Rickettsia prowazekii and Rickettsia conorii

Rickettsia species are obligate intracellular bacteria normally living arthropod hosts (insects or tickes). Vectorized by the arthropods, many Rickettsia species cause human diseases. Rickettsia prowazekii is the agent of epidemic typhus, and Rickettsia conorii is the agent of Mediterranean spotted fever.

Up to now, these bacteria cannot be cultured without eukaryotic cells. Current culture systems of Rickettsia include Vero cell, yolk sac, fibroblast and ticks. The difficulty of culturing these bacteria without eukaryotic cells inhibits the implementation of modern molecular biological technologies such as transcriptomics and proteomics to Rickettsia species.

Complete genome sequences of these two Rickettsia have been determined [Andersson et al., 1998 (PMID: 9823893); Ogata et al., 2001 (PMID: 11557893)]. The analyses of the genome sequences predicted a number of deficiencies in the biosynthetic pathways of small molecules. Remarkably, many genes involved in those metabolism can be detected in the genome sequences as "pseudogenes" or "split genes".

Examples of the genomic features and experimental results that may help the improvement of the culture system:

No de novo biosysnthetic pathways for 20 amino acids, and the presence of several transporters for amino acids and peptides.
No de novo biosynthetic pathways for NTP (e.g. ATP, TTP, GTP, CTP, UTP), while Rickettsia possess 4 copies of ATP/ADP translocase to uptake ATP from hosts.
Lack of glycolysis but the presence of TCA cycle. Rickettsia genus is one of the closest bacterial group to Mitochondria, which is known to possess pyruvate carrier.
The genes for MetK, a biosyntetic enzyme for S-adenosylmethionin (AdoMet), are degradated. Recently, AdoMet transporters were identified in Rickettsia.
There are some methabolic pathways around folate but its biosynthetic pathway is incomplete.

These and other related information can be found in the Metagrowth database.

Tropheryma whipplei

Tropheryma whipplei is the causative agent of Whipple's disease, a rare multisystem chronic infectious disease. T. whipplei is a very fastidious pathogen. It took nearly a century from the first description of the disease in 1907 to the first reproducible propagation of this micro-organism within a fibroblast cell line (HEL) in 2000 [Raoult et al., 2000 (PMID: 10699161].

In 2003, two independent studies determined the complete genome sequences of different strains of T. whipplei, strain Twist [Raoult et al., 2003 (PMID: 12902375], and strain TW08/27 [Bentley et al., 2003 (PMID: 12606174]. T. whipplei is the only known reduced genome species (<1 Mb) within the Actinobacteria [high G+C Gram-positive bacteria].

Despite the small genome size, the sequence analyses and computer modeling of its putative metabolism predicted that this bacterium is well equipped for energy metabolism, nucleotide biosynthesis, and regulatory process compared with other small genome bacteria. However, significant deficiencies were predicted in the biosynthesis of amino acids. With this knowledge, in 2003, the first "cell-free" complex culture medium was successfully designed for T. whipplei growth [Renestro et al., Lancet 2003, (PMID: 12927433; pdf)].

This is the first case of a successful "genome-based" design of cell-free culture medium for "previously-obligate" parasitic bacteria. With the aim of guiding similar studies for other obligate bacteria, we are developping the Metagrowth database.

Further experimental and in silico simulation studies are indispensable to improve the culture conditions (e.g. faster growth rate) as well as to better rasionalize the proccess of "genome-based" design of culture media. In this perspecitve, we continue to provide information about metabolisms and culture conditoins of T. whipplei with the Metagrowth database.

Treponema pallidum

Treponema pallidum is an obligate parasitic spirochete, causing syphilis in human. In human, T. pallidum is initially localized at sexual organs, but rapidly disseminates throughout the body. The bacterium is mesophilic and microaerophilic organism. T. pallidum cells have outer and cytoplasmic membranes, and a thin peptidoglycan layer in between. The outer membrane is mostly composed of lipids with a paucity of proteins. In the laboratory, it is usually cultured in the testes of rabbits.

