bluuebunny/crossref_metadata_2025 huggingface dataset:
α-GlcNAc-1→2-α-Glc, theSalmonellaHomologue of a Conserved Lipopolysaccharide Motif in theEnterobacteriaceae, Elicits Broadly Cross-Reactive Antibodies
Authors: Nnalue Ndubisi Anthony
Published: March 2020

To define cross-reactive epitopes inSalmonellalipopolysaccharide (LPS), antisera designated anti-S, anti-Ra, and anti-Re were generated against smooth (S), complete-core (Ra), and deep-core mutant (Re) strains, respectively, and characterized immunochemically. The reactivities of anti-Ra and anti-S with rough LPS (rLPS) chemotypes in enzyme-linked immunosorbent assays (ELISA) decreased progressively with increasing truncation of the complete-core oligosaccharide (e.g., Ra > Rb1>…Re), while that of anti-Re increased (Ra < Rb1<…Re). Anti-Ra was relatively more reactive with nonhomologous smooth LPS (sLPS) than anti-S, which in turn was more reactive than anti-Re. This order reflected the relative reactivities of these sera with outer-core rLPS but not those with inner-core rLPS, which suggests that the cross-reactivities of all three sera with sLPS were mediated by antibodies which bind outer-core determinants. Anti-Ra, but not anti-S or anti-Re, reacted with molecules substituted by O chains in immunoblots and revealed ladder-like patterns in sLPSs of various serospecificities. Anti-Ra, however, did not react with O-antigen-specific neoglycoconjugates in ELISA, thus demonstrating specificity for core epitopes. Ra and Rb1but not otherSalmonellacore chemotypes inhibited the reactivity of anti-Ra with sLPS in ELISA, which showed that the terminal outer-core disaccharide, α-GlcNAc-1→2-α-Glc (GlcNAc→Glc), was the major epitope of cross-reactive antibodies in the serum. GlcNAc→Glc represents the conserved motif α-hexose-1→2-α-hexose in cores of theEnterobacteriaceae, other homologues of which should likewise be cross-reactive. These results demonstrate that S or Re strains do not elicit cross-reactive antibodies and indicate that immunization with Ra strains may represent a general strategy for eliciting cross-reactive antibodies against LPSs from enteric bacteria.
Role of Immunoglobulin A Monoclonal Antibodies against P23 in Controlling MurineCryptosporidium parvumInfection
Authors: Enriquez F. Javier , Riggs Michael W.
Published: March 2020

Cryptosporidium parvumis an important diarrhea-causing protozoan parasite of immunocompetent and immunocompromised hosts. Immunoglobulin A (IgA) has been implicated in resistance to mucosal infections with bacteria, viruses, and parasites, but little is known about the role of IgA in the control ofC. parvuminfection. We assessed the role of IgA duringC. parvuminfection in neonatal mice. IgA-secreting hybridomas were developed by using Peyer’s patch lymphocytes from BALB/c mice which had been orally inoculated with viableC. parvumoocysts. Six monoclonal antibodies (MAbs) were selected for further study based on indirect immunofluorescence assay reactivity with sporozoite and merozoite pellicles and the antigen (Ag) deposited on glass substrate by gliding sporozoites. Each MAb was secreted in dimeric form and recognized a 23-kDa sporozoite Ag in Western immunoblots. The Ag recognized comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with P23, a previously defined neutralization-sensitive zoite pellicle Ag. MAbs were evaluated for prophylactic or therapeutic efficacy againstC. parvum, singly and in combinations, in neonatal BALB/c mice. A combination of two MAbs given prophylactically prior to and 12 h following oocyst challenge reduced the number of intestinal parasites scored histologically by 21.1% compared to the numbers in mice given an isotype-matched control MAb (P< 0.01). Individual MAbs given therapeutically in nine doses over a 96-h period following oocyst challenge increased efficacy againstC. parvuminfection. Four MAbs given therapeutically each reduced intestinal infection 34.4 to 42.2% compared to isotype-matched control MAb-treated mice (P< 0.05). One MAb reduced infection 63.3 and 72.7% in replicate experiments compared to isotype-matched control MAb-treated mice (P< 0.0001). We conclude that IgA MAbs directed to neutralization-sensitive P23 epitopes may have utility in passive immunization against murineC. parvuminfection.
In Vivo Transduction with Shiga Toxin 1-Encoding Phage
Authors: Acheson David W. K. , Reidl Joachim , Zhang Xiaoping , Keusch Gerald T. , Mekalanos John J. , Waldor Matthew K.
Published: March 2020

To facilitate the study of intestinal transmission of the Shiga toxin 1 (Stx1)-converting phage H-19B, Tn
10d-bla
mutagenesis of an
Escherichia coli
H-19B lysogen was undertaken. Two mutants containing insertions in the gene encoding the A subunit of Stx1 were isolated. The resultant ampicillin-resistant
E. coli
strains lysogenic for these phages produced infectious H-19B particles but not active toxin. These lysogens were capable of transducing an
E. coli
recipient strain in the murine gastrointestinal tract, thereby demonstrating that lysogens of Shiga toxin-converting phages give rise to infectious virions within the host gastrointestinal tract.
Starvation Selection Restores Elastase and Rhamnolipid Production in a
Pseudomonas aeruginosa
Quorum-Sensing Mutant
Authors: Van Delden Christian , Pesci Everett C. , Pearson James P. , Iglewski Barbara H.
Published: March 2020

