David W. Holden

Genes encoding putative effector proteins of the type III secretion system of Salmonella pathogenicity island 2 are required for bacterial virulence and proliferation in macrophages

The type III secretion system of Salmonella pathogenicity island 2 (SPI‐2) is required for systemic infection of this pathogen in mice. Cloning and sequencing of a central region of SPI‐2 revealed the presence of genes encoding putative chaperones and effector proteins of the secretion system. The predicted products of the sseB, sseC and sseD genes display weak but significant similarity to amino acid sequences of EspA, EspD and EspB, which are secreted by the type III secretion system encoded by the locus of enterocyte effacement of enteropathogenic Escherichia coli. The transcriptional activity of an sseA::luc fusion gene was shown to be dependent on ssrA, which is required for the expression of genes encoding components of the secretion system apparatus. Strains carrying non‐polar mutations in sseA, sseB or sseC were severely attenuated in virulence, strains carrying mutations in sseF or sseG were weakly attenuated, and a strain with a mutation in sseE had no detectable virulence defect. These phenotypes were reflected in the ability of mutant strains to grow within a variety of macrophage cell types: strains carrying mutations in sseA, sseB or sseC failed to accumulate, whereas the growth rates of strains carrying mutations in sseE, sseF or sseG were only modestly reduced. These data suggest that, in vivo, one of the functions of the SPI‐2 secretion system is to enable intracellular bacterial proliferation.

Salmonella maintains the integrity of its intracellular vacuole through the action of SifA

A method based on the Competitive Index was used to identify Salmonella typhimurium virulence gene interactions during systemic infections of mice. Analysis of mixed infections involving single and double mutant strains showed that OmpR, the type III secretion system of Salmonella pathogenicity island 2 (SPI‐2) and SifA [required for the formation in epithelial cells of lysosomal glycoprotein (lgp)‐containing structures, termed Sifs] are all involved in the same virulence function. sifA gene expression was induced after Salmonella entry into host cells and was dependent on the SPI‐2 regulator ssrA. A sifA− mutant strain had a replication defect in macrophages, similar to that of SPI‐2 and ompR− mutant strains. Whereas wild‐type and SPI‐2 mutant strains reside in vacuoles that progressively acquire lgps and the vacuolar ATPase, the majority of sifA− bacteria lost their vacuolar membrane and were released into the host cell cytosol. We propose that the wild‐type strain, through the action of SPI‐2 effectors (including SpiC), diverts the Salmonella‐containing vacuole from the endocytic pathway, and subsequent recruitment and maintenance of vacuolar ATPase/lgp‐containing membranes that enclose replicating bacteria is mediated by translocation of SifA.

Identification of Staphylococcus aureus virulence genes in a murine model of bacteraemia using signature‐tagged mutagenesis

Signature‐tagged mutagenesis with transposon Tn917 was used to identify genes of Staphylococcus aureus required for virulence in a murine model of bacteraemia. Screening 1248 mutant strains in pools of 96 resulted in the provisional identification of 50 mutants attenuated in virulence. Subsequent individual analysis of many of these mutants confirmed that they are attenuated in virulence. DNA sequence analysis of regions flanking their transposon insertion points revealed that approximately half of them represent genes with unknown function, while most of the remainder are involved in nutrient biosynthesis and cell surface metabolism. Three mutants were found with transposon insertions in different positions in femA, and one mutant had an insertion in femB. Both femA and femB are involved in the formation of cell wall peptidoglycan pentaglycine cross‐bridges. A further mutation occurred in a previously unknown gene that shares significant similarity to femB. Mutations were also obtained in recA and lsp (encoding the S. aureus prolipoprotein signal peptidase). On the basis of sequence similarities to proteins of known function, the products of other genes are probably involved in the synthesis of diaminopimelic acid (a component of peptidoglycan), maintenance of surface adhesins and cell surface membrane transport, showing that many components of the S. aureus cell surface are critical for the survival and replication of this pathogen in blood.

Functions and effectors of the Salmonella pathogenicity island 2 type III secretion system.

