Bacterial Infections

Allen C. Steere,1 Jenifer Coburn,2 and Lisa Glickstein1

1Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy and Immunology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA. 2Division of Geographic Medicine and Infectious Diseases, Tufts-New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts, USA.

Address correspondence to: Allen C. Steere, Massachusetts General Hospital, 55 Fruit Street, CNY 149/8301, Boston, Massachusetts 02114, USA. Phone: (617) 726-1527; Fax: (617) 726-1544;

E-mail: asteere@partners.org

Abstract

Since its identification nearly 30 years ago, Lyme disease has continued to spread, and there have been increasing numbers of cases in the northeastern and north central US. The Lyme disease agent, Borrelia burgdorferi, causes infection by migration through tissues, adhesion to host cells, and evasion of immune clearance. Both innate and adaptive immune responses, especially macrophage- and antibody-mediated killing, are required for optimal control of the infection and spirochetal eradication. Ecological conditions favorable to the disease, and the challenge of prevention, predict that Lyme disease will be a continuing public health concern.In the late 20th century, Lyme disease, or Lyme borreliosis, was recognized as an important emerging infection (1). It is now the most commonly reported arthropod-borne illness in the US and Europe and is also found in Asia (2). Since surveillance for Lyme disease was begun in the US by the Centers for Disease Control and Prevention, the number of reported cases has increased steadily, and in the year 2000, more than 18,000 cases were reported (3).

Lyme disease was recognized as a separate entity in 1976 because of geographic clustering of children in the Lyme, Connecticut, area who were thought to have juvenile rheumatoid arthritis (4, S1 ). It then became apparent that Lyme arthritis was a late manifestation of an apparently tick-transmitted, multisystem disease, of which some manifestations had been recognized previously in Europe and America (S2–S6 ). In 1981, Burgdorfer and colleagues discovered a previously unidentified spirochetal bacterium, called Borrelia burgdorferi, in a nymphal Ixodes scapularis (also called Ixodes dammini) tick (S7 ). This spirochete was then cultured from patients with early Lyme disease, and patients’ immune responses were linked conclusively with that organism, proving the spirochetal etiology of the infection (S8, S9).

Based on genotyping of isolates from ticks, animals, and humans, the formerly designated B. burgdorferi has now been subdivided into multiple Borrelia species, including three that cause human infection. In the US, the sole cause is B. burgdorferi (S10 ). Although all three species are found in Europe, most of the disease there is due to Borrelia afzelii or Borrelia garinii, and only these two species seem to be responsible for the illness in Asia (S11, S12 ). During the 20th century, conditions evolved in the northeastern US that were especially favorable for enzootic B. burgdorferi infection (5). In this setting, Lyme disease continues to flourish and spread.

Biology of B. burgdorferi

The agents of Lyme borreliosis belong to the eubacterial phylum of spirochetes, which are vigorously motile, corkscrew-shaped bacteria (Figure 1). The spirochetal cell wall consists of a cytoplasmic membrane surrounded by peptidoglycan and flagella and then by a loosely associated outer membrane. The B. burgdorferi (strain B31) genome has been completely sequenced. It has a small linear chromosome that is just under one megabase (6), and nine circular and 12 linear plasmids that constitute 40% of its DNA (7). Some of these plasmids are indispensable and could be thought of as mini-chromosomes. Although it has been difficult to manipulate the B. burgdorferi genome, progress has recently been made using modified selectable markers and shuttle vectors (8, S13–S18 ).

The most remarkable aspect of the B. burgdorferi genome is the large number of sequences encoding predicted or known lipoproteins, including outer-surface proteins (Osp’s) A through F (6). Lipoproteins are found in the outer leaflet of the cytoplasmic membrane, and in both the inner and the outer leaflets of the outer membrane. Some of these proteins are differentially expressed, and one surface-exposed lipoprotein, called VlsE, undergoes extensive antigenic variation (9). In contrast, the genome encodes very few proteins with recognizable biosynthetic activity, and therefore, the organism depends on the host for most of its nutritional requirements. A very unusual feature of B. burgdorferi is that it does not require iron, at least for growth in vitro (10). This may allow the spirochete to circumvent the usual host defense of limiting the availability of iron. Finally, the B. burgdorferi genome encodes no recognizable toxins. Instead, this extracellular pathogen causes infection by migration through tissues, adhesion to host cells, and evasion of immune clearance.

Enzootic cycles of B. burgdorferi infection

The genus Borrelia currently includes three pathogenic species that cause Lyme borreliosis (S10–S12 ) and eight closely related species that rarely if ever cause human infection (S19–S25 ). These spirochetes live in nature in enzootic cycles involving ticks of the Ixodes ricinus complex (also called the Ixodes persulcatus complex) and a wide range of animal hosts (Table 1) (11). These enzootic cycles have evolved somewhat differently in different locations (S26 ). The important vectors of the three pathogenic species of human Lyme borreliosis are the deer tick, I. scapularis, in the northeastern and north central US; Ixodes pacificus in the western US; the sheep tick, I. ricinus, in Europe; and the taiga tick, I. persulcatus, in Asia.

