Pairs of mutants with transposon insertions in the same gene.

<p>(A) Locations of transposon insertion sites. In the pool of mutants used to challenge the animals, we included three pairs of mutants that have insertion sites at different locations in the same gene. The location of both transposon insertions is designated by black arrows in each of the following genes: <i>lic12324</i> (insertion sites: 2806250 and 2806718 in the Fiocruz L1-130 genome), <i>lic12327</i> (insertion sites 2810027 and 2810271) and <i>lic12627</i> (insertion sites: 3177595 and 3178505). (B-G) Fitness of each pair of mutants. The output/input ratios for each of the eight animals were determined for each mutant in blood, kidney and liver. Each ratio is represented by a black diamonds and the median of these ratios by a red line. The dotted line represents fitness of 1.0, which means neutral fitness. The median was compared to 1.0 using the Wilcoxon rank test. The number of asterisks indicates the significance level: * P < 0.05. No statistically significant differences were observed between the pair of <i>lic12327</i> mutants or between the <i>lic12324</i> mutants for any tissue. The comparison of the ratios of these two <i>lic12627</i> mutants confirmed statistically significant differences in kidney (P = 0.0379) and liver (P = 0.0011).</p

Motility assay of <i>L</i>. <i>interrogans</i> control mutants.

<p>0.5% agar EMJH plates were spotted with the wild type strain and four different cultures of (A) the <i>flaA1</i> mutant or (C) the <i>lic20111</i> mutant. (B-D) Growth diameter of each strain was measured after two weeks incubation at 30°C. Three independent experiments were performed. A representative plate is shown from one of them.</p

High-Throughput Parallel Sequencing to Measure Fitness of <i>Leptospira interrogans</i> Transposon Insertion Mutants during Acute Infection

<div><p>Pathogenic species of <i>Leptospira</i> are the causative agents of leptospirosis, a zoonotic disease that causes mortality and morbidity worldwide. The understanding of the virulence mechanisms of <i>Leptospira spp</i> 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 <i>in vivo</i> fitness of a pool of <i>Leptospira interrogans</i> 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 (<i>loa22</i>, <i>ligB</i>, <i>flaA1</i>, and <i>lic20111</i>) 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 (<i>lic12327</i>) that had reduced <i>in vivo</i> fitness in blood, kidney and liver. Both <i>lic12327</i> mutants were attenuated for virulence when tested individually in hamsters. Growth of the control mutants and <i>lic12327</i> mutants in culture medium were similar to that of the wild-type strain. These results demonstrate the feasibility of screening large pools of <i>L</i>. <i>interrogans</i> transposon mutants for those with altered fitness, and potentially attenuated virulence, by transposon sequencing.</p></div

Transposon mutants used in this study.

<p>Transposon mutants used in this study.</p

Relationship between the number of mapped reads and the bacterial load.

<p>Correlation between the log of the total number of mapped reads and the log of the number of bacteria (A) in blood, (B) kidney and (C) liver. In all tissues, there is a significant correlation between the number of reads and the number of bacteria, r² = 0.7963 (P = 0.0029), r² = 0.7818 (P = 0.0036) and r² = 0.9068 (P = 0.0003) in blood, kidney and liver, respectively.</p

Fitness of 42 <i>L</i>. <i>interrogans</i> mutants during acute infection of hamsters.

<p>The 42 transposon mutants were grown independently, quantified, diluted at the concentration of 10⁶/mL and pooled together (input pool). Eight hamsters were challenged intraperitoneally with 1 mL of the pool of mutants. Four days post-challenge, animals were sacrificed, and blood, kidney and liver were collected (output pools). The frequency of each of the 42 mutants in the input pool and output pools was quantified using the Illumina HiSeq 2500. The output/input ratio was determined for each mutant in (A) blood, (B) kidney and (C) liver in each animal. Each ratio is represented by a white circle. For each mutant and each tissue, the median of ratios (red line) was determined and compared to 1.0 using the Wilcoxon rank test. The dotted line represents fitness of 1.0, which means no change in fitness. Mutants whose fitness is significantly affected are marked by asterisks: * P < 0.05; ** P < 0.01.</p

Growth curves of selected <i>L</i>. <i>interrogans</i> mutants.

<p>The <i>loa22</i>::Tn (light blue), <i>ligB</i>::Tn (pink), <i>flaA1</i>::Tn (green), <i>lic20111</i>::Tn (orange), <i>lic12327a</i>::Tn (red), <i>lic12327b</i>::Tn (purple) and the WT (dark blue) strain were cultured at 30°C in EMJH, supplemented with Km as appropriate. Growth was monitored by measuring the OD_420nm. No defect in growth was observed for any of the strains. Data are representative of those from three independent experiments.</p

Fitness of <i>L</i>. <i>interrogans</i> control mutants during acute infection of hamsters.

<p>In the pool of mutants used to challenge the animals, we included mutants whose virulence is known to be affected: (A) <i>loa22</i>::Tn and (D) <i>lic20111</i>::Tn or unaffected: (B) <i>ligB</i>::Tn and (C) <i>flaA1</i>::Tn. The output/input ratio of each mutant was determined for each animal in blood, kidney and liver. Each ratio is represented by a black diamond and the median of these ratios by a red line. The dotted line represents fitness of 1.0, which means neutral fitness. The median was compared to 1 using the Wilcoxon rank test. The number of asterisks indicates the significance level: * P < 0.05.</p

Immunoprotective properties of recombinant LigA and LigB in a hamster model of acute leptospirosis

<div><p>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 <i>Leptospira interrogans</i>. 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 <i>L</i>. <i>interrogans</i> 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 <i>Leptospira</i> 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.</p></div

IgG response to immunization with purified recombinant Lig proteins.

<p>Serum samples were collected from hamsters weekly during the immunization protocol. Anti-LigA7’-13 (A) or anti-LigB0-7 (B) antibody levels were measured in triplicate by ELISA. Each data line represents the IgG response of an individual animal over time; in <i>black</i> are animals that survived the challenge while in <i>red</i> are animals that met the endpoint criteria. Vertical dotted lines indicate immunization days (blue) or challenge with <i>L</i>. <i>interrogans</i> (red). Each data point represents the mean IgG level read at OD₆₅₅ minus pre-bleed read. Error bars indicate standard deviation. There was no significant difference in the IgG response among hamsters that received the same vaccine treatment at all time points (One-way ANOVA, <i>P</i> = 0.9).</p