bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and Expression of the
Moraxella catarrhalis
Lactoferrin Receptor Genes
Authors: Du Run-Pan , Wang Qijun , Yang Yan-Ping , Schryvers Anthony B. , Chong Pele , Klein Michel H. , Loosmore Sheena M.
Published: December 2019

The lactoferrin receptor genes from two strains of
Moraxella catarrhalis
have been cloned and sequenced. The
lfr
genes are arranged as
lbpB
followed by
lbpA
, a gene arrangement found in lactoferrin and transferrin receptor operons from several bacterial species. In addition, a third open reading frame,
orf3
, is located one nucleotide downstream of
lbpA
. The deduced lactoferrin binding protein A (LbpA) sequences from the two strains were found to be 99% identical, the LbpB sequences were 92% identical, and the ORF3 proteins were 98% identical. The
lbpB
gene was PCR amplified and sequenced from a third strain of
M. catarrhalis
, and the encoded protein was found to be 77% identical and 84% similar to the other LbpB proteins. Recombinant LbpA and LbpB proteins were expressed from
Escherichia coli
, and antisera raised to the purified proteins were used to assess antigenic conservation in a panel of
M. catarrhalis
strains. The recombinant proteins were tested for the ability to bind human lactoferrin following gel electrophoresis and electroblotting, and rLbpB, but not rLbpA, was found to bind lactoferrin. Bactericidal antibody activity was measured, and while the anti-rLbpA antiserum was not bactericidal, the anti-rLbpB antisera were found to be weakly bactericidal. Thus, LbpB may have potential as a vaccine candidate.
Characterization of the Thermal Stress Response of
Campylobacter jejuni
Authors: Konkel Michael E. , Kim Bong J. , Klena John D. , Young Colin R. , Ziprin Richard
Published: December 2019

Campylobacter jejuni
, a microaerophilic, gram-negative bacterium, is a common cause of gastrointestinal disease in humans. Heat shock proteins are a group of highly conserved, coregulated proteins that play important roles in enabling organisms to cope with physiological stresses. The primary aim of this study was to characterize the heat shock response of
C. jejuni
. Twenty-four proteins were preferentially synthesized by
C. jejuni
immediately following heat shock. Upon immunoscreening of
Escherichia coli
transformants harboring a
Campylobacter
genomic DNA library, one recombinant plasmid that encoded a heat shock protein was isolated. The recombinant plasmid, designated pMEK20, contained an open reading frame of 1,119 bp that was capable of encoding a protein of 372 amino acids with a calculated molecular mass of 41,436 Da. The deduced amino acid sequence of the open reading frame shared similarity with that of DnaJ, which belongs to the Hsp-40 family of molecular chaperones, from a number of bacteria. An
E. coli dnaJ
mutant was successfully complemented with the pMEK20 recombinant plasmid, as judged by the ability of bacteriophage λ to form plaques, indicating that the
C. jejuni
gene encoding the 41-kDa protein is a functional homolog of the
dnaJ
gene from
E. coli
. The ability of each of two
C. jejuni dnaJ
mutants to form colonies at 46°C was severely retarded, indicating that DnaJ plays an important role in
C. jejuni
thermotolerance. Experiments revealed that a
C. jejuni
DnaJ mutant was unable to colonize newly hatched Leghorn chickens, suggesting that heat shock proteins play a role in vivo.
Regulation of Sucrose-6-Phosphate Hydrolase Activity in
Streptococcus mutans
: Characterization of the
scrR
Gene
Authors: Hiratsuka Koichi , Wang Bing , Sato Yutaka , Kuramitsu Howard
Published: December 2019

Previous results have implicated an important role for the enzyme II
Scr
, the sucrose-specific permease, in the transport of sucrose by cariogenic
Streptococcus mutans
. The product of the
scrB
gene, sucrose-6-phosphate hydrolase (Suc-6PH), is required for the metabolism of phosphorylated sucrose. The results from the utilization of
scrB
::
lacZ
fusions in
S. mutans
GS-5 have suggested that sucrose-grown cells have higher levels of
scrB
gene expression than do cells grown with glucose or fructose. Northern blot analysis of
scrB
transcripts has also confirmed the relative strengths of expression as sucrose>glucose>fructose. Immediately downstream from the
scrB
gene, an open reading frame with homology to regulatory proteins of the GalR-LacI family as well as to ScrR proteins from several other bacteria has been identified. In addition, this gene appears to be transcribed in the same operon as
scrB
. Inactivation of this gene,
scrR
, did not alter the relative expression of the
scrB
gene in the presence of sucrose or fructose but did increase SUC-6PH levels in the presence of glucose to that observed with sucrose. Furthermore, the
S. mutans
ScrR homolog appears to bind to the
scrB
promoter region as determined from the results of gel shift assays. These results suggest that the
scrR
gene is involved in the regulation of
scrB
, and likely
scrA
, expression. However, it is not clear whether sucrose acts as an inducer of expression of these genes or, alternatively, whether glucose and fructose act as repressors.
Induction of Protective Immunity in Rabbits by Coadministration of InactivatedPasteurella multocidaToxin and Potassium Thiocyanate Extract
Authors: Jarvinen L. Z. , Hogenesch H. , Suckow M. A. , Bowersock T. L.
Published: December 2019

