Orientia tsutsugamushi is a Gram-negative bacterium and is a permanent (obligate) parasite in mites. Within a single host cell,
O. tsutsugamushi rapidly divides into many individuals. A unicellular organism, it is oval shaped and measures 0.5 to 0.8 μm wide and 1.2 to 3.0 μm long. Due to similarity, it was previously classified in the genus
Rickettsia among other bacteria, but later assigned a separate genus,
Orientia, which it shares (as of 2010) only with
Candidatus Orientia chuto. It is broader but shorter than other rickettsial bacteria, which are rod shaped and on average measure 0.25 to 0.3 μm wide and 0.8 to 1 μm long. During reproduction, it divides (by
binary fission) into two daughter cells by the process of budding. While undergoing budding, it accumulates on the host cell surface, unlike other bacteria. One complete budding cycle takes 9 to 18 hours. The bacterium is enclosed by a
cell wall on the outside and cell membrane on the inside. The cell covering takes up stains such as
Giemsa and
Gimenez stains. Although its cell wall has a classic bacterial double layer, its outer leaflet is much thicker than the inner one, which is just the opposite in
Rickettsia species. A capsule layer that forms a spherical halo in other bacteria is missing. The cell wall is less rigid due to the absence of peptidoglycan, which is otherwise characteristic of the rigid cell walls of other bacteria. Classic bacterial lipophosphoglycans such as
muramic acid,
glucosamine, hydroxy fatty acids,
heptose, and 2-keto-3-deoxyoctonic acid are also absent in the cell wall. Due to the absence of
peptidoglycan, the bacterium is naturally resistant to all
β-lactam antibiotics (such as
penicillin), to which
Rickettsia species are normally sensitive to. Its genome totally lacks the genes for lipophosphoglycan synthesis, but does contain some for those of peptidoglycan. Important genes essential for peptidoglycan synthesis such as
alr,
dapF and
PBP1 are missing:
alr encodes an enzyme L-alanine racemase, which converts L-alanine to D-alanine in the first step of peptidoglycan synthesis pathway;
dapF encodes diaminopimelate epimerase, which convert LL-2,6-diaminoheptanedioate (L,L-DAP) to meso-diaminoheptanedioate (meso-DAP); and
PBP1 encodes penicillin-binding protein-1 (PBP1), which converts periplasmic lipid II to peptidoglycan. Thus, the bacterium cannot synthesise a typical peptidoglycan cell wall, and instead makes a peptidoglycan-like structure on its surface. The cell membrane is also chemically different in its protein composition, and this difference gives rise to strain variations within the species itself. in purple,
core genes in green, repeat genes in red and
pseudogenes in blue. The innermost line graph shows
GC-content (1000bp windows) with above-median region in green, and below-median regions in red. The bacterium is highly virulent, such that its isolation and cell culture are done only in a laboratory facility with
biosafety level 3. Unlike other bacteria which can easily grow on different culture media, rickettsiales can be cultured only in living cells.
O. tsutsugamushi specifically can be grown only in the yolk sacs of developing chicken embryos and in cultured cell lines such as
HeLa,
BHK,
Vero, and
L929. In contrast to
Rickettsia species which reside in the nucleus of the host cell,
O. tsutsugamushi mostly grows within the cytoplasm of the host cell. Even though adaptation to
obligate intracellular parasitism among bacteria generally results in a reduced genome, it has a genome size of about 2.0–2.7
Mb depending on the strains, which is comparatively larger than those of other rickettsiales – two times larger than that of
Rickettsia prowazekii, the most well-known member. The entire genome is distributed in a single circular chromosome. Whole genome sequences are available only for Ikeda and Boryong strains, both from the Republic of Korea. The genome of the Ikeda strain is 2,008,987 base pairs (bp) long, and contains 1,967 protein-coding genes. The Boryong strain is larger with 2,127,051 bp and 2,179 protein-coding genes. Genome comparison shows only 657 core genes among the different strains. With about 42-47% of repetitive sequences,
O. tsutsugamushi has the most highly repeated bacterial genome sequenced as of 2013. The repeated DNA sequence includes short
repetitive sequences,
transposable elements (including insertion sequence elements, miniature inverted-repeat transposable elements, a
Group II intron), and a greatly amplified
Integrative and Conjugative Element (ICE) called the rickettsial amplified genetic element (RAGE). In rodent and human infections,
Leptotrombidium deliense is the most common vector of
O. tsutsugamushi.
L. pallidum,
L. fletcheri and
L. scutellare are also carriers in many countries. In addition,
L. akamushi is an endemic carrier in Japan,
L. chiangraiensis and
L. imphalum in Thailand,
L. gaohuensis in China, and
L. arenicola in Malaysia and Indonesia. The life cycle of mites consists of egg, prelarva, larva, protonymph, deutonymph, tritonymph, and adult. The larvae, commonly referred to as chiggers, are the only ectoparasitic stage feeding on the body fluids of rodents and other opportunistic mammals. Thus, they are the only stage in the life of mites that transmit the infection. Wild rats of the genus
Rattus are the principal natural hosts of the chiggers. Chiggers feed only once on a mammalian host. The feeding usually takes 2 to 4 days. In contrast to most parasites, they do not feed on blood, but instead on the body fluid through the hair follicles or skin pores. In the process of feeding, they create a
stylostome, which is a tube formed by solidified saliva. Their saliva can dissolve the host tissue around the feeding site, so that they ingest the liquefied tissue.
