bluuebunny/crossref_metadata_2025 huggingface dataset:
Construction and Characterization of
Moraxella catarrhalis
Mutants Defective in Expression of Transferrin Receptors
Authors: Luke Nicole R. , Campagnari Anthony A.
Published: December 2019

We have previously reported the construction of an isogenic mutant defective in expression of OmpB1, the TbpB homologue, in
Moraxella catarrhalis
7169. In this report, we have extended these studies by constructing and characterizing two new isogenic mutants in this clinical isolate. One mutant is defective in expression of TbpA, and the other mutant is defective in expression of both TbpA and TbpB. These isogenic mutants were confirmed by using PCR analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and sequencing. In vitro growth studies, comparing all three mutants, demonstrated that the
tbpA
mutant and the
tbpAB
mutant were severely limited in their ability to grow with human holotransferrin as the sole source of iron. In contrast, the
ompB1
(
tbpB
) mutant was capable of utilizing iron from human transferrin, although not to the extent of the parental strain. While affinity chromatography with human holotransferrin showed that each Tbp was capable of binding independently to transferrin, solid-phase transferrin binding studies using whole cells demonstrated that the
tbpA
mutant exhibited binding characteristics similar to those seen with the wild-type bacteria. However, the
ompB1
(
tbpB
) mutant exhibited a diminished capacity for binding transferrin, and no binding was detected with the double mutant. These data suggest that the
M. catarrhalis
TbpA is necessary for the acquisition of iron from transferrin. In contrast, TbpB is not essential but may serve as a facilitory protein that functions to optimize this process. Together these mutants are essential to provide a more thorough understanding of iron acquisition mechanisms utilized by
M. catarrhalis
.
Enhanced Macrophage Resistance toPseudomonasExotoxin A Is Correlated with Decreased Expression of the Low-Density Lipoprotein Receptor-Related Protein
Authors: Laithwaite James E. , Benn Sally J. , Yamate Jyoji , FitzGerald David J. , LaMarre Jonathan
Published: December 2019

Cellular intoxification by exotoxin A ofPseudomonas aeruginosa(PEA) begins when PEA binds to its cellular receptor, the low-density lipoprotein receptor-related protein (LRP). This receptor is particularly abundant on macrophages. We hypothesize here that inducible changes in cellular expression levels of the LRP represent an important mechanism by which macrophage susceptibility to PEA is regulated by the host. We have examined the effect of lipopolysaccharide (LPS) on LRP expression and PEA sensitivity in the macrophage-like cell line HS-P. Using a [3H]leucine incorporation assay to measure inhibition of protein synthesis, we have demonstrated that HS-P macrophages are highly sensitive to PEA and that PEA toxicity is decreased by the LRP antagonist receptor-associated protein. LPS pretreatment decreases HS-P PEA sensitivity in a time- and dose-dependent manner. The dose of toxin required to inhibit protein synthesis by 50% increased from 11.3 ± 1.2 ng/ml in untreated cells to 25.7 ± 2.0 ng/ml in cells treated with LPS. In pulse experiments, involving brief exposure to saturating concentrations of PEA, [3H]leucine incorporation was more than threefold higher in cells pretreated with LPS than in untreated macrophages. These changes in HS-P PEA sensitivity following LPS treatment were consistently associated with a fivefold decrease in HS-P LRP mRNA expression as measured by Northern blot analysis and a three-and-a-half-fold decrease in HS-P LRP-specific ligand internalization as determined by activated α2-macroglobulin internalization studies. These data demonstrate for the first time that modulation of LRP levels by extracellular signaling molecules can alter cellular PEA sensitivity.
Reactive Nitrogen and Oxygen Species Ameliorate Experimental Cryptosporidiosis in the Neonatal BALB/c Mouse Model
Authors: Leitch Gordon J. , He Qing
Published: December 2019

Four-day-old BALB/c mice were infected by the oral administration of 50,000Cryptosporidium parvumoocysts, and the resulting infection was scored histologically and by counting colonic oocysts. Infection occurred in the ileum and proximal colon (but not duodenum and jejunum), peaked on days 14 to 18, and was cleared between days 24 and 30. Nitric oxide (NO) appeared to play a protective role in this model as evidenced by the facts that plasma nitrite and nitrate levels increased during the period of peak parasitosis; immunohistochemically detected inducible nitric oxide synthase (iNOS) was increased in the ileum and colon enterocytes of infected animals; the NOS inhibitorl-N-iminoethyl lysine orN-nitro-l-arginine methyl ester (L-NAME) decreased the elevated plasma nitrite and nitrate levels while exacerbating the infection and increasing oocyst shedding; administration of a NO donor,S-nitroso-N-penicillamine, reduced oocyst and infection scores; and neonatal iNOS knockout mice exhibited a slightly longer infection than control animals. The oral administration of oocysts to L-NAME-treated BALB/c mice, but not control animals, between 24 and 40 days old resulted in the fecal excretion of oocysts 1 week later. Administration of the antioxidant ascorbic acid also exacerbated theC. parvuminfection, suggesting a protective role for reactive nitrogen and/or reactive oxygen compounds, while administration of the superoxide scavenger superoxide dismutase exacerbated the infection. Taken together these data suggest that both reactive nitrogen and reactive oxygen species play protective roles in experimental cryptosporidiosis.
Shiga Toxin-Producing
Escherichia coli
Can Impair T84 Cell Structure and Function without Inducing Attaching/Effacing Lesions
Authors: Li Zhe , Elliott Elizabeth , Payne Jacqueline , Isaacs Jacqui , Gunning Peter , O'loughlin Edward V.
Published: December 2019

