Effect of iron and haemoglobin on Staphylococcus aureus biofilms

Abstract

The biofilm infection of Staphylococcus aureus is one of its most common and ‘silently’ harmful virulence traits, leading to endocarditis, central line-associated bloodstream infections, ventilator associated pneumonia, implant-related infection and surgical site infections among others. Biofilms are bacterial aggregations embedded in an extracellular matrix with specific gene expression or metabolic networks different from their planktonic counterparts. Biofilms provide a chronic reservoir of infection due to a higher resistance to our immune system and to antibiotics. Iron restriction in the body has been reported as an important signal for biofilm induction. Haemoglobin is the most abundant iron source in the host and S. aureus has the appropriate uptake system to acquire it, hence, this thesis questions the role of haemoglobin in biofilm formation. In this study, it was used a physiological medium (RPMI 1640) to culture biofilms mimicking aspects of the in vivo environment. We used Newman as a model strain, two isogenic strains Newman Δfur and Newman ΔisdB and also include two clinical isolates to represent a broader picture of biofilms in S. aureus and the response to iron, not the strain-specific behavior. Biofilm response to iron was assessed from a phenotypic, microscopic and transcriptional perspective. Microplate assays showed that haemoglobin is sensed as a stimulus in its own, independent of the iron it carries, as it promotes biofilm matrix formation through PIA production whereas the provision of inorganic iron does not. Clinical isolates from endocarditis and bacteraemia infections were not strong biofilm formers under iron restriction; however they both showed induced biofilm formation in the presence of haemoglobin. IsdB, the main haemoglobin receptor, is not essential in the haemoglobin-biofilm response under the conditions used here. Microscopic analysis showed that provision of haemoglobin to biofilms also triggered a characteristic clustered architecture, a response conserved among all tested isolates. The central regulator of iron uptake, Fur, was shown to have a role in regulating cell death (and probably autolysis) under iron restriction, suggesting extracellular DNA has relevance in in the biofilm matrix. The two iron sources, haemoglobin and iron chloride, had different effects on the expression of Agr quorum sensing (QS), a regulator of biofilm dispersal, in a strain-specific manner. In Newman, haemoglobin significantly repressed elements of Agr QS system, whereas inorganic iron induced them. In the endocarditis isolate, provision of haemoglobin induced Agr QS possibly favoring detachment of clusters, allowing travel through bloodstream. In the bacteraemia isolate, repression of Agr QS by haemoglobin suggests preservation of the biofilm; however this was not matched with expression of PSMs, molecules with structuring roles in the biofilm. Overall, we suggest that expression of PSMα and PSMβ involves an extra regulation mechanism besides the established AgrA-dependency. We were unable to correlate expression of PSMs with a structuring role in the biofilm architecture in response to haemoglobin. Haemoglobin/haemin is not toxic to the iron restricted biofilm, suggesting that iron restriction normally found in the body decreases haemtoxicity thresholds. Despite variations among isolates of S. aureus in some situations, the response to haemoglobin as a stimulus to the biofilm is conserved, inducing or repressing regulatory networks or metabolic pathways ultimately affecting biofilm formation or dispersal activities. This knowledge of S. aureus biofilm behavior in the presence of haemoglobin could be useful in targeting expressed molecules or antigens to develop vaccines specifically to prevent this type of biofilm infection.