Haemolysin Sph2 of Leptospira interrogans induces cell apoptosis via intracellular reactive oxygen species elevation and mitochondrial membrane injury.

Leptospira interrogans causes widespread leptospirosis in humans and animals, with major symptoms of jaundice and haemorrhage. Sph2, a member of the sphingomyelinase haemolysins, is an important virulence factor for leptospire. In this study, the function and mechanism of Sph2 in the pathogenesis of leptospirosis were investigated to further understand the pathogenesis of leptospire. Real-time PCR analysis of expression levels during cell invasion showed that sph2 gene expression was transiently induced in human umbilical vein endothelial cells (HUVECs), human embryo liver cells (L02), and human epithelial lung cells (L132), with expression levels reaching a peak after 45 min of infection. Further functional analysis of recombinant Sph2 (rSph2) by LDH assays and confocal microscopy showed that rSph2 can be internalised by cells both by causing cell membrane damage and by a damage-independent clathrin-mediated endocytosis pathway. Subsequently, rSph2 is able to translocate to mitochondria, which led to an increase in the levels of reactive oxygen species (ROS) and a decrease of the mitochondrial membrane potential (ΔΨm ). Further flowcytometry analyses after rSph2 exposure showed that 28.7%, 31%, and 27.3% of the HUVEC, L02, and L132 cells, respectively, became apoptotic. Because apoptosis could be decreased with the ROS inhibitor N-acetyl cysteine, these experiments suggested that rSph2 triggers apoptosis through mitochondrial membrane damage and ROS elevation. The ability of leptospiral haemolysin rSph2 to cause apoptosis likely contributes to the pathogenesis of leptospirosis.

Coaggregation and biofilm formation of Leptospira with Staphylococcus aureus.

It is not known how Leptospira react to wound or a cut infected with microbes, such as pathogenic Staphylococcus, or their common habitat on oral or nasal mucosal membranes. In the present study, Staphylococcus aureus MTCC-737 showed strong co-aggregation with leptospiral strains (>75%, visual score of + 4) in vitro. All tested strains of Leptospira were able to form biofilm with S. aureus. Scanning electron microscopy analysis revealed intertwined networks of attached cells of L. interrogans and S. aureus, thus providing evidence of a matrix-like structure. This phenomenon may have implications in Leptospira infection, which occurs via cuts and wounds of the skin.

Proteomic Analysis of Urine from California Sea Lions ( Zalophus californianus): A Resource for Urinary Biomarker Discovery.

Urinary markers for the assessment of kidney diseases in wild animals are limited, in part, due to the lack of urinary proteome data, especially for marine mammals. One of the most prevalent kidney diseases in marine mammals is caused by Leptospira interrogans, which is the second most common etiology linked to stranding of California sea lions ( Zalophus californianus). Urine proteins from 11 sea lions with leptospirosis kidney disease and eight sea lions without leptospirosis or kidney disease were analyzed using shotgun proteomics. In total, 2694 protein groups were identified, and 316 were differentially abundant between groups. Major urine proteins in sea lions were similar to major urine proteins in dogs and humans except for the preponderance of resistin, lysozyme C, and PDZ domain containing 1, which appear to be over-represented. Previously reported urine protein markers of kidney injury in humans and animals were also identified. Notably, neutrophil gelatinase-associated lipocalin, osteopontin, and epidermal fatty acid binding protein were elevated over 20-fold in the leptospirosis-infected sea lions. Consistent with leptospirosis infection in rodents, urinary proteins associated with the renin-angiotensin system were depressed, including neprilysin. This study represents a foundation from which to explore the clinical use of urinary protein markers in California sea lions.

Quantification of Leptospira interrogans Survival in Soil and Water Microcosms.

