Types Of Hemolysins Research Articles

Polyinfection in Fish Aeromoniasis: A Study of Co-Isolated Aeromonas Species in Aeromonas veronii Outbreaks.

We studied the phenotypic and genomic characteristics related to the virulence and antibiotic resistance of two Aeromonas strains, which were co-isolated before an outbreak of Aeromonas veronii among diseased seabass on Agathonisi Island, Greece, in April 2015. The first strain, AG2.13.2, is a potentially pathogenic mesophilic variant of Aeromonas salmonicida, and the second, AG2.13.5, corresponds to an Aeromonas rivipollensis related to A. rivipollensis KN-Mc-11N1 with an ANI value of 97.32%. AG2.13.2 lacks the type III secretion system just like other mesophilic strains of A. salmonicida. This characteristic has been associated with lower virulence. However, the genome of AG2.13.2 contains other important virulence factors such as type II and type VI secretion systems, and toxins such as rtxA, aerolysin aer/act, and different types of hemolysins. The strain also carries several genes associated with antibiotic resistance such as the tetE efflux pump, and exhibits resistance to tetracycline, ampicillin, and oxolinic acid. In an in vivo challenge test with gilthead seabream larvae, the A. veronii bv sobria strain AG5.28.6 exhibited the highest virulence among all tested strains. Conversely, both A. salmonicida and A. rivipollensis showed minimal virulence when administered alone. Interestingly, when A. veronii bv sobria AG5.28.6 was co-administered with A. rivipollensis, the larvae survival probability increased compared to those exposed to A. veronii bv sobria AG5.28.6 alone. This finding indicates an antagonistic interaction between A. veronii bv sobria AG5.28.6 and A. rivipollensis AG2.13.5. The co-administration of A. veronii bv sobria AG5.28.6 with Aeromonas salmonicida did not yield distinct survival probabilities. Our results validate that the primary pathogen responsible for European seabass aeromoniasis is Aeromonas veronii bv sobria.

Optimized Hemolysin Type 1 Secretion System in Escherichia coli by Directed Evolution of the Hly Enhancer Fragment and Including a Terminator Region.

Type 1 secretion systems (T1SS) have a relatively simple architecture compared to other classes of secretion systems and therefore, are attractive to be optimized by protein engineering. Here, we report a KnowVolution campaign for the hemolysin (Hly) enhancer fragment, an untranslated region upstream of the hlyA gene, of the hemolysin T1SS of Escherichia coli to enhance its secretion efficiency. The best performing variant of the Hly enhancer fragment contained five nucleotide mutations at five positions (A30U, A36U, A54G, A81U, and A116U) resulted in a 2‐fold increase in the secretion level of a model lipase fused to the secretion carrier HlyA1. Computational analysis suggested that altered affinity to the generated enhancer fragment towards the S1 ribosomal protein contributes to the enhanced secretion levels. Furthermore, we demonstrate that involving a native terminator region along with the generated Hly enhancer fragment increased the secretion levels of the Hly system up to 5‐fold.

1186. Cefazolin inoculum effect predicts reduced susceptibility to other antibiotics and patient outcomes in MSSA endovascular infections

