Comparative Analysis of the Properties and Ligand Binding Characteristics of CspZ,a Factor H Binding Protein,Derived from Borrelia burgdorferi Isolates of Human Origin

Borrelia burgdorferi CspZ (BBH06/BbCRASP-2) binds the complement regulatory protein factor H (FH) and additional unidentified serum proteins. The goals of this study were to assess the ligand binding capability of CspZ orthologs derived from an extensive panel of human Lyme disease isolates and to further define the molecular basis of the interaction between FH and CspZ. While most B. burgdorferi CspZ orthologs analyzed bound FH, specific, naturally occurring polymorphisms, most of which clustered in a specific loop domain of CspZ, prevented FH binding in some orthologs. Sequence analyses also revealed the existence of CspZ phyletic groups that correlate with FH binding and with the relationships inferred from ribosomal spacer types (RSTs). CspZ type 1 (RST1) and type 3 (RST3) strains bind FH, while CspZ type 2 (RST2) strains do not. Antibody responses to CspZ were also assessed. Anti-CspZ antibodies were detected in mice by week 2 of infection, indicating that there was expression during early-stage infection. Analyses of sera collected from infected mice suggested that CspZ production continued over the course of long-term infection as the antibody titer increased over time. While antibody to CspZ was detected in several human Lyme disease serum samples, the response was not universal, and the titers were generally low. Vaccination studies with mice demonstrated that while CspZ is immunogenic, it does not elicit an antibody that is protective or that inhibits dissemination. The data presented here provide significant new insight into the interaction between CspZ and FH and suggest that there is a correlation between CspZ production and dissemination. However, in spite of its possible contributory role in pathogenesis, the immunological analyses indicated that CspZ is likely to have limited potential as a diagnostic marker and vaccine candidate for Lyme disease.In mammals, complement is a key component of the innate immune system and represents one of the initial mechanisms of defense against pathogenic organisms (45, 46, 52). Several diverse pathogens, including bacteria, viruses, and parasites, have been demonstrated to bind negative regulators of the complement system to their surfaces as a means of evading complement-mediated destruction (for reviews, see references 29 and 52). Several Borrelia species, including those associated with Lyme disease and relapsing fever, bind members of the factor H (FH) protein family (13-16, 19, 37), which are key regulators of the alternate complement cascade. FH, an abundant ∼150-kDa serum protein, functions as a decay-accelerating factor of the C3 convertase complex and as a cofactor for the factor I-mediated cleavage of C3b (41, 42, 45). In terms of host-pathogen interactions, the binding of FH to the cell surface locally inhibits complement activation and increases the efficiency of C3b cleavage, thereby decreasing opsonophagocytosis (45). The Lyme disease spirochetes, Borrelia burgdorferi, Borrelia afzelii, and Borrelia garinii, differ in serum sensitivity, ranging from highly resistant to highly sensitive, respectively (6, 30, 44). In Borrelia species, serum resistance has been shown to directly correlate with the production of FH binding proteins (3, 7, 13, 40), and consistent with this, B. burgdorferi produces more FH binding proteins than B. afzelii or B. garinii (37). B. burgdorferi FH binding proteins include OspE paralogs (BBL39, BBN38, and BBP38/CRASPs3-5), CspA (BBA68/CRASP-1), and CspZ (BBH06/CRASP-2) (4, 16, 28, 35, 37). CspZ is the most recent of these proteins to be identified. While cspZ has been demonstrated for both B. burgdorferi and B. garinii, it is not clear if this gene is widely distributed among B. afzelii isolates (37, 39). In addition, although B. garinii produces CspZ, the protein lacks FH binding ability (39). However, CspZ appears to have other roles during infection, as suggested by its ability to bind to other, unidentified serum proteins (39). The sequence analyses conducted to date of representative B. burgdorferi and B. garinii cspZ genes have demonstrated that there are species-specific polymorphisms that influence ligand binding (26, 39).The goals of this study were to assess the distribution, phylogeny, expression, and ligand binding properties of CspZ orthologs derived from human B. burgdorferi isolates and to determine if vaccination with CspZ elicits a protective response. The data demonstrate that for CspZ sequences there are distinct phyletic types that are associated with FH binding ability and that correlate with ribosomal spacer type (RST), a genetic marker of invasiveness and dissemination (22, 49). Analysis of the immune response to CspZ during experimental infection in mice revealed that CspZ-specific antibody was produced as early as week 2 of infection. However, the antibody response to CspZ in humans was variable. Vaccination of mice with two different recombinant CspZ (r-CspZ) orthologs also did not elicit a protective response or prevent dissemination.     

