bluuebunny/crossref_metadata_2025 huggingface dataset:
Bone Resorption Caused by Three Periodontal Pathogens In Vivo in Mice Is Mediated in Part by Prostaglandin
Authors: Zubery Yuval , Dunstan Colin R. , Story Beryl M. , Kesavalu Lakshmyya , Ebersole Jeffrey L. , Holt Stanley C. , Boyce Brendan F.
Published: March 2020

Gingival inflammation, bacterial infection, alveolar bone destruction, and subsequent tooth loss are characteristic features of periodontal disease, but the precise mechanisms of bone loss are poorly understood. Most animal models of the disease require injury to gingival tissues or teeth, and the effects of microorganisms are thus complicated by host responses to tissue destruction. To determine whether three putative periodontal pathogens,
Porphyromonas gingivalis
,
Campylobacter rectus
, and
Fusobacterium nucleatum
, could cause localized bone resorption in vivo in the absence of tissue injury, we injected live or heat-killed preparations of these microorganisms into the subcutaneous tissues overlying the calvaria of normal mice once daily for 6 days and then examined the bones histologically. We found that all three microorganisms (both live and heat killed) stimulated bone resorption and that the strain of
F. nucleatum
used appeared to be the strongest inducer of osteoclast activity. Treatment of the mice concomitantly with indomethacin reduced but did not completely inhibit bone resorption by these microorganisms, suggesting that their effects were mediated, in part, by arachidonic acid metabolites (e.g., prostaglandins). Our findings indicate that these potential pathogens can stimulate bone resorption locally when placed beside a bone surface in vivo in the absence of prior tissue injury and support a role for them in the pathogenesis of bone loss around teeth in periodontitis.
Effectiveness of Liposomes Possessing Surface-Linked Recombinant B Subunit of Cholera Toxin as an Oral Antigen Delivery System
Authors: Harokopakis Evlambia , Hajishengallis George , Michalek Suzanne M.
Published: March 2020

Liposomes appear to be a promising oral antigen delivery system for the development of vaccines against infectious diseases, although their uptake efficiency by Peyer’s patches in the gut and the subsequent induction of mucosal immunoglobulin A (IgA) responses remain a major concern. Aiming at targeted delivery of liposomal immunogens, we have previously reported the conjugation via a thioether bond of the GM1ganglioside-binding subunit of cholera toxin (CTB) to the liposomal outer surface. In the present study, we have investigated the effectiveness of liposomes containing the saliva-binding region (SBR) ofStreptococcus mutansAgI/II adhesin and possessing surface-linked recombinant CTB (rCTB) in generating mucosal (salivary, vaginal, and intestinal) IgA as well as serum IgG responses to the parent molecule, AgI/II. Responses in mice given a single oral dose of the rCTB-conjugated liposomes were compared to those in mice given one of the following unconjugated liposome preparations: (i) empty liposomes, (ii) liposomes containing SBR, (iii) liposomes containing SBR and coadministered with rCTB, and (iv) liposomes containing SBR plus rCTB. Three weeks after the primary immunization, significantly higher levels of mucosal IgA and serum IgG antibodies to AgI/II were observed in the rCTB-conjugated group than in mice given the unconjugated liposome preparations, although the latter mice received a booster dose at week 9. The antibody responses in mice immunized with rCTB-conjugated liposomes persisted at high levels for at least 6 months, at which time (week 26) a recall immunization significantly augmented the responses. In general, mice given unconjugated liposome preparations required one or two booster immunizations to develop a substantial anti-AgI/II antibody response, which was more prominent in the group given coencapsulated SBR and rCTB. These data indicate that conjugation of rCTB to liposomes greatly enhances their effectiveness as an antigen delivery system. This oral immunization strategy should be applicable for the development of vaccines against oral, intestinal, or sexually transmitted diseases.
A Cluster of Genes Involved in Polysaccharide Biosynthesis from
Enterococcus faecalis
OG1RF
Authors: Xu Yi , Murray Barbara E. , Weinstock George M.
Published: March 2020

Our previous work identified a cosmid clone containing a 43-kb insert from
Enterococcus faecalis
OG1RF that produced a nonprotein antigen in
Escherichia coli
. In the present work, we studied this clone in detail. Periodate treatment of lysates of the clone confirmed that the antigen was carbohydrate in nature. Analysis of DNA sequences and transposon insertion mutants suggested that the insert contained a multicistronic gene cluster. Database comparison showed that the cluster contained genes similar to genes involved in the biosynthesis of dTDP-rhamnose, glycosyltransferases, and ABC transporters involved in the export of sugar polymers from both gram-positive and gram-negative organisms. Insertions in several genes within the cluster abolished the immunoreactivity of the clone. This is the first report on a gene cluster of
E. faecalis
involved in the biosynthesis of an antigenic polysaccharide.
Immunization against NaturalHelicobacter pyloriInfection in Nonhuman Primates
Authors: Dubois Andre , Lee Cynthia K. , Fiala Nancy , Kleanthous Harry , Mehlman Patrick T. , Monath Thomas
Published: March 2020

