bluuebunny/crossref_metadata_2025 huggingface dataset:
Chlamydia pneumoniae
Infection of Human Endothelial Cells Induces Proliferation of Smooth Muscle Cells via an Endothelial Cell-Derived Soluble Factor(s)
Authors: Coombes Brian K. , Mahony James B.
Published: December 2019

An association of
Chlamydia pneumoniae
with atherosclerosis and coronary heart disease has been determined epidemiologically and by the detection of
C. pneumoniae
organisms in atherosclerotic lesions in both humans and animal models of atherosclerosis. Previously, it has been shown that
C. pneumoniae
is capable of replicating in cell types found within atheromatous lesions, viz., endothelial cells, smooth muscle cells (SMC), and macrophages, yet the role of
C. pneumoniae
in the pathogenesis of atherosclerosis has not been determined. Since intimal thickening is a hallmark of atherosclerosis, we investigated whether
C. pneumoniae
infection of human umbilical vein endothelial cells (HUVEC) could induce the expression of a soluble factor(s) with mitogenic potential for SMC by using [
3
H]thymidine incorporation and direct cell counting. Conditioned medium harvested from HUVEC infected with
C. pneumoniae
stimulated SMC replication in a time- and dose-dependent fashion. Infection studies using various multiplicities of infection (MOIs) ranging from 0.001 to 1 demonstrated a dose-dependent production of the soluble factor(s). At an MOI of 1, SMC stimulation indices were 8.4 (
P
< 0.01) and 12.2 (
P
< 0.01) for conditioned media harvested at 24 and 48 h, respectively. To determine whether viable
C. pneumoniae
was required for production of the soluble factor(s), HUVEC were infected with heat-inactivated
C. pneumoniae
or with viable organisms in the presence of chloramphenicol. Both treatments produced stimulation indices similar to those for live
C. pneumoniae
in the absence of chloramphenicol (
P
> 0.05), indicating that the factor(s) was produced by HUVEC and not by
C. pneumoniae
and that signal transduction events following chlamydia endocytosis may be important in the production of a soluble factor(s). The ability of
C. pneumoniae
to elicit an endothelial cell-derived soluble factor(s) that stimulates SMC proliferation may be important in the pathogenesis of atherosclerosis.
Lipopolysaccharide Complexes withPasteurella haemolyticaLeukotoxin
Authors: Li Jun , Clinkenbeard Kenneth D.
Published: December 2019

The presence of lipopolysaccharide (LPS) in gram-negative bacterial repeats-in-toxin (RTX) toxin preparations, as well as the harsh conditions required to remove it, suggests that LPS may complex with RTX toxins. Concentrated culture supernatant (CCS) preparations of the RTX toxinPasteurella haemolyticaleukotoxin (LKT) contained LKT and LPS as the most prominent components, with LKT and LPS constituting ≈30 and 50% of the density of the silver-stained fraction on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. CCS LKT contained 3.69 ± 0.46 mg of LPS per mg of protein, which was estimated to indicate an LPS/LKT molar ratio of ≈60:1. Subjection of the CCS LKT to preparative SDS-PAGE resulted in separation of LPS from LKT as detected by silver-stained analytical SDS-PAGE; however, the LKT fraction (SDS-PAGE LKT) contained significant endotoxin activity as detected by theLimulusamebocyte lysate assay. Subjection of the SDS-PAGE LKT to a second preparative SDS-PAGE run resulted in a reduction of the LPS/LKT molar ratio to 1:20. The target cell specificity of LKT for bovine leukocytic cells was retained by the SDS-PAGE LKT, and isolated LPS at comparable concentrations to that in CCS LKT exhibited no leukolytic activity. Addition of isolated LPS back to SDS-PAGE LKT resulted in reconstitution of an LPS-LKT complex. Immediately following reconstitution of the LPS-LKT complex, there was minimal change in leukolytic activity of the complex, but following 9.5 h at temperatures from −135 to 37°C, the LPS-LKT complex exhibited increased leukolytic activity and thermal stability compared to SDS-PAGE LKT. Therefore, it appears that LPS complexes with LKT, resulting in enhanced and stabilized leukolytic activity.
Molecular Characterization and Human T-Cell Responses to a Member of a NovelMycobacterium tuberculosis mtb39Gene Family
Authors: Dillon Davin C. , Alderson Mark R. , Day Craig H. , Lewinsohn David M. , Coler Rhea , Bement Teresa , Campos-Neto Antonio , Skeiky Y. A. W. , Orme Ian M. , Roberts Alan , Steen Sean , Dalemans Wilfried , Badaro Roberto , Reed Steven G.
Published: December 2019

We have used expression screening of a genomicMycobacterium tuberculosislibrary with tuberculosis (TB) patient sera to identify novel genes that may be used diagnostically or in the development of a TB vaccine. Using this strategy, we have cloned a novel gene, termedmtb39a, that encodes a 39-kDa protein. Molecular characterization revealed thatmtb39ais a member of a family of three highly related genes that are conserved among strains ofM. tuberculosisandMycobacterium bovisBCG but not in other mycobacterial species tested. Immunoblot analysis demonstrated the presence of Mtb39A inM. tuberculosislysate but not in culture filtrate proteins (CFP), indicating that it is not a secreted antigen. This conclusion is strengthened by the observation that a human T-cell clone specific for purified recombinant Mtb39A protein recognized autologous dendritic cells infected with TB or pulsed with purified protein derivative (PPD) but did not respond toM. tuberculosisCFP. Purified recombinant Mtb39A elicited strong T-cell proliferative and gamma interferon responses in peripheral blood mononuclear cells from 9 of 12 PPD-positive individuals tested, and overlapping peptides were used to identify a minimum of 10 distinct T-cell epitopes. Additionally, mice immunized withmtb39aDNA have shown increased protection fromM. tuberculosischallenge, as indicated by a reduction of bacterial load. The human T-cell responses and initial animal studies provide support for further evaluation of this antigen as a possible component of a subunit vaccine forM. tuberculosis.
Chitinase Secretion by Encysting
Entamoeba invadens
and Transfected
Entamoeba histolytica
Trophozoites: Localization of Secretory Vesicles, Endoplasmic Reticulum, and Golgi Apparatus
Authors: Ghosh Sudip K. , Field Jessica , Frisardi Marta , Rosenthal Benjamin , Mai Zhiming , Rogers Rick , Samuelson John
Published: December 2019