In the northeastern US from Maine to Maryland and in the north central states of Wisconsin and Minnesota, a highly efficient, horizontal cycle of B. burgdorferi transmission occurs among larval and nymphal I. scapularis ticks and certain rodents, particularly white-footed mice and chipmunks (12, S27 ). This cycle results in high rates of infection among rodents and nymphal ticks and many new cases of human Lyme disease during the late spring and early summer months (Figure 2). White-tailed deer, which are not involved in the life cycle of the spirochete, are the preferred host of adult I. scapularis, and they seem to be critical for the survival of the ticks (S28 ).

The vector ecology of B. burgdorferi is quite different on the West Coast in northern California, where the frequency of Lyme disease is low. There, two intersecting cycles are necessary for disease transmission, one involving the dusky-footed wood rat and Ixodes spinipalpis (also called Ixodes neotomae) ticks, which do not bite humans and which maintain the cycle in nature, and the other involving wood rats and I. pacificus ticks, which are less often infected but do bite humans (S29 ). Similarly, in Colorado, wood rats and I. spinipalpis ticks may be infected with Borrelia bissettii, one of the nonpathogenic species, in a cycle that is not known to cause human infection (13). In the southeastern US, nymphal I. scapularis feed primarily on lizards, which are resistant to B. burgdorferi infection because of complement-mediated killing of the spirochete (14). Therefore, Lyme disease is rare in that part of the country.

In Europe, there is still debate about the preferred animal hosts of I. ricinus. These ticks feed on more than 300 animal species, including large and small mammals, birds, and reptiles (15). In Asia, immature I. persulcatus commonly feed on voles, shrews, and birds, and adult ticks feed on virtually all larger animals, including hares, deer, and cattle (16). Because the Borrelia species differ in their resistance to complement-mediated killing, small rodents are important reservoirs for B. afzelii, B. bissettii, and Borrelia japonica, whereas birds are strongly associated with B. garinii, Borrelia valaisiana, and Borrelia turdi (17).

Emergence of human Lyme borreliosis

The earliest known American cases of Lyme disease occurred in Cape Cod in the 1960s (S30 ). However, B. burgdorferi DNA has been identified by PCR in museum specimens of ticks and mice from Long Island dating from the late 19th and early 20th centuries (S31 ), and the infection has probably been present in North America for millennia (2). During the European colonization of North America, woodland in New England was cleared for farming, and deer were hunted almost to extinction (5). However, during the 20th century, conditions improved in the northeastern US for the ecology of Lyme disease. As farmland reverted to woodland, deer proliferated, white-footed mice were plentiful, and the deer tick thrived. Soil moisture and land cover, as found near rivers and along the coast, were favorable for tick survival (18). Finally, these areas became heavily populated with both humans and deer, as more rural wooded areas became wooded suburbs in which deer were without predators and hunting was prohibited.

During the past 40 years, the infection has continued to spread in the northeastern US (19); it has caused focal outbreaks in some coastal areas (S30, S32, S33 ), and it now affects suburban locations near Boston, New York, Philadelphia, and Baltimore, the most heavily populated parts of the country (S34 ). In the year 2000, the overall incidence of reported cases in Connecticut, the state with the highest reported frequency of Lyme disease, was 111 per 100,000 residents (3). However, most of the cases still clustered in foci, particularly in two counties in the southeastern part of the state where the original epidemiologic investigation took place in the town of Lyme (S1 ). In a large, two-year vaccine trial, such high-risk areas had a yearly incidence of the disease of greater than 1 per 100 participants, and the frequency of seropositivity to B. burgdorferi at study entry was as high as 5 per 100 participants (20).

As in America, the European agents of Lyme borreliosis have probably been present there for many thousands of years. They are now known to be widely established in Europe’s remaining forested areas (15). The highest reported frequencies of the disease are in middle Europe, particularly in Germany, Austria, Slovenia, and Sweden (2). In 1995, the yearly incidence of the disease in Slovenia and Austria was estimated to be 120–130 cases per 100,000 residents (2), similar to the frequency in Connecticut.

