20 research outputs found
Reverse Vaccinology: An Approach for Identifying Leptospiral Vaccine Candidates
Leptospirosis is a major public health problem with an incidence of over one million human cases each year. It is a globally distributed, zoonotic disease and is associated with significant economic losses in farm animals. Leptospirosis is caused by pathogenic Leptospira spp. that can infect a wide range of domestic and wild animals. Given the inability to control the cycle of transmission among animals and humans, there is an urgent demand for a new vaccine. Inactivated whole-cell vaccines (bacterins) are routinely used in livestock and domestic animals, however, protection is serovar-restricted and short-term only. To overcome these limitations, efforts have focused on the development of recombinant vaccines, with partial success. Reverse vaccinology (RV) has been successfully applied to many infectious diseases. A growing number of leptospiral genome sequences are now available in public databases, providing an opportunity to search for prospective vaccine antigens using RV. Several promising leptospiral antigens were identified using this approach, although only a few have been characterized and evaluated in animal models. In this review, we summarize the use of RV for leptospirosis and discuss the need for potential improvements for the successful development of a new vaccine towards reducing the burden of human and animal leptospirosis
Conservation of <i>L. interrogans</i> sv. Copenhageni Fiocruz L1-130 differentially-expressed genes among virulent and saprophytic <i>Leptospira</i> spp. Protein sequence similarities were determined using GLSEARCH (v. 34.05).
<p>Genomes used for analysis: <i>L. interrogans</i> sv. Lai strain 56601, <i>L. borgpetersenii</i> sv. Hardjo strain L550, <i>L. santarosai</i> sv. Shermani strain LT821; <i>L. licerasiae</i> sv. Varillal strain VAR010; and <i>L. biflexa</i> sv. Patoc strain Patoc1 Ames, respectively. The color coding used in the heat map is as follows: blue, 95–100% identity; green, 90–94% identity; orange, 85–89%; and yellow, 80–84%.</p
Functional categories of genes differentially-expressed by <i>L. interrogans</i> sv Copenhageni strain Fiocruz L1-130 within DMCs.
<p>Functional categories are based on those of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004004#ppat.1004004-Nascimento1" target="_blank">[11]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004004#ppat.1004004-Nascimento2" target="_blank">[12]</a> and the <i>Leptospira interrogans</i> sv. Copenhageni Genome Project database (<a href="http://aeg.lbi.ic.unicamp.br/world/lic/" target="_blank">http://aeg.lbi.ic.unicamp.br/world/lic/</a>). The number of upregulated (Ups) and downregulated (Down) genes within each category are indicated in red and blue, respectively.</p
Candidate small non-coding RNAs identified by RNA-Seq.
1<p>Predicted sRNAs are annotated according to the genome of <i>L. interrogans</i> sv. Copenhageni (LIC) chromosome number followed by non-coding RNA designation as included in Supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004004#ppat.1004004.s006" target="_blank">Table S2</a>.</p>2<p>Homology to known sRNA families is indicated as is the E-value when transcripts were searched against the Rfam database.</p>3<p>Expression was validated by reverse-transcriptase PCR in <i>L. interrogans</i> sv. Copenhageni strain RJ16441.</p
<i>L. interrogans</i> sv. Copenhageni genes upregulated in DMCs compared to <i>in vitro</i>.
1<p>Gene designations and protein product descriptions are based on those of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004004#ppat.1004004-Nascimento1" target="_blank">[11]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004004#ppat.1004004-Nascimento2" target="_blank">[12]</a> and the <i>L. interrogans</i> sv. Copenhageni Genome Project database (<a href="http://aeg.lbi.ic.unicamp.br/world/lic/" target="_blank">http://aeg.lbi.ic.unicamp.br/world/lic/</a>), except where indicated.</p>2<p>nnotation based on Setubal <i>et al.</i><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004004#ppat.1004004-Setubal1" target="_blank">[47]</a>.</p>3<p>Revised annotation based on bioinformatics.</p
Mapping of RNA-Seq reads.
<p>Percentage of uniquely mapping reads from each biological replicate of leptospires cultivated in DMCs or under standard <i>in vitro</i> growth conditions (30°C in EMJH).</p
Leptospiral genes differentially-expressed within DMCs compared to <i>in vitro</i>.
1<p>Percentage of genes based on the total number of genes in upregulated or downregulated category.</p>2<p>Percentage of genes based on the total number of differentially-expressed genes.</p>3<p>Hypothetical proteins and uncharacterized lipoproteins.</p
Validation of comparative RNA-Seq analysis.
<p>(A) qRT-pCR analysis of representative genes identified by RNA-Seq. Values represent the average transcript copy numbers for each gene normalized per <i>lipL32</i> transcript. Bars indicate the standard error of the mean (SEM). <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004004#s2" target="_blank">Results</a> presented are mean values from at least 3 biologically-independent samples of leptospires for each growth condition. The fold-regulation for each gene determined by RNA-Seq is indicated in parentheses. The fold-regulation between <i>in vitro</i>- (IV) and DMC-cultivated leptospires determined by qRT-PCR are indicated. P values were calculated using an unpaired <i>t</i>-test. (B) Correlation coefficient (R<sup>2</sup>) between RNA-Seq and qRT-PCR data.</p
Virulent leptospires become mammalian host-adapted during growth within dialysis membrane chambers.
<p>Representative whole cell lysates of leptospires cultivated to late-logarithmic phase in EMJH medium at 30°C <i>in vitro</i> (IV) and within dialysis membrane chambers (DMC) implanted into the peritoneal cavities of female Sprague-Dawley rats. (A) Lysates were loaded according to the numbers of leptospires (5×10<sup>6</sup> per lane) or total protein (5 µg per lane) and stained with SYPRO Ruby gel stain. Arrows and asterisks are used to highlight examples of polypeptides whose expression appears to be increased or decreased, respectively, within DMCs compared to <i>in vitro</i>. Molecular mass markers are indicated on the left. (B) Immunoblot analyses using rabbit polyclonal antisera directed against Sph2 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004004#ppat.1004004-Matsunaga2" target="_blank">[34]</a>, LipL32 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004004#ppat.1004004-Haake1" target="_blank">[38]</a> and LipL41 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004004#ppat.1004004-Shang1" target="_blank">[39]</a>. An arrow is used to indicate a band of the predicted molecular mass for SphH, a second, closely-related sphingomyelinase in <i>L. interrogans</i> recognized by antiserum directed against Sph2 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004004#ppat.1004004-Matsunaga2" target="_blank">[34]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004004#ppat.1004004-Carvalho1" target="_blank">[37]</a>.</p
Summary of RNA-Seq mapping data.
1<p>Total number and percentage (in parenthesis) of reads that mapped to the reference genome with 100% accuracy.</p>2<p>Total number and percentage (in parenthesis) of reads that mapped to a single location within the reference genome <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004004#ppat.1004004-Nascimento1" target="_blank">[11]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004004#ppat.1004004-Nascimento2" target="_blank">[12]</a>.</p>3<p>Based on the total of uniquely mapped reads for the corresponding sample.</p