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Objective: To predict readmission in intensive care unit analyzing the first 24 hours data after intensive care unit admission., Methods: The first intensive care unit admission of patients was analyzed from January to May 2009 in a mixed unit. Readmission to the unit was considered those during the same hospital stay or within 3 months after intensive care unit discharge. Deaths during the first admission were excluded. Demographic data, use of mechanical ventilation, and report of stay longer than 3 days were submitted to uni and multivariate analysis for readmission., Results: Five hundred seventy-seven patients were included (33 excluded deaths). The readmission group had 59 patients, while 518 patients were not readmitted. The lead time between the index admission and readmission was 9 (3-28) days (18 were readmitted in less than 3 days), and 10 died. Patients readmitted at least once to the intensive care unit had the differences below in comparison to the control group: older age: 75 (67-81) versus 67 (56-78) years, P<0.01; admission for respiratory insufficiency or sepsis: 33 versus 13%, P<0.01; medical admission: 49 versus 32%, P<0.05; higher SAPS II score: 27 (21-35) versus 23 (18-29) points, P<0.01; Charlson index: 2 (1-2) versus 1 (0-2) points, P<0.01; first ICU stay longer than 3 days: 35 versus 23%, P<0.01. After logistic regression, higher age, Charlson index and admission for respiratory and sepsis were independently associated to readmissions in intensive care unit., Conclusion: Age, comorbidities and respiratory- and/or sepsis-related admission are associated with increased readmission risk in the studied sample.

We hypothesize a potential role for Borrelia burgdorferi OspC in innate immune evasion at the initial stage of mammalian infection. We demonstrate that B. burgdorferi is resistant to high levels (>200 microg/ml) of cathelicidin and that this antimicrobial peptide exhibits limited binding to the spirochetal outer membrane, irrespective of OspC or other abundant surface lipoproteins. We conclude that the essential role of OspC is unrelated to resistance to this component of innate immunity.

Pathogens lacking the enzymatic pathways for de novo purine biosynthesis are required to salvage purines and pyrimidines from the host environment for synthesis of DNA and RNA. Two key enzymes in purine salvage pathways are IMP dehydrogenase (GuaB) and GMP synthase (GuaA), encoded by the guaB and guaA genes, respectively. While these genes are typically found on the chromosome in most bacterial pathogens, the guaAB operon of Borrelia burgdorferi is present on plasmid cp26, which also harbors a number of genes critical for B. burgdorferi viability. Using molecular genetics and an experimental model of the tick-mouse infection cycle, we demonstrate that the enzymatic activities encoded by the guaAB operon are essential for B. burgdorferi mouse infectivity and provide a growth advantage to spirochetes in the tick. These data indicate that the GuaA and GuaB proteins are critical for the survival of B. burgdorferi in the infection cycle and highlight a potential difference in the requirements for purine salvage in the disparate mammalian and tick environments.

Borrelia burgdorferi OspC is required for the spirochete to establish infection in a mammal by tick transmission or needle inoculation. After a brief essential period, the protein no longer is required and the gene can be shut off. Using a system in which spirochetes contain only an unstable wild-type copy of the ospC gene, we can obtain mice persistently infected with bacteria lacking OspC. We implanted pieces of infected mouse skin subcutaneously in naïve mice, using donors carrying wild-type or ospC mutant spirochetes, and found that both could infect mice by this method, with similar numbers of wild-type or ospC mutant spirochetes disseminated throughout the tissues of recipient mice. Recipient mouse immune responses to tissue transfer-mediated infection with wild-type or ospC mutant spirochetes were similar. These experiments demonstrate that mammalian host-adapted spirochetes can infect and disseminate in mice in the absence of OspC, thereby circumventing this hallmark of tick-derived or in vitro-grown spirochetes. We propose a model in which OspC is one of a succession of functionally equivalent, essential proteins that are synthesized at different stages of mammalian infection. In this model, another protein uniquely present on host-adapted spirochetes performs the same essential function initially fulfilled by OspC. The strict temporal control of B. burgdorferi outer surface protein gene expression may reflect immunological constraints rather than distinct functions.

Borrelia burgdorferi is an obligate parasite with a limited genome that severely narrows its metabolic and biosynthetic capabilities. Thus survival of this spirochaete in an arthropod vector and mammalian host requires that it can scavenge amino acids, fatty acids and nucleosides from a blood meal or various host tissues. Additionally, the utilization of ribonucleotides for DNA synthesis is further complicated by the lack of a ribonucleotide reductase for the conversion of nucleoside-5'-diphosphates to deoxynucleosides-5'-diphosphates. The data presented here demonstrate that B. burgdorferi must rely on host-derived sources of purine bases, deoxypurines and deoxypyrimidines for the synthesis of DNA. However, if deoxyguanosine (dGuo) is limited in host tissue, the enzymatic activities of a 2'-deoxyribosyltransferase (DRTase, encoded by bb0426), IMP dehydrogenase (GuaB) and GMP synthase (GuaA) catalyse the multistep conversion of hypoxanthine (Hyp) to dGMP for DNA synthesis. This pathway provides additional biochemical flexibility for B. burgdorferi when it colonizes and infects different host tissues.

In a world that is becoming more multilingual, bilingual aphasia is a clinical problem with a major clinical impact. However, at present we lack causal explanations of the many features of recovery patterns and there is no consensus about the language in which the patient should receive speech therapy. Further advance requires an understanding of the dynamics of recovery. In a novel longitudinal, single-case study, we combine fMRI and dynamic causal modeling to examine the effects of specific language treatment for picture naming on the representation and control of language areas during the course of recovery. Improved performance in the treated language was associated with increased activation in language areas. Consistent with theoretical expectations, causal modeling indicated increased connectedness of the control and language networks for the treated language. This functional approach holds great promise for investigating recovery patterns and the effects of specific language treatment in bilingual aphasic patients.

The therapeutic use of proteins has created an increasing demand for feasible and economical methods for both up- and downstream processes. However, whereas upstream processes have attracted substantial investment and commercial attention, downstream processing has been overlooked, causing a production bottleneck that is shifting the costs of production. This review focuses on the use of aqueous two-phase extraction as an option for the downstream processing of therapeutic proteins. It is a potential and promising liquid-liquid extraction technique for the purification of biomolecules, such as monoclonal antibodies, growth factors and hormones, that combines a high selectivity and biocompatibility with an easy scale-up and continuous operation mode.

