bluuebunny/crossref_metadata_2025 huggingface dataset:
Non-O1 Vibrio cholerae NRT36S produces a polysaccharide capsule that determines colony morphology, serum resistance, and virulence in mice
Authors: Johnson J A , Panigrahi P , Morris J G
Published: January 2020

Non-O1 Vibrio cholerae produced two distinct colony types, designated as opaque and translucent. NRT36S, a clinical isolate shown to be virulent in volunteers, produced predominantly opaque colonies, but translucent colonies appeared on subculture. Opaque variants were recovered exclusively following exposure to normal human serum or animal passage. A nonreverting translucent mutant of NRT36S, JVB52, was isolated following mutagenesis with the transposon Tn5 IS50L::phoA (TnphoA). Only translucent colonies were produced by a nonpathogenic environmental isolate, A5. Electron microscopic examination of the opaque form of NRT36S revealed thick, electron-dense, fibrous capsules surrounding polycationic ferritin-stained cells. The ferritin-stained material around translucent NRT36S or A5 was patchy or absent. JVB52 had a thin but contiguous capsular layer. The amount of ferritin-stained capsular material correlated with the amount of surface polysaccharide determined by phenol-sulfuric acid assay: opaque NRT36S had approximately three times as much polysaccharide as translucent NRT36S or A5 and four times as much as JVB52. The encapsulated, opaque variant of NRT36S was protected from serum bactericidal activity, while translucent non-O1 V. cholerae was readily killed. The encapsulated form also had increased virulence in mice. Our data provide the first indication that non-O1 V. cholerae strains can have a polysaccharide capsule. This capsule may be important in protecting the organism from host defenses and may contribute to the ability of some non-O1 V. cholerae strains to cause septicemia in susceptible hosts.
Growth of Neisseria gonorrhoeae in CMP-N-acetylneuraminic acid inhibits nonopsonic (opacity-associated outer membrane protein-mediated) interactions with human neutrophils
Authors: Rest R F , Frangipane J V
Published: January 2020

Gonococci possessing certain opacity-associated (Opa) outer membrane proteins adhere to and are phagocytosed by human neutrophils in the absence of serum. Recently, it has been shown that serum-sensitive strains of Neisseria gonorrhoeae possessing the appropriate lipooligosaccharide phenotype become serum resistant when grown in the presence of CMP-N-acetylneuraminic acid (CMP-NANA) because of sialylation of their lipooligosaccharide. We investigated whether such sialylation affects nonopsonic (antibody- and complement-independent) interactions of gonococci with human neutrophils in vitro. We grew Opa+ gonococci in the presence of up to 50 micrograms of CMP-NANA per ml, incubated them with neutrophils in vitro, and measured their abilities to adhere to neutrophils, stimulate neutrophil luminol-dependent chemiluminescence (LDCL), and be phagocytically killed by neutrophils. Growth in CMP-NANA dramatically inhibited (in a dose-dependent manner) the ability of Opa+ gonococci to adhere to neutrophils and stimulate neutrophil LDCL. Growth of Opa+ gonococci in 50 micrograms of CMP-NANA per ml appeared to delay, but did not inhibit, their killing by neutrophils. Sialidase treatment of sialylated Opa+ gonococci, i.e., gonococci grown with CMP-NANA, totally restored their abilities to adhere to neutrophils and stimulate neutrophil LDCL. Opa- gonococci grown in the presence of 50 micrograms of CMP-NANA per ml and opsonized with fresh human serum bound to neutrophils only about 30% less efficiently than did Opa- gonococci grown without CMP-NANA and opsonized. The results of our studies show that sialylated Opa+ gonococci have dramatically reduced nonopsonic interactions with neutrophils. Some gonococcal strains may resist killing by human neutrophils in vivo by such a mechanism.
Protein D, the immunoglobulin D-binding protein of Haemophilus influenzae, is a lipoprotein
Authors: Janson H , Hedén L O , Forsgren A
Published: January 2020

Protein D is an immunoglobulin D-binding membrane protein exposed on the surface of the gram-negative bacterium Haemophilus influenzae. Results reported here indicate that protein D is a lipoprotein. The protein is apparently synthesized as a precursor with an 18-residue-long signal sequence modified by the covalent attachment of both ester-linked and amide-linked palmitate to the cysteine residue, which becomes the amino terminus after cleavage of the signal sequence. Globomycin inhibited maturation of protein D in H. influenzae, implying that protein D is exported through the lipoprotein export pathway. A mutant expressing a protein D lacking the cysteine residue was constructed by oligonucleotide site-directed mutagenesis. The mutated protein D molecule was not acylated and partitioned in the aqueous phase after Triton X-114 extraction of intact bacteria, unlike native and recombinant protein D, which partitioned in the detergent phase. The nonacylated protein D molecule was localized to the periplasmic space of Escherichia coli. The hydrophilic protein D molecule will be used in investigations concerning its ability to function as a vaccine component.
Use of restriction endonuclease analysis and ribotyping to study epidemiology of Pasteurella multocida in closed swine herds
Authors: Zhao G , Pijoan C , Murtaugh M P , Molitor T W
Published: January 2020

