It has been more than 100 years since leptospirosis has been known to be caused by spirochetes of the genus Leptospira. However, since then, this upcoming emerging disease has been neglected primarily due to the lack of recognition of its genome. Only in the last 7 years has the first Leptospira genome sequence been published, allowing the understanding of its physiology, pathogenicity and genetics . In this review, the new and approaching information regarding the organisms' genomics are described, focusing on its virulence factors as well as important unanswered questions, still yet to be resolved, with respect to Leptospira pathogenesis.
Leptospirosis is renowned as the most frequent zoonosis disease globally, giving rise to a biphasic set of symptoms [2, 3, 7]. So far there have been over 200 serotypes of Leptospira portrayed which are branched into 25 different serogroups dependent on antigenic structure. Several Leptospira species e.g. Leptospira interrogans, are capable of provoking disease not only in humans but in other animal groups such as birds. Animals infected with leptospirosis act as a readily transmissible reservoir of the disease since they tend to be asymptomatic, discarding Leptospira via their urine . This disease has led to many major public outbreaks and epidemics throughout developing and tropical countries, indicating the need for prevention [2, 5, 8].
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Enclosed within the Leptospira genome are genes encoding various processes such as signal transduction and regulatory mechanisms, underlying the capacity of the organism to react to a number of different inducements. Leptospira physiology can now be rejuvenated to accomplish questions still yet to be solved as a result of the identification and sequencing of genes involved in the biosynthesis of cobalamin and lipopolysaccharide (LPS). Moreover, the organism's pathogenesis can be better explained via the use of distinguishing genes involved in surface proteins, toxins and lipoproteins, and provide new insight into vaccines . This has given scientists a new light in determining key features of the Leptospira spp., marking the establishment of a post-genomic era .
The phylum spirochetes contain firmly coiled organisms known as leptospires . Leptospires can be free-living, saprophytic organisms e.g. Leptospira biflexa, which are incapable of causing disease in animal hosts, conflicting to that of pathogenic leptospires such as Leptospira interrogans. These organisms have a helical structure, and are extremely motile aerobic obligates. They are characterised by their distinctive hook ends, similar in shape to a question mark, seen in figure 1 [5, 10].
Furthermore, leptospires are fairly long, extending from 10 to 20 μm, with a small diameter of approximately 0.15 μm, compared to erythrocytes which have diameter of 7 μm [5, 10]. The propelling movement of the bacterium is reliant on two endoflagella, also known as periplasmic flagella, one located on each end of the organism along the axis . This motility can be expressed in two different fashions: translational and non-translational. Leptospires as well as other spirochetes contain a double membrane, connecting the peptidoglycan layer and cytoplasmic membrane .
Transmission of Leptospirosis
Leptospirosis infection can be obtained via pollution through infected urine of carriers or by direct contact with the contaminated host. Pathogenic strains primarily survive in the proximal renal tubules of hosts, becoming excreted in the urine, allowing contamination of the environment; seen in figure 2. Humans are very rarely chronic hosts since they are an incidental carrier (dead-end host) whose excretion of the pathogen is insufficient for transmission of the disease [5, 6].
Genomics in the Establishment of Chromosome and Genome Features
The use of genomic sequencing in three Leptospira spp., L. borgpetersenii, L. interrogans and L. biflexa, has caused a key progression in the pathogenesis of the bacterium . Each genome contains two chromosomes. The larger replicon displays attributes to that of a bacterial chromosome, containing genes involved in replication, whereas the other smaller replicon accommodates an origin of replication similar to a plasmid, containing genes encoding various processes e.g. amino acid biosynthesis . The key genome features of these strains is summarised in table 1.
Furthermore, out of a possible 3400 (L. interrogans) or 2800 (L. borgpetersenii) coding regions, 656 of them are specific to pathogenic Leptospira spp. whose function is still not known, implying the exclusive virulence applications. L. biflexa shares fewer genes with the other two pathogenic species, 93 with L. interrogans and only 44 with L. borgpetersenii, and therefore contains further environmental sensing genes required for its survival in varied niches. The extensive number of unique L. interrogans genes are necessary for its free-living ascendant and continued existence in its habitat . The similarities and differences in genes between these three genomes can be seen in figure 3.
