A global approach to identify novel broad-spectrum antibacterial targets among proteins of unknown function

Attempted allelic replacement of 144 Streptococcus pneumoniae open reading frames of previously uncharacterized function led to the identification of 36 genes essential for growth under laboratory conditions. Of these, 14 genes (obg, spoIIIJ2, trmU, yacA, yacM, ydiC, ydiE, yjbN, yneS, yphC, ysxC, ytaG, yloI and yxeH4) were also essential in Staphylococcus aureus and Haemophilus influenzae or Escherichia coli, 2 genes (yrrK and ydiB) were only essential in H. influenzae as well as S. pneumoniae and 8 genes were necessary for growth of S.pneumoniae and S. aureus and did not have a homolog in H. influenzae(murD2, ykqC, ylqF, yqeH, ytgP, yybQ) or were not essential in that organism (yqeL, yhcT). The proteins encoded by these genes could represent good targets for novel antibiotics covering different therapeutic profiles. The putative functions of some of these essential proteins, inferred by bioinformatic analysis, are presented. Four mutants, with deletions of loci not essential for in vitro growth, were found to be severely attenuated in a murine respiratory tract infection model, suggesting that not all targets for antibacterial therapeutics are revealed by simple in vitro essentiality testing. The results of our experiments together with those collated from previously reported studies including Bacillus subtilis, E. coli and Mycoplasma sp. demonstrate that gene conservation amongst bacteria does not necessarily indicate that essentiality in one organism can be extrapolated to others. Moreover, this study demonstrates that different experimental procedures can produce apparently contradictory results.     

Large-scale identification of genes required for full virulence of Staphylococcus aureus

Gene products required for in vivo growth and survival of microbial pathogens comprise a unique functional class and may represent new targets for antimicrobial chemotherapy, vaccine construction, or diagnostics. Although some factors governing Staphylococcus aureus pathogenicity have been identified and studied, a comprehensive genomic analysis of virulence functions will be a prerequisite for developing a global understanding of interactions between this pathogen and its human host. In this study, we describe a genetic screening strategy and demonstrate its use in screening a collection of 6,300 S. aureus insertion mutants for virulence attenuation in a murine model of systemic infection. Ninety-five attenuated mutants were identified, reassembled into new pools, and rescreened using the same murine model. This effort identified 24 highly attenuated mutants, each of which was further characterized for virulence attenuation in vivo and for growth phenotypes in vitro. Mutants were recovered in numbers up to 1,200-fold less than wild type in the spleens of systemically infected animals and up to 4,000-fold less than wild type in localized abscess infections. Genetic analysis of the mutants identified insertions in 23 unique genes. The largest gene classes represented by these mutants encoded enzymes involved in small-molecule biosynthesis and cell surface transmembrane proteins involved in small-molecule binding and transport. Additionally, three insertions defined two histidine kinase sensor-response regulator gene pairs important for S. aureus in vivo survival. Our findings extend the understanding of pathogenic mechanisms employed by S. aureus to ensure its successful growth and survival in vivo. Many of the gene products we have identified represent attractive new targets for antibacterial chemotherapy.     

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.     

Differential effect of prior influenza infection on alveolar macrophage phagocytosis of Staphylococcus aureus and Escherichia coli: involvement of interferon-gamma production

The influenza A virus is one of the main causes of respiratory infection. Although influenza virus infection alone can result in pneumonia, secondary bacterial infection combined with the virus is the major cause of morbidity and mortality. Interestingly, while influenza infection increases susceptibility to some bacteria, including Streptococcus pneumoniae, Staphylococcus aureus (S. aureus), and Haemophilus influenzae, other bacteria such as Escherichia coli (E. coli) and Klebsiella pneumoniae are not associated with influenza infection. The reason for this discrepancy is not known. In this study, it was found that prior influenza virus infection inhibits murine alveolar macrophage phagocytosis of S. aureus but not of E. coli. Here, the mechanism for this inhibition is elucidated: prior influenza virus infection strongly increases interferon gamma (IFN-γ) production. Furthermore, it was shown that IFN-γ differentially affects alveolar macrophage phagocytosis of S. aureus and E. coli. The findings of the present study explain how influenza virus infection increases susceptibility to some bacteria, such as S. aureus, but not others, and provides evidence that IFN-γ might be a promising target for protecting the human population from secondary bacterial infection by influenza.     