Entamoeba histolytica
, the protozoan parasite that phagocytoses bacteria and host cells, has a vesicle/vacuole-filled cytosol like that of macrophages. In contrast, the infectious cyst form has four nuclei and a chitin wall. Here, anti-chitinase antibodies identified hundreds of small secretory vesicles in encysting
E. invadens
parasites and in
E. histolytica
trophozoites overexpressing chitinase under an
actin
gene promoter. Abundant small secretory vesicles were also identified with antibodies to the surface antigen Ariel and with a fluorescent substrate of cysteine proteinases. Removal of an N-terminal signal sequence directed chitinase to the cytosol. Addition of a C-terminal KDEL peptide, identified on amebic BiP, retained chitinase in a putative endoplasmic reticulum, which was composed of a few vesicles of mixed sizes. A putative Golgi apparatus, which was Brefeldin A sensitive and composed of a few large, perinuclear vesicles, was identified with antibodies to ADP-ribosylating factor and to ɛ-COP. We conclude that the amebic secretory pathway is similar to those of other eukaryotic cells, even if its appearance is somewhat different.
Variation in the
Bordetella pertussis
Virulence Factors Pertussis Toxin and Pertactin in Vaccine Strains and Clinical Isolates in Finland
Authors: Mooi Frits R. , He Qiushui , van Oirschot Hans , Mertsola Jussi
Published: December 2019

There is evidence that pertussis is reemerging in vaccinated populations. We have proposed, and provided evidence for, one explanation for this phenomenon in The Netherlands: antigenic divergence between vaccine strains and circulating strains. Finland has a pertussis vaccination history very similar to that of The Netherlands, and yet there is no evidence for an increase in the incidence of pertussis to the extent that it was observed in The Netherlands. A comparison of the
Bordetella pertussis
strains circulating in the two countries may shed light on the differences in pertussis epidemiology. Here we investigated whether temporal changes had occurred in pertussis toxin and pertactin types produced by the Finnish
B. pertussis
population. We show that strains isolated before 1964 produced the same pertussis toxin and pertactin variants as the vaccine strains. However, these vaccine types were replaced in later years, and in the 1990s most strains were distinct from the vaccine strains with respect to the two proteins. These trends are similar to those found in the Dutch
B. pertussis
population. An interesting difference between the contemporary Finnish and Dutch
B. pertussis
populations was found in the frequencies of pertactin variants, possibly explaining the distinct epidemiology of pertussis in the two countries.
Stability of
erp
Loci during
Borrelia burgdorferi
Infection: Recombination Is Not Required for Chronic Infection of Immunocompetent Mice
Authors: El Hage Nazira , Lieto Louis D. , Stevenson Brian
Published: December 2019

Borrelia burgdorferi
can persistently infect mammals despite their production of antibodies directed against bacterial proteins, including the Erp lipoproteins. We sequenced
erp
loci of bacteria reisolated from laboratory mice after 1 year of infection and found them to be identical to those of the inoculant bacteria. We conclude that recombination of
erp
genes is not essential for chronic mammalian infection.
Sequence Conservation of Glycerophosphodiester Phosphodiesterase amongTreponema pallidumStrains
Authors: Cameron Caroline E. , Castro Christa , Lukehart Sheila A. , Van Voorhis Wesley C.
Published: December 2019

Previous investigations have demonstrated that immunization withTreponema pallidumsubsp.pallidumglycerophosphodiester phosphodiesterase significantly protects rabbits from subsequent treponeme challenge. In this report, we show that the glycerophosphodiester phosphodiesterase amino acid sequence is conserved among 12 strains from a total of five pathogenic treponemes. The invariant nature of this immunoprotective antigen makes it an attractive candidate for inclusion in a universal subunit vaccine againstT. palliduminfection. In addition, these studies show a silent nucleotide substitution at position 579 of thegpdopen reading frame which is consistently observed in the non-T. pallidumsubsp.pallidumstrains. This sequence alteration introduces aPleI restriction site in the nonsyphilis strains and thus allows genetic differentiation fromT. pallidumsubsp.pallidumstrains.
Effects of Environmental pH on Membrane Proteins in
Borrelia burgdorferi
Authors: Carroll James A. , Garon Claude F. , Schwan Tom G.
Published: December 2019

