bluuebunny/crossref_metadata_2025 huggingface dataset:
Intranasal Immunization with Cytotoxic T-Lymphocyte Epitope Peptide and Mucosal Adjuvant Cholera Toxin: Selective Augmentation of Peptide-Presenting Dendritic Cells in Nasal Mucosa-Associated Lymphoid Tissue
Authors: Porgador Angel , Staats Herman F. , Itoh Yasushi , Kelsall Brian L.
Published: December 2019

We previously reported that cholera toxin (CT) was required as a mucosal adjuvant for the induction of peptide-specific cytotoxic T lymphocytes (CTL) following intranasal immunization with CTL epitope peptides (A. Porgador et al., J. Immunol. 158:834–841, 1997). The present study was performed to identify the site and the antigen-presenting cell (APC) population responsible for the presentation of intranasally administered CTL epitope peptide immunogens and to determine whether CT directly affects antigen presentation by these APCs. For these experiments, C57BL/6 mice were intranasally immunized with the ovalbumin H-2Kb-restricted CTL epitope SIINFEKL with or without CT. Cells were then isolated from the cervical lymph nodes (CLN) and the nasal mucosa-associated lymphoid tissue (NALT) and tested for the ability to stimulate the B3Z T-cell hybridoma, which recognizes SIINFEKL in association with H-2Kb. Dendritic cell (DC)-enriched CLN cells from mice immunized with peptide and CT or peptide only could stimulate B3Z cells, while DC-depleted CLN cells from either group were unable to stimulate B3Z cells. NALT cells of mice immunized with peptide and CT, but not with peptide alone, were able to efficiently stimulate B3Z hybridomas. Depletion of N418-positive DC from these NALT cells resulted in significant reduction of B3Z activation. Our results indicate that DC are the APC responsible for the presentation of CTL epitope peptides following intranasal immunization and that CT augments the ability of dendritic cells in the NALT, but not in the draining CLN, to present CLT epitope peptides. This finding suggests that CT acts locally as a mucosal adjuvant and that NALT DC are the predominant APC involved with the induction of immunity after intranasal immunization with peptide immunogens and CT.
Identification of a 71-Kilodalton Surface-Associated Hsp70 Homologue in
Coxiella burnetii
Authors: Macellaro Anna , Tujulin Eva , Hjalmarsson Karin , Norlander Lena
Published: December 2019

A
Coxiella burnetii
Hsp70 homologue was identified by using an acid activation in vitro system in which protein synthesis has been followed by [
35
S]methionine labeling, autoradiography, and immunoblotting. The protein was one of those predominantly labeled, and the immunoblots revealed that it was recognized by anti-DnaK antibodies. The corresponding gene was isolated, and its nucleotide sequence was determined and analyzed. A single open reading frame (ORF) with a size of 1,968 bp was identified. The ORF encodes a protein containing 656 residues and having a molecular weight of 70,800. The −10 promoter sequence was shown to be identical with the consensus heat shock ς
32
promoter sequence. The base composition at the presumed −35 region revealed an
Eco
RI site in the expected region, which is assumed to be located at the border of the cloned fragment. The gene was expressed in
Escherichia coli
as an intact protein. The
C. burnetii
71-kDa protein sequence has a high degree of homology to sequences of the Hsp70 family. A comparison of sequences revealed that the similarity with Hsp70s from other intracellular bacteria, e.g.,
Legionella pneumophila
and
Francisella tularensis
, as well as
E. coli
DnaK, is more than 80%. The homologous regions are found in the N-terminal and central parts of the protein sequence, and they include the signature patterns of the Hsp70 family of proteins. The presence of the 71-kDa protein in association with the cell wall as well as in the cytoplasm was demonstrated by the use of immunoelectron microscopy. The dual localization was verified by Western blot analysis of proteins in
C. burnetii
cell fractions, using purified antibodies directed to the 71-kDa protein.
Selective Activation of
sar
Promoters with the Use of Green Fluorescent Protein Transcriptional Fusions as the Detection System in the Rabbit Endocarditis Model
Authors: Cheung A. L. , Nast Cynthia C. , Bayer A. S.
Published: December 2019

The global regulatory locus
sar
is composed of three overlapping transcripts initiated from a triple-promoter system (designated P1, P3, and P2). To explore if the individual
sar
promoters are differentially expressed in vitro and in vivo, we constructed a shuttle plasmid (pALC1434) containing a promoterless
gfp
UV
gene (a
gfp
derivative [Clontech]) preceded by a polylinker region. Recombinant shuttle vectors containing individual
sar
promoters upstream of the
gfp
UV
reporter gene were then introduced into
Staphylococcus aureus
RN6390. Northern and immunoblot analysis revealed that P1 is stronger than the P2 and P3 promoters in vitro. Additionally, the levels of the
gfp
UV
transcript driven by individual
sar
promoters also correlated with the growth cycle dependency of these promoters in liquid cultures, thus suggesting the utility of pALC1434 as a vehicle for reporter fusion. Using the rabbit endocarditis model, we examined the expression of these three GFP
UV
fusions in vivo by fluorescence microscopy of infected cardiac vegetations 24 h after initial intravenous challenge. Similar to the in vitro findings, P1 was activated both in the center and on the surface of the vegetations. In contrast, the P3 promoter was silent both in vivo and in vitro as determined by fluorescence microscopy. Remarkably, P2 was silent in vitro but became highly activated in vivo. In particular, the
sar
P2 promoter was activated on the surface of the vegetation but not in the center of the lesion. These data imply that in vivo promoter activation of
sar
differed from that observed in vitro. Moreover, the individual
sar
promoters may be differentially expressed in different areas within the same anatomic niche, presumably reflecting the microbial physiological response to distinct host microenvironments. As the
sar
locus controls the synthesis of both extracellular and cell wall virulence determinants, these promoter-
gfp
UV
constructs should be useful to characterize many aspects of
S. aureus
gene regulation in vivo.
Enteropathogenic
Escherichia coli
Virulence Genes Encoding Secreted Signalling Proteins Are Essential for Modulation of Caco-2 Cell Electrolyte Transport
Authors: Collington Georgina K. , Booth Ian W. , Donnenberg Michael S. , Kaper James B. , Knutton Stuart
Published: December 2019

