bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin-15 Activates Proinflammatory and Antimicrobial Functions in Polymorphonuclear Cells
Authors: Musso Tiziana , Calosso Liliana , Zucca Mario , Millesimo Maura , Puliti Manuela , Bulfone-Paus Silvia , Merlino Chiara , Savoia Dianella , Cavallo Rossana , Ponzi Alessandro Negro , Badolato Raffaele
Published: December 2019

Interleukin-15 (IL-15) is a recently discovered cytokine produced by a wide range of different cell types including fibroblasts, keratinocytes, endothelial cells, and macrophages in response to lipopolysaccharide or microbial infection. This suggests that IL-15 may play a crucial role in the activation of phagocytic cells against pathogens. We studied polymorphonuclear leukocyte (PMN) activation by IL-15, evaluated as enhancement of PMN anti-Candidaactivity as well as IL-8 production, following stimulation with the cytokine. The PMN response to IL-15 depends on binding to the IL-15 receptor. Our experiments show that binding of a biotinylated human IL-15–immunoglobulin G2b IgG2b fusion protein was competed by the addition of human recombinant IL-15 (rIL-15) or of human rIL-2, suggesting that IL-15 binding to PMN might involve the IL-2Rβ and IL-2Rγ chains, which have been shown to be constitutively expressed by PMN. In addition, we show by reverse transcription-PCR and by flow cytometry with a specific anti-IL-15Rα chain monoclonal antibody that PMN express the IL-15Rα chain at the mRNA and protein levels. Incubation with IL-15 activated PMN to secrete the chemotactic factor IL-8, and the amount secreted was increased by costimulation with heat-inactivatedCandida albicans. In addition, IL-15 primed the metabolic burst of PMN in response to formyl-methionyl-leucyl-phenylalanine but was not sufficient to trigger the respiratory burst or to increase the production of superoxide in PMN exposed toC. albicans. IL-15 also increased the ability of PMN to phagocytose heat-killedC. albicansorganisms in a dose-dependent manner, without opsonization by antibodies or complement-derived products. In the same concentration range, IL-15 was as effective as gamma interferon (IFN-γ) and IL-2 in increasing theC. albicansgrowth-inhibitory activity of PMN. Taken together, these results suggest that IL-15 is a potent stimulant of both proinflammatory and antifungal activities of PMN, activating several antimicrobial functions of PMN involved in the cellular response againstC. albicans.
Molecular and Immunological Analyses of the
Mycobacterium avium
Homolog of the Immunodominant
Mycobacterium leprae
35-Kilodalton Protein
Authors: Triccas James A. , Winter Nathalie , Roche Paul W. , Gilpin Andrea , Kendrick Kathleen E. , Britton Warwick J.
Published: December 2019

The analysis of host immunity to mycobacteria and the development of discriminatory diagnostic reagents relies on the characterization of conserved and species-specific mycobacterial antigens. In this report, we have characterized the
Mycobacterium avium
homolog of the highly immunogenic
M. leprae
35-kDa protein. The genes encoding these two proteins were well conserved, having 82% DNA identity and 90% identity at the amino acid level. Moreover both proteins, purified from the fast-growing host
M. smegmatis
, formed multimeric complexes of around 1000 kDa in size and were antigenically related as assessed through their recognition by antibodies and T cells from
M. leprae
-infected individuals. The 35-kDa protein exhibited significant sequence identity with proteins from
Streptomyces griseus
and the cyanobacterium
Synechoccocus
sp. strain PCC 7942 that are up-regulated under conditions of nutrient deprivation. The 67% amino acid identity between the
M. avium
35-kDa protein and SrpI of
Synechoccocus
was spread across the sequences of both proteins, while the homologous regions of the 35-kDa protein and the P3 sporulation protein of
S. griseus
were interrupted in the P3 protein by a divergent central region. Assessment by PCR demonstrated that the gene encoding the
M. avium
35-kDa protein was present in all 30
M. avium
clinical isolates tested but absent from
M. intracellulare
,
M. tuberculosis
, or
M. bovis
BCG. Mice infected with
M. avium
, but not
M. bovis
BCG, developed specific immunoglobulin G antibodies to the 35-kDa protein, consistent with the observation that tuberculosis patients do not recognize the antigen. Strong delayed-type hypersensitivity was elicited by the protein in guinea pigs sensitized with
M. avium.
Indications of the Protective Role of Natural Killer Cells in Human Cutaneous Leishmaniasis in an Area of Endemicity
Authors: Maasho Kerima , Sanchez Fabio , Schurr Erwin , Hailu Asrat , Akuffo Hannah
Published: December 2019

The role of natural versus acquired immunity toLeishmania aethiopicainfection in humans is the focus of our studies. We found in previous studies that mononuclear cells from nonexposed healthy Swedish donors responded toLeishmaniaantigen stimulation by proliferation and gamma interferon production. The main cell type responding was CD3−CD16/56+natural killer (NK) cells. These findings led us to suggest that the potential to produce a rapid, nonacquired NK cell response may be a protective phenotype. In order to test this hypothesis, an area in Ethiopia whereLeishmaniais endemic was selected, and peripheral blood mononuclear cells were obtained from individuals who had lived in the area most of their lives but had no evidence of past or present leishmaniasis. Their responses were compared with those of confirmed leishmaniasis patients from the same region with active lesions or cured leishmaniasis lesions. Cells from these donors were stimulated in vitro withL. aethiopicaantigen. Responses were measured by proliferation, cytokine production, and phenotype analysis by fluorescence-activated cell sorting. The association ofNRAMP1alleles with the studied phenotype and susceptibility toL. aethiopica-induced leishmaniasis was also evaluated. The results show thatLeishmaniaantigens can induce NK cell and CD8+-T-cell responses in vitro. This is clearly seen in proliferating cells from the cured (immune) individuals and the apparently protected controls from the area of endemicity. It contrasted with the reactivity of the patients, where some NK proliferation was coupled with enhanced CD4+-T-cell proliferation. We conclude from these observations that NK cells and CD8+cells proliferating in response toLeishmaniastimulation are involved in protection from and healing of (Ethiopian) cutaneous leishmaniasis; however, such mechanisms appear to be unrelated to theNRAMP1host resistance gene.
Identification and Characterization of a 29-Kilodalton Protein fromMycobacterium tuberculosisCulture Filtrate Recognized by Mouse Memory Effector Cells
Authors: Rosenkrands Ida , Rasmussen Peter Birk , Carnio Markus , Jacobsen Susanne , Theisen Michael , Andersen Peter
Published: December 2019