The 1.13 Mbp genome sequence of T. pallidum was determined in 1998 [Fraser et al., 1998 (PMID: 9665876 )].

Examples of the genomic features and experimental results that may help the improvement of the culture system:

Microaerophilic, requiring a low-level of oxygen concentration. Consistently no genes have been found in the genome for superoxide dismutase, catalase and peroxidase.
Genome sequence analyses revealed that the bacterium probably cannot synthesize fatty acids, many co-factors, nucleotides through de novo pathway.
Treponema pallidum incorporates directory fatty acids into the outer membrane, because of the lack of lipopolysaccharide in the outer membrane. In contrast, Gram-negative bacteria exhibit poor permeability of hydrophobic compounds like fatty acids.
Related bacteria such as T. denticola, T. vincentii, Leptospira species are known to require thiamin or thiamin pyrophosphate (TPP) for their in vitro growth.
Most of the [14C]glucose incorporated into amino acids is in the form of aspartic acids.
Pathogenic Treponema bind host fibronectin to penetrate organs.

These and other related information can be found in the Metagrowth database.

Mycobacterium leprae

Mycobacterium leprae is the agent of leprosy (G.H.A. Hansen 1873). M. leprae is an obligate intracellular parasite, and extremely fastidious bacterium, that is only cultured in animal organs such as foot pads of mice and armadillos (11-13 h of doubling time) with optimal temperature at 27-30 degree C. M. leprae is an aerobic bacterium.

The 3.2 Mb genome sequence of M. leprae was determined in 2001 [Cole et al., 2001 (PMID: 11234002)]. The genome analysis revealed a number of pseudogenes in the genome, and suggested significant deficiencies in a large part of metabolism that are present in related M. tuberculosis.

Examples of the genomic features and experimental results that may help the improvement of the culture system:

Compared with M. tuberculosis, M. leprae lost most of the genes for detoxification of reactive oxygen and nitrogen species such as nitric oxide (NO). Truncated hemoglobins (trHb's) may also explain the susceptibility to the toxic oxygen and nitrogen species.
M. leprae could be auxotrophic for cobinamide because the bacterium possesses the synthetic genes for vitamin B12 from cobinamide but lacks genes for the precursor synthesis.
To resuscitate dormant M. tuberculosis, phospholipids (for example, commercially available phosphatidyl-L-serine, dioleoyl phosphatidyl-L-seine) and peptides derived from Rv1174c is critical (Zhang 2001). In MLE Rv1174c homolog gene has frameshift.

These and other related information can be found in the Metagrowth database.

Coxiella burnetii

Coxiella burnetii is the causative agent of Q-fever, a zoonose exhibiting flu-like symptoms with cyclic fever. C. burnetii is highly infective and defined as a category B bioterrorism agent. It is a Gram-negative, gamma-proteobacteria (order Legionellales). This bacterium is an obligate intracellular acidophile highly adapted for life within the eukaryotic phagolysosome (pH=4.5) of phagocyte, which is rich in hydrolytic enzymes, oxygen, nitrogen radicals. C. burnetii exhibit a high level of resistance to UV, heat, desiccation, pressure, osmotic stress, and oxidative stress. The bacteira is often transmitted by tickes.

The 2.0 Mb with 42% G+C-content genome sequence of C. burnetii was determined in 2003 [Seshadri et al., 2003 (PMID: 12704232)].

Examples of the genomic features and experimental results that may help the improvement of the culture system:

Amino acids appears to be dominant carbon sources for C. burnetii. C. burnetii was predicted to be auxotroph for 11 amino acids. Its genome encodes transporters for amino acid and oligopeptides.
It is known that C. burnetii uptakes guanosine, adenosine, and inosine. It also exhibit limited uptake of thymidine and uridine. But the bacterium is not capable of transport and incorporation of NTPs, CMP, cytosine or uracil.
L-proline transport is optimal at pH 3.0 to 4.5, the same pH range for the host phagolysosome.
Thiamin (vitamin B1) biosynthetic pathway lacks the first part. However, C. burnetii requires thiamin diphosphate as a cofactor of enzymes that it encodes in its genome.

These and other related information can be found in the Metagrowth database.