bluuebunny/crossref_metadata_2025 huggingface dataset:
Complement factor C3 deposition and serum resistance in isogenic capsule and lipooligosaccharide sialic acid mutants of serogroup B Neisseria meningitidis
Authors: Vogel U , Weinberger A , Frank R , Müller A , Köhl J , Atkinson J P , Frosch M
Published: January 2020

Serogroup B meningococci express sialic acids on their surfaces as a modification of the lipooligosaccharide (LOS) and as capsular material consisting of alpha2,8-linked sialic acid homopolymers. The aim of this study was to elucidate the impact of each sialic acid component on the deposition of complement factor C3 and serum resistance. For this purpose, we used isogenic mutants deficient in capsule expression (a polysialyltransferase mutant) or sialylation of the LOS (a galE mutant) or both (a mutant with a deletion of the cps gene locus). Bactericidal assays using 40% normal human serum (NHS) demonstrated that both the capsule and LOS sialic acid are indispensable for serum resistance. By immunoblotting with monoclonal antibody MAb755 that is specific for the C3 alpha-chain, we were able to demonstrate that C3 from 40% NHS was covalently linked to the surface structures of meningococci as C3b and iC3b, irrespective of the surface sialic acid compounds. However, C3b linkage was more pronounced and occurred on a larger number of target molecules in galE mutants with nonsialylated LOS than in meningococci with wild-type LOS, irrespective of the capsule phenotype. C3b deposition was caused by both the classical pathway (CP) and the alternative pathway of complement activation. Use of 10% NHS revealed that at low serum concentrations, C3 deposition occurred via the CP and was detected primarily on nonsialylated-LOS galE mutants, irrespective of the capsular phenotype. Accordingly, immunoglobulin M (IgM) binding to meningococci from heat-inactivated NHS was demonstrated only in both encapsulated and unencapsulated galE mutants. In contrast, inhibition of IgA binding required both encapsulation and LOS sialylation. We conclude that serum resistance in wild-type serogroup B meningococci can only be partly explained by an alteration of the C3b linkage pattern, which seems to depend primarily on the presence of wild-type LOS, since a serum-resistant phenotype also requires capsule expression.
Glycosylphosphatidylinositols are required for the development of Trypanosoma cruzi amastigotes
Authors: Garg N , Postan M , Mensa-Wilmot K , Tarleton R L
Published: January 2020

Induction of a glycosylphosphatidylinositol (GPI) deficiency in Trypanosoma cruzi by the heterologous expression of Trypanosoma brucei GPI-phospholipase C (GPI-PLC) results in decreased expression of major surface proteins (N. Garg, R. L. Tarleton, and K. Mensa-Wilmot, J. Biol. Chem. 272:12482-12491, 1997). To further explore the consequences of a GPI deficiency on replication and differentiation of T. cruzi, the in vitro and in vivo behaviors of GPI-PLC-expressing T. cruzi were studied. In comparison to wild-type controls, GPI-deficient T. cruzi epimastigotes exhibited a slight decrease in overall growth potential in culture. In the stationary phase of in vitro growth, GPI-deficient epimastigotes readily converted to metacyclic trypomastigotes and efficiently infected mammalian cells. However, upon conversion to amastigote forms within these host cells, the GPI-deficient parasites exhibited a limited capacity to replicate and subsequently failed to differentiate into trypomastigotes. Mice infected with GPI-deficient parasites showed a substantially lower rate of mortality, decreased tissue parasite burden, and a moderate tissue inflammatory response in comparison to those of mice infected with wild-type parasites. The decreased virulence exhibited by GPI-deficient parasites suggests that inhibition of GPI biosynthesis is a feasible strategy for chemotherapy of infections by T. cruzi and possibly other intracellular protozoan parasites.
Biotherapeutic effects of probiotic bacteria on candidiasis in immunodeficient mice
Authors: Wagner R D , Pierson C , Warner T , Dohnalek M , Farmer J , Roberts L , Hilty M , Balish E
Published: January 2020

Four species of probiotic bacteria were assessed for their capacities to protect athymic bg/bg-nu/nu and euthymic bg/bg-nu/+ mice from mucosal and systemic candidiasis. Each bacterial species and Candida albicans colonized the gastrointestinal tracts of both strains of mice. The presence of probiotic bacteria (Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus casei GG, or Bifidobacterium animalis) in the gastrointestinal tracts prolonged the survival of adult and neonatal bg/bg-nu/nu mice compared to that of isogenic mice colonized with C. albicans alone. The incidence of systemic candidiasis in bg/bg-nu/nu mice was significantly reduced by each of the four probiotic bacterial species. The numbers of C. albicans present in the alimentary tracts of euthymic bg/bg-nu/+ mice were significantly reduced by L. casei GG and B. animalis. None of the probiotic bacteria species completely prevented mucosal candidiasis, but B. animalis reduced its incidence and severity. Probiotic bacteria also modulated antibody- and cell-mediated immune responses to C. albicans. The prolonged survival of mice, decreased severity of mucosal and systemic candidiasis, modulation of immune responses, decreased number of C. albicans in the alimentary tract, and reduced numbers of orogastric infections demonstrated not only that probiotic bacteria have biotherapeutic potential for prophylaxis against and therapy of this fungal disease but also that probiotic bacteria protect mice from candidiasis by a variety of immunologic (thymic and extrathymic) and nonimmunologic mechanisms in this model.
Haemophilus somnus immunoglobulin binding proteins and surface fibrils
Authors: Corbeil L B , Bastida-Corcuera F D , Beveridge T J
Published: January 2020

The high-molecular-weight (HMW) immunoglobulin binding proteins (IgBPs) of Haemophilus somnus and a 76-kDa surface protein (p76) are found in serum-resistant virulent strains but not in several serum-sensitive strains from asymptomatic carriers. For the first time, p76 was shown to be an IgBP also. This was done by competitive inhibition studies with affinity-purified antidinitrophenol (anti-DNP) and DNP to ensure that binding was not antigen specific. The HMW IgBPs, but not the p76 IgBP, were partially purified from concentrated culture supernatant in detergent by fluid-phase liquid chromatography with a gel filtration column. Membrane extraction studies showed that p76 predominated in the Sarkosyl-soluble fraction of the bacterial cell pellet. Since integral outer membrane (OM) proteins are Sarkosyl insoluble, this is consistent with our previous finding that implicated p76 as a peripheral OM protein. The HMW IgBPs were found predominantly in the Sarkosyl-soluble fraction of the culture supernatant. This suggests that they were not integral membrane proteins and that their presence in the supernatant was not due to OM blebbing. We then showed that two IgBP-positive serum-resistant virulent strains have a surface fibrillar network but that two IgBP-negative serum-sensitive H. somnus strains from asymptomatic preputial carriers do not. Fibrils on the surfaces of IgBP+ strains bound gold-labelled bovine immunoglobulin G2 (IgG2) anti-DNP, indicating that these fibrils have IgG2 binding activity. Therefore, this study shows that H. somnus has two IgBPs, including a peripheral membrane protein and a fibrillar surface network.
Nonopsonic binding of Mycobacterium tuberculosis to complement receptor type 3 is mediated by capsular polysaccharides and is strain dependent
Authors: Cywes C , Hoppe H C , Daffé M , Ehlers M R
Published: January 2020

The choice of host cell receptor and the mechanism of binding (opsonic versus nonopsonic) may influence the intracellular fate of Mycobacterium tuberculosis. We have identified two substrains of M. tuberculosis H37Rv, designated H37Rv-CC and -HH, that differed in their modes of binding to complement receptor type 3 (CR3) expressed in transfected Chinese hamster ovary (CHO-Mac-1) cells: H37Rv-CC bound nonopsonically, whereas H37Rv-HH bound only after opsonization in fresh serum. H37Rv-CC also bound nonopsonically to untransfected CHO cells, whereas H37Rv-HH binding was enhanced by serum and was mediated by the 1D1 antigen, a bacterial adhesin previously identified as a polar phosphatidylinositol mannoside. H37Rv-CC and -HH had identical IS6110 DNA fingerprint patterns. Of five M. tuberculosis clinical isolates examined, four displayed the same binding phenotype as H37Rv-CC, as did the Erdman strain, whereas one isolate, as well as Mycobacterium smegmatis, behaved like H37Rv-HH. Nonopsonic binding of H37Rv-CC to CHO cell-expressed CR3 was apparently to the beta-glucan lectin site, as it was cation independent and inhibited by laminarin (seaweed beta-glucan) and N-acetylglucosamine; laminarin also inhibited the binding of H37Rv-CC to monocyte-derived macrophages. Further, binding of H37Rv-CC to CHO-Mac-1 cells was inhibited by prior agitation of bacteria with glass beads (which strips outer capsular polysaccharides) and by preincubation with amyloglucosidase, as well as by the presence of capsular D-glucan and D-mannan from M. tuberculosis Erdman, but not by Erdman D-arabino-D-mannan, yeast mannan, or capsular components from H37Rv-HH. Analysis of capsular carbohydrates revealed that H37Rv-CC expressed 5-fold more glucose and 2.5-fold more arabinose and mannose than H37Rv-HH. Flow cytometric detection of surface epitopes indicated that H37Rv-CC displayed twofold less surface-exposed phosphatidylinositol mannoside and bound complement C3 less efficiently than H37Rv-HH; these differences were eliminated after treatment of H37Rv-CC with glass beads. Thus, outer capsular polysaccharides mediate the binding of H37Rv-CC to CR3, likely to the beta-glucan site. Moreover, there are strain-dependent differences in the thickness or composition of capsular polysaccharides that determine the mode of binding of M. tuberculosis to mammalian cells.
Molecular cloning and characterization of Dr-II, a nonfimbrial adhesin-I-like adhesin isolated from gestational pyelonephritis-associated Escherichia coli that binds to decay-accelerating factor
Authors: Pham T Q , Goluszko P , Popov V , Nowicki S , Nowicki B J
Published: January 2020

Bacterial adhesins play an important role in the colonization of the human urogenital tract. Escherichia coli Dr family adhesins have been found to be frequently expressed in strains associated with pyelonephritis in pregnant females. The tissue receptor for known Dr adhesins has been localized to the short consensus repeat-3 (SCR-3) domain of decay accelerating factor (DAF), a complement regulatory protein. In this report, we identified and cloned draE2, a gene encoding a novel 17-kDa DAF-binding adhesin, Dr-II, from a strain of E. coli associated with acute gestational pyelonephritis. Despite the significant sequence diversity between Dr-II and Dr family adhesins, the receptor of Dr-II was found to be the SCR-3 domain of DAF. Sequence analysis of the 186-amino-acid Dr-II open reading frame revealed significant diversity from other members of the Dr adhesin family, including Dr, AFA-I, AFA-III, and F1845, but only an 8-amino-acid difference in sequence from that of the 17-kDa nonfimbrial adhesin NFA-I of unknown receptor specificity. N-terminal peptide sequencing of the purified adhesin confirmed the identity of the open reading frame and indicated cleavage of a 28-amino-acid signal peptide. Antibodies raised against purified Dr-II adhesin exhibited little or no cross-reactivity to Dr adhesin. Characterization of the biological properties demonstrated that like the Dr adhesins, Dr-II was associated with the ability of E. coli to bind to tubular basement membranes and Bowman's capsule and to be internalized into HeLa cells.
Comparative analysis of immunoglobulin A1 protease activity among bacteria representing different genera, species, and strains
Authors: Reinholdt J , Kilian M
Published: January 2020