Clinical manifestations and disease pathogenesis

To maintain its complex enzootic cycle, B. burgdorferi must adapt to markedly different environments, the tick and the mammalian or avian host. The spirochete survives in a dormant state in the nymphal tick midgut during the fall, winter, and early spring, where it expresses primarily OspA (21). When the tick feeds in the late spring or early summer, the expression of a number of spirochetal proteins is altered (S35 ). For example, OspA is downregulated, and OspC is upregulated (21). OspC expression is required for infection of the mammalian host (22, 23). In addition, the spirochete binds mammalian plasminogen and its activators, present in the blood meal, which facilitates spreading of the organism within the tick (24). Within the salivary gland, OspC expression predominates, but some organisms express only OspE and OspF; OspA and OspB are absent (25).

After transmission of the spirochete, human Lyme disease generally occurs in stages, with remissions and exacerbations and different clinical manifestations at each stage (4). Early infection consists of stage 1, localized infection of the skin, followed within days or weeks by stage 2, disseminated infection, and months to years later by stage 3, persistent infection. However, the infection is variable; some patients have only localized infection of the skin, while others have only later manifestations of the illness, such as arthritis. Moreover, there are regional variations, primarily between the illness found in America and that found in Europe and Asia (1).

Localized infection

After an incubation period of 3–32 days, a slowly expanding skin lesion, called erythema migrans (EM), forms at the site of the tick bite in 70–80% of cases (26, 27). In the US, the skin lesion is frequently accompanied by flu-like symptoms, such as malaise and fatigue, headache, arthralgias, myalgias, and fever, and by signs that suggest dissemination of the spirochete (28). In about 18% of cases (27), these symptoms are the presenting manifestation of the illness (29). In contrast, EM in Europe is more often an indolent, localized infection, and spirochetal dissemination is less common (30).

In addition to the Lyme disease agent, I. scapularis ticks in the US and I. ricinus ticks in Europe may transmit Babesia microti (a red-blood-cell parasite) or Anaplasma phagocytophilum (formerly referred to as “the agent of human granulocytic ehrlichiosis”) (31–33). In a recent prospective study in the US, 4% of patients with culture-proven EM had coinfection with one of these other two tick-borne agents (34). Although these two infections are usually asymptomatic, coinfection may lead to more severe, acute flu-like illness (35).

In most patients, immune cells first encounter B. burgdorferi at the site of the tick bite. Depending on the Borrelia species and the host, complement-mediated lysis of the organism may be the first line of host defense (Figure 3) (36). On histologic examination, the resulting EM skin lesions consist of mild to marked perivascular infiltrates of lymphocytes, DCs, macrophages, and small numbers of plasma cells (37). As a part of the innate immune response, macrophages engulf and kill spirochetes (38–41). Inflammatory cells within the lesion produce primarily proinflammatory cytokines, including TNF-α and IFN-γ (37, 42). B. burgdorferi–stimulated PBMCs from patients with EM produce Th1 proinflammatory cytokines, especially IFN-γ (43). Within days after disease onset, most patients have an IgM antibody response to OspC or the 41-kDa flagellar protein of the spirochete (44). Thus, both innate and adaptive cellular elements are mobilized to fight the infection.

Disseminated infection

Within days to weeks after disease onset, B. burgdorferi often disseminates widely. During this period, the spirochete has been recovered from blood and cerebrospinal fluid (S7, S36, S37 ), and it has been seen in small numbers in specimens of myocardium, retina, muscle, bone, spleen, liver, meninges, and brain (45). Possible clinical manifestations include secondary annular skin lesions, acute lymphocytic meningitis, cranial neuropathy, radiculoneuritis, atrioventricular nodal block, migratory musculoskeletal pain in joints, bursae, tendon, muscle, or bone, and, rarely, eye manifestations (reviewed in ref. 4). Less often, spirochetal dissemination is asymptomatic.

To disseminate, B. burgdorferi binds certain host proteins and adheres to integrins, proteoglycans, or glycoproteins on host cells or tissue matrices. As in the tick, spreading of the spirochete through tissue matrices may be facilitated by the binding of plasminogen and its activators to the surface of the organism (24). A 47-kDa spirochetal protein (BBK32) binds fibronectin, an ECM protein (46). The sequences of OspC vary considerably among strains, and only a few sequences are associated with disseminated infection (47), probably because they bind as-yet unidentified host structures. A 66-kDa outer-surface protein of the spirochete binds the fibrinogen receptor (αIIbβ3) and the vitronectin receptor (αvβ3) (48), which may allow the organism to establish an initial foothold and disseminate in the vasculature. A 26-kDa Borrelia glycosaminoglycan-binding (GAG-binding) protein, Bgp, binds to the GAG side chains of heparan sulfate on endothelial cells, and to both heparan sulfate and dermatan sulfate on neuronal cells (49, 50). Finally, spirochetal decorin-binding proteins A and B (DbpA and DbpB) bind decorin, a proteoglycan that associates with collagen (51). This may explain the alignment of spirochetes with collagen fibrils in the ECM of the heart, nervous system, or joints (45).

