Reactive Chlorine Species Reversibly Inhibit DnaB Protein Splicing in Mycobacteria.

Intervening proteins, or inteins, are mobile genetic elements that are translated within host polypeptides and removed at the protein level by splicing. In protein splicing, a self-mediated reaction removes the intein, leaving a peptide bond in place. While protein splicing can proceed in the absence of external cofactors, several examples of conditional protein splicing (CPS) have emerged. In CPS, the rate and accuracy of splicing are highly dependent on environmental conditions. Because the activity of the intein-containing host protein is compromised prior to splicing and inteins are highly abundant in the microbial world, CPS represents an emerging form of posttranslational regulation that is potentially widespread in microbes. Reactive chlorine species (RCS) are highly potent oxidants encountered by bacteria in a variety of natural environments, including within cells of the mammalian innate immune system. Here, we demonstrate that two naturally occurring RCS, namely, hypochlorous acid (the active compound in bleach) and N-chlorotaurine, can reversibly block splicing of DnaB inteins from Mycobacterium leprae and Mycobacterium smegmatis in vitro. Further, using a reporter that monitors DnaB intein activity within M. smegmatis, we show that DnaB protein splicing is inhibited by RCS in the native host. DnaB, an essential replicative helicase, is the most common intein-housing protein in bacteria. These results add to the growing list of environmental conditions that are relevant to the survival of the intein-containing host and influence protein splicing, as well as suggesting a novel mycobacterial response to RCS. We propose a model in which DnaB splicing, and therefore replication, is paused when these mycobacteria encounter RCS. IMPORTANCE Inteins are both widespread and abundant in microbes, including within several bacterial and fungal pathogens. Inteins are domains translated within host proteins and removed at the protein level by splicing. Traditionally considered molecular parasites, some inteins have emerged in recent years as adaptive posttranslational regulatory elements. Several studies have demonstrated CPS, in which the rate and accuracy of protein splicing, and thus host protein functions, are responsive to environmental conditions relevant to the intein-containing organism. In this work, we demonstrate that two naturally occurring RCS, including the active compound in household bleach, reversibly inhibit protein splicing of Mycobacterium leprae and Mycobacterium smegmatis DnaB inteins. In addition to describing a new physiologically relevant condition that can temporarily inhibit protein splicing, this study suggests a novel stress response in Mycobacterium, a bacterial genus of tremendous importance to humans.

Enhanced biofilm and extracellular matrix production by chronic carriage versus acute isolates of Salmonella Typhi.

Salmonella Typhi is the primary causative agent of typhoid fever; an acute systemic infection that leads to chronic carriage in 3-5% of individuals. Chronic carriers are asymptomatic, difficult to treat and serve as reservoirs for typhoid outbreaks. Understanding the factors that contribute to chronic carriage is key to development of novel therapies to effectively resolve typhoid fever. Herein, although we observed no distinct clustering of chronic carriage isolates via phylogenetic analysis, we demonstrated that chronic isolates were phenotypically distinct from acute infection isolates. Chronic carriage isolates formed significantly thicker biofilms with greater biomass that correlated with significantly higher relative levels of extracellular DNA (eDNA) and DNABII proteins than biofilms formed by acute infection isolates. Importantly, extracellular DNABII proteins include integration host factor (IHF) and histone-like protein (HU) that are critical to the structural integrity of bacterial biofilms. In this study, we demonstrated that the biofilm formed by a chronic carriage isolate in vitro, was susceptible to disruption by a specific antibody against DNABII proteins, a successful first step in the development of a therapeutic to resolve chronic carriage.

Targeting a bacterial DNABII protein with a chimeric peptide immunogen or humanised monoclonal antibody to prevent or treat recalcitrant biofilm-mediated infections.

