bluuebunny/crossref_metadata_2025 huggingface dataset:
Green Fluorescent Protein as a Marker in
Plasmodium berghei
Transformation
Authors: Sultan Ali A. , Thathy Vandana , Nussenzweig Victor , Ménard Robert
Published: December 2019

We present a new marker that confers both resistance to pyrimethamine and green fluorescent protein-based fluorescence on the malarial parasite
Plasmodium berghei
. A single copy of the cassette integrated into the genome is sufficient to direct fluorescence in parasites throughout the life cycle, in both its mosquito and vertebrate hosts. Erythrocyte stages of the parasite that express the marker can be sorted from control parasites by flow cytometry. Pyrimethamine pressure is not necessary for maintaining the cassette in transformed parasites during their sporogonic cycle in mosquitoes, including when it is borne by a plasmid. This tool should thus prove useful in molecular studies of
P. berghei
, both for generating parasite variants and monitoring their behavior.
Suppression of Platelet Aggregation byBordetella pertussisAdenylate Cyclase Toxin
Authors: Iwaki Masaaki , Kamachi Kazunari , Heveker Nikolaus , Konda Toshifumi
Published: December 2019

The effect ofBordetella pertussisadenylate cyclase toxin (ACT) on platelet aggregation was investigated. This cell-invasive adenylate cyclase completely suppressed ADP (10 μM)-induced aggregation of rabbit platelets at 3 μg/ml and strongly suppressed thrombin (0.2 U/ml)-induced aggregation at 10 μg/ml. The suppression was accompanied by marked increase in platelet intracellular cyclic AMP (cAMP) content and was diminished by the anti-ACT monoclonal antibody B7E11. A catalytically inactive point mutant of ACT did not show the suppressive effect. Since an increase of cAMP content is a known cause of platelet dysfunction, these results indicate that the observed platelet inactivation was due to the catalytic activity of ACT through increase of intracellular cAMP.
Platelet-Activating Factor Induces Nitric Oxide Synthesis inTrypanosoma cruzi-Infected Macrophages and Mediates Resistance to Parasite Infection in Mice
Authors: Aliberti Júlio C. S. , Machado Fabiana S. , Gazzinelli Ricardo T. , Teixeira Mauro M. , Silva João S.
Published: December 2019

Trypanosoma cruzireplicates in nucleated cells and is susceptible to being killed by gamma interferon-activated macrophages through a mechanism dependent upon NO biosynthesis. In the present study, the role of platelet-activating factor (PAF) in the induction of NO synthesis and in the activation of the trypanocidal activity of macrophages was investigated. In vitro, PAF induced NO secretion byT. cruzi-infected macrophages and the secreted NO inhibited intracellular parasite growth. The addition of a PAF antagonist, WEB 2170, inhibited both NO biosynthesis and trypanocidal activity. The inducible NO synthase/l-arginine pathway mediated trypanocidal activity, since it was inhibited by treatment withl-N-monomethyl arginine (l-NMMA), anl-arginine analog. PAF-mediated NO production in infected macrophages appears to be dependent on tumor necrosis alpha (TNF-α) production, since the addition of a neutralizing anti-TNF-α monoclonal antibody mAb inhibited NO synthesis. To test the role of PAF in mediating resistance or susceptibility toT. cruziinfection, infected mice were treated with WEB 2170, a PAF antagonist. These animals had higher parasitemia and earlier mortality than did vehicle-treated mice. Taken together, our results suggest that PAF belongs to a group of mediators that coordinate the mechanisms of resistance to infections with intracellular parasites.
Resistance toCoccidioides immitisin Mice after Immunization with Recombinant Protein or a DNA Vaccine of a Proline-Rich Antigen
Authors: Abuodeh Raed O. , Shubitz Lisa F. , Siegel Erin , Snyder Shannon , Peng Tao , Orsborn Kris I. , Brummer Elmer , Stevens David A. , Galgiani John N.
Published: December 2019

Two inbred strains of mice (BALB/c and C57BL/6) were vaccinated with either recombinant expression protein of aCoccidioides immitisspherule-derived proline-rich antigen (rPRA) in monophosphoryl lipid A-oil emulsion adjuvant or a DNA vaccine based on the same antigen. Four weeks after vaccination, mice were infected intraperitoneally with arthroconidia. By 2 weeks, groups of mice receiving saline or plasmids with no PRA insert exhibited significant weight loss, and quantitative CFUs in the lungs ranged from 5.9 to 6.4 log10. In contrast, groups of mice immunized with either rPRA or DNA vaccine had significantly smaller pulmonary fungal burdens, ranging from 3.0 to 4.5 log10fewer CFUs. In vitro immunologic markers of lymphocyte proliferation and gamma interferon (IFN-γ) release after splenocytes were stimulated with rPRA correlated with protection. Also, plasma concentrations of rPRA-specific total immunoglobulin G (IgG), IgG1, and IgG2a showed increases in vaccinated mice. These studies expand earlier work by demonstrating protection in mice which differ inH-2background, by using an adjuvant that is potentially applicable to human use, and by achieving comparable protections with a DNA-based vaccine. Our in vitro results substantiate a Th1 response as evidenced by IFN-γ release and increased IgG2a. However, IgG1 was also stimulated, suggesting some Th2 response as well. PRA is a promising vaccine candidate for prevention of coccidioidomycosis and warrants further investigation.
A Longitudinal Study of Human Antibody Responses toPlasmodium falciparumRhoptry-Associated Protein 1 in a Region of Seasonal and Unstable Malaria Transmission
Authors: Fonjungo Peter N. , Elhassan Ibrahim M. , Cavanagh David R. , Theander Thor G. , Hviid Lars , Roper Cally , Arnot David E. , McBride Jana S.
Published: December 2019