Immunoglobulin A1 (IgA1) proteases cleaving human IgA1 in the hinge region are produced constitutively by a number of pathogens, including Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, and Streptococcus pneumoniae, as well as by some members of the resident oropharyngeal flora. Whereas IgA1 proteases have been shown to interfere with the functions of IgA antibodies in vitro, the exact role of these enzymes in the relationship of bacteria to a human host capable of responding with enzyme-neutralizing antibodies is not clear. Conceivably, the role of IgA1 proteases may depend on the quantity of IgA1 protease generated as well as on the balance between secreted and cell-associated forms of the enzyme. Therefore, we have compared levels of IgA1 protease activity in cultures of 38 bacterial strains representing different genera and species as well as strains of different pathogenic potential. Wide variation in activity generation rate was found overall and within some species. High activity was not an exclusive property of bacteria with documented pathogenicity. Almost all activity of H. influenzae, N. meningitidis, and N. gonorrhoeae strains was present in the supernatant. In contrast, large proportions of the activity in Streptococcus, Prevotella, and Capnocytophaga species was cell associated at early stationary phase, suggesting that the enzyme may play the role of a surface antigen. Partial release of cell-associated activity occurred during stationary phase. Within some taxa, the degree of activity variation correlated with degree of antigenic diversity of the enzyme as determined previously. This finding may indicate that the variation observed is of biological significance.
Use of a novel approach, termed island probing, identifies the Shigella flexneri she pathogenicity island which encodes a homolog of the immunoglobulin A protease-like family of proteins
Authors: Rajakumar K , Sasakawa C , Adler B
Published: January 2020

The she gene of Shigella flexneri 2a, which also harbors the internal enterotoxin genes set1A and set1B (F. R. Noriega, GenBank accession no. U35656, 1995) encodes a homolog of the virulence-related immunoglobulin A (IgA) protease-like family of secreted proteins, Tsh, EspC, SepA, and Hap, from an avian pathogenic Escherichia coli, an enteropathogenic E. coli, S. flexneri 5, and Haemophilus influenzae, respectively. To investigate the possibility that this locus was carried on a larger deletable element, the S. flexneri 2a YSH6000T she gene was insertionally disrupted by allelic exchange using a Tn10-derived tetAR(B) cassette. Then, to detect loss of the she locus, the tetracycline-resistant derivative was plated onto fusaric acid medium to select for tetracycline-sensitive revertants, which were observed to arise at a frequency of 10(-5) to 10(-6). PCR and pulsed-field gel electrophoresis analysis confirmed loss of the she::tetAR(B) locus in six independent tetracycline-sensitive isolates. Sample sequencing over a 25-kb region flanking she identified four insertion sequence-like elements, the group II intron-like sequence Sf.IntA, and the 3' end of a second IgA protease-like homolog, sigA, lying 3.6 kb downstream and in an orientation inverted with respect to she. The deletion was mapped to chromosomal NotI fragment A and determined to have a size of 51 kb. Hybridization with flanking probes confirmed that at least 17.7 kb of the 51-kb deletable element was unique to the seven she+ strains investigated, supporting the conclusion that she lay within a large pathogenicity island. The method described in this study, termed island probing, provides a useful tool to further the study of pathogenicity islands in general. Importantly, this approach could also be of value in constructing safer live attenuated bacterial vaccines.
Salmonellae activate tumor necrosis factor alpha production in a human promonocytic cell line via a released polypeptide
Authors: Ciacci-Woolwine F , Kucera L S , Richardson S H , Iyer N P , Mizel S B
Published: January 2020

Invasive strains of Salmonella spp. cause both systemic and localized infections in humans. The ability to resist infection and some aspects of the tissue pathology associated with the presence of Salmonella in the gastrointestinal tract have been shown to be mediated in part by the induction of tumor necrosis factor alpha (TNF-alpha), a proinflammatory cytokine produced by activated macrophages and lymphocytes. Recent reports indicate that TNF-alpha is involved in the induction of human immunodeficiency virus replication by Salmonella in the latently infected human promonocytic cell line U1. In the present study, we investigated the effects of Salmonella on TNF-alpha production in U1 cells and a related cell line, U38. Unlike Escherichia coli or Yersinia enterocolitica, salmonellae rapidly induce TNF-alpha expression in these cells through a released factor(s). Time course experiments show that the kinetics of TNF-alpha production by U38 cells stimulated with Salmonella conditioned medium closely resemble those observed in response to live Salmonella. The observation that TNF-alpha levels are elevated by 60 min after exposure to either bacteria or their conditioned medium suggests that the soluble inducer is continuously released or shed by the bacteria and that the signal acts rapidly to increase TNF-alpha production. Furthermore, the ability to produce the TNF-alpha inducer is shared by at least four Salmonella serotypes and does not correlate with the abilities to invade and to survive within phagocytes. Treatment of active conditioned medium with trypsin, but not low pH, high temperature, or urea, significantly inhibits its TNF-alpha-inducing effect on U38 cells, a finding which points to a polypeptide product of Salmonella as the mediator of TNF-alpha production. Gel filtration chromatography of Salmonella conditioned medium reveals two peaks of activity, consistent with molecular masses of approximately 150 and 110 kDa.
Azurophilic granules of human neutrophils contain CD14
Authors: Rodeberg D A , Morris R E , Babcock G F
Published: January 2020

