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* Abstract The capacity of the Brucella spp. to establish and maintain long-term residence in the phagosomal compartment of host macrophages is critical to their ability to produce chronic infections in their mammalian hosts. The RNA binding protein host factor I (HF-I) encoded by the hfq gene is required for the efficient translation of the stationary-phase [sigma] factor RpoS in many bacteria, and a Brucella abortus hfq mutant displays a phenotype in vitro, which suggests that it has a generalized defect in stationary-phase physiology. The inability of the B. abortus hfq mutant to survive and replicate in a wild-type manner in cultured routine macrophages, and the profound attenuation displayed by this strain and its B. melitensis counterpart in experimentally infected animals indicate that stationary-phase physiology plays an essential role in the capacity of the brucellae to establish and maintain long-term intracellular residence in host macrophages. The nature of the Brucella HF-I-regulated genes that have been identified to date suggests that the corresponding gene products contribute to the remarkable capacity of the brucellae to resist the harsh environmental conditions they encounter during their prolonged residence in the phagosomal compartment.
Key Words brucellosis, bacterial pathogenesis, intracellular pathogens, macrophage, bacterial stress response
CONTENTS INTRODUCTION RESISTANCE OF THE BRUCELLAE TO KILLING BY HOST MACROPHAGES IS ESSENTIAL FOR THE ESTABLISHMENT AND MAINTENANCE OF CHRONIC INFECTION IN BOTH NATURAL HOSTS AND HUMANS STATIONARY-PHASE PHYSIOLOGY AND ITS POTENTIAL CONTRIBUTION TO THE SUCCESSFUL ADAPTATION OF THE BRUCELLAE TO THE ENVIRONMENTAL CONDITIONS ENCOUNTERED IN THE PHAGOSOMAL COMPARTMENT OF HOST MACROPHAGES THE IN VIVO PHENOTYPE OF B. ABORTUS AND B. MELITENSIS HFQ MUTANTS SUGGESTS THAT STATIONARY-PHASE PHYSIOLOGY IS ESSENTIAL FOR VIRULENCE IDENTIFICATION OF B. ABORTUS GENES DEPENDENT UPON HF-I FOR OPTIMAL EXPRESSION DURING STATIONARY PHASE HF-I-REGULATED GENES ENCODING DEFENSES AGAINST OXIDATIVE STRESS H-I-REGULATED GENES ENCODING POTENTIAL DEFENSES AGAINST ACID STRESS HF-I-REGULATED GENES POTENTIALLY INVOLVED IN THE SHIFT TO THE MAINTENANCE METABOLISM ASSOCIATED WITH STATIONARY-PHASE PHYSIOLOGY OTHER VIRULENCE-ASSOCIATED GENES IN BRUCELLA WITH LINKS TO STATIONARY-PHASE PHYSIOLOGY HOMOLOGS OF PROKARYOTIC STATIONARY-PHASE GENES IDENTIFIED IN THE B. MELITENSIS 16M AND B. SUIS 1330 GENOME SEQUENCES DOES BRUCELLA POSSESS A BONA FIDE RpoS? ARE QUORUM-SENSING AND STATIONARY-PHASE PHYSIOLOGY LINKED IN BRUCELLA? CONCLUSIONS
The Brucella spp. are gram-negative bacteria that infect a wide variety of mammals (65). In the natural host, infection of the pregnant female often leads to abortion. Infection of the male, on the other hand, leads to orchitis and epididymitis, which can result in infertility in the affected animal (25). The abortion and infertility caused by Brucella melitensis in sheep and goats, by B. abortus in cattle, by B. suis in swine, and B. ovis in sheep can have a profound economic impact upon the agricultural community in areas of the world where these hosts represent important food animals (22, 34, 75-77, 82). For this reason great effort has been expended in many areas to control the incidence of brucellosis in food animals. Infection with B. canis, a natural pathogen of dogs, can also cause serious problems for canine breeders and is the focus of surveillance programs targeted predominately at dogs of significant economic value, such as show and hunting dogs (12).