The pathophysiology of enteropathogenic
Escherichia coli
(EPEC) diarrhea remains uncertain. In vitro, EPEC stimulates a rapid increase in short-circuit current (
I
sc
) across Caco-2 cell monolayers coincident with intimate attaching and effacing (A/E) bacterial adhesion. This study has examined the roles of specific EPEC virulence proteins in this
I
sc
response. EPEC genes encoding EspA, EspB, and EspD, essential for signal transduction in host cells and A/E activity, were also required for modulation of Caco-2 electrolyte transport.
Invasion of Human Oral Epithelial Cells by
Prevotella intermedia
Authors: Dorn Brian R. , Leung K.-P. , Progulske-Fox Ann
Published: December 2019

Invasion of oral epithelial cells by pathogenic oral bacteria may represent an important virulence factor in the progression of periodontal disease. Here we report that a clinical isolate of
Prevotella intermedia
, strain 17, was found to invade a human oral epithelial cell line (KB), whereas
P. intermedia
27, another clinical isolate, and
P. intermedia
25611, the type strain, were not found to invade the cell line. Invasion was quantified by the recovery of viable bacteria following a standard antibiotic protection assay and observed by electron microscopy. Cytochalasin D, cycloheximide, monodansylcadaverine, and low temperature (4°C) inhibited the internalization of
P. intermedia
17. Antibodies raised against
P. intermedia
type C fimbriae and against whole cells inhibited invasion, but the anti-type-C-fimbria antibody inhibited invasion to a greater extent than the anti-whole-cell antibody. This work provides evidence that at least one strain of
P. intermedia
can invade an oral epithelial cell line and that the type C fimbriae and a cytoskeletal rearrangement are required for this invasion.
Towards a Recombinant Vaccine against Diphtheria Toxin
Authors: Lobeck Karin , Drevet Pascal , Léonetti Michel , Fromen-Romano Cécile , Ducancel Frédéric , Lajeunesse Evelyne , Lemaire Caroline , Ménez André
Published: December 2019

Two recombinant fragments of diphtheria toxin (DT) were fused to an engineered tandem repeat of the immunoglobulin (Ig) binding domain of protein A, called ZZ. These fragments are (i) the receptor binding domain (DTR), which comprises amino acids 382 to 535 of DT, and (ii) a linear peptide (DT
168–220
) which comprises residues 168 to 220 of the loop between fragment A and fragment B of DT. The fusion proteins were produced in
Escherichia coli
and purified by affinity chromatography. In vitro experiments showed that the DTR domain is responsible for the capacity of ZZ-DTR to bind to Vero cells and is capable of inhibiting the cytotoxicity of DT for these cells. These findings suggest that DTR binds to the cell surface receptors of DT and hence adopts a conformation that is similar to that of the receptor binding domain of DT. We compared the capacities of ZZ-DTR, ZZ-DT
168–220
, and a chemically detoxified form of DT currently used for vaccination to elicit antibodies in rabbits. The toxoid was more immunogenic than ZZ-DT
168–220
, which in turn was more immunogenic than ZZ-DTR. However, ZZ-DT
168–220
antiserum was poorly efficient at neutralizing DT cytotoxicity on Vero cells, whereas ZZ-DTR antiserum was only 15-fold less potent than anti-DT antisera.
Neutrophil-Activating Protein Mediates Adhesion of
Helicobacter pylori
to Sulfated Carbohydrates on High-Molecular-Weight Salivary Mucin
Authors: Namavar Ferry , Sparrius Marion , Veerman Enno C. I. , Appelmelk Ben J. , Vandenbroucke-Grauls Christina M. J. E.
Published: December 2019

The in vitro binding of surface-exposed material and outer membrane proteins of
Helicobacter pylori
to high-molecular-weight salivary mucin was studied. We identified a 16-kDa surface protein which adhered to high-molecular-weight salivary mucin. This protein binds specifically to sulfated oligosaccharide structures such as sulfo-Lewis a, sulfogalactose and sulfo-
N
-acetyl-glucosamine on mucin. Sequence analysis of the protein proved that it was identical to the N-terminal amino acid sequence of neutrophil-activating protein. Moreover, this adhesin was able to bind to Lewis x blood group antigen.
Differential Effects of Infection with aBabesia-Like Piroplasm, WA1, in Inbred Mice
Authors: Moro Manuel H. , David Chella S. , Magera Jenifer M. , Wettstein Peter J. , Barthold Stephen W. , Persing David H.
Published: December 2019