Leptospira interrogans is the etiological agent of leptospirosis, a globally distributed zoonotic disease. Human infection usually occurs through skin exposure with water and soil contaminated with the urine of chronically infected animals. In this study, we aimed to quantitatively characterize the survival of Leptospira interrogans serovar Copenhageni in environmental matrices. We constructed laboratory microcosms to simulate natural conditions and determined the persistence of DNA markers in soil, mud, spring water and sewage using a quantitative PCR (qPCR) and a propidium monoazide (PMA)-qPCR assay. We found that L. interrogans does not survive at high concentrations in the tested matrices. No net growth was detected in any of the experimental conditions and in all cases the concentration of the DNA markers targeted decreased from the beginning of the experiment following an exponential decay with a decreasing decay rate over time. After 12 and 21 days of incubation the spiked concentration of 106L. interrogans cells/ml or g decreased to approximately 100 cells/ml or g in soil and spring water microcosms, respectively. Furthermore, culturable L. interrogans persisted at concentrations under the limit of detection by PMA-qPCR or qPCR for at least 16 days in soil and 28 days in spring water. Altogether, our findings suggest that the environment is not a multiplication reservoir but a temporary carrier of L. interrogans Copenhageni, although the observed prolonged persistence at low concentrations may still enable the transmission of the disease.IMPORTANCE Leptospirosis is a zoonotic disease caused by spirochetes of the genus Leptospira that primarily affects impoverished populations worldwide. Although leptospirosis is transmitted by contact with water and soil, little is known about the ability of the pathogen to survive in the environment. In this study, we quantitatively characterized the survival of L. interrogans in environmental microcosms and found that although it cannot multiply in water, soil or sewage, it survives for extended time periods (days to weeks depending on the matrix). The survival parameters obtained here may help to better understand the distribution of pathogenic Leptospira in the environment and improve the predictions of human infection risks in areas where such infections are endemic.

Characterizing interactions of Leptospira interrogans with proximal renal tubule epithelial cells.

BACKGROUND: Leptospira interrogans is a pathogenic, spirochetal bacterium that is responsible for leptospirosis, an emerging worldwide zoonosis. Leptospires colonize the renal proximal tubules and chronically infect the kidney. Live bacteria are excreted into urine, contaminating the environment. While it is well known that leptospires can persist in the kidneys without signs of disease for several months, the interactions of leptospires with the proximal renal epithelial tubule cells that allow the chronic renal colonization have not been elucidated yet. In the present study, we compared the interactions between a virulent, low passage (LP) strain and a cultured-attenuated, high passage (HP) strain with renal proximal tubule epithelial cells (RPTECs) to elucidate the strategies used by Leptospira to colonize the kidney. RESULTS: Kinetics analysis of kidney colonization in a mouse model of chronic infection performed by quantitative real-time PCR and immunofluorescence, showed that the LP strain reached the kidney by 3 days post infection (pi) and attached to the basal membrane side of the renal epithelial cells. At 10 days pi, some leptospires were attached to the luminal side of the tubular epithelia and the number of colonizing leptospires gradually increased. On the other hand, the HP strain was cleared during hematogenous dissemination and did not colonize the kidney. Transmission electron microscopy analysis of LP-infected kidneys at 25 days pi showed aggregated leptospires and membrane vesicles attached to the epithelial brush border. Leptospiral kidney colonization altered the organization of the RPTEC brush border. An in vitro model of infection using TCMK-1 cells, showed that leptospiral infection induced a host stress response, which is delayed in LP-infected cells. CONCLUSIONS: After hematogenous dissemination, leptospires create protective and replicative niches in the base membrane and luminal sides of the RPTECs. During the long-term colonization, leptospires attached to the RPTEC brush borders and membrane vesicles might be involved in the formation of a biofilm-like structure in vivo. Our results also suggested that the virulent strain is able to manipulate host cell stress responses to promote renal colonization.

Pathogenic Leptospira interrogans exoproteins are primarily involved in heterotrophic processes.