Enteropathogenic
Escherichia coli
(EPEC) intimately adhere to epithelial cells producing cytoskeletal rearrangement with typical attaching and effacing lesions and altered epithelial barrier and transport function. Since EPEC and Shiga toxin-producing
E. coli
(STEC) share similar genes in the “locus for enterocyte effacement” (LEE) thought to cause these changes, it has been assumed that STEC shares similar pathogenic mechanisms with EPEC. The aims of this study were to compare the effects of EPEC and STEC on bacterial-epithelial interactions and to examine changes in epithelial function. T84 monolayers were infected with STEC O157:H7 (wild strain EDL 933 or non-toxin-producing strain 85/170), EPEC (strain E2348/69), or HB101 (nonpathogenic) and studied at various times after infection. EPEC bound more avidly than EDL 933, but both strains exhibited greater binding than HB101. Attaching and effacing lesions and severe disruption to the actin cytoskeleton were observed in EPEC by 3 h postinfection but not in EDL 933 or HB101 at any time point. EPEC and EDL 933 increased monolayer permeability to [
3
H]mannitol 5- to 10-fold. In contrast to EPEC, EDL 933 completely abolished secretagogue-stimulated anion secretion as assessed under voltage clamp conditions in Ussing chambers. Several other STEC strains induced changes similar to those of EDL 933. In conclusion, STEC impairs epithelial barrier function and ion transport without causing major disruption to the actin cytoskeleton. Pathogenic factors other than products of LEE may be operant in STEC.
Increased Interleukin-1 (IL-1) and Imbalance between IL-1 and IL-1 Receptor Antagonist during Acute Inflammation in Experimental Shigellosis
Authors: Arondel Josette , Singer Monique , Matsukawa Akihiro , Zychlinsky Arturo , Sansonetti Philippe J.
Published: December 2019

Infection by the enteric bacterial pathogenShigellaresults in intense mucosal inflammation and destruction of the colonic and rectal epithelium in infected humans. Initial bacterial translocation occurs through the follicle-associated epithelium. Previous experiments suggest that interleukin-1 (IL-1) is crucial to trigger inflammation, particularly in the follicular zones. During the first 4 hours of infection in a rabbit ligated-loop model of intestinal invasion, there are two salient characteristics: (i) a high concentration of IL-1α and IL-1β, both in infected Peyer's patch tissue and in the corresponding efferent mesenteric blood, and (ii) a very low level of expression of IL-1 receptor antagonist (IL-1ra). These may reflect a combination of regulation of expression and secretion of IL-1α, IL-1β, and IL-1ra by both resident and recruited phagocytes and the induction of mononuclear phagocyte apoptosis byShigella. This low IL-1ra/IL-1 ratio likely accounts for the rapid, uncontrolled inflammation characteristic of shigellosis.
Enterococcus faecalisBearing Aggregation Substance Is Resistant to Killing by Human Neutrophils despite Phagocytosis and Neutrophil Activation
Authors: Rakita Robert M. , Vanek Natalie N. , Jacques-Palaz Karen , Mee Mee , Mariscalco M. Michele , Dunny Gary M. , Snuggs Mark , Van Winkle W. Barry , Simon Scott I.
Published: December 2019

Enterococcus faecalisaggregation substance (AS) mediates efficient bacterium-bacterium contact to facilitate plasmid exchange as part of a bacterial sex pheromone system. We have previously determined that AS promotes direct, opsonin-independent binding ofE. faecalisto human neutrophils (PMNs) via complement receptor type 3 and other receptors on the PMN surface. We have now examined the functional consequences of this bacterium-host cell interaction. AS-bearingE. faecaliswas phagocytosed and internalized by PMNs, as determined by deconvolution fluorescence microscopy. However, these bacteria were not killed by PMNs, and internalized bacteria excluded propidium iodide, indicating intact bacterial membranes. Resistance to killing occurred despite activation of PMNs, as indicated by an increase in both functional and total surface Mac-1 expression, shedding ofl-selectin, and an increase in PMN extracellular superoxide and phagosomal oxidant production. Deconvolution fluorescence microscopy also revealed that phagosomes containing AS-bearing bacteria were markedly larger than phagosomes containing opsonizedE. faecalis, suggesting that some modification of phagosomal maturation may be involved in AS-induced resistance to killing. PMN phagosomal pH was significantly higher after ingestion of nonopsonized AS-bearingE. faecalisthan after that of opsonized bacteria. The novel ability of AS to promote intracellular survival ofE. faecalisinside PMNs suggests that AS may be a virulence factor used by strains ofE. faecalis.
Phenotypic Switching in
Cryptococcus neoformans
Results in Changes in Cellular Morphology and Glucuronoxylomannan Structure
Authors: Fries Bettina C. , Goldman David L. , Cherniak Robert , Ju Rujin , Casadevall Arturo
Published: December 2019

Cryptococcus neoformans
strains exhibit variability in their capsular polysaccharide, cell morphology, karyotype, and virulence, but the relationship between these variables is poorly understood. A hypovirulent
C. neoformans
24067A isolate, which usually produces smooth (SM) colony types, was found to undergo phenotypic switching and to produce wrinkled (WR) and pseudohyphal (PH) colony types at frequencies of approximately 10
−4
to 10
−5
when plated on Sabouraud agar. Cells from these colony types had large polysaccharide capsules and PH morphology, respectively. Scanning electron microscopy showed that different colony types were the result of altered cellular packing in the colony. Phenotypic switching was associated with quantitative and qualitative changes in capsular polysaccharide. Specifically, the glucuronoxylomannan (GXM) of the WR polysaccharide differed in the proportion of structural reporter groups and in increased xylose residue content linked at the 4 to 0 position. The relative virulence of the colony types was WR > PH > SM, as measured by CFU in rat lungs after intratracheal infection. Karyotype instability was observed in strain 24067A and involved primarily two chromosomes. Colonies with an alternative colony type exhibited more karyotype changes, which did not revert to the original karyotype in reverted colonies. In summary, this study revealed that phenotypic switching in
C. neoformans
(i) can produce WR colonies consisting of cells with either large capsule or PH morphology, (ii) is associated with production of structurally different GXM, (iii) is commonly associated with karyotype changes, (iv) can produce cells of PH morphology, and (v) can increase the virulence of a strain. Hence, phenotypic switching is an adaptive mechanism linked to virulence that can generate cell types with very different biological characteristics.
Chlamydia trachomatis(Mouse Pneumonitis Strain) Induces Cardiovascular Pathology following Respiratory Tract Infection
Authors: Fan Yijun , Wang Shuhe , Yang Xi
Published: December 2019