The
las
quorum-sensing system of
Pseudomonas aeruginosa
controls the expression of elastase and rhamnolipid. We report that starvation can select a mutant producing these virulence factors in spite of a
lasR
deletion. Expression of the autoinducer synthase gene
rhlI
was increased in this suppressor mutant, suggesting compensation by the
rhl
system. These data show that
P. aeruginosa
can restore elastase and rhamnolipid production in the absence of a functional
las
quorum-sensing system.
Protective Effect of Vaccination with a Combination of Recombinant Surface Antigen 1 and Interleukin-12 against Toxoplasmosis in Mice
Authors: Letscher-Bru Valerie , Villard Odile , Risse Bernhard , Zauke Michael , Klein Jean-Paul , Kien Truong T.
Published: March 2020

We studied the immune response induced in mice by recombinantToxoplasma gondiisurface antigen 1 (rSAG1) protein, alone or combined with interleukin-12 (IL-12) as an adjuvant, and the protective effect against toxoplasmosis. Immunization with rSAG1 alone induced a specific humoral type 2 immunity and did not protect the animals from infection. In contrast, immunization with rSAG1 plus IL-12 redirected humoral and cellular immunity toward a type 1 pattern and reduced the brain parasite load by 40%.
Increased Capacity for Interleukin-2 Synthesis Parallels Disease Progression in Mice Infected withLeishmania major
Authors: Heinzel Frederick P. , Rerko Ronald M. , Hujer Andrea M. , Maier Richard A.
Published: March 2020

Lymph node cells of BALB/c mice with progressive leishmaniasis produced sixfold more interleukin-2 (IL-2) in culture than those of healing C57BL/6 mice. IL-2 synthesis also increased in C57BL/6 mice made susceptible by IL-12 or gamma interferon deficiency. However, IL-2 mRNA levels in vivo did not reflect IL-2 production in vitro. Because IL-2 contributes to the pathogenesis of progressive leishmaniasis, the functional significance of these findings should be further explored.
Paclitaxel (Taxol)-Induced Killing ofLeishmania majorin Murine Macrophages
Authors: Doherty T. Mark , Sher Alan , Vogel Stefanie N.
Published: March 2020

The antitumor drug paclitaxel (Taxol) has been demonstrated to be a lipopolysaccharide mimetic in murine macrophages. In this study, the capacity of paclitaxel to activate macrophages to become microbicidal forLeishmania majorwas examined. Paclitaxel and gamma interferon synergized to kill intracellularL. majorinLpsn, but notLpsd, macrophages by a nitric oxide (NO·)-dependent mechanism.
E and P Selectins Are Not Required for Resistance to Severe Murine Lyme Arthritis
Authors: Seiler Kathleen Petri , Ma Ying , Weis John H. , Frenette Paul S. , Hynes Richard O. , Wagner Denisa D. , Weis Janis J.
Published: March 2020

Borrelia burgdorferi-induced arthritis in mice is characterized by tendonitis, synovitis, and inflammatory-cell infiltrate, predominantly of neutrophils. Because genetic deficiency in E and P selectins results in delayed recruitment of neutrophils to sites of inflammation, mice with this deficiency were tested for their response to infection withB. burgdorferi. E and P selectins were not required for the control ofB. burgdorferinumbers, nor did deficiency in E and P selectins result in alteration of arthritis severity.
Febrile-Range Temperature Modifies Early Systemic Tumor Necrosis Factor Alpha Expression in Mice Challenged with Bacterial Endotoxin
Authors: Jiang Qingqi , DeTolla Louis , van Rooijen Nico , Singh Ishwar S. , Fitzgerald Bridget , Lipsky Michael M. , Kane Andrew S. , Cross Alan S. , Hasday Jeffrey D.
Published: March 2020

Fever improves survival in acute infections, but the effects of increased core temperature on host defenses are poorly understood. Tumor necrosis factor alpha (TNF-α) is an early activator of host defenses and a major endogenous pyrogen. TNF-α expression is essential for survival in bacterial infections but, if disregulated, can cause tissue injury. In this study, we show that passively increasing core temperature in mice from the basal (36.5 to 37.5°C) to the febrile (39.5 to 40°C) range modifies systemic TNF-α expression in response to bacterial endotoxin (lipopolysaccharide). The early TNF-α secretion rate is enhanced, but the duration of maximal TNF-α production is shortened. We identified Kupffer cells as the predominant source of the excess TNF-α production in the warmer animals. The enhanced early TNF-α production observed at the higher temperature in vivo could not be demonstrated in isolated Kupffer cells or in precision-cut liver slices in vitro, indicating the participation of indirect pathways. Therefore, expression of the endogenous pyrogen TNF-α is regulated by increments in core temperature during fever, generating an enhanced early, self-limited TNF-α pulse.
Microbiological and Inflammatory Effects of Murine Recombinant Interleukin-10 in Two Models of Polymicrobial Peritonitis in Rats
Authors: Montravers Philippe , Maulin Laurence , Mohler Jacqueline , Carbon Claude
Published: March 2020

