Propionibacterium-produced coproporphyrin III induces Staphylococcus aureus aggregation and biofilm formation

Abstract

The majority of bacteria detected in the nostril microbiota of most healthy adults belong to three genera: Propionibacterium, Corynebacterium, and Staphylococcus. Among these staphylococci is the medically important bacterium Staphylococcus aureus. Almost nothing is known about interspecies interactions among bacteria in the nostrils. We observed that crude extracts of cell-free conditioned medium from Propionibacterium spp. induce S. aureus aggregation in culture. Bioassay-guided fractionation implicated coproporphyrin III (CIII), the most abundant extracellular porphyrin produced by human-associated Propionibacterium spp., as a cause of S. aureus aggregation. This aggregation response depended on the CIII dose and occurred during early stationary-phase growth, and a low pH (~4 to 6) was necessary but was not sufficient for its induction. Additionally, CIII induced plasma-independent S. aureus biofilm development on an abiotic surface in multiple S. aureus strains. In strain UAMS-1, CIII stimulation of biofilm depended on sarA, a key biofilm regulator. This study is one of the first demonstrations of a small-molecule-mediated interaction among medically relevant members of the nostril microbiota and the first description of a role for CIII in bacterial interspecies interactions. Our results indicate that CIII may be an important mediator of S. aureus aggregation and/or biofilm formation in the nostril or other sites inhabited by Propionibacterium spp. and S. aureus. Importance: Very little is known about interspecies interactions among the bacteria that inhabit the adult nostril, including Staphylococcus aureus, a potential pathogen that colonizes about a quarter of adults. We demonstrated that coproporphyrin III (CIII), a diffusible small molecule excreted by nostril- and skin-associated Propionibacterium spp., induces S. aureus aggregation in a manner dependent on dose, growth phase, and pH. CIII also induces S. aureus to form a plasma-independent surface-attached biofilm. This report is the first description of a role for CIII in bacterial interspecies interactions at any human body site and a novel demonstration that nostril microbiota physiology is influenced by small-molecule-mediated interactions.

FIG 1

The nostrils (anterior nares) open onto the nasal vestibules, which are lined by skin, complete with sweat and pilosebaceous glands. (A) Inferior view of the nostrils and nasal vestibules; (B) sagittal illustration of the nasal cavity; (C) the limen nasi forms a boundary between the nasal vestibule and posterior aspects of the nasal cavity, which have a mucosal epithelial lining; (D) the nasal vestibule epithelium (skin) contains sweat and pilosebaceous glands and does not secrete mucus.

FIG 2

Extracts of P. acnes KPA171202 conditioned medium (CM) induce S. aureus aggregation. (A) Addition of a crude extract of P. acnes KPA171202 CM to stationary-phase cultures of S. aureus grown in SSD0Fe induced aggregation; (B) HPLC fractions (denoted by elution time) of P. acnes KPA171202 CM were added to S. aureus KPL1845 cultures. The 400-nm (blue line) and 280-nm (orange line) absorbance during HPLC elution are shown. The black arrow indicates the 400-nm-absorbing HPLC peak included in the 10- to 12-min fraction. Histogram bars represent the mean S. aureus aggregation ratio for each elution fraction or sample. All data are from five independent experiments; error bars are standard errors of the means (SEM).

FIG 3

Production of extracellular coproporphyrin I and III by primary nostril isolates (KPL) and standard strains (inset, chemical structures). Extracellular coproporphyrin production estimated from HPLC fractionations. Identification to the species level of the primary nostril isolates (KPL1815, KPL1844, KPL1845, KPL1849, and KPL1855) is described in Text S1, Table S1, and Fig. S7 in the supplemental material. Histogram bars represent the means from three biological replicates; error bars are SEM.

FIG 4

Coproporphyrin III induces S. aureus aggregation in culture. (A) DMF (black symbols) does not induce aggregation; 50 µM CIII (gray symbols) induces aggregation. Data points are the means from five biological replicates of aggregation assays done after 19 h of growth. Error bars are SEM. (Inset) Epifluorescence microscopy of Syto-9-stained UAMS-1 cultures 4 h after addition illustrates lack of aggregation with only DMF (B) and aggregation into multicellular clusters with 50 µM CIII (C).

FIG 5

S. aureus-CIII coaggregation is growth phase dependent. S. aureus UAMS-1 exhibited diauxic growth in SSD0Fe over 24 h (white squares). At each time point, cells were harvested to assess the response to CIII addition; “relative aggregation ratio” (gray squares) is the aggregation ratio with 50 µM CIII over the aggregation ratio with DMF alone 4 h postaddition. The measured pH range of the culture at each time point is depicted above the graph. All values are the means from three biological replicates; error bars are SEM.

FIG 6

S. aureus UAMS-1 cell-free conditioned medium induces CIII aggregation at a low pH. CM from a 19-h UAMS-1 culture was filter sterilized; then the pH was either left unadjusted (black circles) or adjusted to 8.5 with NaOH (gray circles). Fresh SSD0Fe was adjusted to pH 5.0 with HCl (white squares). CIII (FC of 100 µM) was added to each condition and aggregation was measured. Graph points represent the mean aggregation ratio from three experiments, each using medium prepared on a different date; error bars are SEM (inset). A photo taken 72 h post-CIII addition shows CIII visibly aggregated at the bottom of the cuvette with CFCM pH 5.0 but remained in solution in the other three conditions.

FIG 7

CIII induces S. aureus biofilm formation in culture; in strain UAMS-1, this induction requires sarA. (A) For four S. aureus isolates, cell density (biofilm formation) was significantly greater in the presence of 50 µM CIII (gray bars; two-tailed, paired t test with Bonferroni correction: **, P < 0.05; *, P < 0.06) compared to DMF alone (black bars). E. coli MG1655 did not form biofilm under these assay conditions. Data are the means from five biological replicates; error bars represent SEM. (B) Representative photos taken of individual wells after 20 min of 1% crystal violet staining. (C) S. aureus UAMS-1 formed a biofilm in response to culture with SSD0Fe and 50 µM CIII (gray bars) but was unable to form a biofilm in identical conditions when the sarA gene was interrupted. Data represent the means from three biological replicates; error bars are SEM.