Brief heat treatment increases cytotoxicity of Mannheimia haemolytica leukotoxin in an LFA-1 independent manner

Abstract

Mannheimia haemolytica is an important respiratory pathogen in cattle. Its predominant virulence factor is a leukotoxin (LKT) that is a member of the RTX family of exotoxins produced by a variety of gram negative bacteria. LKT binds to the CD18 chain of β2 integrins on bovine leukocytes, resulting in cell death. In this study, we show that brief heat treatment of native LKT (95 °C for 3 min) results in increased cytotoxicity for BL-3 (bovine lymphoblastoid) cells. Similar heat treatment restored the activity of LKT that had been rendered inactive by incubation at 22 °C for 3 days. A hallmark of LKT is that its toxicity is restricted to leukocytes from cattle or other ruminant species. Surprisingly, heat treatment rendered LKT cytotoxic for human, porcine and canine leukocytes. Membrane binding studies suggested that heat-treated LKT binds to membrane proteins other than LFA-1, and is distributed diffusely along the BL3 cell membrane. Circular Dichroism spectroscopy studies indicate that heat treatment induced a small change in the secondary structure of the LKT that was not reversed when the LKT was cooled to room temperature. Thus, we speculate that these structural changes might contribute to the altered biological properties of heat-treated LKT.

Brief heat treatment causes a structural change and enhances cytotoxicity of theEscherichia coliα-hemolysin

“…The cytotoxicity of heat-activated HLY for this cell line ( Figure 2E) was similar to the previously reported results for heatactivated M. haemolytica LKT. (48) Interestingly, brief heat treatment also restored cytotoxicity to HLY that had been inactivated by incubation at room temperature for 3-5 days (white bars in Figure 2A-2D). Preincubation of heat-activated native HLY or inactive HLY with polyclonal anti-hemolysin antisera reduced the cytotoxicity effects of both heat-activated native HLY and inactive HLY (Figure 2A-2E).…”

“…(45,46) It is well recognized that heat treatment (>65°C) for 30-60 min eliminates the cytotoxicity of RTX toxins. (47) However, we recently demonstrated that a brief heat treatment of the M. haemolytica LKT resulted in greater cytotoxicity, (48) which was accompanied by an irreversible change in secondary structure as determined by circular dichroism (CD) spectroscopy. Varied biophysical approaches have identified other proteins (e.g.…”

“…(48) In exploratory studies, HLY was heated to various temperatures, in a heating block for various lengths of time (Figures 1 and 2) and immediately placed into an ice bath until added to target cells. Based on these preliminary studies, standard activation conditions of 1 min at 100°C were used in most other studies of HLY function.…”

“…(48) An Aviv Model 202SF CD spectrophotometer equipped with a Peltier temperature control system (Aviv Biomedical, Inc, Lakewood, NJ) was used to record CD spectra in a 0.1-cm cuvette. Data were recorded at 1-nm steps from 260 to ~ 205 nm with a spectral bandwidth of 1 nm and a 5 s averaging time at each wavelength.…”

“…(48) Therefore, we first examined brief heat treatment of HLY at various temperatures. Compared to unheated HLY, a significant increase in A498 cell cytotoxicity was observed when HLY was heated for 1 min at 90, 100, or 110°C ( Figure 1).…”