bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasmodium falciparum gametocyte adhesion to C32 cells via CD36 is inhibited by antibodies to modified band 3
Authors: Rogers N J , Targett G A , Hall B S
Published: January 2020

Plasmodium falciparum gametocyte-infected erythrocytes are characterized by their ability to sequester in the microvasculature of various organs, primarily the spleen and bone marrow. This phenomenon is thought to play a critical role in the development and survival of the sexual stages. Little is known, however, about ligands on the gametocyte-infected erythrocyte. Infection of erythrocytes with mature asexual stages of P. falciparum (trophozoites and schizonts) has been shown to induce modification of the erythrocyte anion transporter, band 3, and this has been linked to the acquisition of an adherent phenotype. Here, we demonstrate for the first time that immature gametocyte-infected erythrocytes also express modified band 3. In vitro binding assays demonstrate that gametocyte-infected erythrocytes of the 3D7 strain utilize this surface receptor for adhesion to C32 amelanotic melanoma cells via the host cell receptor CD36 (platelet glycoprotein IIIb). Adhesion of gametocyte-infected erythrocytes to CD36-transfected CHO cells is also dependent on modified band 3. However, modified band 3 does not mediate adhesion of gametocyte-infected erythrocytes to intercellular adhesion molecule 1, a second host receptor for gametocytes expressed on C32 cells.
Subcellular localization and functional characterization of Nc-p43, a major Neospora caninum tachyzoite surface protein
Authors: Hemphill A
Published: January 2020

Neospora caninum is a recently identified coccidian parasite which shares many features with, but is clearly distinct from, Toxoplasma gondii. N. caninum tachyzoites infect a wide range of mammalian cells both in vivo and in vitro. The mechanisms by which infection is achieved are largely unknown. Recent evidence has suggested that a receptor-ligand system in which one or several host cell receptors bind to one or several parasite ligands is involved. Parasite cell surface-associated molecules such as the recently identified Nc-p43 antigen are prime suspects for being implicated in this physical interaction. In this study it is shown that invasion of Vero cell monolayers by N. caninum tachyzoites in vitro is impaired on incubation of parasites with subagglutinating amounts of affinity-purified antibodies directed against Nc-p43. Postembedding immunogold labeling with anti-Nc-p43 antibodies demonstrated that Nc-p43 is localized not only on the parasite cell surface but also within dense granules and rhoptries. The fate of Nc-p43 during intracellular proliferation of N. caninum tachyzoites and subsequent maturation of the parasitophorous vacuole was also studied.
Mutants of Streptococcus suis types 1 and 2 impaired in expression of muramidase-released protein and extracellular protein induce disease in newborn germfree pigs
Authors: Smith H E , Vecht U , Wisselink H J , Stockhofe-Zurwieden N , Biermann Y , Smits M A
Published: January 2020

The contribution of muramidase-released protein (MRP) and extracellular factor (EF) to the virulence of Streptococcus suis type 1 and 2 infections was studied. For that aim, we constructed mutants of S. suis types 1 and 2 by inactivating the genes encoding MRP and EF. Moreover, we changed a type 2 strain producing the 110-kDa EF protein into a strain producing a modified protein (EF*) of increased molecular mass. The chromosomally located mrp and epf genes were inactivated by replacement recombination by using nonreplicative plasmids. Newborn germfree pigs were inoculated with pathogenic type 1 and 2 strains and with the isogenic mutant strains. Wild-type as well as mutant strains induced fever, specific signs of disease, and lesions. Moreover, all mutant strains could be reisolated from the central nervous system of infected pigs. These results showed that inactivation or alteration of the mrp and epj genes had no measurable effect on the pathogenicity of S. suis types 1 and 2.
CD14-mediated signal pathway of Porphyromonas gingivalis lipopolysaccharide in human gingival fibroblasts
Authors: Watanabe A , Takeshita A , Kitano S , Hanazawa S
Published: January 2020

Lipopolysaccharide (LPS) induces expression of inflammatory cytokines in monocytes/macrophages via CD14, one of the LPS receptors, which is expressed predominantly in these cells. It has been demonstrated that Porphyromonas gingivalis LPS (P-LPS) also is able to induce inflammatory cytokines in human gingival fibroblasts. Therefore, it is important to determine whether CD14 is expressed in gingival fibroblasts and to define the P-LPS-mediated signal-transducing mechanism in the cells. In this study, we observed unexpectedly by immunohistochemical, Western blotting (immunoblotting), and Northern (RNA) blotting assays that CD14 is expressed at high density in human gingival fibroblasts. P-LPS-induced expression of the monocyte chemoattractant protein 1 (MCP-1) gene in the cells was inhibited markedly by treatment with anti-human CD14 antibody and was completely inhibited by herbimycin A, a potent inhibitor of tyrosine kinase. The inhibitor also dramatically inhibited monocyte chemotactic activity of and MCP-1 production by the cells. Furthermore, P-LPS-induced expression of the MCP-1 gene in the cells also was blocked by inhibitors of two transcription factors, i.e., curcumin, an inhibitor of AP-1, and pyrolidine dithiocarbamate, an inhibitor of NF-kappaB. Both inhibitors inhibited monocyte chemotactic activity in the culture supernatant of P-LPS-treated cells. Gel shift mobility assay showed stimulation of the AP-1 and NF-kappaB contents in P-LPS-treated cells. This study is the first to demonstrate the expression of CD14 in human gingival fibroblasts and to show that the signal-transducing pathway of P-LPS in the cells is mediated by CD14.
Pneumocystis carinii pneumonia in scid mice induced by viable organisms propagated in vitro
Authors: Beck J M , Newbury R L , Palmer B E
Published: January 2020

