Bacteria and their viral predators (bacteriophages) are locked in a constant battle. In order to proliferate in phage-rich environments, bacteria have an impressive arsenal of defence mechanisms, and in response, phages have evolved counter-strategies to evade these antiviral systems. In this Review, we describe the various tactics that are used by phages to overcome bacterial resistance mechanisms, including adsorption inhibition, restriction-modification, CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) systems and abortive infection. Furthermore, we consider how these observations have enhanced our knowledge of phage biology, evolution and phage-host interactions.

For all microorganisms, acquisition of metal ions is essential for survival in the environment or in their infected host. Metal ions are required in many biological processes as components of metalloproteins and serve as cofactors or structural elements for enzymes. However, it is critical for bacteria to ensure that metal uptake and availability is in accordance with physiological needs, as an imbalance in bacterial metal homeostasis is deleterious. Indeed, host defense strategies against infection either consist of metal starvation by sequestration or toxicity by the highly concentrated release of metals. To overcome these host strategies, bacteria employ a variety of metal uptake and export systems and finely regulate metal homeostasis by numerous transcriptional regulators, allowing them to adapt to changing environmental conditions. As a consequence, iron, zinc, manganese, and copper uptake systems significantly contribute to the virulence of many pathogenic bacteria. However, during the course of our experiments on the role of iron and manganese transporters in extraintestinal Escherichia coli (ExPEC) virulence, we observed that depending on the strain tested, the importance of tested systems in virulence may be different. This could be due to the different set of systems present in these strains, but literature also suggests that as each pathogen must adapt to the particular microenvironment of its site of infection, the role of each acquisition system in virulence can differ from a particular strain to another. In this review, we present the systems involved in metal transport by Enterobacteria and the main regulators responsible for their controlled expression. We also discuss the relative role of these systems depending on the pathogen and the tissues they infect.

Roles of the ZnuACB and ZupT transporters were assessed in -and 48-fold reductions, respectively). In addition, in single-strain infection experiments, the ⌬znu and ⌬znu ⌬zupT mutants were reduced in the kidneys (P ‫؍‬ 0.0012 and P < 0.0001, respectively). Complementation of the CFT073 ⌬znu ⌬zupT mutant with the znuACB genes restored growth in Zn-deficient medium and bacterial numbers in the bladder and kidneys. The loss of the zinc transport systems decreased both motility and resistance to hydrogen peroxide, which could be restored by supplementation with zinc. Overall, the results indicate that Znu and ZupT are required for growth in zinc limited-conditions, that Znu is the predominant zinc transporter, and that the loss of Znu and ZupT has a cumulative effect on fitness during UTI, which may in part be due to reduced resistance to oxidative stress and motility.

An operon encoding a member of the family of ATP-binding cassette (ABC) divalent metal ion transporters, homologous to Salmonella enterica SitABCD, has been identified in the avian pathogenic Escherichia coli (APEC) strain χ7122. The sitABCD genes were located on the virulence plasmid pAPEC-1, and were highly similar at the nucleotide level to the chromosomally encoded sitABCD genes present in Shigella spp. A cloned copy of sitABCD conferred increased growth upon a siderophore-deficient E. coli strain grown in nutrient broth supplemented with the chelator 2,2′-dipyridyl. Ion rescue demonstrated that Sit-mediated growth promotion of this strain was due to the transport of iron. SitABCD mediated increased transport of both iron and manganese as demonstrated by uptake of 55Fe, 59Fe or 54Mn in E. coli K-12 strains deficient for the transport of iron (aroB feoB) and manganese (mntH) respectively. Isotope uptake and transport inhibition studies showed that in the iron transport deficient strain, SitABCD demonstrated a greater affinity for iron than for manganese, and SitABCD-mediated transport was higher for ferrous iron, whereas in the manganese transport deficient strain, SitABCD demonstrated greater affinity for manganese than for iron. Introduction of the APEC sitABCD genes into an E. coli K-12 mntH mutant also conferred increased resistance to the bactericidal effects of hydrogen peroxide. APEC strain χ7122 derivatives lacking either a functional SitABCD or a functional MntH transport system were as resistant to hydrogen peroxide as the wild-type strain, whereas a Δsit ΔmntH double mutant was more sensitive to hydrogen peroxide. Overall, the results demonstrate that in E. coli SitABCD represents a manganese and iron transporter that, in combination with other ion transport systems, may contribute to acquisition of iron and manganese, and resistance to oxidative stress.

