bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased protein secretion and adherence to HeLa cells by Shigella spp. following growth in the presence of bile salts
Authors: Pope L M , Reed K E , Payne S M
Published: January 2020

Growth of Shigella spp. in the presence of the bile salt deoxycholate or chenodeoxycholate enhanced the bacterial invasion of HeLa cells. Growth in the presence of other structurally similar bile salts or detergents had little or no effect. Deoxycholate-enhanced invasion was not observed when bacteria were exposed to deoxycholate at low temperatures or when chloramphenicol was added to the growth medium, indicating that bacterial growth and protein synthesis are required. Increased invasion is associated with the presence of an intact Shigella virulence plasmid and is correlated with increased secretion of a set of proteins, including the Ipa proteins, to the outer membrane and into the growth medium. The increased invasion induced by the bile salts appears to be due to increased adherence. The enhanced adherence was specific to Shigella spp., since the enteroinvasive Escherichia coli strains tested did not exhibit the effect in response to growth in bile salts.
Bacterial plasminogen receptors: in vitro evidence for a role in degradation of the mammalian extracellular matrix
Authors: Lähteenmäki K , Virkola R , Pouttu R , Kuusela P , Kukkonen M , Korhonen T K
Published: January 2020

The potential of bacterium-bound plasmin to degrade mammalian extracellular matrix and to enhance bacterial penetration through basement membrane was assessed with the adherent strain SH401-1 of Salmonella enterica serovar Typhimurium. Typhimurium SH401-1 was able to bind plasminogen and to enhance the tissue-type plasminogen activator-mediated activation of the single-chain plasminogen to the two-chain plasmin. The end product, the enzymatically active, bacterium-bound plasmin activity, was also formed in a normal human plasma milieu in the presence of exogenous tissue-type plasminogen activator, indicating that plasmin was protected from the plasminogen activator inhibitors and plasmin inhibitors of plasma. Plasmin bound on Typhimurium cells degraded 125I-labeled laminin as well as 3H-labeled extracellular matrix prepared from the human endothelial cell line EA.hy926. The degradations were not seen with Typhimurium cells without plasminogen and were inhibited by the low-molecular-weight plasmin inhibitor aprotinin. Plasmin bound on Typhimurium cells also potentiated penetration of bacterial cells through the basement membrane preparation Matrigel reconstituted on membrane filters. The results give in vitro evidence for degradation of the mammalian extracellular matrix by bacterium-bound plasmin and for a pathogenetic role for bacterial plasminogen receptors.
Decomplementation by cobra venom factor suppresses Yersinia-induced arthritis in rats
Authors: Gaede K I , Baumeister E , Heesemann J
Published: January 2020

Lewis rats experimentally infected with Yersinia enterocolitica O8 develop Yersinia-induced arthritis (YIA), which resembles very much reactive arthritis in humans. To investigate the involvement of serum complement in induction and maintenance of YIA, we decomplementated Yersinia-infected Lewis rats by treatment with cobra venom factor starting on day 7 after infection (prearthritic state). Reduction of serum complement activity in vivo by cobra venom factor treatment coincided with suppression of YIA clinically and histomorphologically.
Enterohemorrhagic Escherichia coli O157:H7 requires intimin to colonize the gnotobiotic pig intestine and to adhere to HEp-2 cells
Authors: McKee M L , Melton-Celsa A R , Moxley R A , Francis D H , O'Brien A D
Published: January 2020

In a previous study, enterohemorrhagic Escherichia coli (EHEC) O157:H7 with a deletion and insertion in the eaeA gene encoding intimin was used to establish that intimin is required for the organism to attach to and efface microvilli in the piglet intestine (M. S. Donnenberg, S. Tzipori, M. L. McKee, A. D. O'Brien, J. Alroy, and J. B. Kaper, J. Clin. Invest. 92:1418-1424, 1993). However, in the same investigation, a role for intimin in EHEC adherence to HEp-2 cells could not be definitively demonstrated. To analyze the basis for this discrepancy, we constructed an in-frame deletion of eaeA and compared the adherence capacity of this mutant with that of the wild-type strain in vitro and in vivo. We observed a direct correlation between the requisite for intimin in EHEC O157:H7 colonization of the gnotobiotic piglet intestine and adherence of the bacterium to HEp-2 cells. The in vitro-in vivo correlation lends credence to the use of the HEp-2 cell adherence model for further study of the intimin protein.
Regulation of Trypanosoma cruzi infection in mice by gamma interferon and interleukin 10: role of NK cells
Authors: Cardillo F , Voltarelli J C , Reed S G , Silva J S
Published: January 2020

Gamma interferon (IFN-gamma) plays an important role in experimental Trypanosoma cruzi infections, presumably by controlling the early replication of parasites in host macrophages. In this work, we show that NK cells represent an important cell type responsible for the production of most of the IFN-gamma in the early stage of T. cruzi infection and that the in vivo treatment of mice with anti-NK1.1 monoclonal antibody made resistant animals susceptible to the infection. Through in vitro experiments, we demonstrate that normal splenocytes from euthymic or athymic nude mice cultivated for 48 h with live T. cruzi trypomastigotes produced elevated levels of IFN-gamma. In addition, NK-depleted splenocytes show a drastic reduction of IFN-gamma production in response to live T. cruzi trypomastigotes. We also demonstrated that IFN-gamma production is dependent on a factor secreted by adherent cells. Supernatants of spleen cells from athymic nude mice are able to induce IFN-gamma production by normal splenocytes when cultured with trypomastigotes. The addition of anti-interleukin-10 to these cultures resulted in a marked increase in IFN-gamma production. On the other hand, the absence of NK cells led to an increased secretion of interleukin-10 upon in vitro stimulation with T. cruzi. Taken together, these results suggest that NK cells are the major source of IFN-gamma that could be involved in limiting the replication of T. cruzi in host macrophages during the early acute phase of the infection.
Multiple host defense defects in failure of C57BL/6 ep/ep (pale ear) mice to resolve visceral Leishmania donovani infection
Authors: Murray H W , Hariprashad J , McDermott D F , Stoeckle M Y
Published: January 2020

