Roles of Brucella abortus SpoT in morphological differentiation and intramacrophagic replication

Abstract

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 59-diphosphate 39-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 10 6 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. INTRODUCTIONBrucellosis is a major bacterial zoonosis and an important cause of a serious debilitating disease in humans and abortion and sterility in domestic animals. This disease is caused by species of the genus Brucella, classified in the a2 subdivision of Proteobacteria, small Gram-negative and facultative intracellular pathogens that can multiply within professional and non-professional phagocytes (Delrue et al., 2001;Detileux et al., 1990). The genus Brucella consists of seven species according to antigenic variation and primary host: B. melitensis (sheep and goats), B. suis (hogs), B. abortus (cattle), B. ovis (sheep), B. canis (dogs), B. neotomae (wood rats) and B. maris (marine mammals) (Ko & Splitter, 2003). In contrast to other intracellular pathogens, Brucella species do not produce exotoxins, antiphagocytic capsules, thick cell walls, resistance forms or fimbriae and do not show antigenic variation (Finlay & Falkow, 1997).A key aspect of the virulence of Brucella species is their ability to proliferate within professional and nonprofessional phagocytic host cells, therefore successfully bypassing the bactericidal effects of phagocytes. Their virulence and chronic infections are thought to be due to their ability to avoid the killing mechanisms within host cells (Comerci et al., 2001;Ugalde, 1999). Neither virulence factors that allow intracellular survival of Brucella species nor the specific factors that induce such virulence have been elucidated. B. abortus is inte...

ppGpp Conjures Bacterial Virulence

“…In B. abortus, rsh Ba transcription is induced by acid stress as assessed by RT-PCR analysis of broth cultures (104). Consistent with this model, B. abortus rsh Ba mutants are more sensitive to acidic conditions in broth than WT bacteria.…”

“…Both replication and survival in mouse macrophage cultures require Rsh Ba . Likewise, fewer rsh Ba mutant bacteria than WT bacteria are recovered from the spleens of mice 10 days after infection (104).…”

RelA regulates virulence and intracellular survival of Francisella novicida

“…The stringent response has been reported to be involved in intracellular survival in many pathogens, such as Campylobacter jejuni (Gaynor et al, 2005), L. monocytogenes (Taylor et al, 2002) and Brucella abortus (Kim et al, 2005). To examine whether the attenuation observed in vivo is due to a defect in intracellular survival, the ability of U112 DrelA : : cam to infect and replicate within murine macrophages was assessed (Fig.…”

The Molecular Alarmone (p)ppGpp Mediates Stress Responses, Vancomycin Tolerance, and Virulence inEnterococcus faecalis

“…Studies have correlated changes in (p)ppGpp levels to the expression of virulence traits, including stress survival (12,20,25,54), biofilm formation (2,25,46), antibiotic resistance (14,19,20,40), and persistence (5,14,20,21,35,42). In some cases, animal models have been used to provide unequivocal evidence of the role of (p)ppGpp in virulence (6,9,10,16,39,46).…”