The roles of SitABCD, MntH, and FeoB metal transporters in the virulence of avian pathogenic Escherichia coli (APEC) O78 strain 7122 were assessed using isogenic mutants in chicken infection models. In a single-strain infection model, compared to 7122, the ⌬sit strain demonstrated reduced colonization of the lungs, liver, and spleen. Complementation of the ⌬sit strain restored virulence. In a coinfection model, compared to the virulent APEC strain, the ⌬sit strain demonstrated mean 50-fold, 126-fold, and 25-fold decreases in colonization of the lungs, liver, and spleen, respectively. A ⌬mntH ⌬sit strain was further attenuated, demonstrating reduced persistence in blood and mean 1,400-fold, 954-fold, and 83-fold reduced colonization in the lungs, liver, and spleen, respectively. In coinfections, the ⌬feoB ⌬sit strain demonstrated reduced persistence in blood but increased colonization of the liver. The ⌬mntH, ⌬feoB, and ⌬feoB ⌬mntH strains were as virulent as the wild type in either of the infection models. Strains were also tested for sensitivity to oxidative stress-generating agents. The ⌬mntH ⌬sit strain was the most sensitive strain and was significantly more sensitive than the other strains to hydrogen peroxide, plumbagin, and paraquat. sit sequences were highly associated with APEC and human extraintestinal pathogenic E. coli compared to commensal isolates and diarrheagenic E. coli. Comparative genomic analyses also demonstrated that sit sequences are carried on conjugative plasmids or associated with phage elements and were likely acquired by distinct genetic events among pathogenic E. coli and Shigella sp. strains. Overall, the results demonstrate that SitABCD contributes to virulence and, together with MntH, to increased resistance to oxidative stress.

Virulence factors of pathogenic Escherichia coli belonging to a recently emerged and disseminated clonal group associated with urinary tract infection (UTI), provisionally designated clonal group A (CGA), have not been experimentally investigated. We used a mouse model of ascending UTI with CGA member strain UCB34 in order to identify genes of CGA that contribute to UTI. iha was identified to be expressed by strain UCB34 in the mouse kidney using selective capture of transcribed sequences. iha from strain UCB34 demonstrated a siderophore receptor phenotype when cloned in a catecholate siderophore receptor-negative E. coli K-12 strain, as shown by growth promotion experiments and uptake of 55 Fe complexed to enterobactin or its linear 2, 3-dihydroxybenzoylserine (DHBS) siderophore derivatives. Siderophore-mediated growth promotion by Iha was TonB dependent. Growth and iron uptake were more marked with linear DHBS derivatives than with purified enterobactin. The reported phenotype of adherence to epithelial cells conferred by expressing iha from a multicopy cloning vector in a poorly adherent E. coli K-12 host strain was confirmed to be specific to iha, in comparison with other siderophore receptor genes. iha expression was regulated by the ferric uptake regulator Fur and by iron availability, as shown by real-time reverse transcriptase PCR. In a competitive infection experiment using the mouse UTI model, wild-type strain UCB34 significantly outcompeted an isogenic iha null mutant. Iha thus represents a Fur-regulated catecholate siderophore receptor that, uniquely, exhibits an adherence-enhancing phenotype and is the first described urovirulence factor identified in a CGA strain.Urinary tract infections (UTIs) are one of the most frequent bacterial infections in industrialized countries, and Escherichia coli is the major causal agent (26, 61). Many virulence factors associated with extraintestinal pathogenic E. coli (ExPEC) strains, the distinctive strains that cause most UTIs, are important for establishing infection. These include adhesins, toxins, iron acquisition systems, and capsular antigens (11,23,25). Extraintestinal infections, including UTIs, are caused predominantly by E. coli isolates belonging to phylogenetic group B2 (60 to 70%), whereas the remaining cases are caused mostly by strains belonging to phylogenetic group D (8,42,66). Most research into the pathogenic mechanisms of ExPEC has focused on archetype strains, such as CFT073, J96, CP9, and 536, which all belong to group B2. Much less attention has been given to the virulence mechanisms of group D ExPEC strains, which represent the second most important cause of UTI after group B2 strains (8,41,66).Recently, a multidrug-resistant clonal group, termed clonal group A (CGA), was identified as a cause of UTI outbreaks in California, Michigan, and Minnesota (46). It is now known that this clonal group is widespread and quite prevalent throughout the United States and is also widely prevalent, although to a lesser extent, in many other countries (40,4...