Helicobacter pyloriinfection is widespread in some breeding groups of a rhesus monkey colony (71%H. pyloripositive by 1 year), and the rate of seroconversion is also high. As a result, these groups can be used to test the safety and efficacy of an anti-H. pylorivaccine. Nine-month-old female animals were randomized to receive either 8 mg of recombinant urease (rUre) plus 25 μg ofEscherichia coliheat-labile enterotoxin (LT) (n= 26) or placebo plus LT (n= 29), given four times at 1-week intervals followed by a booster 1 month later. Ten months after the start of the immunization, the animals were subjected to endoscopy and biopsy samples were obtained.H. pylorinegativity was defined as noH. pylorigrowth by culture and noH. pyloriobserved at histology. By this criterion, 2 (7%) of 29 animals receiving placebo and 8 (31%) of 26 immunized animals wereH. pylorinegative (P< 0.035). In addition, antral gastritis score was significantly less inH. pylori-negative immunized monkeys than inH. pylori-positive animals, whether they were given rUre plus LT or placebo plus LT (P< 0.02 orP< 0.01, respectively). Interestingly, antral gastritis was also significantly less inH. pylori-positive animals given rUre plus LT than inH. pylori-positive animals given placebo plus LT (P< 0.02). However, quantitative cultures did not demonstrate significant differences between the two latter groups. It is concluded that oral administration of rUre vaccine plus LT significantly protects nonhuman primates againstH. pyloriinfection while not causing undesirable side effects.
Deletion of Repeats in the Alpha C Protein Enhances the Pathogenicity of Group B Streptococci in Immune Mice
Authors: Gravekamp C. , Rosner Bernard , Madoff L. C.
Published: March 2020

The alpha C protein is a protective surface-associated antigen of group B streptococci (GBS). The prototype alpha C protein of GBS (strain A909) contains nine identical tandem repeats, each comprising 82 amino acids, flanked by N- and C-terminal domains. Clinical isolates of GBS show variable numbers of repeats with a normal distribution and a median of 9 to 10 repeats. Here, we show that escape mutants of GBS expressing one-repeat alpha C protein were 100-fold more pathogenic than GBS expressing wild-type nine-repeat alpha C protein in neonatal mice whose dams were immunized with antiserum elicited to nine-repeat alpha C protein (50% lethal doses of 1.6 × 10
3
and 1.8 × 10
5
, respectively;
P
= 0.0073). There was no difference in pathogenicity in nonimmune mice. Enzyme-linked immunosorbent assay inhibition showed that nine-repeat but not one-repeat alpha C protein is readily available for antibody binding on the surface of intact GBS. Immune electron microscopy studies with antibodies to the capsular polysaccharide (CPS) and to the alpha C protein demonstrated localization of the nine-repeat alpha C protein and the CPS at similar distances from the cell wall. The one-repeat alpha C protein was visualized poorly and only in close proximity to the cell wall, thus suggesting that antibody binding to the protein was hindered by CPS or other cell surface components. We concluded that deletion in the repeat region of the alpha C protein enhanced the pathogenicity of GBS in immune mice by (i) loss of a protective (conformational) epitope(s) and (ii) loss of antibody binding to the alpha C protein due to a decrease in antigen size relative to cell wall components and/or CPS.
Isolation and Characterization of Two Proteins fromMoraxella catarrhalisThat Bear a Common Epitope
Authors: McMichael John C. , Fiske Michael J. , Fredenburg Ross A. , Chakravarti Deb N. , VanDerMeid Karl R. , Barniak Vicki , Caplan Jeffrey , Bortell Eric , Baker Steven , Arumugham Rasappa , Chen Dexiang
Published: March 2020

The UspA1 and UspA2 proteins ofMoraxella catarrhalisare potential vaccine candidates for preventing disease caused by this organism. We have characterized both proteins and evaluated their vaccine potential using both in vitro and in vivo assays. Both proteins were purified from the O35E isolate by Triton X-100 extraction, followed by ion-exchange and hydroxyapatite chromatography. Analysis of the sequences of internal peptides, prepared by enzymatic and chemical cleavage of the proteins, revealed that UspA1 and UspA2 exhibited distinct structural differences but shared a common sequence including an epitope recognized by the monoclonal antibody 17C7. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), purified UspA1 exhibited a molecular weight of approximately 350,000 when unheated and a molecular weight of 100,000 after being heated for 10 min at 100°C. In contrast, purified UspA2 exhibited an apparent molecular weight of 240,000 by SDS-PAGE that did not change with the length of time of heating. Their sizes as determined by gel filtration were 1,150,000 and 830,000 for UspA1 and UspA2, respectively. Preliminary results indicate the proteins have separate functions in bacterial pathogenesis. Purified UspA1 was found to bind HEp-2 cells, and sera against UspA1, but not against UspA2, blocked binding of the O35E isolate to the HEp-2 cells. UspA1 also bound fibronectin and appears to have a role in bacterial attachment. Purified UspA2, however, did not bind fibronectin but had an affinity for vitronectin. Both proteins elicited bactericidal antibodies in mice to homologous and heterologous disease isolates. Finally, mice immunized with each of the proteins, followed by pulmonary challenge with either the homologous or a heterologous isolate, cleared the bacteria more rapidly than mock-immunized mice. These results suggest that UspA1 and UspA2 serve different virulence functions and that both are promising vaccine candidates.
Actinomyces naeslundii
Displays Variant
fimP
and
fimA
Fimbrial Subunit Genes Corresponding to Different Types of Acidic Proline-Rich Protein and β-Linked Galactosamine Binding Specificity
Authors: Hallberg K. , Holm C. , Öhman U. , Strömberg N.
Published: March 2020