The single-stage equilibrium aqueous two-phase extraction (ATPE) of human antibodies from a Chinese hamster ovary (CHO) cells supernatant was investigated. The performance of polyethylene glycol 3350 (PEG 3350)/phosphate aqueous two-phase systems (ATPS) was evaluated by studying several experimental conditions, such as pH, ionic strength, volume ratio and initial antibody concentration in the feed stock. The conditions that favoured the extraction of human immunoglobulin G (IgG) were low pH values, high NaCl concentration and high volume ratios. The percentage of contaminants removal did not depend on the pH value and NaCl concentration, increasing, however, as the volume ratio decreased. About 66% of total contaminants were removed when a volume ratio of 0.4 was used. The multi-stage equilibrium ATPE was also investigated by simulating a four stages cross-current operation in test tubes. According to the IgG equilibrium curves and respective McCabe Thiele diagrams, a predicted optimised scheme of a counter-current multi-stage ATPE was described. An IgG recovery yield of 89% and a protein purity of 75% can be achieved using a PEG/phosphate ATPS containing 10% (w/w) NaCl, five stages and a volume ratio of 0.4. This represents significant improvements in the recovery yield and purity when compared to a single-stage trial performed at the same experimental conditions, where a 61% recovery yield and 55% protein purity were attained. Based on the CHO cells supernatant components equilibrium curves, it was observed that IgG can be completely purified from the higher molecular weight (MW) components and partially purified from the lower MW components.

In this paper we explore an alternative process for the purification of human antibodies from a Chinese hamster ovary (CHO) cell supernatant comprising a ligand-enhanced extraction capture step and cation exchange chromatography (CEX). The extraction of human antibodies was performed in an aqueous two-phase system (ATPS) composed of dextran and polyethylene glycol (PEG), in which the terminal hydroxyl groups of the PEG molecule were modified with an amino acid mimetic ligand in order to enhance the partition of the antibodies to the PEG-rich phase. This capture step was optimized using a design of experiments and a central composite design allowed the determination of the conditions that favor the partition of the antibodies to the phase containing the PEG diglutaric acid (PEG-GA) polymer, in terms of system composition. Accordingly, higher recovery yields were obtained for higher concentrations of PEG-GA and lower concentrations of dextran. The highest yield experimentally obtained was observed for an ATPS composed of 5.17% (w/w) dextran and 8% (w/w) PEG-GA. Higher purities were however predicted for higher concentrations of both polymers. A compromise between yield and purity was achieved using 5% dextran and 10% PEG-GA, which allowed the recovery of 82% of the antibodies with a protein purity of 96% and a total purity of 63%, determined by size-exclusion chromatography. ATPS top phases were further purified by cation exchange chromatography and it was observed that the most adequate cation exchange ligand was carboxymethyl, as the sulfopropyl ligand induced the formation of multi-aggregates or denatured forms. This column allowed the elution of 89% of the antibodies present in the top phase, with a protein purity of 100% and a total purity of 91%. The overall process containing a ligand-enhanced extraction step and a cation exchange chromatography step had an overall yield of 73%.

We have evaluated a process incorporating aqueous two-phase extraction, hydrophobic interaction chromatography (HIC) and size-exclusion chromatography (SEC) for the purification of human immunoglobulin G (IgG) from a Chinese hamster ovary (CHO) cell supernatant. These unit operations were chosen not only for allowing the removal of target impurities but also for facilitating the integration of different process units without the need for any conditioning step. Extraction in aqueous two-phase systems (ATPSs), composed of polyethylene glycol (PEG) and sodium citrate, allowed the concentration of the antibodies in the citrate-rich phase and the removal of the most hydrophobic compounds in the PEG-rich phase. An ATPS composed of 10% (w/w) PEG 3350 and 12% (w/w) citrate, at pH 6, allowed the recovery of IgG with a 97% yield, 41% HPLC purity and 72% protein purity. This bottom phase was then directly loaded on a phenyl-Sepharose HIC column. This intermediate purification step allowed the capture of the antibodies using a citrate mobile phase with 99% of the antibody recovered in the elution fractions, with 86% HPLC purity and 91% protein purity. Finally, SEC allowed the final polishing by removing IgG aggregates. HIC-eluted fractions were directly injected in a Superose 6 size-exclusion column affording a 100% pure IgG solution with 90% yield.

The Lyme disease spirochete Borrelia burgdorferi alters the expression of outer surface protein (osp) genes as the bacterium cycles between ticks and mammals. OspA is produced as borreliae enter the tick vector and remains a major surface antigen during midgut colonization. To elucidate the role of OspA in the vector, we created an insertional deletion of ospA in strain B31-A3. The ospA mutant infects mice when it is injected intradermally and is acquired by larval ticks fed on these mice, where it persists through the molt to the nymph stage. Bacterial survival rates in artificially infected tick larvae fed on naïve mice were compared with those in the vector fed on immune mice. The ospA mutant proliferates in larvae if it is exposed to blood from naïve mice, but it declines in density after larval feeding if the blood is from immune mice. When uninfected larvae are fed on B-cell-deficient mice infected with the ospA mutant, larvae show borrelial densities and persistence that are significantly greater than those fed on infected, immunocompetent mice. We conclude that OspA serves a critical antibody-shielding role during vector blood meal uptake from immune hosts and is not required for persistence in the tick vector.

Sentential negation is a universal syntactic feature of human languages that reverses the truth value expressed by a sentence. An intriguing question concerns what brain mechanisms underlie our ability to represent and understand the meaning of negative sentences. We approach this issue by investigating action-related language processing and the associated neural representations. Using functional magnetic resonance imaging we measured brain activity in 18 healthy subjects during passive listening of sentences characterized by a factorial combination of polarity (affirmative vs. negative) and concreteness (action-related vs. abstract). Negation deactivated cortical areas and the left pallidum. Compared to abstract sentences, action-related sentences activated the left-hemispheric action-representation system. Crucially, the polarity by concreteness interactions showed that the activity within the action-representation system was specifically reduced for negative action-related vs. affirmative action-related sentences (compared to abstract sentences). Accordingly, functional integration within this system as measured by Dynamic Causal Modeling was specifically weaker for negative action-related than for affirmative action-related sentences. This modulation of action representations indicates that sentential negation transiently reduces the access to mental representations of the negated information.

