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Leptospirosis is a worldwide re-emerging zoonosis caused by pathogenic Leptospira spp. All vertebrate species can be infected; humans are sensitive hosts whereas other species, such as rodents, may become long-term renal carrier reservoirs. Upon infection, innate immune responses are initiated by recognition of Microbial Associated Molecular Patterns (MAMPs) by Pattern Recognition Receptors (PRRs). Among MAMPs, the lipopolysaccha-ride (LPS) is recognized by the Toll-Like-Receptor 4 (TLR4) and activates both the MyD88-dependent pathway at the plasma membrane and the TRIF-dependent pathway after TLR4 internalization. We previously showed that leptospiral LPS is not recognized by the human-TLR4, whereas it signals through mouse-TLR4 (mTLR4), which mediates mouse resistance to acute leptospirosis. However, although resistant, mice are known to be chronically infected by leptospires. Interestingly, the leptospiral LPS has low endotoxicity in mouse cells and is an agonist of TLR2, the sensor for bacterial lipoproteins. Here, we investigated the signaling properties of the leptospiral LPS in mouse macrophages. Using confocal micros-copy and flow cytometry, we showed that the LPS of L. interrogans did not induce internali-zation of mTLR4, unlike the LPS of Escherichia coli. Consequently, the LPS failed to induce the production of the TRIF-dependent nitric oxide and RANTES, both important antimicro-bial responses. Using shorter LPS and LPS devoid of TLR2 activity, we further found this mTLR4-TRIF escape to be dependent on both the co-purifying lipoproteins and the full-length O antigen. Furthermore, our data suggest that the O antigen could alter the binding of the leptospiral LPS to the co-receptor CD14 that is essential for TLR4-TRIF activation. Overall , we describe here a novel leptospiral immune escape mechanism from mouse macro-phages and hypothesize that the LPS altered signaling could contribute to the stealthiness and chronicity of the leptospires in mice.

Leptospirosis is a zoonosis caused by pathogenic species of bacteria belonging to the genus Leptospira. Most studies infer the epidemiological patterns of a single serogroup or aggregate all serogroups to estimate overall seropositivity, thus not exploring the risks of exposure to distinct serogroups. The present study aims to delineate the demographic, socioeconomic and environmental factors associated with seropositivity of Leptospira serogroup Icterohaemorraghiae and serogroup Cynopteri in an urban high transmission setting for leptospirosis in Brazil. We performed a cross-sectional serological study in five informal urban communities in the city of Salvador, Brazil. During the years 2018, 2020 2021, we recruited 2.808 residents and collected blood samples for serological analysis using microagglutination assays. We used a fixed-effect multinomial logistic regression model to identify risk factors associated with seropositivity for each serogroup. Seropositivity to Cynopteri increased with each year of age (OR 1.03; 95% CI 1.01-1.06) and was higher in those living in houses with unplastered walls (exposed brick) (OR 1.68; 95% CI 1.09-2.59) and where cats were present near the household (OR 2.00; 95% CI 1.03-3.88). Seropositivity to Icterohaemorrhagiae also increased with each year of age (OR 1.02; 95% CI 1.01-1.03) and was higher in males (OR 1.51; 95% CI 1.09-2.10), in those with work-related exposures (OR 1.71; 95% CI 1.10-2.66) or who had contact with sewage (OR 1.42; 95% CI 1.00-2.03). Spatial analysis showed differences in distribution of seropositivity to serogroups Icterohaemorrhagiae and Cynopteri within the five districts where study communities were situated. Our data suggest distinct epidemiological patterns associated with the Icterohaemorrhagiae and Cynopteri serogroups in the urban environment at high risk for leptospirosis and with differences in spatial niches. We emphasize the need for studies that accurately identify the different pathogenic serogroups that circulate and infect residents of low-income areas.