Pneumocystis carinii pneumonia remains a major cause of morbidity and mortality in human immunodeficiency virus-infected individuals, despite the widespread use of prophylaxis and the development of new chemotherapeutic agents. The study of P. carinii and of pulmonary host defenses directed against it has been limited by lack of reliable, reproducible methods to obtain pure populations of organisms in useful quantities. While recent success has been achieved with cultures of rat P. carinii organisms, cultures of mouse P. carinii organisms have not been successful. Experiments were performed to determine whether P. carinii organisms derived from mice could be propagated in vitro. Mouse P. carinii organisms, obtained from the lungs of chronically infected athymic mice, were inoculated into spinner flasks containing HEL299 feeder cells seated on microcarrier beads. The numbers of mouse P. carinii organisms increased significantly over 7 days in culture. To test the viability and pathogenicity of these cultured organisms, P. carinii organisms were harvested after 7 days of culture and were inoculated intratracheally into susceptible scid mice. Four weeks after inoculation, scid mice developed uniformly severe P. carinii pneumonia. These studies demonstrate for the first time that mouse P. carinii organisms can be propagated in vitro. Furthermore, cultured mouse P. carinii organisms maintain their pathogenicity in an in vivo model system. This culture system will have important applications in the study of P. carinii biology and in the study of host defenses directed against this important opportunistic pathogen.
Analysis of complement C3 deposition and degradation on Klebsiella pneumoniae
Authors: Albertí S , Alvarez D , Merino S , Casado M T , Vivanco F , Tomás J M , Benedí V J
Published: January 2020

The majority of Klebsiella pneumoniae serum-resistant strains activate complement and bind C3b, the opsonic fragment of C3, without C5b-9 formation and bacterial killing. The mechanisms leading to C3b deposition without cell death were studied, and the results indicate that serum-resistant strains activate principally the alternative pathway and that serum-sensitive strains activate both the alternative and classical pathways. Bacterial molecules implicated in C3b deposition are the outer membrane porin proteins and smooth and rough lipopolysaccharides. Porins activate both complement pathways, and the rough lipopolysaccharide activates the classical pathway, causing deposition of C3b in serum-sensitive strains. The smooth lipopolysaccharide of serum-resistant strains activates only the alternative pathway, impeding the binding of C1q to porins (S. Albertí, G. Marqués, S. Camprubí, S. Merino, J. M. Tomás, F. Vivanco, and V. J. Benedí, Infect. Immun. 61:852-860, 1993; S. Albertí, F. Rodríguez-Quinónes, T. Schirmer, G. Rummel, J. M. Tomás, J. P. Rosenbusch, and V. J. Benedí, Infect. Immun. 63:903-910, 1995) and rough lipopolysaccharide molecules and thereby preventing activation of the classical pathway. After its deposition, C3b is quickly degraded to iC3b on both types of strains, but the higher-level deposition of C3b on serum-sensitive strains, resulting from activation of both the alternative and classical complement pathways, supports further complement activation and killing of serum-sensitive strains.
Enteropathogenic Escherichia coli markedly decreases the resting membrane potential of Caco-2 and HeLa human epithelial cells
Authors: Stein M A , Mathers D A , Yan H , Baimbridge K G , Finlay B B
Published: January 2020

It is presumed, but not proven, that enteropathogenic Escherichia coli (EPEC) causes secretory diarrhea by altering ion transport in enterocytes. In this study we used the whole-cell, current clamp variant of the patch clamp technique to demonstrate that EPEC infection of HeLa and Caco-2 human epithelial cells reduces cell resting membrane potential. The observed reduction of resting membrane potential in HeLa cells results from EPEC-mediated signal transduction to the host cell but is not dependent upon EPEC-mediated elevation of levels of intracellular free calcium. These findings indicate that EPEC can directly alter the relative distribution of ions across epithelial host cell membranes. This may be relevant to the etiology of diarrhea caused by EPEC infection.
Role of neutrophils in controlling early stages of a Chlamydia trachomatis infection
Authors: Barteneva N , Theodor I , Peterson E M , de la Maza L M
Published: January 2020

Inoculation of mice with the granulocyte-depleting monoclonal antibody RB6-8C5 showed that neutrophils are critical protective effector cells during a Chlamydia trachomatis infection. In addition, administration of monoclonal antibody 2E6 demonstrated that extravasation of neutrophils into the peritoneal cavity in response to inoculation with the C. trachomatis MoPn biovar is dependent on the surface beta-2 integrin molecule CD18.
Helicobacter pylori cytotoxin induces vacuolation of primary human mucosal epithelial cells
Authors: Harris P R , Cover T L , Crowe D R , Orenstein J M , Graham M F , Blaser M J , Smith P D
Published: January 2020

