bacteria – Drug Discovery Opinion

Stomach Bugs Need Vitamin B6

H Pylori electron micrograph — Electron micrograph of *H. pylori* showing multiple flagella. Image: Wikimedia Commons - Yutaka Tsutsumi, M.D. Professor Department of Pathology Fujita Health University School of Medicine

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.

Why Bacteria Turn Bad

bad bacteria — Image: Flickr - beneneuman

The human body is host to a plethora of microorganisms and, for the most part, their presence has no ill effects. Some, particularly intestinal bacteria, even provide benefit. From a microbial perspective, harming the host does not have any obvious survival benefit (unless it enables infection to spread, such as the sneezing induced by the cold virus). So why is it that inoffensive organisms occasionally turn nasty, evolving properties that are damaging or even deadly to us? A study funded by the US Public Health Service and the Wellcome Trust provides one answer to the question.

Since many pathogens interact with their host at mucosal surfaces and have to compete with other microflora, scientists at the University of Pennsylvania School of Medicine and Oxford University used a mouse model of nasal infection to investigate whether competition between microbes promoted virulence. They found that Haemophilus influenzae was able to out-compete Streptococcus pneumoniae by recruiting the host’s immune system. S. pneumoniae is normally harmless and ignored by the immune system, but the immune response stimulated by H. influenzae has unintended consequences. S. pneumoniae strains with polysaccharide capsules that confer resistance to the immune attack are able to survive at the expense of non-resistant strains, resulting in a S. pneumoniae population dominated by the resistant phenotype. Unfortunately, the resistant strains are also more dangerous – if they are able to enter the bloodstream they can multiply unchecked and go on to cause pneumonia, septicaemia and meningitis. So in this battle between S. pneumoniae and H. influenzae, with weapons provided by the host, S. pneumoniae prevails at the expense of the host.

The study is published in Current Biology.

Sibling Rivalry in the Bacterial World

Competing *Paenibacillus dendritiformis* colonies

Credit: Eshel Ben-Jacob

Bacterial colonies cultured on agar avoid each other when forced to compete for nutrients, but the mechanism behind the observed growth inhibition has been unclear. Now a new study by collaborating scientists at UC San Diego, University of Texas and Tel Aviv University has explored the behaviour of Paenibacillus dendritiformis cultures, identifying the factors responsible.

The team found that it was not a shortage of food that halted the growth. They found nutrients in the no-man’s land between the colonies, but also a protein that wasn’t present elsewhere on the dish. When a sample of the purified protein was introduced to a fresh dish inoculated with P. dendritiformis, the bacteria formed a lopsided colony that shied away from the spot. In addition, the bacteria at the edge of the colony closest to the suspect protein were dead.

Analysis of the secretions from P. dendritiformis identified the protease, subtilisin, and a 12 kDa protein, termed sibling lethal factor (Slf). Whilst subtilisin promotes growth and expansion of P. dendritiformis colonies, Slf lyses the bacterial cells in culture. Slf is encoded by a gene belonging to a large family of bacterial genes of unknown function, and the gene is predicted to encode a protein of approximately 20 kDa. The team generated recombinant 20 kDa protein, which was found to be inactive. Exposure to subtilisin, however, resulted in cleavage to the active, 12 kDa form. The experimental results, combined with mathematical modelling, show that subtilisin regulates growth of the colony. Below a threshold concentration subtilisin promotes colony growth and expansion, but once it exceeds a threshold, as occurs at the interface between competing colonies, Slf is then secreted into the medium to rapidly reduce cell density by lysis of the bacterial cells. The presence of genes encoding homologs in other bacterial species suggests that this mechanism for self-regulation of colony growth might not be limited to P. dendritiformis.

The study is published in PNAS.

Eating for How Many?

Soon after birth, the human body is colonised by bacteria. Trillions of bacteria take up residence in the gut and perform a range of useful functions such as helping with digestion and absorption of nutrients, producing vitamins, preventing growth of pathogenic bacteria, and developing the immune system. In 2006, it was shown that the proportion of Bacteroidetes relative to Firmicutes was reduced in the guts of obese people compared with lean individuals and also in the guts of genetically obese mice compared with lean littermates. Researchers at Emory University have now shown that mice engineered to lack toll-like receptor 5 (TLR5) – a component of the innate immune system that is expressed in the gut mucosa and that helps defend against infection – are 20% heavier than normal mice and have elevated triglycerides, cholesterol and blood pressure as well as slightly elevated blood sugar and a decreased response to insulin. TLR5-deficient mice consume about 10% more food than wild type mice and, although they lose weight when food is restricted, they still show insulin resistance. On a high fat diet, TLR5-deficient mice gain more weight than normal mice and develop full-blown diabetes and fatty liver disease, mimicking “metabolic syndrome” which increases the risk of developing heart disease and diabetes in humans.