Despite an active immune response, B. burgdorferi may survive during dissemination by changing or minimizing antigenic expression of surface proteins and by inhibiting certain critical host immune responses. Two linear plasmids (lp’s) seem to be essential, including lp25, which encodes a nicotinamidase (52), and lp28-1, which encodes the VlsE lipoprotein (53), the protein that undergoes antigenic variation. In addition, the spirochete has a number of families of highly homologous, differentially expressed lipoproteins, including the OspE/F paralogs, which further contribute to antigenic diversity (54). B. burgdorferi may downregulate lipoproteins because of host immune pressure (54, 55). For example, in a mouse model, the development of antibody to OspC, a prominent early response, induces downregulation of OspC; and therefore, this antibody response does not completely clear the infection (56). Finally, B. afzelii and, to a lesser degree, B. burgdorferi have complement regulator–acquiring surface proteins that bind complement factor H and factor H–like protein 1 (57). These complement factors inactivate C3b, which protects the organism from complement-mediated killing (57, S38–S41 ). In contrast, B. garinii is efficiently killed by complement (S42 ).

As shown definitively in mouse models, both innate and adaptive immune responses are required for optimal control of disseminated infection (Figure 3). B. burgdorferi lipoproteins, which are B cell mitogens (S39 ), stimulate adaptive T cell–independent B cell responses (58, S43, S44 ). For example, antibody responses to OspC kill spirochetes (59). In addition, humoral immune responses to nonlipidated spirochetal proteins, which are more likely to be T cell–dependent, aid in spirochetal killing (60, 61). The primary role of B. burgdorferi–specific Th1 cells is to prime these T cell–dependent B cell responses (62). The combination of these responses leads to the production of antibodies against many components of the organism (63, 64), which promote spirochetal killing by complement fixation and opsonization (S45 ). Within several weeks to months, these antibody responses, in conjunction with innate immune mechanisms, control widely disseminated infection even without antibiotic treatment, and generalized symptoms resolve.

Persistent infection

After weeks of disseminated infection, the Lyme disease agents may still survive in localized niches for several years. By this time, systemic symptoms are minimal or absent altogether. Although each of the three pathogenic species may spread to the joints, nervous system, or other skin sites, they seem to vary in the frequency of dissemination to these sites and in their ability to persist there. B. burgdorferi, the sole cause of the infection in the US, seems to be the most arthritogenic. Months after the onset of illness, about 60% of untreated patients with this infection experience intermittent attacks of arthritis, primarily of the large joints, especially the knee (65).

As shown in a mouse model, neutrophil extravasation into the infected joint is a key initial step in the development of joint inflammation (66). In the human infection, CD4+ Th cells are of the proinflammatory Th1 subset (S46, S47 ), and B. burgdorferi–specific CD8+ T cells are found as well (S48 ). Within the joint, B. burgdorferi–specific γδ T cells may aid in the regulation of these inflammatory responses (S49, S50 ). Compared with other inbred strains of mice, C57BL/6 mice are protected from severe arthritis by IL-6 and IL-10, despite large numbers of spirochetes in the joint (67, 68). It is unknown, however, whether certain human patients control joint inflammation in this way. Patients with Lyme arthritis have very high antibody responses to many spirochetal proteins, suggestive of hyperimmunization due to recurrent waves of spirochetal growth (63, 64). Even without antibiotic treatment, the number of patients who continue to have attacks of arthritis decreases by about 10–20% each year, and few patients have had attacks for longer than 5 years (65). Thus, these immune mechanisms seem to succeed eventually in eradicating B. burgdorferi from the joint.

In Europe and Asia, B. afzelii may persist in the skin for decades, resulting in acrodermatitis chronica atrophicans, a skin condition that occurs primarily on sun-exposed surfaces of distal extremities in elderly women (S51 ). Compared with EM lesions, infiltrates of T cells and macrophages in acrodermatitis lesions had a restricted cytokine profile, lacking IFN-γ production (37). Consistent with this finding, ultraviolet B irradiation of B. burgdorferi–infected C3H mice decreased the Th1 response (69). Thus, spirochetal persistence in acrodermatitis skin lesions may involve both spirochetal factors and an ineffective local immune response.

B. garinii, which is also found only in Europe and Asia, appears to be the most neurotropic of the three Borrelia species. It may cause an exceptionally wide range of neurologic abnormalities (70), including borrelial encephalomyelitis (S52 ), a multiple sclerosis–like illness. In the US, a rare, late neurologic syndrome has been described, called Lyme encephalopathy or polyneuropathy, which is manifested primarily by subtle cognitive disturbances, spinal radicular pain, or distal paresthesias (71, S53 ). With each of these three late neurologic complications, the possible duration of spirochetal persistence and the pathogenetic mechanisms are unknown.

# posted by Pat O'Connor @ 4:58 AM