Inhibitor profiling of the Pseudomonas aeruginosa virulence factor LasB using N-alpha mercaptoamide template-based inhibitors

We report on the synthesis and biological evaluation of a focussed library of N-alpha mercaptoamide containing dipeptides as inhibitors of the zinc metallopeptidase Pseudomonas aeruginosa elastase (LasB, EC 3.4.24.26). The aim of the study was to derive an inhibitor profile for LasB with regard to mapping the S´1 binding site of the enzyme. Consequently, a focussed library of 160 members has been synthesised, using standard Fmoc-solid phase methods (on a Rink-amide resin), in which a subset of amino acids including examples of those with basic (Lys, Arg), aromatic (Phe, Trp), large aliphatic (Val, Leu) and acidic (Asp, Glu) side-chains populated the P´2 position of the inhibitor sequence and all 20 natural amino acids were incorporated, in turn, at the P´1 position. The study has revealed a preference for aromatic and/or large aliphatic amino acids at P´1 and a distinct bias against acidic residues at P´2. Ten inhibitor sequences were discovered that exhibited sub to low micromolar Ki values.

ZapA, a virulence factor in a rat model of Proteus mirabilis-induced acute and chronic prostatitis.

Our knowledge of pathogenesis has benefited from a better understanding of the roles of specific virulence factors in disease. To determine the role of the virulence factor ZapA, a 54-kDa metalloproteinase of Proteus mirabilis, in prostatitis, rats were infected with either wild-type (WT) P. mirabilis or its isogenic ZapA- mutant KW360. The WT produced both acute and chronic prostatitis showing the typical histological progressions that are the hallmarks of these diseases. Infection with the ZapA- mutant, however, resulted in reduced levels of acute prostatitis, as determined from lower levels of tissue damage, bacterial colonization, and inflammation. Further, the ZapA- mutant failed to establish a chronic infection, in that bacteria were cleared from the prostate, inflammation was resolved, and tissue was seen to be healing. Clearance from the prostate was not the result of a reduced capacity of the ZapA- mutant to form biofilms in vitro. These finding clearly define ZapA as an important virulence factor in both acute and chronic bacterial prostatitis.

Novel Inhibitors of the Pseudomonas aeruginosa Virulence Factor LasB: a Potential Therapeutic Approach for the Attenuation of Virulence Mechanisms in Pseudomonal Infection

Pseudomonas elastase (LasB), a metalloprotease virulence factor, is known to play a pivotal role in pseudomonal infection. LasB is secreted at the site of infection, where it exerts a proteolytic action that spans from broad tissue destruction to subtle action on components of the host immune system. The former enhances invasiveness by liberating nutrients for continued growth, while the latter exerts an immunomodulatory effect, manipulating the normal immune response. In addition to the extracellular effects of secreted LasB, it also acts within the bacterial cell to trigger the intracellular pathway that initiates growth as a bacterial bio?lm. The key role of LasB in pseudomonal virulence makes it a potential target for the development of an inhibitor as an antimicrobial agent. The concept of inhibition of virulence is a recently established antimicrobial strategy, and such agents have been termed “second-generation” antibiotics. This approach holds promise in that it seeks to attenuate virulence processes without bactericidal action and, hence, without selection pressure for the emergence of resistant strains. A potent inhibitor of LasB,N-mercaptoacetyl-Phe-Tyr-amide (Ki 41 nM) has been developed, and its ability to block these virulence processes has been assessed. It has been demonstrated that thes compound can completely block the action of LasB on protein targets that are instrumental in bio?lm formation and immunomodulation. The novel LasB inhibitor has also been employed in bacterial-cell-based assays, to reduce the growth of pseudomonal bio?lms, and to eradicate bio?lm completely when used in combination with conventional antibiotics.

Comprehensive Inhibitor Profiling Of The Proteus Mirabilis Metalloprotease Virulence Factor Zapa (Mirabilysin)

In this study we report for the first time the comprehensive inhibitor profiling of the Proteus mirabilis metalloprotease virulence factor, ZapA (mirabilysin) using a 160 compound focused library of N-alpha mercaptoamide dipeptides, in order to map the S1´ and S2´ binding site preferences of this important enzyme. This study has revealed a preference for the aromatic residues tyrosine and tryptophan in P1´ and aliphatic residues in P2´. From this library, six compounds were identified which exhibited sub- to low micromolar Ki values. The most potent inactivator, SH-CO2-Y-V-NH2 was capable of preventing ZapA-mediated hydrolysis of heat denatured IgA, indicating these inhibitors may be capable of protecting host proteins against ZapA during colonisation and infection.