Actinomyces naeslundii
genospecies 1 and 2 bind to acidic proline-rich proteins (APRPs) and statherin via type 1 fimbriae and to β-linked galactosamine (GalNAcβ) structures via type 2 fimbriae. In addition,
A. naeslundii
displays two types of binding specificity for both APRPs-statherin and GalNAcβ, while
Actinomyces odontolyticus
binds to unknown structures. To study the molecular basis for these binding specificities, DNA fragments spanning the entire or central portions of
fimP
(type 1) and
fimA
(type 2) fimbrial subunit genes were amplified by PCR from strains of genospecies 1 and 2 and hybridized with DNA from two independent collections of oral
Actinomyces
isolates. Isolates of genospecies 1 and 2 and
A. odontolyticus
, but no other
Actinomyces
species, were positive for hybridization with
fimP
and
fimA
full-length probes irrespective of binding to APRPs and statherin, GalNAcβ, or unknown structures. Isolates of genospecies 1 and 2, with deviating patterns of GalNAcβ1-3Galα-
O
-ethyl-inhibitable coaggregation with
Streptococcus oralis
Ss34 and MPB1, were distinguished by a
fimA
central probe from genospecies 1 and 2, respectively. Furthermore, isolates of genospecies 1 and 2 displaying preferential binding to APRPs over statherin were positive with a
fimP
central probe, while a genospecies 2 strain with the opposite binding preference was not. The sequences of
fimP
and
fimA
central gene segments were highly conserved among isolates with the same, but diversified between those with a variant, binding specificity. In conclusion,
A. naeslundii
exhibits variant
fimP
and
fimA
genes corresponding to diverse APRP and GalNAcβ specificities, respectively, while
A. odontolyticus
has a genetically related but distinct adhesin binding specificity.
CD86 (B7-2), but Not CD80 (B7-1), Expression in the Epidermis of Transgenic Mice Enhances the Immunogenicity of Primary CutaneousCandida albicansInfections
Authors: Gaspari Anthony A. , Burns Robert , Nasir Adnan , Ramirez Diana , Barth Richard K. , Haidaris Constantine G.
Published: March 2020

Transgenic (Tg) mice whose epidermal keratinocytes constitutively overexpress either B7-1 (CD80) or B7-2 (CD86) exhibited exaggerated cutaneous delayed type hypersensitivity (DTH) to haptens compared to non-Tg mice. To determine whether enhanced DTH in these Tg mice is seen in response to cutaneous fungal infections, a primary infection withCandida albicanswas established by inoculating this organism on the occluded skin of Tg and non-Tg mice. These infections resolved 7 days after removal of occlusive dressing in all three groups of mice, without evidence of exaggerated inflammation in either the Tg or non-Tg mice. Only B7-2 Tg mice developed enhanced Th1-lymphocyte-mediated immune responses toC. albicansantigens after resolving this infection: enhanced footpad swelling in response to intradermalC. albicansantigens, enhanced production of mRNA encoding Th1 lymphokines in draining lymph nodes, and increased gamma interferon secreted into culture supernatants by lymph node T lymphocytes stimulated withCandidaantigens in vitro. Lastly, Western blotting of sera from mice that had resolved this fungal infection indicated that only B7-2 Tg mice recognized a wide range ofCandida-associated antigens. These data suggest that these two costimulatory molecules, when expressed by keratinocytes, do not deliver identical signals toC. albicansantigen-reactive Th1 lymphocytes. The enhanced immune response in B7-2 Tg mice to a cutaneousC. albicansinfection demonstrates the importance of antigen presentation and costimulation in immune reactivity to fungi. Furthermore, B7-2 Tg mice may be useful in identification of protectiveCandidaantigens.
Binding of Heme-Hemopexin Complexes by Soluble HxuA Protein Allows Utilization of This Complexed Heme by
Haemophilus influenzae
Authors: Cope Leslie D. , Thomas Sharon E. , Hrkal Zbynek , Hansen Eric J.
Published: March 2020

Utilization of heme-hemopexin as a source of heme by
Haemophilus influenzae
type b is dependent on expression by this bacterium of the 100-kDa HxuA protein, which is both present on the bacterial cell surface and released into the culture supernatant (L. D. Cope, R. Yogev, U. Muller-Eberhard, and E. J. Hansen, J. Bacteriol. 177:2644–2653, 1995). Radioimmunoprecipitation analysis showed that the soluble HxuA protein present in
H. influenzae
type b culture supernatant bound heme-hemopexin complexes in solution. An isogenic
H. influenzae
type b
hxuA
mutant was unable to utilize soluble heme-hemopexin complexes for growth in vitro unless soluble HxuA protein was provided exogenously. Soluble HxuA protein secreted by a nontypeable
H. influenzae
strain also allowed growth of this
H. influenzae
type b
hxuA
mutant. These results indicated that the heme present in heme-hemopexin complexes is rendered accessible to
H. influenzae
when these complexes are bound by the soluble HxuA protein.
A Role for Interleukin-6 in Host Defense against MurineChlamydia trachomatisInfection
Authors: Williams Dwight M. , Grubbs Barry G. , Darville Toni , Kelly Kathleen , Rank Roger G.
Published: March 2020

Interleukin-6-deficient (IL-6−/−) knockout mice had significantly increasedChlamydia trachomatislevels in lung tissue and increased mortality compared to B6129F2/J controls early after intranasal infection. Gamma interferon production and chlamydia-specific antibody levels were consistent with a decreased but reversible Th1-like response in IL-6−/−mice. IL-6 is needed for an optimal early host response to this infection.
Construction of a Stable Attenuated
Shigella sonnei
Δ
virG
Vaccine Strain, WRSS1, and Protective Efficacy and Immunogenicity in the Guinea Pig Keratoconjunctivitis Model
Authors: Hartman Antoinette B. , Venkatesan Malabi M.
Published: March 2020

Construction of a stable
Shigella sonnei
vaccine has been complicated by the instability of the virulence phenotype caused by the spontaneous loss of the invasion plasmid. To select a suitable candidate for vaccine construction, 16
S. sonnei
strains were screened for stability of the virulence phenotype. A stable strain,
S. sonnei
Mosely, was selected for further work. pΔvirG2, a deletion derivative of the
virG
gene in the
sacB
suicide vector pCVD442, was used to generate an
S. sonnei virG
deletion strain, WRSS1, which was invasive in HeLa cells but negative in the Sereny test. WRSS1 was found to be both immunogenic and protective in the guinea pig keratoconjunctivitis model.
NF-κB1 (p50) Is Upregulated in Lipopolysaccharide Tolerance and Can Block Tumor Necrosis Factor Gene Expression
Authors: Kastenbauer Stefan , Ziegler-Heitbrock H. W. Löms
Published: March 2020

