The Production of Hlya Toxin by Proteus Penneri Strains
Total Page：16
File Type：pdf, Size：1020Kb
J. Med. Microbiol. - Vol. 39 (1993), 282-289 0 1993 The Pathological Society of Great Britain and Ireland The production of HlyA toxin by Proteus penneri strains B. W. SENIOR Department of Medical Microbiology, University of Dundee Medical School, Ninewells Hospital, Dundee DD I 9SY Summary. Twelve diverse strains of Proteus penneri of clinical origin all produced a calcium- dependent haemolysin, unlike most other Proteus spp. In most strains the haemolysin was secreted into the medium during early exponential growth and lysed not only of a variety of erythrocyte types from several animals including man, but also human neutrophils and human embryo lung fibroblasts. The haemolysin was a protein of 107 kDa, the same size as Escherichia coli HlyA, and it reacted with antiserum to E. coli HlyA. Because of its similarity in size, antigenicity and range of action to the HlyA virulence factor of E. coli, P. penneri HlyA is believed to be an important virulence factor for this organism. It was degradable by an EDTA-sensitive protease-probably the IgA protease-to inactive fragments. The interaction of P. penneri HlyA and IgA protease in vivo and the origin of HlyA, which has now been found in many diverse bacteria, are discussed. Introduction urinary tractg and wounds’O and has been isolated from the faeces of both healthy and sick individuals.” RTX toxins are a family of calcium-dependent, Potential virulence factors already known to be pro- pore-forming, cytolytic toxins produced by diverse duced by P. penneri include urease,” IgA protease13*l4 bacteria that act on different cells in different hosts. and type 3 fimbriae associated with MR/K haemag- Thus Escherichia coli a-haemolysin, Actinobacillus glutinins, of at least two antigenic types.15 The work pleuropneumoniae haemolysin and Bordetella pertussis presented here examined the production of a haemo- adenylate cyclase-haemolysin lyse both erythrocytes lysin antigenically related to the E. coli RTX toxin and a wide range of nucleated cells from many different a-haemolysin (HlyA) that could be an additional animals including man.1-3 On the other hand, the factor to be considered in the virulence of this leucotoxin of Pasteurella haemolytica and A. actino- organism. mycetemcomitans acts only on neutrophils, mono- cytes and macrophages of ruminant animals and Materials and methods selected primates including man, and is without action on erythrocyte^.^,^ These toxins are so named (Repeats Bacteria in ToXin) because, despite the diversity in the suscept- ibility of host cells to them, they have a common The 12 strains of P. penneri studied were clinical structural feature of repeats (usually in tandem) of a isolates from different patients in three countries nine amino acid, glycine-rich sequence (Leu/Ile/Phe- (table). Those prefixed E were from patients with X-Gly-Gly-X-Gly-Asn/Asp-Asp-X)that is thought to diarrhoea and those prefixed H from healthy volun- be involved in the binding of calcium and interaction teers and were donated by Dr H. E. Muller. Strain with target receptors.6 T77 was isolated by Dr. 0. Ang in Istanbul. The RTX toxins are important virulence factors, not Dundee strains were from elderly women with haema- only because they destroy important defence cells of turia. E. coli strain 519, serotype 04, isolated from the body, but also because, at concentrations too low urine, was a known producer of the calcium-dependent to cause cell lysis such as would occur in the body at a-haemolysin, HlyA.16*17P. mirabilis U6450 was a sites distant from the infecting organism, they impair strain from urine that produced the calcium-inde- various functions of host cells involved in the immune pendent cell-bound haemolysin HpmA. l7 P. vulgaris response. strain 76362/79 was isolated from an infected burn Proteus penneri, the bacterium formerly known as graft. It produced both HpmA and HlyA.17-” The strains were characterised by standard biochemical “ indole-negative P. vulgaris” or “P.vulgaris biogroup 1 ”,* although less commonly isolated from clinical testsz0and maintained on Nutrient Agar (Oxoid CM3) specimens than P. mirabilis and P. vulgaris, has, slopes in screw-capped bottles at 4°C. nevertheless, been associated with infections of the Haem olys in product ion Received 28 Jan. 1993; accepted 9 March 1993. An inoculum (100 pl) of a Nutrient Broth (Qxoid 282 HlyA TOXIN IN PROTEUS PENNERI 283 Table. Characteristics of the P. penneri strains studied Proticine Strain production (p) no. /sensitivity (s) Source Origin tY Pe E 46 po/s2, 3, 5 Faeces Germany E 100 POlS2, 3, 5 ,, 3, E 180 Pols4 5, 7 E 587 POIS2, 