Leptospirosis, a neglected zoonotic disease, is caused by pathogenic spirochetes belonging to the genus Leptospira and has one of the highest morbidity and mortality rates worldwide. Vaccination stands out as one of the most effective preventive measures for susceptible populations. Within the outer membrane of Leptospira spp., we find the LIC12287, LIC11711, and LIC13259 lipoproteins. These are of interest due to their surface location and potential immunogenicity. Thorough examination revealed the conservation of these proteins among pathogenic Leptospira spp.; we mapped the distribution of T- and B-cell epitopes along their sequences and assessed the 3D structures of each protein. This information aided in selecting immunodominant regions for the development of a chimeric protein. Through gene synthesis, we successfully constructed a chimeric protein, which was subsequently expressed, purified, and characterized. Hamsters were immunized with the chimeric lipoprotein, formulated with adjuvants aluminum hydroxide, EMULSIGEN®-D, Sigma Adjuvant System®, and Montanide™ ISA206VG. Another group was vaccinated with an inactivated Escherichia coli bacterin expressing the chimeric protein. Following vaccination, hamsters were challenged with a virulent L. interrogans strain. Our evaluation of the humoral immune response revealed the production of IgG antibodies, detectable 28 days after the second dose, in contrast to pre-immune samples and control groups. This demonstrates the potential of the chimeric protein to elicit a robust humoral immune response; however, no protection against challenge was achieved. While this study provides valuable insights into the subject, further research is warranted to identify protective antigens that could be utilized in the development of a leptospirosis vaccine. Key points • Several T- and B-cell epitopes were identified in all the three proteins. • Four different adjuvants were used in vaccine formulations. • Immunization stimulated significant levels of IgG2/3 in vaccinated animals.

Previous studies demonstrated that Leptospira biflexa , a saprophytic species, triggers innate immune responses in the host during early infection. This raised the question of whether these responses could suppress a subsequent challenge with pathogenic Leptospira . We inoculated C3H/HeJ mice with a single or a double dose of L. biflexa before challenge with a pathogenic serovar, Leptospira interrogans serovar Copenhageni FioCruz (LIC). Pre-challenge exposure to L. biflexa did not prevent LIC dissemination and colonization of the kidney. However, it rescued weight loss and mouse survival thereby mitigating disease severity. Unexpectedly, there was correlation between rescue of overall health (weight gain, higher survival, lower kidney fibrosis marker ColA1) and higher shedding of LIC in urine. This stood in contrast to the L. biflexa unexposed LIC challenged control. Immune responses were dominated by increased frequency of effector T helper (CD4+) cells in spleen, as well as significant increases in serologic IgG2a. Our findings suggest that exposure to live saprophytic Leptospira primes the host to develop Th1 biased immune responses that prevent severe disease induced by a subsequent challenge with a pathogenic species. Thus, mice exposed to live saprophytic Leptospira before facing a pathogenic serovar may withstand infection with far better outcomes. Furthermore, a status of homeostasis may have been reached after kidney colonization that helps LIC complete its enzootic cycle.

Serology (the detection of antibodies formed by the host against an infecting pathogen) is frequently used to assess current infections and past exposure to specific pathogens. However, the presence of cross-reactivity among host antibodies in serological data makes it challenging to interpret the patterns and draw reliable conclusions about the infecting pathogen or strain. In our study, we use microscopic agglutination test (MAT) serological data from three host species [California sea lion (Zalophus californianus), island fox (Urocyon littoralis), and island spotted skunk (Spilogale gracilis)] with confirmed infections to assess differences in cross-reactivity by host species and diagnostic laboratory. All host species are known to be infected with the same serovar of Leptospira interrogans. We find that absolute and relative antibody titer magnitudes vary systematically across host species and diagnostic laboratories. Despite being infected by the same Leptospira serovar, three host species exhibit different cross-reactivity profiles to a 5-serovar diagnostic panel. We also observe that the cross-reactive antibody titer against a non-infecting serovar can remain detectable after the antibody titer against the infecting serovar declines below detectable levels. Cross-reactivity in serological data makes interpretation difficult and can lead to common pitfalls. Our results show that the highest antibody titer is not a reliable indicator of infecting serovar and highlight an intriguing role of host species in shaping reactivity patterns. On the other side, seronegativity against a given serovar does not rule out that serovar as the cause of infection. We show that titer magnitudes can be influenced by both host species and diagnostic laboratory, indicating that efforts to interpret absolute titers (e.g., as indicators of recent infection) must be calibrated to the system under study. Thus, we implore scientists and health officials using serological data for surveillance to interpret the data with caution.