A newly identified intraerythrocyticBabesia-like organism, WA1, and its relatives were recently shown to be infectious for humans in the western United States. The purpose of the present study was to determine the susceptibilities of selected mouse genotypes to WA1 infection in an attempt to develop a murine model of the human disease. Several mouse strains were inoculated intraperitoneally with various passages of WA1-parasitized erythrocytes. Parasitemia was evaluated by blood smears and by PCR with blood samples collected at various intervals after inoculation. Hematologic parameters were monitored in blood samples at all intervals. C57BL/6 and C57BL/10 mice exhibited mortality rates of <10%. BALB/cJ, CBAJ, and 129/J mice had higher peak parasitemias than did C57BL mice, with mortality rates of 40, 50, and 50%, respectively. A/J, AKR/N, C3H, and DBA/1J mice also had higher peak parasitemia and mortality rates (>95%). An F1cross of C57BL/6 (resistant) and C3H.RKK (susceptible) mice had a mortality rate similar to that of the resistant parental strain. Histopathology of BALB/cJ and C3H mice at 9 and 14 days after inoculation revealed erythrophagocytosis and deposition of an iron-negative pigment in multiple organs. Morbidly ill C3H mice at 14 days had severe pulmonary edema, hemoglobinuria, and glomerulonephritis.
Mice Are Protected fromHelicobacter pyloriInfection by Nasal Immunization with AttenuatedSalmonella typhimurium phoPcExpressing Urease A and B Subunits
Authors: Corthésy-Theulaz Irène E. , Hopkins Sally , Bachmann Daniel , Saldinger Pierre F. , Porta Nadine , Haas Rainer , Zheng-Xin Yan , Meyer Thomas , Bouzourène Hanifa , Blum André L. , Kraehenbuhl Jean-Pierre
Published: December 2019

LiveSalmonella typhimurium phoPcbacteria were tested as mucosal vaccine vectors to deliverHelicobacter pyloriantigens. The genes encoding the A and B subunits ofH. pyloriurease were introduced intoS. typhimurium phoPcand expressed under the control of a constitutive tac promoter (tac-ureAB) or a two-phase T7 expression system (cT7-ureAB). Both recombinantSalmonellastrains expressed the two urease subunits in vitro and were used to nasally immunize BALB/c mice. The plasmid carrying cT7-ureABwas stably inherited by bacteria growing or persisting in the spleen, lungs, mesenteric or cervical lymph nodes, and Peyer’s patches of immunized mice, while the plasmid carrying tac-ureABwas rapidly lost. Spleen and Peyer’s patch CD4+lymphocytes from mice immunized withS. typhimurium phoPccT7-ureABproliferated in vitro in response to urease, whereas cells from mice givenS. typhimurium phoPcalone did not. Splenic CD4+cells from mice immunized withphoPccT7-ureABsecreted gamma interferon and interleukin 10, while Peyer’s patch CD4+cells did not secrete either cytokine. SpecificH. pylorianti-urease immunoglobulin G1 (IgG1) and IgG2A antibodies were detected following immunization, confirming that both Th1- and Th2-type immune responses were generated by the live vaccine. Sixty percent of the mice (9 of 15) immunized withS. typhimurium phoPccT7-ureABwere found to be resistant to infection byH. pylori, while all mice immunized withphoPctac-ureAB(15 of 15) orphoPc(15 of 15) were infected. Our data demonstrate thatH. pyloriurease delivered nasally by using a vaccine strain ofS. typhimuriumcan trigger Th1- and Th2-type responses and induce protective immunity againstHelicobacterinfection.
Requirement for CD40-CD40 Ligand Interaction for Elimination ofCryptosporidium parvumfrom Mice
Authors: Cosyns Mary , Tsirkin Svetlana , Jones Michelle , Flavell Richard , Kikutani Hitoshi , Hayward Anthony R.
Published: December 2019

Mice with disrupted genes for CD40 and CD40 ligand (CD40L) are unable to clear infection withCryptosporidium parvumand develop cholangitis. Parasites are present in the gut, gall bladder, and biliary tree, and biliary epithelial cells express CD40 on the cell surface. SCID mice infected withC. parvumfor >1 month can clear the infection after reconstitution with spleen cells from CD40, but not CD40L, knockout mice. In an in vitro model,C. parvum-infected HepG2 cells were triggered to apoptosis when incubated with a CD40L-CD8 fusion protein. The requirement for CD40-CD40L interactions for immunity toC. parvumindicated by our results may entail the triggering of apoptosis in infected cells, in addition to the known role of CD40L-CD40 interactions in stimulating cytokine production and promoting T-cell responses.
Anthrax Toxin-Mediated Delivery In Vivo and In Vitro of a Cytotoxic T-Lymphocyte Epitope from Ovalbumin
Authors: Ballard Jimmy D. , Doling Amy M. , Beauregard Kathryn , Collier R. John , Starnbach Michael N.
Published: December 2019

We reported earlier that a nontoxic form of anthrax toxin was capable of delivering a cytotoxic T-lymphocyte (CTL) epitope in vivo, such that a specific CTL response was primed against the epitope. The epitope, of bacterial origin, was fused to an N-terminal fragment (LFn) from the lethal-factor component of the toxin, and the fusion protein was injected, together with the protective antigen (PA) component, into BALB/c mice. Here we report that PA plus LFn is capable of delivering a different epitope—OVA257–264from ovalbumin. Delivery was accomplished in a different mouse haplotype,H-2Kband occurred in vitro as well as in vivo. An OVA257–264-specific CTL clone, GA-4, recognized EL-4 cells treated in vitro with PA plus as little as 30 fmol of the LFn-OVA257–264fusion protein. PA mutants attenuated in toxin self-assembly or translocation were inactive, implying that the role of PA in epitope delivery is the same as that in toxin action. Also, we showed that OVA257–264-specific CTL could be induced to proliferate by incubation with splenocytes treated with PA plus LFn-OVA257–264. These findings imply that PA-LFn may serve as a general delivery vehicle for CTL epitopes in vivo and as a safe, efficient tool for the ex vivo expansion of patient-derived CTL for use in adoptive immunotherapy.
Occurrence of Virulence-Associated Properties inEnterobacter cloacae
Authors: Keller Rogéria , Pedroso Margareth Z. , Ritchmann Rosana , Silva Rosa M.
Published: December 2019

