Masahiro Fukuhara

The Whole-genome Sequencing of the Obligate Intracellular Bacterium Orientia tsutsugamushi Revealed Massive Gene Amplification During Reductive Genome Evolution

Scrub typhus (‘Tsutsugamushi’ disease in Japanese) is a mite-borne infectious disease. The causative agent is Orientia tsutsugamushi, an obligate intracellular bacterium belonging to the family Rickettsiaceae of the subdivision alpha-Proteobacteria. In this study, we determined the complete genome sequence of O. tsutsugamushi strain Ikeda, which comprises a single chromosome of 2 008 987 bp and contains 1967 protein coding sequences (CDSs). The chromosome is much larger than those of other members of Rickettsiaceae, and 46.7% of the sequence was occupied by repetitive sequences derived from an integrative and conjugative element, 10 types of transposable elements, and seven types of short repeats of unknown origins. The massive amplification and degradation of these elements have generated a huge number of repeated genes (1196 CDSs, categorized into 85 families), many of which are pseudogenes (766 CDSs), and also induced intensive genome shuffling. By comparing the gene content with those of other family members of Rickettsiacea, we identified the core gene set of the family Rickettsiaceae and found that, while much more extensive gene loss has taken place among the housekeeping genes of Orientia than those of Rickettsia, O. tsutsugamushi has acquired a large number of foreign genes. The O. tsutsugamushi genome sequence is thus a prominent example of the high plasticity of bacterial genomes, and provides the genetic basis for a better understanding of the biology of O. tsutsugamushi and the pathogenesis of ‘Tsutsugamushi’ disease.

Demonstration of Antigenic and Genotypic Variation in Orientia tsutsugamushi Which Were Isolated in Japan, and Their Classification into Type and Subtype

A total of 40 strains of Orientia tsutsugamushi (34 isolates from patients and trombiculid mites in Japan, and 6 prototype strains of antigenic variants) were examined for classification based on the reactivities with type‐specific monoclonal antibodies in indirect immunofluorescence tests, and on the restriction fragment length polymorphism of a polymerase chain reaction (PCR)‐amplified 56‐kilodalton type‐specific antigenic protein gene. By these methods, several antigenic and genotypic variants were found among the strains, and these variants were classified into types and further into subtypes. These results suggest that there are many variants in O. tsutsugamushi, and the methods used here seem to be useful for the systematic classification of the numerous variants. A strain which may be a new type distinguishable from those identified previously was also found in this study. Furthermore, variety in the degree of pathogenicity in mice related to type and/or subtype classification were observed.

Potassium/Proton Antiport System of Escherichia coli

The intracellular level of potassium (K+) in Escherichia coli is regulated through multiple K+ transport systems. Recent data indicate that not all K+ extrusion system(s) have been identified (15). Here we report that the E. coli Na+ (Ca2+)/H+ antiporter ChaA functions as a K+ extrusion system. Cells expressing ChaA mediated K+ efflux against a K+ concentration gradient. E. coli strains lacking the chaA gene were unable to extrude K+ under conditions in which wild-type cells extruded K+. The K+/H+ antiporter activity of ChaA was detected by using inverted membrane vesicles produced using a French press. Physiological growth studies indicated that E. coli uses ChaA to discard excessive K+, which is toxic for these cells. These results suggest that ChaA K+/H+ antiporter activity enables E. coli to adapt to K+ salinity stress and to maintain K+ homeostasis.

Genome Comparison and Phylogenetic Analysis of Orientia tsutsugamushi Strains

Orientia tsutsugamushi (OT) is an obligate intracellular bacterium belonging to the family Rickettsiaceae and is the causative agent of scrub typhus, or Tsutsugamushi disease. The complete genome sequences of two OT strains (Boryong and Ikeda) have recently been determined. In the present study, we performed a fine genome sequence comparison of these strains. Our results indicate that although the core gene set of the family Rickettsiaceae is highly conserved between the two strains, a common set of repetitive sequences have been explosively amplified in both genomes. These amplified repetitive sequences have induced extensive genome shuffling and duplications and deletions of many genes. On the basis of the results of the genome sequence comparison, we selected 11 housekeeping genes and carried out multilocus sequence analysis of OT strains using the nucleotide sequences of these genes. This analysis revealed for the first time the phylogenetic relationships of representative OT strains. Furthermore, the results suggest the presence of an OT lineage with higher potential for virulence, which may explain the clinical and epidemiological differences between ‘classic’ and ‘new’ types of Tsutsugamushi disease in Japan.

