The
Vibrio cholerae
genome contains a 5.4-kb
pil
gene cluster that resembles the
Aeromonas hydrophila tap
gene cluster and other type IV-A pilus assembly operons. The region consists of five complete open reading frames designated
pilABCD
and
yacE
, based on the nomenclature of related genes from
Pseudomonas aeruginosa
and
Escherichia coli
K-12. This cluster is present in both classical and El Tor biotypes, and the
pilA
and
pilD
genes are 100% conserved. The
pilA
gene encodes a putative type IV pilus subunit. However, deletion of
pilA
had no effect on either colonization of infant mice or adherence to HEp-2 cells, demonstrating that
pilA
does not encode the primary subunit of a pilus essential for these processes. The
pilD
gene product is similar to other type IV prepilin peptidases, proteins that process type IV signal sequences. Mutational analysis of the
pilD
gene showed that
pilD
is essential for secretion of cholera toxin and hemagglutinin-protease, mannose-sensitive hemagglutination (MSHA), production of toxin-coregulated pili, and colonization of infant mice. Defects in these functions are likely due to the lack of processing of N termini of four Eps secretion proteins, four proteins of the MSHA cluster, and TcpB, all of which contain type IV-A leader sequences. Some
pilD
mutants also showed reduced adherence to HEp-2 cells, but this defect could not be complemented in
trans
, indicating that the defect may not be directly due to a loss of
pilD
. Taken together, these data demonstrate the effectiveness of the
V. cholerae
genome project for rapid identification and characterization of potential virulence factors.
Responses of Human Intestinal Microvascular Endothelial Cells to Shiga Toxins 1 and 2 and Pathogenesis of Hemorrhagic Colitis
Authors: Jacewicz Mary S. , Acheson David W. K. , Binion David G. , West Gail A. , Lincicome Lisa L. , Fiocchi Claudio , Keusch Gerald T.
Published: December 2019

Endothelial damage is characteristic of infection with Shiga toxin (Stx)-producingEscherichia coli(STEC). Because Stx-mediated endothelial cell damage at the site of infection may lead to the characteristic hemorrhagic colitis of STEC infection, we compared the effects of Stx1 and Stx2 on primary and transformed human intestinal microvascular endothelial cells (HIMEC) to those on macrovascular endothelial cells from human saphenous vein (HSVEC). Adhesion molecule, interleukin-8 (IL-8), and Stx receptor expression, the effects of cytokine activation and Stx toxins on these responses, and Stx1 and Stx2 binding kinetics and bioactivity were measured. Adhesion molecule and IL-8 expression increased in activated HIMEC, but these responses were blunted in the presence of toxin, especially in the presence of Stx1. In contrast to HSVEC, unstimulated HIMEC constitutively expressed Stx receptor at high levels, bound large amounts of toxin, were highly sensitive to toxin, and were not further sensitized by cytokines. Although the binding capacities of HIMEC for Stx1 and Stx2 were comparable, the binding affinity of Stx1 to HIMEC was 50-fold greater than that of Stx2. Nonetheless, Stx2 was more toxic to HIMEC than an equivalent amount of Stx1. The decreased binding affinity and increased toxicity for HIMEC of Stx2 compared to those of Stx1 may be relevant to the preponderance of Stx2-producing STEC involved in the pathogenesis of hemorrhagic colitis and its systemic complications. The differences between primary and transformed HIMEC in these responses were negligible. We conclude that transformed HIMEC lines could represent a simple physiologically relevant model to study the role of Stx in the pathogenesis of hemorrhagic colitis.
In Vitro Induction of Activation-Induced Cell Death in Lymphocytes from Chronic Periodontal Lesions by Exogenous Fas Ligand
Authors: Sawa Takamasa , Nishimura Fusanori , Ohyama Hideki , Takahashi Keiso , Takashiba Shogo , Murayama Yoji
Published: December 2019