Rhoptry-associated protein 1 (RAP1) ofPlasmodium falciparumis a nonpolymorphic merozoite antigen that is considered a potential candidate for a malaria vaccine against asexual blood stages. In this longitudinal study, recombinant RAP1 (rRAP1) proteins with antigenicity similar to that ofP. falciparum-derived RAP1 were used to analyze antibody responses to RAP1 over a period of 4 years (1991 to 1995) of 53 individuals naturally exposed toP. falciparummalaria. In any 1 year during the study, between 23 and 39% of individuals who had malaria developed immunoglobulin G (IgG) antibodies detectable with at least one rRAP1 protein. However, the anti-RAP1 antibody responses were detected only during or shortly after clinical malarial infections. RAP1 antibody levels declined rapidly (within 1 to 2 months) following drug treatment of the infections. No anti-RAP1 antibodies were usually detected a few months after the end of malaria transmission, during the dry season, or by the start of the next malaria season. Thus, RAP1 IgG responses were very short-lived. The short duration of RAP1 antibody response may explain the apparent lack of response in a surprisingly high proportion of individuals after clinical malarial infections. For some individuals who experienced more than one malarial infection, a higher anti-RAP1 antibody response to subsequent infections than to earlier infections was observed. This suggested secondary responses to RAP1 and thus the development of immunological memory for RAP1.
Effects of Cytotoxic Necrotizing Factor 1 and Lethal Toxin on Actin Cytoskeleton and VE-Cadherin Localization in Human Endothelial Cell Monolayers
Authors: Vouret-Craviari Valérie , Grall Dominique , Flatau Gilles , Pouysségur Jacques , Boquet Patrice , Van Obberghen-Schilling Ellen
Published: December 2019

Integrity of the vascular endothelium is largely dependent on endothelial cell shape and establishment of intercellular junctions. Certain pathogenic bacterial toxins alter the cytoskeletal architecture of intoxicated cells by modulating the GTPase activity of p21 Rho family proteins. In the present study we have analyzed the effect of Rho-directed toxins on the actin cytoskeleton and monolayer integrity of endothelial cells. We report here thatEscherichia colicytotoxic necrotizing factor 1 (CNF1) activates Rho in human umbilical vein endothelial cells (HUVEC). In confluent monolayers, CNF1 treatment induces prominent stress fiber formation without significantly modifying peripheral localization of VE-cadherin, a specific marker of vascular endothelial cell adherens junctions. Further, Rho activation with CNF1 blocks thrombin-induced redistribution of VE-cadherin staining and gap formation in HUVEC monolayers. Inhibition of Rho by prolonged treatment of cells with C3 exoenzyme (Clostridium botulinum) eliminates actin stress fibers without disrupting the continuity of VE-cadherin staining, indicating that Rho-dependent stress fibers are not required for maintaining this adhesion receptor at sites of intercellular contact. Lethal toxin (Clostridium sordellii), an inhibitor of Rac as well as Ras and Rap, potently disrupts the actin microfilament system and monolayer integrity in HUVEC cultures.
Interleukin 5 (IL-5) Is Not Required for Expression of a Th2 Response or Host Resistance Mechanisms during Murine Schistosomiasis Mansoni but Does Play a Role in Development of IL-4-Producing Non-T, Non-B Cells
Authors: Rosa Brunet Laura , Sabin Elizabeth A. , Cheever Allen W. , Kopf Manfred A. , Pearce Edward J.
Published: December 2019

During schistosomiasis, interleukin-5 (IL-5)-dependent eosinophil responses have been implicated in immunopathology, resistance to superinfection, synergistic interactions with chemotherapeutic agents, and the inductive phase of the egg-induced Th2 response. We examined these issues in IL-5-deficient (IL-5−/−) mice. IL-5−/−and wild-type (WT) mice were indistinguishable in terms of susceptibility to primary infections and the ability to resist secondary infections. Moreover, hepatic pathology was similar in both strains apart from a relative lack of eosinophils and, during chronic infection, a significantly larger mast cell component in the granulomas of IL-5−/−mice. Splenocyte cytokine production in response to soluble egg antigen (SEA) or anti-CD3 revealed no significant differences except for heightened tumor necrosis factor alpha production by cells from chronically infected IL-5−/−mice compared to WT animals. In contrast, ionomycin-stimulated non-B, non-T (NBNT) cells from IL-5−/−mice produced significantly smaller IL-4 amounts than did NBNT cells from WT animals. This difference was not apparent following plate-bound anti-immunoglobulin E or SEA stimulation. The absence of IL-5 failed to affect the induction of Th2 responses in naive mice. Peritoneal exudate cells recovered from egg-injected IL-5−/−or WT mice produced equivalent levels of IL-4 following restimulation with SEA or anti-CD3.
Unanticipated Heterogeneity in Growth Rate and Virulence among
Candida albicans AAF1
Null Mutants
Authors: Rieg Günter , Fu Yue , Ibrahim Ashraf S. , Zhou Xiang , Filler Scott G. , Edwards John E.
Published: December 2019

The disruption of a specific gene in
Candida albicans
is commonly used to determine the function of the gene product. We disrupted
AAF1
, a gene of
C. albicans
that causes
Saccharomyces cerevisiae
to flocculate and adhere to endothelial cells. We then characterized multiple heterozygous and homozygous mutants. These null mutants adhered to endothelial cells to the same extent as did the parent organism. However, mutants with presumably the same genotype revealed significant heterogeneity in their growth rates in vitro. This heterogeneity was not the result of the transformation procedure per se, nor was it caused by differences in the expression or function of URA3, a marker used in the process of gene disruption. The growth rate among the different heterozygous and homozygous null mutants was positively correlated with in vivo virulence in mice. It is possible that the variable phenotypes of
C. albicans
were due to mutations outside of the
AAF1
coding region that were introduced during the gene disruption process. These results indicate that careful phenotypic characterization of mutants of
C. albicans
generated through targeted gene disruption should be performed to exclude the introduction of unexpected mutations that may influence pathogenicity in mice.
Green Fluorescent Protein as a Marker in
Rickettsia typhi
Transformation
Authors: Troyer Jill Michelle , Radulovic Suzana , Azad Abdu F.
Published: December 2019