One hundred and sixty-four clinical isolates of Pasteurella multocida recovered from two swine herds in Minnesota were characterized by restriction endonuclease analysis (REA) and rRNA gene restriction fragment length patterns. Bacterial DNA was digested with HpaII and electrophoresed in 0.55% agarose. Restriction fragments were transferred by Southern blot to nylon membranes and then hybridized with digoxigenin-dUTP-labeled Escherichia coli rRNA. Four different REA patterns were observed among the 156 serotype A strains isolated from herds A and B. The two most common REA types (1 and 2) represented 92% of the strains analyzed, while REA types 3 and 4 were observed only in lung samples and accounted for 8% of the isolates. Two different ribotypes were observed for these serotype A isolates. Ribotype I consisted of the most common types, 1 and 2, found by DNA fingerprinting. Ribotype II included REA types 3 and 4. Results from both herds suggest that in closed swine populations, a single strain of P. multocida predominates and causes disease. It is concluded that these genomic fingerprinting techniques were highly discriminatory and that capsular serotyping in combination with REA or ribotyping is an appropriate technique for epidemiological studies of P. multocida of swine origin.
Influence of immunization on Porphyromonas gingivalis colonization and invasion in the mouse chamber model
Authors: Genco C A , Kapczynski D R , Cutler C W , Arko R J , Arnold R R
Published: January 2020

The effects of immunization with invasive or noninvasive Porphyromonas (Bacteroides) gingivalis strains on the pathogenesis of infection in a mouse chamber model were examined. BALB/c mice were immunized by a single injection of heat-killed P. gingivalis invasive strain A7436 or W83 or noninvasive strain 33277, HG405, or 381 directly into subcutaneous chambers. P. gingivalis-specific antibody was detected in chamber fluid 21 days postimmunization, and mice were subsequently challenged by injection of exponential-phase P. gingivalis into chambers. Immunization with A7436 or W83 followed by challenge with A7436 protected mice against secondary abscess formation and death; however, P. gingivalis persisted in chambers for up to 14 days postchallenge. Immunization with noninvasive strain 33277, HG405, or 381 followed by challenge with invasive strain A7436 or W83 protected mice against secondary lesion formation and death. P. gingivalis was cultured from 33277- or HG405-immunized and nonimmunized animals to day 14. All P. gingivalis strains induced an immunoglobulin G response, as measured by an enzyme-linked immunosorbent assay and Western immunoblotting of P. gingivalis whole-cell and outer membrane protein preparations. Western blot analyses indicated that sera from mice immunized with different invasive and noninvasive strains recognized common P. gingivalis antigens. In summary, immunization with invasive P. gingivalis A7436 and W83 or noninvasive P. gingivalis 33277, HG405, and 381 protected mice from secondary lesion formation and death after challenge with invasive P. gingivalis A7436 or W83. P. gingivalis-specific antibody did not, however, inhibit the colonization of P. gingivalis within chambers.
Hydrophobic surface protein masking by the opportunistic fungal pathogen Candida albicans
Authors: Hazen K C , Hazen B W
Published: January 2020

Ultrastructural and biochemical analyses of hydrophobic and hydrophilic yeast cell surface proteins of Candida albicans were performed. Hydrophobic and hydrophilic yeast cells were obtained by growth at 23 and 37 degrees C, respectively. In addition, hydrophilic yeast cells were converted to surface hydrophobicity by treatment with tunicamycin and dithiothreitol. When freeze-etched cells were examined, the temperature-induced hydrophilic cells had long (0.198 micron), compact, evenly distributed fibrils while temperature-induced hydrophobic cells had short (0.085 micron), blunt fibrils. Hydrophobic microsphere attachment to the hydrophobic cells occurred at the basement of and within the short fibril layer. Dithiothreitol-induced hydrophobic cells had the long fibrils removed; tunicamycin-induced hydrophobic cells retained some of the long fibrils, but the fibrils were less compact and more aggregated than the untreated controls. These results suggest that the long fibrils prevent hydrophobic microsphere attachment to the hydrophobic area of the cell surface. This was confirmed by assessing the hydrophobic avidity of hydrophobic yeast cell populations differing in fibril density and arrangement. 125I-labelled surface proteins from hydrophobic and hydrophilic cells were compared after separation by hydrophobic interaction chromatography-high-performance liquid chromatography and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The yeast cell populations had hydrophilic proteins of similar molecular masses (greater than 200 kDa), but the hydrophilic cells possessed at least two additional proteins (ca. 63 and 69 to 71 kDa). Hydrophobic surface proteins appeared to be similar. However, the amount of total radiolabelled hydrophobic proteins was approximately 10-fold higher for the hydrophobic cells than for the hydrophilic cells. This result agrees with the ultrastructural observations which showed that yeast cell surface hydrophobic proteins are masked by hydrophilic high-molecular-mass surface fibrils. Taken together, the data indicate that yeast cell hydrophobicity is not determined by differences in surface hydrophobic proteins but by the presence of hydrophilic, surface fibrils.
Identification of a second hemolysin (HlyII) in Actinobacillus pleuropneumoniae serotype 1 and expression of the gene in Escherichia coli
Authors: Frey J , van den Bosch H , Segers R , Nicolet J
Published: January 2020