Cloning, functional expression and primary characterization of Vibrio parahaemolyticus K+/H+ antiporter genes in Escherichia coli

The regulation of internal Na+ and K+ concentrations is important for bacterial cells, which, in the absence of Na+ extrusion systems, cannot grow in the presence of high external Na+. Likewise, bacteria require K+ uptake systems when the external K+ concentration becomes too low to support growth. At present, we have little knowledge of K+ toxicity and bacterial outward‐directed K+ transport systems. We report here that high external concentrations of K+ at alkaline pH are toxic and that bacteria require K+ efflux and/or extrusion systems to avoid excessive K+ accumulation. We have identified the first example of a bacterial K+(specific)/H+ antiporter, Vp‐NhaP2, from Vibrio parahaemolyticus. This protein, a member of the cation : proton antiporter‐1 (CPA1) family, was able to mediate K+ extrusion from the cell to provide tolerance to high concentrations of external KCl at alkaline pH. We also report the discovery of two V. parahaemolyticus Na+/H+ antiporters, Vp‐NhaA and Vp‐NhaB, which also exhibit a novel ion specificity toward K+, implying that they work as Na+(K+)/H+ exchangers. Furthermore, under specific conditions, Escherichia coli was able to mediate K+ extrusion against a K+ chemical gradient, indicating that E. coli also possesses an unidentified K+ extrusion system(s).

Phylogenetic characterization of Orientia tsutsugamushi isolated in Taiwan according to the sequence homologies of 56-kDa type-specific antigen genes.

Fourteen strains of Orientia tsutsugamushi isolated in Taiwan were characterized by sequencing 56‐kDa type‐specific antigen genes and patterns of restriction fragment length polymorphism (RFLP) predicted by a computer program. The strains showed high varieties in sequence homologies and were classified to 10 types by predicted patterns of RFLP. Furthermore, all Taiwan strains were not identical in typing with strains analyzed previously. These results suggest that there are various types of O. tsutsugamushi in Taiwan that are different from those distributed in other countries.

Epidemiological survey of Orientia tsutsugamushi distribution in field rodents in Saitama Prefecture, Japan, and discovery of a new type.

There are various antigenic variants of Orientia tsutsugamushi which are distinguished by immunological and molecular genetic methods targeted at the antigenic diversity of 56‐kDa type‐specific antigen proteins. The present study was performed to analyze 15 strains successfully isolated from rodents in Saitama Prefecture, Japan, by 56‐kDa gene sequence homologies, reactivities with type‐specific monoclonal antibodies and polymerase chain reaction (PCR) using type‐specific primer‐pairs. We demonstrated the presence of a new type of O. tsutsugamushi among the isolates. This new type, designated as the Saitama type, was located in the branch of Karp type in the phylogenetic tree based on 56‐kDa gene sequences, but distant from the known Karp types, such as Karp, JP‐1 and JP‐2, showing less than 90% homology. Strains of this type could not be distinguished by immunological methods from Karp type strains, but a new primer‐pair for PCR which specifically amplifies the DNA of this new type strain was designed. This primer‐pair may serve to find this strain type in future studies.

Isolation of Orientia tsutsugamushi from patients in shikoku and finding of a strain which grows preferentially at low temperatures

Three strains of Orientia tsutsugamushi were isolated from patients in Anan City, Tokushima Prefecture. The strains were identified as Karp type by analyses of reactivities with type‐specific monoclonal antibodies. One strain, Okazaki, was isolated in L cells cultivated at 31 C, but not in cells at 36 C or in mice. This strain showed better growth at 31 C than 36 C. This is the first report of an O. tsutsugamushi strain which grows preferentially at low temperatures.

Critical involvement of the E373-D434 region in the acid sensitivity of a NhaB-type Na(+)/H(+) antiporter from Vibrio alginolyticus.

It has been well established that VaNhaB, a NhaB-type Na(+)/H(+) antiporter found in Vibrio alginolyticus, exhibits a striking acid sensitivity. However, the molecular basis of the pH-dependent regulatory mechanism of the antiport activity is yet to be investigated. In this study, we generated various chimeric proteins composed of VaNhaB and a pH insensitive ortholog found in Escherichia coli (EcNhaB) and analyzed the pH responses of their Na(+)/H(+) antiport activities to search for the key residues or domains that are involved in the pH sensitivity of VaNhaB. Our results revealed the significant importance of a stretch of amino acid residues within the loop 8-loop 9 regions (E373-D434) responsible for the acid sensitivity of VaNhaB, along with the possible involvement of other unidentified residues that are widely spread in the primary structure of VaNhaB. Moreover, we demonstrated that the E373-D434 region of VaNhaB was able to confer some degree of acid sensitivity on our pH insensitive chimeric antiporter that is mainly composed of EcNhaB except for seven amino acid substitutions at the N-terminal end. This result strongly suggested the possibility that the E373-D434 region is able to act, at least partially, as machinery that diminishes the activity of the NhaB-type antiporter at an acidic pH.