The 3-hydroxy-methylglutaryl coenzyme A lyase HCL1 is required for macrophage colonization by human fungal pathogen Histoplasma capsulatum

Abstract

Histoplasma capsulatum is a fungal respiratory pathogen that survives and replicates within the phagolysosome of macrophages. The molecular factors it utilizes to subvert macrophage antimicrobial defenses are largely unknown. Although the ability of H. capsulatum to prevent acidification of the macrophage phagolysosome is thought to be critical for intracellular survival, this hypothesis has not been tested since H. capsulatum mutants that experience decreased phagosomal pH have not been identified. In a screen to identify H. capsulatum genes required for lysis of bone marrow-derived macrophages (BMDMs), we identified an insertion mutation disrupting the H. capsulatum homolog of 3-hydroxy-methylglutaryl coenzyme A (HMG CoA) lyase (HCL1). In addition to its inability to lyse macrophages, the hcl1 mutant had a severe growth defect in BMDMs, indicating that HMG CoA lyase gene function is critical for macrophage colonization. In other organisms, HMG CoA lyase catalyzes the last step in the leucine catabolism pathway. In addition, both fungi and humans deficient in HMG CoA lyase accumulate acidic intermediates as a consequence of their inability to catabolize leucine. Consistent with observations in other organisms, the H. capsulatum hcl1 mutant was unable to grow on leucine as the major carbon source, caused acidification of its growth medium in vitro, and resided in an acidified vacuole within macrophages. Mice infected with the hcl1 mutant took significantly longer to succumb to infection than mice infected with the wild-type strain. Taken together, these data indicate the importance of Hcl1 function in H. capsulatum replication in the harsh growth environment of the macrophage phagosome.

Fig 1

The FE6-C3 mutant contains an insertion in the HCL1 ORF. (A) Schematic of the HCL1 genomic locus showing the site of the TDNA insertion in the hcl1 mutant, the region of the HCL1 locus that was used to generate the complementation construct, and the region of the HCL1 gene that was used to probe transcript levels. Tick marks are 1 kb apart. (B and C) Total RNA was isolated from wild-type (WT), the hcl1 mutant, and the complemented strain (hcl1+HCL1) grown in broth culture at 37°C. HCL1 mRNA levels were monitored by quantitative RT-PCR (B) and Northern blot analysis (C). Primers for qRT-PCR analysis are described in Materials and Methods and located in the coding sequence of HCL1.

Fig 2

Key catalytic residues are conserved in the H. capsulatum HMG CoA lyase homolog Hcl1. CLUSTAL W alignment of H. capsulatum Hcl1 with HMG CoA lyases from bacterial and mammalian species. Conserved catalytic residues are marked in red. The asterisk symbol indicates that the residues in that column are identical in all sequences in the alignment, the colon indicates that conserved substitutions between the sequences are observed, and the period indicates that semiconserved substitutions are observed.

Fig 3

Hcl1 is required for macrophage colonization and lysis during H. capsulatum infection. (A) BMDMs were mock infected or infected with either wild-type (WT), the hcl1 mutant, or the complemented strain (hcl1+HCL1) at an MOI of 2. At 2, 24, 48, 72, 96, and 120 hpi, supernatants were removed from the infected monolayers and lactate dehydrogenase activity was assessed to monitor BMDM lysis. The average percent BMDM lysis of four measurements ± the standard deviation (from one representative experiment) is shown. (B) BMDMs were infected with wild-type (WT), the hcl1 mutant, or the complemented strain (hcl1+HCL1) at an MOI of 2. At 3, 12, 24, 36, 48, 72, and 144 hpi, BMDMs were osmotically lysed, and the lysates were plated for H. capsulatum CFU. Each measurement is the average of four platings (duplicate infections/duplicate platings) ± the standard deviation from one representative experiment. In some cases, the standard deviation is negligible and is obscured by strain symbols. (C) BMDMs were infected with either wild-type H. capsulatum or the hcl1 mutant at an MOI of 0.1. At 2 and 120 hpi, the infected monolayers were fixed and H. capsulatum yeast cells (arrowhead) were stained with periodic acid-Schiff (PAS) reagent. Macrophage nuclei were counterstained with methyl green. Representative images at ×100 magnification are shown. (D) In vitro growth of wild-type (WT), the hcl1 mutant, and the complemented strain (hcl1+HCL1) was examined in broth culture. Yeast cells were inoculated into HMM at a starting OD₆₀₀ of 0.01 and subsequent OD₆₀₀ was monitored over time. The average of three measurements ± the standard deviation is shown from one representative experiment. In some cases, the standard deviation is negligible and is obscured by strain symbols.

