A multidimensional assessment of in-host fitness costs of drug resistance in the opportunistic fungal pathogen Candida glabrata

Abstract

Drug-resistant microbes typically carry mutations in genes involved in critical cellular functions and may therefore be less fit under drug-free conditions than susceptible strains. Candida glabrata is a prevalent opportunistic yeast pathogen with a high rate of fluconazole resistance (FLZR), echinocandin resistance (ECR), and multidrug resistance (MDR) relative to other Candida. However, the fitness of C. glabrata MDR isolates, particularly in the host, is poorly characterized, and studies of FLZR isolate fitness have produced contradictory findings. Two important host niches for C. glabrata are macrophages, in which it survives and proliferates, and the gut. Herein, we used a collection of clinical and lab-derived C. glabrata isolates to show that FLZR C. glabrata isolates are less fit inside macrophages than susceptible isolates and that this fitness cost is reversed by acquiring ECR mutations. Interestingly, dual-RNAseq revealed that macrophages infected with drug-resistant isolates mount an inflammatory response whereas intracellular drug-resistant cells downregulate processes required for in-host adaptation. Furthermore, drug-resistant isolates were outcompeted by their susceptible counterparts during gut colonization and in infected kidneys, while showing comparable fitness in the spleen. Collectively, our study shows that macrophage-rich organs, such as the spleen, favor the retention of MDR isolates of C. glabrata.

Keywords: Candida glabrata; echinocandin resistant; fitness cost; fluconazole resistant; gut colonization; intracellular replication; multidrug resistant; systemic infection.

Figure 1.

In vitro fitness cost assessment of lab-derived and clinical C. glabrata isolates of various susceptibility profiles. (A) Clinical susceptible and ECR isolates of C. glabrata have relatively high fitness under macrophage-like conditions (see text), whereas FLZR and MDR isolates have relatively high fitness under ER stress. (B) Lab-derived susceptible and ECR isolates of C. glabrata have relatively high fitness under macrophage-like conditions, whereas FLZR and MDR isolates have relatively high fitness under ER stress (at least two biological replicates per each isolate, mean and standard deviation). (C) In the lab-derived strain panel, ECR and susceptible isolates had the highest IR rate in macrophages at 48 h, whereas FLZR and especially MDR isolates had lower IR rates (each dot represents one biological replicate, mean and standard deviation). (D) In the clinical isolate strain panel, susceptible, ECR, and MDR isolates had similar IR rates in macrophages, whereas the FLZR isolates had the lowest IR rate (each dot represents one biological replicate, mean and standard deviation). (E) Lab-derived FLZR isolates had the lowest phagocytosis rate (each dot represents one biological replicate, mean and standard deviation). (F) The four types of clinical isolates were phagocytosed at similar rate (each dot represents one biological replicate, mean and standard deviation). MDR = multidrug resistant, FLZR = fluconazole resistant, ECR = echinocandin resistant. Each data point represents a different biological replicate. * P < .05 ** P < .01 For panels (C) and (D), repeated measures ANOVA was used to investigate intra (within each group) and inter (between groups) variance as well as the interaction of group and time. Additionally, in (C), (D), (E), and (F) pairwise group comparison at individual timepoints was performed using Tukey’s HSD test. No significant differences were found for panel (F).

Figure 2.

Effects of MDR on C. glabrata survival and replication within macrophages. (A) Fks1 and Fks2 hot-spot region sequences showing the sites of equivalent amino acid changes in Fks1 and Fks2 representative of clinically prevalent ECR alleles (each dot represents one biological replicate, mean, and standard deviation). (B) Effects of clinically prevalent mutations in HS1 of FKS1 (S629P and R631G) and equivalent mutations in Fks2 (S663P and R665G) introduced into the FLZR parental background. The resulting MDR isolates had a significantly higher IR rate compared to the FLZR parental strain (each dot represents one biological replicate, mean, and standard deviation). (C) Petite MDR isolates carrying the same Fks mutations as in (B) did not show a difference in IR compared to the parental petite isolates (each dot represents one biological replicate, mean, and standard deviation). MDR = multidrug resistant, FLZR = fluconazole resistant, HS1 = hotspot 1, and IR = intracellular replication. Each data point represents a different biological replicate. * P < .05 ** P < .01 Repeated measures ANOVA was used to investigate intra (within each group) and inter (between groups) group variance as well as the interaction of group and time. Pairwise group comparison at individual timepoints was performed using Tukey’s HSD test.

Figure 3.

Results of intramacrophage competition experiments among susceptible and DR C. glabrata strains. (A) MDR-Fks2^S663P  C. glabrata strain outcompeted its FLZR parental strain in macrophages (each dot represents one biological replicate, mean, and standard deviation). (B) Susceptible C. glabrata strain outcompeted the FLZR strain in macrophages (each dot represents one biological replicate, mean, and standard deviation). (C) MDR-Fks2^S663P  C. glabrata strain outcompeted the MDR-Fks1^S629P strain in macrophages (each dot represents one biological replicate, mean, and standard deviation). (D) Susceptible C. glabrata strain had equivalent intramacrophage fitness with the MDR-Fks2^S663P strain. (E) Susceptible C. glabrata strain outcompeted the MDR-Fks1^S629P strain in macrophages (each dot represents one biological replicate, mean, and standard deviation). MDR = multidrug resistant, FLZR = fluconazole resistant, HS1 = hotspot 1, and IR = intracellular replication. Two biological replicates were tested for each inoculum and six biological replicates were tested for each timepoint. * P < .05 ** P < .01 Two-way ANOVA was used to investigate intra (within each group) and inter (between groups) group variance as well as the interaction of group and time. Pairwise group comparison at individual timepoints was performed using Tukey’s HSD test.

