Low Concentration of the Neutrophil Proteases Cathepsin G, Cathepsin B, Proteinase-3 and Metalloproteinase-9 Induce Biofilm Formation in Non-Biofilm-Forming Staphylococcus epidermidis Isolates

Abstract

Neutrophils play a crucial role in eliminating bacteria that invade the human body; however, cathepsin G can induce biofilm formation in a non-biofilm-forming Staphylococcus epidermidis 1457 strain, suggesting that neutrophil proteases may be involved in biofilm formation. Cathepsin G, cathepsin B, proteinase-3, and metalloproteinase-9 (MMP-9) from neutrophils were tested on the biofilm induction in commensal (skin isolated) and clinical non-biofilm-forming S. epidermidis isolates. From 81 isolates, 53 (74%) were aap+, icaA−, icaD− genotype, and without the capacity of biofilm formation under conditions of 1% glucose, 4% ethanol or 4% NaCl, but these 53 non-biofilm-forming isolates induced biofilm by the use of different neutrophil proteases. Of these, 62.3% induced biofilm with proteinase-3, 15% with cathepsin G, 10% with cathepsin B and 5% with MMP -9, where most of the protease-induced biofilm isolates were commensal strains (skin). In the biofilm formation kinetics analysis, the addition of phenylmethylsulfonyl fluoride (PMSF; a proteinase-3 inhibitor) showed that proteinase-3 participates in the cell aggregation stage of biofilm formation. A biofilm induced with proteinase-3 and DNAse-treated significantly reduced biofilm formation at an early time (initial adhesion stage of biofilm formation) compared to untreated proteinase-3-induced biofilm (p < 0.05). A catheter inoculated with a commensal (skin) non-biofilm-forming S. epidermidis isolate treated with proteinase-3 and another one without the enzyme were inserted into the back of a mouse. After 7 days of incubation period, the catheters were recovered and the number of grown bacteria was quantified, finding a higher amount of adhered proteinase-3-treated bacteria in the catheter than non-proteinase-3-treated bacteria (p < 0.05). Commensal non-biofilm-forming S. epidermidis in the presence of neutrophil cells significantly induced the biofilm formation when multiplicity of infection (MOI) 1:0.01 (neutrophil:bacteria) was used, but the addition of a cocktail of protease inhibitors impeded biofilm formation. A neutrophil:bacteria assay did not induce neutrophil extracellular traps (NETs). Our results suggest that neutrophils, in the presence of commensal non-biofilm-forming S. epidermidis, do not generate NETs formation. The effect of neutrophils is the production of proteases, and proteinase-3 releases bacterial DNA at the initial adhesion, favoring cell aggregation and subsequently leading to biofilm formation.

THE Staphylococcus epidermidis BIOFILM MAY EVADE THE NEUTROPHIL IMMUNOLOGICAL RESPONSE

“…In addition, another study showed that cathepsin G, cathepsin B, metalloproteinase 9, and neutrophil proteinase 3 induce biofilm formation in S. epidermidis isolates with a non-biofilm-producing phenotype. It was also shown that S. epidermidis with a non-biofilmproducing phenotype in the presence of neutrophil, the biofilm is induced by the release of the proteases mentioned above (Gómez-Alonso et al, 2022). These observations strongly suggest that S. epidermidis has taken advantage of releasing neutrophil granule contents as neutrophil proteases cleave Aap protein for biofilm formation.…”

Non-biofilm-forming Staphylococcus epidermidis planktonic cell supernatant induces alterations in osteoblast biological function

“…We have reported that S. epidermidis isolates from PJI are more resistant to biofilm production under various in vitro biofilm induction conditions and that most commensal isolates do not form biofilms 42 . Furthermore, commensal isolates from healthy skin can induce biofilm formation when exposed to neutrophil proteases, such as cathepsin G, cathepsin B, proteinase-3 and metalloproteinase-9 53 . In this study, we demonstrated that both the clinical isolate Se353 and commensal isolate HS145 affected osteoblast function, suggesting that their influence on osteoblast function is independent of biofilm status, which is surprising because it is well known that commensal isolates are not potentially virulent.…”

Interactions of Neutrophils with the Polymeric Molecular Components of the Biofilm Matrix in the Context of Implant-Associated Bone and Joint Infections

“…Both genes are required for Aap-dependent S. epidermidis biofilm formation. Indeed, in a recent paper [83], Gomez-Alonso et al demonstrated the induction of biofilm production by neutrophil proteases in non-biofilm-forming commensal isolates of S. epidermidis. More precisely, commensal isolates with icaA−, icaD− and aap+, sepA+ genotypes, which had previously been recognized as non-biofilm-forming, produced biofilms in the presence of neutrophilic proteases such as cathepsin G, cathepsin B, proteinase-3 and MM-9 in neutrophils.…”

“…More precisely, commensal isolates with icaA−, icaD− and aap+, sepA+ genotypes, which had previously been recognized as non-biofilm-forming, produced biofilms in the presence of neutrophilic proteases such as cathepsin G, cathepsin B, proteinase-3 and MM-9 in neutrophils. Neutrophils do not generate NETs against commensal bacteria but produce proteases that help commensal bacteria in survival [83]. Aap contributes both to the initial attachment phase and to the intercellular connections, with each cell stretching twisted fibrils of truncated Aap to interact with the neighboring cells.…”