Papel de Peroxirredoxina 6 (PRDX6) humana en proliferación, migración e invasión en líneas celulares de hepatocarcinoma

Reactive oxygen species (ROS) have been considered toxic waste of cellular metabolism and enzymatic activities. However, nowadays it is well-known their tight regulation and the wide range of specific targets that they have in biological systems. Mainly, ROS are responsible for reversible oxidative modifications in proteins leading to appropriate cell signaling in aerobic cells. ROS balance, between production and removal by antioxidant cellular defense, is maintained under normal conditions, for the correct functionality of cellular processes. However, uncontrolled production of ROS generates oxidative stress, one of the main factors involved in cancer disease. Tumorigenesis is described as the gaining of malignant properties which dysregulate cell signaling maintaining a sustained proliferation, evading cell death, and doting cells with new characteristics. Tumoral cells can reactivate the developmental epithelium-mesenchymal transition (EMT) process, and it is here when tumoral cells carry out the “cadherin switch”, gain motility capacity and develop extracellular matrix degrader properties through metalloproteinases (MMP) resulting in metastasis. It is widely assumed that malignant cells exhibit higher levels of ROS than normal cells, contributing to all these protumoral properties. Surprisingly, these uncontrolled levels of ROS that are harmful to normal cells do not have cytotoxic effects on tumor cells but instead lead to exacerbated cell signaling. This is only possible due to the increase of antioxidants in tumoral cells that not only controls the exacerbated induction of ROS but allow to achieve protumorigenic signaling and at the same time avoid cell death. In fact, the antioxidant master regulator NRF2 is a potential contributor to all described cancer hallmarks. Consequently, downregulation of antioxidant defenses has been postulated as a very promising antitumor strategy in cancer, alone or in combination with conventional treatments. Peroxiredoxins are important enzymes of cellular antioxidant defense whose function consists of reducing H2O2, peroxynitrite, and alkyl hydroperoxides. Within them, PRDX6, through its peroxidase activity, is the only one that also reduces phospholipids hydroperoxides derived from lipid peroxidation. Moreover, PRDX6 through its calcium independent phospholipase A2 (aiPLA2) and lisophosphatidylcholine acyl transferase (LPCAT) activities is involved in repairing cell membranes. In addition, PRDX6 is overexpressed in cancer cells. Although its role in tumors remains unclear, PRDX6 seems to protect against cytotoxic effects of ROS as well as contribute to the synthesis of some tumoral-related lipokines. Moreover, PRDX6 is described to support prooxidant activity of NOX1 whose effects seem to be related to the malignant phenotype of certain tumoral cells. Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, sorafenib and other drugs represent the only treatment at advanced stages, but they have reduced effects since hepatoma cells rapidly acquire resistance to these drugs leading to the urgent necessity of finding new therapeutic options. The aim of this thesis is to decipher protumoral functions of PRDX6 in HCC using epithelioid HepG2 and mesenchymal SNU475 cell lines. For that, cells were knocked out for PRDX6 through CRISPR/Cas9 technology. Moreover, PRDX6 was overexpressed in conjunction with NOX1 system in SNU475 cells to explore the malignant effects of this partnership in HCC. After PRDX6 removal, both cell lines showed a redox status disruption, increased ROS, lack of NRF2 induction and lower Grx1 levels. Redox proteomic analysis detected 218 peptides more oxidized and 36 more reduced in HepG2 cells after PRDX6 deprivation. Among them, the two extra cysteines of Grx1, Cys79 and Cys83 were more oxidized in knockout cells which can have drastic consequences for Grx1 activity. In both cell lines, Seahorse analysis showed impaired mitochondrial and glycolytic functionality when PRDX6 was absent, leading to metabolic reprogramming. Furthermore, a decreased proliferative rate without apoptotic effects was exhibited in those cell lines. Interestingly, cell cycle studies identified a G2/M arrest as the cause of slow proliferation in the absence of PRDX6, with involvement of PCNA and GSK3b. The proliferation protein PCNA was repressed