Brucella abortus bacA mutant induces greater pro-inflammatory cytokines than the wild-type parent strain

Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003, USA.
Microbes and Infection (Impact Factor: 2.86). 02/2007; 9(1):55-62. DOI: 10.1016/j.micinf.2006.10.008
Source: PubMed


The inner-membrane protein BacA affects Brucella LPS structure. A bacA deletion mutant of Brucella abortus, known as KL7 (bacA(mut)-KL7), is attenuated in BALB/c mice and protects against challenge. Thus, bacA mutation was a candidate for incorporation into live attenuated vaccines. We assessed bacA(mut)-KL7 in 2 additional mouse strains: the more resistant C57BL/6 that produces interferon-gamma throughout the infection and the highly susceptible interferon-gamma-deficient C57BL/6 in which brucellae exhibit continual exponential growth. While it was hypothesized that bacA(mut)-KL7 would exhibit even greater attenuation relative to its parent strain B. abortus 2308 in C57BL/6 mice than it did in BALB/c mice, this was not the case. Moreover, it was more pathogenic in C57BL/6 interferon-gamma-deficient mice than 2308 causing abscesses and wasting even though the splenic loads of bacA(mut)-KL7 were significantly lower. These 2 observations were correlated, respectively, with an ability of IFNgamma-activated macrophages to equivalently control strains 2308 and bacA(mut)-KL7 and the ability of bacA(mut)-KL7 organism and its LPS to induce greater amounts of pro-inflammatory cytokines than 2308. We conclude that attenuation properties of bacA mutation are dependent upon the nature of the host but more importantly that bacterial gene deletion can result in increased host pathology without an increase in bacterial load, crucial considerations for vaccine design.

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Available from: Radhika Goenka
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    • "This decrease seems to be necessary for intracellular adaptation of lon mutant from media culture condition. Recently, other groups have reported similar findings in virulent Brucella strain [22,23]. The recovery mechanisms and metabolic pathway proteins to establish intracellular Brucella adaptation were elucidated by a comprehensive analysis of its proteomes. "
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    ABSTRACT: The objective of this study was to isolate a Brucella lon mutant and to analyze the cytokine response of B. lon mutant during macrophage infection. A wild-type Brucella abortus strain was mutagenized by Tn5 transposition. From the mouse macrophage J774.A1 cells, total RNA was isolated at 0 hours, 6 hours, 12 hours, and 24 hours after infection with Brucella. Using mouse cytokine microarrays, we measured transcriptional levels of the cytokine response, and validated our results with a reverse transcriptase-polymerase chain reaction (RT-PCR) assay to confirm the induction of cytokine messenger RNA (mRNA). In host J774.A1 macrophages, mRNA levels of T helper 1 (Th1)-type cytokines, including tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin-2 (IL-2), and IL-3, were significantly higher in the lon mutant compared to wild-type Brucella and the negative control. TNF-α levels in cell culture media were induced as high as 2 μg/mL after infection with the lon mutant, a greater than sixfold change. In order to understand the role of the lon protein in virulence, we identified and characterized a novel B. lon mutant. We compared the immune response it generates to the wild-type Brucella response in a mouse macrophage cell line. We demonstrated that the B. lon mutants induce TNF-α expression from the host J774.A1 macrophage.
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    • "The strategy of B. abortus is to evade the innate immune system and persist in the host long enough to be transmitted. The bacterium contains an unusual lipid A composing the LPS molecule, which is important for evading the host immune system during the early stages of infection (Parent et al., 2007). In addition, when entering the host intracellular space, B. abortus displays several strategies to avoid the cellular killing mechanism. "
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    • "evade sensing via TLR4 by producing a poorly recognized form of Lipid A. Brucella Lipid A contains a much longer fatty acid residue (C 28) compared with enterobacterial LPS (C12–C16), and this modification greatly diminishes its endotoxic properties by reducing TLR4 agonist activity (Fig. 1; Lapaque et al., 2006). A B. abortus bacA mutant, which fails to add the C28 acyl chain to Lipid A, induces more inflammation than wild-type bacteria , which likely contributes to its reduced infectivity in macrophages and in BALB/c mice (Ferguson et al., 2004; Parent et al., 2007). While in Yersinia pestis, a change in the LPS that increased its TLR4 agonist activity decreased its virulence, comparable studies to demonstrate definitively the crucial role of the non-stimulatory Lipid A in Brucella infection have not been reported (Montminy et al., 2006). "
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