Salmonella enterica uses two functionally distinct type III secretion systems encoded on the pathogenicity islands SPI‐1 and SPI‐2 to transfer effector proteins into host cells. A major function of the SPI‐1 secretion system is to enable bacterial invasion of epithelial cells and the principal role of SPI‐2 is to facilitate the replication of intracellular bacteria within membrane‐bound Salmonella ‐containing vacuoles (SCVs). Studies of mutant bacteria defective for SPI‐2‐dependent secretion have revealed a variety of functions that can be attributed to this secretion system. These include an inhibition of various aspects of endocytic trafficking, an avoidance of NADPH oxidase‐dependent killing, the induction of a delayed apoptosis‐like host cell death, the control of SCV membrane dynamics, the assembly of a meshwork of F‐actin around the SCV, an accumulation of cholesterol around the SCV and interference with the localization of inducible nitric oxide synthase to the SCV. Several effector proteins that are translocated across the vacuolar membrane in a SPI‐2‐dependent manner have now been identified. These are encoded both within and outside SPI‐2. The characteristics of these effectors, and their relationship to the physiological functions listed above, are the subject of this review. The emerging picture is of a multifunctional system, whose activities are explained in part by effectors that control interactions between the SCV and intracellular membrane compartments.

The classical pathway is the dominant complement pathway required for innate immunity to Streptococcus pneumoniae infection in mice

The complement system is an important component of the innate immune response to bacterial pathogens, including Streptococcus pneumoniae. The classical complement pathway is activated by antibody–antigen complexes on the bacterial surface and has been considered predominately to be an effector of the adaptive immune response, whereas the alternative and mannose-binding lectin pathways are activated directly by bacterial cell surface components and are considered effectors of the innate immune response. Recently, a role has been suggested for the classical pathway during innate immunity that is activated by natural IgM or components of the acute-phase response bound to bacterial pathogens. However, the functional importance of the classical pathway for innate immunity to S. pneumoniae and other bacterial pathogens, and its relative contribution compared with the alternative and mannose-binding lectin pathways has not been defined. By using strains of mice with genetic deficiencies of complement components and secretory IgM we have investigated the role of each complement pathway and natural IgM for innate immunity to S. pneumoniae. Our results show that the proportion of a population of S. pneumoniae bound by C3 depends mainly on the classical pathway, whereas the intensity of C3 binding depends on the alternative pathway. Furthermore, the classical pathway, partially targeted by the binding of natural IgM to bacteria, is the dominant pathway for activation of the complement system during innate immunity to S. pneumoniae, loss of which results in rapidly progressing septicemia and impaired macrophage activation. These data demonstrate the vital role of the classical pathway for innate immunity to a bacterial pathogen.

A functional genomic analysis of type 3 Streptococcus pneumoniae virulence

Streptococcus pneumoniae remains a serious cause of morbidity and mortality in humans, but relatively little is known about the molecular basis of its pathogenesis. We used signature‐tagged mutagenesis together with an analysis of S. pneumoniae genome sequence to identify and characterize genes required for pathogenesis. A library of signature‐tagged mutants was created by insertion–duplication mutagenesis, and 1786 strains were analysed for their inability to survive and replicate in murine models of pneumonia and bacteraemia. One hundred and eighty‐six mutant strains were identified as attenuated, and 56 were selected for further genetic characterization based on their ability to excise the integrated plasmid spontaneously. The genomic DNA inserts of the plasmids were cloned in Escherichia coli and sequenced. These sequences were subjected to database searches, including the S. pneumoniae genome sequence, which allowed us to examine the chromosomal regions flanking these genes. Most of the insertions were in probable operons, but no pathogenicity islands were found. Forty‐two novel virulence loci were identified. Five strains mutated in genes involved in gene regulation, cation transport or stress tolerance were shown to be highly attenuated when tested individually in a murine respiratory tract infection model. Additional experiments also suggest that induction of competence for genetic transformation has a role in virulence.

Internalization of Salmonella by Macrophages Induces Formation of Nonreplicating Persisters

Persistent Survival The role of persister cells—dormant cells that survive multidrug treatment—in the context of bacterial pathogenesis has not been explored in depth. Using a single-cell fluorescent dilution technique, Helaine et al. (p. 204) examined Salmonella Typhimurium persister-cell formation in vitro and in infections in mice. Within 30 min after phagocytosis by macrophages, Salmonella cells follow one of two fates, either to replication and generation of virulence effectors or to remaining viable but become nonreplicating persisters. Salmonella living within a macrophage vacuole are exposed to potentially stressful conditions that induce the expression of 14 Type II toxin-antidote loci in a ppGpp/lon protease-dependent manner, and this system appears to play a role in both virulence factor induction and persister-cell formation. The nonreplicating bacteria represent at least four distinct subpopulations, as defined by their ability to resume growth and their metabolic activity, but different phenotypes are observed in different pathogens and Escherichia coli persisters are distinct from Salmonella persisters. Upon internalization, intracellular Salmonella choose between replication or a form of quiescence known as persistence. Many bacterial pathogens cause persistent infections despite repeated antibiotic exposure. Bacterial persisters are antibiotic-tolerant cells, but little is known about their growth status and the signals and pathways leading to their formation in infected tissues. We used fluorescent single-cell analysis to identify Salmonella persisters during infection. These were part of a nonreplicating population formed immediately after uptake by macrophages and were induced by vacuolar acidification and nutritional deprivation, conditions that also induce Salmonella virulence gene expression. The majority of 14 toxin-antitoxin modules contributed to intracellular persister formation. Some persisters resumed intracellular growth after phagocytosis by naïve macrophages. Thus, the vacuolar environment induces phenotypic heterogeneity, leading to either bacterial replication or the formation of nonreplicating persisters that could provide a reservoir for relapsing infection.