A protective effect of interleukin-10 (IL-10) against the development of lethal shock has been demonstrated in various animal models. In contrast, the immunosuppressant properties of this mediator have been minimally evaluated in low-mortality models of infections. The clinical, microbiological, and inflammatory effects of murine recombinant IL-10 (mrIL-10) therapy were evaluated in two models of peritonitis in rats, which differed in the degree of severity of peritoneal inflammation 3 days after inoculation ofEscherichia coliandBacteroides fragiliswith or withoutEnterococcus faecalis. The severity of the disease remained unchanged compared to that in control animals. A dose-related decrease in the peritoneal phagocyte count was observed in the treated groups compared to the counts in control animals. The subsequent experiments were performed exclusively in the mixed gram-positive–gram negative model, which exhibits an intense and prolonged inflammatory response with similar criteria. The early effects of mrIL-10 (evaluated 6 h after inoculation), repeated injections of mrIL-10 (four doses injected from 0 to 9 h after bacterial challenge), and pretreatment (two doses injected 6 and 3 h before inoculation) were evaluated. The clinical and microbiological parameters remained unchanged in the treated animals. Decreases in the peritoneal phagocyte count and the peritoneal concentration of tumor necrosis factor were observed following repeated injections of mrIL-10. In summary, our data suggest that mrIL-10 does not worsen the manifestations of sepsis. However, these results need to be confirmed in clinical practice.
Antigen-Specific B-Cell Unresponsiveness Induced by ChronicMycobacterium aviumsubsp.paratuberculosisInfection of Cattle
Authors: Waters W. R. , Stabel J. R. , Sacco R. E. , Harp J. A. , Pesch B. A. , Wannemuehler M. J.
Published: March 2020

Mycobacterium aviumsubsp.paratuberculosisinfection of cattle results in a chronic granulomatous enteritis. Clinical disease (i.e., cachexia, diarrhea, and high fecal bacterial counts) is preceded by a lengthy subclinical stage of disease. The immunologic mechanisms associated with the progression of infected cattle from subclinical to clinical disease are unclear. In this study, a cell proliferation assay was used in combination with flow cytometry to compare peripheral blood lymphocyte responses of cattle with subclinical paratuberculosis to responses of cattle with clinical paratuberculosis. B cells from cattle with subclinical disease proliferated vigorously upon stimulation withM. aviumsubsp.paratuberculosisantigen, with up to 12.4% of the total B cells responding. However, B cells from cattle with clinical disease did not proliferate upon antigen stimulation despite good proliferation in response to concanavalin A stimulation. In addition, these animals had high percentages of peripheral blood B cells. B cells from noninfected animals did not proliferate uponM. aviumsubsp.paratuberculosisantigen stimulation. Thus, it appears that B-cell proliferation is a sensitive indicator of subclinical Johne’s disease. Furthermore, the immunologic mechanisms responsible for the antigen-specific unresponsiveness of peripheral blood B cells may be significant in the eventual progression from subclinical to clinical Johne’s disease in cattle.
PhoP-PhoQ-Regulated Loci Are Required for Enhanced Bile Resistance in
Salmonella
spp
Authors: van Velkinburgh Jennifer C. , Gunn John S.
Published: March 2020

As enteric pathogens,
Salmonella
spp. are resistant to the actions of bile.
Salmonella typhimurium
and
Salmonella typhi
strains were examined to better define the bile resistance phenotype. The MICs of bile for wild-type
S. typhimurium
and
S. typhi
were 18 and 12%, respectively, and pretreatment of log-phase
S. typhimurium
with 15% bile dramatically increased bile resistance. Mutant strains of
S. typhimurium
and
S. typhi
lacking the virulence regulator PhoP-PhoQ were killed at significantly lower bile concentrations than wild-type strains, while strains with constitutively active PhoP were able to survive prolonged incubation with bile at concentrations of >60%. PhoP-PhoQ was shown to mediate resistance specifically to the bile components deoxycholate and conjugated forms of chenodeoxycholate, and the protective effect was not generalized to other membrane-active agents. Growth of both
S. typhimurium
and
S. typhi
in bile and in deoxycholate resulted in the induction or repression of a number of proteins, many of which appeared identical to PhoP-PhoQ-activated or -repressed products. The PhoP-PhoQ regulon was not induced by bile, nor did any of the 21 PhoP-activated or -repressed genes tested play a role in bile resistance. However, of the PhoP-activated or -repressed genes tested, two (
prgC
and
prgH
) were transcriptionally repressed by bile in the medium independent of PhoP-PhoQ. These data suggest that salmonellae can sense and respond to bile to increase resistance and that this response likely includes proteins that are members of the PhoP regulon. These bile- and PhoP-PhoQ-regulated products may play an important role in the survival of
Salmonella
spp. in the intestine or gallbladder.
Defensins Impair Phagocytic Killing by Neutrophils in Biomaterial-Related Infection
Authors: Kaplan S. S. , Heine R. P. , Simmons R. L.
Published: March 2020

The implantation of foreign material carries a risk of infection which frequently is resistant to all treatment short of removing the implant. We have previously shown that these materials activate neutrophils by contact, leading to production of oxygen free radicals accompanied by release of granule products. Such activation further results in depletion of local host defenses, including the capacity of biomaterial-activated neutrophils to kill bacteria. Among the granule products released from neutrophils are small cationic antibacterial peptides (human neutrophil peptides [HNP]) known as defensins. Here we tested the hypothesis that defensins, released from activated neutrophils onto the surface of biomaterials, might play a role in the deactivation of subsequent neutrophil populations. Incubation of neutrophils with purified HNP resulted in a dose-related impairment of stimulus-induced oxygen radical production and of phagocytic killing. Furthermore, fresh neutrophils added to biomaterial-associated neutrophils exhibited impaired phagocytic killing. This impairment could be abrogated by antibody to HNP but not by an irrelevant antibody. Taken together, these observations support the idea that neutrophils activated at a material surface can create, by means of HNP release, an environment hostile to their microbicidal function and that of their infiltrating brethren.
The p47phox−/−Mouse Model of Chronic Granulomatous Disease Has Normal Granuloma Formation and Cytokine Responses toMycobacterium aviumandSchistosoma mansoniEggs
Authors: Segal Brahm H. , Doherty T. Mark , Wynn Thomas A. , Cheever Allen W. , Sher Alan , Holland Steven M.
Published: March 2020

