Histoplasma capsulatum and stress during pathogenesis

During the pathogenesis of Histoplasma capsulatum infection, partly resulting from the dimorphic transition of this mammalian fungal pathogen, it encounters significant environmental stress during growth before and after ingestion by neutrophils and activated macrophages of the host. Some stress experienced during growth includes elevated temperature, oxidative stress due to both decoupling of fungal electron transport (resulting in a hyperoxidative state) and the host cell antimicrobial oxidative burst, and the toxic environmental conditions of the phagolysosome. In spite of the considerable toxicity of the host cell defensive response H. capsulatum is still able to grow and propagate within the host immune cell.

Using various screening methods, we have isolated and characterized a number of H. capsulatum genes we believe to be involved in promoting pathogenesis. Expression of these genes is necessary for the degradation of hydrogen peroxide (CATA, CATB, and CATP), for an alternate pathway for biosynthesis of ATP (AOX1), during the initial steps of the glycosylation pathway (GDT1), during the citric acid cycle (IDH1), and during cell wall biosynthesis (chs1, chs2, and chs3). We have investigated the expression of these genes, using northern blot analysis, during a number of growth conditions that mimic those of the defensive mechanisms of the host immune cell during infection.

We have found a complex order of regulated expression for a number of these genes that indicates considerable redundancy of protective mechanisms operating at the earliest stages of and throughout the infection process. In response to oxidative stress, a number of catalase genes are expressed in either a housekeeping constitutive manner (CATB and CATP), while another catalase gene is only expressed in response to oxidative stress as added protection (CATA). In response to increased metabolic needs, due to electron transport decoupling during the dimorphic transition or as a result of oxidative stress that occurs during infection, the alternative oxidase gene shows a complex set of variations in mRNA levels. Furthermore, genes involved in such housekeeping functions, such as glycosylation and the citric acid cycle (GDT1, IDH1), show altered levels of mRNA while the pathogen is grown under conditions of stress. Finally, genes involved in cell wall biosynthesis show altered mRNA levels during stress (chs1, chs2, chs3, chs4, and chs5), indicating they play a role in pathogen virulence. Many of these alterations in the mRNA levels of these genes show they are responding in a dose dependent manner.

Taken in total, our results show that H. capsulatum has evolved considerable defensive mechanisms in order to counter the host antimicrobial response. These defensive mechanisms likely result in the expression of a wide array of fungal genes in a complex cascade in order to offer protection from the host antimicrobial mechanisms during an ongoing infection. Our present results have been collected using in vitro growth conditions but we are pursuing confirmation of these results using in vivo cell culture and animal model systems. We are also in the process of isolating possible knockout strains for these various genes to confirm their significance to the virulence of this fungal pathogen.