Approximately half of the world’s population is infected with Helicobacter pylori, the bacterium that causes peptic ulcers and some forms of stomach cancer. Although ‘triple therapy’ with a proton pump inhibitor and two antibiotics – selected from a very limited number – can eradicate H.pylori, an increasing number of people are found to be infected with antibiotic-resistant bacteria. Scientists in Australia, New Zealand and France have now shown that H.pylori needs vitamin B6 to establish and maintain chronic infection, and have identified two genes in the vitamin B6 biosynthesis pathway as potential targets for new antibiotics.
The team used an established technique known as in vitro attenuation to create variants of a mouse-colonising strain of H.pylori with low infectivity and then compared the gene expression profiles of the attenuated bacteria with the original highly virulent strain. The most significant changes were found to be in the genes that encode homologues of the Escherichia coli vitamin B6 biosynthesis enzymes, PdxA and PdxJ, which catalyse sequential steps in the pathway. In vitro, H. pylori PdxA mutants could only be recovered when pyridoxal-5’-phosphate, the bioactive form of vitamin B6, was added to the growth medium whereas it was not possible to produce viable bacteria with mutated PdxJ. PdxA was also shown to be necessary for H. pylori to establish a chronic infection in mice.
Further studies showed that, in addition to its well known metabolic roles, vitamin B6 is needed for the synthesis of glycosylated flagella and for flagellum-based motility in H. pylori. The study, which is published in the new open access journal mBio™, suggests that Pdx enzymes, which are present in a number of human pathogens, but not in mammalian cells, may present attractive targets for new antibiotic medicines.
Methicillin-resistant Staphylococcus aureus (MRSA) infections are a particular problem in hospitals and other healthcare environments. MRSA can survive on normal surfaces and fabrics but researchers at Rensselaer Polytechnic Institute have now developed a coating that kills MRSA on contact. The coating contains lysostaphin linked by a short flexible polymer to carbon nanotubes and can be applied to surgical equipment, hospital walls, door handles and other surfaces. Lysostaphin is an example of a bacteriocin, a defensive bacterial antimicrobial agent that kills other, often closely related, bacteria. Lysostaphin, a cell wall-degrading enzyme, is produced by non-pathogenic strains of Staphylococcus bacteria and is very effective against Staphylococcus aureus, including MRSA, but completely harmless to humans and other organisms.
The lysostaphin-nanotube conjugate can be mixed with a wide range of surface finishes – in the present study, latex house paint was used. In tests, 100% of MRSA were killed within 20 minutes of contact with the paint. Treated surfaces can be washed repeatedly without losing their effectiveness and the team believe that the new coating is likely to prove superior to coatings that release biocides or those that ‘spear’ bacteria using amphipathic polycations and antimicrobial peptides. The team also believe that is unlikely that Staphylococcus aureus will be able to develop resistance to lysostaphin.
The bacterium responsible for tuberculosis (TB), mycobacterium tuberculosis (Mtb), is notoriously difficult to kill. The most commonly used antibiotics, rifampicin and isoniazid, need to be used for extended periods of time (typically 6-24 months) to effectively eliminate infection. In addition, emergence of antibiotic-resistant strains is an increasing problem.
Researchers at Albert Einstein College of Medicine of Yeshiva University have now identified a new biochemical pathway in Mtb and two novel ways to kill the bacterium. The pathway involves four enzymatic steps in the conversion of the disaccharide, trehalose, to α-glucan mediated by TreS, Pep2, GlgE (which has been identified as a maltosyltransferase that uses maltose 1-phosphate) and GlgB. Focusing on GlgE, the researchers found that blocking the enzyme induced toxic accumulation of maltose-1-phosphate, killing the bacteria in vitro and in a mouse model of infection. Inhibition of another enzyme in the pathway was non-lethal until combined with inactivation of Rv3032, a glucosyltransferase involved in a distinct α-glucan pathway. Inhibition of Rv3032 alone was also non-lethal to the bacteria.
The research validates inhibition of GlgE as therapy for TB but also highlights the potential for targeting two α-glucan pathways – a strategy that potentially leads to reduced incidence of resistance. Both approaches are also distinct from the mechanisms of currently used antibiotics.
Multidrug-resistant Gram-negative bacteria are a particular problem in both hospital and community settings and treatment is rendered more difficult by low intrinsic permeability to antibacterial compounds and by the presence of multidrug efflux pumps. The small number of molecular targets against which antibacterial compounds are directed is thought to have contributed to the emergence of multidrug-resistance but, despite much effort, only two new classes of antibiotics have reached the clinic in the past forty years. Connecting phenotype with mechanism in cell-based screening programmes has proved a significant challenge, but scientists at McMaster University have used high copy suppression to determine the cellular targets of new antibacterial leads identified by high-throughput screening. In this approach, the abundance of an essential target at high copy is believed to exceed the amount of available compound, leading to a suppression of a growth-inhibitory phenotype. The team used an array of E. coli clones over-expressing essential genes to screen for those that were able to suppress the activity of high-throughput screening actives. As well as discovering new chemical genetic interactions for some known antibiotics, the team identified MAC13243, a new antibacterial compound that interferes with the bacterial lipoprotein targeting pathway. MAC13243 inhibits the function of LolA, a key component of this pathway, which is present in Gram-negative (but not Gram-positive) bacteria, and is the first compound that has been shown to act via this mechanism.