Chronic granulomatous disease (CGD) is a genetic disorder of NADPH oxidase in which phagocytes are defective in generating reactive oxidants. CGD patients suffer from recurrent infections and exuberant and persistent tissue granuloma formation. We hypothesized that abnormal granulomata in CGD may result from aberrant T-cell-mediated cytokine responses. To assess Th-1-type cytokine responses and granulomata, we challenged p47phox−/−and wild-type mice with avirulent (SmD) or virulent (SmT) variants ofMycobacterium avium2-151. To assess Th-2-type cytokine responses and granulomata, we usedSchistosoma mansonieggs (SME). Mononuclear cells were harvested, and cytokine responses were determined by enzyme-linked immunosorbent assay or reverse transcriptase PCR. Following SmD or SmT challenge, splenocytes from p47phox−/−and wild-type mice generated similar polar Th-1 responses (increased levels of gamma interferon and basal levels of interleukin 4 [IL-4] and IL-5). By 8 weeks after SmT challenge, exuberant splenic granulomata developed in p47phox−/−and wild-type mice. After SME challenge, thoracic lymph node mononuclear cells from p47phox−/−and wild-type mice generated similar mixed Th-1 and Th-2 cytokine responses to SME antigen and concanavalin A. Peak lung granuloma sizes and rates of regression were similar in p47phox−/−and wild-type mice. These results suggest that exuberant granulomatous inflammation in CGD is probably not the result of skewing of T-cell responses toward the Th-1 or Th-2 pole. Appropriate regression of established tissue granulomata in p47phox−/−mice challenged with SME suggests that abnormal granuloma formation in CGD is stimulus dependent and is not an invariant feature of the disease.
Intracellular Tryptophan Pool Sizes May Account for Differences in Gamma Interferon-Mediated Inhibition and Persistence of Chlamydial Growth in Polarized and Nonpolarized Cells
Authors: Kane Colleen D. , Vena Renee M. , Ouellette Scot P. , Byrne Gerald I.
Published: March 2020

Gamma interferon (IFN-γ) is an important factor in the modulating inhibition of intracellular chlamydial growth and persistence. In human epithelial cells and macrophages, this inhibition is the result of depletion of the essential amino acid tryptophan via the IFN-γ-induced enzyme indoleamine 2,3-dioxygenase. Under these conditions, chlamydiae must successfully compete with the host cell for limited resources in order to maintain viability. We provide evidence to support the hypothesis that the host cell polarization state influences the host-pathogen interplay and outcome of IFN-γ-mediated inhibition. In polarized cells, intracellular soluble tryptophan pools were larger than those in nonpolarized cells despite only small differences in the initial uptake rate of this amino acid compared to that in nonpolarized cells. Furthermore, inChlamydia trachomatis-infected cells, the amounts of tryptophan consumed by the organisms were similar for cells grown in either state. We propose that intracellular tryptophan pool sizes can account for differences in IFN-γ-mediated chlamydial persistence and growth inhibition in polarized and nonpolarized cells. Collectively, these results argue that polarized cell models, which more accurately reflect the conditions in vivo, may be more relevant than conventionally cultured cells in the study of intimate intracellular host-parasite interactions.
AnArcanobacterium(Actinomyces)pyogenesMutant Deficient in Production of the Pore-Forming Cytolysin Pyolysin Has Reduced Virulence
Authors: Jost B. H. , Songer J. G. , Billington S. J.
Published: March 2020

Pyolysin (PLO), the hemolytic exotoxin expressed byArcanobacterium(Actinomyces)pyogenes, is a member of the thiol-activated cytolysin family of bacterial toxins. Insertional inactivation of theplogene results in loss of expression of PLO with a concomitant loss in hemolytic activity. Theplomutant, PLO-1, has an approximately 1.8-log10reduction in the 50% infectious dose compared to that for wild-typeA. pyogenesin a mouse intraperitoneal infection model. Studies involving cochallenge of wild-type and PLO-1 bacteria resulted in recovery of similar numbers of both strains, suggesting that PLO production is required for survival in vivo. Recombinant, His-tagged PLO (His-PLO) is cytotoxic for mouse peritoneal macrophages and J774 cells in a dose-dependent manner. Protection against challenge withA. pyogenescould be afforded by vaccination with formalin-inactivated His-PLO, suggesting that PLO is a host-protective antigen, as well as a virulence determinant.
Lipopolysaccharides (LPS) of Oral Black-Pigmented Bacteria Induce Tumor Necrosis Factor Production by LPS-Refractory C3H/HeJ Macrophages in a Way Different from That ofSalmonellaLPS
Authors: Kirikae Teruo , Nitta Toshimasa , Kirikae Fumiko , Suda Yasuo , Kusumoto Shoichi , Qureshi Nirofer , Nakano Masayasu
Published: March 2020

