Leptospires Are Killed In Vitro by Both Oxygen-Dependent and -Independent Reactions

Abstract

This study reports for the first time that leptospires are killed by H 2 O 2 and by low-molecular-weight primary granule components, which are agents normally released by neutrophils upon stimulation. Although both pathogenic and nonpathogenic strains were sensitive to H 2 O 2 -mediated killing, nonpathogenic organisms were found to be more susceptible. In addition, the killing of leptospires by H 2 O 2 was found to be independent of the presence of the neutrophil primary granule component myeloperoxidase and therefore not a consequence of halogenation reactions. We have also determined that leptospires are significantly sensitive only to primary granule components and, among those, to proteins and/or peptides of less than 30 kDa.Leptospires are the etiological agents of leptospirosis, a zooanthroponosis widespread throughout the world. The course of human leptospirosis ranges from mild to severe; some forms can be fatal, depending on the serovar of Leptospira involved and its virulence.Although we are far from having a complete picture of the Leptospira pathogenic factors, it is clear that these organisms are invasive and that the success of their invasions is mainly due to their ability to survive and grow in tissues by escaping natural defense mechanisms such as phagocytic cells, the complement system, and pharmacologically active tissue peptides and mediators. In fact, pathogenic leptospires can survive in the nonimmune host by evading complement-mediated killing (6) and (probably) phagocytosis. Studies have shown that pathogenic leptospires bind nonopsonically to and are slowly internalized by guinea pig macrophages and human polymorphonuclear leucocytes (1,(5)(6)(7)20). Although nonpathogenic spirochetes are rapidly engulfed and killed by human polymorphonuclear phagocytes, there is no clear evidence of the killing of pathogenic spirochetes other than that mediated by specific antibodies directed against some of their surface components (5). Moreover, live pathogenic leptospires can partially escape uptake by Kupffer cells from perfused rat liver (16). Overall, these data indicate that survival of leptospires is due primarily to scarce uptake by phagocytes. However, whether those leptospires that have been engulfed through nonopsonic phagocytosis are susceptible to intracellular killing is not known.The bactericidal systems activated after uptake by phagocytes include the generation of oxygen metabolites (O 2 -dependent killing) and the release of granule components such as proteins and peptides with microbicidal activity (10, 15) into the phagocytic vacuole (O 2 -independent killing) and combinations of the two. Since it is not known which among the killing mechanisms of phagocytes are effective toward leptospires, we have investigated the in vitro sensitivity of pathogenic and nonpathogenic strains of leptospires to the O 2 -dependent and -independent killing machinery of neutrophils.The oxygen-dependent chlorinating system constitutes an important microbicidal strategy employed by neutrophils (13), and ...

Leptospira interrogans Catalase Is Required for Resistance to H 2 O 2 and for Virulence

“…Here we showed that L. interrogans serovars Manilae and Pomona are more resistant to high H 2 O 2 concentrations than the saprophyte L. biflexa, a finding in agreement with data from a previous report (38). Additionally, we demonstrated that leptospiral resistance to an H 2 O 2 -rich environment is mediated by KatE and that the loss of KatE expression leads to an attenuation of virulence for hamsters.…”

“…The pathogenic species L. interrogans displays catalase activity, while the saprophytic species L. biflexa displays predominantly peroxidatic activity (7), despite the fact that a katG homolog is present in L. biflexa (65). L. interrogans can degrade H 2 O 2 at concentrations 50-fold higher than those tolerated by L. biflexa (8), consistent with a greater susceptibility of L. biflexa to H 2 O 2 -mediated killing (38). This suggests that L. interrogans has evolved an extensive H 2 O 2 detoxification system which is absent from L. biflexa.…”

Leptospira and Leptospirosis

“…PMNs are able to kill both non-pathogenic and pathogenic strains of Leptospira by oxygen-dependent and -independent mechanisms. L. biflexa and L. interrogans are both killed by hydrogen peroxide (H 2 O 2 ) and by primary granules of PMNs in vitro, although pathogenic leptospires are more resistant than non-pathogenic leptospires [131]. This resistance could be associated with the expression of catalase (KatE), an enzyme involved in the resistance to oxidative killing, which is produced only by pathogenic strains [40À42].…”

Functional thioredoxin reductase from pathogenic and free-living Leptospira spp.

“…The functionality of different proteins involved in the resistance against oxidative stress has been already evidenced; for example, catalase (a hemeprotein that catalyze the decomposition of H 2 O 2 to H 2 O and O 2 ) [11,12] and a typical 2-Cys peroxiredoxin (AhpC) (a thiolperoxidase, which are defined by their ability to reduce H 2 O 2 , and other organic hydroperoxides) [13]. On the other hand, the saprophytic species L. biflexa is more susceptible to exogenous hydrogen peroxide in vitro than L. interrogans [14], indicating that some antioxidant proteins in the pathogenic species could play an important role in the infection. The redox metabolic pathways for these organisms have been poorly studied.…”