The Brucella HtrA protease contributes to resistance to oxidative stress

Brucella are gram-negative, facultative intracellular, bacterial pathogens of animals and man. Brucella species cause an abortigenic disease as well as a chronic infection in animals while human brucellosis is a chronic debilitating illness characterized by fever, headache, and arthritis. The capacity of the brucellae to survive and replicate for prolonged periods in host macrophages is believed to be key to disease production, especially during a chronic infection. Although the biology of the Brucella-macrophage interaction is poorly understood, the ability of Brucella to resist oxidative killing has been shown to be important for intracellular survival.;The high-temperature requirement A (HtrA) protease is a periplasmic stress response protein that degrades damaged proteins before they accumulate to toxic levels within the bacterial cell. Genetic and biochemical evidence suggests that HtrA can degrade proteins damaged by oxygen radicals. This function could be particularly important when the brucellae are exposed to the products of the oxidative burst of host phagocytes. To determine the role of the Brucella HtrA protease in pathogenesis, B. abortus and B. melitensis htrA mutants were constructed and characterized. Brucella htrA mutants are more sensitive to oxidative killing in vitro when compared to the wildtype strain. Brucella abortus htrA mutants also show decreased survival in cultured murine macrophages and increased sensitivity to killing by polymorphonuclear phagocytes compared to the virulent parental strain. However, htrA mutants are still capable of intracellular replication in cultured macrophages after an initial period of killing. Consistent with the capacity of Brucella htrA mutants to replicate within cultured murine macrophages, Brucella htrA mutants are also able to produce a chronic spleen and liver infection in the BALB/c mouse model of brucellosis. Together, these results suggest that the Brucella HtrA has a biological role that is similar to that of previously described bacterial HtrA proteins. However, unlike the complete attenuation observed in two other intracellular pathogens, Salmonella and Yersinia, Brucella htrA mutants are able to produce chronic disease in experimentally infected animals suggesting that the Brucella HtrA protease is not essential for virulence.