in HepG2PRDX6-/- or SNU475PRDX6-/- cell lines by oxidative changes or by lower expression, respectively. Importantly, SNU475PRDX6-/- cells presented a GSK3b inactivation in Ser9 produced by phosphorylation of AKT. Successfully, these PCNA levels and GSK3b activation were recovered once PRDX6 was again present in SNU475PRDX6-/- cell line. The malignant phenotype was also affected since both knockout cells presented a reduced expression of EMT markers through the “cadherin switch”. Additionally, SNU475 cells exhibited repressed intracellular and extracellular MMP2 enzymatic activities with lower migratory and invasive capacities after PRDX6 deficiency. The malignant effects of prooxidant capacity of PRDX6 through NOX1 were also evaluated in SNU475 cell line. Surprisingly, PRDX6 supported NOX1 activity through a possible stabilization of NOXA1 component. Thus, PRDX6 main activities, phospholipase and peroxidase, were essential to generate higher NOX1-ROS derived production which actively contributed to an increased expression of EMT markers, migratory and invasive capacities in SNU475 cells. All these changes supported the role of PRDX6 in tumor development that was demonstrated in xenograft models since its absence reduced the tumor formation capacity of these HCC cell lines. Consequently, these results point to PRDX6 as a good candidate for antitumoral strategies. In fact, preliminary data from the use of specific miRNA against PRDX6 contained in nanoliposomes showed lower tumoral formation capacity of HepG2 cells in xenograft models. Thus, this strategy of targeting PRDX6 in vivo could be a very promising antitumoral treatment alone or in presence of drugs as sorafenib in HCC patients.Las especies reactivas de oxígeno (EROS) han sido consideradas el desecho tóxico del metabolismo celular y de diversas actividades enzimáticas. Sin embargo, recientemente se han identificado las múltiples dianas que estas especies coordinan y su estricta regulación en sistemas biológicos. Principalmente, EROS son responsables de modificaciones oxidativas en las proteínas las cuales son esenciales para señalización celular en células aeróbicas. El equilibrio de EROS entre su producción y su eliminación por los sistemas antioxidantes es mantenido bajo condiciones celulares normales y posibilita el correcto funcionamiento de procesos celulares. Sin embargo, una producción descontrolada de EROS genera estrés oxidativo, uno de los principales factores implicados en la enfermedad del cáncer. El proceso tumorigénico es descrito como una ganancia de características malignas las cuales desregulan la señalización celular incrementando proliferación, evadiendo muerte celular y dotando a células tumorales con nuevas características. Estas células pueden reactivar el proceso de transición epitelio-mesénquima (TEM) esencial para el desarrollo del organismo. Así que aquí es cuando las células tumorales llevan a cabo “el cambio en cadherinas”, ganan motilidad y desarrollan propiedades de degradación de matriz extracelular a través de metaloproteinasas (MMP) lo cual genera metástasis. Se conoce que las células malignas de cáncer poseen mayores niveles de EROS que células con un fenotipo normal ya que esto contribuye a la generación de cualidades protumorales. Sorprendentemente, estos descontrolados niveles de EROS que son dañinos para las células normales no conllevan efectos citotóxicos en células tumorales y participan en una activación de múltiples cascadas de señalización. Esto es solo posible debido a un incremento de antioxidantes en células tumorales controlando EROS y permitiendo alcanzar señalización protumorigénica y al mismo tiempo impidiendo la muerte celular. De hecho, el gran regulador de la defensa antioxidante NRF2 es descrito como un potencial contribuidor a todas las cualidades relacionadas con el fenotipo tumoral. Por tanto, la represión de estos sistemas de defensa antioxidante ha sido planteada como una estrategia antitumoral muy prometedora en cáncer y también en combinación con los tratamientos convencionales. Peroxirredoxinas son enzimas importantes en la defesa antioxidante celular cuya función consiste en la reducción de H2O2, peroxinitrito e hidroperóxidos acilados. Dentro de ellas se encuentra PRDX6 que a través de su actividad peroxidasa es la única que reduce hidroperóxidos de fosfolípidos derivados de peroxidación lipídica. Además, PRDX6 a través de sus actividades fosfolipasa A2 independiente de calcio (aiPLA2) y lisofosfatidilcolina acil transferasa (LPCAT) está involucrada en la reparación de membranas celulares. PRDX6 está sobreexpresada en cáncer. Aunque su papel en esta enfermedad queda aún por descifrar, PRDX6 protege contra efectos citotóxicos de EROS y contribuye en la síntesis de lipoquinas protumorales. Además, PRDX6 participa en la actividad prooxidante de NOX1 cuyos efectos parecen estar relacionados con la capacidad metastásica de ciertas células tumorales. El carcinoma hepatocelular (CHC) es el tipo más común entre los cánceres de hígado primarios donde sorafenib y otros fármacos representan los únicos tratamientos disponibles en estadios avanzados, pero cuentan con efectos reducidos ya que las células tumorales hepáticas rápidamente adquieren resistencia a estos fármacos lo cual muestra la necesidad urgente de encontrar nuevas opciones terapéuticas. El propósito de esta tesis es descifrar funciones protumorales de PRDX6 en CHC usando las líneas celulares epitelioide HepG2 y mesenquimal SNU475. Para ello, PRDX6 fue eliminada de estas líneas utilizadas a través de la metodología CRISPR/Cas9. Además, PRDX6 fue sobreexpresada en conjunción con el sistema NOX1 en células SNU475 para explorar los efectos metastásicos de esta asociación de proteínas en CHC. Después de la eliminación de PRDX6, ambas líneas celulares mostraron una disrupción del estatus redox celular, incrementaron los niveles de EROS, sin una inducción de NRF2 y los niveles de Grx1 fueron más bajos. El análisis del proteoma redox detectó 218 péptidos más oxidados y 36 más reducidos en células HepG2 después de la ausencia de PRDX6. Entre ellos, las dos cisteínas extras de Grx1, Cys79 y Cys83, que estuvieron más oxidadas lo cual puede tener consecuencias drásticas para la actividad de Grx1. En ambas líneas celulares sin PRDX6, el análisis “Seahorse“ mostró disfunción mitocondrial y glucolítica resultando en una reprogramación metabólica. Además, la eliminación de PRDX6 conllevó una disminución en proliferación celular sin efectos apoptóticos en ambas líneas celulares. Estudios de ciclo celular identificaron una parada en G2/M como la causa de proliferación ralentizada en ausencia de PRDX6 donde proteínas como PCNA y GSK3b podrían estar involucradas. La proteína de proliferación PCNA fue reprimida en las líneas celulares HepG2PRDX6-/- y SNU475PRDX6-/- por cambios oxidativos o por una expresión más baja, respectivamente. Las células SNU475PRDX6-/- presentaron una inactivación de GSK3b a través de la fosforilación de su residuo Ser9 producido por fosforilación de AKT. Estos niveles de PCNA y la activación GSK3b fueron recuperados de forma exitosa una vez que PRDX6 estuvo presente otra vez en la línea celular SNU475PRDX6-/-. El grado de malignidad de ambas líneas tumorales fue también afectado con la falta de PRDX6 presentando una reducida expresión de marcadores del proceso TEM a través del “cambio en cadherinas”. Adicionalmente, la línea celular SNU475 mostró una represión intracelular y extracelular de la actividad enzimática de MMP2 con bajas capacidades migratorias e invasivas después de la eliminación de PRDX6. Los efectos metastásicos de la actividad prooxidante de PRDX6 a través de NOX1 fueron también evaluados en la línea celular SNU475. Sorprendentemente, PRDX6 incrementó la actividad del sistema NOX1 a través de un posible mecanismo de estabilización del componente NOXA1. También se observó que las principales actividades de PRDX6, fosfolipasa y peroxidasa, fueron esenciales en la generación de EROS derivadas de NOX1 lo cual contribuyó activamente a un incremento de la expresión de marcadores del proceso TEM y las capacidades migratorias e invasivas de las células SNU475. Todos estos cambios apoyaron el papel de PRDX6 en el desarrollo tumoral lo cual fue demostrado en modelos de ratón a través de xenoinjertos ya que su ausencia redujo la capacidad de formación tumoral de estas líneas celulares en dichos modelos. Como consecuencia, estos resultados apuntan a PRDX6 como un buen candidato en estrategias antitumorales. De hecho, datos preliminares del uso de microARNs específicos contra PRDX6 encapsulados en nanopartículas mostraron una menor capacidad de formación tumoral de células HepG2 en dichos modelos de ratón. De este modo, esta estrategia contra los niveles de PRDX6 in vivo podría servir como un tratamiento antitumoral bastante prometedor o también combinando sus efectos con sorafenib en pacientes de CHC