Comparative analysis of Klebsiella pneumoniae genomes identifies a phospholipase D family protein as a novel virulence factor

BACKGROUND: Klebsiella pneumoniae strains are pathogenic to animals and humans, in which they are both a frequent cause of nosocomial infections and a re-emerging cause of severe community-acquired infections. K. pneumoniae isolates of the capsular serotype K2 are among the most virulent. In order to identify novel putative virulence factors that may account for the severity of K2 infections, the genome sequence of the K2 reference strain Kp52.145 was determined and compared to two K1 and K2 strains of low virulence and to the reference strains MGH 78578 and NTUH-K2044.

RESULTS: In addition to diverse functions related to host colonization and virulence encoded in genomic regions common to the four strains, four genomic islands specific for Kp52.145 were identified. These regions encoded genes for the synthesis of colibactin toxin, a putative cytotoxin outer membrane protein, secretion systems, nucleases and eukaryotic-like proteins. In addition, an insertion within a type VI secretion system locus included sel1 domain containing proteins and a phospholipase D family protein (PLD1). The pld1 mutant was avirulent in a pneumonia model in mouse. The pld1 mRNA was expressed in vivo and the pld1 gene was associated with K. pneumoniae isolates from severe infections. Analysis of lipid composition of a defective E. coli strain complemented with pld1 suggests an involvement of PLD1 in cardiolipin metabolism.

CONCLUSIONS: Determination of the complete genome of the K2 reference strain identified several genomic islands comprising putative elements of pathogenicity. The role of PLD1 in pathogenesis was demonstrated for the first time and suggests that lipid metabolism is a novel virulence mechanism of K. pneumoniae.

Klebsiella pneumoniae AcrAB efflux pump contributes to antimicrobial resistance and virulence

Respiratory infections caused by Klebsiella pneumoniae are characterized by high rates of mortality and morbidity. Management of these infections is often difficult, due to the high frequency of strains that are resistant to multiple antimicrobial agents. Multidrug efflux pumps play a major role as a mechanism of antimicrobial resistance in Gram-negative pathogens. In the present study, we investigated the role of the K. pneumoniae AcrRAB operon in antimicrobial resistance and virulence by using isogenic knockouts deficient in the AcrB component and the AcrR repressor, both derived from the virulent strain 52145R. We demonstrated that the AcrB knockout was more susceptible, not only to quinolones, but also to other antimicrobial agents, including beta-lactams, than the wild-type strain and the AcrR knockout. We further showed that the AcrB knockout was more susceptible to antimicrobial agents present in human bronchoalveolar lavage fluid and to human antimicrobial peptides than the wild-type strain and the AcrR knockout. Finally, the AcrB knockout exhibited a reduced capacity to cause pneumonia in a murine model, in contrast to the wild-type strain. The results of this study suggest that, in addition to contributing to the multidrug resistance phenotype, the AcrAB efflux pump may represent a novel virulence factor required for K. pneumoniae to resist innate immune defense mechanisms of the lung, thus facilitating the onset of pneumonia.

Detection of the icaADBC gene cluster and biofilm formation in Staphylococcus epidermidis isolates from catheter-related urinary tract infections.

Cho SH, Naber K, Hacker J, Ziebuhr W. Institut für Molekulare Infektionsbiologie, Röntgenring 11, D-97070 Würzburg, Germany. Biofilm production in Staphylococcus epidermidis is an important virulence factor that is mediated by the expression of the icaADBC operon. In this study 41 S. epidermidis isolates obtained from catheter-related urinary tract infections were analyzed for the presence of the icaADBC operon and biofilm formation. Eighteen of 41 isolates (44%) were shown to carry ica-specific DNA, but only 11 isolates (27%) produced biofilms spontaneously under normal growth conditions. Upon induction by external stress or antibiotics, biofilm formation could be stimulated in five of seven ica-positive, biofilm-negative isolates, indicating that the icaADBC expression was down-regulated in these strains. Genetic analyses of the ica gene clusters of the remaining two ica-positive, biofilm-negative strains revealed a spontaneous ICAC::IS256 insertion in one strain. Insertion of the element caused a target site duplication of seven base pairs and a biofilm-negative phenotype. After repeated passages the insertion mutant was able to revert to a biofilm-forming phenotype which was due to the precise excision of IS256 from the icaC gene. The data show that icaC::IS256 integrations occur during S. epidermidis polymer-related infections and the results highlight the biological relevance of the IS256-mediated phase variation of biofilm production in S. epidermidis during an infection.