We investigated whether Helicobacter pylori cytotoxin induces vacuolation in primary epithelial cells from normal human mucosa. Epithelial cells purified by enzyme digestion and elutriation were evaluated for vacuolation in a blinded protocol by light and electron microscopy before and after incubation with culture supernatant (CS) from H. pylori 60190, which has vacuolating activity for HeLa cells (Tox+), and isogenic H. pylori mutant 60190-v1, which lacks this activity (Tox-). Primary epithelial cells (>98% pure) exposed to CS from Tox+ H. pylori exhibited marked vacuolation (52% +/- 5% of cells) compared with epithelial cells exposed to either CS from Tox- H. pylori (23% +/- 3.2%) or uninoculated control broth (23% +/- 3.7%) (P < 0.05) by light microscopy, which was confirmed by electron microscopy and antibody inhibition studies. These are the first data to show that H. pylori cytotoxin causes vacuolation of primary human mucosal epithelial cells.
Schistosoma mansoni infection in mice augments the capacity for interleukin 3 (IL-3) and IL-9 production and concurrently enlarges progenitor pools for mast cells and granulocytes-macrophages
Authors: Khalil R M , Luz A , Mailhammer R , Moeller J , Mohamed A A , Omran S , Dörmer P , Hültner L
Published: January 2020

Mast cells and granulocytes-macrophages (GM) are components of the host defense system against worm infections, including schistosomiasis. Here we report the kinetics of changes in the number of colony-forming cells (CFC) for mast cells and GM during the course of a primary experimental infection of mice with Schistosoma mansoni cercariae over a period of 24 weeks postinfection (p.i.). Concurrently, we measured known myelopoietic and/or mast cell-stimulating cytokines (i.e., interleukin 3 [IL-3] and IL-9) in pokeweed mitogen-activated spleen cell-conditioned medium. Our results show that during the acute phase of the hepatic granulomatous reaction, the numbers of both mast-CFC and GM-CFC were significantly elevated in bone marrow. However, while femoral GM-CFC numbers had returned to normal control values at week 16 p.i., femoral and splenic mast-CFC numbers remained significantly elevated until week 20 p.i., which corresponds to the chronic fibrotic phase of hepatic granulomatous inflammation. Increased GM-CFC numbers correlated with elevated IL-3 levels, while increased mast-CFC numbers paralleled the increased IL-9 concentrations in spleen cell-conditioned medium. By the reverse transcription-PCR method, enhanced expression of IL-3 and IL-9 transcripts was found in RNA samples obtained from livers and spleens of infected mice. Our data demonstrate that during the course of infection of mice with S. mansoni, the coordinate need for mast cells and GM is at least partly regulated at the stage of progenitor cell commitment in the bone marrow and spleen. It appears that IL-3 and IL-9 help to promote at this stage the ultimate generation of mature effector cells.
Candidacidal activity of recombinant human salivary histatin-5 and variants
Authors: Tsai H , Raj P A , Bobek L A
Published: January 2020

Human salivary histatins possess fungicidal and bactericidal activities. The current investigation evaluates the structure-function relationship of histatins with regard to their candidacidal activity by using recombinant histatin-5 and its variants produced in Escherichia coli. The purified recombinant histatins were examined for their candidacidal activity and secondary structure. The m21 (with Lys-13 replaced by Thr [Lys-13-->Thr]) and m71 (Lys-13-->Glu) variants are significantly less effective than recombinant histatin-5 in killing Candida albicans, suggesting that Lys-13 is critical for candidacidal activity. The m68 (Lys-13-->Glu and Arg-22-->Gly) variant is significantly less potent than the recombinant histatin-5 as well as m71, indicating that Arg-22 is crucial for the cidal activity. The candidacidal activities of m1 (Arg-12-->Ile), m2 (Arg-12-->Ile and Lys-17-->Asp), m12 (Arg-12-->Lys and His-21-->Leu), and m70 (His-19-->Pro and His-21-->Arg) variants, however, are comparable to that of recombinant histatin-5, indicating that Arg-12, Lys-17, His-19, and His-21 are not functionally important. The conformational preferences of histatin-5 and variants were determined by circular dichroism. The results indicate that all proteins have a strong tendency to adopt alpha-helical conformation in trifluoroethanol. Previously, we have shown that the alpha-helical conformation is one of the important structural requirements for eliciting appreciable candidacidal activity. Collectively, the data suggest that in addition to the helical conformation, specific residues such as Lys-13 and Arg-22 in the sequence of histatin-5 are, indeed, important for candidacidal activity.
Characterization of the fim2 and fim3 fimbrial subunit genes of Bordetella bronchiseptica: roles of Fim2 and Fim3 fimbriae and flagella in adhesion
Authors: Savelkoul P H , de Kerf D P , Willems R J , Mooi F R , van der Zeijst B A , Gaastra W
Published: January 2020

With DNA probes derived from the fimbrial subunit genes fim2 and fim3 of Bordetella pertussis, two homologous subunit genes of Bordetella bronchiseptica were identified and cloned. The nucleotide sequences of these genes were determined. Comparison of these nucleotide sequences with the B. pertussis fimbrial fim2 and fim3 subunit genes showed a pronounced homology. Therefore, the B. bronchiseptica genes were also designated fim2 and fim3. Expression of the two B. bronchiseptica genes was demonstrated by Western blotting (immunoblotting) with polyclonal antiserum directed against the Fim2 and Fim3 fimbrial subunit proteins of B. pertussis and by enzyme-linked immunosorbent assay with monoclonal antibodies. After growth of B. bronchiseptica in the presence of MgSO4, no expression of both fimbrial subunit genes was observed. While no fimbriae were expressed, expression of flagella was observed under these circumstances. A longer C-stretch (up to 19 cytosine residues) than the one in front of the fim2 and fim3 genes of B. pertussis is present in front of the B. bronchiseptica fimbrial genes. In adherence experiments, fimbriated (Bvg+) as well as flagellated (Bvg-) B. bronchiseptica bacteria were able to adhere to HeLa cells, whereas nonflagellated B. pertussis did not. This suggests that fimbriae as well as other factors (possibly flagella) contribute to adherence of B. bronchiseptica to eukaryotic cells.
Role of phagocytosis in activation of the coagulation system in Streptococcus sanguis endocarditis
Authors: Bancsi M J , Veltrop M H , Bertina R M , Thompson J
Published: January 2020