CD14, the leukocyte receptor for lipopolysaccharide (LPS), is important in the response of human polymorphonuclear leukocytes (PMNs) to infection with gram-negative bacteria. The level of CD14 on the PMN surface increases after exposure to some inflammatory stimuli such as N-formyl-methionyl-leucyl-phenylalanine (fMLP). These newly expressed CD14 molecules probably come from an intracellular pool of preformed receptors. We sought to further characterize PMN CD14 expression, upregulation, and shedding and to define the intracellular location of CD14 molecules. Our results demonstrate that both LPS and fMLP significantly increased CD14 cell surface expression; however, neither phorbol myristate acetate (PMA) or A23187 increased receptor levels on the PMN surface. Neither fMLP, PMA, or A23187 stimulated the release of soluble CD14 from PMNs. Intracellular CD14 was observed in >90% of PMNs examined by flow cytometry and confocal microscopy. Additional analyses using CD14 enzyme-linked immunosorbent assays and electron microscopy studies, examining PMN granules separated by discontinuous sucrose or Percoll gradients, showed that CD14 was present in both the plasma membrane-secretory vesicle fractions and azurophilic granules.
The major surface glycoprotein of Pneumocystis carinii induces release and gene expression of interleukin-8 and tumor necrosis factor alpha in monocytes
Authors: Benfield T L , Lundgren B , Levine S J , Kronborg G , Shelhamer J H , Lundgren J D
Published: January 2020

Recent studies suggest that interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-alpha) may play a central role in host defense and pathogenesis during Pneumocystis carinii pneumonia. In order to investigate whether the major surface antigen (MSG) of human P. carinii is capable of eliciting the release of IL-8 and TNF-alpha, human monocytes were cultured in the presence of purified MSG. MSG-stimulated cells released significant amounts of IL-8 within 4 h, and at 20 h, cells stimulated with MSG released 45.5 +/- 9.3 ng of IL-8/ml versus 3.7 +/- 1.1 ng/ml for control cultures (P = 0.01). In a similar fashion, MSG elicited release of TNF-alpha. Initial increases were also seen at 4 h, and at 20 h, TNF-alpha levels reached 6.4 +/- 1.1 ng/ml, compared to 0.08 +/- 0.01 ng/ml for control cultures (P < 0.01). A concentration-dependent increase in IL-8 and TNF-alpha secretion was observed at 20 h with 0.2 to 5 microg of MSG/ml (P < 0.01). Secretion of IL-8 and TNF-alpha from MSG-stimulated monocytes at 20 h was inhibited by 60 and 86%, respectively, after coincubation with soluble yeast mannan (P = 0.01). With an RNase protection assay, increases in steady-state mRNA levels for IL-8 and TNF-alpha were detectable at 4 h. These data show that recognition of MSG by monocytes involves a mannose-mediated mechanism and results in the release of the proinflammatory cytokines IL-8 and TNF-alpha.
Role of tumor necrosis factor alpha in induction of murine CD14 gene expression by lipopolysaccharide
Authors: Fearns C , Loskutoff D J
Published: January 2020

We previously demonstrated CD14 gene expression in myeloid and epithelial cells of the mouse and showed that expression of the CD14 gene in both is modulated by lipopolysaccharide (LPS). Here we test the hypothesis that the induction of CD14 in these cells is an indirect effect of LPS, one mediated by tumor necrosis factor alpha (TNF-alpha). TNF-alpha induced a transient increase in levels of CD14 in plasma with a peak at 6 to 8 h, and this increase in levels of CD14 antigen in plasma was accompanied by increased levels of CD14 mRNA in lung, liver, and kidney. Moreover, in situ hybridization studies revealed that CD14 mRNA was induced in both myeloid cells and epithelial cells, the same cells that respond to LPS. Pretreatment of mice with anti-TNF antiserum reduced the LPS-mediated increase in levels of CD14 in plasma and significantly reduced the level of induction of CD14 mRNA in selected epithelial cells in the kidney and liver. The antiserum did not appear to block LPS-mediated induction in myeloid cells in the tissues examined. In C3H/HeJ mice, the epithelial response to LPS was markedly attenuated whereas the response to TNF-alpha was normal. Thus, regulation of CD14 gene expression by LPS differs in epithelial and myeloid cells, with the epithelial responses in kidney and liver being mediated, in part, by TNF-alpha.
Helicobacter bilis-induced inflammatory bowel disease in scid mice with defined flora
Authors: Shomer N H , Dangler C A , Schrenzel M D , Fox J G
Published: January 2020

Helicobacter bilis has been isolated from aged inbred mice with multifocal chronic hepatitis and from scid mice with diarrhea, proliferative typhlitis, and colitis. To determine the pathogenic potential of H. bilis, we inoculated 4-week-old female Tac:Icr:Ha(ICR)-scidfDF mice by intraperitoneal injection of approximately 10(8) CFU of H. bilis in phosphate-buffered saline (PBS) (n = 15) or PBS alone (n = 10) and necropsied them at 7 weeks postinfection. Sham-inoculated mice had no significant gross or histopathological findings. In contrast, all 15 experimentally inoculated mice (confirmed to be H. bilis-colonized by culture and PCR of cecal contents) exhibited varying degrees of inflammatory bowel disease (IBD). Proliferative typhlocolitis was characterized by focal to segmental areas of crypt hyperplasia and a predominantly histiocytic inflammatory cell infiltrate. Labeling indices for 5-bromo-2'-deoxyuridine incorporation were increased approximately 2.5-fold in the ceca and colons of H. bilis-inoculated mice. This is the first study to demonstrate experimentally that infection with H. bilis causes IBD in scid mice with defined flora. This result both confirms a pathogenic role for H. bilis in mice and provides a new model relating a specific microbial agent and IBD.
Plasmodium falciparum- and merozoite surface protein 1-specific antibody isotype balance in immune Senegalese adults
Authors: Nguer C M , Diallo T O , Diouf A , Tall A , Dieye A , Perraut R , Garraud O
Published: January 2020