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Table 1 indicates the huge difference in pseudogene number between L. borgpetersenii and L. interrogans (368 and 41 respectively), suggesting a continuing specialisation development of L. borgpetersenii [6, 12]. The smaller genome size and reduced CDS of L. borgpetersenii compared with L. interrogans, indicates the gradual decay of its genome via the exploitation of insertion sequences. Gene function loss is connected to transport and utilisation of metabolites and solutes as well as sensing of the environment, implying L. borgpetersenii is currently evolving to depend on strict integration of the pathogen within the host [6, 13]. Moreover, the three sequenced Leptospira genomes share 2052 genes, seen in figure 3, providing evidence that once a common ancestor existed for both pathogenic species and L. biflexa. Since then, these organisms have evolved towards their specific environment and different way of life such as modes of transmission [5, 6, 13].
In addition to the two chromosomes common to all sequenced Leptospira spp. genomes, L. biflexa has an extra 74 kb circular replicon (p74), whose exact function is unknown, shown in figure 4 [11, 12]. It may be vital for the organism's endurance or just an additional replicon constituent, but is believed to function for L. biflexa survival .
The virulence factors concerning the genus Leptospira are not well understood primarily due to the lack of knowledge, until of late, regarding the genetic properties of the bacterium. However, those that have been identified, mostly surface-exposed proteins, are believed to provoke the interaction between this organism and its host [5, 6]. It is only the pathogenic strains of Leptospira that are capable of attaching and penetrating the host's cell membrane . The genes thought to be involved in Leptospira virulence are summarised in table 2 .
Many of the potential virulence factors of Leptospira spp., can be seen in the diagram of its cell wall, figure 5, consisting of an inner and outer membrane (IM and OM respectively). Such components include LipL32 and LPS.
Motility and Chemotaxis
The Leptospira genome contains 79 highly conserved genes related to chemotaxis and motility, each associated with the attachment and violation of animal host cells [3, 12, 17]. Bacterial motility could be a significant feature in the organism's science, regardless of this element being present in both pathogenic and non-pathogenic species, and is commonly accepted as a virulence factor . The exclusive nature and motility offers these organism's supplementary means of causing a successful infection . Within L. interrogans, FlaA constitutes the outer sheath protein, whereas the flagger core protein is composed of FlaB . The rapid, 'corkscrew' motility permits Leptospira spp. to be able to swim through highly vicious, gel-like mediums, but the mechanism is still not yet fully understood. Mutations in the FlaB protein within L. biflexa cause the endoflagellar to become ineffective [3, 5].
The acknowledgement of 12 protein-coding sequences, associated with methyl-accepting chemotaxis in Leptospira spp., implies that chemotactic responses arise due to a range of repellents and attractants. Moreover, it mirrors the huge variety of different environmental stimuli that this obligate bacterium can come across. Hence, chemotaxis may serve as a key part in Leptospira virulence as indicated by the positive chemotaxis response by L. interrogans to haemoglobin, absent in saprophytic species [3, 5].
Several hemolysins have been identified to take part in harming the endothelial cells lining the blood vessels . The L. interrogans genome contains 9 genes associated with alleged hemolysins which contain SphH, a protein involved in the formation of pores, as well as a sphingomyelinase; these constituents are absent in L. biflexa [5, 6]. The rich glycosphingolipid structure of erythrocytes causes them to remain a vulnerable target for attack by these proteins . Hemolysins acting as phospholipase toxins can introduce lysis of the erythrocytes, as observed in contaminated calves .
Furthermore, the role of other Sph sphingomyelinases with regard to Leptospira virulence is still unknown. Nevertheless, genomic sequencing has permitted more in-depth knowledge of the genome properties. For instance, the lack of sphingomyelinases in the non-pathogenic Leptospira spp. L. biflexa indicates that these genes are involved in the virulence of the bacterium in preference to the view that they are concerned with survival within their environment .
Surface proteins are critical in the establishment and endurance of virulent Leptospira species within their carrier host . Given that pathogenic strains of Leptospira attach to mammalian host surfaces, the bacterium's cell wall functions as an interaction between the two cells suggesting the surface proteins are highly associated with virulence .