Hemolysin genes of the reference strains of Actinobacillus pleuropneumoniae serotypes 1 and 2 were identified, cloned, and expressed in Escherichia coli by using polymerase chain reaction amplification with oligonucleotides derived from the DNA sequence of the corresponding appA gene from A. pleuropneumoniae serotype 5. The three genes from serotypes 1, 2, and 5 have identical restriction maps and appear to encode a hemolysin which was previously identified in serotype 2 and designated HlyII. Gene appA is different from hlyIA encoding the major hemolysin type I (HlyI) which was identified earlier in serotype 1. Polymerase chain reaction amplification with oligonucleotides derived from the DNA sequence of hlyIA of serotype 1 showed that the gene encoding HlyI is present in serotype 1 but not in serotype 2, in contrast to the gene encoding HlyII that was present in both serotypes. This was confirmed by Western blot (immunoblot) experiments using monoclonal antibodies specific for either recombinant HlyI or recombinant HlyII, which showed that A. pleuropneumoniae serotype 1 strain 4074 produces both HlyI and HlyII, whereas serotype 2 strain S1536 produces only HlyII. The expression of both hemolysins was investigated in all serotypes by the use of monoclonal antibodies. HlyI was shown to be expressed by the reference strains of serotypes 1, 5a, 5b, 9, 10, and 11, whereas HlyII was shown to be expressed by the reference strains of all 12 serotypes tested except serotype 10. A. pleuropneumoniae serotype 1 strain 4074 is the first bacterium which has been shown to contain two different actively expressed RTX toxin genes. Comparison of our data with those from other groups shows that the originally described strongly hemolytic hemolysin type I (HlyI) corresponds to cytolysin I (ClyI) which was recently described by others, while the weakly hemolytic hemolysin type II (HlyII) seems to be identical to ClyII and AppA.
Consequences of microbial attachment: directing host cell functions with adhesins
Authors: Hoepelman A I , Tuomanen E I
Published: January 2020

We take the view that adherence is not just a static process of holding hands but rather elicits a response in the targeted cell. From this point of view, adherence is an active process with an outcome. This outcome or fate is predictable only when several parameters of the host cell-adhesin interaction are known: is the adhesin acting alone or in series with other products, is the receptor up- or down-regulated at the time of ligation, which domain of the receptor is bound, and finally, which intracellular response circuits are connected to the receptor in the cell type targeted? Variations in these parameters are the basis for the ability of the adhesins of pathogens to orchestrate outcomes as disparate as simple address recognition versus actin nucleation, cytokine induction, activation of plasmin, derangement of leukocyte migration, or deposition of antibody on host cell membranes. The recognition of the relatedness of some eukaryotic and prokaryotic adhesive domains and the shared use of existing eukaryotic cell-cell interaction systems between host and pathogen suggest that the cellular interactions of interest in eukaryotic cell biology can be revealed by taking clues from the pathogens, which have studied and adapted to them the longest.
A protective role for endogenous tumor necrosis factor in Toxoplasma gondii infection
Authors: Johnson L L
Published: January 2020

The involvement of tumor necrosis factor (TNF) in resistance to Toxoplasma gondii infection was examined by means of experiments in which mice were treated with anti-TNF antibodies prior to infection with ME49, a low-virulence Toxoplasma strain. In (BALB/cBy x C57BL/6J)F1 (CB6F1) mice, which are highly resistant to intraperitoneal (i.p.) infection with T. gondii ME49, 10(4) neutralizing units of anti-TNF caused a significant increase in trophozoite numbers in the peritoneal cavities of infected mice and transient signs of illness but no deaths. i.p. infection of anti-TNF-treated C57BL/6J (B6) mice, which are more susceptible to T. gondii and develop a chronic progressive toxoplasmosis, resulted in death for some of the mice. If the mice were infected perorally, however, and treated with anti-TNF, mortality was extensive in B6 mice but not in CB6F1 mice. Although it was not detected in their sera, TNF was found in the peritoneal fluids of i.p.-infected CB6F1 and B6 mice. Endogenously produced TNF thus appears to be an important mediator of resistance to T. gondii.
Molecular characterization of an RTX toxin determinant from Actinobacillus suis
Authors: Burrows L L , Lo R Y
Published: January 2020

RTX cytolysins are a family of calcium-dependent, pore-forming, secreted toxins found in a variety of gram-negative bacteria. The prototypical member of this family is the alpha-hemolysin of Escherichia coli. The RTX genetic determinants from seven members of the family Pasteurellaceae, Pasteurella haemolytica, Actinobacillus actinomycetemcomitans, and A. pleuropneumoniae serotypes 1,5,7, and 9 were previously cloned and sequenced. Using the leukotoxin determinant from P. haemolytica serotype A1 as a probe, we detected the presence of RTX-type determinants in Actinobacillus suis, A. equuli, and A. lignieresii of the family Pasteurellaceae. All three species elaborate proteins of approximately 104 to 110 kDa that are recognized by polyclonal antisera against the 104-kDa hemolysin of A. pleuropneumoniae serotype 1. An RTX determinant of A. suis isolate 3714 was cloned and sequenced and was found to be almost identical to the RTX determinant of A. pleuropneumoniae serotypes 5 and 9. In addition, the determinant is not composed of four contiguous genes, as had been reported for most other RTX determinants; instead, the genes encoding the two proteins responsible for secretion of the toxin are at a locus distinct from that containing the toxin structural and activation genes.
Gene block encoding production of cytotoxic necrotizing factor 1 and hemolysin in Escherichia coli isolates from extraintestinal infections
Authors: Falbo V , Famiglietti M , Caprioli A
Published: January 2020