Leptospirosis is a life-threatening and emerging zoonotic disease with a worldwide annual occurrence of more than 1 million cases. Leptospirosis is caused by spirochetes belonging to the genus Leptospira. The mechanisms of disease manifestation in the host remain elusive, and the roles of leptospiral exoproteins in these processes have yet to be determined. Our aim in this study was to assess the composition and quantity of exoproteins of pathogenic Leptospira interrogans and to construe how these proteins contribute to disease pathogenesis. Label-free quantitative mass spectrometry of proteins obtained from Leptospira spirochetes cultured in vitro under conditions mimicking infection identified 325 exoproteins. The majority of these proteins are conserved in the nonpathogenic species Leptospira biflexa, and proteins involved in metabolism and energy-generating functions were overrepresented and displayed the highest relative abundance in culture supernatants. Conversely, proteins of unknown function, which represent the majority of pathogen-specific proteins (presumably involved in virulence mechanisms), were underrepresented. Characterization of various L. interrogans exoprotein mutants in the animal infection model revealed host mortality rates similar to those of hosts infected with wild-type L. interrogans. Collectively, these results indicate that pathogenic Leptospira exoproteins primarily function in heterotrophic processes (the processes by which organisms utilize organic substances as nutrient sources) to maintain the saprophytic lifestyle rather than the virulence of the bacteria. The underrepresentation of proteins homologous to known virulence factors, such as toxins and effectors in the exoproteome, also suggests that disease manifesting from Leptospira infection is likely caused by a combination of the primary and potentially moonlight functioning of exoproteins.

Usage of a selective media (EMJH-STAFF) in primary culturing of pathogenic leptospires from bovine clinical samples.

UNLABELLED: Isolation of local strains is mandatory for the success of control programs. However, clinical samples are typically contaminated by other bacteria, which impair leptospires growth. The purpose of this study was to evaluate the use of a previously reported EMJH-STAFF media in the recovery of pathogenic leptospires from bovine clinical samples, namely urine (n = 123) and vaginal fluid-VF (n = 102). EMJH-STAFF presented less contamination than EMJH (<0·005), which was more evident in VF culture tubes. Nine pure leptospires cultures were obtained, six from urine (4·9%) and three from VF (2·9%). From those, seven grew on EMJH-STAFF, one on EMJH and one in both media. All the isolates were confirmed as pathogenic leptospires by lipL32-PCR, and sequencing of partial rrs showed them to belong to Leptospira noguchii, Leptospira santarosai and Leptospira interrogans species. EMJH-STAFF media was an important tool in the recovery of leptospires from bovine clinical samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The slow growth of leptospires and overgrowth of co-existing micro-organisms from environmental and microbiota are the major difficult to recovery Leptospira from animal clinical samples. Implementing an efficient control programme is essential to determine circulating leptospires in the region and their reservoirs. This study evaluated the relationship of a selective media (EMJH-STAFF) on the recovery of pathogenic leptospires (Leptospira noguchii, Leptospira santarosai and Leptospira interrogans), from bovine clinical samples (urine and vaginal fluid). EMJH-STAFF seems to be an important tool in obtaining local strains for epidemiological and control purposes.

A putative regulatory genetic locus modulates virulence in the pathogen Leptospira interrogans.

Limited research has been conducted on the role of transcriptional regulators in relation to virulence in Leptospira interrogans, the etiological agent of leptospirosis. Here, we identify an L. interrogans locus that encodes a sensor protein, an anti-sigma factor antagonist, and two genes encoding proteins of unknown function. Transposon insertion into the gene encoding the sensor protein led to dampened transcription of the other 3 genes in this locus. This lb139 insertion mutant (the lb139(-) mutant) displayed attenuated virulence in the hamster model of infection and reduced motility in vitro. Whole-transcriptome analyses using RNA sequencing revealed the downregulation of 115 genes and the upregulation of 28 genes, with an overrepresentation of gene products functioning in motility and signal transduction and numerous gene products with unknown functions, predicted to be localized to the extracellular space. Another significant finding encompassed suppressed expression of the majority of the genes previously demonstrated to be upregulated at physiological osmolarity, including the sphingomyelinase C precursor Sph2 and LigB. We provide insight into a possible requirement for transcriptional regulation as it relates to leptospiral virulence and suggest various biological processes that are affected due to the loss of native expression of this genetic locus.

Rapid isolation and susceptibility testing of Leptospira spp. using a new solid medium, LVW agar.