Transformation of
rickettsiae
is a recent accomplishment, but utility of this technique is limited due to the paucity of selectable markers suitable for use in this intracellular pathogen. We chose a green fluorescent protein variant optimized for fluorescence under UV lights (GFP
UV
) as a fluorometric marker and transformed
Rickettsia typhi
with an
rpoB
-GFP
UV
fusion construct. The rickettsiae were subsequently grown in Vero cells, and cultures were screened by PCR and restriction fragment length polymorphism (RFLP) to confirm incorporation of the
rpoB
-GFP
UV
construct. Cultures were then analyzed by flow cytometry for detection of GFP
UV
expression, and transformed
R. typhi
were isolated in a fluorescence-activated cell sorter. This is the first report of transformation of rickettsiae with a nonrickettsial (GFP
UV
) gene.
A Novel Means of Self-Protection, Unrelated to Toxin Activation, Confers Immunity to the Bactericidal Effects of the
Enterococcus faecalis
Cytolysin
Authors: Coburn Phillip S. , Hancock Lynn E. , Booth Mary C. , Gilmore Michael S.
Published: December 2019

Enterococcus faecalis
has become a pervasive clinical problem due to the emergence of resistance to most antibiotics. The cytolysin of
E. faecalis
is a novel bacterial toxin that contributes to the severity of disease. It consists of two structural subunits, which together possess both hemolytic and bactericidal activity. Both toxin subunits are encoded in a complex operon frequently harbored on pheromone-responsive plasmids.
E. faecalis
strains lacking such plasmids are susceptible to the bactericidal effects of the cytolysin. A novel cytolysin immunity determinant at the 3′ end of the pAD1 cytolysin operon is described in the present study. Deletion analysis and specific mutagenesis isolated the immunity function to a single open reading frame. Specific mutagenesis experiments demonstrate that cytolysin immunity is unrelated to cytolysin activator (CylA) expression as previously proposed. Cytolysin immunity is, however, encoded on the same transcript as and 3′ to CylA, and previous associations between immunity and CylA can be ascribed to the polar behavior of Tn
917
insertion.
Role of Superoxide Dismutase Activity in the Physiology of
Porphyromonas gingivalis
Authors: Lynch Michael C. , Kuramitsu Howard K.
Published: December 2019

Porphyromonas gingivalis
is a gram-negative, obligate anaerobe strongly associated with chronic adult periodontitis. A previous study has demonstrated that this organism requires superoxide dismutase (SOD) for its modest aerotolerance. In this study, we have constructed a mutant deficient in SOD activity by insertional inactivation as well as a
sod
::
lacZ
reporter translational fusion construct to study the regulation of expression of this gene. We have confirmed that SOD is essential for tolerance to atmospheric oxygen but does not appear to be protective against hydrogen peroxide or exogenously generated reactive oxygen species. Furthermore, the
sod
mutant appeared to be no more sensitive to killing by neutrophils than the parental strain 381. SOD appears to be protective against oxygen-dependent DNA damage as measured by increased mutation to rifampin resistance by the
sod
mutant. Use of the
sod
::
lacZ
construct confirmed that SOD expression is maximal at mid-log phase and is influenced by oxygen, temperature, and pH. However, expression does not appear to be significantly affected by iron depletion, osmolarity, or nutrient depletion. The transcription start site of the
sod
gene was determined to be 315 bp upstream of the
sod
start codon and to be within an upstream open reading frame. Our studies demonstrate the essential role that SOD plays in aerotolerance of this organism as well as the selective induction of this enzyme by environmental stimuli.
Mycoplasmal Lipopeptide MALP-2 Induces the Chemoattractant Proteins Macrophage Inflammatory Protein 1α (MIP-1α), Monocyte Chemoattractant Protein 1, and MIP-2 and Promotes Leukocyte Infiltration in Mice
Authors: Deiters Ursula , Mühlradt Peter F.
Published: December 2019

Natural as well as experimental infections with pathogenic mycoplasmas lead to cellular responses characterized by early polymorphonuclear leukocyte influx, which in turn is followed by infiltration of macrophages. Since some of the most potent leukocyte chemoattractants are macrophage products, we investigated whether the 2-kDa macrophage-activating lipopeptide (MALP-2) fromMycoplasma fermentanswas capable of inducing chemoattractant chemokines and initiating an in vivo inflammatory effect. MALP-2 was a potent in vitro inducer of the chemokines macrophage inflammatory protein 1α (MIP-1α), monocyte chemoattractant protein 1 (MCP-1), and MIP-2, yielding a maximal response at 0.1 ng/ml (5 × 10−11M). Leukocyte infiltration was determined after intraperitoneal injection of MALP-2, liposome-encapsulated MALP-2, and heat-killed mycoplasmas. There was a steady increase in the number of peritoneal cells over 72 h in response to these agents. Polymorph counts were maximal by 24 to 48 h, decreasing thereafter. Monocytes/macrophages had significantly increased after 3 days. MIP-1α, MCP-1, and MIP-2 levels in serum or peritoneal lavage fluid were determined. MIP-1α and MCP-1 levels were elevated by 2 to 6 h after injection and were still above control values after 24 h. In contrast, MIP-2 levels reached their maximum at 2 h, dropping to control values after 24 h. We conclude that macrophage-stimulating mycoplasmal lipoproteins, exemplified by MALP-2, play an important role in the late phase of phagocyte recruitment at sites of infection and that this is affected by leukoattractive chemokines.
Induction of Lethal Shock and Tolerance byPorphyromonas gingivalisLipopolysaccharide ind-Galactosamine-Sensitized C3H/HeJ Mice
Authors: Tanamoto Ken-Ichi
Published: December 2019