Monocytes respond to lipopolysaccharide (LPS) stimulation with a rapid expression of the tumor necrosis factor (TNF) gene. Upon repeated LPS stimulation there is, however, little production of TNF mRNA and protein; i.e., the cells are tolerant to LPS. Analysis of NF-κB proteins in gel shift assays demonstrated that the DNA binding activity that is induced by LPS stimulation in tolerant cells consists mainly of p50-p50 homodimers. Since p50 can bind to DNA but lacks a transactivation domain, this may explain the blockade of TNF gene expression. We now show that in the monocytic cell line Mono Mac 6, this inability to respond can be largely ascribed to NF-κB, since a reporter construct directed by a trimeric NF-κB motif is strongly transactivated by LPS stimulation of naive cells whereas LPS-tolerant cells exhibit only low activity. Also, Western blot analyses of proteins extracted from purified nuclei showed mobilization of threefold-higher levels of p50 protein in tolerant compared to naive cells, while mobilization of p65 was unaltered. Overexpression of p50 in HEK 293 cells resulted in a strong reduction of p65-driven TNF promoter activity at the levels of both luciferase mRNA and protein. These data support the concept that an upregulation of p50 is instrumental in LPS tolerance in human monocytes.
Interleukin-10 and Antigen-Presenting Cells Actively Suppress Th1 Cells in BALB/c Mice Infected with the Filarial ParasiteBrugia pahangi
Authors: Osborne Julie , Devaney Eileen
Published: March 2020

Infection with the third-stage larvae (L3) of the filarial nematodeBrugiaresults in a Th2-biased immune response in mice and humans. Previously we have shown that the production of interleukin 4 (IL-4) is critical for down-regulating polyclonal Th1 responses in L3-infected mice. However, the in vitro neutralization of IL-4 did not fully recover the defective polyclonal Th1 responses, nor did it result in the production of any antigen (Ag)-specific Th1 cytokines, suggesting that perhaps infection with L3 does not result in priming of Th1 cells in vivo. In this study, we analyzed the role of IL-10 and Ag-presenting cells (APCs) in the spleen as additional factors controlling the Th2 bias in infected mice. Our data show that IL-10 and APCs also contribute to the suppression of mitogen-driven Th1 responses of spleen cells from infected mice. In addition, the neutralization of IL-10 or the replacement of the resident APC population from spleen cell cultures resulted in the production of Ag-specific Th1 cytokines. Irradiated spleen cells from either L3-infected or uninfected mice were able to restore Ag-specific Th1 responses in vitro. Therefore, it appears thatBrugia-reactive Th1 cells are primed following infection with L3, but are actively suppressed in vivo by a mechanism that involves IL-10 and the resident APC population, but not IL-4. These results indicate that a complex interplay of cytokines and cell populations underscores the Th2-polarized response in L3-infected mice.
Lipoteichoic Acid Acts as an Antagonist and an Agonist of Lipopolysaccharide on Human Gingival Fibroblasts and Monocytes in a CD14-Dependent Manner
Authors: Sugawara Shunji , Arakaki Rieko , Rikiishi Hidemi , Takada Haruhiko
Published: March 2020

CD14 has been implicated as a receptor of lipoteichoic acid (LTA) and other bacterial components as well as lipopolysaccharide (LPS). Since the structures of LTAs from various gram-positive bacteria are heterogeneous, we analyzed the effects of LTAs on the secretion of interleukin-8 (IL-8) by high- and low-CD14-expressing (CD14highand CD14low) human gingival fibroblasts (HGF). WhileBacillus subtilisLTA had an IL-8-inducing effect on CD14highHGF which was considerably weaker than that of LPS,Streptococcus sanguisandStreptococcus mutansLTAs had practically no effect on the cells.B. subtilisLTA had only a weak effect on CD14lowHGF, as did LPS.S. sanguisandS. mutansLTAs at a 1,000-fold excess each completely inhibited the IL-8-inducing activities of both LPS and a synthetic lipid A on CD14highHGF. The effect of LPS was also inhibited by the presence of an LPS antagonist, synthetic lipid A precursor IVA(LA-14-PP), with a 100-fold higher potency thanS. sanguisandS. mutansLTAs and by anti-CD14 monoclonal antibody (MAb).S. sanguisandS. mutansLTAs, LA-14-PP, and anti-CD14 MAb had no significant effect on phorbol myristate acetate-stimulated IL-8 secretion by HGF. These LTAs also inhibited the IL-8-inducing activity ofB. subtilisLTA on CD14highHGF, as did LA-14-PP and anti-CD14 MAb. The antagonistic and agonistic functions of LTAs were also observed with human monocytes. Binding of fluorolabeled LPS to human monocytes was inhibited byS. sanguisLTA, although the inhibition was 100 times weaker than that of LPS itself, and anti-CD14 MAb inhibited fluorolabeled LPS andS. sanguisLTA binding. Binding of LTAs to CD14 was also observed with nondenaturing polyacrylamide gel electrophoresis. These results indicate that LTAs act as antagonists or agonists via a CD14-dependent mechanism, probably due to the heterogeneous structure of LTAs, and that an antagonistic LTA might be a useful agent for suppressing the periodontal disease caused by gram-negative bacteria.
Immunization of Mice with DNA-Based Pfs25 Elicits Potent Malaria Transmission-Blocking Antibodies
Authors: Lobo Cheryl Ann , Dhar Ravi , Kumar Nirbhay
Published: March 2020

