SummaryGroup A Streptococcus (GAS, Streptococcus pyogenes) is a Gram-positive human pathogen responsible for several acute diseases and autoimmune sequelae that account for half a million deaths worldwide every year. GAS infections require the capacity of the pathogen to adhere to host tissues and assemble in cell aggregates. Furthermore, a role for biofilms in GAS pathogenesis has recently been proposed. Here we investigated the role of GAS pili in biofilm formation. We demonstrated that GAS pilusnegative mutants, in which the genes encoding either the pilus backbone structural protein or the sortase C1 have been deleted, showed an impaired capacity to attach to a pharyngeal cell line. The same mutants were much less efficient in forming cellular aggregates in liquid culture and microcolonies on human cells. Furthermore, mutant strains were incapable of producing the typical three-dimensional layer with bacterial microcolonies embedded in a carbohydrate polymeric matrix. Complemented mutants had an adhesion and aggregation phenotype similar to the wild-type strain. Finally, in vivo expression of pili was indirectly confirmed by demonstrating that most of the sera from human patients affected by GASmediated pharyngitis recognized recombinant pili proteins. These data support the role of pili in GAS adherence and colonization and suggest a general role of pili in all pathogenic streptococci.

Daptomycin is a bactericidal antibiotic of last resort for serious infections caused by methicillin-34resistant Staphylococcus aureus (MRSA) 1,2 . Although resistance is rare, treatment failure can occur 35 in >20% of cases 3,4 and so there is a pressing need to identify and mitigate factors that contribute 36 to poor therapeutic outcomes. Here, we show that loss of the Agr quorum-sensing system, which 37 frequently occurs in clinical isolates, enhances S. aureus survival during daptomycin treatment. 38Wild-type S. aureus was killed rapidly by daptomycin but Agr-defective mutants survived 39 antibiotic exposure by releasing membrane phospholipid, which bound and inactivated the 40 antibiotic. Although wild-type bacteria also released phospholipid in response to daptomycin, Agr-41 triggered secretion of small cytolytic toxins, known as phenol soluble modulins, prevented 42 antibiotic inactivation. Phospholipid shedding by S. aureus occurred via an active process and was 43 inhibited by the β-lactam antibiotic oxacillin, which slowed inactivation of daptomycin and 44 enhanced bacterial killing. In conclusion, S. aureus possesses a transient defence mechanism that 45 protects against daptomycin, which can be compromised by Agr-triggered toxin production or an 46 existing therapeutic antibiotic. 47 S. aureus encodes multiple virulence factors, many of which are controlled by Agr 5,6 , a 48 quorum-sensing system encoded by a 4 gene operon (agrBDCA) and a gene encoding a regulatory 49 RNA (RNAIII). However, invasive S. aureus infections often give rise to Agr-defective mutants, 50 typically involving agrA or agrC, hypothesised to provide a selective advantage in the presence of 51 antibiotics [7][8][9][10][11][12][13][14] . To test this hypothesis, we determined the killing kinetics of wild-type S. aureus or agr 52 mutants by clinically-relevant antibiotics. 53Agr status did not affect the rate of staphylococcal killing by vancomycin, oxacillin or 54 gentamicin ( Supplementary Fig. 1, 2). By contrast, whilst wild-type S. aureus was killed by 55 daptomycin, loss of quorum-sensing components of Agr (AgrA or AgrC) enabled S. aureus strains 56 USA300 or SH1000 to survive in the presence of daptomycin during the first 8 hours of exposure (Fig. 57 1a,b). A mutant lacking the regulatory RNAIII component of agr was killed as efficiently as the wild-58 type (Fig. 1a), as were agrA or agrC mutants complemented with the relevant genes on plasmids 59 3 ( Supplementary Fig. 3). After the initial period of killing, CFU counts of both wild-type and agr-60 mutant S. aureus recovered to similar levels by 24 h, without the acquisition of resistance, explaining 61 why all strains had identical daptomycin MIC and MBC values (Fig. 1c, Supplementary Supplementary Fig. 4). This biphasic killing and subsequent recovery profile is similar to several 63 previously reported daptomycin killing assays, although the contribution of Agr to this phenomenon 64 was unknown [15][16][17] . 65In addition to agr-deletion mutants, clinical isolat...

The difficulty in successfully treating infections caused by methicillin-resistant Staphylococcus aureus (MRSA) has led to them being referred to as highly virulent or pathogenic. In our study of one of the major healthcare-associated MRSA (HA-MRSA) clones, we show that expression of the gene responsible for conferring methicillin resistance (mecA) is also directly responsible for reducing the ability of HA-MRSA to secrete cytolytic toxins. We show that resistance to methicillin induces changes in the cell wall, which affects the bacteria's agr quorum sensing system. This leads to reduced toxin expression and, as a consequence, reduced virulence in a murine model of sepsis. This diminished capacity to cause infection may explain the inability of HA-MRSA to move into the community and help us understand the recent emergence of community-associated MRSA (CA-MRSA). CA-MRSA typically express less penicillin-binding protein 2a (encoded by mecA), allowing them to maintain full virulence and succeed in the community environment.

Entry of Staphylococcus aureus into the bloodstream can lead to metastatic abscess formation and infective endocarditis. Crucial to the development of both these conditions is the interaction of S. aureus with endothelial cells. In vivo and in vitro studies have shown that the staphylococcal invasin FnBPA triggers bacterial invasion of endothelial cells via a process that involves fibronectin (Fn) bridging to α5β1 integrins. The Fn-binding region of FnBPA usually contains 11 non-identical repeats (FnBRs) with differing affinities for Fn, which facilitate the binding of multiple Fn molecules and may promote integrin clustering. We thus hypothesized that multiple repeats are necessary to trigger the invasion of endothelial cells by S. aureus. To test this we constructed variants of fnbA containing various combinations of FnBRs. In vitro assays revealed that endothelial cell invasion can be facilitated by a single high-affinity, but not low-affinity FnBR. Studies using a nisin-inducible system that controlled surface expression of FnBPA revealed that variants encoding fewer FnBRs required higher levels of surface expression to mediate invasion. High expression levels of FnBPA bearing a single low affinity FnBR bound Fn but did not invade, suggesting that FnBPA affinity for Fn is crucial for triggering internalization. In addition, multiple FnBRs increased the speed of internalization, as did higher expression levels of FnBPA, without altering the uptake mechanism. The relevance of these findings to pathogenesis was demonstrated using a murine sepsis model, which showed that multiple FnBRs were required for virulence. In conclusion, multiple FnBRs within FnBPA facilitate efficient Fn adhesion, trigger rapid bacterial uptake and are required for pathogenesis.

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