BackgroundMSSA Infective endocarditis (IE) is inherently a high-burden infection with up to a 30% mortality rate. Cefazolin is an appealing treatment option for IE with low toxicity and a favorable dosing scheme. However, cefazolin has been associated with treatment failure in IE, attributed to an inoculum effect. The specific mechanism underlying the cefazolin inoculum effect (CIE) remains undetermined, but CIE has been linked to both blaZ expression and agr dysfunction. This study aims to determine whether CIE is linked to reduced susceptibility to other antibiotics and worse outcomes regardless of therapy in MSSA endovascular infections.MethodsSixty-four MSSA strains were collected from patients with endovascular infections not treated with cefazolin. To determine CIE phenotype, strains were cultured and MICs assayed for cefazolin, nafcillin, and vancomycin at 107 CFU/mL for high-inocula (HI) and 105 CFU/mL for standard-inocula (SI). This study defined CIE as a ≥ 4-fold increase in MIC at HI compared to SI, with at least an MIC of 4 mg/L at HI. Nitrocefin disks identified blaZ expression, and beta lysin disks were used to determine hemolysin type and agr function. Patient outcomes of mortality and bacteremia duration were assessed across cohorts.ResultsTwenty-four strains exhibit a CIE (38%), with 10 strains having an MIC of ≥ 32mg/L at HI. Nafcillin and vancomycin also had an inoculum effect, uncoupled from the CIE and occurring at a lower frequency and amplitude at HI. Presence of CIE had a greater association with blaZ expression (71% vs 25%) than agr dysfunction (38% vs 20%). 50% (9/18) of CIE infections were cleared within 48 hours while 77% (20/26) of CIE-negative infections were cleared within 48 hours (P=0.106). However, presence of CIE was not associated with increased mortality (25% CIE-positive vs 35%; P=0.578)ConclusionPrevious studies for CIE failed to enrich for isolates from endovascular sources, where inocula are known to be high. This study presents one of the largest endovascular source cohorts for CIE evaluation. It identifies that CIE prevalence (38%) is higher than reports from diverse infection sources (10-36%). CIE appears to predict bacteremia duration with other MSSA treatment options, suggesting mechanisms independent of blaZ and agr function for this phenomenon.Disclosures Warren Rose, PharmD, MPH, Merck (Grant/Research Support)Paratek (Grant/Research Support)

Identification of Hemolysine Genes and their Association with Antimicrobial Resistance Pattern among Clinical Isolates of Staphylococcus aureus in West of Iran

Background:Staphylococcus aureus is expressing a broad range of different hemolysins enhancing its ability to establish and maintain infection in humans. The aim of this study was to identify the types of hemolysins in different clinical isolates of S. aureus and their association with antibiotic resistance patterns.Materials and Methods:In this cross-sectional and descriptive study, clinical isolates of S. aureus were collected from Hamedan's hospitals during an 11-month period from June 2016 to January 2017 and identified by using biochemical tests. To determine the antibiotic resistance pattern, disk diffusion method and minimum inhibitory concentration (MIC) were conducted. Genomic DNA was extracted using extraction kit. The polymerase chain reaction was done with specific primers for identification of hla, hlb, hld, and hld genes.Results:Among a total of 389 clinical samples, 138 isolates (35.45%) of S. aureus were identified, which 87 isolates (63.04%) were cefoxitin MIC of >4 μg/ml and resistant to methicillin. The highest frequency of antibiotic resistance was observed against erythromycin in 108 isolates (78.26%) and penicillin in 133 isolates (96.37%) and the lowest resistance was against gatifloxacin in 50 isolates (36.23%) and Cefazolin in 11 isolates (97.7%). Furthermore, the hla, hlb, hld, and hlg genes were detected among 11 (7.97%), 7 (5.07%), 16 (11.59%), and 4 (2.89%) isolates, respectively. There was a significant relationship between the presence of alpha and delta hemolysin-encoding genes and the antibiotic resistance pattern of isolates (P < 0.05).Conclusion:The results exhibited that the association between the presence of the hemolysin genes and the antibiotic resistance pattern can be considered as a serious issue.

Cytolytic and systemic toxic effects induced by the aqueous extract of the fire coral Millepora alcicornis collected in the Mexican Caribbean and detection of two types of cytolisins.

