bluuebunny/crossref_metadata_2025 huggingface dataset:
Release of lipopolysaccharide from intracellular compartments containing Salmonella typhimurium to vesicles of the host epithelial cell
Authors: Garcia-del Portillo F , Stein M A , Finlay B B
Published: January 2020

The biological effects of bacterial lipopolysaccharide (LPS) on eucaryotic cells have traditionally been characterized following extracellular challenge of LPS on susceptible cells. In this study, we report the capacity of Salmonella typhimurium to release LPS once it is located in the intracellular environment of cultured epithelial cells. LPS is liberated from vacuolar compartments, where intracellular bacteria reside, to vesicles present in the host cell cytosol. The vesicle-associated LPS is detected in infected cells from the time when invading bacteria enter the host cell. Release of LPS is restricted to S. typhimurium-infected cells, with no LPS observed in neighboring uninfected cells, suggesting that dissemination of LPS occurs entirely within the intracellular environment of the infected cell. The amount of LPS present in host vesicles reaches a maximum when intracellular S. typhimurium cells start to proliferate, a time at which the entire host cell cytosol is filled with numerous vesicles containing LPS. All these data support the concept that intracellular bacterial pathogens might signal the host cell from intracellular locations by releasing bioactive bacterial components such as LPS.
Cryptococcus neoformans and cryptococcal glucuronoxylomannan, galactoxylomannan, and mannoprotein induce different levels of tumor necrosis factor alpha in human peripheral blood mononuclear cells
Authors: Chaka W , Verheul A F , Vaishnav V V , Cherniak R , Scharringa J , Verhoef J , Snippe H , Hoepelman I M
Published: January 2020

Tumor necrosis factor alpha (TNF-alpha) release by peripheral blood mononuclear cells (PBMC) during disseminated infection by Cryptococcus neoformans may initiate and amplify the immune response of the host, leading to elimination of the fungus. The ability to induce TNF-alpha in PBMC by four clinical strains of C. neoformans, a laboratory strain (NIH 37), and the purified cryptococcal components glucuronoxylomannan (GXM), galactoxylomannan (GalXM), and mannoproteins (MP1 and MP2) were investigated under different opsonic conditions. In the absence of serum, the levels of TNF-alpha induced by all strains and cryptococcal components were not above background levels. Normal human serum (NHS) enhanced TNF-alpha induction by whole cryptococci and the different cryptococcal components, with MP2 being the most potent TNF-alpha inducer. Inactivation of complement (HI NHS) almost abrogated the ability of whole cryptococci and the GXMs to induce TNF-alpha. In contrast, when MP1, MP2, and GalXM were incubated with HI NHS, 48, 71, and 44%, respectively, of the original TNF-alpha levels remained. MPs incubated with heat-inactivated immunoglobulin G (IgG)-depleted serum still induced 50% of the levels of TNF-alpha induced by components incubated with HI NHS. Both these sera contained the same very low levels of anti-MP IgG antibodies, indicating the opsonic effect of a heat-stable factor other than antibody. Two anti-CD14 monoclonal antibodies (60BCA and 3C10) inhibited the production of TNF-alpha induced by MP2. The results indicate that (i) induction of TNF-alpha by C. neoformans and GXMs strongly depends on complement, (ii) MP1 and MP2 induction of TNF-alpha is facilitated by a heat-stable serum factor other than Ig, and (iii) CD14 may be involved in the induction of TNF-alpha by MP2.
Identification of CD14 residues involved in specific lipopolysaccharide recognition
Authors: Shapiro R A , Cunningham M D , Ratcliffe K , Seachord C , Blake J , Bajorath J , Aruffo A , Darveau R P
Published: January 2020

CD14 is a key molecule responsible for the innate host inflammatory response to microbial infection. It is able to bind a wide variety of microbial ligands and facilitate the activation of both myeloid and nonmyeloid cells. However, its specific contribution to the innate recognition of bacteria is not known. Presently there is no information on the contribution of individual CD14 residues to Escherichia coli lipopolysaccharide (LPS) binding or on the molecular basis of the interaction between CD14 and LPS from other bacteria. LPS obtained from Porphyromonas gingivalis, a bacterium associated with chronic inflammatory disease, binds CD14 and activates myeloid cells but does not facilitate the activation of nonmyeloid cells. The transfer and binding of these two LPS species to soluble CD14 recombinant globulin proteins with single point mutations was examined. Functional activity of the mutant proteins was monitored by E-selectin expression on human umbilical cord endothelial cells. The analysis identified a charge reversal mutation in a single residue, E47, that demonstrated selective binding to E. coli LPS but not to P. gingivalis LPS. E-selectin activation assays indicated that proteins with mutations at position E47 maintained their structural integrity. Other mutations, including a charge reversal mutation of residue E58, did not significantly reduce the binding of either LPS ligand or the ability of the molecule to facilitate E-selectin activation. These data demonstrate that CD14 can selectively recognize different LPS ligands.
Infection by Mycobacterium tuberculosis promotes human alveolar macrophage apoptosis
Authors: Keane J , Balcewicz-Sablinska M K , Remold H G , Chupp G L , Meek B B , Fenton M J , Kornfeld H
Published: January 2020