In geographic regions where brucellosis is endemic in food animals, the Brucella spp., particularly B. melitensis, B. abortus, and B. suis, also represent a significant public health concern (65). This is because of the relative ease with which these pathogens can be transmitted to humans, via contact with either infected animals or their products, Brucellosis in humans, also known as undulant fever, is a chronic, debilitating febrile illness that can last for months and is characteristically difficult to treat with antimicrobial therapy (102-104). Interestingly, although orchitis and epididymitis can occur in males during human infection, abortion in infected females is not considered a primary manifestation of human brucellosis. In a natural setting brucellosis is strictly a zoonotic disease in humans; it is not transmitted person to person. The brucellae are, however, highly infectious in a laboratory setting, and for this reason they are classified as Biosafety Level 3 organisms (97) and must be handled ha appropriately equipped laboratories.
With regard to their virulence in humans B. melitensis, B. suis, and B. abortus possess several properties that have made them attractive in the past as potential agents of biowarfare (16, 29, 47). These properties include their low infectious dose via the aerosol route, the chronic, debilitating nature of the disease they produce, the difficulty encountered in treating these infections with antibiotics, and the fact that no safe and effective vaccine exists that can be used to prevent human brucellosis. Because of the threat of these bacteria to the agricultural community and their potential use as agents of biowarfare or bioterrorism, the Brucella spp. fall in the category of "select agents" (96) and the possession and shipment of these organisms are now tightly regulated in many areas of the world.
RESISTANCE OF THE BRUCELLAE TO KILLING BY HOST MACROPHAGES IS ESSENTIAL FOR THE ESTABLISHMENT AND MAINTENANCE OF CHRONIC INFECTION IN BOTH NATURAL HOSTS AND HUMANS
The Brucella spp. are intracellular pathogens in their mammalian hosts (3, 35). They do not exist as commensals nor are they found free-living in the environment. Following entry into the host, the brucellae are taken up by macrophages, where they display a remarkable resistance to killing by these phagocytes. Indeed, the capacity of these bacteria to maintain long-term residence within macrophages serves as the basis for their ability to establish and maintain chronic infection. When the brucellae infect their natural hosts during the latter stages of pregnancy, these bacteria can also proliferate extensively in the placental trophoblasts that surround the developing fetus (59, 88). The resulting loss of placental integrity that results from this massive intracellular replication of the brucellae is thought to play an important role in the abortion that occurs during these infections (25).
Although the placental trophoblasts represent a key host cell for a limited time during pregnancy, the major site of residence of the brucellae in both the natural host and humans is the macrophage. Numerous experimental studies using cultured macrophages from a variety of hosts including cattle (36), humans (23), and mice (46) have shown a clear correlation between the ability of the brucellae to resist killing by these host phagocytes and virulence. Within host macrophages the brucellae remain enclosed in the phagosomal compartment (51). The extent to which the Brucella-containing phagosomes mature along the endosomal-lysosomal pathway in host macrophages is a matter of contention and presently an active area of investigation (35). Nevertheless, there is considerable experimental evidence indicating that the brucellae withstand harsh environmental conditions, including exposure to reactive oxygen intermediates (ROIs) (37, 46), acidic pH (71, 74), and nutrient deprivation (1, 18, 28), during their long-term residence in the phagosoreal compartment of host macrophages. Not unexpectedly, it also appears that the brucellae are well adapted from a physiologic standpoint to meet the challenges encountered in their natural ecological niche (50, 52, 80).
STATIONARY-PHASE PHYSIOLOGY AND ITS POTENTIAL CONTRIBUTION TO THE SUCCESSFUL ADAPTATION OF THE BRUCELLAE TO THE ENVIRONMENTAL CONDITIONS ENCOUNTERED IN THE PHAGOSOMAL COMPARTMENT OF HOST MACROPHAGES
When bacteria are presented with unlimited nutrients, and in the case of respiring bacteria with an optimal level of terminal electron accepter, they typically display exponential or "balanced" growth. In contrast, when nutrients become limiting, they enter a physiologic state known as stationary phase (38). Upon transition into stationary phase, the bacteria switch from a state of active growth geared toward the production of cell mass and new daughter cells to a maintenance metabolism oriented toward maintaining cell viability over prolonged periods with little or no net increase in cell number (44). Coincident with entry into stationary-phase physiology bacteria also become increasingly resistant to a wide variety of environmental stresses including exposure to ROIs, acidic pH, and nutrient deprivation.
The capacity of the brucellae to withstand these environmental stresses likely plays a critical role in their ability to resist killing by macrophages and ultimately produce disease in the host. With this in mind, it seems obvious that stationary-phase physiology would be of great potential benefit to the brucellae with regard to their ability to successfully adapt to the harsh environmental conditions encountered in the phagosomal compartment. In fact, there are several lines of …