Enterobacter cloacaeis not a primary human pathogen but has been considered to be an important cause of nosocomial infections. Even so, there are almost no reports on its ability to produce recognized virulence-associated properties. In this study, we show that most of theE. cloacaestrains examined were resistant to serum bactericidal activity and were able to produce aerobactin and mannose-sensitive hemagglutinin, and all of them could adhere to and invade HEp-2 cells. SinceE. cloacaeis part of the normal intestinal floras of many individuals, we believe that infectious disease due to endogenousE. cloacaemight be a result of both host predisposing factors and the bacterial virulence determinants that we have detected in this survey.
Comparative Analysis of
Haemophilus influenzae hifA
(Pilin) Genes
Authors: Clemans Daniel L. , Marrs Carl F. , Patel Mayuri , Duncan Michelle , Gilsdorf Janet R.
Published: December 2019

Adherence of
Haemophilus influenzae
to epithelial cells plays a central role in colonization and is the first step in infection with this organism. Pili, which are large polymorphic surface proteins, have been shown to mediate the binding of
H. influenzae
to cells of the human respiratory tract. Earlier experiments have demonstrated that the major epitopes of
H. influenzae
pili are highly conformational and immunologically heterogenous; their subunit pilins are, however, immunologically homogenous. To define the extent of structural variation in pilins, which polymerize to form pili, the pilin genes (
hifA
) of 26 type a to f and 16 nontypeable strains of
H. influenzae
were amplified by PCR and subjected to restriction fragment length polymorphism (RFLP) analysis with
Alu
I and
Rsa
I. Six different RFLP patterns were identified. Four further RFLP patterns were identified from published
hifA
sequences from five nontypeable
H. influenzae
strains. Two patterns contained only nontypeable isolates; one of these contained
H. influenzae
biotype aegyptius strains F3031 and F3037. Another pattern contained predominantly
H. influenzae
type f strains. All other patterns were displayed by a variety of capsular and noncapsular types. Sequence analysis of selected
hifA
genes confirmed the 10 RFLP patterns and showed strong identity among representatives displaying the same RFLP patterns. In addition, the immunologic reactivity of pili with antipilus antisera correlated with the groupings of strains based on
hifA
RFLP patterns. Those strains that show greater reactivity with antiserum directed against
H. influenzae
type b strain M43 pili tend to fall into one RFLP pattern (pattern 3); while those strains that show equal or greater reactivity with antiserum directed against
H. influenzae
type b strain Eagan pili tend to fall in a different RFLP pattern (pattern 1). Sequence analysis of representative HifA pilins from typeable and nontypeable
H. influenzae
identified several highly conserved regions that play a role in bacterial pilus assembly and other regions with considerable amino acid heterogeneity. These regions of HifA amino acid sequence heterogeneity may explain the immunologic diversity seen in intact pili.
Encapsulation ofCryptococcus neoformanswith Glucuronoxylomannan Inhibits the Antigen-Presenting Capacity of Monocytes
Authors: Retini Cinzia , Vecchiarelli Anna , Monari Claudia , Bistoni Francesco , Kozel Thomas R.
Published: December 2019

This report examines the effect of the major capsular polysaccharide ofCryptococcus neoformans, glucuronoxylomannan (GXM), on the antigen-presenting capability of human monocytes treated with acapsular cells ofC. neoformans. We found that pretreatment of acapsular cryptococci with GXM downregulates, in a dose-dependent manner, the antigen-presenting capacity of monocytes, leading to reduced proliferative T-lymphocyte responses. Similar levels of suppression occurred when monocytes were exposed to encapsulated cryptococci or acapsular cryptococci that were pretreated with GXM. The magnitude of the T-cell response correlated with the ability of monocytes to ingest the yeast. Supernatant fluids from cocultures of monocytes and T cells cultured with encapsulated cryptococci contained higher levels of interleukin-10 (IL-10) than supernatant fluids of cells with acapsular cryptococci. Addition of anti-IL-10 monoclonal antibodies to the incubation medium of monocytes and T cells cultured with encapsulated cryptococci restored proliferative T-cell responses to levels observed during culture with acapsular cryptococci. Finally, treatment of monocytes with encapsulated cryptococci or GXM-treated acapsular cryptococci suppressed expression of class II major histocompatibility complex (MHC) molecules in a manner consistent with previous reports of IL-10-mediated suppression of class II MHC molecules and suppression of proliferative T-cell responses. These results suggest a link between GXM encapsulation, increased IL-10 synthesis by monocytes, decreased expression of class II MHC molecules on monocytes, and reduced proliferative T-cell responses.
Proteolytic Activation of the Interleukin-1β Precursor byCandida albicans
Authors: Beauséjour Annie , Grenier Daniel , Goulet Jean-Paul , Deslauriers Noëlla
Published: December 2019

Chronic inflammation rather than invasion is characteristic of some forms of superficial candidiasis such as denture stomatitis. We hypothesized thatCandida albicansmay play a critical role in the pathogenesis of inflammatory lesions observed in chronic candidiasis by activating the proinflammatory cytokine interleukin-1β (IL-1β) from epithelial stores of the precursor. The aim of this study was therefore to demonstrate the proteolytic cleavage and activation of the inactive precursor of IL-1β (pro-IL-1β) byC. albicans. After incubation of either blastospores or hyphae with the inactive precursor, proteolytic cleavage was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis Western immunoblotting analysis, and the biological activity of the cleavage products was tested in a bioassay. We report here that late-stationary-growth-phase blastospores as well as hyphae ofC. albicans, but not exponentially growing cells, can efficiently cleave pro-IL-1β to yield fragments of molecular masses compatible with mature biologically active IL-1β (17 to 19 kDa). Assays conducted in the presence of selected proteinase inhibitors suggest that the cleavage of pro-IL-1β involves the participation of one or more aspartyl proteinases. Cleavage products showed a dose-dependent IL-1β-like activity in a thymocyte proliferation bioassay, which was inhibited by anti-IL-1β neutralizing antibodies. The present data thus suggest a role forC. albicansproteinases in the activation and maintenance of the inflammatory response at epithelial surfaces.
Environmental Regulation of
Salmonella typhi
Invasion-Defective Mutants
Authors: Leclerc Guy J. , Tartera Carmen , Metcalf Eleanor S.
Published: December 2019