Lipopolysaccharide (LPS) obtained fromPorphyromonas gingivaliswas found to exhibit marked lethal toxicity in galactosamine-sensitized C3H/HeJ mice. Although no lethality was observed in mice intraperitoneally challenged with 1 mg ofP. gingivalisLPS without galactosamine, when they were sensitized with 30 mg of galactosamine, challenge with 1 and 10 μg of LPS resulted in 67 and 100% lethality, respectively. The lethal dose of LPS was almost the same in LPS-responsive C57BL/6 mice and non-LPS-responsive C3H/HeJ mice. Furthermore, when 1 μg ofP. gingivalisLPS was administered to each mouse 90 min before the challenge with the same LPS with galactosamine, tolerance to the lethal action of LPS was induced, and the mice were completely protected from death, even at a dose 100-fold greater than the lethal dose of LPS. Neither a lethal effect nor induction of tolerance to the lethality ofP. gingivalisLPS was exhibited bySalmonellaLPS in galactosamine-sensitized C3H/HeJ mice. A protein-LPS complex derived fromPseudomonas aeruginosa, which exhibited strong lethality and induced tolerance to a subsequent challenge with a lethal dose of LPS in galactosamine-sensitized LPS-responsive mice, did not exhibit lethal toxicity in galactosamine-sensitized C3H/HeJ mice and failed to induce tolerance in these mice to the lethality ofP. gingivalisLPS. These results indicate thatP. gingivalisLPS plays the central role in the activation of non-LPS-responsive C3H/HeJ mice.
Analysis of a Capsular Polysaccharide Biosynthesis Locus ofBacteroides fragilis
Authors: Comstock Laurie E. , Coyne Michael J. , Tzianabos Arthur O. , Pantosti Annalisa , Onderdonk Andrew B. , Kasper Dennis L.
Published: December 2019

A major clinical manifestation of infection withBacteroides fragilisis the formation of intra-abdominal abscesses, which are induced by the capsular polysaccharides of this organism. Transposon mutagenesis was used to locate genes involved in the synthesis of capsular polysaccharides. A 24,454-bp region was sequenced and found to contain a 15,379-bp locus (designatedwcf) with 16 open reading frames (ORFs) encoding products similar to those encoded by genes of other bacterial polysaccharide biosynthesis loci. Four genes encode products that are similar to enzymes involved in nucleotide sugar biosynthesis. Seven genes encode products that are similar to sugar transferases. Two gene products are similar toO-acetyltransferases, and two products are probably involved in polysaccharide transport and polymerization. The product of one ORF, WcfH, is similar to a set of deacetylases of the NodB family. Deletion mutants demonstrated that thewcflocus is necessary for the synthesis of polysaccharide B, one of the two capsular polysaccharides ofB. fragilis9343. The virulence of the polysaccharide B-deficient mutant was comparable to that of the wild type in terms of its ability to induce abscesses in a rat model of intra-abdominal infection.
Genetic Basis for Lipopolysaccharide O-Antigen Biosynthesis in Bordetellae
Authors: Preston Andrew , Allen Andrew G. , Cadisch Joanna , Thomas Richard , Stevens Kim , Churcher Carol M. , Badcock K. L. , Parkhill Julian , Barrell Bart , Maskell Duncan J.
Published: December 2019

Bordetella bronchiseptica
and
Bordetella parapertussis
express a surface polysaccharide, attached to a lipopolysaccharide, which has been called O antigen. This structure is absent from
Bordetella pertussis
. We report the identification of a large genetic locus in
B. bronchiseptica
and
B. parapertussis
that is required for O-antigen biosynthesis. The locus is replaced by an insertion sequence in
B. pertussis
, explaining the lack of O-antigen biosynthesis in this species. The DNA sequence of the
B. bronchiseptica
locus has been determined and the presence of 21 open reading frames has been revealed. We have ascribed putative functions to many of these open reading frames based on database searches. Mutations in the locus in
B. bronchiseptica
and
B. parapertussis
prevent O-antigen biosynthesis and provide tools for the study of the role of O antigen in infections caused by these bacteria.
Secretion of Functional Salivary Peptide by
Streptococcus gordonii
Which Inhibits Fimbria-Mediated Adhesion of
Porphyromonas gingivalis
Authors: Kataoka Kosuke , Amano Atsuo , Kawabata Shigetada , Nagata Hideki , Hamada Shigeyuki , Shizukuishi Satoshi
Published: December 2019

Porphyromonas gingivalis
, a putative periodontopathogen, can bind to human salivary components with its fimbriae. We have previously shown that fimbriae specifically bind to a peptide domain shared by a major salivary component, i.e., proline-rich (glyco)proteins (PRPs). The synthetic domain peptide PRP-C (pPRP-C) significantly inhibits the fimbrial binding to PRPs. In this study, a recombinant strain of
Streptococcus gordonii
secreting pPRP-C was generated as a model of a possible approach to prevent the oral colonization by the pathogen. A duplicate DNA fragment (
prpC
) encoding pPRP-C was obtained by self-complementary annealing of synthetic oligonucleotides.
prpC
was connected downstream to a promoter and a gene encoding a signal peptide of
Streptococcus downei
glucosyltransferase I in frame. The linked fragments were inserted into the plasmid pMNK-4 derived from pVA838. The constructed plasmid was inserted to produce the transformant
S. gordonii
G9B, which then successfully secreted recombinant pPRP-C (r-pPRP-C) of the expected size. The concentrated bacterial culture supernatant containing r-pPRP-C inhibited the binding of
P. gingivalis
cells and fimbriae to PRP1 in a dose-dependent manner up to 72 and 77%, respectively. The r-pPRP-C concentrate also inhibited the coaggregation of
P. gingivalis
with various streptococcal strains as effectively as synthetic pPRP-C in a dose-dependent manner. Collectively, pPRP-C was found to be able to prevent
P. gingivalis
adherence to salivary receptor protein and plaque-forming bacteria. These results suggest that this recombination approach with a nonperiodontopathic bacterium may be suitable for the therapeutic prevention of
P. gingivalis
adherence to the oral cavity.
In Vitro Expansion of T-Cell-Receptor Vα2.3+CD4+T Lymphocytes in HLA-DR17(3), DQ2+Individuals upon Stimulation withMycobacterium tuberculosis
Authors: Esin Semih , Batoni Giovanna , Saruhan-Direskeneli Güher , Harris Robert A. , Grunewald Johan , Pardini Manuela , Svenson Stefan B. , Campa Mario , Wigzell Hans
Published: December 2019