A small array of tetrahydotriazines was then prepared to explore structure-activity relationships around MAC13243. Key findings were that a Cl- or Br-substituent in the para-position of the thiobenzyl moiety is needed for good activity, and that potency is maintained if the dimethoxy-phenethyl group is truncated to a methyl group (Compound 19). Both MAC13243 and Compound 19 showed good activity against a number of Gram-negative bacteria, including clinical isolates, but had no impact on Gram-positive organisms, confirming their proposed mechanism of action. The effectiveness of MAC13243 was not impaired by over-expression of acrB, which encodes the most pervasive multidrug efflux system, suggesting that the compound may represent an excellent starting point for the development of new treatments for infections caused by multidrug-resistant Gram-negative organisms.
Since the late 1980s, when nitric oxide (NO) was first shown to play a physiological role in mammals, this small molecule has been found to be a key mediator of an extraordinary variety of biological processes including blood pressure regulation, learning and memory, penile erection, digestion and the fighting of infection and cancer. Researchers at NYU School of Medicine have shown that bacteria also use NO to protect against oxidative stress and a new study by the same team shows that a broad spectrum of antibacterial drugs kill bacteria (at least in part) by inducing oxidative stress and that this effect is opposed by NO.
As well as opposing the antibacterial effects of oxidative stress, NO also reduced the effectiveness of antibiotics by chemical modifications that resulted in detoxification. Antibiotics were found to be more potent when the NO-mediated bacterial defence was eliminated, suggesting that co-administration of an inhibitor of NO-synthase could increase the effectiveness of existing antibiotics and might make antibiotic-resistant bacteria such as MRSA and anthrax more sensitive to available drugs during acute infection.
The study was published on September 11th in the journal Science.
Brain infections such as bacterial meningitis and encephalitis can cause death or serious disability and are difficult to treat with conventional antibiotics because of poor CNS-penetration and bacterial drug-resistance. Small cationic antimicrobial peptides form an important part of the host defence system and have potential as therapeutic agents. Unlike conventional antibiotics, antimicrobial peptides appear to be bacteriocidal rather than bacteriostatic, require a short contact time to induce killing, and do not easily induce resistance. Most small antimicrobial peptides form α-helices or β-sheet-like structures that can insert into, and subsequently disrupt, negatively charged bacterial cell surfaces.
Scientists at Singapore’s Institute of Bioengineering and Nanotechnology have described novel antimicrobial peptide nanoparticles that are able to readily cross the blood brain barrier. The nanoparticles are self-assembled from an amphiphilic oligopeptide containing a motif that promotes cell penetration. The nanoparticles were effective against a range of bacteria, yeasts and fungi in vitro and were also more effective against Staphylococcus aureus infection in mice than their unassembled peptide counterparts. Importantly, the nanoparticles were also able to cross the blood brain barrier and suppress bacterial growth in rabbits with Staphylococcus aureus meningitis. The study showed that antimicrobial nanoparticles are effective against bacterial brain infections in animals and do not damage red blood cells, liver or kidneys at the tested doses. The next challenge will be to develop nanoparticles that can be used in people.
Although the ubiquitination pathway in eukaryotes was characterised in the early 1980s, it has only recently been recognised that bacteria also tag proteins to determine their fate. Ubiquitination controls a variety of cellular processes, but one of its major roles is to label proteins for degradation by the proteasome. In Mycobacterium tuberculosis (Mtb), the small ubiquitin-like protein that targets other proteins for proteasomal degradation is called prokaryotic ubiquitin-like protein (Pup) and, because proteasome function is essential for the Mtb virulence, interruption of the Pup pathway could potentially be a target for anti-tuberculosis drugs. Writing in Nature Structural & Molecular Biology, researchers at the Institute of Molecular Biology & Biophysics of ETH Zurich have now described an enzyme called Pup deaminase (Dop) that is involved in the Pupylation of proteins in Mtb. Dop modifies Pup by deaminating the C-terminal glutamine to glutamate and the Pup ligase, proteasome accessory factor A (PafA), then couples the modified Pup to the ε-amino group of lysine residues in the target proteins. This formation of an isopeptide bond requires hydrolysis of ATP to ADP, suggesting that the C-terminal glutamate of the modified Pup is activated for attachment via phosphorylation.
Since the Pupylation pathway differs from the ubiquitination pathway, drugs targeting the bacterial Pup pathway may offer the potential for safe and effective treatments for Mtb.
