Ramakrishnan Srikumar

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 ΔmexCD-oprJ deletion demonstrated significantly reduced invasiveness compared with WT. In particular, a ΔmexAB-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 ΔmexB and ΔmexXY mutants, were intermediate between WT and the ΔmexAB-oprM mutant in invasiveness and murine virulence. Invasiveness was restored to the ΔmexAB-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.

Multidrug Efflux in Pseudomonas aeruginosa: Components, Mechanisms and Clinical Significance

Pseudomonas aeruginosa is an opportunistic human pathogen characterized by an intrinsic resistance to multiple antimicrobial agents and the ability to develop high-level (acquired) multidrug resistance during antibiotic therapy. Much of this resistance is promoted by highly homologous three-component efflux systems of broad substrate specificity, of which four have been identified to date. These include MexA-Mexs-OprM and MexX-MexY-OprM, which are expressed constitutively in wild type cells and, thus, provide for intrinsic multidrug resistance, and MexC-MexD-OprJ and MexE-MexF-OprN, whose expression so far has only been seen in acquired multidrug resistant mutant strains. Additional homologues of these efflux systems are identifiable in the recently released genome sequence, though their roles, if any, in antimicrobial efflux are unknown. These tripartite pumps are composed of an integral cytoplasmic membrane drug-proton antiporter of the resistance-nodulation-cell division (RND) family of exporters, a channel-forming outer membrane efflux protein (or outer membrane factor [OMF]) and a periplasmic membrane fusion protein (MFP) that links the other two. In addition to a number of antimicrobials of clinical significance, these pumps also export dyes, detergents, disinfectants, organic solvents and acylated homoserine lactones involved in quorum-sensing. While the natural functional of these pumps remains undefined, the fact that they contribute to antimicrobial resistance in P. aeruginosa makes them reasonable targets for therapeutic intervention.

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.

Monoclonal antibodies specific to porin of Haemophilus influenzae type b: localization of their cognate epitopes and tests of their biological activities

The major outer membrane protein of Haemophilus influenzae type b (Hib) is porin (Mr 38000, 341 amino acids). To identify antigenic determinants on Hib porin that might be exposed at the bacterial ceil surface, seven mouse monoclonal anti‐Hib porin antibodies were generated. The monoclonal antibodies were tested for their binding to intact cells by flow cytometry; all but one bound to the cell surface. Digestions of Hib porin with cyanogen bromide, hydroxylamine or trypsin generated fragments, the identities of which were confirmed by microsequencing of the amino termini. Following electrophoresis and immunoblotting of the fragments, the specificities of the monoclonal antibodies for their cognate sequences were determined. The porin gene ompP2 was expressed in the baculovirus expression vector system; the recombinant porin was recognized by all of the monoclonal antibodies. Deletions were created by omega mutagenesis of ompP2, generating proteins truncated after amino acids 139, 174, 182, and 264. These deletion proteins were tested for reactivities with the monoclonal antibodies, thereby establishing the boundaries of three antigenic determinants that were recognized by the monoclonals: domain (i), amino acids 104–139; domain (ii) amino acids 162–174; and domain (iii), amino acids 267–341. The biological activities of monoclonal antibodies that were representative of these three classes were tested for their bactericidal activity in complement‐mediated tysis of whole cells. The monoclonal antibodies were also tested for their immunoprotective properties in the infant rat model of bacteraemia. Although the monoclonal antibodies were surface‐binding, they were neither bactericidal nor protective.

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.

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

ABSTRACT 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.

PURIFICATION AND REFOLDING OF RECOMBINANT HAEMOPHILUS INFLUENZAE TYPE B PORIN PRODUCED IN BACILLUS SUBTILIS

The major diffusion channel in the outer membrane of Haemophilus influenzae type b (Hib) is porin (341 amino acids; M r 37 782). The Hib porin gene was cloned and overexpressed in Bacillus subtilis. Recombinant Hib porin (Bac porin), having aggregated into inclusion bodies, was purified under denaturing conditions and subsequently refolded. To compare Bac porin that is intrinsically devoid of lipooligosaccharides versus native Hib porin, the properties of Bac porin were assessed by the following four criteria: circular dichroism spectroscopy, channel formation in planar bilayers, resistance to trypsin digestion and formation of the conformational epitope recognized by an anti‐Hib porin monoclonal antibody. We conclude that in the absence of lipooligosaccharides, Bac porin was refolded into a functional form which closely resembled the structure of Hib porin.

Erratum: 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 (Molecular Microbiology (2006) 62 (1132-1143))

Adam Belley,1 Mario Callejo,1 Francis Arhin,1 Mohammed Dehbi,1 Ibtihal Fadhil,1 Jing Liu,1 Geoffrey McKay,1 Ramakrishnan Srikumar,1 Pascale Bauda,1 Dominique Bergeron,1 Nhuan Ha,1 Michael DuBow,2 Philippe Gros,3 Jerry Pelletier3 and Greg Moeck1* Targanta Therapeutics, St-Laurent, QC, Canada. Université Paris Sud, Orsay, France. Department of Biochemistry and McGill Cancer Center, McGill University, Montreal, QC, Canada.