Zinc Uptake System (znuA Locus) of Brucella abortus is Essential for Intracellular Survival and Virulence in Mice

Abstract

ABSTRACT. Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in professional and nonprofessional phagocytes, and cause abortion in domestic animals and undulant fever in humans. The mechanism and factors of virulence are not fully understood. High-affinity zinc uptake system protein mutant (znuA mutant) showed reduced growth in zinc chelated medium, and failed to replicate in HeLa cells and mouse bone marrow-derived macrophages. Transformation of znuA mutant with a shuttle vector pBBR1MCS-4 containing znuA gene restored the growth in zinc chelated medium and intracellular replication in HeLa cells and macrophages to a level comparable to that of wild-type strain. Bacterial internalization into HeLa cells and macrophages and co-localization with either late endosomes or lysosomes of znuA mutant were not different from those of wild-type strain. These results suggest that znuA does not contribute to intracellular trafficking of B. abortus, but contributes to utilization of zinc required for intracellular growth. KEY WORDS: Brucella abortus, intracellular growth, zinc, znuA.J. Vet. Med. Sci. 66(9): 1059-1063, 2004 Brucella abortus, a facultative, gram-negative, intracellular pathogen, is the etiologic agent of brucellosis, a widely distributed zoonosis [22]. The establishment of chronic infection depends on the ability of brucellae to survive within phagocytes [9]. In contrast to other intracellular pathogens, Brucella species do not produce exotoxins, antiphagocytic capsules or thick cell walls, resistance forms or fimbriae and do not show antigenic variation [7]. Intracellular survival and replication are key virulence features of Brucella because mutants defective for these attributes are also avirulent [10,14]. A key aspect of the virulence of Brucella successfully bypasses the bactericidal effects of phagocytes, and their virulence and chronic infections are thought to be due to their ability to avoid the killing mechanisms within host cells [5,20].The genetic basis of Brucella virulence is still poorly understood. The VirB type IV secretion system of Brucella has been identified recently [16]. This operon is composed of 13 open reading frames (ORFs) that share homology with other bacterial type IV secretion systems in the intracellular trafficking of pathogens. Deletion or polar and non-polar mutations of these ORFs were not able to replicate and survive within phagocytes [19,21], and internalization and uptake pathway in phagosome trafficking between wildtype and virB mutant showed different patterns [11]. Thus, the VirB proteins of B. abortus are thought to be constituents of the secretion apparatus. The genus Brucella does not contain plasmids naturally, and it is therefore possible that the proteins encoded by virB genes are involved in protein secretion rather than conjugation. A possible role in virulence is to inject effector molecules, which help in the establishment of replication niche into the host cell.Zinc plays an important role in living organism...

Interleukin-6 regulates the zinc transporter Zip14 in liver and contributes to the hypozincemia of the acute-phase response

“…Similarly, a biological role for the depression of plasma zinc during infections has not yet been experimentally defined. Zinc is needed for microbial pathogenicity (5,35), and a hypozincemia may limit zinc availability. Zn 2ϩ mobilization induced by nitric oxide may also be an explanation for the up-regulation of Zip14 during inflammation and infection (24).…”

“…A beneficial role for corresponding hypozincemia has not been deduced. Zinc availability for hepatic synthesis of acute-phase proteins, regulation of gluconeogenesis, control of reactive species (e.g., nitric oxide or hydroxyl radicals), and control of microbial growth are among the postulated functions (4,5).…”

Deletion of znuA Virulence Factor Attenuates Brucella abortus and Confers Protection against Wild-Type Challenge

“…The aromatic amino acid pathway aroC mutant of B. suis (14), the B. abortus lipid A fatty acid-transporting gene bacA mutant (12,26), the type IV secretion virB mutant (11), and the ferrochelatase hemH mutant (2) showed highly attenuated virulence, but their protective efficacy has yet to be reported. B. abortus nicotinamidase pncA (22) and exopolysaccharide transporter exsA (33) mutants showed substantial protection, but these vaccine strains still colonized the host even after 6 weeks. One promising live brucellae vaccine candidate was shown with the cyclic ␤-1,2-glucan B. abortus cgs mutant of S19, which effectively cleared within 4 weeks and conferred complete protection against low-dose, not high-dose, challenge with wild-type B. abortus 2308 (6).…”

Structure and metal binding properties of ZnuA, a periplasmic zinc transporter from Escherichia coli

“…In addition, these cells are unable to take up 65 Zn 2+ [3]. Mutants deficient in the expression of ZnuABC in pathogens such as Hemophilus influenzae and Brucella abortus exhibit decreased virulence as well as retarded transformation and growth rates [4,5]. Thus, ZnuABC might play an important role in bacterial survival during host infection.…”