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Loa22. This lipoprotein remains the only gene to obey the four criteria of Koch's postulates, establishing the connection between this protein and leptospirosis . This gene contains a C-terminal domain, OmpA, which is upregulated during infection causing it to be identified by the sera of individuals infected with leptospirosis. The exact function of this lipoprotein is unknown but it is highly conserved in virulent Leptospira species. It is believed that in Gram-negative bacteria such as E. coli (as is Leptospira), the lipoprotein OmpA is thought to operate in various functions in both the virulence and physiology of the bacterium .
LipL32. This conserved prominent protein also known as Hap1, dominates the cell surface, taking up approximately 75% of the total protein content expressed by Leptospira, found exclusively in virulent strains [5, 6, 11]. It has been believed for a long time that this protein is associated with Leptospira pathogenesis but its function is unknown, and as with Loa22, seems to be upregulated during host infection [5, 6]. LipL32 is now known to attach to various host components such as fibronectin, laminin and collagen .
Leptospira Immunoglobulin (Ig)-like proteins. Leptospira express LigA and LigB, as well as LigC of the Ig-like protein superfamily [5, 19, 20]. These Lig surface-exposed proteins, unique to pathogenic Leptospira spp., contain at least 12 Ig-like repeat domains [5, 19]. Physiological osmolarity stimulates the upregulation of LigA and LigB which are also capable of adhering to fibronectin . As are LipL32 proteins, the Lig proteins are also thought to be associated with Leptospira pathogenesis i.e. a putative virulent element . From experiments carried out on hamsters, it has been shown that loss of LigA and LigB in Leptospira, represses the pathogenesis of the bacterium so much that it can no longer cause disease, i.e. LigA and LigB are essential for Leptospira virulence .
The LPS molecules are known to be the partial reason behind Leptospira pathogenesis, a distinctive property of the outer membrane [3, 7]. The Leptospira genome encloses a locus in which genes critical for LPS biosynthesis are found. Observations have identified that instead of the LPS normally using the TLR4 (toll-like receptor 4); virulent strains use TLR2 for use in human signalling within cells . The diversity of non-pathogenic Leptospira spp. is believed to be due to genetic mutations within the genes associated with LPS biosynthesis .
In addition, genes encoding the enzymes involved in the polysaccharide portion of the LPS are located in the O-antigen gene (rfb) cluster . This rfb locus, spanning approximately 40kb, of L. interrogans has almost certainly been obtained via horizontal gene transfer. The diversity of the virulent Leptospira strains and the huge range of mammalian hosts that the bacterium can infect, is thought to be caused by alterations in LPS biosynthesis .
Once inside the host cell Leptospira needs to take up specific supplements, including iron, crucial for growth and survival. However, this nourishment can be difficult to obtain due to high affinity bacterial iron-binding proteins tightly bound to the ion . Hence, embedded within the Leptospira cell wall are a number of iron uptake mechanisms such as the TonB-dependent receptor . Virulent Leptospira species need to acquire haem oxygenase (via the hemO gene), to destroy the tetrapyrrole ring, allowing the ferrous iron to be dispensed. Hamster studies have shown that degradation in L. interrogans of the hemO gene diminishes the pathogenesis within the mammalian host .
The humoral immune response acts primarily towards leptospirosis immunity, and is somewhat dependent on serovar type [5, 6, 9]. The LPS element of the cell wall is often the chief target site for protective antibody activity against Leptospira . Antibodies in transferred sera and monoclonal antibodies, both acting against the LPS, implement passive immunity against leptospirosis in animal hosts . However, the latest studies have indicated that cell-mediated immunity also functions in protection against the disease [5, 22].
Avoidance of leptospirosis would be hard to implement without vaccination use . However, problems arise since protection is only provided against the same or antigenically similar serovars. Following the 1920s, vaccines have been put into action in mammalian hosts; the majority being killed-vaccines composed of dead cells of Leptospira for instance through formalin . Current vaccines are produced by growth in a protein-free medium, rarely causing undesirable side effects . However, the majority of these vaccines only provide protection for a restricted amount of time, and therefore boosters are necessary every/every other year. Despite the adverse effects of leptospiral killed-vaccines, they have still been effective in certain countries such as China where leptospirosis risk is high [9, 23].
