Porenbildende Toxine und mucosale Integrität

Abstract

This short communication focuses on the role of hemolysins from barrier-breaking bacteria in mucosal damaging. Pore-forming toxins (PFTs), produced by hemolytic bacteria, are secreted proteins that integrate into the membrane of host cells and form pores, which are frequently cytolytic. But beyond these cytolytic effects, PFTs can induce a variety of defined pathogenic processes. Depending on the toxin, intestinal epithelial function can be impaired by e.g. secretion, induction of lesions or tight junction disturbance. The latter was shown as leak flux mechanism in diarrhea, by which water and small solutes can cross the epithelial barrier. Here, the action of PFTs on the intestinal epithelial barrier and underlying pathomechanisms are reviewed.

hemolysin – enterotoxin – pore – focal leaks – chloride secretion – transepithelial resistance – tight junctions – barrier dysfunction – leak flux – diarrhea

Zusammenfassung

Diese Kurzbeschreibung konzentriert sich auf die Rolle von Hämolysinen barrierebrechender Bakterien bei der Mucosaschädigung. Porenbildende Toxine (PFTs), die durch hämolytische Bakterien erzeugt werden, sind sezernierte Proteine, die in die Membran von Wirtszellen integrieren. Diese Poren sind oft zytolytisch, aber neben zytolytischen Effekten können PFTs eine Vielfalt von definierten pathogenen Prozessen induzieren. Abhängig vom Toxin kann die epitheliale Darmfunktion durch z.B. Sekretion, Induktion von Läsionen oder Tight-Junction-Störungen verschlechtert werden. Letzteres wurde als Leck-Fluss-Mechanismus bei Diarrhö bezeichnet, dabei können Wasser und kleinere Solute die epitheliale Barriere durchqueren. Hier wird die Wirkung von PFTs auf die epitheliale Darmbarriere und die zugrunde liegenden Pathomechanismen untersucht.

Introduction

Hemolysins are traditionally characterized on blood-agar plates. Furthermore, the hemolytic activity correlates to the amount of PFTs produced by a bacterium. But the ability to lyse erythrocytes does not reflect the function of these virulence factors in the intestine in vivo, abstract away from acquired hemolytic anemia. In the bacterial kingdom a vast diversity of PFTs are known. Zoonotic germs harbor certain PFTs; as e.g. listeriolysin-O from Listeria monocytogenes or α-hemolysins from Escherichia coli and others. Not all PFTs are solely cytotoxic, different reactions on different toxins can lead to numerous cellular response mechanisms; including ion flux imbalance, active chloride secretion, membrane permeation to ions and/or larger solutes, vacuolation, cytoskeleton depolarization etc. Lysis or programmed cell death can subsequently occur, even with low hemolytic activities. Current investigations aim on these toxins and their impact on the intestinal mucosa.

Pore-forming toxins

Among the hemolysins, the pore-forming toxins are widespread and can be divided into different categories, depending on their structural similarities; (i) Small PFTs, (ii) binary toxins, (iii) cholesterol-dependent cytolysins (CDCs), and (iv) beta-barrel-PFTs (β-PFTs).

The group of the CDCs form relatively large pores in the host cell membrane. These pores consist of 30 to 40 monomers and have an inner pore diameter of approximately 260 Å, which allow ions and small solutes to cross the cell membrane. Listeriolysin-O is one representative of this group. Another group of PFTs this article focuses on are the β-PFTs. These are mushroom-shaped structures composed of seven monomers that form small, cation-permeable pores with an inner diameter between 10 and 20 Å. E. coli α-hemolysin (HlyA) or aerolysin (AerA) from Aeromonas hydrophila belong to this group, sharing the structure of a heptameric β-barrel pore, but showing different actions on the intestinal mucosa.