Fig 4

H. capsulatum HCL1 is required for growth on leucine as the major carbon source. (A) Four-day cultures of wild-type H. capsulatum (WT), the hcl1 mutant, and the complemented strain (hcl1+HCL1) were pelleted, washed with PBS, and resuspended in PBS. Tenfold serial dilutions were then made on the indicated medium. In the case of 3M-glu+leu, medium was supplemented with 10 mM leucine. Plates were incubated at 37°C and 5% CO₂ for 16 days. For the HMM plates, the 10⁻³ to 10⁻⁶ dilutions are shown (due to the enhanced growth on this rich medium). On all other plates, undiluted, 10⁻¹, and 10⁻² dilutions are shown. (B to D) Four-day cultures of wild-type H. capsulatum (WT), the hcl1 mutant, and the complemented strain (hcl1+HCL1) were pelleted, washed with PBS, and resuspended in either minimal media (3M) (B), minimal medium without glucose (3M-glu) (C), or minimal media without glucose, supplemented with 5 mM leucine (3M-glu+leu). At the indicated time points, the OD₆₀₀ was measured in triplicate to monitor growth. The average OD and standard deviation from one representative experiment are plotted, but the standard deviation is negligible and is obscured by strain symbols.

Fig 5

Hcl1 is required for maintenance of neutral pH in culture. Four-day cultures of wild-type H. capsulatum (WT), the hcl1 mutant, and the complemented strain (hcl1+HCL1) were pelleted, washed, resuspended in PBS, and diluted into 30 ml of unbuffered pH-HMM at OD₆₀₀ of 2.0. The pH of the culture medium was measured at the indicated time points. The average of three pH measurements ± the standard deviation is shown from one representative experiment. In most cases, the standard deviation is negligible and is obscured by strain symbols.

Fig 6

Hcl1 is required for maintenance of near-neutral pH in the macrophage phagosome. (A and B) BMDMs were infected with either wild-type (WT), fixed wild-type cells, the hcl1 mutant, or the complemented strain (hcl1+HCL1) at an MOI of 2. BMDMs were incubated with LysoSensor Green DND-189 30 min prior to fixation at 3, 6, 12, and 24 hpi. Control experiments were performed in the presence of 50 nM bafilomycin A1. (A) At 3 hpi, the acidic pH of the phagosomes was revealed by bright green fluorescence (LysoSensor; white arrows), which surrounded and impregnated the yeast cells (blue). LAMP-1 staining is shown in red. hcl1 mutant cells, but not wild-type cells, maintained localization with LysoSensor at later time points such as 24 hpi. Scale bars, 5 μm. (B) Quantification of the percentage of yeast cells that colocalized with LysoSensor fluorescence within LAMP-1-positive phagosomes. Fixed wild-type cells were degraded by the macrophage after 6 hpi and thus could not be quantified after that time point. Their absence is indicated by an asterisk. At least 200 yeast cells were counted per sample and standard deviation is shown. (C) BMDMs were infected with either the wild-type strain (WT) or the WU15 G217B ura5Δ strain at an MOI of 2 in the absence or presence of bafilomycin as described above. Quantification of the percentage of yeast cells that colocalized with LysoSensor fluorescence within LAMP-1-positive phagosomes was performed. At least 200 yeast cells were counted per sample and the standard deviation is shown.

Fig 7

The hcl1 mutant exhibits decreased virulence in vivo. Female C57BL/6 mice were infected intranasally with 1.25 × 10⁶ CFU of either the wild-type (WT) (n = 15), the hcl1 mutant (hcl1) (n = 15), or the complemented strain (hcl1+HCL1) (n = 15) strain. (A) Kaplan-Meir plots/survival curve. P < 0.0001 (log rank test, WT versus hcl1). (B) In the case of all three cohorts of mice, lungs were harvested from infected mice and lung homogenates were plated to enumerate H. capsulatum CFU at 4 hpi and at 7 days postinfection, which was time of death for mice infected with the wild-type strain. Lung CFU were also enumerated at time of death (10 days postinfection) for mice infected with the hcl1 mutant. Average CFU/group is plotted. (C) Spleen CFU were enumerated from all three cohorts of mice at 7 days postinfection. The average CFU/group is plotted.