This study shows markedly different isotype distributions of antibodies to asexual blood stages of Plasmodium falciparum and to merozoite surface protein 1 in clinically immune Senegalese adults depending on the study site. The relationships between immunoglobulin M (IgM) and IgG and between IgG3 and IgG1 antibodies differed in settings where transmission is perennial compared to settings where it is seasonal. This suggests a role for antibody class and/or subclass production and utilization in the regulation of protective immunity to such antigens.
Natural proteoglycan receptor analogs determine the dynamics of Opa adhesin-mediated gonococcal infection of Chang epithelial cells
Authors: van Putten J P , Hayes S F , Duensing T D
Published: January 2020

Many bacterial pathogens possess a complex machinery for the induction and/or secretion of factors that promote their uptake by mammalian cells. We searched for the molecular basis of the 60- to 90-min lag time in the interaction of Neisseria gonorrhoeae carrying the heparin-binding Opa adhesin with Chang epithelial cells. Infection assays in the presence of chloramphenicol demonstrated that the Opa-mediated gonococcal infection of Chang cells required bacterial protein synthesis when the microorganisms were derived from GC agar but not when grown in liquid media. Further analysis indicated that contact with agar ingredients rather than the growth state of the microorganisms determined the infection dynamics. DEAE chromatography of GC agar extracts and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and testing of collected fractions in infection assays identified negatively charged high-molecular-weight polysaccharides in the agar as inhibitors of the cellular infection. Electron microscopy showed that agar-grown gonococci were surrounded by a coat of alcian blue-positive material, probably representing accreted polysaccharides. Similar antiphagocytic material was isolated from bovine serum, indicating that in biological fluids gonococci producing the heparin-binding Opa adhesin may become covered with externally derived polysaccharides as well. Binding assays with gonococci and epithelial proteoglycan receptors revealed that polysaccharides derived from agar or serum compete with the proteoglycans for binding of the heparin-binding Opa adhesin and thus act as receptor analogs. Growth of gonococci in a polysaccharide-free environment resulted in optimal proteoglycan receptor binding and rapid bacterial entry into Chang cells. The recognition that gonococci with certain phenotypes can recruit surface polysaccharides that determine in vitro infection dynamics adds a different dimension to the well-recognized biological significance of genetic variation for this pathogen.
Characterization of a new region required for macrophage killing by Legionella pneumophila
Authors: Segal G , Shuman H A
Published: January 2020

In a previous study, a collection of 55 Legionella pneumophila mutants defective for macrophage killing was isolated by transposon mutagenesis. In this study, nine of these mutants that belong to the same DNA hybridization group (group 3) were characterized. A wild-type DNA fragment that covers this DNA hybridization group was cloned and sequenced. This region was found to contain six new genes (designated icmT, icmS, icmR, icmQ, icmP, and icmO), five of which contain at least one transposon insertion. No transposon insertion was found in icmS. However, this gene was found to be required for macrophage killing, since a kanamycin resistance cassette introduced into icmS by gene replacement resulted in a mutant that was attenuated for macrophage killing. A plasmid containing the DNA fragment that covers this region complements all the mutants for macrophage killing, although various levels of complementation were observed for mutants in different genes. Complementation tests were also performed with plasmids containing one or two of these genes, as well as with plasmids containing nonpolar in-frame deletions. The results from these complementation tests indicated that all six genes located in this region are needed for macrophage killing and that they are probably arranged as two transcriptional units (icmTS and icmPO) and two genes (icmR and icmQ). A region upstream of the coding sequence of several icm genes may contain a potential promoter and/or regulatory site. Homology searches show that icmP and icmO bear significant homology to the trbA and trbC genes from the Salmonella R64 plasmid, respectively. The sequences of the other four genes do not show significant homology with any entries in sequence databases.
Escherichia coli cytolethal distending toxin blocks the HeLa cell cycle at the G2/M transition by preventing cdc2 protein kinase dephosphorylation and activation
Authors: Comayras C , Tasca C , Pérès S Y , Ducommun B , Oswald E , De Rycke J
Published: January 2020

Cytolethal distending toxins (CDT) constitute an emerging heterogeneous family of bacterial toxins whose common biological property is to inhibit the proliferation of cells in culture by blocking their cycle at G2/M phase. In this study, we investigated the molecular mechanisms underlying the block caused by CDT from Escherichia coli on synchronized HeLa cell cultures. To this end, we studied specifically the behavior of the two subunits of the complex that determines entry into mitosis, i.e., cyclin B1, the regulatory unit, and cdc2 protein kinase, the catalytic unit. We thus demonstrate that CDT causes cell accumulation in G2 and not in M, that it does not slow the progression of cells through S phase, and that it does not affect the normal increase of cyclin B1 from late S to G2. On the other hand, we show that CDT inhibits the kinase activity of cdc2 by preventing its dephosphorylation, an event which, in normal cells, triggers mitosis. This inhibitory activity was demonstrated for the three partially related CDTs so far described for E. coli. Moreover, we provide evidence that cells exposed to CDT during G2 and M phases are blocked only at the subsequent G2 phase. This observation means that the toxin triggers a mechanism of cell arrest that is initiated in S phase and therefore possibly related to the DNA damage checkpoint system.
A mutation of F47 to A in staphylococcus enterotoxin A activates the T-cell receptor Vbeta repertoire in vivo
Authors: Rosendahl A , Hansson J , Antonsson P , Sékaly R P , Kalland T , Dohlsten M
Published: January 2020