Treating TLR5-deficient mice with antibiotics to kill most of the bacteria in the intestine reduced their metabolic abnormalities and, conversely, transfer of intestinal bacteria from TLR5-deficient mice to germ-free wild type mice transferred many of the characteristics of metabolic syndrome, including increased appetite, obesity, elevated blood sugar, and insulin resistance. Although earlier studies had shown that greater numbers of Firmicutes bacteria lead to more calories being extracted from the diet, the TLR5-deficient mice had normal proportions of Firmicutes and Bacteroidetes but differed in the composition of bacterial species in the two families. The new study shows that, as well as influencing how well energy is absorbed from food, gut flora can also influence appetite and may contribute to human obesity and metabolic disease.

The study is published in Science Express.

Into The Looking Glass World of Bacteria

Although some scientists have suggested that carbon-based life forms could exist with either chirality, life has seemingly emerged ‘left-handed’ and all living organisms use predominantly L-amino acids as building blocks. The incorporation of D-Ala and D-Glu into bacterial cell wall peptidoglycans is one exception, with specific amino acid racemases to convert the L-forms to the D-isomers. Incorporation of D-Ala and D-Glu residues into peptidoglycan cross-linkages is believed to confer resistance to degradation by enzymes that selectively hydrolyse linkages between L-amino acids.

Newer research is suggesting broader roles for D-amino acids and a team led by researchers at Harvard Medical School has now shown that bacteria use a more diverse set of D-amino acids than previously thought. They found that a mutant form of Vibrio cholera, the bacterium which causes cholera, produces D-Met and D-Leu, along with smaller amounts of D-Val and D-Ile. These amino acids, when present in the culture medium, were found to stimulate a shape change from rods to spheres, consistent with cell wall remodelling and reduced peptidoglycan synthesis during the stationary phase. A broad-spectrum racemase capable of generating the four D-amino acids was identified in V. cholera and a variety of other bacterial species were found to encode putative racemases and to produce D-amino acids. The specific amino acids identified in stationary phase supernatants varied among bacterial species, with Bacillus subtilis producing both D-Phe and D-Tyr. The team also found that D-Met was incorporated into peptidoglycans of Escherichia Coli and that physiological concentrations of D-amino acids down-regulated peptidoglycan synthesis in B. subtilis, a Gram-positive bacterium that is highly divergent from V. cholera. The study, which is published in the journal Science, shows that D-amino acids released during the stationary phase allow populations of bacteria to synchronise cell wall assembly and composition and may enable coordination of metabolic slowing as nutrients are depleted and toxic products accumulate. Release of D-amino acids can also influence cell wall composition of nearby bacteria and may allow interspecies regulation between bacteria and other organisms that coexist or compete for the same resources.

Intestinal Flora may Trigger Colon Cancer

Just as Helicobacter pylori, which causes stomach ulcers and gastric cancers, is being eradicated from many developed countries, Johns Hopkins scientists have shown that bacteria that cause diarrhoea may also lead to some colon cancers. Enterotoxigenic strains of Bacteroides fragilis (ETBF) asymptomatically colonise a proportion of the human population but can also cause inflammatory diarrhoea in both children and adults. An earlier study in Turkey had linked ETBF infection to colon cancer and, to further understand this association, the Johns Hopkins team have carried out a study in multiple intestinal neoplasia (Min) mice. These animals carry mutations in the APC (adenomatosis polyposis coli) gene and spontaneously develop multiple small intestinal adenomas as well as more sporadic colonic adenomas. Mutations in the tumour-suppressing APC gene are also associated with human colon cancer. The present study showed that, although both ETBF and nontoxigenic B. fragilis (NTBF) chronically colonise mice, only ETBF causes diarrhoea and inflammation and induces colonic tumours. The diarrhoea resolved quickly but the mice developed colitis within 7 days and, after 4 weeks, had numerous colonic tumours. ETBF was found to strongly activate Stat3 in the colon, leading to a dramatic (100-fold higher than normal) and selective T_H17 response. Blocking IL-17 as well as the receptor for IL-23, a key cytokine amplifying T_H17 responses, inhibited the colitis, colonic hyperplasia and tumour formation triggered by ETBF. The study, which is published in the August 23^rd issue of Nature Medicine, provides new mechanistic insights into the development of human colon cancers and may lead to the development of vaccines or improved therapies.

GERD Linked to Changes in Oesophageal Microbiome

The incidence of gastroesophageal reflux disease (GERD) has increased significantly in the United States since the 1970s. The chronic inflammation associated with GERD can lead to the development of Barrett’s oesophagus, a precancerous condition that, in rare cases, leads to oesophageal adenocarcinoma. Despite extensive epidemiological investigation, the cause of GERD and the reasons underlying the increase in prevalence remain unclear. Researchers at the University of New York Langone Medical Center have now shown, however, that the condition is linked to a global alteration of the microbiome in the oesophagus. The team collected and sequenced bacteria from the oesophagus of patients with oesophagitis or Barrett’s oesophagus and compared these with samples from healthy individuals. Although it wasn’t possible to obtain a detailed picture of species present in low abundance, they found that streptococci predominated in healthy patients whereas samples from patients with oesophagitis or Barrett’s oesophagus were more diverse and contained more Gram-negative bacteria.