Pasteurella multocidais a bacterial pathogen that causes rhinitis (snuffles), pneumonia, otitis media, septicemia, metritis, and death in domestic rabbits. Currently, there are no effective vaccines to prevent infection by this organism. Subcutaneous (s.c.) immunization with either exotoxin or thiocyanate extracts ofP. multocidainduces partial protection in rabbits. Since disease begins at mucosal sites, induction of local immunity may be important in preventing systemic disease. Little is known concerning the efficacy of intranasal (i.n.) administration of these antigens in inducing protective mucosal immunity toP. multocidain rabbits. The purpose of this study was twofold: (i) to investigate the effectiveness of vaccination with purifiedP. multocidatoxin (PMT) and a potassium thiocyanate extract ofP. multocida(CN) in combination and (ii) to evaluate the efficacy of administration of these antigens i.n. versus s.c. Forty-eight rabbits were randomly divided into eight different treatment groups. Rabbits received either one or both antigens by either s.c. or i.n. administration. Following vaccination, each group received an i.n. challenge ofP. multocida. Rabbits vaccinated with both antigens i.n. or s.c. had a 100% survival rate, few or no bacteria in the liver and lungs, high serum immunoglobulin G (IgG) and IgM antibody titers, and significant numbers of IgG antibody-secreting cells (ASC) in the spleen and tracheobronchial lymph node. Rabbits vaccinated i.n. had significant nasal and bronchoalveolar lavage IgA antibody levels. Rabbits vaccinated with only one antigen, either PMT or CN, had lower antibody titers, moderate to severe liver and lung infections, and fewer ASC compared to rabbits receiving both antigens. Rabbits in the control groups had moderate to severe liver and lung infections. This study indicates that i.n. immunization with both PMT and CN induces an effective response against homologousP. multocidachallenge.
Molecular Evolution of a Pathogenicity Island from Enterohemorrhagic
Escherichia coli
O157:H7
Authors: Perna Nicole T. , Mayhew George F. , Pósfai György , Elliott Simon , Donnenberg Michael S. , Kaper James B. , Blattner Frederick R.
Published: December 2019

We report the complete 43,359-bp sequence of the locus of enterocyte effacement (LEE) from EDL933, an enterohemorrhagic
Escherichia coli
O157:H7 serovar originally isolated from contaminated hamburger implicated in an outbreak of hemorrhagic colitis. The locus was isolated from the EDL933 chromosome with a homologous-recombination-driven targeting vector. Recent completion of the LEE sequence from enteropathogenic
E. coli
(EPEC) E2348/69 afforded the opportunity for a comparative analysis of the entire pathogenicity island. We have identified a total of 54 open reading frames in the EDL933 LEE. Of these, 13 fall within a putative P4 family prophage designated 933L. The prophage is not present in E2348/69 but is found in a closely related EPEC O55:H7 serovar and other O157:H7 isolates. The remaining 41 genes are shared by the two complete LEEs, and we describe the nature and extent of variation among the two strains for each gene. The rate of divergence is heterogeneous along the locus. Most genes show greater than 95% identity between the two strains, but other genes vary more than expected for clonal divergence among
E. coli
strains. Several of these highly divergent genes encode proteins that are known to be involved in interactions with the host cell. This pattern suggests recombinational divergence coupled with natural selection and has implications for our understanding of the interaction of both pathogens with their host, for the emergence of O157:H7, and for the evolutionary history of pathogens in general.
Differential Regulation of the Interleukin-12 Receptor during the Innate Immune Response toLeishmania major
Authors: Jones Douglas , Elloso M. Merle , Showe Louise , Williams Donna , Trinchieri Giorgio , Scott Phillip
Published: December 2019

Previous studies have shown the central role of interleukin 12 (IL-12) in the development of resistance toLeishmania majorinfection in C3H mice. We now show that during the innate immune response the lymph node cells ofL. major-infected C3H mice upregulate the IL-12 receptor on CD4+, CD8+, and B220+cells. An increase in the ability of the lymph node cells to bind IL-12 correlates with 9.3- and 4.6-fold increases in the mRNA expression levels of the IL-12Rβ1 and -β2 subunits, respectively. In contrast, BALB/c mice, which are susceptible toL. majorinfection, have no increase in the ability of the lymph node cells to bind IL-12 and correspondingly smaller increases in the mRNA expression levels of the IL-12Rβ1 and -β2 subunits of 2- and 1.5-fold, respectively. Neutralizing IL-4 and the administration of exogenous IL-12 upregulate IL-12R expression in BALB/c mice, while the neutralization of IL-12 in C3H mice blocks increased IL-12 receptor expression. These experiments reveal an important role for the regulation of the IL-12 receptor during the innate immune response after infection of mice with a pathogen.
Production of Stabilized Virulence Factor-Negative Variants by Group A Streptococci during Stationary Phase
Authors: Leonard B. A. B. , Woischnik M. , Podbielski A.
Published: December 2019

Many of the virulence factors associated with fulminant group A streptococci (GAS) infection are expressed under in vitro exponential growth conditions. However, the survival of GAS in tissue and intracellularly, as well as colonization of asymptomatic carriers, has been reported for GAS. The bacteria associated with these niches may encounter high-density, low-nutrient-flowthrough conditions that may more closely mimic in vitro stationary-phase conditions than exponential growth. Therefore, the behavior of GAS in stationary-phase culture was examined. We observed that after 24 h in stationary phase, GAS serotypes M49 and M2 developed a unstable colony dimorphism of typical large and atypical small colonies. Between days 4 and 5, we isolated stabilized atypical small colonies which remained stable for up to nine passages (approximately 200 generations) on fresh medium before fully reverting to the large-colony phenotype. Upon analysis, the small colonies showed no difference in cell number per colony, growth rate, survival in prolonged stationary-phase culture, or antibiotic sensitivity. However, the small colonies showed decreased transcription of hyaluronic acid capsule, the global positive virulence factor regulator gene
mga
, the
mga
-regulated
emm
mRNA (M-protein structural gene), and
speB
(cysteine protease). Accordingly, the small colonies were completely sensitive in a traditional phagocytosis assay. The production of virulence factors and phagocytosis resistance of the small-colony isolates was recovered when, after several passages on fresh medium, the colony morphology began to revert.
Hemolysin-Positive Enteroaggregative and Cell-DetachingEscherichia coliStrains Cause Oncosis of Human Monocyte-Derived Macrophages and Apoptosis of Murine J774 Cells
Authors: Fernandez-Prada Carmen , Tall Ben D. , Elliott Simon E. , Hoover David L. , Nataro James P. , Venkatesan Malabi M.
Published: December 2019

Infection of human monocyte-derived macrophages (HMDM) and J774 cells (murine macrophage cell line) with several enteroaggregative and cytodetachingEscherichia coli(EAggEC and CDEC, respectively) strains demonstrated that some strains could induce macrophage cell death accompanied by release of lactate dehydrogenase activity and interleukin 1β (IL-1β) into culture supernatants. The mode of cell death differed in the two types of macrophages. Damage to macrophage plasma membrane integrity without changes in nuclear morphology resulted in cytolysis of HMDM. This mechanism of cell death has been previously described for virulentShigellainfection of HMDM and is termed oncosis. In contrast, infection of J774 cells by EAggEC and CDEC strains resulted in apoptosis. The presence of α-hemolysin (Hly) in EAggEC and CDEC strains appears to be critical for both oncosis in HMDM and apoptosis in J774 cells. Bacteria lacking Hly, including Hly−EAggEC strains as well as enterotoxigenic, enteropathogenic, and enterohemorrhagicE. colistrains, behaved like avirulentShigella flexneriin that the macrophage monolayers were intact, with no release of lactate dehydrogenase activity or IL-1β into the culture supernatants.
Heterogeneous Antibody Responses in Tuberculosis
Authors: Lyashchenko Konstantin , Colangeli Roberto , Houde Michel , Al Jahdali Hamdan , Menzies Dick , Gennaro Maria Laura
Published: December 2019

Antibody responses during tuberculosis were analyzed by an enzyme-linked immunosorbent assay with a panel of 10 protein antigens of
Mycobacterium tuberculosis
. It was shown that serum immunoglobulin G antibodies were produced against a variety of
M. tuberculosis
antigens and that the vast majority of sera from tuberculosis patients contained antibodies against one or more
M. tuberculosis
antigens. The number and the species of serologically reactive antigens varied greatly from individual to individual. In a given serum, the level of specific antibodies also varied with the antigen irrespective of the total number of antigens recognized by that particular serum. These findings indicate that person-to-person heterogeneity of antigen recognition, rather than recognition of particular antigens, is a key attribute of the antibody response in tuberculosis.
Constitutive and Inducible Green Fluorescent Protein Expression inBartonella henselae
Authors: Lee Anthea K. , Falkow Stanley
Published: December 2019

The green fluorescent protein (GFP) gene was expressed on a plasmid inB. henselae, and GFP-expressing bacteria were visualized by fluorescence microscopy. HEp-2 cells infected with GFP-expressing bacteria were separated from uninfected cells with a fluorescence activated cell sorter. Promoter fusions ofB. henselaechromosomal DNA togfpwere examined by flow cytometry, and aB. henselae groELpromoter fusion which induced expression at 37°C was isolated.
Binding of theHelicobacter pyloriVacuolating Cytotoxin to Target Cells
Authors: Massari Paola , Manetti Roberto , Burroni Daniela , Nuti Sandra , Norais Nathalie , Rappuoli Rino , Telford John L.
Published: December 2019

The vacuolating cytotoxin ofHelicobacter pylori, VacA, enters the cytoplasm of target cells and causes vacuolar degeneration by interfering with late stages of endocytosis. By using indirect immunofluorescence and flow cytometry, we have demonstrated that VacA binds to specific high-affinity cell surface receptors and that this interaction is necessary for cell intoxication.
A Chimeric Influenza Virus Expressing an Epitope of Outer Membrane Protein F ofPseudomonas aeruginosaAffords Protection against Challenge withP. aeruginosain a Murine Model of Chronic Pulmonary Infection
Authors: Staczek J. , Gilleland H. E. , Gilleland L. B. , Harty R. N. , García-Sastre A. , Engelhardt O. G. , Palese P.
Published: December 2019

The ability of a chimeric influenza virus containing, within the antigenic B site of its hemagglutinin, an 11-amino-acid (AEGRAINRRVE) insert from the peptide 10 epitope of outer membrane (OM) protein F ofPseudomonas aeruginosato serve as a protective vaccine againstP. aeruginosawas tested by using the murine chronic pulmonary infection model. Mice immunized with the chimeric virus developed antibodies that reacted in an enzyme-linked immunosorbent assay with peptide 10, with purified protein F, and with whole cells of various immunotype strains ofP. aeruginosabut failed to react with a protein F-deficient strain ofP. aeruginosa. The chimeric-virus antisera reacted specifically with protein F alone when immunoblotted against proteins extracted from cell envelopes of each of the seven Fisher-Devlin immunotype strains and had significantly greater in vitro opsonic activity forP. aeruginosathan did antisera from wild-type influenza virus-immunized mice. Subsequent to intratracheal challenge with agar-encased cells ofP. aeruginosa, chimeric-virus-immunized mice developed significantly fewer severe lung lesions than did control mice immunized with the wild-type influenza virus. Furthermore, the chimeric influenza virus-immunized group had a significantly smaller percentage of mice with >5 × 103CFU ofP. aeruginosain their lungs upon bacterial quantitation than did the control group. These data indicate that chimeric influenza viruses expressing epitopes of OM protein F warrant continued development as vaccines to prevent pulmonary infections caused byP. aeruginosa.
Comparison of Surface Proteins ofAnaplasma marginaleGrown in Tick Cell Culture, Tick Salivary Glands, and Cattle
Authors: Barbet A. F. , Blentlinger R. , Yi Jooyoung , Lundgren A. M. , Blouin E. F. , Kocan K. M.
Published: December 2019

