A peptidoglycan storm caused by β-lactam antibiotic’s action on host microbiota drives Candida albicans infection

The commensal fungus Candida albicans often causes life-threatening infections in patients who are immunocompromised with high mortality. A prominent but poorly understood risk factor for the C. albicans commensal‒pathogen transition is the use of broad-spectrum antibiotics. Here, we report that β-lactam antibiotics cause bacteria to release significant quantities of peptidoglycan fragments that potently induce the invasive hyphal growth of C. albicans. We identify several active peptidoglycan subunits, including tracheal cytotoxin, a molecule produced by many Gram-negative bacteria, and fragments purified from the cell wall of Gram-positive Staphylococcus aureus. Feeding mice with β-lactam antibiotics causes a peptidoglycan storm that transforms the gut from a niche usually restraining C. albicans in the commensal state to promoting invasive growth, leading to systemic dissemination. Our findings reveal a mechanism underlying a significant risk factor for C. albicans infection, which could inform clinicians regarding future antibiotic selection to minimize this deadly disease incidence.


Supplementary Information
of the main text). Resistant bacteria release significantly less PGN and hypha-inducing activity than sensitive strains in response to β-lactam antibiotics. a C. albicans were grown side-by-side on LB plates with MSSA, MRSA, Amp-sensitive (AmpS-Ec) or resistant E. coli (AmpR-Ec) strains in the presence or absence of the indicated antibiotics at 30°C for 4 days as described in Figure 1a. Scale bar, 250 µm. b Quantification of filamentous growth. The heatmap was generated as described in Figure 1c, which compares the degree of filamentous growth of the cultures shown in a. c Average hyphal length of the cultures shown in a. Images of two representative areas of filamentous growth of each C. albicans patch were analyzed, and 10 filaments (n = 10) of each image were measured. P-values were determined using two-tailed unpaired t test. Bars are means ± SEM. ns, not significant. Figure 2d and e of the main text). Identification of a muropeptide in the supernatant S. aureus culture treated with Cef or Amp.

Supplementary Figure 3 (related to
To identify the active PGN subunits released after β-lactam antibiotic treatment of S. aureus cultures, HPLC fraction F13, which is descriebd in Figure 2d and e, were subjected to liquid chromatography-mass spectrometry (LC-MS) analysis. F13 of Amp and Cef-treated cultures showed similar UV and MS spectra (a and b), and the peaks were identified to have a molecular mass of 1251.56 [M+H] + and 628.26 [M+H] 2+ (c). The same muropeptide was also identified from purified S. aureus sacculi digested with lysozyme (Supplementary Figure 4, compound e). The black line is TIC (total ion chromatogram), and the blue one is the EIC (Extract ion chromatogram). Supplementary Figure 4 (related to Figure 4 of the main text). a, LC/MS analysis of isolated muropeptides to confirm the purifty and structure identity. The six muropeptides described in Figure 3 were obtained by enzymatic digestion of isolated bacterial sacculi (E. coli for compound a-c; S. aureus for compound d-e), followed by HPLC purification. The identities and purifities of the purified compounds were confirmed by LC/MS. Left: total ion chromatogram (TIC) of the sample indicates the relative purity of each compound (identified by the major peak); right: mass spectra of the major peak in TIC which gives the desired m/z, confirming the identity of the compound.
b, c, Tabulation of C. albicans hyphal growth induced by different muropeptides.
Muropeptides were added to HBBS at the indicated concentrations, and hyphal induction was done at 37℃ for 2h. Fifty C. albicans cells (n = 50) in each test were analyzed to calculate the percentage and average length of hyphae. Fetal bovine serum (5%) was included as the positive control. Error bars, means±SD. Pair-wise comparison was made and significance was determined by two-tailed unpaired t test. ns, not signifcant. To minimize the inter-individual variations due to different drinking behaviors, each mouse (7-8 weeks old; n = 5) was orally administered 0.5 mL of an antibiotic solution (8 mg/mL for all antibiotics used) twice on day 1 with an 8 h interval. The drinking water also contained the same antibiotic. At 24 h of the antibiotic treatment, fresh feces were collected, weighed, resuspended in sterile PBS to a final concentration of 1 g/mL, and serially diluted 10-fold before spreading onto LB plates for incubation at 37°C for one day. Then, the CFUs on plates were counted. Three mice were used for each antibiotic treatment. P-values were calculated using two-tailed unpaired t test. Error bars: means±SEM. Balb/c mice (7-8 weeks old; n = 3) were administered orally with 0.5 mL of streptomycin solution (8 mg/mL) twice per day on day 1 and given drinking water supplemented with streptomycin at the same concentration throughout the experiment. After 4 days of streptomycin treatment, each mouse was gavaged with 1 × 10 8 C. albicans yeast cells. At 48 h, mice were gavaged with 0.5 mL of PGN solution (4 mg in 0.5 mL) or PBS. Feces were collected at 3 and 7 h to examine C. albicans morphology. The percentage of hyphae was calculated, and the hyphal length was measured (n = 50). PGN polymers were prepared from S. aureus and digested with lysozyme as described in Methods. Scale bar, 5µm.

Supplementary
Supplementary Figure 8. WT C. albicans and the hgc1Δ/Δ mutant showed comparable ability to colonize mouse kidneys.
BALB/c mice (8-10 weeks old; n = 3) were used for this experiment. WT C. albicans (SC5314) and hgc1Δ/Δ cells (+ARG4+HIS1+URA3) were grown in YPD at 30 °C overnight before harvesting by centrifugation. Cells were resuspended in PBS at a density of 5 × 10 6 cells / mL. Each mouse was inoculated via the tail vein with 200 μl of the cell suspension. At 48 h, all mice were sacrificed to harvest the kidney. The kidneys were homogenized in ice-cold PBS and 10-fold serially diluted before spreading aliquots onto YPD plates. CFU was counted after two days of incubation at 30 °C . Error bars, means ± SEM Supplementary Figure 9. Recovery of bacterial growth during Amp treatment.
Each mouse (n = 2) was orally administered 0.5 mL of Amp (8 mg/mL) twice on day 1 with an 8 h interval. The drinking water also contained Amp (4 mg/mL). At the indicated time points, fresh feces were collected, weighed, resuspended in sterile PBS to a final concentration of 1 g/mL, and serially diluted 10-fold before spreading onto LB plates for incubation at 37°C for one day. The CFUs on plates were counted. We only counted aerobic bacteria. The everage CFU at 0 h was treated as 100% and that at 24 h as 0. The experiment was repeated three times independently with similar results.
Supplementary Figure 10. Morphology of C. albicans in the feces of mice treated with the mixture of penicillin and streptomycin.
Three Balb/c female mice (7-8 weeks) were orally administered 0.5 mL of penicillin (2000 u/ mL)+streptomycin (2 mg/mL) solution twice on day 1. The same antibiotics were also added to the drinking water throughout the experiment, as described in Figure 4a. At 24 h, 1×108 wild-type C. albicans yeast cells were orally inoculated into mice. After 24 h, fresh feces were collected and resuspended in distilled water to examine C. albicans morphology. We detected only yeast cells, like in the feces of untreated mice (Figure 4a). The experiment was repeated three times independently with similar results.