Pathogenic Leptospira spp., the causative agents of leptospirosis, are slow-growing Gram-negative spirochetes. Isolation of Leptospira from clinical samples and testing of antimicrobial susceptibility are difficult and time-consuming. Here, we describe the development of a new solid medium that facilitates more-rapid growth of Leptospira spp. and the use of this medium to evaluate the Etest's performance in determining antimicrobial MICs to drugs in common use for leptospirosis. The medium was developed by evaluating the effects of numerous factors on the growth rate of Leptospira interrogans strain NR-20157. These included the type of base agar, the concentration of rabbit serum (RS), and the concentration and duration of CO(2) incubation during the initial period of culture. The highest growth rate of NR-20157 was achieved using a Noble agar base supplemented with 10% RS (named LVW agar), with an initial incubation at 30°C in 5% CO(2) for 2 days prior to continuous culture in air at 30°C. These conditions were used to develop the Etest for three species, L. interrogans (NR-20161), L. kirschnerii (NR-20327), and L. borgpetersenii (NR-20151). The MICs were read on day 7 for all samples. The Etest was then performed on 109 isolates of pathogenic Leptospira spp. The MIC(90) values for penicillin G, doxycycline, cefotaxime, ceftriaxone, and chloramphenicol were 0.64 units/ml and 0.19, 0.047, 0.5, and 2 µg/ml, respectively. The use of LVW agar, which enables rapid growth, isolation of single colonies, and simple antimicrobial susceptibility testing for Leptospira spp., provides an opportunity for new areas of fundamental and applied research.

Inactivation of clpB in the pathogen Leptospira interrogans reduces virulence and resistance to stress conditions.

Leptospira interrogans is the causative agent of leptospirosis, which is an emerging zoonotic disease. Resistance to stress conditions is largely uncharacterized for this bacterium. We therefore decided to analyze a clpB mutant that we obtained by random transposon mutagenesis. The mutant did not produce any of the two isoforms of ClpB. The clpB mutant exhibited growth defects at 30° and 37°C and in poor nutrient medium and showed increased susceptibility to oxidative stress, whereas the genetically complemented strain was restored in ClpB expression and in vitro wild-type growth. We also showed that the clpB mutant was attenuated in virulence in an animal model of acute leptospirosis. Our findings demonstrate that ClpB is involved in the general stress response. The chaperone is also necessary, either directly or indirectly, for the virulence of the pathogen L. interrogans.

TLR4- and TLR2-mediated B cell responses control the clearance of the bacterial pathogen, Leptospira interrogans.

Leptospirosis is a widespread zoonosis caused by pathogenic Leptospira interrogans that are transmitted by asymptomatic infected rodents. Leptospiral lipoproteins and LPS have been shown to stimulate murine cells via TLRs 2 and 4. Host defense mechanisms remain obscure, although TLR4 has been shown to be involved in clearing Leptospira. In this study, we show that double (TLR2 and TLR4) knockout (DKO) mice rapidly died from severe hepatic and renal failure following Leptospira inoculation. Strikingly, the severe proinflammatory response detected in the liver and kidney from Leptospira-infected DKO mice appears to be independent of MyD88, the main adaptor of TLRs. Infection of chimeric mice constructed with wild-type and DKO mice, and infection of several lines of transgenic mice devoid of T and/or B lymphocytes, identified B cells as the crucial lymphocyte subset responsible for the clearance of Leptospira, through the early production of specific TLR4-dependent anti-Leptospira IgMs elicited against the leptospiral LPS. We also found a protective tissue compartmentalized TLR2/TLR4-mediated production of IFN-gamma by B and T lymphocytes, in the liver and kidney, respectively. In contrast, the tissue inflammation observed in Leptospira-infected DKO mice was further characterized to be mostly due to B lymphocytes in the liver and T cells in the kidney. Altogether these findings demonstrate that TLR2 and TLR4 play a key role in the early control of leptospirosis, but do not directly trigger the inflammation induced by pathogenic Leptospira.

Transcriptional response of Leptospira interrogans to iron limitation and characterization of a PerR homolog.