Cloning and characterization of the parC and parE genes of Streptococcus pneumoniae encoding DNA topoisomerase IV: role in fluoroquinolone resistance.

DNA topoisomerase IV mediates chromosome segregation and is a potential target for antibacterial agents including new antipneumococcal fluoroquinolones. We have used hybridization to a Staphylococcus aureus gyrB probe in concert with chromosome walking to isolate the Streptococcus pneumoniae parE-parC locus, lying downstream of a putative new insertion sequence and encoding 647-residue ParE and 823-residue ParC subunits of DNA topoisomerase IV. These proteins exhibited greatest homology respectively to the GrlB (ParE) and GrlA (ParC) subunits of S. aureus DNA topoisomerase IV. When combined, whole-cell extracts of Escherichia coli strains expressing S. pneumoniae ParC or ParE proteins reconstituted a salt-insensitive ATP-dependent decatenase activity characteristic of DNA topoisomerase IV. A second gyrB homolog isolated from S. pneumoniae encoded a 648-residue protein which we identified as GyrB through its close homology both to counterparts in S. aureus and Bacillus subtilis and to the product of the S. pneumoniae nov-1 gene that confers novobiocin resistance. gyrB was not closely linked to gyrA. To examine the role of DNA topoisomerase IV in fluoroquinolone action and resistance in S. pneumoniae, we isolated mutant strains stepwise selected for resistance to increasing concentrations of ciprofloxacin. We analysed four low-level resistant mutants and showed that Ser-79 of ParC, equivalent to resistance hotspots Ser-80 of GrlA and Ser-84 of GyrA in S. aureus, was in each case substituted with Tyr. These results suggest that DNA topoisomerase IV is an important target for fluoroquinolones in S. pneumoniae and establish this organism as a useful gram-positive system for resistance studies.     

Evaluation of Staphylococcus aureus virulence factors using a silkworm model

Previous studies have indicated that the silkworm model is useful for identifying virulence genes of Staphylococcus aureus, a human pathogenic bacterium. Here we examined the scope of S.?aureus virulence factors that can be evaluated using the silkworm model. Gene-disrupted mutants of the agr locus, arlS gene and saeS gene, which regulate the expression of cell surface adhesins and hemolysins, exhibited attenuated virulence in silkworms. Mutants of the hla gene encoding α-hemolysin, the hlb gene encoding β-hemolysin, and the psmα and psmβ operons encoding cytolysins, however, showed virulence in silkworms indistinguishable from that of the parent strain. Thus, these S.?aureus cytolysins are not required for virulence in silkworms. In contrast, the gene-disrupted mutants of clfB, fnbB and sdrC, which encode cell-wall-anchored proteins, attenuated S.?aureus virulence in silkworms. In addition, the mutant of the srtA gene encoding sortase A, which anchors cell-wall proteins, showed attenuated virulence in silkworms. These findings suggest that the silkworm model can be used to evaluate S.?aureus cell-wall proteins and regulatory proteins as virulence factors.     

Role of Staphylococcus aureus catalase in niche competition against Streptococcus pneumoniae

Nasal colonization by Staphylococcus aureus is a major predisposing factor for subsequent infection. Recent reports of increased S. aureus colonization among children receiving pneumococcal vaccine implicate Streptococcus pneumoniae as an important competitor for the same niche. Since S. pneumoniae uses H2O2 to kill competing bacteria, we hypothesized that oxidant defense could play a significant role in promoting S. aureus colonization of the nasal mucosa. Using targeted mutagenesis, we showed that S. aureus expression of catalase contributes significantly to the survival of this pathogen in the presence of S. pneumoniae both in vitro and in a murine model of nasal cocolonization.     

