The role of the immune response in influencing leptospirosis clinical outcomes is not yet well understood. We hypothesized that acute-phase serum cytokine responses may play a role in disease progression, risk for death, and severe pulmonary hemorrhage syndrome (SPHS).
We performed a case-control study design to compare cytokine profiles in patients with mild and severe forms of leptospirosis. Among patients hospitalized with severe disease, we compared those with fatal and nonfatal outcomes. During active outpatient and hospital-based surveillance we prospectively enrolled 172 patients, 23 with mild disease (outpatient) and 149 with severe leptospirosis (hospitalized). Circulating concentrations of pro- and anti-inflammatory cytokines at the time of patient presentation were measured using a multiplex bead array assay. Concentrations of IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-17A, and TNF-α were significantly higher (P<0.05) in severe disease compared to mild disease. Among severe patients, levels of IL-6 (P<0.001), IL-8 (P = 0.0049) and IL-10 (P<0.001), were higher in fatal compared to non-fatal cases. High levels of IL-6 and IL-10 were independently associated (P<0.05) with case fatality after adjustment for age and days of symptoms. IL-6 levels were higher (P = 0.0519) among fatal cases who developed SPHS than among who did not.
This study shows that severe cases of leptospirosis are differentiated from mild disease by a “cytokine storm” process, and that IL-6 and IL-10 may play an immunopathogenic role in the development of life-threatening outcomes in human leptospirosis.
Leptospirosis is a tropical bacterial disease that is transmitted to humans from infected animals. Leptospirosis symptoms can range from mild fever to fatal disease forms, such as massive bleeding into the lungs, called Severe Pulmonary Hemorrhage Syndrome (SPHS). It is not known what determines the severity of leptospirosis, but we hypothesized that it may be influenced by differences in the type and concentration of signaling proteins called cytokines that are produced by the immune system in response to infection. We collected blood from patients with mild and severe leptospirosis, and compared the concentration of eight different cytokines circulating in the blood. We found that patients with severe leptospirosis had higher levels of most cytokines. Among patients who had severe forms, higher levels of specific cytokines called IL-6 and IL-8 were predictive of death even after statistical adjustment for age and number of days of symptoms prior to hospitalization. IL-6 was higher in patients who died from SPHS compared to those who died of other leptospirosis complications. This knowledge suggests that severe forms of leptospirosis may be due to a specific kind of immune response, which may lead to targeted therapies to reduce the impact of this disease.
Relapsing fever spirochetes are global yet neglected pathogens causing recurrent febrile episodes, chills, nausea, vomiting, and pregnancy complications. Given these nonspecific clinical manifestations, improving diagnostic assays for relapsing fever spirochetes will allow for identification of endemic foci and expedite proper treatment. Previously, an antigen designated the Borrelia immunogenic protein A (BipA) was identified in the North American species Borrelia hermsii. Thus far, BipA appears unique to relapsing fever spirochetes. The antigen remains unidentified outside of these pathogens, while interspecies amino acid identity for BipA in relapsing fever spirochetes is only 24–36%. The current study investigated the immunogenicity of BipA in Borrelia turicatae, a species distributed in the southern United States and Latin America.
bipA was amplified from six isolates of Borrelia turicatae, and sequence analysis demonstrated that the gene is conserved among isolates. A tick transmission system was developed for B. turicatae in mice and a canine, two likely vertebrate hosts, which enabled the evaluation of serological responses against recombinant BipA (rBipA). These studies indicated that BipA is antigenic in both animal systems after infection by tick bite, yet serum antibodies failed to bind to B. hermsii rBipA at a detectable level. Moreover, mice continued to generate an antibody response against BipA one year after the initial infection, further demonstrating the protein's potential toward identifying endemic foci for B. turicatae.
These initial studies support the hypothesis that BipA is a spirochete antigen unique to a relapsing fever Borrelia species, and could be used to improve efforts for identifying B. turicatae endemic regions.
Undiagnosed febrile illnesses continue to afflict those in resource poor countries. Relapsing fever spirochetes are one such pathogen causing a significant health burden, yet the pathogenesis, ecology, and distribution of B. turicatae is understudied. To address these shortcomings, we analyzed the amino acid sequence of the Borrelia immunogenic protein A (BipA) in isolates of B. turicatae. Mice and a canine were also infected by tick bite and transmission and serological responses were evaluated in these two likely mammalian hosts. B. turicatae was visualized within the blood of both animals and antibody responses generated against recombinant BipA indicated that the antigen that may be unique to infections caused by B. turicatae. Moreover, mice continued to generate antibodies a year after tick bite, suggesting a persistent infection. Our results indicate that the immune responses generated against BipA could identify additional vertebrate hosts, define endemic foci for B. turicatae, and increase the awareness of the disease to improve healthcare.
P66 is a Borrelia burgdorferi surface protein with β3 integrin binding and channel forming activities. In this study, the role of P66 in mammalian and tick infection was examined. B. burgdorferiΔp66 strains were not infectious in wild-type, TLR2−/− or MyD88−/− deficient mice. Strains with p66 restored to the chromosome restored near wild-type infectivity, while complementation with p66 on a shuttle vector did not restore infectivity. Δp66 mutants are cleared quickly from the site of inoculation, but analyses of cytokine expression and cellular infiltrates at the site of inoculation did not reveal a specific mechanism of clearance. The defect in these mutants cannot be attributed to nutrient limitation or an inability to adapt to the host environment in vivo as Δp66 bacteria were able to survive as well as wild-type in dialysis membrane chambers in the rat peritoneum. Δp66 bacteria were able to survive in ticks through the larva to nymph molt, but were non-infectious in mice when delivered by tick bite. Independent lines of evidence do not support any increased susceptibility of the Δp66 strains to factors in mammalian blood. This study is the first to define a B. burgdorferi adhesin as essential for mammalian, but not tick infection.