Borrelia burgdorferi BmpA Is a Laminin-Binding Protein

The Borrelia burgdorferi BmpA outer surface protein plays a significant role in mammalian infection by the Lyme disease spirochete and is an important antigen for the serodiagnosis of human infection. B. burgdorferi adheres to host extracellular matrix components, including laminin. The results of our studies indicate that BmpA and its three paralogous proteins, BmpB, BmpC, and BmpD, all bind to mammalian laminin. BmpA did not bind mammalian type I or type IV collagens or fibronectin. BmpA-directed antibodies significantly inhibited the adherence of live B. burgdorferi to laminin. The laminin-binding domain of BmpA was mapped to the carboxy-terminal 80 amino acids. Solubilized collagen inhibited BmpA-laminin binding, suggesting interactions through the collagen-binding domains of laminin. These results, together with previous data, indicate that BmpA and its paralogs are targets for the development of preventative and curative therapies for Lyme disease.Early during the course of Lyme disease, humans frequently produce antibodies directed against a Borrelia burgdorferi antigen originally described as “P39” (66). Antibodies recognizing P39 are considered to be specific and diagnostic for Lyme disease spirochete infection (5, 18, 30, 62, 64). The antigenic protein was subsequently identified as BmpA (Borrelia membrane protein A) (65). The bmpA gene is located on the main borrelial chromosome, adjacent to three paralogous genes named bmpB, bmpC, and bmpD, which together form a complex operon (3, 4, 28, 32, 55, 56, 65). These other Bmp proteins are also often antigenic in infected humans (14). In addition to the serological data described above, examination of B. burgdorferi within skin and joint tissues confirmed the production of BmpA protein during mammalian infection (21, 49). BmpA is located in the borrelial outer membrane (46), where it is exposed to the external environment and can be a target of bactericidal antibodies (49, 63; F. Cabello, personal communication). BmpA and its paralogs have been implicated as playing roles in some symptoms of Lyme disease (49, 72). B. burgdorferi mutants in which bmpA or bmpB is specifically deleted are unable to persist in mouse joint tissues (49), indicating an important role for these proteins in the maintenance of mammalian infection. Despite the extensive research conducted on these important antigens, functions for the Bmp proteins had not been determined previously.B. burgdorferi is an extracellular organism, frequently found associated with its hosts'' connective tissues (6-9, 16, 17, 24, 26, 31, 36, 39, 48). In the laboratory, B. burgdorferi shows affinity for various host extracellular matrix (ECM) components, such as type I collagen, fibronectin, and decorin (16, 33, 34, 50, 74). We recently determined that B. burgdorferi also adheres to mammalian laminin, an important component of many mammalian ECMs (13). Ligand affinity blot analyses of a B. burgdorferi cell fraction enriched for outer membrane components revealed that the type strain, B31, can produce several distinct laminin-binding proteins, one of which we previously identified as being the surface-exposed outer membrane lipoprotein ErpX (11, 13, 69). We now present data indicating that BmpA and its paralogs are also laminin-binding proteins.     

Borrelia burgdorferi Infection-Associated Surface Proteins ErpP,ErpA, and ErpC Bind Human Plasminogen

Host-derived plasmin plays a critical role in mammalian infection by Borrelia burgdorferi. The Lyme disease spirochete expresses several plasminogen-binding proteins. Bound plasminogen is converted to the serine protease plasmin and thereby may facilitate the bacterium''s dissemination throughout the host by degrading extracellular matrix. In this work, we demonstrate plasminogen binding by three highly similar borrelial outer surface proteins, ErpP, ErpA, and ErpC, all of which are expressed during mammalian infection. Extensive characterization of ErpP demonstrated that this protein bound in a dose-dependent manner to lysine binding site I of plasminogen. Removal of three lysine residues from the carboxy terminus of ErpP significantly reduced binding of plasminogen, and the presence of a lysine analog, ɛ-aminocaproic acid, inhibited the ErpP-plasminogen interaction, thus strongly pointing to a primary role for lysine residues in plasminogen binding. Ionic interactions are not required in ErpP binding of plasminogen, as addition of excess NaCl or the polyanion heparin did not have any significant effect on binding. Plasminogen bound to ErpP could be converted to the active enzyme, plasmin. The three plasminogen-binding Erp proteins can also bind the host complement regulator factor H. Plasminogen and factor H bound simultaneously and did not compete for binding to ErpP, indicating separate binding sites for both host ligands and the ability of the borrelial surface proteins to bind both host proteins.Lyme disease is the most commonly reported arthropod-borne disease in the United States (8). Borrelia burgdorferi, the causative agent of Lyme disease, is transmitted to its hosts through the bites of infected Ixodes ticks. In the earliest stage of Lyme disease, a bull''s-eye-shaped rash, erythema migrans, occurs as the spirochete spreads outward from the site of the tick bite. If left untreated, serious clinical outcomes can occur, including arthritis, neuropathies, and carditis (48).The bacterium disseminates from the bite site to other host tissues. B. burgdorferi can traverse the epithelium and invade vascular walls but is rarely abundant in blood (1). In addition, B. burgdorferi can pass through the blood-brain barrier to enter the central nervous system (58). The spirochete, unlike many invasive pathogens, lacks surface protease activities (12, 26). Therefore, binding of host proteases to the surface of the bacterium may aid in the spirochete''s dissemination. Indeed, B. burgdorferi binds plasminogen, a component of the host''s fibrinolytic system (12, 19). Plasminogen circulates in the plasma as an inactive proenzyme and is activated by tissue-type plasminogen activator and urokinase-type plasminogen activator (uPA) to plasmin (55). Plasminogen binding is an important virulence factor for invasive pathogens such as group A streptococci and Staphylococcus, as well as Borrelia species (10, 43, 55). The binding of plasminogen to bacteria and its subsequent activation allow bacteria to degrade the host''s extracellular matrix and basement membranes either through the direct protease activity of plasmin or by plasmin''s activation of host matrix metalloproteases (MMPs). B. burgdorferi has previously been shown to bind plasminogen, which is rapidly converted to active plasmin in the presence of host plasminogen activator (11). In vitro, plasmin-coated B. burgdorferi is able to penetrate endothelial cell monolayers (12). Surface-associated plasmin on B. burgdorferi can directly degrade fibronectin, a major component of the extracellular matrix, as well as laminin and vitronectin (11, 19). B. burgdorferi induces the release of MMP-9 (gelatinase) and MMP-1 (collagenase) from human cells, and plasmin-coated B. burgdorferi activates pro-MMP-9 (20), initiating a cascade that leads to degradation of basement membranes. Plasminogen has previously been shown to be important in B. burgdorferi pathogenesis. Although not strictly required for infection, plasminogen was required for efficient dissemination in ticks, and its absence decreased spirochetemia in plasminogen-deficient mice (10).Plasminogen-binding proteins of B. burgdorferi have previously been identified, including the outer-surface lipoprotein OspA (19). A role for OspC in plasminogen binding has also been suggested (31). However, OspA is generally not expressed during human infection, and OspC production ceases within the first few days of mammalian infection (13, 24, 25, 34, 42). Other, unidentified plasminogen-binding proteins have been observed in B. burgdorferi, including a protein(s) with an approximate molecular mass of 20 kDa, which is close to the size of several Erp proteins (12, 19). The members of the Erp family of outer-surface lipoproteins are expressed at high levels during mammalian infection (15, 23, 38-41).Lyme disease spirochetes contain numerous DNA elements, including the main chromosome as well as linear and circular plasmids (6). Infectious isolates carry several distinct yet homologous elements called cp32s, circular prophages of approximately 32 kb (54). All cp32 elements encode one or two Erp proteins, which can vary widely in amino acid sequence (50). However, all erp loci are preceded by nearly identical promoter regions (36, 53). Hence, most of the erp genes analyzed follow the same pattern of expression, being repressed in the tick vector but synthesized during mammalian infection (15, 21, 23, 35, 37-41). Roles for most of the Erp proteins have yet to be defined. ErpX has been demonstrated to bind host laminin (our unpublished results and reference 3). Three Erp proteins bind the host complement regulator factor H and factor H-related protein 1: ErpP, ErpC, and ErpA (22, 28, 29). Some factor H binding proteins of other human pathogens have been demonstrated to bind multiple ligands, including plasminogen (30, 47). These data, and the presence of unidentified plasminogen-binding proteins in B. burgdorferi, prompted us to examine if Erp proteins are able to bind plasminogen.     