Some lipopolysaccharide (LPS) preparations from S- or R-form members of the familyEnterobacteriaceaeand oral black-pigmented bacteria (Porphyromonas gingivalisandPrevotella intermedia) are known to activate LPS-refractory C3H/HeJ macrophages. When contaminating proteins are removed from R-form LPS ofEnterobacteriaceaeby repurification, however, this ability is lost. In the present study, we investigated the capacity of LPS fromP. gingivalis,P. intermedia,Salmonella minnesota, andSalmonella abortusequito induce production of tumor necrosis factor (TNF) in gamma interferon-primed C3H/HeJ macrophages before and after repurification.P. abortusequiS-LPS was fractionated by centrifugal partition chromatography into two LPS forms: SL-LPS, having homologous long O-polysaccharide chains, and SS-LPS having short oligosaccharide chains. Prior to repurification, all LPS forms except SL-LPS induced TNF production in both C3H/HeJ and C3H/HeN macrophages. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that repurification removed contaminating protein from the preparations, and repurified SS-LPS andS. minnesotaRa-LPS no longer stimulated TNF production in C3H/HeJ macrophages, although C3H/HeN macrophages remained responsive. In contrast, repurified oral bacterial LPS retained the capacity to induce TNF production in C3H/HeJ macrophages. Oral bacterial LPS preparations also were not antagonized by excess inactive, repurified SL-LPS; Ra-LPS;Rhodobacter sphaeroideslipid A, a competitive LPS antagonist, or paclitaxel, an LPS agonist, and they were comparatively resistant to polymyxin B treatment. Nevertheless, oral bacterial LPS was less toxic tod-galactosamine-treated C3H/HeN mice than was LPS fromSalmonella. These findings indicate that the active molecule(s) and mode of action of LPS fromP. gingivalisandP. intermediaare quite different from those of LPS fromSalmonella.
Strain Variation in Glycosaminoglycan Recognition Influences Cell-Type-Specific Binding by Lyme Disease Spirochetes
Authors: Parveen Nikhat , Robbins Douglas , Leong John M.
Published: March 2020

Lyme disease, a chronic multisystemic disorder that can affect the skin, heart, joints, and nervous system is caused by
Borrelia burgdorferi
sensu lato. Lyme disease spirochetes were previously shown to bind glycosaminoglycans (GAGs). In the current study, the GAG-binding properties of eight Lyme disease strains were determined. Binding by two high-passage HB19 derivatives to Vero cells could not be inhibited by enzymatic removal of GAGs or by the addition of exogenous GAG. The other six strains, which included a different high-passage HB19 derivative (HB19 clone 1), were shown to recognize both heparan sulfate and dermatan sulfate in cell-binding assays, but the relative efficiency of binding to these two GAGs varied among the strains. Strains N40, CA20-2A, and PBi bound predominantly to heparan sulfate, PBo bound both heparan sulfate and dermatan sulfate roughly equally, and VS461 and HB19 clone 1 recognized primarily dermatan sulfate. Cell binding by strain HB19 clone 1 was inhibited better by exogenous dermatan sulfate than by heparin, whereas heparin was the better inhibitor of binding by strain N40. The GAG-binding preference of a Lyme disease strain was reflected in its cell-type-specific binding. Strains that recognized predominantly heparan sulfate bound efficiently to both C6 glioma cells and EA-Hy926 cells, whereas strains that recognized predominantly dermatan sulfate bound well only to the glial cells. The effect of lyase treatment of these cells on bacterial binding was consistent with the model that cell-type-specific binding was a reflection of the GAG-binding preference. We conclude that the GAG-binding preference varies with the strain of Lyme disease spirochete and that this variation influences cell-type-specific binding in vitro.
Listeria monocytogenesPhospholipase C-Dependent Calcium Signaling Modulates Bacterial Entry into J774 Macrophage-Like Cells
Authors: Wadsworth Sandra J. , Goldfine Howard
Published: March 2020

Listeria monocytogenessecretes several proteins that have been shown to contribute to virulence. Among these is listeriolysin O (LLO), a pore-forming hemolysin that is absolutely required for virulence. Two other virulence factors are phospholipases: a phosphatidylinositol-specific phospholipase C (PI-PLC [plcA]) and a broad-range PLC (plcB). Although mutations inplcAorplcBresulted in small increases in mouse 50% lethal dose (LD50), deletions in both genes resulted in a 500-fold increase in LD50. We have examined the role of these secreted proteins in host intracellular signaling in the J774 macrophage-like cell line. Measurements of cytosolic free calcium ([Ca2+]i) have revealed a rapid spike upon exposure of these cells to wild-typeL. monocytogenes. This is followed by a second peak at 5 min and a third prolonged peak with a maximal [Ca2+]iof 800 to 1,000 nM. The pattern of calcium changes was greatly altered by deletion of any of the three virulence factors. An LLO mutant produced none of these elevations in [Ca2+]i; however, a transient elevation was observed whenever these bacteria entered the cell. A PI-PLC mutant produced a diminished single elevation in [Ca2+]iat 15 to 30 min. A broad-range PLC mutant produced only the first calcium spike. Studies with inhibitors suggested that the first elevation arises from influx of calcium from the extracellular medium through plasma membrane channels and that the second and third elevations come from release of Ca2+from intracellular stores. We observed that internalization of wild-type bacteria and the broad-range PLC mutant was delayed for 5 to 10 min, but the LLO and PI-PLC mutants were internalized rapidly upon infection. Inhibitors that affected calcium signaling changed the kinetics of association of wild-type bacteria with J774 cells, the kinetics of entry, and the efficiency of escape from the primary phagosome.
Mapping of Staphylococcal Enterotoxin A Functional Binding Sites and Presentation by Monoclonal Antibodies and Fusion Proteins
Authors: Mahana Wahib
Published: March 2020