BACKGROUND: Chronic and recurrent bacterial diseases are recalcitrant to treatment due to the ability of the causative agents to establish biofilms, thus development of means to prevent or resolve these structures are greatly needed. Our approach targets the DNABII family of bacterial DNA-binding proteins, which serve as critical structural components within the extracellular DNA scaffold of biofilms formed by all bacterial species tested to date. DNABII-directed antibodies rapidly disrupt biofilms and release the resident bacteria which promote their subsequent clearance by either host immune effectors or antibiotics that are now effective at a notably reduced concentration. METHODS: First, as a therapeutic approach, we used intact IgG or Fab fragments against a chimeric peptide immunogen designed to target protective epitopes within the DNA-binding tip domains of integration host factor to disrupt established biofilms in vitro and to mediate resolution of existing disease in vivo. Second, we performed preventative active immunisation with the chimeric peptide to induce the formation of antibody that blocks biofilm formation and disease development in a model of viral-bacterial superinfection. Further, toward the path for clinical use, we humanised a monoclonal antibody against the chimeric peptide immunogen, then characterised and validated that it maintained therapeutic efficacy. FINDINGS: We demonstrated efficacy of each approach in two well-established pre-clinical models of otitis media induced by the prevalent respiratory tract pathogen nontypeable Haemophilus influenzae, a common biofilm disease. INTERPRETATION: Collectively, our data revealed two approaches with substantive efficacy and potential for broad application to combat diseases with a biofilm component. FUNDING: Supported by R01 DC011818 to LOB and SDG.

Conditional DnaB Protein Splicing Is Reversibly Inhibited by Zinc in Mycobacteria.

Inteins, as posttranslational regulatory elements, can tune protein function to environmental changes by conditional protein splicing (CPS). Translated as subdomains interrupting host proteins, inteins splice to scarlessly join flanking sequences (exteins). We used DnaB-intein1 (DnaBi1) from a replicative helicase of Mycobacterium smegmatis to build a kanamycin intein splicing reporter (KISR) that links splicing of DnaBi1 to kanamycin resistance. Using expression in heterologous Escherichia coli, we observed phenotypic classes of various levels of splicing-dependent resistance (SDR) and related these to the insertion position of DnaBi1 within the kanamycin resistance protein (KanR). The KanR-DnaBi1 construct demonstrating the most stringent SDR was used to probe for CPS of DnaB in the native host environment, M. smegmatis We show here that zinc, important during mycobacterial pathogenesis, inhibits DnaB splicing in M. smegmatis Using an in vitro reporter system, we demonstrated that zinc potently and reversibly inhibited DnaBi1 splicing, as well as splicing of a comparable intein from Mycobacterium leprae Finally, in a 1.95 Å crystal structure, we show that zinc inhibits splicing through binding to the very cysteine that initiates the splicing reaction. Together, our results provide compelling support for a model whereby mycobacterial DnaB protein splicing, and thus DNA replication, is responsive to environmental zinc.IMPORTANCE Inteins are present in a large fraction of prokaryotes and localize within conserved proteins, including the mycobacterial replicative helicase DnaB. In addition to their extensive protein engineering applications, inteins have emerged as environmentally responsive posttranslational regulators of the genes that encode them. While several studies have shown compelling evidence of conditional protein splicing (CPS), examination of splicing in the native host of the intein has proven to be challenging. Here, we demonstrated through a number of measures, including the use of a splicing-dependent sensor capable of monitoring intein activity in the native host, that zinc is a potent and reversible inhibitor of mycobacterial DnaB splicing. This work also expands our knowledge of site selection for intein insertion within nonnative proteins, demonstrating that splicing-dependent host protein activation correlates with proximity to the active site. Additionally, we surmise that splicing regulation by zinc has mycobacteriocidal and CPS application potential.

Monoclonal antibodies against DNA-binding tips of DNABII proteins disrupt biofilms in vitro and induce bacterial clearance in vivo.

The vast majority of chronic and recurrent bacterial diseases are attributed to the presence of a recalcitrant biofilm that contributes significantly to pathogenesis. As such, these diseases will require an innovative therapeutic approach. We targeted DNABII proteins, an integral component of extracellular DNA (eDNA) which is universally found as part of the pathogenic biofilm matrix to develop a biofilm disrupting therapeutic. We show that a cocktail of monoclonal antibodies directed against specific epitopes of a DNABII protein is highly effective to disrupt diverse biofilms in vitro as well as resolve experimental infection in vivo, in both a chinchilla and murine model. Combining this monoclonal antibody cocktail with a traditional antibiotic to kill bacteria newly released from the biofilm due to the action of the antibody cocktail was highly effective. Our results strongly support these monoclonal antibodies as attractive candidates for lead optimization as a therapeutic for resolution of bacterial biofilm diseases.

Pharmacokinetics-pharmacodynamics of the helicase-primase inhibitor pritelivir following treatment of wild-type or pritelivir-resistant virus infection in a murine herpes simplex virus 1 infection model.