Chlamydia, especiallyChlamydia pneumoniae, infection is closely associated with human cardiovascular diseases. Thus far, however, few experimental studies have been carried out to investigate whether naturalC. trachomatisinfection can induce cardiovascular pathological changes. In this article, we report that pulmonary infection withC. trachomatismouse pneumonitis strain (MoPn) can induce myocardial and perivascular inflammation and fibrosis in C57BL/6 mice. The pulmonary MoPn infection appeared to be disseminated systemically, because chlamydial antigens were readily detectable in multiple organs including the cardiovascular tissues. In addition, gamma interferon gene knockout mice with a C57BL/6 genetic background showed significant endocarditis and pancarditis characterized by vegetation in aortic valves, interstitial and pericardial inflammatory cellular infiltration, and growth of the organisms in the heart following respiratory tract MoPn infection. The results indicate thatC. trachomatiscan induce cardiovascular diseases following respiratory tract infection and suggest that murine MoPn respiratory tract infection may be a useful experimental model for investigating cardiovascular diseases caused by chlamydial infection.
Natural Immunity toAscaris lumbricoidesAssociated with Immunoglobulin E Antibody to ABA-1 Allergen and Inflammation Indicators in Children
Authors: McSharry Charles , Xia Yu , Holland Celia V. , Kennedy Malcolm W.
Published: December 2019

Children putatively immune to the large roundwormAscaris lumbricoideswere identified in an area of Nigeria where infection is hyperendemic. Immunity was associated with higher levels of serum ferritin, C-reactive protein, and eosinophil cationic protein, indicating ongoing acute phase or inflammatory processes. In contrast, children who were susceptible to the infection had little serological evidence of inflammation despite their high parasite burdens. Immunoglobulin G (IgG) antibody activity in all subclasses was present in high titer in most children but appeared to have no protective function. Despite exceptionally high total IgE levels, there was no evidence that atopic responses to local common allergens was associated with natural immunity toAscaris. Among those individuals who produced IgG antibody to recombinant ABA-1 allergen ofAscaris, the naturally immune group had significantly more IgE antibody to the allergen than did those susceptible to the infection. IgE antibody responses in conjunction with innate inflammatory processes therefore appear to associate with natural immunity to ascariasis.
Deamidation of Cdc42 and Rac by
Escherichia coli
Cytotoxic Necrotizing Factor 1: Activation of c-Jun N-Terminal Kinase in HeLa Cells
Authors: Lerm M. , Selzer J. , Hoffmeyer A. , Rapp U. R. , Aktories K. , Schmidt G.
Published: December 2019

Recently,
Escherichia coli
cytotoxic necrotizing factor 1 (CNF1) was shown to activate the low-molecular-mass GTPase RhoA by deamidation of Gln63, thereby inhibiting intrinsic and GTPase-activating protein (GAP)-stimulated GTPase activities (G. Schmidt, P. Sehr, M. Wilm, J. Selzer, M. Mann, and K. Aktories, Nature 387:725–729, 1997; G. Flatau, E. Lemichez, M. Gauthier, P. Chardin, S. Paris, C. Fiorentini, and P. Boquet, Nature 387:729–733, 1997). Here we report that in addition to RhoA, Cdc42 and Rac also are targets for CNF1 in vitro and in intact cells. Treatment of HeLa cells with CNF1 induced a transient formation of microspikes and formation of membrane ruffles. CNF1 caused a transient 10- to 50-fold increase in the activity of the c-Jun N-terminal kinase. Tryptic peptides of Cdc42 obtained from CNF1-treated cells by immunoprecipitation exhibited an increase in mass of 1 Da compared to control peptides, indicating the deamidation of glutamine 61 by the toxin. The same increase in mass was observed with the respective peptides obtained from CNF1-modified recombinant Cdc42 and Rac1. Modification of recombinant Cdc42 and Rac1 by CNF1 inhibited intrinsic and GAP-stimulated GTPase activities and retarded binding of 2′(3′)- O-(N -methylanthraniloyl)GDP. The data suggest that recombinant as well as cellular Cdc42 and Rac are substrates for CNF1.
Receptor-Dependent Immune Responses in Pigs after Oral Immunization with F4 Fimbriae
Authors: Van den Broeck Wim , Cox Eric , Goddeeris Bruno M.
Published: December 2019