Analysis of the transcriptome of Borrelia burgdorferi, the causative agent of Lyme disease, during infection has proven difficult due to the low spirochete loads in the mammalian tissues. To overcome this challenge, we have developed an In Vivo Expression Technology (IVET) system for identification of B. burgdorferi genes expressed during an active murine infection. Spirochetes lacking linear plasmid (lp) 25 are non-infectious yet highly transformable. Mouse infection can be restored to these spirochetes by expression of the essential lp25-encoded pncA gene alone. Therefore, this IVET-based approach selects for in vivo-expressed promoters that drive expression of pncA resulting in the recovery of infectious spirochetes lacking lp25 following a three week infection in mice. Screening of approximately 15,000 clones in mice identified 289 unique in vivo-expressed DNA fragments from across all 22 replicons of the B. burgdorferi B31 genome. The in vivo-expressed candidate genes putatively encode proteins in various functional categories including antigenicity, metabolism, motility, nutrient transport and unknown functions. Candidate gene bbk46 on essential virulence plasmid lp36 was found to be highly induced in vivo and to be RpoS-independent. Immunocompetent mice inoculated with spirochetes lacking bbk46 seroconverted but no spirochetes were recovered from mouse tissues three weeks post inoculation. However, the bbk46 gene was not required for B. burgdorferi infection of immunodeficient mice. Therefore, through an initial IVET screen in B. burgdorferi we have identified a novel in vivo-induced virulence factor critical for the ability of the spirochete to evade the humoral immune response and persistently infect mice.
Lyme disease is caused by tick-bite transmission of the pathogenic spirochete Borrelia burgdorferi. An increased understanding of how B. burgdorferi survives throughout its infectious cycle is critical for the development of innovative diagnostic and therapeutic protocols to reduce the incidence of Lyme disease. One of the major difficulties blocking this effort has been genome-wide identification of the B. burgdorferi genes that are expressed in the mammalian host environment. Using in vivo expression technology (IVET) in B. burgdorferi for the first time, we have identified B. burgdorferi genes that are expressed during an active murine infection. We demonstrate that candidate gene bbk46, encoded on essential linear plasmid 36, is highly expressed in vivo and, unlike some other known B. burgdorferi in vivo-induced genes, is not RpoS regulated. Spirochetes lacking bbk46 establish an infection in mice and elicit an antibody response but are undetectable in mouse tissues three weeks post inoculation. The bbk46 is not required for spirochete infection of mice lacking a functional immune system. In sum, development of an IVET-based approach in B. burgdorferi has identified a novel virulence gene critical for the spirochete's ability to evade the mammalian adaptive immune response.
Hard ticks subvert the immune responses of their vertebrate hosts in order to feed for much longer periods than other blood-feeding ectoparasites; this may be one reason why they transmit perhaps the greatest diversity of pathogens of any arthropod vector. Tick-induced immunomodulation is mediated by salivary components, some of which neutralise elements of innate immunity or inhibit the development of adaptive immunity. As dendritic cells (DC) trigger and help to regulate adaptive immunity, they are an ideal target for immunomodulation. However, previously described immunoactive components of tick saliva are either highly promiscuous in their cellular and molecular targets or have limited effects on DC. Here we address the question of whether the largest and globally most important group of ticks (the ixodid metastriates) produce salivary molecules that specifically modulate DC activity. We used chromatography to isolate a salivary gland protein (Japanin) from Rhipicephalus appendiculatus ticks. Japanin was cloned, and recombinant protein was produced in a baculoviral expression system. We found that Japanin specifically reprogrammes DC responses to a wide variety of stimuli in vitro, radically altering their expression of co-stimulatory and co-inhibitory transmembrane molecules (measured by flow cytometry) and their secretion of pro-inflammatory, anti-inflammatory and T cell polarising cytokines (assessed by Luminex multiplex assays); it also inhibits the differentiation of DC from monocytes. Sequence alignments and enzymatic deglycosylation revealed Japanin to be a 17.7 kDa, N-glycosylated lipocalin. Using molecular cloning and database searches, we have identified a group of homologous proteins in R. appendiculatus and related species, three of which we have expressed and shown to possess DC-modulatory activity. All data were obtained using DC generated from at least four human blood donors, with rigorous statistical analysis. Our results suggest a previously unknown mechanism for parasite-induced subversion of adaptive immunity, one which may also facilitate pathogen transmission.
Dendritic cells (DC) are specialised cells of the vertebrate immune system. DC can sense different types of infectious agents and parasites, and both trigger and help regulate the specific types of immunity needed to eliminate them. We have discovered that the largest and globally most important group of hard ticks produce a unique family of proteins in their saliva that selectively targets DC, radically altering functions that would otherwise induce robust immune responses; these proteins also prevent DC developing from precursor cells. The production of these salivary molecules may help to explain two highly unusual features of these hard ticks compared with other blood-feeding parasites: their ability to feed continuously on their vertebrate hosts for considerable lengths of time (7 days or more) without eliciting potentially damaging immune responses, and their capacity to transmit possibly the greatest variety of pathogens of any type of invertebrate.
Lipid rafts in eukaryotic cells are sphingolipid and cholesterol-rich, ordered membrane regions that have been postulated to play roles in many membrane functions, including infection. We previously demonstrated the existence of cholesterol-lipid-rich domains in membranes of the prokaryote, B. burgdorferi, the causative agent of Lyme disease [LaRocca et al. (2010) Cell Host & Microbe 8, 331–342]. Here, we show that these prokaryote membrane domains have the hallmarks of eukaryotic lipid rafts, despite lacking sphingolipids. Substitution experiments replacing cholesterol lipids with a set of sterols, ranging from strongly raft-promoting to raft-inhibiting when mixed with eukaryotic sphingolipids, showed that sterols that can support ordered domain formation are both necessary and sufficient for formation of B. burgdorferi membrane domains that can be detected by transmission electron microscopy or in living organisms by Förster resonance energy transfer (FRET). Raft-supporting sterols were also necessary and sufficient for formation of high amounts of detergent resistant membranes from B. burgdorferi. Furthermore, having saturated acyl chains was required for a biotinylated lipid to associate with the cholesterol-lipid-rich domains in B. burgdorferi, another characteristic identical to that of eukaryotic lipid rafts. Sterols supporting ordered domain formation were also necessary and sufficient to maintain B. burgdorferi membrane integrity, and thus critical to the life of the organism. These findings provide compelling evidence for the existence of lipid rafts and show that the same principles of lipid raft formation apply to prokaryotes and eukaryotes despite marked differences in their lipid compositions.
Specialized domains (“lipid rafts”) rich in specific membrane lipids (sphingolipids and cholesterol) have been proposed to form in the cell membranes of higher organisms, and to be of functional importance. We recently found that domains can be detected in the membranes of the bacterium that causes Lyme disease, Borrelia burgdorferi. In this report it is shown that, despite a lack of sphingolipids in B. burgdorferi, these domains have all the characteristic properties of lipid rafts, and can be detected in living B. burgdorferi. This shows that true lipid rafts can form in bacteria. In addition, it is shown that sterols having a structure that promotes lipid raft formation are necessary and sufficient for those sterols to maintain B. burgdorferi membrane integrity. This is suggestive of a role for membrane domains in B. burgdorferi membrane integrity. Therefore, interfering with lipid raft formation may have biomedical applications in combatting B. burgdorferi infections.
