THE ROLE OF G6PD IN MALARIA RESISTANCE

Malaria, resulting from infection by the Plasmodium falciparum and Plasmodium vivax parasites, is the leading cause of death in the global human population. Each year 500 million people suffer from malaria, resulting in approximately 2 million deaths annually. During the course of human evolution in regions where malaria is prevalent, naturally occurring genetic defense mechanisms have evolved for resisting infection by the Plasmodium parasite. Most of the human genes that are thought to provide resistance against malarial infection are expressed in red blood cells or play a role in the immune system (Hill, 2001). These loci include: HLA, α- and β-globins, Duffy factor (FY), tumor necrosis factor (TNF), and glucose-6-phosphate dehydrogenase (G6PD).

G6PD is an important “housekeeping” enzyme in the glycolytic pathway for glucose metabolism. G6PD also plays a critical role in maintaining the balance of NADPH, a necessary cofactor for cell detoxification. Although several enzymes can recycle this essential cofactor, G6PD is the sole generator of NADPH in the red blood cells and alone may prevent oxidative damage and severe anemia. Because of its role in preventing oxidative stress within the cell, G6PD may also play a role in longevity (Ho et al., 2000).

G6PD deficiency is the most common enzymopathy of humans, affecting an estimated 400 million people worldwide (Vulliamy et al., 1992).

Although the Gram-negative bacterium Escherichia coli is normally considered to be a harmless commensal of the gastrointestinal flora, there are some exceptions to this rule. In the past few decades it has become increasingly evident that there are serotypes of E. coli that may cause disease in susceptible hosts. Disease states range from the invasive infections of the urinary tract caused by uropathogenic E. coli (UPEC) to the more typical diarrheal disease caused by several groups of E. coli serotypes, including enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC). There is an increasing realization that bacteria–host interactions with pathogenic E. coli are far more complex and intricate than originally imagined. Studying the ways that bacteria such as EHEC and EPEC are able to subvert host cell functions to their own ends can be thought of as a “window” through which we are able to view the inner workings of the eukaryotic cell. In this chapter we examine some of the mechanisms by which EHEC and EPEC are able to coerce their host; we also examine the sequelae of these interactions.

EPEC usually refers to E. coli serotypes O55:[H6], O86:H34, O111:[H2], O114:H2, O119:[H6], O127:H6, O142:H6, and O142:H34, where square brackets indicate the occurrence of nonmotile strains (Nataro and Kaper, 1998). Infection with EPEC typically causes a chronic watery diarrhea, accompanied by a low-grade fever and nausea.

Few microbiologists are likely to forget the moment when the sheer scale and diversity of the microbial world became apparent to them. Similarly, it is a sobering thought that, in (or more accurately on) the human body, bacteria outnumber human cells by at least 10 to 1. Fortunately, most of these bacteria behave as good guests should, and they are content to remain on the other side of the physical barriers that separate us from the outside world. It is inevitable that at such a large gathering, some guests, the pathogens, will misbehave, and worse, start a fight. For many years it was thought that the battle between host and pathogen at the mucosal membranes was fought at arm's length. The bacteria lobbed toxins and other noxious agents at the host and the host returned the favor with antibodies. Bacteria that ventured too close were rapidly dispatched by the professional killing machines, the phagocytic cells. Some bacteria, such as Mycobacterium tuberculosis, however, proved inconveniently recalcitrant to intracellular killing and could become permanent guests within macrophages. Nonetheless, despite the appreciation that mitochondria originate from intracellular bacteria, the notion that mucosal pathogens could be intimately involved with nonprofessional phagocytes is relatively new.

We now know that a wide variety of organisms are capable of directing their own entry into epithelial cells and other host nonphagocytic cells. These bacteria engage in a remarkably sophisticated molecular dialogue with host cells in order to manipulate signal transduction pathways and effectuate bacterial entry.