F4 receptor-positive (F4R
+
) and F4 receptor-negative (F4R
−
) pigs were orally vaccinated with purified F4 fimbriae of enterotoxigenic
Escherichia coli
(ETEC). Serum immunoglobulin G (IgG) and IgA responses were readily detected in F4R
+
animals, whereas immune responses were not detected in F4R
−
animals. Even after a subsequent oral infection with virulent F4
+
ETEC and a booster immunization with F4, the F4R
−
animals remained F4 seronegative whereas the unvaccinated F4R
+
pigs exhibited clear IgA and IgG responses. These results clearly demonstrate that F4Rs are a prerequisite for an immune response following oral immunization. Furthermore, indications that oral F4 vaccination can induce mucosal protection were obtained, since the experimental ETEC infection did not induce a systemic booster response or fecal ETEC excretion in orally vaccinated F4R
+
pigs, in contrast to the clear immune response and ETEC excretion of unvaccinated F4R
+
animals. F4-specific IgA antibodies could be found in the feces of the vaccinated F4R
+
pigs. They are secreted at the intestinal mucosal surface and appear to prevent ETEC infection. The F4R-dependent induction of a mucosal immune response can be used as a model to better understand mucosal immunization and mucosal immune responses and can contribute to the development of oral vaccines in veterinary as well as in human medicine.
Serum Antitoxin Antibodies Mediate Systemic and Mucosal Protection from
Clostridium difficile
Disease in Hamsters
Authors: Giannasca Paul J. , Zhang Zhen-xi , Lei Wen-de , Boden James A. , Giel Mary Ann , Monath Thomas P. , Thomas William D.
Published: December 2019

Clostridium difficile
is the bacterial pathogen identified as the cause of pseudomembranous colitis and is principally responsible for nosocomial antibiotic-associated diarrhea and colitis. The pathologic findings associated with this infection are believed to be caused by two large (∼300-kDa) exotoxins, toxins A and B. Because of the mucosal nature of this infection, vaccination strategies aimed at providing prophylactic or therapeutic immune protection have included immunization by mucosal routes. Using the hamster model of
C. difficile
infection, we examined the protective efficacy of inactivated toxin (toxoid) vaccine formulations prepared as either culture filtrate or partially purified toxoid. We compared combination parenteral and mucosal vaccination regimens involving intranasal, intragastric, or rectal routes of immunization and found that rectal immunization in conjunction with intramuscular (i.m.) vaccination provided full protection of hamsters from death and diarrhea while the other mucosal routes did not. Protection was associated with high levels of toxin-neutralizing antibodies in serum. The requirement for adjuvants for protection was assessed by using sequential i.m. and rectal or i.m. vaccination regimens. Unexpectedly, i.m. immunization without adjuvant conferred the highest protection from death and diarrhea; this regimen elicited the highest serum anti-toxin B titers as well as toxin B neutralizing titers. Passive transfer of mouse antitoxin antibodies protected hamsters in a dose-dependent manner, demonstrating the principal role of circulating antitoxin antibodies in immunity from this toxin-mediated mucosal disease. These results suggest that prophylactic parenteral vaccination or intravenous immunotherapy could provide protection from
C. difficile
disease in humans.
TheURA5Gene Is Necessary forHistoplasma capsulatumGrowth during Infection of Mouse and Human Cells
Authors: Retallack Diane M. , Heinecke Elizabeth L. , Gibbons Reta , Deepe George S. , Woods Jon P.
Published: December 2019

TheHistoplasma capsulatum URA5gene, which has recently been cloned and disrupted by allelic replacement, encodes orotidine-5′-monophosphate pyrophosphorylase. Inactivation ofURA5by either targeted or UV mutagenesis results in disruption of the pyrimidine biosynthetic pathway and uracil auxotrophy. We examined the effect of uracil auxotrophy due to aura5mutation onH. capsulatumvirulence in both cell culture and whole-animal models. Uracil auxotrophs of twoH. capsulatumrestriction fragment length polymorphism classes were found to be avirulent in cultured murine and human cells, as well as in mice. Moreover, virulence could be restored either by supplying a functionalURA5gene in trans or by supplying exogenous uracil during infection in vitro. These experiments demonstrate that the pyrimidine biosynthetic pathway is essential forH. capsulatumgrowth and virulence.
Protective Immunization with a Novel Membrane Protein ofPlasmodium yoelii-Infected Erythrocytes
Authors: Burns James M. , Adeeku Eric K. , Dunn Patricia D.
Published: December 2019

Immunization with a particulate fraction of blood-stage antigens was shown previously to protect mice againstPlasmodium yoeliimalaria. To identify antigens inducing the protective response, sera from immunized mice were used to screen aP. yoeliicDNA expression library. Sequence analysis of one 2.6-kb cDNA clone indicated that the identified gene,pypag-1, encoded a novel plasmodial antigen. Two nonoverlapping regions ofpypag-1were expressed inEscherichia coli. The first recombinant antigen, pAg-1N, contained the N-terminal 337 residues, which included a putative transmembrane domain and a region relatively rich in tryptophan residues. The second recombinant antigen, pAg-1C, contained the remaining C-terminal 211 residues, which included 31 copies of a 5-amino-acid degenerative repeat. Immunoblot studies using rabbit antiserum raised against recombinant pAg-1N showed that the native pypAg-1 protein migrated at approximately 98 kDa, considerably slower than its predicted molecular mass of 66 kDa. Immunofluorescence studies localized the expression of the native pypAg-1 protein both to the cytoplasm and at the surface ofP. yoelii-infected erythrocytes. Immunization with either pAg-1N or pAg-1C induced a four- to sevenfold reduction inP. yoeliiblood-stage parasitemia. As such, pypAg-1 appears to contain at least two distinct protective epitopes. To our knowledge, this is the first characterization of a protective antigen ofP. yoeliithat is associated with the erythrocyte membrane.
Involvement of Mitogen-Activated Protein Kinase Pathways in Interleukin-8 Production by Human Monocytes and Polymorphonuclear Cells Stimulated with Lipopolysaccharide orMycoplasma fermentansMembrane Lipoproteins
Authors: Marie Christelle , Roman-Roman Sergio , Rawadi Georges
Published: December 2019