Herpes simplex virus (HSV) infections can cause considerable morbidity. Transmission of HSV-2 has become a major health concern, since it has been shown to promote transmission of other sexually transmitted diseases. Pritelivir (AIC316, BAY 57-1293) belongs to a new class of HSV antiviral compounds, the helicase-primase inhibitors, which have a mode of action that is distinct from that of antiviral nucleoside analogues currently in clinical use. Analysis of pharmacokinetic-pharmacodynamic parameters is a useful tool for the selection of appropriate doses in clinical trials, especially for compounds belonging to new classes for which no or only limited data on therapeutic profiles are available. For this purpose, the effective dose of pritelivir was determined in a comprehensive mouse model of HSV infection. Corresponding plasma concentrations were measured, and exposures were compared with efficacious concentrations derived from cell cultures. The administration of pritelivir at 10 mg/kg of body weight once daily for 4 days completely suppressed any signs of HSV infection in the animals. Associated plasma concentrations adjusted for protein binding stayed above the cell culture 90% effective concentration (EC90) for HSV-1 for almost the entire dosing interval. Interestingly, by increasing the dose 6-fold and prolonging the treatment duration to 8 days, it was possible to treat mice infected with an approximately 30-fold pritelivir-resistant but fully pathogenic HSV-1 virus. Corresponding plasma concentrations exceeded the EC90 of this mutant for <8 h, indicating that even suboptimal exposure to pritelivir is sufficient to achieve antiviral efficacy, possibly augmented by other factors such as the immune system.

Coumarin-based inhibitors of Bacillus anthracis and Staphylococcus aureus replicative DNA helicase: chemical optimization, biological evaluation, and antibacterial activities.

The increasing prevalence of drug-resistant bacterial infections demands the development of new antibacterials that are not subject to existing mechanisms of resistance. Previously, we described coumarin-based inhibitors of an underexploited bacterial target, namely the replicative helicase. Here we report the synthesis and evaluation of optimized coumarin-based inhibitors with 9-18-fold increased potency against Staphylococcus aureus (Sa) and Bacillus anthracis (Ba) helicases. Compounds 20 and 22 provided the best potency, with IC(50) values of 3 and 1 µM, respectively, against the DNA duplex strand-unwinding activities of both B. anthracis and S. aureus helicases without affecting the single strand DNA-stimulated ATPase activity. Selectivity index (SI = CC(50)/MIC) values against S. aureus and B. anthracis for compound 20 were 33 and 66 and for compound 22 were 20 and 40, respectively. In addition, compounds 20 and 22 demonstrated potent antibacterial activity against multiple ciprofloxacin-resistant MRSA strains, with MIC values ranging between 0.5 and 4.2 µg/mL.

Probing intein-catalyzed thioester formation by unnatural amino acid substitutions in the active site.

Inteins are single-turnover catalysts that splice themselves out of a precursor polypeptide chain. For most inteins, the first step of protein splicing is the formation of a thioester through an N-S acyl shift at the upstream splice junction. However, the mechanism by which this reaction is achieved and the impact of mutations in and close to the active site remain unclear on the atomic level. To investigate these questions, we have further explored a split variant of the Ssp DnaB intein by introducing substitutions with unnatural amino acids within the short synthetic N-terminal fragment. A previously reported collapse of the oxythiazolidine anion intermediate into a thiazoline ring was found to be specificially dependent on the methyl side chain of the flanking Ala(-1). The stereoisomer d-Ala and the constitutional isomers ß-Ala and sarcosine did not lead to this side reaction but rather supported splicing. Substitution of the catalytic Cys1 with homocysteine strongly inhibited protein splicing; however, thioester formation was not impaired. These results argue against the requirement of a base to deprotonate the catalytic thiol group prior to the N-S acyl shift, because it should be misaligned for optimal proton abstraction. A previously described mutant intein evolved for more general splicing in different sequence contexts could even rather efficiently splice with this homocysteine. Our findings show the large impact of some subtle structural changes on the protein splicing pathway, but also the remarkable tolerance toward other changes. Such insights will also be important for the biotechnological exploitation of inteins.

Inhibition of Klebsiella pneumoniae DnaB helicase by the flavonol galangin.