Host cell invasion by apicomplexan parasites: the junction conundrum

No abstract available

BMC Biology BMC Biology The toxoplasma-host cell junction is anchored to the cell cortex to sustain parasite invasive force

International audienceBackgroundThe public health threats imposed by toxoplasmosis worldwide and by malaria in sub-Saharan countries are directly associated with the capacity of their closely related causative agents Toxoplasma and Plasmodium, respectively to colonize and expand inside host cells. Therefore, deciphering how these two Apicomplexan protozoan parasites access their hosting cells has been highlighted as a high priority research with the relevant perspective of designing anti-invasive molecules to prevent diseases. Central to the mechanistic base of invasion for both genera is mechanical force, which is thought to be applied by the parasite at the interface between the two cells following assembly of a unique cell junction but this model lacks direct evidence and has been challenged by recent genetic and cell biology studies. In this work, using parasites expressing the fluorescent core component of this junction, we analyse characteristic features of the kinematics of penetration of more than 1000 invasion events.ResultsThe majority of invasion events occur with a typical forward rotational progression of the parasite through a static junction into a vacuole formed from the invaginating host cell plasma membrane, in which the parasite subsequently replicates. However, if parasites encounter resistance and if the junction is not strongly anchored to the host cell cortex, as when parasites do not secrete the toxofilin protein and therefore are unable to locally remodel the cortical actin cytoskeleton, the junction is capped backwards and travels retrogradely with the host cell membrane along the parasite surface as it is enclosed within a functional vacuole. Kinetic measurements of the invasive trajectories strongly support a similar parasite driven force in both static and capped junctions, both of which lead to successful invasion. However about 20% of toxofilin mutants fail to enter and eventually disengage from the host cell membrane while the secreted RON2 molecules are capped at the posterior pole before being cleaved and released in the medium. By contrast in cells characterized by low cortex tension and high cortical actin dynamics, junction capping and entry failure are drastically reduced.ConclusionThis kinematic analysis of pre-invasive and invasive T. gondii tachyzoite behaviors newly highlights that to invade cells, parasites need to engage their motor with the junction molecular complex where force is efficiently applied only upon proper anchorage to the host cell membrane and cortex

Loss of PRDX6 aborts proliferative and migratory signaling in hepatocarcinoma cell lines