Interleukin-8 (IL-8) is a chemokine that belongs to the α-chemokine or CXC subfamily and is produced by a wide variety of human cells, including monocytes and polymorphonuclear cells (PMN). IL-8 is secreted in response to inflammatory stimuli, notably bacterial products such as lipopolysaccharide (LPS), but little is known about the mechanisms by which these agents mediate IL-8 induction. In this report, we show thatMycoplasma fermentanslipid-associated membrane proteins (LAMPf) induce the production of high levels of IL-8 by THP-1 (human monocyte) cells and PMN at the same extent as LPS. It was previously demonstrated that stimulation of monocytic cells with either LPS or LAMPf led to a series of common downstream signaling events, including the activation of protein tyrosine kinase and of mitogen-activated protein kinase cascades. By using PD-98059 and SB203580, two potent and selective inhibitors of MEK1 (a kinase upstream of ERK1/2) and p38, respectively, we have demonstrated that both ERK1/2 and p38 cascades play a key role in the production of IL-8 by monocytes and PMN stimulated with bacterial fractions.
Differential Posttranslational Processing Confers Intraspecies Variation of a Major Surface Lipoprotein and a Macrophage-Activating Lipopeptide ofMycoplasma fermentans
Authors: Calcutt Michael J. , Kim Mary F. , Karpas Arthur B. , Mühlradt Peter F. , Wise Kim S.
Published: December 2019

Themalpgene ofMycoplasma fermentansis shown to occur in single copy but to encode two discrete translated forms of lipid-modified surface protein that can be differentially expressed on isolates within this species: MALP-2, a 14-amino-acid (2-kDa) lipopeptide with potent macrophage-stimulatory activity (P. F. Mühlradt, M. Kiess, H. Meyer, R. Süssmuth, and G. Jung, J. Exp. Med. 185:1951–1958, 1997), and MALP-404, an abundant, full-length (404-amino-acid) surface lipoprotein of 41 kDa, previously designated P41 (K. S. Wise, M. F. Kim, P. M. Theiss, and S.-C. Lo, Infect. Immun. 61:3327–3333, 1993). The sequences, transcripts, and translation products ofmalpwere compared between clonal isolates of strains PG18 (known to express P41) and II-29/1 (known to express high levels of MALP-2). Despite conservedmalpDNA sequences containing full-length open reading frames and expression of full-length monocistronic transcripts in both isolates, Western blotting using a monoclonal antibody (MAb) to the N-terminal MALP-2 peptide revealed marked differences in the protein products expressed. Whereas PG18 expressed abundant MALP-404 with detectable MALP-2, II-29/1 revealed no MALP-404 even in samples containing a large comparative excess of MALP-2. Colony immunoblots with the MAb showed uniform surface expression of MALP-2 in II-29/1 populations. A second MAb to an epitope of MALP-404 outside the MALP-2 sequence predictably failed to stain II-29/1 colonies but uniformly stained PG18 populations. Collectively, these results provide evidence for novel posttranscriptional (probably posttranslational) processing pathways leading to differential intraspecies expression of a major lipoprotein, and a potent macrophage-activating lipopeptide, on the surface ofM. fermentans. In the course of this study, a striking conserved motif (consensus, TD-G--DDKSFNQSAWE--), designated SLA, was identified in MALP-404; this motif is also distributed among selected lipoproteins and species from diverse bacterial genera, includingBacillus,Borrelia,Listeria,Mycoplasma, andTreponema. In addition,malpwas shown to flank a chromosomal polymorphism. In eight isolates ofM. fermentansexamined,malpoccurred upstream of an operon encoding the phase-variable P78 ABC transporter; but, in three of these isolates, a newly discovered insertion sequence, IS1630(of the IS30class), was located between these genes.
Strategy for Cross-Protection amongShigella flexneriSerotypes
Authors: Noriega Fernando R. , Liao Fang Ming , Maneval David R. , Ren Shuxun , Formal Samuel B. , Levine Myron M.
Published: December 2019

Based upon the lipopolysaccharide (LPS) structure and antigenicity ofShigellagroup B, a strategy for broad cross-protection against 14Shigella flexneriserotypes was designed. This strategy involves the use of twoS. flexneriserotypes (2a and 3a), which together bear the all of the major antigenic group factors of this group. The novel attenuated strains used in these studies wereS. flexneri2a strain CVD 1207 (ΔguaB-A ΔvirG Δset1 Δsen) andS. flexneri3a strain CVD 1211 (ΔguaB-A ΔvirG Δsen). Guinea pigs were immunized with an equal mixture of these strains and later challenged (Sereny test) with a wild-typeS. flexneriserotype 1a, 1b, 2b, 4b, 5b, Y, or 6 strain of demonstrated virulence in the same model. Guinea pigs that were immunized with these two vaccine strains produced serum and mucosal antibodies that cross-reacted with all theS. flexneriserotypes tested (except ofS. flexneriserotype 6) as assessed by enzyme-linked immunosorbent assay, immunoblotting, and slide agglutination. Furthermore, the combination vaccine conferred significant protection against challenge withS. flexneriserotypes 1b, 2b, 5b, and Y but not with serotypes 1a, 4b, or (as predicted) 6.
Functional and Immunogenic Characterization of Two Cloned Regions of
Streptococcus mutans
Glucosyltransferase I
Authors: Jespersgaard Christina , Hajishengallis George , Greenway Terrence E. , Smith Daniel J. , Russell Michael W. , Michalek Suzanne M.
Published: December 2019