BackgroundMillepora alcicornis is a branching hydrocoral common throughout the Caribbean Sea. Like other members of this genus, this species is capable of inducing skin eruptions and blisters with severe pain after contact. In the present study, we investigated the toxicity of the M. alcicornis aqueous extract on several animal models. Considering that some cnidarian hemolysins have been associated to local tissue damage, since they also induce lysis of other cell types, we also made a partial characterization of the hemolytic activity of M. alcicornis aqueous extract. This information is important for understanding the defense mechanisms of the “fire corals”.MethodsThe effects of pH, temperature, and some divalent cations on the hemolytic activity of the extract were assayed, followed by a zymogram analysis to detect the cytolysins and determine their approximate molecular weight. The toxicity of the aqueous extract was assayed in mice, by intravenous administration, and histopathological changes on several tissues were analyzed by light microscopy. The toxicity of the extract was also tested in Artemia salina nauplii, and the damages caused on the crustaceans were analyzed by transmission and scanning electron microscopy.ResultsThe hemolytic activity of the hydrocoral extract was enhanced in the presence of Ca2+ (≥2 mM), Mg2+ (≥6 mM), and Ba2+ (≥0.1 mM); however, it was reduced in the presence of Cu2+ (≥0.1 mM), Zn2+ (≥6 mM), and EDTA (≥0.34 mM). Differences in the pH did not affect the hemolytic activity, but it was temperature-sensitive, since preincubation at ≥ 50 °C sharply reduced hemolysis. The zymogram showed the presence of two types of hemolysins: ~ 28–30 kDa proteins with phospholipase A2 activity and ~ 200 kDa proteins that do not elicit enzymatic activity. The aqueous extract of this cnidarian was lethal to mice (LD50 = 17 μg protein/g), and induced kidney, liver, and lung damages. Under denaturing conditions, the aqueous extract completely lost its toxic and hemolytic activities.ConclusionsThe results showed that the M. alcicornis aqueous extract contains two types of thermolabile hemolysins: proteins of approximately 28–30 kDa with PLA2 activity, while the others are larger proteins of approximately 200 kDa, which do not possess PLA2 activity. Those thermolabile cytolysins, which are stable to pH changes and whose activity is calcium dependent, are capable of inducing damage in lung, kidney and liver tissues, resulting in a slow death of mice. M. alcicornis cytolysins also provoke tissue dissociation in Artemia salina nauplii that might be attributed to pore forming mechanisms.

Identification of a Novel Calcium Binding Motif Based on the Detection of Sequence Insertions in the Animal Peroxidase Domain of Bacterial Proteins

Proteins of the animal heme peroxidase (ANP) superfamily differ greatly in size since they have either one or two catalytic domains that match profile PS50292. The orf PP_2561 of Pseudomonas putida KT2440 that we have called PepA encodes a two-domain ANP. The alignment of these domains with those of PepA homologues revealed a variable number of insertions with the consensus G-x-D-G-x-x-[GN]-[TN]-x-D-D. This motif has also been detected in the structure of pseudopilin (pdb 3G20), where it was found to be involved in Ca2+ coordination although a sequence analysis did not reveal the presence of any known calcium binding motifs in this protein. Isothermal titration calorimetry revealed that a peptide containing this consensus motif bound specifically calcium ions with affinities ranging between 33–79 µM depending on the pH. Microcalorimetric titrations of the purified N-terminal ANP-like domain of PepA revealed Ca2+ binding with a KD of 12 µM and stoichiometry of 1.25 calcium ions per protein monomer. This domain exhibited peroxidase activity after its reconstitution with heme. These data led to the definition of a novel calcium binding motif that we have termed PERCAL and which was abundantly present in animal peroxidase-like domains of bacterial proteins. Bacterial heme peroxidases thus possess two different types of calcium binding motifs, namely PERCAL and the related hemolysin type calcium binding motif, with the latter being located outside the catalytic domains and in their C-terminal end. A phylogenetic tree of ANP-like catalytic domains of bacterial proteins with PERCAL motifs, including single domain peroxidases, was divided into two major clusters, representing domains with and without PERCAL motif containing insertions. We have verified that the recently reported classification of bacterial heme peroxidases in two families (cd09819 and cd09821) is unrelated to these insertions. Sequences matching PERCAL were detected in all kingdoms of life.