Cytotoxic necrotizing factors (CNFs) are Escherichia coli protein toxins causing cell multinucleation and enlargement in tissue cultures and necrosis in rabbit skin. In E. coli isolates causing urinary tract infections in humans, the production of CNF1 is closely associated with hemolysin production. In this study, we obtained data suggesting that this phenotypic association is due to the genetic linkage of the determinants of the two toxins on the chromosome of uropathogenic E. coli. The genes encoding hemolysin and CNF1 were shown to be closely linked in a 37-kb cloned DNA fragment from an E. coli urinary tract isolate of serotype O4:K12:H5 (E-B35). A DNA region encoding CNF1 production but not hemolysin production was further subcloned as a 12-kb SalI-EcoRI fragment and used as a CNF1-specific gene probe. DNA hybridization experiments indicated that the CNF1 and hemolysin determinants were closely linked on the chromosomes of isolate E-B35 and six additional extraintestinal isolates belonging to serogroups O2, O4, O6, O22, O75, and O85.
Novel invasion determinant of enteropathogenic Escherichia coli plasmid pLV501 encodes the ability to invade intestinal epithelial cells and HEp-2 cells
Authors: Fletcher J N , Embaye H E , Getty B , Batt R M , Hart C A , Saunders J R
Published: January 2020

An Escherichia coli K-12 transformant carrying 96.5-kb plasmid pLV501 from enteropathogenic E. coli (EPEC) strain K798 is able to produce the same characteristic attaching-effacing lesions in a rabbit ileal biopsy explant model as its parent strain. Cloned EcoRI-SalI DNA restriction fragments from this plasmid failed to reproduce the attaching-effacing lesions, but one recombinant plasmid, pLV527, containing 4.5 kb of pLV501 DNA, conferred on E. coli DH1 transformants the ability to invade enterocytes in the rabbit explant model. DH1(pLV527) was also able to adhere to and invade HEp-2 cells. The relative invasive ability of DH1(pLV527) was quantified by recovery of internalized bacteria following gentamicin treatment of infected HEp-2 monolayers. DH1(pLV527) was 1,000-fold more invasive than DH1 carrying pBR322 or a recombinant plasmid which had no physiological effect on ileal biopsy explants but was less invasive than an enteroinvasive E. coli strain or a transformant carrying the cloned invasion genes of Shigella flexneri. Invasion by DH1(pLV501) could also be detected but occurred at a level 30 times lower than that by DH1(pLV527). Colony-hybridization of the pLV527 insert against a panel of 49 EPEC and related strains revealed that only 11 contained pLV527-hybridizing sequences; thus, the invasion determinant is not an essential component of the attachment-effacement pathogenic mechanism. One pLV527-hybridizing strain displayed both attachment-effacement and invasiveness in the rabbit ileal biopsy explant model. No significant hybridization was observed to non-EPEC invasive pathogenic enteric bacteria, indicating that the invasion determinant encoded on pLV527 is distinct from those used by these organisms.
Immune responses of systemic and mucosal lymphoid organs to Pnu-Imune vaccine as a function of age and the efficacy of monophosphoryl lipid A as an adjuvant
Authors: Garg M , Subbarao B
Published: January 2020

A murine model system was established to study immune responses to the Pnu-Imune vaccine, which is made up of 23 different pneumococcal capsular polysaccharides. In this animal model, antibody-forming cell responses to 21 of 23 individual polysaccharides in the vaccine were detected. The Pnu-Imune vaccine elicited good antibody responses from the spleens and mesenteric lymph nodes (MLN) of young mice, whereas a variety of other peripheral lymph nodes were unresponsive. The immunoglobulin M plaque-forming cell (PFC) response in the spleen to the Pnu-Imune vaccine (given intraperitoneally or subcutaneously) decreased dramatically with increasing age. However, the spleen and MLN differed in their susceptibility to an age-associated decline in immune function. While the PFC responses in the spleen declined with age, the PFC response in the mucosa-associated MLN did not decline with age but instead remained constant over the entire age span of 4 to 28 months studied. These studies showed that the spleen, peripheral lymph nodes, and MLN did not demonstrate parallel age-associated defects in antibody responses to pneumococcal polysaccharides when the antigen was administered systematically. Also, the deficient splenic antibody response to Pnu-Imune vaccine in aged mice could be enhanced by injecting a combination of Pnu-Imune vaccine and the nontoxic adjuvant monophosphoryl lipid A. Moreover, an immunoglobulin G response was induced when the immunogen was a mixture of vaccine and adjuvant.
Common antigenic domains in transferrin-binding protein 2 of Neisseria meningitidis, Neisseria gonorrhoeae, and Haemophilus influenzae type b
Authors: Stevenson P , Williams P , Griffiths E
Published: January 2020

There is now considerable evidence to show that in the Neisseria and Haemophilus species, membrane receptors specific for either transferrin or lactoferrin are involved in the acquisition of iron from these glycoproteins. In Neisseria meningitidis, the transferrin receptor appears to consist of two proteins, one of which (TBP 1) has an M(r) of 95,000 and the other of which (TBP 2) has an M(r) ranging from 68,000 to 85,000, depending on the strain; TBP 2 binds transferrin after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting, but TBP 1 does not do so. The relative contributions of these two proteins to the binding reaction observed with intact cells and to iron uptake are presently unknown. However, they are being considered as potential components of a group B meningococcal vaccine. Analogous higher- and lower-molecular-weight proteins associated with transferrin binding have been found in N. gonorrhoeae and Haemophilus influenzae. Previous work with polyclonal antibodies raised in mice with whole cells of iron-restricted N. meningitidis showed that the meningococcal TBP 2 exhibits considerable antigenic heterogeneity. Here, we report that antiserum against purified TBP 2 from one strain of N. meningitidis cross-reacts on immunoblotting with the TBP 2 of all meningococcal isolates examined, as well as with the TBP 2 of N. gonorrhoeae. This antiserum also cross-reacted with the TBP 2 of several strains of H. influenzae type b, thus showing the presence of common antigenic domains among these functionally equivalent proteins in different pathogens; no cross-reaction was detected with a purified sample of the human transferrin receptor.
Protective effects of a leukotriene inhibitor and a leukotriene antagonist on endotoxin-induced mortality in carrageenan-pretreated mice
Authors: Ogata M , Matsumoto T , Kamochi M , Yoshida S I , Mizuguchi Y , Shigematsu A
Published: January 2020