The T-cell receptor (TCR) Vα/β gene product expression upon in vitro stimulation with mycobacteria was investigated to assess whether T-cell proliferation was associated with any specific TCR V gene usage. T-cell-enriched populations from peripheral blood ofMycobacterium bovisBCG-vaccinated healthy blood donors were stimulated in vitro with live or killedM. tuberculosisor with a soluble extract thereof. TCR Vα/β repertoire analysis of reactive CD4+and CD8+T cells revealed a selective HLA-DR17(3), DQ2-restricted expansion of Vα2.3+CD4+T cells upon stimulation with liveM. tuberculosisor its soluble extract. Third-complementarity-determining-region (CDR3) length analysis of the expanded Vα2.3+T cells indicated an oligoclonal pattern with short CDR3 lengths in six of seven HLA-DR17(3), DQ2+individuals tested. In addition, Vα/Vβ repertoire analysis of T lymphocytes from a DR17(3), DQ2+donor before and after BCG vaccination revealed that positivity of skin test reactivity was associated with expansion of Vα2.3+CD4+T lymphocytes with preferential use of a short CDR3 peak length after in vitro stimulation. Separation ofM. tuberculosissoluble extract by fast protein liquid chromatography (FPLC) purification indicated that fractions corresponding to molecular masses of 60 to 70 and 15 to 25 kDa were particularly effective in eliciting Vα2.3+CD4+T-cell expansion.
Antigenic Variation of the Class I Outer Membrane Protein in Hyperendemic
Neisseria meningitidis
Strains in The Netherlands
Authors: Bart Aldert , Dankert Jacob , van der Ende Arie
Published: December 2019

Since 1980, the number of cases of meningococcal disease caused by serogroup B isolates with the P1.4 serosubtype has greatly increased in The Netherlands. Screening for this serosubtype in the strain collection of The Netherlands Reference Laboratory for Bacterial Meningitis revealed that a low number of P1.4 strains had been present in the Dutch meningococcal population since 1965. Genotyping of P1.4 strains showed that one cluster of strains, the hyperendemic lineage III (D. A. Caugant et al., J. Infect. Dis. 162:867–874, 1990), is responsible for the increase since 1980. The diversity of the
porA
genes, which encode the P1 protein on which serosubtyping is based, was studied for genotypically different P1.4 strains and for lineage III strains expressing antigenically different P1 proteins. Sequence analysis showed that
porA
genes of genotypically distinct strains that express antigenically indistinguishable P1 proteins are identical only in the epitope-encoding region, suggesting that this region has spread through the meningococcal population via horizontal gene transfer. Analysis of
porA
genes of lineage III strains showed that both horizontal gene transfer and partial deletion of the epitope-encoding region may contribute to the different antigenic properties for P1 of these strains. Phase variation of expression of the
porA
gene seems to account for most nonreacting strains. These results show that serosubtyping may underestimate the rise of a hyperendemic clone.
Functional Analysis of the
Staphylococcus aureus
Collagen Adhesin B Domain
Authors: Snodgrass James L. , Mohamed Nehal , Ross Julia M. , Sau Subrata , Lee Chia Y. , Smeltzer Mark S.
Published: December 2019

The
Staphylococcus aureus
collagen adhesin (CNA) occurs in at least four forms that differ in the number (one, two, three, or four) of B domains. The B domains contain 187 amino acids and are located between the domains that anchor CNA to the cell envelope and the ligand-binding A domain. To determine whether a B domain is required for functional expression of CNA, we cloned the 2B
cna
gene from
S. aureus
strain Phillips and then eliminated both B domains by overlapping PCR. The absence of a B domain did not affect processing of the collagen adhesin to the cell surface or the ability to bind collagen. Based on our recent demonstration that the capsule can mask CNA on the surface of
S. aureus
cells (A. F. Gillaspy et al., Infect. Immun. 66:3170–3178, 1998), we also investigated the possibility that multiple B domains can extend the ligand-binding A domain outward from the cell surface and thereby overcome the inhibitory effect of the capsule. Specifically, we cloned the naturally occurring 4B CNA variant from
S. aureus
UAMS-639 and, by successive elimination of B domains, generated 1, 2, and 3B variants that are isogenic with respect to the 4B clone. After introducing each variant into microencapsulated and heavily encapsulated strains of
S. aureus
and growing cells under conditions known to affect capsule production (e.g., growth on Columbia agar), we correlated capsule production with exposure of CNA on the cell surface and the ability to bind collagen. Under no circumstance was the masking effect of the capsule reduced by the presence of multiple B domains. These results indicate that the B domains do not extend the ligand-binding A domain outward in a fashion that can overcome the inhibition of collagen binding associated with capsule production.
Decay-Accelerating Factor and Cytoskeleton Redistribution Pattern in HeLa Cells Infected with RecombinantEscherichia coliStrains Expressing Dr Family of Adhesins
Authors: Goluszko Pawel , Selvarangan Rangaraj , Popov Vsevolod , Pham Tuan , Wen Julie W. , Singhal Jyotsana
Published: December 2019