Peroxiredoxin 6 (PRDX6), the only mammalian 1-Cys member of the peroxiredoxin family, has peroxidase, phospholipase A2 (PLA2), and lysophosphatidylcholine (LPC) acyltransferase (LPCAT) activities. It has been associated with tumor progression and cancer metastasis, but the mechanisms involved are not clear. We constructed an SNU475 hepatocarcinoma cell line knockout for PRDX6 to study the processes of migration and invasiveness in these mesenchymal cells. They showed lipid peroxidation but inhibition of the NRF2 transcriptional regulator, mitochondrial dysfunction, metabolic reprogramming, an altered cytoskeleton, down-regulation of PCNA, and a diminished growth rate. LPC regulatory action was inhibited, indicating that loss of both the peroxidase and PLA2 activities of PRDX6 are involved. Upstream regulators MYC, ATF4, HNF4A, and HNF4G were activated. Despite AKT activation and GSK3β inhibition, the prosurvival pathway and the SNAI1-induced EMT program were aborted in the absence of PRDX6, as indicated by diminished migration and invasiveness, down-regulation of bottom-line markers of the EMT program, MMP2, cytoskeletal proteins, and triggering of the “cadherin switch”. These changes point to a role for PRDX6 in tumor development and metastasis, so it can be considered a candidate for antitumoral therapies

Peroxiredoxin 6 Down-Regulation Induces Metabolic Remodeling and Cell Cycle Arrest in HepG2 Cells

Peroxiredoxin 6 (Prdx6) is the only member of 1-Cys subfamily of peroxiredoxins in human cells. It is the only Prdx acting on phospholipid hydroperoxides possessing two additional sites with phospholipase A2 (PLA2) and lysophosphatidylcholine-acyl transferase (LPCAT) activities. There are contrasting reports on the roles and mechanisms of multifunctional Prdx6 in several pathologies and on its sensitivity to, and influence on, the redox environment. We have down-regulated Prdx6 with specific siRNA in hepatoblastoma HepG2 cells to study its role in cell proliferation, redox homeostasis, and metabolic programming. Cell proliferation and cell number decreased while cell volume increased; import of glucose and nucleotide biosynthesis also diminished while polyamines, phospholipids, and most glycolipids increased. A proteomic quantitative analysis suggested changes in membrane arrangement and vesicle trafficking as well as redox changes in enzymes of carbon and glutathione metabolism, pentose-phosphate pathway, citrate cycle, fatty acid metabolism, biosynthesis of aminoacids, and Glycolysis/Gluconeogenesis. Specific redox changes in Hexokinase-2 (HK2), Prdx6, intracellular chloride ion channel-1 (CLIC1), PEP-carboxykinase-2 (PCK2), and 3-phosphoglycerate dehydrogenase (PHGDH) are compatible with the metabolic remodeling toward a predominant gluconeogenic flow from aminoacids with diversion at 3-phospohglycerate toward serine and other biosynthetic pathways thereon and with cell cycle arrest at G1/S transition

Three Toxoplasma gondii dense granule proteins are required for induction of Lewis rat macrophage pyroptosis

Upon invasion of Lewis rat macrophages, Toxoplasma rapidly induces programmed cell death (pyroptosis), which prevents Toxoplasma replication, possibly explaining the resistance of the Lewis rat to Toxoplasma Using a chemical mutagenesis screen, we identified Toxoplasma mutants that no longer induced pyroptosis. Whole-genome sequencing led to the identification of three Toxoplasma parasitophorous vacuole-localized dense granule proteins, GRA35, GRA42, and GRA43, that are individually required for induction of Lewis rat macrophage pyroptosis. Macrophage infection with Δgra35, Δgra42, and Δgra43 parasites led to greatly reduced cell death rates and enhanced parasite replication. Lewis rat macrophages infected with parasites containing a single, double, or triple deletion of these GRAs showed similar levels of cell viability, suggesting that the three GRAs function in the same pathway. Deletion of GRA42 or GRA43 resulted in GRA35 (and other GRAs) being retained inside the parasitophorous vacuole instead of being localized to the parasitophorous vacuole membrane. Despite having greatly enhanced replication in Lewis rat macrophages in vitro, Δgra35, Δgra42, and Δgra43 parasites did not establish a chronic infection in Lewis rats. Toxoplasma did not induce F344 rat macrophage pyroptosis, but F344 rats infected with Δgra35, Δgra42, and Δgra43 parasites had reduced cyst numbers. Thus, these GRAs determined parasite in vivo fitness in F344 rats. Overall, our data suggest that these three Toxoplasma dense granule proteins play a critical role in establishing a chronic infection in vivo, independently of their role in mediating macrophage pyroptosis, likely due to their importance in regulating protein localization to the parasitophorous vacuole membrane.IMPORTANCE Inflammasomes are major components of the innate immune system and are responsible for detecting various microbial and environmental danger signals. Upon invasion of Lewis rat macrophages, the parasite rapidly activates the NLRP1 inflammasome, resulting in pyroptosis and elimination of the parasite's replication niche. The work reported here revealed that Toxoplasma GRA35, GRA42, and GRA43 are required for induction of Lewis rat macrophage pyroptosis. GRA42 and GRA43 mediate the correct localization of other GRAs, including GRA35, to the parasitophorous vacuole membrane. These three GRAs were also found to be important for parasite in vivo fitness in a Toxoplasma-susceptible rat strain, independently of their role in NLRP1 inflammasome activation, suggesting that they perform other important functions. Thus, this study identified three GRAs that mediate the induction of Lewis rat macrophage pyroptosis and are required for pathogenesis of the parasite