The leukotrienes and tumor necrosis factor (TNF) play an important role in the pathophysiology of septic shock, in which hypotension, leukopenia, thrombocytopenia, and hemoconcentration are observed. This study was performed to examine the effects of a 5-lipoxygenase inhibitor (AA-861), a selective leukotriene receptor antagonist (ONO-1078), and a cyclooxygenase inhibitor (indomethacin) on endotoxin-induced mortality and TNF production in mice. Mice were injected intraperitoneally with carrageenan (5 mg per mouse), which we previously reported as an effective priming agent for lipopolysaccharide (LPS)-induced TNF production and mortality (M. Ogata, S. Yoshida, M. Kamochi, A. Shigematsu, and Y. Mizuguchi, Infect. Immun. 59:679-683, 1991). The indicated doses of AA-861, ONO-1078, indomethacin, or controls were administrated subcutaneously 30 min before LPS (50 micrograms per mouse) provocation. The mortality of mice was significantly decreased by pretreatment with AA-861 (P less than 0.001) or ONO-1078 (P less than 0.01) but not by pretreatment with indomethacin. The 50% lethal dose of LPS in the mice treated with dimethyl sulfoxide or ethanol was 32 or 33 micrograms, respectively, and it increased to 83 micrograms with AA-861 or 59 micrograms with ONO-1078, respectively. Neither AA-861 nor ONO-1078 suppressed LPS-induced TNF production in sera. Treatment with AA-861 significantly decreased the leukopenia and thrombocytopenia, and ONO-1078 significantly decreased the hemoconcentration and thrombocytopenia. The role of endogenous TNF was also examined in the carrageenan-pretreated mice. Treatment with 2 x 10(5) U of rabbit anti TNF-alpha antibody intravenously 2 h before LPS challenge significantly suppressed the LPS-induced TNF activity and decreased the mortality. Therefore, both leukotrienes and TNF play important roles in endotoxin-induced shock and mortality.
Characterization of the immunoglobulin A protease of Ureaplasma urealyticum
Authors: Spooner R K , Russell W C , Thirkell D
Published: January 2020

Ureaplasma urealyticum strains of all serotypes express a specific human immunoglobulin A1 protease that cleaves immunoglobulin A1 to produce intact Fab and Fc fragments. The use of a variety of inhibitors suggests that the enzyme is a serine protease. N-terminal sequencing of the Fc digestion product showed that the enzyme cleaves between the proline and threonine residues 235 and 236 in the hinge region of the heavy chain of immunoglobulin A1.
The coccidioidal complement fixation and immunodiffusion-complement fixation antigen is a chitinase
Authors: Johnson S M , Pappagianis D
Published: January 2020

Culture filtrates and autolysates of Coccidioides immitis have provided suitable crude antigens for the serodiagnosis and prognosis of coccidioidomycosis. One of these, a heat-labile antigen which participates in the immunodiffusion reaction corresponding to the complement fixation reaction (IDCF), has been characterized as a 110-kDa native protein that, when subjected to reducing conditions and heat, yields a 48-kDa component. The present report provides serologic and biochemical evidence that this antigen is a chitinase. This chitinase, isolated from 48-h culture filtrate of the spherule-endospore-phase C. immitis by affinity adsorption to chitin, formed a line of identity with the IDCF reference antigen and participated in the complement fixation reaction with human serum. It lost its enzymatic as well as antigenic activity when heated, but when not heated it retained its enzymatic activity even when precipitated with coccidiodal antibody present in human serum. This chitinase represents a significant serodiagnostic substance and may be important in the morphogenesis of C. immitis.
Capacity of listeriolysin O, streptolysin O, and perfringolysin O to mediate growth of Bacillus subtilis within mammalian cells
Authors: Portnoy D A , Tweten R K , Kehoe M , Bielecki J
Published: January 2020

The Listeria monocytogenes hemolysin listeriolysin O (LLO) plays a major role in mediating the escape of L. monocytogenes from a vacuolar compartment. In a previous report, it was shown that Bacillus subtilis expressing LLO could escape from a host vacuolar compartment and grow in the cytoplasm (J. Bielecki, P. Youngman, P. Connelly, and D. A. Portnoy, Nature [London] 345:175-176, 1990). In the present study, two related thiol-activated hemolysins, streptolysin O (SLO) and perfringolysin O (PFO), were expressed in B. subtilis and their ability to mediate intracellular growth was monitored by visual inspection and by assaying for CFU. Like LLO, PFO was active within the vacuolar environment, whereas SLO showed negligible activity. However, expression of PFO seemed to damage the host cells. The pH of the vacuole probably had little to do with these results, since all three hemolysins showed full or enhanced activity at pH 5.5, although LLO showed greatly reduced activity at pH 7. In addition, neutralization of the pH within host vacuoles by using weak bases had little effect on the lysis of the vacuole. The lack of SLO activity is probably caused by its lower specific activity; the purified protein had 10-fold less activity on a molar basis. These results suggest that LLO is not unique in its capacity to mediate intracellular growth of B. subtilis.
Role of T-lymphocyte subsets in Rhodococcus equi infection
Authors: Nordmann P , Ronco E , Nauciel C
Published: January 2020