Borrelia burgdorferi, the agent of Lyme disease, has cholesterol and cholesterol-glycolipids that are essential for bacterial fitness, are antigenic, and could be important in mediating interactions with cells of the eukaryotic host. We show that the spirochetes can acquire cholesterol from plasma membranes of epithelial cells. In addition, through fluorescent and confocal microscopy combined with biochemical approaches, we demonstrated that B. burgdorferi labeled with the fluorescent cholesterol analog BODIPY-cholesterol or 3H-labeled cholesterol transfer both cholesterol and cholesterol-glycolipids to HeLa cells. The transfer occurs through two different mechanisms, by direct contact between the bacteria and eukaryotic cell and/or through release of outer membrane vesicles. Thus, two-way lipid exchange between spirochetes and host cells can occur. This lipid exchange could be an important process that contributes to the pathogenesis of Lyme disease.
Lyme disease, the most prevalent arthropod-borne disease in North America, is caused by the spirochete Borrelia burgdorferi. Cholesterol is a significant component of the B. burgdorferi membrane lipids, and is processed to make cholesterol-glycolipids. Our interest in the presence of cholesterol in B. burgdorferi recently led to the identification and characterization of eukaryotic-like lipid rafts in the spirochete. The presence of free cholesterol and cholesterol-glycolipids in B. burgdorferi creates an opportunity for lipid-lipid interactions with constituents of the lipid rafts in eukaryotic cells. We present evidence that there is a two-way exchange of lipids between B. burgdorferi and epithelial cells. Spirochetes are unable to synthesize cholesterol, but can acquire it from the plasma membrane of epithelial cells. In addition, free cholesterol and cholesterol-glycolipids from B. burgdorferi are transferred to epithelial cells through direct contact and through outer membrane vesicles. The exchange of cholesterol between spirochete and host could be an important aspect of the pathogenesis of Lyme disease.
Treponema pallidum is a highly invasive pathogen that undergoes rapid dissemination to establish widespread infection. Previous investigations identified the T. pallidum adhesin, pallilysin, as an HEXXH-containing metalloprotease that undergoes autocatalytic cleavage and degrades laminin and fibrinogen. In the current study we characterized pallilysin's active site, activation requirements, cellular location, and fibrin clot degradation capacity through both in vitro assays and heterologous treponemal expression and degradation studies. Site-directed mutagenesis showed the pallilysin HEXXH motif comprises at least part of the active site, as introduction of three independent mutations (AEXXH [H198A], HAXXH [E199A], and HEXXA [H202A]) abolished pallilysin-mediated fibrinogenolysis but did not adversely affect host component binding. Attainment of full pallilysin proteolytic activity was dependent upon autocatalytic cleavage of an N-terminal pro-domain, a process which could not occur in the HEXXH mutants. Pallilysin was shown to possess a thrombin cleavage site within its N-terminal pro-domain, and in vitro studies confirmed cleavage of pallilysin with thrombin generates a truncated pallilysin fragment that has enhanced proteolytic activity, suggesting pallilysin can also exploit the host coagulation process to facilitate protease activation. Opsonophagocytosis assays performed with viable T. pallidum demonstrated pallilysin is a target of opsonic antibodies, consistent with a host component-interacting, surface-exposed cellular location. Wild-type pallilysin, but not the HEXXA mutant, degraded fibrin clots, and similarly heterologous expression of pallilysin in the non-invasive spirochete Treponema phagedenis facilitated fibrin clot degradation. Collectively these results identify pallilysin as a surface-exposed metalloprotease within T. pallidum that possesses an HEXXH active site motif and requires autocatalytic or host-mediated cleavage of a pro-domain to attain full host component-directed proteolytic activity. Furthermore, our finding that expression of pallilysin confers upon T. phagedenis the capacity to degrade fibrin clots suggests this capability may contribute to the dissemination potential of T. pallidum.
Syphilis, caused by the spirochete Treponema pallidum, is a chronic sexually transmitted disease which infects 12 million people annually. Treponema pallidum is highly invasive and undergoes widespread dissemination via the circulatory system. Similar to other invasive pathogens, T. pallidum has been shown to express a host-component-degrading protease, pallilysin, that binds and degrades human fibrinogen and laminin, suggesting a role for pallilysin in bacterial dissemination. Here we identify pallilysin active site residues using mutagenesis and show that, unlike wild-type, mutants fail to degrade fibrinogen. We show that pallilysin is converted into a highly proteolytically active form via truncation of a pro-domain through either autocatalytic cleavage or host-derived, thrombin-mediated cleavage. We also demonstrate that recombinant pallilysin enables clot dissolution and that pallilysin expressed on the surface of the non-invasive spirochete Treponema phagedenis confers the ability to degrade fibrin clots. Further, we show that pallilysin is present on the surface of T. pallidum and thus resides in a cellular location that facilitates direct contact with host components. Our study provides insight into the mechanism of interaction between pallilysin and two important coagulation system proteins, fibrinogen and thrombin, and suggests a novel mechanism that T. pallidum may utilize for dissemination during infection.
Pseudomonas aeruginosa, an important opportunistic pathogen of man, exploits numerous factors for initial attachment to the host, an event required to establish bacterial infection. In this paper, we rigorously explore the role of two major bacterial adhesins, type IV pili (Tfp) and flagella, in bacterial adherence to distinct host receptors at the apical (AP) and basolateral (BL) surfaces of polarized lung epithelial cells and induction of subsequent host signaling and pathogenic events. Using an isogenic mutant of P. aeruginosa that lacks flagella or utilizing beads coated with purified Tfp, we establish that Tfp are necessary and sufficient for maximal binding to host N-glycans at the AP surface of polarized epithelium. In contrast, experiments utilizing a P. aeruginosa isogenic mutant that lacks Tfp or using beads coated with purified flagella demonstrate that flagella are necessary and sufficient for maximal binding to heparan sulfate (HS) chains of heparan sulfate proteoglycans (HSPGs) at the BL surface of polarized epithelium. Using two different cell-free systems, we demonstrate that Tfp-coated beads show highest binding affinity to complex N-glycan chains coated onto plastic plates and preferentially aggregate with beads coated with N-glycans, but not with single sugars or HS. In contrast, flagella-coated beads bind to or aggregate preferentially with HS or HSPGs, but demonstrate little binding to N-glycans. We further show that Tfp-mediated binding to host N-glycans results in activation of phosphatidylinositol 3-kinase (PI3K)/Akt pathway and bacterial entry at the AP surface. At the BL surface, flagella-mediated binding to HS activates the epidermal growth factor receptor (EGFR), adaptor protein Shc, and PI3K/Akt, and induces bacterial entry. Remarkably, flagella-coated beads alone can activate EGFR and Shc. Together, this work provides new insights into the intricate interactions between P. aeruginosa and lung epithelium that may be potentially useful in the development of novel treatments for P. aeruginosa infections.