The effect of Mycobacterium tuberculosis infection on the viability of healthy (control) human alveolar macrophages was evaluated by staining with ethidium homodimer and calcein to discriminate live from dead cells. Infection with M. tuberculosis H37Ra or H37Rv increased macrophage mortality at 6 days from the control level of 3.8% +/- 0.7% to 28.7% +/- 6.9% or 12.6% +/- 3.1%, respectively (P < 0.001 for comparisons of all conditions). A role for tumor necrosis factor alpha (TNF-alpha) in the M. tuberculosis-induced cytolysis of alveolar macrophages was demonstrated by increased cytotoxicity following the addition of exogenous TNF-alpha to the cultures and by enhancement of macrophage survival when M. tuberculosis-infected alveolar macrophages were treated with pentoxifylline or anti-TNF-alpha antibody. The cytolytic mechanism was determined to be apoptosis by the demonstration of a characteristic internucleosomal ladder of genomic DNA by agarose gel electrophoresis, by finding nuclear fragmentation and condensation by electron microscopy, and by in situ terminal transferase-mediated nick end labeling of fragmented DNA in alveolar macrophages infected with M. tuberculosis in vitro. The latter technique was employed to reveal extensive apoptosis within caseating granulomas from lung tissue samples from clinical tuberculosis cases. The induction of apoptosis in alveolar macrophages by M. tuberculosis may play a role in the macrophage-pathogen interaction of tuberculosis in vivo.
Expression and identification of a laminin-binding protein in Aspergillus fumigatus conidia
Authors: Tronchin G , Esnault K , Renier G , Filmon R , Chabasse D , Bouchara J P
Published: January 2020

Adhesion of Aspergillus fumigatus, the causative agent of human aspergillosis, to the extracellular matrix protein laminin has been previously demonstrated. This study investigated the expression of laminin receptors during swelling of conidia, a step leading to germination and subsequent colonization of tissues. Scanning electron microscopy showed that the laminin binding sites were distributed over the external rodlet layer of resting conidia. During swelling, the characteristic rodlet layer progressively disintegrated and conidia surrounded by a smooth cell wall layer appeared. Flow cytometry using fluorescein isothiocyanate-conjugated laminin demonstrated that expression of laminin receptors at the surface of conidia was swelling dependent. Resting conidia expressed high levels of laminin receptors on their surface. A gradual decrease of laminin binding was then observed as swelling occurred, reaching a minimum for 4-h-swollen conidia. This correlated with a loss of adherence of swollen conidia to laminin immobilized on microtiter plates. Trypsin pretreatment of conidia reduced laminin binding. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ligand blotting with laminin identified in a cell wall extract a major 72-kDa cell wall glycoprotein which binds laminin. Thus, one of the initial events in the host colonization may be the recognition of basement membrane laminin by this 72-kDa cell wall surface component.
Secretion of Ipa proteins by Shigella flexneri: inducer molecules and kinetics of activation
Authors: Bahrani F K , Sansonetti P J , Parsot C
Published: January 2020

The type III Mxi-Spa secretion machinery of Shigella flexneri is responsible for secretion of Ipa proteins, which are involved in the entry of bacteria into epithelial cells. Ipa proteins accumulate within bacteria growing in laboratory media, and their secretion is activated upon contact of bacteria with eukaryotic cells. In this study, we have identified a group of chemical compounds, including Congo red, Evans blue, and direct orange, which are able to induce secretion of Ipa proteins by bacteria suspended in phosphate-buffered saline. Parameters of kinetics of activation of Ipa secretion by Congo red were determined by measuring by enzyme-linked immunosorbent assay the amount of IpaC secreted and by investigating the increase in susceptibility of Ipa proteins to proteinase K degradation. Ipa secretion occurred at 37 degrees C, was obtained with 5 to 10 microM Congo red, and was complete within 30 min. In addition, activation of Ipa secretion by Congo red was observed with bacteria harvested throughout the exponential phase of growth but not with bacteria in the stationary phase. The interactions of Congo red and Congo red-related compounds with the Mxi-Spa secretion apparatus might be specific hydrophobic interactions similar to those involved in binding of Congo red to amyloid proteins.
Differential stimulation of interleukin-12 (IL-12) and IL-10 by live and killed Helicobacter pylori in vitro and association of IL-12 production with gamma interferon-producing T cells in the human gastric mucosa
Authors: Haeberle H A , Kubin M , Bamford K B , Garofalo R , Graham D Y , El-Zaatari F , Karttunen R , Crowe S E , Reyes V E , Ernst P B
Published: January 2020

The objective of these experiments was to examine the ability of Helicobacter pylori to stimulate interleukin-10 (IL-10) or IL-12 and select for either Th1 or Th2 cells. Gastric biopsy specimens were collected from patients who were categorized with respect to the presence of H. pylori and gastric disease as well as their age, gender, medications, and other factors. As Th1 and Th2 cells are selected by IL-12 and IL-10, respectively, biopsy specimens were screened for mRNA and protein for these cytokines. Although mRNA for IL-12 and IL-10 was detected in biopsy specimens obtained from both infected and uninfected patients, IL-12 protein predominated. Levels of IL-10 and IL-12 in gastric tissue did not change in response to infection. Moreover, gamma interferon (IFN-gamma)-producing T cells were found in both the infected and the uninfected gastric mucosa. Stimulation of peripheral blood leukocytes from either infected or uninfected donors with various concentrations of live or killed H. pylori induced immunoreactive IL-12 and IL-10. After stimulation with live H. pylori, IL-12 levels increased more than 30-fold, whereas IL-10 levels increased only 2- to 5-fold, compared to cells stimulated with medium alone. Interestingly, killed H. pylori induced significantly more IL-10 (P < 0.05) than live H. pylori, while recombinant urease only induced IL-10. These results demonstrate that live H. pylori selectively stimulates the induction of IL-12 and Th1 cells that produce IFN-gamma, whereas preparations used in oral vaccines induce more IL-10 and may favor Th2 cell responses.
HLA-B27 modulates the survival of Salmonella enteritidis in transfected L cells, possibly by impaired nitric oxide production
Authors: Virtala M , Kirveskari J , Granfors K
Published: January 2020