Characterization of Unique Regions of Borrelia burgdorferi Surface-Located Membrane Protein 1

The pathogen of Lyme disease, Borrelia burgdorferi, produces a putative surface protein termed “surface-located membrane protein 1” (Lmp1). Lmp1 has been shown previously to assist the microbe in evasion of host-acquired immune defenses and in the establishment of persistent infection of mammals. Here, we show that Lmp1 is an integral membrane protein with surface-exposed N-terminal, middle, and C-terminal regions. During murine infection, antibodies recognizing these three protein regions were produced. Separate immunization of mice with each of the discrete regions exerted differential effects on spirochete survival during infection. Notably, antibodies against the C-terminal region primarily interfered with B. burgdorferi persistence in the joints, while antibodies specific to the N-terminal region predominantly affected pathogen levels in the heart, including the development of carditis. Genetic reconstitution of lmp1 deletion mutants with the lmp1 N-terminal region significantly enhanced its ability to resist the bactericidal effects of immune sera and also was observed to increase pathogen survival in vivo. Taken together, the combined data suggest that the N-terminal region of Lmp1 plays a distinct role in spirochete survival and other parts of the protein are related to specific functions corresponding to pathogen persistence and tropism during infection that is displayed in an organ-specific manner. The findings reported here underscore the fact that surface-exposed regions of Lmp1 could potentially serve as vaccine targets or antigenic regions that could alter the course of natural Lyme disease.Lyme disease is caused by the spirochete Borrelia burgdorferi, which is transmitted by ticks belonging to the Ixodes scapularis complex (2, 31). Upon introduction to the host dermis, spirochetes disseminate from the site of infection to distal cutaneous areas and navigate to a variety of internal host organs (6). The colonization of the pathogen in a select set of infected host organs eventually leads to robust inflammatory responses resulting in multiple clinical complications, such as arthritis, carditis, and neurological disorders (31, 48). In mammalian hosts, B. burgdorferi infection may persist in diverse tissues despite the development of a vigorous immune response (5, 8, 13, 16, 24, 44). Understanding the mechanism by which spirochetes selectively persist in certain tissues and induce a severe inflammatory response is important for the development of preventive and therapeutic strategies against Lyme borreliosis.B. burgdorferi survives in a complex enzootic life cycle. The varied metabolic and immune host environments have been shown to dramatically influence spirochete gene expression (11, 14, 26, 29, 32-34, 41, 43, 49, 51). Microbial antigens that are produced in a time- or tissue-specific manner might assist B. burgdorferi to overcome host defenses and to persist in local environments. Differentially expressed gene products, particularly surface antigens, could directly participate in host-pathogen interaction or host immune evasion, contributing to microbial survival and organ-specific pathogenesis (36, 51). In recent years, a few spirochete gene products have been identified that are either indispensable or contribute significantly to host or vector infectivity and transmission through the tick-mouse infection cycle (9, 23, 25, 26, 28, 37, 39, 40, 45, 47, 52, 53). However, in most cases, the genes identified encoded proteins that lack orthologs in other bacteria; therefore, their molecular functions in spirochete biology or infectivity remain unclear.Recently, a protein termed Lmp1, a chromosomally encoded antigen with an approximate molecular mass of 128 kDa, was shown to be induced in infected murine tissues, especially at early phases of infection in the heart (51). Lmp1 has been suggested to be integral to pathogen persistence and to be involved in evading the host adaptive immune response during infection (51). The antigen is localized to the microbial surface, is immunogenic during animal or human infection (3, 51), and is conserved across orthologs in other B. burgdorferi sensu lato isolates. Computer algorithms suggest that Lmp1 contains a typical type I leader peptide, although whether the signal sequence is cleaved remains unknown. Lmp1 contains three possible separate functional regions located at the N-terminal, middle, and C-terminal portions of the protein. Although the overall structure of Lmp1 is unrelated to known proteins, the middle region of the protein contains several peptide repeats which may be related to adhesins (3). The C-terminal region contains several tetratricopeptide repeats (TPRs), which are motifs that are well documented to play important roles in protein-protein interactions (17, 22, 42). Despite earlier studies, the molecular function of Lmp1 and the possible unique role(s) of its individual protein regions with regard to B. burgdorferi virulence and Lyme disease pathogenesis remain unclear. Characterization of functional protein regions of novel spirochete virulence determinants, such as Lmp1, will likely shed further light into how Lmp1 could potentially serve as a vaccine target or how antibodies against antigenic regions of Lmp1 could alter the course of a natural Lyme disease infection.     