Staphylococcus aureus genetic loci impacting growth and survival in multiple infection environments

The Gram-positive bacterium Staphylococcus aureus infects diverse tissues and causes a wide spectrum of diseases, suggesting that it possesses a repertoire of distinct molecular mechanisms promoting bacterial survival in disparate in vivo environments. Signature-tag transposon mutagenesis screening of a 1520-member library identified numerous S. aureus genetic loci affecting growth and survival in four complementary animal infection models including mouse abscess, bacteraemia and wound and rabbit endocarditis. Of a total of 237 in vivo attenuated mutants identified by the murine models, less than 10% showed attenuation in all three models, emphasizing the advantage of screening in diverse disease environments. The largest gene class identified by these analyses encoded peptide and amino acid transporters, some of which were important for S. aureus survival in all animal infection models tested. The identification of staphylococcal loci affecting growth, persistence and virulence in multiple tissue environments provides insight into the complexities of human infection and on the molecular mechanisms that could be targeted by new antibacterial therapies.     

Essentiality, expression, and characterization of the class II 3-hydroxy-3-methylglutaryl coenzyme A reductase of Staphylococcus aureus

Sequence comparisons have implied the presence of genes encoding enzymes of the mevalonate pathway for isopentenyl diphosphate biosynthesis in the gram-positive pathogen Staphylococcus aureus. In this study we showed through genetic disruption experiments that mvaA, which encodes a putative class II 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is essential for in vitro growth of S. aureus. Supplementation of media with mevalonate permitted isolation of an auxotrophic mvaA null mutant that was attenuated for virulence in a murine hematogenous pyelonephritis infection model. The mvaA gene was cloned from S. aureus DNA and expressed with an N-terminal His tag in Escherichia coli. The encoded protein was affinity purified to apparent homogeneity and was shown to be a class II HMG-CoA reductase, the first class II eubacterial biosynthetic enzyme isolated. Unlike most other HMG-CoA reductases, the S. aureus enzyme exhibits dual coenzyme specificity for NADP(H) and NAD(H), but NADP(H) was the preferred coenzyme. Kinetic parameters were determined for all substrates for all four catalyzed reactions using either NADP(H) or NAD(H). In all instances optimal activity using NAD(H) occurred at a pH one to two units more acidic than that using NADP(H). pH profiles suggested that His378 and Lys263, the apparent cognates of the active-site histidine and lysine of Pseudomonas mevalonii HMG-CoA reductase, function in catalysis and that the general catalytic mechanism is valid for the S. aureus enzyme. Fluvastatin inhibited competitively with HMG-CoA, with a K(i) of 320 microM, over 10(4) higher than that for a class I HMG-CoA reductase. Bacterial class II HMG-CoA reductases thus are potential targets for antibacterial agents directed against multidrug-resistant gram-positive cocci.     

Effects of an immunomodulatory feed additive on the development of mastitis in a mouse infection model using four bovine-origin isolates

The goal of this study was to examine the ability of a commercially available feed additive (OmniGen-AF) to reduce mammary infections caused by a single strain of mastitic pathogens (Streptococcus uberis, Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae) and to examine the effects of the additive on markers of mammary immunity. Four experiments were completed using a murine model of bovine mastitis. Infection progression was examined using Sybr-green- and TaqMan-based quantitative PCR assays of 16S ribosomal DNA. Infection of the mammary gland with all pathogens caused rapid (24 to 48 h) appearance of pathogen DNA in mammary tissue. Provision of the feed additive for 2 weeks before infection significantly (P < 0.05) reduced the extent of pathogen DNA accumulation in models of S. uberis, E. coli and S. aureus infection. The additive was ineffective in reducing mammary infections caused by K. pneumoniae. We examined mechanisms of action of the additive through assessment of mammary concentrations of mammary myeloperoxidase (MPO), major histocompatibility complex 2 class II (MHC) and macrophage inflammatory protein-1α (MIP) messenger RNA (mRNA) concentrations and by examining serum complement C3 concentration. Infection of the mammary gland increased concentrations of MPO and MHC mRNAs (P < 0.05). Ability of the pathogen to elicit changes in mammary MPO and MHC gene expression was enhanced by the provision of the additive for 2 weeks before infection. These data imply that the additive increased the mammary inflammatory response and increased antigen presentation during a mammary infection. Value of the additive in preventing mastitis in cattle awaits additional studies using a bovine model and further evaluation of additional strains of the pathogens used in this study.     