Cloning, expressing, and hemolysis of tdh, trh and tlh genes of Vibrio parahaemolyticus

Vibrio parahaemolyticus (VP) is one of the pathogenic vibrios endangering net-cage cultured Pseudosciaena crocea, Fennerpenaeus chinensis, and shellfish in coastal areas of China. Several types of hemolysins produced by Vp have been characterized as major virulence factors. They are thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH) and thermolabile hemolysin (TLH). In this study, we cloned tdh, trh, and tlh genes from the genome DNA of VP by polymerase chain reaction (PCR). We ligated the three genes into prokaryotic expression vector pET-28a (+), and transformed the recombinant plasmids into Escherichia coli BL21 (DE3). The expression of recombinant proteins was induced by isopropyl-β-D-thiogalacto-pyranoside (IPTG). The recombinant proteins were expressed in a form of inclusion bodies and thus purified with Ni-NTA affinity chromatography. Western blotting results showed that recombinant proteins, TDH, TRH and TLH, could be recognized by rabbit anti-VP serum. The three purified proteins were renatured by gradient dialysis. The renatured proteins exhibited hemolytic activity except for TLH in the presence of phosphatidylcholine. These results not only are helpful for better understanding these genes’ functions under a single factor level, but also provide evidence for VP vaccine engineering.

The Rate of Folding Dictates Substrate Secretion by the Escherichia coli Hemolysin Type 1 Secretion System

Secretion of the Escherichia coli toxin hemolysin A (HlyA) is catalyzed by the membrane protein complex HlyB-HlyD-TolC and requires a secretion sequence located within the last 60 amino acids of HlyA. The Hly translocator complex exports a variety of passenger proteins when fused N-terminal to this secretion sequence. However, not all fusions are secreted efficiently. Here, we demonstrate that the maltose binding protein (MalE) lacking its natural export signal and fused to the HlyA secretion signal is poorly secreted by the Hly system. We anticipated that folding kinetics might be limiting secretion, and we therefore introduced the "folding" mutation Y283D. Indeed this mutant fusion protein was secreted at a much higher level. This level was further enhanced by the introduction of a second MalE folding mutation (V8G or A276G). Secretion did not require the molecular chaperone SecB. Folding analysis revealed that all mutations reduced the refolding rate of the substrate, whereas the unfolding rate was unaffected. Thus, the efficiency of secretion by the Hly system is dictated by the folding rate of the substrate. Moreover, we demonstrate that fusion proteins defective in export can be engineered for secretion while still retaining function.

Detection of Urovirulence Genes (eae,E-hly,α-hly) of Uropathogenic Escherichia coli by Specific PCR

Urinary tracts infection remains a common troublesome health problem in the world. Although E.coli is normal intestinal flora but considered as the main important opportunistic active uropathogene because of it’s pathogenesity which referred to it’s different virulence factors like hemolysin, biofilm, enterotoxins, shiga like toxins, cytotoxic necrotizing factor and others. Seventy seven E.coli isolates which isolated from 125 midstream urine samples of patients suffering from different types of UTIs. The results showed that 44 isolates 57% were produced hemolysin. These isolates were differed in the efficiency of erythrocyte lysis because which depend on type of hemolysin and source of blood. Seventy two isolates 90.9% were formed biofilm with variety in thickness of biofilm layer. Each isolate produce hemolysin also formed biofilm. Presence of genes encoded for virulence traits of UPEC (hemolysin, biofilm) were examined by PCR with specific primers. The results showed that the percentage of α-hly gene encoded for α-hemolysin 50% while percentage of eae gene encoded for intimin and E-hly gene encoded for enterohemolysin were 40% and 20% respectively.

