Non-culturable Legionella pneumophila associated with Acanthamoeba castellanii: detection of the bacterium using DNA amplification and hybridization.

The intracellular localization of Legionella pneumophila serogroup 1 within Acanthamoeba castellanii rendered the bacteria non-culturable on supplemented BCYE agar. DNA amplification, using two 19-mer primers, and hybridization using a 25-mer oligonucleotide probe, permitted detection of Leg. pneumophila in approximately 81% (29/36) of samples where the bacteria could not be detected using culture. A combination of co-cultivation of samples with Leg. pneumophila-naive A. polyphaga or Hartmannella vermiformis, incubation in a defined liquid medium or use of catalase indicated that approximately 31% (9/29) of the samples contained Leg. pneumophila which were viable although not culturable.

Comparison of outer-membrane proteins of Pasteurella haemolytica expressed in vitro and in vivo in cattle.

Outer-membrane protein (OMP) profiles of two serotype A1 isolates of Pasteurella haemolytica were compared by SDS-PAGE and Western blotting with bovine convalescent serum after growth (a) in vitro under iron-sufficient and -deficient conditions, (b) in vivo in the lungs of experimentally infected calves and (c) in vivo in diffusion chambers implanted into the peritoneal cavities of calves. Lung-grown bacteria differed from iron-sufficient in vitro-grown bacteria in having enhanced expression of the previously recognized 71, 77 and 100 kDa iron-regulated proteins, reduced expression of 18, 31, 39.5 and 50 kDa proteins, and expression of a 19 kDa protein. Differences were also apparent in the Western blot profiles of OMPs of in vitro- and lung-grown bacteria. These included the apparent lack of recognition of the 100 kDa protein in the lung-grown bacteria, but not in the in vitro-grown bacteria, and more intense staining of a 47 kDa protein in in vitro-grown bacteria, but not in lung-grown bacteria. The OMP profiles of the chamber-grown bacteria resembled those of the lung-grown bacteria in that expression of the 18, 19, 31 and 39.5 kDa proteins was similar. These similarities demonstrated that the chamber-grown bacteria had adapted to the in vivo environment, and that growth conditions within the chambers resembled, but not perfectly, those within the lungs. For example, expression of the three iron-regulated OMPs was very low in the chamber-grown bacteria compared to the lung-grown bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of an intraperitoneal implant chamber for the study of in vivo-grown Pasteurella haemolytica in cattle.

An intraperitoneal implant chamber was developed for the study of the in vivo growth of Pasteurella haemolytica in calves. The chamber had a volume of approximately 100 ml and featured an external sampling port which allowed multiple and sequential sampling of the chamber contents. A single polycarbonate diffusion membrane with a pore size of 0.22 micron allowed host peritoneal fluid to diffuse into the chamber and maintained the bacterial population free of white blood cells. Chambers were implanted into the peritoneal cavities of four five-month-old dairy-cross calves, demonstrated to be sero-negative by indirect haemagglutination assay. Three days later, four different P. haemolytica isolates, of serotypes A1 or A2, were inoculated into the chambers. In all cases, there was a slow decline in the viable bacterial numbers within the chambers. Western blot analysis of the antibody content of the chamber fluids revealed IgG antibodies to P. haemolytica OMPs in the fluid prior to inoculation and both 9 and 15 days after inoculation. Furthermore, there was no significant change in the IgG antibody content of the chamber fluid, either quantitatively or qualitatively, during the course of the experiment. Analysis of the bactericidal activity of pre-inoculation chamber fluid against the corresponding bacterial isolate suggested that an antibody-dependent complement-mediated process was not responsible for the decline in bacterial numbers. Overall, the chamber design was demonstrated to be extremely effective for in vivo studies of P. haemolytica in calves, allowing easy and regular sampling of the chamber contents and maintaining bacteria free of white blood cells. Although there was a slow decline in bacterial numbers over time, sufficient numbers of cells could be obtained for analysis of cell-surface antigens.

A new assay for the invasive adenylate cyclase toxin of Bordetella pertussis based on its morphological effects on the fibronectin-stimulated spreading of BHK21 cells.

When baby hamster kidney (BHK) cells are allowed to spread on fibronectin-coated substrata in the absence of serum and the presence of agents which elevate intracellular 3':5'-cyclic AMP (cAMP) levels they adopt an abnormal, stellated morphology. To determine whether the invasive adenylate cyclase (AC) toxin of Bordetella pertussis induced the same response, cell extracts were prepared from several B. pertussis strains. They were characterized for AC toxin production by enzymic assay and by immunoblotting with an AC-toxin-specific monoclonal antibody. Extracts of strains producing AC toxin induced elevated levels of intracellular cAMP in BHK cells and promoted a stellation response during cell spreading. Extracts prepared from strains defective in AC toxin production showed no effect. Using image analysis to quantify the morphological change, we have demonstrated that the effect of AC toxin on cell spreading is dose dependent. This technique is a rapid and sensitive assay for the invasive AC toxin.