Culture filtrate proteins fromMycobacterium tuberculosisinduce protective immunity in various animal models of tuberculosis. Two molecular mass regions (6 to 10 kDa and 24 to 36 kDa) of short-term culture filtrate are preferentially recognized by Th1 cells in animal models as well as by patients with minimal disease. In the present study, the 24- to 36-kDa region has been studied, and the T-cell reactivity has been mapped in detail. Monoclonal antibodies were generated, and one monoclonal antibody, HYB 71-2, with reactivity against a 29-kDa antigen located in the highly reactive region below the antigen 85 complex was selected. The 29-kDa antigen (CFP29) was purified fromM. tuberculosisshort-term culture filtrate by thiophilic adsorption chromatography, anion-exchange chromatography, and gel filtration. In its native form, CFP29 forms a polymer with a high molecular mass. CFP29 was mapped in two-dimensional electrophoresis gels as three distinct spots just below the antigen 85 complex component MPT59. CFP29 is present in both culture filtrate and the membrane fraction fromM. tuberculosis, suggesting that this antigen is released from the envelope to culture filtrate during growth. Determination of the N-terminal amino acid sequence allowed cloning and sequencing of thecfp29gene. The nucleotide sequence showed 62% identity to the bacteriocin Linocin fromBrevibacterium linens. Purified recombinant histidine-tagged CFP29 and native CFP29 had similar T-cell stimulatory properties, and they both elicited the release of high levels of gamma interferon from mouse memory effector cells isolated during the recall of protective immunity to tuberculosis. Interspecies analysis by immunoblotting and PCR demonstrated that CFP29 is widely distributed in mycobacterial species.
Characterization of Mannose Receptor-Dependent Phagocytosis Mediated byMycobacterium tuberculosisLipoarabinomannan
Authors: Kang Byoung K. , Schlesinger Larry S.
Published: December 2019

The macrophage mannose receptor (MR) along with complement receptors mediates phagocytosis of theM. tuberculosisvirulent strains Erdman and H37Rv. We have determined that the terminal mannosyl units of theM. tuberculosissurface lipoglycan, lipoarabinomannan (LAM), from the Erdman strain serve as ligands for the MR. The biology of the MR (receptor binding and trafficking) in response to phagocytic stimuli is not well characterized. This study analyzes the MR-dependent phagocytosis mediated by Erdman LAM presented on a 1-μm-diameter phagocytic particle. Erdman LAM microspheres exhibited a time- and dose-dependent rapid increase in attachment and internalization by human monocyte-derived macrophages (MDMs). In contrast, internalization of LAM microspheres by monocytes was minimal. Microsphere internalization by MDMs was visualized and quantitated by immunofluorescence and confocal and electron microscopy and resembled conventional phagocytosis. Phagocytosis of LAM microspheres by MDMs was energy, cytoskeleton, and calcium dependent and was mannan inhibitable. Trypsin treatment of MDMs at 37°C, which depleted surface and recycling intracellular pools of the MR, reduced the subsequent attachment of LAM microspheres. Trypsin treatment at 4°C allowed for subsequent recovery of LAM microsphere phagocytosis at 37°C by recycled MRs. Pretreatment of MDMs with cycloheximide influenced LAM microsphere phagocytosis to only a small extent, indicating that MR-dependent phagocytosis of the microspheres was occurring primarily by preformed recycled receptors. This study characterizes the requirements for macrophage phagocytosis of a LAM-coated particle mediated by the MR. This model will be useful in further characterization of the intracellular pathway taken by phagocytic particles coated with different LAM types in macrophages following ingestion.
The Abilities of aStaphylococcus epidermidisWild-Type Strain and Its Slime-Negative Mutant To Induce Endocarditis in Rabbits Are Comparable
Authors: Perdreau-Remington Francoise , Sande Merle A. , Peters Georg , Chambers Henry F.
Published: December 2019

The abilities of a parent and mutant pair ofStaphylococcus epidermidisstrains, the slime-producing parent RP62A and its slime-negative mutant, to establish endocarditis in a rabbit model of aortic valve endocarditis and to accumulate and adhere to surfaces in vitro were compared. Vegetation titer and infection rate depended on the presence or absence of a catheter (P= 0.020) and on inoculum size (P< 0.001) but not on the infecting strain. The ability of the parent strain vis-à-vis its mutant to accumulate in vitro on surfaces as demonstrated in a slime test did not correlate with any enhancement in the development of endocarditis in the rabbit model. In vitro initial adherence rates were identical. Both isolates accumulated to the same reduced extent in vitro in the presence of serum, albumin, or gelatin. Adhesion was equally promoted by addition of fibronectin. These data suggest that the in vitro phenomenon of accumulation described as slime production in the absence of serum may not be an important virulence determinant in vivo.
Characterization of Protective Epitopes in a Highly ConservedPlasmodium falciparumAntigenic Protein Containing Repeats of Acidic and Basic Residues
Authors: Sharma Pawan , Kumar Anil , Singh Balwan , Bharadwaj Ashima , Sailaja V. Naga , Adak T. , Kushwaha Ashima , Malhotra Pawan , Chauhan V. S.
Published: December 2019