Pseudomonas aeruginosa is one of the most virulent nosocomial opportunistic pathogens that is associated with a broad spectrum of life-threatening infections. Antibiotic resistance is widespread and attributable mortality remains near 50%. Complex binding to epithelial cells is a key first step for this potent pathogen to unleash its armamentarium of virulence factors. Polarized epithelium has distinct apical (AP) and basolateral (BL) surface, composed of different glycosylated molecules, and P. aeruginosa can potentially employ different adhesins to bind to these receptors. Using isogenic mutants as well as in vitro cell-free assays, we demonstrate that bacterial type IV pili are necessary and sufficient to mediate AP interactions with N-glycans whereas bacterial flagella interact with heparan sulfate chains of proteoglycans on the BL surface. These interactions induce specific host signaling pathways that lead to subsequent pathogenic events, such as bacterial entry into host epithelium. Moreover, we show that flagella alone are sufficient to activate the epidermal growth factor receptor and the adaptor protein on the BL surface. These studies reveal new information about key players in the versatile interactions of P. aeruginosa with the host and provide appealing targets for blocking early binding steps essential for establishment of P. aeruginosa infections.
While the roles of rpoSBb and RpoS-dependent genes have been studied extensively within the mammal, the contribution of the RpoS regulon to the tick-phase of the Borrelia burgdorferi enzootic cycle has not been examined. Herein, we demonstrate that RpoS-dependent gene expression is prerequisite for the transmission of spirochetes by feeding nymphs. RpoS-deficient organisms are confined to the midgut lumen where they transform into an unusual morphotype (round bodies) during the later stages of the blood meal. We show that round body formation is rapidly reversible, and in vitro appears to be attributable, in part, to reduced levels of Coenzyme A disulfide reductase, which among other functions, provides NAD+ for glycolysis. Our data suggest that spirochetes default to an RpoS-independent program for round body formation upon sensing that the energetics for transmission are unfavorable.
Lyme disease, caused by the spirochetal pathogen Borrelia burgdorferi, is the most prevalent arthropod-borne infection in the United States. In order to maintain itself in nature, B. burgdorferi must cycle between its arthropod vector, Ixodes ticks, and a mammalian reservoir, usually a small rodent. Previous studies have demonstrated that the alternative sigma factor RpoS is essential for B. burgdorferi to infect a mammalian host, whereas a role within the tick has never been examined. In this study, we determined that one or more RpoS-dependent genes are required for B. burgdorferi to disseminate through the tick. Using a combination of microscopy techniques, we show that RpoS-deficient organisms are confined to the lumen of the tick midgut during nymphal feeding where they form round bodies, while wild-type spirochetes remain elongated and traverse the midgut to enter the hemolymph and salivary glands en route to the mammalian host.
Borrelia burgdorferi, an agent of Lyme disease, establishes persistent infection in immunocompetent animals and humans. Although the infection in humans can be cleared by antibiotic therapy, persistence in reservoir animals is necessary for the maintenance of the bacterium in the natural reservoir host⇔tick vector infectious cycle. B. burgdorferi binds to β1- and β3-chain integrins, and the P66 outer membrane protein is responsible for at least some of the integrin binding activity of the spirochete. Because integrins are transmembrane, bidirectional signaling molecules, integrin binding may alter the nature of the host response to the bacteria. We used isogenic B. burgdorferi p66+ and Δp66 strains to analyze the responses of cultured human cells to P66-integrin interaction during infection. Microarray results suggest that the response differs according to the cell type, infection time, and experimental conditions. Clusters of genes in functionally related categories that showed significant changes included proteins involved in cell-extracellular matrix interactions, actin dynamics, stress response, and immune responses. Integrin binding by P66 may therefore help B. burgdorferi establish infection by facilitating tissue invasion and modulating the activation of the immune system to other components of the bacteria, e.g., lipoproteins. These results provide insight into how B. burgdorferi is able to establish infection in immunocompetent hosts.
A significant number of environmental microorganisms can cause serious, even fatal, acute and chronic infections in humans. The severity and outcome of each type of infection depends on the expression of specific bacterial phenotypes controlled by complex regulatory networks that sense and respond to the host environment. Although bacterial signals that contribute to a successful acute infection have been identified in a number of pathogens, the signals that mediate the onset and establishment of chronic infections have yet to be discovered. We identified a volatile, low molecular weight molecule, 2-amino acetophenone (2-AA), produced by the opportunistic human pathogen Pseudomonas aeruginosa that reduces bacterial virulence in vivo in flies and in an acute mouse infection model. 2-AA modulates the activity of the virulence regulator MvfR (multiple virulence factor regulator) via a negative feedback loop and it promotes the emergence of P. aeruginosa phenotypes that likely promote chronic lung infections, including accumulation of lasR mutants, long-term survival at stationary phase, and persistence in a Drosophila infection model. We report for the first time the existence of a quorum sensing (QS) regulated volatile molecule that induces bistability phenotype by stochastically silencing acute virulence functions in P. aeruginosa. We propose that 2-AA mediates changes in a subpopulation of cells that facilitate the exploitation of dynamic host environments and promote gene expression changes that favor chronic infections.
P. aeruginosa causes acute as well as chronic infections in humans. In this paper we report the identification of a P. aeruginosa small molecule, 2-AA, that modulates this pathogen's virulence to promote chronic infections. We show that the synthesis of 2-AA, responsible for the grape-like odor of P. aeruginosa cultures and of wound infections, is controlled by the multiple virulence factor regulator (MvfR) important for virulence in acute infections. 2-AA reduces the production of MvfR-regulated acute virulence factors, and attenuates acute virulence by negatively fine-tuning the MvfR regulon activity. Moreover, we show that 2-AA adapts P. aeruginosa for chronic infections by promoting mutations in a key acute virulence gene (lasR) and by prolonging bacterial survival. The findings presented here reveal the function of a new MvfR-regulated molecule, and highlight MvfR's importance as a highly promising target for the development of inhibitors that can simultaneously halt acute and chronic infections caused by P. aeruginosa, and possibly by other pathogenic bacteria. This study uncovers insights that paradigmatically pave the way for the search of 2-AA-like small volatile molecules that promote pathogen adaptation and establishment of chronic infections caused by foreboding human pathogens.