Anaplasma marginale, a tick-borne rickettsial pathogen of cattle, infects bovine erythrocytes, resulting in mild to severe hemolytic disease that causes economic losses in domestic livestock worldwide. Recently, the Virginia isolate ofA. marginalewas propagated in a continuous tick cell line, IDE8, derived from embryonicIxodes scapularis. Development ofA. marginalein cell culture was morphologically similar to that described previously in ticks. In order to evaluate the potential of the cell culture-derived organisms for use in future research or as an antigen for serologic tests and vaccines, the extent of structural conservation of the major surface proteins (MSPs) between the cell culture-derivedA. marginaleand the bovine erythrocytic stage, currently the source ofA. marginaleantigen, was determined. Structural conservation on the tick salivary-gland stage was also examined. Monoclonal and monospecific antisera against MSPs 1 through 5, initially characterized against erythrocyte stages, also reacted withA. marginalefrom cell culture and tick salivary glands. MSP1a among geographicA. marginaleisolates is variable in size because of different numbers of a tandemly repeated 28- or 29-amino-acid peptide. The cell culture-derivedA. marginalemaintained the same-size MSP1a as that found on the Virginia isolate ofA. marginalein bovine erythrocytes and tick salivary glands. Although differences were observed in the polymorphic MSP2 antigen between culture and salivary-gland stages, MSP2 did not appear to vary, by two-dimensional gel electrophoresis, during continuous passage in culture. These data show that MSPs of erythrocyte-stageA. marginaleare present on culture stages and may be structurally conserved during continuous culture. The presence of all current candidate diagnostic and vaccine antigens suggests that in vitro cultures are a valuable source of rickettsiae for basic research and for the development of improved diagnostic reagents and vaccines against anaplasmosis.
Expression of the EspB Protein of Enteropathogenic
Escherichia coli
within HeLa Cells Affects Stress Fibers and Cellular Morphology
Authors: Taylor Kathleen A. , Luther Paul W. , Donnenberg Michael S.
Published: December 2019

The EspB protein of enteropathogenic
Escherichia coli
(EPEC) is essential for the signaling events that lead to the accumulation of actin beneath intimately attached bacteria, a process that is known as the attaching and effacing effect. EspB is targeted to the host cell cytoplasm by a type III secretion apparatus. To determine the effect of intracellular EspB on the host cell cytoskeleton, we transfected HeLa cells with a plasmid containing the
espB
gene under the control of an inducible eukaryotic promoter. A HeLa cell clone that expressed
espB
mRNA and EspB protein after induction was selected for further study. The expression of EspB in these cells caused a dramatic change in cell morphology and a marked reduction in actin stress fibers. Cells expressing EspB were significantly impaired in their ability to support invasion by EPEC and
Salmonella typhimurium
. However, the expression of EspB within host cells could not compensate for the lack of EspB expression by an
espB
mutant strain of EPEC to restore attaching and effacing activity. These studies suggest that EspB is a cytoskeletal toxin that is translocated to the host cell cytoplasm, where it causes a redistribution of actin.
The Immunoglobulin (IgG) Antibody Response to OspA and OspB Correlates with Severe and Prolonged Lyme Arthritis and the IgG Response to P35 Correlates with Mild and Brief Arthritis
Authors: Akin Evren , McHugh Gail L. , Flavell Richard A. , Fikrig Erol , Steere Allen C.
Published: December 2019

In an effort to implicate immune responses to specificBorrelia burgdorferiproteins that may have a role in chronic Lyme arthritis, we studied the natural history of the antibody response toB. burgdorferiin serial serum samples from 25 patients monitored throughout the course of Lyme disease. In these patients, the immunoglobulin G (IgM) and IgG antibody responses to 10 recombinantB. burgdorferiproteins, determined during early infection, early arthritis, and maximal arthritis, were correlated with the severity and duration of maximal arthritis. The earliest responses were usually to outer surface protein C (OspC), P35, P37, and P41; reactivity with OspE, OspF, P39, and P93 often developed weeks later; and months to years later, 64% of patients had responses to OspA and OspB. During early infection and early arthritis, the levels of IgG antibody to P35 correlated inversely with the subsequent severity or duration of maximal arthritis. In contrast, during periods of maximal arthritis, the levels of IgG antibody to OspA and OspB, especially to a C-terminal epitope of OspA, correlated directly with the severity and duration of arthritis. Thus, the higher the IgG antibody response to P35 earlier in the infection, the milder and briefer the subsequent arthritis, whereas during maximal arthritis, the higher the IgG response to OspA and OspB, the more severe and prolonged the arthritis.
Mechanisms for Induction of L-Selectin Loss from T Lymphocytes by a Cryptococcal Polysaccharide, Glucuronoxylomannan
Authors: Dong Zhao Ming , Jackson Lydgia , Murphy Juneann W.
Published: December 2019