Glucosyltransferase (GTF) enzymes of mutans streptococci are considered virulence factors due to their ability to synthesize adhesive glucans, which facilitate cell-to-cell adherence and accumulation. In this study we report the cloning, expression, and characterization of the catalytic (CAT) and glucan-binding (GLU) domains of
S. mutans
GTF-I encoded by
gtfB
. The CAT and GLU polypeptides represent amino acid residues 253 to 628 and 1183 to 1473, respectively, of
S. mutans
GTF-I. Antibodies to recombinant CAT and GLU were generated in rabbits and purified by affinity chromatography. Purified anti-CAT antibodies significantly inhibited water-insoluble glucan synthesis by
S. mutans
and
S. sobrinus
GTFs (
P
< 0.0001 and
P
< 0.05, respectively). The purified anti-GLU antibodies significantly inhibited both water-insoluble and water-soluble glucan synthesis by
S. mutans
GTFs (
P
< 0.0001 and
P
< 0.05, respectively). These results demonstrate that anti-CAT and anti-GLU antibodies are capable of inhibiting a variety of GTF activities. Since antibodies to
S. mutans
in saliva are implicated in protection against disease, we next assessed the ability of CAT and GLU polypeptides to induce mucosal antibody responses in mice. Intranasal (i.n.) immunization of mice with CAT showed significantly (
P
< 0.005) elevated levels of specific immunoglobulin G (IgG) antibody activity in serum and specific IgA antibody activity in serum, saliva, vaginal washes, and fecal samples. GLU immunized animals showed significantly (
P
< 0.005) elevated levels of specific IgA antibody activity in serum and vaginal secretions. Taken together, these results demonstrate that the recombinant CAT and GLU polypeptides are effective in inducing both mucosal and systemic immune responses. The ability of these polypeptides to induce a mucosal IgA immune response in mice after i.n. immunization supports their use as subunit vaccine candidates in the development of an anticaries vaccine.
Isolation and Characterization of Rugose Form of
Vibrio cholerae
O139 Strain MO10
Authors: Mizunoe Yoshimitsu , Wai Sun Nyunt , Takade Akemi , Yoshida Shin-Ichi
Published: December 2019

An extracellular exopolysaccharide (slime) is produced by
Vibrio cholerae
O139 MO10 in response to nutrient starvation. The presence of this slime layer on the cell surface and its subsequent release have been shown to be associated with biofilm formation and the change from a normal smooth colony morphology to a rugose one. An immunoelectron microscopic examination demonstrated that there is an epitope common to the exopolysaccharide antigen of
V. cholerae
O1 and that of O139 MO10.
Interleukin-5 Transgenic Mice Show Enhanced Resistance to Primary Infections withNippostrongylus brasiliensisbut Not Primary Infections withToxocara canis
Authors: Dent Lindsay A. , Daly Christine M. , Mayrhofer Graham , Zimmerman Trudy , Hallett Ann , Bignold Leon P. , Creaney Jenette , Parsons Jim C.
Published: December 2019

In this study, interleukin-5 (IL-5) transgenic mice with lifelong eosinophilia were assessed for resistance to primary infections with two tissue-invading nematodes,Nippostrongylus brasiliensisandToxocara canis. Relative to nontransgenic littermates, three lines of IL-5 transgenic mice with varying degrees of eosinophilia all displayed enhanced resistance toN. brasiliensis. Although the timing of final worm expulsion was similar in transgenic and nontransgenic hosts, intestinal worms in transgenic mice were fewer in number throughout infection, failed to increase in size over the course of the infection, and were much less fecund. In contrast,T. canislarvae were recovered in similar numbers from tissues of transgenic mice with “low” or “high” eosinophilia and from nontransgenic mice. These results and other data suggest that eosinophils can contribute to host resistance to some parasite species. Parasite transit time through the host may correlate with relative sensitivity to eosinophils.
Role of Nitric Oxide in Lipopolysaccharide-Induced Hepatic Injury ind-Galactosamine-Sensitized Mice as an Experimental Endotoxic Shock Model
Authors: Morikawa Akiko , Kato Yutaka , Sugiyama Tuyoshi , Koide Naoki , Chakravortty Dipshika , Yoshida Tomoaki , Yokochi Takashi
Published: December 2019

The role of nitric oxide (NO) in lipopolysaccharide (LPS)-induced hepatic injury was studied ind-galactosamine (d-GalN)-sensitized mice. The inducible isoform of NO synthase (iNOS) was immunohistochemically detected on hepatocytes around blood vessels in livers of mice injected withd-GalN and LPS not on hepatocytes in mice injected withd-GalN or LPS alone, although mRNA for iNOS was found in those mice. Nitrotyrosine (NT) was also found in livers of mice injected withd-GalN and LPS. The localization of NT was consistent with that of iNOS, and the time courses of NT and iNOS expression were almost the same. Expression of iNOS and NT was detected exclusively in the hepatic lesions of mice injected withd-GalN and LPS. Anti-tumor necrosis factor alpha neutralizing antibody inhibited iNOS and NT expression and hepatic injury. The results suggested that NO from iNOS may play a role in LPS-induced hepatic injury ond-GalN-sensitized mice as an experimental endotoxic shock model.
Generation of CD8+T-Cell Responses toMycobacterium bovisand Mycobacterial Antigen in Experimental Bovine Tuberculosis
Authors: Liébana Ernesto , Girvin Robert M. , Welsh Michael , Neill Sydney D. , Pollock John M.
Published: December 2019