Leptospirosis is a globally significant zoonosis caused by Leptospira spp. Iron is essential for growth of most bacterial species. Since iron availability is low in the host, pathogens have evolved complex iron acquisition mechanisms to survive and establish infection. In many bacteria, expression of iron uptake and storage proteins is regulated by Fur. L. interrogans encodes four predicted Fur homologs; we have constructed a mutation in one of these, la1857. We conducted microarray analysis to identify iron-responsive genes and to study the effects of la1857 mutation on gene expression. Under iron-limiting conditions, 43 genes were upregulated and 49 genes were downregulated in the wild type. Genes encoding proteins with predicted involvement in inorganic ion transport and metabolism (including TonB-dependent proteins and outer membrane transport proteins) were overrepresented in the upregulated list, while 54% of differentially expressed genes had no known function. There were 16 upregulated genes of unknown function which are absent from the saprophyte L. biflexa and which therefore may encode virulence-associated factors. Expression of iron-responsive genes was not significantly affected by mutagenesis of la1857, indicating that LA1857 is not a global regulator of iron homeostasis. Upregulation of heme biosynthetic genes and a putative catalase in the mutant suggested that LA1857 is more similar to PerR, a regulator of the oxidative stress response. Indeed, the la1857 mutant was more resistant to peroxide stress than the wild type. Our results provide insights into the role of iron in leptospiral metabolism and regulation of the oxidative stress response, including genes likely to be important for virulence.

Expanding the genetic toolbox for Leptospira species by generation of fluorescent bacteria.

Our knowledge of the genetics and molecular basis of the pathogenesis associated with Leptospira, in comparison to those of other bacterial species, is very limited. An improved understanding of pathogenic mechanisms requires reliable genetic tools for functional genetic analysis. Here, we report the expression of gfp and mRFP1 genes under the control of constitutive spirochetal promoters in both saprophytic and pathogenic Leptospira strains. We were able to reliably measure the fluorescence of Leptospira by fluorescence microscopy and a fluorometric microplate reader-based assay. We showed that the expression of the gfp gene had no significant effects on growth in vivo and pathogenicity in L. interrogans. We constructed an expression vector for L. biflexa that contains the lacI repressor, an inducible lac promoter, and gfp as the reporter, demonstrating that the lac system is functional in Leptospira. Green fluorescent protein (GFP) expression was induced by the addition of isopropyl-ß-d-thiogalactopyranoside (IPTG) in L. biflexa transformants harboring the expression vector. Finally, we showed that GFP can be used as a reporter to assess promoter activity in different environmental conditions. These results may facilitate further advances for studying the genetics of Leptospira spp.

Evaluation of the expression and protective potential of Leptospiral sphingomyelinases.

Leptospirosis is a zoonotic disease of global distribution, which affects both animals and humans. Pathogenic leptospires, the bacteria that cause this disease, require iron for their growth, and these spirochetes probably use their hemolysins, such as the sphingomyelinases, as a way to obtain this important nutrient from host red blood cells during infection. We expressed and purified the leptospiral sphingomyelinases Sph1, Sph2, Sph4, and SphH in a heterologous system. However, the recombinant proteins were not able to lyse sheep erythrocytes, despite having regular secondary structures. Transcripts for all sphingomyelinases tested were detected by RT-PCR analyses, but only Sph2 and SphH native proteins could be detected in Western blot assays using Leptospira whole extracts as well as in renal tubules of infected hamsters. Moreover, antibodies present in the serum of a human patient with laboratory-confirmed leptospirosis recognized Sph2, indicating that this sphingomyelinase is expressed and exposed to the immune system during infection in humans. However, in an animal challenge model, none of the sphingomyelinases tested conferred protection against leptospirosis.

Survival of Burkholderia pseudomallei and Pathogenic Leptospira in Cola, Beer, Energy Drinks, and Sports Drinks.

