SUMMARY: The genusLegionellacontains more than 50 species, of which at least 24 have been associated with human infection. The best-characterized member of the genus,Legionella pneumophila, is the major causative agent of Legionnaires' disease, a severe form of acute pneumonia.L. pneumophilais an intracellular pathogen, and as part of its pathogenesis, the bacteria avoid phagolysosome fusion and replicate within alveolar macrophages and epithelial cells in a vacuole that exhibits many characteristics of the endoplasmic reticulum (ER). The formation of the unusualL. pneumophilavacuole is a feature of its interaction with the host, yet the mechanisms by which the bacteria avoid classical endosome fusion and recruit markers of the ER are incompletely understood. Here we review the factors that contribute to the ability ofL. pneumophilato infect and replicate in human cells and amoebae with an emphasis on proteins that are secreted by the bacteria into theLegionellavacuole and/or the host cell. Many of these factors undermine eukaryotic trafficking and signaling pathways by acting as functional and, in some cases, structural mimics of eukaryotic proteins. We discuss the consequences of this mimicry for the biology of the infected cell and also for immune responses toL. pneumophilainfection.

Colonizing enteric bacteria are shown to inhibit the antimicrobial process of host cell apoptosis through the action of NleB1, a type III secretion system effector with N-acetylglucosamine transferase activity, which can bind and modify eukaryotic death-domain-containing proteins. Previous studies identified a group of effectors from enteropathogenic Escherichia coli that can inhibit host nuclear factor-κB (NF-κB) signalling, yet only one of them, known as NleB, is required for bacterial virulence in vivo. Two papers published in this issue demonstrate the unique mechanism of action of NleB. It directly targets death receptor signalling complexes, binding to the death domains (DDs) of multiple DD-containing proteins including the TNF receptor, FAS, RIPK1, TRADD and FADD. NleB is shown to function as an N-acetylglucosamine (GlcNAc) transferase that modifies a conserved DD arginine, blocking the receptor-adapter interaction. These findings suggest that GlcNAc modification is essential for bacterial virulence and can regulate death receptor signalling. Successful infection by enteric bacterial pathogens depends on the ability of the bacteria to colonize the gut, replicate in host tissues and disseminate to other hosts. Pathogens such as Salmonella, Shigella and enteropathogenic and enterohaemorrhagic (EPEC and EHEC, respectively) Escherichia coli use a type III secretion system (T3SS) to deliver virulence effector proteins into host cells during infection that promote colonization and interfere with antimicrobial host responses1,2,3. Here we report that the T3SS effector NleB1 from EPEC binds to host cell death-domain-containing proteins and thereby inhibits death receptor signalling. Protein interaction studies identified FADD, TRADD and RIPK1 as binding partners of NleB1. NleB1 expressed ectopically or injected by the bacterial T3SS prevented Fas ligand or TNF-induced formation of the canonical death-inducing signalling complex (DISC) and proteolytic activation of caspase-8, an essential step in death-receptor-induced apoptosis. This inhibition depended on the N-acetylglucosamine transferase activity of NleB1, which specifically modified Arg 117 in the death domain of FADD. The importance of the death receptor apoptotic pathway to host defence was demonstrated using mice deficient in the FAS signalling pathway, which showed delayed clearance of the EPEC-like mouse pathogen Citrobacter rodentium and reversion to virulence of an nleB mutant. The activity of NleB suggests that EPEC and other attaching and effacing pathogens antagonize death-receptor-induced apoptosis of infected cells, thereby blocking a major antimicrobial host response.

Proteins containing membrane attack complex/perforin (MACPF) domains play important roles in vertebrate immunity, embryonic development, and neural-cell migration. In vertebrates, the ninth component of complement and perforin form oligomeric pores that lyse bacteria and kill virus-infected cells, respectively. However, the mechanism of MACPF function is unknown. We determined the crystal structure of a bacterial MACPF protein, Plu-MACPF from Photorhabdus luminescens , to 2.0 angstrom resolution. The MACPF domain reveals structural similarity with poreforming cholesterol-dependent cytolysins (CDCs) from Gram-positive bacteria. This suggests that lytic MACPF proteins may use a CDC-like mechanism to form pores and disrupt cell membranes. Sequence similarity between bacterial and vertebrate MACPF domains suggests that the fold of the CDCs, a family of proteins important for bacterial pathogenesis, is probably used by vertebrates for defense against infection.

Staphylococcus aureus frequently invades the human bloodstream, leading to life threatening bacteremia and often secondary foci of infection. Failure of antibiotic therapy to eradicate infection is frequently described; in some cases associated with altered S. aureus antimicrobial resistance or the small colony variant (SCV) phenotype. Newer antimicrobials, such as linezolid, remain the last available therapy for some patients with multi-resistant S. aureus infections. Using comparative and functional genomics we investigated the molecular determinants of resistance and SCV formation in sequential S. aureus isolates from a patient who had a persistent and recurrent S. aureus infection, after failed therapy with multiple antimicrobials, including linezolid. Two point mutations in key staphylococcal genes dramatically affected clinical behaviour of the bacterium, altering virulence and antimicrobial resistance. Most strikingly, a single nucleotide substitution in relA (SACOL1689) reduced RelA hydrolase activity and caused accumulation of the intracellular signalling molecule guanosine 3′, 5′-bis(diphosphate) (ppGpp) and permanent activation of the stringent response, which has not previously been reported in S. aureus. Using the clinical isolate and a defined mutant with an identical relA mutation, we demonstrate for the first time the impact of an active stringent response in S. aureus, which was associated with reduced growth, and attenuated virulence in the Galleria mellonella model. In addition, a mutation in rlmN (SACOL1230), encoding a ribosomal methyltransferase that methylates 23S rRNA at position A2503, caused a reduction in linezolid susceptibility. These results reinforce the exquisite adaptability of S. aureus and show how subtle molecular changes cause major alterations in bacterial behaviour, as well as highlighting potential weaknesses of current antibiotic treatment regimens. The treatment of serious infections caused by Staphylococcus aureus is complicated by the development of antibiotic resistance, and in some cases the appearance of more persistent bacteria that have a reduced growth rate resulting in small colony variants (SCV). Here we have shown using whole genome sequencing and gene replacement experiments on sequential S. aureus isolates obtained from a patient with a serious bloodstream infection, how S. aureus evolved into a multi-antibiotic resistant, persistent and almost untreatable SCV. Specifically we show that a minor DNA change in a S. aureus gene encoding an enzyme called RelA causes an accumulation of a small signalling molecule called (p)ppGpp, which in turn leads to persistent activation of the important bacterial stress response known as the stringent response. This is the first report of the involvement of the stringent response in S. aureus SCV formation and its association with persistent infection. Additionally, we have uncovered a novel mechanism of resistance to the new antimicrobial linezolid, caused by a mutation in a gene encoding a 23S rRNA methyltransferase. This study highlights the exquisite adaptability of this important pathogen in the face of antimicrobial treatment.