Congenital neck lesions reflect abnormal embryogenesis in head and neck development. A thorough knowledge of embryology and anatomy is critical in the diagnosis and treatment of these lesions. The appropriate diagnosis of these lesions is necessary to provide appropriate treatment and long-term follow up, because some of these lesions may undergo malignant transformation or be harbingers of malignant disease.

The purification of human immunoglobulin G (IgG) from a Chinese hamster ovary (CHO) cells supernatant was studied using an aqueous two-phase system (ATPS) composed of ethylene oxide/propylene oxide (UCON) and dextran. In UCON/dextran systems IgG partitions preferentially to the less hydrophobic dextran-rich phase (Kp<1). The addition of triethylene glycol-diglutaric acid (TEG-COOH) shifted the IgG partition into the upper phase showing significant improvements in both the recovery yields and purity. The purification of IgG from a CHO cell supernatant with UCON 2000/dextran/TEG-COOH system was optimised using a central composite design. Using an ATPS composed of 8% UCON, 6% dextran and 20% TEG-COOH, IgG was purified, in two steps, with a global yield of 85% and 88% purity. Statistical valid models were obtained to predict the effect of the experimental conditions on the IgG yield and purity, for both extraction and back-extraction steps. A system composed of 10% UCON, 5.5% dextran and 20% TEG-COOH was identified as the best compromise between final purity and yield.

The spirochete Borrelia burgdorferi is a tick-borne obligate parasite whose normal reservoir is a variety of small mammals. Although infection of these natural hosts does not lead to disease, infection of humans can result in Lyme disease as a consequence of the human immunopathologic response to B burgdorferi. Consistent with the pathogenesis of Lyme disease, bacterial products that allow B burgdorferi to replicate and survive seem to be primarily what is required for the bacterium to cause disease in a susceptible host. This article describes the basic biology of B burgdorferi and reviews some of the bacterial components required for infection of and survival in the mammalian and tick hosts.

Borrelia burgdorferi synthesizes a variety of differentially regulated outer surface lipoproteins in the tick vector and in vertebrate hosts. Among these is OspD, a protein that is highly induced in vitro by conditions that mimic the tick environment. Using genetically engineered strains in which ospD is deleted, we demonstrate that this protein is not required for B. burgdorferi survival and infectivity in either the mouse or the tick. However, examination of both transcript levels and protein expression indicates that OspD expression is limited to a discrete window of time during B. burgdorferi replication within the tick. This time frame corresponds to tick detachment from the host following feeding, and expression of OspD continues during tick digestion of the blood meal but is low or undetectable after the tick has molted. The high level of OspD production correlates to the highest cell densities that B. burgdorferi is known to reach in vivo. Although OspD is nonessential to the infectious cycle of B. burgdorferi, the tight regulation of expression suggests a beneficial contribution of OspD to the spirochete during bacterial replication within the tick midgut.

Borrelia burgdorferi, the causative agent of Lyme disease, is an obligate parasite that cycles between vertebrate hosts and tick vectors. Attempts to understand the genetic factors that allow B. burgdorferi to sense, adapt to, and survive in different environments have been limited by a relatively low transformation rate. Here, we describe a mariner-based transposon system that achieves saturating levels of random mutagenesis in B. burgdorferi. In comparison with allelic exchange, which targets a single locus, transposon mutagenesis can create libraries of mutants encompassing disruptions of all genes. Suitably designed screens or selections of such a library permit the recovery of mutants exhibiting a desired phenotype. The system described here allows rapid identification of the genetic locus responsible for the mutant phenotype. With appropriate modifications, this mariner-based transposon can be adapted to other spirochetes and bacteria with inefficient genetic transformation methods.

The genome of Borrelia burgdorferi is composed of one linear chromosome and approximately 20 linear and circular plasmids. Although some plasmids are required by B. burgdorferi in vivo, most plasmids are dispensable for growth in vitro. However, circular plasmid (cp) 26 is present in all natural isolates and has never been lost during in vitro growth. This plasmid carries ospC, which is critical for mammalian infection. We previously showed that cp26 encodes essential functions, including the telomere resolvase, ResT, and hence cannot be displaced. Here we identify two additional essential genes on cp26, bbb26 and bbb27, through a systematic attempt to inactivate each open reading frame (ORF). Furthermore, an incompatible plasmid carrying resT, bbb26 and bbb27 could displace cp26. Computational and experimental analyses suggested that both BBB26 and BBB27 are membrane-associated, periplasmic proteins. These data indicate that bbb26 and bbb27 encode essential but possibly redundant functions and that one or the other of these cp26 genes, in addition to resT, is required for bacterial viability. We conclude that the genetic linkage of critical physiological and virulence functions on cp26 is pertinent to its stable maintenance throughout the evolution of B. burgdorferi.

The purification of human antibodies in an aqueous two-phase system (ATPS) composed of polyethylene glycol (PEG) 6000 and phosphate was optimised by surface response methodology. A central composite design was used to evaluate the influence of phosphate, PEG and NaCl concentration and of the pH on the purity and extraction yield of IgG from a simulated serum medium. The conditions that maximise the partition of IgG into the upper phase were determined to be high concentrations of NaCl and PEG, low concentrations of phosphate and low pH values. An ATPS composed of 12% PEG, 10% phosphate, 15% NaCl at pH 6 was further used to purify human monoclonal antibodies from a Chinese Hamster Ovary (CHO) concentrated cell culture supernatant with a recovery yield of 88% in the upper PEG-rich phase and a purification factor of 4.3. This ATPS was also successfully used to purify antibodies from a hybridoma cell culture supernatant with a recovery yield of 90% and a purification factor of 4.1.