Evaluation of different methods for the detection of outer membrane proteins and lipopolysaccharides of Pasteurella haemolytica by immunoblotting.

The optimal conditions for the detection of outer membrane proteins (OMPs) and lipopolysaccharide (LPS) of Pasteurella haemolytica by immunoblotting were evaluated. The variables examined included the equilibration time of the gels before transfer, composition of the transfer buffer, type of blotting membrane, blocking agent, effect of the zwitterionic detergent Empigen BB on protein renaturation, and the development reagent. The composition of the transfer buffer and time of gel equilibration significantly affected the efficiency of transfer of both OMPs and LPS. However, the optimal conditions for the transfer of OMPs were not the same as those for LPS. Thus, optimal transfer of OMPs occurred in Tris-glycine buffer, with prior equilibration of the gels to allow for expansion, whereas optimal transfer of LPS was achieved in Tris-glycine-methanol buffer with no equilibration of the gels. In Tris-glycine-methanol buffer, gel equilibration resulted in a significantly reduced transfer of both OMPs and LPS, probably due to the removal of SDS from these components. The use of Zeta-Probe blotting membrane which, unlike nitrocellulose, does not require methanol for optimal protein binding, did not result in improved binding of OMPs or LPS in the absence of methanol and, even after prolonged blocking (> 2 h), gave higher background staining than did nitrocellulose. Effective blocking of nitrocellulose was achieved with 3% (w/v) gelatin, 2.5% (w/v) skimmed milk or 0.3% (v/v) Tween 20, whereas increased background staining occurred with 1% (w/v) bovine serum albumin or 1% (w/v) ovalbumin. The incorporation of Empigen BB in the primary antibody buffer did not improve antibody recognition of proteins as a result of their renaturation. For the horseradish-peroxidase enzyme development system, the substrate 3,3'-diaminobenzidine tetrahydrochloride was more sensitive, and developed more quickly, than 4-chloro-1-naphthol, but faded more rapidly after drying of the membrane. 4-chloro-1-naphthol was more suitable for identifying OMPs because less background staining occurred, whereas 3,3'-diaminobenzidine tetrahydrochloride was more suitable for the detection of LPS due to its greater sensitivity.

Heat-labile and heat-stable haemolysins of Campylobacter jejuni.

During studies on the virulence mechanisms of Campylobacter jejuni clinical isolates it became apparent that some strains produced one or more haemolysins and some did not. There was no great difference between Group C (cholera-like) strains and Group D (dysentery-like) strains. The protein haemolysin(s) showed a spectrum of activity against erythrocytes from different animals; with maximum activity against rabbit and minimal activity against chicken erythrocytes. The results suggested a two-stage activation mechanism for haemolysis which involved a multi-hit lytic activity. It was concluded that the C. jejuni haemolysins were not identical to those described in other organisms and they may be involved in iron acquisition in vivo.

Outer-membrane protein and lipopolysaccharide variation in Pasteurella haemolytica serotype A1 under different growth conditions.

Growth characteristics, as well as outer-membrane protein (OMP) and lipopolysaccharide (LPS) profiles in SDS-polyacrylamide gels, of two serotype A1 isolates of Pasteurella haemolytica were examined under different in vitro growth conditions. The two isolates were chosen as representatives of disease (S/C 82/1) and non-disease (W/D 83/4) isolates, respectively. The growth rates and final cell densities of both isolates increased as the degree of aeration increased. In particular, the final cell densities varied significantly according to the degree of aeration. Under anaerobic conditions, however, both the growth rate and final cell density were significantly reduced. There was reduced expression of a 40.5 kDa protein under anaerobic conditions in both isolates, whereas in S/C 82/1 expression of the 71, 77 and 100 kDa iron-regulated proteins increased as aeration decreased. There were also differences in low-molecular-mass components of LPS between cells grown anaerobically and those grown aerobically. Growth in the presence of 5% CO2 did not significantly alter the growth rate and had little, if any, affect on OMPs or LPS. Differences in the expression of certain proteins occurred as growth progressed from the exponential to the stationary phase. Growth in the presence of the iron chelators 2,2'-dipyridyl, ethylenediamine-dihydroxyphenylacetic acid (EDDA), desferrioxamine mesylate (desferal), ovotransferrin (conalbumin) and bovine transferrin was inhibited within a very narrow concentration range. In the presence of 2,2'-dipyridyl, EDDA or desferal, 71 and 100 kDa iron-regulated OMPs increased in both isolates whereas a 77 kDa protein increased in isolate S/C 82/1 only. In the presence of ovotransferrin or bovine transferrin there was, in both isolates, increased expression of the 71 kDa protein, a slight increase in expression of the 100 kDa protein but no expression of the 77 kDa protein; there was also increased production of the 40.5 kDa protein, and synthesis of two additional proteins of 23 and 26 kDa. Other differences occurred after growth in foetal and newborn calf sera. In foetal calf serum there was enhanced expression of the 71 but not of the 100 kDa protein. In newborn calf serum there was no enhanced expression of the 71, 77 or 100 kDa proteins, but expression of novel proteins of 97 and 98 kDa as well as a high-molecular-mass protein occurred. There was also slight quantitative differences in the LPS profiles of cells grown in foetal or newborn calf sera compared to those of cells grown in other media.(ABSTRACT TRUNCATED AT 400 WORDS)