The formation of vegetations consisting of fibrin, cellular elements, humoral factors, and bacteria is the central event in the pathogenesis of bacterial endocarditis. Fibrin formation occurs on the vegetation, the coagulation system being activated locally via the expression of tissue factor (TF) on fibrin-adherent monocytes. This study was performed to assess the importance of phagocytosis of fibrin-adherent Streptococcus sanguis in the stimulation of TF expression on fibrin-adherent monocytes, as well as a role for "frustrated" phagocytosis. With the latter process, these cells are unable to remove bacteria from the fibrin surface but nonetheless might be activated to generate TF. We found that serum was not required for the stimulation of TF expression by fibrin-adherent monocytes in the presence of S. sanguis in an in vitro model for bacterial endocarditis. The bacterial adhesin dextran did not influence the TF activity (TFA) of fibrin-adherent monocytes: TFA was the same after stimulation with a dextran-positive streptococcus as with its dextran-negative mutant. Furthermore, dextran did not influence the TFA of endocardial vegetations, which was the same for vegetations isolated from rabbits infected either with dextran-positive S. sanguis or its dextran-negative mutant. These results do not support the hypothesis that in bacterial endocarditis (frustrated) phagocytosis significantly contributes to TF expression on vegetation-adherent monocytes. Fibronectin, however, although not influencing the fibrin binding of the streptococci, did enhance the TFA of monocytes in a concentration-dependent manner. We conclude that although streptococci do enhance expression of TFA on monocytes, phagocytosis and bacterial adhesins do not play a major role in this process. Stimulation of monocyte TFA may be more dependent on interactions between monocytes and the vegetational surface via fibronectin receptors, such as VLA 4 and VLA 5 (very late antigens 4 and 5).
Tumor necrosis factor mediates lung antibacterial host defense in murine Klebsiella pneumonia
Authors: Laichalk L L , Kunkel S L , Strieter R M , Danforth J M , Bailie M B , Standiford T J
Published: January 2020

Tumor necrosis factor (TNF) is a proinflammatory cytokine which has recently been shown to have beneficial effects in the setting of acquired host immunity. However, the role of TNF in innate immune responses, as in the setting of bacterial pneumonia, has been incompletely characterized. To determine the role of TNF in gram-negative bacterial pneumonia, CBA/J mice were challenged with 10(2) CFU of Klebsiella pneumoniae intratracheally, resulting in the time-dependent expression of TNF MRNA and protein within the lung. Passive immunization of animals with a soluble TNF receptor-immunoglobulin (Ig) construct (sTNFR:Fc) intraperitoneally 2 h prior to K. pneumoniae inoculation resulted in a significant reduction in bronchoalveolar lavage neutrophils, but not macrophages, at 48 h, as compared with animals receiving control IgG1. Furthermore, treatment with sTNFR:Fc resulted in 19.6- and 13.5-fold increases in K. pneumoniae CFU in lung homogenates and plasma, respectively, as compared with animals receiving control IgG1. Finally, treatment of Klebsiella-infected mice with sTNFR:Fc markedly decreased both short- and long-term survival of these animals. In conclusion, our studies indicate that endogenous TNF is a critical component of antibacterial host defense in murine Klebsiella pneumonia.
Binding of extracellular matrix proteins to Aspergillus fumigatus conidia
Authors: Gil M L , Peñalver M C , Lopez-Ribot J L , O'Connor J E , Martinez J P
Published: January 2020

As detected by confocal immunofluorescence microscopy, binding of fibronectin and laminin appeared to be associated with the protrusions present on the outer cell wall layer of resting Aspergillus fumigatus conidia. Flow cytometry confirmed that binding of laminin to conidia was dose dependent and saturable. Laminin binding was virtually eliminated in trypsin-treated organisms, thus suggesting the protein nature of the binding site. Conidia were also able to specifically adhere to laminin immobilized on microtiter plates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) with laminin and antilaminin antibody of whole conidial homogenates allowed identification, among the complex array of protein and glycoprotein species, of one polypeptide with an apparent molecular mass of 37 kDa which specifically interacts with laminin. The fact that binding of conidia to soluble or immobilized laminin or fibronectin was inhibited by fibronectin or laminin, respectively, suggests the existence of common binding sites for both ligands on the surface of conidia. Intact conidia were also able to adhere to type I and IV collagen immobilized on microtiter plates; adhesion was found to be dose dependent and saturable. Adhesion to immobilized type I and IV collagen was markedly inhibited by laminin and weakly inhibited by fibronectin. Coincubation of conidia with Arg-Gly-Asp (RGD) peptides caused a dose-dependent decrease in binding of cells to immobilized or soluble fibronectin, yet interaction of cells with soluble or immobilized laminin and type I and IV collagen remained unaffected. Interactions described here could be important in mediating attachment of the fungus to host tissues, thus playing a role in the establishment of the disease.
Sequence heterogeneity of PsaA, a 37-kilodalton putative adhesin essential for virulence of Streptococcus pneumoniae
Authors: Berry A M , Paton J C
Published: January 2020