Differential fluorescence induction reveals Streptococcus pneumoniae loci regulated by competence stimulatory peptide

Differential fluorescence induction (DFI) in Streptococcus pneumoniae was used as a method for the discovery of genes activated in specific growth environments. Competence stimulatory peptide (CSP) was used as the model inducing system to identify differentially expressed genes. To identify CSP-induced promoters, a plasmid library was constructed by inserting random pieces of S. pneumoniae chromosomal DNA upstream of the promoterless gfpmut2 gene in an Escherichia coli/S. pneumoniae shuttle vector. S. pneumoniae carrying the library were induced with CSP and enriched for green fluorescent protein (GFP)-expressing bacteria using fluorescence-activated cell sorting. A total of 886 fluorescent clones was screened, and 12 differentially activated promoter elements were identified. Sequence analysis of these clones revealed that three were associated with novel competence loci, one of which we show is essential for DNA uptake, and six are known CSP-inducible promoters. We also explored whether competence proteins have a role in virulence and found that mutations in three CSP-inducible genes resulted in attenuated virulence phenotypes in either of two murine infection models. These results demonstrate the utility of DFI as a method for identifying differentially expressed genes in S. pneumoniae and the potential utility of applying DFI to other Gram-positive bacteria.     

Activation of XerCD-dif recombination by the FtsK DNA translocase

The FtsK translocase pumps dsDNA directionally at ～5 kb/s and facilitates chromosome unlinking by activating XerCD site-specific recombination at dif, located in the replication terminus of the Escherichia coli chromosome. We show directly that the γ regulatory subdomain of FtsK activates XerD catalytic activity to generate Holliday junction intermediates that can then be resolved by XerC. Furthermore, we demonstrate that γ can activate XerCD-dif recombination in the absence of the translocase domain, when it is fused to XerCD, or added in isolation. In these cases the recombination products are topologically complex and would impair chromosome unlinking. We propose that FtsK translocation and activation of unlinking are normally coupled, with the translocation being essential for ensuring that the products of recombination are topologically unlinked, an essential feature of the role of FtsK in chromosome segregation.     

A novel vertebrate model of Staphylococcus aureus infection reveals phagocyte-dependent resistance of zebrafish to non-host specialized pathogens

With the emergence of multiply resistant Staphylococcus aureus, there is an urgent need to better understand the molecular determinants of S. aureus pathogenesis. A model of staphylococcal pathogenesis in zebrafish embryos has been established, in which host phagocytes are able to mount an effective immune response, preventing overwhelming infection from small inocula. Myeloid cell depletion, by pu.1 morpholino-modified antisense injection, removes this immune protection. Macrophages and neutrophils are both implicated in this immune response, phagocytosing circulating bacteria. In addition, in vivo phagocyte/bacteria interactions can be visualized within transparent embryos. A preliminary screen for bacterial pathogenesis determinants has shown that strains bearing mutations in perR, pheP and saeR are attenuated. perR and pheP mutants are deficient in growth in vivo, and their virulence is not fully restored by myeloid cell depletion. On the other hand, saeR mutants are able to grow in vivo, and are completely restored to virulence by myeloid cell depletion. Thus specific pathogen gene function can be matched with particular facets of host response. Zebrafish are a new addition to the tools available for the study of S. aureus pathogenesis, and may provide insights into the interactions of bacterial and host genomes in determining the outcome of infection.     