A Streptococcus pneumoniae pathogenicity island encoding an ABC transporter involved in iron uptake and virulence

Restricted iron availability is a major obstacle to growth and survival of pathogenic bacteria during infection. In contrast to Gram‐negative pathogens, little is known about how Gram‐positive pathogens obtain this essential metal. We have identified two Streptococcus pneumoniae genetic loci, pit1 and pit2, encoding homologues of ABC iron transporters that are required for iron uptake by this organism. S. pneumoniae strains containing disrupted copies of either pit1 or pit2 had decreased sensitivity to the iron‐dependent antibiotic streptonigrin, and a strain containing disrupted copies of both pit1 and pit2 was unable to use haemoglobin as an iron source and had a reduced rate of iron uptake. The pit2− strain was moderately and the pit1−/pit2− strain strongly attenuated in virulence in mouse models of pulmonary and systemic infection, showing that the pit loci play a critical role during in vivo growth of S. pneumoniae. The pit2 locus is contained within a 27 kb region of chromosomal DNA that has several features of Gram‐negative bacterial pathogenicity islands. This probable pathogenicity island (PPI‐1) is the first to be described for S. pneumoniae, and its acquisition is likely to have played a significant role in the evolution of this important human pathogen.

Complementary activities of SseJ and SifA regulate dynamics of the Salmonella typhimurium vacuolar membrane

The Salmonella pathogenicity island 2 (SPI‐2) type III secretion system (TTSS) of Salmonella typhimurium is required for bacterial replication within host cells. It acts by translocating effector proteins across the membrane of the Salmonella‐containing vacuole (SCV). The SifA effector is required to maintain the integrity of the SCV membrane, and for the formation in epithelial cells of Salmonella‐induced filaments (Sifs), which are tubular extensions of SCVs. We have investigated the role in S. typhimurium virulence of the putative SPI‐2 effector genes sifB, srfJ, sseJ and sseI. An S. typhimurium strain carrying a mutation in sseJ was mildly attenuated for systemic virulence in mice, but strains carrying mutations in either srfJ, sseI or sifB had very little or no detectable virulence defect after intraperitoneal inoculation. Expression of SseJ in HeLa cells resulted in the formation of globular membranous compartments (GMCs), the composition of which appears to be similar to that of SCV membranes and Sifs. The formation of GMCs was dependent on the serine residue of the predicted acyltransferase/lipase active site of SseJ. Transiently expressed SseJ also inhibited Sif formation by wild‐type bacteria, and was found to associate with Sifs, SCV membranes and simultaneously expressed SifA. Intracellular vacuoles containing sseJ mutant bacteria appeared normal but, in contrast to a sifA mutant, a sifA sseJ double mutant strain did not lose its vacuolar membrane, indicating that loss of vacuolar membrane around sifA mutant bacteria requires the action of SseJ. Collectively, these results suggest that the combined action of SseJ and SifA regulate dynamics of the SCV membrane in infected cells.

Intracellular replication of Salmonella typhimurium strains in specific subsets of splenic macrophages in vivo

We used flow cytometry and confocal immunofluorescence microscopy to study the localization of Salmonella typhimurium in spleens of infected mice. Animals were inoculated intragastrically or intraperitoneally with S. typhimurium strains, constitutively expressing green fluorescent protein. Independently of the route of inoculation, most bacteria were found in intracellular locations 3 days after inoculation. Using a panel of antibodies that bound to cells of different lineages, including mononuclear phagocyte subsets, we have shown that the vast majority of S. typhimurium bacteria reside within macrophages. Bacteria were located in red pulp and marginal zone macrophages, but very few were found in the marginal metallophilic macrophage population. We have demonstrated that the Salmonella SPI‐2 type III secretion system is required for replication within splenic macrophages, and that sifA− mutant bacteria are found within the cytosol of these cells. These results confirm that SifA and SPI‐2 are involved in maintenance of the vacuolar membrane and intracellular replication in vivo.