The delineation of putatively protective and immunogenic epitopes in vaccine candidate proteins constitutes a major research effort towards the development of an effective malaria vaccine. By virtue of its role in the formation of the immune clusters of merozoites, its location on the surface of merozoites, and its highly conserved nature both at the nucleotide sequence level and the amino acid sequence level, the antigen which contains repeats of acidic and basic residues (ABRA) of the human malaria parasitePlasmodium falciparumrepresents such an antigen. Based upon the predicted amino acid sequence of ABRA, we synthesized eight peptides, with six of these (AB-1 to AB-6) ranging from 12 to 18 residues covering the most hydrophilic regions of the protein, and two more peptides (AB-7 and AB-8) representing its repetitive sequences. We found that all eight constructs bound an appreciable amount of antibody in sera from a large proportion ofP. falciparummalaria patients; two of these peptides (AB-1 and AB-3) also elicited a strong proliferation response in peripheral blood mononuclear cells from all 11 human subjects recovering from malaria. When used as carrier-free immunogens, six peptides induced a strong, boostable, immunoglobulin G-type antibody response in rabbits, indicating the presence of both B-cell determinants and T-helper-cell epitopes in these six constructs. These antibodies specifically cross-reacted with the parasite protein(s) in an immunoblot and in an immunofluorescence assay. In another immunoblot, rabbit antipeptide sera also recognized recombinant fragments of ABRA expressed in bacteria. More significantly, rabbit antibodies against two constructs (AB-1 and AB-5) inhibited the merozoite reinvasion of human erythrocytes in vitro up to ∼90%. These results favor further studies so as to determine possible inclusion of these two constructs in a multicomponent subunit vaccine against asexual blood stages ofP. falciparum.
In Vivo Binding of Immunoglobulin M to the Surfaces ofBabesia bigemina-Infected Erythrocytes
Authors: Echaide Ignacio E. , Hines Stephen A. , McElwain Terry F. , Suarez Carlos E. , McGuire Travis C. , Palmer Guy H.
Published: December 2019

Babesia bigeminainfection of mature bovine erythrocytes results in new proteins specifically exposed on the parasitized cell surface. Monoclonal antibody (MAb) 64/32 binds a protein, designated p94, onB. bigemina-infected erythrocytes but not on either uninfected orB. bovis-parasitized erythrocytes. However, p94 was not encoded byB. bigeminaand was not a parasite-modified erythrocyte membrane protein. In contrast, we showed that p94 could be eluted from the infected erythrocyte surface and was identified as specifically bound immunoglobulin M (IgM) heavy chain for the following reasons: (i) MAb 64/32 bound a reduced molecule of 94 kDa in both infected erythrocyte lysates and normal bovine serum; (ii) MAb 64/32 bound a 94-kDa molecule in reduced preparations of purified IgM; (iii) an anti-bovine μ heavy-chain MAb, BIg73, reacted specifically with the surface of infected erythrocytes and bound the 94-kDa molecule in lysates of infected erythrocytes, normal bovine serum, and purified IgM; and (iv) immunoprecipitation of infected erythrocyte lysates with MAb 64/32 depleted the 94-kDa antigen bound by anti-μ MAb BIg73 and vice versa. Binding of IgM to the infected erythrocyte surface was detected in vivo early in acute parasitemia and occurred during both the trophozoite and merozoite stages of intraerythrocytic parasitism. The common feature of IgM binding to the parasitized erythrocyte surface among otherwise genetically and antigenically distinctB. bigeminastrains is suggestive of an advantageous role in parasite survival in vivo.
Multiple Adhesive Phenotypes Linked to Rosetting Binding of Erythrocytes inPlasmodium falciparumMalaria
Authors: Fernandez Victor , Treutiger Carl Johan , Nash Gerard B. , Wahlgren Mats
Published: December 2019

The cerebral form of severe malaria is associated with excessive intravascular sequestration ofPlasmodium falciparum-infected erythrocytes (PRBC). Retention and accumulation of PRBC may lead to occlusion of brain microvessels and direct the triggering of acute pathologic changes. Here we report that by selection, cloning, and subcloning, we have identified rareP. falciparumparasites expressing a pan-adhesive phenotype linked to erythrocyte rosetting, a previously identified correlate of cerebral malaria. Rosetting PRBC not only bound uninfected erythrocytes but also formed autoagglutinates, adhered to endothelial cells, and bound to CD36, immunoglobulins, and the blood group A antigen. The linkage of rosetting, autoagglutination, and cytoadherence involved the coexpression on a single PRBC of ligands with multiple specificities and the binding to two or more receptors on erythrocytes and to at least two other cell adhesion molecules, including a new endothelial cell receptor forP. falciparum-infected erythrocytes. Limited proteolysis that differentially cleaved the rosetting ligand PfEMP1 from the PRBC surface abrogated all the binding phenotypes of these parasites, implicating the variant antigen PfEMP1 as a carrier of multiple ligand specificities. The results encourage the further study of pan-adhesion as a potentially important parasite phenotype in the pathogenesis of severeP. falciparummalaria.
Identification and Molecular Analysis oflbpBA, Which Encodes the Two-Component Meningococcal Lactoferrin Receptor
Authors: Lewis L. A. , Rohde K. , Gipson M. , Behrens B. , Gray E. , Toth S. I. , Roe B. A. , Dyer D. W.
Published: December 2019