Rhodococcus equi, a facultative intracellular gram-positive bacterium, can induce life-threatening infections in immunocompromised patients, especially those with AIDS. We have studied the mechanism of acquired immunity to this pathogen in a murine model. Protective immunity was induced by live but not killed bacteria. Adoptive transfer of resistance was obtained with spleen cells but not immune serum from mice immunized intravenously 30 days earlier with live bacteria. In normal mice, an intravenous challenge of 5 x 10(6) CFU of R. equi was cleared from the spleen, liver, and lungs within 3 weeks, whereas athymic nude mice were unable to clear the bacteria. In vivo depletion with monoclonal antibodies showed that both CD4+ and CD8+ T-cell subsets participate in the clearance of bacteria and that CD8+ T cells play the major role.
Interleukin-8 in sepsis: relation to shock and inflammatory mediators
Authors: Hack C E , Hart M , van Schijndel R J , Eerenberg A J , Nuijens J H , Thijs L G , Aarden L A
Published: January 2020

Because of its neutrophil-activating properties, interleukin-8 (IL-8) may play an important role in the pathophysiology of sepsis. We measured circulating IL-8 levels in 47 patients with clinical sepsis. Levels on admission were elevated in 42 of the 47 patients (89%) and were comparable in patients with gram-positive or gram-negative infections. Patients with shock had significantly higher IL-8 levels than normotensive patients (P = 0.0014, Wilcoxon-Mann-Whitney test), whereas no differences in IL-8 levels were found between patients with or without adult respiratory distress syndrome. Patients who died had higher IL-8 levels on admission than the patients who survived. The largest differences in IL-8 levels between survivors and nonsurvivors was found when only patients with positive cultures were considered (P = 0.0342). IL-8 levels appeared to correlate significantly with lactate levels and inversely with leukocyte and platelet numbers and mean arterial pressure. In addition, the IL-8 level in the sepsis patients was found to correlate significantly with levels of IL-6, elastase-alpha 1-antitrypsin, and C3a. Serial observations revealed that in most patients IL-8 levels decreased, irrespective of the outcome. Thus, our results demonstrate that IL-8 levels are increased in most patients with sepsis and correlate with some important clinical, biochemical, and inflammatory parameters. These findings suggest a role for IL-8 in the pathophysiology of sepsis.
Interaction of Mycoplasma dispar with bovine alveolar macrophages
Authors: Almeida R A , Wannemuehler M J , Rosenbusch R F
Published: January 2020

The capacity to avoid phagocytosis and the activation of bovine alveolar macrophages (BAM) by encapsulated Mycoplasma dispar or purified M. dispar capsule was investigated. Encapsulated and unencapsulated M. dispar were cocultured with BAM in the presence or absence of antisera prepared against unencapsulated M. dispar or purified capsule antiserum. Unopsonized mycoplasmas resisted phagocytosis, while only anti-capsule antibodies enhanced the phagocytosis of encapsulated mycoplasmas. BAM were cultured in the presence of purified M. dispar capsule or either live or heat-killed encapsulated or unencapsulated M. dispar. These BAM were then activated with Escherichia coli endotoxin or left without further activation. The supernatants of these cultures were assayed for tumor necrosis factor, interleukin 1, and glucose consumption as indicators of macrophage activation. Tumor necrosis factor and interleukin 1 were produced by BAM stimulated with unencapsulated M. dispar but not when encapsulated M. dispar or its purified capsule was used. Similarly, glucose consumption was increased in the presence of unencapsulated M. dispar, but not when BAM were cocultured with encapsulated M. dispar or purified capsule. When BAM were treated with purified capsule or encapsulated mycoplasmas, they could not be subsequently activated by endotoxin. These results indicate that encapsulated M. dispar or purified capsule exerts an inhibitory effect on the activity of BAM and prevents the activation of these cells.
Histopathological and serological progression of experimental Staphylococcus aureus arthritis
Authors: Bremell T , Abdelnour A , Tarkowski A
Published: January 2020

In a newly developed mouse model of Staphylococcus aureus arthritis the kinetics of joint destruction and serological manifestations as well as the clinical course of arthritis and osteitis were studied. Almost all mice developed histopathological signs of arthritis upon a single intravenous injection of 10(7) S. aureus LS-1 cells. There was rapid joint destruction, with synovial hypertrophy already visible, within 24 h after injection of the bacteria. Cartilage and/or bone erosions were seen in a majority of the mice within 72 h. Extra-articular manifestations, especially signs of bone infection, were also found soon after inoculation of the bacteria. Tail osteitis was frequent (50% of the mice) but appeared later than arthritis. Polymorphonuclear cells prevailed in the early joint lesions and were also common in the extra-articular manifestations. Within 3 days, mononuclear cells were also seen in the inflamed synovium, gaining a dominant position 3 weeks after the start of the disease. Serum interleukin-6 levels were already increased within 6 h after bacterial injection and remained elevated throughout the course of arthritis. Serum tumor necrosis factor levels were increased within 24 h. There was a tremendous induction of immunoglobulin production, especially of the immunoglobulin G1 isotype. This was paralleled by the production of specific antibodies to S. aureus (cell walls and toxin), as well as autoantibodies (rheumatoid factors and anti-single-stranded DNA antibodies), all predominantly of the immunoglobulin G isotype. The type and magnitude of the immunoglobulin G response together with the elevated interleukin-6 levels speak in favor of both antigen-specific and polyclonal B-cell activation during S. aureus arthritis. This study points out important similarities between our new model of S. aureus arthritis and human S. aureus arthritis. This resemblance will enable controlled studies of pathogenetic mechanisms of septic arthritis as well as therapeutic and prophylactic approaches.
Identification of eNAP-1, an antimicrobial peptide from equine neutrophils
Authors: Couto M A , Harwig S S , Cullor J S , Hughes J P , Lehrer R I
Published: January 2020