Periodontitis is a chronic inflammatory disease which gradually destroys the supporting tissues of the teeth, leading to tooth loss in adults. The lesions are characterized by a persistence of inflammatory cells in gingival and periodontal connective tissues. To understand what mechanisms are involved in the establishment of chronic lesions, we hypothesized that infiltrating lymphocytes might be resistant to apoptosis. However, both Bcl-2 and Bcl-xL were weakly detected in lymphocytes from the lesions, compared with those from peripheral blood, suggesting that these cells are susceptible to apoptosis. Nevertheless, very few apoptotic cells were observed in tissue sections from the lesions. Lymphocytes from the lesions expressed mRNA encoding Fas, whereas Fas-ligand mRNA was very weakly expressed in lymphocytes from the lesions and in periodontal tissues. Since the results indicated that lymphocytes in the lesions might be susceptible to Fas-mediated apoptosis but lack the death signal, we next investigated if these lymphocytes actually undergo apoptosis by the addition of anti-Fas antibodies in vitro. Fas-positive lymphocytes from the lesions underwent apoptosis by these antibodies, but Fas-negative lymphocytes and Fas-positive peripheral lymphocytes did not undergo apoptosis by these antibodies. These results indicate that lymphocytes in the lesions are susceptible to activation-induced cell death and are induced to die by apoptosis after the addition of exogenous Fas ligand.
Specificity and Function of Immunogenic Peptides from the 35-Kilodalton Protein ofMycobacterium leprae
Authors: Wilkinson Robert John , Wilkinson Katalin Andrea , Jurcevic Stipo , Hills Adrian , Sinha Sudhir , Sengupta Utpal , Lockwood Diana N. J. , Katoch Kiran , Altman Daniel , Ivanyi Juraj
Published: December 2019

We identified a T-cell determinant of the 35-kDa antigen ofMycobacterium lepraewhich is discriminatory against cross-sensitization by its closely related homologue inMycobacterium avium. From synthetic peptides covering the entire sequence, those with the highest affinity and permissive binding to purified HLA-DR molecules were evaluated for the stimulation of proliferation of peripheral blood mononuclear cells (PBMCs) from leprosy patients and healthy sensitized controls. Responses to the peptide pair 206–224, differing by four residues betweenM. lepraeandM. avium, involved both species-specific and cross-reactive T cells. Lymph node cell proliferation in HLA-DRB1*01 transgenic mice was reciprocally species specific, but only the response to theM. lepraepeptide in the context of DR1 was immunodominant. Of the cytokines in human PBMC cultures, gamma interferon production was negligible, while interleukin 10 (IL-10) responses in both patients and controls were more pronounced. IL-10 was most frequently induced by the shared 241–255 peptide, indicating that environmental cross-sensitization may skew the response toward a potentially pathogenic cytokine phenotype.
Legionella pneumophila
Utilizes the Same Genes To Multiply within
Acanthamoeba castellanii
and Human Macrophages
Authors: Segal Gil , Shuman Howard A.
Published: December 2019

In previous reports we described a 22-kb
Legionella pneumophila
chromosomal locus containing 18 genes. Thirteen of these genes (
icmT
, -
R
, -
Q
, -
P
, -
O
, -
M
, -
L
, -
K
, -
E
, -
C
, -
D
, -
J
, and -
B
) were found to be completely required for intracellular growth and killing of human macrophages. Three genes (
icmS
, -
G
, and -
F
) were found to be partially required, and two genes (
lphA
and
tphA
) were found to be dispensable for intracellular growth and killing of human macrophages. Here, we analyzed the requirement of these genes for intracellular growth in the protozoan host
Acanthamoeba castellanii
, a well-established important environmental host of
L. pneumophila
. We found that all the genes that are completely required for intracellular growth in human macrophages are also completely required for intracellular growth in
A. castellanii
. However, the genes that are partially required for intracellular growth in human macrophages are completely required for intracellular growth in
A. castellanii
. In addition, the
lphA
gene, which was shown to be dispensable for intracellular growth in human macrophages, is partially required for intracellular growth in
A. castellanii
. Our results indicate that
L. pneumophila
utilizes the same genes to grow intracellularly in both human macrophages and amoebae.
Immunogenicity of aSalmonella typhimurium aroA aroDVaccine Expressing a Nontoxic Domain ofClostridium difficileToxin A
Authors: Ward Stephen J. , Douce Gill , Figueiredo Dayse , Dougan Gordon , Wren Brendan W.
Published: December 2019