Possible Role of Fungal Hemolysins in Sick Building Syndrome

Publisher Summary The chapter first reviews fungal hemolysins and compares them with bacterial hemolysins. Hemolysins are molecules that have the ability to lyse red blood cells (RBCs). There are primarily two types of hemolysins: alpha (α) and beta (β). Alpha hemolysins cause a partial lysis of the RBCs, resulting in a darkening of the media around a colony on sheep's blood agar (SBA). Beta hemolysins produce a complete lysis of the RBCs, resulting in a clearing around the colony growing on SBA. The chapter discusses the biochemistry of hemolysins. Hemolysins are produced by many of the common indoor fungi. Fungal hemolysins might be useful as biomarkers of exposure to indoor fungi because they can be measured in bodily fluids and environmental samples. Fungal hemolysins are generally composed of smaller-molecular-weight monomers than most bacterial hemolysins. They usually have more acidic isoelectric points and contain more cysteine amino acids than bacterial hemolysins. Fungal hemolysins are remarkably slow acting as compared with bacterial hemolysins. However, like most bacterial hemolysins, fungal hemolysins are aggregating proteins that create pores in membranes.

Demonstration and characterization of simultaneous production of a thermostable direct hemolysin (TDH/I) and a TDH-related hemolysin (TRHx) by a clinically isolated Vibrio parahaemolyticus strain, TH3766

Simultaneous production of a thermostable direct hemolysin (TDH)-like toxin (TDHx) and a TDH-related hemolysin (TRH)-like toxin (TRHx) by a clinical isolate (strain TH3766) of Kanagawa phenomenon-positive Vibrio parahaemolyticus was demonstrated and characterized. The two hemolysins were differentially purified by column chromatography on hydroxyapatite and immunoaffinity columns. The molecular weight of the two hemolysins were estimated to be 23,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE). The purified TDHx was indistinguishable from the previously reported TDH/I (from strain TH012) but was different from the authentic TDH of a Kanagawa phenomenon-positive strain (T4750) physicochemically. The mobility of TRHx in nondenaturing PAGE differed from all the known TDHs and TRHs. The genes (tdhX and trhX) coding for TDHx and TRHx were cloned and sequenced. Homologies of nucleotide sequences of the coding regions between tdhX and tdhA (a gene for the authentic TDH) and between trhX and trh (a gene for the authentic TRH) were 98.1 and 99.1%, respectively, and homology between tdhX and trhX was 68.1%. At the amino acid level, TdhX was completely identical to TDH/I, although two base differences were found in the nucleotide sequences between tdhX and tdh/I. Two amino acid differences were observed between TrhX and Trh. Thus, these findings suggest that the TH3766 strain produces two types of hemolysins simultaneously. This is the first evidence that a strain of V. parahaemolyticus produces two types of toxins of the TDH-TRH family at the same time.

BACILLUS CEREUS PATHOGENICITY: A REVIEW

Pathogenic Bacillus cereus can be routinely isolated and identified in the laboratory from foods and other sources. Typing of B. cereus strains implicated in food poisoning outbreaks is helpful for confirmation of the origin of the outbreak and for epidemiological studies. Data concerning vegetative growth and spores are given. Different types of toxin are produced by B. cereus in the course of its growth: a so‐called diarrheal enterotoxin and an emetic heat‐stable toxin; their biochemical characteristics and the systems used for their detection are reviewed. Different types of hemolysins and phospholipases C are also produced and may play a role in pathogenicity. Nongastrointestinal infections were also traced to this species.

Identification of a second hemolysin (HlyII) in Actinobacillus pleuropneumoniae serotype 1 and expression of the gene in Escherichia coli.

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.

Purification and characterization of a hemolysin of Vibrio mimicus that relates to the thermostable direct hemolysin of Vibrio parahaemolyticus

A hemolysin (designated Vm-rTDH) from Vibrio mimicus (AQ0915-E13) was purified by ammonium sulfate fractionation and successive column chromatography with DEAE-Sephadex A-25, hydroxyapatite, Mono Q, Superose 12 and Phenyl-Superose. The Mr of the subunit was estimated to be about 22,000 by sodium dodecyl sulfate-slab gel electrophoresis. The isoelectric point of Vm-rTDH was approximately pH 4.9. The hemolytic activity of Vm-rTDH was stable upon heating at 100 degrees C for 10 min, similar to that of the thermostable direct hemolysin (Vp-TDH) of V. parahaemolyticus. Vm-rTDH also showed lytic activities similar to those of Vp-TDH. Immunological cross-reactivity between Vp-TDH and Vm-rTDH was demonstrated by the Ouchterlony double-diffusion test. Thus we conclude that V. mimicus produces a newly discovered type of hemolysin (Vm-rTDH) which is similar to Vp-TDH.

