Leptospira interrogans is a thin, P1+, motile, gram-negative, obligate aerobic spirochaete bacterium that belongs to the genus Leptospira in the phylum Spirochaetota. L. interrogans cells are long, helical, and tightly coiled like a corkscrew with hooked and spiraled ends. L. interrogans has many properties that ensure its optimal survival in specific conditions, including two periplasmic flagella located within its outer sheath, enabling the bacteria to move rapidly through a variety of mediums such as water, soil, and host tissue. These flagella enable L. interrogans to more easily access and infect mammalian tissues. The outer membrane of L. interrogans contains unique lipopolysaccharides (LPS) and surface-exposed proteins that play key roles in environmental survival, adhesion, and interaction with host organisms. It is able to metabolize long-chain fatty acids through ꞵ-oxidation as an energy source and uses oxygen as a terminal electron acceptor.
L. interrogans is mainly found in warmer tropical regions. The bacteria can live for weeks to months in the ground or water. Leptospira is one of the genera of the spirochaete phylum that causes severe mammalian infections. Although many Leptospira species can cause infection, most of the serious infections are caused by L. interrogans. This species is pathogenic to some wild and domestic animals, including pet dogs. It can also spread to humans through abrasions on the skin with contaminated water or soil, where infection can cause flu-like symptoms with kidney and liver damage.
L. interrogans has a genome that consists of two circular chromosomes. Together, these chromosomes encode genes involved in motility, nutrient acquisition, stress response, and adaptation to different ecological niches.
History
Identification and isolation While illnesses consistent with a leptospirosis diagnosis had been described in ancient medical journals as “rice field jaundice” or “autumn fever”, the modern history began with the identification of Weil’s disease, or leptospirosis, in Adolf Weil in 1886 wherein he had been experiencing “a particular type of jaundice accompanied by splenomegaly, renal dysfunction, conjunctivitis, and skin rashes”. The specific causative agent had yet to be identified, but higher rates of infection were seen in those who worked outdoor occupations and came into contact with water, with epidemics common in sewer workers, rice-field workers, and coal miners.
Leptospira were isolated independently and almost simultaneously in Japan and Europe, though the earliest identification of leptospires was done by A.M. Stimson in 1907 using Levaditi silver deposition staining in the kidney tissue of an infected cadaver. Stimson dubbed the organism Spirocheta interrogans because of its resemblance to a question mark due to the characteristic hooked ends of the leptospires. In 1915, Japanese researchers Ryukichi Inada and Yutaka Ido identified the causative agent of Weil’s disease by observing spiral-shaped bacteria in the blood and organs of infected patients and experimentally transmitting the disease to guinea pigs, proving it was caused by a spirochete. They were able to cultivate spirochetes in vitro using a medium made from emulsified guinea pig kidney and developed the first successful antiserum for treatment. This discovery allowed for rapid advancements in this research area as they defined transmissibility, routes of infection, pathological changes, tissue distribution, urinary excretion, leptospiral filterability, morphology, and motility.
In 1917, Inada and Ido identified rats as renal carriers of Leptospira by finding spirochetes in the kidneys of healthy field mice and house rats which was the first observation of an asymptomatic carrier. In the following decades, as advancements in the field occurred, leptospirosis was recognized as an infectious disease affecting almost all mammalian species with ranging severity from mild influenza-like illness to severe, fatal infections with liver and kidney failure. By the 1950s, the number of identified serovars and susceptible host species had grown considerably, and by the 1980s, leptospirosis was firmly established as a veterinary disease of significant economic impact in dogs, cattle, pigs, horses, and possibly sheep.
Nomenclature and classification The genus Leptospira was first proposed in 1918 to differentiate the Leptospirosis spirochete from others based on morphology. As serovars were isolated, they were given species status and species were grouped into serogroups based on related antigens. When it was clear that a vast number of serovars existed, the subcommittee on the Taxonomy of Leptospira accepted the idea of two species existing, L. biflexa and L. interrogans, with former containing saprophytic serovars and the latter containing pathogenic serovars. Additional advances in molecular taxonomy led to major revisions in Leptospira classification, with DNA–DNA analysis dividing the former single species L. interrogans into seven distinct species. Leptospira are also classified into serogroups and serovars based on antigenic differences in the LPS surface molecules, with there being at least 25 serogroups and over 300 different serovars. Significant serogroups include Icterohaemorrhagiae, Australis, Canicola, Pomona, and Sejroe and each serogroup is subdivided into multiple serovars.
Microbiology
Morphology L. interrogans cells are gram-negative, tightly coiled, motile spirochetes, with two periplasmic flagella. One flagellum is inserted at each end of the bacterium. The cells are thin, about 0.15 μm, and long, between 6-20 μm, with a corkscrew shaped body with spiral or hooked ends. The hooked ends often resemble a question mark, and this is where the name 'interrogans' comes from. The periplasmic flagella are crucial to the bacteria's ability to move and survive in specific host cells. The leptospires reveal two unique forms of movement, translational and non translational.