Disseminated cryptococcosis is accompanied by cryptococcal polysaccharides in the serum and the lack of cellular infiltrates in infected tissues. Cryptococcal polysaccharides given intravenously to mice inhibit the influx of T lymphocytes into the sites of cell-mediated immune response. The focus here was to determine whether cryptococcal polysaccharides modulate the expression of molecules, such as L-selectin, that are important in extravasation of T cells. Cryptococcal glucuronoxylomannan (GXM), but not galactoxylomannan or mannoprotein, was found to cause loss of L-selectin from freshly isolated human T cells of both CD4 and CD8 subsets and from Jurkat cells. With the signaling-pathway inhibitors staurosporine (which inhibits protein kinase C) and herbimycin A (which inhibits protein tyrosine kinases), we showed that GXM or the cryptococcal culture filtrate antigen CneF directly induces L-selectin loss from CD4+and CD8+T cells via a herbimycin A-sensitive pathway(s) presumably involving one or more protein tyrosine kinases but not via a pathway involving protein kinase C. Loss of L-selectin from the T cells before the T cells have a chance to bind to L-selectin ligands on endothelial cells would be expected to prevent T-cell migration into inflamed tissues and/or lymph organs.
Existing Antilisterial Immunity Does Not Inhibit the Development of aListeria monocytogenes-Specific Primary Cytotoxic T-Lymphocyte Response
Authors: Bouwer H. G. Archie , Shen Hao , Fan Xin , Miller Jeff F. , Barry Ronald A. , Hinrichs David J.
Published: December 2019

Infection of BALB/c mice withListeria monocytogenesstimulates an antilisterial immune response evident by the appearance of H2-Kd-restricted CD8+cytotoxic T lymphocytes (CTLs) specific for the nanomer peptides amino acids (aa) 91 to 99 of listeriolysin O (LLO 91–99) and aa 217 to 225 of the p60 molecule (p60 217–225). We have introduced point mutations at anchor residues within LLO 91–99 (92F) or p60 217–225 (218F), and BALB/c mice infected withL. monocytogenesstrains containing these point mutations do not develop CTLs specific for LLO 91–99 or p60 217–225, respectively. We have used these strains to test whether primary CTL responses againstL. monocytogenes-derived determinants can be stimulated within an environment of existing antilisterial immunity. We found that the development of a primaryL. monocytogenes-specific CTL response is not altered by existing immunity toL. monocytogenes. For example, primary immunization with the p60 218F strain ofL. monocytogenesfollowed by a secondary immunization with wild-typeL. monocytogenesresults in stimulation of p60 217–225-specific CTLs at primary response levels and LLO 91–99-specific effectors at levels consistent with a memory CTL response. Similarly, primary immunization with the 92F strain ofL. monocytogenesfollowed by a secondary immunization with wild-typeL. monocytogenesresults in stimulation of LLO 91–99-specific CTLs at primary response levels and p60 217–225-specific effectors at levels consistent with a memory CTL response. These results provide additional support for the use ofL. monocytogenesas a recombinant vaccine vector and show that antivector immunity does not inhibit the development of a primary CTL response when the epitope is delivered byL. monocytogenesas the vaccine strain.
Establishment of Unipolar Localization of IcsA in
Shigella flexneri
2a Is Not Dependent on Virulence Plasmid Determinants
Authors: Sandlin Robin C. , Maurelli Anthony T.
Published: December 2019

Unipolar localization of IcsA on the surface of
Shigella flexneri
is required for efficient formation of actin tails and protrusions in infected eucaryotic cells. Lipopolysaccharide (LPS) mutations have been demonstrated to affect either the establishment or the maintenance of IcsA in a unipolar location, although the mechanism is unknown. In order to analyze the contribution of virulence plasmid determinants on the unipolar localization of IcsA, we examined the localization of IcsA expressed from a cloned plasmid copy in two different genetic backgrounds. The localization of IcsA was first examined in a virulence plasmid-cured derivative of the wild-type
S. flexneri
2a isolate 2457T. This approach examined the contribution of virulence plasmid-borne factors, including the previously identified virulence plasmid-borne protease that is responsible for cleaving IcsA in the outer membrane and releasing the 95-kDa secreted form from the cell surface. IcsA localization in a related but nonpathogenic
Escherichia coli
strain expressing LPS of the O8 serotype was also examined. IcsA surface presentation in both of these genetic backgrounds continued to be unipolar, demonstrating that virulence plasmid-borne determinants are not responsible for unipolar localization of IcsA. The unipolar localization of IcsA in the
E. coli
background suggests that a common pathway that allows IcsA to be spatially restricted to one pole on the bacterial cell surface exists in
Shigella
and
E. coli
.
Recombinant Expression and Localization of
Schistosoma mansoni
Cathepsin L1 Support Its Role in the Degradation of Host Hemoglobin
Authors: Brady Ciaran P. , Dowd Andrew J. , Brindley Paul J. , Ryan Thecla , Day Sharon R. , Dalton John P.
Published: December 2019

Cysteine proteinases expressed by schistosomes appear to play key roles in the digestion of host hemoglobin, the principal source of amino acid nutrients utilized by these parasites. We have shown previously that the predominant cysteine proteinase activity in soluble extracts and excretory/secretory (ES) products of adults of
Schistosoma mansoni
and
S. japonicum
is cathepsin L-like in its substrate specificity. However, biochemical analysis of the cathepsin L activity in extracts and ES products of schistosomes has been complicated by the presence of at least two distinct forms of schistosome cathepsin L, termed SmCL1 and SmCL2. We now report the purification and enzyme characteristics of active, recombinant SmCL1 which was obtained by transforming
Saccharomyces cerevisiae
with an expression plasmid encoding the preproenzyme of SmCL1. Recombinant SmCL1 was secreted by the transformed yeast into the culture media from which it was purified by gel filtration and ion-exchange chromatography. The purified enzyme exhibited substrate specificity against synthetic peptidyl substrates (e.g., Boc-Val-Leu-Lys-NHMec and Z-Phe-Arg-NHMec;
k
cat
/