Characterization of a Conditional bosR Mutant in Borrelia burgdorferi

Borrelia burgdorferi, the etiological agent of Lyme disease, adapts to unique host environments as a consequence of its complex life cycle that spans both arthropod and mammalian species. In this regard, B. burgdorferi must adapt to various environmental signals, pHs, temperatures, and O₂ and CO₂ levels to establish infectious foci. We hypothesize that the BosR protein functions as a global regulator that is required for both borrelial oxidative homeostasis and pathogenesis. To assess the role of BosR in B. burgdorferi, we constructed an IPTG (isopropyl-β-d-thiogalactopyranoside)-regulated bosR strain. The selective decrease of bosR resulted in a change in growth when cells were cultured either anaerobically or microaerobically; however, a distinct growth defect was observed for anaerobically grown B. burgdorferi relative to the growth attenuation observed for microaerobically grown B. burgdorferi. B. burgdorferi cells in which BosR levels were reduced were more sensitive to hydrogen peroxide and produced lower levels of NapA (Dps) and SodA, proteins involved in the oxidative stress response. In addition, the levels of OspC and DbpA were also induced coincident with increased BosR levels, suggesting that BosR interfaces with the RpoS regulatory cascade, which is known to modulate virulence gene expression in B. burgdorferi. Taken together, these results indicate that BosR is involved in the resistance of B. burgdorferi to oxidative stressors and affects the expression of genes, either directly or indirectly, whose products are important in borrelial pathogenesis.Infection with Borrelia burgdorferi, the etiologic agent of Lyme disease, is the leading arthropod-borne infection in the United States and contributes to extensive morbidity in areas of endemicity, where the chronic phase of disease generally presents as arthritis. One characteristic of B. burgdorferi is its ability to adapt to both arthropod and mammalian hosts. Several studies demonstrated that B. burgdorferi responds to a number of environmental signals, including temperature, pH, and O₂ and CO₂ levels, as well as uncharacterized host factors (1, 2, 8, 13, 15-17, 20, 30, 36, 39, 40, 43, 46, 47, 51, 52, 55, 57). However, specific details regarding how these signals are integrated into a regulatory response are poorly understood.The genome sequence of B. burgdorferi predicted only a few regulatory proteins (14, 22); one such regulator was annotated as Fur. However, Posey and Gherardini demonstrated that B. burgdorferi has no requirement for iron (42), suggesting that the assignment of a Fur protein in B. burgdorferi was inaccurate. In fact, examination of the primary amino acid sequence showed that borrelial Fur was most similar to PerR, an oxidative stress regulator that represses genes involved in the oxidative stress response in Bacillus spp. (9, 23, 27, 38). However, unlike PerR, BosR appears to activate expression of target borrelial genes involved in the oxidative stress response, including napA (dps) and a coenzyme A (CoA) disulfide reductase gene designated cdr (5, 7). Thus, although BosR is similar to PerR, it appears to function more like OxyR, an activator that promotes the expression of genes involved in the oxidative stress response in Escherichia coli (24, 53, 60). BosR binds in vitro to sequences upstream of genes such as napA (dps), cdr, the superoxide dismutase gene (sodA), bosR, bb0646, and oppA4, providing further support that BosR regulates the expression of many unlinked genes within the B. burgdorferi genome (5, 7, 31, 37, 48).In this report, we describe the isolation and characterization of a conditional bosR mutant in infectious B. burgdorferi, using an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible hybrid E. coli-B. burgdorferi promoter (25) linked to bosR (P_flac-bosR). Cells with reduced BosR have a delayed growth phenotype under all conditions but exhibit restricted cell density under microaerobic conditions. B. burgdorferi cells that do not make BosR protein exhibit a modest but significant increase in sensitivity to hydrogen peroxide, suggesting that BosR is needed for maximal resistance to oxidative stressors. Along these lines, when bosR is induced with IPTG, the levels of SodA and NapA increase, consistent with the prior contentions that BosR activates expression of napA (7) and that these proteins are important in borrelial oxidative stress homeostasis. Furthermore, BosR production coincides with the increased synthesis of OspC and DbpA, suggesting that BosR may directly or indirectly interface with the Rrp2/RpoN/RpoS regulatory machinery. These results show that in addition to affecting the expression of genes involved in the oxidative stress response, BosR may alter the production of proteins that affect the pathogenic potential of B. burgdorferi.     

Variation in Susceptibility of Bloodstream Isolates of Candida glabrata to Fluconazole According to Patient Age and Geographic Location in the United States in 2001 to 2007

We examined the susceptibilities to fluconazole of 642 bloodstream infection (BSI) isolates of Candida glabrata and grouped the isolates by patient age and geographic location within the United States. Susceptibility of C. glabrata to fluconazole was lowest in the northeast region (46%) and was highest in the west (76%). The frequencies of isolation and of fluconazole resistance among C. glabrata BSI isolates were higher in the present study (years 2001 to 2007) than in a previous study conducted from 1992 to 2001. Whereas the frequency of C. glabrata increased with patient age, the rate of fluconazole resistance declined. The oldest age group (≥80 years) had the highest proportion of BSI isolates that were C. glabrata (32%) and the lowest rate of fluconazole resistance (5%).Candidemia is without question the most important of the invasive mycoses (6, 33, 35, 61, 65, 68, 78, 86, 88). Treatment of candidemia over the past 20 years has been enhanced considerably by the introduction of fluconazole in 1990 (7, 10, 15, 28, 29, 31, 40, 56-58, 61, 86, 90). Because of its widespread usage, concern about the development of fluconazole resistance among Candida spp. abounds (2, 6, 14, 32, 47, 53, 55, 56, 59, 60, 62, 80, 86). Despite these concerns, fluconazole resistance is relatively uncommon among most species of Candida causing bloodstream infections (BSI) (5, 6, 22, 24, 33, 42, 54, 56, 65, 68, 71, 86). The exception to this statement is Candida glabrata, of which more than 10% of BSI isolates may be highly resistant (MIC ≥ 64 μg/ml) to fluconazole (6, 9, 15, 23, 30, 32, 36, 63-65, 71, 87, 91). Suboptimal fluconazole dosing practices (low dose [<400 mg/day] and poor indications) may lead to an increased frequency of isolation of C. glabrata as an etiological agent of candidemia in hospitalized patients (6, 17, 29, 32, 35, 41, 47, 55, 60, 68, 85) and to increased fluconazole (and other azole) resistance secondary to induction of CDR efflux pumps (2, 11, 13, 16, 43, 47, 50, 55, 69, 77, 83, 84) and may adversely affect the survival of treated patients (7, 10, 29, 40, 59, 90). Among the various Candida species, C. glabrata alone has increased as a cause of BSI in U.S. intensive care units since 1993 (89). Within the United States, the proportion of fungemias due to C. glabrata has been shown to vary from 11% to 37% across the different regions (west, midwest, northeast, and south) of the country (63, 65) and from <10% to >30% within single institutions over the course of several years (9, 48). It has been shown that the prevalence of C. glabrata as a cause of BSI is potentially related to many disparate factors in addition to fluconazole exposure, including geographic characteristics (3, 6, 63-65, 71, 88), patient age (5, 6, 25, 35, 41, 42, 48, 63, 82, 92), and other characteristics of the patient population studied (1, 32, 35, 51). Because C. glabrata is relatively resistant to fluconazole, the frequency with which it causes BSI has important implications for therapy (21, 29, 32, 40, 41, 45, 56, 57, 59, 80, 81, 86, 90).Previously, we examined the susceptibilities to fluconazole of 559 BSI isolates of C. glabrata and grouped the isolates by patient age and geographic location within the United States over the time period from 1992 to 2001 (63). In the present study we build upon this experience and report the fluconazole susceptibilities of 642 BSI isolates of C. glabrata collected from sentinel surveillance sites throughout the United States for the time period from 2001 through 2007 and stratify the results by geographic region and patient age. The activities of voriconazole and the echinocandins against this contemporary collection of C. glabrata isolates are also reported.     