Tick-borne diseases and co-infection: Current considerations

Over recent years, a multitude of pathogens have been reported to be tick-borne. Given this, it is unsurprising that these might co-exist within the same tick, however our understanding of the interactions of these agents both within the tick and vertebrate host remains poorly defined. Despite the rich diversity of ticks, relatively few regularly feed on humans, 12 belonging to argasid and 20 ixodid species, and literature on co-infection is only available for a few of these species. The interplay of various pathogen combinations upon the vertebrate host and tick vector represents a current knowledge gap. The impact of co-infection in humans further extends into diagnostic challenges arising when multiple pathogens are encountered and we have little current data upon which to make therapeutic recommendations for those with multiple infections. Despite these short-comings, there is now increasing recognition of co-infections and current research efforts are providing valuable insights into dynamics of pathogen interactions whether they facilitate or antagonise each other. Much of this existing data is focussed upon simultaneous infection, however the consequences of sequential infection also need to be addressed. To this end, it is timely to review current understanding and highlight those areas still to address

Population-based study of diagnostic assays for Borrelia infection: comparison of purified flagella antigen assay (Ideia™, Dako Cytomation) and recombinant antigen assay (Liaison®, DiaSorin)

<p>Abstract</p> <p>Background</p> <p>Testing for <it>Borrelia</it>-specific IgM and IgG-antibodies are often performed on a variety of poorly defined symptoms, and isolated IgM results are a frequent finding, which results in diagnostic uncertainty and further testing. We wanted to test the hypothesis that Borrelia-specific assays using recombinant antigens perform differently from assays based on purified flagella antigen.</p> <p>Methods</p> <p>We compared the use of recombinant antigens (LIAISON^®DiaSorin, Saluggia, Italy) and purified flagella antigen (IDEIA™ Borrelia, DakoCytomation, Glostrup, Denmark) in the assay for <it>Borrelia</it>-specific IgM and IgG-antibodies. The assays were tested on an unselected population of serum samples submitted from general practice. A total of 357 consecutive samples for analysis of <it>Borrelia </it>IgM and IgG antibodies. Furthermore, we analysed 540 samples for <it>Borrelia</it>-specific IgM or IgG antibodies first by the IDEIA™ and, if they were positive, the samples were further analysed using the LIAISON^®assay. To verify the correctness of the patient's serological status, discrepant samples were analysed by line blots (EcoLine, Virotech).</p> <p>Results</p> <p>In the consecutive series of 357 samples, the IgM assays detected 308 negative and 3 positive samples with concordant results. Compared with the line blot, the IDEIA™ system produced 21 false-positive IgM results, whereas the LIAISON^®system produced only one false-positive IgM result. The IgG assays showed 1 positive and 328 negative concordant results. The LIAISON^®system produced 9 true IgG-positive samples that were not detected by the IDEIA™ system, but the former produced 4 positive IgG results that were negative by line blot.</p> <p>Conclusion</p> <p>Diagnostic assays based on flagella antigen seem to show more false-positive IgM and false-negative IgG results than assays based on recombinant antigens. The latter may reduce the number of presumably false-positive IgM results and identify more IgG-positive subjects, but this system also produces more false-positive IgG results.</p

