H. capsulatum is an intracellular fungal pathogen that colonizes the macrophage phagolysosome, ultimately triggering host cell lysis. Here, we used a forward genetic approach to identify an enzyme, HMG CoA lyase, which is required for growth within macrophages and host cell lysis. Interestingly, due to the metabolic defects that ensue in response to HMG CoA lyase deficiency, it is indirectly required for maintenance of phagolysosomal pH. Studies in other organisms have shown that HMG CoA lyase catalyzes the final step in leucine catabolism; thus, HMG CoA lyase deficiency results in the inability of microbes to grow when leucine is the sole carbon source. In addition, both microbes and humans with HMG CoA lyase deficiency accumulate acidic species that arise due to the block in leucine metabolism. In the case of H. capsulatum, we observed that Hcl1 is dispensable for growth in standard laboratory medium supplemented with glucose but required for growth on minimal medium when leucine is substituted for glucose as the major carbon source. In addition, growth of H. capsulatum in unbuffered medium revealed that Hcl1 is required to prevent the accumulation of acidic species in the culture supernatant. During colonization of the phagolysosome of macrophages, the hcl1 mutant is present in an acidified compartment, suggesting that these acidic species can lower the pH of the phagolysosome, which, in turn, could potentially activate lysosomal hydrolases and restrict microbial growth.
These studies highlight the challenges faced by intracellular microbes that grow within the phagosome of host cells (reviewed in reference 1
). The initiation of phagocytosis of microbes by macrophages is normally accompanied by a superoxide burst catalyzed by the NADPH complex. Subsequently, the normal course of phagosome maturation involves transient interactions between the phagosome and a variety of intracellular organelles, ultimately culminating in fusion with lysosomes. As this maturation process occurs, phagosomal pH drops and the phagosome acquires lysosomal hydrolases that are active at acidic pH. H. capsulatum
counters normal phagosome function and maturation in at least two key ways. First, H. capsulatum
fails to trigger a superoxide burst in resting macrophages (3
) and produces an extracellular superoxide dismutase for protection against the reactive oxygen species generated upon phagocytosis by activated macrophages and polymorphonuclear leukocytes (24
). Second, although the H. capsulatum
phagosome undergoes fusion with the lysosome (in murine macrophages), the fungus uses unknown means to block acidification of phagolysosomes (4
). It has been assumed that the ability of H. capsulatum
to inhibit phagolysosome acidification is critical for survival of the fungus within host cells. Our data demonstrate that the hcl1
mutant is competent to neutralize acidic pH in vitro
but cannot maintain neutral pH as it grows, presumably due to the production of acidic species. Correspondingly, in the macrophage we observed that the hcl1
mutant, unlike wild-type cells, is present in an acidified phagosome, suggesting that the phagosome containing the mutant cells may be more hydrolytically competent than the phagosome containing wild-type H. capsulatum
. Consistent with this model, the hcl1
mutant is clearly restricted for growth within macrophages. We hoped to determine whether neutralization of the phagosome with the weak base chloroquine (7
) could rescue the intracellular growth defect of the hcl1
mutant; if so, these results would strongly suggest that the intracellular growth deficiency of the mutant is due to acidification of the phagosome. However, these experiments were not possible in BMDMs for technical reasons (data not shown).
The inability of the hcl1
mutant to grow within macrophages may also reflect the nutritional environment of the phagosome. Studies of other phagosomal pathogens such as Leishmania major
and Mycobacterium tuberculosis
have shown that enzymes required for gluconeogenesis or the glyoxylate shunt, respectively, are required for colonization or persistence in macrophages and mice, suggesting that the phagosome is a glucose-poor environment (25
). Similarly, the fungal pathogen Candida albicans
upregulates its expression of isocitrate lyase, a key glyoxylate cycle enzyme, after phagocytosis by host cells and requires this enzyme for full virulence in the mouse model of pathogenesis (27
). HMG CoA lyase in other organisms is required for growth when leucine is the sole carbon source, since HMG CoA lyase is a key enzyme in leucine catabolism. A similar result was observed for H. capsulatum
, since the hcl1
mutant could not grow in vitro
when leucine was substituted for glucose. The levels of leucine or other amino acids in the Histoplasma
-containing phagolysosome is unknown, although the inability of the hcl1
mutant to grow within macrophages leads us to speculate that the phagolysosome is a glucose-poor, leucine-replete environment. Interestingly, leucine catabolism generates acetyl-CoA, which can be used as a carbon source when assimilated through the glyoxylate cycle (bypassing the catabolic steps of the standard tricarboxylic acid cycle). Thus, the requirement for Hcl1 in macrophage colonization and virulence may correlate with its link to carbohydrate metabolism.
Whether Hcl1 affects other metabolic processes in the H. capsulatum
cell is unknown. In vertebrates, HMG CoA lyase plays additional roles in energy transfer within and between cells. For example, HMG CoA lyase is required to link fatty acid oxidation in the mitochondria to ketogenesis, which is a critical mode of energy transport in higher eukaryotes (28
). Fatty acid oxidation produces acetoacetyl CoA, which is then converted into HMG CoA by mitochondrial HMG CoA synthase. HMG CoA lyase then converts HMG CoA into acetoacetate, which is a “ketone body” that is produced in the liver and used to transport energy to other organs when glucose is not available. However, lower eukaryotes lack mitochondrial HMG synthase, and thus there is no known direct link between fatty acid oxidation and HMG CoA lyase-dependent metabolism in fungi.
Despite the profound growth defect of the hcl1 mutant in BMDMs and the J774 macrophage-like cell line, the mutant was able to replicate in the mouse model of pathogenesis. However, mice infected with the hcl1 mutant took significantly longer to succumb to infection than wild-type mice, indicating that the mutant is partially attenuated for virulence. Interestingly, the fungal burden in the lungs of mice infected with the wild-type strain at time of death (day 7, ) was indistinguishable from the fungal burden in the lungs of mice infected with the hcl1 mutant at the same day postinfection (), but mice in the hcl1-infected cohort did not succumb to infection for another 3 days (). In addition, there was no significant increase in CFU between day 7 and day 10 for mice infected with the hcl1 mutant (). Finally, fungal burden in the spleens of mice infected with either wild-type H. capsulatum or the hcl1 mutant at day 8 was not significantly different (P = 0.4, ), indicating that the mutant does not have a gross dissemination defect. These data evoke the hypothesis that an aspect of infection other than fungal burden, such as the host inflammatory response, could differ in mice infected with the wild-type versus mutant strains. Alternatively, it could be the case that in vivo growth kinetics of the wild-type and mutant strains differ at earlier time points in infection not examined in this study, which could affect subsequent symptomatology and disease progression. In addition, these data suggest that despite its profound growth defect in BMDMs, the hcl1 mutant replicates well within an unknown cell type in vivo, such as alveolar macrophages and/or inflammatory monocytes. Future studies of the role in Hcl1 in fungal pathogenesis will continue to utilize the mutant as an informative tool to dissect the nutritional and metabolic requirements of life in the Histoplasma phagosome, as well as the relationship between intracellular growth and immune response during infection of the host.