The genus Yersinia contains three species of Gram-negative bacteria that are pathogenic for humans: Y. pestis, the agent of bubonic plague; Y. pseudotuberculosis, causing mesenteric adenitis and septicemia; and Y. enterocolitica, causing gastrointestinal syndromes (enteritis and mesenteric lymphadenitis). Bacteria from these three species have a tropism for lymphoid tissues and share the common capacity to resist the innate immune response. Whereas Y. pestis is generally inoculated by a fleabite or aerosol, Y. enterocolitica and Y. pseudotuberculosis are foodborne pathogens, which gain access to the underlying lymphoid tissue (e.g., Peyer's patches) of the intestinal mucosa through M cells (Fig. 3.1; see Autenrieth and Firsching, 1996; Perry and Fetherston, 1997). Once Yersinia has entered the lymphoid system, it overcomes the primary immune response of the host by using the type III secretion system (TTSS) (Cornelis et al., 1998; Cornelis, 2002). TTSS is a sophisticated virulence mechanism by which Gram-negative pathogens inject effector proteins directly into host cells. Currently, more than 20 different TTSSs have been described in animal, plant, and even insect pathogens (Hueck, 1998; Galan and Collmer, 1999; Cornelis, 2000; Buttner and Bonas, 2002).

Depending on the effectors injected, the employment of the TTSS will have a different outcome. Some, like the Mxi–Spa system of Shigella flexneri or the Salmonella pathogenicity island 1 (SPI-1) system of Salmonella enterica, make use of the innate immune system of the host to enhance the proinflammatory response and to trigger phagocytosis by normally nonphagocytic cells, whereas others, such as the pathogenic Yersinia Ysc–Yop system, essentially paralyze the innate immune response of the host (Galan, 2001; Sansonetti, 2001; Cornelis, 2002; Juris et al., 2002).

Studies focused on the Gram-positive, facultative intracellular bacterial path-ogen Listeria monocytogenes have provided valuable insights into many facets of biology, including cell-mediated immunity, cell physiology, and bacterial pathogenesis. The bacterium invades and replicates within a wide variety of cell types, and is capable of infecting an astonishing diversity of hosts, including mammals, fish, and insects (Gray and Killinger, 1966). The well-established use of murine and tissue culture models of infection, the ease of growing L. monocytogenes within the laboratory, and the existence of numerous genetic tools for the generation and analysis of bacterial mutants have helped to make L. monocytogenes a powerful model system for the exploration of the molecular basis of host–pathogen interactions. Because of its ubiquity within the environment and robust survival skills, this important foodborne pathogen remains a constant concern for public health departments and the food industry.

Listeriae are noncapsulated, nonspore-forming, facultative anaerobic bacilli. They are 0.4 μm by 1 to 1.5 μm in size and are motile at 10°C to 25°C through the expression of polar flagellae (Farber and Peterkin, 1991; Lorber, 1997; Vazquez-Boland et al., 2001b). There are six species in the Listeria genus: L. monocytogenes, L. ivanovii, L. innocua, L. seeligeri, L. welshimeri, and L. grayi (Collins et al., 1991; Sheehan et al., 1994). Only L. monocytogenes and L. ivanovii are considered pathogenic and are responsible for the disease known as listeriosis.

Neisseria gonorrhoeae, the gonococcus, is the causative agent of gonorrhea, one of the oldest human diseases on record. Biblical references to gonorrhea in Leviticus (15:1–15:19) showed that the infectious nature of the disease was recognized even at that time. Probably, the best description of gonorrhea in a man in the preantibiotic era can be found in the writings of Boswell, who described in detail each of his 19 episodes of infection (Ober, 1970). These descriptions also allude to the asymptomatic nature of the disease in women, as many of the contacts from whom he acquired the infection were without symptoms of disease. Today, it is estimated that greater than 1 million cases of N. gonorrhoeae infection occur in the United States, and 60 million cases are reported annually worldwide. Hence, N. gonorrhoeae infection remains prevalent in the general population, despite the fact that antibiotic therapy is readily available. The high incidence of this disease remains a major concern in lower socioeconomic groups in the United States; however, the highest incidence of infection and of complications resulting from infection occur in developing countries. In underdeveloped nations it has been shown that patients with gonorrhea are at much higher risk for contracting human immunodeficiency virus (HIV). It is proposed that this increased susceptibility to HIV infection results from the inflammatory response generated by infecting gonococci with the subsequent disruption and shedding of the mucosal epithelium.

INVASION

Shigella invasion and the host inflammatory responses

Shigella cause bacillary dysentery (shigellosis), a disease provoking a severe inflammatory diarrhea in humans and primates. In tropical areas of developing countries, shigellosis is endemic and a major killer of children under 5 years of age. Shigellosis occurs following ingestion of a very small number (100–1000) of bacteria, thus permitting easy spread of the disease by person-to-person contact as well as by the drinking of contaminated water.

