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Roles of Brucella abortus SpoT in morphological differentiation and intramacrophagic replication

¹ Department of Applied Veterinary Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan
² Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan
³ Department of Developmental and Medical Technology, Graduate School of Medicine, The University of Tokyo, Hongo, Tokyo 113, Japan

Correspondence
Masahisa Watarai
watarai{at}obihiro.ac.jp

The essential mechanisms and virulence factors enabling Brucella species to survive and replicate inside host macrophages are not fully understood. The authors previously reported that a putative guanosine 5'-diphosphate 3'-diphosphate (ppGpp) mutant (spoT mutant) of Brucella abortus failed to replicate in HeLa cells. The present study showed that the pattern of surface proteins and morphological change of the spoT mutant were different from B. abortus wild-type. B. abortus wild-type changed its morphology upon treatment with ppGpp synthetase I activation inhibitor. In various tests under stress conditions, including nutrient starvation, nitric oxide resistance, acid resistance and antibiotic resistance, the spoT mutant had a lower stress resistance than B. abortus wild-type. Although the spoT mutant has the same smooth phenotype and LPS profile as B. abortus wild-type, it had a higher rate of adherence to macrophages but lower internalization and intracellular replication within macrophages. The spoT mutant did not co-localize with either late endosomes or lysosomes and was almost cleared from the spleens of mice after 10 days, without splenomegaly. RT-PCR was used to detect spoT mRNA from around 10⁶ cells incubated in low-pH enriched medium; it showed that the expression of spoT increased after 30 min incubation. The data suggest that SpoT does not contribute to intracellular trafficking of B. abortus, but contributes to the maintenance of bacterial morphology and the physiological adaptation required for intracellular replication.