BBK07, a Dominant In Vivo Antigen of Borrelia burgdorferi,Is a Potential Marker for Serodiagnosis of Lyme Disease

One of the recently identified Borrelia burgdorferi immunogens, BBK07, is characterized for its expression in the spirochete infection cycle and evaluated for its potential use as a serodiagnostic marker for Lyme disease. We show that the BBK07 gene is expressed at extremely low levels in vitro and in ticks but is dramatically induced by spirochetes once introduced into the host and is highly expressed throughout mammalian infection. In contrast, the expression of BBK12, a paralog of BBK07 with 87% amino acid identity, although expressed in vitro, remained undetectable in vivo throughout murine infection and in ticks. BBK07 is localized in the outer membrane, and the amino-terminal domain of the antigen is exposed on the microbial surface. A truncated BBK07 protein representing the amino-terminal domain is able to effectively detect antibodies to B. burgdorferi, both in experimentally infected mice and in humans. Further characterization of the immunodominant antigens of B. burgdorferi, such as BBK07, could contribute to the development of novel serodiagnostic markers for detection of Lyme disease.Since the identification of Borrelia burgdorferi as the causative agent of Lyme disease (LD) over 25 years ago, the number of reported cases of LD has increased steadily (4, 49). In some U.S. counties, the incidence is more than 500 cases per 100,000 individuals, and more than 20,000 cases in the United States are diagnosed each year (4). Difficulties in diagnosis have long complicated the treatment of LD, as the bite of an infected tick may go unnoticed by the patient, and the clinical manifestations of LD can significantly vary among diagnosed patients (47). Common symptoms, such as fever, malaise, and arthritis, can resemble those caused by other conditions, further complicating diagnosis. Antibiotic therapy is highly effective, especially if administered in the early stages of LD; however, serious complications can result from false diagnoses and inappropriate treatment (9, 17, 40, 50, 51). There is no commercially available vaccine for human LD, so the development of accurate, sensitive laboratory diagnostics is an important goal of LD research.While many laboratory methods have been used to assess B. burgdorferi infection, direct detection of the bacterium is difficult, due to the low pathogen load in clinical samples (2, 24). Likewise, the extremely slow growth of B. burgdorferi, the high cost, and the labor-intensive procedure needed to culture this bacterium have limited the effectiveness of culture as a diagnostic tool (34, 46). PCR detection is possible (44), but not widely used for diagnosis, due primarily to low sensitivity in tissues, such as cerebrospinal fluid and blood (2). Instead, the primary means used to detect B. burgdorferi exposure is serodiagnosis (2). Immunodetection has been performed using whole-cell antigens, as well as recombinant proteins or peptide fragments (2). Whole-cell lysate provides a wide variety of antigens for detection, but is difficult to standardize due to variations in protein expression by culture growth phase (42). False-positive results are also an issue, as antibodies against other bacteria can cross-react with conserved B. burgdorferi proteins (5, 13, 21, 29).To reduce cross-reactivity, several recombinant B. burgdorferi antigens and various fragments thereof have been evaluated as serodiagnostic markers for LD, including OspC (35), BmpA (45), VlsE (27), BBK32 (22), L25 (33), P37 (31), and DbpA (20). OspC is exposed on the B. burgdorferi surface, is produced during early infection, and is highly immunogenic (1, 13, 16, 35). A peptide fragment termed pepC10, containing a conserved immunogenic epitope, has been developed for serodiagnosis (32). BmpA, another surface-exposed protein, has also been studied for use in diagnosis (10, 45). Though immunogenic, significant protein sequence heterogeneity exists among B. burgdorferi isolates, constituting several serotypes, which limit the effectiveness of both OspC (14) and BmpA as serodiagnostic markers (43). VlsE is a dominant surface-exposed antigen of B. burgdorferi, a lipoprotein that undergoes antigenic variation by genetic recombination with silent vls cassettes (53). Expressed throughout late infection, VlsE and C6, a conserved peptide fragment of VlsE, have been evaluated as serodiagnostic markers for LD (15, 27, 28). These studies suggest that while the use of recombinant proteins can reduce cross-reactivity, thereby enhancing specificity, the use of only select antigens can reduce the sensitivity of the diagnostic test (30). A promising sensitivity in such tests was reported by Bacon et al. (3). Using kinetic enzyme-linked immunosorbent assay (ELISA), the combined detection of immunoglobulin M (IgM) against pepC10 and IgG against C6 provided 78% sensitivity in all tested samples. While assays using only recombinant antigens show promise, the identification and inclusion of more immunodominant antigens could improve the sensitivity of these tests.In an effort to more completely catalogue antigens produced during infection, a recent study by Barbour et al. used synthetic protein arrays to test the immunogenicity of the majority of B. burgdorferi open reading frames (6). Though most open reading frames were not measurably immunogenic, they identified several novel antigens, including BBK07 and BBK12, putative lipoproteins from the linear plasmid lp36. These proteins are extremely similar in sequence, though BBK07 is slightly larger than BBK12 (250 and 232 amino acids, respectively) (18). The genes are members of paralogous family 59, and their products are 87% identical in their overlapping amino acid sequences. While both BBK07 and BBK12 were identified as immunogens and potential antigenic markers, a detailed characterization of their expression and the resulting immune response was not explored. We sought to characterize the expression, surface localization, and immune response against BBK07 to further evaluate its inclusion as a diagnostic marker to improve the accuracy and sensitivity of LD serodiagnosis.     