Optimal conditions for the analysis of Pasteurella haemolytica lipopolysaccharide by sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

The optimal conditions for the analysis of the lipopolysaccharide (LPS) of two serotype A1 isolates and a serotype A2 isolate of Pasteurella haemolytica by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining were determined. The LPS of the A1 isolates possessed O side chains, consisting of high molecular mass bands with the appearance of a ladder-like pattern, as well as a low molecular mass core-oligosaccharide region; the LPS of the A2 isolate consisted only of the core-oligosaccharide region. Furthermore, the LPS of the two A1 isolates differed in the core-oligosaccharide region. Optimal resolution of low molecular mass LPS components was obtained in a 15% acrylamide resolving gel containing 4 M urea whereas optimal resolution of high molecular mass components was obtained when urea was omitted. Conventional silver staining resulted in excellent visualisation of LPS bands, whereas a modified staining method did not detect additional bands, as has been demonstrated with the LPS of Pseudomonas aeruginosa. Proteinase K digestion of outer membranes gave more clearly defined LPS profiles than did similar digestions of whole cells, and more closely resembled the profiles of purified LPS. With the exception of slight variation in the average molecular mass of a group of O side chains between logarithmic and stationary phases there were no differences in LPS profiles at various stages of the growth cycle; freezing and thawing of LPS samples had no effect on the profiles.

Assay of Bordetella pertussis heat-labile toxin with human embryonic lung cells.

An assay has been developed for Bordetella pertussis heat-labile toxin (HLT) based on morphological alterations in certain human embryonic lung (HEL) cell lines. Eighteen cell lines from human and other sources were tested but only two, MRC-5 and HELu2, were responsive to HLT. Confluent monolayers of the cells contracted within 24 h of exposure to the toxin, but without loss of viability during incubation for a further 3 days. The effect of HLT was quantitated by scoring the extent of morphological change, and by the decrease in Giemsa staining of the cell monolayers, as measured on an ELISA plate reader. This cell culture assay for HLT was more sensitive than lethality titration in mice but the dose-response curve had a lower slope. The specificity of the response was established by comparing unheated HLT with HLT heated at 56 degrees C, and with extracts from transposon-insertion mutants of B. pertussis which were deficient in HLT. Purified preparations of pertussis toxin and B. pertussis lipopolysaccharide gave no morphological response even at high doses.

A colourimetric, microplate assay for the leucotoxin of Pasteurella haemolytica.

Culture supernates of Pasteurella haemolytica, which contain leucotoxin, inhibited the reduction of nitroblue tetrazolium (NBT) by bovine and ovine but not rabbit leucocytes in response to phorbol 12-myristate 13-acetate (PMA). Culture supernates of P. multocida, which contain no leucotoxin, had no inhibitory effect on the response of leucocytes from any species. The inhibition of NBT reduction was assessed visually or spectrophotometrically in the wells of microplates and used as a simple assay for leucotoxin. It was as sensitive as the trypan blue dye-exclusion method and did not require the use of radioisotopes. In addition, sera from P. haemolytica-infected calves inhibited leucotoxin activity in the microplate assay. Thus, inhibition of NBT reduction after stimulation of ruminant leucocytes with PMA can be used as a simple, specific assay for leucotoxin and for anti-leucotoxin antibodies.

Delta-haemolysin from Staphylococcus aureus and model membranes. A solid-state 2H-NMR and 31P-NMR study.