Borrelia burgdorferi, the spirochetal agent of Lyme disease, is a vector-borne pathogen that cycles between a mammalian host and tick vector. This complex life cycle requires that the spirochete modulate its gene expression program to facilitate growth and maintenance in these diverse milieus. B. burgdorferi contains an operon that is predicted to encode proteins that would mediate the uptake and conversion of glycerol to dihydroxyacetone phosphate. Previous studies indicated that expression of the operon is elevated at 23°C and is repressed in the presence of the alternative sigma factor RpoS, suggesting that glycerol utilization may play an important role during the tick phase. This possibility was further explored in the current study by expression analysis and mutagenesis of glpD, a gene predicted to encode glycerol 3-phosphate dehydrogenase. Transcript levels for glpD were significantly lower in mouse joints relative to their levels in ticks. Expression of GlpD protein was repressed in an RpoS-dependent manner during growth of spirochetes within dialysis membrane chambers implanted in rat peritoneal cavities. In medium supplemented with glycerol as the principal carbohydrate, wild-type B. burgdorferi grew to a significantly higher cell density than glpD mutant spirochetes during growth in vitro at 25°C. glpD mutant spirochetes were fully infectious in mice by either needle or tick inoculation. In contrast, glpD mutants grew to significantly lower densities than wild-type B. burgdorferi in nymphal ticks and displayed a replication defect in feeding nymphs. The findings suggest that B. burgdorferi undergoes a switch in carbohydrate utilization during the mammal to tick transition. Further, the results demonstrate that the ability to utilize glycerol as a carbohydrate source for glycolysis during the tick phase of the infectious cycle is critical for maximal B. burgdorferi fitness.
Borrelia burgdorferi is the vector-borne pathogen that causes Lyme disease. It has a complex life cycle that involves growth in a tick vector and a mammalian host — two diverse environments that present B. burgdorferi with alternative carbohydrate sources for support of growth. Previous studies suggested that glycerol may be an important nutrient in the tick vector. Here we show that genes predicted to be involved in glycerol metabolism have significantly elevated expression during all tick stages. Repression of expression in the mammalian host is dependent on the alternative sigma factor, RpoS. A mutant that cannot convert glycerol into dihydroxyacetone phosphate to support glycolysis was able to infect mice. In contrast, the mutant was present at significantly lower levels in nymphal ticks, its replication was delayed during nymphal feeding and longer feeding times were required for transmission from nymph to mouse. The results demonstrate that the ability to utilize glycerol as a carbohydrate source for glycolysis during the tick phase of the infectious cycle is critical for maximal B. burgdorferi fitness.
Leptospirosis, the most widespread zoonosis in the world, is an emerging public health problem, particularly in large urban centers of developing countries. Several pathogenic species of the genus Leptospira can cause a wide range of clinical manifestations, from a mild, flu-like illness to a severe disease form characterized by multiorgan system complications leading to death. However, the mechanisms of pathogenesis of Leptospira are largely unknown. This article will address the animal models of acute and chronic leptospire infections, and the recent developments in the genetic manipulation of the bacteria, which facilitate the identification of virulence factors involved in pathogenesis and the assessment of their potential values in the control and prevention of leptospirosis.
epidemiology; leptospirosis; pathogenesis; virulence
Cyclic dimeric GMP (c-di-GMP) is a bacterial second messenger that modulates many biological processes. Although its role in bacterial pathogenesis during mammalian infection has been documented, the role of c-di-GMP in a pathogen's life cycle within a vector host is less understood. The enzootic cycle of the Lyme disease pathogen Borrelia burgdorferi involves both a mammalian host and an Ixodes tick vector. The B. burgdorferi genome encodes a single copy of the diguanylate cyclase gene (rrp1), which is responsible for c-di-GMP synthesis. To determine the role of c-di-GMP in the life cycle of B. burgdorferi, an Rrp1-deficient B. burgdorferi strain was generated. The rrp1 mutant remains infectious in the mammalian host but cannot survive in the tick vector. Microarray analyses revealed that expression of a four-gene operon involved in glycerol transport and metabolism, bb0240-bb0243, was significantly downregulated by abrogation of Rrp1. In vitro, the rrp1 mutant is impaired in growth in the media containing glycerol as the carbon source (BSK-glycerol). To determine the contribution of the glycerol metabolic pathway to the rrp1 mutant phenotype, a glp mutant, in which the entire bb0240-bb0243 operon is not expressed, was generated. Similar to the rrp1 mutant, the glp mutant has a growth defect in BSK-glycerol medium. In vivo, the glp mutant is also infectious in mice but has reduced survival in ticks. Constitutive expression of the bb0240-bb0243 operon in the rrp1 mutant fully rescues the growth defect in BSK-glycerol medium and partially restores survival of the rrp1 mutant in ticks. Thus, c-di-GMP appears to govern a catabolic switch in B. burgdorferi and plays a vital role in the tick part of the spirochetal enzootic cycle. This work provides the first evidence that c-di-GMP is essential for a pathogen's survival in its vector host.
The Lyme disease pathogen Borrelia burgdorferi has two sets of two-component systems, Hk1-Rrp1 and Hk2-Rrp2. The Hk2-Rrp2 signaling system has been shown to modulate differential expression of numerous surface lipoprotein genes and to play an essential role in spirochete transformation from a tick colonizer to a mammalian host-adapted state. In this study, we show that Rrp1, the only diguanylate cyclase in B. burgdorferi, is not required for mammalian infection but is essential for spirochete survival in the tick vector. We identify over 39 genes whose expression is influenced by this c-di-GMP signaling system. We further demonstrate that one set of the Rrp1-dependent genes, the glp operon for glycerol transport and metabolism, plays an important role in the spirochete adaptation to tick environment and partially accounts for the essentiality of c-di-GMP for B. burgdorferi survival in ticks.