Escherichia colistrains expressing Dr fimbriae are able to enter epithelial cells by interacting with a complement-regulatory protein, decay-accelerating factor. This model of bacterial internalization, with a well-characterized bacterial ligand and host receptor, provides a unique opportunity to investigate the early stages of invasion. We used immunofluorescence staining techniques to examine the distribution of receptor and cytoskeletal proteins in HeLa cells infected withE. colirecombinant strains that expressed Dr family of adhesins: Dr, Dr-II, F1845, AFA-I, and AFA-III. A major rearrangement of decay-accelerating factor was found at the adherence sites of recombinant strains expressing Dr, Dr-II, and F1845 adhesins. The changes in the distribution of receptor were significantly smaller on HeLa cells infected withE. colibearing AFA-I or AFA-III afimbrial adhesins. Receptor aggregation was associated with the redistribution of cytoskeleton-associated proteins such as actin, α-actinin, ezrin, and occasionally tropomyosin. Purified Dr fimbriae coated on polystyrene beads were capable of triggering clustering of receptor and accumulating actin at the adhesion sites of beads to HeLa cells. Using scanning and transmission electron microscopic techniques, we have shown that beads coated with Dr fimbriae, as opposed to beads coated with bovine serum albumin, were enwrapped by cellular microvilli and ultimately internalized into HeLa cells. This indicates that interaction of Dr fimbriae with decay-accelerating factor is associated with redistribution of receptor and is sufficient to promote bacterial internalization.
Cloning, Expression, and Immunological Evaluation of Two Putative Secreted Serine Protease Antigens ofMycobacterium tuberculosis
Authors: Skeiky Yasir A. W. , Lodes Michael J. , Guderian Jeffrey A. , Mohamath Raodoh , Bement Teresa , Alderson Mark R. , Reed Steven G.
Published: December 2019

Culture filtrate proteins (CFP) ofMycobacterium tuberculosishave been shown to contain immunogenic components that elicit at least partial protective immunity againstMycobacteriuminfection. To clone genes encoding some of the immunogenic proteins, we made a high-titer rabbit anti-CFP serum and used it to screen anM. tuberculosisgenomic expression library inEscherichia coli. In this paper, we describe the molecular cloning of two new protein components of CFP and identified them as members of the serine protease gene family. Their open reading frames contain N-terminal hydrophobic secretory signals consistent with their detection in CFP. The predicted molecular masses of the mature, fully processed forms of both antigens are ∼32 kDa, in agreement with their observed sizes on immunoblots of CFP probed with polyclonal rabbit antisera made to the recombinant proteins. Thus, these proteins have been designated MTB32A and MTB32B. Interestingly, and despite 66% amino acid sequence homology between the two proteins, polyclonal rabbit antisera made to each of the recombinant proteins were found to be specific for the respective immunizing antigens. The recombinant proteins were also evaluated in in vitro assays with donor peripheral blood mononuclear cells (PBMC) from healthy purified protein derivative (PPD)-positive individuals of diverse ethnic backgrounds. MTB32A but not MTB32B stimulated PBMC from healthy PPD-positive donors but not from PPD-negative donors to proliferate and secrete gamma interferon. MTB32A is encoded by a single-copy gene which is present in both virulent and avirulent strains of theM. tuberculosiscomplex and the BCG strain ofMycobacterium bovisbut absent in the environmental mycobacterial species tested. In addition, nucleotide sequence comparison ofmtb32aof the avirulent H37Ra strain and the virulent Erdman strain, as well as with the corresponding sequences (identified in the databases) of strain H37Rv and the clinical isolate CSU93, revealed 100% identity. MTB32A, therefore, represents a candidate for inclusion in subunit vaccine development. Finally, the possible role of MTB32 serine proteases as a virulence factor(s) duringMycobacteriumspp. infection is discussed.
Roles of CD4+T Cells and Gamma Interferon in Protective Immunity againstBabesia microtiInfection in Mice
Authors: Igarashi Ikuo , Suzuki Reiko , Waki Seiji , Tagawa Yoh-Ichi , Seng Seyha , Tum Sothyra , Omata Yoshitaka , Saito Atsushi , Nagasawa Hideyuki , Iwakura Yohichiro , Suzuki Naoyoshi , Mikami Takeshi , Toyoda Yutaka
Published: December 2019

Babesia microtiproduces a self-limiting infection in mice, and recovered mice are resistant to reinfection. In the present study, the role of T cells in protective immunity against challenge infection was examined. BALB/c mice which recovered from primary infection showed strong protective immunity against challenge infection. In contrast, nude mice which failed to control the primary infection and were cured with an antibabesial drug did not show protection against challenge infection. Treatment of immune mice with anti-CD4 monoclonal antibody (MAb) diminished the protective immunity against challenge infection, but treatment with anti-CD8 MAb had no effect on the protection. Transfer of CD4+T-cell-depleted spleen cells resulted in higher parasitemia than transfer of CD8+T-cell-depleted spleen cells. A high level of gamma interferon (IFN-γ), which was produced by CD4+T cells, was observed for the culture supernatant of spleen cells from immune mice, and treatment of immune mice with anti-IFN-γ MAb partially reduced the protection. Moreover, no protection against challenge infection was found in IFN-γ-deficient mice. On the other hand, treatment of immune mice with MAbs against interleukin-2 (IL-2), IL-4, or tumor necrosis factor alpha did not affect protective immunity. These results suggest essential requirements for CD4+T cells and IFN-γ in protective immunity against challenge infection withB. microti.
Heat-Shocked Monocytes Are Resistant toStaphylococcus aureus-Induced Apoptotic DNA Fragmentation due to Expression of HSP72
Authors: Guzik Krzysztof , Bzowska Małgorzata , Dobrucki Jerzy , Pryjma Juliusz
Published: December 2019