Purification and characterization of a hemolysin ofVibrio mimicusthat relates to the thermostable direct hemolysin ofVibrio parahaemolyticus

A hemolysin (designated Vm-rTDH) from Vibrio mimicus (AQ0915-E13) was purified by ammonium sulfate fractionation and successive column chromatography with DEAE-Sephadex A-25, hydroxyapatite, Mono Q, Superose 12 and Phenyl-Superose. The Mr of the subunit was estimated to be about 22,000 by sodium dodecyl sulfate-slab gel electrophoresis. The isoelectric point of Vm-rTDH was approximately pH 4.9. The hemolytic activity of Vm-rTDH was stable upon heating at 100 degrees C for 10 min, similar to that of the thermostable direct hemolysin (Vp-TDH) of V. parahaemolyticus. Vm-rTDH also showed lytic activities similar to those of Vp-TDH. Immunological cross-reactivity between Vp-TDH and Vm-rTDH was demonstrated by the Ouchterlony double-diffusion test. Thus we conclude that V. mimicus produces a newly discovered type of hemolysin (Vm-rTDH) which is similar to Vp-TDH.

Pore-Forming Bacterial Protein Hemolysins (Cytolysins)

Protein toxins forming pores in biological membranes occur frequently in Gram-positive and Gram-negative bacteria. They kill either bacteria or eukaryotic cells (at most, a few seem to act on both groups of organisms). Most of the toxins affecting eukaryotes have clearly been shown to be related to the pathogenicity of the producing organisms. Toxin formation frequently involves a number of genes which encode the toxin polypeptide as well as proteins for toxin activation and secretion. Regulation of toxin production is usually coupled with that of the synthesis of a number of other virulence factors. Iron is the only known environmental factor that regulates transcription of a number of toxin genes by a Fur repressor-type mechanism, as has been originally described in Escherichia coli. Interestingly, the thiol-activated hemolysins (cytolysins) of Gram-positive bacteria contain a single cysteine which can be replaced by alanine without affecting the cytolytic activity. The Gram-negative hemolysins (cytolysins) are usually synthesized as precursor proteins, then covalently modified to yield an active hemolysin and secreted via specific export systems, which differ for various types of hemolysins.

Close association of verotoxin (Shiga-like toxin) production with enterohemolysin production in strains of Escherichia coli.

Sixty-four verotoxin-producing (VT+) Escherichia coli strains were analyzed for VT1- and VT2-specific DNA sequences and for production of hemolysin. Strains of human origin were of the following serotypes: O157:H7 or H-, O111:H8 or H-, O26:H11, O114:H4, and rough:H7. Strains of serotypes O157:H7, O113:H21, O116:H21, and rough:H- were from cattle, while those of serotype O139:K12:H1 were from pigs. All 64 isolates carried either VT1 or VT2 or both genes. Sixty of the strains (93.8%) were hemolytic (Hly+). The three O139:K12:H1 strains examined produced alpha-hemolysin, as shown by their reaction with the alpha-hemolysin-specific monoclonal antibody h2A and by DNA hybridization with an alpha-hly gene probe. The remaining 57 Hly+ strains (95%) produced a different type of hemolysin (enterohemolysin), which is genetically and serologically unrelated to alpha-hemolysin. The two types of hemolysin are further distinguished by the appearance of the lysis zone on blood agar and by the time interval for the detection of hemolysis. In contrast to alpha-hemolysin, enterohemolysin can be detected only on blood plates containing washed erythrocytes. The frequent association of enterohemolysin with verotoxin production (89%) makes it useful as an epidemiological marker for rapid and simple detection of potential VT+ E. coli.