Reactive arthritis is triggered by certain microbes that cause primary infections mainly on the gastrointestinal or urogenital mucosa. The disease is strongly associated with HLA-B27. Long persistence of causative microbes or their structures in the body has been thought to have an important role in the pathogenesis of reactive arthritis. This suggests that the elimination of the microbes causing reactive arthritis is ineffective or disturbed in HLA-B27-positive individuals developing this complication. We examined the role of the HLA-B27 antigen in microbe-host interaction in vitro by monitoring the invasion and intracellular survival of Salmonella enteritidis in mouse fibroblasts transfected with HLA-B27, HLA-B7, or beta2-microglobulin only. S. enteritidis invaded into all the three transfectants with the same efficiency. However, at 6 and 10 days after incubation, there were more living intracellular Salmonella organisms in HLA-B27 transfectants than in the other transfected cell lines (P < 0.05), suggesting that the bactericidal effect is impaired in these cells. Impaired NO production in HLA-B27-transfected cells was indicated as a possible mechanism, since the amount of nitrite in the supernatants of the Salmonella-infected HLA-B27-transfected cells was smaller than that in the supernatants of the Salmonella-infected HLA-B7- or beta2-microglobulin-transfected cells (P < 0.001). The inhibition of NO synthesis by N-monomethyl-L-arginine resulted in impaired elimination of Salmonella also in HLA-B7and beta2-microglobulin-transfected cells. The inverse correlation between intracellular survival of Salmonella and the amount of nitrite detected in culture supernatants supports the hypothesis that the L-arginine-dependent NO pathway plays an important role in the murine fibroblast response against Salmonella. We suggest that a major histocompatibility complex class I antigen, HLA-B27, may contribute to the intracellular persistence of Salmonella by a mechanism which involves the NO pathway.
Arabinose assimilation defines a nonvirulent biotype of Burkholderia pseudomallei
Authors: Smith M D , Angus B J , Wuthiekanun V , White N J
Published: January 2020

Two distinct types of Burkholderia pseudomallei, differentiated by the ability to assimilate L-arabinose but with similar morphologies and antigenicities, can be isolated from soil in Thailand. Approximately 25% of soil isolates from northeast Thailand were arabinose assimilators (Ara+), but in 1,200 sequentially studied patients, only arabinose "nonassimilators" (Ara-) caused melioidosis (P < 0.0001). In a murine model, there was a striking difference in virulence between Ara- and Ara+ B. pseudomallei. The mean (standard deviation) 50% lethal dose (LD[50]) inoculum for Ara- isolates was 182 (111) CFU/mouse compared with approximately 10(9) CFU/mouse for Ara+ soil isolates. There was no significant difference between the LD(50)s for clinical and soil Ara- isolates. All attempts to convert the biochemical phenotype by selective culture failed, which suggests that the biotype is stable.
Sialylation of Neisseria meningitidis lipooligosaccharide inhibits serum bactericidal activity by masking lacto-N-neotetraose
Authors: Estabrook M M , Griffiss J M , Jarvis G A
Published: January 2020

Exogenous sialylation of gonococcal lipooligosaccharide causes resistance to serum bactericidal activity. The aim of this study was to determine how lipooligosaccharide sialylation affects the serum sensitivities of group C Neisseria meningitidis strains. The relationship between the degree of sialylation or expression of the lipooligosaccharide sialic acid acceptor, lacto-N-neotetraose (LNnT), of nine meningococcal strains and their sensitivities to a pool of normal human sera was assessed. All strains expressed LNnT that was variously endogenously sialylated. Susceptibility to serum bactericidal activity ranged from extremely sensitive to resistant in 50% serum. For endogenously sialylated strains, the amount of killing correlated with the amount of free LNnT above a threshold of expression; strains that expressed less than the threshold survived in 25% serum. All strains added more sialic acid when they were grown in medium that contained cytidine monophospho-N-acetylneuraminic acid. Exogenous sialylation reduced the expression of free LNnT and significantly increased serum resistance. Exogenous sialylation affected killing through both classical and alternative complement pathways. The killing of exogenously sialylated strains also correlated with the amount of free LNnT. The amounts of endogenous, exogenous, and total sialic acid bound to LNnT did not correlate with the resistance of strains to serum bactericidal activity; rather, the loss of free LNnT expression by sialylation was associated with resistance. In conclusion, the expression of free LNnT by group C meningococcal strains is directly associated with the amount of killing of organisms in pooled human sera. Both endogenous and exogenous lipooligosaccharide sialylation are associated with increased serum resistance by masking LNnT.
Th1 response in Salmonella typhimurium-infected mice with a high or low rate of bacterial clearance
Authors: Pie S , Truffa-Bachi P , Pla M , Nauciel C
Published: January 2020