Shigella, a Gram-negative bacillus, comprises four species – S. dysenteriae, S. flexneri, S. boydii, and S. sonnei (Pupo et al., 2000; Lan and Reeves, 2002). Shigella is now recognized as a member of Escherichia coli; however, the group of bacteria causing shigellosis is idiomatically called Shigella in this chapter. Shigellosis is also caused by enteroinvasive E. coli (EIEC), a pathogenic E. coli. Shigella and EIEC possess a large 210- to 230-kb plasmid on which the major virulence functions are encoded. Because Shigella has neither adhesins for upper GI tract cells nor flagella, after infection by means of the fecal–oral route the bacteria reach the colon and rectum directly, where they translocate through the epithelial barrier by means of the M cells overlaying the solitary lymphoid nodules (Fig. 2.1; also see Wassef et al., 1989; Sansonetti et al., 1991, 1996). Once they have reached the underlying M cells, Shigella infect the resident macrophages and multiply.

Porphyromonas gingivalis cells are Gram-negative, anaerobic, nonmotile short rods that produce black pigmented colonies on blood agar. The taxonomy of the species dates back to 1921 when Oliver and Wherry isolated an organism from a variety of oral and nonoral sites that they were to designate Bacterium melaninogenicum. This heterogeneous grouping was later subdivided into nonfermenters, weak fermenters, and strong fermenters. After a number of status changes within the genus Bacteroides, asaccharolytic oral isolates were assigned to the taxon P. gingivalis. The primary ecological niche of P. gingivalis is in the subgingival crevice, the gap between the surfaces of the tooth and the gingiva (gum); however, the organism can be found elsewhere in the mouth, including supragingival (above the gum) tooth surfaces, the tongue, tonsils, and buccal (cheek) mucosa. Although the species has been associated with odontogenic abscesses and nonoral infections (discussed later), the primary pathogenic potential of P. gingivalis is in periodontal disease. The periodontal tissues include the gingiva, periodontal ligament, and alveolar bone, and they constitute the supporting tissues of the teeth. Chronic destruction of the periodontium, such as occurs in periodontal diseases, can eventually lead to exfoliation of teeth and is the most common cause of tooth loss in adults. Periodontal diseases vary in severity and age of onset, and P. gingivalis is associated, either alone or in combination with other bacteria, with the most severe manifestations.

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that causes gastroenteritis in humans and a systemic disease similar to typhoid fever in mice. Following oral ingestion, bacteria colonize the intestinal tract and then penetrate the lymphatic and blood circulation systems. Passage of eukaryotic organisms through the intestinal epithelium is thought to be initiated by bacterial invasion into M cells and enterocytes. The process of epithelial cell invasion can be studied experimentally because S. enterica serovar Typhimurium invades cultured epithelial cells in vitro. Many of the genes required for epithelial invasion have been found within eukaryotic pathogenicity island 1 (SPI-1), which is a contiguous 40-kb region at centrosome 63 of the chromosome. SPI-1 genes encode a bacterial type III secretion apparatus and several effectors, which contribute to pathogenesis through an interaction with eukaryotic proteins. The type III secretion apparatus is a multiprotein complex that is thought to build a contiguous channel across both the bacterial and epithelial cell membranes, resulting in efficient translocation of bacterial effectors directly into the cytosol of epithelial cells. The secreted effectors are thought to interact with eukaryotic proteins to activate signal transduction pathways and rearrange the actin cytoskeleton, leading to membrane ruffling and engulfment of the bacterium. This chapter discusses the mechanism by which S. typhimurium enter into host cells.

CLINICAL DESCRIPTION

S. enterica, gram-negative bacteria of the family Enterobacteriaceae, cause a variety of diseases in humans and other animal hosts.

