Hemolytic and hemoxidative activities in bacteria

1.4.1 Hemolysis and hemoxidation

The bacterial struggle for iron is often accompanied by hemolysis. Hemolysis describes a process in which red blood cells (RBCs) are destroyed due to the action of lytic compounds. This is a convenient effect, since the bacteria gain access to a lot of nutrients which are released from the lysed erythrocyte.

Most importantly, they gain access to iron which is bound inside the hemoglobin molecules of the red blood cell (Fig. 1.2 A).

A

B

Bacterial hemolysis can be divided into three major types, which are illustrated in Fig. 1.2 B.: alpha-, beta-, and gamma hemolysis. Bacteria which exhibit gamma hemolysis do not have hemolytic activity.

In contrast, beta-hemolysis describes the process of complete lysis of blood cells leading to a clear yellow halo around the colonies in which no intact red blood cells containing hemoglobin are present anymore. Finally, alpha hemolysis is a process in which the red blood cells are not destroyed, but the hemoglobin is modified. One possibility is, that the Fe²⁺ bound in the middle of the tetrapyrrole ring in

Fig. 1.2. Illustration of heme as part of hemoglobin which then again is part of a red blood cell (A) and Types of bacterial hemolysis (B). A. Hemoglobin (1GZX) is made up of four subunits, each of which contains a polypeptide chain called globin and a heme group. Red blood cells (erythrocytes) consist of about 270 million hemoglobin molecules which makes circa 97% (w/w) dry weight (Weed et al., 1963). (modified from www.chem.ucla.edu, www.rcsb.org and Toumey, 2011) B.

Blood agar plate with alpha-hemolytic (α), beta-hemolytic (β) and non-hemolytic (γ) streptococci recorded with transmission light (www.microbelibrary.org.)

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the heme molecule is oxidized to Fe³⁺ resulting in a different form of hemoglobin: methemoglobin (metHb). This process of hemoxidation leads to a brownish discoloration of the blood around the bacterial colonies on the plate. In vivo, oxidized hemoglobin has a reduced binding affinity towards oxygen and also the release of oxygen is hindered. However, this process is reversible. Similar to oxididation, hemoglobin can also be sulfenylated resulting in so called sulfhemoglobin (sulfHb), which appears as a greenish-brownish discoloration of blood (Chatfield and La Mar, 1992). As a result, hemoglobin loses its ability to bind oxygen in a non-reversible manner. Both, the formation of sulfHb and metHb are forms of alpha hemolysis. For simplification, the term “hemoxidation” will in this work be referred to as any kind of alpha-hemolysis, whereas the term “hemolysis” is only used for beta-hemolysis.

1.4.2 Hemolysins and hemolytic toxins

Beta-hemolysis is usually induced by the action of proteins which destroy the phospholipid bilayer of the RBC’s membrane. This can be mediated by (i) enzymes like phospholipases which hydrolyze the membrane phospholipids, (ii) toxins which exhibit a detergent-like (surfactant) activity that results in membrane solubilization and (or) partial insertion into the hydrophobic regions of target membranes, or (iii) pore-forming toxins which, after their secretion, build oligomers inserting into eukaryotic cell membranes, thereby causing their leakage (Titball, 1993; Braun and Focareta, 1991). The hemolytic actions of phospholipases A and C have been described for several pathogenic bacteria like Borrelia, Staphyloccocci, Clostridia or Listeria (Williams and Austin, 1992; Smith and Price, 1938; van Heyningen, 1941; Geofflroy et al., 1991). Examples for phospholipases C in gram-positive bacteria are phosphatidylinositol phospholipases C, e.g. PLC-A from L. monocytogenes, sphingomyelinases as the beta-hemolysin from Staphylococcus aureus or Zinc-metalloenzymes like the alpha-toxin of Clostridium perfringens. These enzymes have different preferences concerning their targeted phospholipid: Zinc-dependent phosphilopases C preferentially degrade phosphatidylcholine, whereas sphingomyelinases prefer sphingomyelin (Nakamura et al., 1988; Maheswaran and Lindorfer, 1967;

Titball, 1993).