Streptococcus pneumoniae causes several diseases, including pneumonia, septicemia, and meningitis. Phage Dp-1 is one of the very few isolated virulent S. pneumoniae bacteriophages, but only a partial characterization is currently available. Here, we confirmed that Dp-1 belongs to the family Siphoviridae. Then, we determined its complete genomic sequence of 56,506 bp. It encodes 72 open reading frames, of which 44 have been assigned a function. We have identified putative promoters, Rho-independent terminators, and several genomic clusters. We provide evidence that Dp-1 may be using a novel DNA replication system as well as redirecting host protein synthesis through queuosine-containing tRNAs. Liquid chromatography-mass spectrometry analysis of purified phage Dp-1 particles identified at least eight structural proteins. Finally, using comprehensive yeast two-hybrid screens, we identified 156 phage protein interactions, and this intraviral interactome was used to propose a structural model of Dp-1.Streptococcus pneumoniae (pneumococcus) is a low-GC-content, Gram-positive bacterium belonging to the mitis group of streptococci (40). It is a facultative human pathogen, colonizing the mucosal surface of the upper respiratory tract and causing invasive infections such as pneumonia, meningitis, and sepsis (97). S. pneumoniae diseases are associated with severe morbidity and a high rate of mortality, especially among young children and the elderly. Antibiotics are commonly used to control and cure the invasive pneumococcal diseases, but this high exposure to antibiotics has led to multiresistant S. pneumoniae strains worldwide (79). Vaccination is efficient in preventing invasive diseases (103), but pneumococcal clones expressing capsular polysaccharides with serotypes not included in the current vaccines' formulations exist, and the incidence of invasive diseases induced by these vaccine-escaping clones is increasing (34). Replacement of the vaccine-included clones by the vaccine-escaping ones is possible (91).The appearance of antibiotic-resistant and vaccine-escaping clones is partly due to the high genome plasticity of S. pneumoniae. This genome plasticity is supported by the ability of the pneumococcus to acquire DNA through natural competence and by genome rearrangements facilitated by the mobile elements present in its genome (15,43,93). S. pneumoniae is also able to colonize many sites inside the human host using phase variation of cell surface components such as polysaccharide capsule, lipoteichoic acid, and cell-surface expressed proteins (42,78,101). The complexity of physiology and genetics of S. pneumoniae complicates the search for novel therapeutic approaches needed to counter increasing antibiotic resistance and emergence of the vaccine-escaping clones.One of the novel therapeutic approaches toward antibioticresistant strains is the use of bacteriophage proteins to lyse pneumococcus cells. The feasibility of this approach was first demonstrated when bacteriophage endolysin enzymes were used successfully t...

Mass spectrometry analysis of Streptococcus pneumoniae bacteriophage Cp-1 identified a total of 12 proteins, and proteome-wide yeast two-hybrid screens revealed 17 binary interactions mainly among these structural proteins. On the basis of the resulting linkage map, we suggest an improved structural model of the Cp-1 virion.