psaA encodes a 37-kDa putative pneumococcal surface adhesin. Although its complete nucleotide sequence has been determined, its contribution to the pathogenicity of Streptococcus pneumoniae has not previously been assessed. In this study, we used a PCR-amplified internal fragment of the psaA gene from S. pneumoniae type 2 strain D39 cloned in pVA891, to direct the construction of D39 derivatives in which the psaA gene had been specifically interrupted, by insertion-duplication mutagenesis. Two independent D39 psaA mutants (PsaA-(1) and PsaA-(2)) were significantly less virulent (as judged by intranasal or intraperitoneal challenge of mice) than either the wild-type D39 strain or a derivative of PsaA-(1) in which the psaA gene had been reconstituted by back-transformation with an intact copy of the cloned gene. pVA891-directed mutagenesis of an open reading frame (designated ORF3) immediately 3' to psaA or insertion of pVA891 between psaA and ORF3 had no impact on intranasal virulence. However, a small but significant difference in virulence was observed between these two derivatives and the parental D39 strain in a low-dose intraperitoneal challenge model, suggesting that the ORF3 product may also contribute to pathogenesis. Adherence of PsaA-(1) to A549 cells (type II pneumocytes) was only 9% of that for D39, while the ORF3-negative strain exhibited intermediate adherence (23%). This is the first functional evidence that PsaA is an adhesin. Sequence analysis of the psaA gene from D39 indicated significant deviation from that previously published for the homolog from S. pneumoniae R36A. The deduced amino acid sequences of mature PsaA from the two strains had only 81% homology, with the bulk of the variation occurring in the amino-terminal portion.
Outer membrane protein of Neisseria meningitidis as a mucosal adjuvant for lipopolysaccharide of Brucella melitensis in mouse and guinea pig intranasal immunization models
Authors: Van De Verg L L , Hartman A B , Bhattacharjee A K , Tall B D , Yuan L , Sasala K , Hadfield T L , Zollinger W D , Hoover D L , Warren R L
Published: January 2020

A mucosal vaccine against brucellosis consisting of the lipopolysaccharide (LPS) of Brucella melitensis complexed with the outer membrane protein (GBOMP) of group B Neisseria meningitidis was tested in small-animal models of intranasal immunization. Mice given two doses of the vaccine developed high levels of immunoglobulin G (IgG) and IgA antibodies specific for B. melitensis LPS in lung lavages and specific IgG and IgA antibody-secreting cells in the lungs and spleen. Similarly, in guinea pigs immunized twice intranasally, IgG and IgA LPS-specific antibodies were detected in lung lavages, and specific antibody-secreting cells were isolated from the spleen and cervical nodes. In mice immunized with LPS only, pulmonary responses consisted mostly of IgM antibodies, while guinea pigs given LPS alone developed local antibody of all three isotypes, but at lower levels compared to animals given the complex vaccine. Both mice and guinea pigs also developed high levels of serum IgG and moderate levels of IgA as a result of intranasal immunization with the complex vaccine. The serum antibodies in both cases were found to cross-react with the LPS of B. abortus, which shares an immunogenic epitope with B. melitensis LPS. In mice given the complex vaccine, there was a prominent serum IgG1 response that was absent in the mice given LPS alone. In conclusion, the N. meningitidis GBOMP was an effective mucosal adjuvant for secretory IgA and IgG responses in the lungs of both mice and guinea pigs. The IgG1 subclass response in mice suggests that GBOMP may have favored a Th2 type of response to the LPS. A vaccine capable of stimulating high levels of antibody at local sites has the potential to protect against brucellae, since these pathogens gain entry to the host via mucosal routes.
Construction of a flagellum-negative mutant of Proteus mirabilis: effect on internalization by human renal epithelial cells and virulence in a mouse model of ascending urinary tract infection
Authors: Mobley H L , Belas R , Lockatell V , Chippendale G , Trifillis A L , Johnson D E , Warren J W
Published: January 2020

To examine the role of flagella in pathogenesis of urinary tract infection caused by Proteus mirabilis, we constructed a nonmotile, nonswarming flagellum mutant of strain WPM111 (an hpmA hemolysin mutant of strain BA6163, chosen because of its lack of in vitro cytotoxicity in renal epithelial cell internalization studies). A nonpolar mutation was introduced into the flaD gene, which encodes the flagellar cap protein. This mutation does not affect the synthesis of flagellin but rather prevents the assembly of an intact flagellar filament. In in vitro assays, the genetically characterized nonmotile mutant was found to be internalized by cultured human renal proximal tubular epithelial cells in numbers less than 1% of those of the flagellated parent strain. Internalization of the nonmotile mutant was increased significantly (14- to 21-fold) by centrifugation onto the monolayer. To assess virulence in vivo, CBA mice were challenged transurethrally with 10(7) CFU of P. mirabilis BA6163 (wild type) (n = 16), WPM111 (hpmA mutant) (n = 46), or BB2401 (hmpA flaD mutant) (n = 46). Differences in quantitative cultures between the parent strain and the hemolysin-negative mutant were not significant. However, the hpmA flaD mutant was recovered in numbers approximately 100-fold lower than those of the hmpA mutant or the wild-type parent strain and thus was clearly attenuated. We conclude that while hemolysin does not significantly influence virulence, flagella contribute significantly to the ability of P. mirabilis to colonize the urinary tract and cause acute pyelonephritis in an experimental model of ascending urinary tract infection.
Intranasal immunization induces long-term protection in mice against a Chlamydia trachomatis genital challenge
Authors: Pal S , Peterson E M , de la Maza L M
Published: January 2020