Prevention of leptospirosis also requires immunisation of livestock to lessen the chance of urinary viral shedding. Modern commercial inactivated vaccines for leptospirosis protection are also accessible for use in livestock, pigs and domestic animals. However, these vaccines are only moderately efficient, partly because of the limited number of serovars the vaccine can defend against. Hence, efficient vaccines are required to contain the specific serovars present in the population at the time of immunisation. Most vaccines are usually comprised of more than one pathogenic serovar .
Present research is aimed at establishing the surface-exposed proteins believed to be involved in leptospiral virulent serovars. Despite these studies, preparing vaccines associated with these virulence factors has proved difficult due to low efficacy in animal testing. The use of Lig proteins in vaccines is looking encouraging, since they have been shown in both hamsters and mice to provide almost full protection (cross-protection) against leptospirosis [5, 22]. The development of genome sequencing has allowed fresh and upcoming possibilities in the progress of vaccines and aims to generate a vaccine capable of providing protection against a diverse range of virulent serovars. However, before a truly successful vaccine can be produced, the identification and mechanisms of the virulent factors needs to be established .
Moreover, other factors are involved in the control and prevention of leptospirosis such as the regulation of disease carriers and high level of sanitation . These aspects include coating abrasions of the skin, and dressing appropriately when handling possible disease carriers, especially rodents, such as wearing protective gloves . Since rodents are the prime reservoir for leptospirosis, controls need to be implemented primarily in those regions where leptospirosis risk is high . Journeys to certain countries of the world where risk of becoming infected is high, restrains concerning contact with water should be implemented . Furthermore, the amount of time spent exposed to animal urine should also be restricted as well as contact with water or mud in flooded areas. Lastly, the key factor that is vital in prohibiting the spread of the disease is to generate knowledge and realisation of the true impact the disease can have on the world through the use of education .
Unanswered Questions and Future Directions
Despite the new technique of genome sequencing, many key questions need to be addressed in order for the true understanding of the biology and pathogenesis of Leptospira. Little is known about the pathogenic mechanisms or the biology of the contributing factors involved in causing disease [12, 15]. Only a limited number of alleged virulence factors have been distinguished; approximately 40% of the genome is composed of proteins whose true role is not yet understood which could possibly function in Leptospira virulence [12, 15]. The use of different advances such as proteomics, are required to establish the traits of the bacterium and correlate these findings with Leptospira virulence in both human and animal hosts .
Only the surface protein Loa22 has so far obeyed Koch's postulates and the lack of others is again due to a deficiency in gene manipulation . General techniques used in other bacteria i.e. the use of the model organism Escherichia. coli, to enter DNA into virulent Leptospira spp. are extremely complex. Even the saprophytic L. biflexa shares a number of genes with the pathogenic species, and the use of targeted gene inactivation has still remained unsuccessful, therefore, like E. coli, L. biflexa could possibly act as a model prokaryotic organism in order to establish the role of these shared genes [5, 11]. Moreover, the huge genetic distinctions among the different species of Leptospira, provides the goal of reflecting these differences in biological deviations. Other factors yet to be established include the correlation between the genomes, virulence and survival within the environment .
Clarification of virulent factor functions will assist in the control and prevention of leptospirosis [5, 12]. These factors need to be implemented quickly in order to avoid the spread of leptospirosis and leptospiral infections, especially in developing countries where prevalence is elevated. Immunity to this zoonotic disease involves the use of vaccines, but potential developments in the near future involve seeing if infection with this neglected emerging disease provides consequent defence against re-infection among those most at risk. The next obstacle needed to be overcome concerns gaining insight into the regulation of gene expression as well as leptospiral interactions through the use of experimental rodents and microarrays .
The origin and role of leptospirosis in affecting the health of the population has been well known for a long time, allowing schemes to be undertaken in order to try and prohibit the spread and prevalence of leptospirosis and to create global awareness. The disease is vastly spread and is of prime importance in impoverished countries, especially where rain fall is high, but is still of economic importance in developed countries . It is now the biology and virulence mechanisms of Leptospira that are preventing the development and understanding of the organism. This has been caused by a deficiency in genetic machinery for use in leptospiral mutagenesis and transformation, which has otherwise been possible in other virulent bacterial organisms. The genome structure and function has revealed distinctive elements involved in causing disease which are yet to be understood . The growth and expansion of techniques such as transposon mutagenesis presents a greater outlook on improving and developing current knowledge, to eventually understand how the virulent species are able to adhere with their hosts and cause an infection [6, 9].