High-throughput Parallel Sequencing to Measure Fitness of <em>Leptospira interrogans</em> Transposon Insertion Mutants During Golden Syrian Hamster Infection

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness ...

Veterans Affairs Greater Los Angeles Healthcare System

David Geffen School of Medicine at University of California Los Angeles

Pathogenic members of the genus Leptospira are the causative agents of leptospirosis, a neglected disease of public and veterinary health concern. Leptospirosis is a systemic disease that in its severest forms leads to renal insufficiency, hepatic dysfunction, and pulmonary failure. Many strains of Leptospira produce hemolytic and sphingomyelinase activities, and a number of candidate leptospiral hemolysins have been identified based on sequence similarity to well-characterized bacterial hemolysins. Five of the putative hemolysins are sphingomyelinase paralogs. Although recombinant forms of the sphingomyelinase Sph2 and other hemolysins lyse erythrocytes, none have been demonstrated to contribute to the hemolytic activity secreted by leptospiral cells. In this study, we examined the regulation of sph2 and its relationship to hemolytic and sphingomyelinase activities produced by several L. interrogans strains cultivated under the osmotic conditions found in the mammalian host. The sph2 gene was poorly expressed when the Fiocruz L1-130 (serovar Copenhageni), 56601 (sv. Lai), and L495 (sv. Manilae) strains were cultivated in the standard culture medium EMJH. Raising EMJH osmolarity to physiological levels with sodium chloride enhanced Sph2 production in all three strains. In addition, the Pomona subtype kennewicki strain LC82-25 produced substantially greater amounts of Sph2 during standard EMJH growth than the other strains, and sph2 expression increased further by addition of salt. When 10% rat serum was present in EMJH along with the sodium chloride supplement, Sph2 production increased further in all strains. Osmotic regulation and differences in basal Sph2 production in the Manilae L495 and Pomona strains correlated with the levels of secreted hemolysin and sphingomyelinase activities. Finally, a transposon insertion in sph2 dramatically reduced hemolytic and sphingomyelinase activities during incubation of L. interrogans at physiologic osmolarity. Complementation of the mutation with the sph2 gene partially restored production of hemolytic and sphingomyelinase activities. These results indicate that the sph2 gene product contributes to the hemolytic and sphingomyelinase activities secreted by L. interrogans and most likely dominates those functions under the culture condition tested.

Role of sph2 Gene Regulation in Hemolytic and Sphingomyelinase Activities Produced by Leptospira interrogans.

Pathogenic species of Leptospira are the causative agents of leptospirosis, a zoonotic disease that causes mortality and morbidity worldwide. The understanding of the virulence mechanisms of Leptospira spp is still at an early stage due to the limited number of genetic tools available for this microorganism. The development of random transposon mutagenesis in pathogenic strains a decade ago has contributed to the identification of several virulence factors. In this study, we used the transposon sequencing (Tn-Seq) technique, which combines transposon mutagenesis with massive parallel sequencing, to study the in vivo fitness of a pool of Leptospira interrogans mutants. We infected hamsters with a pool of 42 mutants (input pool), which included control mutants with insertions in four genes previously analyzed by virulence testing (loa22, ligB, flaA1, and lic20111) and 23 mutants with disrupted signal transduction genes. We quantified the mutants in different tissues (blood, kidney and liver) at 4 days post-challenge by high-throughput sequencing and compared the frequencies of mutants recovered from tissues to their frequencies in the input pool. Control mutants that were less fit in the Tn-Seq experiment were attenuated for virulence when tested separately in the hamster model of lethal leptospirosis. Control mutants with unaltered fitness were as virulent as the wild-type strain. We identified two mutants with the transposon inserted in the same putative adenylate/guanylate cyclase gene (lic12327) that had reduced in vivo fitness in blood, kidney and liver. Both lic12327 mutants were attenuated for virulence when tested individually in hamsters. Growth of the control mutants and lic12327 mutants in culture medium were similar to that of the wild-type strain. These results demonstrate the feasibility of screening large pools of L. interrogans transposon mutants for those with altered fitness, and potentially attenuated virulence, by transposon sequencing.

High-Throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants during Acute Infection.

Leptospirosis is the most widespread zoonosis and is considered a major public health problem worldwide. Currently, there is no widely available vaccine against leptospirosis for use in humans. A purified, recombinant subunit vaccine that includes the last six immunoglobulin-like (Ig-like) domains of the leptospiral protein LigA (LigA7'-13) protects against lethal infection but not renal colonization after challenge by Leptospira interrogans. In this study, we examined whether the addition of the first seven Ig-like domains of LigB (LigB0-7) to LigA7'-13, can enhance immune protection and confer sterilizing immunity in the Golden Syrian hamster model of acute leptospirosis. Hamsters were subcutaneously immunized with soluble, recombinant LigA7'-13, LigB0-7, or a combination of LigA7'-13 and LigB0-7 in Freund's adjuvant. Immunization with Lig proteins generated a strong humoral immune response with high titers of IgG that recognized homologous protein, and cross-reacted with the heterologous protein as assessed by ELISA. LigA7'-13 alone, or in combination with LigB0-7, protected all hamsters from intraperitoneal challenge with a lethal dose of L. interrogans serovar Copenhageni strain Fiocruz L1-130. However, bacteria were recovered from the kidneys of all animals. Of eight animals immunized with LigB0-7, only three survived Leptospira challenge, one of which lacked renal colonization and had antibodies to native LigB by immunoblot. In addition, sera from two of the three LigB0-7 immunized survivors cross-reacted with LigA11-13, a region of LigA that is sufficient for protection. In summary, we confirmed that LigA7'-13 protects hamsters from death but not infection, and immunization with LigB0-7, either alone or in combination with LigA7'-13, did not confer sterilizing immunity.

Kristel Lourdault

Veterans Affairs Greater Los Angeles Healthcare System

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection