Secretion and mechanism of action of the hole-forming toxin aerolysin.

Aeromonas hydrophila exports aerolysin as a protoxin which is activated by proteolysis after release. Aerolysin binds to the eucaryotic cell receptor glycophorin and oligomerizes, forming holes in the membrane. Important regions of the molecule have been identified by site-directed mutagenesis, and channel formation has been studied in planar lipid bilayers.

Functional analysis of exotoxin A-related protein of Pseudomonas aeruginosa lacking residues 225-412.

The crystal structure of the exotoxin A (ETA) of Pseudomonas aeruginosa showed that this protein is folded into three distinct domains. Domain I (Ia and Ib), the amino-terminal domain, is the receptor-binding domain of ETA and domain III, the carboxy-terminal domain, is responsible for the ADP-ribosyl transferase activity of the toxin. To elucidate the function(s) of domains 1b and II in the intoxication process and to define the region of the domain III necessary for ADP-ribosylating activity, a defined deletion in the structural gene of P. aeruginosa ETA encompassing residues 225-412 was constructed and an ETA-related product DeID, (from which all of domains II and Ib were deleted) was expressed. The ETA-related protein did not penetrate sensitive cells, but retained the same specific activity to ADP-ribosylate elongation factor-2 as wild-type toxin. This suggests that domain II is necessary to allow toxin internalization by sensitive cells and that the absence of domain Ib does not interfere with enzymic activity. The domain strictly involved in ADP-ribosylation activity encompasses residues 412-613.

Shiga toxin: purification, structure, and function.

Shiga toxin is a potent toxin produced by Shigella dysenteriae type 1 strains. The toxin has three biologic activities--cytotoxicity, enterotoxicity, and neurotoxicity--and one known biochemical effect: inhibition of protein synthesis. It consists of two polypeptide chains, an A chain (molecular weight, 32,225) and a B chain (molecular weight, 7,691). These two peptides associate with a stoichiometry of one A and five B subunits to form the holotoxin. The A chain is responsible for the biochemical effect of the holotoxin: cleavage of the N-glycosidic bond of adenine at nucleotide position 4324 in the 28S rRNA of the 60S ribosomal subunit. The B chain mediates binding of toxin to cell surface receptors. Shiga toxin is the prototype of a family of toxin molecules that have been termed Shiga-like in terms of both structural and functional analysis.

[What are toxins to microbes? (the role of toxins in bacterial ecology)]. / Zachem mikrobam toskiny? (o roli toksinov v ékologii bakterii).

The author attempts to answer two questions: whether the toxins, in particular the toxins having their specificity connected with enzymatic activity, are needed for microbial cell physiology and their significance for bacteria that are not the obligate parasites for warm blooded animals. The analysis of literary data supposes the toxins to be essential cellular metabolites since many of them participate in energy acquiring. Besides that a number of toxins is shown to be relevant to microbial life and to affect the micropredators, especially the monocellular organisms feeding the microbes. In connection with the above mentioned, special attention is paid to extrachromosomal location of many toxins genes relating them to bacteriocins. The possibility is not excluded that in the future the new toxins might come to be found having the enzymatic activities.

Aerolysin of Aeromonas sobria: evidence for formation of ion-permeable channels and comparison with alpha-toxin of Staphylococcus aureus.

Aerolysin from Aeromonas sobria AB3 was isolated and purified. The pure toxin formed sodium dodecyl sulfate-insoluble oligomers in a lipidic environment. The addition of aerolysin to the aqueous phase bathing lipid bilayer membranes resulted in the formation of ion-permeable channels which had a single-channel conductance of about 70 pS in 0.1 M KCl. This defines the toxin as a channel-forming component similar to other toxins but without any indication for an association-dissociation reaction, since the channels had a long lifetime at low voltages. At voltages higher than 50 mV, the aerolysin channel switched into a closed state with a low residual conductance. The single-channel conductance was a linear function of the total aqueous conductance, which suggested that the toxin oligomers formed aqueous channels with an estimated minimal diameter of about 0.7 nm. The aerolysin pores were found to be slightly anion selective. The pore-forming properties of aerolysin were compared with those of alpha-toxin of Staphylococcus aureus. Both aerolysin and alpha-toxin share secondary structure features, must oligomerize to form pores in lipid bilayer membranes, and form channels with similar properties.

Overview of the virulence attributes of the HAP-group of bacteria.

The pathogenicity requirements of the HAP bacteria include colonization of mucous surfaces, invasion of the host tissues, survival and multiplication in the host, interference with the defences of the host, and damage to the host. For these purposes the bacteria possess adhesion structures, capsular polysaccharides, surface structures such as outer membrane proteins, and lipopolysaccharides. They also secrete extracellular products, including exotoxins. The information available in this context for the major pathogens of the HAP group is reviewed.

Influence of a glycine or proline substitution on the functional properties of a 14-amino-acid analog of Escherichia coli heat-stable enterotoxin.

Analogs of Escherichia coli heat-stable enterotoxin (ST) differing in chain length or the presence of turn-forming residues were assessed for binding to receptors, activation of particulate guanylate cyclase, and stimulation of secretion in suckling mice. These analogs included the native 18-amino-acid peptide (ST), the 14-amino-acid carboxy terminus of this native peptide with a proline at position 12 (ST[5-18]proline), and the 14-amino-acid carboxy terminus in which the proline at position 12 was substituted with glycine (ST[5-18]glycine). Each analog bound to the receptor in a dose-dependent fashion, completely displacing [125I]ST in competitive binding assays. However, their potencies differed significantly: ST demonstrated the highest affinity (inhibition constant [Ki], 10(-9) M), followed by ST[5-18]proline (Ki, 10(-7) M) and ST[5-18]glycine (Ki, 10(-6) M). Similarly, these peptides maximally activated particulate guanylate cyclase and stimulated intestinal secretion in suckling mice. Their rank order of potency in these assays was similar to that described for receptor binding: ST greater than ST[5-18]proline greater than ST[5-18]glycine. These data demonstrate that the full peptide structure is not absolutely required for pharmacological, biochemical, or biological activity. However, the four amino-terminal residues contribute significantly to the potency of these peptides. In addition, the turn imposed by the proline residue at position 12 is not absolutely required for receptor occupancy or activation of the biochemical cascade that results in intestinal secretion. However, it significantly increases the potency of the toxin. These data illustrate the importance of primary and secondary structures to the biochemical, pharmacological, and physiological activities of the ST produced by E. coli.

Roles of alpha-toxin and beta-toxin in virulence of Staphylococcus aureus for the mouse mammary gland.

Mutants of Staphylococcus aureus which fail to express alpha-toxin (Hly), beta-toxin (Hlb), or both have been constructed by site-specific mutagenesis. The virulence of the mutants was compared with that of wild-type toxigenic strains by intramammary inoculation of lactating mice. A bovine strain, M60, and a laboratory strain, 8325-4, caused acute mastitis and death within 48 h for 60% of the mice inoculated. Animals inoculated with Hly mutants also developed acute mastitis, but no deaths occurred. Comparisons of Hly- or Hlb-positive strains with the double mutation Hly Hlb showed that both toxins led to a significantly higher recovery of S. aureus from the gland 48 h postinfection. Histopathological examination of mammary glands showed that phagocytosis of bacteria occurred irrespective of toxigenicity, but toxigenic strains, particularly those which were Hly+, continued to multiply, invaded the interalveolar tissues, and produced severe lesions. Stimulation of an inflammatory response by inoculation of the mammary gland with endotoxin prior to challenge with S. aureus reduced recovery of the bacteria 10- to 100-fold and, under these conditions, neither alpha-toxin nor beta-toxin contributed significantly to growth and survival.

Common themes in microbial pathogenicity.

A bacterial pathogen is a highly adapted microorganism which has the capacity to cause disease. The mechanisms used by pathogenic bacteria to cause infection and disease usually include an interactive group of virulence determinants, sometimes coregulated, which are suited for the interaction of a particular microorganism with a specific host. Because pathogens must overcome similar host barriers, common themes in microbial pathogenesis have evolved. However, these mechanisms are diverse between species and not necessarily conserved; instead, convergent evolution has developed several different mechanisms to overcome host barriers. The success of a bacterial pathogen can be measured by the degree with which it replicates after entering the host and reaching its specific niche. Successful microbial infection reflects persistence within a host and avoidance or neutralization of the specific and nonspecific defense mechanisms of the host. The degree of success of a pathogen is dependent upon the status of the host. As pathogens pass through a host, they are exposed to new environments. Highly adapted pathogenic organisms have developed biochemical sensors exquisitely designed to measure and respond to such environmental stimuli and accordingly to regulate a cascade of virulence determinants essential for life within the host. The pathogenic state is the product of dynamic selective pressures on microbial populations.

Virulence determinants in nonsporeforming anaerobic bacteria.

The literature dealing with adherence, host-protective mechanisms and tissues damaging products of nonsporeforming anaerobes is reviewed. The adherence mechanisms are poorly understood. There is evidence for that encapsulation plays a role in the pathogenicity of Bacteroides fragilis and black-pigmented bacteroides. A leukocidin is produced by Fusobacterium necrophorum, and Ig protease, collagenase and a trypsin-like enzyme by some Bacteroides species. Some Bacteroides fragilis strains produce an enterotoxin. The pathogenetic role of endotoxin is unclear.

Demonstration of an outer membrane protein with antiphagocytic activity from Pasteurella multocida of avian origin.

A strain of Pasteurella multocida of avian origin was found to inhibit phagocytosis of Candida albicans by mononuclear phagocytes in vitro. Whole-cell lysates of P. multocida showed this effect, as did a 50-kilodalton (kDa) protein eluted from sodium dodecyl sulfate-polyacrylamide gels obtained by electrophoresis of whole-cell lysates. Heat, digestion with trypsin, and antibody specific for this 50-kDa protein neutralized the antiphagocytic effects of P. multocida, of the whole-cell lysates, and of the 50-kDa protein itself. Evidence that this protein was in the outer membrane of the bacterial cell included the findings that (i) treatment of encapsulated or unencapsulated P. multocida with trypsin reduced the antiphagocytic effect; (ii) whole-cell lysates prepared from trypsinized, unencapsulated P. multocida had reduced antiphagocytic activity; and (iii) antibody to outer membrane proteins neutralized the antiphagocytic effect. Turkeys given antibodies specific for the 50-kDa outer membrane protein were protected against lethal challenge with P. multocida.