The bacterial superantigen staphylococcal enterotoxin A (SEA) binds with high affinity to major histocompatibility complex (MHC) class II molecules and subsequently activates T cells bearing particular T-cell receptor (TCR) Vbeta chains. Structural and mutational studies have defined two distinct MHC class II binding sites located in the N-terminal and C-terminal domains of SEA. The N-terminal F47 amino acid is critically involved in a low-affinity interaction to the MHC class II alpha-chain, while the C-terminal residues H187, H225, and D227 coordinate a Zn2+ ion and bind with moderate affinity to the beta-chain. In order to analyze whether the SEA-MHC class II alpha-chain interaction plays a role in dictating the in vivo repertoire of T-cell subsets, we studied distinct Vbeta populations after stimulation with wild-type SEA [SEA(wt)] and SEA with an F47A mutation [SEA(F47A)]. Injections of SEA(wt) in C57BL/6 mice induced cytokine release in serum, strong cytotoxic T-lymphocyte activity, expansion of T-cell subsets, and modulated expression of the T-cell activation antigens CD25, CD11a, CD44, CD62L, and CD69. SEA-reactive TCR Vbeta3+ and Vbeta11+ T cells were activated, while TCR Vbeta8+ T cells remained unaffected. The SEA(F47A) mutant protein induced a weaker T-cell response and failed to induce substantial interleukin-6 production compared to SEA(wt). Notably, SEA(F47A) failed to activate TCR Vbeta11+ T cells, whereas in vivo expansion and modulation of T-cell activation markers on TCR Vbeta3+ T cells were similar to those for SEA(wt). A similar response to SEA(F47A) was seen among CD4+ and CD8+ T cells. Activation of TCR Vbeta3+ and TCR Vbeta11+ T-cell hybridomas confirmed that SEA(F47A) activates TCR Vbeta3+ but not TCR Vbeta11+ T cells. The data support the view that the SEA-N-terminal MHC class II alpha-chain interaction defines a topology that is required for engagement of certain TCR Vbeta chains in vivo.
Contact-dependent disruption of the host cell membrane skeleton induced by Trichomonas vaginalis
Authors: Fiori P L , Rappelli P , Addis M F , Mannu F , Cappuccinelli P
Published: January 2020

This report presents evidence showing that the pathogenetic process of the protozoan parasite Trichomonas vaginalis involves degradation of the target cell membrane skeleton; spectrin, the most representative protein within this structure, has been identified as the main molecular target. Degradation of the target cell spectrin is accomplished only upon contact with the parasite, and immunochemical and immunofluorescence studies performed with the erythrocyte as a model demonstrate that degradation of the protein takes place before target cell lysis. A preliminary characterization of the effectors involved has led to the identification of a nonsecreted 30-kDa proteinase which is characterized by a high specificity for spectrin. This molecule is suggested as the main effector responsible for cytoskeletal disruption.
Systemic infection of mice by wild-type but not Spv- Salmonella typhimurium is enhanced by neutralization of gamma interferon and tumor necrosis factor alpha
Authors: Gulig P A , Doyle T J , Clare-Salzler M J , Maiese R L , Matsui H
Published: January 2020

The spv genes of the virulence plasmid of Salmonella typhimurium and other nontyphoidal serovars of S. enterica are involved in systemic infection by increasing the replication rate of the bacteria in host tissues beyond the intestines. We considered the possibility that the Spv virulence function is to evade suppression by the host response to infection. To examine this possibility, gamma interferon (IFN-gamma) and/or tumor necrosis factor alpha (TNF-alpha) were neutralized in BALB/c mice by intraperitoneal administration of monoclonal antibodies. Neutralization of IFN-gamma and/or TNF-alpha resulted in increased splenic infection with wild-type salmonellae after oral inoculation; however, Spv- salmonellae were defective at increasing splenic infection in cytokine-depleted mice. The use of a temperature-sensitive marker plasmid, pHSG422, indicated that neutralization of IFN-gamma caused less killing of wild-type S. typhimurium, while neutralization of TNF-alpha resulted in an increased in vivo replication rate for wild-type salmonellae. These results demonstrate that the Spv virulence function is not to evade suppression of bacterial infection normally mediated by IFN-gamma or TNF-alpha.
CD8+ T cells are activated during the early Th1 and Th2 immune responses in a murine Lyme disease model
Authors: Dong Z , Edelstein M D , Glickstein L J
Published: January 2020

T-helper responses following Borrelia burgdorferi infection in mice determine susceptibility to Lyme arthritis. The ratio of interleukin 4-positive, CD4+ to gamma interferon (IFN-gamma)-positive, CD4+ T cells was significantly greater in infected BALB/cJ mice than in infected C3H/HeJ mice. Increased numbers of IFN-gamma-producing cells predicted greater arthritis severity, and CD8+ T cells were the main source of IFN-gamma in both strains.
Interference of peptides and specific antibodies with the function of the Actinobacillus pleuropneumoniae transferrin-binding protein
Authors: Strutzberg K , Franz B , Gerlach G F
Published: January 2020