Lymphadenopathy is a hallmark of acute infection with Borrelia burgdorferi, a tick-borne spirochete and causative agent of Lyme borreliosis, but the underlying causes and the functional consequences of this lymph node enlargement have not been revealed. The present study demonstrates that extracellular, live spirochetes accumulate in the cortical areas of lymph nodes following infection of mice with either host-adapted, or tick-borne B. burgdorferi and that they, but not inactivated spirochetes, drive the lymphadenopathy. The ensuing lymph node response is characterized by strong, rapid extrafollicular B cell proliferation and differentiation to plasma cells, as assessed by immunohistochemistry, flow cytometry and ELISPOT analysis, while germinal center reactions were not consistently observed. The extrafollicular nature of this B cell response and its strongly IgM-skewed isotype profile bear the hallmarks of a T-independent response. The induced B cell response does appear, however, to be largely antigen-specific. Use of a cocktail of recombinant, in vivo-expressed B. burgdorferi-antigens revealed the robust induction of borrelia-specific antibody-secreting cells by ELISPOT. Furthermore, nearly a quarter of hybridomas generated from regional lymph nodes during acute infection showed reactivity against a small number of recombinant Borrelia-antigens. Finally, neither the quality nor the magnitude of the B cell responses was altered in mice lacking the Toll-like receptor adaptor molecule MyD88. Together, these findings suggest a novel evasion strategy for B. burgdorferi: subversion of the quality of a strongly induced, potentially protective borrelia-specific antibody response via B. burdorferi's accumulation in lymph nodes.
Acute Lyme Disease is one of the most important emerging diseases in the US. People with acute Lyme disease often develop swollen lymph nodes, or lymphadenopathy, but we do not know why this happens or what effect it has on the course of the disease. We show here that when mice are infected with live Borrelia burgdorferi spirochetes (the bacteria that cause Lyme disease), live spirochetes collect in the lymph nodes. These lymph nodes then swell up and start producing large numbers of antibody-producing cells. Although many of these antibodies can recognize the bacteria, they apparently lack the quality to clear the infection. We hypothesize that by moving into the lymph node, usually a site in which strong immune responses are induced, Borrelia evades the immune response: it goes to the lymph nodes and tricks the immune system into making a very strong but inadequate response.
The Borrelia burgdorferi surface lipoprotein OspC is a critical virulence factor, but its precise role in the establishment of B. burgdorferi infection remains unclear. To determine whether OspC affects the host response at the site of inoculation of the bacterium, the recruitment of macrophages and neutrophils and the production of cytokines were examined at the site of infection by wild-type, ospC mutant, and complemented mutant B. burgdorferi strains. Of the 21 cytokines tested, monocyte chemoattractant protein 1 (MCP-1), keratinocyte-derived chemokine (KC, CXCL1), and vascular endothelial growth factor (VEGF) were found at increased levels at the site of inoculation of B. burgdorferi, and the levels varied with the production of OspC at one or more time points over the 1-week course of infection. The kinetics of expression and the dependence on OspC production by B. burgdorferi varied among the cytokines. The production of KC and MCP-1, and the appearance of monocytic infiltrates, correlated with the presence of the bacteria rather than with OspC specifically. In contrast, VEGF production was not correlated simply to the presence of the bacteria and is influenced by the presence of OspC. In in vitro assays, OspC and B. burgdorferi expressing OspC stimulated the growth of endothelial cells more than did the controls. These data suggest the possibility of a novel role for OspC in the life of B. burgdorferi at the interface of its mammalian and tick hosts.
In Borrelia burgdorferi (Bb), the Lyme disease spirochete, the alternative σ factor σ54 (RpoN) directly activates transcription of another alternative σ factor, σS (RpoS) which, in turn, controls the expression of virulence-associated membrane lipoproteins. As is customary in σ54-dependent gene control, a putative NtrC-like enhancer-binding protein, Rrp2, is required to activate the RpoN-RpoS pathway. However, recently it was found that rpoS transcription in Bb also requires another regulator, BosR, which was previously designated as a Fur or PerR homolog. Given this unexpected requirement for a second activator to promote σ54-dependent gene transcription, and the fact that regulatory mechanisms among similar species of pathogenic bacteria can be strain-specific, we sought to confirm the regulatory role of BosR in a second virulent strain (strain 297) of Bb. Indeed, BosR displayed the same influence over lipoprotein expression and mammalian infectivity for strain Bb 297 that were previously noted for Bb strain B31. We subsequently found that recombinant BosR (rBosR) bound to the rpoS gene at three distinct sites, and that binding occurred despite the absence of consensus Fur or Per boxes. This led to the identification of a novel direct repeat sequence (TAAATTAAAT) critical for rBosR binding in vitro. Mutations in the repeat sequence markedly inhibited or abolished rBosR binding. Taken together, our studies provide new mechanistic insights into how BosR likely acts directly on rpoS as a positive transcriptional activator. Additional novelty is engendered by the facts that, although BosR is a Fur or PerR homolog and it contains zinc (like Fur and PerR), it has other unique features that clearly set it apart from these other regulators. Our findings also have broader implications regarding a previously unappreciated layer of control that can be involved in σ54–dependent gene regulation in bacteria.
Lyme disease, caused by the bacterium Borrelia burgdorferi (Bb), remains the most common arthropod-borne illness in the United States. A critical strategy for Bb to maintain its presence in nature is adaptation to its diverse tick and mammalian (mouse) hosts. To accomplish this, Bb encodes a potential gene regulator, BB0647 (BosR). Herein, we confirmed that BosR is essential for Bb to establish mammalian infection. We then found that purified recombinant BosR bound to the promoter DNA (regulatory region) of rpoS, suggesting that BosR directly controls the expression of the rpoS gene. This study has revealed a new mechanism of bacterial gene control. The discovery that BosR governs Bb's virulence may lead to new strategies to interrupt the bacterium's complex life cycle.
The opportunistic pathogen Pseudomonas aeruginosa can establish life-long chronic infections in the airways of cystic fibrosis (CF) patients. Persistent lifestyle is established with P. aeruginosa patho-adaptive variants, which are clonal with the initially-acquired strains. Several reports indicated that P. aeruginosa adapts by loss-of-function mutations which enhance fitness in CF airways and sustain its clonal expansion during chronic infection. To validate this model of P. aeruginosa adaptation to CF airways and to identify novel genes involved in this microevolution, we designed a novel approach of positive-selection screening by PCR-based signature-tagged mutagenesis (Pos-STM) in a murine model of chronic airways infection. A systematic positive-selection scheme using sequential rounds of in vivo screenings for bacterial maintenance, as opposed to elimination, generated a list of genes whose inactivation increased the colonization and persistence in chronic airways infection. The phenotypes associated to these Pos-STM mutations reflect alterations in diverse aspects of P. aeruginosa biology which include lack of swimming and twitching motility, lack of production of the virulence factors such as pyocyanin, biofilm formation, and metabolic functions. In addition, Pos-STM mutants showed altered invasion and stimulation of immune response when tested in human respiratory epithelial cells, indicating that P. aeruginosa is prone to revise the interaction with its host during persistent lifestyle. Finally, sequence analysis of Pos-STM genes in longitudinally P. aeruginosa isolates from CF patients identified signs of patho-adaptive mutations within the genome. This novel Pos-STM approach identified bacterial functions that can have important clinical implications for the persistent lifestyle and disease progression of the airway chronic infection.
