Changes in membrane fluid state and heat shock response cause attenuation of virulence

Abstract

So far attenuation of pathogens has been mainly obtained by chemical or heat treatment of microbial pathogens. Recently, live attenuated strains have been produced by genetic modification. We have previously demonstrated that in several prokaryotes as well as in yeasts and mammalian cells the heat shock response is controlled by the membrane physical state (MPS). We have also shown that in Salmonella enterica serovar Typhimurium LT2 (Salmonella Typhimurium) overexpression of a Delta(12)-desaturase gene alters the MPS, inducing a sharp impairment of transcription of major heat shock genes and failure of the pathogen to grow inside macrophage (MPhi) (A. Porta et al., J. Bacteriol. 192:1988-1998, 2010). Here, we show that overexpression of a homologous Delta(9)-desaturase sequence in the highly virulent G217B strain of the human fungal pathogen Histoplasma capsulatum causes loss of its ability to survive and persist within murine MPhi along with the impairment of the heat shock response. When the attenuated strain of H. capsulatum was injected in a mouse model of infection, it did not cause disease. Further, treated mice were protected when challenged with the virulent fungal parental strain. Attenuation of virulence in MPhi of two evolutionarily distant pathogens was obtained by genetic modification of the MPS, suggesting that this is a new method that may be used to produce attenuation or loss of virulence in both other intracellular prokaryotic and eukaryotic pathogens. This new procedure to generate attenuated forms of pathogens may be used eventually to produce a novel class of vaccines based on the genetic manipulation of a pathogen's membrane fluid state and stress response.

FIG. 1.

Growth curve of yeast form of H. capsulatum G217B and HcD3 strains at 37°C. The values of the graph represent one of four independent growth curves.

FIG. 2.

Hsp70 gene expression in the H. capsulatum HcD3 strain. Yeast cells of H. capsulatum G217B and of HcD3 strains were grown at 34° and 37°C and heat shocked for 30 min at different temperatures. The Hsp70 gene was highly expressed in strain G217B (from 34° to 37°C, from 34° to 42°C, and from 37° to 42°C), whereas in strain HcD3 Hsp70 transcription was detectable only from 34° to 42°C.

FIG. 3.

Murine MΦ infection with H. capsulatum. Yeast phase cells were grown at 34°C before infection. (A) Yeast phase cells of strain HcD3 as well as the control G217B strain were internalized inside MΦ. (B) Yeast cells recovered from MΦ lysed at different time points of infection were counted. G217B grew inside MΦ while no growth was detectable for the HcD3 strain. Statistical differences were evaluated by a two-tailed Student's t test. P values less than 0.05 were considered significant. Each experiment in duplicate was performed at least three times. The figure shows one of such experiments. The pictures were taken after the macrophages were washed. Arrows point to internalized yeasts cells.

FIG. 4.

Mouse infection with H. capsulatum. (A) Mice infected with 10⁶ yeast cells of strain G217B died within 40 days after infection, whereas all mice infected with strain HcD3 survived. (B) About 10⁷ G217B yeast cells killed all mice after 23 days, whereas all mice infected with 10⁷ HcD3 yeast cells survived up to 30 days after infection. (C) All mice infected with 5 × 10⁷ G217B yeast cells died within 6 days, while 7 out of 10 mice infected with the same inoculum of HcD3 survived up to 45 days after infection. (D) Surviving mice were challenged with a lethal dose (5 × 10⁷ yeast cells) of the virulent strain G217B. After 20 days (60 days from the initial infection with attenuated HcD3 strain) six mice still survived.