The partitioning of human immunoglobulin (IgG) in a polymer-polymer and polymer-salt aqueous two-phase system (ATPS) in the presence of several functionalised polyethylene glycols (PEGs) was studied. As a first approach, the partition studies were performed with pure IgG using systems in which the target protein remained in the bottom phase when the non-functionalised systems were tested. The effect of increasing functionalised PEG concentration and the type of ligand were studied. Afterwards, selectivity studies were performed with the most successful ligands first by using systems containing pure proteins and an artificial mixture of proteins and, subsequently, with systems containing a Chinese hamster ovary (CHO) cells supernatant. The PEG/phosphate ATPS was not suitable for the affinity partitioning of IgG. In the PEG/dextran ATPS, the diglutaric acid functionalised PEGs (PEG-COOH) displayed great affinity to IgG, and all IgG could be recovered in the top phase when 20% (w/w) of PEG 150-COOH and 40% (w/w) PEG 3350-COOH were used. The selectivity of these functionalised PEGs was evaluated using an artificial mixture of proteins, and PEG 3350-COOH did not show affinity to IgG in the presence of typical serum proteins such as human serum albumin and myoglobin, while in systems with PEG 150-COOH, IgG could be recovered with a yield of 91%. The best purification of IgG from the CHO cells supernatant was then achieved in a PEG/dextran ATPS in the presence of PEG 150-COOH with a recovery yield of 93%, a purification factor of 1.9 and a selectivity to IgG of 11. When this functionalised PEG was added to the ATPS, a 60-fold increase in selectivity was observed when compared to the non-functionalised systems.

Borrelia burgdorferi, the aetiological agent of Lyme disease, follows a life cycle that involves passage between the tick vector and the mammalian host. To investigate the role of the 36 kb linear plasmid, lp36 (also designated the B. burgdorferi K plasmid), in the infectious cycle of B. burgdorferi, we examined a clone lacking this plasmid, but containing all other plasmids known to be required for infectivity. Our results indicated that lp36 was not required for spirochete survival in the tick, but the clone lacking lp36 demonstrated low infectivity in the mammal. Restoration of lp36 to the mutant strain confirmed that the infectivity defect was due to loss of lp36. Moreover, spirochetes lacking lp36 exhibited a nearly 4-log increase in ID(50) relative to the isogenic lp36(+) clone. The infectivity defect of lp36-minus spirochetes was localized, in part, to loss of the bbk17 (adeC) gene, which encodes an adenine deaminase. This work establishes a vital role for lp36 in the infectious cycle of B. burgdorferi and identifies the bbk17 gene as a component of this plasmid that contributes to mammalian infectivity.

The partition of human antibodies in aqueous two-phase systems (ATPSs) of polyethylene glycol (PEG) and phosphate was systematically studied using first pure proteins systems and then an artificial mixture of proteins containing 1mg/ml human immunoglobulin G (IgG), 10mg/ml serum albumin and 2mg/ml myoglobin. Preliminary results obtained using pure proteins systems indicated that the PEG molecular weight and concentration, the pH value and the salts concentration had a pronounced effect on the partitioning behaviour of all proteins. For high ionic strengths and pH values higher than the isoelectric point (pI) of the contaminant proteins, IgG could be selectively recovered on the top phase. According to these results, a face centred composite design was performed in order to optimise the purification of IgG from the mixture of proteins. The optimal conditions for the isolation of IgG were observed for high concentrations of NaCl and low concentrations of both phase forming components. The best purification was achieved using an ATPS containing 8% (w/w) PEG 3350, 10% (w/w) phosphate pH 6 and 15% (w/w) NaCl. A recovery yield of 101+/-7%, a purity of 99+/-0% and a yield of native IgG of 97+/-4% were obtained. Back extraction studies of IgG to a new phosphate phase were performed and higher yields were obtained using 10% phosphate buffer at pH 6. The total extraction yield was 76% and the purity 100%.

Lyme disease Borrelia organisms are highly invasive spirochetes that alternate between vertebrate and arthropod hosts and that establish chronic infections and elicit inflammatory reactions in mammals. Although progress has been made in the targeted mutagenesis of individual genes in infectious Borrelia burgdorferi, the roles of the vast majority of gene products in pathogenesis remain unresolved. In this study, we examined the feasibility of using transposon mutagenesis to identify infectivity-related factors in B. burgdorferi. The transformable, infectious strain 5A18 NP1 was transformed with the spirochete-adapted Himar1 transposon delivery vector pMarGent to create a small library of 33 insertion mutants. Single mouse inoculations followed by culture of four tissue sites and serology were used to screen the mutants for infectivity phenotypes. Mutants that appeared attenuated (culture positive at some sites) or noninfectious (negative at all sites) and contained the virulence-associated plasmids lp25 and lp28-1 were examined in more extensive animal studies. Three of these mutants (including those with insertions in the putative fliG-1-encoded flagellar motor switch protein and the guaB-encoded IMP dehydrogenase) were noninfectious, whereas four clones appeared to exhibit reduced infectivity. Serological reactivity in VlsE enzyme-linked immunosorbent assays correlated with the assignment of mutants to the noninfectious or attenuated-infectivity groups. The results of this study indicate that random transposon mutagenesis of infectious B. burgdorferi is feasible and will be of value in studying the pathogenesis of Lyme disease Borrelia.

Causative agents of Lyme disease and relapsing fever, including Borrelia burgdorferi and Borrelia hermsii, respectively, are unusual among bacteria in that they possess a segmented genome with linear DNA molecules terminated by hairpin ends, known as telomeres. During replication, these telomeres are processed by the essential telomere resolvase, ResT, in a unique biochemical reaction known as telomere resolution. In this study, we report the identification of the B. hermsii resT gene through cross-species hybridization. Sequence comparison of the B. hermsii protein with the B. burgdorferi orthologue revealed 67% identity, including all the regions currently known to be crucial for telomere resolution. In vitro studies, however, indicated that B. hermsii ResT was unable to process a replicated B. burgdorferi type 2 telomere substrate. In contrast, in vivo cross-species complementation in which the native resT gene of B. burgdorferi was replaced with B. hermsii resT had no discernible effect, even though B. burgdorferi strain B31 carries at least two type 2 telomere ends. The B. burgdorferi ResT protein was also able to process two telomere spacing mutants in vivo that were unresolvable in vitro. The unexpected differential telomere processing in vivo versus in vitro by the two telomere resolvases suggests the presence of one or more accessory factors in vivo that are normally involved in the reaction. Our current results are also expected to facilitate further studies into ResT structure and function, including possible interaction with other Borrelia proteins.