Figure 4.

Gene expression changes in susceptible and DR C. glabrata strains upon macrophage infection. (A) PCA plot of all studied C. glabrata samples across studied conditions. The plot is based on vst-transformed read count data generated by DESeq2. Labels on the data points correspond to drug susceptibility profiles of each strain: S—susceptible, FR—fluconazole resistant, and MDR—multidrug resistant. Percentages on PC1 and PC2 axes indicate the total amount of variance described by each axis. (B) GO term enrichment analysis (category “Biological Process”) of upregulated genes at a given comparison of C. glabrata strains shown on the X axis. (C) GO term enrichment analysis (category “Biological Process”) of downregulated genes at a given comparison of C. glabrata strains shown on the x-axis. For (B) and (C), the numbers underneath the comparisons correspond to the “counts” of clusterProfiler (i.e. total number of genes assigned to GO categories). GeneRatio corresponds to the ratio between the number of input genes assigned to a given GO category and “counts”. Only significant (padj < .05) enrichments are shown. Adjustment of P-values is done by Benjamini–Hochberg procedure. Three biological replicates were analysed for every condition.

Figure 5.

Gene expression changes in macrophages infected with susceptible and DR C. glabrata strains. (A) PCA plot of all studied macrophage samples. The plot is based on vst-transformed read count data generated by DESeq2. Labels on the data points correspond to drug susceptibility profiles of infecting C. glabrata strains: S—susceptible, FR—fluconazole resistant, and MDR—multidrug resistant. Percentages on PC1 and PC2 axes indicate the total amount of variance described by each axis. (B) GO term enrichment analysis (category “Biological Process”) of upregulated genes of macrophages infected with C. glabrata strains (as depicted on x-axis) compared to uninfected macrophages. (C) GO term enrichment categories (when available) of upregulated genes of macrophages infected with different C. glabrata strains (see the x-axis for specific comparisons). (D) GO term enrichment categories (when available) of downregulated genes of macrophages infected with different C. glabrata strains (see the x-axis for specific comparisons). For (B), (C), and (D), the numbers underneath the comparisons correspond to the “counts” of clusterProfiler (i.e. total number of genes assigned to GO categories). GeneRatio corresponds to the ratio between the number of input genes assigned to a given GO category and “counts”. Only significant (padj < .05) enrichments are shown. Adjustment of P-values is done by Benjamini–Hochberg procedure. (E) Gene Set Enrichment Analysis (Barr et al. 2021) depicting enrichment of the “classically” activated macrophage transcripts in the FLZR or MDR-FKS1-R631G strain-infected macrophages in comparison to their CBS138- (top panel) and MDR-FKS2-S663P-infected (bottom panel) macrophage counterparts. Plots depict enrichment of the “classically” activated macrophage transcriptional module (Bolger et al. 2014) for macrophages infected with the indicated strains at 24 hpi. Normalized enrichment score and adjusted P-values are shown in the inset. Three biological replicates were analysed for every condition.

Figure 6.

Results of GI tract colonization competition experiments among susceptible and DR C. glabrata strains. (A) Susceptible strain readily outcompeted the FLZR strain (each dot represents one mouse, mean to max). (B) Susceptible strain outcompeted the MDR-Fks2^S663P strain (each dot represents one mouse, mean to max). (C) FLZR isolate outcompeted the MDR-Fks2^S663P strain (each dot represents one mouse, mean to max). MDR = multidrug resistant, FLZR = fluconazole resistant, and GFP = green fluorescent protein. Four biological replicates (mice) were examined at every timepoint. * P < .05 ** P < .01 Two-way ANOVA was used to investigate intra (within each group) and inter (between groups) group variance as well as the interaction of group and time. Pairwise group comparison at individual timepoints was performed using Tukey’s HSD test. Error bars denote standard deviation.

Figure 7.

Results of in vivo systemic infection competition experiments. (A) Susceptible isolate readily outcompeted the FLZR isolate in the kidney (each dot represents one mouse, mean to max). (B) Susceptible isolate readily outcompeted the MDR-Fks2^S663P isolate in the kidney (each dot represents one mouse, mean to max). (C) MDR-Fks2^S663P was outcompeted by the FLZR isolate in the kidney (each dot represents one mouse, mean to max). (D) FLZR isolate showed comparable fitness in the spleen to the susceptible strain (each dot represents one mouse, mean to max). (E) MDR-Fks2^S663P isolate showed comparable fitness in the spleen to the susceptible strain (each dot represents one mouse, mean to max). (F) MDR-Fks2^663P and FLZR showed similar fitness in the spleen (each dot represents one mouse, mean to max). MDR = multidrug resistant and FLZR = fluconazole resistant. Four biological replicates (mice) were examined at each timepoint. * P < .05 ** P < .01. Two-way ANOVA was used to investigate intra (within each group) and inter (between groups) group variance as well as the interaction of group and time. Pairwise group comparison at individual timepoints was performed using Tukey’s HSD test. Error bars denote standard deviation.