Human peripheral blood monocytes became apoptotic following phagocytosis ofStaphylococcus aureus. The consequences of heat stress for monocytes were studied with regard to the effect onS. aureus-induced apoptosis. Exposure of monocytes to 41.5°C for 1 h resulted in HSP72 expression and had no influence on phagocytosis of bacteria; moreover, phagocytosis ofS. aureusimmediately or shortly after heat shock had no effect on theS. aureus-induced monocyte apoptosis, as evidenced by DNA fragmentation assay. In contrast, cells which recovered from heat shock for 18 to 24 h, although active as phagocytes, were resistant to theS. aureus-induced apoptosis. The observed protective effect was related to the induction of HSP72, since blocking of HSP72 synthesis by an antisense oligomer abolished the protective effect of heat shock on bacterium-induced monocyte apoptosis.
Helicobacter pyloriInduces Gastric Epithelial Cell Apoptosis in Association with Increased Fas Receptor Expression
Authors: Jones Nicola L. , Day Andrew S. , Jennings Hilary A. , Sherman Philip M.
Published: December 2019

The mechanisms involved in mediating the enhanced gastric epithelial cell apoptosis observed during infection withHelicobacter pyloriin vivo are unknown. To determine whetherH. pyloridirectly induces apoptosis of gastric epithelial cells in vitro and to define the role of the Fas-Fas ligand signal transduction cascade, human gastric epithelial cells were infected withH. pylorifor up to 72 h under microaerophilic conditions. As assessed by both transmission electron microscopy and fluorescence microscopy, incubation with acagA-positive,cagE-positive, VacA-positive clinicalH. pyloriisolate stimulated an increase in apoptosis compared to the apoptosis of untreated AGS cells (16.0% ± 2.8% versus 5.9% ± 1.4%,P< 0.05) after 72 h. In contrast, apoptosis was not detected following infection withcagA-negative,cagE-negative, VacA-negative clinical isolates or aCampylobacter jejunistrain. In addition to stimulating apoptosis, infection withH. pylorienhanced Fas receptor expression in AGS cells to a degree comparable to that of treatment with a positive control, gamma interferon (12.5 ng/ml) (148% ± 24% and 167% ± 24% of control, respectively). The enhanced Fas receptor expression was associated with increased sensitivity to Fas-mediated cell death. Ligation of the Fas receptor with an agonistic monoclonal antibody resulted in an increase in apoptosis compared to the apoptosis of cells infected with the bacterium alone (38.5% ± 7.1% versus 16.0% ± 2.8%,P< 0.05). Incubation with neutralizing anti-Fas antibody did not prevent apoptosis ofH. pylori-infected cells. Taken together, these findings demonstrate that the gastric pathogenH. pyloristimulates apoptosis of gastric epithelial cells in vitro in association with the enhanced expression of the Fas receptor. These data indicate a role for Fas-mediated signaling in the programmed cell death that occurs in response toH. pyloriinfection.
Identification of Genes Encoding Two-Component Lantibiotic Production in
Staphylococcus aureus
C55 and Other Phage Group II
S. aureus
Strains and Demonstration of an Association with the Exfoliative Toxin B Gene
Authors: Navaratna Maduwe A. D. B. , Sahl Hans-Georg , Tagg John R.
Published: December 2019

The production of exfoliative toxin B (ET-B), but not ET-A, was shown to be specifically associated with production of a highly conserved two-component lantibiotic peptide system in phage group II
Staphylococcus aureus
. Two previously studied but incompletely characterized
S. aureus
bacteriocins, staphylococcins C55 and BacR1, were found to be members of this lantibiotic system, and considerable homology was also found with the two-component
Lactococcus lactis
bacteriocin, lacticin 3147.
sacαA
and
sacβA
, the structural genes of the lantibiotics staphylococcins C55α and C55β and two putative lantibiotic processing genes,
sacM1
and
sacT
, were localized together with the ET-B structural gene to a single 32-kb plasmid in strain C55. Irreversible loss of both ET-B and two-component lantibiotic production occurs during laboratory passage of ET-B-positive
S. aureus
strains, particularly at elevated temperatures.
Avirulence of
Candida albicans CaHK1
Mutants in a Murine Model of Hematogenously Disseminated Candidiasis
Authors: Calera José Antonio , Zhao Xiao-Jiong , De Bernardis Flavia , Sheridan Michael , Calderone Richard
Published: December 2019

Deletion of both alleles of the
Candida albicans CaHK1
gene, which causes cells to flocculate when grown at pH 7.5, a pH comparable to that of mammalian blood, abolishes the ability of the yeast to establish a successful infection in a murine model of hematogenously disseminated candidiasis. Within 72 h all mice inoculated with the parental
C. albicans
strain had died. The mice infected with either the heterozygote or revertant strain, either of which harbors only one functional
CaHK1
allele, also succumbed to the infection, although survivors were observed for up to 16 days postinfection. However, mice inoculated with the
Δcahk1
null strain survived for the course of the infection. These results indicate that
CaHK1
is required for the virulence of
C. albicans
in a murine model of hematogenously disseminated candidiasis. In contrast,
CaHK1
is not required for the virulence of
C. albicans
in a rat model of vaginal candidiasis.
Fibrinogen Cleavage by the
Streptococcus pyogenes
Extracellular Cysteine Protease and Generation of Antibodies That Inhibit Enzyme Proteolytic Activity
Authors: Matsuka Yury V. , Pillai Subramonia , Gubba Siddeswar , Musser James M. , Olmsted Stephen B.
Published: December 2019