Euthymic C57BL/L ep/ep (pale ear [PE]) mice halt the visceral replication of intracellular Leishmania donovani but fail to properly resolve infection. A previous study identified an isolated defect in tissue granuloma formation in these mice; CD4+ and CD8+ cell number, gamma interferon (IFN-gamma) production, and macrophage antimicrobial activity in vitro were all intact. New in vivo results reported here suggest a considerably more complex immune defect, with evidence indicating (i) enhanced control over L. donovani after transfer of normal C57BL/6 spleen cells, (ii) a partially suppressive Th2 cell-associated response mediated by interleukin-4 (IL-4) but not reversed by CD4+ cell depletion, (iii) absent responses to endogenous Th1 cell lymphokines (IFN-gamma and IL-2) but preserved responsiveness to endogenous tumor necrosis factor alpha, (iv) absent responses to exogenous treatment with recognized antileishmanial cytokines (IFN-gamma, IL-2, IL-12, and granulocyte-macrophage colony-stimulating factor [GM-CSF]) not corrected by transfer of C57BL/6 spleen cells, and (v) a deficient response to antimony chemotherapy. Defective hepatic granuloma formation was not corrected by transfer of C57BL/6 spleen cells or by anti-IL-4 administration. While treatment with IL-2 and GM-CSF modified the tissue reaction and induced selected effector cells to encase tissue macrophages, no antileishmanial activity resulted. Together, these observations suggest that the failure of PE mice to resolve visceral L. donovani infection likely represents expression of multiple suboptimal immune responses and/or partial defects, probably involving a combination of T-cell dysfunction, a Th2 cell response, and target cell (macrophage) hyporesponsiveness.
Further characterization of delta aroA delta virG Shigella flexneri 2a strain CVD 1203 as a mucosal Shigella vaccine and as a live-vector vaccine for delivering antigens of enterotoxigenic Escherichia coli
Authors: Noriega F R , Losonsky G , Wang J Y , Formal S B , Levine M M
Published: January 2020

The use of attenuated delta aroA delta virG Shigella flexneri 2a strain CVD 1203 as a live vector for enterotoxigenic Escherichia coli (ETEC) antigens is reported. CVD 1203 alone or expressing colonization factor antigen fimbriae and CS3 fibrillae of ETEC was given to guinea pigs and mice, orogastrically (o.g.) or intranasally (i.n.). CVD 1203 given i.n. elicited high titers of antilipopolysaccharide (anti-LPS) immunoglobulin A (IgA) and was protective in guinea pigs against a homologous conjunctival challenge. Whereas a strong IgA response against colonization factor antigen CS3, and Shigella LPS was detected in tears and serum of guinea pigs after o.g. or i.n. immunization, the i.n. route elicited significantly higher antibody titers. A strong serum IgG response was also observed against the ETEC antigens, although no serum anti-LPS IgG response was detected. The immune response in mice followed a pattern similar to that in guinea pigs, and the difference between the responses after o.g. and i.n. administration was even more remarkable.
Characterization of Tn917 insertion mutants of Staphylococcus epidermidis affected in biofilm formation
Authors: Heilmann C , Gerke C , Perdreau-Remington F , Götz F
Published: January 2020

Biofilm formation is thought to result from the concerted action of primary attachment to a specific surface and accumulation in multilayered cell clusters. Here we describe the isolation and characterization of transposon (Tn917) mutants of Staphylococcus epidermidis O-47 which were biofilm negative in the polystyrene microtiter plate assay. Among 5,000 Tn917 insertion mutants, 4 biofilm-negative mutants were isolated. Each mutant carried one copy of Tn917. The mutants were divided into two phenotypic classes: class A (mut1 and mut1a) and class B (mut2 and mut2a). Mutants of phenotypic class A lacked four cell surface proteins, were less hydrophobic, and were affected in primary attachment to polystyrene, but were still able to form multilayered cell clusters. They were able to form a biofilm on a glass surface, a trait that was even more pronounced than in the wild-type stain O-47. Loss of several surface proteins might have led to the reduced surface hydrophilic structures, thus favoring primary attachment to a glass surface and leading to subsequent biofilm formation. Mutants of phenotype class B were able to attach to polystyrene but were unable to form multilayered cell clusters, had unchanged cell surface proteins and hydrophobicity, and were unable to form a biofilm on a glass surface, mut1 and mut2 could be complemented by wild-type DNA fragments containing the Tn917 insertion sites of mut1 and mut2, respectively. The complemented biofilm-positive clone mut1 (pRC20) produced a 60-kDa protein which is postulated to function as the adhesin for binding to plastic. The traits of binding to polystyrene and the ability to form multilayered cell clusters are phenotypically and genetically distinct.
Interactions of human mannose-binding protein with lipoteichoic acids
Authors: Polotsky V Y , Fischer W , Ezekowitz R A , Joiner K A
Published: January 2020