Pseudomonas aeruginosa chronic infections cause persistent respiratory symptoms and decline of the lung functions in patients with cystic fibrosis (CF). Despite the continuous immune response of the host defense and the aggressive antibiotics treatment, bacterial persistence is anyhow established after an acute infection stage. P. aeruginosa establishes a permanent and detrimental relationship with the host by pathogenic variants different from the initially acquired strain. Currently, much is known about the bacterial factors needed for acute infections while the mechanisms involved in the colonization and persistence in chronic airways infection remain mostly unknown. The purpose of this study was to design a novel approach of genomics-based method for in vivo high-throughput screening to directly identify bacterial functions whose inactivation promotes airways long-term chronic infection. These studies may be relevant to the design of future drugs acting against chronic infections.
Since the 1980s, the incidence of severe pulmonary hemorrhage caused by Leptospira spp. infection has increased. The mild, non-specific symptoms or the more classical form of severe disease with hepatorenal manifestations, Weil’s syndrome, predominate world-wide. However, several regions of the world have seen increases in numbers of patients with pulmonary hemorrhage attributed to leptospirosis. The reasons behind the emergence of this syndrome, which carries a high mortality rate, are not known. Several avenues for future research may shed light on the mechanisms involved in development of pulmonary hemorrhage, and inform targeted therapeutics to improve outcomes. Possibilities to consider include: (1) emergence of new bacterial strains, (2) acquisition of virulence traits by strains in the endemic regions, (3) changes in underlying health of the affected human populations, and (4) increased recognition of the syndrome and better record keeping by the medical and veterinary communities. Determining the causes of emerging clinical manifestations presents challenges and opportunities for potentially life-saving research into the pathogenesis of a number of infectious diseases, including leptospirosis.
leptospirosis; Leptospira; pulmonary hemorrhage
Leptospirosis is a widespread zoonotic infection that primarily affects residents of tropical regions, but causes infections in animals and humans in temperate regions as well. The agents of leptospirosis comprise several members of the genus Leptospira, which also includes non-pathogenic, saprophytic species. Leptospirosis can vary in severity from a mild, non-specific illness to severe disease that includes multi-organ failure and widespread endothelial damage and hemorrhage. To begin to investigate how pathogenic leptospires affect endothelial cells, we compared the responses of two endothelial cell lines to infection by pathogenic versus non-pathogenic leptospires. Microarray analyses suggested that pathogenic L. interrogans and non-pathogenic L. biflexa triggered changes in expression of genes whose products are involved in cellular architecture and interactions with the matrix, but that the changes were in opposite directions, with infection by L. biflexa primarily predicted to increase or maintain cell layer integrity, while L. interrogans lead primarily to changes predicted to disrupt cell layer integrity. Neither bacterial strain caused necrosis or apoptosis of the cells even after prolonged incubation. The pathogenic L. interrogans, however, did result in significant disruption of endothelial cell layers as assessed by microscopy and the ability of the bacteria to cross the cell layers. This disruption of endothelial layer integrity was abrogated by addition of the endothelial protective drug lisinopril at physiologically relevant concentrations. These results suggest that, through adhesion of L. interrogans to endothelial cells, the bacteria may disrupt endothelial barrier function, promoting dissemination of the bacteria and contributing to severe disease manifestations. In addition, supplementing antibiotic therapy with lisinopril or derivatives with endothelial protective activities may decrease the severity of leptospirosis.
Leptospirosis is a widespread zoonotic infection that primarily affects residents of tropical regions, but is seen occasionally in temperate regions as well. Leptospirosis can vary in severity from a mild, non-specific illness to severe disease that includes multi-organ failure and widespread endothelial damage and hemorrhage. To investigate how pathogenic leptospires affect endothelial cells, we compared the responses of two endothelial cell lines to infection by pathogenic versus non-pathogenic leptospires. Our analyses suggested that pathogenic L. interrogans and non-pathogenic L. biflexa caused changes in expression of genes whose products are involved in cellular architecture and interactions with the matrix, but that the changes were in opposite directions, with infection by L. biflexa primarily maintaining cell layer integrity, while L. interrogans disrupted cell layers. In fact, L. interrogans caused significant disruption of endothelial cell layers, but this damage could be abrogated by the endothelial protective drug lisinopril. Our results suggest that L. interrogans binds to endothelial cells and disrupts endothelial barrier function, which may promote dissemination of the bacteria and contribute to severe disease manifestations. This disruption may be slowed by endothelial-protective drugs to decrease damage in leptospirosis.
Ticks are distributed worldwide and affect human and animal health by transmitting diverse infectious agents. Effective vaccines against most tick-borne pathogens are not currently available. In this study, we characterized a tick histamine release factor (tHRF) from Ixodes scapularis and addressed the vaccine potential of this antigen in the context of tick engorgement and B. burgdorferi transmission. Results from western blotting and quantitative Reverse Transcription-PCR showed that tHRF is secreted in tick saliva, and upregulated in Borrelia burgdorferi-infected ticks. Further, the expression of tHRF was coincident with the rapid feeding phase of the tick, suggesting a role for tHRF in tick engorgement and concomitantly, for efficient B. burgdorferi transmission. Silencing tHRF by RNA interference (RNAi) significantly impaired tick feeding and decreased B. burgdorferi burden in mice. Interfering with tHRF by actively immunizing mice with recombinant tHRF, or passively transferring tHRF antiserum, also markedly reduced the efficiency of tick feeding and B. burgdorferi burden in mice. Recombinant tHRF was able to bind to host basophils and stimulate histamine release. Therefore, we speculate that tHRF might function in vivo to modulate vascular permeability and increase blood flow to the tick bite-site, facilitating tick engorgement. These findings suggest that blocking tHRF might offer a viable strategy to complement ongoing efforts to develop vaccines to block tick feeding and transmission of tick-borne pathogens.
Ticks are distributed worldwide and affect human and animal health by transmitting diverse infectious agents. Safe and effective vaccines against most tick-borne pathogens are not currently available. Typical vaccines target microbes directly, using extracts of the organism, or recombinant antigens as the immunogen; the transmission of tick-borne pathogens can also theoretically be prevented by interfering with the ability of ticks to feed on a mammalian host. In this study, we have characterized a putative histamine release factor (tHRF) from I. scapularis ticks, the predominant vector of B. burgdorferi, the agent of Lyme disease in North America. Our results suggested that tHRF is presented in tick saliva and critical for tick feeding; blocking tHRF markedly reduced the efficiency of tick feeding, and reduced the B. burgdorferi burden in mice. This finding provides novel insights into the molecular mechanisms of tick feeding and provides a potential vaccine target to block tick feeding and pathogen transmission.