Salmonella typhi
is the etiologic agent of human typhoid. During infection,
S. typhi
adheres to and invades epithelial and M cells that line the distal ileum. To survive in the human host,
S. typhi
must overcome numerous complex extracellular and intracellular environments. Since relatively little is known about
S. typhi
pathogenesis, studies were initiated to identify
S. typhi
genes involved in the early steps of interaction with the host and to evaluate the environmental regulation of these genes. In the present study, Tn
phoA
mutagenesis was used to study these early steps. We isolated 16
Salmonella typhi
Tn
phoA
mutants that were defective for both adherence and invasion of the human small intestinal epithelial cell line Int407. Twelve of sixteen mutations were identified in genes homologous to the
S. typhimurium invG
and
prgH
genes, which are known to be involved in the type III secretion pathway of virulence proteins. Two additional insertions were identified in genes sharing homology with the
cpxA
and
damX
genes from
Escherichia coli
K-12, and two uncharacterized invasion-deficient mutants were nonmotile. Gene expression of Tn
phoA
fusions was examined in response to environmental stimuli. We found that the
cpxA
,
invG
, and
prgH
genes were induced when grown under conditions of high osmolarity (0.3 M NaCl). Expression of
invG
and
prgH
genes was optimal at pH 6.5 and strongly reduced at low pH (5.0). Transcription of both
invG
and
prgH
Tn
phoA
gene fusions was initiated during the late logarithmic growth phase and was induced under anaerobic conditions. Finally, we show that both
invG
and
prgH
genes appear to be regulated by DNA supercoiling, a mechanism influenced by environmental factors. These results are the first to demonstrate that in
S. typhi
, (i) the
prgH
and
cpxA
genes are osmoregulated, (ii) the
invG
gene is induced under low oxygen conditions, (iii) the
invG
gene is pH regulated and growth phase dependent, and (iv) the
prgH
gene appears to be regulated by DNA supercoiling. Since our experimental conditions were designed to mimic the in vivo environmental milieu, our results suggest that specific environmental conditions act as signals to induce the expression of
S. typhi
invasion genes.
Investigation of the Roles of Toxin-Coregulated Pili and Mannose-Sensitive Hemagglutinin Pili in the Pathogenesis of
Vibrio cholerae
O139 Infection
Authors: Tacket Carol O. , Taylor Ronald K. , Losonsky Genevieve , Lim Yu , Nataro James P. , Kaper James B. , Levine Myron M.
Published: December 2019

In this study, adult volunteers were fed
tcpA
and
mshA
deletion mutants of
V. cholerae
O139 strain CVD 112 to determine the role of toxin-coregulated pili (TCP) and mannose-sensitive hemagglutinin (MSHA) in intestinal colonization. Eight of 10 volunteers who received CVD 112 or CVD 112 Δ
mshA
shed the vaccine strains in their stools; the geometric mean peak excretion for both groups was 1.4 × 10
5
CFU/g of stool. In contrast, only one of nine recipients of CVD 112 Δ
tcpA
shed vibrios in his stool (
P
< 0.01); during the first 24 h after inoculation, 3 × 10
2
CFU/g was recovered from this volunteer. All recipients of CVD 112 and 8 (80%) of the recipients of CVD 112 Δ
mshA
developed at least a fourfold rise in vibriocidal titer after immunization. In contrast, only one (11%) of the nine recipients of CVD 112 Δ
tcpA
developed a fourfold rise in vibriocidal titer (
P
< 0.01). We conclude that TCP are an important colonization factor of
V. cholerae
O139 and probably of El Tor
V. cholerae
O1. In contrast, MSHA does not appear to promote intestinal colonization in humans.
Induction of a Gradual, Reversible Morphogenesis of Its Host’s Epithelial Brush Border by
Vibrio fischeri
Authors: Lamarcq Laurence H. , McFall-Ngai Margaret J.
Published: December 2019

Bacteria exert a variety of influences on the morphology and physiology of animal cells whether they are pathogens or cooperative partners. The association between the luminous bacterium
Vibrio fischeri
and the sepiolid squid
Euprymna scolopes
provides an experimental model for the study of the influence of extracellular bacteria on the development of host epithelia. In this study, we analyzed bacterium-induced changes in the brush borders of the light organ crypt epithelia during the initial hours following colonization of this tissue. Transmission electron microscopy of the brush border morphology in colonized and uncolonized hosts revealed that the bacteria effect a fourfold increase in microvillar density over the first 4 days of the association. Estimates of the proportions of bacterial cells in contact with host microvilli showed that the intimacy of the bacterial cells with animal cell surfaces increases significantly during this time. Antibiotic curing of the organ following colonization showed that sustained interaction with bacteria is essential for the retention of the induced morphological changes. Bacteria that are defective in either light production or colonization efficiency produced changes similar to those by the parent strain. Conventional fluorescence and confocal scanning laser microscopy revealed that the brush border is supported by abundant filamentous actin. However, in situ hybridization with β-actin probes did not show marked bacterium-induced increases in β-actin gene expression. These experiments demonstrate that the
E. scolopes-V. fischeri
system is a viable model for the experimental study of bacterium-induced changes in host brush border morphology.
Analysis of the Immune Responses of Mice to Infection withLeishmania braziliensis
Authors: DeKrey Gregory K. , Lima Hermenio C. , Titus Richard G.
Published: December 2019