We explored the interaction of human recombinant mannose-binding protein and lipoteichoic acids (LTAs) by enzyme-linked immunosorbent assay. The best ligand was Micrococcus luteus lipomannan, followed by Enterococcus spp. LTA containing mono-, di-, and oligoglucosyl substituents. LTAs lacking terminal sugars (those of Streptococcus pyogenes and Staphylococcus aureus) or containing galactosyl substituents (those of Listeria spp. and Lactococcus spp.) were poor ligands. These results are consistent with known structural requirements for binding through the mannose-binding protein carbohydrate recognition domain.
In vivo regulation of nitric oxide production by tumor necrosis factor alpha and gamma interferon, but not by interleukin-4, during blood stage malaria in mice
Authors: Jacobs P , Radzioch D , Stevenson M M
Published: January 2020

We investigated whether gamma interferon (IFN-gamma; a Th1 cytokine), tumor necrosis factor alpha (TNF-alpha), and interleukin-4 (IL-4; a Th2 cytokine) modulate nitric oxide (NO) production in vivo during blood stage infection with Plasmodium chabaudi AS. Treatment of resistant C57BL/6 mice, which resolve infection with P. chabaudi AS and produce increased levels of IFN-gamma, TNF-alpha, and NO early during infection, with anti-IFN- gamma plus anti-TNF-alpha monoclonal antibodies (MAbs) resulted in a reduction of both splenic inducible NO synthase mRNA and serum NO3- levels by 50 and 100%, respectively. Treatment with the anti-TNF-alpha MAb alone reduced only serum NO3- levels by 35%, and treatment with the anti-IFN-gamma MAb alone had no effect on NO production by these mice during infection. Susceptible A/J mice, which succumb to infection with P. chabaudi AS and produce increased levels of IL-4 but low levels of IFN-gamma, TNF-alpha, and NO early during infection, were treated with an anti-IL-4 MAb. The latter treatment had no effect on NO production by this mouse strain during infection. In addition, our results also demonstrate that treatment of resistant C57BL/6 mice with anti-IFN-gamma plus anti-TNF-alpha MAbs affects, in addition to NO production, other traits of resistance to P. chabaudi AS malaria such as the peak level of parasitemia and the development of splenomegaly. Furthermore, the change in spleen weight was shown to be an IFN-gamma-independent effect of TNF-alpha. Treatment of susceptible A/J mice during infection with an anti IL-4 MAb had no effect on these markers of resistance. Thus, these results demonstrate that TNF-alpha and IFN-gamma are critical in the regulation of NO production and other traits of resistance during P. chabaudi AS malaria in C57BL/6 mice. These data also indicate that treatment with an anti-IL-4 antibody alone is not able to induce NO production or confer resistance to A/J mice against P. chabaudi AS malaria.
Synthesis, characterization, and immunologic properties of detoxified lipooligosaccharide from nontypeable Haemophilus influenzae conjugated to proteins
Authors: Gu X X , Tsai C M , Ueyama T , Barenkamp S J , Robbins J B , Lim D J
Published: January 2020

Nontypeable Haemophilus influenzae (NTHi) is an important cause of otitis media in children and of pneumonitis in adults with depressed resistance. Lipooligosaccharide (LOS) is a major surface antigen of NTHi and elicits bactericidal and opsonic antibodies. We prepared detoxified LOS (dLOS) protein conjugates from NTHi for use as experimental vaccines. LOS from NTHi 9274 was treated with anhydrous hydrazine and had its toxicity reduced to clinically acceptable levels. dLOS was bound to tetanus toxoid (TT) or high- molecular-weight proteins (HMPs) from NTHi through a linker of adipic acid dihydrazide to form dLOS-TT or dLOS-HMP. The molar ratio of the dLOS to protein carriers ranged from 26:1 to 50:1. The antigenicity of the conjugates was similar to that of the LOS alone as determined by double immunodiffusion. Subcutaneous or intramuscular injection of the conjugates elicited a 28- to 486-fold rise in the level of immunoglobulin G antibodies in mice to the homologous LOS after two or three injections and a 169- to 243-fold rise in the level of immunoglobulin G antibodies in rabbits after two injections. The immunogenicity of the conjugates in mice and rabbits was enhanced by formulation with monophosphoryl lipid A plus trehalose dimycolate. In rabbits, conjugate-induced LOS antibodies induced complement-mediated bactericidal activity against the homologous strain 9274 and prototype strain 3189. These results indicate that a detoxified LOS-protein conjugate is a candidate vaccine for otitis media and pneumonitis caused by NTHi.
Invasion of respiratory epithelial cells by Burkholderia (Pseudomonas) cepacia
Authors: Burns J L , Jonas M , Chi E Y , Clark D K , Berger A , Griffith A
Published: January 2020