Bacterin That Induces Anti-OspA and Anti-OspC Borreliacidal Antibodies Provides a High Level of Protection against Canine Lyme Disease

Groups of 15 laboratory-bred beagles were vaccinated and boosted with either a placebo or adjuvanted bivalent bacterin comprised of a traditional Borrelia burgdorferi strain and a unique ospA- and ospB-negative B. burgdorferi strain that expressed high levels of OspC and then challenged with B. burgdorferi-infected Ixodes scapularis ticks. The vaccinated dogs produced high titers of anti-OspA and anti-OspC borreliacidal antibodies, including borreliacidal antibodies specific for an epitope within the last seven amino acids at the OspC carboxy terminus (termed OspC7) that was conserved among pathogenic Borrelia genospecies. In addition, spirochetes were eliminated from the infected ticks that fed on the bacterin recipients, B. burgdorferi was not isolated from the skin or joints, and antibody responses associated specifically with canine infection with B. burgdorferi were not produced. In contrast, B. burgdorferi was recovered from engorged ticks that fed on 13 (87%) placebo-vaccinated dogs (P < 0.0001), skin biopsy specimens from 14 (93%) dogs (P < 0.0001), and joint tissue specimens from 8 (53%) dogs (P = 0.0022). In addition, 14 (93%) dogs developed specific antibody responses against B. burgdorferi proteins, including 11 (73%) with C6 peptide antibodies (P < 0.0001). Moreover, 10 (67%) dogs developed Lyme disease-associated joint abnormalities (P < 0.0001), including 4 (27%) dogs that developed joint stiffness or lameness and 6 (40%) that developed chronic joint inflammation (synovitis). The results therefore confirmed that the bacterin provided a high level of protection against Lyme disease shortly after immunization.Dogs with Lyme disease rarely develop acute illness (26); but the infection reliably causes chronic subclinical polyarthritis and/or periarteritis (43) and occasionally causes frank recurrent arthritis with myalgia, fever, anorexia, and lethargy (41, 42); renal failure (11); heart block (24); or neurologic disease (9). In addition, the severity of the illness appears to be influenced by the species and the age of the dog. For example, beagle puppies are prone to oligoarthropathy (2, 39), while adults are more likely to develop asymptomatic synovitis (2, 7, 43). Moreover, Labrador retrievers, golden retrievers, and Shetland sheepdogs appear to be more susceptible to kidney nephropathy (11).Several commercial dog vaccines are currently available, and each provides protection primarily by inducing the production of anti-OspA borreliacidal antibodies that stimulate complement to form a membrane attack complex (33) that kills Borrelia burgdorferi in the tick midgut as the infected vectors ingest blood (12, 16). The approach has been effective (8, 10, 31, 40), but the vaccines may also fail (23) because the expression of OspA is downregulated immediately after the infected tick begins acquiring a blood meal (36), borreliacidal antibodies specific for OspA are genospecies specific (28, 49), and ticks can be infected with variant OspA-negative Lyme disease spirochetes (15).Another viable target for antibody-mediated immunity is OspC (18), especially since, in contrast to OspA, the Lyme disease spirochetes upregulate the expression of OspC as the tick begins feeding (36) and express OspC during the early stages of a mammalian infection (45). However, vaccines that provide protection by inducing anti-OspC antibodies have not been pursued aggressively, likely because the extreme heterogeneity, even among B. burgdorferi isolates from the same geographic area (44, 47), suggested that the protection afforded by anti-OspC antibodies would not be comprehensive.However, researchers (21) recently identified an epitope within the surface-exposed 7 amino acids of the carboxy terminus of OspC (hereafter referred to as OspC7) recognized by anti-OspC borreliacidal antibodies. More significantly, the epitope within the OspC7 region is conserved among the pathogenic Borrelia genospecies, including B. afzelii and B. garinii. Therefore, anti-OspC borreliacidal antibodies formed against the OspC7 region should provide comprehensive protection and should be effective against spirochetes in the tick and during the early stages of mammalian infection. Moreover, in contrast to the use of vaccination to induce anti-OspA borreliacidal antibodies, vaccination with proteins such as OspC that are also expressed during mammalian infection provides the possibility of producing an effective anamnestic immunologic memory response. We therefore developed a bivalent bacterin that induced both anti-OspA and anti-OspC borreliacidal antibodies, including borreliacidal antibodies specific for the conserved epitope within the OspC7 region, and evaluated the ability of immunization to provide protection against challenge from B. burgdorferi-infected ticks.(This study was presented in part at the 25th American College of Veterinary Internal Medicine Forum, Seattle, WA, 6 to 9 June 2007 [22a, 22b].)     