Immunological intervention, in addition to vector control and malaria chemotherapy, will be needed to stop the resurgence of malaria, a disease with a devastating impact on the health of 300 to 500 million people annually. We have pursued a vaccination strategy, based on DNA immunization in mice with genes encoding two antigens present on the sexual stages ofPlasmodium falciparum, Pfs25 and Pfg27, to induce biologically important antibodies that can block development of the parasite in theAnophelesmosquito and thus transmission of the disease. DNA encoding Pfs25 when administered by the intramuscular route, either alone or with DNA encoding Pfg27, had the most potent transmission-blocking effects, resulting in up to a 97% decrease in oocyst numbers in mosquito midguts and a 75% decrease in rate of infection. Immunization with DNA encoding a Pfg27-Pfs25 fusion protein was less effective and DNA encoding Pfg27 elicited antibodies in sera that had only modest effects on the infectivity of the parasite. These results show for the first time that DNA vaccination can result in potent transmission-blocking antibodies in mice and suggest that the Pfs25 gene should be included as part of a multicomponent DNA vaccine.
A Novel 62-Kilodalton Egg Antigen fromSchistosoma mansoniInduces a Potent CD4+T Helper Cell Response in the C57BL/6 Mouse
Authors: Asahi Hiroko , Hernandez Hector J. , Stadecker Miguel J.
Published: March 2020

In infection withSchistosoma mansoni, hepatic granuloma formation is mediated by CD4+T helper (Th) cells sensitized to schistosomal egg antigens. There is considerable variation among infected individuals with respect to both severity of disease and the T-cell response to egg antigens. In the BL/6 mouse, the egg granulomas are relatively small and the relevant sensitizing egg antigens are largely unknown. We investigated the CD4+Th cell response of infected BL/6 mice to egg antigens fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and found a prominent lymphoproliferative response to be directed against a 62-kDa component. With the aid of a specific T-cell hybridoma, 4E6, the 62-kDa antigen was isolated; following partial digestion with endoproteinase Glu-C, an internal amino acid sequence was found to be identical with one present in the enzyme phosphoenolpyruvate carboxykinase (PEPCK) of the organismsCaenorhabditis elegansandTreponema pallidumand to differ by one residue from PEPCK of various other species. In CD4+Th cells from 7.5- 8.5-week-infected BL/6 mice, the purified 62-kDa molecule elicited a potent proliferative response which, based on cytokine analysis, was of a mixed Th-1 and Th-2 type. Our results reveal a novel egg antigen of particular prominence in the BL/6 mouse and suggest that the immune response in schistosomiasis is a product of sensitization to egg antigens that may vary considerably in immunogenicity from strain to strain.
Identification and Characterization of thecpsLocus ofStreptococcus suisSerotype 2: the Capsule Protects against Phagocytosis and Is an Important Virulence Factor
Authors: Smith Hilde E. , Damman Marloes , van der Velde Joeke , Wagenaar Frans , Wisselink Henk J. , Stockhofe-Zurwieden Norbert , Smits Mari A.
Published: March 2020

To study the role of the capsule ofStreptococcus suisserotype 2 in virulence, we generated two isogenic mutants disturbed in capsule production. For that purpose, we first cloned and characterized a major part of the capsular polysaccharide biosynthesis (cps) locus ofS. suisserotype 2. Based on the established sequence, 14 open reading frames (ORFs), designated Orf2Z, Orf2Y, Orf2X, and Cps2A to Cps2K, were identified. Twelve ORFs belonged to a single transcriptional unit. The gene products of 11 of these ORFs showed similarity to proteins involved in polysaccharide biosynthesis of other gram-positive microorganisms. Nonencapsulated isogenic mutants were generated in thecps2Bandcps2EFgenes by insertional mutagenesis. In contrast to the wild-typeS. suisserotype 2 strain, the nonencapsulated strains were highly sensitive to ingestion by porcine alveolar lung macrophages in vitro. More importantly, the nonencapsulated mutant strains were completely avirulent in young germfree pigs after intranasal inoculation. These observations indicate that the capsule ofS. suisserotype 2 plays an essential role in the pathogenesis ofS. suisserotype 2 infections.
Anion-Coordinating Residues at Binding Site 1 Are Essential for the Biological Activity of the Diphtheria Toxin Repressor
Authors: Goranson-Siekierke Joanne , Pohl Ehmke , Hol Wim G. J. , Holmes Randall K.
Published: March 2020

The homodimeric diphtheria toxin repressor (DtxR) uses Fe2+as a corepressor, binds to iron-regulated promoters, and negatively regulates the syntheses of diphtheria toxin, corynebacterial siderophore, and several otherCorynebacterium diphtheriaeproducts. The crystal structure of DtxR shows that the second domain of each monomer has two binding sites for Fe2+or certain other divalent metal ions. In addition, site 1 binds a sulfate or phosphate anion, suggesting that phosphate may function intracellularly as a co-corepressor. The effects of alanine substitutions for selected residues in sites 1 and 2 were determined by measuring the β-galactosidase activities of atoxoperator/promoter-lacZreporter construct inEscherichia colistrains expressing each DtxR variant. Our studies demonstrated that single alanine substitutions for the anion-binding residues in site 1 (R80A, S126A, or N130A) caused severely decreased DtxR activity, similar to the effects of alanine substitutions for metal-binding residues in site 2 (C102A, E105A, or H106A) and greater than the effects of alanine substitutions for metal-binding residues in site 1 (H79A, E83A, or H98A) reported previously by other investigators. Various combinations of alanine substitutions for site 1 and site 2 residues were also analyzed to further elucidate the roles of these cation- and anion-binding ligands in DtxR activity. Furthermore, the interaction between residue E20 in the DNA binding domain and R80 in anion/cation binding site 1 was analyzed, and the E20A variant of DtxR was shown to have a phenotype indistinguishable from that of the R80A variant. Our data demonstrated for the first time that the anion-binding residues R80, S126, and N130 at site 1 are essential for DtxR activity. The data also showed that the interaction of E20 in domain 1 with R80 in domain 2, first revealed by X-ray crystallography in apo-DtxR and holo-DtxR, is a structural feature of DtxR that is important for its repressor activity.
Targeted Disruption of Fibronectin-Integrin Interactions in Human Gingival Fibroblasts by the RI Protease of
Porphyromonas gingivalis
W50
Authors: Scragg M. A. , Cannon S. J. , Rangarajan M. , Williams D. M. , Curtis M. A.
Published: March 2020