Solid-state 2H NMR and 31P NMR of 2H-enriched chains and polar head groups, respectively, of dipalmitoylglycerophosphatidylcholine/water dispersions were undertaken to investigate the action of delta-haemolysin from Staphylococcus aureus on biomembranes. When the lipid/toxin molar ratio, Ri, is greater than or equal to 10, the gel-phase 2H powder patterns and the temperature of the gel-fluid phase transition, tc, are unchanged by the presence of the toxin whereas the 31P powder spectra of polar head groups are perturbed. At t greater than tc, a detailed analysis of methylene ordering indicates that delta-haemolysin orders the lipid chains near tc and disorders them for t much greater than tc. These findings are interpreted in terms of peptide location with regard to the membrane and suggest that the position of the toxin depends on the temperature relative to tc. Experiments carried out at Ri = 4 exhibit sharp, isotropic 2H-NMR lines, at t greater than tc, indicating that delta-haemolysin promotes the appearance of very small objects undergoing fast isotropic reorientation which average to zero the quadrupolar interaction. Below tc, one observes gel-phase powder patterns which indicate that the bacterial toxin is unable to form such small structures with ordered dipalmitoylglycerophosphocholine phospholipids. From comparison of the action of delta-haemolysin with that of melittin on same lipids [Dufourc et al. (1986) Biochemistry 25, 6448-6455] it results that both toxins perturb similarly fluid-phase lipids at elevated temperature, but they behave differently with gel-phase lipids, the former toxin being less efficient in membrane restructuring than the latter.

A plasmid which can be transferred between Escherichia coli and Pasteurella haemolytica by electroporation and conjugation.

Three broad-host-range plasmids (pRK290, pSa4 and pKT230) and one native Pasteurella haemolytica plasmid (pPH33) were used in transformation experiments with P. haemolytica strains T179 (serotype A1), Y216 (serotype A2) and its capsular-deficient variant Y216/NS1. No transformants were detected with either heat-shock or freeze-thaw techniques. However, by electroporation, all P. haemolytica strains were transformed by pPH33 but not by pRK290 or pSa4. The highest frequency obtained was 91 x 10(4) transformants per microgram of pPH33 DNA with P. haemolytica strain Y216/NS1. Although pPH33 itself was non-transmissible by conjugation, it could be mobilized from Escherichia coli, using the transfer function of the IncP plasmid pRK2013, into P. haemolytica at a frequency of 0.3-2.2 x 10(-3) per recipient cell.

Interaction of Bordetella pertussis virulence components with neutrophils: effect on chemiluminescence induced by a chemotactic peptide and by intact bacteria.

The effect of secreted virulence components of Bordetella pertussis on chemiluminescence (CL) of rabbit peritoneal neutrophils was determined with the chemotactic peptide N'-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) or intact B. pertussis as the stimulus. Pertussis toxin (PT) inhibited the response to fMLP in a dose-dependent manner, although only after the neutrophils had been exposed to the toxin for greater than 15 min. Both filamentous haemagglutinin (FHA) and lipopolysaccharide (LPS) markedly enhanced the CL response to fMLP after greater than or equal to 15 min incubation with the neutrophils. Similar effects to those of B. pertussis LPS were also seen with smooth and rough LPS from Salmonella minnesota. With the lowest dose of each component which elicited a maximal effect on CL, the inhibitory effect of PT overrode the enhancing effect of FHA and B. pertussis LPS. Pre-incubation of neutrophils with PT, FHA or B. pertussis LPS caused a slight reduction in the subsequent CL response to virulent B. pertussis Tohama. Virulent (phase I, or X-mode) organisms of B. pertussis 18334 and B. pertussis Tohama induced greater neutrophil CL than their avirulent (C-mode) derivatives. There appeared to be an inverse correlation between bacterial hydrophilicity and the ability to induce neutrophil CL: X-mode bacteria were significantly less hydrophilic than C-mode organisms. Three mutants, the adenylate cyclase (AC)- and haemolysin (HLY)-deficient B. pertussis BP348, the FHA-deficient B. pertussis BP353, and the PT-deficient B. pertussis BP357, generated similar levels of CL and had similar hydrophilicity values. The hydrophilicity value of the avirulent mutant B. pertussis BP347 (deficient in AC, HLY, FHA and PT) and the CL induced by this strain were similar to those of B. pertussis C-mode organisms. Thus, the interaction of B. pertussis with neutrophils appears to be complex, reflecting both the alteration of leucocyte function by secreted virulence components of the organism and, in the absence of opsonins, the surface properties of the bacterium.

A sensitive chemiluminescence assay for pertussis toxin and for evaluation of cell-free pertussis toxoids.