Previous studies have shown that the capacity to clear an attenuated strain of Salmonella typhimurium after the second week of infection varies widely among mouse strains. Bacterial clearance is mediated by CD4+ T cells and is regulated in part by the H-2 complex. The aim of the present study was to compare the patterns of cytokine mRNA expression in the spleens of C57BL/6 (H-2b) and CBA (H-2k) mice, which exhibit a low and a high rate of bacterial clearance, respectively. A transient increase in interleukin-12 (IL-12) mRNA levels was found in both mouse strains. Gamma interferon (IFN-gamma) gene expression was higher and more sustained in C57BL/6 than in CBA mice. No increase in IL-4 mRNA was detected. A transient increase in IL-10 mRNA was found in C57BL/6 mice. Separation of spleen cells into CD4+ and CD4- fractions showed that CD4+ T cells produced the bulk of IFN-gamma in both mouse strains and of IL-10 in C57BL/6 mice. Infection of H-2 congenic mice induced a higher level of IFN-gamma mRNA expression by CD4+ T cells in mice with a low rate of clearance (H-2b) than in mice with a high rate of clearance (H-2q). Treatment of infected C57BL/6 mice with anti-IFN-gamma or anti-CD4 monoclonal antibodies indicated that IFN-gamma participates in resistance in the early phase of infection, but not in bacterial clearance, and that CD4+ T cells mediate bacterial clearance during the 3rd week of infection. Taken together, these results suggest that defective bacterial clearance in H-2b mice is not linked to defective IFN-gamma production and that CD4+ T cells mediate bacterial clearance by an IFN-gamma-independent mechanism.
Response of Chlamydia trachomatis serovar E to iron restriction in vitro and evidence for iron-regulated chlamydial proteins
Authors: Raulston J E
Published: January 2020

Iron is a well-established mediator of virulence in several bacterial pathogens, yet little is known about the role of iron in infectious disease processes caused by obligate intracellular bacterial pathogens. In this study, the effect of iron limitation was examined for the sexually transmitted infectious agent Chlamydia trachomatis in an in vitro model of human genital infection using the intracellular iron-chelating reagent deferoxamine mesylate (Desferal). Iron restriction caused a significant reduction in infectivity of C. trachomatis elementary bodies (EB) harvested from Desferal-exposed polarized epithelial cells when compared to that of EB harvested from iron-sufficient control cell cultures. Replacement of the Desferal exposure medium with medium containing iron-saturated transferrin restored chlamydial infectivity, whereas replacement with growth medium alone had no effect. The following three prominent morphological features were observed by electron microscopic examination of chlamydia-infected cells exposed to Desferal: (i) inclusions containing chlamydiae greatly delayed in maturation, (ii) substantial blebbing within chlamydial inclusions, and (iii) electron-dense material surrounding inclusions. Protein analyses of highly purified EB by two-dimensional polyacrylamide gel electrophoresis revealed that there were at least 19 candidate iron-repressible proteins in C. trachomatis and at least one protein which was iron inducible. One putative iron-repressible protein was confirmed by Western blot (immunoblot) analysis to be the chlamydial heat shock protein 60 (hsp60). The enhanced production of this antigen by chlamydiae as a result of iron limitation is of particular importance since there is a well-documented association between chlamydial hsp60 and destructive immunopathological sequelae in infected patients.
Nucleotide sequence of the Porphyromonas gingivalis W83 recA homolog and construction of a recA-deficient mutant
Authors: Fletcher H M , Morgan R M , Macrina F L
Published: January 2020

Degenerate oligonucleotide primers were used in PCR to amplify a region of the recA homolog from Porphyromonas gingivalis W83. The resulting PCR fragment was used as a probe to identify a recombinant lambda DASH phage (L10) carrying the P. gingivalis recA homolog. The recA homolog was localized to a 2.1-kb BamHI fragment. The nucleotide sequence of this 2.1-kb fragment was determined, and a 1.02-kb open reading frame (341 amino acids) was detected. The predicted amino acid sequence was strikingly similar (90% identical residues) to the RecA protein from Bacteroides fragilis. No SOS box, characteristic of LexA-regulated promoters, was found in the 5' upstream region of the P. gingivalis recA homolog. In both methyl methanesulfonate and UV survival experiments the recA homolog from P. gingivalis complemented the recA mutation of Escherichia coli HB101. The cloned P. gingivalis recA gene was insertionally inactivated with the ermF-ermAM antibiotic resistance cassette to create a recA-deficient mutant (FLL33) by allelic exchange. The recA-deficient mutant was significantly more sensitive to UV irradiation than the wild-type strain, W83. W83 and FLL33 showed the same level of virulence in in vivo experiments using a mouse model. These results suggest that the recA gene in P. gingivalis W83 plays the expected role of repairing DNA damage caused by UV irradiation. However, inactivation of this gene did not alter the virulence of P. gingivalis in the mouse model.
Phase variation and conservation of lipooligosaccharide epitopes in Haemophilus somnus
Authors: Inzana T J , Hensley J , McQuiston J , Lesse A J , Campagnari A A , Boyle S M , Apicella M A
Published: January 2020