We previously demonstrated that outer surface protein C (OspC) of Borrelia burgdorferi is essential for establishing mammalian infection. However, the role of OspC in mammalian infection is unknown. Here, we report experiments designed to distinguish between two models of OspC function in the mammalian host: (i) OspC fulfills an essential physiological role for growth and host adaptation or (ii) OspC provides a protective role for evasion of components of the innate immune response. We found that a B. burgdorferi ospC mutant, previously demonstrated to be noninfectious in both immunocompetent and SCID mice, could survive in the relatively immune-privileged environment of dialysis membrane chambers implanted within the peritoneum of a rat. The ospC mutant also adapts to the mammalian environment, as determined by the protein profiles of the chamber-cultivated spirochetes. Therefore, OspC does not appear to provide a physiological function for the survival of B. burgdorferi within the mammalian host. The second model, evasion of the innate immune system, was tested by assessing the infectivity of the ospC mutant in mice deficient for myeloid differentiation protein 88 (MyD88). Recent studies have shown that B. burgdorferi is prevented from reaching high cell numbers in the mammalian host by MyD88-dependent signaling pathways. The ospC mutant was incapable of infecting MyD88-deficient mice, suggesting that the role of OspC cannot be related solely to evasion of MyD88-mediated innate immunity. These results reiterate the importance of OspC in mammalian infection and eliminate simple models of function for this enigmatic protein.

The spirochaetes that cause tick-borne relapsing fever and Lyme disease are closely related human pathogens, yet they differ significantly in their ecology and pathogenicity. Genome sequencing of two species of relapsing fever spirochaetes, Borrelia hermsii and Borrelia turicatae, identified a chromosomal open reading frame, designated bhpA, not present in the Lyme disease spirochaete Borrelia burgdorferi. The predicted amino acid sequence of bhpA was homologous with the HtrA serine proteases, which are involved with stress responses and virulence in other bacteria. B. hermsii produced an active serine protease that was recognized by BhpA antibodies and the recombinant BhpA protein-degraded beta-casein. bhpA was transcribed in vitro at all growth temperatures and transcription levels were slightly elevated at higher temperatures. These results correlated with the synthesis of BhpA during B. hermsii infection in mice. With the exception of Borrelia recurrentis, the bhpA gene, protein and enzymatic activity were found in all relapsing fever spirochaetes, but not in Lyme disease or related spirochaetes. Heterologous expression of bhpA in B. burgdorferi increased the spirochaete's resistance to both oxidative stress and killing by human neutrophils. Therefore, we propose that bhpA encodes a unique and functional serine protease in relapsing fever spirochaetes. This periplasmic enzyme may prevent the accumulation of proteins damaged by the innate immune response and contribute to the ability of the relapsing fever spirochaetes to achieve high cell densities in blood.

Maintenance in nature of Borrelia burgdorferi, the pathogenic bacterium that causes Lyme disease, requires transmission through an infectious cycle that includes a tick vector and a mammalian host. The genetic requirements for persistence in these disparate environments have not been well defined. B. burgdorferi has a complex genome composed of a chromosome and >20 plasmids. Previous work has demonstrated that B. burgdorferi requires two plasmids, lp25 and lp28-1, in the mammalian host. To investigate the requirement for these same two plasmids during tick infection, we experimentally infected larval ticks with B. burgdorferi lacking either lp25 or lp28-1 and then assessed the spirochete load in ticks at different points of the infection. Whereas plasmid lp28-1 was dispensable in ticks, plasmid lp25 was essential for tick infection. Furthermore, we investigated the requirement in ticks for the lp25 gene bbe22, which encodes a nicotinamidase that is necessary and sufficient for mammalian infection by B. burgdorferi clones lacking lp25. This gene was also sufficient in ticks to restore survival of spirochetes lacking lp25. This is the first study to investigate the requirement for specific plasmids by B. burgdorferi within the tick vector, and it begins to establish the genomic components required for persistence of this pathogen throughout its natural infectious cycle.

Previous studies have shown that a sigma54-sigma(S) cascade regulates the expression of a few key lipoproteins in Borrelia burgdorferi, the agent of Lyme disease. Here, we demonstrate that these sigma factors, both together and independently, regulate a much more extensive number of genes and cellular processes. Microarray analyses of sigma54 and sigma(S) mutant strains identified 305 genes regulated by sigma54 and 145 regulated by sigma(S), whereas the sigma54-sigma(S) regulatory cascade appears to control 48 genes in B. burgdorferi. In silico analyses revealed that nearly 80% of genes with altered expression in the sigma54 mutant were linked to potential sigma54-dependent promoters. Many sigma54-regulated genes are expressed in vivo, and through genetic complementation of the mutant, we demonstrated that sigma54 was required by B. burgdorferi to infect mammals. Surprisingly, sigma54 mutants were able to infect Ixodes scapularis ticks and be maintained for at least 24 wk after infection, suggesting the sigma54-sigma(S) regulatory network was not involved in long-term survival in ticks. However, sigma54 mutants did not enter the salivary glands during tick feeding, indicating that sigma54-regulated genes were involved in the transmission process.

Lyme disease is the most commonly reported vector-borne disease in North America and Europe, yet we know little about which components of the causative agent, Borrelia burgdorferi, are critical for infection or virulence. Molecular genetics has provided a powerful means by which to address these topics in other bacterial pathogens. Certain features of B. burgdorferi have hampered the development of an effective system of genetic analysis, but basic tools are now available and their application has begun to provide information about the identities and roles of key bacterial components in both the tick vector and the mammalian host. Increased genetic analysis of B. burgdorferi should advance our understanding of the infectious cycle and the pathogenesis of Lyme disease.

The spirochete Borrelia burgdorferi, the causative agent of Lyme disease, has an unusual genome comprised of a linear chromosome and the largest plasmid complement of any characterized bacterium. Certain plasmid-encoded elements are required for virulence and viability, both in vitro and in vivo. The genetic tools to manipulate B. burgdorferi are sufficiently developed for precise molecular genetic investigations. B. burgdorferi now represents a prime system with which to address basic questions of plasmid biology and plasmid contributions to bacterial virulence and disease pathogenesis.