Burkholderia pseudomallei and pathogenic Leptospira in contaminated drinking water can cause melioidosis and leptospirosis, respectively. Here, we evaluated their survival in beverages. We mixed six isolates (three isolates per organism) in four beverages (Coca-Cola®, Red Bull®, Singha® beer, and Gatorade®) and distilled water as the control at two final concentrations (1 × 107 colony-forming units [CFU]/mL and 1 × 103 CFU/mL). The solution was kept at two temperatures (37°C and 4°C). At 4°C and at the high concentration, pathogenic Leptospira survived in Coca-Cola® up to 3 minutes and in Singha, Red Bull®, and Gatorade up to 15 minutes, whereas B. pseudomallei survived in these beverages up to 8 hours, and 14, 14, and 28 days, respectively. The survival time of both organisms was shorter at 37°C (P = 0.01) and at the lower concentration (P = 0.001). In conclusion, Leptospira can survive in some beverages for up to 15 minutes, whereas B. pseudomallei can survive in some beverages for up to 4 weeks.

Cultivation of Leptospira interrogans Within Rat Peritoneal Dialysis Membrane Chambers.

In order to sustain its zoonotic lifecycle, leptospires must adapt to growth within the host milieu. Signals encountered within the mammal also trigger regulatory programs required by Leptospira for the expression of virulence-related gene products. The complex transcriptional, antigenic, and physiological changes leptospires undergo within the mammal are collectively referred to as "host adaptation." In this chapter, we describe the procedures for the generation of host-adapted Leptospira spp. by cultivation within dialysis membrane chambers (DMCs) implanted in rat peritoneal cavities. In this model, Leptospira spp. diluted in EMJH medium are sequestered within sterile dialysis membrane tubing closed at both ends. The chamber then is surgically implanted within the peritoneal cavity of a rat and incubated for 7-10 days. During this period, leptospires are exposed to many, if not all, of the physiological and nutritional cues required for host adaptation while at the same time protected from clearance by host innate and adaptive immune defenses.

Isolation and propagation of leptospires at 37 °C directly from the mammalian host.

The causative agent of leptospirosis includes multiple serovars and species of pathogenic leptospires that are excreted via urine from reservoir hosts of infection. Primary isolation takes weeks to months, and is limited to semi-solid media at 28-30 °C. Here we present an alternative media formulation, HAN, compared to commercially available EMJH and the more specialized T80/40/LH media formulations, in semi-solid and liquid compositions, for the primary isolation of two diverse species and serovars of pathogenic leptospires directly from host kidney tissue. All three media types supported the isolation and propagation of L. interrogans serovar Copenhageni strain IC:20:001 in semi-solid media at 29 °C. However, only HAN and T80/40/LH supported the growth of L. borgpetersenii serovar Hardjo strain HB15B203 at 29 °C. In addition, HAN supported primary isolation at 37 °C. Both T80/40/LH and HAN supported primary isolation of strain IC:20:001 in liquid media at 29 °C but only HAN supported growth of strain HB15B203 in liquid media, at both 29 and 37 °C. HAN media supports the primary isolation of fastidious pathogenic leptospires directly from infected host tissue at either 29 or 37 °C: this formulation represents a more defined media for the continued optimization of growth factors required to support the primary isolation of the large and diverse range of species and serovars within the genus Leptospira circulating within domestic and wild animal populations.

Plasminogen acquisition and activation at the surface of leptospira species lead to fibronectin degradation.

Pathogenic Leptospira species are the etiological agents of leptospirosis, a widespread disease of human and veterinary concern. In this study, we report that Leptospira species are capable of binding plasminogen (PLG) in vitro. The binding to the leptospiral surface was demonstrated by indirect immunofluorescence confocal microscopy with living bacteria. The PLG binding to the bacteria seems to occur via lysine residues because the ligation is inhibited by addition of the lysine analog 6-aminocaproic acid. Exogenously provided urokinase-type PLG activator (uPA) converts surface-bound PLG into enzymatically active plasmin, as evaluated by the reaction with the chromogenic plasmin substrate d-Val-Leu-Lys 4-nitroanilide dihydrochloridein. The PLG activation system on the surface of Leptospira is PLG dose dependent and does not cause injury to the organism, as cellular growth in culture was not impaired. The generation of active plasmin within Leptospira was observed with several nonvirulent high-passage strains and with the nonpathogenic saprophytic organism Leptospira biflexa. Statistically significant higher activation of plasmin was detected with a low-passage infectious strain of Leptospira. Plasmin-coated virulent Leptospira interrogans bacteria were capable of degrading purified extracellular matrix fibronectin. The breakdown of fibronectin was not observed with untreated bacteria. Our data provide for the first time in vitro evidence for the generation of active plasmin on the surface of Leptospira, a step that may contribute to leptospiral invasiveness.