Borrelia burgdorferi
, the causative agent of Lyme disease, alternates between the microenvironments of the tick vector,
Ixodes scapularis
, and a mammalian host. The environmental conditions the spirochete encounters during its infectious cycle are suspected to differ greatly in many aspects, including available nutrients, temperature, and pH. Here we identify alterations in the membrane protein profile, as determined by immunoblotting and two-dimensional nonequilibrium pH gradient gel electrophoresis (2D-NEPHGE), that occur in virulent
B. burgdorferi
B31 as the pH of the medium is altered. Initial comparisons of cultures incubated at pHs 6.0, 7.0, and 8.0 yielded alterations in the expression of seven membrane proteins as determined by probing with hyperimmune rabbit serum. Six of these membrane proteins (54, 45, 44, 43, 35, and 24 kDa) were either present in increased amounts in or solely expressed by cultures incubated at pHs 6.0 and 7.0. The 24-kDa protein that decreased in expression at pH 8.0 was identified as outer surface protein C (OspC). In addition, a 42-kDa membrane protein increased in amount in cultures incubated at pH 8.0. Similar changes were observed with serum from a mouse infected by tick bite, with the recognition of two additional bands (48 and 46 kDa) unique to pHs 6.0 and 7.0. When membrane fractions were analyzed by 2D-NEPHGE, at least 37 changes in the membrane protein profile between cells incubated at pHs 6.0, 7.0, and 8.0 were observed by immunoblotting and silver staining. Environmental cues such as pH may prove important in the regulation of virulence determinants and factors necessary for the adaptation of
B. burgdorferi
to the tick or mammalian microcosm.
Pili Binding to Asialo-GM1 on Epithelial Cells Can Mediate Cytotoxicity or Bacterial Internalization by
Pseudomonas aeruginosa
Authors: Comolli James C. , Waite Leslie L. , Mostov Keith E. , Engel Joanne N.
Published: December 2019

The interaction of
Pseudomonas aeruginosa
type IV pili and the glycosphingolipid asialo-GM1 (aGM1) can mediate bacterial adherence to epithelial cells, but the steps subsequent to this adherence have not been elucidated. To investigate the result of the interaction of pili and aGM1, we used polarized epithelial monolayers of Madin-Darby canine kidney (MDCK) cells in culture, which contained little detectable aGM1 on their apical surface but were able to incorporate exogenous aGM1. Compared to an untreated monolayer,
P. aeruginosa
PA103 displayed an eightfold increase in association with and fivefold more cytotoxicity toward MDCK cells pretreated with aGM1. Cytotoxicity of either carrier-treated or aGM1-treated monolayers required the type III secreted protein ExoU. Asialo-GM1 pretreatment of MDCK monolayers likewise augmented bacterial internalization of an isogenic invasive strain approximately fourfold. These increases were not seen in monolayers treated with GM1, the sialyated form of the glycolipid, and were inhibited by treatment with an antibody to aGM1. Also, the aGM1-mediated adhesion, cytotoxicity, and internalization required intact type IV pili since nonpiliated PA103 mutants were unaffected by aGM1 pretreatment of MDCK cells. These results demonstrate that epithelial cell injury and bacterial internalization can proceed from the same adhesin-receptor interaction, and they indicate that
P. aeruginosa
exoproducts solely determine the steps subsequent to adhesion.
Moesin Functions as a Lipopolysaccharide Receptor on Human Monocytes
Authors: Tohme Ziad N. , Amar Salomon , Van Dyke Thomas E.
Published: December 2019

Bacterial endotoxin (lipopolysaccharide [LPS]), a glycolipid found in the outer membranes of gram-negative bacteria, induces the secretion of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), and IL-6 by monocytes/macrophages. The secretion of these biologically active compounds leads to multiple pathological conditions, such as septic shock. There is substantial evidence that chronic exposure to LPS mediates, at least in part, the tissue destruction associated with gram-negative infection. CD14, a 55-kDa protein, has been identified as an LPS receptor. In conjunction with a serum protein, LPS binding protein (LBP), LPS-CD14 interactions mediate many LPS functions in the inflammatory response. However, CD14 lacks a cytoplasmic domain, or any known signal transduction sequence motif, suggesting the existence of another cell surface domain capable of transducing signals. In this paper, we report a second, CD14-independent LPS binding site, which, based on biological activity, appears to be a functional LPS receptor. Cross-linking experiments were performed to identify LPS binding sites. Two molecules were identified: a 55-kDa protein (CD14) and a second, 78-kDa band. Sequencing of the 78-kDa protein by mass spectroscopic analysis revealed 100% homology with moesin (membrane-organizing extension spike protein). Antibody to CD14 induced partial blocking of the LPS response. However, antimoesin monoclonal antibody completely blocked the LPS-induced TNF-α response in human monocytes, without blocking CD14 binding of LPS. Irrelevant isotype controls had no effect. Additional experiments were performed to evaluate the specificity of the antimoesin blocking. Separate experiments evaluated antimoesin effects on monocyte chemotaxis, IL-1 production in response to IL-1 stimulation, and TNF-α secretion in response toStaphylococcus aureusstimulation. Antimoesin blocked only LPS-mediated events. The data suggest that moesin functions as an independent LPS receptor on human monocytes. The role of moesin in transduction of CD14-mediated signals is discussed.
T-Cell Hyporesponsiveness Induced by Activated Macrophages through Nitric Oxide Production in Mice Infected withMycobacterium tuberculosis
Authors: Nabeshima Shigeki , Nomoto Mari , Matsuzaki Goro , Kishihara Kenji , Taniguchi Hatsumi , Yoshida Shin-ichi , Nomoto Kikuo
Published: December 2019

In active tuberculosis, T-cell response toMycobacterium tuberculosisis known to be reduced. In the course ofMycobacterium tuberculosisinfection in mice, we observed that T-cell proliferation in response toM. tuberculosispurified protein derivative (PPD) reached the maximum level on day 7, then declined to the minimal level on day 14, and persisted at a low level through day 28 postinfection. The frequency of PPD-specific CD4 T cells in the spleen on day 28 decreased to one-sixth on day 7. To further investigate the mechanism of this T-cell hyporesponsiveness, we next analyzed the suppressive activity of spleen macrophages on T-cell function. The nonspecific proliferative response of naive T cells and the PPD-specific proliferative response of T cells were suppressed by day 28 macrophages, but not by day 7 macrophages or naive macrophages. This reduction of proliferative response was restored by addition of nitric oxide synthesis inhibitor, NG-monoethyl-l-arginine monoacetate, but not by monoclonal antibody against interleukin 10 or transforming growth factor β. These data indicate that the macrophages from mice chronically infected withM. tuberculosissuppress T-cell response through production of nitric oxide, suggesting that nitric oxide-induced elimination mediated by activated macrophages may reduce the T-cell response and the number of mycobacterium-specific CD4 T cells in vivo.
Purification, Characterization, and Sequence Analysis of a Potential Virulence Factor from
Porphyromonas gingivalis
, Peptidylarginine Deiminase
Authors: McGraw Walker T. , Potempa Jan , Farley David , Travis James
Published: December 2019