The bovine-specific pathogen Haemophilus somnus is capable of undergoing structural and antigenic phase variation in its lipooligosaccharide (LOS) components after in vivo and in vitro passage. However, commensal isolates from the reproductive tract have not been observed to vary in phase (T. J. Inzana, R. P. Gogolewski, and L. B. Corbeil, Infect. Immun. 60:2943-2951, 1992). We now report that specific monoclonal antibodies (MAbs) to the LOSs of Haemophilus aegyptius, Neisseria gonorrhoeae, and Haemophilus influenzae, as well as H. somnus, reacted with some phase-variable epitopes in H. somnus LOS. All reactive MAbs bound to LOS components of about 4.3 kDa in the same H. somnus isolates, including a non-phase-varying strain. Following in vitro passage of a clonal variant of strain 738 that was nonreactive with the MAbs, 11.8% of young colonies shifted to a reactive phenotype. A digoxigenin-labelled 5'-CAATCAATCAATCAATCAATCAATCAAT-3' oligonucleotide probe hybridized to genomic DNA from strain 738 but did not react with DNA from a non-phase-varying strain. Sequence analysis of the gene containing 5'-CAAT-3' tandem sequences revealed 48% amino acid homology with the lex-2B gene-encoded protein of H. influenzae type b. Our results indicate that some LOS epitopes are conserved between H. somnus and other Haemophilus and Neisseria species, that LOS phase variation may occur at a high rate in some strains of H. somnus, and that phase variation may, in part, be due to 5'-CAAT-3' tandem sequences present in H. somnus genes.
Transcription of genes encoding iron and heme acquisition proteins of Haemophilus influenzae during acute otitis media
Authors: Whitby P W , Sim K E , Morton D J , Patel J A , Stull T L
Published: January 2020

Unencapsulated Haemophilus influenzae is the second most common etiologic agent of otitis media in children. H. influenzae requires heme for aerobic growth in vitro and is able to utilize hemoglobin and complexes of heme-hemopexin, heme-albumin, and hemoglobin-haptoglobin and ferritransferrin as sources of iron and heme in vitro. Several of the acquisition mechanisms have been characterized and been shown to be heme repressible in vitro. However, little is known about the expression of heme and/or iron acquisition mechanisms during infections in the middle ear. This study was performed to determine if the genes encoding heme and iron acquisition proteins are transcribed during in vivo growth and to compare these findings with those for samples grown in vitro. Reverse transcriptase PCR (RT-PCR) was used to analyze total RNA fractions derived from in vitro- and in vivo-grown H. influenzae. Genes encoding the transferrin-binding proteins TbpA and TbpB, the 100-kDa hemopexin-binding protein HxuA, and the hemoglobin-binding protein HgpA were transcribed during otitis media. Twelve middle ear fluid samples were analyzed by blind RT-PCR to determine the transcriptional status of these genes in H. influenzae during otitis media. Five isolates had transcripts corresponding to tbpA, tbpB, and hxuA. The presence of hgpA transcripts was variable, depending on the presence of hgpA in the genome of the H. influenzae isolate. Samples without H. influenzae gene transcripts contained other etiologic agents commonly causing otitis media. These data demonstrate that H. influenzae iron and/or heme acquisition genes are transcribed during otitis media and suggest that the microenvironment during acute otitis media starves H. influenzae of heme.
Utilization of similar mechanisms by Legionella pneumophila to parasitize two evolutionarily distant host cells, mammalian macrophages and protozoa
Authors: Gao L Y , Harb O S , Abu Kwaik Y
Published: January 2020

The Legionnaires' disease bacterium, Legionella pneumophila, is an intracellular pathogen of humans that is amplified in the environment by intracellular multiplication within protozoa. Within both evolutionarily distant hosts, the bacterium multiplies in a rough endoplasmic reticulum-surrounded phagosome that is retarded from maturation through the endosomal-lysosomal degradation pathway. To gain an understanding of the mechanisms utilized by L. pneumophila to invade and replicate within two evolutionarily distant hosts, we isolated a collection of 89 mini-Tn10::kan insertion mutants that exhibited defects in cytotoxicity, intracellular survival, and replication within both U937 macrophage-like cells and Acanthamoeba polyphaga. Interestingly, the patterns of defects in intracellular survival and replication of the mutants within both host cells were highly similar, and thus we designated the defective loci in these mutants pmi (for protozoan and macrophage infectivity loci). On the basis of their ability to attach to host cells and their growth kinetics during the intracellular infection, the mutants were grouped into five groups. Groups 1 and 2 included 41 mutants that were severely defective in intracellular survival and were completely or substantially killed during the first 4 h of infection in both host cells. Three members of group 1 were severely defective in attachment to both U937 cells and A. polyphaga, and another four mutants of group 1 exhibited severe defects in attachment to A. polyphaga but only a mild reduction in their attachment to U937 cells. Four members of groups 1 and 2 were serum sensitive. Intracellular replication of mutants of the other three groups was less defective than that of mutants of groups 1 and 2, and their growth kinetics within both host cells were similar. The mutants were tested for several other phenotypes in vitro, revealing that 14 of the pmi mutants were resistant to NaCl, 3 had insertions in dot or icm, 3 were aflagellar, 12 were highly intolerant to a hyperosmotic medium, and one failed to grow in a minimal medium. Our data indicated that similar mechanisms are utilized by L. pneumophila to replicate within two evolutionarily distant hosts. Although some mechanisms of attachment to both host cells were similar, other distinct mechanisms were utilized by L. pneumophila to attach to A. polyphaga. Our data supported the hypothesis that preadaptation of L. pneumophila to infection of protozoa may play a major role in its ability to replicate within mammalian cells and cause Legionnaires' disease.
Display of a PorA peptide from Neisseria meningitidis on the bacteriophage T4 capsid surface
Authors: Jiang J , Abu-Shilbayeh L , Rao V B
Published: January 2020