Interruption of the cydB locus in Brucella abortus attenuates intracellular survival and virulence in the mouse model of infection

Brucellosis is characterized by abortion in ruminants and a protracted undulant fever in humans, which often results in severe pathological manifestations. Scant information exists about the molecular mechanisms employed by Brucella abortus to combat host defenses or to persist and replicate within host cells. Transposon (Tn5) mutagenesis of B. abortus and the subsequent screening of mutants for sensitivity to killing in murine macrophages and in the mouse model led to the identification of mutants which were severely attenuated for intracellular survival. One group of mutants was interrupted in cydB, a gene that is part of the cydAB operon encoding cytochrome bd oxidase, which catalyzes an alternate terminal electron transport step in bacterial respiration. The elevated affinity for molecular oxygen of this enzyme in Escherichia coli has suggested that it is involved in the protection of sensitive enzymatic activities such as those of hydrogenases and nitrogenases from damage. B. abortus cydB::Tn5 strains exhibited heightened sensitivity to the respiratory inhibitors zinc and azide, highly reactive oxygen species such as hydrogen peroxide, low pH, and attenuated virulence in the mouse model of infection. Virulence was restored by an intact copy of cydAB or by B. abortus genes encoding the oxidative radical-scavenging enzyme Cu/Zn superoxide dismutase or catalase. These results suggest a bifunctional role for the products of the cydAB operon, both in preventing the buildup of oxidative free radicals and in detoxifying the intracellular compartment, thus indicating the importance of these products in preventing intracellular destruction. Intracellular conditions that favor expression of the cydAB operon are under investigation and may be linked to the acid sensitivity also observed in this strain.     

Role of histone-like proteins H-NS and StpA in expression of virulence determinants of uropathogenic Escherichia coli

The histone-like protein H-NS is a global regulator in Escherichia coli that has been intensively studied in nonpathogenic strains. However, no comprehensive study on the role of H-NS and its paralogue, StpA, in gene expression in pathogenic E. coli has been carried out so far. Here, we monitored the global effects of H-NS and StpA in a uropathogenic E. coli isolate by using DNA arrays. Expression profiling revealed that more than 500 genes were affected by an hns mutation, whereas no effect of StpA alone was observed. An hns stpA double mutant showed a distinct gene expression pattern that differed in large part from that of the hns single mutant. This suggests a direct interaction between the two paralogues and the existence of distinct regulons of H-NS and an H-NS/StpA heteromeric complex. hns mutation resulted in increased expression of alpha-hemolysin, fimbriae, and iron uptake systems as well as genes involved in stress adaptation. Furthermore, several other putative virulence genes were found to be part of the H-NS regulon. Although the lack of H-NS, either alone or in combination with StpA, has a huge impact on gene expression in pathogenic E. coli strains, its effect on virulence is ambiguous. At a high infection dose, hns mutants trigger more sudden lethality due to their increased acute toxicity in murine urinary tract infection and sepsis models. At a lower infectious dose, however, mutants lacking H-NS are attenuated through their impaired growth rate, which can only partially be compensated for by the higher expression of numerous virulence factors.     

Molecular cloning and genetic analysis of the determinant for gamma-lysin, a two-component toxin of Staphylococcus aureus

The gamma-lysin determinant of Staphylococcus aureus strain Smith 5R has been cloned in phage lambda and plasmid vectors in Escherichia coli. Genetic evidence is presented which demonstrates that gamma-lysin requires the co-operative action of two polypeptides expressed by the closely linked hlgA and hlgB genes. Recombinants expressed haemolytic activity in agarose medium but not in agar, a known property of gamma-lysin. Haemolysis was inhibited by antiserum raised against the 32 kDa component of gamma-lysin, but not by anti-alpha-, anti-beta- or anti-delta-lysin serum. Subcloning and transposon Tn5 mutagenesis identified a 3.5 kb region which was necessary for gamma-lysin expression in E. coli. Two genes (hlgA and hlgB) were mapped and their polypeptide products identified. Non-haemolytic Tn5 mutants fell into two groups based upon complementation tests done between extracts of mutants in vitro and also between extracts of mutants and components of gamma-lysin purified from S. aureus culture supernates. Immunoblotting showed that some mutants in group A (defective in expression of hlgA) did not express a 32 kDa polypeptide which was synthesized by the parental haemolytic recombinant and by mutants in group B. Minicell analysis suggested that the products of the hlgB gene were proteins of 38 kDa and 36 kDa. The smaller molecule co-migrates with a protein in a fraction of the S. aureus culture supernate containing component B of gamma-lysin. The 38 kDa polypeptide is probably an unprocessed precursor. Southern hybridization demonstrated that the hlgA and hlgB genes are closely linked in the chromosome of several strains of S. aureus.     

