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.

Role of glucosyltransferase R in biofilm interactions between Streptococcus oralis and Candida albicans.

Streptococcal glucosyltransferases (Gtf) synthesize α-glucan exopolymers which contribute to biofilm matrix. Streptococcus oralis interacts with the opportunistic pathogen Candida albicans to form hypervirulent biofilms. S. oralis 34 has a single gtf gene (gtfR). However, the role of gtfR in single and mixed species biofilms with C. albicans has never been examined. A gtfR deletion mutant, purified GtfR, and recombinant GtfR glucan-binding domain were tested in single and mixed biofilms on different substrata in vitro. A mouse oral infection model was also used. We found that in single species biofilms growing with sucrose on abiotic surfaces S. oralis gtfR increased biofilm matrix, but not bacterial biomass. In biofilms with C. albicans, S. oralis encoding gtfR showed increased bacterial biomass on all surfaces. C. albicans had a positive effect on α-glucan synthesis, and α-glucans increased C. albicans accretion on abiotic surfaces. In single and mixed infection of mice receiving sucrose S. oralis gtfR enhanced mucosal burdens. However, sucrose had a negative impact on C. albicans burdens and reduced S. oralis burdens in co-infected mice. Our data provide new insights on the GtfR-mediated interactions between the two organisms and the influence of biofilm substratum and the mucosal environment on these interactions.

Discovery of Novel Leptospirosis Vaccine Candidates Using Reverse and Structural Vaccinology.

Leptospira spp. are diderm (two membranes) bacteria that infect mammals causing leptospirosis, a public health problem with global implications. Thousands of people die every year due to leptospirosis, especially in developing countries with tropical climates. Prophylaxis is difficult due to multiple factors, including the large number of asymptomatic hosts that transmit the bacteria, poor sanitation, increasing numbers of slum dwellers, and the lack of an effective vaccine. Several leptospiral recombinant antigens were evaluated as a replacement for the inactivated (bacterin) vaccine; however, success has been limited. A prospective vaccine candidate is likely to be a surface-related protein that can stimulate the host immune response to clear leptospires from blood and organs. In this study, a comprehensive bioinformatics approach based on reverse and structural vaccinology was applied toward the discovery of novel leptospiral vaccine candidates. The Leptospira interrogans serovar Copenhageni strain L1-130 genome was mined in silico for the enhanced identification of conserved ß-barrel (ßb) transmembrane proteins and outer membrane (OM) lipoproteins. Orthologs of the prospective vaccine candidates were screened in the genomes of 20 additional Leptospira spp. Three-dimensional structural models, with a high degree of confidence, were created for each of the surface-exposed proteins. Major histocompatibility complex II (MHC-II) epitopes were identified, and their locations were mapped on the structural models. A total of 18 ßb transmembrane proteins and 8 OM lipoproteins were identified. These proteins were conserved among the pathogenic Leptospira spp. and were predicted to have epitopes for several variants of MHC-II receptors. A structural and functional analysis of the sequence of these surface proteins demonstrated that most ßb transmembrane proteins seem to be TonB-dependent receptors associated with transportation. Other proteins identified included, e.g., TolC efflux pump proteins, a BamA-like OM component of the ßb transmembrane protein assembly machinery, and the LptD-like LPS assembly protein. The structural mapping of the immunodominant epitopes identified the location of conserved, surface-exposed, immunogenic regions for each vaccine candidate. The proteins identified in this study are currently being evaluated for experimental evidence for their involvement in virulence, disease pathogenesis, and physiology, in addition to vaccine development.

A Universal Vaccine against Leptospirosis: Are We Going in the Right Direction?