Metabolism and physiology L. interrogans displays neutralophilic properties, growing in a pH range of 7.2 - 7.6, with an optimal pH of 7.4. The bacteria also display mesophilic growth properties and grow at a temperature range of 28 °C to 30 °C. The optimal growth of the obligate aerobe L. interrogans occurs in simple media containing vitamins, salts, and specific long chain fatty acids. Leptospira require ammonium salts as well as long-chain fatty acids for metabolism.
The major energy and carbon source of this organism is the beta-oxidation of long chain fatty acids. L. interrogans must obtain the long chain fatty acids from its surroundings to metabolize them as an energy source. Unique to the metabolic characteristics of L. interrogans, long chain unsaturated fatty acids are required for the bacterium to grow, as saturated fatty acids can only be metabolized in these conditions. L. interrogans contains genes that code for the use of the TCA cycle in its metabolism. L. interrogans transforms fatty acids into acetyl-CoA, then uses acetyl-CoA to produce some ATP through the TCA cycle. The TCA cycle produces the electron carriers necessary for oxidative phosphorylation. L. interrogans main ATP production comes through oxidative phosphorylation. Oxygen serves as the terminal electron acceptor in this beta-oxidation. Hydrogen peroxide (H2O2) may also serve as a terminal electron acceptor, with catalase activity needed for survival of L. interrogans during infection. L. interrogans has only one glucose uptake system, known as the glucose sodium symporter. L. interrogans cannot use glycolysis because it cannot use glucose as it main carbon source. Hence, fatty acid β-oxidation is the microbe's main source of carbon and energy.
L. interrogans has its own Glutathione (GSH) enzymatic pathway and uses the produced GSH as an antioxidant. L. interrogans synthesizes GSH using the enzymes γ-glutamylcysteine synthetase (GCL) and glutathione synthetase (GS).Natalia Sasoni, Danisa M.L. Ferrero, Sergio A. Guerrero, Alberto A. Iglesias, Diego G. Arias, First evidence of glutathione metabolism in Leptospira interrogans, Free Radical Biology and Medicine, Volume 143, 2019, Pages 366-374, ISSN 0891-5849, https://doi.org/10.1016/j.freeradbiomed.2019.08.028. GSH protects the cell from oxidative stress by preventing oxidation and the creation of free radicals.
Genomics The L. interrogans genome consists of two circular chromosomes composed of a total of almost 4.7 Mbp. The larger chromosome has a total genome of 4.3 Mbp, and the smaller chromosome has a size of 350 Kbp. It has a G+C content of 35% and contains 3,400-3,700 protein-coding genes, depending on the strain. The genes on the large chromosome encodes mostly housekeeping functions. Unlike most other bacteria, where the rRNA genes are clustered, in Leptospira the 16S, 23S, and 5S rRNA genes are scattered on the large chromosome. Genes specifically encoding for long-chain fatty-acid usage, the TCA cycle, and electron transport chain have also been identified in L. interrogans. The detection of such genes confirms the use of oxidative phosphorylation as the primary metabolic pathway of L. interrogans. A large amount of genes related to eukaryotic cell invasion, cell attachment, and motility have been discovered. L. interrogans also has a complex set of genes associated with chemotaxis, more so than other pathogenic bacteria such as B. burgdorferi and T. palladium. Such genes able L. interrogans to be such a successful pathogen.
To have the energy necessary for growth and to take over host functions, the bacterium employs methods such as oxidative stress. Stress responses seen in L. interrogans include the up-regulation expression of genes encoding proteins such as chaperone proteins including clpA, heat shock proteins including GroEL, and endoflagellar proteins including flgA.
Molecular pathogenesis The loa22 gene has been classified as a virulence factor.
LipL32 is the most abundant protein in L. interrogans. Although LipL32 binds to a number of extracellular matrix components in test tube experiments, there is doubt regarding where this protein is located. One study suggests that it is a subsurface membrane lipoprotein on the inner leaflet of the outer membrane. Some outer membrane proteins, such as OmpL1, aid in the infection process of L. interrogans by allowing adherence to host cells's surface molecules.
As L. interrogans is an obligate aerobe, reactive oxygen species (ROS) must be avoided during metabolism. The perRA and perRB genes encode peroxide responsive regulators, and these regulators promote host adaptation as they contain approximately 17 genes which aid in signaling. L. interrogans also has a rather complex chemotaxis system compared to other pathogenic microbes, contributing to its effectiveness as a pathogen. Virulence is also related to the leptospiral LPS, which is known to uniquely activate macrophages in response to infection.
The bacterial chaperone ClpB is a major driver in the overall virulence of L. interrogans, as it aids in survival inside the host, the control of stress responses, and the unique role of protein disaggregation.