The exterior of bacteriophage T4 capsid is coated with two outer capsid proteins, Hoc (highly antigenic outer capsid protein; molecular mass, 40 kDa) and Soc (small outer capsid protein; molecular mass, 9 kDa), at symmetrical positions on the icosahedron (160 copies of Hoc and 960 copies of Soc per capsid particle). Both these proteins are nonessential for phage infectivity and viability and assemble onto the capsid surface after completion of capsid assembly. We developed a phage display system which allowed in-frame fusions of foreign DNA at a unique cloning site in the 5' end of hoc or soc. A DNA fragment corresponding to the 36-amino-acid PorA peptide from Neisseria meningitidis was cloned into the display vectors to generate fusions at the N terminus of Hoc or Soc. The PorA-Hoc and PorA-Soc fusion proteins retained the ability to bind to the capsid surface, and the bound peptide was displayed in an accessible form as shown by its reactivity with specific monoclonal antibodies in an enzyme-linked immunosorbent assay. By employing T4 genetic strategies, we show that more than one subtype-specific PorA peptide can be displayed on the capsid surface and that the peptide can also be displayed on a DNA-free empty capsid. Both the PorA-Hoc and PorA-Soc recombinant phages are highly immunogenic in mice and elicit strong antipeptide antibody titers even with a weak adjuvant such as Alhydrogel or no adjuvant at all. The data suggest that the phage T4 hoc-soc system is an attractive system for display of peptides on an icosahedral capsid surface and may emerge as a powerful system for construction of the next generation multicomponent vaccines.
Chlamydia pneumoniae infection induces inflammatory changes in the aortas of rabbits
Authors: Laitinen K , Laurila A , Pyhälä L , Leinonen M , Saikku P
Published: January 2020

Chlamydia pneumoniae, a common human respiratory pathogen, has been associated with atherosclerosis in several seroepidemiological studies. Moreover, its presence in lesions of vessel walls has been demonstrated by culture, immunohistochemistry, PCR, and electron microscopy. In this study, we infected intranasally with C. pneumoniae New Zealand White rabbits which had been fed a normal diet. Reinfection was given 3 weeks later. Six of the nine reinfected animals showed inflammatory changes consisting of intimal thickening or fibroid plaques resembling atherosclerosis in 2 to 4 weeks after reinfection. One rabbit had calcified lesions. Immunohistochemistry for C. pneumoniae was strongly positive in the three older affected animals. No lesions were seen in the controls. The results suggest that C. pneumoniae infection is capable of inducing inflammatory atherosclerosis-like changes in the aortas of infected rabbits.
A gene homologous to Saccharomyces cerevisiae SNF1 appears to be essential for the viability of Candida albicans
Authors: Petter R , Chang Y C , Kwon-Chung K J
Published: January 2020

The SNF1 gene of Saccharomyces cerevisiae (ScSNF1) is essential for the derepression of catabolic repression. We report here the isolation and characterization of an SNF1 homolog from Candida albicans (CaSNF1) which is apparently essential for the viability of this organism. The putative amino acid sequence of CaSNF1 has 68% identity with that of ScSNF1 and can restore the S. cerevisiae snf1 delta mutant's ability to utilize sucrose. Disruption of one of the CaSNF1 alleles resulted in morphological changes and decreased growth rates but did not modify the carbon source utilization pattern. Repetitive unsuccessful attempts to generate a snf1/snf1 homozygote by disruption of the second allele, using various vectors and approaches, suggest the lethal nature of this mutation. Integration into the second allele was possible only when a full-length functional SNF1 sequence was reassembled, further supporting this hypothesis and indicating that the indispensability of Snf1p prevented the isolation of snf1/snf1 mutants. The mutant bearing two disrupted SNF1 alleles and the SNF1 functional sequence maintained its ability to utilize sucrose and produced stellate colonies with extensive hyphal growth on agar media. It was demonstrated that in a mouse model, the virulences of this mutant and the wild-type strain are similar, suggesting that hyphal growth in vitro is not an indicator for higher virulence.
Therapeutic intragastric vaccination against Helicobacter pylori in mice eradicates an otherwise chronic infection and confers protection against reinfection
Authors: Ghiara P , Rossi M , Marchetti M , Di Tommaso A , Vindigni C , Ciampolini F , Covacci A , Telford J L , De Magistris M T , Pizza M , Rappuoli R , Del Giudice G
Published: January 2020

Chronic infection of the gastroduodenal mucosae by the gram-negative spiral bacterium Helicobacter pylori is responsible for chronic active gastritis, peptic ulcers, and gastric cancers such as adenocarcinoma and low-grade gastric B-cell lymphoma. The success of eradication by antibiotic therapy is being rapidly hampered by the increasing occurrence of antibiotic-resistant strains. An attractive alternative approach to combat this infection is represented by the therapeutic use of vaccines. In the present work, we have exploited the mouse model of persistent infection by mouse-adapted H. pylori strains that we have developed to assess the feasibility of the therapeutic use of vaccines against infection. We report that an otherwise chronic H. pylori infection in mice can be successfully eradicated by intragastric vaccination with H. pylori antigens such as recombinant VacA and CagA, which were administered together with a genetically detoxified mutant of the heat-labile enterotoxin of Escherichia coli (referred to as LTK63), in which the serine in position 63 was replaced by a lysine. Moreover, we show that therapeutic vaccination confers efficacious protection against reinfection. These results represent strong evidence of the feasibility of therapeutic use of VacA- or CagA-based vaccine formulations against H. pylori infection in an animal model and give substantial preclinical support to the application of this kind of approach in human clinical trials.
Protection against Pneumocystis carinii pneumonia by antibodies generated from either T helper 1 or T helper 2 responses
Authors: Garvy B A , Wiley J A , Gigliotti F , Harmsen A G
Published: January 2020