The study examined samples from only 34 individuals and it is not yet known whether the changes in bacterial populations seen in GERD patients are cause or effect, but if the changes in the microbiome can be shown by further studies to play a causal role in pathogenesis, it may be possible to design new treatments to treat this increasingly common disease.

The findings are reported in the August 1^st issue of Gastroenterology.

Discovery Could Lead to Better Treatment for Bacterial Meningitis

meninges Meningitis – infection of the cerebrospinal fluid and protective membranes surrounding the brain and spinal cord – can be caused by infection with either viruses or bacteria. Viral meningitis is typically relatively mild and self-limiting whereas bacterial meningitis is much more serious and can result in severe brain damage or even death. Bacterial meningitis in children is almost exclusively caused by infection with one of three bacterial strains: Streptococcus pneumoniae, Neisseria meningitidis, or Haemophilus influenza. Exactly how these bacteria are able to breach the blood-brain barrier and cause infection was not understood, but researchers from the University of Nottingham and St. Jude Children’s Research Hospital have now discovered that all three pathogens use the same receptor on human cerebrovascular endothelial cells to begin the process of crossing the barrier. Bacteria need to have some way of fixing their position and becoming established and use attachment molecules – known as adhesins – to achieve this. streptococcus pneumoniae Some bacteria take the attachment process a stage further and use the adhesins as a first step towards gaining entry into host cells. It was known that the three bacteria responsible for most cases of meningitis share the same strategy for the second step of crossing endothelial cells and the new study has shown that they also use the same host cell receptor, the laminin receptor, for initial attachment. Other infectious agents known to use the laminin receptor to gain access to the CNS include prions and some neurotropic viruses, although the interaction of the laminin receptor with bacteria appears to be somewhat different to that of other pathogens.

The study suggests that blocking the interaction between bacterial adhesins and the laminin receptor might offer broad protection against bacterial meningitis and may lead to better treatments and prevention strategies. The study is published in full in the Journal of Clinical Investigation.

MTAN Inhibitors Disrupt Bacterial Quorum Sensing

E coli O157 Bacterial resistance – often arising as a result of over, or inappropriate, use of antibiotics – is a major obstacle to the treatment of many bacterial infections. Recently, interference with quorum sensing has emerged as a strategy for the development of new antibiotics which minimises the evolution of drug-resistant strains. Quorum sensing is a process used by bacteria to coordinate gene expression according to local population densities. The bacteria secrete signalling molecules, known as autoinducers, and have receptors that specifically recognize the signalling molecules released by other bacteria of the same or different species. Bacterial cell density and concentration of autoinducers control factors such as expression of virulence factors, pathogenicity and biofilm formation.

BuT-DADMe-Immucillin-A Writing in the journal Nature Chemical Biology, researchers from Albert Einstein College of Medicine of Yeshiva University have recently described the effectiveness of 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) inhibitors against Vibrio cholera and Escherichia coli O157:H7. MTAN plays a key role in the synthesis of autoinducers essential for bacterial quorum sensing and the absence of the nucleosidase in mammals suggests that it is likely to be an attractive target for antimicrobial drug design. Three transition state analogue inhibitors of MTAN were found to be highly potent at blocking quorum sensing, bacterial virulence and biofilm formation. Importantly, the effect persisted for several generations.

Key Role for Bicarbonate Transporter in Anthrax Virulence

Anthrax is caused by the Gram-positive bacterium, Bacillus anthracis. The disease mainly affects herbivorous mammals which ingest or inhale the spores while grazing, but can also be passed to humans by contact with infected animal products. Once within the host, the bacteria begin to multiply and infection typically proves lethal within a few days or weeks. Virulence requires expression of both the anthrax toxin and capsule genes, and one of the first factors found to be important in controlling virulence was elevated levels of CO₂/bicarbonate which are thought to signal the presence of a mammalian host environment. bacillus anthracis It has been difficult to unravel the precise mechanism of virulence control because of the equilibrium between CO₂, H₂CO₃, HCO₃^–, and CO₃^2-, but a study by scientists at the Scripps Research Institute published in the journal PLos Pathogens has demonstrated that expression of a specific bicarbonate transporter is critical for virulence. Deletion of the genes for the transporter strongly decreased the rate of bicarbonate uptake ex vivo and abolished induction of toxin gene expression. Importantly, the strain lacking the transporter was avirulent in a mouse model of anthrax infection, demonstrating the importance of this pathway for recognition of the host environment and pathogenesis.

The identification of an essential bicarbonate transporter may be of relevance to other pathogens, such as Staphylococcus aureus, that also regulate expression of virulence factors in response to CO₂/bicarbonate levels, and suggests a novel target for antibacterial intervention. Similar transporters have been identified and characterized in photosynthetic bacteria, and the availability of 3-dimensional structures of the bicarbonate binding domain of the Synechococcus transporter may help with the design of new inhibitors.