The initiation and progression of adult-onset periodontitis has been associated with infection of the gingival sulcus by
Porphyromonas gingivalis
. This organism utilizes a multitude of virulence factors to evade host defenses as it establishes itself as one of the predominant pathogens in periodontal pockets. A feature common to many other oral pathogens is the production of ammonia due to its protective effect during acidic cleansing cycles in the mouth. Additionally, ammonia production by
P. gingivalis
has been proposed as a virulence factor due to its negative effects on neutrophil function. In this study, we describe the first purification of a peptidylarginine deiminase (PAD) from a prokaryote. PAD exhibits biochemical characteristics and properties that suggest that it may be a virulence agent. PAD deiminates the guanidino group of carboxyl-terminal arginine residues on a variety of peptides, including the vasoregulatory peptide-hormone bradykinin, to yield ammonia and a citrulline residue. The soluble protein has an apparent mass of 46 kDa, while the DNA sequence predicts a full-length protein of 61.7 kDa. PAD is optimally active at 55°C, stable at low pH, and shows the greatest activity above pH 9.0. Interestingly, in the presence of stabilizing factors, PAD is resistant to limited proteolysis and retains significant activity after short-term boiling. We propose that PAD, acting in concert with arginine-specific proteinases from
P. gingivalis
, promotes the growth of the pathogen in the periodontal pocket, initially by enhancing its survivability and then by assisting the organism in its circumvention of host humoral defenses.
Comparative Analysis and Immunological Characterization of theBorreliaBdr Protein Family
Authors: Zückert Wolfram R. , Meyer Jürg , Barbour Alan G.
Published: December 2019

Multiple circular and linear plasmids of Lyme disease and relapsing feverBorreliaspirochetes carry genes for members of the Bdr (Borreliadirect repeat) protein family. To define their common and divergent attributes, we first comprehensively compared the known homologs. Bdr proteins with predicted sizes ranging from 10.7 to 30.6 kDa formed five homology groups, based on variable numbers of short direct repeats in a central domain and diverse N- and C-terminal domains. In a further characterization, Western blots were probed with rabbit antisera raised against either of two purified recombinant Bdr proteins fromBorrelia burgdorferiB31. The results showed that antibodies cross-react and several Bdr paralogs 19.5 to 30.5 kDa in size are expressed by cultured strain B31 in a temperature-independent manner. In situ proteolysis, immunofluorescence, and growth inhibition assays indicated that Bdr proteins are not surface exposed. Distinct patterns of cross-reacting proteins of 17.5 to 33 kDa were also detected in otherB. burgdorferi,Borrelia garinii, andBorrelia afzeliistrains as well as in relapsing fever spirochetesBorrelia hermsiiandBorrelia turicatae. Last, we examined whether these proteins are antibody targets during Lyme disease. Analysis of 47 Lyme disease patient sera by immunoblotting and enzyme-linked immunosorbent assays showed that 24 (51%) and 20 (43%), respectively, had detectable antibodies to one or more of the Bdr proteins. Together, these data indicate that Bdr proteins constitute a family of cross-reactiveBorreliaproteins which are expressed in the course of Lyme disease and in vitro.
Role of Hemolysin BL in the Pathogenesis of Extraintestinal
Bacillus cereus
Infection Assessed in an Endophthalmitis Model
Authors: Callegan Michelle C. , Jett Bradley D. , Hancock Lynn E. , Gilmore Michael S.
Published: December 2019

Bacillus cereus
is a rare cause of serious human infection but, paradoxically, causes one of the most severe posttraumatic or endogenous infections of the eye, endophthalmitis, which frequently results in blindness. The virulence of
B. cereus
endophthalmitis historically has been attributed to toxin production. We therefore sought to examine the contribution of the dermonecrotic toxin, hemolysin BL, to the pathogenesis of
B. cereus
infection in an endophthalmitis system that is highly amenable to study. The pathogenesis of infection resulting from intravitreal injection of 10
2
CFU of either a clinical ocular isolate of
B. cereus
producing hemolysin BL (HBL
+
) or an isogenic mutant in this trait (HBL
−
) was assessed bacteriologically and by slit lamp biomicroscopy, electroretinography, histology, and inflammatory cell enumeration. Both HBL
+
and HBL
−
strains evoked severe intraocular inflammatory responses as early as 12 h postinfection, with complete loss of retinal responsiveness by 12 h. The infections caused by both strains spread of the infection to adjacent tissues by 18 h. No significant differences in intraocular bacterial growth (
P
≥ 0.21) or inflammatory changes (
P
≥ 0.21) were observed in eyes infected with either HBL
+
or HBL
−
strains during the course of infection. The level of retinal responsiveness was greater in HBL
−
infected eyes than in HBL
+
-infected eyes at 6 h only (
P
= 0.01). These results indicate that hemolysin BL makes no essential contribution to the severe and rapid course of infection in the endophthalmitis model.
Genomic Loci of the
Porphyromonas gingivalis
Insertion Element IS
1126
Authors: Dong Hong , Chen Tsute , Dewhirst Floyd E. , Fleischmann Robert D. , Fraser Claire M. , Duncan Margaret J.
Published: December 2019