Pulmonary infections caused by Burkholderia (Pseudomonas) cepacia are an important cause of morbidity and mortality in cystic fibrosis (CF) patients. Several features suggestive of cellular invasion and intracellular sequestration of B. cepacia in CF are persistence of infection in the face of antibiotic therapy to which the organism demonstrates in vitro susceptibility and a propensity to cause bacteremic infections in patients with CF. Epithelial cell invasion was demonstrated in vitro in A549 cells by a modified gentamicin protection assay. The kinetics of invasion appear to be saturable. Electron microscopy of invaded monolayers showed intracytoplasmic bacteria enclosed by membrane-bound vacuoles. No lysosomal fusion with these vacuoles was observed. Intraepithelial cell replication was suggested by electron microscopy and confirmed by both a quantitative assay and a visual assay. Cytochalasin D, but not colchicine, inhibited invasion, suggesting a role for microfilaments but not microtubules. The invasion phenotype in B. cepacia may be an important virulence factor for CF infections.
Attachment of Toxoplasma gondii to host cells is host cell cycle dependent
Authors: Grimwood J , Mineo J R , Kasper L H
Published: January 2020

The initial attachment of Toxoplasma tachyzoites to target host cells is an important event in the life cycle of the parasite and hence critical in the pathogenesis of this infection. The efficiency of Toxoplasma attachment to synchronized populations of Chinese hamster ovary cells and bovine kidney cells was investigated by using a glutaraldehyde-fixed host cell assay system. For both cell lines, parasite attachment increased as the synchronized host cells proceeded from the G1 phase to the mid-S phase and then decreased as the cells entered the G2-M boundary. Postulating that these differences in attachment reflect the upregulation of a specific receptor, polyclonal antibodies were generated against whole MDBK antigen at 0 and 4 h into the S phase. Both antisera were shown to inhibit parasite attachment to both synchronous and asynchronous host cell populations. However, the attachment blockade observed with the 4-h antiserum was significantly greater than that with the 0-h antiserum, completely abolishing the cell cycle-dependent increase in attachment found in control samples. These findings suggest that Toxoplasma tachyzoites bind specifically to a host cell receptor which is upregulated in the mid-S phase of the cell cycle.
Production of interleukin-10 and transforming growth factor beta by peripheral blood mononuclear cells in Q fever endocarditis
Authors: Capo C , Zaffran Y , Zugun F , Houpikian P , Raoult D , Mege J L
Published: January 2020

The pathophysiology of Q fever endocarditis is characterized by the suppression of antigen-specific cell-mediated immune responses. We investigated the production of interleukin-10 (IL-10) and transforming growth factor beta (TGF-beta), known to interfere with the development of protective cell immunity. IL-10 was markedly released by unstimulated peripheral blood mononuclear cells (PBMC) from patients with Q fever endocarditis. This release resulted from the upregulation of IL-10 gene transcription. Similarly, the release of TGF-beta1 and TGF-beta2 was significantly higher in patient PBMC than in control cells, but the expression of their respective mRNA was not enhanced in patient cells. In contrast, lipopolysaccharide-stimulated transcription and release of IL-10 and TGF-beta were similar in patients and controls. The release of IL-10 by PBMC but not that of TGF-beta was correlated with the clinical status of the patients. First, IL-10 production was correlated with specific antibody levels. Second, IL-10 release remained elevated in patients prone to relapse. Taken together, our results suggest that the release of IL-10 and TGF-beta is upregulated in Q fever endocarditis. IL-10 might be considered as a marker of disease relapses and might be instrumental in monitoring the efficiency of the treatment.
In vitro complement-dependent binding and in vivo kinetics of pneumococcal polysaccharide TI-2 antigens in the rat spleen marginal zone and follicle
Authors: Harms G , Hardonk M J , Timens W
Published: January 2020

For a better understanding of the spleen-dependent induction of the humoral immune response against thymus-independent type 2 antigens, we have studied the in vitro and in vivo localization of different capsular pneumococcal polysaccharides (PPSs) in the rat spleen. In this study, we found that in vitro binding of PPS types 3, 4, 6B, 9N/V, 14, and 23F was dependent on complement (probably a C3 fragment) and that the localization was predominantly restricted to the marginal-zone B lymphocytes and the follicular dendritic cells. In vivo, we observed with increase of time a shift of localized antigens. Shortly after injection, all PPS types localized in the marginal-zone B lymphocytes, then localized in the outer follicular mantle, and finally were found to be diffuse in the complete follicle and follicle corona. PPS types 3 and 9N/V and later also PPS type 23F localized additionally in red pulp macrophages. In particular, the localization in the marginal zone is important since the low flow in this area in combination with strongly CD21+ B cells, which are activated early, gives a maximum opportunity for the induction of a primary humoral immune response with subsequent differentiation into plasma cells or migration to the germinal center. In addition, the localization of PPSs at follicular dendritic cells should be considered important in the induction of an ongoing immune response not restricted to the spleen.
Altered regulation of inducible nitric oxide synthase expression in macrophages from senescent mice
Authors: Chen L C , Pace J L , Russell S W , Morrison D C
Published: January 2020