Staphylococal enterotoxins (SE) bind with high affinity to major histocompatibility complex (MHC) class II proteins and stimulate large number of T cells via the Vβ region of the T-cell receptor (TCR). To map the epitopes of SE type A (SEA) involved in MHC binding and cell proliferation, 20 specific anti-SEA monoclonal antibodies (MAbs) and two large glutathioneS-transferase fusion proteins corresponding to the amino and carboxy termini, respectively, of SEA were used. The functionality of these antibodies was tested, by MHC binding inhibition, interleukin-2 production, and T-cell proliferation assays. Moreover, I studied the ability of the MAbs to present SEA in vitro to human and murine cells and their reactivity with the two fusion proteins. This study showed that all of the MAbs have a defined effect on one or both immunological properties of SEA and were able to present SEA to human and murine cells. However, one MAb (4H8) recognized SEA but without any interference with its biological activities. When the MAbs were tested to react with the two fusion proteins representing the SEA molecule, all of the MAbs were negative except for two. These results confirmed the presence of two functionally different binding sites of SEA with MHC class II molecules and the importance of the disulfide loop for the mitogenic activity of SEA. I further demonstrated that MAbs can present SEA to immune cells independent of the site recognized by the antibody and that the integrity of the SEA molecule is very important for its functions.
Transformation and Expression of a Cloned
fimA
Gene in
Porphyromonas gingivalis
Authors: Takahashi Yusuke , Kato Daisuke , Hamada Nobushiro , Yoshimoto Hisashi , Umemoto Toshio
Published: March 2020

The
Porphyromonas gingivalis
fimbria is an important virulence factor involved in the adherence and colonization of the organism in the oral cavity. In this study, we transformed this organism with a gene,
fimA
381
, encoding the fimbrial subunit of
P. gingivalis
381 (fimbrillin) by using the host-vector system that we developed previously and examined expression of the cloned
fimA
381
gene. The recombinant plasmid pYHF2 was constructed by ligating a fragment containing the
fimA
381
gene into the plasmid vector pYH420 and transformed into the restriction-deficient
P. gingivalis
host YH522. pYHF2 was autonomously maintained in YH522 cells, and the fimbrillin polypeptide (recombinant fimbrillin) was fully expressed. The molecular mass of the recombinant fimbrillin was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as 41 kDa, which was identical to that of the native fimbrillin of strain 381. The amino acid sequences of the 20 amino-terminal residues of the recombinant fimbrillin and the native fimbrillin of the strain 381 were identical. In addition, characteristic long and thin fimbrial structures (recombinant fimbriae) that were distinguishable from the host’s native fimbriae when examined by immunogold electron microscopy were observed around the cell surface of the transformants containing the
fimA
381
gene. These results suggested that transformation of
fimA
gene from a different strain of
P. gingivalis
followed by accumulation of the mature fimbrial subunit protein was sufficient for production of fimbrial structures that were observable by electron microscopy.
Characterization of an Immunogenic Glycocalyx on the Surfaces of
Cryptosporidium parvum
Oocysts and Sporozoites
Authors: Nanduri Jayasri , Williams Selvi , Aji Toshiki , Flanigan Timothy P.
Published: March 2020

Ruthenium red staining of
Cryptosporidium parvum
oocysts revealed the presence of a carbohydrate matrix on their outer bilayers that is characteristic of a glycocalyx. Surface labeling of intact oocysts identified material of high molecular weight (>10
6
) that reacted positively with sera from cryptosporidium-infected patients and with immunoglobulin A monoclonal antibodies.
Clonal Expansion of Antigen-Specific CD4 T Cells following Infection withSalmonella typhimuriumIs Similar in Susceptible (Itys) and Resistant (Ityr) BALB/c Mice
Authors: Chen Zong-ming , Jenkins Marc K.
Published: March 2020

The results show that CD4 T cells specific for a recombinant antigen expressed inSalmonella typhimuriumproliferate normally in mice that express the susceptible form of theItygene at early times after infection but do not retain the capacity to produce gamma interferon later in the infection.
Expanded Safety and Immunogenicity of a Bivalent, Oral, Attenuated Cholera Vaccine, CVD 103-HgR Plus CVD 111, in United States Military Personnel Stationed in Panama
Authors: Taylor David N. , Sanchez José L. , Castro José M. , Lebron Carlos , Parrado Carlos M. , Johnson David E. , Tacket Carol O. , Losonsky Genevieve A. , Wasserman Steven S. , Levine Myron M. , Cryz Stanley J.
Published: March 2020

To provide optimum protection against classical and El Tor biotypes of
Vibrio cholerae
O1, a single-dose, oral cholera vaccine was developed by combining two live, attenuated vaccine strains, CVD 103-HgR (classical, Inaba) and CVD 111 (El Tor, Ogawa). The vaccines were formulated in a double-chamber sachet; one chamber contained lyophilized bacteria, and the other contained buffer. A total of 170 partially-immune American soldiers stationed in Panama received one of the following five formulations: (a) CVD 103-HgR at 10
8
CFU plus CVD 111 at 10
7
CFU, (b) CVD 103-HgR at 10
8
CFU plus CVD 111 at 10
6
CFU, (c) CVD 103-HgR alone at 10
8
CFU, (d) CVD 111 alone at 10
7
CFU, or (e) inactivated
Escherichia coli
placebo. Among those who received CVD 111 at the high or low dose either alone or in combination with CVD 103-HgR, 8 of 103 had diarrhea, defined as three or more liquid stools. None of the 32 volunteers who received CVD 103-HgR alone or the 35 placebo recipients had diarrhea. CVD 111 was detected in the stools of 46% of the 103 volunteers who received it. About 65% of all persons who received CVD 103-HgR either alone or in combination had a fourfold rise in Inaba vibriocidal titers. The postvaccination geometric mean titers were comparable among groups, ranging from 450 to 550. Ogawa vibriocidal titers were about twice as high in persons who received CVD 111 as in those who received CVD 103-HgR alone (600 versus 300). The addition of CVD 111 improved the overall seroconversion rate and doubled the serum Ogawa vibriocidal titers, suggesting that the combination of an El Tor and a classical cholera strain is desirable. While CVD 111 was previously found to be well tolerated in semiimmune Peruvians, the adverse effects observed in this study indicate that this strain requires further attenuation before it can be safely used in nonimmune populations.
The
Staphylococcus aureus
Alternative Sigma Factor ς
B
Controls the Environmental Stress Response but Not Starvation Survival or Pathogenicity in a Mouse Abscess Model
Authors: Chan Pan F. , Foster Simon J. , Ingham Eileen , Clements Mark O.
Published: March 2020