The
Porphyromonas gingivalis
genome contains multiple copies of insertion element IS
1126
. When chromosomal DNA digests of different strains were probed with IS
1126
, between 25 and 35 hybridizing fragments per genome were detected, depending on the strain. Unrelated strains had very different restriction fragment length polymorphism (RFLP) patterns. When different laboratory copies of a specific strain were examined, the IS
1126
RFLP patterns were very similar but small differences were observed, indicating that element-associated changes had occurred during laboratory passage. Within the next year, genome sequencing, assembly, and annotation for
P. gingivalis
W83 will be completed. Because repetitive elements complicate the assembly of randomly sequenced DNA fragments, we isolated and sequenced the flanking regions of IS
1126
copies in strain W83. We also isolated and sequenced the flanking regions of IS
1126
copies in strain ATCC 33277 in order to compare insertion sites in phylogenetically divergent strains. We identified 37 new sequences flanking IS
1126
from strain ATCC 33277 and 30 from strain W83. The insertion element was found between genes except where it transposed into another insertion element. Examination of identifiable flanking genes or open reading frames indicated that the insertion sites were different in the two strains, except that both strains possess an insertion adjacent to the Lys-gingipain gene (J. P. Lewis and F. L. Macrina, Infect. Immun. 66:3035–3042, 1998). Most of the genes or sequences flanking IS
1126
in ATCC 33277 were present in W83 but were contiguous and not insertion element associated. Thus, where genes were identified in both strains, their order was maintained, indicating that the two genomes are organized similarly, but the loci of IS
1126
are different. In both strains, insertion element-associated duplicated target sites were lost from several copies of IS
1126
, providing evidence of homologous recombination between elements. Larger organizational differences between the genomes, such as deletions and inversions, may result from insertion element-mediated recombination events.
Identification and Characterization of TspA, a Major CD4+T-Cell- and B-Cell-StimulatingNeisseria-Specific Antigen
Authors: Kizil Goksel , Todd Ian , Atta Mustafa , Borriello S. Peter , Ait-Tahar Kamel , Ala’Aldeen Dlawer A. A.
Published: December 2019

In search for novel T-cell immunogens involved in protection against invasive meningococcal disease, we screened fractionated proteins ofNeisseria meningitidis(strain SD, B:15:P1.16) by using peripheral blood mononuclear cells (PBMCs) and specific T-cell lines obtained from normal individuals and patients convalescing fromN. meningitidisinfection. Proteins of iron-depleted meningococci produced higher PBMC proliferation indices than proteins of iron-replete organisms, indicating that iron-regulated proteins are T-cell immunogens. Insoluble proteins of the iron-depleted cells, which produced better T-cell stimulation than soluble ones, were fractionated by using sodium dodecyl sulfate-polyacrylamide gels and recovered as five fractions (F1 to F5) corresponding to decreasing molecular weight ranges. The proteins were purified (by elution and precipitation) or electroblotted onto nitrocellulose membranes (dissolved and precipitated) before use in further T-cell proliferation assays. One of the fractions (F1), containing high-molecular-mass proteins (>130 kDa), consistently showed the strongest T-cell proliferation responses in all of the T-cell lines examined. F1 proteins were subdivided into four smaller fractions (F1A to F1D) which were reexamined in T-cell proliferation assays, and F1C induced the strongest responses in patients’ T-cell lines. Rabbit polyclonal antibodies to F1C components were used to screen a genomic expression library ofN. meningitidis. Two major clones (C1 and C24) of recombinant meningococcal DNA were identified and fully sequenced. Sequence analysis showed that C24 (1,874 bp) consisted of a single open reading frame (ORF), which was included in clone C1 (2,778 bp). The strong CD4+T-cell-stimulating effect of the polypeptide product of this ORF (named TspA) was confirmed, using a patient T-cell line. Immunogenicity for B cells was confirmed by showing that convalescent patients’ serum antibodies recognized TspA on Western blots. Additional genetic sequence downstream of C24 was obtained from the meningococcal genomic sequence database (Sanger Centre), enabling the whole gene of 2,761 bp to be reconstructed. The DNA and deduced amino acid sequence data fortspAfailed to show significant homology to any known gene, except for a corresponding (uncharacterized) gene inNeisseria gonorrhoeaegenome sequences, suggesting thattspAis unique to the genusNeisseria. The DNA and deduced amino acid sequence of the second ORF of clone C1 showed significant homology togloA, encoding glyoxalase I enzyme, ofSalmonella typhimuriumandEscherichia coli. Thus, we have identified a novel neisserial protein (TspA) which proved to be a strong CD4+T-cell- and B-cell-stimulating immunogen with potential as a possible vaccine candidate.
Role of CD40 Ligand inMycobacterium aviumInfection
Authors: Hayashi Tomoko , Rao Savita P. , Meylan Pascal R. , Kornbluth Richard S. , Catanzaro Antonino
Published: December 2019