We investigated the capacity of mouse macrophages obtained from senescent animals to respond in vitro to microbial stimuli. Significant hypersecretion of nitric oxide (NO) was observed in thioglycolate-elicited macrophages from senescent mice compared with those obtained from young mice in response to lipopolysaccharide (LPS). In contrast, both cell populations manifested equivalent responses to LPS with respect to tumor necrosis factor alpha secretion. Further, macrophages from senescent animals also showed potentiated responses to both zymosan and heat-killed Staphylococcus aureus, as assessed by NO production. Both cell populations were equivalently inhibited by a competitive inhibitor of NO synthase NG-monomethyl-L-arginine. Since endogenous beta interferon (IFN-beta) is recognized as an essential cofactor for LPS-induced NO production by macrophages, we investigated the role of IFN-beta in enhancing the capacity of both macrophage populations for LPS-induced NO production. Macrophages from young mice were minimally activated by LPS alone to express inducible NO synthase (iNOS), and the response was significantly potentiated by the addition of IFN-beta. These findings were confirmed by immunocytochemical staining of iNOS in which the frequency of iNOS-positive cells in response to LPS was enhanced in the presence of IFN-beta. Reverse transcription-PCR analyses revealed that macrophages from senescent animals produced larger amounts of iNOS mRNA in response to LPS. Further, exogenous IFN-beta potentiated iNOS mRNA expression in macrophages from young mice. In contrast, the frequency of LPS-activated macrophages for iNOS expression was markedly increased during senescence and addition of IFN-beta did not significantly change this frequency. These results correlated with reverse transcription PCR data showing high levels of iNOS mRNA in LPS-stimulated macrophages from senescent mice. LPS-induced NO production in macrophages from both young and senescent mice was inhibited by neutralizing antibody to either IFN-beta or IFN-gamma. Mixed cultures of macrophages from young and senescent mice stimulated with LPS manifested significantly enhanced NO production relative to that which would be predicted from an additive response of the two macrophage populations stimulated separately. The differential responsiveness of NO production observed with thioglycolate-elicited macrophages from young and senescent mice was also observed in resident macrophages but, interestingly, not in bone marrow culture-derived macrophages. These results suggest that environmental factors may be responsible for the potentiated NO responses of macrophages from senescent mice. Collectively, these data suggest that macrophages from senescent animals manifest an altered mechanism for regulation of macrophage function in NO production and iNOS expression by constitutive and/or induced expression of autoregulatory cytokines.
The gamma/delta T-cell response to Plasmodium falciparum malaria in a population in which malaria is endemic
Authors: Hviid L , Kurtzhals J A , Dodoo D , Rodrigues O , Rønn A , Commey J O , Nkrumah F K , Theander T G
Published: January 2020

Frequencies and absolute numbers of peripheral gamma/delta T cells have been reported to increase after episodes of Plasmodium falciparum malaria in adults with limited or no previous malaria exposure. In contrast, little is known about the gamma/delta T-cell response to malaria in children from areas where malaria is endemic, who bear the burden of malaria-related morbidity and mortality. We investigated the gamma/delta T-cell response in 19 Ghanaian children from an area of hyperendemic, seasonal malaria transmission. The children presented with cerebral malaria (n = 7), severe malarial anemia (n = 5), or uncomplicated malaria (n = 7) and were monitored from admission until 4 weeks later. We found no evidence of increased frequencies of gamma/delta T cells in any of the patient groups, whereas one adult expatriate studied in Ghana and three adults admitted to the hospital in Copenhagen, Denmark, all with uncomplicated, primary P. falciparum malaria, showed increased gamma/delta T-cell frequencies similar to those previously reported. All patients had lowered absolute numbers of peripheral gamma/delta T cells at admission, changing to increased numbers by days 7 to 14 and then returning to normal levels. The study raises questions regarding age and degree of previous exposure as determinants of malaria-induced gamma/delta T-cell responses.
The dsbB gene product is required for protease production by Burkholderia cepacia
Authors: Abe M , Nakazawa T
Published: January 2020

Burkholderia cepacia KF1, isolated from a pneumonia patient, produces a 37-kDa extracellular metalloprotease. A protease-deficient and lipase-proficient mutant, KFT1007, was complemented by a clone having an open reading frame coding for a 170-amino-acid polypeptide which showed significant homology to Escherichia coli DsbB. KFT1007, a presumed dsbB mutant, also failed to show motility, and both protease secretion and motility were restored by the introduction of the cloned dsbB gene of B. cepacia. The mutant KFT1007 excreted a 43-kDa polypeptide that is immunologically related to the 37-kDa mature protease. These results suggested that the dsbB mutant secretes a premature and catalytically inactive form of protease and that disulfide formation is required for the production of extracellular protease by B. cepacia.
Monocyte chemoattractant protein 1 and interleukin-8 production in mononuclear cells stimulated by oral microorganisms
Authors: Jiang Y , Russell T R , Graves D T , Cheng H , Nong S H , Levitz S M
Published: January 2020

Chemokines are a family of low-molecular-weight proinflammatory cytokines that stimulate recruitment of leukocytes. The chemokines interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) are relatively specific chemoattractants for neutrophils and monocytes, respectively. Chemokine expression contributes to the presence of different leukocyte populations observed in normal and pathologic states. In the present studies, peripheral blood mononuclear cells (PBMC) were stimulated by microbes (Candida albicans, Streptococcus mutans, Porphyromonas gingivalis, and Actinobacillus actinomycetemcomitans) selected based upon their importance as oral pathogens. IL-8 and MCP-1 gene expression and protein release were determined by Northern blot (RNA blot) analysis and enzyme-linked immunosorbent assay. C. albicans, P. gingivalis, and A. actinomycetemcomitans induced high levels of production of both MCP-1 and IL-8. S. mutans was a strong inducer of MCP-1, but it did not stimulate significant production of IL-8. C. albicans, S. mutans, and A. actinomycetemcomitans were 500 to 5,000 times more potent than P. gingivalis in terms of MCP-1 production. In general, the microbe-to-PBMC ratios required for maximum gene expression of MCP-1 were lower than those for IL-8. However, for actual protein release of MCP-1 versus IL-8, differences in the effects of various microbe concentrations were observed only for A. actinomycetemcomitans. These results demonstrate that different oral pathogens induce specific dose-dependent patterns of chemokine gene expression and release. Such patterns may help explain the immunopathology of oral infections, particularly with regard to inflammatory leukocyte recruitment.
Rapid clearance of the bacterial superantigen staphylococcal enterotoxin B in vivo
Authors: Vabulas R , Bittlingmaier R , Heeg K , Wagner H , Miethke T
Published: January 2020