Role of galectin-3 in leukocyte recruitment in a murine model of lung infection by Streptococcus pneumoniae

Pneumonia can be caused by a variety of pathogens, among which Streptococcus pneumoniae causes one of the most common forms of community-acquired pneumonia. Depending on the invading pathogen, the elements of the immune response triggered will vary. For most pathogens, such as Escherichia coli, neutrophil recruitment involves a well-described family of adhesion molecules, beta(2)-integrins. In the case of streptococcal pneumonia, however, neutrophil recruitment occurs mainly through a beta(2)-integrin-independent pathway. Despite decades of research on this issue, the adhesion molecules involved in neutrophil recruitment during lung infection by S. pneumoniae have not been identified. We have previously shown that galectin-3, a soluble mammalian lectin, can be found in lungs infected by S. pneumoniae, but not by E. coli, and can mediate the adhesion of neutrophils on the endothelial cell layer, implying its role in the recruitment of neutrophils to lungs infected with S. pneumoniae. In this study, using galectin-3 null mice, we report further evidence of the involvement of this soluble lectin in the recruitment of neutrophils to S. pneumonia-infected lungs. Indeed, in the absence of galectin-3, lower numbers of leukocytes, mainly neutrophils, were recruited to the infected lungs during infection by S. pneumoniae. In the case of beta(2)-integrin-dependent recruitment induced by lung infection with E. coli, the number of recruited neutrophils was not reduced. Thus, taken together, our data suggest that galectin-3 plays a role as a soluble adhesion molecule in the recruitment of neutrophils to lungs infected by S. pneumoniae, which induces beta(2)-integrin-independent migration.     

DNA binding ligands with in vivo efficacy in murine models of bacterial infection: optimization of internal aromatic amino acids

DNA binding ligands with potent antimicrobial activity against Gram-positive bacteria were further optimized by variation of the internal aromatic amino acids. This modification led to compounds with improved in vivo efficacy in lethal murine models of peritonitis (methicillin-resistant S. aureus, MRSA) and lung infection (S. pneumoniae).     

Truncated forms of PspA that are secreted from Streptococcus pneumoniae and their use in functional studies and cloning of the pspA gene.

Insertion-duplication mutagenesis was used to generate mutants of Streptococcus pneumoniae that produced truncated forms of PspA (pneumococcal surface protein A). The truncated products, representing from 20 to 80% of the complete PspA molecule, were all secreted from the cell and could be detected in unconcentrated culture medium. Analysis of the truncated molecules showed that the antigenic variability known to be associated with PspA is located in the alpha-helical N-terminal half of the molecule. This region was also found to contain immunogenic and protection-eliciting epitopes and to define the maximum region of the molecule that is likely to be surface exposed. The apparent molecular weight variability seen for PspA molecules of different S. pneumoniae strains was localized to both the N- and C-terminal halves of the protein. Attachment of PspA to S. pneumoniae was found to require regions located carboxy to the fifth repeat unit in the C-terminal end of the molecule. From the insertion-duplication mutants, the complete pspA gene was cloned and expressed in Escherichia coli. Differences in apparent molecular weight were observed when the same cloned product was expressed in E. coli and S. pneumoniae, suggesting that PspA is modified differently in the two hosts.