The splanchnic organs as the source of toxic mediators in shock.

The splanchnic organs, and especially the pancreas and the small intestine, are susceptible to injury during ischemia and shock and the following reperfusion. This particular tissue injury is associated with a release of cardiotoxic and other toxic mediators which further aggravate the shock condition, leading to further tissue injury etc. Thereby a vicious circle is created, which is likely to contribute to collapse and death.

In vivo emergence of enterotoxigenic Escherichia coli variants lacking genes for K99 fimbriae and heat-stable enterotoxin.

Neonatal pigs were inoculated with porcine enterotoxigenic Escherichia coli 431, which carries genes for K99 fimbriae and STaP enterotoxin. Colonies of strain 431 were recovered from feces of pigs for up to 17 days after inoculation and tested for hybridization with gene probes for K99 and STaP. Variants of strain 431 that did not hybridize with the probes were considered to have lost the genes. Variants were recovered from 10 of 13 suckling pigs that survived the infection. Only 0.4% of the isolates recovered during the first 2 days after inoculation were variants. Of the isolates recovered 3 to 5 days after inoculation, 20 to 36% were variants. Variant colonies were detected more frequently among pigs in some litters than in others. The litter with the highest number of variant-shedding pigs had the dam with the highest titer of K99 antibody in her colostrum. Variants also occurred in colostrum-deprived, artificially reared pigs. However, the number of variants detected was lower and they occurred later in the course of the infection in colostrum-deprived pigs than in suckling pigs. More variants were detected and they were detected earlier in colostrum-deprived pigs fed anti-K99 monoclonal antibody than in controls fed anti-K88 monoclonal antibody. Loss of STaP appeared to be secondary to loss of K99 in that some variants lacked only K99 (K99- STaP+) and some lacked both genes (K99- STaP-), but none was of the K99+ STaP- type. Our results confirmed reports of gene loss from enterotoxigenic E. coli during infection. They are consistent with the hypothesis that variants emerge under in vivo selection pressure of K99 antibody and with the speculation that gene loss may be an important component of protection in vaccinated populations. However, the emergence of variants did not appear to play a major role in the recovery of individual pigs from clinical disease.

T84 cell receptor binding and guanyl cyclase activation by Escherichia coli heat-stable toxin.

Escherichia coli heat-stable enterotoxin (STa) induces intestinal secretion by binding to enterocyte receptors and activating the guanylate cyclase-guanosine 3',5'-cyclic monophosphate (cGMP) system. The intermediate steps between binding of STa and secretion are poorly understood, due in part to the lack of a convenient system to study the effects of STa at the cellular level. To establish such a model, we investigated the binding of 125I-STa, STa activation of guanylate cyclase, and STa-induced increase in cGMP production in a well-characterized human colonic cell line, T84. Binding was specific, linear with cell number, and time, temperature and pH dependent, and reversible. ST may also be internalized by these cells. Addition of unlabeled STa competitively inhibited binding of 125I-STa. These parameters closely resemble those described in intact rat enterocytes and cell-free membrane preparations. STa stimulated guanylate cyclase and cGMP production in a dose-related manner. The similar dose-response relationships for binding, guanylate cyclase stimulation by STa, and cGMP production suggest that the guanylate cyclase-cGMP system is coupled to ST occupancy of specific receptors. These data, together with the fact that STa induces chloride secretion from T84 cells suggest that T84 cells are a suitable and convenient system to study the cellular mechanism of action of STa.

Sudden infant death syndrome. The role of putrefactive toxins in respiratory paralysis and cerebral coma.

The Sudden Infant Death Syndrome (SIDS) is responsible for the death of 10,000 infants annually in the U.S.A. The cause of these deaths is not known; all SIDS infants studied died during sleep in a silent fashion and there are no specific lesions observable post mortem. It may be of significance, however, that nearly half of SIDS infants had a respiratory tract infection in the last two weeks of life while forty percent had bloody froth over their mouths when found, presumably pulmonary oedema fluid. It is hypothesis that improper feeding or diarrhoea may likewise be involved in SIDS and that enterotoxins from putrefactive products are the precipitating event in this syndrome.

Subgingival microorganisms and bacterial virulence factors in periodontitis.

Considerable information has come forth in recent years on the pathogenic organisms in human periodontitis and the sequence of events by which they produce periodontal disease. Important periodontopathogens include Bacteroides gingivalis, Bacteroides intermedius and Actinobacillus actinomycetemcomitans. Virulence factors of B. gingivalis and B. intermedius may mainly involve enzymes with potential to interfere with host defenses and to disintegrate periodontal tissues. Pathogenic properties of A. actinomycetemcomitans appear predominantly to be exerted by leukotoxin and other noxious products.