The most intensely investigated and best described hemolysins are the numerous pore-forming toxins.

Important examples from gram-negative bacteria are the α-hemolysin of E. coli, which is encoded by the gene hlyA, and the similar hemolysin CyaA of Bordetella pertussis (Cavalieri et al., 1984; Hackett et

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al., 1994). HlyA from E. coli is a 107 kDa protein that induces hemolysis by creating about 2-nm-wide pores in the erythrocyte membrane. Those pores are thought to increase the permeability thereby producing cell swelling, which ends up in RBC rupture (Bhakdi et al., 1986).

The α-toxin of S. aureus is probably the most famous hemolysin from gram-positive bacteria. It is a small β-barrel pore-forming toxin which is secreted as a monomer, but oligomerizes into a heptameric structure when binding the host-cell membrane. This binding leads to formation of a 1-3 nm membrane-perforating barrel pore that allows the efflux of Ca²⁺, K⁺, ATP and low-molecular weight molecules with a maximum size of 4 kDa (Bhakdi and Tranum-Jensen, 1991). Another group of pore-forming toxins which is present in several genera of gram-positive bacteria are the thiol-activated hemolysins. These include the listeriolysin O (L. monocytogenes), the pneumolysin (Streptococcus pneumoniae), the perfringolysin (C. perfringens) and the streptolysin O from Streptococcus pyogenes.

All these toxins are rapidly inactivated in the presence of oxygen but can be activated again after addition of sulfhydryl compunds. Streptolysin O has been shown to insert into cholesterol-containing membranes, where up to 100 monomers aggregate and assemble as a superstructure forming a transmembrane channel with up to 7.5 nm width (Bhakdi et al., 1985).

In contrast to the hemolytic toxins evoking beta-hemolysis, alpha-hemolysis is not induced by proteins. Secretion of hydrogen peroxide or hydrogen sulfide is the main cause for bacterial oxidation or sulfenylation of hemoglobin. Alpha-hemolysis following production of hydrogen peroxide is used for typing of bacterial species and typically seen in Streptococci like S. pneumoniae and S. mutans (Duane et al., 1993; Hamada and Slade, 1981).

1.4.3 Hydrogen sulfide

In the recent years, hemoglobin alteration and hemolysis as result of hydrogen sulfide production has been studied in several oral pathogens. Among them, the “cystalysin” of Treponema denticola and its hemoxidative and hemolytic activity have been elaborately studied. Cystalysin is a 46 kDa, pyridoxal-5-phosphate (PLP) dependent L-cysteine desulfhydrase, which is homologous to aminotransferases and is able to produce ammonia, pyruvate and H2S from L-cysteine (Chu et al., 1995; Chu et al., 1997).

Heterologous expression of cystalysin in E. coli lead to high hemoxidation and hemolysis rates which became apparent in clear halos around the respective E. coli colonies (Chu et al., 1995). Detailed investigation of human erythrocytes incubated with the purified enzyme revealed strong

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methemoglobin and sulfhemoglobin formation which was attributed to the production of hydrogen sulfide (Kurzban et al., 1999). The production of hydrogen sulfide is a prevalent feature of oral pathogenic bacteria and responsible for periodontal diseases and oral malodor (Tonzetich, 1971). The genera Fusobacterium, Prevotella and Porphyromonas are amongst the predominant H²S producers (Persson et al., 1990). For H2S formation, these bacteria possess PLP dependent βC-S lyases which catalyze the α,β-elimination of L-cysteine. Hemolytic activity correlating with H2S production has not only been demonstrated for T. denticola, but also for Fusobacterium nucleatum, Streptococcus anginosus, Streptococcus intermedius and Prevotella intermedia (Fukamachi et al., 2002; Yoshida et al., 2002; Ito et al., 2008; Yano et al., 2009).