Protective immunity against tuberculosis is considered to be essentially cell mediated, and an important role for CD8+T lymphocytes has been suggested by several studies of murine and human infections. The present work, using an experimental model of infection withMycobacterium bovisin cattle, showed that liveM. boviselicits the activation of CD8+T cells in vitro. However, a sonic extract prepared fromM. bovis(MBSE) and protein purified derivative (PPDb) also induced a considerable degree of activation of the CD8+T cells. Analysis of proliferative responses of peripheral blood mononuclear cells, purified CD8+T cells, and CD8+T-cell clones toM. bovisand to soluble antigenic preparations (MBSE, PPDb) showed that the responses of all three types of cells were always superior for live mycobacteria but that strong responses were also obtained with complex soluble preparations. Furthermore, while cytotoxic capabilities were not investigated, the CD8+T cells were found to produce and release gamma interferon in response to antigen (live and soluble), which indicated one possible protective mechanism for these cells in bovine tuberculosis. Finally, it was demonstrated by metabolic inhibition with brefeldin A and cytochalasin D at the clonal level that an endogenous pathway of antigen processing is required for presentation to bovine CD8+cells and that presentation is also dependent on phagocytosis of the antigen.
Borrelia burgdorferiSpirochetes Induce Mast Cell Activation and Cytokine Release
Authors: Talkington Jeffrey , Nickell Steven P.
Published: December 2019

The Lyme disease spirochete,Borrelia burgdorferi, is introduced into human hosts via tick bites. Among the cell types present in the skin which may initially contact spirochetes are mast cells. Since spirochetes are known to activate a variety of cell types in vitro, we tested whetherB. burgdorferispirochetes could activate mast cells. We report here that freshly isolated rat peritoneal mast cells or mouse MC/9 mast cells cultured in vitro with live or freeze-thawedB. burgdorferispirochetes undergo low but detectable degranulation, as measured by [5-3H] hydroxytryptamine release, and they synthesize and secrete the proinflammatory cytokine tumor necrosis factor alpha (TNF-α). In contrast to findings in previous studies, whereB. burgdorferi-associated activity was shown to be dependent upon protein lipidation, mast cell TNF-α release was not induced by either lipidated or unlipidated recombinant OspA. This activity was additionally shown to be protease sensitive and surface expressed. Finally, comparisons of TNF-α-inducing activity in known low-, intermediate-, and high-passageB. burgdorferiB31 isolates demonstrated passage-dependent loss of activity, indicating that the activity is probably plasmid encoded. These findings document the presence in low-passageB. burgdorferispirochetes of a novel lipidation-independent activity capable of inducing cytokine release from host cells.
Dysregulated Production of Interleukin-8 in Individuals Infected with Human Immunodeficiency Virus Type 1 andMycobacterium tuberculosis
Authors: Meddows-Taylor Stephen , Martin Desmond J. , Tiemessen Caroline T.
Published: December 2019

Interleukin-8 (IL-8) production in vivo was monitored in four study groups: normal blood donors, patients with pulmonary tuberculosis (TB), patients with human immunodeficiency virus type 1 (HIV-1) infection, and dually infected (HIV/TB) patients. We show that whereas there was evidence of detectable levels of cell-associated IL-8 (mRNA and protein) in peripheral cells of healthy individuals, this was largely lost in the disease states studied. Coupled with this finding was significantly increased circulating levels of IL-8 in HIV-1-infected individuals with or without concomitant pulmonary TB (P< 0.001). On the other hand, the capacity of peripheral mononuclear cells to produce IL-8 spontaneously ex vivo was enhanced in HIV-1 and TB patients (P< 0.05) and many of the HIV/TB group, but their corresponding capacities to respond to various stimuli, in particular phytohemagglutinin, were significantly diminished compared to those of normal donors (P< 0.05). Circulating levels of IL-8 in a group of HIV/TB patients were significantly positively correlated with the percentage of polymorphonuclear leukocytes (PMN) in the peripheral circulation (r= 0.65;P= 0.01), the proportions of IL-8 receptor A (IL-8RA)-expressing (r= 0.86;P< 0.01) and IL-8RB-expressing (r= 0.77;P< 0.01) PMN, and the capacity of PMN to migrate in response to IL-8 as chemoattractant (r= 0.68;P< 0.01). IL-8RB fluorescence intensity, however, was negatively correlated with plasma IL-8 levels (r= −0.73;P< 0.01). Our results suggest that altered regulation of IL-8 in HIV-1 may have important implications for antimicrobial defenses and for normal immune processes.
Functional Activities and Epitope Specificity of Human and Murine Antibodies against the Class 4 Outer Membrane Protein (Rmp) of
Neisseria meningitidis
Authors: Rosenqvist Einar , Musacchio Alexis , Aase Audun , Høiby E. Arne , Namork Ellen , Kolberg Jan , Wedege Elisabeth , Delvig Alexei , Dalseg Rolf , Michaelsen Terje E. , Tommassen Jan
Published: December 2019

Antibodies against the class 4 outer membrane protein (OMP) from
Neisseria meningitidis
have been purified from sera from vaccinees immunized with the Norwegian meningococcal group B outer membrane vesicle vaccine. The human sera and purified antibodies reacted strongly with the class 4 OMP in immunoblots, whereas experiments with whole bacteria showed only weak reactions, indicating that the antibodies mainly reacted with parts of the class 4 molecule that were not exposed. The purified human anti-class 4 OMP antibodies and the monoclonal antibodies (MAbs) were neither bactericidal nor opsonic against live meningococci. Three new MAbs against the class 4 OMP were generated and compared with other, previously described MAbs. Three linear epitopes in different regions of the class 4 OMP were identified by the reaction of MAbs with synthetic peptides. The MAbs showed no blocking effect on bactericidal activity of MAbs against other OMPs. However, one of the eight purified human anti-class 4 OMP antibody preparations, selected from immunoblot reactions among sera from 27 vaccinees, inhibited at high concentrations the bactericidal effect of a MAb against the class 1 OMP. However, these antibodies were not vaccine induced, as they were present also before vaccination. Therefore, this study gave no evidence that vaccination with a meningococcal outer membrane vesicle vaccine containing the class 4 OMP induces blocking antibodies. Our data indicated that the structure of class 4 OMP does not correspond to standard β-barrel structures of integral OMPs and that no substantial portion of the OmpA-like C-terminal region of this protein is located at the surface of the outer membrane.
Expression of the Plague Plasminogen Activator inYersinia pseudotuberculosisandEscherichia coli
Authors: Kutyrev V. , Mehigh R. J. , Motin V. L. , Pokrovskaya M. S. , Smirnov G. B. , Brubaker R. R.
Published: December 2019