Klebsiella pneumoniae is a ubiquitous opportunistic pathogen that colonizes at the mucosal surfaces in humans and causes severe diseases. Many clinical strains of K. pneumoniae are highly resistant to antibiotics. Here, we used fluorescence quenching to show that the flavonols galangin, myricetin, quercetin, and kaempferol, bearing different numbers of hydroxyl substituent on the aromatic rings, may inhibit dNTP binding of the primary replicative DnaB helicase of K. pneumoniae (KpDnaB), an essential component of the cellular replication machinery critical for bacterial survival. The binding affinity of KpDnaB to dNTPs varies in the following order: dCTP ~ dGTP > dTTP > dATP. Addition of 10 µM galangin significantly decreased the binding ability of KpDnaB to dATP, whereas the binding affinity of KpDnaB to dGTP that was almost unaffected. Our analyses suggest that these flavonol compounds may be used in the development of new antibiotics that target K. pneumoniae and other bacteria.

Drug targets in severe acute respiratory syndrome (SARS) virus and other coronavirus infections.

Coronaviruses are important human and animal pathogens of the order Nidovirales. Several new members were discovered following the emergence of SARS-CoV in human populations, including two human coronaviruses and several animal coronaviruses. They cause respiratory and gastrointestinal illnesses and have been found in the brains of patients with multiple sclerosis. The high mortality of SARS, the identification of a natural reservoir, and the well-founded fear of provoking antibody-enhanced disease as a result of vaccination fueled the ongoing efforts in anti-coronavirus drug discovery. This review presents the results of current research.

Other inhibitors of viral enzymes and functions.

Until the end of the 1970s, the mainstays of antiviral chemotherapy were nucleoside analogues that targeted virus polymerase, in particular, the herpesvirus DNA polymerase. The scourge of HIV triggered an unprecedented commitment to identify novel antivirals, and these efforts transformed antiviral therapy into the modern, sophisticated treatment form described in this book, with targets such as the reverse transcriptase and the protease as well as the entry of the human immunodeficiency virus. As the regulation of human pathogenic virus growth cycles became more understandable, the realisation grew that these pathogens had more than one Achilles heel that might be suitable targets for small molecules with antiviral activity. This chapter addresses those "other" targets as well as other approaches to the tried and tested polymerase inhibitors, the so-called non-nucleoside inhibitors of reverse transcriptase.

Myricetin inhibits Escherichia coli DnaB helicase but not primase.

Primase and DnaB helicase play central roles during DNA replication initiation and elongation. Both enzymes are drug targets because they are essential, persistent among bacterial genomes, and have different sequences than their eukaryotic equivalents. Myricetin is a ubiquitous natural product in plants that is known to inhibit a variety of DNA polymerases, RNA polymerases, reverse transcriptases, and telomerases in addition being able to inhibit kinases and helicases. We have shown that myricetin inhibits Escherichia coli DnaB helicase according to a mechanism dominated by noncompetitive behavior with a K(i) of 10.0+/-0.5 microM. At physiological ATP concentration, myricetin inhibits E. coli DnaB helicase with an inhibitory concentration at 50% maximal (IC(50)) of 11.3+/-1.6 microM. In contrast, myricetin inhibited E. coli primase at least 60-fold weaker than DnaB helicase and far weaker than any other polymerase.

Inactivation of the DnaB helicase leads to the collapse and degradation of the replication fork: a comparison to UV-induced arrest.

Replication forks face a variety of structurally diverse impediments that can prevent them from completing their task. The mechanism by which cells overcome these hurdles is likely to vary depending on the nature of the obstacle and the strand in which the impediment is encountered. Both UV-induced DNA damage and thermosensitive replication proteins have been used in model systems to inhibit DNA replication and characterize the mechanism by which it recovers. In this study, we examined the molecular events that occur at replication forks following inactivation of a thermosensitive DnaB helicase and found that they are distinct from those that occur following arrest at UV-induced DNA damage. Following UV-induced DNA damage, the integrity of replication forks is maintained and protected from extensive degradation by RecA, RecF, RecO, and RecR until replication can resume. By contrast, inactivation of DnaB results in extensive degradation of the nascent and leading-strand template DNA and a loss of replication fork integrity as monitored by two-dimensional agarose gel analysis. The degradation that occurs following DnaB inactivation partially depends on several genes, including recF, recO, recR, recJ, recG, and xonA. Furthermore, the thermosensitive DnaB allele prevents UV-induced DNA degradation from occurring following arrest even at the permissive temperature, suggesting a role for DnaB prior to loading of the RecFOR proteins. We discuss these observations in relation to potential models for both UV-induced and DnaB(Ts)-mediated replication inhibition.