In an attempt to confer long-term protective immunity, BALB/c female mice were immunized intranasally with 10(4) inclusion-forming units (IFU) of the Chlamydia trachomatis mouse pneumonitis biovar (MoPn). Animals were subsequently challenged in the ovarian bursa with 10(5) C. trachomatis MoPn IFU at 60, 120, or 180 days post-intranasal immunization. Two control groups were included in the study. One control was sham immunized and mock challenged, and another group was sham immunized and challenged with 10(5) C. trachomatis MoPn IFU. Vaginal cultures were collected at regular intervals following the intrabursal challenge. In comparison with the sham-immunized mice, the animals that were intranasally immunized with C. trachomatis had significant protection, as shown by a reduction in the number of animals that had positive vaginal cultures and by a decrease in the intensity and length of the shedding. Furthermore, histopathological characterization of the genital tract following challenge, in the three groups of mice, showed a minimal inflammatory infiltrate in the C. trachomatis-immunized animals, when compared with the sham-immunized control group. Subsequently, the three groups of female mice that were challenged at 60, 120 and 180 days postimmunization were mated at 6 weeks following the challenge. Overall, in the mice intranasally immunized with C. trachomatis the fertility rates and the number of embryos were similar to those in the sham-immunized and mock-challenged group. In contrast, there was a significant increase in infertility in the groups of mice that were sham immunized and C. trachomatis challenged. In conclusion, intranasal immunization with C. trachomatis induces long-term protection against a genital challenge as shown by a decrease in the infection and infertility rates when compared with sham-immunized animals. Thus, this model may help to characterize the parameters of the immune response that are important in maintaining long-term protection and may aid in identifying the antigenic determinants involved in eliciting protection.
Distribution of pathogenicity islands in Salmonella spp
Authors: Ochman H , Groisman E A
Published: January 2020

We investigated the phylogenetic distribution of the SPI-1 and SPI-2 pathogenicity islands in Salmonella spp. SPI-1 was present in representatives of all eight subspecific groups, but no SPI-2-hybridizing sequences were detected in group V (S. bongori). Our data suggest that SPI-2 was acquired by S. enterica after its split from S. bongori.
Acquisition of iron from host proteins by the group A streptococcus
Authors: Eichenbaum Z , Muller E , Morse S A , Scott J R
Published: January 2020

To identify mammalian iron-binding proteins that can serve as iron sources for Streptococcus pyogenes, the group A streptococcus (GAS), we used a plate assay. Ferritin, hemin, hemoglobin, myoglobin, and catalase can support growth of GAS on iron-depleted medium. However, growth was not detected when iron was provided as iron-saturated transferrin or lactoferrin or bound to cytochrome c. Therefore, it appears that GAS can use the intracellular iron sources available in the human body, which is consistent with its ability to cause tissue destruction during infection.
Antibody responses to Brucella abortus 2308 in cattle vaccinated with B. abortus RB51
Authors: Stevens M G , Olsen S C
Published: January 2020

Cattle vaccinated with Brucella abortus rough strain RB51 (SRB51) produced small amounts of serum immunoglobulin G (IgG) but no IgM antibody to smooth strain 2308 (S2308) bacteria and produced no IgG or IgM antibody to S2308 lipopolysaccharide (LPS). Western immunoblot analysis revealed that antiserum from SRB51-vaccinated cattle contained IgG antibody that reacted with S2308 proteins of 84 to <20 kDa. However, antiserum from the vaccinated cattle did not contain agglutinating B. abortus antibody in the tube agglutination test for brucellosis. These results suggest that SRB51-vaccinated cattle produced no antibody to S2308 LPS, although they did produce nonagglutinating IgG antibody that reacted with S2308 bacteria and bacterial proteins of 84 to <20 kDa.
Cysteine protease of Porphyromonas gingivalis 381 enhances binding of fimbriae to cultured human fibroblasts and matrix proteins
Authors: Kontani M , Ono H , Shibata H , Okamura Y , Tanaka T , Fujiwara T , Kimura S , Hamada S
Published: January 2020

It has been shown that Porphyromonas gingivalis 381, a suspected periodontopathogen, possesses fimbriae on its cell surface. The organism is also known to produce proteases which can degrade the host cell surface matrix proteins. In this study, we investigated the effect of protease on the binding of the purified P. gingivalis fimbriae to cultured fibroblasts or matrix proteins. A protease that can hydrolyze benzoyl-L-arginine p-nitroanilide was obtained from P. gingivalis 381 cells by sonication in phosphate-buffered 0.2% Triton X-100 and was purified by column chromatography. The molecular size of the protease was estimated to be 55 kDa by gel filtration or 47 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The enzyme activity was markedly inhibited by sulfhydryl reagents, antipain, and leupeptin. The protease degraded various host proteins, including collagen and fibronectin, and cleaved the COOH terminus of the arginine residue in peptides such as benzoyl-L-arginine p-nitroanilide. However, P. gingivalis fimbriae were not degraded by protease activity. The enzyme activity was enhanced in the presence of reducing agents or CaCl2. When cultured fibroblasts were partially treated with the protease, the binding of the purified P. gingivalis fimbriae to the fibroblast monolayer was increased significantly. However, this enhancing effect was suppressed upon the addition of antipain and leupeptin. Similarly, binding of the fimbriae to the collagen or fibronectin immobilized on the microtiter wells was also enhanced. Addition of these host matrix proteins efficiently inhibited the binding of fimbriae to the fibroblast monolayer. The binding assay of fimbriae using dipeptidyl ligand affinity column chromatography demonstrated a clear interaction between fimbriae and the arginine residue. Taken together, these results indicate that the P. gingivalis protease at least partially degrades the host matrix proteins, which, in turn, may lead to an increased exposure of the cryptic ligands that can result in enhanced fimbria-mediated binding of this organism to periodontal tissues.
Blood group glycolipids as epithelial cell receptors for Candida albicans
Authors: Cameron B J , Douglas L J
Published: January 2020