Pertussis toxin (PT) inhibited luminol-enhanced chemiluminescence induced in rabbit peritoneal neutrophils by N'-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) at doses as low as 0.8 ng.ml-1, even in the presence of a 10-fold higher concentration of filamentous haemagglutinin (FHA). A cell-free extract of Bordetella pertussis, containing predominantly PT and FHA, suppressed the neutrophil response to fMLP. After toxoiding with carbodiimide, the inhibitory activity of the extract was abolished and an enhancement of neutrophil chemiluminescence was observed due to FHA activity. Abrogation of the chemiluminescent response of neutrophils to fMLP is proposed as a sensitive, in vitro assay for pT, and may be useful for monitoring the residual toxin activity in pertussis toxoids and for determining the anti-toxic effects of anti-PT antibodies.

High-resolution 1H NMR study of the solution structure of delta-hemolysin.

The 26-residue toxin from Staphylococcus aureus, delta-hemolysin, is thought to act by traversing the plasma membrane. The structure of this peptide, in methanol solution, has been investigated by using high-resolution NMR in combination with molecular dynamics calculations. The 1H NMR spectrum has been completely assigned, and it is shown that residues 2-20 form a relatively stable helix while the residues at the C-terminal end appear to be more flexible. The structures were calculated only from nuclear Overhauser effect data and standard bond lengths. It is shown that the results are consistent with 3JNH-alpha CH coupling constants and amide hydrogen exchange rates.

Release of pertussis toxin and its interaction with outer-membrane antigens.

The absence of subunit S3 in cell-associated pertussis toxin (PT) from a mutant of Bordetella pertussis which failed to produce cell-free toxin suggested that this subunit was involved in the release of PT into the culture medium. The addition of methylated beta-cyclodextrin (MCD) to the culture medium caused a small but consistent increase in the release of lipopolysaccharide (LPS) by four wild-type strains of B. pertussis. Since previous studies have shown that MCD also enhances the levels of PT in culture supernates, it seemed probable that the increased shedding of outer-membranes vesicles (OMV) may explain the increased levels of both cell-free PT and LPS. Release of PT was inhibited in media buffered with HEPES but was unaffected in Tris/HC1 buffer. This suggested that in addition to shedding of the outer membrane, increased permeability and greater destabilization of the outer membrane, as caused by Tris/HC1 buffer, may be important in the release of PT. Our data do not support the idea that PT is packaged into OMV because only an insignificant proportion (0.01%) of the total cell-free PT was associated with LPS. The association of PT with small micelles derived from outer-membrane amphiphiles may be more important since the LPS content of PT purified from culture supernates (containing no large OMV) was nearly 18% by weight.

Accumulation of a precursor of subunit S1 of pertussis toxin in cell envelopes of Bordetella pertussis in response to the membrane perturbant phenethyl alcohol.

Exponential cultures of Bordetella pertussis strain 18334 were treated with the membrane-perturbing agent phenethyl alcohol which, at a concentration of 0.075% v/v, blocked the synthesis of mature subunit S1 of pertussis toxin as revealed by Western blotting. It also caused the accumulation of a precursor, pS1, with an estimated mol. wt of 32 X 10(3), that was located in the cytoplasmic membrane. These findings suggested that subunit S1 of pertussis toxin was exported in a signal peptide-dependent manner.

Antigenic heterogeneity in subunit S1 of pertussis toxin.

Culture supernates containing pertussis toxin (PT) from four strains of Bordetella pertussis were examined for both immunological reactivity and biological activity. PT from all four strains sensitized mice to histamine and toxin was detectable in supernates of all strains when examined by Western blotting with polyclonal antiserum to PT. In supernates of three of the four strains, PT was detectable by an enzyme-linked immunosorbent assay (ELISA) using mouse monoclonal antibody to subunit S1 of PT as the third antibody layer. However, supernates from one strain, 18323, failed to react in ELISA. Electroblots probed with the monoclonal antibody labelled subunit S1 of PT from all strains except that of strain 18323. PT of strain 18323, whilst retaining histamine-sensitizing activity, differed antigenically from that of other strains.

One-dimensional peptide mapping of the subunits of pertussis toxin.

Pertussis toxin (pertussigen) purified from the cytoplasmic fraction of Bordetella pertussis strain 18334, phase 1, consisted of five subunits which included an additional subunit (S1a) not previously reported. Subunits S1, S1a and S2 showed extensive structural homology when analysed by one-dimensional peptide mapping, indicating that the latter two were probably derived from proteolytic cleavage of the largest subunit, S1. Subunits S3 and S4,5 generated only a limited number of peptides following chemical and enzymic degradation, but these subunits differed structurally from each other and from those showing structural homology.