Multiple-antigenic peptides (MAPs) containing transferrin-binding domains of the Actinobacillus pleuropneumoniae serotype 7-derived transferrin-binding protein (TfbA) (K. Strutzberg, L. von Olleschik, B. Franz, C. Pyne, M. A. Schmidt, and G.-F. Gerlach, Infect. Immun. 63:3846-3850, 1995) were constructed. It was found that the MAPs inhibited transferrin binding of the recombinant TfbA protein, whereas antibodies directed against transferrin-binding domains failed to do so.
Characterization of lipoprotein IRP1 from Corynebacterium diphtheriae, which is regulated by the diphtheria toxin repressor (DtxR) and iron
Authors: Schmitt M P , Talley B G , Holmes R K
Published: January 2020

The Corynebacterium diphtheriae irp1 gene is negatively regulated by DtxR and iron. The nucleotide sequence of irp1 revealed that it has homology with genes involved in iron acquisition. Expression of the irp1 gene showed that it encodes a lipoprotein (IRP1) with a predicted size of 38 kDa. Northern blot experiments indicated that transcription from the irp1 promoter is repressed in high-iron medium and suggested that irp1 is part of an iron-regulated operon.
Role of gamma interferon in the host immune and inflammatory responses to Pneumocystis carinii infection
Authors: Garvy B A , Ezekowitz R A , Harmsen A G
Published: January 2020

The role of gamma interferon (IFN-gamma) in host defense to Pneumocystis carinii was investigated by use of three different murine models of infection. C57BL/6 scid/scid (severe combined immunodeficient [SCID]) mice were given intratracheal inoculations of P. carinii and reconstituted with splenocytes from either mice with disrupted IFN-gamma genes (IFN-gamma-/- mice) or homozygous wild-type (IFN-gamma+/+) mice. Unreconstituted SCID mice had log10 7.08 +/- 0.13 P. carinii nuclei in their lungs at day 22 postinfection, whereas SCID mice reconstituted with splenocytes from either wild-type or IFN-gamma-/- mice had cleared the infection. However, there was a prolonged and exacerbated inflammatory response in the lungs of SCID mice reconstituted with IFN-gamma-/- splenocytes which was characterized by interstitial pneumonia, eosinophilia, and multinucleated giant cell formation. Similar results were found in C.B17 SCID mice reconstituted with CD4+ cells from P. carinii-immunized donors treated with neutralizing anti-IFN-gamma monoclonal antibody (MAb). These mice resolved their P. carinii infections; however, they also exhibited exacerbated lung pathology compared with mice treated with a control MAb. Finally, IFN-gamma-/- mice challenged intratracheally with P. carinii resolved their infection within 56 days as did IFN-gamma+/- mice. Furthermore, depletion of T cells in vivo with a MAb resulted in IFN-gamma-/- mice becoming susceptible to P. carinii infection. Together, these data indicate that IFN-gamma is not required for resolution of P. carinii infection; however, in the absence of IFN-gamma, there is a prolonged and exacerbated P. carinii-driven interstitial pneumonia characterized by eosinophilia and formation of multinucleated giant cells.
Examination of diarrheagenicity of cytolethal distending toxin: suckling mouse response to the products of the cdtABC genes of Shigella dysenteriae
Authors: Okuda J , Fukumoto M , Takeda Y , Nishibuchi M
Published: January 2020

Some strains of Escherichia coli, Shigella spp., and Campylobacter spp. that have been implicated in diarrheal disease produce cytolethal distending toxin (CDT). CDT induces unique morphological changes in Chinese hamster ovary cells, but its association with diarrheal disease is unclear. We studied the diarrheagenicity of CDT using the cdt genes that we originally cloned from Shigella dysenteriae. The cdt genes were subcloned into a high-copy-number plasmid in E. coli JM109 to achieve high-level CDT production into the culture supernatant. An isogenic CDT- derivative was constructed by deletion of the 0.9-kb sequence internal to the cdt genes. A suckling mouse model was established, in which the intragastrically administered culture supernatant of the CDT+ E. coli strain induced excretion of loose and/or watery feces more often than did that of the CDT- strain in 24 h. The partially purified CDT preparation induced profuse watery diarrhea by 12 h in this model. High-level intestinal fluid accumulation in 4 h appeared to be related to the watery diarrhea. The results indicate that CDT is diarrheagenic to suckling mice and suggest that diarrheagenicity is dependent on CDT level. The preparations containing wild-type CDT induced tissue damage (necrosis and reparative hyperplasia) in the descending colon, whereas the tissues of the small intestines remained apparently intact. Association between the colonic damage and diarrhea is unclear and needs further investigation.
Ferric iron reduction by Cryptococcus neoformans
Authors: Nyhus K J , Wilborn A T , Jacobson E S
Published: January 2020

The pathogenic yeast Cryptococcus neoformans must reduce Fe(III) to Fe(II) prior to uptake. We investigated mechanisms of reduction using the chromogenic ferrous chelator bathophenanthroline disulfonate. Iron-depleted cells reduced 57 nmol of Fe(III) per 10(6) cells per h, while iron-replete cells reduced only 8 nmol of Fe(III). Exponential-phase cells reduced the most and stationary-phase cells reduced the least Fe(III), independent of iron status. Supernatants from iron-depleted cells reduced up to 2 nmol of Fe(III) per 10(6) cells per h, while supernatants from iron-replete cells reduced 0.5 nmol of Fe(III), implying regulation of the secreted reductant(s). One such reductant is 3-hydroxyanthranilic acid (3HAA), which was found at concentrations up to 29 microM in iron-depleted cultures but <2 microM in cultures supplemented with iron. Moreover, when washed and resuspended in low iron medium, iron-depleted cells secreted 20.4 microM 3HAA, while iron-replete cells secreted only 4.5 microM 3HAA. Each mole of 3HAA reduced 3 mol of Fe(III), and increasing 3HAA concentrations correlated with increasing reducing activity of supernatants; however, 3HAA accounted for only half of the supernatant's reducing activity, indicating the presence of additional reductants. Finally, we found that melanized stationary-phase cells reduced 2 nmol of Fe(III) per 10(6) cells per h--16 times the rate of nonmelanized cells--suggesting that this redox polymer participates in reduction of Fe(III).
Safety of live oral Salmonella typhi vaccine strains with deletions in htrA and aroC aroD and immune response in humans
Authors: Tacket C O , Sztein M B , Losonsky G A , Wasserman S S , Nataro J P , Edelman R , Pickard D , Dougan G , Chatfield S N , Levine M M
Published: January 2020

