Competition of bacteriophage polypeptides with native replicase proteins for binding to the DNA sliding clamp reveals a novel mechanism for DNA replication arrest in Staphylococcus aureus.

Bacteriophages have evolved specific mechanisms that redirect bacterial metabolic pathways to the bacteriophage reproduction cycle. In this study, we characterized the bactericidal mechanism of two polypeptides from bacteriophages Twort and G1 that target the DNA sliding clamp of Staphylococcus aureus. The DNA sliding clamp, which tethers DNA polymerase to its template and thereby confers processivity upon the enzyme, was found to be essential for the viability of S. aureus. Expression of polypeptides TwortORF168 and G1ORF240 in S. aureus selectively inhibited DNA replication which in turn resulted in cell death. Both polypeptides specifically inhibited the S. aureus DNA replicase that was reconstituted in vitro but not the corresponding replicase of Streptococcus pyogenes. We demonstrated that inhibition of DNA synthesis is multifaceted and occurs via binding the DNA sliding clamp: TwortORF168 and G1ORF240 bound tightly to the DNA sliding clamp and prevented both its loading onto DNA and its interaction with DNA polymerase C. These results elucidate the impact of bacteriophage polypeptide expression upon DNA replication in the growing cell.

A new class of small molecule RNA polymerase inhibitors with activity against rifampicin-resistant Staphylococcus aureus.

The RNA polymerase holoenzyme is a proven target for antibacterial agents. A high-throughput screening program based on this enzyme from Staphylococcus aureus had previously identified a 2-ureidothiophene-3-carboxylate as a low micromolar inhibitor. An investigation of the relationships between the structures of this class of compounds and their inhibitory- and antibacterial activities is described here, leading to a set of potent RNA polymerase inhibitors with antibacterial activity. Characterization of this bioactivity, including studies of the mechanism of action, is provided, highlighting the power of the reverse chemical genetics approach in providing tools to inhibit the bacterial RNA polymerase.

Fhua and HgbA, outer membrane proteins of Actinobacillus pleuropneumoniae: their role as virulence determinants.

For the recently described serotype 15 of biotype I and serotypes 13 and 14 of biotype II of Actinobacillus pleuropneumoniae, fhuA and hgbA were detected by polymerase chain reaction and DNA sequencing. To determine the substrate specificity of the iron receptors FhuA and HgbA and to study their role in the virulence of A. pleuropneumoniae, we used two isogenic A. pleuropneumoniae serotype 1 deletion mutants of fhuA and hgbA. Different sources of iron and siderophores were tested in growth promotion assays. FhuA and HgbA are specific for their ligands ferrichrome and hemoglobin, respectively. The virulence of the two deletion mutant strains was evaluated in experimental infections using specific pathogen-free piglets. While the fhuA mutant (DG02) was as highly virulent as the parental strain S4074, the virulence of the hgbA mutant (DeltahgbA) was reduced. Our data indicate that both FhuA and HgbA are conserved among all serotypes and biotypes of A. pleuropneumoniae and that HgbA, the receptor for porcine hemoglobin, may play a role in virulence.

MexAB-OprM hyperexpression in NalC-type multidrug-resistant Pseudomonas aeruginosa: identification and characterization of the nalC gene encoding a repressor of PA3720-PA3719.

MexAB-OprM is a multidrug efflux system that contributes to intrinsic and acquired multidrug resistance in Pseudomonas aeruginosa, the latter as a result of mutational hyperexpression of the mexAB-oprM operon. While efflux gene hyperexpression typically results from mutations in the linked mexR repressor gene, it also occurs independently of mexR mutations in so-called nalC mutants that demonstrate more modest mexAB-oprM expression and, thus, more modest multidrug resistance than do mexR strains. Using a transposon insertion mutagenesis approach, nalC mutant strains were selected and the disrupted gene, PA3721, identified. Amplification and sequencing of this gene from previously isolated spontaneous nalC mutants revealed the presence of mutations in all instances and as such, PA3721 has been renamed nalC. PA3721 (nalC) encodes a probable repressor of the TetR/AcrR family and occurs upstream of an apparent two-gene operon, PA3720-PA3719, whose expression was negatively regulated by PA3721. Thus, PA3720-PA3719 was hyperexpressed in transposon insertion and spontaneous nalC mutants. The loss of PA3719 but not of PA3720 expression in a spontaneous nalC mutant reduced MexAB-OprM expression to wild-type levels and compromised multidrug resistance, an indication that hyperexpression of PA3719 only was necessary for the nalC phenotype. Introduction of PA3719 into wild-type P. aeruginosa on a multicopy plasmid was, in fact, sufficient to promote elevated MexAB-OprM expression and multidrug resistance characteristic of a nalC strain. Thus, the nalC (PA3721) mutation serves only to enhance PA3720-PA3719 expression, with expression of PA3719 (encodes a 53 amino acid protein of predicted pI 10.4) directly or indirectly impacting MexAB-OprM expression. Intriguingly, nalC strains produce markedly elevated levels of stable MexR protein suggesting that PA3720-PA3719 hyperexpression somehow modulates MexR repressor activity. The deduced products of PA3720-PA3719 show no homology to sequences presently in the GenBank databases, however, and as such provide no clues as to how this might occur.