We identifiedlbpB, encoding the lipoprotein component of the meningococcal lactoferrin receptor. An LbpB mutant was unable to acquire Fe from lactoferrin and exhibits decreased surface binding to lactoferrin. Primer extension and reverse transcription-PCR analysis indicate thatlbpBandlbpAare cotranscribed on a polycistronic Fe-repressible mRNA.
Expression of
Legionella pneumophila
Virulence Traits in Response to Growth Conditions
Authors: Byrne Brenda , Swanson Michele S.
Published: December 2019

In nature,
Legionella pneumophila
replicates exclusively as an intracellular parasite of amoebae, but it also persists in the environment as a free-living microbe. Studies of how this opportunistic pathogen recognizes and responds to distinct extracellular and intracellular environments identified a link between the growth phase and expression of traits previously correlated with virulence. When cultured in broth, only post-exponential-phase
L. pneumophila
was sodium sensitive, cytotoxic, osmotically resistant, competent to evade macrophage lysosomes, infectious, and motile. Likewise, the
L. pneumophila
phenotype changed during growth in macrophages. During the intracellular replication period, this bacterium was sodium resistant and lacked flagella; concomitant with macrophage lysis,
L. pneumophila
became sodium sensitive and flagellated. Expression of the virulent phenotype was a response to starvation, since exponential-phase
L. pneumophila
became cytotoxic, sodium sensitive, and motile after incubation in broth from stationary-phase cultures, except when it was supplemented with amino acids. Together, these data indicate that while nutrients are plentiful, intracellular
L. pneumophila
organisms are dedicated to replication; when amino acids become limiting, the progeny express virulence factors to escape the spent host, to disperse and survive in the aquatic environment, and to reestablish a protected intracellular niche favorable for growth.
Comparison ofEscherichia coliStrains Recovered from Human Cystitis and Pyelonephritis Infections in Transurethrally Challenged Mice
Authors: Johnson David E. , Lockatell C. Virginia , Russell Robert G. , Hebel J. Richard , Island Michael D. , Stapleton Ann , Stamm Walter E. , Warren John W.
Published: December 2019

Urinary tract infection, most frequently caused byEscherichia coli, is one of the most common bacterial infections in humans. A vast amount of literature regarding the mechanisms through whichE. coliinduces pyelonephritis has accumulated. Although cystitis accounts for 95% of visits to physicians for symptoms of urinary tract infections, few in vivo studies have investigated possible differences betweenE. colirecovered from patients with clinical symptoms of cystitis and that from patients with symptoms of pyelonephritis. Epidemiological studies indicate that cystitis-associated strains appear to differ from pyelonephritis-associated strains in elaboration of some putative virulence factors. With transurethrally challenged mice we studied possible differences using three each of the most virulent pyelonephritis and cystitisE. colistrains in our collection. The results indicate that cystitis strains colonize the bladder more rapidly than do pyelonephritis strains, while the rates of kidney colonization are similar. Cystitis strains colonize the bladder in higher numbers, induce more pronounced histologic changes in the bladder, and are more rapidly eliminated from the mouse urinary tract than pyelonephritis strains. These results provide evidence that cystitis strains differ from pyelonephritis strains in this model, that this model is useful for the study of the uropathogenicity of cystitis strains, and that it would be unwise to use pyelonephritis strains to study putative virulence factors important in the development of cystitis.
Oral Vaccination against Tetanus: Comparison of the Immunogenicities ofSalmonellaStrains Expressing Fragment C from thenirBandhtrAPromoters
Authors: Roberts Mark , Li Jingli , Bacon Andrew , Chatfield Steven
Published: December 2019

We have found the in vivo-regulatednirBpromoter (PnirB) to be effective for directing expression of a number of antigens in salmonella in vivo. We wished to determine if other in vivo-regulated promoters have utility for antigen expression in salmonella and to compare the effectiveness of these promoters with that of PnirB. To this end, we have devised a scheme that allows the promoter element of the PnirB-fragment C plasmid pTETnir15 to be swapped with other promoters of interest. We demonstrate the usefulness of this system by replacing PnirBwith PhtrAto create plasmid pTEThtrA1.htrAis a stress response gene that is required for virulence of salmonella in mice and survival within macrophages. Expression of fragment C inSalmonella typhimuriumBRD509 (aroA aroD) harboring pTEThtrA1 (strain BRD937) correlated with growth temperature in vitro. A comparison was made of the immune responses to fragment C elicited in mice immunized orally with BRD937 or BRD847 (BRD509/pTETnir15) or subcutaneously with purified fragment C plus alhydrogel. High levels of anti-fragment C antibodies that persisted for at least 12 weeks were present in all groups of mice. Vaccination with BRD937 was the most effective means of immunization: the serum immunoglobulin G (IgG), IgA, and IgM anti-fragment C titers were higher in the BRD937-immunized mice throughout the duration of the study than in mice in the other groups. The kinetics of the serum anti-fragment C responses were different in different groups. The response was most rapid in the BRD937 group, with the titers almost at peak levels at 2 weeks postimmunization. Only the mice immunized with BRD937 or BRD847 developed an intestinal IgA response to fragment C. Again, the response was superior in the BRD937 group. The peak of the intestinal response was delayed with respect to the serum response. Analysis of the IgG subtype response to fragment C revealed a dominant IgG2a response in the salmonella-immunized mice, indicating a type 1 helper T-cell response to fragment C, whereas the major subtype in the group parenterally immunized with fragment C plus alhydrogel was IgG1. The IgG1/IgG2a ratio was much higher in sera of BRD937-immunized mice than in sera of BRD847-immunized mice. At 15 to 20 weeks after immunization, the mice immunized with BRD937 or BRD847 were solidly immune to tetanus toxin and salmonella. The immune responses to fragment C seen in mice immunized with BRD937 are the strongest we have observed and indicate that thehtrApromoter may be very useful for expressing foreign antigens in salmonella vaccine strains.
Inhibition of Murine Splenic and Mucosal Lymphocyte Function by Enteric Bacterial Products
Authors: Malstrom Carolyn , James Stephen
Published: December 2019