Leptospirosis is the most widespread zoonosis in the world and a neglected tropical disease estimated to cause severe infection in more than one million people worldwide every year that can be combated by effective immunization. However, no significant progress has been made on the leptospirosis vaccine since the advent of bacterins over 100 years. Although protective against lethal infection, particularly in animals, bacterin-induced immunity is considered short term, serovar restricted, and the vaccine can cause serious side effects. The urgent need for a new vaccine has motivated several research groups to evaluate the protective immune response induced by recombinant vaccines. Significant protection has been reported with several promising outer membrane proteins, including LipL32 and the leptospiral immunoglobulin-like proteins. However, efficacy was variable and failed to induce a cross-protective response or sterile immunity among vaccinated animals. As hundreds of draft genomes of all known Leptospira species are now available, this should aid novel target discovery through reverse vaccinology (RV) and pangenomic studies. The identification of surface-exposed vaccine candidates that are highly conserved among infectious Leptospira spp. is a requirement for the development of a cross-protective universal vaccine. However, the lack of immune correlates is a major drawback to the application of RV to Leptospira genomes. In addition, as the protective immune response against leptospirosis is not fully understood, the rational use of adjuvants tends to be a process of trial and error. In this perspective, we discuss current advances, the pitfalls, and possible solutions for the development of a universal leptospirosis vaccine.

Lectin I from Bauhinia variegata (BVL-I) expressed by Pichia pastoris inhibits initial adhesion of oral bacteria in vitro.

Lectins are non-immune proteins that reversibly bind to carbohydrates in a specific manner. Bauhinia variegata lectin I (BVL-I) is a Gal/GalNAc-specific, single-chain lectin isolated from Bauhinia variegata seeds that has been implicated in the inhibition of bacterial adhesion and the healing of damaged skin. Since the source of the native protein (nBVL) is limited, this study aimed to produce recombinant BVL-I in Pichia pastoris (rBVL-Ip). The coding sequence for BVL-I containing preferential codons for P. pastoris was cloned into the pPICZαB plasmid. A single expressing clone was selected and fermented, resulting in the secretion and glycosylation of the protein. Fed-batch fermentation in 7L-scale was performed, and the recombinant lectin was purified from culture supernatant, resulting in a yield of 1.5mg/L culture. Further, rBVL-Ip was compared to nBVL and its recombinant version expressed in Escherichia coli BL21 (DE3) (rBVL-Ie). Although it was expressed as a monomer, rBVL-Ip retained its biological activity since it was able to impair the initial adhesion of Streptococcus mutans and S. sanguinis in an in vitro model of biofilm formation and bacterial adhesion. In summary, rBVL-Ip produced in Pichia pastoris represents a viable alternative to large-scale production, encouraging further biological application studies with this lectin.

Evaluation of the Leptospira interrogans Outer Membrane Protein OmpL37 as a Vaccine Candidate.

The identification of potential vaccine candidates against leptospirosis remains a challenge. However, one such candidate is OmpL37, a potentially surface-exposed antigen that has the highest elastin-binding ability described to date, suggesting that it plays an important role in host colonization. In order to evaluate OmpL37's ability to induce a protective immune response, prime-boost, DNA and subunit vaccine strategies were tested in the hamster model of lethal leptospirosis. The humoral immune response was evaluated using an indirect ELISA test, and the cytokine profile in whole blood was determined by quantitative real-time PCR. Unlike the DNA vaccine, the administration of recombinant OmpL37 induced a strong IgG antibody response. When individually administrated, both formulations stimulated a TNF-α mediated inflammatory response. However, none of the OmpL37 formulations or vaccination strategies induced protective immunity. Further studies are required towards the identification of new vaccine targets against leptospirosis.

Generation of Mammalian Host-adapted Leptospira interrogans by Cultivation in Peritoneal Dialysis Membrane Chamber Implantation in Rats.