Molecular cloning of haemoglobin-binding protein HgbA in the outer membrane of Actinobacillus pleuropneumoniae.

From the porcine pathogen Actinobacillus pleuropneumoniae cultivated in iron-deficient or haem-deficient media, haemoglobin (Hb)-agarose affinity purification was exploited to isolate an outer-membrane protein of approximately 105 kDa, designated HgbA. Internal peptide sequences of purified HgbA were used to design oligonucleotide primers for PCR amplification, yielding amplicons that showed partial sequences with homology to hgbA of Pasteurella multocida. Upon screening two genomic libraries of A. pleuropneumoniae serotype 1 strain 4074, positive clones were assembled into an ORF of 2838 bp. HgbA (946 aa) includes a signal peptide of 23 aa and the deduced HgbA sequence (104 890 Da) also demonstrated a possible Ton box. The promoter region of hgbA from A. pleuropneumoniae serotype 1 showed consensus for -35 and -10 sequences and a putative Fur-binding site. RT-PCR confirmed that hgbA of A. pleuropneumoniae is upregulated in response to diminished levels of iron in the culture medium. While an internally deleted hgbA mutant was unable to use pig Hb as sole source of iron for growth, flow cytometry confirmed its Hb binding; the internally deleted sequences may not be required for Hb binding, but appear necessary for the iron supply from Hb. HgbA is required for growth of A. pleuropneumoniae in the presence of Hb as sole iron source.

Antimicrobial drug discovery through bacteriophage genomics.

Over evolutionary time bacteriophages have developed unique proteins that arrest critical cellular processes to commit bacterial host metabolism to phage reproduction. Here, we apply this concept of phage-mediated bacterial growth inhibition to antibiotic discovery. We sequenced 26 Staphylococcus aureus phages and identified 31 novel polypeptide families that inhibited growth upon expression in S. aureus. The cellular targets for some of these polypeptides were identified and several were shown to be essential components of the host DNA replication and transcription machineries. The interaction between a prototypic pair, ORF104 of phage 77 and DnaI, the putative helicase loader of S. aureus, was then used to screen for small molecule inhibitors. Several compounds were subsequently found to inhibit both bacterial growth and DNA synthesis. Our results suggest that mimicking the growth-inhibitory effect of phage polypeptides by a chemical compound, coupled with the plethora of phages on earth, will yield new antibiotics to combat infectious diseases.

fhuA of Actinobacillus pleuropneumoniae encodes a ferrichrome receptor but is not regulated by iron.

The swine pathogen Actinobacillus pleuropneumoniae possesses a 75-kDa outer membrane protein (OMP), FhuA, the receptor for ferrichrome, a hydroxamate-type siderophore. Polyclonal serum to FhuA reacted with OMP preparations from 12 serotypes of A. pleuropneumoniae under conditions of iron repletion and restriction. Reverse transcription-PCR confirmed that A. pleuropneumoniae fhuA expression is not upregulated in response to low iron levels. An A. pleuropneumoniae fhuA deletion mutant was generated and showed abolishment of ferrichrome uptake.

Multidrug efflux systems play an important role in the invasiveness of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an important opportunistic human pathogen. Certain strains can transmigrate across epithelial cells, and their invasive phenotype is correlated with capacity to cause invasive human disease and fatal septicemia in mice. Four multidrug efflux systems have been described in P. aeruginosa, however, their contribution to virulence is unclear. To clarify the role of efflux systems in invasiveness, P. aeruginosa PAO1 wild-type (WT) and its efflux mutants were evaluated in a Madin-Darby canine kidney (MDCK) epithelial cell monolayer system and in a murine model of endogenous septicemia. All efflux mutants except a deltamexCD-oprJ deletion demonstrated significantly reduced invasiveness compared with WT. In particular, a deltamexAB-oprM deletion strain was compromised in its capacity to invade or transmigrate across MDCK cells, and could not kill mice, in contrast to WT which was highly invasive (P < 0.0006) and caused fatal infection (P < 0.0001). The other mutants, including deltamexB and deltamexXY mutants, were intermediate between WT and the deltamexAB-oprM mutant in invasiveness and murine virulence. Invasiveness was restored to the deltamexAB-oprM mutant by complementation with mexAB-oprM or by addition of culture supernatant from MDCK cells infected with WT. We conclude that the P. aeruginosa MexAB-OprM efflux system exports virulence determinants that contribute to bacterial virulence.

The mexR repressor of the mexAB-oprM multidrug efflux operon in Pseudomonas aeruginosa: characterization of mutations compromising activity.

Mutations in mexR yield a multidrug resistance phenotype in nalB mutants of Pseudomonas aeruginosa as a result of derepression of the mexAB-oprM multidrug efflux operon. MexR produced by several nalB strains carried single amino acid changes that compromised MexR stability or its ability to dimerize. Changes at residues L95 and R21, however, produced a stable MexR protein capable of dimerization and, thus, likely compromised DNA binding.