Previously we showed that lysates of enteropathogenicEscherichia coli(EPEC) inhibit lymphokine production by mitogen-activated human peripheral blood and lamina propria mononuclear cells. The aims of the present study were to determine whether EPEC-inhibitory factors have similar effects on murine lymphoid populations in order to further delineate the mechanisms of alteration of cytokine production. Preexposure to EPEC lysates inhibited mitogen-stimulated interleukin-2 (IL-2), IL-4, and gamma interferon (IFN-γ) production by murine spleen cells, but IL-10 production was increased. The inhibition was not due to increased apoptosis and was not blocked by neutralizating antibodies against IL-10 or transforming growth factor β (TGF-β). EPEC lysates also inhibited mitogen-stimulated IL-2 and IFN-γ production by CD11b-depleted spleen cells, IL-2 and IL-4 production by intraepithelial and Peyer’s patch lymphocytes, IL-2 production by the human T-cell line Jurkat, and antigen-stimulated IL-2 production by murine spleen cells. Lysates obtained from Shiga-like toxin-producingE. coli,E. coliRDEC-1,Citrobacter rodentium, and an EPECespBinsertion mutant all inhibited IL-2 and IL-4 production by mitogen-stimulated lymphoid cells. In conclusion, lysates of EPEC and related bacteria directly inhibit cytokine production by lymphoid cells from multiple sites by a mechanism that does not increase apoptosis or result from secondary effects of IL-10 or TGF-β.
In Vitro Effects of a High-Molecular-Weight Heat-Labile Enterotoxin from Enteroaggregative
Escherichia coli
Authors: Navarro-García Fernando , Eslava Carlos , Villaseca Jorge M. , López-Revilla Rubén , Czeczulin John R. , Srinivas S. , Nataro James P. , Cravioto Alejandro
Published: December 2019

The pathogenic mechanisms of enteroaggregative
Escherichia coli
(EAggEC) infection are not fully elucidated. In this work we show that an ammonium sulfate precipitate of culture supernatant of EAggEC strain 049766 increased the potential difference (PD) and the short-circuit current (Isc) in rat jejunal preparations mounted in Ussing chambers. The precipitate contained two major proteins of 108 and 116 kDa, which were partially copurified by chromatography in DEAE-cellulose. This chromatographic fraction (peak I) increased jejunal PD and Isc in a dose-dependent manner, accompanied by a decrease in tissue electrical resistance. These effects were inhibited by incubation of peak I at 75°C for 15 min or for 1 h with proteinase K at 37°C. Rabbit polyclonal antibodies against peak I containing both the 108- and 116-kDa proteins inhibited the enterotoxic effect. Specific polyclonal antibodies raised against the 108-kDa but not against the 116-kDa protein inhibited the enterotoxic effect, suggesting that the 108-kDa protein is the active toxic species. Moreover, another EAggEC strain (065126) producing the 116-kDa protein but not the 108-kDa protein had no effect on rat jejunal mucosa in the Ussing chamber. The >100-kDa fraction derived from prototype EAggEC strain 042, which also expressed both 108- and 116-kDa proteins, also produced an enterotoxic effect on rat jejunal preparations in Ussing chambers; however, the same strain cured of its 65-MDa adherence plasmid did not. A subclone derived from the 65-MDa plasmid expressing the 108-kDa toxin (and not the 116-kDa protein) elicited rises in Isc. Tissue exposed to any preparation containing the 108-kDa toxin exhibited similar histopathologic changes, characterized by increased mucus release, exfoliation of cells, and development of crypt abscesses. Our data suggest that some EAggEC strains produce a ca. 108-kDa enterotoxin/cytotoxin which is encoded on the large virulence plasmid.
Mucosal Delivery of Murine Interleukin-2 (IL-2) and IL-6 by Recombinant Strains ofLactococcus lactisCoexpressing Antigen and Cytokine
Authors: Steidler Lothar , Robinson Karen , Chamberlain Lisa , Schofield Karin M. , Remaut Erik , Le Page Richard W. F. , Wells Jeremy M.
Published: December 2019

Lactococcus lactisis a nonpathogenic and noncolonizing bacterium which is being developed as a vaccine delivery vehicle for immunization by mucosal routes. To determine whether lactococci can also deliver cytokines to the immune system, we have constructed novel constitutive expression strains ofL. lactiswhich accumulate a test antigen, tetanus toxin fragment C (TTFC), within the cytoplasmic compartment and also secrete either murine interleukin-2 (IL-2) or IL-6. When mice were immunized intranasally with various different expression strains ofL. lactis, the anti-TTFC antibody titers increased more rapidly and were substantially higher in mice immunized with the bacterial strains which secreted IL-2 or IL-6 in addition to their production of TTFC. This adjuvant effect was lost when the recombinant strains ofL. lactiswere killed by pretreatment with mitomycin C and could therefore be attributed to the secretion of IL-2 or IL-6 by the recombinant lactococci. These results provide the first example of the use of a cytokine-secreting, noninvasive experimental bacterial vaccine vector to enhance immune responses to a coexpressed heterologous antigen and point the way to experiments which will test the possible therapeutic efficacy of this mode of cytokine delivery.
Bioluminescence as a Reporter of Intracellular Survival of
Bordetella bronchiseptica
in Murine Phagocytes
Authors: Forde Catrina B. , Parton Roger , Coote John G.
Published: December 2019

