Date Published: October 18, 2013
Publisher: Public Library of Science
Author(s): Sarah Veloso Nogueira, Brian T. Backstedt, Alexis A. Smith, Jin-Hong Qin, Elsio A. Wunder, Albert Ko, Utpal Pal, Janakiram Seshu.
Leptospira interrogans is the agent for leptospirosis, an important zoonosis in humans and animals across the globe. Surface proteins of invading pathogens, such as L. interrogans, are thought to be responsible for successful microbial persistence in vivo via interaction with specific host components. In particular, a number of invasive infectious agents exploit host proteolytic pathways, such as one involving plasminogen (Pg), which aid in efficient pathogen dissemination within the host. Here we show that L. interrogans serovar Lai binds host Pg and that the leptospiral gene product LA1951, annotated as enolase, is involved in this interaction. Interestingly, unlike in related pathogenic Spirochetes, such as Borrelia burgdorferi, LA1951 is not readily detectable in the L. interrogans outer membrane. We show that the antigen is indeed secreted extracellularly; however, it can reassociate with the pathogen surface, where it displays Pg-binding and measurable enzymatic activity. Hamsters infected with L. interrogans also develop readily detectable antibody responses against enolase. Taken together, our results suggest that the L. interrogans enolase has evolved to play a role in pathogen interaction with host molecules, which may contribute to the pathogenesis of leptospirosis.
Leptospirosis is a systemic disease of humans and domestic animals. In fact, it is regarded as one of the most widespread zoonotic illnesses caused by pathogenic spirochetes of the genus Leptospira [1-5]. While these organisms are extremely motile, they are slow-growing obligate aerobes with an optimal in vitro growth temperature of 30°C and can be distinguished from other spirochetes on the basis of their unique hook-shaped ends . Leptospira interrogans constitutes the major pathogenic leptospiral species that is responsible for human infection. L. interrogans can readily penetrate abraded skin and mucous membrane barriers to establish a systemic infection via haematogenous dissemination and subsequently colonizes multiple organs, particularly the kidneys and liver. While wild rodents serve as natural reservoirs, humans and a few other domesticated animals are accidental hosts in the transmission cycle of leptospirosis [3,6]. As L. interrogans are shed in the urine of reservoir hosts and can survive in the environment, such as in water or soil for weeks to months, proper sanitation is a key intervention in reducing the transmission of leptospirosis [7,8]. Moreover, the disease has emerged as a global health threat in impoverished populations, particularly in developing countries and tropical regions where inadequate sanitation has produced the perfect conditions for this rodent-borne disease . The incidence of human infection is generally higher in the tropics than in temperate regions, but transmission to humans can occur in both industrialized and developing countries . Over the past decade, a number of factors, including unexpected outbreaks during sporting events, adventure tourism, and natural disasters, have underscored the ability of leptospirosis to become a public health problem even in nontraditional settings . Incidence is thought to be significantly underestimated because of the lack of awareness as well as relatively imprecise diagnosis . Due to a wide diversity of clinical symptoms and manifestations shared with many other diseases, diagnosis of leptospirosis is particularly challenging and depends on a variety of laboratory assays . Spirochetes can be detected in cultures of infected urine or tissue samples, and diagnostics usually employ methods based on direct detection of spirochetes or their antigens using dark-field microscopy, immunostaining, or PCR, as well as indirect approaches based on host immune responses [1,9-11].
The Pg-binding property of many pathogenic spirochetes facilitates their invasiveness, thereby supporting bacterial survival in the host [19,35,38,47-51]. Interaction of host Pg with a specific microbial surface ligand can lead to the activation of plasmin, which mediates degradation of intravascular clots and extracellular proteolysis, thus influencing a wide variety of physiological and pathological processes [9,29,30]. Here we show that L. interrogans enolase specifically interacts with recombinant Pg and that the native protein is secreted extracellularly by L. interrogans. The exact mechanism by which enolase is secreted by spirochetes remains enigmatic. As described in studies using other bacteria [46,52], enolase secretion might not be a consequence of cell lysis or membrane shedding but rather through a process in which protein structure, such as a hydrophobic α-helical domain of enolase, is a contributing factor . Involvement of a secretion system also remains a possibility, as the L. interrogans genome encodes for type I and II secretion-like genes [2,37,53]. In either case, our data suggest that once secreted by a yet-unknown mechanism, enolase probably localizes on the bacterial surface by reassociation. Although the nature of secreted enolase binding to the L. interrogans surface and the identity of the cellular receptor remain interesting subjects of future investigation, a recent study involving Streptococcus pyogenes raises an intriguing possibility that cell surfaces play a role in enolase-Pg interaction . The interaction of enolase with the cell surface is thought to produce a conformation of enolase capable of binding to host plasminogen. Despite its ability to interact with a ligand, L. interrogans enolase, either in recombinant form or as the native surface-associated protein, retains measurable enzymatic activities; this is an expected finding, as ClustalW analyses of enolase sequences from various microorganisms (data not shown) also show that L. interrogans enolase retains the motif ‘SHRSGETED’ integral to its catalytic properties . Whether L. interrogans uses the glycolytic pathway as a source of energy [37,55] or how the enzymatic activity of enolase contributes to leptospiral physiology, however, remains unknown. Although the biological significance of enolase-Pg interaction in leptospiral virulence remains to be studied, our data showing the generation of enolase-specific antibody responses in infected hosts as well as extracellular or microbial surface-associated localization of enolase suggest that the protein may facilitate the pathogen’s infection in the host.