Legionella pneumophila, a Gram-negative bacillus that is ubiquitous in aquatic environments, is responsible for Legionnaires' disease. It is a facultative intracellular pathogen that can replicate within eukaryotic host cells such as protozoan and macrophages. In water, L. pneumophila grows within protozoan hosts. There are at least 13 species of amoebae and 2 species of ciliated protozoa that support intracellular replication of L. pneumophila (Fields, 1996). Among the most predominant amoebae in water sources are hartmannellae and acanthamoebae, which have also been isolated from water sources associated with Legionnaires' disease outbreaks (Fields, 1996). Interaction between L. pneumophila and protozoa is considered to be central to the pathogenesis and ecology of L. pneumophila (Rowbotham, 1986; Harb et al., 2000). In humans, L. pneumophila reaches the lungs after inhalation of contaminated aerosol droplets (Fields, 1996; Fliermans, 1996; also see Fig. 5.1). The main sources of contaminated water droplets are hot water and air-conditioning systems, but the bacteria have been isolated from fountains, spas, pools, dental and hospital units, and other man-made water systems (Fliermans, 1996; also see Fig. 5.1). No person-to-person transmission has been described. Once in the lungs, L. pneumophila are ingested in alveolar macrophages, the major site of bacterial replication. This results in an acute and severe pneumonia. In addition to Legionnaires' disease, L. pneumophila also causes Pontiac fever, which is a self-limiting flu-like illness that is not well understood but is not lethal. Approximately one half of the 48 species of Legionella have been associated with human disease.

ACTINOBACILLUS ACTINOMYCETEMCOMITANS: ADHERENCE MECHANISMS REQUIRED FOR INVASION

Colony phase variation

A. actinomycetemcomitans produces three distinct colonial morphologies on solid medium. A rough colony phenotype is typically generated by organisms upon isolation from the gingiva. These are small (~0.5–1 mm in diameter), translucent circular colonies with rough surfaces and irregular edges (Fig. 9.1). An internal star-shaped or crossed cigar morphology that embeds the agar is a distinguishing characteristic that gives A. actinomycetemcomitans its name (Zambon, 1985). In liquid culture, the rough colony phenotype cells form aggregates on the vessel walls, resulting in a clear medium (Fig. 9.1). Repeated subculture on agar of rough phenotypic isolates yields two distinct colonial variants; one is smooth surfaced and transparent, and the other is smooth surfaced and opaque (Slots, 1982; Scannapieco et al., 1987; Rosan et al., 1988; Inouye et al., 1990). The transparent smooth-surfaced variants appear to be an intermediate between the transparent rough-surfaced and opaque smooth-surfaced types (Inouye et al., 1990). In broth, the smooth-surfaced opaque type grows as a turbid homogeneous suspension, whereas the smooth-surfaced transparent type aggregates and adheres to the vessel walls (Inouye et al., 1990). In general, isolates undergo a rough-to-smooth variant transition soon after culture in vitro. In contrast, a smooth-to-rough variant transition that appears to be associated with nutritional requirements occurs only rarely during in vitro culture (Inouye et al., 1990; Meyer et al., 1991; Meyer, unpublished observation).

Group A streptococci (GAS), Streptococcus pyogenes, are beta-hemolytic, Gram-positive, pyogenic cocci that usually grow in chains. S. pyogenes is classified as group A based on serological reactions with its C carbohydrate, which consists of polymers of rhamnose substituted with N-acetylglucosamine (Lancefield, 1933). For epidemiological purposes, GAS are further classified into more than 100 different types based on serological reactions with the variable domains of M proteins, or, more recently, based on 5′ emm gene sequences (Beall et al., 1996). Other typing schemes based on serological reactions with serum opacity factor, T proteins, and R proteins are also used (Johnson and Kaplan, 1993).

S. pyogenes is almost exclusively associated with humans and commonly causes a variety of diseases, including pharyngotonsillitis, impetigo, scarlet fever, and more severe infections, such as puerperal sepsis, myositis, necrotizing fasciitis, and toxic shock syndrome. Among several of the nonsuppurative complications of group A streptococcal infections are acute rheumatic fever and acute glomerulonephritis, which are usually preceded by infections of the throat and skin, respectively. These sequelae are thought to be due to autoimmune T-and B-cell responses induced by streptococcal products. Accumulating evidence also suggests that group A streptococcal infections may lead to other autoimmune diseases, such as obsessive compulsive disorders, or they may exacerbate others such as guttate psoriasis (reviewed by Cunningham, 2000).

ADHESION: PRELUDE TO INVASION?

To establish these infections, the streptococcus must first attach to the epithelium of the host.