The spirochete Borrelia burgdorferi is the causative agent of Lyme disease, the leading vector-borne illness in the United States. Many of the genetic factors affecting spirochete morphology and physiology are unknown due to the limited genetic tools available and the large number of open reading frames with unknown functions. By adapting a mariner transposon to function in B. burgdorferi, we have developed a random mutagenesis system that tags the mutated locus for rapid identification. Transposition occurs at saturating levels in B. burgdorferi and appears to be random, targeting both linear and circular replicons. By combining the transposon system with a screen for factors affecting growth rate, mutations were readily identified in genes putatively involved in cell division and chemotaxis and a hypothetical open reading frame involved in outer membrane integrity. The successful adaptation of a mariner transposon to function in B. burgdorferi should aid in identifying virulence factors and novel gene products related to spirochete physiology.

Borrelia burgdorferi, which causes Lyme disease in humans, has an unusual genome composed of a linear chromosome and up to 21 extrachromosomal elements. Experimental data suggest that two of these elements, linear plasmids lp25 and lp28-1, play essential roles for infectivity in mice. In this study, we prove the essential natures of these two plasmids by selectively displacing lp25 or lp28-1 in an infectious wild-type clone with incompatible shuttle vectors derived from the native plasmids, rendering the respective transformants noninfectious to mice. Conversely, restoration of plasmid lp25 or lp28-1 in noninfectious clones that naturally lack the corresponding plasmid reestablished infectivity in mice. This approach establishes the ability to manipulate the plasmid content of strains by eliminating or introducing entire plasmids in B. burgdorferi and will be valuable in assessing the roles of plasmids even in unsequenced B. burgdorferi strains.

Environmentally responsive synthesis of surface proteins represents a hallmark of the infectious cycle of the Lyme disease agent, Borrelia burgdorferi. Here we created and analyzed a B. burgdorferi mutant lacking outer-surface protein C (OspC), an abundant Osp that spirochetes normally synthesize in the tick vector during the blood meal and down-regulate after transmission to the mammal. We demonstrate that B. burgdorferi strictly requires OspC to infect mice but not to localize or migrate appropriately in the tick. The induction of a spirochetal virulence factor preceding the time and host in which it is required demonstrates a developmental sequence for transmission of this arthropod-borne pathogen.

Surface-exposed lipoproteins of relapsing fever (RF) and Lyme borreliosis Borrelia spirochetes mediate certain interactions of the bacteria with their arthropod and vertebrate hosts. RF spirochetes such as Borrelia hermsii serially evade the host's antibody response by multiphasic antigenic variation of Vsp and Vlp proteins. Furthermore, the expression of Vsp1 and Vsp2 by Borrelia turicatae is associated with neurotropism and higher blood densities, respectively. In contrast to RF Borrelia species, the Lyme borreliosis spirochete Borrelia burgdorferi is amenable to genetic manipulation. To facilitate structure-function analyses of RF surface lipoproteins, we used recombinant plasmids to introduce full-length vsp1 and vsp2 as well as two representative vlp genes into B. burgdorferi cells. Recombinant B. burgdorferi cells constitutively expressed the proteins under the control of the B. burgdorferi flaB promoter. Antibody and protease accessibility assays indicated proper surface exposure and folding. Expression of Vsp1 and Vsp2 conferred glycosaminoglycan binding to recombinant B. burgdorferi cells that was similar to that observed with purified recombinant proteins and B. turicatae expressing native Vsp. These data demonstrate that the lipoprotein modification and export mechanisms in the genus Borrelia are conserved. They also validate the use of recombinant B. burgdorferi in studies of surface lipoprotein structure-function and the biogenesis of spirochete membranes.

Borrelia burgdorferi causes Lyme disease in humans. The genome of the sequenced type strain B31 MI consists of a linear chromosome, 12 linear plasmids, and 9 circular plasmids. Previous studies by other investigators indicated that some of these plasmids are essential for the survival of the spirochetes in vivo but not in vitro. We have studied plasmid stability during in vitro growth at 23 and 35 degrees C, conditions that approximate the temperatures of the tick vector and the mammalian host, respectively. Starting with two clones that have all 21 plasmids, we investigated plasmid maintenance within the population and on a clonal level. After three passages (27 generations), the cultures were no longer homogeneous and some derivative clones had already lost multiple plasmids. Despite this, one of six clones analyzed after 25 passages (225 generations) retained all but one plasmid (cp9) and was able to complete the mouse-tick-mouse infectious cycle. We analyzed protein composition and regulation of gene expression of clones differing in plasmid content after serial passages. All clones tested exhibited temperature-regulated expression of several proteins, including OspC. In addition, analysis of cultures inoculated from frozen stocks suggests that freezing and/or thawing contributes to heterogeneity in the outgrowth population with respect to plasmid content. Our investigations show that in vitro propagation of a clone leads to a heterogeneous population but that virulent clones can persist through extended passage. We therefore conclude that isogenicity of clones must be confirmed irrespective of their in vitro passage history.

In this report we describe two distinct approaches to develop new antibiotic resistance cassettes that allow for efficient selection of Borrelia burgdorferi transformants. The first approach utilizes fusions of borrelial flagellar promoters to antibiotic resistance markers from other bacteria. The AACC1 gene, which encodes a gentamicin acetyltransferase, conferred a high level of gentamicin resistance in B. Burfdorferi when expressed from these promoters. No cross-resistance occurred between this cassette and the kanamycin resistance cassette, which was previously developed in an analogous fashion. A second and different approach was taken to develop an efficient selectable marker that confers resistance to the antibiotic coumermycin A1. A synthetic gene was designed from the GYRB301 allele of the coumermycin-resistant B. Burgdorferi strain B31-NGR by altering the coding sequence at the wobble position. The resulting gene, GYRB(SYN), encodes a protein identical to the product of GYRB301, but the genes share only 66% nucleotide identity. The nucleotide sequence of GYRB(SYN)is sufficiently divergent from the endogenous B. Burgdorferi GYRB gene to prevent recombination between them. The cassettes described in this paper improve our repertoire of genetic tools in B. Burgdorferi. These studies also provide insight into parameters governing recombination and gene expression in B. Burgdorferi., (Copyright 2003 S. Karger AG, Basel)

Clonal Borrelia burgdorferi N40 (cN40) passaged 75 times in vitro (N40-75) infects mice but does not cause disease. N40-75 passaged 45 times further in vitro (N40-120) was no longer infectious and lacked genes encoded on linear plasmids 38 and 28-1, among other differences. These data suggest that B. burgdorferi cN40, N40-75, and N40-120 have distinct phenotypes that can be used to dissect the genetic elements responsible for pathogenicity and infectivity.