The role of glycosphingolipids as possible epithelial cell receptors for Candida albicans was examined by investigating the binding of biotinylated yeasts to lipids extracted from human buccal epithelial cells and separated on thin-layer chromatograms. Binding was visualized by the addition of 125I-streptavidin followed by autoradiography. Five C. albicans strains thought from earlier work to have a requirement for fucose-containing receptors all bound to the same three components in the lipid extract. A parallel chromatogram overlaid with biotinylated Ulex europaeus lectin, which is a fucose-binding lectin with a specificity for the H blood group antigen, showed that two of these glycosphingolipids carried this antigenic determinant. Preparations of crude and purified adhesin (a protein with a size of 15.7 kDa which lacked cysteine residues) from one of the strains also bound to these same two components. The third glycosphingolipid, which bound whole cells but neither preparation of adhesin, was recognized by Helix pomatia lectin, indicating that it contained N-acetylgalactosamine, possibly in the form of the A blood group antigen. Overlay assays with a sixth strain of C. albicans (GDH 2023) revealed a completely different binding pattern of four receptors, each of which contained N-acetylglucosamine. These results confirm earlier predictions about the receptor specificity of the strains made on the basis of adhesion inhibition studies and indicate that blood group antigens can act as epithelial cell receptors for C. albicans.
Effects of interleukin-10 on human peripheral blood mononuclear cell responses to Cryptococcus neoformans, Candida albicans, and lipopolysaccharide
Authors: Levitz S M , Tabuni A , Nong S H , Golenbock D T
Published: January 2020

Deactivation of mononuclear phagocytes is critical to limit the inflammatory response but can be detrimental in the face of progressive infection. We compared the effects of the deactivating cytokine interleukin 10 (IL-10) on human peripheral blood mononuclear cell (PBMC) responses to lipopolysaccharide (LPS), Cryptococcus neoformans, and Candida albicans. IL-10 effected dose-dependent inhibition of tumor necrosis factor alpha (TNF-alpha) release in PBMC stimulated by LPS and C. neoformans, with significant inhibition seen with 0.1 U/ml and greater than 90% inhibition noted with 10 U/ml. In contrast, even at doses as high as 100 U/ml, IL-10 inhibited TNF-alpha release in response to C. albicans by only 50%. IL-10 profoundly inhibited release of IL-1beta from PBMC stimulated by all three stimuli. TNF-alpha mRNA and release was inhibited even if IL-10 was added up to 8 h after cryptococcal stimulation. In contrast, inhibition of IL-1 beta mRNA was of lesser magnitude and occurred only when IL-10 was added within 2 h of cryptococcal stimulation. IL-10 inhibited translocation of NF-kappaB in response to LPS but not the fungal stimuli. All three stimuli induced IL-10 production in PBMC, although over 10-fold less IL-10 was released in response to C. neoformans compared with LPS and C. albicans. Thus, while IL-10 has deactivating effects on PBMC responses to all three stimuli, disparate stimulus- and response-specific patterns of deactivation are seen. Inhibition by IL-10 of proinflammatory cytokine release appears to occur at the level of gene transcription for TNF-alpha and both transcriptionally and posttranscriptionally for IL-1beta.
Reactivity of mouse T-cell hybridomas expressing human Vbeta gene segments with staphylococcal and streptococcal superantigens
Authors: Fleischer B , Necker A , Leget C , Malissen B , Romagne F
Published: January 2020

A panel of 15 mouse T-cell hybridomas, each expressing a different human Vbeta gene segment (hVbeta) in an otherwise mouse T-cell receptor (i.e., mouse alpha chain and CD3 complex), was constructed by transfection of hVbeta/mouse Cbeta chimeric T-cell receptor (TCR)-beta genes into a mouse T-cell hybridoma recipient lacking the endogenous TCR-beta chain. Several qualities that are conferred by the hVbeta chain of the TCR are retained in the chimeric human-mouse TCR complex: a large panel of hVbeta-specific antibodies specifically stained the hVbeta expressed by the mouse T-cell hybridomas. Moreover, hVbeta-transfected mouse cells could readily produce interleukin 2 when stimulated by superantigens presented by antigen-presenting cells. These characteristics made it possible to refine the reactivity of 17 superantigen preparations with the available transfected Vbetas. Each superantigen gave a characteristic pattern of reactivity on the transfectants. Positive reactivities with some of these transfectants, which differ only by the expressed hVbeta, demonstrate unambiguously the superantigenic character of a protein or fraction and its potential to react with the corresponding Vbetas. Therefore, these hVbeta-transfected cells constituted a valuable tool for determining "specificity fingerprints" of known or putative superantigens. First, commonly used, commercially available superantigens such as staphylococcal enterotoxin B and toxic shock syndrome toxin-1 (TSST-1) showed additional Vbeta reactivities, compared with those of their recombinant counterparts. This stresses the importance of using defined preparations of superantigens for the definition of Vbeta specificities. Second, the stimulatory pattern of a strain of Streptococcus pyogenes demonstrated that this strain, unlike others, produces a potent Vbeta 8-specific superantigen that is an yet undefined at the molecular level.
Role of the acid tolerance response in virulence of Salmonella typhimurium
Authors: Wilmes-Riesenberg M R , Bearson B , Foster J W , Curtis R
Published: January 2020