First evidence of glutathione metabolism in Leptospira interrogans.

BACKGROUND: Glutathione (GSH) plays a role as a main antioxidant metabolite in all eukaryotes and many prokaryotes. Most of the organisms synthesize GSH by a pathway involving two enzymatic reactions, each one consuming one molecule of ATP. In a first step mediated by glutamate-cysteine ligase (GCL), the carboxylate of l-glutamic acid reacts with l-cysteine to form the dipeptide γ-glutamylcysteine (γ-GC). The second step involves the addition of glycine to the C-terminal of γ-GC catalyzed by glutathione synthetase (GS). In many bacteria, such as in the pathogen Leptospira interrogans, the main intracellular thiol has not yet been identified and the presence of GSH is not clear. METHODS: We performed the molecular cloning of the genes gshA and gshB from L. interrogans; which respectively code for GCL and GS. After heterologous expression of the cloned genes we recombinantly produced the respective proteins with high degree of purity. These enzymes were exhaustively characterized in their biochemical properties. In addition, we determined the contents of GSH and the activity of related enzymes (and proteins) in cell extracts of the bacterium. RESULTS: We functionally characterized GCL and GS, the two enzymes putatively involved in GSH synthesis in L. interrogans serovar Copenhageni. LinGCL showed higher substrate promiscuity (was active in presence of l-glutamic acid, l-cysteine and ATP, and also with GTP, l-aspartic acid and l-serine in lower proportion) unlike LinGS (which was only active with γ-GC, l-glycine and ATP). LinGCL is significantly inhibited by γ-GC and GSH, the respective intermediate and final product of the synthetic pathway. GSH showed inhibitory effect over LinGS but with a lower potency than LinGCL. Going further, we detected the presence of GSH in L. interrogans cells grown under basal conditions and also determined enzymatic activity of several GSH-dependent/related proteins in cell extracts. CONCLUSIONS: and General Significance. Our results contribute with novel insights concerning redox metabolism in L. interrogans, mainly supporting that GSH is part of the antioxidant defense in the bacterium.

The preventable efficacy of ß-glucan against leptospirosis.

Leptospirosis, caused by pathogenic Leptospira species, has emerged as an important neglected zoonotic disease. Few studies have reported the preventable effects of immunoregulators, except for antibiotics, against leptospirosis. Generally, immunostimulatory agents are considered effective for enhancing innate immune responses. Many studies have found that beta-glucan (ß-glucan) could be a potent and valuable immunostimulant for improving immune responses and controlling diseases. In this study, we investigated the preventable role of ß-glucan against Leptospira infection in hamsters. First, ß-glucan was administered 24 h prior to, during and after infection. The results showed that ß-glucan increased the survival rate to 100%, alleviated tissue injury, and decreased leptospire loads in target organs. Additionally, we found using quantitative real-time PCR that application of ß-glucan significantly enhanced the expression of Toll-like receptor (TLR) 2, interleukin (IL)-1ß and iNOS at 2 dpi (days post infection) and reduced the increase of TLR2, IL-1ß and iNOS induced by Leptospira at 5 dpi. Furthermore, to induce memory immunity, ß-glucan was administered 5 days prior to infection. ß-Glucan also significantly increased the survival rates and ameliorated pathological damage to organs. Moreover, we demonstrated that ß-glucan-trained macrophages exhibited elevated expression of proinflammatory cytokines (IL-1ß and IL-6) in vitro, indicating that ß-glucan induces an enhanced inflammatory response against Leptospira infection. These results indicate that administration of ß-glucan and other immunostimulants could be potential valuable options for the control of Leptospira infection.