Lysyl-tRNAs are essential for protein biosynthesis by ribosomal mRNA translation in all organisms. They are synthesized by lysyl-tRNA synthetases (EC 6.1.1.6), a group of enzymes composed of two unrelated families. In bacteria and eukarya, all known lysyl-tRNA synthetases are subclass IIc-type aminoacyl-tRNA synthetases, whereas some archaea have been shown to contain an unrelated class I-type lysyl-tRNA synthetase. Examination of the preliminary genomic sequence of the bacterial pathogen Borrelia burgdorferi, the causative agent of Lyme disease, indicated the presence of an open reading frame with over 55% similarity at the amino acid level to archaeal class I-type lysyl-tRNA synthetases. In contrast, no coding region with significant similarity to any class II-type lysyl-tRNA synthetase could be detected. Heterologous expression of this open reading frame in Escherichia coli led to the production of a protein with canonical lysyl-tRNA synthetase activity in vitro. Analysis of B. burgdorferi mRNA showed that the lysyl-tRNA synthetase-encoding gene is highly expressed, confirming that B. burgdorferi contains a functional class I-type lysyl-tRNA synthetase. The detection of an archaeal-type lysyl-tRNA synthetase in B. burgdorferi and other pathogenic spirochetes, but not to date elsewhere in bacteria or eukarya, indicates that the gene that encodes this enzyme has a common origin with its orthologue from the archaeal kingdom. This difference between the lysyl-tRNA synthetases of spirochetes and their hosts may be readily exploitable for the development of anti-spirochete therapeutics.

The first four strains of Borrelia burgdorferi isolated in Slovakia from ticks and mice were studied using monoclonal antibodies, the polymerase chain reaction (PCR) with 16S rRNA specific primers and plasmid profiles. Two tick isolates were typed as Borrelia garinii, one strain isolated from Apodemus flavicollis was found to be B. afzelii and the fourth tick isolate reacted as a mixed culture of B. garinii and B. afzelii. All four strains harboured several plasmids ranging from 6-50 kbp including a plasmid with a size of approximately 41 kbp.

This article reviews the natural history, taxonomy, physical structure, growth requirements, and molecular structure of Borrelia burgdorferi sensu lato, the causative agent of Lyme disease. These spirochetal bacteria are maintained in nature through an infectious cycle between wild mammals and ticks. Borreliae are fastidious, slow-growing bacteria, found only in association with their arthropod or mammalian hosts in nature, and propagatable in the laboratory in a rich growth medium. The characteristic shape of borreliae is imposed by periplasmic flagella, located beneath the outer membrane and attached to the protoplasmic cylinder. The outer membrane of borreliae contains a number of abundant lipoproteins that are of serodiagnostic utility and currently under consideration as vaccine targets. The borrelial genome is unique in structure, organization, and copy number. Recent experiments demonstrate the feasibility of specific gene inactivation as a means with which to study the biology of borreliae and the pathogenesis of Lyme disease.

We have characterized seven different 32-kb circular plasmids carried by Borrelia burgdorferi isolate B31. Restriction endonuclease recognition site mapping and partial sequencing of these plasmids indicated that all seven are probably closely related to each other throughout their lengths and have substantial relationships to cp8.3, an 8.3-kb circular plasmid of B. burgdorferi sensu lato isolate Ip21. With the addition of the seven 32-kb plasmids, this bacterial strain is known to carry at least 10 linear and 9 circular plasmids. Variant cultures of B. burgdorferi B31 lacking one or more of the 32-kb circular plasmids are viable and, at least in some cases, infectious. We have examined a number of different natural isolates of Lyme disease borreliae and found that all of the B. burgdorferi sensu stricto isolates and most of the B. burgdorferi sensu lato isolates tested appear to carry multiple 32-kb circular plasmids related to those of B. burgdorferi B31. The ubiquity of these plasmids suggests that they may be important in the natural life cycle of these organisms. They may be highly conjugative plasmids or prophage genomes, which could prove to be useful in genetically manipulating B. burgdorferi.

We have identified four loci in Borrelia burgdorferi B31 that contain open reading frames capable of encoding six proteins that are related to the antigenic proteins OspE and OspF. We have designated these proteins Erp, for OspEF-related protein, and named their respective genes erp. The erpA and erpB genes are linked, as are erpC and erpD, and the pairs probably constitute two operons. The erpG and erpH genes appear to be monocistronic. The ErpA and ErpC proteins are expressed by B. burgdorferi B31 in culture and are recognized by a polyclonal antiserum raised against the OspE protein of B. burgdorferi N40. The four erp loci are each located on different 32-kb circular plasmids that contain additional DNA sequences that are homologous to each other and to an 8.3-kb circular plasmid of B. burgdorferi sensu lato Ip2l. All four 32-kb plasmids can be maintained within a single bacterium, which may provide a model for the study of plasmid replication and segregation in B. burgdorferi.

Previous studies have demonstrated that Borrelia burgdorferi in the midguts of infected ticks shows increased expression of the antigenic outer surface protein OspC after the ticks have ingested a blood meal. This differential expression is at least partly due to a change in temperature, as an increase in OspC levels is also observed when cultures are shifted from 23 to 35 degrees C. Immunoblotting of bacterial lysates with sera from infected mice indicated that the levels of several additional antigens were also increased in bacterial cultures shifted to 35 degrees C; we have identified one antigen as OspE. We have also observed differential expression of OspF, which has been proposed to be coexpressed in an operon with the gene encoding OspE.