The C-terminal repeat domain ofClostridium difficiletoxin A harbors toxin-neutralizing epitopes and is considered to be a candidate component of a vaccine againstC. difficile-associated disease (CDAD). Fourteen of the 38 C-terminal toxin A repeats (14CDTA) were cloned into pTECH-1 in frame with the immunogenic fragment C of tetanus toxin (TETC) to generate plasmid p56TETC. Expression of the TETC-14CDTA fusion protein was driven from the anaerobically induciblenirBpromoter within attenuatedSalmonella typhimuriumBRD509 (aroA aroD). The TETC-14CDTA fusion protein was purified and shown to bind to known toxin A receptors found on the surface of rabbit erythrocytes. Intranasal (i.n.) and intragastric (i.g.) immunization with 107and 1010CFU, respectively, of BRD509(p56TETC) generated significant (P< 0.05) anti-toxin A serum responses after a single dose. Antibody titers were elevated following a boosting dose with either live vaccine or a subcutaneous injection of 0.5 μg of purified 14CDTA protein. Importantly, serum from mice immunized with BRD509(p56TETC) neutralized toxin A cytotoxicity. Both i.n. and i.g. immunizations also generated toxin A-specific immunoglobulin A on the pulmonary and intestinal mucosa, respectively. Intranasal vaccination induced consistently higher serum and mucosal anti-toxin A antibody responses. Significant anti-tetanus toxoid serum and mucosal antibodies were also generated by both immunization routes. The availability of live attenuatedSalmonella typhifor human use may allow the development of a multivalent mucosal vaccine against CDAD, tetanus, and typhoid.
Transient Transcriptional Activation of the
Vibrio cholerae
El Tor Virulence Regulator ToxT in Response to Culture Conditions
Authors: Medrano Ana I. , DiRita Victor J. , Castillo Gabriela , Sanchez Joaquin
Published: December 2019

Vibrio cholerae
El Tor require special in vitro culture conditions, consisting of an initial static growth period followed by shift to shaking (AKI conditions), for expression of cholera toxin (CT) and toxin coregulated pili (TCP). ToxT, a regulator whose initial transcription depends on the ToxR regulator, positively modulates expression of CT and TCP. To help understand control of CT and TCP in El Tor vibrios, we monitored
ctxAB
and ToxR-dependent
toxT
transcription by time course primer extension assays. AKI conditions stimulated CT synthesis with an absence of
ctxAB
transcription during static growth followed by induction upon shaking. ToxR-dependent
toxT
transcription was induced at the end of the static growth period but was transient, stopping shortly after shaking was initiated but, interestingly, also if the static phase was prolonged. Immunoblot assays showed that ToxR protein levels were not coincidentally transient, implying a protein on/off switch mechanism for ToxR. Despite the transient activation by ToxR, transcription of
ctxAB
was maintained during shaking. This finding suggested continued
toxT
expression, possibly through relay transcription from another promoter. The 12.6-kb distant upstream
tcpA
promoter responsible for expression of the TCP operon has been proposed to provide an alternate
toxT
message by readthrough transcription. Activation of the
tcpA
promoter is supported by increased expression of TcpA protein during the shaking phase of the culture. Readthrough transcription of
toxT
from
tcpA
would be compatible with reverse transcription-PCR evidence for a
toxT
mRNA at times when ToxR-dependent transcription was no longer detectable by primer extension.