Endogenous, cysteine-rich antimicrobial peptides known as defensins are prominent components of human, rabbit, and rat neutrophils, yet little is known about their occurrence in other mammalian species. Although we did not detect mature (i.e., processed) defensins in equine neutrophil granules, we found that these granules contained small amounts of other cysteine-rich peptides with antimicrobial activity. One of these, eNAP-1, was purified by a combination of gel permeation and reversed-phase high-performance liquid chromatography from acid extracts prepared from the cytoplasmic granules of equine neutrophils. The molecular mass of eNAP-1 was approximately 7.2 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Amino acid analysis revealed that eNAP-1 had an unusually high cysteine content and that it was relatively enriched in alanine, glycine, lysine, and proline residues. The partial (N-terminal) amino acid sequence of eNAP-1 was DVQCGEGHFCHDXQTCCRASQGGXACCPYSQGVCCADQRHCCPVGF. Thirty-six of these residues (78.3%) were identical to those of a recently cloned human neutrophil peptide of unknown function and belonging to the granulin family. Homologous peptides have also been noted in rat bone marrow cells and rat kidney epithelins. We tested the ability of eNAP-1 to kill several equine uterine pathogens. Streptococcus zooepidemicus was killed most effectively, sustaining a greater than 99.8% decrease in CFU per milliliter after a 2-h exposure to 100 micrograms of eNAP-1 per ml (approximately 15 microM). Escherichia coli and Pseudomonas aeruginosa were somewhat less susceptible, manifesting 87.0 and 87.1% mean decreases in CFU per milliliter, respectively, after incubation for 2 h with 200 micrograms of eNAP-1 per ml. Klebsiella pneumoniae numbers were not significantly reduced after exposure to eNAP-1. These antimicrobial properties suggest that eNAP-1 may contribute to phagocyte-mediated host defense against equine infections.
Adhesion of Salmonella typhimurium to porcine intestinal epithelial surfaces: identification and characterization of two phenotypes
Authors: Isaacson R E , Kinsel M
Published: January 2020

Salmonella typhimurium 798 is known to persistently colonize swine. A key step required to initiate colonization of intestines is adhesion of the organism to the intestinal epithelium. However, S. typhimurium 798 initially failed to attach to porcine enterocytes in vitro. An enrichment procedure was used to select adhesive S. typhimurium, and when cells of one colony type were grown in tryptone phosphate broth they were adhesive. Cells from a colony with a different morphology were not adhesive. Adhesion was time dependent, with maximal adhesion occurring at 1 h. As determined by electron microscopy, cells of the adhesive phenotype had pili while none of the cells with the nonadhesive phenotype produced pili. The pili on the adhesive cells were morphologically similar to type 1 pili. Mannose (0.5%) did not affect adhesion, suggesting that the adhesin on strain 798 did not recognize mannose as a receptor. An analysis of envelope proteins from cells of both phenotypes showed that the adhesive-phenotype cells expressed at least 10 unique proteins ranging in size from 20 to 60 kDa. Absorbed antiserum against cells of the adhesive phenotype agglutinated adhesive cells and was used to detect unique surface antigens on the cells of the adhesive phenotype by Western blots (immunoblots). These antigens were in the range of 30 kDa in size. An envelope extract competitively inhibited the binding of S. typhimurium to enterocytes, as did Fab fragments prepared from the absorbed serum. Cells of both phenotypes contained two plasmids, and each had identical restriction digestion patterns. Cells of the adhesive phenotype consistently were found to be more readily phagocytosed by pig leukocytes, and once in the phagocytes they survived better than cells of the nonadhesive phenotype.
Salivary receptors for GalNAc beta-sensitive adherence of Actinomyces spp.: evidence for heterogeneous GalNAc beta and proline-rich protein receptor properties
Authors: Strömberg N , Borén T , Carlén A , Olsson J
Published: January 2020

The receptors for GalNAc beta 1-3Gal alpha Oethyl (GalNAc beta)-sensitive adherence of Actinomyces strains to salivary pellicles were investigated. Parotid and submaxillary saliva from one individual was size fractionated and utilized in hydroxyapatite adherence assays with Actinomyces naeslundii 12104 and A. viscosus 19246 and LY7 with and without GalNAc beta. Three parotid salivary fractions, the high-molecular-weight, acidic proline-rich protein (PRP), and statherin fractions, promote GalNAc beta-sensitive adherence of strain 12104, whereas only the high-molecular-weight fraction of submaxillary saliva promotes such adherence. In contrast, strain LY7, possessing a variant GalNAc beta specificity, shows GalNAc beta-sensitive adherence to the leading and trailing regions of the submaxillary PRP fractions but less distinct adherence to the parotid and submaxillary high-molecular-weight fractions. In addition, the PRP and statherin fractions promote adherence of strains LY7 and 19246 that is not inhibited by GalNAc beta. However, whereas strain LY7 binds more strongly to the PRP fraction than to the statherin fraction, strain 19246 binds preferentially to the statherin fractions of parotid and submaxillary saliva. These salivary protein fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunostained to detect glycosylated proteins. The different salivary receptor properties are paralleled by different glycosylation patterns. The variable GalNAc beta specificities may have evolved to match different salivary glycosylation patterns, and PRP and statherin binding properties seem to be heterogeneous among the Actinomyces strains.
Treponema denticola induces actin rearrangement and detachment of human gingival fibroblasts
Authors: Baehni P C , Song M , McCulloch C A , Ellen R P
Published: January 2020

