HrpA, a new ribosome-associated protein which appears in heat-stressed Mycobacterium bovis bacillus Calmette-Guérin

Abstract

A novel 18-kDa heat shock protein, HrpA, has been identified from Mycobacterium bovis BCG. HrpA was rapidly synthesized in membrane and ribosome fractions but not in the cytoplasmic fraction under heat shock stress. HrpA bound tightly to 70S ribosomes, mainly in 30S subunits. HrpA might be involved in the initiation step of translation at high temperature.Bacillus Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis, is the most widely used live vaccine against tuberculosis in the world, but the efficacy of this vaccine varies considerably (5). For more effective prevention and treatment of tuberculosis, new strategies and a deeper understanding of the bacterium, including the host-pathogen interactions, are needed. For mycobacteria, the intracellular environment of the macrophage is one of the most hostile environments. Some reports provide evidence that mycobacteria respond during infection to macrophages of the host with increased synthesis of proteins (1, 9). Major proteins induced by mycobacteria within macrophages are heat shock proteins (HSPs).Recently, we found that the 65-and the 12-kDa HSPs associated with ribosomes obtained from BCG cells grown under favorable culture conditions (11). To define the relationship between HSPs and ribosomes, we first investigated the localization of HSPs in BCG cells grown at 37°C and under heat stress conditions. In the course of this study, we found a new ribosome binding protein.Induction of heat shock response. M. bovis BCG substrain Tokyo was used. Culture was carried out aerobically in Middlebrook 7H9 medium supplemented with albumin-dextrose-catalase (ADC; Difco, Detroit, Mich.) and 0.05% Tween 80 at 37°C with shaking (160 rpm). Temperature shifts were performed by transferring the culture grown to an optical density at 590 nm of 0.6 to other water baths kept at the appropriate temperatures (42 to 48°C). Bacteria were labeled with 2 Ci of L-[ 35 S]methionine and L-[35 S]cysteine ml Ϫ1 [specific activity, 1,000 Ci mmol Ϫ1 (Pro-mix TM ; Amersham, Little Chalfont, England)] for the last 30 min of the heat pulse. Labeling was continued for a further 90 min during recovery at 37°C to allow a total labeling period of 2 h. Control cells were labeled at 37°C for 2 h. The labeled cells were harvested and washed with ice-cold TMNSH buffer [10 mM Tris-HCl (pH 7.5), 10 mM (CH 3 COO) 2 Mg, 60 mM NH 4 Cl, 6 mM 2-mercaptoethanol]. Protein synthesis was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (8) and autoradiography. When cultures were shifted to high temperature, proteins with apparent molecular masses of 90, 70, 65, 32, 22, and 18 kDa were synthesized prominently (Fig. 1A). The 65-kDa protein was strongly induced only when the temperature was shifted to 42°C (data not shown). Maximal synthesis of 90-, 70-, 32-, 22-, and 18-kDa proteins occurred at 45°C (data not shown). The 18-kDa protein was not induced at 37°C but was strongly induced when cells were heat shocked for 45 min at 45°C (Fig. 1A).Intracellular localization of ...

Evaluation of Humoral Immunity toMycobacterium tuberculosis-Specific Antigens for Correlation with Clinical Status and Effective Vaccine Development

“…Although these experiments were performed using mouse models, they suggested that humoral immunity could at least modify or reduce the growth of Mtb [ 31 , 32 ]. HrpA was identified by Ohara et al in 1997 [ 60 ] with a 30% homology to Acr, which increases to 40% when comparing Acr core residues [ 61 ]. It was later demonstrated that the expression of HrpA is upregulated by heat-shock, nitrate, or macrophage engulfment, suggesting that HrpA is one of the early immune targets of Mtb antigens [ 62 ].…”

Identification of a Novel DNA‐Binding Protein from Mycobacterium bovis Bacillus Calmette‐Guérin

“…4A). Second, each subcellular fraction from BCG was prepared as described previously (20). These samples were electron-transferred on the membrane and reacted with the and-MDPI antibody (Fig.…”

Antigen 85A and mycobacterial DNA‐binding protein 1 are targets of immunoglobulin G in individuals with past tuberculosis

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