A single-dose, oral Salmonella typhi vaccine strain has been sought as a carrier or vector of cloned genes encoding protective antigens of other pathogens. Such a hybrid vaccine, administered orally, would stimulate immune responses both at the mucosal surface and in the systemic compartment and would potentially provide protection against multiple pathogens. S. typhi CVD 908 and CVD 906, which harbor deletions in aroC and aroD, were further engineered by deletion in htrA to produce strains CVD 908-htrA and CVD 906-htrA, which are unable to sustain growth and are severely impaired in their ability to survive in host tissues. These strains were fed to humans at doses of 5 x 10(7) to 5 x 10(9) CFU with buffer, and safety and immune responses were assessed. CVD 908-htrA and CVD 906-htrA were well tolerated in volunteers; mild diarrhea in 3 of 36 volunteers and mild fever in 1 volunteer were the only notable adverse responses. The vaccine strains were not detected in blood cultures and only transiently detected in stool. Serum immune responses to S. typhi lipopolysaccharide and H antigens were observed in 75 to 100% of volunteers who received 5 x 10(8) to 5 x 10(9) CFU, and cells secreting S. typhi-specific antibodies were found in all volunteers after ingestion of either strain. Sixty-three percent to 83% of volunteers developed lymphoproliferative responses to S. typhi flagellar and particulate antigens after the higher doses. These studies demonstrate the potential of CVD 908-htrA as a live vector for the delivery of heterologous genes, and a clinical trial of such a construct is planned.
Purification and characterization of Campylobacter rectus surface layer proteins
Authors: Nitta H , Holt S C , Ebersole J L
Published: January 2020

Campylobacter rectus is a putative periodontopathogen which expresses a proteinaceous surface layer (S-layer) external to the outer membrane. S-layers are considered to play a protective role for the microorganism in hostile environments. The S-layer proteins from six different C. rectus strains (five human isolates and a nonhuman primate [NHP] isolate) were isolated, purified, and characterized. The S-layer proteins of these strains varied in molecular mass (ca. 150 to 166 kDa) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They all reacted with monospecific rabbit antiserum to the purified S-layer of C. rectus 314, but a quantitative enzyme-linked immunosorbent assay demonstrated a strong antigenic relationship among the five human strains, while the NHP strain, 6250, showed weaker reactivity. Amino acid composition analysis showed that the S-layers of four C. rectus strains contained large proportions of acidic amino acids (13 to 27%) and that >34% of the amino acid residues were hydrophobic. Amino acid sequence analysis of six S-layer proteins revealed that the first 15 amino-terminal amino acids were identical and showed seven residues of identity with the amino-terminal sequence of the Campylobacter fetus S-layer protein SapA1. CNBr peptide profiles of the S-layer proteins from C. rectus 314, ATCC 33238, and 6250 confirmed that the S-layer proteins from the human strains were similar to each other and somewhat different from that of the NHP isolate (strain 6250). However, the S-layer proteins from the two human isolates do show some structural heterogeneity. For example, there was a 17-kDa fragment unique to the C. rectus 314 S-layer. The amino-terminal sequence of this peptide had homology with the C. rectus 51-kDa porin and was composed of nearly 50% hydrophobic residues. Thus, the S-layer protein from C. rectus has structural heterogeneity among different human strains and immunoheterogeneity with the NHP strain.
Identification, characterization, and immunogenicity of the lactoferrin-binding protein from Helicobacter pylori
Authors: Dhaenens L , Szczebara F , Husson M O
Published: January 2020

Iron acquisition plays an important role in bacterial virulence. Different studies have been initiated to define the mechanism by which Helicobacter pylori acquires iron. We had previously demonstrated that human lactoferrin (HLf) supported full growth of the bacteria in media lacking other iron sources. The ability of H. pylori to use HLf as an iron source had been found to be dependent on cell-to-protein contact. Since lactoferrin has been found in significant amounts in human stomach resection specimens from patients with superficial or atrophic gastritis, the iron uptake of H. pylori via a specific HLf receptor may play a major role in the virulence of H. pylori infection. In this study, by using affinity chromatography with biotinylated HLf and streptavidin-agarose, we identified a 70-kDa lactoferrin-binding protein (Lbp) from outer membrane proteins of H. pylori. This Lbp was only present when H. pylori was grown in an iron-starved medium, suggesting that it serves in iron uptake. Direct binding assays with increasing concentrations of biotinylated HLf demonstrated that the lactoferrin interaction with the outer membrane of H. pylori grown in iron-restricted medium was saturable. Competitive binding experiments with bovine and human lactoferrin and with transferrin of horse, bovine, and human origin indicated that this Lbp appeared highly specific for HLf. A number of other studies have focused on the importance of transferrin and lactoferrin receptors in pathogenic bacteria and their specificity with the host species. This observation might explain the very strict human specificity of H. pylori.