3, 5 3, 2, E 1125 po/s 12 5, 3 E 1152 po/s 12 't 3, E 1263 po/s 12 9, 9, H 1209 PO/S2,3, 5 9 9, H 1360 po/s 12 31 1, T 77 po/s9, 12 Thorax wound Istanbul, Turkey 05665V po/s4, 12 Urine Dundee, UK 168032 POISO 9, 3, CM67) culture of each strain, which had been incu- for 30 min through an equal volume of Lymphoprep bated overnight at 37"C, was added to 10ml of (Nycomed, Oslo ;specific gravity 1-077)lying above an nutrient broth and incubated with shaking at 37°C. At equal volume of Ficoll- hypaque (specific gravity intervals, samples of culture were removed for de- 1.119). The neutrophils which collected at the termination of growth (absorbance at 550nm). In Lymphoprep-Ficoll-hypaque interface were collected, addition 5Opl of the culture, or the supernate after washed and resuspended to lo6 cells/ml in Tris- centrifugation of the culture sample for 2min at buffered saline (TBS; NaClO.85 YOin 50 mM Tris-HC1; 11 600 9, was added to a microcentrifuge tube holding pH 7.5). The average purity of the preparations was 1 ml of a washed suspension of erythrocytes (usually > 95% neutrophils. horse erythrocytes), 2 YOv/v in saline with and without For the test, 200-pl amounts of active haemolysin 20mM CaC1,. After inverting the tubes to mix the from the supernate of a nutrient-broth culture of P. contents and incubating them in a water bath at 37°C penneri strain E 1125 incubated for 3 h was added to for 15 min, the tubes were centrifuged at 11 600 g for 2004 amounts of neutrophils in TBS in the 30 s to pellet the erythrocytes and bacteria. The presence (test reaction) and absence (control reaction) amount of free haemoglobin in the supernate was of 20 mM CaCl,. The mixtures were incubated in a measured by its absorbance at 540 nm against a blank water bath at 37°C. At timed intervals, samples of of water and this indicated the haemolytic activity of equal volume of a control and test reaction were the sample. removed and added simultaneously to separate cu- vettes each holding 600 pl of TBS to which had been Efect of culture supernates on human embryo lung added 3Opl of a fresh solution of reduced j3-nico- (HEL)jibroblasts tinamide adenine dinucleotide @-NADH ; 3-5 mg/ml in TBS) and 3Opl of fresh sodium pyruvate solution At intervals, 0.5 ml of the culture of each strain, (1 1 mg/ml in TBS) and the 340 nm absorption of the growing under the conditions as for haemolysin reaction in each cuvette was recorded with time. A production, was centrifuged at 11 600 g for 2 min. A further enzyme control reaction consisted of 200 p1 of 50-p1 portion of the clear supernate was removed and neutrophils in TBS treated with 200 pl of Triton XlOO assayed for haemolytic activity as above and 50 p1 of (2% in TBS) for 5 min at room temperature to cause the supernate was also added to the wells of micro- total lysis of neutrophils. The amount of lactate titration plates bearing a confluent growth of HEL dehydrogenase from this reaction was measured as fibroblasts in Eagle's maintenance medium. The plates above. were incubated at 37°C and examined under a plate microscope for cytopathic effects after 24 and 48 h. SDS-PAGE of P. penneri haemolysins Efect of haemolysin on neutrophils Strains were cultured as for haemolysin production Haemolysin-induced damage to neutrophils was and, when found to be actively producing haemolysin, examined by assaying for lactate dehydrogenase re- 1-ml samples were removed and centrifuged at 11 600 g leased from damaged neutrophils. Lactate dehydro- for 2 min. The clear supernate was transferred to a genase causes the following reaction to take place: microcentrifuge tube and lysozyme, as a carrier pro- pyruvate +/3NADH++ H+ + lactate +mAD+. The tein, was added to it to a final concentration of formation of WAD+ can be followed by measuring 100 pg/ml. Trichloroacetic acid (TCA) was added to the decrease in absorption at 340 nm. the mixture to 10 YOw/v to precipitate proteins. After Neutrophils were isolated from the heparinised 10 min, the mixture was centrifuged at 11 600 g for blood of healthy volunteers by centrifugation at 400 g 2min, the supernate was discarded and the tube 284 B. W. SENIOR contents were drained well. Twenty-five p1 of 1 M Tris glucose and maltose; however, none was able to was added to the precipitate to neutralise contami- produce ornithine decarboxylase or indole nor to nating residual acid. The precipitate was resuspended ferment lactose, mannose, mannitol, adonitol, inositol and dissolved after the further addition of 25 pl of or salicin, nor to degrade aesculin. sample buffer (0-125mM Tris-HC1, pH 6.8, SDS 4 YO, glycerol 20 YO,mercaptoethanol 10 YO and a trace of Kinetics of haemolysin production bromophenol blue dye).