SummaryHigh levels of copper are toxic and therefore bacteria must limit free intracellular levels to prevent cellular damage. In this study, we show that a number of pneumococcal genes are differentially regulated by copper, including an operon encoding a CopY regulator, a protein of unknown function (CupA) and a P1-type ATPase, CopA, which is conserved in all sequenced Streptococcus pneumoniae strains. Transcriptional analysis demonstrated that the cop operon is induced by copper in vitro, repressed by the addition of zinc and is autoregulated by the copperresponsive CopY repressor protein. We also demonstrate that the CopA ATPase is a major pneumococcal copper resistance mechanism and provide the first evidence that the CupA protein plays a role in copper resistance. Our results also show that copper homeostasis is important for pneumococcal virulence as the expression of the cop operon is induced in the lungs and nasopharynx of intranasally infected mice, and a copA -mutant strain, which had decreased growth in high levels of copper in vitro, showed reduced virulence in a mouse model of pneumococcal pneumonia. Furthermore, using the copA -mutant we observed for the first time in any bacteria that copper homeostasis also appears to be required for survival in the nasopharynx.

Staphylococcus aureus is a versatile pathogen capable of causing life-threatening infections. Many of its cell wall and exoproduct virulence determinants are controlled via the accessory gene regulator (agr). Although considered primarily as an extracellular pathogen, it is now recognized that S. aureus can be internalized by epithelial and endothelial cells. Traditional experimental approaches to investigate bacterial internalization are extremely time-consuming and notoriously irreproducible. We present here a new reporter gene method to assess intracellular growth of S. aureus in MAC-T cells that utilizes a gfp-luxABCDE reporter operon under the control of the Bacillus megaterium xylA promoter, which in S. aureus is expressed in a growth-dependent manner. This facilitates assessment of the growth of internalized bacteria in a nondestructive assay. The dual gfplux reporter cassette was also evaluated as a reporter of agr expression and used to monitor the temporal induction of agr during the MAC-T internalization process. The data obtained suggest that agr induction occurs prior to endosomal lysis and that agr-regulated exoproteins appear to be required prior to the release and replication of S. aureus within the infected MAC-T cells.Staphylococcus aureus is the etiologic agent of numerous infections in humans and domesticated animals and has been implicated in a multitude of diseases, ranging from minor wound infections to more serious diseases, including endocarditis, osteomyelitis, and septic shock (reviewed by Projan and Novick [34]). The expression of many S. aureus virulence factors is under the control of the accessory gene regulator (agr) which, on entering post-exponential phase, downregulates the production of cell surface-associated proteins and upregulates the expression of secreted toxins and extracellular enzymes (28,33,38). The role of the agr regulon is supported by in vivo studies, which show that agr mutants are greatly attenuated in several animal models, including intramammary infections (13), arthritis in mice (1), and endocarditis in rabbits (7). The agr locus is a quorum-sensing-regulated system activated by autoinducing peptide pheromone (AIP) (21, 25). The agr locus consists of two divergent transcriptional units, RNAII and RNAIII, which are under the control of the P2 and P3 promoters, respectively (reviewed by Novick and Muir [30]). RNAII is a polycistronic mRNA that encodes the agrB and agrD genes required for the synthesis of the AIP and also the two component signal transduction proteins, AgrA and AgrC, which are responsible for sensing and responding to the AIP. RNAIII is the effector molecule in the agr regulon acting primarily at the level of gene transcription. Different S. aureus strains produce AIPs with distinct structures, and strains can be grouped on this basis since they will activate the agr response of strains within the same group and inhibit the agr response of strains from different groups by competitive inhibition (21,30). This inhibitory action of AIPs has identi...

Staphylococcus aureus biofilm formation is induced in iron-restricted growth conditions in vitro. In this study, we showed that Emp and Eap play important roles in low-iron-induced biofilm formation of S. aureus Newman. Eap and Emp are secreted proteins which are non-covalently attached to the S. aureus cell surface and have previously been implicated in a number of aspects of S. aureus pathogenesis. We showed here that the transcription of these important virulence factors is induced by growth in low-iron medium, reflective of the in vivo environment. Our results show that iron regulation of Eap and Emp is Fur independent. However, Fur is required for full induction of eap and emp expression in low-iron conditions. In this study, we demonstrated that in addition to Fur, low-iron-induced biofilm formation requires Sae, Agr, and SarA. In iron-restricted growth conditions, Sae and Agr are essential for Emp and Eap expression and hence for biofilm formation, whereas SarA appears to have a less-significant role. We also showed that expression of the ica operon is required for biofilm formation in iron-restricted growth conditions. We demonstrated that in fact, ica is required for the expression of the important multifunctional virulence determinants eap and emp.

Copper is an important cofactor for many enzymes; however, high levels of copper are toxic. Therefore, bacteria must ensure there is sufficient copper for use as a cofactor but, more importantly, must limit free intracellular levels to prevent toxicity. In this study, we have used DNA microarray to identify Staphylococcus aureus copper-responsive genes. Transcriptional profiling of S. aureus SH1000 grown in excess copper identified a number of genes which fall into four groups, suggesting that S. aureus has four main mechanisms for adapting to high levels of environmental copper, as follows: (i) induction of direct copper homeostasis mechanisms; (ii) increased oxidative stress resistance; (iii) expression of the misfolded protein response; and (iv) repression of a number of transporters and global regulators such as Agr and Sae. Our experimental data confirm that resistance to oxidative stress and particularly to H 2 O 2 scavenging is an important S. aureus copper resistance mechanism. Our previous studies have demonstrated that Eap and Emp proteins, which are positively regulated by Agr and Sae, are required for biofilm formation under low-iron growth conditions. Our transcriptional analysis has confirmed that sae, agr, and eap are repressed under high-copper conditions and that biofilm formation is indeed repressed under high-copper conditions. Therefore, our results may provide an explanation for how copper films can prevent biofilm formation on catheters.