Cytoplasmic and extracellular expression of pharmaceutical-grade mycobacterial 65-kDa heat shock protein in Lactococcus lactis

Abstract

ABSTRACT. Lactic acid bacteria (LAB) are an attractive and safe alternative for the expression of heterologous proteins, as they are nonpathogenic and endotoxin-free organisms. Lactococcus lactis, the LAB model organism, has been extensively employed in the biotechnology field for large-scale production of heterologous proteins, and its use as a "cell factory" has been widely studied. We have been particularly interested in the use of L. lactis for production of heat shock proteins (HSPs), which reportedly play important roles in the initiation of innate and adaptive immune responses. However, this activity has been questioned, as LPS contamination appears to be responsible for most, if not all, immunostimulatory activity of HSPs. In order to study the effect of pure HSPs on the immune system, we constructed recombinant L. lactis strains able to produce and properly address the Mycobacterium leprae 65-kDa HSP (Hsp65) to the cytoplasm or to the extracellular medium, using a xylose-induced expression system. Expression of mycobacterial Hsp65 in Lactococcus lactisApproximately 7 mg/L recombinant Hsp65 was secreted. Degradation products related to lactococcal HtrA activity were not observed, and the Limulus amebocyte lysate assay demonstrated that the amount of LPS in the recombinant Hsp65 preparations was 10-100 times lower than the permitted levels established by the U.S. Food and Drug Administration. These new L. lactis strains will allow investigation of the effects of M. leprae Hsp65 without the interference of LPS; consequently, they have potential for a variety of biotechnological, medical and therapeutic applications.

Hsp65-producing Lactococcus lactis prevents experimental autoimmune encephalomyelitis in mice by inducing CD4+LAP+ regulatory T cells

“…Azevedo and coworkers showed that L. lactis expressed Hsp65 preparations contained less endotoxin (lypopolysaccharide, LPS) than the limit set by the Food and Drug Administration (FDA) [35]. …”

“…As described elsewhere [35], a recombinant L. lactis strain NCDO2118 able to secrete M. leprae Hsp65, using a xylose-inducible expression system (XIES), was constructed. The constructed vector (pSEC: hsp65 ) directed the expression of Hsp65 to the extracellular medium.…”

“…Daily, a fresh L. lactis total culture (bacteria plus supernatant obtained as described in item 2.3)was offered to mice. Since each mouse drank about 5 ml of culture per day (data no shown) containing 7 µg/ml [35] of M. leprae Hsp65, the total dose of bacteria per mouse was estimated to be 5 × 10 9 CFU and the total daily dose of M. leprae Hsp65-was about 35 µg per mouse. However, since live bacteria are fed, the effective dose is larger than that.…”

Overexpression of Arabidopsis HsfA1a enhances diverse stress tolerance by promoting stress-induced Hsp expression

“…The molecular basis underlying the role of Hsfs in stress tolerance is still not clear. Expression of Hsps as chaperones, including Hsp18.2 and Hsp70, protects cells against injuries associated with various stressors (Simões-Araújo et al, 2003;de Azevedo et al, 2012;Magdalena et al 2012). On the other hand, HsfA1a can bind various promoter targets that are not limited to classical Hsp genes and also include other genes (Guo et al, 2008), implying a broad physiological basis for the stress tolerance role of HsfA1a.…”

Previous Ingestion ofLactococcus lactisby Ethanol-Treated Mice Preserves Antigen Presentation Hierarchy in the Gut and Oral Tolerance Susceptibility

“…When required, chloramphenicol (10 lg/ml) was added to the media. Hsp65-producer L. lactis (Hsp65 LL; NCDO 2118 strain) which is able to secrete Mycobacterium leprae Hsp65 upon xylose-inducible expression system, was constructed as described elsewhere (de Azevedo et al, 2012). Briefly, a single colony of Hsp65 LL was grown at 30°C, without agitation, in 5 ml of GM17, containing chloramphenicol (10 lg/ml).…”