Mycobacterium aviumis a common opportunistic pathogen in immunocompromised patients such as those infected with human immunodeficiency virus. AlthoughM. aviumis an intracellular organism replicating predominantly in macrophages, disseminatedM. aviuminfection is seen in AIDS patients with CD4+cell counts of <50 cells/μl, suggesting a possible involvement of a T cell-macrophage interaction for the elimination ofM. avium. To determine whether CD40-CD40 ligand (CD40L) interactions play a role inM. aviuminfection, we studied the ability of CD40L to restrictM. aviumreplication in human monocyte-derived macrophages (MDM) in vitro. MDM were infected withM. aviumand cocultured with CD40L-transfected 293 cells for 7 days. Intracellular growth ofM. aviumin these MDM was assessed by colony counting. CD40L-expressing cells inhibited growth ofM. aviumin MDM by 86.5% ± 4.2% compared to MDM cultured with control cells. These findings were verified by assays using purified, soluble recombinant human CD40L (CD40LT). CD40LT (5 μg/ml) inhibited intracellular growth ofM. aviumby 76.9% ± 18.0% compared to cells treated with medium alone. Inhibition by CD40LT was reduced by monoclonal antibodies (MAbs) against CD40 and CD40L. The inhibitory effect of CD40LT was not accompanied by enhancement of interleukin-12 (IL-12) production byM. avium-infected MDM, while CD40L-expressing cells stimulated IL-12 production by these cells. Treatment ofM. avium-infected mice with MAb against murine CD40L resulted in recovery of larger numbers of organisms (0.8 to 1.0 log) from the spleens, livers, and lungs of these animals compared to infected mice which received normal immunoglobulin G. These results indicate that CD40-CD40L signaling may be an important step in host immune response againstM. aviuminfection.
Differential Regulation of Immune Responses by Highly and Weakly VirulentCryptococcus neoformansIsolates
Authors: Blackstock Rebecca , Buchanan Kent L. , Adesina Adekunle M. , Murphy J. W.
Published: December 2019

Early inflammatory responses, delayed-type hypersensitivity (DTH) responses, and cytokine profiles were studied in mice infected by the pulmonary route with either a highly virulent isolate (NU-2) or a weakly virulent isolate (184A) ofCryptococcus neoformans. After infection, NU-2 remained in the lungs and the capsule became more pronounced during the first 24 h, whereas 184A induced an immediate inflammatory reaction and was rapidly cleared from the lungs. Cryptococcal antigen (GXM) appeared in sera early after infection with NU-2 and increased over the entire observation period. There was no detectable GXM in sera from 184A-infected mice. BothC. neoformansisolates induced anticryptococcal cell-mediated immune responses, but the responses had different profiles. DTH in NU-2-infected mice appeared at day 15 after infection and waned by day 21, whereas DTH in 184A-infected mice was present by day 5 and continued to increase. T helper 1 (Th1) cytokines (interleukin 2 [IL-2] and gamma interferon) were made by spleen cells early after infection with either isolate. NU-2-infected mice lost their ability to produce these cytokines, but 184A-infected mice retained it. IL-4, a Th2 cytokine, was not detected in infected mice. The regulatory cytokine IL-10 was made by spleen cells early but not later after infection with the highly virulent isolate and was not produced by spleen cells from 184A-infected mice. IL-10-deficient mice survived an NU-2 infection significantly longer than wild-type mice, suggesting that IL-10 is important in down-regulating the protective immune response. The induction of anergy appears to be responsible for the inability of NU-2-infected mice to control aC. neoformansinfection.
Salmonella typhiFlagella Are Potent Inducers of Proinflammatory Cytokine Secretion by Human Monocytes
Authors: Wyant Timothy L. , Tanner Michael K. , Sztein Marcelo B.
Published: December 2019

The cytokine production patterns of human peripheral blood mononuclear cells (PBMC) in response toSalmonella typhiflagella (STF) were examined in culture supernatants of PBMC stimulated with STF. Consistent with previous findings in volunteers vaccinated witharoC aroDdeletion mutants ofS. typhi, PBMC from volunteers immunized with the licensed live Ty21aS. typhivaccine secreted gamma interferon following exposure to STF. Stimulation with STF induced rapid de novo synthesis of tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β), followed by IL-6 and IL-10. Trypsin treatment of STF abrogated their effects, while polymyxin B had no effect. Intracellular cytokine measurements of STF-stimulated PBMC revealed the existence of monocyte subpopulations that produce only TNF-α, IL-1β or both cytokines. Moreover, STF markedly decreased the percentage of CD14+cells. These data demonstrate that STF are powerful monocyte activators which may have important implications for vaccine development and for understanding the pathogenesis ofS. typhiinfection.
Pseudomonas aeruginosa
Gene Products PilT and PilU Are Required for Cytotoxicity In Vitro and Virulence in a Mouse Model of Acute Pneumonia
Authors: Comolli James C. , Hauser Alan R. , Waite Leslie , Whitchurch Cynthia B. , Mattick John S. , Engel Joanne N.
Published: December 2019

Type IV pili of the opportunistic pathogen
Pseudomonas aeruginosa
mediate twitching motility and act as receptors for bacteriophage infection. They are also important bacterial adhesins, and nonpiliated mutants of
P. aeruginosa
have been shown to cause less epithelial cell damage in vitro and have decreased virulence in animal models. This finding raises the question as to whether the reduction in cytotoxicity and virulence of nonpiliated
P. aeruginosa
mutants are primarily due to defects in cell adhesion or loss of twitching motility, or both. This work describes the role of PilT and PilU, putative nucleotide-binding proteins involved in pili function, in mediating epithelial cell injury in vitro and virulence in vivo. Mutants of
pilT
and
pilU
retain surface pili but have lost twitching motility. In three different epithelial cell lines,
pilT
or
pilU
mutants of the strain PAK caused less cytotoxicity than the wild-type strain but more than isogenic, nonpiliated
pilA
or
rpoN
mutants. The
pilT
and
pilU
mutants also showed reduced association with these same epithelial cell lines compared both to the wild type, and surprisingly, to a
pilA
mutant. In a mouse model of acute pneumonia, the
pilT
and
pilU
mutants showed decreased colonization of the liver but not of the lung relative to the parental strain, though they exhibited no change in the ability to cause mortality. These results demonstrate that pilus function mediated by PilT and PilU is required for in vitro adherence and cytotoxicity toward epithelial cells and is important in virulence in vivo.
The Nature of the Attenuation ofSalmonella typhimuriumStrains Expressing Human Papillomavirus Type 16 Virus-Like Particles Determines the Systemic and Mucosal Antibody Responses in Nasally Immunized Mice
Authors: Benyacoub Jalil , Hopkins Sally , Potts Alexandra , Kelly Sandra , Kraehenbuhl Jean-Pierre , Curtiss Roy , De Grandi Pierre , Nardelli-Haefliger Denise
Published: December 2019