The Effects of PspC on Complement-Mediated Immunity to Streptococcus pneumoniae Vary with Strain Background and Capsular Serotype

Streptococcus pneumoniae may evade complement activity by binding of factor H (FH), a negative regulator of the alternative pathway, to the surface protein PspC. However, existing data on the effects of FH binding to PspC on complement activity are conflicting, and there is also considerable allelic variation in PspC structure between S. pneumoniae strains that may influence PspC-dependent effects on complement. We have investigated interactions with complement for several S. pneumoniae strains in which the gene encoding PspC has been deleted. The degree of FH binding varied between strains and was entirely dependent on PspC for seven strains. Data obtained with TIGR4 strains expressing different capsular serotypes suggest that FH binding is affected by capsular serotype. Results of immunoblot analysis for C3 degradation products and iC3b deposition assays suggested that FH bound to PspC retained functional activity, but loss of PspC had strikingly varied effects on C3b/iC3b deposition on S. pneumoniae, with large increases on serotype 4, 6A, 6B, and 9V strains but only small increases or even decreases on serotype 2, 3, 17, and 23F strains. Repeating C3b/iC3b assays with TIGR4 strains expressing different capsular serotypes suggested that differences in the effect of PspC on C3b/iC3b deposition were largely independent of capsular serotype and depend on strain background. However, data obtained from infection in complement-deficient mice demonstrated that differences between strains in the effects of PspC on complement surprisingly did not influence the development of septicemia.Streptococcus pneumoniae is a common cause of invasive diseases such as pneumonia, meningitis, and septicemia even in immunocompetent subjects. One important component of host immunity to S. pneumoniae is the complement system, a series of approximately 30 serum and cell surface proteins organized into three enzyme cascades termed the classical, alternative, and mannan binding lectin (MBL) pathways (44). Infection experiments using complement-deficient mice have demonstrated that both the classical and alternative pathways are important for immunity to S. pneumoniae (2, 11, 15, 23, 43), and the high incidence of S. pneumoniae infections in patients with complement deficiencies confirms the relevance of complement for preventing disease in humans (3, 21). Both pathways lead to the formation of a C3 convertase that cleaves the central complement component C3, resulting in deposition of C3b on the surface of the pathogen that is further processed to iC3b. C3b and iC3b are opsonins, (44), and coating of S. pneumoniae with C3b/iC3b is vital for efficient phagocytosis of this organism by neutrophils (49). The importance of complement for immunity to S. pneumoniae is emphasized by the identification of several mechanisms by which the bacteria can inhibit complement activity. The choline binding surface protein PspA and the capsule prevent C3b/iC3b deposition on S. pneumoniae by mechanisms that remain unclear, whereas the secreted toxin pneumolysin seems to divert classical pathway activity away from the bacteria (1, 30, 32, 35, 39, 43, 48).PspC is another choline binding surface protein that is related to PspA and is important for virulence in models of nasopharyngeal colonization, septicemia, and pneumonia (16, 22, 33, 35, 38). PspC can bind to factor H (FH), a negative regulator of the alternative pathway (8, 13, 18). PspC and the closely related proteins encoded at the same genetic locus in different strains (termed SpsA, CbpA, PbcA, and Hic) could, therefore, prevent alternative pathway complement activity against S. pneumoniae by one of three potential mechanisms (7). First, FH may speed up the degradation of C3b by promoting the factor I-dependent cleavage of C3b bound to the bacterial surface to iC3b; second, FH causes the dissociation of factor B from the alternative pathway C3 convertase C3bBb, decreasing C3b deposition on the bacteria; and third, FH may also inhibit the formation on the bacterial surface of the C3 convertase C3bBb by preferentially binding C3b, thus preventing C3b binding to factor B. However, although the affinity of PspC for FH has been clearly demonstrated and the binding sites have been identified (6, 12, 13, 17, 18, 28, 38), the effects of this interaction on complement-mediated immunity to S. pneumoniae is relatively poorly defined. Loss of PspC can cause reduced iC3b (compatible with reduced processing of C3b to iC3b) (28) and increased C3b/iC3b (compatible with reduced C3 convertase activity) deposition on a capsular serotype 2 (ST2) strain D39 or an unencapsulated mutant derived from a capsular ST3 strain (19, 35). However, in contrast to Quin et al., Lu et al. and Li et al. found little effect on total C3 bound to a ΔpspC mutant derived from the D39 strain compared to the wild type unless this mutation was combined with mutation of pspA (25, 28, 35).The role of PspC on complement-mediated immunity in different strains could also be affected by the marked allelic variation in the structure of PspC. Ianelli et al. sequenced pspC from 43 different strains, and although the derived protein sequences had the same domain organization, each protein had a unique sequence that could be divided into 11 subgroups (17). The majority of PspC alleles contain a C-terminal cell wall choline binding motif (similar to PspA), but 17 of the allelic variants contain a cell wall anchor LPTXG motif instead. FH binding requires only the N-terminal 89 amino acids of PspC from the D39 strain and is dependent on a 12-amino-acid motif which is conserved between PspC alleles from different S. pneumoniae strains (28). However, this domain is not enough for full FH binding capacity and requires additional flanking amino acids that vary with allelic variation of PspC. Indeed, it is known that the ability of S. pneumoniae to bind to FH varies between strains (36). This variation in FH binding between strains seems to be independent of serotype but is increased on strains isolated from the blood or cerebrospinal fluid (CSF), under which conditions PspC expression is increased (29, 34). Overall, the existing data suggest that the effects of PspC on complement deposition on S. pneumoniae is not clear and could vary between strains. Furthermore, several other functions have been ascribed to PspC, including direct binding to C3 (which might counteract any FH-mediated reduction in C3b deposition) (4, 40) and aiding S. pneumoniae adhesion to host cells and translocation across epithelial layers (12, 13, 37, 38, 50), which could also affect virulence independent of any effect of PspC on complement-mediated immunity. Further clarification of the effects of PspC on opsonization of S. pneumoniae with C3b/iC3b is important for a better understanding of how PspC aids S. pneumoniae virulence. To address this question, in this study we have assessed the effects of loss of PspC on C3b/iC3b deposition on a range of S. pneumoniae strains representing different capsular STs.     

