Coculture of Staphylococcus aureus with Pseudomonas aeruginosa Drives S. aureus towards Fermentative Metabolism and Reduced Viability in a Cystic Fibrosis Model

Abstract

The airways of patients with cystic fibrosis are colonized with diverse bacterial communities that change dynamically during pediatric years and early adulthood. Staphylococcus aureus is the most prevalent pathogen during early childhood, but during late teens and early adulthood, a shift in microbial composition occurs leading to Pseudomonas aeruginosa community predominance in ϳ50% of adults. We developed a robust dual-bacterial in vitro coculture system of P. aeruginosa and S. aureus on monolayers of human bronchial epithelial cells homozygous for the ⌬F508 cystic fibrosis transmembrane conductance regulator (CFTR) mutation to better model the mechanisms of this interaction. We show that P. aeruginosa drives the S. aureus expression profile from that of aerobic respiration to fermentation. This shift is dependent on the production of both 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) and siderophores by P. aeruginosa. Furthermore, S. aureus-produced lactate is a carbon source that P. aeruginosa preferentially consumes over medium-supplied glucose. We find that initially S. aureus and P. aeruginosa coexist; however, over extended coculture P. aeruginosa reduces S. aureus viability, also in an HQNO-and P. aeruginosa siderophore-dependent manner. Interestingly, S. aureus small-colony-variant (SCV) genetic mutant strains, which have defects in their electron transport chain, experience reduced killing by P. aeruginosa compared to their wild-type parent strains; thus, SCVs may provide a mechanism for persistence of S. aureus in the presence of P. aeruginosa. We propose that the mechanism of P. aeruginosa-mediated killing of S. aureus is multifactorial, requiring HQNO and P. aeruginosa siderophores as well as additional genetic, environmental, and nutritional factors. IMPORTANCEIn individuals with cystic fibrosis, Staphylococcus aureus is the primary respiratory pathogen during childhood. During adulthood, Pseudomonas aeruginosa predominates and correlates with worse patient outcome. The mechanism(s) by which P. aeruginosa outcompetes or kills S. aureus is not well understood. We describe an in vitro dual-bacterial species coculture system on cystic fibrosis-derived airway cells, which models interactions relevant to patients with cystic fibrosis. Further, we show that molecules produced by P. aeruginosa additively induce a transition of S. aureus metabolism from aerobic respiration to fermentation and eventually lead to loss of S. aureus viability. Elucidating the molecular mechanisms of P. aeruginosa community predominance can provide new therapeutic targets and approaches to impede this microbial community transition and subsequent patient worsening. C omplex polymicrobial communities colonize the airways of cystic fibrosis (CF) patients within the first month of life (1). Culture-independent studies have revealed the simultaneous presence of numerous bacterial taxa, fungi, and viruses in respiratory samples from CF patients at all stages of life (2-8). This abundance of microbes colonizing the respiratory ...

Iron-Mediated Control of Pseudomonas aeruginosa-Staphylococcus aureus Interactions in the Cystic Fibrosis Lung

“…This feat is accomplished via sabotaging S. aureus's aerobic metabolism with a combination of factors, including the excretion of the potent staphylococcal respiratory chain inhibitor 2-heptyl-4-hydroxyquinoline N-oxide along with iron-scavenging siderophores. The shift proves detrimental to S. aureus growth rates and fitness, while these conditions are more than hospitable for P. aeruginosa, which preferentially feeds on the lactate that its victim produces (7). Relevant to this model, Nguyen et al also demonstrate that free iron levels regulate P. aeruginosa's inhibition of S. aureus in coculture (9).…”

“…In contrast, iron levels have been found to be high in CF sputum and correlate negatively with patient outcomes (6). Additionally, pathogen lung colonization in CF patients displays a conserved pattern of succession, with S. aureus predominating early on before being displaced by P. aeruginosa (7). Although it has been previously demonstrated that P. aeruginosa harvests iron via killing of S. aureus (8), the exact dynamics of this hostile takeover remain unclear.…”

Increased rates of genomic mutation in a biofilm co-culture model of Pseudomonas aeruginosa and Staphylococcus aureus

“…As an aside, it 548 was found that P. aeruginosa PA21 still contained genes encoding the siderophores 549 pyoverdine and pyochelin, pvdA and pchE, respectively. The presence of these 550 genes has previously been associated with increased killing of S. aureus in co-551 culture 9 . As P. aeruginosa PA21 was less lethal to S. aureus than P. aeruginosa 552 PAO1, it suggests these genes are not implicated and the improved survival is a 553 result of a different mechanism.…”

Iron acquisition in the cystic fibrosis lung and potential for novel therapeutic strategies

“…Staphylococcus aureus, however, can also compete with P. aeruginosa for free iron (Mashburn et al, 2005;Harrison et al, 2008). Nguyen & Oglesby-Sherrouse (2015) recently reported that iron depletion increases the ability of P. aeruginosa to suppress the growth of Staphylococcus aureus, and subsequently Filkins et al (2015) demonstrated that P. aeruginosa requires both of its major siderophores to kill Staphylococcus aureus in a CF bronchial epithelial co-culture model (Filkins et al, 2015) -all evidence that iron plays a central role in modulating interactions between P. aeruginosa and Staphylococcus aureus in the CF lung. Evidence also suggests that complex bacterial-fungal interactions occur in the CF lung.…”