We have recently shown by using a recombinantSalmonella typhimuriumPhoPcstrain in mice the feasibility of using aSalmonella-based vaccine to prevent infection by the genital human papillomavirus type 16 (HPV16). Here, we compare the HPV16-specific antibody responses elicited by nasal immunization with recombinantS. typhimuriumstrains harboring attenuations that, in contrast to PhoPc, are suitable for human use. For this purpose, χ4989 (Δcya Δcrp) and χ4990 [ΔcyaΔ(crp-cdt)] were constructed in the ATCC 14028 genetic background, and comparison was made with the isogenic PhoPcand PhoP−strains. Although the levels of expression of HPV16 virus-like particle (VLP) were similar in all strains, only PhoPcHPV16 induced sustained specific antibody responses after nasal immunization, while all strains induced high antibody responses with a single nasal immunization when an unrelated viral hepatitis B core antigen was expressed. The level of the specific antibody responses induced did not correlate with the number of recombinant bacteria surviving in various organs 2 weeks after immunization. Our data suggest that the immunogenicity of attenuatedSalmonellavaccine strains does not correlate with either the number of persisting bacteria after immunization or the levels of in vitro expression of the antigen carried. Rather, the PhoPcphenotype appears to provide the unique ability inSalmonellato induce immune responses against HPV16 VLPs.
Mucosal and Systemic Immune Responses in Humans after Primary and Booster Immunizations with Orally Administered Invasive and Noninvasive Live Attenuated Bacteria
Authors: Viret Jean-Francois , Favre Didier , Wegmüller Bernhard , Herzog Christian , Que John U. , Cryz Stanley J. , Lang Alois B.
Published: December 2019

The mucosal and systemic immune responses after primary and booster immunizations with two attenuated live oral vaccine strains derived from a noninvasive (Vibrio cholerae) and an invasive (Salmonella typhi) enteric pathogen were comparatively evaluated. Vaccination withS. typhiTy21a elicited antibody-secreting cell (ASC) responses specific forS. typhiO9, 12 lipopolysaccharide (LPS), as well as significant increases in levels of immunoglobulin G (IgG) and IgA antibodies to the same antigen in serum. A strong systemic CD4+T-helper type 1 cell-mediated immune (CMI) response was also induced. In contrast to results with Ty21a, no evidence of a CMI response was obtained after primary immunization withV. choleraeCVD 103-HgR in spite of the good immunogenicity of the vaccine. Volunteers who received a single dose of CVD 103-HgR primarily developed an IgM ASC response against whole vaccine cells and purifiedV. choleraeInaba LPS, and seroconversion of serum vibriocidal antibodies occurred in four of five subjects. Serum IgG anti-cholera toxin antibody titers were of lower magnitude. For both live vaccines, the volunteers still presented significant local immunity 14 months after primary immunization, as revealed by the elevated baseline antibody titers at the time of the booster immunization and the lower ASC, serum IgG, and vibriocidal antibody responses after the booster immunization. These results suggest that local immunity may interfere with colonization of the gut by both vaccine strains at least up to 14 months after basis immunization. Interestingly, despite a low secondary ASC response, Ty21a was able to boost both humoral (anti-LPS systemic IgG and IgA) and CMI responses. Evidence of a CMI response was also observed for one of three volunteers given a cholera vaccine booster dose. The direct comparison of results with two attenuated live oral vaccine strains in human volunteers clearly showed that the capacity of the vaccine strain to colonize specific body compartments conditions the pattern of vaccine-induced immune responses.
Identification of Functional Domains of
Bordetella
Dermonecrotizing Toxin
Authors: Kashimoto Takashige , Katahira Jun , Cornejo Willian R. , Masuda Minako , Fukuoh Atsushi , Matsuzawa Takeshi , Ohnishi Takahiro , Horiguchi Yasuhiko
Published: December 2019

Bordetella
dermonecrotizing toxin (DNT) stimulates the assembly of actin stress fibers and focal adhesions by deamidating Gln63 of the small GTPase Rho. To clarify the functional and structural organization of DNT, we cloned and sequenced the DNT gene and examined the functions of various DNT mutants. Our analyses of the nucleotide and amino acid sequences revealed that the start codon of the DNT gene is a GTG triplet located 39 bp upstream of the reported putative initiation ATG codon; consequently, DNT contains an additional 13 amino acids at its N-terminal end. All of the N-terminally truncated mutants were found to modify Rho. The shortest fragment of DNT possessing the Rho modification activity consists of amino acids from Ile1176 to the C-terminal end. This fragment overlaps the region homologous to
Escherichia coli
cytotoxic necrotizing factors (CNFs), which show activity similar to that of DNT. The introduction of a mutation at Cys1305 located in the highly conserved region between CNFs and DNT eliminated the activity, indicating that this domain is the catalytic center of DNT. The N-terminal fragment (1 to 531) of DNT failed to modify Rho but reduced the DNT-induced polynucleation in MC3T3-E1 cells when simultaneously added with the holotoxin, suggesting competitive inhibition in the receptor-binding or internalizing step. Our finding that DNT consists of an N-terminal receptor-binding and/or internalizing domain and a C-terminal catalytically active domain may facilitate analysis of the overall action of the toxin on the mammalian target cells.
Analysis of the Immunological Responses to Transferrin and Lactoferrin Receptor Proteins from
Moraxella catarrhalis
Authors: Yu Rong-hua , Bonnah Robert A. , Ainsworth Samuel , Schryvers Anthony B.
Published: December 2019