Pathogenic spp. are the causative agents of leptospirosis, a significant zoonotic disease that has emerged as a crucial public health concern. This study aims to evaluate the interactions of two proteins, LIC_10499 and LIC_12339, with host components as well as with endothelial and epithelial cells. The coding sequences (CDSs) for LIC_10499 and LIC_12339 were cloned, expressed in , and successfully purified from inclusion bodies. Both recombinant proteins demonstrated interactions with fibronectin, fibrinogen, and plasminogen (PLG). Notably, these proteins were capable of sequestering PLG from normal human serum (NHS). In the presence of an activator, the bound PLG is converted to plasmin (PLA), a broad-spectrum protease involved in pathogen invasion and immune evasion. Additionally, LIC_10499 and LIC_12339 were found to bind to complement system regulators, including factor H and C4b-binding protein, as well as to components C7, C8, and C9. We observed that the formation of C9 complexes was inhibited in the presence of recombinant proteins, and a higher survival rate of was noted when the proteins were incubated with NHS. The protein rLIC_10499 was able to bind to both monolayer and suspension cells of HMEC, Ea.hy926, and HEK293T, whereas rLIC_12339 only bound to HEK293T suspension cells. A significant production of IFN-γ was detected after 24 h when HEK293T epithelial cells were incubated with rLIC_10499, while a modest production of IL-6 and IL-8 was observed. No cytokine production occurred when HEK293T cells were stimulated with rLIC_12339. Collectively, these findings suggest that these proteins play a role in leptospiral immune evasion and have the potential to induce an inflammatory response in host cell monolayers.

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 (β-glucan) could be a potent and valuable immunostimulant for improving immune responses and controlling diseases. In this study, we investigated the preventable role of β-glucan against Leptospira infection in hamsters. First, β-glucan was administered 24 h prior to, during and after infection. The results showed that β-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 β-glucan significantly enhanced the expression of Toll-like receptor (TLR) 2, interleukin (IL)-1β and iNOS at 2 dpi (days post infection) and reduced the increase of TLR2, IL-1β and iNOS induced by Leptospira at 5 dpi. Furthermore, to induce memory immunity, β-glucan was administered 5 days prior to infection. β-Glucan also significantly increased the survival rates and ameliorated pathological damage to organs. Moreover, we demonstrated that β-glucan-trained macrophages exhibited elevated expression of proinflammatory cytokines (IL-1β and IL-6) in vitro, indicating that β-glucan induces an enhanced inflammatory response against Leptospira infection. These results indicate that administration of β-glucan and other immunostimulants could be potential valuable options for the control of Leptospira infection.