K
m

= 17.25 and 6.24 mM
−1
s
−1
, respectively) and against gelatin and hemoglobin, characteristic of cathepsin L. Immunoblot analysis using antiserum raised against recombinant SmCL1 demonstrated that native SmCL1 of 33 kDa was present in ES products and soluble extracts of
S. mansoni
. Using this antiserum and thin tissue sections, we localized the native SmCL1 to the gastrodermis and to the tegument of adult schistosomes. Recombinant SmCL1 was capable of degrading human hemoglobin at pH 4.0 to 4.5 but not higher, suggesting that denaturation of hemoglobin by low pH, as found in the cecum of the adult schistosome, may be necessary for its catalysis by cathepsin L and other gut-associated proteinases. Together, these results support a role for SmCL1 in the degradation of host hemoglobin within the gut of the schistosome.
Tonsillar Application of Formalin-Killed Cells ofStreptococcus sobrinusReduces Experimental Dental Caries in Rabbits
Authors: Fukuizumi Takaki , Inoue Hiromasa , Tsujisawa Toshiyuki , Uchiyama Choji
Published: December 2019

LivingStreptococcus sobrinuscells were orally inoculated into nonimmune rabbits and rabbits immunized with formalin-killed cells ofS. sobrinusthrough tonsillar application to examine the anticaries potential of this method of immunization. The livingS. sobrinuscell numbers and the caries areas in the rabbits immunized by tonsillar application decreased to a level one-fifth of that in nonimmune rabbits.
High-Level Expression of
Plasmodium vivax
Apical Membrane Antigen 1 (AMA-1) in
Pichia pastoris
: Strong Immunogenicity in
Macaca mulatta
Immunized with
P. vivax
AMA-1 and Adjuvant SBAS2
Authors: Kocken Clemens H. M. , Dubbeld Martin A. , Van Der Wel Annemarie , Pronk Jack T. , Waters Andrew P. , Langermans Jan A. M. , Thomas Alan W.
Published: December 2019

The apical membrane antigen 1 (AMA-1) family is a promising family of malaria blood-stage vaccine candidates that have induced protection in rodent and nonhuman primate models of malaria. Correct conformation of the protein appears to be essential for the induction of parasite-inhibitory responses, and these responses appear to be primarily antibody mediated. Here we describe for the first time high-level secreted expression (over 50 mg/liter) of the
Plasmodium vivax
AMA-1 (PV66/AMA-1) ectodomain by using the methylotrophic yeast
Pichia pastoris
. To prevent nonnative glycosylation, a conservatively mutagenized PV66/AMA-1 gene (PV66Δglyc) lacking N-glycosylation sites was also developed. Expression of the PV66Δglyc ectodomain yielded similar levels of a homogeneous product that was nonglycosylated and was readily purified by ion-exchange and gel filtration chromatographies. Recombinant PV66Δglyc
43–487
was reactive with conformation-dependent monoclonal antibodies. With the SBAS2 adjuvant,
Pichia
-expressed PV66Δglyc
43–487
was highly immunogenic in five rhesus monkeys, inducing immunoglobulin G enzyme-linked immunosorbent assay titers in excess of 1:200,000. This group of monkeys had a weak trend showing lower cumulative parasite loads following a
Plasmodium cynomolgi
infection than in the control group.
Killing of
Borrelia burgdorferi
by Antibody Elicited by OspA Vaccine Is Inefficient in the Absence of Complement
Authors: Nowling Jena M. , Philipp Mario T.
Published: December 2019

A Lyme disease vaccine, based on the
Borrelia burgdorferi
lipoprotein OspA, has recently undergone phase III trials in humans. The results of one of these trials indicate that vaccine efficacy positively correlates with anti-OspA antibody titer. Spirochete killing within the tick vector midgut, upon which vaccine efficacy appears to depend, may occur chiefly via a mechanism that involves antibody alone, as it has been reported that complement is degraded by tick saliva decomplementing factors. We compared the in vitro killing efficiencies of anti-OspA antibody elicited in rhesus monkeys by the OspA vaccine, in the presence and in the absence of monkey complement. Killing in the absence of complement was between 14 and 3,800 times less efficient than with complement present, depending on the spirochete strain. The relative inefficiency of the complement-independent killing mechanism by anti-OspA antibody may explain why OspA vaccine efficacy is critically dependent on antibody titer.
Murine γδ T Lymphocytes Elicited duringPlasmodium yoeliiInfection Respond toPlasmodiumHeat Shock Proteins
Authors: Kopacz Jeffrey , Kumar Nirbhay
Published: December 2019

γδ T cells accumulate duringPlasmodiuminfections in both murine and human malarias. The biological role of these cells and the antigens that they recognize are not clearly understood, although recent findings indicate that γδ T cells in general influence both innate and antigen-specific adaptive host responses. We examined the accumulation of γδ T cells elicited during infection with virulent and avirulentPlasmodium yoeliiparasites in relatively susceptible and resistant strains of mice. Our results indicated that in nonlethal malaria infections, γδ T cells comprise a larger proportion of splenic T cells than in lethal infections and that only a live infection is capable of inducing an increase in the percentage of γδ T cells in vivo. Furthermore, we demonstrate that γδ T cells elicited during aP. yoeliiinfection respond by proliferation in vitro toP. falciparumheat shock proteins (HSPs) of 60 and 70 kDa, suggesting a possible immunological involvement of parasite HSPs in this arm of the cellular immune response during malarial infection in mice.
Campylobacter jejuni
-Stimulated Secretion of Interleukin-8 by INT407 Cells
Authors: Hickey Thomas E. , Baqar Shihida , Bourgeois A. Louis , Ewing Cheryl P. , Guerry Patricia
Published: December 2019