Lyme disease spirochetes, Borrelia burgdorferi sensu lato, are maintained in zoonotic cycles involving ticks and small mammals. In unfed ticks, the spirochetes produce one outer surface protein, OspA, but not OspC. During infection in mammals, immunological data suggest that the spirochetes have changed their surface, now expressing OspC but little or no OspA. We find by in vitro growth experiments that this change is regulated in part by temperature; OspC is produced by spirochetes at 32-37 degrees C but not at 24 degrees C. Furthermore, spirochetes in the midgut of ticks that have fully engorged on mice now have OspC on their surface. Thus two environmental cues, an increase in temperature and tick feeding, trigger a major alteration of the spirochetal outer membrane. This rapid synthesis of OspC by spirochetes during tick feeding may play an essential role in the capacity of these bacteria to successfully infect mammalian hosts, including humans, when transmitted by ticks.

The Lyme disease spirochete Borrelia burgdorferi must survive in both its tick vector and its mammalian host to be maintained in nature. We have identified the B. burgdorferi guaA gene encoding GMP synthetase, an enzyme involved in de novo purine biosynthesis that is important for the survival of bacteria in mammalian blood. This gene encodes a functional product that will complement an Escherichia coli GMP synthetase mutant. The gene is located on a 26-kb circular plasmid, adjacent to and divergent from the gene encoding the outer surface protein C (OspC). The guaB gene homolog encoding IMP dehydrogenase, another enzyme in the purine biosynthetic pathway, is adjacent to guaA. In Borrelia hermsii, a tick-borne relapsing fever spirochete, the guaA and guaB genes are located on a linear plasmid. These are the first genes encoding proteins of known function to be mapped to a borrelial plasmid and the only example of genes encoding enzymes involved in the de novo purine biosynthesis pathway to be mapped to a plasmid in any organism. The unique plasmid location of these and perhaps other housekeeping genes may be a consequence of the segmented genomes in borreliae and reflect the need to adapt to both the arthropod and mammalian environments.

Synthesis of the Borrelia burgdorferi outer surface protein C (OspC) is quite variable. We have cloned and sequenced the ospC gene from B. burgdorferi isolate CA-11.2A, a clone in which ospC expression varies. The 5' flanking region of the gene contains at least two consensus promoter regions, as well as two large overlapping inverted repeats. Sequence comparison to other OspC proteins indicated that the CA-11.2A OspC is as closely related to OspC from two different genospecies of Lyme disease spirochetes as it is to OspC from the prototype B. burgdorferi strain, B31. Comparisons of the OspC amino acid (aa) sequence with those in aa sequence databases revealed partial identity with the variable major proteins Vmp3 and Vmp24 of B. hermsii, a causative agent of tick-borne relapsing fever. An ospC probe hybridized to B. hermsii restriction fragments and linear plasmids that also were recognized by the vmp3 and vmp24 probes. OspC and these Vmp appear to be related, but their synthesis is regulated differently in the two species of spirochetes. This represents a fascinating example of the evolution of the number, position, regulation and perhaps function of homologous genes in two related pathogens. These parameters may relate to characteristic properties of the pathogens and their separate tick vectors.

An escape variant of Borrelia burgdorferi, selected with a monoclonal antibody to OspB, expressed a truncated form of OspB, the result of point mutations in the ospB gene leading to a premature termination codon. A single amino acid position in the C terminus of OspB was critical for monoclonal antibody recognition. The variations in the ospB gene suggest a mechanism for the evasion of the immune response by these organisms and may also have implications for current diagnostic and vaccine efforts.

Previous studies describing the occurrence and molecular characteristics of Lyme disease spirochetes, Borrelia burgdorferi, from California have been restricted primarily to isolates obtained from the north coastal region of this large and ecologically diverse state. Our objective was to look for and examine B. burdorferi organisms isolated from Ixodes pacificus ticks collected from numerous regions spanning most parts of California where this tick is found. Thirty-one isolates of B. burgdorferi were examined from individual or pooled I. pacificus ticks collected from 25 counties throughout the state. One isolate was obtained from ticks collected at Wawona Campground in Yosemite National Park, documenting the occurrence of the Lyme disease spirochete in an area of intensive human recreational use. One isolate from an Ixodes neotomae tick from an additional county was also examined. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot analysis, agarose gel electrophoresis, Southern blot analysis, and the polymerase chain reaction were used to examine the molecular and genetic determinants of these uncloned, low-passage-number isolates. All of the isolates were identified as B. burgdorferi by their protein profiles and reactivities with monoclonal and polyclonal antibodies, and all the isolates were typed by the polymerase chain reaction as North American-type spirochetes (B. burgdorferi sensu stricto). Although products of the ospAB locus were identified in protein analyses in all of the isolates, several isolates contained deleted forms of this locus that would result in the expression of chimeric OspA-OspB proteins. The analysis of OspC demonstrated that this protein was widely conserved among the isolates but was also quite variable in its molecular mass and the amount of it that was expressed.

Most Borrelia burgdorferi strains have two major surface proteins, OspA and OspB. In the present study, we selected from a clonal population of infectious B. burgdorferi an OspB escape mutant, identified the genetic basis for this phenotype, and evaluated its functional activities. Selection with the anti-OspB antibody H614 was performed in vitro in medium and extended in vivo in scid mice. Mutants with a truncated OspB protein were selected at a frequency of 1 x 10(-5) to 3 x 10(-5). After no major rearrangements in DNA were detected, sequence analysis of the mutant's ospAB locus revealed a single base change in the consensus ribosomal binding sequence for ospB and a single nucleotide deletion in the ospB gene itself. The effect of these mutations was reduced expression of a truncated OspB protein. When functional abilities of the wild type and mutant were compared, the mutant had a threefold-lower capacity to penetrate a human endothelium umbilical vein cell monolayer. Infectivity of wild-type and mutant cells for scid mice was evaluated by culturing different organs, and the median infectious dose was calculated. The inoculum of mutant cells for infecting the mice was 30- to 300-fold higher than that of wild-type cells. This study shows that reduced size and expression of OspB are associated with lowered virulence of B. burgdorferi. Selection of mutants that to some degree remain infectious is one approach to defining the role of different surface proteins in the pathogenesis of Lyme disease.