Morphology-Independent Virulence of Candida Species during Polymicrobial Intra-abdominal Infections with Staphylococcus aureus

Abstract

Intra-abdominal polymicrobial infections cause significant morbidity and mortality. An experimental mouse model of Candida albicans-Staphylococcus aureus intra-abdominal infection (IAI) results in 100% mortality by 48 to 72 h postinoculation, while monomicrobial infections are avirulent. Mortality is associated with robust local and systemic inflammation without a requirement for C. albicans morphogenesis. However, the contribution of virulence factors coregulated during the yeast-to-hypha transition is unknown. This also raised the question of whether other Candida species that are unable to form hyphae are as virulent as C. albicans during polymicrobial IAI. Therefore, the purpose of this study was to evaluate the ability of non-albicans Candida (NAC) species with various morphologies and C. albicans transcription factor mutants (efg1/efg1 and cph1/cph1) to induce synergistic mortality and the accompanying inflammation. Results showed that S. aureus coinoculated with C. krusei or C. tropicalis was highly lethal, similar to C. albicans, while S. aureus-C. dubliniensis, S. aureus-C. parapsilosis, and S. aureus-C. glabrata coinoculations resulted in little to no mortality. Local and systemic interleukin-6 (IL-6) and prostaglandin E2 (PGE2) levels were significantly elevated during symptomatic and/or lethal coinfections, and hypothermia strongly correlated with mortality. Coinoculation with C. albicans strains deficient in the transcription factor Efg1 but not Cph1 reversed the lethal outcome. These results support previous findings and demonstrate that select Candida species, without reference to any morphological requirement, induce synergistic mortality, with IL-6 and PGE2 acting as key inflammatory factors. Mechanistically, signaling pathways controlled by Efg1 are critical for the ability of C. albicans to induce mortality from an intra-abdominal polymicrobial infection.

FIG 1

Survival following coinoculation with NAC species and S. aureus. Mice (n = 5 mice/group) were injected intraperitoneally with 8 × 10⁷ CFU of S. aureus and 3.5 × 10⁷ CFU of C. albicans, C. krusei, C. tropicalis, C. dubliniensis, C. parapsilosis, or C. glabrata. Mice inoculated with S. aureus and 3.5 × 10⁷ CFU of S. cerevisiae or UV-killed C. albicans served as negative controls. Mice were monitored for 10 days postinoculation (p.i.). Data are cumulative from two repeat experiments. Mortality compared to that of C. albicans coinfection was measured by log-rank (Mantel-Cox) test. *, P < 0.001; **, P < 0.0001.

FIG 2

Microbial burden in peritoneal lavage fluid during NAC species-S. aureus coinfections. Mice were infected with S. aureus and NAC species as described in the legend to Fig. 1. Mice (n = 5 mice/group) were sacrificed at 24 h p.i. to quantify fungal and bacterial burden in peritoneal lavage fluid. Results are expressed as the median CFU. Shown are cumulative data from two repeat experiments. Fungal and bacterial burden from C. albicans (CA), C. krusei (CK), and C. tropicalis (CT) coinfections were grouped and individually compared to C. dubliniensis (CD), C. parapsilosis (CP), C. glabrata (CG), and S. cerevisiae (SC) coinfections. Data were analyzed using the Mann-Whitney U test.

FIG 3

Local immune mediators during peritoneal coinfections with NAC species and S. aureus. Mice (n = 5 mice/group) were infected with S. aureus and NAC species as described in the legend to Fig. 1. At 4 or 24 h p.i., peritoneal lavage fluid was collected and analyzed for PGE₂ (A and E), IL-6 (B and F), IL-1β (C), and TNF-α (D). Results are expressed as the mean cytokine or eicosanoid concentration ± standard errors of the means (SEM). Shown are cumulative data from two repeat experiments. Cytokine or eicosanoid concentrations from C. albicans (CA), C. krusei (CK), and C. tropicalis (CT) coinoculations were individually compared to those of C. dubliniensis (CD), C. parapsilosis (CP), C. glabrata (CG), S. cerevisiae (SC), and UV-killed C. albicans (UV-killed CA) coinoculations or those of naive mice. Data were analyzed using the unpaired Student's t test. n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Graphs in which multiple groups are significantly different are labeled with an asterisk, representing the least significant comparison.

FIG 4

Systemic immune mediators during peritoneal coinfections with NAC species and S. aureus. Mice (n = 5 mice/group) were inoculated with S. aureus and NAC species as described in the legend to Fig. 1. At 24 h p.i., serum was collected and analyzed for IL-6 (A), IL-1β (B), and TNF-α (C). Results are expressed as the mean cytokine concentrations ± SEM. Shown are cumulative data from two repeat experiments. Cytokine concentrations from C. albicans (CA), C. krusei (CK), and C. tropicalis (CT) coinoculations were individually compared to those of C. dubliniensis (CD), C. parapsilosis (CP), C. glabrata (CG), S. cerevisiae (SC), and UV-killed C. albicans (UV-killed CA) coinoculations. Data were analyzed using the unpaired Student's t test. n.s., not significant; *, P < 0.05; **, P < 0.01. Graphs in which multiple groups are significantly different are labeled with an asterisk, representing the least significant comparison.

FIG 5

Murine core temperature during NAC species-S. aureus coinfections. Mice (n = 5 to 10 mice/group) were coinoculated with S. aureus and C. albicans, C. krusei, C. tropicalis, C. dubliniensis, or C. parapsilosis. Survival and rectal body temperature were monitored daily for 10 days p.i. Core temperature at 4 (A) and 24 (B) h p.i. was compared between mice that succumbed to infection and mice that survived infection to day 10 p.i. Shown are representative results from two experiments. Body temperatures were analyzed using the Mann-Whitney U test. *, P < 0.05; ***, P < 0.001.

FIG 6

Morphogenetic signaling in C. albicans is crucial for synergistic mortality during polymicrobial infection with S. aureus. Mice (n = 5 mice/group) were inoculated with 8 × 10⁷ CFU of S. aureus and 3.5 × 10⁷ CFU of wild-type (WT) C. albicans or the efg1/efg1 cph1/cph1, cph1/cph1, efg1/efg1, or efg1/efg1 EFG1 mutant. Survival was monitored for 5 days p.i. Results are cumulative from two repeat experiments, except for those for the efg1/efg1 EFG1 plus S. aureus experiment, which was performed once.