Nuclease modulates biofilm formation in community-associated methicillin-resistant Staphylococcus aureus

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is an emerging contributor to biofilm-related infections. We recently reported that strains lacking sigma factor B (sigB) in the USA300 lineage of CA-MRSA are unable to develop a biofilm. Interestingly, when spent media from a USA300 sigB mutant was incubated with other S. aureus strains, biofilm formation was inhibited. Following fractionation and mass spectrometry analysis, the major anti-biofilm factor identified in the spent media was secreted thermonuclease (Nuc). Considering reports that extracellular DNA (eDNA) is an important component of the biofilm matrix, we investigated the regulation and role of Nuc in USA300. The expression of the nuc gene was increased in a sigB mutant, repressed by glucose supplementation, and was unaffected by the agr quorum-sensing system. A FRET assay for Nuc activity was developed and confirmed the regulatory results. A USA300 nuc mutant was constructed and displayed an enhanced biofilm-forming capacity, and the nuc mutant also accumulated more high molecular weight eDNA than the WT and regulatory mutant strains. Inactivation of nuc in the USA300 sigB mutant background partially repaired the sigB biofilm-negative phenotype, suggesting that nuc expression contributes to the inability of the mutant to form biofilm. To test the generality of the nuc mutant biofilm phenotypes, the mutation was introduced into other S. aureus genetic backgrounds and similar increases in biofilm formation were observed. Finally, using multiple S. aureus strains and regulatory mutants, an inverse correlation between Nuc activity and biofilm formation was demonstrated. Altogether, our findings confirm the important role for eDNA in the S. aureus biofilm matrix and indicates Nuc is a regulator of biofilm formation.     

Staphylococcus aureus accessory regulators: expression within biofilms and effect on adhesion.

Many of the genes encoding the virulence factors for Staphylococcus aureus are controlled by the accessory gene regulator (agr) and staphylococcal accessory regulator (sar). This regulation may be affected by the environment in which the organisms are grown. In the majority of ecosystems, bacteria grow attached to surfaces and form biofilms. We used S. aureus strains containing mutations inactivating agr and sar to determine whether the presence of these genes influences the attachment of the bacterium to a surface. We also used strains harbouring reporter constructs of the agr and sar operons to determine their expression in biofilms. The attachment study results showed that the sarA mutant strain adhered better to glass than did the agrA mutant or the wild type. There was an increased adherence to fibronectin-coated glass for all three strains compared to glass. Thus, these adhesion studies demonstrate that agr and sar have pleiotrophic effects on the surface expression of molecules responsible for binding to different substrata. In the biofilms higher numbers of bacteria and the greatest expression were observed at the base, but there were no observable differences between the reporter constructs. Expression of the agr and sar reporter fusions was significantly higher in the deepest layers of the biofilms where the greatest numbers of bacteria were also observed, perhaps as one might expect for genes that are regulated in a cell density dependent fashion.     

Biofilm formation by Staphylococcus epidermidis depends on functional RsbU, an activator of the sigB operon: differential activation mechanisms due to ethanol and salt stress

Staphylococcus epidermidis is a common pathogen in medical device-associated infections. Its major pathogenetic factor is the ability to form adherent biofilms. The polysaccharide intercellular adhesin (PIA), which is synthesized by the products of the icaADBC gene cluster, is essential for biofilm accumulation. In the present study, we characterized the gene locus inactivated by Tn917 insertions of two isogenic, icaADBC-independent, biofilm-negative mutants, M15 and M19, of the biofilm-producing bacterium S. epidermidis 1457. The insertion site was the same in both of the mutants and was located in the first gene, rsbU, of an operon highly homologous to the sigB operons of Staphylococcus aureus and Bacillus subtilis. Supplementation of Trypticase soy broth with NaCl (TSB(NaCl)) or ethanol (TSB(EtOH)), both of which are known activators of sigB, led to increased biofilm formation and PIA synthesis by S. epidermidis 1457. Insertion of Tn917 into rsbU, a positive regulator of alternative sigma factor sigma(B), led to a biofilm-negative phenotype and almost undetectable PIA production. Interestingly, in TSB(EtOH), the mutants were enabled to form a biofilm again with phenotypes similar to those of the wild type. In TSB(NaCl), the mutants still displayed a biofilm-negative phenotype. No difference in primary attachment between the mutants and the wild type was observed. Similar phenotypic changes were observed after transfer of the Tn917 insertion of mutant M15 to the independent and biofilm-producing strain S. epidermidis 8400. In 11 clinical S. epidermidis strains, a restriction fragment length polymorphism of the sigB operon was detected which was independent of the presence of the icaADBC locus and a biofilm-positive phenotype. Obviously, different mechanisms are operative in the regulation of PIA expression in stationary phase and under stress induced by salt or ethanol.     

Multiple virulence factors are required for Staphylococcus aureus-induced apoptosis in endothelial cells

Staphylococcus aureus infections can result in sepsis and septic shock associated with vascular damage and multiple organ failure. Apoptosis appears to play a key role during sepsis, and the ability of S. aureus to induce apoptosis in endothelial cells might contribute to metastatic infection. In contrast to leukocytes, in human umbilical vein endothelial cells and two endothelial cell lines neither purified alpha-toxin nor staphylococcal supernatants were sufficient to induce apoptosis. Apoptosis induction instead required staphylococcal invasion as well as signals from metabolically active intracellular staphylococci. Only strongly haemolytic and invasive staphylococci, but not non-invasive strains induced apoptosis that was caspase-dependent but Fas-independent. However, only a subgroup of clinical isolates with an invasive and haemolytic phenotype induced apoptosis. Expression of alpha-toxin in a non-haemolytic strain partially restored apoptosis induction, suggesting a role of alpha-toxin as a trigger of apoptosis. Furthermore, infection of endothelial cells with isogenic mutants of various regulator genes revealed that apoptosis induction was dependent on the global regulator agr and the alternative sigma factor sigB, but not influenced by sarA. Together, our results indicate that the ability of S. aureus to induce apoptosis in endothelial cells is determined by multiple virulence factors.     

Staphylococcus aureus develops an alternative, ica-independent biofilm in the absence of the arlRS two-component system

The biofilm formation capacity of Staphylococcus aureus clinical isolates is considered an important virulence factor for the establishment of chronic infections. Environmental conditions affect the biofilm formation capacity of S. aureus, indicating the existence of positive and negative regulators of the process. The majority of the screening procedures for identifying genes involved in biofilm development have been focused on genes whose presence is essential for the process. In this report, we have used random transposon mutagenesis and systematic disruption of all S. aureus two-component systems to identify negative regulators of S. aureus biofilm development in a chemically defined medium (Hussain-Hastings-White modified medium [HHWm]). The results of both approaches coincided in that they identified arlRS as a repressor of biofilm development under both steady-state and flow conditions. The arlRS mutant exhibited an increased initial attachment as well as increased accumulation of poly-N-acetylglucosamine (PNAG). However, the biofilm formation of the arlRS mutant was not affected when the icaADBC operon was deleted, indicating that PNAG is not an essential compound of the biofilm matrix produced in HHWm. Disruption of the major autolysin gene, atl, did not produce any effect on the biofilm phenotype of an arlRS mutant. Epistatic experiments with global regulators involved in staphylococcal-biofilm formation indicated that sarA deletion abolished, whereas agr deletion reinforced, the biofilm development promoted by the arlRS mutation.