The extracellular cysteine protease from
Streptococcus pyogenes
is a virulence factor that plays a significant role in host-pathogen interaction. Streptococcal protease is expressed as an inactive 40-kDa precursor that is autocatalytically converted into a 28-kDa mature (active) enzyme. Replacement of the single cysteine residue involved in formation of the enzyme active site with serine (C192S mutation) abolished detectable proteolytic activity and eliminated autocatalytic processing of zymogen to the mature form. In the present study, we investigated activity of the wild-type (wt) streptococcal protease toward human fibrinogen and bovine casein. The former is involved in blood coagulation, wound healing, and other aspects of hemostasis. Treatment with streptococcal protease resulted in degradation of the COOH-terminal region of fibrinogen α chain, indicating that fibrinogen may serve as an important substrate for this enzyme during the course of human infection. Polyclonal antibodies generated against recombinant 40- and 28-kDa (r40- and r28-kDa) forms of the C192S streptococcal protease mutant exhibited high enzyme-linked immunosorbent assay titers but demonstrated different inhibition activities toward proteolytic action of the wt enzyme. Activity of the wt protease was readily inhibited when the reaction was carried out in the presence of antibodies generated against r28-kDa C192S mutant. Antibodies produced against r40-kDa C192S mutant had no significant effect on proteolysis. These data suggest that the presence of the NH
2
-terminal prosegment prevents generation of functionally active antibodies and indicate that inhibition activity of antibodies most likely depends on their ability to bind the active-site region epitope(s) of the protein.
Host Responses to Recombinant Hemagglutinin B ofPorphyromonas gingivalisin an Experimental Rat Model
Authors: Katz Jannet , Black Kelley P. , Michalek Suzanne M.
Published: December 2019

Porphyromonas gingivalis, a gram-negative, black-pigmented anaerobe, is among the microorganisms implicated in the etiology of adult periodontal disease. This bacterium possesses a number of factors, including hemagglutinins, of potential importance in virulence. Several hemagglutinin genes have been identified, cloned, and expressed inEscherichia coli. The purpose of this study was to characterize host responses to purified recombinant hemagglutinin B (rHag B), using the conventional Fischer rat as the experimental animal model. The effectiveness of immunization with rHag B on protection against experimental periodontal bone loss following infection withP. gingivaliswas also evaluated. Groups of rats were immunized by the subcutaneous route with rHag B in complete Freund’s adjuvant, immunized with rHag B and orally infected withP. gingivalis, nonimmunized and noninfected, or orally infected withP. gingivalisonly. Serum and saliva samples were collected throughout the experiment and evaluated for serum immunoglobulin G (IgG) and IgM and salivary IgA antibody activity by enzyme-linked immunosorbent assay. No salivary IgA anti-Hag B activity was detected in the various groups of rats. A slight serum IgM response similar to that seen in preimmune samples was observed. Serum IgG antibody activity to Hag B was detected only in samples from rats immunized with rHag B. This response was primarily of the IgG1 and IgG2a subclasses, followed by IgG2b and low levels of IgG2c. Supernatants from rHag B-stimulated splenic lymphoid cell cultures from immunized rats contained high levels of gamma interferon, followed by interleukin-2 (IL-2), IL-10, and then IL-4. These results are consistent with the induction of T helper type 1 (Th1)- and Th2-like responses. Western blot analysis of sera derived from rHag B-immunized rats reacted with trichloroacetic acid (TCA) precipitates ofP. gingivalis33277, 381, A7A1-28, and W50, revealing a 50-kDa band reflective of Hag B. However, sera derived from rats immunized withP. gingivaliswhole cells or from rats infected withP. gingivalisonly did not react with rHag B but did react with TCA precipitates ofP. gingivalisstrains. Finally, radiographic measurements of periodontal bone loss indicated that rats immunized with rHag B had less bone loss than those infected withP. gingivalisonly. These results demonstrate the effectiveness of purified rHag B in inducing a protective immune response and support the potential usefulness of this component ofP. gingivalisin the development of a vaccine against adult periodontitis.
Characterization of the Interaction between
Yersinia enterocolitica
Biotype 1A and Phagocytes and Epithelial Cells In Vitro
Authors: Grant Travis , Bennett-Wood Vicki , Robins-Browne Roy M.
Published: December 2019

Yersinia enterocolitica
strains of biotype 1A are increasingly being recognized as etiological agents of gastroenteritis. However, the mechanisms by which these bacteria cause disease differ from those of highly invasive, virulence plasmid-bearing
Y. enterocolitica
strains and are poorly understood. We have investigated several biotype 1A strains of diverse origin for their ability to resist killing by professional phagocytes. All strains were rapidly killed by polymorphonuclear leukocytes but persisted within macrophages (activated with gamma interferon) to a significantly greater extent (survival = 40.5% ± 17.4%) than did
Escherichia coli
HB101 (9.3% ± 0.7%;
P
= 0.0001). Strains isolated from symptomatic patients were significantly more resistant to killing by macrophages (survival = 48.9% ± 19.5%) than were strains obtained from food or the environment (survival = 32.1% ± 10.3%;
P
= 0.04). Some strains which had been ingested by macrophages or HEp-2 epithelial cells showed a tendency to reemerge into the tissue culture medium over a period lasting several hours. This phenomenon, which we termed “escape,” was observed in 14 of 15 strains of clinical origin but in only 3 of 12 nonclinical isolates (
P
= 0.001). The capacity of bacteria to escape from cells was not directly related to their invasive ability. To determine if escape was due to host cell lysis, we used a variety of techniques, including lactate dehydrogenase release, trypan blue exclusion, and examination of infected cells by light and electron microscopy, to measure cell viability and lysis. These studies established that biotype 1A
Y. enterocolitica
strains were able to escape from macrophages or epithelial cells without causing detectable cytolysis, suggesting that escape was achieved by a process resembling exocytosis. The observations that biotype 1A
Y. enterocolitica
strains of clinical origin are significantly more resistant to killing by macrophages and significantly more likely to escape from host cells than are strains of nonclinical origin suggest that these properties may account for the virulence of these bacteria.