Impact on the intestinal mucosa

In general, hemolytic bacteria can induce several pathological effects in the intestine, including malabsorption [1], secretory dysfunction [2], or epithelial lesions by necrosis or apoptosis induction [3]. A well-known effect of PFTs is induction of cell death by necrosis or apoptosis. An increased epithelial apoptotic rate can contribute to gross lesions. Epithelial lesions contribute to functional barrier defects that can be accompanied by an inflammatory response. Our group could discover a novel mechanism by which epithelial lesions can be induced or exacerbated.

Results

Lesions

α-hemolysin from uropathogenic E. coli O4 induced focal leaks in epithelial cell monolayers as shown in Fig. 10.1 [4]. An accumulation of E. coli was observed within the induced lesion. Moreover, supernatants from these HlyA-containing E. coli O4 delayed epithelial restitution after induction of artificial lesions (single-cell lesions) in human intestinal epithelium [5]. The underlying cellular response mechanisms, that mediate these effects, were not resolved so far.

Fig. 10.1 Confocal laser-scanning microscopy of focal leaks in monolayers of the human intestinal epithelial cell line HT-29/B6 after infection with E. coli O4 (green) [4]. The tight junction protein occludin is marked red. Bar 30 µm.

Secretion

The CDC listeriolysin-O (LLO) as well as the β-PFT aerolysin (AerA) were shown to induce active chloride secretion in intestinal epithelial cells. In Figs. 10.2 and 10.3 short-circuit current (I_SC) is increased as an indication for opening of chloride channels. Concomitant to the AerA response on I_SC, a reduction in transepithelial resistance was observed. However, the permanent drop in resistance cannot be explained by a transient induction of active chloride secretion or by the pore-formation alone. Here, an involvement of the tight junctions is supposable.

Fig. 10.2 Short-circuit current (I_SC) is increased after stimulation of HT-29/B6 monolayers with listeriolysin-O (LLO) [8].

Fig. 10.3 I_SC of HT-29/B6 monolayers increased after treatment with aerolysin-containing preparations from recombinant E. coli (DH5aerA), while transepithelial resistance (R^t) decreased [9].

Discussion

Barrier function of the intestinal epithelium is maintained for the most part by the tight junction (TJ) strands. In diarrhea several mechanisms can contribute to the barrier impairment; one main reason is disturbance of TJs. Members of the claudin protein family, which are present in defined compositions in the TJ strands, act as major sealing component. But the role of PFTs on the action on TJ stability is rather underestimated. Claudins can be a target of pathogenic influence by a β-PFT. The enterotoxin from Clostridium perfringens (CPE) has a binding site for claudin-3 and -4, removes these claudins from the TJ strands and leads to rapid cell death [6,7]. For other PFTs no claudin binding was described, but a cellular reaction on integration of a PFT into the host cell membrane could be TJ disturbance, which is not described so far.

The induction of active chloride secretion by LLO was shown to be mediated by calcium signaling, as a Ca²⁺-induced Ca²⁺-release opens Ca²⁺-dependent chloride channels [8]. In case of AerA a PKC-dependent activation of chloride channels was shown [9]. In addition, both studies showed an increase in epithelial permeability in mannitol flux measurements. From these experiments it was deduced that the paracellular pathway could also be affected. Furthermore, the described signaling pathways are also involved in TJ assembly/disassembly. Hence, this suggests that the TJ can subsequently be regulated by PFTs. However, such effects depend on the concentration of each toxin, its cell type specificity and its cellular target structure. Thereby, protective mechanisms of the epithelium as e.g. internalization of PFTs or TJ expression-changes, to rinse off noxious agents from the mucosa, deserve closer attention.

Epithelial gross lesions by induction of necrosis or apoptosis as well as by induction of focal leaks, as shown for HlyA [4], can on the one hand contribute to intestinal barrier impairment in diarrhea, on the other hand the entry of antigens or bacteria into the organism triggers intestinal inflammation. Here, the underlying cell signaling pathways and morphological changes, e.g. by cytoskeleton alterations, are of high interests. The cellular response to hemolysins including cytoskeletal and TJ disturbance is the purpose of current investigations of our group [10].

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Correspondence
Prof. Dr. Jörg-Dieter Schulzke
[e-mail]