Moraxella catarrhalis
expresses surface receptor proteins that specifically bind host transferrin (Tf) and lactoferrin (Lf) in the first step of the iron acquisition pathway. Acute- and convalescent-phase antisera from a series of patients with
M. catarrhalis
pulmonary infections were tested against Tf and Lf receptor proteins purified from the corresponding isolates. After the purified proteins had been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, we observed strong reactivity against Tf-binding protein B (TbpB; also called OMP1) and Lf-binding protein B (LbpB) but little or no reactivity against Tf-binding protein A (TbpA) or Lf-binding protein A (LbpA), using the convalescent-phase antisera. Considerable antigenic heterogeneity was observed when TbpBs and LbpBs isolated from different strains were tested with the convalescent-phase antisera. Comparison to the reactivity against electroblotted total cellular proteins revealed that the immune response against LbpB and TbpB constitutes a significant portion of the total detectable immune response to
M. catarrhalis
proteins. Preparations of affinity-isolated TbpA and LbpA reacted with convalescent-phase antisera in a solid-phase binding assay, but blocking with soluble TbpB, soluble LbpB, or extracts from an LbpA
−
mutant demonstrated that this reactivity was attributed to contaminants in the TbpA and LbpA preparations. These studies demonstrate the immunogenicity of
M. catarrhalis
TbpB and LbpB in humans and support their potential as vaccine candidates.
Genetic Characterization of a Tn5-Disrupted Glycosyltransferase Gene Homolog inBrucella abortusand Its Effect on Lipopolysaccharide Composition and Virulence
Authors: McQuiston J. R. , Vemulapalli R. , Inzana T. J. , Schurig G. G. , Sriranganathan N. , Fritzinger D. , Hadfield T. L. , Warren R. A. , Snellings N. , Hoover D. , Halling S. M. , Boyle S. M.
Published: December 2019

We constructed a rough mutant ofBrucella abortus2308 by transposon (Tn5) mutagenesis. Neither whole cells nor extracted lipopolysaccharide (LPS) from this mutant, designated RA1, reacted with aBrucellaO-side-chain-specific monoclonal antibody (MAb), Bru-38, indicating the absence of O-side-chain synthesis. Compositional analyses of LPS from strain RA1 showed reduced levels of quinovosamine and mannose relative to the levels in the parental, wild-type strain, 2308. We isolated DNA flanking the Tn5insertion in strain RA1 by cloning a 25-kbXbaI genomic fragment into pGEM-3Z to create plasmid pJM6. Allelic exchange of genomic DNA inB. abortus2308 mediated by electroporation of pJM6 produced kanamycin-resistant clones that were not reactive with MAb Bru-38. Southern blot analysis of genomic DNA from these rough clones revealed Tn5in a 25-kbXbaI genomic fragment. A homology search with the deduced amino acid sequence of the open reading frame disrupted by Tn5revealed limited homology with various glycosyltransferases. ThisB. abortusgene has been namedwboA. Transformation of strain RA1 with a broad-host-range plasmid bearing the wild-typeB. abortus wboAgene resulted in the restoration of O-side-chain synthesis and the smooth phenotype.B. abortusRA1 was attenuated for survival in mice. However, strain RA1 persisted in mice spleens for a longer time than theB. abortusvaccine strain RB51, but as expected, neither strain induced antibodies specific for the O side chain.
The Cysteine-Rich Region of theEntamoeba histolyticaAdherence Lectin (170-Kilodalton Subunit) Is Sufficient for High-Affinity Gal/GalNAc-Specific Binding In Vitro
Authors: Pillai Dylan R. , Wan Peter S. K. , Yau Yvonne C. W. , Ravdin Jonathan I. , Kain Kevin C.
Published: December 2019

Adherence ofEntamoeba histolyticatrophozoites to colonic mucin, epithelium, and other target cells is mediated by the amebic Gal/GalNAc lectin. We constructed in vitro expression vectors containing full-length (residues 1 to 1280), cysteine-poor (1 to 353 and 1 to 480), and cysteine-rich (356 to 1143 and 480 to 900) fragments of the gene encoding the heavy subunit of the adherence lectin,hgl2. In vitro transcription followed by translation using a nuclease-treated rabbit reticulocyte lysate system was carried out. Immunoreactivity of in vitro-translated Hgl2 was confirmed by immunoprecipitation with lectin-specific monoclonal antibodies (MAbs) 1G7 and 8A3, which recognize linear epitopes. Protein disulfide isomerase (PDI) refolding of Hgl2 enhanced immunoreactivity (P< 0.05) with the conformationally dependent MAb 3F4. Binding of PDI-refolded full-length (P< 0.001) and cysteine-rich (P= 0.005) Hgl2 to CHO cells was galactose dependent and competitively inhibited by native hololectin (50% inhibitory concentration of 39.6 ng/ml). The cysteine-poor region (1 to 353) did not bind CHO cells. Both full-length (1 to 1280) and cysteine-rich (356 to 1143) Hgl2 bound the glyconeoconjugate GalNAc19BSA in a GalNAc-specific manner. The smaller cysteine-rich fragment (480 to 900) also exhibited GalNAc-specific binding but to a lesser extent (P< 0.05) than residues 1 to 1280 and 356 to 1143. Neither the cysteine-poor fragment (1 to 480), luciferase (protein control), nor control translation reactions (withouthgl2lectin mRNA) bound GalNAc19BSA. Binding to GalNAc19BSA was shown to be dependent on the concentration of GalNAc19BSA coated in each well or35S-lectin added (KD= 0.85 ± 0.37 pM). Binding was competitively inhibited by the terminal GalNAc-containing glycoprotein asialofetuin (P< 0.005). Taken together, these data provide direct evidence that the cysteine-rich region of the Gal/GalNAc lectin heavy subunit contains one or more carbohydrate-binding domains.