Binding and degradation of fibrinogen by Bacteroides fragilis and characterization of a 54 kDa fibrinogen-binding protein

Bacteroides fragilis is a bacterium that resides in the normal human gastro-intestinal tract; however, it is also the most commonly isolated Gram-negative obligate anaerobe from human clinical infections, such as intra-abdominal abscesses, and the most common cause of anaerobic bacteraemia. Abscess formation is important in bacterial containment, limiting dissemination of infection and bacteraemia. In this study, we investigated B. fragilis binding and degradation of human fibrinogen, the major structural component involved in fibrin abscess formation. We have shown that B. fragilis NCTC9343 binds human fibrinogen. A putative Bacteroides fragilis fibrinogen-binding protein, designated BF-FBP, identified in the genome sequence of NCTC9343, was cloned and expressed in Escherichia coli. The purified recombinant BF-FBP bound primarily to the human fibrinogen Bß-chain. In addition, we have identified fibrinogenolytic activity in B. fragilis exponential phase culture supernatants, associated with fibrinogenolytic metalloproteases in NCTC9343 and 638R, and cysteine protease activity in YCH46. All nine clinical isolates of B. fragilis examined degraded human fibrinogen; with eight isolates, initial A-chain degradation was observed, with varying Bß-chain and -chain degradation. With one blood culture isolate, Bß-chain and -chain degradation occurred first, followed by subsequent A-chain degradation. Our data raise the possibility that the fibrinogen-binding protein of B. fragilis, along with a variety of fibrinogenolytic proteases, may be an important virulence factor that facilitates dissemination of infection via reduction or inhibition of abscess formation.

Burkholderia cenocepacia requires a periplasmic HtrA protease for growth under thermal and osmotic stress and for survival in vivo

Burkholderia cenocepacia, a member of the B. cepacia complex, is an opportunistic pathogen that causes serious infections in patients with cystic fibrosis. We identified a six-gene cluster in chromosome 1 encoding a two-component regulatory system (BCAL2831 and BCAL2830) and an HtrA protease (BCAL2829) hypothesized to play a role in the B. cenocepacia stress response. Reverse transcriptase PCR analysis of these six genes confirmed they are cotranscribed and comprise an operon. Genes in this operon, including htrA, were insertionally inactivated by recombination with a newly created suicide plasmid, pGPOmegaTp. Genetic analyses and complementation studies revealed that HtrA(BCAL2829) was required for growth of B. cenocepacia upon exposure to osmotic stress (NaCl or KCl) and thermal stress (44 degrees C). In addition, replacement of the serine residue in the active site with alanine (S245A) and deletion of the HtrA(BCAL2829) PDZ domains demonstrated that these areas are required for protein function. HtrA(BCAL2829) also localizes to the periplasmic compartment, as shown by Western blot analysis and a colicin V reporter assay. Using the rat agar bead model of chronic lung infection, we also demonstrated that inactivation of the htrA gene is associated with a bacterial survival defect in vivo. Together, our data demonstrate that HtrA(BCAL2829) is a virulence factor in B. cenocepacia.

Regulatory network of lipopolysaccharide O-antigen biosynthesis in Yersinia enterocolitica includes cell envelope-dependent signals

Lipopolysaccharide (LPS) is a glycolipid present in the outer membrane of all Gram-negative bacteria, and it is one of the signature molecules recognized by the receptors of the innate immune system. In addition to its lipid A portion (the endotoxin), its O-chain polysaccharide (the O-antigen) plays a critical role in the bacterium-host interplay and, in a number of bacterial pathogens, it is a virulence factor. We present evidence that, in Yersinia enterocolitica serotype O:8, a complex signalling network regulates O-antigen expression in response to temperature. Northern blotting and reporter fusion analyses indicated that temperature regulates the O-antigen expression at the transcriptional level. Promoter cloning showed that the O-antigen gene cluster contains two transcriptional units under the control of promoters P(wb1) and P(wb2). The activity of both promoters is under temperature regulation and is repressed in bacteria grown at 37 degrees C. We demonstrate that the RosA/RosB efflux pump/potassium antiporter system and Wzz, the O-antigen chain length determinant, are indirectly involved in the regulation mainly affecting the activity of promoter P(wb2). The rosAB transcription, under the control of P(ros), is activated at 37 degrees C, and P(wb2) is repressed through the signals generated by the RosAB system activation, i.e. decreased [K+] and increased [H+]. The wzz transcription is under the control of P(wb2), and we show that, at 37 degrees C, overexpression of Wzz downregulates slightly the P(wb1) and P(wb2) activities and more strongly the P(ros) activity, with the net result that more O-antigen is produced. Finally, we demonstrate that overexpression of Wzz causes membrane stress that activates the CpxAR two-component signal transduction system.