Recent research has shown that gene expression can be regulated at the level of mRNA by riboswitches. A riboswitch is an aptamer region on an mRNA molecule that can specifically bind a small effector molecule, causing changes in the structure of the expression platform and so regulating the activity of the mRNA. Riboswitches most usually switch off the ability of mRNA to carry out protein synthesis but can also switch it on. A variety of riboswitch classes have been identified, with most of the examples being discovered in bacteria including E. coli and streptococcus as well as bacteria causing anthrax, gonorrhoea, meningitis and dysentry.
Researchers at the University of Rochester Medical Center have now solved the crystal structure of the smallest known riboswitch, the preQ1 riboswitch. The preQ1 riboswitch controls the ability of bacteria to produce queuosine (Q), a molecule which enables accurate gene expression by overcoming an inbuilt defect in the mRNA-ribosome-tRNA system known as tRNA wobble, and which is essential for the survival of many important pathological bacteria. The preQ1 riboswitch ‘senses’ the level of preQ1, a precursor to Q. If too much preQ1 is present, genes responsible for producing preQ1, or for its transport, are shut down. The preQ1 precursor, known as preQ0, has the same effect in reducing production of Q. One gene that is regulated by the preQ1 riboswitch is that which codes for the enzyme queF, which converts preQ0 into preQ1.
The structure of the preQ1 riboswitch from Thermoanaerobacter tengcongensis complexed with preQ0 shows preQ0 bound in a buried pocket. The structure also reveals how the first base of the mRNA ribosome binding site binds to a loop of the riboswitch, and how the loop end of the preQ1 riboswitch aptamer domain binds to preQ0. Binding of the preQ1 aptamer loop to the first base in the ribosome binding site was found to be mediated by a standard G to C base pairing interaction. The preQ1 aptamer (34 nucleotides) is about 2.5-fold shorter than functionally related riboswitches that recognize similar metabolites.
An understanding of how bacterial species sequester their ribosome binding sites using divergent preQ1 riboswitch aptamers could lead to the design of a new class of antibiotics. There is evidence that some existing antibiotics act – in part at least – by targeting riboswitches and, since riboswitches have not yet been found in human cells, the hope is that antibiotics acting on riboswitches will have a low propensity for side effects. The study is published in the Journal of Biological Chemistry.
Whereas mammalian fatty acid synthase (FASI) is a multidomain, multifunctional homodimeric protein which carries out all of the enzymatic steps needed for de novo synthesis of long chain fatty acids, bacterial fatty acid synthesis is carried out by a number of discrete enzymes, collectively known as FASII. This difference between FASI and FASII has led to the identification of FASII as a target for antibiotic therapy. The hypothesis is strengthened by the activities of the antiseptic, triclosan, the anti-Mycobacterium tuberculosis agent, isoniazid, and the antifungal antibiotic, cerulenin, which are believed to act primarily by inhibiting steps in the FASII pathway. The natural products, platensimycin and platensin, have also been shown to exhibit broad spectrum Gram-positive antibacterial activity and to inhibit fatty acid biosynthesis.
A recently published letter in the journal Nature, however, suggests that inhibition of FASII as an approach to antimicrobial therapy may be fundamentally flawed. The new study showed that major Gram-positive pathogens, such as streptococci, enterococci and staphylococci, were able to grow in the presence of FASII pathway inhibitors, cerulenin and triclosan, if supplied with exogenous fatty acids at levels that would be present in human serum. Using Streptococcus agalactiae – an opportunistic pathogen that can cause serious meningitis in newborns – as a model, the authors demonstrated that the unsaturated fatty acids, linoleic acid and oleic acid, but not the saturated fatty acids, palmitic acid and stearic acid, were able to overcome the inhibitory effect of cerulenin treatment.
The authors also demonstrated that when S agalactiae is grown in the presence of serum, there is an overall decrease in FASII gene expression. In further experiments, deletion mutants were used to demonstrate that FASII enzymes are dispensable in vivo during S agalactiae infection. Growth of all deletion mutants was severely restricted in standard Todd Hewitt (TH) medium, but all grew comparably with wild type strains in serum or in the presence of added oleic acid or linoleic acid. The mutant strains were also as virulent as wild type strains in animal models, even if the animals were treated with the hypolipidemic agent, fenofibrate. The authors believe that drugs targeting FASII would be ineffective in natural infections unless the bacteria had a requirement for specific fatty acids not present in serum.
Bacterial resistance to available antibiotics is becoming an increasing problem; methicillin-resistant Staphylococcus aureus (MRSA), which is broadly resistant to penicillins and cephalosporins, is a particular problem in hospital settings.
A recent report in the journal Science describes new compounds effective against MRSA. The compounds target FtsZ, a bacterial homologue of mammalian β-tubulin, which is essential for bacterial cell division. One of the compounds, PC190723, has been shown to have potent in vitro bactericidal activity against staphylococci, including MRSA, and also to cure mice infected with a lethal dose of MRSA.
The binding site for PC190723 has been mapped to a region of FtsZ that is analogous to the paclitaxel -binding site of tubulin. The activity of PC190723 further validates FtsZ as an antibacterial target, and provides the basis for optimisation to provide new treatments for MRSA.