The uptake and persistence of
Bordetella bronchiseptica
was characterized in murine phagocytes by using a novel bioluminescence-based reporter system. A mini-Tn
5
promoter probe carrying the intact
lux
operon from the terrestrial bacterium
Photorhabdus luminescens
which allowed measurement of light output without the addition of exogenous substrate was constructed. It was used to create a pool of bioluminescent fusion strains of
B. bronchiseptica
. The internalization and persistence in murine macrophages of a constitutive bioluminescent strain of
B. bronchiseptica
was monitored by luminometry and by fluorescence and electron microscopy. The number of bacteria internalized, in a microfilament-dependent process, by a mouse macrophage-like cell line after 2 h was approximately 1% of the inoculum for several different multiplicities of infection (MOI). At an MOI of <500:1 (bacteria to macrophages), viable numbers of intracellular bacteria declined over a 4-day period. However, at an MOI of ≥500:1, long-term survival was enhanced, with viable bacteria recovered up to 4 days postinfection with little decline in numbers, indicating that a critical population size may have been essential for intracellular persistence. No evidence of macrophage killing by intracellular bacteria was detected over the 4-day period. Intracellular bioluminescent
B. bronchiseptica
organisms in mouse peritoneal cells were detected at 24 and 48 h after intraperitoneal injection of mice. Bioluminescence is shown to act as a convenient real-time technique for monitoring of intracellular survival of
B. bronchiseptica
in vitro and may provide a suitable means for examining the role of long-term intracellular survival of the bacterium in the host.
Expansion of a Novel Pulmonary CD3−CD4+CD8+Cell Population in Mice duringChlamydia pneumoniaeInfection
Authors: Penttilä Jenni M. , Pyhälä Reijo , Sarvas Matti , Rautonen Nina
Published: December 2019

A new pulmonary T-cell-like lymphocyte population with the phenotype CD3−CD4+CD8+was discovered in mice. CD4+CD8+but CD3+cells among murine intestinal intraepithelial lymphocytes have previously been described. We describe herein a dramatic expansion of the CD3−CD4+CD8+cell population in response to experimental respiratory infection. After intranasalChlamydia pneumoniaeinfection, CD4+CD8+cells became transiently the dominant lymphocyte type (maximum of 87% of all lymphocytes) in the lungs of NIH/S mice but remained virtually undetectable in spleen and blood. The enrichment of these cells was not aC. pneumoniae-specific event, since infection of NIH/S mice with influenza A virus also resulted in an increase in the number of CD4+CD8+cells (maximum of 42% of all lymphocytes). In addition to outbred NIH/S mice, two other mouse strains were studied: BALB/c (H-2d) and C57BL/6 (H-2b).C. pneumoniae-infected BALB/c mice responded with an intermediate increase in the number of CD4+CD8+cells in lungs, whereas C57BL/6 mice did not respond. The double-positive CD4+CD8+cells lacked a major part of the T-cell receptor complex, being both CD3−and TCR αβ−. However, when they were stimulated in vitro with a T-cell mitogen, they responded by proliferation but did not secrete gamma interferon. The dramatic expansion of this cell population at the infection site suggests an active role for them in respiratory infection, but the specification of this requires further study.
Differential Kinetics and Distribution of Antibodies in Serum and Nasal and Vaginal Secretions after Nasal and Oral Vaccination of Humans
Authors: Rudin Anna , Johansson Eva-Liz , Bergquist Charlotta , Holmgren Jan
Published: December 2019

Although nasal vaccination has emerged as an interesting alternative to systemic or oral vaccination, knowledge is scarce about the immune responses after such immunization in humans. In the present study, we have compared the kinetics and organ distribution of the antibody responses after nasal and oral vaccination. We immunized female volunteers nasally or orally with cholera toxin B subunit (CTB) and determined the specific antibody levels in serum and nasal and vaginal secretions, as well as the number of circulating antibody-secreting cells, before immunization and 1, 2, 3, 6, and 26 weeks thereafter. Nasal vaccination induced 9-fold CTB-specific immunoglobulin A (IgA) and 56-fold specific IgG antibody increases in nasal secretions, whereas no significant IgA increase was seen after oral vaccination. Both oral and nasal vaccination resulted in 5- to 6-fold CTB-specific IgA and 20- to 30-fold specific IgG increases in vaginal secretions. Strong serum responses to CTB were also induced by both routes of vaccination. A notable difference between nasal and oral vaccination was that the nasal route elicited a specific antibody response with a later onset but of much longer duration than did the oral route. We conclude from this study that the nasal route is superior to the oral route for administering at least nonliving vaccines against infections in the upper respiratory tract, whereas either oral or nasal vaccination might be used for eliciting antibody responses in the female genital tract.
Low Genetic Diversity of
Haemophilus influenzae
Type b Compared to Nonencapsulated
H. influenzae
in a Population in Which
H. influenzae
Is Highly Endemic
Authors: Smith-Vaughan Heidi C. , Sriprakash Kadaba S. , Leach Amanda J. , Mathews John D. , Kemp David J.
Published: December 2019