Cell surface integrins mediate interactions between cells and their extracellular matrix and are frequently exploited by a range of bacterial pathogens to facilitate adherence and/or invasion. In this study we examined the effects of
Porphyromonas gingivalis
proteases on human gingival fibroblast (HGF) integrins and their fibronectin matrix. Culture supernatant from the virulent strain W50 caused considerably greater loss of the β
1
integrin subunit from HGF in vitro than did that of the beige-pigmented strain W50/BE1. Prior treatment of the W50 culture supernatant with the protease inhibitor Nα-
p
-tosyl-
l
-lysine chloromethyl ketone (TLCK) blocked its effects on cultured cells, indicating that this process is proteolytically mediated. Purified arginine-specific proteases from
P. gingivalis
W50 were able to mimic the effects of the whole-culture supernatant on loss of β
1
integrin expression. However purified RI, an α/β heterodimer in which the catalytic chain is associated with an adhesin chain, was 12 times more active than RIA, the catalytic monomer, in causing loss of the α
5
β
1
integrin (fibronectin receptor) from HGF. No effect was observed on the α
V
β
3
integrin (vitronectin receptor). The sites of action of RI and RIA were investigated in cells exposed to proteases pretreated with TLCK to inactivate the catalytic component. Use of both monoclonal antibody 1A1, which recognizes only the adhesin chain of RI, and a rabbit antibody against
P. gingivalis
whole cells indicated localization of RI on the fibroblasts in a clear, linear pattern typical of that seen with fibronectin and α
5
β
1
integrin. Exact colocalization of RI with fibronectin and its α
5
β
1
receptor was confirmed by double labeling and multiple-exposure photomicroscopy. In contrast, RIA bound to fibroblasts in a weak, patchy manner, showing only fine linear or granular staining. It is concluded that the adhesin component of RI targets the
P. gingivalis
arginine-protease to sites of fibronectin deposition on HGF, contributing to the rapid loss of both fibronectin and its main α
5
β
1
integrin receptor. Given the importance of integrin-ligand interactions in fibroblast function, their targeted disruption by RI may represent a novel mechanism of damage in periodontal disease.
Examination of
Listeria monocytogenes
Intracellular Gene Expression by Using the Green Fluorescent Protein of
Aequorea victoria
Authors: Freitag Nancy E. , Jacobs Kathleen E.
Published: March 2020

The ActA protein of
Listeria monocytogenes
is an essential virulence factor and is required for intracellular bacterial motility and cell-to-cell spread.
plcB
, cotranscribed with
actA
, encodes a broad-specificity phospholipase C that contributes to lysis of host cell vacuoles and cell-to-cell spread. Construction of a transcriptional fusion between
actA-plcB
and the green fluorescent protein gene of
Aequorea victoria
has facilitated the detailed examination of patterns of
actA/plcB
expression within infected tissue culture cells.
actA/plcB
expression began approximately 30 min postinfection and was dependent upon entry of
L. monocytogenes
into the host cytosol.
L. monocytogenes Δhly
mutants, which are unable to escape from host cell vacuoles, did not express
actA/plcB
at detectable levels within infected tissue culture cells; however, complementation of the
hly
defect allowed entry of the bacteria into the host cytoplasm and subsequent
actA/plcB
expression. These results emphasize the ability of
L. monocytogenes
to sense the different host cell compartment environments encountered during the course of infection and to regulate virulence gene expression in response.
Oligomerization of Anthrax Toxin Protective Antigen and Binding of Lethal Factor during Endocytic Uptake into Mammalian Cells
Authors: Singh Yogendra , Klimpel Kurt R. , Goel Seema , Swain Prabodha K. , Leppla Stephen H.
Published: March 2020

The protective antigen (PA) protein of anthrax toxin binds to a cellular receptor and is cleaved by cell surface furin to produce a 63-kDa fragment (PA63). The receptor-bound PA63 oligomerizes to a heptamer and acts to translocate the catalytic moieties of the toxin, lethal factor (LF) and edema factor (EF), from endosomes to the cytosol. In this report, we used nondenaturing gel electrophoresis to show that each PA63 subunit in the heptamer can bind one LF molecule. Studies using PA immobilized on a plastic surface showed that monomeric PA63 is also able to bind LF. The internalization of PA and LF by cells was studied with radiolabeled and biotinylated proteins. Uptake was relatively slow, with a half-time of 30 min. The number of moles of LF internalized was nearly equal to the number of moles of PA subunit internalized. The essential role of PA oligomerization in LF translocation was shown with PA protein cleaved at residues 313-314. The oligomers formed by these proteins during uptake into cells were not as stable when subjected to heat and detergent as were those formed by native PA. The results show that the structure of the toxin proteins and the kinetics of proteolytic activation, LF binding, and internalization are balanced in a way that allows each PA63 subunit to internalize an LF molecule. This set of proteins has evolved to achieve highly efficient internalization and membrane translocation of the catalytic components, LF and EF.
Capsular Sialic Acid Limits C5a Production on Type III Group B Streptococci
Authors: Takahashi Shinji , Aoyagi Youko , Adderson Elisabeth E. , Okuwaki Yoshiyuki , Bohnsack John F.
Published: March 2020