The role of ς
B
, an alternative sigma factor of
Staphylococcus aureus
, has been characterized in response to environmental stress, starvation-survival and recovery, and pathogenicity. ς
B
was mainly expressed during the stationary phase of growth and was repressed by 1 M sodium chloride. A
sigB
insertionally inactivated mutant was created. In stress resistance studies, ς
B
was shown to be involved in recovery from heat shock at 54°C and in acid and hydrogen peroxide resistance but not in resistance to ethanol or osmotic shock. Interestingly,
S. aureus
acquired increased acid resistance when preincubated at a sublethal pH 4 prior to exposure to a lethal pH 2. This acid-adaptive response resulting in tolerance was mediated via
sigB
. However, ς
B
was not vital for the starvation-survival or recovery mechanisms. ς
B
does not have a major role in the expression of the global regulator of virulence determinant biosynthesis, staphylococcal accessory regulator (
sarA
), the production of a number of representative virulence factors, and pathogenicity in a mouse subcutaneous abscess model. However, SarA upregulates
sigB
expression in a growth-phase-dependent manner. Thus, ς
B
expression is linked to the processes controlling virulence determinant production. The role of ς
B
as a major regulator of the stress response, but not of starvation-survival, is discussed.
Localization of Cell Division Protein FtsQ by Immunofluorescence Microscopy in Dividing and Nondividing Cells of
Escherichia coli
Authors: Buddelmeijer Nienke , Aarsman Mirjam E. G. , Kolk Arend H. J. , Vicente Miguel , Nanninga Nanne
Published: March 2020

The localization of cell division protein FtsQ in
Escherichia coli
wild-type cells was studied by immunofluorescence microscopy with specific monoclonal antibodies. FtsQ could be localized to the division site in constricting cells. FtsQ could also localize to the division site in
ftsQ1
(Ts) cells grown at the permissive temperature. A hybrid protein in which the cytoplasmic domain and the transmembrane domain were derived from the γ form of penicillin-binding protein 1B and the periplasmic domain was derived from FtsQ was also able to localize to the division site. This result indicates that the periplasmic domain of FtsQ determines the localization of FtsQ, as has also been concluded by others for the periplasmic domain of FtsN. Noncentral FtsQ foci were found in the area of the cell where the nucleoid resides and were therefore assumed to represent sites where the FtsQ protein is synthesized and simultaneously inserted into the cytoplasmic membrane.
Rpb4, a Subunit of RNA Polymerase II, Enables the Enzyme To Transcribe at Temperature Extremes In Vitro
Authors: Rosenheck Sonia , Choder Mordechai
Published: March 2020

Rpb4 is a subunit of
Saccharomyces cerevisiae
RNA polymerase II (Pol II). It associates with the polymerase preferentially in stationary phase and is essential for some stress responses. Using the promoter-independent initiation and chain elongation assay, we monitored Pol II enzymatic activity in cell extracts. We show here that Rpb4 is required for the polymerase activity at temperature extremes (10 and 35°C). In contrast, at moderate temperature (23°C) Pol II activity is independent of Rpb4. These results are consistent with the role previously attributed to Rpb4 as a subunit whose association with Pol II helps Pol II to transcribe during extreme temperatures. The enzymatic inactivation of Pol II lacking Rpb4 at the nonoptimal temperature was prevented by the addition of recombinant Rpb4 produced in
Escherichia coli
prior to the in vitro reaction assay. This finding suggests that modification of Rpb4 is not required for its functional association with the other Pol II subunits. Sucrose gradient and immunoprecipitation experiments demonstrated that Rpb4 is present in the cell in excess over the Pol II complex during all growth phases. Nevertheless, the rescue of Pol II activity at the nonoptimal temperature by Rpb4 is possible only when cell extracts are obtained from postlogarithmic cells, not from logarithmically growing cells. This result suggests that Pol II molecules should be modified in order to recruit Rpb4; the portion of the modified Pol II molecules is small during logarithmic phase and becomes predominant in stationary phase.
In Vitro Repair of Gaps in Bacteriophage T7 DNA
Authors: Lai Ying-Ta , Masker Warren
Published: March 2020

An in vitro system based upon extracts of
Escherichia coli
infected with bacteriophage T7 was used to study the mechanism of double-strand break repair. Double-strand breaks were placed in T7 genomes by cutting with a restriction endonuclease which recognizes a unique site in the T7 genome. These molecules were allowed to repair under conditions where the double-strand break could be healed by (i) direct joining of the two partial genomes resulting from the break, (ii) annealing of complementary versions of 17-bp sequences repeated on either side of the break, or (iii) recombination with intact T7 DNA molecules. The data show that while direct joining and single-strand annealing contributed to repair of double-strand breaks, these mechanisms made only minor contributions. The efficiency of repair was greatly enhanced when DNA molecules that bridge the region of the double-strand break (referred to as donor DNA) were provided in the reaction mixtures. Moreover, in the presence of the donor DNA most of the repaired molecules acquired genetic markers from the donor DNA, implying that recombination between the DNA molecules was instrumental in repairing the break. Double-strand break repair in this system is highly efficient, with more than 50% of the broken molecules being repaired within 30 min under some experimental conditions. Gaps of 1,600 nucleotides were repaired nearly as well as simple double-strand breaks. Perfect homology between the DNA sequence near the break site and the donor DNA resulted in minor (twofold) improvement in the efficiency of repair. However, double-strand break repair was still highly efficient when there were inhomogeneities between the ends created by the double-strand break and the T7 genome or between the ends of the donor DNA molecules and the genome. The distance between the double-strand break and the ends of the donor DNA molecule was critical to the repair efficiency. The data argue that ends of DNA molecules formed by double-strand breaks are typically digested by between 150 and 500 nucleotides to form a gap that is subsequently repaired by recombination with other DNA molecules present in the same reaction mixture or infected cell.
Myxococcus xanthus sasN
Encodes a Regulator That Prevents Developmental Gene Expression during Growth
Authors: Xu Di , Yang Chun , Kaplan Heidi B.
Published: March 2020