Leishmania majorandLeishmania braziliensisboth cause cutaneous leishmaniasis, but the former kills BALB/c mice while the latter is killed by the mice. This killing ofL. braziliensisoccurred by a gamma interferon-dependent mechanism, potentially made possible by the observed lack of high interleukin-4 production.
Role of Tumor Necrosis Factor Alpha in the Host Response of Mice to Bacteremia Caused by Pneumolysin-DeficientStreptococcus pneumoniae
Authors: Benton Kimberly A. , VanCott John L. , Briles David E.
Published: December 2019

Pneumolysin-deficient mutant strains ofStreptococcus pneumoniaeare known to cause less-severe sepsis than wild-type pneumococcal strains that produce pneumolysin. This difference is associated with greater host resistance in mice infected with the pneumolysin-deficient strains. These studies show that the host resistance developed during the first 1 to 2 days after infection with a pneumolysin-deficient mutant strain is dependent on tumor necrosis factor alpha but is apparently independent of interleukin 1β (IL-1β) or IL-6. Survival beyond 5 days appeared to depend on the ability of the mice to produce IL-1β.
Enteric β-Defensin: Molecular Cloning and Characterization of a Gene with Inducible Intestinal Epithelial Cell Expression Associated with
Cryptosporidium parvum
Infection
Authors: Tarver Alan P. , Clark Douglas P. , Diamond Gill , Russell John P. , Erdjument-Bromage Hediye , Tempst Paul , Cohen Kenneth S. , Jones Douglas E. , Sweeney Ray W. , Wines Mary , Hwang Shirley , Bevins Charles L.
Published: December 2019

A growing body of evidence suggests that endogenous antibiotics contribute to the innate defense of mammalian mucosal surfaces. In the cow, β-defensins constitute a large family of antibiotic peptides whose members have been previously isolated from the respiratory and oral mucosa, as well as circulating phagocytic cells. A novel bovine genomic clone with sequence related to those of these α-defensins was isolated and characterized. The corresponding cDNA was isolated from a small intestinal library; its open reading frame predicts a deduced sequence of a novel β-defensin, which we designate enteric β-defensin (EBD). Northern blot analysis of a variety of bovine tissues revealed that EBD mRNA is highly expressed in the distal small intestine and colon, anatomic locations distinct from those for previously characterized β-defensins. EBD mRNA was further localized by in situ hybridization to epithelial cells of the colon and small intestinal crypts. Infection of two calves with the intestinal parasite
Cryptosporidium parvum
induced 5- and 10-fold increases above control levels of EBD mRNA in intestinal tissues. An anchored-PCR strategy was used to identify other β-defensin mRNAs expressed in the intestine. In addition to that of EBD, several low-abundance cDNAs which corresponded to other β-defensin mRNAs were cloned. Most of these clones encoded previously characterized β-defensins or closely related isoforms, but two encoded a previously uncharacterized prepro-β-defensin. Northern blot evidence supported that all of these other β-defensin genes are expressed at levels lower than that of the EBD gene in enteric tissue. Furthermore, some of these β-defensin mRNAs were abundant in bone marrow, suggesting that in enteric tissue their expression may be in cells of hematopoietic origin. Extracts of small intestinal mucosa obtained from healthy cows have numerous active chromatographic fractions as determined by an antibacterial assay, and one peptide was partially purified. The peptide corresponded to one of the low-abundance cDNAs. This study provides evidence of β-defensin expression in enteric tissue and that the mRNA encoding a major β-defensin of enteric tissue, EBD, is inducibly expressed in enteric epithelial cells. These findings support the proposal that β-defensins may contribute to host defense of enteric mucosa.
Purification and Expression of the Tf190 Adhesin inTritrichomonas foetus
Authors: Shaia Carl I. , Voyich Jovanka , Gillis Shaun J. , Singh B. N. , Burgess Donald E.
Published: December 2019

Bovine trichomoniasis is a sexually transmitted disease caused byTritrichomonas foetusand characterized by early embryo loss. The mechanism of this loss is not known, although the parasite is known to cause inflammation and to have the ability to kill host cells by a contact-dependent cytotoxic mechanism. Antibody specific for a 190,000-Da surface complex (Tf190) was previously shown to inhibit this adhesion. In this study we used immunoaffinity chromatography to purify Tf190 fromT. foetusin order to analyze its composition and examine its expression. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified Tf190 followed by silver staining revealed three components of Tf190. Western blotting and antibody-binding experiments showed that the 140- and 60-kDa bands were immunogenic. By using a battery of monoclonal antibodies (MAbs) periodate-sensitive epitopes were identified on Tf190, suggesting that these epitopes contained carbohydrate structures. Analyses of affinity-purified Tf190 by high-performance liquid chromatography and gas-liquid chromatography demonstrated the presence of the monosaccharides and lipids known to be prominent constituents of the lipophosphoglycan (LPG) ofT. foetus. Flow cytometry experiments on several isolates ofT. foetuswith Tf190-specific antibodies revealed that Tf190 was present on subpopulations of all isolates but that not all epitopes were present on every isolate. This pattern of reactivities on the different parasite isolates was confirmed by Western blots of whole-parasite extracts probed with MAbs and antiserum. These results suggest that although variation in the expression of epitopes of Tf190 occurs in different strains ofT. foetus, the Tf190 adhesion complex is widespread in different populations of the parasite. The data further suggest that immunogenic structures, important in the adhesion ofT. foetusto mammalian cells, are located in the LPG-like component of Tf190.
Endotoxin Binding and Elimination by Monocytes: Secretion of Soluble CD14 Represents an Inducible Mechanism Counteracting Reduced Expression of Membrane CD14 in Patients with Sepsis and in a Patient with Paroxysmal Nocturnal Hemoglobinuria
Authors: Hiki Naoki , Berger Dieter , Prigl Claus , Boelke Edwin , Wiedeck Heidemarie , Seidelmann Manuela , Staib Ludger , Kaminishi Michio , Oohara Takeshi , Beger Hans G.
Published: December 2019