Key Points Leptospira spp. belong to the bacterial phylum Spirochaetes, which contains evolutionary and structurally unique bacteria. The genus Leptospira is composed of saprophytic and pathogenic bacterial species that are fastidious and slow growing. Leptospirosis is an emerging zoonotic disease that has a worldwide distribution, with more than a million cases reported annually. Compared with other bacterial species, the genetic data for leptospires and the determination of the molecular basis of their pathogenesis are in their infancy. Pathogenic leptospires are the bacterial agents of leptospirosis, which is an emerging zoonotic disease that affects both animals and humans worldwide. In this Review, the recent advances in our understanding of the epidemiology, taxonomy, genomics and the molecular basis of virulence in leptospires, and of how these properties contribute to the pathogenesis of leptospirosis, are discussed. Pathogenic leptospires are the bacterial agents of leptospirosis, which is an emerging zoonotic disease that affects animals and humans worldwide. The success of leptospires as pathogens is explained by their spiral shape and endoflagellar motility (which enable these spirochetes to rapidly cross connective tissues and barriers), as well as by their ability to escape or hijack the host immune system. However, the basic biology and virulence factors of leptospires remain poorly characterized. In this Review, we discuss the recent advances in our understanding of the epidemiology, taxonomy, genomics and the molecular basis of virulence in leptospires, and how these properties contribute to the mechanism of pathogenesis of leptospirosis.

Leptospirosis is a widespread systemic zoonosis, considered as reemerging in certain developing countries. Although the cross agglutinin absorption test is still considered the standard method for Leptospira identification, it presents several disadvantages. The aim of this study was to characterize Leptospira spp. isolated from various hosts by genotyping and broth microdilution susceptibility testing in an attempt to differentiate Leptospira species, serogroups and serovars. Forty-seven isolates were studied. They were previously serotyped, and species confirmation was performed by 16S rRNA sequencing. Single-enzyme amplified fragment length polymorphism (SE-AFLP) and pulsed-field gel electrophoresis (PFGE) analysis enabled the distinction of L. interrogans from L. santarosai, L. meyeri and L. borgpetersenii in two main clusters. Among L. interrogans, it was possible to differentiate into two new clusters the serogroup Icterohaemorrhagiae from the serogroups Canicola and Pomona. L. santarosai isolates presented higher genetic variation than the other species in both techniques. Interestingly, the minimum inhibitory concentration (MIC) cluster analysis also provided Leptospira serogroup differentiation. Further studies are necessary regarding serovar Bananal isolates, as they presented the highest MIC values for most of the antimicrobials tested. All studied techniques successfully distinguished Leptospira species and serogroups. Despite being library-dependent methods, these approaches are less labor intensive and more economically viable, particularly SE-AFLP, and can be implemented in most reference laboratories worldwide to enable faster Leptospira typing.

Key Points Leptospira spp. belong to the bacterial phylum Spirochaetes, an evolutionarily and structurally unique group of bacteria. The genus Leptospira is composed of saprophytic and pathogenic species that are all fastidious and slow-growing bacteria. Leptospirosis is a zoonotic disease with a worldwide distribution. More than half a million cases are reported annually. Acute disease and chronic colonization can be reproduced in experimental animal models. Determination and comparison of the genome sequences of saprophytic and pathogenic strains could shed light on the virulence determinants involved in disease. Compared to other bacterial species, work to gather genetic data for leptospires and to elucidate the molecular basis of the pathogenesis of these organisms is in its infancy. Further development of genetic tools should allow a better understanding of the virulence and survival mechanisms that are used by these bacteria to ensure their persistence in different ecological niches. Leptospirosis is a neglected disease that has emerged as a widespread problem in impoverished populations in developing countries and tropical regions. Here, Picardeau and colleagues discuss the progress that has been made in our understanding of the pathogenesis of leptospirosis as a result of the recent availability of complete genome sequences for Leptospira spp. and the development of genetic tools for the analysis of this zoonotic species. Leptospirosis is a zoonotic disease that has emerged as an important cause of morbidity and mortality among impoverished populations. One hundred years after the discovery of the causative spirochaetal agent, little is understood about Leptospira spp. pathogenesis, which in turn has hampered the development of new intervention strategies to address this neglected disease. However, the recent availability of complete genome sequences for Leptospira spp. and the discovery of genetic tools for their transformation have led to important insights into the biology of these pathogens and their pathogenesis. We discuss the life cycle of the bacterium, the recent advances in our understanding and the implications for the future prevention of leptospirosis.