If insufficiently treated, Lyme borreliosis can evolve into an inflammatory disorder affecting skin, joints, and the CNS. Early innate immunity may determine host responses targeting infection. Thus, we sought to characterize the immediate cytokine storm associated with exposure of PBMC to moderate levels of live Borrelia burgdorferi. Since Th17 cytokines are connected to host defense against extracellular bacteria, we focused on interleukin (IL)-17 and IL-22. Here, we report that, despite induction of inflammatory cytokines including IL-23, IL-17 remained barely detectable in response to B. burgdorferi. In contrast, T cell-dependent expression of IL-22 became evident within 10 h of exposure to the spirochetes. This dichotomy was unrelated to interferon-γ but to a large part dependent on caspase-1 and IL-1 bioactivity derived from monocytes. In fact, IL-1β as a single stimulus induced IL-22 but not IL-17. Neutrophils display antibacterial activity against B. burgdorferi, particularly when opsonized by antibodies. Since neutrophilic inflammation, indicative of IL-17 bioactivity, is scarcely observed in Erythema migrans, a manifestation of skin inflammation after infection, protective and antibacterial properties of IL-22 may close this gap and serve essential functions in the initial phase of spirochete infection.
Lyme borreliosis displays multifaceted clinical manifestations caused by the Borrelia burgdorferi sensu lato complex. If insufficiently treated, infection may proceed to inflammatory complications of chronic infection. Th17-like cytokines, foremost IL-17 and IL-22, are crucial for host defense against extracellular bacteria. IL-17/IL-22 secretion by human leukocytes exposed to live Borreliae has not been analyzed. Here we report that B. burgdorferi-activated PBMC lack immediate IL-17 expression despite being highly activated and robust T cell-dependent production of IL-22 that to a large part is mediated by monocyte-derived IL-1. Early innate immunity may shape dermal infection, thus likely affecting bacterial dissemination. Specifically, insufficient neutrophil recruitment/function, supposedly due to insufficient early IL-17 production along with a lack of opsonizing antibodies, may favor the spread of B. burgdorferi. Indeed, neutrophilic inflammation, indicative of IL-17 bioactivity, is scarcely observed in Erythema migrans, a manifestation of skin inflammation after infection. Production of IL-22 may fill this gap. Current knowledge on the role of IL-22 in epithelial biology in fact supports the hypothesis that IL-22 may serve as protection, particularly under conditions of inadequate neutrophil-driven host defense as seen early in B. burgdorferi infection.
Borrelia burgdorferi, the Lyme disease spirochete, dramatically alters its transcriptome and proteome as it cycles between the arthropod vector and mammalian host. During this enzootic cycle, a novel regulatory network, the Rrp2-RpoN-RpoS pathway (also known as the σ54–σS sigma factor cascade), plays a central role in modulating the differential expression of more than 10% of all B. burgdorferi genes, including the major virulence genes ospA and ospC. However, the mechanism(s) by which the upstream activator and response regulator Rrp2 is activated remains unclear. Here, we show that none of the histidine kinases present in the B. burgdorferi genome are required for the activation of Rrp2. Instead, we present biochemical and genetic evidence that supports the hypothesis that activation of the Rrp2-RpoN-RpoS pathway occurs via the small, high-energy, phosphoryl-donor acetyl phosphate (acetyl∼P), the intermediate of the Ack-Pta (acetate kinase-phosphate acetyltransferase) pathway that converts acetate to acetyl-CoA. Supplementation of the growth medium with acetate induced activation of the Rrp2-RpoN-RpoS pathway in a dose-dependent manner. Conversely, the overexpression of Pta virtually abolished acetate-induced activation of this pathway, suggesting that acetate works through acetyl∼P. Overexpression of Pta also greatly inhibited temperature and cell density-induced activation of RpoS and OspC, suggesting that these environmental cues affect the Rrp2-RpoN-RpoS pathway by influencing acetyl∼P. Finally, overexpression of Pta partially reduced infectivity of B. burgdorferi in mice. Taken together, these findings suggest that acetyl∼P is one of the key activating molecule for the activation of the Rrp2-RpoN-RpoS pathway and support the emerging concept that acetyl∼P can serve as a global signal in bacterial pathogenesis.
Borrelia burgdorferi, the causative agent of Lyme disease, is maintained in nature in a complex enzootic cycle involving Ixodes ticks and mammals. A novel regulatory network, the Rrp2-RpoN-RpoS pathway, which governs differential expression of numerous genes of B. burgdorferi, is essential for this complex life cycle. In this study, we provide evidence showing that the activation of the Rrp2-RpoN-RpoS pathway is modulated, not by the predicted histidine kinase for Rrp2, but rather by acetyl phosphate (acetyl∼P), the intermediate of the Ack-Pta (acetate kinase-phosphate acetyltransferase) metabolic pathway. Based on our findings, we propose that during the enzootic cycle of B. burgdorferi, changes in environmental cues and nutrient conditions lead to an increase in the intracellular acetyl∼P pool in B. burgdorferi, which in turn modulates the activation of the Rrp2-RpoN-RpoS pathway.
Leptospirosis is a global public health problem, primarily in the tropical developing world. The pathogenic mechanisms of the causative agents, several members of the genus Leptospira, have been underinvestigated. The exception to this trend has been the demonstration of the binding of pathogenic leptospires to the extracellular matrix (ECM) and its components. In this work, interactions of Leptospira interrogans bacteria with mammalian cells, rather than the ECM, were examined. The bacteria bound more efficiently to the cells than to the ECM, and a portion of this cell-binding activity was attributable to attachment to glycosaminoglycan (GAG) chains of proteoglycans (PGs). Chondroitin sulfate B PGs appeared to be the primary targets of L. interrogans attachment, while heparan sulfate PGs were much less important. Inhibition of GAG/PG-mediated attachment resulted in partial inhibition of bacterial attachment, suggesting that additional receptors for L. interrogans await identification. GAG binding may participate in the pathogenesis of leptospirosis within the host animal. In addition, because GAGs are expressed on the luminal aspects of epithelial cells in the proximal tubules of the kidneys, this activity may play a role in targeting the bacteria to this critical site. Because GAGs are shed in the urine, GAG binding may also be important for transmission to new hosts through the environment.