Little is known about the role of peripheral blood mononuclear cells (PBMCs) in lipopolysaccharide (LPS) elimination. We studied the endotoxin elimination capacities (EEC) of PBMCs of 15 healthy volunteers, 13 patients with sepsis, and 1 patient suffering from paroxysmal nocturnal hemoglobinuria (PNH). Although expression of CD14, the best-characterized receptor for LPS to date, was reduced from 93.6% ± 0.8% in healthy subjects to 50.5% ± 6.5% in patients with sepsis and was 0.3% in a patient with septic PNH, EEC were found to be unchanged. There was no difference in the amount of tumor necrosis factor alpha (TNF-α) released by PBMCs of healthy donors and patients with sepsis. Anti-CD14 antibodies (MEM-18) completely suppressed EEC, binding of fluorescein isothiocyanate-labeled LPS to monocytes as determined by FACScan analysis, and TNF-α release in all three groups studied. The concentrations of soluble CD14 (sCD14) secreted by endotoxin-stimulated PBMCs from healthy donors and patients with sepsis amounted to 4.5 ± 0.4 and 20.1 ± 1.8 ng/ml, respectively. Based on our results, we suggest that PBMCs eliminate LPS by at least two different mechanisms; in healthy subjects, the membrane CD14 (mCD14) receptor is the most important factor for LPS elimination, while in patients with sepsis (including the septic state of PNH), increased sCD14 participates in LPS elimination. Secretion of sCD14 is strongly enhanced under conditions of low expression of mCD14 in order to counteract the reduction of mCD14 and maintain the function of monocytes. This sCD14 may substitute the role of mCD14 in LPS elimination during sepsis. The elimination of LPS by PBMCs correlates with the binding reaction and the secretion of TNF-α.
Relationship of Impairment of Schistosome 28-Kilodalton GlutathioneS-Transferase (GST) Activity to Expression of Immunity toSchistosoma mattheeiin Calves Vaccinated with RecombinantSchistosoma bovis28-Kilodalton GST
Authors: Grzych Jean-Marie , De Bont Jan , Liu Jinli , Neyrinck Jean-Loup , Fontaine Josette , Vercruysse Jozef , Capron André
Published: December 2019

Sera from calves vaccinated with the recombinantSchistosoma bovis-derived 28-kDa glutathioneS-transferase (28GST) and subsequently naturally or experimentally exposed toSchistosoma mattheeiwere studied for their content of specific immunoglobulin G (IgG) and IgA antibodies to recombinantS. bovis28GST as well as for their capacity to inhibit the enzymatic activity of the antigen. The results were analyzed in regard to the presence (natural infection) or absence (experimental infection) of a protective effect(s) (reductions in worm burden, egg load, fecal egg counts, and excretion of viable eggs) towardS. mattheeichallenge. Under such conditions, no differences in the IgG- and IgA-specific antibodies to recombinantS. bovis28GST or in the ability to block the catalytic function of the antigen between the two groups were recorded. Nevertheless, correlation analysis between the specific antibody responses to recombinantS. bovis28GST and the inhibition of GST activity suggested an association with IgG in experimentally infected vaccinated animals, while in naturally infected vaccinated calves, the inhibitory activity appeared to be linked to a greater degree with IgA. These results suggest that in contrast to schistosomiasis in humans, IgG antibodies in calves with schistosomiasis may exhibit inhibitory functions toward GST enzymatic activity or have a modulatory effect on IgA antibody properties. Furthermore, sera from animals immunized with recombinantS. bovis28GST recognized the nativeS. mattheei28GST and achieved comparable levels of inhibition of activity of recombinantS. bovis28GST andS. matthei28GST, indicating the presence of cross-reactive epitopes on these two molecules.
Expression of Virulence ofMycobacterium tuberculosiswithin Human Monocytes: Virulence Correlates with Intracellular Growth and Induction of Tumor Necrosis Factor Alpha but Not with Evasion of Lymphocyte-Dependent Monocyte Effector Functions
Authors: Silver Richard F. , Li Qing , Ellner Jerrold J.
Published: December 2019

We assessed the applicability of an in vitro model of low-level infection of human monocytes to the characterization of the virulence of strains of theMycobacterium tuberculosisfamily. Peripheral blood monocytes were infected at a 1:1 ratio with the virulentM. tuberculosisstrain H37Rv, the avirulentM. tuberculosisstrain H37Ra, and the attenuatedM. bovisstrain BCG. Both the percentages of cells infected by the three strains and the initial numbers of intracellular organisms were equivalent, as were levels of monocyte viability up to 7 days following infection. Intracellular growth reflected virulence, as H37Rv replicated in logarithmic fashion throughout the assay, BCG growth reached a plateau at 4 days, and H37Ra did not grow at all. The same patterns of growth were observed following infection of human alveolar macrophages with H37Rv and H37Ra. Monocyte production of tumor necrosis factor alpha was significantly higher following infection with virulent H37Rv than with either BCG or H37Ra. In contrast, there was no clear correlation of interleukin 10 production with virulence. Nonadherent cells of purified-protein-derivative-positive donors mediated equivalent degrees of reduction of the intracellular growth of H37Rv, BCG, and H37Ra. Low-level infection of human monocytes with H37Rv, BCG, and H37Ra thus provides an in vitro model for assessment of the virulence of theseM. tuberculosisfamily strains. Furthermore, it is suggested that the virulence of these strains is expressed primarily by their differing abilities to adapt to the intracellular environment of the mononuclear phagocyte.
Expression ofAnaplasma marginaleMajor Surface Protein 2 Variants during Persistent Cyclic Rickettsemia
Authors: French Dorothy M. , McElwain Terry F. , McGuire Travis C. , Palmer Guy H.
Published: December 2019