The majority of type III group B streptococcus (GBS) human neonatal infections are caused by a genetically related subgroup called III-3. We have proposed that a bacterial enzyme, C5a-ase, contributes to the pathogenesis of neonatal infections with GBS by rapidly inactivating C5a, a potent pro-inflammatory molecule, but many III-3 strains do not express C5a-ase. The amount of C5a produced in serum following incubation with representative type III strains was quantitated in order to better understand the relationship between C5a production and C5a-ase expression. C5a production following incubation of bacteria with serum depleted of antibody to the bacterial surface was inversely proportional to the sialic acid content of the bacterial capsule, with the more heavily sialylated III-3 strains generating less C5a than the less-virulent, less-sialylated III-2 strains. The amount of C5a produced correlated significantly with C3 deposition on each bacterial strain. Repletion with type-specific antibody caused increased C3b deposition and C5a production through alternative pathway activation, but C5a was functionally inactivated by strains that expressed C5a-ase. The increased virulence of III-3 strains compared to that of III-2 strains results at least partially from the higher sialic acid content of III-3 strains, which inhibits both opsonophagocytic killing and C5a production in the absence of type-specific antibody. We propose that C5a-ase is not necessary for III-3 strains to cause invasive disease because the high sialic acid content of III-3 strains inhibits C5a production.
Humoral Immunity to Commensal Oral Bacteria in Human Infants: Salivary Secretory Immunoglobulin A Antibodies Reactive with
Streptococcus mitis
biovar 1,
Streptococcus oralis
,
Streptococcus mutans
, and
Enterococcus faecalis
during the First Two Years of Life
Authors: Cole Michael F. , Bryan Stacey , Evans Mishell K. , Pearce Cheryl L. , Sheridan Michael J. , Sura Patricia A. , Wientzen Raoul L. , Bowden George H. W.
Published: March 2020

Secretory immunoglobulin A (SIgA) antibodies reactive with the pioneer oral streptococci
Streptococcus mitis
biovar 1 and
Streptococcus oralis
, the late oral colonizer
Streptococcus mutans
, and the pioneer enteric bacterium
Enterococcus faecalis
in saliva samples from 10 human infants from birth to age 2 years were analyzed. Low levels of salivary SIgA1 and SIgA2 antibodies reactive with whole cells of all four species were detected within the first month after birth, even though
S. mutans
and
E. faecalis
were not recovered from the mouths of the infants during the study period. Although there was a fivefold increase in the concentration of SIgA between birth and age 2 years, there were no differences between the concentrations of SIgA1 and SIgA2 antibodies reactive with the four species over this time period. When the concentrations of SIgA1 and SIgA2 antibodies reactive with all four species were normalized to the concentrations of SIgA1 and SIgA2 in saliva, SIgA1 and SIgA2 antibodies reactive with these bacteria showed a significant decrease from birth to 2 years of age. Adsorption of each infant’s saliva with cells of one species produced a dramatic reduction of antibodies recognizing the other three species. Sequential adsorption of saliva samples removed all SIgA antibody to the bacteria, indicating that the SIgA antibodies were directed to antigens shared by all four species. The induction by the host of a limited immune response to common antigens that are likely not involved in adherence may be among the mechanisms that commensal streptococci employ to persist in the oral cavity.
Molecular Cloning, Characterization, and Expression of the M Antigen of
Histoplasma capsulatum
Authors: Zancopé-Oliveira Rosely M. , Reiss Errol , Lott Timothy J. , Mayer Leonard W. , Deepe George S.
Published: March 2020

The major diagnostic antigens of
Histoplasma capsulatum
are the H and M antigens, pluripotent glycoproteins that elicit both humoral and T-cell-mediated immune responses. These antigens may play a role in the pathogenesis of histoplasmosis. M antigen is considered immunodominant because antibodies against it are the first precipitins to arise in acute histoplasmosis and are commonly present during all phases of infection. The biological activity of monomolecular M antigen and its ability to elicit a protective immune response to
H. capsulatum
are largely unknown. A molecular approach was used to identify the biological nature of M antigen, including its purification from histoplasmin, partial digestion with proteinases, and reverse-phase high-performance liquid chromatography to separate the released peptides. The amino acid sequences of the purified peptides were obtained by Edman degradation, and using degenerate oligonucleotide primers for PCR, a 321-bp fragment of the gene encoding the M antigen was amplified from genomic
H. capsulatum
DNA. This fragment was used to screen an
H. capsulatum
genomic DNA library, leading to the isolation, cloning, and sequencing of the full-length gene. The M gene consists of 2,187-bp DNA encoding a protein of 80,719 Da, which has significant homology to catalases from
Aspergillus fumigatus
,
Aspergillus niger
, and
Eimericella nidulans
. A cDNA was generated by reverse transcription-PCR and cloned into the expression vector pQE40. The identity of the cloned, expressed protein was confirmed by Western blotting. The recombinant fusion protein was immunoreactive with monoclonal antibodies raised against M antigen, with polyclonal mouse anti-M antiserum, and with a serum sample from a patient with histoplasmosis. The gene encoding the major immunodominant M antigen of
H. capsulatum
is a presumptive catalase, and the recombinant protein retains serodiagnostic activity.
Vaccination and Protection of Pigs against Pleuropneumonia with a Vaccine Strain of
Actinobacillus pleuropneumoniae
Produced by Site-Specific Mutagenesis of the ApxII Operon
Authors: Prideaux C. T. , Lenghaus C. , Krywult J. , Hodgson A. L. M.
Published: March 2020