Serotype-related differences in production and type of heat-labile hemolysin and heat-labile cytotoxin of Actinobacillus (Haemophilus) pleuropneumoniae

Reference strains of serotypes 1 to 12 of Actinobacillus (Haemophilus) pleuropneumoniae were cultured in Eagle minimal essential medium with 10% Serum Plus. Culture supernatants were examined for cytotoxicity to alveolar macrophages and for the ability to hemolyze sheep erythrocytes. All strains except the reference strain of serotype 6 produced cytotoxin, whereas only serotypes 1, 5, 9, 10, and 11 produced hemolysin. Both cytotoxin and hemolysin appeared to be heat labile. Antisera were raised against cytotoxin- and hemolysin-containing culture supernatants of serotypes 1 to 11. Cross-neutralization studies revealed that the hemolysins were serologically homogeneous. In contrast, four serologically different cytotoxins were distinguished. One cytotoxin was produced by serotypes 1, 5, 9, and 11, and a second was produced by serotypes 2, 3, 4, and 8. A third cytotoxin was produced by serotypes 7 and 12; this cytotoxin was related to the cytotoxins of serotypes 1, 2, 4, 5, 9, and 11. A fourth cytotoxin, produced by serotype 10, was related to the cytotoxin of serotypes 1, 5, 9, and 11. Seventy field strains belonging to serotypes 2, 3, 7, 8, 9, and 11 were also tested for production of cytotoxin and hemolysin. All strains belonging to serotypes 9 and 11 produced hemolysin and cytotoxin, whereas all strains of serotypes 2, 3, 7, and 8 produced only cytotoxin. Hemolysins and cytotoxins of both the field strains and the corresponding serotype reference strains were comparably neutralized. These findings strongly suggest that the observed differences in production and type of hemolysin and cytotoxin were related to serotype and not to strain.

Enterohemolysin, a new type of hemolysin produced by some strains of enteropathogenic E. coli (EPEC)

42 Escherichia coli O26 strains which had been isolated at geographically different places and over a long time period were examined for hemolysin synthesis. 17 of these were found to be hemolysin-negative, nine strains were found to produce plasmid encoded alpha-hemolysin and 16 strains were shown to produce a phenotypically different hemolysin. This new type of hemolysin was called enterohemolysin and found to be genetically and immunologically non-related with the already described E. coli alpha-hemolysin. Enterohemolytic E. coli were not found in feces of 200 healthy infants under the age of two years. However, four E. coli O111 strains, one O121:H- and one O25:K5:H- strain, all from infants with diarrhoea were also enterohemolysin producers.

Comparison of four hemolysin-producing organisms (Escherichia coli, Serratia marcescens, Aeromonas hydrophila, and Listeria monocytogenes) for release of inflammatory mediators from various cells

We investigated the role of various hemolysin-producing strains (Escherichia coli, Serratia marcescens, Aeromonas hydrophila, and Listeria monocytogenes) in induction of inflammatory mediators, e.g., histamine release from rat mast cells as well as the chemiluminescence response and the release of lipoxygenase transformation products from human polymorphonuclear neutrophils. Our data show that the hemolysin-positive bacteria as well as the hemolysin-positive culture supernatants were active in inducing the chemiluminescence response, leukotriene (LTB4 and LTC4) release from human granulocytes, and histamine release from rat mast cells. The degree of leukotriene release was dependent on the hemolysin type and on the expression of hemolysin activity. The E. coli alpha-hemolysin and the aerolysin-producing A. hydrophila were the most potent stimuli whether washed bacteria or bacterial supernatant was used. Bacteria expressing the S. marcescens hemolysin and the listeriolysin were only poor inducers of leukotriene generation. In contrast to leukotriene generation, all hemolysin-positive strains induced nearly the same histamine release in a dose-dependent manner. Our data suggest a potent role for various hemolysins as virulence factors in inducing the release of inflammatory mediators.