To determine whether different antibody isotypes associated with T helper 1 (Th1) or Th2 responses are protective against Pneumocystis carinii, mice with disrupted interleukin 4 genes (IL-4(-/-) mice) or gamma interferon genes (IFN-gamma(-/-) mice) along with wild-type C57BL/6 mice were immunized intratracheally against P. carinii, depleted of T cells in vivo by use of monoclonal antibodies, and rechallenged intratracheally with 10(7) viable P. carinii organisms. Nearly all immunized mice resolved their lung P. carinii infections (limit of detection, log10 4.06) within 21 days of challenge even though they were depleted of T cells. Unimmunized mice depleted of T cells had significant lung infections (>log10 5.5) at day 21 post-P. carinii challenge. IFN-gamma(-/-) and wild-type mice developed P. carinii-specific immunoglobulin primarily of the immunoglobulin G1 (IgG1) subclass with relatively little P. carinii-specific IgG2a, IgG2b, or IgG3 in their sera, characteristic of a Th2-type response. In contrast, IL-4(-/-) mice had primarily an IgG2b P. carinii-specific antibody response in their sera with very little IgG1. Although IgG2b was the predominant isotype in IL-4(-/-) mice, optical density values of IgG2a and IgG3 were significantly higher in these mice (two and three times, respectively) than in IFN-gamma(-/-) mice, characteristic of a Th1-type response. Together, these data indicate that resolution of P. carinii infection can be mediated by specific antibody responses and that the antibody response can be either a predominantly Th1 or Th2 type. Furthermore, although wild-type mice mounted a Th2-like antibody response, IL-4(-/-) mice could resolve P. carinii pneumonia, indicating that resistance to P. carinii can occur in the absence of IL-4.
Prohibitin, a putative negative control element present in Pneumocystis carinii
Authors: Narasimhan S , Armstrong M , McClung J K , Richards F F , Spicer E K
Published: January 2020

Little is known about the molecules involved in the regulation of Pneumocystis carinii replication and development in vitro and in vivo. We describe in this report the identification of a P. carinii gene encoding the P. carinii prohibitin protein. In mammals, the prohibitin gene product has been shown to negatively regulate cell proliferation. A cDNA clone encoding the P. carinii prohibitin gene was isolated from a P. carinii cDNA library and identified on the basis of amino acid sequence homology with prohibitin from mammalian sources. Southern blot analysis confirmed that the prohibitin cDNA clone was of P. carinii origin. Western blot analysis of total P. carinii protein indicated that the prohibitin gene is transcribed and translated in vivo. The P. carinii prohibitin gene was expressed in vivo in human fibroblasts and shown to arrest the cell cycle in the G1 phase. The results obtained suggest a potential role for P. carinii prohibitin in the regulation of P. carinii proliferation and development.
Invasion of dentinal tubules by oral streptococci is associated with collagen recognition mediated by the antigen I/II family of polypeptides
Authors: Love R M , McMillan M D , Jenkinson H F
Published: January 2020

Cell surface proteins SspA and SspB in Streptococcus gordonii and SpaP in Streptococcus mutans are members of the antigen I/II family of polypeptides produced by oral streptococci. These proteins are adhesins and mediate species-specific binding of cells to a variety of host and bacterial receptors. Here we show that antigen I/II polypeptides are involved in the attachment of oral streptococci to collagen and that they also determine the ability of these bacteria to invade human root dentinal tubules. Wild-type S. gordonii DL1 (Challis) cells showed heavy invasion of tubules to a depth of approximately 200 microm, whereas the abilities of cells of isogenic mutant strains OB220 (sspA) and OB219 (sspA sspB) to invade were 50 and >90% reduced, respectively. Likewise, wild-type S. mutans NG8 cells invaded dentinal tubules, whereas cells of isogenic mutant strain 834 (spaP) did not. The invasive abilities of strains OB220 and OB219 were restored by heterologous expression of S. mutans SpaP polypeptide in these strains. The extents of tubule invasion by various wild-type and mutant strains correlated with their levels of adhesion to type I collagen, a major component of dentin. Furthermore, S. gordonii DL1 cells exhibited a growth response to collagen by forming long chains. This was not shown by ssp mutants but was restored by the expression of SpaP in these cells. The production of SspA polypeptide by S. gordonii DL1, but not production of SspB polypeptide by strain OB220 (sspA), was enhanced in the presence of collagen. These results are the first to demonstrate that antigen I/II family polypeptides bind collagen and mediate a morphological growth response of streptococci to collagen. These antigen I/II polypeptide activities are critical for intratubular growth of streptococci and thus for establishment of endodontic infections.
Mapping of antigenic determinant regions of the Bor56 protein of Orientia tsutsugamushi
Authors: Seong S Y , Park S G , Huh M S , Jang W J , Kim H R , Han T H , Choi M S , Chang W H , Kim I S
Published: January 2020