In Vitro Identification of Novel Plasminogen-Binding Receptors of the Pathogen Leptospira interrogans

Background: Leptospirosis is a multisystem disease caused by pathogenic strains of the genus Leptospira. We have reported that Leptospira are able to bind plasminogen (PLG), to generate active plasmin in the presence of activator, and to degrade purified extracellular matrix fibronectin. Methodology/Principal Findings: We have now cloned, expressed and purified 14 leptospiral recombinant proteins. The proteins were confirmed to be surface exposed by immunofluorescence microscopy and were evaluated for their ability to bind plasminogen (PLG). We identified eight as PLG-binding proteins, including the major outer membrane protein LipL32, the previously published rLIC12730, rLIC10494, Lp29, Lp49, LipL40 and MPL36, and one novel leptospiral protein, rLIC12238. Bound PLG could be converted to plasmin by the addition of urokinase-type PLG activator (uPA), showing specific proteolytic activity, as assessed by its reaction with the chromogenic plasmin substrate, D-Val-Leu-Lys 4-nitroanilide dihydrochloride. The addition of the lysine analog 6-aminocaproic acid (ACA) inhibited the protein-PLG interaction, thus strongly suggesting the involvement of lysine residues in plasminogen binding. The binding of leptospiral surface proteins to PLG was specific, dose-dependent and saturable. PLG and collagen type IV competed with LipL32 protein for the same binding site, whereas separate binding sites were observed for plasma fibronectin. Conclusions/Significance: PLG-binding/activation through the proteins/receptors on the surface of Leptospira could help the bacteria to specifically overcome tissue barriers, facilitating its spread throughout the host.FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo)CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico)Fundacao Butantan, BrazilFAPESP (Brazil

Evidence That Two ATP-Dependent (Lon) Proteases in Borrelia burgdorferi Serve Different Functions

The canonical ATP-dependent protease Lon participates in an assortment of biological processes in bacteria, including the catalysis of damaged or senescent proteins and short-lived regulatory proteins. Borrelia spirochetes are unusual in that they code for two putative ATP-dependent Lon homologs, Lon-1 and Lon-2. Borrelia burgdorferi, the etiologic agent of Lyme disease, is transmitted through the blood feeding of Ixodes ticks. Previous work in our laboratory reported that B. burgdorferi lon-1 is upregulated transcriptionally by exposure to blood in vitro, while lon-2 is not. Because blood induction of Lon-1 may be of importance in the regulation of virulence factors critical for spirochete transmission, the clarification of functional roles for these two proteases in B. burgdorferi was the object of this study. On the chromosome, lon-2 is immediately downstream of ATP-dependent proteases clpP and clpX, an arrangement identical to that of lon of Escherichia coli. Phylogenetic analysis revealed that Lon-1 and Lon-2 cluster separately due to differences in the NH2-terminal substrate binding domains that may reflect differences in substrate specificity. Recombinant Lon-1 manifested properties of an ATP-dependent chaperone-protease in vitro but did not complement an E. coli Lon mutant, while Lon-2 corrected two characteristic Lon-mutant phenotypes. We conclude that B. burgdorferi Lons -1 and -2 have distinct functional roles. Lon-2 functions in a manner consistent with canonical Lon, engaged in cellular homeostasis. Lon-1, by virtue of its blood induction, and as a unique feature of the Borreliae, may be important in host adaptation from the arthropod to a warm-blooded host