Following bolus injection of the superantigen Staphylococcus enterotoxin B (SEB) into mice, ligand-reactive T cells are triggered to release toxic amounts of lymphokines. Subsequently, within 6 to 10 h ligand-reactive Vbeta8+ T cells become anergic and clonally expand and thereafter are deleted via apoptosis. Since the binding affinities of SEB to major histocompatibility complex (MHC) class II molecules are as low as 1.7 microM, the concentration of SEB in the fluid phase dictates the presentation via MHC class II molecules. Here, we study the pharmacokinetics of SEB in vivo and correlate pharmacokinetics with immunogenicity. We describe here how after a bolus injection of SEB, the superantigen becomes systemically distributed, with peak levels within 5 to 30 min in blood and in lymph nodes. Clearance occurs within 10 to 24 h, with the kidneys being a major route. To induce T-cell activation in vivo, SEB must be present in concentrations above 10(-4) microg/ml. These concentrations exist for only 15 h. Manifestation of functional outcomes such as anergy, clonal expansion, and clonal deletion begins after 24 h. We conclude that the SEB model system can be used to separate the phase of T-cell receptor ligation from the phase of manifestation of functional outcomes.
Biological activity of serum antibodies to a nonacylated form of lipoprotein D of Haemophilus influenzae
Authors: Akkoyunlu M , Janson H , Ruan M , Forsgren A
Published: January 2020

Protein D, a surface-exposed 42-kDa membrane lipoprotein, is well conserved among both type b and nontypeable Haemophilus influenzae strains, and it is considered a vaccine against H. influenzae infections. Here, we report the large-scale purification of a nonacylated form of protein D (PDm) from the periplasmic space of Escherichia coli overexpressing PDm. Screening of human sera for levels of antibodies to PDm demonstrated that the immunoglobulin G (IgG) antibody level is above background levels in infants less than 6 months of age. Following a drop to background values in the age group 6 months to 1 year, IgG antibody levels start to increase, together with IgA antibody levels, after 1 year of age. The first appearance of serum IgM antibodies is in 6-month- to 1-year-old infants whose IgG antibody levels have dropped to the postnatal background level. Affinity-purified antibodies from humans and from PDm-immunized rats detected epitopes of protein D which are normally exposed on the bacterial surface. Affinity-isolated human anti-PDm antibodies eluted in acidic buffer were not bactericidal against H. influenzae. Loss of bactericidal activity may occur in this buffer, as was demonstrated in pooled human sera with high bactericidal activity after incubation in the same buffer. Hyperimmunization of rats with PDm induced high levels of serum IgG and IgA antibodies against PDm and significant bactericidal activity against homologous and heterologous H. influenzae strains.
Association of RTX toxins with erythrocytes
Authors: Bauer M E , Welch R A
Published: January 2020

A critical step in the target cell attack by RTX cytotoxins is their association with target cells. A binding assay was used to study the association of the Escherichia coli hemolysin protein (HlyA) with erythrocytes. Several parameters required for lysis by HlyA were tested for their effects on its initial association with erythrocytes. The results demonstrate that HlyA binding to target cells is independent of several structural components of the active toxin, including the N-terminal hydrophobic region, the glycine-rich repeat region, and the HlyC-dependent acylation of HlyA. Further, the association with erythrocytes was independent of Ca2+ concentration or temperature, while the lytic event is both Ca2+ dependent and temperature dependent. The association of two other RTX toxin proteins, the Pasteurella haemolytica leukotoxin (LktA) and the enterohemorrhagic E. coli toxin (EhxA), were also examined; these toxins bound to erythrocytes much less efficiently than did HlyA. The association of HlyA with erythrocytes occurred rapidly, within 12 s of incubation, and demonstrated no measurable dissociation. HlyA bound to erythrocytes with a maximum of approximately 2,000 molecules per cell. Competition between active HlyA and unacylated HlyA demonstrated no inhibition of binding by unacylated HlyA; rather, active HlyA appeared to displace unacylated HlyA on the cell surface. These data demonstrate that binding and lysis by HlyA are separable events and challenge the concept of nonspecific binding to the cell surface by RTX toxins.
Fine mapping of outer membrane protein P2 antigenic sites which vary during persistent infection by Haemophilus influenzae
Authors: Duim B , Vogel L , Puijk W , Jansen H M , Meloen R H , Dankert J , van Alphen L
Published: January 2020