Incubation of INT407 cells with various clinical isolates of
Campylobacter jejuni
resulted in secretion of interleukin-8 (IL-8) at levels ranging from 96 to 554 pg/ml at 24 h. The strains which produced the highest levels of IL-8 secretion were 81-176 and BT44. Induction of IL-8 secretion required live cells of 81-176 and was dependent on de novo protein synthesis. Site-specific mutants of 81-176, which were previously shown to be defective in adherence and invasion, resulted in reduced levels of secretion of IL-8, and
cheY
mutants of strains 81-176 and 749, which are hyperadherent and hyperinvasive, resulted in higher levels of IL-8 secretion. Another mutant of 81-176, which adheres at about 43% of the wild-type levels but is noninvasive, also showed marked reduction in IL-8 levels, suggesting that invasion is necessary for high levels of IL-8 secretion. When gentamicin was added to INT407 cells at 2 h after infection with 81-176, IL-8 secretion 22 h later was equivalent to that of controls without gentamicin, suggesting that the events which trigger induction and release of IL-8 occur early in the interactions of bacteria and eukaryotic cells.
Roles of Glycoproteins and Oligosaccharides Found in Human Vaginal Fluid in Bacterial Adherence
Authors: Rajan Nithya , Cao Qing , Anderson Byron E. , Pruden Denise L. , Sensibar Julia , Duncan James L. , Schaeffer Anthony J.
Published: December 2019

Adherence of type 1-piliatedEscherichia colito carbohydrate structures of vaginal mucosa plays a major role in the pathogenesis of ascending urinary tract infections in women. Colonization of the vaginal introitus is influenced by interactions between pathogens, vaginal fluid, and vaginal epithelium. In this study, the type and amount of carbohydrates and glycoproteins present in vaginal fluid were determined. Free and protein-bound oligosaccharides in vaginal fluid specimens were analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE) and high-pressure liquid chromatography (HPLC). Two-dimensional electrophoretic separations of vaginal fluid glycoproteins were performed together with bacterial overlay assays. The results of FACE showed that the majority of the oligosaccharides are in the free state and the bound oligosaccharides are undetectable. HPLC analysis of free sugars revealed glucose as the major sugar (3.3 ± 0.3 mM), and the concentrations of mannose and glucosamine were 0.065 ± 0.04 and 0.02 ± 0.001 mM, respectively. RadiolabeledE. colibound three vaginal fluid glycoproteins with the following molecular masses and pIs: 82 kDa and pI 5.5, 55 kDa and pI 4.5, and 55 kDa and pI 6.5. The binding was inhibited by mannose and by deglycosylation of the proteins prior to the overlay assay. One of these putative receptors was identified to be the heavy chain of secretory IgA (S-IgA). These data suggest that the free mannose in the fluid is less than that required to affectE. coli-epithelial cell binding interactions and that S-IgA may bindE. coliin the vaginal introitus.
Humoral and Cellular Immune Responses in Mice Immunized with RecombinantMycobacterium bovisBacillus Calmette-Guérin Producing a Pertussis Toxin-Tetanus Toxin Hybrid Protein
Authors: Abomoelak B. , Huygen K. , Kremer L. , Turneer M. , Locht C.
Published: December 2019

The development of combined vaccines constitutes one of the priorities in modern vaccine research. One of the most successful combined vaccines in use is the diphtheria-pertussis-tetanus vaccine. However, concerns about the safety of the pertussis arm have led to decreased acceptance of the vaccine but also to the development of new, safer, and effective acellular vaccines against pertussis. Unfortunately, the production cost of these new vaccines is significantly higher than that of previous vaccines. Here, we explore the potential of live recombinantMycobacterium bovisBCG producing the hybrid protein S1-TTC, which contains the S1 subunit of pertussis toxin fused to fragment C of tetanus toxin, as an alternative to the acellular vaccines. S1-TTC was produced in two different expression systems. In the first system its production was under the control of the 85A antigen promoter and signal peptide, and in the second system it was under the control of thehsp60promoter. Although expression of the hybrid antigen was obtained in both cases, only the second expression system yielded a recombinant BCG strain able to induce both a specific humoral immune response and a specific cellular immune response. The antibodies generated were directed against the TTC part and neutralized toxin activity in an in vivo challenge model, whereas interleukin-2 production was specific for both parts of the molecule. Since protection against tetanus is antibody mediated and protection against pertussis may be cell mediated, this constitutes a first promising step towards the development of a cost-effective, protective, and safe combined vaccine against pertussis, tetanus, and tuberculosis.
Genomic Subtractive Hybridization and Selective Capture of Transcribed Sequences Identify a NovelSalmonella typhimuriumFimbrial Operon and Putative Transcriptional Regulator That Are Absent from theSalmonella typhiGenome
Authors: Morrow Brian J. , Graham James E. , Curtiss Roy
Published: December 2019

Salmonella typhi, the etiologic agent of typhoid fever, is adapted to the human host and unable to infect nonprimate species. The genetic basis for host specificity inS. typhiis unknown. The avirulence ofS. typhiin animal hosts may result from a lack of genes present in the broad-host-range pathogenSalmonella typhimurium. Genomic subtractive hybridization was successfully employed to isolateS. typhimuriumgenomic sequences which are absent from theS. typhigenome. These genomic subtracted sequences mapped to 17 regions distributed throughout theS. typhimuriumchromosome. A positive cDNA selection method was then used to identify subtracted sequences which were transcribed byS. typhimuriumfollowing macrophage phagocytosis. A novel putative transcriptional regulator of the LysR family was identified as transcribed by intramacrophageS. typhimurium. This putative transcriptional regulator was absent from the genomes of the human-adapted serovarsS. typhiandSalmonella paratyphiA. Mutations within this gene did not alter the level ofS. typhimuriumsurvival within macrophages or virulence within mice. A subtracted genomic fragment derived from the ferrichrome operon also hybridized to the intramacrophage cDNA. Nucleotide sequence analysis ofS. typhimuriumandS. typhichromosomal sequences flanking the ferrichrome operon identified a novelS. typhimuriumfimbrial operon with a high level of similarity to sequences encodingProteus mirabilismannose-resistant fimbriae. The novel fimbrial operon was absent from theS. typhigenome. The absence of specific genes may have allowedS. typhito evolve as a highly invasive, systemic human pathogen.
Exposure ofN-Formyl-l-Methionyl-l-Leucyl-l-Phenylalanine-Activated Human Neutrophils to thePseudomonas aeruginosa-Derived Pigment 1-Hydroxyphenazine Is Associated with Impaired Calcium Efflux and Potentiation of Primary Granule Enzyme Release
Authors: Ramafi Grace , Anderson Ronald , Theron Annette , Feldman Charles , Taylor Graham W. , Wilson Robert , Cole Peter J.
Published: December 2019