The 56-kDa protein (Bor56) of Orientia tsutsugamushi is an immunoprotective antigen and is the target molecule of neutralizing antibodies. This antigen is recognized by almost all of the serum antibodies produced by patients in the convalescence phase of scrub typhus. We expressed the Bor56 open reading frame in Escherichia coli and generated from it a series of deletion constructs as MalE fusion proteins. Antibody-binding domains were characterized by using patient sera, mouse monoclonal antibodies (MAbs), and Bor56-immunized-mouse sera. None of the antibodies bound to a fusion protein containing the carboxy-terminal 140 amino acids (aa) of the Bor56 protein, suggesting that the carboxy-terminal domain of Bor56 is not exposed on the surface of the molecule. Human immunoglobulin M (IgM) antibodies predominantly bound to antigenic domain I (AD I; amino acids [aa] 19 to 113) and AD III (aa 243 to 328). Human IgG antibodies also showed preferential binding to AD I. The epitope recognized by strain-specific MAb (KI4) or group-specific MAb (KI57) was mapped to AD II (aa 142 to 203). Mouse serum antibodies, elicited by immunization with deletion mutants, consistently bound to AD III. Moreover, the carboxy-terminal 140 aa of the Bor56 protein did not elicit an antibody response in C3H/HeDub mice. A model of the antigenic structure of Bor56 is presented and discussed. These results suggest that antigenic fragments from AD I and AD III are useful in the induction of humoral immunity against O. tsutsugamushi. These antigenic analyses provide an important foundation for further analyses of the neutralizing-antibody responses generated during rickettsial infections. They also provide potential peptide substrates for diagnostic assays and vaccine strategies.
The role of Legionella pneumophila-infected Hartmannella vermiformis as an infectious particle in a murine model of Legionnaire's disease
Authors: Brieland J K , Fantone J C , Remick D G , LeGendre M , McClain M , Engleberg N C
Published: January 2020

Legionella pneumophila is a bacterial parasite of many species of freshwater protozoa and occasionally an intracellular pathogen of humans. While protozoa are known to play a key role in the persistence of L. pneumophila in the environment, there has been limited research addressing the potential role of L. pneumophila-infected protozoa in the pathogenesis of human infection. In this report, the potential role of an L. pneumophila-infected amoeba as an infectious particle in replicative L. pneumophila lung infection was investigated in vivo with the amoeba Hartmannella vermiformis, a natural reservoir of L. pneumophila in the environment. L. pneumophila-infected H. vermiformis organisms were prepared by coculture of the amoebae and virulent L. pneumophila cells in vitro. A/J mice, which are susceptible to replicative L. pneumophila lung infection, were subsequently inoculated intratracheally with L. pneumophila-infected H. vermiformis organisms (10(6) amoebae containing 10(5) bacteria), and intrapulmonary growth of the bacteria was assessed. A/J mice inoculated intratracheally with L. pneumophila-infected H. vermiformis organisms developed replicative L. pneumophila lung infections. Furthermore, L. pneumophila-infected H. vermiformis organisms were more pathogenic than an equivalent number of bacteria or a coinoculum of L. pneumophila cells and uninfected amoebae. These results demonstrate that L. pneumophila-infected amoebae are infectious particles in replicative L. pneumophila infections in vivo and support the hypothesis that inhaled protozoa may serve as cofactors in the pathogenesis of pulmonary disease induced by inhaled respiratory pathogens.
A role for B cells in resistance to Cryptococcus neoformans in mice
Authors: Aguirre K M , Johnson L L
Published: January 2020

The role of B cells in immunity to Cryptococcus neoformans was investigated. Genetically targeted, B-cell-deficient mice (mu Mt) examined at various times after intravenous infection with C. neoformans 184 had lung and brain yeast burdens that were equivalent to tissue burdens in control B-cell-sufficient mice. Both B-cell-deficient and B-cell-sufficient control mice were effectively vaccinated by a sublethal intratracheal instillation of strain 184 yeast against a systemic infection with the C. neoformans strain carrying ura5; vaccinated control and vaccinated B-cell-deficient mice had equivalent brain and lung burdens of the ura5 strain 10 days after intravenous rechallenge. Additionally, B-cell-deficient and B-cell-sufficient vaccinated mice survived an intravenous rechallenge with a dose of yeast cells which is normally lethal for unimmunized mice. In further studies of the role of B cells in murine cryptococcosis, SCID mice were reconstituted with lymphocytes from B-cell-deficient and B-cell-sufficient mice. SCID mice reconstituted with lymphocytes from vaccinated B-cell-deficient animals failed to express effective adoptive immunity to C. neoformans brain infection. In contrast, SCID mice reconstituted with lymphocytes from vaccinated B-cell-sufficient mice had 10-fold fewer yeast cells in their brains than did uninfused SCID controls. However, SCID mice given lymphocytes from B-cell-deficient immune donors had fewer yeast cells in their lungs than did uninfused controls. Fewer CD4+ lymphocytes were recovered at 7 and 11 days after infection from the peripheral blood and spleens of SCID mice reconstituted with lymphocyte suspensions from B-cell-deficient animals than from the peripheral blood and spleens of SCID mice reconstituted with suspensions from B-cell-sufficient control donors. These data suggest that B cells can play an important role in host defense against Cryptococcus in the brain under conditions in which T-cell-mediated immunity is impaired.