Immunization with
Haemophilus influenzae
type b (Hib) conjugate polysaccharide vaccines has dramatically reduced Hib disease worldwide. As in other populations, nasopharyngeal carriage of Hib declined markedly in Aboriginal infants following vaccination, although carriage has not been entirely eliminated. In this study, we describe the genetic characteristics and the carriage dynamics of longitudinal isolates of Hib, characterized by using several typing methods. In addition, carriage rates of nonencapsulated
H. influenzae
(NCHi) are high, and concurrent colonization with Hib and NCHi is common; we also observed NCHi isolates which were genetically similar to Hib. There is a continuing need to promote Hib immunization and monitor
H. influenzae
carriage in populations in which the organism is highly endemic, not least because of the possibility of genetic exchange between Hib and NCHi strains in such populations.
Neisseria gonorrhoeae
Induces Focal Polymerization of Actin in Primary Human Urethral Epithelium
Authors: Giardina Peter C. , Williams Richard , Lubaroff David , Apicella Michael A.
Published: December 2019

The pathogenic
Neisseria
species induce cytoskeletal reorganization in immortalized cell lines. In Chang conjunctival epithelium and T84 intestinal epithelium, focal cytoskeletal rearrangements in which bacteria contacted the epithelial surface were observed. We show that actin footprints are induced in gonococcus-challenged primary urethral epithelium. Moreover, the microbes induced microvillus extension from the epithelial cell surface. Our results indicate that formation of actin footprints is not an artifact of commonly used immortalized cell lines.
Roles of Innate and Adaptive Immunity in Respiratory Mycoplasmosis
Authors: Cartner Samuel C. , Lindsey J. Russell , Gibbs-Erwin Julie , Cassell Gail H. , Simecka Jerry W.
Published: December 2019

Current evidence suggests that host defense in respiratory mycoplasmosis is dependent on both innate and humoral immunity. To further delineate the roles of innate and adaptive immunity in antimycoplasmal defenses, we intranasally infected C3H/HeSnJ-
scid/scid
(C3H-SCID), C3H/HeSnJ (C3H), C57BL/6J-
scid/scid
(C57-SCID), and C57BL/6N (C57BL) mice with
Mycoplasma pulmonis
and at 14 and 21 days postinfection performed quantitative cultures of lungs and spleens, quantification of lung lesions, and histopathologic assessments of all other major organs. We found that numbers of mycoplasmas in lungs were associated with genetic background (C3H susceptible, C57BL resistant) rather than functional state of adaptive immunity, indicating that innate immunity is the main contributor to antimycoplasmal defense of the lungs. Extrapulmonary dissemination of mycoplasmas with colonization of spleens and histologic lesions in multiple organs was a common occurrence in all mice. The absence of adaptive immune responses in severe combined immunodeficient (SCID) mice resulted in increased mycoplasmal colonization of spleens and lesions in extrapulmonary sites, particularly spleens, hearts, and joints, and also reduced lung lesion severity. The transfer of anti-
M. pulmonis
serum to infected C3H-SCID mice prevented extrapulmonary infection and disease, while the severity of lung lesions was restored by transfer of naive spleen cells to infected C3H-SCID mice. Collectively, our results strongly support the conclusions that innate immunity provides antimycoplasmal defense of the lungs and humoral immunity has the major role in defense against systemic dissemination of mycoplasmal infection, but cellular immune responses may be important in exacerbation of mycoplasmal lung disease.
Evaluation of the Proline-Rich Antigen of
Coccidioides immitis
as a Vaccine Candidate in Mice
Authors: Kirkland Theo N. , Finley Fred , Orsborn Kris I. , Galgiani John N.
Published: December 2019

We have expressed the proline-rich antigen (PRA) from
Coccidioides immitis
in
Escherichia coli
and evaluated its potential as a vaccine candidate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the recombinant protein (rPRA) revealed two bands, which exhibited virtually identical primary amino acid sequences. T cells from rPRA-immunized BALB/c mice showed a significant in vitro proliferative response to rPRA. A small but statistically significant proliferative response was also induced by rPRA in T cells from mice immunized with whole-cell coccidioidal vaccines. BALB/c mice immunized with rPRA and challenged intraperitoneally with virulent
C. immitis
had a greatly reduced fungal burden in their lungs and spleens compared to unvaccinated mice. The number of organisms in the lungs was reduced 500-fold, and similar reductions were observed in the spleens of immunized mice. These studies support the continued development of rPRA as a candidate vaccine for prevention of coccidioidomycosis.
Pathogenicity and Immunogenicity of aListeria monocytogenesStrain That Requiresd-Alanine for Growth
Authors: Thompson Robert J. , Bouwer H. G. Archie , Portnoy Daniel A. , Frankel Fred R.
Published: December 2019

Listeria monocytogenesis an intracellular bacterial pathogen that elicits a strong cellular immune response following infection and therefore has potential use as a vaccine vector. However, while infections byL. monocytogenesare fairly rare and can readily be controlled by a number of antibiotics, the organism can nevertheless cause meningitis and death, particularly in immunocompromised or pregnant patients. We therefore have endeavored to isolate a highly attenuated strain of this organism for use as a vaccine vector.d-Alanine is required for the synthesis of the mucopeptide component of the cell walls of virtually all bacteria and is found almost exclusively in the microbial world. We have found inL. monocytogenestwo genes that control the synthesis of this compound, an alanine racemase gene (dal) and ad-amino acid aminotransferase gene (dat). By inactivating both genes, we produced an organism that could be grown in the laboratory when supplemented withd-alanine but was unable to grow outside the laboratory, particularly in the cytoplasm of eukaryotic host cells, the natural habitat of this organism during infection. In mice, the double-mutant strain was completely attenuated. Nevertheless, it showed the ability, particularly under conditions of transient suppression of the mutant phenotype, to induce cytotoxic T-lymphocyte responses and to generate protective immunity against lethal challenge by wild-typeL. monocytogenesequivalent to that induced by the wild-type organism.
Receptor-Mediated Recognition and Uptake of Iron from Human Transferrin by
Staphylococcus aureus
and
Staphylococcus epidermidis
Authors: Modun Belinda , Evans Robert W. , Joannou Christopher L. , Williams Paul
Published: December 2019

