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A Three-Dimensional Lung Cell Model to Leptospira Virulence Investigations

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Abstract

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.

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Acknowledgements

I would like to thank to Dr Carlos Jared to provide the confocal microscopy equipment (Grant 175 FINEP-IBUINFRA 01.12.0175.00) and Alexsander S. de Souza for the confocal images’ technical services. We thank Dr. Marcelo L. Santoro from Instituto Butantan to provide the quantitative PCR equipment and expertise.

Funding

This work received grants from Fundação de Amparo a Pesquisa do Estado de São Paulo-FAPESP (Da Silva JB Grant 2013/16451-1 and 2017/20903-6), and Fundação Butantan (Brazil). Campos CL received scholarship from Instituto Butantan-Secretaria da Saúde do Estado de São Paulo. Funding bodies did not have any role in study design, data collection and analysis, decision to publish, or writing the manuscript.

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Author notes

  1. Renata M. A. Da Costa

    Present address: Global Antibiotics Research and Development Partnership (GARDP), Chemin Louis-Dunant 15, 1202, Geneva, Switzerland

Authors and Affiliations

  1. Laboratório de Bacteriologia, Instituto Butantan, São Paulo, Brazil

    Camila L. Campos, Renata M. A. Da Costa & Josefa B. Da Silva

  2. Laboratório de Ciclo Celular-Center for Research on Toxins, Immune-Response and Cell Signaling (CeTICS), Instituto Butantan, São Paulo, Brazil

    Luciana R. Gomes

  3. Facultad de Ciencias de la Salud, Escuela de Tecnología Médica, Universidad San Sebastian, Concepción, Chile

    Ambart E. Covarrubias

  4. Laboratorio de Fisiopatologia, Instituto Butantan, São Paulo, Brazil

    Ellen E. Kato

  5. Laboratório de Zoonoses Bacterianas, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, Brazil

    Gisele G. Souza, Silvio A. Vasconcellos & Marcos B. Heinemann

  6. Laboratório de Biológicos Recombinantes, Instituto Butantan, São Paulo, Brazil

    Elizabeth A. L. Martins

  7. Divisão BioIndustrial, Instituto Butantan, São Paulo, Brazil

    Paulo L. Ho

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  1. Camila L. Campos
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Contributions

JBS and RMAC contributed to the study conception, formal analysis, and writing. EALM contributed to formal analysis and writing. LRG provide the initial growth of the 3D cell culture. CLC performed the experiments, data collection and first draft preparation. AEC edited the figures. All authors contributed to data analysis and wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to Josefa B. Da Silva.

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Campos, C.L., Gomes, L.R., Covarrubias, A.E. et al. A Three-Dimensional Lung Cell Model to Leptospira Virulence Investigations. Curr Microbiol 79, 57 (2022). https://doi.org/10.1007/s00284-021-02720-5

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