The effects of pathologically relevant concentrations (0.38 to 12.5 μM) of the proinflammatory,Pseudomonas aeruginosa-derived pigment 1-hydroxyphenazine (1-hp) on Ca2+metabolism and intracellular cyclic AMP (cAMP) inN-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP; 1 μM)-activated human neutrophils, as well as on the release of myeloperoxidase (MPO) and elastase from these cells, have been investigated in vitro. Ca2+fluxes were measured by the combination of a fura-2/AM-based spectrofluorimetric method and radiometric procedures, which together enable distinction between net efflux and influx of the cation, while radioimmunoassay and colorimetric methods were used to measure cAMP and granule enzymes, respectively. Coincubation of neutrophils with 1-hp did not affect intracellular cAMP levels or the FMLP-activated release of Ca2+from intracellular stores but did retard the subsequent decline in the chemoattractant-induced increase in the concentration of cytosolic free Ca2+. These effects of 1-hp on the clearance of Ca2+from the cytosol of activated neutrophils were associated with decreased efflux of the cation from the cells and increased release of MPO and elastase, while the delayed store-operated influx of the cation into the cells was unaffected by the pigment. The plasma membrane Ca2+-ATPase rather than a Na+-Ca2+exchanger appeared to be the primary target of 1-hp. These observations suggest that the proinflammatory interactions of 1-hp with activated human neutrophils are a consequence of interference with the efflux of cytosolic Ca2+from these cells.
Streptococcus parasanguis pepOEncodes an Endopeptidase with Structure and Activity Similar to Those of Enzymes That Modulate Peptide Receptor Signaling in Eukaryotic Cells
Authors: Froeliger Eunice H. , Oetjen Joyce , Bond Jeffrey P. , Fives-Taylor Paula
Published: December 2019

Studies in our laboratory have identified two fimbria-associated adhesins, FimA and Fap1, ofStreptococcus parasanguisFW213. In this study, we isolated and sequenced DNA fragments linked tofimAto determine if they contained additional factors associated with adherence, virulence, or survival in the host. An open reading frame just upstream and divergently transcribed from thefimAoperon was identified and namedpepO. Northern hybridization indicated thatpepOis transcribed as a monocistronic message.pepOencodes a predicted 631-amino-acid protein with a molecular mass of approximately 70.6 kDa. PepO contains the essential motif HEXXH, typical of many zinc-dependent metalloproteases and metallopeptidases. PepO has significant sequence identity to mammalian metallopeptidases, including endothelin-converting enzyme, which converts a potent vasoconstrictor into its active form, and neutral endopeptidase (NEP), which is involved in terminating the activity of opioid peptides. The opioid peptide metenkephalin is a natural substrate of NEP. Cell extracts of FW213 cleaved metenkephalin at the same site as does NEP, while an extract from an insertionally inactivatedpepOmutant did not. These results indicate that FW213pepOencodes an enzyme with activity similar to that of known mammalian endopeptidases. Phylogenetic analysis of PepO and its homologues suggests lateral genetic exchange between bacteria and eukaryotes.
CD40-CD40 Ligand Interactions Augment Survival of Normal Mice, but Not CD40 Ligand Knockout Mice, Challenged Orally withSalmonella dublin
Authors: Marriott Ian , Thomas Elaine K. , Bost Kenneth L.
Published: December 2019

Interactions between CD40 expressed on macrophages and CD40 ligand expressed on T lymphocytes can be an important signal for optimal macrophage activation. Previous studies have demonstrated that the optimal response against certain intracellular pathogens (e.g.,CrytosporidiumandLeishmaniaspp.) by macrophages requires CD40-CD40 ligand interactions. However, this finding is not universal, since two recent reports utilizing CD40 knockout mice have shown no such contribution to the protective immune response againstMycobacterium tuberculosisorHistoplasma capsulatum. We demonstrate here that CD40-CD40 ligand interactions are significant events in the protective response against the intracellular pathogenSalmonella dublinin normal mice but not for animals genetically deficient in CD40 ligand expression. Treating BALB/c mice exogenously with a CD40 agonist (i.e., soluble trimeric CD40 ligand) increased resistance against a lethal, orally administered dose ofS. dublin. Conversely, in vivo administration of a monoclonal antibody against CD40 ligand to block endogenous CD40-CD40 ligand interactions resulted in a decreased resistance to salmonellosis. In contrast, CD40 ligand knockout mice demonstrated no increased susceptibility to salmonellosis. In vitro treatment ofSalmonella-infected macrophages from BALB/c mice with soluble trimeric CD40 ligand resulted in an elevated production of interleukin 12p70 by these cells, suggesting a mechanism whereby CD40-CD40 ligand interactions might enhance protective immune responses to this pathogen. Taken together, these studies strongly suggest that CD40-CD40 ligand interactions in normal mice play an important protective role in immune responses against the gram-negative, intracellular pathogenS. dublin.