During its life cycle, Salmonella typhimurium is exposed to a variety of acidic conditions. Survival in the acidic environments within the host may require the adaptive acid tolerance response (ATR), which is characterized by the induction of several Salmonella proteins upon exposure to mildly acidic conditions. These induced proteins protect the bacterium from death under severe acid challenge. The goal of this study was to examine the role of ATR in Salmonella pathogenesis. Initially, we observed that differences exist between the virulent S. typhimurium strains and the laboratory S. typhimurium strain LT2 with respect to their ATR. Mutations affecting the ATR of S. typhimurium LT2, including atrB, atrC (polA), atrD, atbR, and fur, were crossed into virulent Salmonella strains, and the resultant transductants were screened for virulence in mice and acid sensitivity. Surprisingly, with the exception of the fur mutation, none of the muatations had a major effect on acid resistance or virulence in the pathogenic strains. The fur mutants showed a 1-to 3-log increase in the 50% lethal dose; however, the magnitude of its effect was dependent on the strain background. Strains containing two or three different atr mutations were constructed, and these were also examined for acid sensitivity and virulence. The double and triple mutants that contained an atrC mutation no longer displayed an ATR. Those mutants which were more acid sensitive were also highly attenuated, suggesting a strong correlation between the ability to mount and ATR and virulence in S. typhimurium. Comparison of the ability of the various atr single, double, and triple mutants to survive within macrophages showed that strains containing an atrC mutation survived much less than the wild type in bone marrow-derived macrophages. No difference in survival within J774 macrophage like cells were detected.
Effect of cell polarization and differentiation on entry of Listeria monocytogenes into the enterocyte-like Caco-2 cell line
Authors: Gaillard J L , Finlay B B
Published: January 2020

The entry of Listeria monocytogenes into the enterocyte-like Caco-2 cell line was studied as a function of cell polarization and differentiation. L. monocytogenes entered through the entire surface of nonpolarized cells and, predominantly, through the basolateral surface of polarized cells based on the following observations: (i) sites of L. monocytogenes invasion paralleled the distribution of the transferrin receptor, a well-known basolateral marker of polarization; (ii) numbers of internalized bacteria decreased dramatically when Caco-2 monolayers cultured beyond confluency were used (about 0.1% of the inoculated bacteria versus 1 to 2% with nonconfluent monolayers); and (iii) L. monocytogenes entry into postconfluent monolayers was greatly enhanced by treating cells with Ca(2)+ -free medium, a procedure that disrupts intercellular junctions and thus exposes the basolateral surface to bacteria. Ethylene glycol-bis (beta-aminoethyl ether)-N, N, N',N' -tetraacetic acid (EGTA) had contradictory effects on L. monocytogenes entry as this reagent opened intercellular junctions but inhibited binding and internalization of bacteria. Finally, the role of the inlAB locus in L. monocytogenes entry was confirmed because and inlAB mutant was 50- to 100-fold less invasive than the parental strain regardless of the monolayer's age. However, the inlAB mutant was still able to enter cells and to induce intracellular actin polymerization. Entry of inlAB bacteria into Caco-2 cells was not inhibited by EGTA.
Resistance to platelet microbicidal protein results in increased severity of experimental Candida albicans endocarditis
Authors: Yeaman M R , Soldan S S , Ghannoum M A , Edwards J E , Filler S G , Bayer A S
Published: January 2020

Thrombin-induced platelet microbicidal protein (tPMP) exerts potent in vitro microbicidal activity against pathogens commonly found in the bloodstream, including Staphylococcus aureus, Staphylococcus epidermidis, and Candida albicans. Localized platelet release of tPMP may be important in defense against infections involving the vascular endothelium caused by tPMP-susceptible organisms. In contrast, pathogens capable of surviving in the presence of tPMP could then exploit the platelet as an adhesive surface for attachment to damaged endothelium. To examine these hypotheses, we derived a tPMP-resistant (tPMP(r)) C. albicans strain from its tPMP-sensitive (tPMP(s)) parental strains were equivalent in vitro as assessed by genotyping (electrophoretic karyotype and restriction endonuclease analysis of genomic DNA), biotyping, germination, platelet aggregation, adherence to vascular endothelial cells, and growth characteristics. In addition, the tPMP(r) phenotype was stable following multiple in vitro and in vivo passages. We then investigated the in vivo relevance of tPMP susceptibility on endovascular infection using a rabbit model of endocarditis and hematogenous dissemination. Rabbits with transaortic catheters (n = 15 in each group) were challenged with either the tPMP(s) or tPMP(r) C. albicans strain. All rabbits developed C. albicans-induced endocarditis, as determined by the presence of infected vegetations. In rabbits challenged with tPMP(s) strain (P < 0.001). These results were seen in the absence of differences in either initial adherence of strains to cardiac valves or vegetation weights. Furthermore, although these C. albicans strains induced equivalent rates and extent of hematogenous renal infection, only the tPMP(r) strain disseminated hematogenously to the spleen (15 of 15 rabbits) versus 0 of 15 [tpmp(s) strain]; P < 0.0001). Thus, tPMP(r) C. albicans caused more-severe endocarditis and produced greater metastatic sequelae than the tPMP(s) counterpart.