Anaplasma marginaleis an intraerythrocytic rickettsial pathogen of cattle in which infection persists for the life of the animal. PersistentA. marginaleinfection is characterized by repetitive rickettsemic cycles which we hypothesize reflect emergence ofA. marginaleantigenic variants. In this study, we determined whether variants of major surface protein 2 (MSP-2), a target of protective immunity encoded by a polymorphic multigene family, arise during persistent rickettsemia. By using a quantitative competitive PCR to identify rickettsemic cycles,msp-2transcripts expressed in vivo were isolated from peak rickettsemia of sequential cycles. Cloning and sequencing ofmsp-2cDNA revealed that genetic variants of MSP-2 emerge representing a minimum of four genetic variant types in each cycle during persistent infection. Two-color immunofluorescence using variant-specific antibody showed that emergence of MSP-2 variants resulted in expression of a minimum of three antigenic types of MSP-2 within one rickettsemic cycle. Therefore immune control of each cycle would require responses to an antigenically diverseA. marginalepopulation. These findings demonstrate that polymorphic MSP-2 variants emerge during cyclic rickettsemia in persistentA. marginaleinfection and suggest that emergent variants play an important role in persistence.
Transforming Growth Factor β and Immunosuppression in Experimental Visceral Leishmaniasis
Authors: Rodrigues Virmondes , Santana da Silva João , Campos-Neto Antonio
Published: December 2019

Hamsters infected withLeishmania donovanidevelop a disease similar to human kala-azar. They present hypergammaglobulinemia, and their T cells do not respond to parasite antigens. This unresponsiveness has been primarily ascribed to defects in antigen-presenting cells (APCs), because these cells are unable to stimulate proliferation of parasite-specific T cells from immunized animals. In this study, we show that APCs (adherent spleen cells) fromL. donovani-infected hamsters produce high levels of the inhibitory cytokine transforming growth factor β (TGF-β). Immunohistochemical studies with an anti-TGF-β monoclonal antibody (MAb) showed that this cytokine is abundantly produced in vivo by the spleen cells of infected animals. In addition, high levels of TGF-β are produced in vitro by infected hamster cells, either spontaneously or after stimulation with parasite antigen or lipopolysaccharide. Furthermore, in vivo-infected adherent cells obtained from spleens ofL. donovani-infected hamsters caused profound inhibition of the in vitro antigen-induced proliferative response of lymph node cells from hamsters immunized with leishmanial antigens. Moreover, this inhibition was totally abrogated by the anti-TGF-β MAb. These results suggest that the immunosuppression observed in visceral leishmaniasis is, at least in part, due to the abundant production of TGF-β during the course of the infection.
GroEL Heat Shock Protein of
Haemophilus ducreyi
: Association with Cell Surface and Capacity To Bind to Eukaryotic Cells
Authors: Frisk A. , Ison C. A. , Lagergård T.
Published: December 2019

The
Haemophilus ducreyi
homolog of GroEL, a 58.5-kDa heat shock protein (Hsp), is a dominant protein produced not only in response to heat stress but also under in vitro growth conditions. Extracellular localization of the 58.5-kDa Hsp was investigated by whole-cell enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy and in supernatants of washed bacteria by immunoblotting with a
Haemophilus ducreyi
GroEL-specific mouse monoclonal antibody (BB11). To investigate binding of the Hsp to eukaryotic cells, the 58.5-kDa Hsp was purified by ion-exchange and size exclusion chromatography; incubated with HEp-2 cells, HeLa cells, and human fibroblasts; and then analyzed by immunoblotting. Direct involvement of the 58.5-kDa Hsp in the adherence of
H. ducreyi
to HEp-2 cells was investigated by using an inhibition assay. An epitope of the 58.5-kDa Hsp was detected by whole-cell ELISA on all of the strains tested, suggesting that it is associated with the cell surface. This was also supported by immunoelectron microscopy results. In supernatants of washed bacteria, the 58.5-kDa Hsp was detected by immunoblotting after 10 h of cultivation. The 58.5-kDa Hsp bound to the eukaryotic cells tested but exerted only limited (about 20%) inhibition of
H. ducreyi
adherence to HEp-2 cells. These results demonstrate that the 58.5-kDa Hsp of
H. ducreyi
is associated with the bacterial surface, binds to eukaryotic cells, and partially influences
H. ducreyi
adherence to HEp-2 cells, indicating possible involvement of the 58.5-kDa Hsp in the attachment of bacteria to host cells and to each other.
Sulfated Polysaccharides and a Synthetic Sulfated Polymer Are Potent Inhibitors of
Chlamydia trachomatis
Infectivity In Vitro but Lack Protective Efficacy in an In Vivo Murine Model of Chlamydial Genital Tract Infection
Authors: Su Hua , Caldwell Harlan D.
Published: December 2019

Heparin, dextran sulfate, pentosan polysulfate, and a sulfated synthetic copolymer of acrylic acid and vinyl alcohol were shown to be potent inhibitors of
Chlamydia trachomatis
infectivity for cultured human epithelial cells. Despite their potent antichlamydial activity in vitro, neither heparin nor dextran sulfate was effective in inhibiting the infectivity of
C. trachomatis
in a murine model of chlamydial infection of the female genital tract.