BB0844, an RpoS-regulated protein, is dispensable for Borrelia burgdorferi infectivity and maintenance in the mouse-tick infectious cycle

The genome of Borrelia burgdorferi, the causative agent of Lyme disease, is comprised of a large linear chromosome and numerous smaller linear and circular plasmids. B. burgdorferi exhibits substantial genomic variation, and previous studies revealed genotype-specific variation at the right chromosomal telomere. A correlation has also been established between genotype and invasiveness. The correlation between chromosome length and genotype and between genotype and invasiveness suggested that a gene(s) at the right chromosome telomere may be required for virulence. Of particular interest was bb0844, an RpoS-regulated gene at the right telomere, the expression of which is induced when the spirochete undergoes adaptation to the mammalian host. The structure of the right chromosomal telomere was examined in 53 B. burgdorferi clinical isolates of various genotypes. Four distinct patterns were observed for bb0844: (i) chromosomal localization, (ii) plasmid localization, (iii) presence on both chromosome and plasmid, and (iv) complete absence. These patterns correlated with the B. burgdorferi genotype. On the basis of available sequence data, we propose a mechanism for the genomic rearrangements that accounts for the variability in bb0844 genomic localization. To further explore the role of BB0844 in the spirochete life cycle, a bb0844 deletion mutant was constructed by allelic exchange, and the viability of wild-type and bb0844 deletion mutants was examined in an experimental mouse-tick infection model. The bb0844 mutant was fully infectious in C3H/HeJ mice by either needle inoculation or tick transmission with B. burgdorferi-infected Ixodes scapularis larvae. Naïve larval ticks acquired both wild-type and mutant spirochetes with equal efficiency from B. burgdorferi-infected mice. The results demonstrate that BB0844 is not required for spirochete viability, pathogenicity, or maintenance in the tick vector or the mammalian host. At present, a defined role for BB0844 in B. burgdorferi cannot be ascertained.Borrelia burgdorferi, the Lyme disease pathogen, has a complex life cycle in nature which involves an arthropod vector and diverse mammalian hosts (6, 46). The type strain, B31, has a complex genome comprised of a linear chromosome and at least 21 linear and circular plasmids (12, 19). The linear replicons have covalently closed hairpin telomeric ends (11, 24). The chromosome of B. burgdorferi strain B31 is 910,725 bp long and includes 853 putative open reading frames (ORFs) (19). A majority of the chromosomal sequence and gene order is conserved among B. burgdorferi strains; however, the chromosomes vary in length. These differences were shown to be the result of variability in the right telomeric region (10, 11) and have been confirmed by sequencing of additional B. burgdorferi genomes (40). It has been proposed that linear plasmid fragments were added to the right chromosomal telomere by means of nonhomologous recombination, after which portions of the transposed plasmid sequences underwent mutational decay (12, 13, 25).Numerous approaches have been employed for typing of B. burgdorferi isolates (56). For example, North American B. burgdorferi isolates can be classified into three rRNA gene spacer types (RST) based on the rRNA gene spacer sequence (32) and 17 types based on the ospC sequence (41). Importantly, a correlation between genotype and pathogenicity has been observed. Isolates with an RST1 genotype were more likely than RST3 isolates to produce disseminated infection in humans or mice (55, 58, 59). Similarly, OspC type A, B, I, H, and K isolates were most likely to disseminate (41, 58). Comparative genome hybridization analysis of 16 B. burgdorferi isolates revealed that ORFs bb0843.1 to bb0853.1 were absent from the chromosomes of most of the RST3 isolates analyzed (11, 49). Overall, the length of the right chromosomal telomere correlated with the RST; RST1 and RST2 isolates contained longer right chromosomal telomeric regions than most RST3 strains, whose chromosomes end with ORF bb0843 (11, 25, 49). The right telomere region of strain B31MI contains only two intact ORFs, bb0844 and bb0852 (19). bb0844 encodes a hypothetical protein of 323 amino acid residues with a predicted molecular mass of 37.5 kDa. The encoded sequence contains a signal peptidase II recognition signal and is predicted to be a lipoprotein. It should be noted that in the recent reannotation of the B. burgdorferi B31MI genome sequence on the Institute for Genomic Research (TIGR) comprehensive microbial resource (http://cmr.jcvi.org/cgi-bin/CMR/CmrHomePage.cgi), bb0844 has been renamed bb0860; the gene name remains as bb0844 in GenBank. In this report, bb0844 is used throughout.Regulation of gene expression is a key strategy that B. burgdorferi employs in response to diverse environmental signals encountered in arthropod and mammalian hosts. Central to the process of differential gene expression is the alternative sigma factor RpoS, which governs differential expression of various B. burgdorferi genes during its enzootic life cycle (8, 27, 60). RpoS appears to serve a “gatekeeper” function; its induction during nymphal feeding (i.e., the RpoS-on state) results in repression of tick phase genes and the expression of genes whose products are required for transmission to the mammal or establishment and maintenance of mammalian infection. Expression of genes repressed by RpoS during mammalian infection resumes when spirochetes are acquired by naïve larvae (i.e., the RpoS-off state) (9, 36). bb0844 was reported to be an RpoS-regulated gene with an expression pattern identical to that of ospC and dbpA, two well-characterized B. burgdorferi virulence factors (3, 9, 21, 43, 48, 51, 57). bb0844 is induced under mammalian-host-like conditions (5, 9, 52). A proteome-wide array analysis revealed that BB0844 elicits an antibody response in humans and laboratory-infected mice, indicating that BB0844 is expressed during mammalian infection (2).Given the evidence for expression of bb0844 by spirochetes infecting mammalian hosts and the variable presence of this gene among clinical isolates, which appears to correlate with virulence, we hypothesized that bb0844 might contribute to B. burgdorferi infectivity. To explore this possibility, we analyzed the right chromosomal telomeric regions of 53 B. burgdorferi clinical isolates and studied the effect of bb0844 deletion on the ability of B. burgdorferi to infect mice and to survive throughout an experimental mouse-tick-mouse cycle.