Myxococcus xanthus
multicellular fruiting body development is initiated by nutrient limitation at high cell density. Five clustered point mutations (
sasB5
, -
14
, -
15
, -
16
, and -
17
) can bypass the starvation and high-cell-density requirements for expression of the
4521
developmental reporter gene. These mutants express
4521
at high levels during growth and development in an
asgB
background, which is defective in generation of the cell density signal, A signal. A 1.3-kb region of the
sasB
locus cloned from the wild-type chromosome restored the SasB
+
phenotype to the five mutants. DNA sequence analysis of the 1.3-kb region predicted an open reading frame, designated SasN. The N terminus of SasN appears to contain a strongly hydrophobic region and a leucine zipper motif. SasN showed no significant sequence similarities to known proteins. A strain containing a newly constructed
sasN
-null mutation and Ω4521 Tn
5lac
in an otherwise wild-type background expressed
4521
at a high level during growth and development. A similar
sasN
-null mutant formed abnormal fruiting bodies and sporulated at about 10% the level of wild type. These data indicate that the wild-type
sasN
gene product is necessary for normal
M. xanthus
fruiting body development and functions as a critical regulator that prevents
4521
expression during growth.
Identification and Characterization of SpcU, a Chaperone Required for Efficient Secretion of the ExoU Cytotoxin
Authors: Finck-Barbançon Viviane , Yahr Timothy L. , Frank Dara W.
Published: March 2020

In recent studies, we have shown that
Pseudomonas aeruginosa
strains that are acutely cytotoxic in vitro damage the lung epithelium in vivo. Genetic analysis indicated that the factor responsible for acute cytotoxicity was controlled by ExsA and therefore was part of the exoenzyme S regulon. The specific virulence determinant responsible for epithelial damage in vivo and cytotoxicity in vitro was subsequently mapped to the
exoU
locus. The present studies are focused on a genetic characterization of the
exoU
locus. Northern blot analyses and complementation experiments indicated that a region downstream of
exoU
was expressed and that the expression of this region corresponded to increased ExoU secretion. DNA sequence analysis of a region downstream of
exoU
identified several potential coding regions. One of these open reading frames, SpcU (specific
Pseudomonas
chaperone for ExoU), encoded a small 15-kDa acidic protein (137 amino acids [pI 4.4]) that possessed a leucine-rich motif associated with the Syc family of cytosolic chaperones for the
Yersinia
Yops. T7 expression analysis and nickel chromatography of histidine-tagged proteins indicated that ExoU and SpcU associated as a noncovalent complex when coexpressed in
Escherichia coli
. The association of ExoU and SpcU required amino acids 3 to 123 of ExoU. In
P. aeruginosa
, ExoU and SpcU are coordinately expressed as an operon that is controlled at the transcriptional level by ExsA.
Characterization of CorR, a Transcriptional Activator Which Is Required for Biosynthesis of the Phytotoxin Coronatine
Authors: Peñaloza-Vázquez Alejandro , Bender Carol L.
Published: March 2020

Coronatine (COR) is a plasmid-encoded phytotoxin synthesized by several pathovars of phytopathogenic
Pseudomonas syringae
. The COR biosynthetic gene cluster in
P. syringae
pv. glycinea PG4180 is encoded by a 32-kb region which contains both the structural and regulatory genes needed for COR synthesis. The regulatory region contains three genes:
corP
,
corS
, and
corR. corS
is thought to function as a histidine protein kinase, whereas
corP
and
corR
show relatedness to response regulators of the two-component regulatory paradigm. In the present study, we investigated whether CorR is a positive activator of COR gene expression. We also studied whether CorR specifically binds the DNA region located upstream of
cfl
, a gene located at the 5′ end of the gene cluster encoding coronafacic acid, the polyketide portion of COR. Complementation analysis with a
corR
mutant, PG4180.P2, and transcriptional fusions to a promoterless glucuronidase gene (
uidA
) indicated that CorR functions as a positive regulator of COR gene expression. Deletion analysis of the 5′ end of the
cfl
upstream region was used to define the minimal region required for COR gene expression. A 360-bp DNA fragment located over 500 bp upstream from the
cfl
transcriptional start site was used in DNase I protection assays to define the specific bases bound by CorR. An area extending from −704 to −650 with respect to the
cfl
transcriptional start site was protected by DNase I footprinting, indicating a rather large area of protection. This area was also conserved in the promoter region for
cmaA
, which encodes a transcript containing genes for coronamic acid synthesis, another intermediate in the COR biosynthetic pathway. The results obtained in the current study suggest that both the coronafacic acid and the coronamic acid structural genes are controlled by CorR, a positive activator of COR gene expression.