O. tsutsugamushi is present in the
salivary glands of mites and is released into the host tissue during this feeding.
Cellular invasion Orientia tsutsugamushi initially attacks the
myelocytes (young white blood cells) in the area of inoculation, and then the
endothelial cells lining the
vasculature. In the blood circulation, it targets professional
phagocytes, white blood cells) such as
dendritic cells and
macrophages in all organs as the secondary targets. The parasite first attaches itself to the target cells using surface proteoglycans present on the host cell and bacterial surface proteins such as type specific protein 56 (or type specific antigen, TSA56) and surface cell antigens (ScaA and ScaC, which are membrane transporter proteins). These proteins interact with the host
fibronectin to induce
phagocytosis (the process of ingesting the bacterium). The ability to actually enter the host cell depends on
integrin-mediated signaling and reorganisation of the
actin cytoskeleton.
Orientia tsutsugamushi has a special adaptation for surviving in the host cell by evading the host immune reaction. Once it interacts with the host cells, it causes the host cell membrane to form a transportation bubble called a
clathrin-coated vesicle by which it gets transported into the cytoplasm. Inside the cytoplasm, it makes an exit from the vesicle (now known as an
endosome) before the endosome is destroyed (in the process of cell-eating called
autophagy) by the
lysosomes. It then moves towards the nucleus, specifically at the perinuclear region, where it starts to grow and multiply. Unlike other closely related bacteria which use actin-mediated processes for movement in the cytoplasm (called
intracellular trafficking or transport),
O. tsutsugamushi is unusual in using
microtubule-mediated processes similar to those employed by viruses such as
adenoviruses and
herpes simplex viruses. Further, the escape (
exocytosis) from an infected host cell is also unusual. It forms another vesicle using the host cell membrane, gives rise to a small bud, and releases itself from the host cell surface while still enclosed in the vesicle. The membrane-bound bacterium is formed by interaction between cholesterol-rich lipid rafts as well as HtrA, a 47-kDa protein on the bacterial surface. However, the process of budding and importance of the membrane-bound bacterium are not yet understood.
Strains Orientia tsutsugamushi is a diverse species of bacteria.
Ida A. Bengtson of the
United States Public Health Service was the first to note the existence of different strains using antigen-antibody interaction (
complement fixation test) in 1944. She observed that different strains had varying degree of virulence, and that the antibodies in the blood sera of patients cross-react to different strains. By 1946, she established that there were three principal strains (serotypes), namely Karp (from New Guinea), Gilliam (from India) and Seerangay (from British Malaya). Akira Shishido described the Kato strain, in addition to Gilliam and Karp, in Japan in 1958. Since then, six basic antigenic strains are recognised, namely Gilliam, Karp, Kato, Shimokoshi, Kawasaki, and Kuroki. Karp is the most abundant strain, accounting for about 50% of all infections. As of 2009, more than 20 different strains have been established in humans based on
antigenic variation using serological tests such as complement fixation and immunofluorescence assay. Another study in 1996 reported 40 strains. Genetic methods have revealed even greater complexity than had been previously described (for example, Gilliam is further divided into Gilliam and JG types). Due to immunological differences of the serotypes, simultaneous and repeated infection with different strains is possible.
Antigenic variation Orientia tsutsugamushi has four major surface-membrane proteins (
antigens) having molecular weights 22 kDa, 47 kDa, 56 kDa and 110 kDa. A 56-kDa type specific antigen (TSA56) is the most important because it is not produced by any other bacteria, and is responsible for making the genetic diversity in different strains. It accounts for about 10–15% of the total cell proteins. The 22-kDa, 47-kDa or 110-kDa antigens are not strain specific so that TSA56 is the main target in sophisticated diagnostic tests such as immunoblotting, ELISA and DNA analysis. The protein assists the adhesion and entry of the bacterium into host cells, as well as evasion of the host's immune reaction. It varies in size from 516 to 540 amino acid residues between different strains, and its gene is approximately 1,550 base pairs long. Its gene contains four hypervariable regions, indicating that it synthesises many antigenically different proteins. GroES and GroEL are
heat shock proteins belonging to the family of
molecular chaperones in bacteria. DNA analyses have shown that the
GroES and
GroEL genes are indeed present in
O. tsutsugamushi with slight variation in different strains, and they produce the 11-kDa and 60-kDa proteins. ==Disease==