A Three-Dimensional Lung Cell Model to Leptospira Virulence Investigations.

Leptospirosis is a worldwide zoonosis and a serious public health threat in tropical and subtropical areas. The etiologic agents of leptospirosis are pathogenic spirochetes from the genus Leptospira. In severe cases, patients develop a pulmonary hemorrhage that is associated with high fatality rates. Several animal models were established for leptospirosis studies, such as rodents, dogs, and monkeys. Although useful to study the relationship among Leptospira and its hosts, the animal models still exhibit economic and ethical limitation reasons and do not fully represent the human infection. As an attempt to bridge the gap between animal studies and clinical information from patients, we established a three-dimensional (3-D) human lung cell culture for Leptospira infection. We show that Leptospira is able to efficiently infect the cell lung spheroids and also to infiltrate in deeper areas of the cell aggregates. The ability to infect the 3-D lung cell aggregates was time-dependent. The 3-D spheroids infection occurred up to 120 h in studies with two serovars, Canicola and Copenhageni. We standardized the number of bacteria in the initial inoculum for infection of the spheroids and we also propose two alternative culture media conditions. This new approach was validated by assessing the expression of three genes of Leptospira related to virulence and motility. The transcripts of these genes increased in both culture conditions, however, in higher rates and earlier times in the 3-D culture. We also assessed the production of chemokines by the 3-D spheroids before and after Leptospira infection, confirming induction of two of them, mainly in the 3-D spheroids. Chemokine CCL2 was expressed only in the 3-D cell culture. Increasing of this chemokine was observed previously in infected animal models. This new approach provides an opportunity to study the interaction of Leptospira with the human lung epithelium in vitro.

Phagocytosis of Leptospira by leukocytes from mice with different susceptibility to leptospirosis and possible role of chemokines.

BACKGROUND: Leptospirosis is a widespread zoonosis caused by pathogenic prokaryotic microbes of the genus Leptospira. Although there are several reports in the literature, host-pathogen interaction is still poorly understood. The role of chemokine expression is important on the chemotaxis, activation and regulation of immune cells. Recent studies have shown that their expression profiles play an important role on the severity of leptospirosis outcome. We evaluated the phagocytosis of Leptospira by spleens cells from C3H/HeJ, C3H/HePas and BALB/c mouse strains, respectively susceptible, intermediate and resistant to leptospirosis, and by RAW 264.7 macrophages. Besides, we evaluated the effects of CCL2 treatment on the phagocytosis. The cells were incubated with or without CCL2 chemokine, and infected with virulent L. interrogans sv Copenhageni. Cells and culture supernatants were collected for subsequent analysis. RESULTS: The number of leptospires was higher in BALB/c cells, CCL2 pre-treated or only infected groups, when compared to C3H/HeJ and C3H/HePas cells. Indeed, CCL2 activation did not interfere in the phagocytosis of Leptospira. Expression of chemokines CXCL5 and CCL8 levels were significantly inhibited in infected BALB/c cells when compared to the non-infected control. CONCLUSIONS: Higher ability to phagocytosis and early modulation of some chemokines correlated with the resistance to leptospirosis disease. Exposure to CCL2 did not interfere on phagocytosis of Leptospira in our experimental conditions, but acted in the modulation of chemokines expression during Leptospira infection.

Genomic survey and expression analysis of DNA repair genes in the genus Leptospira.

Leptospirosis is an emerging zoonosis with important economic and public health consequences and is caused by pathogenic leptospires. The genus Leptospira belongs to the order Spirochaetales and comprises saprophytic (L. biflexa), pathogenic (L. interrogans) and host-dependent (L. borgpetersenii) members. Here, we present an in silico search for DNA repair pathways in Leptospira spp. The relevance of such DNA repair pathways was assessed through the identification of mRNA levels of some genes during infection in animal model and after exposition to spleen cells. The search was performed by comparison of available Leptospira spp. genomes in public databases with known DNA repair-related genes. Leptospires exhibit some distinct and unexpected characteristics, for instance the existence of a redundant mechanism for repairing a chemically diverse spectrum of alkylated nucleobases, a new mutS-like gene and a new shorter version of uvrD. Leptospira spp. shares some characteristics from Gram-positive, as the presence of PcrA, two RecQ paralogs and two SSB proteins; the latter is considered a feature shared by naturally competent bacteria. We did not find a significant reduction in the number of DNA repair-related genes in both pathogenic and host-dependent species. Pathogenic leptospires were enriched for genes dedicated to base excision repair and non-homologous end joining. Their evolutionary history reveals a remarkable importance of lateral gene transfer events for the evolution of the genus. Up-regulation of specific DNA repair genes, including components of SOS regulon, during infection in animal model validates the critical role of DNA repair mechanisms for the complex interplay between host/pathogen.

Leptospira interrogans serovar copenhageni harbors two lexA genes involved in SOS response.

Bacteria activate a regulatory network in response to the challenges imposed by DNA damage to genetic material, known as the SOS response. This system is regulated by the RecA recombinase and by the transcriptional repressor lexA. Leptospira interrogans is a pathogen capable of surviving in the environment for weeks, being exposed to a great variety of stress agents and yet retaining its ability to infect the host. This study aims to investigate the behavior of L. interrogans serovar Copenhageni after the stress induced by DNA damage. We show that L. interrogans serovar Copenhageni genome contains two genes encoding putative LexA proteins (lexA1 and lexA2) one of them being potentially acquired by lateral gene transfer. Both genes are induced after DNA damage, but the steady state levels of both LexA proteins drop, probably due to auto-proteolytic activity triggered in this condition. In addition, seven other genes were up-regulated following UV-C irradiation, recA, recN, dinP, and four genes encoding hypothetical proteins. This set of genes is potentially regulated by LexA1, as it showed binding to their promoter regions. All these regions contain degenerated sequences in relation to the previously described SOS box, TTTGN 5CAAA. On the other hand, LexA2 was able to bind to the palindrome TTGTAN10TACAA, found in its own promoter region, but not in the others. Therefore, the L. interrogans serovar Copenhageni SOS regulon may be even more complex, as a result of LexA1 and LexA2 binding to divergent motifs. New possibilities for DNA damage response in Leptospira are expected, with potential influence in other biological responses such as virulence.

Resistance to ultraviolet-induced apoptosis in DNA repair deficient growth arrested human fibroblasts is not related to recovery from RNA transcription blockage.

The impact of ultraviolet (UV-C) photoproducts on apoptosis induction was investigated in growth arrested (confluent) and proliferating human primary fibroblasts. Confluent fibroblasts were more resistant to UV-C-induced apoptosis than proliferating cells, and this was observed for normal human cells and for cells from patients with Cockayne and trichothiodystrophy syndromes, deficient in transcription coupled repair. This resistance was sustained for at least seven days and was not due to DNA repair efficiency, as the removal of CPDs in the genome was similar under both growth conditions. There was no correlation between reduced apoptosis and RNA synthesis recovery. Following UV-C treatment, proliferating and confluent fibroblasts showed a similar level of RNA synthesis inhibition and recovery from transcription blockage. These results support the hypothesis that the decrease of DNA replication, in growth arrested cells, protects cell from UV-C-induced apoptosis, even in the presence of DNA lesions.