Spirochetes are associated with destructive periodontal diseases, and one cultivatable oral species, Treponema denticola, binds to mammalian cells and perturbs metabolism. To evaluate the cytoskeletal responses and attachment functions of human gingival fibroblasts (HGF) exposed to T. denticola, monolayers of HGF were incubated with T. denticola strains ATCC 35405, e, and e' in serum-free medium. HGF retracted pseudopods, rounded up, and ultimately detached from the substratum. Scanning electron microscopy showed spirochetes in close contact with HGF surfaces; occasionally, bacteria were partially submerged between folds in the HGF membrane. Blebbing and numerous microvilli formed on the cell surface as the HGF retracted. By confocal microscopy, spirochetes were detected in contact with the HGF surface but were never found on the ventral surface of fibroblasts between the substratum and cell. Morphological alterations were associated with and preceded by actin assembly, as measured by microscopic fluorimetry: there was a 263% increase in actin fluorescence over controls within 30 min. Detachment of fibroblasts from the substratum was related to incubation time and was dependent on the concentration of T. denticola. Detachment was observed for all strains tested and was also dependent on the viability of T. denticola: UV light, heat, and metronidazole treatment markedly reduced the HGF detachment response. Detachment was also significantly reduced by the protease inhibitor phenylmethylsulfonyl fluoride. HGF viability was not significantly affected by coincubation with spirochetes, as measured by lactate dehydrogenase release. Thus, T. denticola induces rapid cytoskeletal remodelling followed by cell detachment, which might be stimulated by a bacterially associated protease but is not likely directly mediated by proteolytic degradation at the cell-substratum adhesive contact points.
Isolation, characterization, and nucleotide sequence of the Streptococcus mutans mannitol-phosphate dehydrogenase gene and the mannitol-specific factor III gene of the phosphoenolpyruvate phosphotransferase system
Authors: Honeyman A L , Curtiss R
Published: January 2020

Streptococcus mutans, the causative agent of dental caries, utilizes carbohydrates by means of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). The PTS facilitates vectorial translocation of metabolizable carbohydrates to form the corresponding sugar-phosphates, which are subsequently converted to glycolytic intermediates. The PTS consists of both sugar-specific and sugar-independent components. Complementation of an Escherichia coli mtlD mutation with a streptococcal recombinant DNA library allowed isolation of the mannitol-1-phosphate dehydrogenase gene (mtlD) and the adjacent sugar-specific mannitol factor III gene (mtlF) from S. mutans. Subsequent transposon mutagenesis of the complementing DNA fragment with Tn5seq1 defined the region that encodes the mtlD-complementing activity, the streptococcal mtlD gene. Nucleotide sequence analysis of this region revealed two complete open reading frames (ORFs) from within the streptococcal mannitol PTS operon. One ORF encodes the mtlD gene product, a 43.0-kDa protein which exhibits similarity to the E. coli and Enterococcus faecalis mannitol-1-phosphate dehydrogenases. The second ORF encodes a 15.8-kDa protein which exhibits similarity to mannitol factor III proteins from several bacterial species. In vitro transcription-translation assays were used to produce proteins of the sizes predicted by the streptococcal ORFs. These data indicate that the S. mutans mannitol PTS utilizes an enzyme II-factor III complex similar to the mannitol system found in other gram-positive organisms, as opposed to that of E. coli, which utilizes an independent enzyme II system.
Mouse neutrophils lack defensins
Authors: Eisenhauer P B , Lehrer R I
Published: January 2020

Defensins are broad-spectrum antimicrobial peptides that are abundant in human, rat, and rabbit neutrophils. We now report that neutrophils from nine strains of mice lacked appreciable defensin content. Mice may therefore be imperfect experimental surrogates for humans or rats in models of infection in which neutrophil function is significant.
Granulocyte-macrophage colony-stimulating factor increases the infectivity of Leishmania amazonensis by protecting promastigotes from heat-induced death
Authors: Barcinski M A , Schechtman D , Quintao L G , Costa D de A , Soares L R , Moreira M E , Charlab R
Published: January 2020

We have studied the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the infectivity of promastigotes of Leishmania amazonensis, an obligate intramacrophage parasite. We measured the capacity of the promastigotes to infect macrophages after preincubation at different temperatures (28, 34, and 37 degrees C) with recombinant murine GM-CSF, as well as the effect of an anti-murine GM-CSF antibody on the in vitro and in vivo infectivity of the parasite. GM-CSF increases the capacity of the promastigotes to infect cells when preincubated at 34 and 37 degrees C, whereas the anti-GM-CSF antibody exerts the opposite effect: it decreases the internalization rate and the progression of infection in macrophage cultures and slows the growth of the lesion in infected BALB/c mice. Neither of the described effects were observed when the in vitro and in vivo infections were made with amastigotes. Promastigotes die in a time-dependent manner when incubated at temperatures higher than 28 degrees C in the absence of GM-CSF. They are protected from this heat-induced death by incubation with the recombinant hormone. Our interpretation of these data is that the increase in the infectivity of promastigotes when incubated with GM-CSF at the temperatures at which infection occurs (34 and 37 degrees C) is due to the larger number of surviving forms within the infecting population. The decrease in infectivity when they are incubated with the antibody is due to inhibition of the protection conferred by the GM-CSF produced by the macrophages during the in vitro and in vivo infections.