Antigenic drift of the major outer membrane protein (MOMP) P2 of nonencapsulated Haemophilus influenzae as observed during persistent infections in patients with chronic bronchitis was mimicked in a rabbit model in which H. influenzae persisted in subcutaneous cages. The antigenic drift resulted from amino acid substitutions in potentially surface-exposed loops of MOMP P2. Since in a rabbit model the appearance of antigenic variants was associated with the presence of strain-specific bactericidal antibodies (L. Vogel, B. Duim, F. Geluk, P. Eijk, H. Jansen, J. Dankert, and L. van Alphen, Infect. Immun. 64:980-986, 1996), we determined the epitope specificities of these bactericidal antibodies. The eight loops of MOMP P2 of H. influenzae d1 were separately expressed as fusion proteins with glutathione S-transferase. Sera of rabbits persistently infected with H. influenzae reacted with the loop 5 and loop 6 fusion proteins in immunoblotting and enzyme-linked immunosorbent assay. For fine mapping of the epitopes with pepscan analysis, overlapping synthetic peptides consisting of 12 amino acids were made. Rabbit sera contained antibodies reacting with peptides derived from loop 5 and peptides containing amino acids of the side of loop 6. In addition, MOMP P2 variant-specific reactions with the amino acids located at the tip of loop 6 were detected. The rabbit sera showed variant-specific complement-dependent bactericidal activities, which were eliminated by affinity chromatography with fusion proteins of loop 6 but not of loop 5. We conclude that, during persistence of H. influenzae in rabbits, variant-specific bactericidal antibodies are elicited to the variable tip of MOMP P2 loop 6.
Immunogenicity and efficacy against lethal aerosol staphylococcal enterotoxin B challenge in monkeys by intramuscular and respiratory delivery of proteosome-toxoid vaccines
Authors: Lowell G H , Colleton C , Frost D , Kaminski R W , Hughes M , Hatch J , Hooper C , Estep J , Pitt L , Topper M , Hunt R E , Baker W , Baze W B
Published: January 2020

Staphylococcal enterotoxin B (SEB), a primary cause of food poisoning, is also a superantigen that can cause toxic shock after traumatic or surgical staphylococcal wound [correction of would] infections or viral influenza-associated staphylococcal superinfections or when aerosolized for use as a potential biologic warfare threat agent. Intranasal or intramuscular (i.m.) immunization with formalinized SEB toxoid formulated with meningococcal outer membrane protein proteosomes has previously been shown to be immunogenic and protective against lethal respiratory or parenteral SEB challenge in murine models of SEB intoxication. Here, it is demonstrated that immunization of nonhuman primates with the proteosome-SEB toxoid vaccine is safe, immunogenic, and protective against lethal aerosol challenge with 15 50% lethal doses of SEB. Monkeys (10 per group) were primed i.m. and given booster injections by either the i.m. or intratracheal route without adverse side effects. Anamnestic anti-SEB serum immunoglobulin G (IgG) responses were elicited in all monkeys, but strong IgA responses in sera and bronchial secretions were elicited both pre- and post-SEB challenge only in monkeys given booster injections intratracheally. The proteosome-SEB toxoid vaccine was efficacious by both routes in protecting 100% of monkeys against severe symptomatology and death from aerosolized-SEB intoxication. These data confirm the safety, immunogenicity, and efficacy in monkeys of parenteral and respiratory vaccination with the proteosome-SEB toxoid, thereby supporting clinical trials of this vaccine in humans. The safety and enhancement of both bronchial and systemic IgA and IgG responses by the proteosome vaccine delivered by a respiratory route are also encouraging for the development of mucosally delivered proteosome vaccines to protect against SEB and other toxic or infectious respiratory pathogens.
Isolation and characterization of a minor fimbria from Porphyromonas gingivalis
Authors: Hamada N , Sojar H T , Cho M I , Genco R J
Published: January 2020

We have discovered two distinctly different fimbriae expressed by the same Porphyromonas gingivalis strain. The construction of a fimA mutant of P. gingivalis ATCC 33277 has previously been reported by N. Hamada et al. (Infect. Immun. 62:1696-1704, 1994). Expression of fimbriae on the surface of the fimA mutant and the wild-type strain, ATCC 33277, were investigated by electron microscopy. The wild-type strain produced long fimbrial structures extending from the cell surface, whereas those structures were not observed on the fimA mutant. However, short fimbrial structures were seen on the surface of the fimA mutant. The short fimbrial protein was purified from the fimA mutant by selective protein precipitation and chromatography on DEAE Sepharose CL-6B. We have found that the second fimbrial structure of P. gingivalis ATCC 33277 is distinct from the 41-kDa (43-kDa) major fimbrial protein (FimA). We provisionally call this protein minor fimbriae. The molecular mass of the minor fimbriae is 67 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions after boiling at 100 degrees C. The component shows a ladder-like pattern at 80 degrees C under nonreducing conditions, suggesting a tendency to aggregate or polymerize. In immunoblotting analysis, anti-minor fimbria serum reacted with both the 100 degrees C- and the 80 degrees C-treated minor fimbriae. The anti-minor fimbria serum also reacts with the same-molecular-size fimbrial preparation from the wild-type strain. Immunogold electron microscopy showed that the anti-minor fimbria serum bound to the minor fimbria on the cell surface of the wild-type strain. This is the first report on the identification of the minor fimbria produced by P. gingivalis. These results suggest that the minor fimbriae appearing on the fimA mutant strain are produced together with numerous long major fimbriae on the wild-type strain. Moreover, the minor fimbriae are different in size and antigenicity from the earlier-reported FimA, a major 41-kDa fimbrial component of P. gingivalis.
Cloning, nucleotide sequence, and expression of the gene coding for a ribosome releasing factor-homologous protein of Brucella melitensis
Authors: Vizcaíno N , Cloeckaert A , Dubray G , Zygmunt M S
Published: January 2020