The production of toxin (Apx)-neutralizing antibodies during infection plays a major role in the induction of protective immunity to
Actinobacillus pleuropneumoniae
reinfection. In the present study, the gene encoding the ApxII-activating protein,
apxIIC
, was insertionally inactivated on the chromosome of a serovar 7 strain, HS93. Expression of the structural toxin, ApxIIA, and of the two genes required for its secretion,
apxIB
and
apxID
, still occurs in this strain. The resulting mutant strain, HS93C
−
Amp
r
, was found to secrete the unactivated toxin. Pigs vaccinated with live HS93C
−
Amp
r
via the intranasal route were protected against a cross-serovar challenge with a virulent serovar 1 strain of
A. pleuropneumoniae
. This is the first reported vaccine strain of
A. pleuropneumoniae
which can be delivered live to pigs and offers cross-serovar protection against porcine pleuropneumonia.
The Mxi-Spa Type III Secretory Pathway ofShigella flexneriRequires an Outer Membrane Lipoprotein, MxiM, for Invasin Translocation
Authors: Schuch Raymond , Maurelli Anthony T.
Published: March 2020

Invasion of epithelial cells byShigella flexneriis mediated by a set of translocated bacterial invasins, the Ipa proteins, and its dedicated type III secretion system, called Mxi-Spa. We show here thatmxiM, part of themxi-spalocus in theS. flexnerivirulence plasmid, encodes an indispensable type III secretion apparatus component, required for both Ipa translocation and tissue culture cell invasion. We demonstrated that mature MxiM, first identified as a putative lipoprotein, is lipidated in vivo. Consistent with features of known lipoproteins, MxiM (i) can be labeled with [3H]palmitate and [2-3H]glycerol, (ii) is associated with the cell envelope, (iii) is secreted independently of the type III pathway, and (iv) requires an intact lipoprotein modification and processing site for full activity. The lipidated form of MxiM was detected primarily in the outer membrane, where it establishes a peripheral association with the inner leaflet. Through analysis of subcellular Ipa distribution in amxiMnull mutant background, MxiM was found to be required for the assembly and/or function of outer, but not inner, membrane regions of Mxi-Spa. This function probably requires interactions with other Mxi-Spa subunits within the periplasmic space. We discuss implications of these findings with respect to the function of MxiM and the structure of Mxi-Spa as a whole.
Alkaline Conditions Accelerate Polymorphonuclear Leukocyte Apoptosis In Vitro
Authors: Leblebicioglu Binnaz , Walters John
Published: March 2020

Apoptosis was monitored in polymorphonuclear leukocytes (PMNs) cultured under mildly acidic, neutral, and alkaline conditions. Within 3 h, 9.0% of the PMNs underwent apoptosis at pH 6.7, as did 12% at pH 7.2, 38% at pH 7.7, and 60% at pH 8.2. Inhibitors of serine proteases, caspase-1, or caspase-3 significantly inhibited PMN apoptosis at pH 8.2, suggesting an involvement by these enzymes.
Characterization of Exochelins of the
Mycobacterium bovis
Type Strain and BCG Substrains
Authors: Gobin Jovana , Wong Diane K. , Gibson Bradford W. , Horwitz Marcus A.
Published: March 2020

Pathogenic mycobacteria must acquire iron in the host in order to multiply and cause disease. To do so, they release abundant quantities of siderophores called exochelins, which have the capacity to scavenge iron from host iron-binding proteins and deliver it to the mycobacteria. In this study, we have characterized the exochelins of
Mycobacterium bovis
, the causative agent of bovine and occasionally of human tuberculosis, and the highly attenuated descendant of
M. bovis
, bacillus Calmette-Guérin (BCG), widely used as a vaccine against human tuberculosis. The
M. bovis
type strain, five substrains of
M. bovis
BCG (Copenhagen, Glaxo, Japanese, Pasteur, and Tice), and two strains of virulent
Mycobacterium tuberculosis
all produce the same set of exochelins, although the relative amounts of individual exochelins may differ. Among these mycobacteria, the total amount of exochelins produced is greatest in
M. tuberculosis
, intermediate in
M. bovis
, and smallest in
M. bovis
BCG.
Characterization of
Proteus mirabilis
Precocious Swarming Mutants: Identification of
rsbA
, Encoding a Regulator of Swarming Behavior
Authors: Belas Robert , Schneider Rachel , Melch Michael
Published: March 2020

Proteus mirabilis
swarming behavior is characterized by the development of concentric rings of growth that are formed as cyclic events of swarmer cell differentiation, swarming migration, and cellular differentiation are repeated during colony translocation across a surface. This cycle produces the bull’s-eye colony often associated with cultures of
P. mirabilis
. How the cells communicate with one another to coordinate these perfectly synchronized rings is presently unknown. We report here the identification of a genetic locus that, when mutated, results in a precocious swarming phenotype. These mutants are defective in the temporal control of swarming migration and start swarming ca. 60 min sooner than wild-type cells. Unlike the wild type, precocious swarming mutants are also constitutive swarmer cells and swarm on minimal agar medium. The defects were found to be localized to a 5.4-kb locus on the
P. mirabilis
genome encoding RsbA (regulator of swarming behavior) and the
P. mirabilis
homologs to RcsB and RcsC. RsbA is homologous to membrane sensor histidine kinases of the two-component family of regulatory proteins, suggesting that RsbA may function as a sensor of environmental conditions required to initiate swarming migration. Introduction of a
rsbA
mutation back into the wild type via allelic-exchange mutagenesis reconstructed the precocious swarming phenotype, which could be complemented in trans by a plasmid-borne copy of
rsbA
. Overexpression of RsbA in wild-type cells resulted in precocious swarming, suggesting that RsbA may have both positive and negative functions in regulating swarming migration. A possible model to describe the role of RsbA in swarming migration is discussed.