Staphylococcus aureus
and
Staphylococcus epidermidis
both recognize and bind the human iron-transporting glycoprotein, transferrin, via a 42-kDa cell surface protein receptor. In an iron-deficient medium, staphylococcal growth can be promoted by the addition of human diferric transferrin but not human apotransferrin. To determine whether the staphylococcal transferrin receptor is involved in the removal of iron from transferrin, we employed 6 M urea–polyacrylamide gel electrophoresis, which separates human transferrin into four forms (diferric, monoferric N-lobe, and monoferric C-lobe transferrin and apotransferrin).
S. aureus
and
S. epidermidis
but not
Staphylococcus saprophyticus
(which lacks the transferrin receptor) converted diferric human transferrin into its apotransferrin form within 30 min. During conversion, iron was removed sequentially from the N lobe and then from the C lobe. Metabolic poisons such as sodium azide and nigericin inhibited the release of iron from human transferrin, indicating that it is an energy-requiring process. To demonstrate that this process is receptor rather than siderophore mediated, we incubated (i) washed staphylococcal cells and (ii) the staphylococcal siderophore, staphyloferrin A, with porcine transferrin, a transferrin species which does not bind to the staphylococcal receptor. While staphyloferrin A removed iron from both human and porcine transferrins, neither
S. aureus
nor
S. epidermidis
cells could promote the release of iron from porcine transferrin. In competition binding assays, both native and recombinant N-lobe fragments of human transferrin as well as a naturally occurring human transferrin variant with a mutation in the C-lobe blocked binding of
125
I-labelled transferrin. Furthermore, the staphylococci removed iron efficiently from the iron-loaded N-lobe fragment of human transferrin. These data demonstrate that the staphylococci efficiently remove iron from transferrin via a receptor-mediated process and provide evidence to suggest that there is a primary receptor recognition site on the N-lobe of human transferrin.
Site-Directed Mutagenesis of the 19-Kilodalton Lipoprotein Antigen Reveals No Essential Role for the Protein in the Growth and Virulence of
Mycobacterium intracellulare
Authors: Mahenthiralingam Eshwar , Marklund Britt-Inger , Brooks Lucy A. , Smith Debbie A. , Bancroft Gregory J. , Stokes Richard W.
Published: December 2019

The mycobacterial 19-kilodalton antigen (19Ag) is a highly expressed, surface-associated glycolipoprotein which is immunodominant in infected patients and has little homology with other known proteins. To investigate the pathogenic significance of the 19Ag, site-directed mutagenesis of the
Mycobacterium intracellulare
19Ag gene was carried out by using a suicide vector-based strategy. Allelic replacement of the 19Ag gene of a mouse-avirulent M. intracellulare strain, 1403, was achieved by double-crossover homologous recombination with a gentamicin resistance gene-mutated allele. Unfortunately, an isogenic 19Ag was not achievable in the mouse-virulent strain, D673. However, a 19Ag mutant was successfully constructed in
M. intracellulare
FM1, a chemically mutagenized derivative of strain D673. FM1 was more amenable to genetic manipulation and susceptible to site-directed mutagenesis of the 19Ag gene yet retained the virulent phenotype of the parental strain. No deleterious effects of 19Ag gene mutation were observed during in vitro growth of
M. intracellulare
. Virulence assessment of the isogenic 19Ag mutants in a mouse infection model demonstrated that the antigen plays no essential role in the growth of
M. intracellulare
in vivo. Site-directed mutagenesis of the 19Ag gene demonstrated that it plays no essential role in growth and pathogenicity of
M. intracellulare
; however, the exact nature of its biological function remains unknown.
Regulation of
hly
Expression in
Listeria monocytogenes
by Carbon Sources and pH Occurs through Separate Mechanisms Mediated by PrfA
Authors: Behari Jaideep , Youngman Philip
Published: December 2019

Expression of the PrfA-controlled virulence gene
hly
(encoding the pore-forming cytolysin listeriolysin) is under negative regulation by readily metabolized carbon sources in
Listeria monocytogenes
. However, the hyperhemolytic strain NCTC 7973 exhibits deregulated
hly
expression in the presence of repressing sugars, raising the possibility that a defect in carbon source regulation is responsible for its anomalous behavior. We show here that the activity of a second glucose-repressed enzyme, α-glucosidase, is 10-fold higher in NCTC 7973 than in 10403S. Using
hly-gus
fusions, we show that the
prfA
allele from NCTC 7973 causes deregulated
hly-gus
expression in the presence of sugars in either the wild-type or the NCTC 7973 background, while the 10403S
prfA
allele restores carbon source regulation. However, the
prfA
genotype does not affect the regulation of α-glucosidase activity by repressing sugars. Of the two mutational differences in PrfA, only a Gly145Ser change is important for regulation of
hly-gus
. Therefore, NCTC 7973 and 10403S have genetic differences in at least two loci: one in
prfA
that affects carbon source regulation of virulence genes and another in an unidentified gene(s) that up-regulates α-glucosidase activity. We also show that the decrease in pH associated with utilization of sugars negatively regulates
hly-gus
expression, although sugars can affect
hly-gus
expression by another mechanism that is independent of pH.