The gene coding for a Brucella melitensis cytosoluble protein (CP24) that is immunogenic in infected sheep and a major component of brucellin INRA was cloned and sequenced. As in Brucella cells, CP24 was located in the cytoplasm of recombinant Escherichia coli. The amino acid sequence predicted from the cloned gene revealed 48 and 46% identity with the ribosome releasing factor, a protein factor required for release of the 70S ribosome from the mRNA, of E. coli and Haemophilus influenzae Rd, respectively. Sera from naturally infected sheep and sheep experimentally infected with B. melitensis H38 showed antibody reactivity against recombinant CP24.
Bactericidal activity of a synthetic peptide (CG 117-136) of human lysosomal cathepsin G is dependent on arginine content
Authors: Shafer W M , Hubalek F , Huang M , Pohl J
Published: January 2020

The importance of individual amino acids in mediating the broad-spectrum bactericidal action of a 20-mer amphipathic, cationic peptide (CG 117-136) of human lysosomal cathepsin G was determined by using a single amino acid replacement strategy. This strategy revealed an important role for arginine because loss of any of the four arginine residues in CG 117-136 due to substitution with alanine, citrulline, or lysine residues resulted in a reduction of its bactericidal activity against both Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 33593. However, the replacement of a single alanine residue in CG 117-136 with arginine, but not glutamic acid, enhanced the activity of CG 117-136 against both P. aeruginosa and S. aureus. The importance of certain bulky, nonpolar amino acids for the bactericidal activity of CG 117-136 was also evident, since their substitutions by alanine diminished bactericidal activity. Accordingly, contributions of hydrophobic amino acids and structural considerations of the guanidinium side chain of arginine are major determinants in the broad-spectrum antimicrobial action of CG 117-136.
Role of complement in C-reactive-protein-mediated protection of mice from Streptococcus pneumoniae
Authors: Szalai A J , Briles D E , Volanakis J E
Published: January 2020

Expression of a human C-reactive protein (CRP) transgene has been shown to protect mice from lethal Streptococcus pneumoniae infection. In the present study, we used cobra venom factor-induced decomplementation to investigate the role of complement in this CRP-mediated protection. An intact complement system significantly reduced pneumococcal bacteremia at 24 h postinfection and extended median survival time of both CRP-transgenic and nontransgenic mice. However, mortality was significantly lowered only for CRP transgenic mice. The transgene significantly reduced bacteremia for both normocomplementemic and decomplemented mice, but it resulted in a significantly longer median survival time and lower mortality only for normocomplementemic mice. These data suggest that in vivo complement and CRP amplify each other's protective capacity, particularly during the early course of infection.
Evidence for apoptosis of human macrophage-like HL-60 cells by Legionella pneumophila infection
Authors: Müller A , Hacker J , Brand B C
Published: January 2020

Legionella pneumophila, the causative agent of Legionnaires' disease and Pontiac fever, replicates within and eventually kills human macrophages. In this study, we show that L. pneumophila is cytotoxic to HL-60 cells, a macrophage-like cell line. We demonstrate that cell death mediated by L. pneumophila occurred at least in part through apoptosis, as shown by changes in nuclear morphology, an increase in the proportion of fragmented host cell DNA, and the typical ladder pattern of DNA fragmentation indicative of apoptosis. We further sought to determine whether potential virulence factors like the metalloprotease and the macrophage infectivity potentiator of L. pneumophila are involved in the induction of apoptosis. None of these factors are essential for the induction of apoptosis in HL-60 cells but may be involved in other cytotoxic mechanisms that lead to accidental cell death (necrosis). The ability of L. pneumophila to promote cell death may be important for the initiation of infection, bacterial survival, and escape from the host immune response. Alternatively, the triggering of apoptosis in response to bacterial infection may have evolved as a means of the host immune system to reduce or inhibit bacterial replication.
Effect of Porphyromonas gingivalis on epithelial cell MMP-9 type IV collagenase production
Authors: Fravalo P , Ménard C , Bonnaure-Mallet M
Published: January 2020

Porphyromonas gingivalis is reportedly capable of stimulating the expression of host cell matrix metalloproteinases (MMP), contributing to tissue destruction. However, the impact of this bacterium on specific molecules remains to be determined. In this study, we evaluate the effect of P. gingivalis on regulation of MMP-9 expression in human gingival epithelial cells (HGEC). Various inocula of P. gingivalis were added to cultures of HGEC. The effects of live bacteria, heat-killed bacteria, and outer membrane extract were analyzed. MMP-9 secretion by HGEC was evaluated by enzyme-linked immunosorbent assay. For inocula smaller than one bacterium per cell, the quantity of MMP-9 secreted by HGEC was increased in comparison to control conditions. For inocula from 2.5 to 250 bacteria per cell, an inhibition of MMP-9 secretion in a dose-response fashion was observed, with a maximum reduction (ranging from 80 to 95% in five experiments) at 50 bacteria per cell. Gelatin zymograms confirmed the decrease in MMP-9 secretion. A band of 83 kDa, corresponding to activated enzyme, was present for inocula of 0.5 to 50 bacteria. Inhibition took place without any alteration of epithelial cell viability. Heat-killed bacteria and outer membrane extract also provoked proenzyme activation but did not inhibit MMP-9 secretion. These results demonstrate a direct effect of P. gingivalis on HGEC, suggesting a specific action on the collagen renewal process at the interface between the epithelium and connective tissue.