Leptospira interrogans can infect a myriad of mammalian hosts, including humans (Bharti et al., 2003; Ko et al., 2009). Following acquisition by a suitable host, leptospires disseminate via the bloodstream to multiple tissues, including the kidneys, where they adhere to and colonize the proximal convoluted renal tubules (Athanazio et al., 2008). Infected hosts shed large number of spirochetes in their urine and the leptospires can survive in different environmental conditions before transmission to another host. Differential gene expression by Leptospira spp. permits adaption to these new conditions. Here we describe a protocol for the cultivation of Leptospira interrogans within Dialysis Membrane Chambers (DMCs) implanted into the peritoneal cavities of Sprague-Dawley rats (Caimano et al., 2014). This technique was originally developed to study mammalian adaption by the Lyme disease spirochete, Borrelia burgdorferi (Akins et al., 1998; Caimano, 2005). The small pore size (8,000 MWCO) of the dialysis membrane tubing used for this procedure permits access to host nutrients but excludes host antibodies and immune effector cells. Given the physiological and environmental similarities between DMCs and the proximal convoluted renal tubule, we reasoned that the DMC model would be suitable for studying in vivo gene expression by L. interrogans. In a 20 to 30 min procedure, DMCs containing virulent leptospires are surgically-implanted into the rat peritoneal cavity. Nine to 11 days post-implantation, DMCs are explanted and organisms recovered. Typically, a single DMC yields ~109 mammalian host-adapted leptospires (Caimano et al., 2014). In addition to providing a facile system for studying the transcriptional and physiologic changes pathogenic L. interrogans undergo within the mammal, the DMC model also provides a rationale basis for selecting new targets for mutagenesis and the identification of novel virulence determinants. Caution: Leptospira interrogans is a BSL-2 level pathogen and known to be excreted in the urine of infected animals. Animals should be handled and disposed of using recommended Animal Biosafety Levels (ABSL) for infectious agents using vertebrate animal guidelines. Note: All protocols using live animals must conform to governmental regulations regarding the care and use of laboratory animals. The success of this protocol is dependent on the proper use of aseptic techniques during all stages of both dialysis membrane chamber preparation and animal surgery.

Highly virulent Leptospira borgpetersenii strain characterized in the hamster model.

Abstract. A recent study by our group reported the isolation and partial serological and molecular characterization of four Leptospira borgpetersenii serogroup Ballum strains. Here, we reproduced experimental leptospirosis in golden Syrian hamsters (Mesocricetus auratus) and carried out standardization of lethal dose 50% (LD50) of one of these strains (4E). Clinical disease features and histopathologic analyses of tissue lesions were also observed. As results, strain 4E induced lethality in the hamster model with inocula lower than 10 leptospires, and histopathological examination of animals showed typical lesions found in severe leptospirosis. Gross pathological findings were peculiar; animals that died early had more chance of presenting severe jaundice and less chance of presenting pulmonary hemorrhages (P < 0.01). L. borgpetersenii serogroup Ballum has had a considerable growth in human leptospirosis cases in recent years. This strain has now been thoroughly characterized and can be used in more studies, especially evaluations of vaccine candidates.

B subunit of Escherichia coli heat-labile enterotoxin as adjuvant of humoral immune response in recombinant BCG vaccination.

The B subunit of Escherichia coli heat-labile enterotoxin (LTB), a nontoxic molecule with potent biological properties, is a powerful mucosal and parenteral adjuvant that induces a strong immune response against co-administered or coupled antigens. In this paper, the effect of LTB on the humoral immune response to recombinant BCG (rBCG) vaccination was evaluated. Isogenic mice were immunized with rBCG expressing the R1 repeat region of the P97 adhesin of Mycoplasma hyopneumoniae alone (rBCG/R1) or fused to LTB (rBCG/LTBR1). Anti-R1 systemic antibody levels (IgG1, IgG2a, IgG2b, IgG3, IgM, and IgA) were measured by ELISA using recombinant R1 as antigen. With the exception of IgM, LTB doubled the anti-R1 antibody levels in rBCG vaccination. The IgG1/IgG2a mean ratio showed that both rBCG/LTBR1 and rBCG/R1 induced a mixed Th1/Th2 immune response. Interestingly, anti-R1 serum IgA was induced only by rBCG/LTBR1. These results demonstrate that LTB has an adjuvant effect on the humoral immune response to recombinant antigens expressed in BCG.