Enteropathogenic yersiniae (Yersinia pseudotuberculosisandYersinia enterocolitica) typically cause chronic disease as opposed to the closely relatedYersinia pestis, the causative agent of bubonic plague. It is established that this difference reflects, in part, carriage byY. pestisof a unique 9.6-kb pesticin or Pst plasmid (pPCP) encoding plasminogen activator (Pla) rather than distinctions between shared ∼70-kb low-calcium-response, or Lcr, plasmids (pCD inY. pestisand pYV in enteropathogenic yersiniae) encoding cytotoxic Yops and anti-inflammatory V antigen. Pla is known to exist as a combination of 32.6-kDa (α-Pla) and slightly smaller (β-Pla) outer membrane proteins, of which at least one promotes bacterial dissemination in vivo and degradation of Yops in vitro. We show here that only α-Pla accumulates inEscherichia coliLE392/pPCP1 cultivated in enriched medium and that either autolysis or extraction of this isolate with 1.0 M NaCl results in release of soluble α and β forms possessing biological activity. This process also converted cell-bound α-Pla to β-Pla and smaller forms inY. pestisKIM/pPCP1 andY. pseudotuberculosisPB1/+/pPCP1 but did not promote solubilization. Pla-mediated posttranslational hydrolysis of pulse-labeled Yops inY. pseudotuberculosisPB1/+/pPCP1 occurred more slowly than that inY. pestisbut was otherwise similar except for accumulation of stable degradation products of YadA, a pYV-mediated fibrillar adhesin not encoded in frame by pCD. Carriage of pPCP byY. pseudotuberculosisdid not significantly influence virulence in mice.
Surface Expression of a Protective Recombinant Pertussis Toxin S1 Subunit Fragment in
Streptococcus gordonii
Authors: Lee Song F. , March Robert J. , Halperin Scott A. , Faulkner Gary , Gao Lingqiu
Published: December 2019

In this study, the expression of the
Bordetella pertussis
S1 subunit was tested in
Streptococcus gordonii
, a commensal oral bacterium which has the potential to be a live oral vaccine vehicle. The DNA fragment encoding the N-terminal 179 amino acids of the S1 subunit was ligated into the middle part of
spaP
, the surface protein antigen P1 gene originating from
Streptococcus mutans
. The resulting construct, carried on the
Escherichia coli-Streptococcus
shuttle vector pDL276, was introduced into
S. gordonii
DL-1 by natural transformation. One of the transformants (RJMIII) produced a 187-kDa protein (the predicted size of the SpaP-S1 fusion protein) which was recognized by both the anti-pertussis toxin (anti-PT) and anti-SpaP antibodies, suggesting that an in-frame fusion had been made. Results from immunogold-electron microscopic studies and cellular fractionation studies showed that the fusion protein was surface localized and was mainly associated with the cell wall of RJMIII, indicating that SpaP was able to direct the fusion protein to the cell surface. A rabbit antiserum raised against heat-killed
S. gordonii
RJMIII recognized the native S1 subunit of PT in Western blotting and showed a weak neutralization titer to PT by the Chinese hamster ovary cell-clustering assay. BALB/c mice immunized with the heat-killed
S. gordonii
RJMIII were protected from the toxic effect of PT in the leukocytosis-promoting and histamine sensitization assays. In conclusion, a fragment of the S1 subunit of PT was successfully surface expressed in
S. gordonii
; the recombinant S1 fragment was found to be immunogenic and could induce protection against the toxic effect of PT in mice.
Identification of thePlasmodium chabaudiHomologue of Merozoite Surface Proteins 4 and 5 ofPlasmodium falciparum
Authors: Black Casilda G. , Wang Lina , Hibbs Alan R. , Werner Ekkehard , Coppel Ross L.
Published: December 2019

Previous studies ofPlasmodium falciparumhave identified a region of chromosome 2 in which are clustered three genes for glycosylphosphatidylinositol (GPI)-anchored merozoite surface proteins, MSP2, MSP5, and MSP4, arranged in tandem.MSP4andMSP5both encode proteins 272 residues long that contain hydrophobic signal sequences, GPI attachment signals, and a single epidermal growth factor (EGF)-like domain at their carboxyl termini. Nevertheless, the remainder of their protein coding regions are quite dissimilar. The locations and similar structural features of these genes suggest that they have arisen from a gene duplication event. Here we describe the identification of the syntenic region of the genome in the murine malaria parasite,Plasmodium chabaudi adamiDS. Only one open reading frame is present in this region, and it encodes a protein with structural features reminiscent of both MSP4 and MSP5, including a single EGF-like domain. Accordingly, the gene has been designatedPcMSP4/5. The homologue of theP. falciparum MSP2gene could not be found inP. chabaudi; however, the amino terminus of the PcMSP4/5 protein shows similarity to that of MSP2. ThePcMSP4/5gene encodes a protein with an apparent molecular mass of 36 kDa, and this protein is detected in mature stages of the parasite. The protein partitions in the detergent-enriched phase after Triton X-114 fractionation and is localized to the surfaces of trophozoites and developing and free merozoites. ThePcMSP4/5gene is transcribed in both ring and trophozoite stages but appears to be spliced in a stage-specific manner such that the central intron is spliced from the mRNA in the parasitic stage in which the protein is expressed.